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Veterans and Agent Orange: Update 1998 (1999)

Chapter: 3 Toxicology

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Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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3
Toxicology

As in Veterans and Agent Orange (VAO) and Veterans and Agent Orange: Update 1996 (Update 1996), this review summarizes the experimental data that serves as a scientific basis for assessment of the biologic plausibility of health outcomes reported in epidemiologic studies. Efforts to establish the biologic plausibility of effects due to herbicide exposure in the laboratory strengthen the evidence for the herbicide effects suspected to occur in humans. Differences in chemical levels, frequency of administration, single or combined exposures, preexisting health status, genetic factors, and routes of exposure significantly influence toxicity outcomes. Thus, any attempt to extrapolate from experimental studies to human exposure must carefully consider such variables before conclusions are made.

Multiple chemicals were used for various purposes in Vietnam. Four herbicides documented in military records were of particular concern and are addressed here: 2,4-dichlorophenoxyacetic acid (2,4-D); 2,4,5-trichlorophenoxyacetic acid (2,4,5-T); picloram; and cacodylic acid. In addition, the toxicologic properties of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin), a contaminant of 2,4,5-T, are discussed. This chapter focuses to a large extent on the toxicological effects of TCDD, because considerably more information is available on TCDD than on the herbicides.

SUMMARY

Toxicokinetics

New information on the distribution of 2,4-D and the metabolism of cacodylic acid has improved understanding of how the body handles these sub-

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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stances. 2,4-D enters the brain, but only to a limited extent, and its uptake by the brain appears to be an energy-dependent process. Cacodylic acid is one of the major metabolic products of ingested arsenic in mammals. Studies using skin taken from mice report that the absorption of cacodylic acid is influenced by the substance in which it is dissolved and the length of time that cacodylic acid remains in contact with the skin.

TCDD, unlike the herbicides, stays in the body for a long time. In humans about half is eliminated every 8.5 years. It is removed from the body as it is metabolized to less toxic forms that are more easily eliminated in the urine than TCDD itself. The length of time that TCDD remains in the body increases with increasing body fat.

New evidence based on animal models suggests that rats and humans tend to handle TCDD in body tissues in similar ways. However, rats tend to excrete TCDD more quickly. Rats are most likely to absorb TCDD through food and air and this fact may carry over to humans. However, the types of TCDD and other dioxins that accumulate in the body may differ markedly between humans and rodents.

Mechanisms of Toxic Action

Little is known about the way in which the herbicides produce toxic effects in animals. Recent studies have focused on the mechanisms of cellular toxicity of 2,4,5-T. For example, some studies using animal tissues suggest that 2,4,5-T may alter nerve and muscle function by interacting with chemicals that participate in nervous system function. 2,4,5-T may induce mutations at different stages of cell development. Finally, it may alter the cellular process involved in the elimination of harmful carcinogens.

To date, the consensus is that TCDD is not directly toxic to the body's genetic material. However, it may affect enzymes and hormone levels, which in turn may produce adverse effects.

Recent studies confirm earlier findings that most of the toxic effects of TCDD are caused by its binding to a protein called the aryl hydrocarbon receptor (AhR). The binding of TCDD to this protein triggers various events that result in toxic sequelae. However, some tests suggest that other events, in addition to the binding of TCDD to the AhR, are involved. Studies of the AhR and its partner protein Arnt (aryl hydrocarbon nuclear translocator protein) indicate that similar proteins exist in different species and interact with a number of other proteins to produce an effect. Researchers have recently bred mice that lack the AhR protein. It is anticipated that these mice will allow more informative studies of the way TCDD reacts with the AhR to produce a toxic effect.

Disease Outcomes

Disease outcomes associated with herbicide exposures continue to be debated. Some cellular-level effects have been identified, although it is not clear

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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what impact these may have on living organisms. Other studies suggest disease effects including neurotoxicity and kidney, liver, and muscle damage at certain high dose levels in particular animal species; however, translating these results to the exposures experienced by veterans and others remains problematic.

2,4-D appears to affect the membrane sheath around nerve cells. Other studies support the view that 2,4-D may disrupt cellular processes in the liver, and reports of kidney and muscle damage have been published. Results from studies indicate that high doses of 2,4-D are necessary to produce these effects. A case-control study of dogs exposed to 2,4-D in addition to other pesticides used in yard work, reported an increase in lymphomas associated with exposure.

The limited evidence published during the past two years suggest that cacodylic acid may promote cancer in rats.

Several recent studies have examined the role of TCDD in producing certain disease outcomes in animals, including acute toxicity, dermal toxicity, liver toxicity, neurotoxicity, immunotoxicity, reproductive and developmental toxicity, and cancer.

A prominent symptom of the acute toxicity of TCDD is the loss of fat tissue and body weight, a phenomenon known as wasting syndrome. Several mechanisms are under investigation including inhibition by TCDD of sugar transport activity, effects on fat cell differentiation, and effects on certain receptors and enzymes. There is some evidence to suggest that gender differences exist in the response of fat cells to TCDD.

TCDD has also been shown to affect the development of skin cells by binding to the AhR. This effect is antagonized by retinoids.

Liver enlargement has been shown to occur following high doses of TCDD. The mechanism by which TCDD affects the liver is still under investigation. Recently, it has been shown to inhibit DNA synthesis of liver cells, decrease certain receptors in liver cell membranes, and inhibit liver enzymes.

Animal and test-tube studies continue to emphasize the importance of alterations in neurological systems as underlying mechanisms of TCDD-induced behavioral dysfunction. TCDD can affect the metabolism of serotonin, a substance in the brain that can modulate food intake. This biochemical change is consistent with observations of progressive weight loss and anorexia in experimental animals exposed to TCDD. In certain brain cells, there is evidence that TCDD may increase the uptake of calcium.

It is known that TCDD exposure causes a broad range of immunologic effects in experimental animals. Recent studies support earlier data that TCDD decreases immunity and host resistance to pathogenic microorganisms. Despite considerable laboratory research, the mechanisms underlying the immunotoxic effects of TCDD are still unclear. TCDD immunotoxicity appears to be mediated primarily through the AhR, but some components of immunosuppression have been shown to act independently of this receptor.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
×

Low doses of TCDD administered to experimental animals alter reproductive development and fertility of the offspring. When TCDD is administered to pregnant rats, malformations of the external genitalia are observed in female offspring. Functional reproductive alterations in female offspring are also observed after TCDD exposure, including decreased fertility rates and reduced fecundity.

Studies in male rats and hamsters have shown that decreased daily sperm production is one of the most sensitive effects of exposure to TCDD in the womb and through breast milk. Results also suggest that TCDD exposures selectively impair rat prostate growth and development.

TCDD has been shown to affect blood serum hormone levels. This outcome is thought to be due partially to the action of TCDD on the pituitary gland.

Several reports published during the reference period focused on the mechanism by which TCDD induces cleft palates in experimental animals. Evidence suggests that this effect involves the AhR. There have also been reports of developmental defects in the cardiovascular system of TCDD-treated animals. Evidence suggests that cells lining the blood vessels are a primary target of TCDD-induced developmental cardiovascular toxicity.

Studies continue to focus on the mechanism by which TCDD induces cancer in animals. Although there is considerable evidence that TCDD-induced cancer is mediated by the AhR, it does not appear to be solely responsible. There is also evidence that the mechanism by which TCDD induces tumor promotion may involve reactive molecules containing oxygen, which are known as oxygen radicals. It is hypothesized that a release of oxygen radicals by TCDD causes DNA damage that could lead to mutation and cancer. There is also evidence that TCDD tumor promotion may be due to its ability to interfere with intercellular communications.

Inconsistencies reported in the molecular basis of dioxin' s actions reflect the degree of tissue, cell, and gene specificity that characterizes the toxic response.

Relevance To Human Health

Exposure to 2,3,7,8-TCDD, a contaminant in some of the herbicides used in Vietnam, has been associated with both cancer and noncancer end points in animals. Studies in animals indicate that TCDD effects are mediated through the AhR. Although structural differences in the AhR have been identified, it operates in a similar manner in animals and humans, and a connection between TCDD exposure and human health effects is, in general, considered biologically plausible. Evidence has also begun to accumulate for non-AhR mediated effects. Animal research indicates that TCDD can both cause cancers or tumors and enhance the incidence of certain cancers or tumors in the presence of known carcinogens. However, experimental animals greatly differ in their susceptibility to TCDD-induced effects, and the sites at which tumors are induced also varies

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
×

from species to species. Other noncancer health effects vary according to dose and to the animal exposed. Controversy exists over whether the effects of TCDD and other exposures are threshold dependent, that is, whether some exposure levels may be too low to induce any effect.

Limited information is available on the biologic plausibility of herbicide health effects not connected with TCDD. Although concerns have been raised about non-dioxin contaminants of herbicides, far too little is known about the ubiquitousness and concentration of these compounds in the formulations used in Vietnam to draw conclusions about their impact.

Considerable uncertainty remains about how to apply this information to evaluation of the potential health effects in Vietnam veterans of herbicide or dioxin exposure. Scientists disagree over the extent to which information derived from animal and cellular studies predicts human health outcomes and the extent to which health effects resulting from high-dose exposure are comparable to those resulting from low-dose exposure. Research on biological mechanisms is burgeoning, and subsequent VAO updates may have more and better information on which to base conclusions.

VAO AND UPDATE 1996—OVERVIEW

Chapter 4 of VAO and Chapter 3 of Update 1996 review the results of animal and test-tube studies published until 1995 that investigated the toxicokinetics, mechanism of action, and disease outcomes of TCDD and herbicides. According to these earlier reviews, TCDD elicits a diverse spectrum of biological sex-, strain-, age-, and species-specific effects, including carcinogenicity, immunotoxicity, reproductive and developmental toxicity, hepatotoxicity, neurotoxicity, chloracne, and loss of body weight. The scientific consensus is that TCDD is not genotoxic and that its ability to influence the carcinogenic process is mediated via epigenetic events such as enzyme induction, cell proliferation, apoptosis, and intracellular communication. The toxicity of the herbicides used in Vietnam has been poorly studied. In general, the herbicides 2,4-D, 2,4,5-T, cacodylic acid, and picloram have not been identified as particularly toxic substances since high concentrations are often required to modulate cellular and biochemical processes. A comprehensive description of the toxicological literature published until 1995 can be found in VAO and Update 1996.

UPDATE OF THE SCIENTIFIC LITERATURE—OVERVIEW

Toxicokinetics

A limited number of studies have been published since Update 1996 that examine the biologic and toxic effects of 2,4-D. Toxicokinetic studies using rabbits suggest that uptake of 2,4-D by the brain is restricted by the developing,

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
×

as well as the mature, blood-brain barrier. In hamsters, cellular 2,4-D uptake appears to be an energy-dependent process.

During the reference period since the publication of Update 1996, the disposition of TCDD in humans has been investigated in two studies. Based on multiple serum measurements collected over a 10-year period from 213 veterans of Operation Ranch Hand, the mean decay rate of TCDD was estimated to be 0.0812 per year, with a corresponding half-life estimate of 8.532 years. In these veterans, half-life increased significantly with increasing body fat, but not with age or relative changes in the percentage of body fat. In another human study, the impact of breastfeeding on the body burden of dioxin-like chemicals in Arctic Inuit people was investigated. Toxicokinetic modeling revealed that breast feeding strongly influences the body burden of TCDD during childhood but not after 20 years of age. In addition, liver and adipose tissue concentrations in adults greater than 20 years of age appeared to be lower than those associated with cancer and adverse reproductive effects in laboratory animals.

Using a physiologically based model that describes the distribution kinetics of dioxin-like chemicals in various mammalian species, the kinetic profile of TCDD was found to be similar in rats and humans, although the half-lives differ considerably between species. The half-life of TCDD in rats and humans is measured in weeks and years, respectively. Comparative studies of the systemic absorption of TCDD in rats following oral and inhalation exposures indicate that both exposures are significant routes of absorption—an observation that is of relevance to humans given the similarities in kinetic profiles between rats and humans. In addition, for a given body burden, the adipose tissue concentrations have been found to vary in an inversely proportional manner to the mass of adipose tissues. Despite similarities in the toxicokinetic profile of rats and humans, some data suggest that humans may bioaccumulate higher levels of certain dioxins than mice due to interspecies metabolic differences.

Results from another model of the disposition of TCDD in the rat indicate that TCDD increases the enzymatic activity of UDP-glucuronosyltransferase (UGT) and the levels of blood thyroid-stimulating hormone (TSH). Calculated increases in blood TSH levels are consistent with prolonged stimulation of the thyroid and may represent an early stage in the induction of thyroid tumors identified in previous two-year bioassays. This suggests that increases in UGT activity may be a useful biomarker for tumorigenic changes in hormone levels after TCDD exposure. However, certain noncancer end points may be more significant in assessing human health risks to TCDD than cancer end points. For instance, immune suppression and enzymatic induction have been found to occur at lower doses and under conditions more relevant to general population exposure conditions. In assessing the risk of humans to dioxins, it should also be noted that recent data suggest that toxic equivalency factors (TEFs) derived from short-term assays may not adequately predict the relative potencies of this class of compounds following chronic exposure.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
×

Mechanisms of Toxic Action

The mechanisms of cellular toxicity of 2,4,5-T have been the focus of a number of recent studies. One study presents compelling evidence that 2,4,5-T interacts with choline to generate false cholinergic messengers that alter neuronal and muscular function. Another study found that 2,4,5-T can induce mutations at different germ cell stages. Finally, there is some evidence that 2,4,5-T modulates cellular metabolism to alter the expression of membrane pumps and drug-metabolizing enzymes involved in the disposition of chemical carcinogens.

Dimethylarsinic acid (cacodylic acid, DMA) is one of the major methylated metabolites of ingested arsenicals in mammals. During the reference period, toxicokinetic studies reported that the rates of in vitro dermal absorption of DMA can be influenced by both the vehicle of administration and the duration of exposure.

Scientific reports published during the past two years continue to focus on the mechanism by which TCDD exerts its effects. Structural and functional studies of the AhR and Arnt indicate that both proteins are highly conserved, are found in diverse vertebrate groups, and interact with a large number of proteins to influence nuclear events. In vitro studies have confirmed in vivo findings regarding the functional binding domains of mouse AhR that interact with the heat shock protein (hsp90). Other results continue to support the view that TCDD influences patterns of gene expression by modulating transcriptional and post-transcriptional events. Such responses are often mediated by the AhR but exhibit considerable tissue and cell specificity. From a toxicologic perspective, the development of AhR knockout mice has been an important advance because it has helped establish a definitive association between the AhR and TCDD-mediated toxicities. Some studies suggest that specific patterns of Arnt expression differ in certain tissues from those of the AhR and that Arnt may have roles in normal embryonic development independent of the AhR. The recent discovery that the oxygen-regulated transcription factor HIF-1α and the AhR share a common heterodimerization partner Arnt (HIF-1β) has fueled intensive investigation into the possible crosstalk between oxygen and dioxin signal transduction pathways.

Disease Outcomes

While disease outcomes associated with 2,4-D exposures continue to be debated, neurotoxic effects have been reported in rats administered high acute doses, possibly as a result of neuronal demyelination. Studies on rats continue to support the view that the hepatotoxic effects of 2,4-D may involve disruption of thiol homeostasis. Reports of kidney and muscle damage have also been published. A case-control study of dogs exposed to 2,4-D in addition to other pesticides used in yard work, reported an increase in lymphomas associated with exposure. Although 2,4-D induced significant numbers of mutations in a Droso-

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
×

phila cell line and increased mRNA levels of multidrug resistance (mdr) genes in mouse liver, cancer bioassays show no carcinogenic effect. Results from chronic and subchronic toxicity studies indicate that 2,4-D is relatively nontoxic.

Limited research has been conducted on the offspring of male animals exposed to herbicides. A study of male mice fed varying concentrations of simulated Agent Orange mixtures concluded there were no adverse effects in offspring. A statistically significant excess of fused sternebra in the offspring of the two most highly exposed groups was attributed to an anomalously low rate of the defect in controls. Another study reported an increase in the incidence of mal-formed offspring of male mice exposed to subacute levels of a mixture of 2,4-D and picloram in drinking water. However, the paternal toxicity observed in the high dosage levels used and inconsistent dose-response pattern are of concern.

Limited evidence presented during the past two years suggests that DMA acts as a promoter of urinary bladder, kidney, liver, and thyroid gland carcinogenesis in rats. DMA induces apoptosis and sensitizes DNA to oxidative injury.

TCDD has been shown to adversely affect a number of organ systems that have been or may be linked to a variety of disease outcomes. TCDD lethality has been associated with changes in brain serotonin metabolism. However, the wide interspecies differences in TCDD-induced lethality cannot be explained by changes in tryptophan metabolism or carbohydrate homeostasis.

A prominent symptom of the acute toxicity of TCDD is the loss of adipose tissue and body weight, a phenomenon known as wasting syndrome. Several mechanisms are under investigation including inhibition by TCDD of glucose transport activity and hepatic phosphoenolpyruvate carboxykinase (PEPCK, the rate-limiting enzyme of hepatic gluconeogenesis); the effects of TCDD on adipocyte differentiation; and the effects of TCDD on epidermal growth factor receptor and protein-tyrosine kinase. There is some evidence to suggest that gender differences exist in the response of cells to TCDD. Glucose uptake and lipoprotein lipase activity were significantly decreased in adipose tissue in vitro after intraperitoneal (ip) injection of TCDD in male guinea pigs. No significant effect was observed in females. In addition, radiolabeled-TCDD binding affinity studies in adipose explant tissues showed that tissues from male guinea pigs and monkeys had a higher binding capacity for TCDD than female tissues.

TCDD has been shown to induce differentiation in human keratinocytes, which may be initiated by TCDD binding to the AhR. This effect is antagonized by retinoids and may involve interactions between TCDD and retinoids in the regulation of epithelial differentiation.

The mechanism by which TCDD induces hepatotoxicity is still under investigation. TCDD has been shown to inhibit hepatocyte DNA synthesis; decrease hepatic plasma membrane epidermal growth factor receptor; inhibit hepatic pyruvate carboxylase activity as a consequence of a reduction in pyruvate carboxylase mRNA levels (this effect was ten-fold greater than in congenic Ahb/b mice, suggesting that a competent AhR is required); and induce cytochrome P4501A1

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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(CYP1A1) in fish and chick embryo hepatocyte cultures, resulting in porphyrin accumulation. Studies have been conducted to examine the short-and long-term effects of TCDD on rat ethoxyresorufin o-deethylase (EROD) activity and liver enzymes. Four days after oral dosing, EROD activity was considerably elevated. Hepatic PEPCK and glutamyl transpeptidase activities were inhibited and stimulated, respectively. Ninety days after dosing, liver EROD activity and PEPCK activity revealed considerable reversibility, whereas glutamyl transpeptidase activity remained elevated. Hepatomegaly has been shown to occur following high subchronic doses.

Using Mardin Darvey canine kidney cells, TCDD has been shown to stimulate transcription of the PGHS-2 gene. It has been suggested that PGHS-2 expression may be involved in toxic reactions that involve inappropriate cellular growth, such as tumor promotion.

Animal studies and in vitro mechanistic studies continue to emphasize the importance of alterations in neurotransmitter systems as underlying mechanisms of TCDD-induced behavioral dysfunction. Lethal doses of TCDD administered to rats affect the metabolism of serotonin, a neurotransmitter in the brain able to modulate food intake. This biochemical change is consistent with observations of progressive weight loss and anorexia in experimental animals exposed to TCDD. In primary cultures of rat hippocampal neuronal cells, there is evidence that TCDD may increase the uptake of intracellular calcium. This concentration-dependent increase in calcium is associated with a decrease in mitochondrial membrane potentiation and activation of β-protein kinase C (β-PKC).

TCDD and structurally related halogenated aromatic hydrocarbons cause a broad range of immunologic effects in experimental animals. Recent studies support earlier data that TCDD decreases innate immunity and host resistance to pathogenic microorganisms; impairs cell-mediated immune responses, such as the generation and lytic activity of cytotoxic T cells; and suppresses humoral immunity by inhibiting B-lymphocyte differentiation into antibody-producing cells. Despite considerable laboratory research, the mechanisms underlying the immunotoxic effects of TCDD are still unclear. TCDD immunotoxicity appears to be mediated primarily through AhR-dependent processes, but some components of immunosuppression have been shown to act independently of the AhR.

Low doses of TCDD administered to experimental animals alter reproductive development and fertility of the progeny. Studies in male rats and hamsters have shown that decreased daily sperm production and cauda epididymal sperm number are some of the most sensitive effects of in utero and lactational TCDD exposure. However, in utero and lactational TCDD exposure does not appear to alter radiolabeled sperm transit time through the whole epididymis. Studies have been conducted to determine whether in utero and lactational TCDD exposure decreases male rat accessory sex organ weights during postnatal development and whether this effect involved decreases in testicular androgen production or changes in peripheral androgen metabolism. Results suggest that in utero and

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
×

lactational TCDD exposure selectively impairs rat prostate growth and development without inhibiting testicular androgen production or consistently decreasing prostate dihydrotestosterone (DHT) concentration. Male mice treated with a mixture of 2,4-D, 2,4,5-T, and TCDD exhibited dose-related liver and thymus toxicity and reduced weight gain, although no significant effects were observed on sperm function, reproductive outcomes, survival of offspring, or neonatal development.

In female rats, a single dose of TCDD administered on gestational day (GD) 15 results in malformations of the external genitalia in Long Evans (LE) and Holtzman rats. There was complete to partial clefting of the phallus. Treatment on GD 8 was more effective in inducing functional reproductive alterations in female progeny (e.g., decreased fertility rate, reduced fecundity, cystic endometrial hyperplasia, increased incidence of constant estrus).

TCDD administered by gastric intubation altered serum hormone levels in immature female rats. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), and gonadotropin levels were increased. This effect is due partially to the action of TCDD on the pituitary and is calcium dependent.

After water-borne exposure of newly fertilized eggs to TCDD, the toxicity and histopathology of TCDD in zebrafish revealed that TCDD did not increase egg mortality or affect time to hatching. However, pericardial edema and craniofacial malformations were observed in zebrafish larvae. Reports indicate that in ovo TCDD exposure of the domestic chicken, domestic pigeon, and great blue heron adversely affected the body and skeletal growth and hatchability of the domestic pigeon but had no effect on the domestic chicken or great blue heron.

Studies involving human luteinizing granulose cells have shown that glucose transporting activity can be used as a sensitive biomarker to detect the very early response to TCDD in these steroid-producing cells and that the effect of TCDD on progesterone is mediated through cyclic adenosine 5'-monophosphate (cAMP)-dependent protein kinase.

TCDD-induced cleft palate and hydronephrosis involve mechanisms that are AhR mediated. There are data to suggest that TCDD interacts with other signaling pathways in inducing cleft palate. For example, cross-regulation of the receptors is believed to be important in the synergistic interaction between TCDD and hydrocortisone. When female mice are treated with TCDD and retinoic acid simultaneously, palatal clefts can be observed in 100 percent of offspring at dose levels far lower than those required for either agent to produce clefting if given alone. This synergy suggests that the pathways controlled by these agents converge at one or more points in cells of the developing palate.

The effects of TCDD on the estrogen-signaling system during fetal and perinatal development of peripubertal female rats has been investigated. The mechanism for the reduction in female fertility that accompanies in utero and lactational exposure to TCDD remains unknown, although it could be linked to estrogenic effects such as clefting of the phallus and hypospadias.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
×

Several reports published during the reference period describe developmental deficits in the cardiovascular system of TCDD-treated animals. Evidence suggests that the endothelial lining of blood vessels is a primary target site of TCDD-induced cardiovascular toxicity. CYP1A1 is induced in mammalian endothelial cells in culture. The vascular endothelium in lake trout is also uniquely sensitive to induction of CYP1A1 by TCDD in developing animals. CYP1A1 induction in the endothelium may be linked to early lesions that result in TCDD-induced vascular derangements leading to the yolk sac, pericardial, and meningial edema associated with lake trout sac fry mortality. CYP1A1 induction has also been observed in adult quail aortic tissue.

The cardiotoxicity induced by TCDD was examined in chick embryo. The spatial and temporal expression of AhR and Arnt suggests that the developing myocardium and cardiac septa are potential targets of TCDD-induced teratogenicity, and such targets are also consistent with cardiac hypertrophy and septal defects observed following TCDD exposure.

DNA damage and consequent cell death in the embryonic vasculature are key physiological mediators of TCDD-induced embryotoxicity in medaka (a small Japanese freshwater fish [Oryzias latipes]). Treatment of TCDD-exposed medaka embryos with an antioxidant provides significant protection against TCDD-induced embryotoxicity and suggests that reactive oxygen species may participate in the teratogenic effects of TCDD.

TCDD has been shown to significantly induce CYP1A1 mRNA levels and EROD activity in several human cancer cells. Experiments involving several strains of mice provide evidence that a functional Ah receptor is required for TCDD induction of CYP1A1 and liver tumor promotion. However, the AhR does not appear to be exclusively responsible. CYP1A1 induction in various mice strains was not directly related to the degree of tumor-promoting capability, which suggests that other undefined genetic factors may play an important role.

Studies comparing liver induction in TCDD-responsive (C57BL/6J) and less responsive (DBA/2J) mice indicate that induction of CYP1B1 and CYP1A1 mRNA content is more pronounced in the former. CYP1A1 was more responsive to TCDD that CYP1B1 in both strains, suggesting that CYP1B1 mRNA expression is less inducible by TCDD but that both genes are AhR regulated. Other studies indicate that the expression of CYP1A1 and CYP1B1 is highly cell specific even though each is regulated through the AhR. However, each P450 exhibits a surprising similar pattern of hormonal regulation even though expressed in different cell types.

Studies conducted to compare AhR in cultured fetal cells and adult liver tumors from TCDD-responsive (C57BL/6J) and less responsive (DBA/2J) mice indicate that the responsiveness of fetal cells is likely mediated by the AhR and is not due to a different allelic form of AhR ligand binding subunit in fetal versus adult cells.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
×

There is evidence that the mechanism by which TCDD induces tumor promotion may involve oxygen radicals since scavengers of hydroxyl radicals or antioxidants hinder the tumor-promoting effects of TCDD in transformed mice fibroblasts. In support of this, other studies have shown that TCDD induction of CYP1A1 in hepatoma 1c1c7 cells appears to lead to a release of oxygen radicals and subsequent oxidative DNA damage that could result in mutation and cancer. This is also evidence that tumor promotion by TCDD may be due to its ability to interfere with gap junctional intercellular communications.

In human CYP1A1 genes from MCF-7 cells, there is some evidence for cell-specific autoregulation of CYP1A1 tumor promotion. Other studies indicate that topoisomerase I activity is necessary for the primary CYP1A1 induction response and that the decreased expression of CYP1A1 in high-passage rat epidermal cells may be mediated by altered negative regulatory DNA (NeRD) binding factors present in these cells. TCDD induction of CYP1A1 in rainbow trout hepatocytes does not appear to depend on protein kinase activity.

TOXICITY PROFILE UPDATES

This section updates the toxicity profiles of the five substances discussed in VAO and Update 1996: (1) 2,4-D, (2) 2,4,5-T, (3) picloram, (4) cacodylic acid, and (5) TCDD (dioxin). The chemical nature of these substances is discussed in more detail in Chapter 6 of VAO.

Each profile update contains a review of experimental studies published during 1995-1997. Information in this literature update is organized under the topics (1) toxicokinetics, (2) mechanisms of toxic action, (3) disease outcomes, and if applicable, (4) estimating potential health risks and factors influencing toxicity.

Toxicity Profile Update of 2,4-D

Toxicokinetics

Kim et al. (1996) constructed a physiologically based pharmacokinetic (PBPK) model describing the kinetics of 2,4-D in developing fetal rabbit brain. Pregnant rabbits were administered 2,4-D intravenously (1, 10, or 40 mg/kg). The concentrations of 2,4-D in maternal and fetal brain, maternal and fetal plasma, and amniotic fluid were examined over time. Results indicated that the uptake of 2,4-D was membrane limited by the blood-brain barrier, with saturable clearance from the cerebrospinal fluid into the venous blood observed in both fetus and mother. In related studies, Sandberg et al. (1996) measured the relationship between 2,4-D concentration in maternal and fetal tissues following intravenous administration of radiolabeled 2,4-D (1, 10, and 40 mg/kg) to pregnant New Zealand rabbits (GD 28-30). The highest levels of 2,4-D accumulated in mater-

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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nal kidney and uterus and the lowest in maternal and fetal brain. At levels between 10 and 40 mg/kg, the maternal plasma binding capacity of 2,4-D became saturated and fetal levels increased more than four-fold. The organic anion transporter of the brain barrier system is functional in the late-gestational phase of the fetal rabbit. But its development is probably not complete because higher brain tissue-to-plasma ratios of 2,4-D were found in the fetus than in the dam.

At the cellular level, Bergesse and Balegro (1995) have shown that 2,4-D influx into Chinese hamster ovary cells is an energy-dependent process. These cells take up 2,4-D against a concentration gradient but do not metabolize its undissociated form. The uptake process is inhibited by sodium azide and dinitrophenol, but not ouabain, indicating that (Na+/K+) adenosine triphosphatase (AT-Pase) is not involved. Although pH less than 4.5 favors the occurrence of the undissociated form of 2,4-D (pKa 2.9), a decrease in cellular uptake is observed under these conditions. Alterations at the carrier level induced by changes in electrical charge at the cell membrane are believed to favor movement of the dissociated form of 2,4-D through the membrane.
Mechanisms of Toxic Action

Studies published since Update 1996 continue to support the view that the mechanism of toxic action of 2,4-D involves disruption of thiol homeostasis. Palmeira et al. (1995a) showed that the viability of freshly isolated rat hepatocytes decreases significantly when incubated with 10 mM 2,4-D for 60 minutes. Basal calcium ion levels increased only slightly with concentrations of 2,4-D ranging from 1 to 10 mM, suggesting that cell death in hepatocytes was not related to early increases in calcium ion concentration. In a related study, Palmeira et al. (1995b) demonstrated that the metabolism of 2,4-D rapidly depletes glutathione (GSH) and protein thiols and induces lipid peroxidation, suggesting that 2,4-D is hepatotoxic by a mechanism related to disruption of GSH homeostasis.

Kale et al. (1995) evaluated the mutagenicity of several pesticides. 2,4-D induced significant numbers of mutations in at least one of the cell types tested. Because these results differed from earlier studies, it was hypothesized that different germ cell stages and treatment regimens might account for the observed inconsistencies. Treatment with 2,4,5-T led to similar results which are reported later in this chapter.

A recent report by Miranda et al. (1997) concluded that to a small extent, 2,4-D increases mRNA levels of mdr genes in mouse liver. The study also reported effects due to 2,4,5-T exposure, which are discussed later.

Disease Outcomes

Lethality In studies by Paulino et al. (1996), rats were exposed acutely (600 mg/kg), subchronically (200 mg/kg per day for 30 days), and chronically (200

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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mg/kg per day for 180 days) to 2,4-D by the oral route. Macroscopic or histopathological lesions were not observed following acute, subchronic, or chronic exposures. However, acute exposure to 2,4-D was associated with decreased locomotor activity and ataxia, sedation, muscular weakness (mainly of the hind quarters), and gasping for breath; increased aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (AP), and amylase activities and elevated creatine levels; decreased total protein (TP) and glucose levels; and increased hematocrit values. Subchronic herbicide exposure increased AST activity and albumin and hematocrit values. Chronic exposure increased AST, AP, and LDH activities; decreased amylase and glucose levels; but induced no change in hematocrit values. Chromatographic analysis of the serum of chronically exposed rats suggested that 2,4-D does not accumulate in the body.

In related studies, Paulino and Palermo-Neto (1995) evaluated the acute toxicity of 2,4-D (100-600 mg/kg) in cattle by monitoring levels of serum AST, ALT, AP, O-glutamyl transferase (O-GT), and creatine kinase (CK); LDH activity and urea, creatinine, glucose, total protein, and albumin levels. The lowest dose (100 mg/kg) did not affect any of the biochemical parameters studied, whereas 300 mg/ kg decreased AST, O-GT, and CK activities and increased urea glucose levels. Increased LDH and CK activities and protein, urea, creatine, and glucose levels were observed in animals treated with 600 mg/kg. These changes were time and dose dependent as well as reversible. Collectively, the results indicate that acute 2,4-D intoxication disrupts serum levels of several enzymes and blood components that reflect kidney and muscle damage induced by this herbicide.

Charles et al. (1996c) conducted subchronic toxicity studies in dogs comparing three forms of 2,4-D: (1) the parent form, 2,4-D acid; (2) 2,4-D dimethylamine salt; and (3) 2,4-D 2-ethylhexyl ester. Groups of four dogs of each sex received in their food, on an acid equivalent basis, either 0, 0.5 (parent form only), 1.0, 3.75, or 7.5 mg/kg daily for 13 weeks. The dogs exhibited reductions in body weight gain and food consumption, and minor increases in blood urea nitrogen, creatinine, and ala-nine aminotransferase as a function of chemical treatment. A no-observed-adverse-effect-level (NOAEL) of 1.0 mg/kg per day was identified for all three forms indicating the comparable and generally low toxicity of different forms of 2,4-D.

Charles et al. (1996b) reported that, in Fischer rats, 2,4-D decreases red cell mass, 3,5,3'-triiodothyronine (T3) and 3,5,3',5'-tetraiodothyronine (thyroxine, T4) levels, and ovary and testes weights, and increases liver, kidney, and thyroid weights, and cataracts and retinal degeneration (high-dose females). These data suggest that the three forms of 2,4-D acid evaluated exert comparable low toxicities and support a subchronic no-observed-effect level (NOEL) of 15 mg/kg per day for all three forms.

Neurotoxicity Using a gas-liquid chromatographic method for determination of 2,4-D levels in serum and brain tissue of rats, Oliveira and Palermo-Neto (1995)

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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showed that the toxic effects of 2,4-D in rats were observed within one-half hour after its oral administration and correlated with signs and symptoms of central nervous system (CNS) depression. The data were interpreted to suggest that the toxic mechanism of 2,4-D is related to an action on the central nervous system.

According to Duffard et al. (1996), 2,4-D renders the developing rat nervous system vulnerable by hindering the process of myelination in the brain. Investigators examined rat pups following 100 mg/kg administration of 2,4-D to dams during the period of rapid demyelination (day 15 to 25 postnatal). Brains of both male and female pups showed a significant diminution of myelin markers, such as monohexosylceramide, phospholipids, and free fatty acids, and an increase of cholesteryl esters.

Immunotoxicity Blakley (1997) reported that female CD-1 mice exposed to Tordon 202C (a mixture of 2,4-D and picloram) in the drinking water for 26 days at concentrations ranging from 0 to 0.42 mg/kg significantly reduced antibody production in response to inoculation with sheep red blood cells. Although the individual component responsible for this effect was not identified, it is important to note that these levels of exposure are only marginally higher than the levels encountered following recommended application of the herbicide.

Reproductive Or Developmental Toxicity In 1980, Lamb et al. conducted studies to evaluate the effects of a mixture of 2,4-D (20-40 mg/kg), 2,4,5-T (20-40 mg/kg), and the herbicide contaminant TCDD (0.16-2.4 μg/kg) on the reproduction and fertility of male C57BL/6 mice. These mixtures were intended to simulate Agent Orange. Dose-related liver and thymus toxicity and reduced weight gain were observed in treated animals. No significant effects were observed on sperm function (concentration, motility, or abnormalities) or reproductive outcomes (including litter size, number of dead fetuses, and sex ratio). Survival of offspring and neonatal development were unaffected by paternal exposure to the mixture. No increase in most congenital anomalies was found. A statistically significant excess of fused sternebra in the offspring of the two most highly exposed groups was observed, but this was attributed to an anomalously low rate of the defect in the controls.

Blakley et al. (1989) exposed male CD-1 mice to Tordon 202C (a mixture of 2,4-D and picloram) in drinking water at concentrations of 0.21-0.84 mg/kg for 60 days prior to mating with untreated females. Symptoms of paternal toxicity were observed in all exposure groups. Pregnancy failure was increased. Fetal weight and crown-rump length were reduced in offspring of the highest-dosage group, and various malformations (including ablepharon, cleft palate, unilateral agenesis of the testes, extra ribs, and incomplete ossification) were observed in the offspring of mice at all three dosage levels. However, the paternal toxicity observed at the high dosage levels used and the inconsistent dose-response pattern are of concern.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Carcinogenicity Charles et al. (1996a) evaluated the carcinogenicity of 2,4-D in a two-year rodent bioassay. Groups of 50 rats per sex per dose group received varying doses (0, 5, 75, and 150 mg/kg per day) of 2,4-D for two years. Groups of 50 mice per sex per dose group were administered 0, 5, 62.5, and 125 mg/kg of 2,4-D per day for two years. Clinical chemistry, hematology, and urinalysis were performed at six month intervals in both species. No evidence of astrocytomas was observed in rats or mice, even at concentrations equaling the maximum tolerated dose. No other oncogenic effects were reported in either test species.

Hayes et al. (1991, 1995) conducted a retrospective case-control study of malignant lymphoma in dogs. Researchers gathered data from 492 dogs diagnosed with lymphomas from three veterinary medical teaching hospitals in Minnesota, Indiana, and Colorado. There were two control groups, dogs with cancers other than lymphomas and dogs without cancer. Owners of the dogs were questioned about the frequency and duration of various pesticides used in yardwork. The authors reported a statistically significant association between the risk of canine malignant lymphoma and 2,4-D exposure. However, it must be emphasized that the dogs were concomittantly exposed to other pesticides in addition to 2,4-D.

Toxicity Profile Update of 2,4,5-T

Toxicokinetics

No toxicokinetic studies were identified for the reference period.

Mechanisms of Toxic Action

Sastry et al. (1997) examined the in vitro formation of 2,4,5-T-acetylcoenzyme A by acetylcoenzyme A synthetase and 2,4,5-trichlorophenoxyacetylcholine (2,4,5-T-Ach) by human placental choline acetyltransferase. The measured reaction rates for both endogenous and exogenous substrates were comparable. Low concentrations of synthetic 2,4,5-T-Ach decreased contraction heights of the rat phrenic nerve hemidiaphragm when the nerve or muscle was electrically stimulated. Collectively, these results suggest that 2,4,5-T can enter cellular metabolic pathways involving acetylcoenzyme A, resulting in altered metabolism and cholinergic transmission.

Kale et al. (1995) evaluated the mutagenicity of several pesticides. 2,4,5-T induced significant numbers of mutations in at least one of the cell types tested. Because these results differed from earlier studies, it was hypothesized that different germ cell stages and treatment regimens may account for the observed inconsistencies. Similar results were shown for 2,4-D treatment.

Miranda et al. (1997) concluded that 2,4,5-T increases mRNA levels of mdr

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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genes in mouse liver. At the protein level, P-glycoprotein traffic ATPase content increased in the canalicular domain of hepatocytes treated with 2,4,5-T. This response appeared to be mediated at the transcriptional level. Similar results were shown for 2,4-D treatment.

Voskoboinik et al. (1997) reported that the formation of DNA adducts by cytochrome P450-derived metabolites of benzo[a]pyrene (BP) increased in hepatocytes isolated from rats treated with 2,4,5-T for 14 days. The activity of CYP1A1 was increased, whereas the activity of glutathione S-transferase (GST) was decreased by the herbicide, suggesting that the increased formation of DNA adducts, following carcinogen treatment of 2,4,5-T treated rats, is due to increased metabolic activation to reactive intermediates coupled with reduced detoxification. The latter effects are particularly interesting since they are strikingly similar to those elicited by dioxin.

Disease Outcomes

Results from in vitro mechanistic studies suggest that 2,4,5-T may acutely affect neuronal and muscular function by altering cellular metabolism and cholinergic transmission. Mechanistic studies also suggest that repeated exposures to 2,4,5-T may be associated with modulation of xenobiotic metabolizing enzymes that alter the disposition of chemical carcinogens, such as BP. These alterations coupled with the ability of 2,4,5-T to induce mutations under certain conditions can influence the carcinogenic process.

Toxicity Profile Update of Cacodylic Acid

Toxicokinetics

Hughes et al. (1995) evaluated the in vitro dermal absorption of radiolabeled DMA. Discs of preclipped dorsal skin were cut from adult female B6C3F1 mice, mounted in flow-through diffusion cells, and then challenged for 24 hours with DMA at doses of 10, 100, and 500 μg using solid compound and aqueous solution (20, 100, and 250 μl) and soil (23 μg/cm2) as vehicles. Absorption of the compound into the skin and receptor fluid ranged from < 1 to 40 percent in all exposure scenarios examined. The rank order of DMA absorption into the skin was 20, μl water > 100 μl water > solid > 250 μl water > soil. No dose or pH effects were observed. There was also no pH effect on the partitioning of DMA between 1-octanol and buffer. Short-term (1-hour) exposure of DMA in water followed by wash of the skin resulted in < 1 percent of the dose being absorbed. Although the implications of this work for human risk assessment remain to be established, the studies suggest that vehicles and duration of exposure are important factors in the in vitro dermal absorption of DMA.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Mechanisms of Toxic Action

Ochi et al. (1996) investigated the ability of certain arsenic compounds, including DMA, to induce apoptosis or programmed cell death in cultured human HL-60 cells. Of the agents examined, DMA was the most potent inducer of apoptosis. Depletion of cell glutathione following inhibition of γ-glutamylcys-teine synthetase by L-buthionine-SR-sulfoximine enhanced the cytotoxicity of arsenite, arsenate, and methylarsonic acid, but suppressed the toxicity of DMA. The implications of these findings are not clear.

According to Rin et al. (1995), paraquat, a superoxide-generating agent, enhances DMA-induced DNA single-strand breaks in cultured alveolar cells. This enhancement occurs following washing of cells treated with DMA, suggesting that DMA-induced DNA modifications are recognized by active forms of oxygen leading to single-strand breaks. This interpretation is consistent with ultraviolet (UV) irradiation and electron spin resonance studies showing that superoxide produced by paraquat in DMA-exposed cells was more efficiently consumed than in nonexposed cells. Collectively, DMA may induce DNA modifications that sensitize it to free-radical injury.

DMA treatment (50, 100, 200, or 400 mg/kg) in the drinking water of male rats initiated by sequential treatment with nitrosamines (diethylnitrosamine [100 mg/kg, i.p., single dose]; N-methyl-N-nitrosourea [20 mg/kg, i.p., four times, on days 5, 8, 11, and 14]; 1,2-dimethylhydrazine [40 mg/kg, subcutaneously, 4 times, on days 18, 22, 26, and 30]; N-butyl-N-(4-hydroxybutyl)nitrosamine [0.05 mg/kg in drinking water, during weeks 1 and 2]; and N-bis(2-hydroxypropyl)-nitrosamine [BBN; 0.1 mg/kg in drinking water, during weeks 3 and 4]) showed that DMA significantly enhanced tumor induction in the urinary bladder, kidney, liver, and thyroid gland (Yamamoto et al., 1997). Induction of preneoplastic lesions as reflected by GST-positive foci in the liver and atypical tubules in the kidney, respectively, was also significantly increased in animals treated with 100 or 400 parts per million (ppm) of DMA alone. Ornithine decarboxylase activity in the kidneys of rats treated with 100 ppm DMA was significantly increased compared to control values (p < .001). From these studies it was concluded that DMA is a promoter of urinary bladder, kidney, liver, and thyroid gland carcinogenesis in rats.

In related studies, Wanibuchi et al. (1996) examined the promotion potential of DMA for rat urinary bladder carcinogenesis. Six-week-old male F344 rats were treated with 0.05 mg/kg BBN for four weeks and then given DMA in their drinking water (0, 2, 10, 25, 50, and 100 mg/kg) for 32 weeks. The development of preneoplastic lesions and tumors (papillomas and carcinomas) in the urinary bladder was enhanced by treatment with DMA in a dose-dependent manner. A significant increase in the multiplicity of tumors (papillomas and carcinomas) was observed even at a low concentration of DMA (10 mg/kg). On the other hand, no preneoplastic lesions and tumors were observed in rats treated with

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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DMA alone. Administration of DMA alone (0, 10, 25, and 100 ppm) in drinking water for eight weeks without prior initiation was associated with a significant increase in the 5-bromo-2'-deoxyuridine labeling index and alteration of the surfaces of urinary bladder epithelial cells. These results suggest that the potential of DMA to promote rat urinary bladder carcinogenesis may be related to its ability to stimulate cell proliferation in the urinary bladder epithelium.

Disease Outcomes

The limited evidence presented during the past two years suggests that DMA acts as a multiorgan promoter of carcinogenesis.

Toxicity Profile Update of Picloram

No studies published since Update 1996 have reported on the toxicity of picloram alone. As described earlier, Blakley (1997) reported that female CD-1 mice exposed to Tordon 202C (a mixture of 2,4-D and picloram) in the drinking water for 26 days at concentrations ranging from 0 to 0.42 mg/kg significantly reduced antibody production in response to sheep red blood cell inoculation. Although the individual component responsible for this effect was not identified, it is important to note that these levels of exposure are only marginally above the levels encountered following recommended application of the herbicide.

Toxicity Profile Update of TCDD

Toxicokinetics

Michalek and coworkers (1996a, 1997) examined TCDD disposition in veterans of Operation Ranch Hand, the Air Force unit responsible for aerial spraying of Agent Orange in Vietnam. The half-life of TCDD in 213 veterans was estimated based on multiple serum measurements collected over a 10-year period. Researchers took into account the potential influence of age, percentage of body fat, and changes in the percentage of body fat. In agreement with previous estimates, the mean decay rate of TCDD for these veterans is 0.0812 per year; with a corresponding half-life estimate of 8.532 years. Half-life increased significantly with increasing body fat, but not with age or relative changes in percentage of body fat. Investigators warned that serum dioxin measurements should not be used when the first measurement exceeds 50 mg/kg due to low reliability (Michalek et al., 1996b).

Ayotte et al. (1996) estimated the impact of breastfeeding on the body burden of dioxin-like chemicals in Arctic Inuit people from birth to age 75. Toxicokinetic modeling in the Inuit revealed that breastfeeding strongly influences body burden during childhood but not after age 20. Liver and adipose tissue

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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concentrations appeared to be lower than those associated with cancer and adverse reproductive effects in laboratory rats. On the other hand, a substantial proportion of Inuit women may have adipose tissue concentrations close to or higher than those associated with increased incidence of endometriosis in rhesus monkey.

Recent studies have also addressed the toxicokinetics of TCDD in experimental animals. For example, Diliberto et al. (1996) compared the absorption, tissue distribution, and elimination of TCDD in laboratory rats. Researchers exposed male rats to radiolabeled-TCDD (0.32 mg/kg) through inhalation or oral treatment. They also injected rats directly with TCDD because this exposure route serves as a standard for evaluating the others. Injection eliminates problems with absorption. Three days after exposure, fecal excretion accounted for 22, 26, and 32 percent of the injected, inhaled, and orally administered doses, respectively. Urinary excretion accounted for only 2.2, 1.3, and 1.4 percent, respectively. Thus, 95 percent of the administered dose was absorbed by inhalation, whereas 88 percent was absorbed after oral administration. After absorption, the liver and fat serve as major TCDD depots. Following injection, 37 percent and 21 percent of the absorbed dose were distributed to the liver and fat, respectively. After inhalation, the numbers were 35 percent and 16 percent. After oral treatment, 28-30 percent of the absorbed dose was distributed to both liver and fat. These results indicate that both the oral and the inhalation routes are important for the systemic absorption of dioxins. Carrier et al. (1995 a,b) proposed a physiologically based model that describes the distribution kinetics of polychlorinated dibenzodioxins or polychlorinated dibenzofurans (PCDD/Fs) in various mammalian species. Simulations were in agreement with published data on the distribution kinetics of PCDD/Fs in rodents, monkeys, and humans. The model takes into account intercellular diffusion, PCDD/F-receptor and PCDD/F-protein binding, and PCDD/F-dependent enzyme induction in the liver. Investigators formulated nonlinear differential equations, with anatomically and biochemically relevant parameters to predict functional dependencies between the fraction of total PCDD/F body burden contained in liver and adipose tissues and the overall body concentration at any time. The liver fraction of the total body burden decreases as a function of the overall body concentration. Since elimination of these chemicals occurs principally via the liver, this results in slower elimination rates and longer half-lives. The kinetic profiles were also found to be similar for rats and humans. However, the half-lives differed considerably, with rats calculated in weeks but humans in years. For a given body burden, adipose tissue concentrations vary in inverse proportion to the mass of adipose tissues, an observation that is relevant to humans.

Aozasa et al. (1995) evaluated the EROD activity and liver accumulation of dioxin-like chemicals (i.e., 2,3,7,8-chlorine substituted PCDD/F) in two species of mice (C57BL/6 and DBA/2) administered these chemicals orally for 28 days. The C57BL/6 mice with high EROD induction or DBA/2 mice with low EROD

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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induction did not accumulate high levels of the chemicals. However, C57BL/6 mice accumulated larger amounts in their liver than DBA/2 mice. A remarkable difference in congener levels was observed when comparing the ratios in human tissue and in food. The tissue-food ratio of octachlorodibenzo-p-dioxin (OCDD) level was 20 times higher than that of octachlorodibenzofuran (OCDF), despite the same number of chlorine substituents in each compound. Taken collectively, the finding that in humans, OCDD accumulates more readily than OCDF is potentially significant because it suggests the possibility that the high bioaccumulation of OCDD in humans may be caused by metabolic pathways different from those in mice.

During the reference period, Kohn et al. (1996) extended a model of the physiological disposition of TCDD in the rat (Kohn et al., 1993) to include the distribution of blood among major vessels and tissue capillary beds; resorption of TCDD released from the liver into the gut lumen as a consequence of cell lysis; and compartments for thyroid and thyroxine-sensitive tissues (e.g., pituitary, kidney, brown fat) that take into consideration secretion and tissue uptake of thyroid hormones; the binding of T3 and T4 to proteins in blood and tissues; the deiodination of iodothyronines; and TCDD induction of T4 glucuronidation by hepatic UGT. The extended model fit the observed dose-response of P450 isozymes and Ah and estrogen receptors reported in the previous model after repeated oral doses. The extended model provided a better fit with respect to liver and fat TCDD levels after single and repeated oral and subcutaneous doses. Predicted liver TCDD concentrations at very low doses were verified experimentally. In addition, the model reproduced the responses observed for blood T3, T4, and TSH after 31 weeks of biweekly oral dosing of rats with TCDD. The model also predicted the responses of UGT mRNA and UGT enzymatic activity observed in experiments with TCDD-treated rats. Based on the model, TCDD increases UGT enzymatic activity and blood TSH levels. Calculated increases in blood TSH levels are consistent with prolonged stimulation of the thyroid and may represent an early stage in the induction of thyroid tumors identified in previous two-year bioassays. This suggests that increases in UGT activity may be a useful biomarker of tumorigenic changes in hormone levels following TCDD exposure.

Analysis of linear, sigmoid-Emax, and power law functions by McGrath et al. (1995) suggests that the use of a wide dose range may bias the interpretation of low-dose phenomena. This interpretation was based on the change in slope observed from low-to high-dose subsets for thymic atrophy, immune suppression, BP hydroxylase activity, and EROD activity. These investigators suggest that the power law function can provide a more accurate and biologically relevant assessment of risk and that noncancer end points may be more significant than cancer end points in assessing human health risks from TCDD. For example, immune suppression and enzyme induction occur at lower doses and under conditions more relevant to general population exposure conditions.

DeVito and Birbaum (1995) studied the pharmacokinetic factors that can

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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influence the relative potency of TCDD and tetrachorinated dibenzofuran (TCDF), in vivo. TCDD and TCDF were administered to female B6C3F1 mice for 4 and 13 weeks and EROD activity, a useful marker of CYP1A1 gene expression, was measured in liver and skin. Mice received either 150 ng TCDD/kg or 1,500 ng TCDF/kg daily, 5 days per week, for either 4 or 13 weeks, a regimen designed to provide equipotent doses. At four weeks, hepatic EROD was induced eleven-and seven-fold by TCDD and TCDF, respectively, suggesting that published TEFs accurately estimate the relative potency of TCDD and TCDF after four weeks of treatment. In marked contrast, the induction of hepatic EROD after 13 weeks was elevated 41- and 5-fold for TCDD and TCDF, respectively. Investigators suggested that the inability of TEFs to predict the relative potency of these compounds after 13 weeks of treatment may be due in part to differences in the pharmacokinetic properties of each congener since the half-lives of TCDF and TCDD are approximately 2 and 15 days, respectively. As such, steady-state levels of TCDD were not attained by 4 weeks, which is reflected in the increase in hepatic EROD between 4 and 13 weeks, whereas steady-state levels of TCDF were reached within 4 weeks. Similar results were observed for skin EROD activity. These findings are potentially significant since they suggest that TEFs derived from short-term assays may not adequately predict the relative potencies of this class of compounds following chronic exposure.

Mechanisms of Toxic Action

The biochemical and toxic effects of TCDD are in large measure mediated by the AhR, a cytoplasmic protein that binds several classes of xenobiotics including polycyclic aromatic hydrocarbons (PAHs), benzoflavones, heterocyclic amines, and halogenated aromatic hydrocarbons. The AhR is ubiquitously expressed in almost every organ and cell in the body. The receptor is normally found in association with two heat shock proteins (hsp90), which are thought to aid in stabilization of the receptor protein. Ligand binding (e.g., with TCDD) transforms the receptor to a form that exhibits increased DNA binding affinity leading to translocation to the nucleus where it dimerizes with a partner protein, Arnt. The AhR-Arnt complex binds to specific sequences in the regulatory region of target genes called aryl hydrocarbon-responsive elements (AhREs) to influence patterns of gene expression. Based on extensive studies of the CYP1A1 gene, binding of the AhR to the promoter region alters chromatin structure, leading to increases in the rate of gene transcription. Although several other genes are known to be influenced via a comparable mechanism, the net influence of AhR binding to DNA is dictated by the nature of the molecular interactions governing the regulation of the particular gene. Scientific reports published during the past two years have identified several genes that are involved in the regulation of development, growth, and differentiation of mammalian tissues as targets of TCDD action. As such, the ability of TCDD to influence the expression

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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of these ''target genes" may mediate at least in part the health outcomes associated with herbicide exposures. Other recent studies summarized below discuss the structural and functional aspects of the AhR and Arnt, DNA binding and transcriptional events, biological consequences associated with activation, significant and insignificant interactions and interspecies, and interindividual differences in sensitivity. The section ends with a discussion of methods used to estimate the potential health risk associated with exposure to TCDD and related compounds.

Abbott et al. (1996) examined the disposition of TCDD in pregnant C57BL/ 6N mice 24 hours after oral administration on GD 12. TCDD was detected in maternal blood, liver, and fat and in the placenta, embryonic liver, and palate within 30 minutes of dosing on GD 12. The levels peaked in blood and placenta at 3 hours and in other tissues at 8 hours. The early peak level found in maternal blood is in agreement with findings for distribution of this class of compounds in adult rats and mice. In general, dioxins and furans are rapidly absorbed from the gastrointestinal tract, and tissue distribution in the first hour parallels blood levels and reflects blood flow and tissue size. During this period the embryo grows rapidly (e.g., liver weight almost doubles from GD 12 to 13). This suggests that although the embryo continues to accumulate TCDD, increasing mass results in a relatively constant tissue concentration.

Structural and Functional Aspects of Ahr and Arnt Both the AhR and Arnt are members of the basic helix-loop-helix (bHLH) superfamily of gene regulatory proteins. A conserved domain of 200-350 amino acids, designated PAS (Per, Arnt, AhR, Sim), defines this superfamily. The AhR is a highly conserved protein found in diverse vertebrate groups. For example, in the PAS domain, the N-terminus of human AhR shows 87 percent and 86 percent amino acid identity with mouse and rat AhR, respectively. Recently, Hahn and Karchner (1995) have shown that the amino acid sequence of the PAS domain of a teleost AhR is 62-64 percent identical to the PAS domains of mammalian AhR, and Brown et al. (1995) detected the presence of two proteins (28 and 39 kDa) in the cytosols of the hard-shell clam species, Mercenaria Mercenaria, that bind a chemical analogue of TCDD. The latter studies reported that these proteins are homologous to the AhR.

Arnt, like the AhR, also shows a high degree of conservation between vertebrate groups. Two cDNAs encoding bHLH-PAS proteins with similarity to Arnt protein have been isolated from RTG-2 rainbow trout gonad cells (Pollenz et al., 1996). The deduced proteins, termed rtARNTa and rtARNTb, are identical over the first 533 amino acids and contain a bHLH domain that is 100 percent identical to human Arnt.

Both the AhR and Arnt have been shown to interact with a large number of proteins to influence receptor function. Of particular significance from a biochemical and toxicological perspective has been the recent demonstration that

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Arnt interacts in vivo with HIF-1α to form the active HIF-1 transcription factor complex. This complex appears responsible for mediating the cellular response to hypoxia by transcriptional regulation of genes such as erythropoietin and other mediators of cellular oxygenation. Five new members of the PAS superfamily that interact with the AhR or Arnt were identified by Hogenesch et al. (1997). Two of these proteins (MOP1 and MOP2) dimerize with Arnt and form complexes that are transcriptionally active, an interaction suggesting that cellular pathways mediated by MOP1 and MOP2 may influence or respond to the dioxin signaling pathway.

In general, the AhR and Arnt proteins appear to be coexpressed; however, pronounced differences in relative expression levels exist between the two dimeric partners in some tissues, suggesting that additional dimerization partners and signaling pathways may be present. Arnt is able to homodimerize, as well as heterodimerize, with Sim and Per. Identification of a human Sim homologue that may play a causal role in Down's syndrome (trisomy 21) has recently been demonstrated. This human Sim protein is found in embryonic CNS and facial tissues, where it may interact with Arnt or an Arnt-like protein to regulate important developmental processes.

Abbott and Probst (1995) characterized the expression of Arnt in mouse embryos during GDs 10-16. On GD 10-11, embryos showed the highest levels of Arnt in neuroepithelial cells of the neural tube, visceral arches, otic and optic placodes, and preganglionic complexes. The heart also had significant expression of Arnt with strong nuclear localization. After GD 11, expression in heart and brain declined. On GD 12-13, embryonic expression was highest in the liver, where expression increased from GD 12 to 16. On GD 15-16, the highest levels of Arnt occurred in adrenal gland and liver, although it was also detected in submandibular gland, ectoderm, tongue, bone, and muscle. In all of these tissues, Arnt was cytoplasmic as well as nuclear, except in some of the cortical adrenal cells in which it was strongly cytoplasmic with little or no nuclear localization. These specific patterns of Arnt expression differ in certain tissues from those of the AhR, suggesting that Arnt may have roles in normal embryonic development that are independent of the AhR. In Arnt-deficient cells, the AhR can still translocate to the nucleus in vivo, a process therefore independent of Arnt.

The PAS domain of the AhR also harbors the contact region for association with hsp90. Interestingly, this region colocalizes with a domain previously identified as a repression domain on AhR signaling (Dolwick et al., 1993). It has been shown that in a purified system the AhR-hsp90 complex is not dissociated by the addition of ligand (McGuire et al., 1994) and that addition of a cellular fraction from Hepa cells, but not Arnt-deficient Hepa mutants, can promote hsp90 dissociation. These results suggest that the Arnt protein plays an active role in this process.

Studies by Coumailleau et al. (1995) defined a minimal ligand binding domain of the AhR within the central PAS region that interacts with hsp90 in vitro. The minimal ligand binding domain maintains the quantitative and qualitative

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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aspects of ligand binding exhibited by the full-length receptor. These studies are consistent with in vitro findings by Whitelaw et al. (1995).

In contrast to the results of McGuire et al. (1994), Fukunaga et al. (1995) found that ligand binding was localized to a region encompassing the PAS B repeat. One hsp90 molecule appears to bind within the PAS region, whereas the other appears to require interaction over the bHLH region. In addition, ligand-mediated dissociation of AhR from hsp90 did not require Arnt. These results also suggested that equivalent regions of the AhR and Arnt associate with each other since both the first and the second helices of the bHLH motif and the PAS region are required for dimerization. Finally, Fukunaga et al. (1995) suggested that the carboxyl-terminal half of the AhR plays a more prominent role in transcriptional activation of the CYP1A1 gene than the corresponding region of Arnt. Deletion of the carboxyl-terminal half of AhR did not affect dimerization or AhRE binding, but did eliminate biological activity as assessed by an in vivo transcriptional activation assay. Deletion of the carboxyl-terminal half of Arnt did not affect biological activity in the same assay system.

In contrast to the findings of Fukunaga et al. (1995), Li et al. (1994) have reported that the activation of an AhRE-driven reporter plasmid by AhR-Amt is dependent on the transactivation domain (TAD) of Arnt. In vivo, the TADs of the AhR and Amt may synergize, because removal of the Q-rich region of Arnt did not impair AhR-Arnt dimerization but diminished transactivation of a AhRE-driven CAT reporter gene in Arnt-defective Hepa cells.

The C-terminal 34 amino acids of Arnt harbor a TAD that functions independently of other sequences in the AhR complex (Corton et al., 1996). The strength of the Arnt TAD is cell-type specific since Arnt and herpes simplex virus VP16 TAD were equally strong in COS-1 cells, but the Arnt TAD had weak activity in an Arnt-deficient mouse hepatoma cell line and was not needed for restoration of CYP1A1 activation. These results imply that for CYP1A1 activation the AhR provides the dominant activation function for the heterodimer in hepatoma cells. This may not be the case for other genes or for different transcription factor complexes.

Analyses of AhR cDNA deletion mutants from C57BL/6 mouse liver indicate that the carboxyl half of AhR contains several TADs that function independently of the domains that mediate TCDD recognition, DNA binding, and heterodimerization with Arnt (Ma and Whitlock, 1996). The transactivation domains function independently of each other, display different levels of activity, and act synergistically when linked.

In addition to dimerizing with bHLH-PAS proteins, the AhR interacts with other cellular proteins involved in the regulation of rabbit liver cell function. Dunn et al. (1996) identified two active protein fractions in ranging in molecular weight from 12 to 14 kDa that bind to the AhR. Of particular significance was the identification of the protein histone H4, which is known to interact with transcription factors in a variety of systems.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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In other studies, a yeast two-hybrid system was used to identify proteins that interact with the AhR (Li and Dougherty, 1997). These investigators cloned a mouse cDNA that encodes a novel 37 kDa protein that binds to the AhR. The amino acid sequence of this protein exhibits homology with proteins required for cell cycle control and RNA synthesis and is related to steroid receptor binding immunophilins. The 37 kDa protein is thought to be cytoplasmic and to associate with unliganded AhR and with hsp90. TCDD treatment disrupts the protein-protein interaction, whereas overexpression of the AhR interacting protein augments the response of the CYP1A1 gene to TCDD.

The regulation of AhR gene expression has not been studied in as much detail as other aspects of receptor function. To study tissue-specific regulation of the mouse AhR gene, Fitzgerald et al. (1996) transfected chimeric deletion constructs containing the AhR 5'-flanking region and the firefly luciferase reporter gene into five established mouse cell lines: Hepa 1c1c7 (derived from hepatoma), JB6-C1 41-5a (epidermis), MLE-12 (lung epithelium), F9 (embryonal carcinoma), and NIH/3T3 (fibroblasts). In all cell lines except F9 cells, maximal constitutive expression occurred with constructs containing 78 base pairs of AhR promoter sequences. This region includes several putative binding sites for the transcription factor Sp1. It is interesting that in F9 cells, other transcription factors appear to be important in AhR gene expression since up to 174 promoter sequences were required for induction. Results suggests that regulation of the AhR gene occurs in a tissue-and cell-type specific manner.

Although the ability of TCDD to modulate gene expression often involves transcriptional mechanisms, posttranscriptional events may also be important in the regulation of gene expression (Gaido and Maness, 1995). Treatment of SCC-12F cells with 10 nM TCDD increases urokinase-plasminogen activator (u-PA) mRNA. Transcription of u-PA was not altered by TCDD; instead, induction of u-PA occurred as a result of a stabilization of the u-PA mRNA. Tissue-plasminogen activator and plasminogen activator inhibition (PAI-1) expression were not altered by TCDD. Thus, TCDD acts through different mechanisms in SCC-12F cells to induce both a plasminogen activator and a specific inhibitor of plasminogen activation.

Of perhaps greatest significance during the past two years has been the development of AhR-deficient mice (AhR-/-). Using gene targeting, Gonzalez and coworkers (1995) developed AhR-deficient mice by inactivation of the first exon of the AhR. In a separate study, Schmidt et al. (1996) used gene targeting to delete exon 2, which encodes the bHLH DNA binding and dimerization domain. These knockout mice do not express receptor protein, and as expected, transcriptional activation of AhR target genes by TCDD is abolished. The results from knockout experiments present compelling evidence that the AhR plays a fundamental role in cell and organ physiology and homeostasis. If ligand activation is indeed required for receptor function, an endogenous ligand must be present in most cells. From a toxicologic perspective, AhR knockout mice have been impor-

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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tant since they helped establish a definitive association of the AhR with TCDD-mediated toxicity.

Peters and Wiley (1995b) have shown that culturing embryos in medium with an AhR antisense oligodeoxynucleotide reduces the incidence of blastocyst formation as well as mean embryo cell number, suggesting that AhR may function in embryonic cell differentiation and proliferation independent of its known function in mediating TCDD toxicity.

AhR-/- mice are relatively unaffected by doses of TCDD (2,000 mg/kg) tenfold higher than those found to induce severe toxic and pathologic effects in littermates expressing a functional AhR. The resistance of AhR-/- mice to TCDD-induced thymic atrophy and cortical lymphocyte depletion appears restricted to processes involving AhR since the corticosteroid dexamethasone, which induces thymic atrophy by a pathway unrelated to the AhR, rapidly and efficiently induces cortical depletion in both AhR-/- and normal littermate control mice. It is important to note, however, that at high doses of TCDD, AhR-/- mice display limited vasculitis and scattered single-cell necrosis in lung and liver, respectively. The mechanism(s) responsible for these apparently receptor-independent processes remain unclear but may involve novel, alternative pathways for TCDD-induced toxicity.

Evidence also continues to accumulate that ligand-independent events can mediate activation of the receptor. Benzimidazole derivatives are potent CYP1A1 inducers in rabbit and human liver cells, but do not bind the AhR, suggesting that ligand-independent mechanisms may activate the AhR. Lesca et al. (1995) showed that benzimidazoles bind early and transiently to an unknown protein in rabbit liver cells, causing a depletion of AhR in a time-and dose-dependent manner. In contrast, benzimidazoles are unable to induce CYP1A1 mRNA in specific mouse liver cells and to deplete the high-affinity AhR form from these cells. A signal transduction pathway similar to that involved in the ligand-independent activation of steroid receptors may activate the low-affinity forms of AhR present in rabbit and human cells, but not in mouse cells.

DNA Binding and Transcriptional Interference As described previously, modulation of gene expression by TCDD is dependent on its ability to bind DNA to influence transcriptional events. Recent studies have focused on characterizing the nature of TCDD-induced ligand binding (e.g., transactivation domains) and transcriptional events (e.g., conformational characteristics and protein-DNA complexes).

Weiss et al. (1996) evaluated transient and stable AhR expression in AhR-deficient clones. The AhR that was transiently expressed into receptor-deficient variants exhibited high basal transactivation activity on promoters containing AhR binding sites compared to wild-type cells. Hybrid receptors also showed high basal activity in the absence of exogenous TCDD in AhR-deficient variant cells, indicating that endogenous AhR activating signal acts directly on the recep-

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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tor. Stable expression of AhR in variant cell clones fully reconstituted TCDD responsiveness, including target gene induction and delay of cell cycle progression. These AhR-reconstituted cells, like wild-type cells that contain AhR, showed low basal activity of the transiently expressed AhR hybrid. The increased basal activity in AhR-deficient cells suggests a negative feedback control of AhR activity compatible with the existence of an endogenous AhR ligand.

Bacsi et al. (1995) completed studies to evaluate TCDD-induced binding of AhR-Arnt to the asymmetric AhRE in nuclear extracts of Hepa 1c1c7 cells. Covalent cross-linking analysis and immunoprecipitation with antibodies specific for AhR or Arnt demonstrated that Arnt directly contacts the 3'-most thymine position, the AhR directly contacts the second thymine position, and neither protein contacts the 5'-most thymine position. The thymine position contacted by Arnt lies within a three-nucleotide sequence (5'-GTG-3') identical to a half-site of an E-box element (5'-CACGTG-3') recognized by other bHLH transcription factors, whereas AhR binds to a portion of the AhRE that does not resemble an E-box. In this study, evidence was also presented that neither protein loops over to contact residues located beyond the other's binding site.

In studies to characterize the binding of transformed guinea pig hepatic AhR to DNA, Bank et al. (1995) identified two distinct TCDD-inducible protein-AhRE complexes, but only a single high-affinity binding site. The formation of both DNA-binding complexes exhibited nucleotide specificity for the AhR complex.

Santostefano and Safe (1996) investigated ligand-dependent differences in molecular properties of the transformed cytosolic and nuclear AhR. For several different AhR ligands including TCDD, TCDF, 1,2,7,8-TCDF, and α-naphtho-flavone, the pattern of proteolytic protein-DNA products using transformed cytosolic or nuclear AhR complexes was comparable. In contrast, significant differences were observed in the pattern of degraded protein-DNA products using nuclear AhR complexes derived from mouse Hepa 1c1c7 cells treated with TCDD or 6-methyl-l,3,8-trichlorodibenzofuran (MCDF). Such differences may be related to conformational characteristics induced by TCDD, a potent AhR agonist, relative to MCDF, a partial AhR agonist and antagonist.

Yamaguchi and Kuo (1995) have demonstrated that ligand-free AhR has no transactivating properties in yeast. However, the C-terminal portion (amino acid residues 580-797) of the AhR, including the Q-rich domain, confers transactivation activity in the same system, suggesting that the N-terminal portion of the AhR contains transcription repression properties. In contrast, the 75 C-terminal amino acids of Arnt, including the Q-rich domain, exhibited full transactivation function in yeast and mammalian cells, suggesting that structural organizations of the transactivation properties differ between AhR and Arnt, although both contain transactivation domains at the C-termini.

Lindebro et al. (1995) have shown that Arnt interacts with the AhR via the PAS domain. The PAS domain of Per could dimerize with both AhR and Arnt in

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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vitro and disrupt the ability of these subunits to form a DNA binding heterodimer. Ectopic expression of Per blocked dioxin signaling in mammalian cells. Thus, the PAS domains of the dioxin receptor and Arnt are novel dimerizing regions critical in the formation of a functional AhR-Arnt complex, whereas the PAS domain in Per is a potential negative regulator of bHLH-PAS function. Evidence was also presented that hsp90 may modulate dioxin receptor function by directing correct folding of the ligand binding domain, interference with Arnt heterodimerization, and folding of a DNA binding conformation of the bHLH domain.

Swanson et al. (1995) examined the DNA recognition and pairing of several bHLH-PAS families of proteins. The AhR-Arnt complex exhibits a preference for the sequence commonly found in dioxin-responsive enhancers in vivo (GCGTG). As discussed previously, the Arnt protein is capable of forming a homodimer with a binding preference for the palindromic E-box sequence (CACGTG). These studies revealed that Arnt has a broader range of interactions than other members of the bHLH-PAS proteins.

Dioxin-induced binding of the AhR-Arnt heterodimer to enhancer chromatin is associated with a localized alteration in chromatin structure is manifest by increased accessibility of the DNA (Okino and Whitlock, 1995). These changes likely reflect disruption of a nucleosome by AhR-Arnt. However, in the CYP1A1 promoter, such changes must occur by a different, more indirect mechanism because they are induced from a distance and do not reflect a local effect of AhR-Arnt binding. Dose-response experiments indicate that changes in chromatin structure at the enhancer and promoter are graded and correlated with the graded induction of CYP1A1 transcription by dioxin. Thus, TCDD induces a shift in equilibrium between nucleosomal and nonnucleosomal chromatin configurations.

Certain ellipticine derivatives have been reported to bind the AhR and inhibit the ability of TCDD to transform it to a form that recognizes an AhRE upstream of the CYP1A1 gene. Gasiewicz et al. (1996) examined more than 30 ellipticine derivatives and structurally related compounds for their ability to bind the AhR, activate it to a dioxin response element (DRE) binding form, induce the luciferase gene under the control of a DRE containing enhancer, and block activation of the AhR by TCDD. The ability of ellipticine derivatives to inhibit TCDD-elicited DRE binding and TCDD-induced luciferase activity was inversely related to their ability to stimulate these responses. Their antagonistic activity was related to their binding affinity for the AhR as predicted by their van der Waals dimensions and the presence of an electron-rich ring nitrogen at or near a relatively unsubstituted x-axis terminal position.

The dioxin-inducible transcriptional control mechanism for the mouse CYP1A1 gene in its native chromosomal context was recently evaluated by Ko et al. (1996). The C-terminal segment of the AhR was shown to contain latent transactivation capability and to communicate the induction signal from enhancer to promoter. Heterodimerization activates the latent transactivation function of the AhR and silences that of Arnt since removal of Arnt's transactivation domain

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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does not affect dioxin-induced CYP1A1 transcription in vivo. Dioxin-induced changes in chromatin structure at the CYP1A1 enhancer and promoter may occur by unique mechanisms.

Rowlands et al. (1996) used a series of fusion proteins with a heterologous DNA binding domain to study the trans activating function of the human AhR and Arnt proteins in yeast. The results of these studies confirmed that human AhR and Arnt both contain carboxyl-terminal TADs. The AhR has a complex TAD composed of multiple segments that function independently, exhibit varying levels of activation, and cooperate to induce synergistic activation of transcription. In the absence of a DNA binding domain, the AhR and Arnt TAD probably inhibit activated and basal transcription because of selective binding of basal transcription factors, the TATA-binding protein and TFIIF. Thus, activation of target gene expression by AhR-Arnt may involve direct interactions with basal transcription factors. However, Henry et al. (1997a) published data suggesting that a purified TCDD-AhR complex retains both specific DNA binding and transcriptional activities in the absence of other factors. Purified and partially purified receptors gave a similar footprint of interaction with G-residues within the AhRE consensus sequence and were able to stimulate transcription from a AhRE-containing template in a cell-free system in the presence of HeLa cell nuclear extract.

Biological Consequences of Activation The ability of TCDD to influence patterns of gene expression involves modulation of transcriptional and posttranscriptional events. Such responses are often mediated by the AhR and thus exhibit considerable tissue and cell specificity. However, evidence continues to accumulate to suggest that some actions of TCDD may be independent of the AhR. Hoffer et al. (1996) examined mouse hepatoma Hepa-1 cells to analyze the mechanism of fos/jun activation by TCDD. Their results suggested that TCDD induces expression of the immediate early-response genes fos and jun by activation of three separate signal transduction pathways, at least one of which does not require a functional AhR complex. The serum response elements (SRE) mediate the response of c-fos to TCDD in serum in a dose-dependent manner independent of the AhR. The SRE also mediates c-fos induction by growth factors, cytokines, UV irradiation, oxidants, and other stimuli that activate mitogen-activated protein kinases (MAPKs). It was proposed that activation of the SRE ternary complex by TCDD may be initiated by a signal triggered at the cell surface that precedes binding of TCDD to the cytosolic AhR.

Selmin et al. (1996) used the differential display technique to identify genes regulated by TCDD. Sequencing of one of the differentially expressed clones showed 71 percent homology with the transmembrane domain of the precursor for the interleukin-6 receptor and a conserved consensus sequence found in the cytokine growth factor-prolactin receptor superfamily, respectively. TCDD appears to modulate cytokine expression and function in multiple systems and with

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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some degree of selectivity. The effect of TCDD on a series of TGF-β2 (transforming growth factor β2) gene promoter deletions ranging from 1,391 to 64 base pairs upstream of the transcription start site was investigated by Lee et al. (1996) to identify the region necessary for down-regulation by TCDD. The effect appears to be localized to the TATA box and is dose dependent, with saturation kinetics maximal by 10 nM and complete by 24 hours. TCDD can modulate gene transcription by acting on the transcription initiation complex via a tyrosine kinase-dependent pathway. These data support an alternative mechanism by which TCDD can alter gene expression, an indirect route mediated by the AhR complex through a tyrosine kinase-dependent pathway affecting the transcription initiation complex. Signaling interactions between AhR and other growth pathways are described below.

Ma and Whitlock (1996) reported that AhR-deficient Hepa 1c1c7 cells exhibited a different morphology, decreased albumin synthesis, and a prolonged doubling time relative to wild-type counterparts. Introduction of AhR cDNA into deficient cells by stable transfection induced acquisition of the wild-type phenotype. Conversely, introduction of antisense AhR cDNA into wild-type cells induced the AhR-deficient phenotype. Flow cytometric and biochemical analyses suggest that the slow growth rate of AhR-deficient cells reflects prolongation of G1, suggesting a link between AhR and the G1 phase of the Hepa 1c1c7 cell cycle. Also of significance was the fact that the effects of AhR occurred in the absence of TCDD and thus may represent responses to an endogenous AhR ligand.

AhR Signaling Interactions Evidence has continued to accumulate during the past two years that AhR interactions with other signal transduction pathways are complex and often redundant in nature. The complexity of AhR signaling interactions in evident from the large number of proteins that interact with the AhR to influence growth and differentiation, redox signaling, modulation of kinase activities, and estrogen receptor signaling. New data with respect to these areas are discussed below.

Growth and Differentiation Signaling During the reference period, evidence continued to lend support to the fact that the AhR signal transduction pathway is involved in the regulation of development and growth. Enan and Matsamura (1996) described the ability of TCDD to increase protein-tyrosine kinase activity in the cytosol of male guinea pig adipose tissue, an effect believed to be AhR dependent. The protooncogene c-Src, involved in growth control, was reported to be associated with the AhR-protein complex in cytosol from adipose tissue as well as in liver of guinea pig and C57BL/6J mice and NIH 3T3 mouse fibroblasts. The c-Src protein is functionally attached to the AhR and specifically activated on ligand binding.

Wanner et al. (1996) have shown that the transcript levels of the AhR and Arnt increase as a function of differentiation in a human keratinocyte cell line. In

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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situ hybridization studies established that in normal human skin, AhR expression is absent in proliferating basal cells and increases in the upper cell layers as differentiation progresses. In agreement with these correlations, AhR expression in differentiation-deficient hyperproliferative psoriatic skin is markedly decreased. When keratinocytes were continuously treated with retinoic acid (RA), the upregulation of AhR and Arnt mRNA levels was inhibited as was keratin 4 expression, a marker of keratinocyte differentiation. In contrast, treatment of already differentiated cells with RA did not down-regulate transcript levels. The mRNA levels of retinoic acid receptors in keratinocytes, RAR gamma and RXR α, were not influenced by the process of differentiation or by the addition of RA. These data suggest that the regulation of AhR, Arnt, and keratin 4 expression by RA is mediated via an indirect mechanism. To examine interactions between retinoid and AhR signaling, Fiorella et al. (1995) evaluated RA metabolism in microsomes from four retinoid-responsive tissues in male Sprague-Dawley rats three days after a single exposure to TCDD (80 μg/kg, i.p.). Microsomes from all four tissues catalyzed increased rates of RA metabolism, with a rank order of induction of liver > lung = kidney = testis. These data were interpreted to suggest that one aspect of TCDD toxicity involves alterations in the metabolism of RA.

Significant interactions between TCDD and TGF-β1 have been described by several investigators. Dohr et al. (1997) reported that basal mRNA expression of CYP1A1, CYP1B1, and AhR, as well as inducible CYP1A1 expression, is down-regulated by TGF-β1 in cells treated with TCDD. In contrast, mRNA expression of the AhR partner protein Arnt was not influenced. Treatment of cells with cycloheximide led to superinduction of TCDD-induced CYP1A1 and CYP1B1 mRNA expression and abolished the inhibitory effect of TGF-β1 on basal as well as TCDD-induced CYP1 and AhR mRNA expression. These results suggest that TGF-β1 induces rapid transcription and translation of an as-yet-unknown negative regulatory factor or factors that may directly regulate expression of the AhR and genes of the Ah gene battery at the transcriptional level.

TCDD and related chemicals also interact with insulin, a hormone involved in the growth regulation of several cells. Lu et al. (1996b) reported that TCDD and TCDF inhibit insulin-induced cell proliferation and DNA synthesis in MCF-7 cells, a response blocked by α-naphthoflavone, a partial AhR antagonist. TCDD alone did not affect Kd and Bmax values for binding of insulin to the receptor. However, the insulin-induced Kd value for insulin receptor ligand binding was decreased and the Bmax value was increased by TCDD cotreatment. TCDD elevated insulin receptor mRNA levels and inhibited several other insulin-induced responses including c-fos protooncogene expression, phosphorylation of the insulin receptor, and a 185 kDa protein in MCF-7 cells.

Redox Signaling The discovery that the oxygen-regulated transcription factor HIF-1α and the AhR share a common heterodimerization partner Arnt (HIF-1β) has fueled intensive investigation into the possible cross-talk between

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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oxygen and dioxin signal transduction pathways. HIF-lα is activated following changes in O2 concentration and translocates to the nucleus to form a heterodimer with Arnt. This complex activates gene expression by binding to a hypoxia-responsive element. All of these transcription factors (HIF-1α AhR, Arnt-HIF-1β) share an N-terminal bHLH region followed by a PAS domain, as described previously for the AhR and Arnt. Gradin et al. (1996) demonstrated that HIF-1α required Arnt for DNA binding in vitro and functional activity in vivo. Both the bHLH and the PAS motifs of Arnt were critical for dimerization with HIF-lα. Strikingly, HIF-1α exhibited very high affinity for Arnt in coimmunoprecipitation assays in vitro, resulting in competition with the ligand-activated AhR for recruitment of Arnt. These findings have implications for risk assessment in that TCDD itself may induce alterations in the redox status of cells.

Consistent with these observations, activation of HIF-1α function in vivo or overexpression of HIF-1α inhibited ligand-dependent induction of DNA binding activity and AhR function on minimal reporter gene constructs. However, HIF-lα-and AhR-mediated signaling pathways were not mutually exclusive since activation of AhR did not impair HIF-lα-dependent induction of target gene expression. Both HIF-lα and Arnt mRNAs are expressed constitutively in a large number of human tissues and cell lines, and steady-state expression levels is not affected by exposure to hypoxia. HIF-lα is associated with hsp90. Given the critical role of hsp90 for ligand binding activity and activation of the AhR, it is therefore possible that HIF-1α is regulated by a similar mechanism, possibly by binding an as-yet-unknown class of ligands.

Gassmann et al. (1997) showed that Arnt is indispensable for hypoxia-inducible HIF-1 DNA binding as well as for oxygen-regulated reporter gene activity medicated by the hypoxia response element present in the 3' enhancer of the erythropoietin gene (EPO 3'). Hypoxic induction of the vascular endothelial growth factor (VEGF) gene, however, was only partially abrogated in Hepa 1 C4 cells, suggesting that an HIF-1-independent oxygen signaling pathway is present. De novo translation, phosphorylation, and redox processes seem to be involved in hypoxic HIF-1α and probably also Arnt activation.

Yao et al. (1995) studied the effect of TCDD and BP on the transient expression of a chloramphenicol acetyltransferase (CAT) reporter gene linked to the promoter sequences in the long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1). Induction of a functional CYP1A1 monooxygenase by TCDD stimulates a pathway that generates thiol-sensitive reactive oxygen intermediates, which in turn are responsible for the TCDD-dependent activation of genes linked to the LTR. NFκB and an adjacent AhRE are required for TCDD-dependent CAT expression. In addition, mutation of the NFAT/AP-1 binding sites in the negative regulatory region of the promoter increases the magnitude of the TCDD effect. In related studies, the inducibility of three phase II genes in the mouse dioxin-inducible Ah battery: Nmol [encoding reduced nicotinamideadenine dinucleotide [NAD(P)H]: menadione oxidoreductase], Ahd4 (encoding

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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the cytosolic aldehyde dehydrogenase ALDH3c), and Ugtl*06 (encoding UGT) were evaluated by Vasiliou et al. (1996). Increases in Ah phase II gene expression in the 14CoS/14CoS mouse correlated with the electrophile response element (EpRE) found in the 5'-flanking regulatory regions of these genes. Gel mobility shift assays with a synthetic oligonucleotide probe corresponding to the EpRE showed that EpRE binding proteins are more than twice as abundant in 14CoS/ 14CoS than in the wild-type ch/ch nuclear extracts. Competition studies of EpRE-specific binding with an excess of EpRE, mutated EpRE, AP-1, AhRE3, mutated AhRE3, and C/EBP α-oligonucleotides suggest that several common transcriptional factors bind to the EpRE and AhRE3 motifs. Two monospecific antibodies to the AhR protein block formation of an EpRE-specific complex on gel mobility electrophoresis, suggesting that AhR or an AhR-related protein might be an integral part of the EpRE binding transcriptional complex associated with the oxidative stress response.

Protein Kinases Reports linking TCDD to the modulation of kinase activities continued to appear during the reference period. These interactions are reciprocal since evidence was also published that various kinases directly modulate AhR function. Enan and Matsamura (1995b) reported that TCDD stimulates nuclear protein phosphorylation in explant tissue cultures within 10 minutes, a response that is followed by a substantial decrease in the level of total protein phosphorylation activity. Manganese-stimulated protein kinase was found to be the predominant type of nuclear protein phosphorylating activity affected by TCDD, with 60 percent of the total activity due to heparin-sensitive casein kinase II (CK II). TCDD was also found to increase protein-kinase C and microtubule associated protein 2 kinase activities as early as 15 minutes after treatment in isolated adipose tissue in culture. Changes in kinase activities are of biological significance since DNA binding activity of the transcriptional factor AP-1 increases, while c-Myc DNA binding activity decreases. Genistein, a specific protein-tyrosine kinase inhibitor, abolished the stimulatory effect of TCDD on the AP-1 binding activity, but not the DNA binding activity of c-Myc. Although TPA (12-O-tetradecanoylphorbol 13-acetate), a phorbol ester, increased the binding activity of AP-1 and c-Myc, TCDD in combination with TPA caused a slight reduction in the binding activity of both transcriptional factors. In the presence of forskolin, the stimulatory effect of TCDD on AP-1 binding and the inhibitory effect on c-Myc were still apparent. Okadaic acid almost abolished the binding activity of c-Myc, whereas in combination with TCDD a stimulatory effect was found. Thus, TCDD regulates the DNA binding activity of AP-1 and c-Myc by modulating their phosphorylation status through alterations in protein kinase and phosphatase activities. Incubation of a nuclear-free subcellular homogenate of guinea pig adipose tissue with TCDD results in a significant elevation of protein kinase activity within 1-10 minutes (Enan and Matsamura, 1995b). The kinetics of this response is not consistent with the classic transcriptional mechanism of

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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action for TCDD, but interestingly, the actions of TCDD were blocked by AhR blockers. TCDD-induced increases in protein phosphorylation occurred mainly in cytosolic preparations devoid of nucleus, microsomes, and plasma membranes and were still observed in the presence of inhibitors of protein phosphatases. Furthermore, TCDD caused a rise in protein-tyrosine kinase activity in a purified AhR preparation, as well as in an isolated hsp complex preparation containing the AhR. This activation is unrelated to de novo protein synthesis. Evidence was also presented that this action of TCDD triggers the protein kinase-mediated growth factor signal transduction pathway (e.g., stimulation of mitogen-activated protein kinase 2 and tyrosine kinase activity). These results support the view that the TCDD-induced activation of protein kinases operates via a mechanism different from its conventional pathway involving changes in gene transcription in the nucleus. Finally, the association of c-Src protein kinase with AhR was described earlier (Enan and Matsamura, 1996).

Several earlier studies suggested that AhR phosphorylation may be critical in activation of the AhR to a DNA binding state. To further evaluate the functional role of AhR phosphorylation, Mahon and Gasiewicz (1995) investigated whether TCDD binding altered total AhR phosphorylation and identified phosphorylated regions in the receptor based on chemical cleavage patterns. The total level of AhR phosphorylation was not affected by ligand binding. The shortest regions of overlap determined by the chemical cleavage patterns localized phosphorylation sites to two regions in the C-terminal half of the AhR. One region was centrally located between amino acids 368 and 605 and within or adjacent to a DNA binding repressor domain. The other region was located at the glutamine-rich carboxyl terminus between amino acids 636 and 759. These data suggest that total AhR phosphorylation is not altered by ligand-induced transformation of the receptor, but that phosphorylation nevertheless plays an important role in the ability of an active AhR-Arnt complex to associate with cis-acting regulatory elements. This interpretation is consistent with studies showing that the activity of the AhR-Arnt dimer can be decreased by treatments that cause the down-regulation of protein kinase C and decrease nuclear accumulation of the receptor.

A reporter plasmid containing two xenobiotic responsive elements (XREs) was used to investigate the effects of phosphatase inhibitors on TCDD-dependent transcription by the Hepa-1 mouse liver cell line (Li and Dougherty, 1997). The inhibitors calyculin A and okadaic acid caused two-to threefold increases in TCDD-dependent transcription at concentrations capable of selectively inhibiting protein phosphatase 1 and protein phosphatase 2A. The inhibitor cyclosporin A doubled TCDD-dependent transcription at a concentration capable of selectively inhibiting protein phosphatase 2B. All three of the phosphatase inhibitors increased TCDD-dependent transcription without affecting transcription in the absence of TCDD. Nuclear extracts were prepared from cells treated with concentrations of okadaic acid or cyclosporin A that substantially stimulated TCDD-dependent transcription. Neither of the inhibitors significantly increased the level

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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of TCDD-dependent XRE binding in extracts. GAL4-Arnt fusion proteins were used to further investigate whether the phosphatase inhibitors affected a step other than DNA binding. Okadaic acid treatment specifically increased the ability of a GAL4 fusion protein containing the Arnt-PAS and transactivation domains to stimulate transcription. These results suggest that serine-or threonine-specific protein phosphatases can act at a level subsequent to XRE binding to inhibit the ability of the AhR-Arnt dimer to stimulate transcription.

Chen and Tukey (1996) examined the effects of phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C, on the ligand-induced transcriptional activation of the CYP1A1 gene and cellular function of the AhR in human HepG2 101L cells. Pretreatment of cells with PMA enhanced ligand-induced CYP1A1 gene expression two-to threefold. Inhibition of PKC activity blocked the transcriptional activation and transactivation of the CYP1A1 gene, indicating a role for PKC in the AhR-mediated transcriptional activation process. However, DNA binding activities of the in vitro activated and the induced nuclear AhR were not affected when CYP1A1 transcription was inhibited, indicating that the action of PKC is a nuclear event that works in concert with or precedes AhR binding to the gene. The effects of TCDD on growth factor-coupled activation of nuclear protein kinase C (nPKC) and on the subcellular distribution of PKC activity in rat splenocytes were investigated by Zorn et al. (1995). Seven days after a single injection of TCDD (50 fg/kg body weight), cytosolic and particulate PKC activities were elevated in splenocytes from TCDD-treated rats or pair-fed control rats compared to ad libitum-fed animals. Growth factor-stimulated nPKC activation was attenuated in splenic nuclei from TCDD-treated rats compared to vehicle-treated controls. Thus, TCDD may uncouple growth factor receptors linked to PKC activation at the level of the nucleus.

Estrogen Receptor Signaling Considerable research activity during the past two years has focused on reciprocal interactions between estrogen receptor signal transduction pathways and the AhR. These studies were fueled in part by the early observation that TCDD is a more potent hepatocarcinogen in female than in male or ovariectomized rats. Measurement of 8-oxodeoxyguanosine (8-oxo-dG), a marker for oxidative DNA damage, in livers of intact and ovariectomized Sprague-Dawley rats chronically treated with TCDD (125 ng/kg per day) with or without diethylnitrosamine as an initiator showed elevated levels of 8-oxo-dG in intact compared to ovariectomized TCDD-treated rats (Tritscher et al., 1996). Expression of CYP1B1 mRNA, a newly identified cytochrome P450 with estrogen hydroxylase activity, was highly induced in these animals by TCDD, suggesting that the increased metabolism of endogenous estrogens to catechols caused by TCDD-induced enzymes may lead to increased oxidative DNA damage and hepatocarcinogenicity in female rats.

TCDD and related hydrocarbons have also been identified as potent anti-estrogens, and this effect involves reciprocal interactions between the estrogen

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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and the AhR signal transduction pathway. For instance, Kharat and Saatcioglu (1996) have shown that the TCDD-mediated decrease in estradiol-inducible gene products, such as the cathepsin D gene (cat D), is due to a decline in mRNA accumulation despite changes in estrogen receptor (ER) mRNA levels. The decline in cat D mRNA levels likely involves decreased transcription rates since TCDD blocks the ability of ER to bind DNA and transactivate from an estrogen response element. Interestingly, TCDD does not function as an antiestrogen in mutant cells lacking a functional AhR. Likewise, estradiol treatment blocked TCDD-induced accumulation of CYP1A1 mRNA and AhR-mediated activation of the CYP1A1 promoter, due to the ability of liganded ER to interfere with the binding of AhR to the xenobiotic response element. Liu and Safe (1996) and Lu et al. (1996b) have shown that treatment of MCF-7 human breast cancer cells with TCDD decreases prolactin receptor (PRLR) mRNA levels within 12 hours after treatment, and for up to 48 hours, while PRLR binding is not affected. The effects of TCDD on PRLR mRNA levels were inhibited by the AhR antagonist α-naphthoflavone and were not observed in Ah-nonresponsive MCF-7 cells. TCDD antagonizes 17β-estradiol-induced increases in PRLR mRNA levels. Using MCF-7 or mouse Hepa 1c1c7 cells transiently transfected with E2-responsive Vit A2 gene 5'-promoter constructs, Nodland et al. (1997) showed that there was a correlation between the antiestrogenic activity of AhR ligands in MCF-7 cells and their rank order binding affinity for the AhR. A role for the AhR is suggested by the finding that α-naphthoflavone inhibited the antiestrogenic activity of TCDD in MCF-7 cells and TCDD inhibited E2-induced CAT activity in Ah-responsive wild-type, but not in Ah-nonresponsive, class 2 mutant Hepa 1c1c7 cells. The antiestrogenic activity of TCDD was also observed in cells that transiently overexpressed human ER, suggesting that the mechanism does not involve down-regulation of the ER by TCDD. In other studies, Krishnan et al. (1995) presented compelling evidence that AhR-mediated inhibition of estrogen-induced cat D gene expression is affected by disruption of the ER-Sp1 complex by targeted interaction with an overlapping XRE.

The interaction between TCDD and estrogen was evaluated by White et al. (1995) in weanling females Sprague-Dawley rats. Estrogen (10 μg/kg per day at days 21 and 22) increased relative uterine weight and induced keratinization of the vaginal epithelium. Estrogen reduced uterine ER protein levels and serum FSH levels. None of these parameters were affected by pretreatment with 20, 40, or 80 μg/kg TCDD on day 19. Given that other signs of TCDD toxicity were reproducibly observed in these rats, it was concluded that weanling female Sprague-Dawley rats are not sensitive to the antiestrogenic effects of TCDD at doses that cause overt toxicity. Collectively, these data suggest that the antiestrogenic effects of TCDD are species, strain, and age dependent.

Dohr et al. (1995) reported that TCDD inhibits cell growth and induces CYP1A1-associated EROD activity in MCF-7 cells that express the ER, but not in ER-negative MDA-MB 231 cells. Transcripts of CYP1B1 were detected in

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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both cell lines, and mRNA content was enhanced eight-and thirtyfold in MCF-7 and MDA-MB 231 cells treated with TCDD, respectively. In the gel mobility shift assay, a stronger signal of DNA binding AhR was observed in MDA-MB 231 than in MCF-7 cells treated with TCDD. A fortyfold higher AhR mRNA content was observed in untreated MDA-MB 231 than in MCF-7 cells, while the mRNA of the AhR nuclear translocator was expressed in a similar range of magnitude. Treatment of cells with TCDD did not change mRNA expression of either gene. Analysis of NADPH: quinone oxidoreductase (NMO-1) and PAI-2 mRNA expression revealed a dose-dependent induction of both genes in MDA-MB 231 cells after TCDD treatment. These studies strengthened the view that AhR-mediated transactivation is not impaired in ER-negative cells, but expression of ER is important for regulation of CYP1A1 induction after TCDD treatment in these human breast cancer cell lines. It was interesting to note, however, that ER does not appear to have a function in TCDD-induced mRNA expression of CYP1B1, NMO-1, and PAI-2 in MDA-MB 231 cells.

In summary, evidence published during the past two years indicates that the mechanisms of interaction between the TCDD-and E2-induced signaling pathways are highly complex. Some of the inhibitory effects of TCDD may involve 5'-flanking inhibitory AhREs present in target genes.

Significant Interactions Recent data suggest that TCDD and related hydrocarbons are not the only ubiquitous AhR agonists encountered by human populations. Several AhR agonists of dietary origin have been identified. For example, Kleman and Gustafsson (1996) described the formation of procarcinogenic heterocyclic aromatic amines, following cooking of protein-rich foods, capable of activating the AhR to a form that interacts with AhRE in vitro. Another group of putative dioxin receptor ligands of dietary origin involves the indolocarbazoles, produced in vivo from precursor molecules in cruciferous plants. Indolocarbazoles are potent regulators of the expression of a reporter gene driven by a minimal AhRE in both mouse and human hepatoma cells. The indolocarbazole-induced human receptor appeared to form more stable complexes with AhRE in vitro relative to those generated by the dioxin-activated receptor. Indolo-3-carbinol (I3C), a major component of Brassica vegetables, and its metabolite diindolylmethane (DIM) have been identified as partial AhR antagonists (Chen et al., 1996). Both compounds competitively bind to the AhR with low affinity. In Ah-responsive T47D human breast cancer cells, I3C and DIM do not induce EROD activity or CYP1A1 mRNA levels. However, cotreatment with TCDD plus different concentrations of I3C or DIM reduced the TCDD-induced response at high concentrations. In T47D cells cotreated with TCDD alone or in combination with I3C or DIM, there was a marked reduction in the formation of the nuclear AhR.

The induction kinetics and CYP1A1 mRNA half-life by the diet-derived indole derivative, indolo[3,2-b]carbazole (ICZ) are concentration dependent and transient due to rapid clearance of ICZ (Chen et al., 1995). TCDD and ICZ

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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displayed equal efficacies in the activation of a TCDD-responsive CAT (chloramphenicol acetyltransferase) reporter construct in Hepa 1 cells. ICZ is also a potent and selective noncompetitive inhibitor of EROD activity.

Significant interactions between TCDD-related AhR signaling and other environmental chemicals have long been described. In a recent study, the mechanism by which an ambient level of aged and diluted sidestream cigarette smoke (ADSS) induces CYP1A1 was investigated in C57BL/6N and DBA/2N mice, strains that exhibit high- and low-affinity forms of the AhR, respectively (Gebremichael et al., 1996). Induction of CYP1A1-associated EROD activity was observed in the lungs of C57BL/6N mice, whereas no induction occurred in DBA/ 2N mice. ADSS also induced EROD in wild-type mouse hepatoma (Hepa 1c1c7) cells (Hepa 1), but not in variant Hepa 1 cells defective in the Arnt protein. ADSS exposure of recombinant Hepa 1 cells stably transfected with a reporter plasmid containing the luciferase gene under the control of several dioxin-responsive enhancers resulted in a time- and exposure-dependent induction of luciferase activity. ADSS-mediated induction of luciferase activity was inhibited by α-naphthoflavone, an AhR antagonist. Exposure to ADSS induced transformation and DNA binding of the AhR complex. Collectively, these results indicate a role for the AhR in mediating the induction of CYP1A1 by ADSS and suggest that environmentally relevant levels of ADSS contain AhR ligands at sufficient concentrations to activate gene expression in an AhR-dependent manner.

Using 1- and 2-aminonaphthalene as model substrates, the influence of a second amino group on mutagenicity, binding to the cytosolic AhR, and CYP1A inducibility relative to the effects of 3,3'-diaminobenzidine and 1-naphthylethylenediamine were examined by Cheung et al. (1997). 1,5- and 1,8-Diaminonaphthalene were effective inducers of CYP1A activity and more potent than 1-aminonaphthalene. 2,3-Diaminonaphthalene was also an inducer of CYP1A, but the effect was similar to that elicited by 2-aminonaphthalene. In contrast, 3,3'-diaminobenzidine and 1-naphthylethylenediamine did not induce CYP1A activity. All aminonaphthalenes displaced radiolabeled-TCDD from the AhR, whereas 3,3'-diaminobenzidine and 1-naphthylethylenediamine failed to do so. The latter two compounds did not elicit a mutagenic response in the Ames test. Introduction of a second amino group at the 3-position of 2-aminonaphthalene did not modulate its mutagenicity. In the case of the nonmutagenic 1-aminonaphthalene, introduction of a second amino group at position 5 had no effect, but when it was incorporated at position 8, mutagenic potential was conferred on the molecule. The ability of substituted flavones to modulate AhR signal transduction in MCF-7 human breast cancer cells was evaluated by Lu et al. (1996b). The 4'-methoxy-3'-nitro- and 3'-amino-4'-methoxyflavones were characterized as AhR agonists and inducers of CYP1A1 gene expression, whereas the 3-methoxy-substituted flavones (3'-methoxy-4'-nitro- and 4'-amino-3'-methoxy-) were inactive. All four compounds inhibited induction of EROD activity by TCDD. These results were interpreted to suggest that two forms of the nuclear AhR complex exist in MCF-

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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7 cells and that 3-methoxy-substituted flavones inhibit nuclear uptake of the transcriptionally active form.

Disease Outcomes

Lethality TCDD lethality has been associated with changes in brain serotonin (5-HT) metabolism (Unkila et al., 1995b). This response was recently examined in the most TCDD-susceptible and TCDD-resistant species, guinea pigs and hamsters, respectively. Body weight gain of guinea pigs exposed to TCDD (0.2-2.7 μg/kg) diminished dose dependently, while the effect was marginal in hamsters (900-4,600 μg/kg). 5-Hydroxyindoleacetic acid (the primary metabolite of 5-HT), brain tryptophan (the precursor amino acid of 5-HT), and plasma-free and total tryptophan were not affected at any dose in guinea pigs. In contrast, four days after exposure, the levels of plasma-free and total tryptophan were increased in hamsters and, along with brain tryptophan, remained elevated ten days after exposure. TCDD did not affect plasma glucose level in either species. Liver glycogen was decreased in a dose-dependent manner in TCDD-treated guinea pigs, as well as in their pair-fed controls, on day 10. There was no change in liver glycogen in hamsters. The activity of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase was depressed only in hamsters by all doses of TCDD. Changes in tryptophan metabolism or in carbohydrate homeostasis cannot explain the wide interspecies differences in susceptibility to the acute lethality of TCDD, although they may correlate with some aspects of its toxicity in certain species.

Cells harvested six to eight days after TCDD intubation from pair-fed rats contained significantly more fat and higher levels of glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity. The mRNA for lipoprotein lipase (LPL) and GPDH genes was also higher for cells from pair-fed rats, suggesting that TCDD inhibits the differentiation of fat cells (Brodie et al., 1996). TCDD treatment in vivo inhibits the increase of mRNA for the PPAR (2, aP2 and C/EBP) during differentiation of isolated preadipocytes. C/EBPβ and CHOP mRNAs were unaffected. 3T3-L1 cells appear to provide a good model to study adipogenesis and the inhibition of this process by TCDD (Brodie et al., 1997).

Fan and Rozman (1995) completed studies to examine short-and long-term effects of TCDD in female LE rats. Female rats were dosed orally with either 5.3, 12, 18, and 60 μg TCDD/kg and sacrificed four days after dosing or 27, 40, and 60 μg TCDD/kg and sacrificed 90 days after dosing. Four days after dosing, EROD activity was fully induced at all doses studied, hepatic PEPCK and γ-glutamyl transpeptidase activities were reduced dose dependently, and hepatic tryptophan 2,3-dioxygenase (TdO) activity was stimulated at low doses but decreased at high doses. Serum total T4 (TT4) levels were dose dependently decreased, whereas serum total T3 (TT3) and tryptophan levels were unaffected. The short-term effects of TCDD examined indicate only small differences in the

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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response to TCDD of female LE rats compared to males. Ninety days after dosing, liver EROD activity revealed considerable reversibility although it was still elevated compared to controls. Hepatic PEPCK activity at this time was no different from controls. In contrast to four days after dosing, serum TT3, TT4, and hepatic glutamyl transpeptidase activity were elevated dose dependently at 90 days.

Male Sprague-Dawley rats were administered orally a total dose of 0, 0.2, 2.3, 11.5, 35, 70, or 115 μg/kg of TCDD over 10 weeks at 4 ml/kg of vehicle. Results show that dose-responses for the induction of EROD activity and the reduction of serum TT4 occurred at much lower doses than those for decreased TdO and PEPCK activities or elevated tryptophan levels and mortality. After a six-week recovery period, PEPCK and TdO activities in liver, as well as tryptophan in serum, returned to near-control values, whereas EROD activity and serum TT4 still displayed a dose-dependent induction and reduction, respectively, albeit both shifted to the right in accordance with toxicokinetics. These data support the interpretation that subchronic dose-responses of TCDD are similar to acute dose-responses when corrected for toxicokinetics and that at least some TCDD-induced effects are reversible (Li and Rozman, 1995).

In male mice treated with TCDD, body weights and feed intake were not much affected until day 8 after exposure (Weber et al., 1995). Hepatomegaly developed at doses greater than 3 and 97.5 μg/kg in C57 and DBA mice, respectively. EROD activity was induced in liver with a median effective dose (ED50) of 1.1 and 16 μg/kg, and in kidney with an ED50 of 65 and 380 μg/kg in C57 and DBA mice, respectively. The PEPCK in livers of both mouse strains was reduced over the entire dose range, displaying a plateau in the dose response at the onset of acute toxicity of TCDD. This enzyme activity was decreased by as much as 80 percent at the respective lethal doses. PEPCK activity in kidney was not affected. Glucose-6-phosphatase activity (G-6-Pase) in liver was altered only in the lethal dose range, with a maximum reduction of about 50 percent. Serum glucose concentration was reduced over the entire dose range, but the reduction was significant only at doses in which G-6-Pase activity was affected, reaching levels as low as 3 mmol per liter in DBA mice. Tryptophan-2,3,-dioxygenase activity was not lowered at any dose of TCDD in either mouse strain, and no increase in serum tryptophan levels was observed. Serum levels of T4 and T3 were decreased dose dependently over most of the dose range administered, with T3 levels exactly paralleling T4 levels in both mouse strains. These data were interpreted to suggest that TCDD causes acute toxicity in male C57 and DBA mice by a severe reduction of gluconeogenesis but, in contrast to rats, does not affect tryptophan homeostasis. After administration of TCDD, serum T3 levels in the mouse appear to correlate with T4 levels, whereas in the rat they are independent of each other.

Li et al. (1997) examined the effect of single doses (0.03-30 μg/kg) of TCDD administered orally by gastric intubation on serum hormone levels in female rats (22 days old). Two distinct peaks for LH and FSH were detected, the

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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first of which was seen at 1 hour and appeared to be a nonspecific response to the vehicle, and a second at 24 hours that appeared to be induced by TCDD. Gonadotropin levels in these animals were dose dependently elevated. In cultured pituitary halves and primary pituitary cell cultures exposed to gonadotropinreleasing hormone (GnRH) and/or TCDD, TCDD caused a dose-dependent release of LH from pituitary halves with an ED50 of about 0.1 nM. This effect was abolished in calcium-free medium but was not attenuated by a GnRH antagonist. In primary pituitary cell cultures, although the cells responded to GnRH, no effect of up to 100 nM TCDD on the release of gonadotropins was detected. These results suggest that TCDD dose dependently induces a brief release of gonadotropins in immature female rats. This effect is at least partially due to an effect of TCDD on the pituitary. Increased release of gonadotropins as a result of TCDD treatment depends on the action of calcium but does not occur via activation of GnRH receptors. However, cells in a primary pituitary culture do not respond to TCDD with increased release of gonadotropins, suggesting that the effect of TCDD in the pituitary is mediated by a factor present in pituitary halves but not in primary cell culture.

Dermal Toxicity Wanner et al. (1995) reported on the relative levels of AhR mRNA and TCDD-induced CYP1A1 mRNA and their modulation by retinoic acid in the human keratinocyte cell line HaCaT. AhR mRNA was present already in proliferating keratinocytes and increased eightfold in the course of differentiation. Addition of 10 nM TCDD did not alter the level of AhR transcripts. In contrast, addition of 1 μM retinoic acid (RA) maintained the amount of AhR mRNA at the basal level only in proliferating keratinocytes. The transcription of CYP1A1 was dependent on TCDD-treatment and increased fivefold in more differentiated cells compared to proliferating cells. Simultaneous addition of RA revealed only a twofold increase. These results indicate that expression of the AhR depends on the state of differentiation of keratinocytes and seems to be affected by retinoic acid.

As discussed previously, TCDD strongly induces a switch from proliferation to terminal differentiation in keratinocytes, which can be antagonized effectively by retinoic acid and retinol. As parameters for differentiation, the [35S]methionine incorporation into cross-linked envelopes (revealing the total CLE) was quantified by. TCDD is a potent inducer of both CLE biomass and number with an EC50 of 1.4 nM. Both effects were dependent on Ca2+ and increased with elevated cell density, being optimal in postconfluent cultures. Retinoic acid dose dependently decreased the effect of 10-8 M TCDD, 10-6M having a nearly complete antagonistic action. Retinyl palmitate and etretinate were not effective as TCDD antagonists. Supplementation of hydrocortisone suppressed TCDD-induced keratinocyte differentiation (REF). Reduced increase in cell number and diminished cell biomass are the result of early withdrawal of proliferating cells from the cell cycle due to premature and accelerated induction of differentiation. TCDD-induced

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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differentiation in human keratinocytes may be initiated by TCDD binding to the AhR. The exact nature of the interaction between retinoids and TCDD is not clear, but may involve interactions in the regulation of epithelial differentiation.

Cardiovascular Toxicity Several reports appeared during the reference period describing developmental deficits in the cardiovascular system of TCDD-treated animals. Endothelium is a single-cell layer lining that could be a primary site of chemical effects in the cardiovasculature and systemically.

CYP1A expression and activity in cultured porcine aortic endothelial cells (PAEC) exposed to the AhR agonists TCDD, 3,3',4,4'-tetrachlorobiphenyl (TCB), BP, or β-naphthoflavone (BNF) were evaluated by Stegeman et al. (1995). CYP1A1 was induced in cultures exposed to TCDD, TCB, BP, or BNF. Gene induction was observed at intermediate concentrations (0.1 or 1.0 μM) of TCB, BP, or BNF but inhibited at higher concentrations. The suppression response was not due to generalized cytotoxicity. Immunohistochemical analysis showed that CYP1A1 induction in PAEC was not present in all cells. PAEC exhibited a typical complement of microsomal electron transport components. NADPH-cytochrome P450 reductase showed comparable rates in induced and control cultures, and the addition of purified rat reductase to PAEC microsomes increased EROD rates threefold. EROD rates in intact cells maximally induced by BP, TCB, or TCDD ranged from 15 to 30 pmol/mg per minute of whole-cell protein. Methoxyresorufin O-demethylase activity induced by TCDD was 2 pmol/ mg per minute (i.e., < 10 percent of EROD activity). In cultures in which CYP1A1 was strongly induced, CYP1A2 was not detectably expressed. The CYP1A2 inducer acenaphthylene did not induce EROD or methoxyresorufin O-demethylase in intact cells. Results show that CYP1A1 but not CYP1A2 is strongly induced in mammalian endothelial cells in culture and that CYP1A1 is active in intact cells, although the catalytic rates are low.

A recent study by Guiney et al. (1997) examined CYP1A induction in endothelium and its possible association with mortality due to the edema and vascular effects of TCDD in lake trout early life stages. Lake trout eggs were injected at 24-50 hours postfertilization with 0.2 μl of 50 mM phosphatidylcholine liposomes or liposomes containing TCDD to give seven doses ranging from 11 to 176 pg TCDD/g egg. Doses of TCDD greater than 44 pg/g egg elicited hemorrhages; yolk sac, pericardial, and meningial edema; craniofacial malformations; regional ischemia; growth retardation; and mortality at the sac fry stage of development. Expression of CYP1A was assessed at four developmental stages, by immunohistochemical analysis of serial sections of individual fish with monoclonal antibody 1-12-3 to teleost CYP1A. CYP1A staining occurred in endothelial cells of many organs of TCDD-exposed but not vehicle-exposed embryos at one week prehatch and sac fry at two weeks posthatch. Earlier developmental stages examined were negative for CYP1A expression at any dose of TCDD. The strongest response occurred in sac fry at TCDD doses greater than 88 pg TCDD/g egg but

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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was detected at doses as low as 22 pg TCDD/g egg. CYP1A staining in endothelium appeared at lower doses and was stronger than in other cell types in both prehatch embryos and posthatch sac fry. Thus, the vascular system is a major initial site affected by TCDD in lake trout early life stages, and the vascular endothelium is a cell type uniquely sensitive to induction of CYP1A in these developing animals. Based on an index of immunohistochemical staining of CYP1A, endothelial CYP1A induction in sac fry by TCDD occurred with an ED50 of 64-69 pg TCDD/g egg, similar to the dose-response for mortality occurring during the sac fry stage of development (LD50 [median lethal dose] = 47 pg TCDD/g egg). These correlations suggest that CYP1A or AhR in the endothelium may be linked to early lesions that result in TCDD-induced vascular derangements leading to yolk sac, pericardial, and meningial edema associated with lake trout sac fry mortality, but the precise mechanism remains to be determined.

Cantrell et al. (1996) characterized embryotoxicity in medaka (Oryzias latipes) by TCDD. DNA degradation in cells of the embryonic vasculature and loss of functional integrity of the medial yolk vein were demonstrated in TCDD-exposed embryos. Piperonyl butoxide inhibited TCDD-induced DNA degradation, restored the functional integrity of the medial yolk vein, and protected against the embryotoxicity of TCDD. Treatment of TCDD-exposed embryos with the antioxidant N-acetylcysteine also provided significant protection against the embryotoxicity of TCDD. These results demonstrate that DNA damage and consequent cell death in the embryonic vasculature are key physiological mediators of TCDD-induced embryotoxicity and implicate oxidative mechanisms in this response.

Celander and colleagues (1997) examined CYP1A1 induction in cultures of porcine aortic endothelial cells and human aortic endothelial cells exposed to TCDD with or without the glucocorticoid receptor (GR) agonist cortisol or dexamethasone. In porcine cells exposed to 0.1 nM TCDD and 10 μM cortisol, the level of CYP1A1 protein and the degree of EROD activity induction were two-to threefold higher than with 0.1 nM TCDD alone. A similar enhancement of EROD induction was obtained when 0.1 or 1 nM TCDD was added together with 0.1, l, or 10 M dexamethasone in the media. Human cell counterparts also showed potentiated EROD induction when 1 nM TCDD was coadministered with 10 μM dexamethasone. This potentiation caused by dexamethasone was abolished by the addition of 10 μM of the GR antagonist RU-38486, suggesting that potentiation of CYP1A1 induction in endothelial cells proceeds by a GR-dependent mechanism. The implications of these data have not been fully defined but demonstrate that vascular cells are responsive to TCDD.

Renal Toxicity Kraemer and colleagues (1996) examined the molecular mechanisms for TCDD-stimulated prostaglandin synthesis in Mardin Darvey canine kidney (MDCK) cells. TCDD stimulated prostaglandin synthesis in these cells, at least in part by elevation of prostaglandin endoperoxide H2 synthase-2 (PGHS-2)

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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levels. TCDD-stimulated transcription of the PGHS-2 gene was maximal (six-fold) within two hours and resulted in a hundredfold increase in PGHS-2 mRNA and a twenty-five-fold increase in PGHS-2 protein levels by four hours. Transient transfection experiments using luciferase-reporter plasmids demonstrated that the control element(s) responsible for TCDD activation of the murine PGHS-2 promoter in MDCK cells are located in the first 965 nucleotides upstream from the PGHS-2 transcriptional initiation site. A canonical xenobiotic response element, similar to those that control transcription of other well-known TCDD-sensitive genes, is present at position -157, but does not appear to be sufficient for halogenated aromatic hydrocarbon activation of the PGHS-2 promoter. TCDD failed to stimulate transcription from the PGHS-2 promoter when reporter plasmids were transfected into Hepa 1c1c7 cells, a line that contains the functional AhR. It seems likely that inappropriate expression of PGHS-2 may contribute to the toxic effects of TCDD and other halogenated aromatic hydrocarbons (HAHs). In particular, PGHS-2 expression may affect those toxic reactions that involve inappropriate cellular growth, such as dermal hyperplasia and tumor formation. It is also likely that elevated synthesis of prostaglandins, which are potent regulators of immune function, could play a role in the immunotoxicity associated with HAH exposure.

Hepatotoxicity Schuetz et al. (1995) conducted studies to determine if aromatic hydrocarbons induce the mdr gene product P-glycoprotein and whether this induction involves the AhR. Induction of mdr mRNA was compared to the induction of CYP1A1 mRNA in Ah-treated cultures of primary human hepatocytes. Hepatocytes from all 15 individuals tested responded to 3-methylcholanthrene (MC) or TCDD with induction of CYP1A1 mRNA. However, only 62 percent and 55 percent of the preparations responded to treatment with MC and TCDD, respectively, with induction of mdr mRNA. Indeed, in some individuals, mdr mRNA was suppressed by MC and TCDD despite robust CYP1A1 induction. These studies suggest not only that individual variations exist in mdr induction by AH but that aryl hydrocarbons regulate mdr in humans by a novel mechanism distinguishable from the classical AhR pathway.

In a 13-week feeding study in female Sprague-Dawley rats by Van Birgelen et al. (1995), diets were supplemented with 0, 0.2, 0.4, 0.7, 5, or 20 fg TCDD/kg diet. The estimated daily intakes were calculated to be 0, 14, 26, 47, 320, or 1,024 ng TCDD/kg body weight per day. The lowest estimated daily intake associated with increased liver weights was 320 ng TCDD/kg, while a daily intake of 47 ng TCDD/kg resulted in decreased plasma thyroid hormone concentrations and decreased body weight gain. Decreases in relative thymus weights, loss of hepatic retinoids, and induction of CYP1A1 and CYP1A2 activities were found at 14 ng/ kg, the lowest dose used. For increases in CYP1A1 and CYP1A2 activities, the right critical values for the 95 percent confidence intervals for the no effect levels (CNELs) ranged from 0.7 to 4 ng TCDD/kg per day. Based on hepatic TCDD

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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residue levels, these right critical values for CNELs ranged from 0.06 to 0.4 ng TCDD/g liver (wet weight). The CNELs in this study are consistent with the NOAELs reported before in chronic and reproductive studies with rats and TCDD (i.e., 1 ng/kg per day).

The basis for the regional specificity of TCDD in the modulation of hepatocyte proliferation that results in enhanced proliferation in the periportal region, but reduced proliferation in the remainder of the hepatic lobule, is not known. Hushka and Greenlee (1995) have shown that TCDD caused a dose-dependent inhibition of DNA synthesis in primary hepatocytes isolated from either male or female Sprague-Dawley rats in the presence or absence of known hepatocyte mitogens (epidermal growth factor [EGF], hepatocyte growth factor, and TGF-α). No change in DNA synthesis was observed at TCDD concentrations less than 1 pM. Initial characterization of the EGF response system in these cells revealed that TCDD did not alter the specific binding of EGF, or the levels of EGF receptor protein measured in intact cells or cell lysates. TCDD-dependent inhibition of DNA synthesis occurred independently of the suppression observed with TGF-β1. Estradiol did not alter DNA synthesis in the presence or absence of TCDD. Taken together, these findings indicate that TCDD suppresses DNA synthesis via a novel pathway that is nonresponsive to estradiol, indepeéndent of TGF-β, and does not involve a decreased ability of hepatocytes to recognize (bind) EGF, a prototype mitogen.

Zhao and Ramos (1995) arrived at the same conclusions using primary cultured rat hepatocytes. Scheduled DNA synthesis in control cultures peaked at 64 hours and was negligible by 72 hours after initial seeding of freshly isolated hepatocytes. A concentration-dependent inhibition of DNA synthesis was observed in one-day-old hepatocyte cultures treated with BP (0.3-30 μM) for up to 28 hours, and comparable inhibitory responses were observed in cultures treated for 24 hours with TCDD (0.01 nM) or TCDF (0.01-1 nM) but not in cultures treated with perylene (0.01-100 nM) or benzo[e]pyrene (1-1,000 nM). EROD activity was highly inducible in hepatocytes challenged for 24 hours with BP (0.3-3 μM) or TCDD (0.1-100 nM), with peak induction at 12 or 36 hours after chemical challenge, respectively. To assess the role of the AhR in this response, the interactions of α-naphthoflavone (α-NF) and ellipticine (ET) with BP and TCDD in this cell system were evaluated. Pretreatment with α-NF (10 nM) for 24 hours prevented the inhibitory effects of both BP (3 μM) and TCDD (1 nM), whereas ET (0.01 nM) pretreatment selectively antagonized the effects of BP (3 μM). Pretreatment of hepatocytes with TCDD or TCDF (1 nM) for 24 hours before the onset of DNA synthesis, followed by challenge with BP (3 μM), partially antagonized the inhibitory response to BP. These data implicate AhR-related signal transduction in the inhibition of hepatocyte DNA synthesis by BP and related agents, and suggest that in the case of BP, metabolism by cytochrome P450 to toxic intermediates contributes to the inhibitory response.

Administration of TCDD to rats results in a dose-dependent decrease in

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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hepatic plasma membrane epidermal growth factor receptor (EGFR). Sewall et al. (1995a) monitored alterations in hepatic EGFR levels in female Sprague-Dawley rats seven days after a single oral gavage dose of TCDD (0, 1, 5, 25, and 50 fg/kg). The level of hepatic EGFR was significantly decreased at a dose of TCDD as low as 1 fg/kg. Thus, TCDD decreased total EGFR protein and maximum binding capacity without altering ligand binding affinity (Kd). The results demonstrated that ligand-induced autophosphorylation capacity and basal phosphotyrosine residues of plasma membrane EGFR both decreased parallel with the decrease in EGFR protein, suggesting no TCDD-related alteration in the inherent functional ability of the receptor to undergo activation. Furthermore, it was found that the dose-response curve for EGFR protein level determined by Western blot analysis was similar for both male and female Sprague-Dawley rats.

In other studies, Ilian et al. (1996) observed that day 8 post-TCDD treatment is associated with a dose-dependent reduction of hepatic pyruvate carboxylase (PC) mRNA levels in Ahb/b mice. This response was tenfold greater than in congenic Ahb/b mice, suggesting that previously reported reduction in PC activity by TCDD treatment of mice is a consequence of a reduction in PC mRNA levels and that the effect requires a competent AhR. TCDD inhibits normal accumulation of vitamin A in hepatic stellate cells, the main storage site for vitamin A. TCDD-induced inhibition of hepatic vitamin A accumulation does not seem to involve a reduction in the number of stellate cells or cell transformation (Hanberg et al., 1996). TCDD acts as a promoter of lesions initiated either spontaneously or by vinyl carbamate. TCDD overrides the intrinsic resistance of both male and female C57BL/6 mice to liver tumor formation (Watson et al., 1995).

Xiao et al. (1995) tested the effects of glucocorticoids on the expression of a number of genes under the control of the AhR in cultured primary rat hepatocytes. Treatment of cultured hepatocytes with 1.0 gM dexamethasone potentiated the induction of CYP1A1, glutathione S-transferase Ya subunit (GSTYa), and UDP-glucuronosyltransferase gene expression by polycyclic aromatic hydrocarbons, whereas the glucocorticoid agonist suppressed PAH induction of NAD(P)H: quinone oxidoreductase (QOR) subunit and aldehyde dehydrogenase 3C gene expression. Two of these rat genes, GSTYa and QOR are also induced by electrophilic agents, such as tert-butylhydroquinone. In the presence of tert-butyl hydroquinone, dexamethasone caused a similar level of potentiation of GSTYa subunit expression and suppression of QOR subunit expression as observed with the PAH 1,2-benzanthracene. Studies using the glucocorticoid receptor antagonist RU-38486 demonstrated that the modulation of PAH induction by glucocorticoids of CYP1A1 and QOR activity apparently depends on the action of the glucocorticoid receptor. These results suggest that the positive and negative changes observed are the result of specific alterations in the rates of transcription of these genes because of the action of the glucocorticoid receptor, thereby affecting regulation of GSTYa and QOR by both AhR-dependent and AhR-independent mechanisms.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Although hepatic uroporphyria is induced by HAHs in mammalian and avian systems, attempts to produce uroporphyria in vertebrate (mammalian) hepatoma lines have been unsuccessful. Dose-dependent accumulation of porphyrins was observed in cells treated for 48 hours with TCDD or 3,3',4,4'-tetrachlorobiphenyl when the heme precursor δ-aminolevulinic acid (ALA) was present during the last 5 hours of treatment (Hahn and Chandran, 1996). Uroporphyria did not occur in cells treated with TCDD or TCB in the absence of added ALA. ALA-dependent porphyrin accumulation was also seen following treatment of PLHC-1 cells with TCDF or with the non-ortho-substituted chlorobiphenyls 3,4,4',5-tetrachlorobiphenyl and 3,3',4,4',5-pentachlorobiphenyl. The EC50 values for porphyrin accumulation were similar to, or slightly higher than, the concentrations at which peak EROD activities were obtained, suggesting a relationship between the decline in EROD activity and enhanced porphyrin accumulation. α-Naphthofla-vone inhibited TCDD-induced EROD activity and porphyrin accumulation, providing further evidence for the involvement of a fish CYP1A in the mechanism of this porphyria. Addition of TCB to TCDD-treated cells also inhibited EROD activity, but enhanced porphyrin accumulation, suggesting that an interaction between the halogenated inducer and the induced CYP1A is necessary for the porphyrogenic response.

Lorenzen et al. (1997) observed concentration-dependent induction of CYP1A and intracellular porphyrin accumulation following treatment of chicken embryo hepatocyte cultures with TCDD and related chemicals. Maximal CYP1A activity (measured as EROD activity) and immunodetectable protein were observed at concentrations coincident with those at which porphyrin accumulation became evident. These results are consistent with a role of CYP1A induction and/or AhR activation in porphyrin accumulation mediated by HAHs with a planar configuration.

Münzel et al. (1996) studied the modulation of DNA synthesis by TCDD in primary cultures of hepatocytes and rat liver epithelial cells (WB-F344). In hepatocytes, TCDD either positively or negatively modulated EGF-stimulated DNA synthesis. In the presence of ethinylestradiol, 10-12 M TCDD moderately increased EGF-stimulated DNA synthesis (approximately 30 percent). In contrast, in the absence of ethinylestradiols 10-9 M TCDD decreased DNA synthesis (approximately 30 percent). The response of ''early genes" of the jun/fos family and the corresponding proteins was also studied under these two conditions. In agreement with DNA synthesis data, the level of c-Jun was increased or decreased in nuclear extracts. Furthermore, DNA binding of Jun/Fos proteins, including c-Jun and Fra-1, was decreased under conditions of mitoinhibition, while the level of Fra-1 in nuclear extracts was increased. In WB-F344 cells, TCDD treatment for 44 hours increased DNA synthesis two-to threefold compared to controls, based on measuring radiolabeled thymidine incorporation into DNA or on determining the nuclear labeling index with bromodeoxyuridine. This effect is probably due to the inhibition of high-density growth arrest by TCDD.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Wasting Syndrome A prominent symptom of the acute toxicity of TCDD is the loss of adipose tissue and body weight, a phenomenon known as the wasting syndrome. The effect of TCDD on glucose transport in mice was examined by Liu and Matsamura (1995). A single i.p. dose of TCDD (116 μg/kg) resulted in a time-dependent decrease in transport activity in adipose tissue and brains of C57BL/6 mice. Reduction of transport occurred within 24 hours in both tissues. In adipose tissue, a slight recovery was observed by 30 days, but in the brains of treated animals, glucose transport was significantly decreased even at the latest time. A comparison of dose-response relationships for several tissues between C57BL/6 (TCDD responsive) and DBA/2J (TCDD-nonresponsive) mice resulted in parallel curves, with C57BL/6 animals showing a ten-to twentyfold greater sensitivity. The estimated ED50 values for reduction of transport in adipose tissue were 50 and 800 μg/kg for the C57BL/6 and DBA/2J strains, respectively. Immunoblotting for the adipose-type (type 4) glucose transporter (GLUT) showed a 40 percent decrease in the membrane fraction of adipose tissue from C57BL/6 mice treated with 116 μg/kg TCDD for 40 hours. A similar decrease in brain-type GLUT1 was observed in the plasma membrane fraction of brain tissues isolated from the same animals. Analysis of RNA for the corresponding GLUT4 and GLUT 1 genes showed a dramatic decrease in GLUT4 mRNA as early as 24 hours after treatment. In contrast, the level of GLUT1 mRNA increased slightly in the brains of treated mice. Based on these data it was concluded that regulation by TCDD of glucose transport activity in mice is an AhR-dependent process and that adipose-type GLUT4 appears to be regulated at the mRNA level, whereas brain-type GLUT1 is affected mainly at the protein level.

Enan and colleagues (1996) investigated the involvement of the EGFR and protein-tyrosine kinase (PTK) in TCDD-induced toxicity. Up-regulation in radiolabeled EGF binding to EGFR was measured after 24 hours of TCDD treatment, whereas down-regulation in EGFR binding was measured after 72 hours of TCDD treatment. Up-regulation of EGFR binding was associated with a significant decrease in postnuclear (7,000 × g supernatant) PTK activity, but this activity was stimulated after 72 hours of TCDD treatment. TCDD altered the level of tyrosine phosphorylation in proteins with molecular weights of 34, 40, 43, 45, 60, and > 205 kDa. TCDD caused a significant increase in postnuclear cAMP-dependent protein kinase A (PKA) after 24 hours of treatment. The action of TCDD on protein kinases was partially blocked by the protein synthesis inhibitor cycloheximide. TCDD increased nuclear PTK and decreased nuclear PKA activity. Estradiol (E2) inhibited the postnuclear and nuclear activity of both PTK and PKA in control samples, but did not affect the action of TCDD on either postnuclear or nuclear PTK activity. However, E2 abolished the stimulatory effect of TCDD on PKA activity in postnuclear protein. In the presence of insulin, TCDD did not induce any additional changes in postnuclear or nuclear PTK. Forskolin alone inhibited postnuclear PTK activity and stimulated its nuclear activity. Addition of TCDD 20 minutes after forskolin

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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resulted in an increase in postnuclear PTK, but there was little change in nuclear PTK compared to the effect of forskolin alone. The stimulatory effect of TCDD on postnuclear PKA activity was enhanced by insulin, and TCDD reversed the negative effect of forskolin, but there was no effect of either insulin or forskolin on the inhibition by TCDD of nuclear PKA activity. TCDD decreased the activity of MAP2 kinase and reduced the binding activity of AP-1 DNA when given alone; it also blocked E2 stimulation of MAP2K. These findings suggest that TCDD may interrupt the endocrine function of human luteinized granulosa cells through the blockage of the mitotic signal directly or indirectly through the interaction of PTK-MAP2K and PKA signaling.

It had been reported that TCDD dose dependently reduces the activity of PEPCK, the rate-limiting enzyme of hepatic gluconeogenesis. To further investigate the mechanism by which TCDD decreases PEPCK activity, Stahl (1995) investigated the effect of TCDD on PEPCK activity in primary cultured rat hepatocytes. Cells were pretreated with dexamethasone (100 nM) 8 hours before PEPCK induction was initiated by the addition of glucagon (10 nM) and concurrent withdrawal of insulin. This hormonal treatment induced twofold elevation of PEPCK activity in control cells within 8 hours. Using this induction regimen, experiments were conducted in which rats were pretreated with TCDD (125 μg/ kg in corn oil by gastric intubation) four days prior to isolation of primary rat hepatocytes (PRH). This resulted in a complete block of the glucagon-dependent induction of PEPCK in PRH from TCDD-pretreated animals. In another experiment, TCDD (100 nM) was added directly to the PRH either 24 or 48 hours prior to the induction regimen. Incubation of PRG with TCDD 24 hours prior to initiation of the induction regimen resulted in a slight decrease in the degree of PEPCK induction compared to controls. However, treatment of PRH with TCDD 48 hours prior to initiation of the induction regimen almost completely blocked PEPCK induction. It is, therefore, suggested that the effect of TCDD on liver PEPCK activity is due to a direct effect on liver cells and is not mediated by factors outside the liver.

Viluksela et al. (1995) analyzed the toxicological significance of reduced gluconeogenesis by studying dose-responses and time courses of effects of TCDD on the activity of PEPCK in liver and two other tissues with high specific activity, kidney and brown adipose tissue. Liver PEPCK activity was significantly decreased from 1 to 32 days after oral dosing (60 μg/kg). A clear dose-response was present 8 days after dosing, beginning at a dose of 1 μg/kg. In contrast to liver, TCDD treatment increased PEPCK activity in kidney and brown adipose tissue, but only at the two highest doses administered (30 and 60 μg/kg). PEPCK activity in kidney began to increase slowly, reaching a maximum on day 16 and declining thereafter, whereas in brown adipose tissue the activity was significantly increased on day 1 and maximally day 4 after dosing. A likely explanation for these tissue-specific effects is related partly to toxicokinetics and partly to homeostatic responses of the organism to the toxic insult of TCDD. High concen-

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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trations of TCDD in liver and brown adipose tissue combined with early responses (one day after dosing) suggest a direct effect in these organ or tissues, whereas very low concentrations and delayed responses in kidney indicate an indirect effect. This interesting enzymatic constellation suggests that the reduction in gluconeogenesis due to decreased PEPCK activity in liver is partially counterbalanced by increased gluconeogenesis in kidney as a result of induction of PEPCK in this organ. Induction of PEPCK in brown adipose tissue (BAT), where it is a glyceroneogenic enzyme, provides for the first time a plausible explanation for the initial accumulation of fat in brown adipose tissue of TCDD-treated rats.

Tuomisto et al. (1995) performed portocaval anastomosis and vagotomy in LE and Han/Wistar (HW) rats to elucidate the mechanism of anorexia induced by TCDD. TCDD-sensitive LE rats were given a sublethal (5 μg/kg) or lethal (20 μg/kg) dose by gavage five to eight weeks after portacaval anastomosis. TCDD-resistant HW rats were given a nonlethal dose (500 or 7,200 μg/kg). The shunt operation did not reduce the lethality of TCDD. The effect on wasting of the marginally toxic dose of 5 μg/kg in LE rats was potentiated by the portacaval operation, and the lethal dose was effective in both shunted and sham-operated LE rats. TCDD failed to decrease food intake and body weight in shunted HW rats at either dose level although it did so in sham-operated controls. The absence of an effect may be due to the already reduced weight of shunted rats at the time of TCDD dosing. TCDD anorexia was not explained by changes in histamine or serotonin turnover in the brain. Vagotomy did not influence the lethality of TCDD, although reduction in food intake was somewhat blunted in HW rats. The results were interpreted to suggest that the anorectic effect of TCDD is modified when portal blood bypasses the liver. The results are not consistent with the suggestion that the liver plays a role as the major initiator of TCDD anorexia. Little evidence was found to support a crucial role of vagal afferent input.

The fact that TCDD toxicity in adipose tissue causes severe wasting suggests that TCDD could have effects on adipocyte differentiation. Using 3T3-L1, cells Phillips et al. (1995) demonstrated that when cells were treated with 10 nM TCDD before differentiation or during the first two days of induction in the presence of dexamethasone and isobutylmethylxanthine (IBMX), the number of fat cell colonies measured seven to ten days later decreased. Researchers observed an accompanying reduction in the amounts of mRNA encoding several adipocyte markers. In contrast, when TCDD was added after differentiation, it had no effect on maintenance of the adipose phenotype. Dose-response and structure-activity relationships were consistent with a process mediated by the interaction of TCDD with the AhR. TCDD did not interfere with glucocorticoid-inducible transcription. Treatment of cells with TCDD augmented the increase in protein kinase A activity elicited by either IBMX or forskolin, suggesting that if TCDD disrupts the cAMP signaling pathway, interference occurs after activation of PKA.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Enan et al. (1996c) presented evidence that gender differences in the response of nonreproductive cells to TCDD exist and that some of these differences involve differential effects in the cytoplasmic and nuclear compartments of the cell. Glucose uptake by adipose tissue in vitro was decreased significantly in male guinea pigs within 1 day of i.p. injection of TCDD, but there was no significant effect in females, even 28 days after treatment. A similar difference between male and female guinea pigs was detected in the effect of TCDD on lipoprotein lipase (LPL) activity, except that a significant decrease in LPL activity was observed 28 days after treatment. Experiments with adipose tissue explants from untreated guinea pigs and macaques revealed similar gender differences in the effect of TCDD in vitro on glucose uptake and LPL activity. Both time course and dose-response studies with TCDD in vitro confirmed the greater sensitivity of male tissues to TCDD toxicity. TCDD induced lipid peroxidation in the adipose tissues of male guinea pigs, but had no effect on females. Radiolabeled-TCDD binding affinity studies in adipose explant tissues showed that tissues from male guinea pigs and monkeys had a higher binding capacity for TCDD than female tissues. TCDD induced a significant reduction in nuclear protein phosphorylation and an increase in cytosolic protein phosphorylation in adipose tissue from male guinea pigs; the effects in female tissues were the opposite: nuclear protein phosphorylation increased and cytosolic protein phosphorylation decreased. In a cell-free system in the absence of the nucleus, adipose tissues from male guinea pigs and monkeys responded to TCDD with a rapid stimulation of tyrosine kinase activity, but female tissues from both species had a significantly lower and slower response. TCDD induced the DNA binding of AP-1 in adipose tissues of male guinea pigs, but in female tissues, TCDD reduced the DNA binding of AP-1.

Endocrine Effects Sewall et al. (1995b) reported follicular hyperplasia and hypertrophy of the thyroid gland in rats administered 0.1-125 ng/kg TCDD per day via oral gavage biweekly for 30 weeks. TCDD induction of UGT1 resulted in increased excretion of T3 glucuronide. The observed hyperplasia and hypertrophy are consistent with elevated TSH levels. Results suggest that TCDD induces alterations in thyroid hormone function, probably as a result of chronic perturbations of the liver-pituitary-thyroid axis.

Enan and colleagues (1996a) examined the effects of TCDD on cellular glucose uptake, cAMP-dependent PKA, and progesterone production in human luteinizing granulosa cells (LGCs) in culture. Treatment of human LGCs with TCDD produced a time-and dose-dependent decrease in cellular uptake of glucose. The Vmax and Km of glucose transport were decreased by TCDD treatment. Furthermore, cytochalasin B, a specific inhibitor of facilitative glucose transporter proteins, totally abolished the portion of glucose transport activity that is sensitive to TCDD. Pretreatment of cells with the AhR blockers 4,7-phenanthroline and α-naphthoflavone antagonized the effect of TCDD on [3H]Me-glucose

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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uptake. Structure-activity relationship studies with TCDD and three dioxin congeners revealed a rank order that is consistent with their previously determined biological activity. Treatment of cells for 48 hours with 10 nM TCDD substantially reduced PKA and progesterone production. The inhibitory effect of TCDD on progesterone production was more pronounced in the presence of insulin (10 μg/ml) and D-glucose (13.3 μM). However, cytochalasin B abolished the effect of TCDD on progesterone production. Forskolin (an adenylate cyclase activator) abolished the effect of TCDD on glucose uptake and progesterone production but did not alter its effect on PKA activity. A relationship between glucose transporting activity and progesterone production in human LGCs treated with TCDD is suggested by the finding that cytochalasin B down-regulated glucose transporting activity and progesterone production, insulin plus D-glucose down-regulated glucose uptake and amplified the negative effect of TCDD on progesterone production, and forskolin abolished the negative effect of TCDD on glucose transporting activity and on progesterone production. From these data it was concluded that glucose transporting activity can be used as a sensitive biomarker to detect the very early response to TCDD in human steroid-producing cells and that the effect of TCDD on steroid production is mediated through the cAMP-dependent protein kinase.

Neurotoxicity The behavioral signs exhibited by animals exposed to TCDD (progressive anorexia and loss of body weight) suggest a role for the CNS in TCDD toxicity. At lethal doses, TCDD affects the metabolism of serotonin, a neurotransmitter that can modulate food intake in the brain, and this effect is associated with elevated concentrations of free tryptophan in the plasma (Unkila et al., 1995a). No major changes in catecholaminergic neurotransmitter systems were observed in TCDD-treated rats. Cytochrome P450-related enzyme activities are induced by TCDD in the brain. As in the liver, this induction does not correlate with susceptibility to TCDD lethality in rats.

Hanneman et al. (1996) examined the effects of TCDD and related compounds on the uptake of intracellular calcium in primary cultures of rat hippocampal neuronal cells. Treatment of cell cultures with 2,3,7,8-TCDD (10-100 nM) resulted in a rapid, concentration-dependent increase in calcium associated with a decrease in mitochondrial membrane potential and activation of α-protein kinase C. In contrast, 1,2,3,4-TCDD, a weak AhR agonist, had no effect on calcium at concentrations as high as 10 μM, and similar results were observed with TCB. Maximal calcium concentrations were observed within 30 seconds after addition of 2,3,7,8-TCDD and remained elevated above resting levels for the duration of the experiment. This rapid increase in calcium was blocked by addition of ethylenediamine tetraacetic acid (EDTA) (2 μM) to the external medium or by pretreatment of cells with the calcium channel antagonist nifedipine (10 μM). However, the pretreatment of cells with 100 μM cycloheximide failed to block calcium uptake in neuronal cells. These data indicate that rat hippocam-

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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pal neuronal cells are responsive to 2,3,7,8-TCDD; however, the mechanism is not associated with altered gene transcription and may involve cellular targets. Animal studies and in vitro mechanistic studies continue to emphasize the importance of alterations in neurotransmitter systems and thyroid function as underlying mechanisms of behavioral dysfunction caused by TCDD and related chemicals (Golub and Jacobson, 1995).

Immunotoxicity TCDD and structurally related halogenated aromatic hydrocarbons have a broad range of immunologic effects in experimental animals, including effects on host resistance and innate, cell-mediated, and humoral immune responses (Kerkvliet, 1995). As discussed in VAO and Update 1996, thymic atrophy is the most consistent biological effect found in laboratory animals treated with TCDD and is believed to be mediated primarily through the T-cell arm of the immune system. TCDD prevents the maturation of thymocytes to mature T cells by inducing differentiation of thymic epithelial cells. Suppression of humoral immunity by TCDD results in an inhibition of B-lymphocyte differentiation into antibody-producing cells. Summarized below are recent studies that support and expand on these findings.

The thymus plays an important role in generating the ability of the immune system to distinguish self from nonself, thereby avoiding autoimmune responses. The potential of TCDD to disrupt self-nonself discrimination was evaluated using the popliteal lymph node (PLN) assay (Fan et al. 1995). Male Sprague-Dawley rats were injected subcutaneously with either 10 μg/kg TCDD or 5 mg per 50 μl chlorpromazine (CPZ), a structural analogue of TCDD (dissolved in dimethylsulfoxide), into the right hind footpad. Vehicle was injected into the contralateral footpad of treated animals, as well as into both hind footpads of control rats. When the animals were sacrificed on day 7, the weight ratio of right PLN over left PLN was significantly higher in both CPZ-and TCDD-treated rats than in controls. Mild follicular hyperplasia of the PLN with no evidence of an acute inflammatory response was found in both groups. These results indicate that TCDD has the potential to induce or exacerbate autoimmune-like reactions.

Fan et al. (1996) studied the effect of TCDD on delayed-type hypersensitivity reaction as a measure of cell-mediated immunity in Sprague-Dawley rats. A time-course evaluation demonstrated that the greatest effect on cell-mediated immunity occurred when TCDD treatments were administered five days before immunization with the antigen keyhole limpet hemocyanin (KLH). A dose-response experiment of the effect of 1, 3, 10, 20, 30, 40, and 90 μg/kg TCDD on delayed-type hypersensitivity to KLH showed an inverted U-shaped dose-response curve, indicating that low doses enhanced and high doses suppressed this immune function.

The effects of TCDD on another measure of cell-mediated immunity, cytotoxic T-lymphocyte (CTL) activity, has also been evaluated (De Krey and Kerkvliet, 1995). When mice were administered single oral doses of 2.5-40 μg/kg

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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TCDD, a dose-dependent suppression of CTL activity was observed. In contrast, plasma corticosterone (CS) levels were not significantly altered at doses lower than 40 μg/kg TCDD, suggesting that TCDD-induced CTL suppression is not dependent on CS elevation. The direct effect of TCDD on CTL generation was tested in vitro by adding 10-13 to 10-9 M TCDD to cultures of mixed lymphocyte-tumor cells. No alteration of CTL activity was observed after five days of culture at any of the doses tested. In contrast, CS alone significantly suppressed CTL activity. CS-induced CTL suppression in vitro was neither enhanced nor inhibited by the presence of TCDD, which suggests that TCDD causes CTL suppression in vivo by a mechanism that does not involve CS.

In a study with nonhuman primates, Neubert et al. (1995) vaccinated marmosets with tetanus toxoid and administered a second booster in conjunction with 100 ng/kg TCDD. The proliferative response of lymphocytes to recall antigen was measured in vitro in blood samples. No reduction in lymphocyte response was observed, but when the ratio of the responses between the first and second booster was compared, a slight but statistically significant increase in this ratio was observed in the lymphocytes of TCDD-treated marmosets compared to controls.

Based on the observed suppressive effects of TCDD on T-cell activity, reports of increased susceptibility of laboratory animals to pathogenic microorganisms that interact primarily with cell-mediated immunity are not surprising. Recently, Burleson et al. (1996) found that a single oral dose of 0.01, 0.05, or 0.10 μg/kg TCDD increased mortality in mice when they were subsequently infected with influenza A/Hong Kong/8/68 (H3N2) virus. There was no effect on the virus-enhanced increase in lung weight-to-body ratio or the virus-induced decrease in thymus weight. Thus, TCDD-augmented mortality did not appear to be due to additive or synergistic effects of TCDD and virus on pulmonary edema or thymic atrophy. However, another study reported minimal effects of TCDD on Trichinella spiralis infection in rats (Luebke et al., 1995), which was markedly different from the increased persistence of infection observed in an earlier study with mice (Luebke et al., 1994). Researchers suggested that this difference was a clear indication of differential species sensitivity and underscored the need to determine which species more closely reflects the potential outcome of human exposure to TCDD (Luebke et al., 1995).

The toxic action of TCDD on the thymus of rats and humans has been compared by treating Wistar rats and SCID-ra and SCID-hu mice (engrafted with fetal rat or human thymus, respectively) with 1, 5, or 25 μg/kg TCDD (de Heer et al., 1995a). Four days after exposure, the thymuses were removed, weighed, and examined histopathologically. There was a dose-dependent decrease in the relative size of the cortex of both normal rat thymus and grafted human thymus; the decrease was significant in the highest-dose group. Only limited data were obtained from grafted rat thymus because of a cutaneous graft-versus-host reaction, but they were consistent with those in normal rat and grafted human thymus.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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TCDD tissue concentrations in normal rat thymus and grafted human thymus were similar. Thus, it appears that the human thymus and the Wistar rat thymus display a comparable sensitivity to the toxic action of TCDD.

To determine if TCDD interferes with intrathymic negative selection processes, de Heer et al. (1995b) exposed Mls-1a DBA/2 mice to a single thymotoxic dose of 75 or 225 μg/kg TCDD and evaluated the emergence of Vβ6+ cells in thymus, spleen, and mesenteric lymph nodes during the subsequent recovery of TCDD-induced thymic atrophy. In addition, the extrathymic differentiation of T lymphocytes in the liver was studied. TCDD exposure resulted in severe thymic atrophy and an increase in hepatic mononuclear cells. However, no evidence of potentially autoreactive Vβ6+ cells, differentiated either intrathymically or extrathymically, in TCDD-exposed DBA/2 mice was observed.

Fan et al. (1996) examined the effects of TCDD primary antibody response to sheep red blood cells (SRBCs), as an end point of the effects of TCDD on humoral immunity, in studies with male rats. At doses of 10, 20, and 40 μg/kg TCDD, enzyme-linked immunosorbent assay (ELISA) revealed that serum immunoglobulin M (IgM) levels measured seven or fourteen days after immunization were not affected by TCDD compared to controls. In contrast, serum IgG levels were elevated in a dose-dependent manner at both times. In a related study, the involvement of cytokines (interleukin 1 [IL-1] and tumor necrosis factor [TNF]) in mediating the enhanced IgG response and delayed-type hypersensitivity reaction to 1, 2, 10, 30, and 90 μg/kg TCDD was investigated (Fan et al., 1997). Levels of mRNA for IL-1β were elevated in all dose groups, with a fivefold increase above controls in the 90 μg/kg TCDD group. The mRNA levels of TNF were also significantly elevated, beginning at 30 μg/ kg TCDD. These results suggest that at low doses of TCDD, increased levels of IL-1β may account for immune function stimulation, whereas at high doses, greatly elevated TNF and IL-1β levels might exacerbate or mediate acute toxicity such as immune suppression and related biochemical effects. A time course study using 60 μg/kg TCDD without immunization indicated that mRNA levels of TNF in the liver were significantly elevated starting at 24 hours, and reached a maximum at 48 hours. This change was accompanied by a transient increase in mRNA levels of IL-1β at day 4. Thus, TCDD alone and without immunization can cause transient increases in mRNA levels of TNF and IL-1β in liver.

Smialowicz et al. (1996) studied the effect of TCDD on the antibody plaque-forming cell (PFC) response to the T-cell-independent antigen trinitrophenyllipopolysaccharide (TNP-LPS) in female B6C3F1 mice and F344 rats. The animals were injected i.p. with a single dose of 1-30 μg/kg TCDD seven days prior to immunization with TNP-LPS. In mice, thymus weights were decreased at 10 and 30 μg/kg TCDD, whereas spleen weights were decreased and liver weights increased at 3, 10, and 30 μg/kg. Mice treated with 10 and 30 μg/kg TCDD also had suppressed PFC responses and serum hemagglutination titers. In rats, thymus

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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weights were decreased and liver weights increased at 3, 10, and 30 μg/kg TCDD; however, the PFC response and serum hemagglutination titers to TNP-LPS were suppressed only at 30 μg/kg. No effects on splenic lymphocyte subsets were observed. Collectively, these data suggest that TCDD suppresses the T-cell-independent antibody response to TNP-LPS in both B6C3F1 mice and F344 rats and that mice are more sensitive to immune suppression by TCDD than rats.

In addition to immune suppression, TCDD has been shown to promote inflammatory responses. This effect could be a result of upregulation of the production of inflammatory cytokines, such as TNF and IL-1. Recently, Moos and Kerkvliet (1995) examined the effects of exogenous TNF and the effects of blocking TNF activity with a soluble TNF receptor (rhuTNFR:Fc) on antibody production to SRBCs in control and TCDD-exposed C57BL/6 mice. Their results indicated that increased TNF can suppress antibody production to SRBC, but that TNF itself does not appear to mediate TCDD-induced antibody suppression.

Investigation into the effect of TCDD on cytokine production was conducted using a novel in vitro model based on injection of hamster monoclonal antibody to the CD3 epsilon portion of the mouse T-cell receptor (Prell et al., 1995). T-cell activation resulted in the release of several cytokines, including TNF, interferon (IFN), IL-2, IL-3, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In an in vivo study, administration of 15 μg/kg TCDD to mice followed by an injection of anti-CD3 two days later significantly reduced plasma levels of IFN and elevated plasma levels of IL-6 and GM-CSF, suggesting that increased IL-6 and GM-CSF contributed to the toxic effects of TCDD.

Kerkvliet et al. (1996) characterized changes in CTL, alloantibody, and cytokine responses to the P815 tumor allograft in mice treated with 15 μg/kg TCDD. TCDD suppressed CTL activity as well as cytotoxic antibody responses, and suppression correlated with a reduced percentage of CD8+ T cells. The cytokine profile of TCDD-treated rats was markedly different from that of control animals, which showed increases in IL-1 and TNF on day 5 and IL-2 on day 6, followed by peak induction of IL-6, IL-7, IL-2, IFN, TNF, and IL-1 on subsequent days. In contrast, cytokine production in TCDD-treated mice showed early increases in IFN, IL-2, and TNF up to day 5 but failed to increase normally thereafter; the production of IL-1, IL-4, or IL-6 was unaffected by TCDD. This differential effect of TCDD on cytokine production was reflected in the degree of suppression of cytotoxic antibody isotypes. TCDD abrogated the production of IgG2a (generally associated with IFN production) but had much less effect on the level of IgG (associated with IL-4).

The effects of TCDD on the production of cytokines IL-1 and IL-2 were evaluated by Badesha et al. (1995). Young adult male Leeds rats fed a total dose of 3 μg/kg TCDD showed a duration-dependent reduction of in vitro lipopolysaccharide-induced production of IL-1 by splenic macrophages within 30 days of exposure. A prolonged, 180-day exposure was required before a significant suppression in the generation of IL-2 by activated splenic T cells was measured.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Thymic atrophy has been shown to be influenced by prostanoid metabolites of arachidonic acid. Olnes et al. (1996) reported that TCDD inhibits prostaglandin G/H synthase, one of the important enzymes in the cyclooxygenase pathway from arachidonic acid to prostaglandin H2. It was reported that incubation of thymocytes with TCDD resulted in inhibition of synthase gene expression in a concentration-dependent manner.

Kraemer et al. (1996) studied TCDD-stimulated prostaglandin synthesis in canine kidney cell cultures. TCDD stimulated prostaglandin synthesis in these cells, at least in part, by elevating PGHS-2 levels. This enzyme is believed to be responsible for producing inflammatory prostaglandins and, indirectly, to modulate cytokines such as TGF and IL-1β. Results suggest that inappropriate expression of synthase may contribute to the diverse immunotoxic effects of TCDD.

Despite extensive laboratory research, the mechanism of TCDD-mediated immunotoxicity remains uncertain. This is due, in part, to unsuccessful attempts to demonstrate a direct effect of TCDD on immune function in vitro. As discussed in earlier reports, the immunotoxic effects of TCDD and related substances appear to be mediated predominantly through binding to the Ah receptor. However, AhR-independent mechanisms also appear to be involved.

Recently, Fernandez-Salguero et al. (1995) demonstrated that AhR-deficient mice are relatively unaffected by 2,000 μg/kg TCDD, a dose that is tenfold higher than that found to cause severe pathologic effects on the thymus of littermates expressing the functional receptor. These results suggest that thymic involution by TCDD is mediated entirely by the AhR. However, a number of other pathological effects, such as vasculitis and scattered single cell necrosis of the lung and liver, were present in receptor-deficient mice, suggesting that some effects of TCDD may be AhR-independent.

Thymic atrophy is a prominent effect of TCDD exposure. In the presence of TCDD, the distribution of CD4/CD8 thymocyte subsets is strongly skewed toward CD4-CD8+ single positives. The primary target of TCDD action appears to be stroma cells, which have an important role in thymocyte maturation and in the selection of thymocytes bearing T-cell receptors specific for foreign antigen in the context of self. Using staphylococcus enterotoxin B as a superantigen, Kremer et al. (1995) investigated whether the effects of TCDD on thymocyte differentiation and maturation had further consequences for the selection process by analyzing the repertoire of Vβ genes as a measure of negative selection and the expression of CD69 and bcl-2 by thymocytes as a measure of positive selection. TCDD had no effect on negative selection but did increase the parameters for positive selection. Researchers suggested that these effects on thymocyte maturation are mediated through the action of TCDD on the thymus stroma via the AhR. This hypothesis is strengthened by the results of Germolec et al. (1996), who reported an increased expression of CYP1A1 in thymus cells from rats exposed to TCDD. This pattern of induction was related to the expression of the AhR on macro-phages or other stromal cells of the thymus.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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The role of the AhR in the immunosuppressive effect of TCDD on B lymphocytes has also been investigated. In order to identify the genes potentially regulated by TCDD in B lymphocytes, Masten and Shiverick (1995) searched the published data on genes important in B-cell function for DNA sequences that have homology to the consensus AhR binding site. This approach identified a subset of DNA binding sites for the transcription factor B-cell lineage-specific activator protein (BSAP), which resembles the consensus binding site of the receptor. BSAP expression is essential for B-cell development, and DNA binding sites for BSAP occur in regulatory regions of the immunoglobulin heavy-chain gene locus. The BSAP binding sites were localized in the promoter region of the CD19 gene. CD19 is a cell surface signal-transducing protein expressed exclusively on B lymphocytes at early stages of development. This evidence, therefore, suggests a role for BSAP in the regulation of CD19 gene expression and further suggests that binding of TCDD to the AhR could interfere with transcription by competing with BSAP for binding to this site.

Masten and Shiverick (1996) also compared TCDD responsiveness and AhR complex formation in a cultured human hepatoma cell line and two human B-cell lines. The B lymphocytes were found to express the AhR as well as the Arnt, and gel mobility shift analysis demonstrated that the AhR complex in B cells was functional with respect to TCDD activity. Furthermore, TCDD treatment induced CYP1A1 activity in one of the two B-cell lines. The lack of response in the other B-cell line was probably due to a relatively low level of AhR expression.

AhR-independent responses have been reported in certain strains of mice immunized with SRBCs and exposed to TCDD or polychlorinated biphenyls. These results, however, may have been due to the particular method of cultivating B cells in vitro using fetal bovine serum. Using a standard in vitro culture method for spleen cells of B6C3F1 female mice, Harper et al. (1995) reported that B lymphocytes cultured with mouse serum showed a dose-dependent suppression of plaque-forming activity when exposed to TCDD. There was, furthermore, excellent correlation between the immunosuppressive activity of a number of halogenated aromatic hydrocarbons and their binding affinity for the AhR. These results support the role of the AhR in mediating TCDD-induced humoral immunosuppression.

Macrophages are important cellular components of the innate immune response. Although TCDD exerts profound immunosuppressive effects on B and T lymphocytes, it appears to have much less activity against macrophages. TCDD does not alter macrophage-mediated antigen presentation, phagocytosis, or tumor cytolysis and cytostasis. However, there is some evidence suggesting that TCDD treatment may stimulate macrophage-generated inflammatory cytokines and reactive oxygen species. The toxic effects of TCDD and related compounds may require activation to toxic metabolites by drug-metabolizing enzymes, such as CYP1A1 and alcohol dehydrogenase (ALDH). Germolec et al. (1995) compared the induction of these enzymes in various macrophage populations following

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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treatment of F344 rats with TCDD. Kupfer cells, alveolar macrophages, and splenic macrophages from TCDD-treated animals expressed elevated levels of inducible CYP1A1 compared to other macrophage subpopulations or to cells from control rats. In contrast, CYP1A1 induction was not detectable in resident peritoneal macrophages or peripheral blood monocytes. Examination of AhR levels in macrophage populations indicated that the ability of TCDD to induce metabolic enzymes in specific cell types correlated well with AhR expression. In a related study, Germolec et al. (1996) followed the induction of CYP1A1 and ALDH in various lymphoid tissues from F344 rats exposed to TCDD. They found that macrophages of the spleen and liver were the primary sites for generation of the metabolic enzymes. Other cells, such as thymocytes, showed enzyme induction only if previously stimulated by mitogens. These effects correlated with increased expression of the AhR and indicate that TCDD-induced increases in these enzymes are related to the level of expression of the AhR in different populations of immune cells.

Evidence for AhR-independent immunologic effects is supported by a recent study in which the distribution and behavior of the AhR in isolated spleen T lymphocytes and T-cell clones derived from Ah-responsive mouse strains were evaluated (Lawrence et al., 1996). Western immunoblot were used to determine the presence of the AhR in whole-cell extracts of resting and activated splenic lymphocytes and T-cell clones. Increased EROD activity was observed in T-cell clones and spleen cells, and the level of induction was about a hundredfold less than in Hepa cells. The AhR was detected in all cell types examined, but the it translocated to the nucleus only in activated, TCDD-treated T cells. Whereas AhR derived from TCDD-treated wild-type Hepa cells bound specifically to a dioxin response element, no binding was detected when an identical amount of AhR obtained from activated T cells was used. The inability to detect binding of the T-cell nuclear AhR complex to a consensus response element, combined with difficulties of reproducing in vivo immunotoxic effects of TCDD in vitro suggests that T cells may lack one or more factors required for AhR binding to a DRE or may contain a suppressor factor that inhibits AhR binding to DNA. Based on these data, it was suggested that TCDD affects T-cell function via an indirect mechanism.

Rhile et al. (1996) studied the role of Fas (CD95), an important molecule involved in the induction of apoptosis, and major histocompatibility complex (MHC) genes in TCDD-mediated immunotoxicity. When TCDD was orally administered to different strains of C57B1 mice at doses of 0.1, 1.0, or 5.0 μg/kg for 11 days, it was less toxic to thymocytes from C57BL/6 lpr/lpr mice (Ah responsive, Fas-) than to those from C57BL/6+/+ mice (Ah responsive, Fas+). Similar results were obtained when peripheral T-cell responsiveness to antigenic challenge with conalbumin was studied in these mice. When mice that differ only at the MHC were compared for immunotoxic effects of TCDD, it was noted that B 10.D2 mice (Ah responsive H-2d) were more sensitive to TCDD-mediated ef-

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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fects than B10.A mice (Ah responsive H-2b). In all TCDD-sensitive strains tested, thymic atrophy was accompanied by a uniform depletion in all four subsets of T cells (CD4+, CD4+CD8+, CD4-CD8-, and CD8+) and the proportion of the subsets was not altered. In these strains, TCDD suppressed the antigen-specific peripheral T-cell responsiveness, but not the responsiveness of naive resting T cells to mitogens. It was also demonstrated that TCDD directly affected T cells responding to conalbumin, but not antigen-presenting cells. Thus, although the Ah locus has the primary role in determining the toxicity of TCDD to T cells, secondary factors such as the expression of Fas or the MHC phenotype might also play an important role in TCDD-mediated immunotoxicity.

The effects of TCDD on the expression of costimulatory molecules B7-1 and B7-2 in P815 allograft immunity were evaluated in C57BL/6 mice (Prell and Kerkvliet, 1997). Expression of B7-2, but not B7-1, was up-regulated in splenic B220+ and Mac-1+ cells in P815-challenged mice. Exposure to TCDD significantly decreased the expression of B7-2 on B220+ and Mac-1+ cells in P815-challenged mice. Providing exogenous B7-mediated costimulation, in the form of B7-transfected P815 tumor cells, induced CTL activity in TCDD-treated mice by a mechanism that was independent of CD4+ T cells. In contrast, B7-transfected P815 cells did not restore the cytotoxic alloantibody response in TCDD-treated mice. Based on these results, it was suggested that MHC class II B7-transfected P815 tumor cells can directly activate CD8+ CTL precursors, but cannot directly stimulate CD4+ T-helper cells required for B-cell activation. In addition, these results demonstrated that CTL precursors in TCDD-treated mice are functional and able to differentiate into effector CTL provided they receive adequate costimulation via B7. Thus, defective costimulation, through reduced B7-2 expression, may play a role in TCDD-induced immunotoxicity. In support of this hypothesis, evidence was presented that blocking B7-2/CD28 interactions, and to a lesser degree B7-1/CD28 interactions, suppressed the alloimmune responses to P815 tumor cells.

To determine the basis for TCDD-induced suppression of the humoral immune response, Karras et al. (1996) examined the effects of TCDD using in vitro models of T-independent (antibody directed against surface IgM) and T-dependent (activated T-helper cells beating CD40 ligand) B-cell maturation. TCDD suppressed murine B-cell IgM secretion induced by anti-IgM, but did not affect IgM secretion stimulated by activated T cells through the CD40 pathway. Because mobilization of calcium has been shown to be an integral event in the stimulation of proliferation via the antigen receptor in B cells, the effect of TCDD exposure on B-cell intracellular calcium concentration and mobilization was examined. TCDD suppressed calcium mobilization in B cells, whereas stimulation by activated T cells was unaffected. The results support a role for the disruption of calcium homeostasis as another AhR-independent mechanism for TCDD toxicity.

Reproductive Or Developmental Toxicity The effects of TCDD on reproductive development and fertility of the progeny have been investigated in a number

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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of studies. Results are presented below for male and female mammals (specifically rodents) and for other nonmammalian species (i.e., fish and birds).

Low doses of TCDD in pregnant rats alter the reproductive development and fertility of the progeny. In comparative reproductive studies, Gray et al. (1995) administered TCDD to LE hooded rats and pregnant Syrian hamsters, a species relatively insensitive to the lethal effects of TCDD. When the rats and hamsters were dosed on GDs 15 and 11, respectively, puberty was delayed by about three days, ejaculated sperm counts were reduced by at least 58 percent and epididymal sperm storage was reduced by 38 percent. Testicular sperm production was less affected. The accessory sex glands were also reduced in size in rat offspring treated on GD 15 despite the fact that serum testosterone (T), T production by the testis in vitro, and androgen receptor (AR) levels were not reduced. Some reproductive measures, such as anogenital distance and male sex behavior, were altered by TCDD treatment in rat but not hamster offspring. Since T and AR levels appeared normal in the accessory sex glands and the epididymis following perinatal TCDD exposure, alterations in these tissues are not likely to have resulted from an alteration of the androgenic status of the male offspring.

Roman et al. (1995) recently completed studies to determine whether in utero and lactational TCDD exposure decreases male rat accessory sex organ weights during postnatal development and whether this effect involves decreases in testicular androgen production or changes in peripheral androgen metabolism. Pregnant rats were administered a single dose of TCDD on GD 15, and offspring were exposed via placental and subsequent lactational transfer until weaning on postnatal day (PND) 21. No significant differences were observed between PNDs 21 and 63 in circulating androgen concentrations and intratesticular androgen content. In vitro human chorionic gonadotropin-stimulated testosterone production from TCDD-exposed animals did not differ from control, although 5-androstane-3,17α-diol production was decreased on PNDs 32 and 49 and increased on PND 63. Thus, in utero and lactational TCDD exposure can cause subtle decreases in testicular androgen production. These observed reductions, however, do not correlate temporally with one another or with decreases in androgen-dependent male accessory sex organ weights. Of the male accessory sex organs, the ventral prostate (VP) and dorsolateral prostate (DLP) were the most severely affected. Between PNDs 21 and 63, relative VP and DLP weights were decreased to 65-84 percent and 57-80 percent of control, respectively, and the magnitude of observed decreases was greatest at early times. In contrast, relative weights of the seminal vesicle and coagulating gland ranged from 80 to 104 percent of control, and the magnitude of observed decreases was greatest at later times. The sensitivity of the prostate to TCDD could not be explained by tissue-specific decreases in dihydrotestosterone concentrations. Although VP DHT concentration was decreased to 63 percent of control on PND 21, DHT concentration was not decreased in the VP between PNDs 32 and 63 or in the DLP at any time. These results suggest that in utero and lactational TCDD exposure selectively

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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impairs rat prostate growth and development without inhibiting testicular androgen production or consistently decreasing prostate DHT concentration.

Decreased daily sperm production (DSP) and cauda epididymal sperm number are some of the most sensitive effects of in utero and lactational TCDD exposure. To determine if TCDD exposure increases the rate of sperm transit through the excurrent duct system, thereby decreasing the number of sperm in the system at any given time, pregnant Holtzman rats were administered a single dose of TCDD (1.0 fg/kg, p.o.) on GD 15 and offspring were weaned on PND 21 (Sommer et al., 1996). On PND 50, testicular sperm were labeled with radiolabeled thymidine in five males per litter from control and TCDD-exposed litters. On PNDs 92-93, TCDD exposure significantly decreased DSP and testis, corpus and cauda epididymis, vas deferens, and ejaculated sperm numbers by 28, 30, 36, 39, and 46 percent, respectively. Decreases in sperm number in the distal excurrent duct system were greater than the decrease in DSP, consistent with the hypothesis that TCDD exposure has an effect other than decreased DSP that reduces epididymal and ejaculated sperm numbers. However, in utero and lactational TCDD exposure did not alter radiolabeled sperm transit time through the whole epididymis (15 days). With TCDD exposure causing no obvious alteration in sperm transit rate, a plausible explanation for sperm loss is an increase in sperm phagocytosis in the excurrent duct system.

The reproductive alterations in female progeny after gestational administration of TCDD were evaluated by Gray et al. (1995). In these experiments, LE hooded rats were given a single dose of 1 fg TCDD/kg by gavage on GD 8 (i.e., a period that includes major organogenesis) or GD 15 (i.e., a period prior to sex differentiation and a dosing regime that alters sex differentiation of the male LE rat). In a second experiment, Holtzman rats were dosed with TCDD at 1 fg/kg on GD 15 to determine if the progeny of this strain displayed malformations of the external genitalia and vaginal orifice as did LE rats. TCDD-treated female LE offspring displayed a number of unusual reproductive alterations. In the GD 15 group, puberty was delayed, more than 65 percent of the female offspring displayed complete to partial clefting of the phallus, and 80 percent displayed a permanent thread of tissue across the opening of the vagina. In the GD 8 treatment group, 25 percent displayed partially cleft phallus and 14 percent had a vaginal thread. GD 15 TCDD administration also induced a high incidence of malformations in Holtzman female progeny (100 percent clefting and 83 percent with a vaginal thread). At necropsy (>550 days old), ovarian weight was significantly reduced by 23 percent in both rat strains. In the LE rat, vaginal and behavioral estrous cyclicity, estrous cycle-mediated running wheel activity, and female sexual behavior at proestrus (darting and lordosis to mount ratios) were not affected by GD 15 TCDD treatment. However, untreated stud males had difficulty attaining intromission and took longer to ejaculate, and vaginal bleeding was displayed during mating by GD 15 TCDD-exposed female offspring. GD 8 TCDD-treated female offspring displayed enhanced incidences of constant

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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estrus (CE) (47 percent CE versus 16 percent CE in the control and GD 15 groups at middle age) and cystic endometrial hyperplasia. In addition, in the GD 8 group fertility rate declined significantly faster than in controls and fecundity was reduced by 38 percent. These data suggest that administration of a single dose of 1 fg TCDD/kg on GD 15 results in malformations of the external genitalia in female LE and Holtzman rats. Although treatment on GD 15 is generally more toxic to the offspring than treatment on GD 8 with respect to growth, viability, male reproductive effects, and malformations of the external genitalia in female progeny, treatment on GD 8 is more effective in inducing functional reproductive alterations in female progeny.

Twenty-one days prior to induction of surgery to produce endometriosis, female Sprague-Dawley rats and B6C3F1 mice were pretreated with at 0, 3, or 10 fg TCDD/kg. Animals were treated again at the time of surgery and at three, six, and nine weeks following surgery. TCDD produced a dose-dependent increase in endometriotic site diameter when all time points were pooled within each dose in rats and a dramatic increase in site diameter in mice at 9 and 12 weeks (Cummings et al., 1996). In rats but not mice, ovarian weight was decreased at 9 and 12 weeks. The occurrence of persistent vaginal estrus was increased at these times, and histological evaluation of the ovaries revealed ovulatory arrest at 12 weeks. In both species, thymic atrophy and hepatomegaly were also observed. Histological evaluations of endometriotic sites revealed fibrosis in control rats, necrotic and inflammatory changes in sites from TCDD-treated rats, and predominantly fibrotic changes in sites from TCDD-treated mice. Differences observed between rat and mouse with respect to the magnitude of changes in endometrial site diameter (rat < mice), ovarian function (rat > mice) and immune response suggest that the mechanisms mediating the promotion of endometriosis by TCDD are complex and may differ in rats and mice. Endometriosis in the rhesus monkey, which bears a close parallel to the human disease, is exacerbated by TCDD (Rier et al., 1993).

Treatment of pregnant female Sprague-Dawley rats on GD 15 with a single oral dose of TCDD (0.5, 1.0, or 2.0 μg/kg) or indole-3-carbinol (I3C; 1.0 or 100 μg/kg), an AhR agonist found in cruciferous vegetables, resulted in reproductive abnormalities in male offspring (Wilker et al., 1996). Anogenital distance and crown-to-rump length were altered by both compounds; however, the timing of the effects (day 1 or 5) was variable and the responses were not necessarily dose dependent. In 62-day-old offspring, seminal vesicle, prostate, testicular parenchymal, and epididymal weight were decreased by one or more doses of TCDD. The total number of sperm in the epididymis was significantly decreased in rats perinatally exposed to TCDD due to a decreased number of sperm in the tail of the epididymis. Perinatal exposure to I3C did not affect any of these parameters. TCDD did not affect the transit time of sperm through the complete epididymis at any doses (0.5-2.0 fg/kg). However, at the two highest doses (1.0 and 2.0 fg/kg), TCDD increased the transit rate of sperm through the tail of the epididymis. In

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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contrast, primarily due to the decreased transit rate of sperm through the head plus body of the epididymis, I3C (1 mg/kg) significantly increased total epididymal transit time by 31 percent. The authors concluded that perinatal exposure of pregnant rats to I3C causes reproductive abnormalities in male offspring but that, relative to TCDD, both common and different responses are present.

After water-borne exposure of newly fertilized eggs to TCDD (35-2,100 ng per liter), Henry et al. (1997b) characterized the toxicity and histopathology of TCDD in zebrafish during early life stages from 12 to 240 hours postfertilization (hpf). TCDD did not increase egg mortality (0-48 hpf), nor did it affect time to hatching (48-96 hpf). Eggs exposed to 1.5 ng or more of radiolabeled TCDD per gram of egg elicited toxic responses in zebrafish larvae. Pericardial edema and craniofacial malformations were first observed at 72 hpf, followed by the onset of yolk sac edema (96 hpf) and mortality (132 hpf). The LD50, determined at 240 hpf, was 2.5 ng TCDD/g egg. Severe hemodynamic changes, observed as slowed blood flow in vascular beds of the trunk, head, and gills and decreased heart rate, occurred in TCDD-treated zebrafish prior to or coincident with the onset of gross signs of toxicity. Histological examination of TCDD-treated zebrafish revealed a variety of epithelial tissue lesions including arrested gill development and ballooning degeneration and/or necrosis of the renal tubules, hepatocytes, pancreas, and all major brain regions. Mesenchymal tissue lesions included subcutaneous edema in the head, trunk, and yolk sac; edema of the pericardium and skeletal muscle; and underdevelopment of the swim bladder.

Using a TCDD photoaffinity analogue, Brown et al. (1995) detected the presence of two proteins (28 and 39 kDa) in the cytosol of the hard-shell clam, Mercenaria Mercenaria that bind to this chemical. Expression of these proteins is tissue dependent, with the highest concentrations observed in gill and gonadal tissue. Gonadal tissue also exhibited gender-specific expression, with female clams exhibiting higher levels of the 39 kDa protein. The varying concentrations in different tissues suggest that these proteins are not proteolytic fragments of a larger precursor, although they could be homologous to the AhR.

Janz and Bellward (1996a) evaluated the effects of in ovo TCDD exposure on perinatal plasma thyroid hormone concentrations (total T3, total T4) and body and skeletal growth in the domestic chicken, domestic pigeon, and great blue heron. EROD activity in the liver was employed as an enzymatic marker of CYP1A1 induction by TCDD. Although the EROD activity was induced 13 to 43 times above control values in chickens treated with TCDD, there was no effect on hatchability, body growth, subcutaneous edema, or plasma thyroid hormone levels. In pigeons exposed to TCDD, EROD was induced significantly, hatchability was decreased, liver-to-body weight ratio was elevated, and body and skeletal growth decreased (p < .01); however, there was no effect on plasma thyroid hormone levels. In heron, EROD activity was induced two-to threefold above control birds; however, no effect was observed on plasma thyroid hormone levels or body growth. Thus, in ovo TCDD exposure adversely affected the body and

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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skeletal growth, and hatchability of the domestic pigeon, but had no effect on the domestic chicken or great blue heron. Collectively, these results suggest that perinatal plasma thyroid hormone levels cannot be used as a relatively noninvasive biomarker of TCDD exposure during embryonic development in these species.

In another set of experiments, Janz and Bellward (1996b) reported no effect of in ovo TCDD exposure on liver ER levels or plasma estradiol concentrations in female chickens and pigeons exposed early in incubation. In female pigeons exposed during the latter third part of incubation to a TCDD dose that would cause high embryo lethality if injected early in incubation, hepatic ER concentrations were elevated (p < .001) and plasma estradiol concentrations were decreased (p < .01) at hatch. There was no effect of TCDD exposure on plasma estradiol levels in male pigeons. In herons, TCDD exposure had no effect on hepatic ER levels or plasma estradiol and testosterone concentrations at either time. Based on these results it was concluded that in chicken, pigeon, and great blue heron hatchlings exposed early in incubation to low doses of TCDD, hepatic ER levels and plasma estradiol concentrations are not altered.

Several studies have been published during the reference period on the developmental effects of TCDD in mice, rats, chicks, and medaka. These are discussed below. Effects discussed include cleft palate, hydronephrosis, cardiotoxicity, and angiogenesis. Advances in the understanding of the mechanisms underlying these effects are also discussed.

Developing mice seem to be sensitive to TCDD, which acts to alter the proliferation and differentiation of epithelial, as well as mesenchymal cells. A mouse line deficient in TGF-β3 exhibits cleft palate remarkably similar to that seen with TCDD, suggesting that the AhR may be involved, directly or indirectly, in the regulation of TGF-β3 in developing palate. TCDD exposure would increase the formation AhR-Arnt dimers, decreasing the amount of Arnt available for other interactions and resulting in decreased TGF-β3 expression. Decreased concentrations of free Arnt owing to recruitment by liganded AhR may shift the balance of this general dimeric partner away from HIF-1α or other partners.

Structural defects following dioxin exposure have been reported in the mouse at doses that do not cause either maternal or fetal toxicity, the best described of which is cleft palate. There is a critical window for the induction of this defect, with peak incidence following exposure on day 11 or 12 of gestation (Couture et al., 1990a). In contrast, the induction of hydronephrosis does not appear to have a peak window of sensitivity during organogenesis and can even be induced lactationally (Couture et al., 1990b). Interestingly, hydronephrosis is a more sensitive response to TCDD than cleft palate.

When EGF, TGF-α, EGFR, and the TGF-βs are considered as a combinatorial, interacting set of regulators, TCDD and the synthetic glococorticoid hydro-cortisone (HC) each produce a unique pattern of increased and/or decreased expression of these genes (Abbott, 1995). HC in combination with TCDD pro-

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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duced increased expression of both receptors; this pattern would produce HC-like clefts since the GR-mediated responses would result in small palatal shelves. The observed cross-regulation of the receptors is believed to be important in the synergistic interaction between TCDD and HC for the induction of cleft palate.

Peters and Wiley (1995a) investigated the developmental expression of Arnt mRNA with the goal of identifying the mechanisms by which AhR functions during preimplantation embryo development. Blastocyst-stage preimplantation mouse embryos were collected after 72 hours of in vitro culture. Hepa 1c1c7 cells served as a positive control. Arnt was detected in blastocyst-stage embryos as well as in positive controls. Southern analysis with a human Arnt cDNA probe confirmed that the detected reverse transcription (RT) polymerase chain reaction (PCR) product in blastocysts was similar in sequence to human Arnt mRNA. These data suggest that the AhR-Arnt pathway may function during embryonic development. When mice are treated with TCDD and RA simultaneously, palatal clefts can be observed in 100 percent of offspring of mothers at dose levels far lower than those required for either agent to produce clefting if given alone (Weston et al., 1995). This synergy suggests that the pathways controlled by these agents converge at one or more points in cells of the developing palate. The effects of TCDD on induction of the type II cellular RA binding protein and the RA receptor β by RA in murine embryonic palate mesenchymal cells were also examined. Although TCDD alone had no effect on basal levels of expression of either gene, the induction of both genes by RA was strongly inhibited by TCDD. These results represent the first evidence for a direct molecular interaction between the RA and TCDD-mediated signaling pathways.

Recent reports have investigated TCDD-induced cardiotoxicity. Walker et al. (1997) injected chicken eggs with TCDD (1.0 pmol/g) prior to incubation and collected them after cardiac development was complete. Relative to controls, TCDD increased heart wet weight (27.2 ± 0.5 mg versus 36.6 ± 1.3 mg, p < .001) and dry weight (2.7 ± 0.1 mg versus 3.1 ± 0.1 mg, p < .01) and tended to increase heart myosin content (3.5 ± 0.6 μg versus 6.3 ± 2.5 μg, p < .07), suggesting an increase in cardiac muscle mass and edema. Histologic and morphometric analyses revealed that TCDD-exposed hearts exhibited enlarged right and left ventricles, thickened ventricular septum, and a thinner left ventricular wall with increased trabeculation, and some exhibited ventricular septal defects compared to controls. The AhR was expressed ubiquitously in cardiac myocytes, whereas Arnt expression was restricted to myocytes overlying developing septa: the atrioventricular canal, outflow tract, and atrial and ventricular septa. Both proteins were absent from endocardium and endocardial-derived mesenchyme. In addition, cardiac expression of an AhR-Arnt target CYP1A1 was restricted to myocardium coexpressing AhR and Amt. Thus, the spatial and temporal expression of AhR and Arnt suggests that the developing myocardium and cardiac septa are potential targets of TCDD-induced teratogenicity, and such targets are also consistent with cardiac hypertrophy and septal defects observed after TCDD exposure.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Hassoun et al. (1995) exposed pregnant mice to TCDD (30 μg/kg) on the twelfth day of gestation and observed 1.8- and 2.3-fold increases in DNA single-strand breaks in fetal and placental nuclei, respectively. They also observed increases in lipid peroxidation in placental and fetal tissues. TCDD administration produced increases in amniotic fluid levels of the lipid metabolites malondialdehyde, formaldehyde, acetaldehyde, and acetone. Altogether, reactive oxygen species may participate in the teratogenic effects of TCDD.

TCDD produces dose-dependent decreases in fetal weight, fetal thymic weight, and placental weight, and dose-dependent increases in fetolethality, cleft palate formation, and hydronephrosis at doses of 10-30 and 30-60 μg/kg body weight in C57BL/6J and DBA/2J mice, respectively (Hassoun and Stohs, 1996). Based on these response patterns it has been suggested that TCDD-induced cleft plate and hydronephrosis involve mechanisms that are AhR mediated. However, the letotoxic effects appear to involve mechanisms not related to the AhR since endrin and lindane exhibited comparable responses. Finally, it has been shown that high levels of PCBs, PCDDs, and PCDFs in breast milk were related to reduced neonatal neurological optimality. These results are consistent with the suggestion of the neurotoxic effects of these compounds on the developing brain of newborn infants (Huisman et al., 1995a).

Chaffin et al. (1996) investigated the effects of TCDD exposure during fetal and perinatal development on the estrogen-signaling system in peripubertal female rats. Pregnant rats were given 1 μg/kg TCDD on GD 15. Body weights were reduced, although not significantly, on postnatal day 21. Estrogen receptor mRNA increased in the hypothalamus, uterus, and ovary and decreased in the pituitary. The results of DNA binding assays paralleled the mRNA profile of the uterus, whereas DNA binding activity was decreased in the hypothalamus and unchanged in ovarian protein extracts. Circulating concentrations of estrogen were significantly lower in TCDD-exposed rats, suggesting that the decrease in serum estrogen may be a cause of the alterations in ER mRNA. However, changes in ER DNA binding activity are suggestive of alterations in translation or posttranslational events. The mechanism of the reduction in female fertility that accompanies in utero and lactational exposure to TCDD remains unknown, although it could be linked to the estrogenic effects observed by Gray and Ostby (1995), such as clefting of the phallus and hypospadias.

In 1988, Henshel et al. (1995) started to cull wild heron eggs from contaminated areas of British Columbia and hatch them in the laboratory. Hatchling brains exhibited a high frequency of intercerebral asymmetry, which decreased in subsequent years as TCDD levels decreased. This frequency correlated with the level of TCDD and TCDD toxicity equivalence factors (TEQs) in eggs taken from the same nest. The yolk-free body weight correlated negatively and the brain somatic index correlated positively with TCDD levels in such pair-matched eggs. These results indicate that gross brain morphology, and specifically interce-

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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rebral asymmetry, may be useful as a biomarker for the developmental neurotoxic effects of PCDDs and related chemicals.

Carcinogenicity During the reference period, studies were conducted to examine the role of the AhR in TCDD enzyme induction and tumor promotion. The mechanism by which TCDD induces tumor promotion was also under investigation.

Wang et al. (1995) determined the Ah responsiveness of numerous human cancer cell lines (T-47D, Hep G2, LS 180, MCF-7, A431, C-4II, and MDA-MB-231) on induction of CYP1A1 mRNA levels and EROD activity. With the exception of the MDA-MB-231 breast cancer cell line, TCDD significantly induced CYP1A1 mRNA levels and EROD activity in the remaining six cell lines. However, EC50 values for EROD induction in all cell lines were not consistent for the nuclear AhR complex. The nuclear AhR complex varied from 175 kDa (for the MDA-MB-231 cells) to 221 kDa. Altogether, the molecular properties and levels of the nuclear AhR complex from seven different human cancer cell lines do not predict Ah responsiveness.

Males of the C57BL/6, DBA/2, or F1 strain were initiated with a single i.p. dose of N-nitrosodiethylamine (14, 21, and 21 percent respectively) (Beebe et al., 1995). Although TCDD did not induce CYP1A or promote liver tumors in DBA/ 2 mice, in all other strains results indicate that a functional Ah receptor is required for liver tumor promotion. However, CYP1A1 induction was not directly related to the degree of tumor-promoting capability, suggesting that other genetic factors must play a role in mediating the final tumor outcome.

Abel et al. (1996) studied the dose-response relationship of cytochrome P4501B1 (CYP1B1) and CYP1A1 induction in livers of two strains of TCDD-treated female mice (C57BL/6J and DBA/2J). For both strains, CYP1B1 and CYP1A1 mRNA content increased after TCDD exposure (24 hours) in a dose-dependent manner (0.001-50 μg/kg). These effects were more pronounced in TCDD-responsive C57BL/6J mice than in the less responsive DBA/2J mice. CYP1A1 was more responsive to TCDD than CYP1B1 in both strains, suggesting that CYP1B1 mRNA expression is less inducible by TCDD than CYP1A1 but that both genes are highly AhR regulated.

Huang et al. (1995) conducted studies to compare AhR in cultured fetal cells and adult livers from TCDD-responsive (C57BL/6J) and nonresponsive (DBA/ 2J) mice. In each strain, the molecular mass of the AhR from fetal cells is identical to that from adult liver. The AhR in DBA/2J fetal cells was able to activate a transfected chloramphenicol acetyltransferase linked to a dioxin-responsive element nucleotide sequence. These data suggest that the responsiveness of fetal cells from ''nonresponsive" mice is likely mediated by the AhR but is not due to expression of a different allelic form of AhR ligand binding subunit in fetal cells versus adult liver.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Hakkola and colleagues (1997) studied the expression of the AhR-regulated CYP1B1 gene in human adult and fetal tissues and cell cultures. In adults, CYP1B1 mRNA was detected in lymphocytes and cells of bronchoalveolar lavage, uterine endometrium, and liver, but not lung. The level of expression was very low in adult liver, and only three of six fetal livers expressed CYP1B1. Fetal tissue other than the liver, especially brain and kidney, expressed high levels of CYP1B1. CYP1B1 mRNA was detected at a low level in first-trimester and full-term placental samples. CYP1B1 mRNA was not induced in placenta by maternal cigarette smoking.

The group also studied the inducibility of CYP1B1 by TCDD in primary fibroblasts and a carcinoma cell line (JEG-3) having different CYP1A1 induction properties. The inducibility of CYP1B1 was found to be regulated independently of CYP1A1. In carcinoma cells, CYP1A1 mRNA was induced up to 9,000-fold, while the expression of CYP1B1 was not affected. Expression of the AhR and Arnt was determined in human placenta and in the carcinoma cell line. Expression of these transcription factors was found to be neither coregulated nor affected by AhR ligands. This study provides evidence that in addition to the AhR complex, other cell-specific factors modulate the response of CYP1B1 and CYP1A1 to AhR ligands. The level of complexity of CYP gene induction continues to increase.

The exposure of two hepatoma cell lines Hep G2 and Hepa-1 to moderate hydrodynamic shear, in microcarrier-attached suspension cultures, resulted in the transient induction of CYP1A1 (Mufti et al., 1995). Both cell lines have been characterized with respect to their AhR concentrations and induce CYP1A1 in response to exposure to xenobiotics such as TCDD. Using an AhR antagonist, α-naphthoflavone, and a protein kinase C inhibitor, staurosporine (ST), in the Hep G2 cell line, the induced CYP1A1 activity was modulated in the same manner as when cells were coexposed to TCDD and either α-NF or ST. Exposure of the Hep G2 cell line to TCDD and shear resulted in enhancement of both the induced CYP1A1 activity and a competitive response. Finally, using wild-type and AhR-defective Hepa-1 cell lines, it was demonstrated that a functional AhR was required for shear-induced CYP1A1 expression. The data obtained in three cell lines indicate a role for the AhR in the induction of CYP1A1 by shear in agitated microcarrier cultures.

Gilday et al. (1996) reported the cloning and sequencing of cDNAs for two catalytically distinct TCDD-induced CYP enzymes in chick embryo liver. One mediates classic CYP1A1 activities, whereas the other has some CYP1A2-like activity and is also responsible for TCDD-induced arachidonic acid epoxygenation. Amino acid sequence analysis shows that although each chick enzyme can be classified in the CYP1A family, both are more like CYP1A1 than CYP1A2, and neither can be said to be directly orthologous to CYP1A1 or CYP1A2. Phylogenetic analysis shows that the two chick enzymes form a separate branch in the CYP1A family tree distinct from mammalian CYP1A1 and CYP1A2 and

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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from fish CYP1A enzymes. The findings suggest that CYP1A progenitors split independently in evolutionary lines into two CYP enzymes with some parallel functions, which offers evidence for convergent evolution in the CYP1A family. Northern analysis shows that the chick enzymes have a different tissue distribution of CYP1A1 and CYP1A2. PRC and in situ hybridization data show that both chick enzymes are expressed in response to TCDD even before organ morphogenesis. The findings were interpreted to suggest that beyond their role in activating carcinogens, CYP1A enzymes have conferred evolutionary and developmental advantage, perhaps as defenses in maintaining homeostatic responses to toxic chemicals.

According to Christou et al. (1995), rat mammary cells express both CYP1A1 and CYP1B1 in response to PAHs and TCDD exposure, depending on cell type. CYP1B1 protein was scarcely detected in rat mammary cell but was surprisingly active as a participant in 7,12-dimethylbenz[ a]anthracene (DMBA) metabolism. CYP1B1 was selectively expressed in the stromal fibroblast population of rat mammary cells to the exclusion of CYP1A1. In rat mammary fibroblasts, CYP1B1 protein and associated activity were each present at low levels and were highly induced by benz[a]anthracene (BA) to a greater extent than by TCDD (twelve-versus sixfold). However, BA (10 μM) and TCDD (10 nM) stimulated the 5.2-kilobase CYP1B1-specific mRNA equally. These increases are consistent with the involvement of the AhR in the transcription of the CYP1B1 gene and with the additional stabilization of CYP1B1 protein by BA, as previously observed in embryo fibroblasts. The constitutive expression and PAH inducibility of CYP1B1 and CYP1A1 proteins in rat mammary fibroblasts and epithelial cells, respectively, were each decreased approximately 75 percent by a hormonal mixture of 17β-estradiol (0.2 μM), progesterone (1.5 μM), cortisol (1.5 μM) and prolactin (5 μg/ml). Progesterone and cortisol, added singly to fibroblasts suppressed CYP1B1 protein expression in both untreated and BA-induced cells, whereas cortisol also suppressed CYP1B1 mRNA. In contrast, 17β-estradiol stimulated constitutive expression of CYP1B1 protein (50-75 percent) and mRNA level (two-and threefold) but did not affect CYP1B1 expression in BA-treated fibroblasts. The expression of CYP1A1 and CYP1B1 is therefore highly cell specific even though each is regulated through the AhR. Each cytochrome P450 exhibits a surprisingly similar pattern of hormonal regulation even though expressed in different cell types.

In MCF-7 human breast cancer cells, E2 induction of cat D gene expression is associated with formation of an ER-Sp1 complex within the promoter region (-199/-165) of this gene. E2-induced cat D gene expression is inhibited by TCDD within 30 minutes in MCF-7 cells. Moreover, using a series of synthetic oligonucleotides, which include the wild-type ER-Sp1 and various mutants, it was shown the nuclear AhR complex binds to an imperfect DRE located between the ER and Sp1 binding sequences. This interaction results in disruption of the ER-Sp1 complex and inhibition of E2-induced gene expression. These results are

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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among the first to illustrate that the nuclear AhR complex also exhibits activity as a negative transcription factor via a mechanism similar to that reported for AhR-mediated induction of gene expression.

Wolfle and Marquardt (1996) studied the tumor-promoting activity of TCDD on mouse fibroblasts transformed by certain known carcinogenic chemicals. The promoting effect of TCDD was maximal at a very low concentration (1.5 pM) and comparable to another well-studied tumor promoter TPA (0.25 μg/ml). Chemicals containing reactive oxygen (e.g., scavengers of hydroxyl radicals or antioxidants) hindered the tumor-promoting effect of both TCDD and TPA, suggesting that the promotional effects may involve oxygen radicals.

Induction of CYP1A1 in the hepatoma Hepa 1c1c7 cell line results in elevation of the excretion rate of 8-oxoguanine (oxo8Gua), a biomarker of oxidative DNA damage, and the major repair product of DNA residues 8-oxo-2'-deoxyguanosine (oxo8dG) (Park et al., 1996). Treatment of this cell line with TCDD and ICZ, a metabolite of a natural pesticide found in cruciferous vegetables, induces CYP1A1 activity and elevates excretion rate of oxo8Gua or α-naphtho-flavone. An inhibitor of CYP1A1 activity and an antagonist of the AhR reduced the excretion rate of oxo8Gua. The essential role of AhR in this response is shown by the inability of TCDD to induce CYP1A1 and to increase excretion of oxo8Gua in AhR-defective c4 mutant cells. Although there was a significant sevenfold increase over two days in the excretion rate of oxo8Gua into the growth medium of TCDD-treated Hepa 1c1c7 cells compared to controls, no significant increase was detected in the steady-state level of oxo8dG in the DNA presumably due to efficient DNA repair. Thus, the induction of CYP1A1 appears to result in a leak of oxygen radicals and consequent oxidative DNA damage that could lead to mutation and cancer.

Studies by Baker et al. (1995) examined the effect of TCDD in primary cultures of rat hepatocytes. At noncytolethal doses, TCDD inhibited gap junctional intercellular communication (GJIC) in a time-and concentration (10-8-10-14 M) dependent manner. This inhibition occurred within 4 hours of treatment at doses of 10-8-10-12 M TCDD and persisted up to 48 hours, despite removal of TCDD. Treatment of rat hepatocytes with TCDD resulted in a decrease in hepatocyte connexin 32 mRNA but had no apparent effect on connexin 26 mRNA. Coincubation of rat hepatocytes with TCDD and α-NF abolished down-regulation of GJIC by TCDD. Similarly, co-treatment with a cAMP analogue (8-bromoadenosine 3',5'-cyclic monophosphate) prevented down-regulation of GJIC by TCDD. Results of this investigation suggest that TCDD inhibits GJIC through the AhR. In addition, this study showed that the inhibition of GJIC by TCDD may be due to transcriptional down-regulation or stability of the connexin 32 gap junction mRNA. In other studies, Wamgard et al. (1996) investigated the function, expression, and phosphorylation of different connexins in vitro and in vivo. A good correlation between the ability of TCDD to act as a tumor promoter and to interfere with gap junctional intercellular communication was also reported.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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P450RAP protein, a novel adrenocorticotropic hormone-inducible cytochrome P450, is encoded by a rat CYP1B1 gene orthologous to the mouse CYP1B1 gene (Bhattacharyya et al., 1995). Alignment of rat CYP1B1 amino acid sequences with rat CYP1A1 (39 percent identical) indicated eight regions of high identity for each (60-78 percent), interspersed with extensive regions of less than 30 percent similarity. CYP1B1 mRNA was elevated by two-day adrenocorticotropic hormone treatment but much less than CYP11A1 (cytochrome P450 side chain cleavage) mRNA (twofold versus fourfold). Lower levels of the 5.2-kilobase mRNA in other steroidogenic cells (ovary) were consistent with the amount of immunodetectable CYP1B1 protein, and unlike the adrenal, expression in the ovary was stimulated fivefold by β-naphthoflavone, an AhR agonist, in parallel with CYP1A1 induction. In several other tissues (liver > lung > uterus >> kidney), CYP1B1 mRNA and protein were constitutively undetectable but highly induced by β-naphthoflavone, although at much lower levels than CYP1A1. Thus, rat CYP1B1 exhibits regulation through hormonal signaling and the AhR in a cell-specific manner.

In a recent study, Jorgensen and Autrup (1996) used HepG2 and MCF-7 cell lines to examine a possible cell-specific autoregulation of CYP1A1 promotor function. In HepG2 cells coexpression of increasing amounts of CYP1A1 cDNA significantly down-regulated constitutive as well as TCDD-induced CYP1A1 promoter-driven chloramphenicol acetyltransferase (CAT) activity. In contrast, cotransfection of MCF-7 cells with a threefold molar excess of CYP1A1 cDNA relative to the CYP1A1-CAT reporter construct caused a similar twofold increase in TCDD-induced CAT activity, whereas no effect was observed on constitutive promoter activity. This autoregulatory mechanism of the human CYP1A1 gene product was independent of specific 5'-flanking promoter segments tested. RT-PCR analyses did not indicate any changes in mRNA level of AhR and Arnt in the cotransfection studies. Thus, these studies show that the human CYP1A1 gene is exposed to cell-specific autoregulation, probably achieved via different functions of trans-acting factors.

Weiss et al. (1996) described the results of studies in which transient and stable AhR expression analysis in AhR-deficient subclones was carded out. Transiently expressed AhR has a high basal activity on promoters containing AhR binding sites when transfected into receptor-deficient variant cells compared to wild-type cells. Single-and double-hybrid analysis dissociates AhR ligand responsiveness, transactivation, and heterodimerization with Arnt from receptor binding to a xenobiotic response element (XRE). Hybrid receptors also show high basal activity in the absence of exogenous TCDD in AhR-deficient variant cells, indicating that the endogenous AhR activating signal acts directly on the receptor rather than on the XRE-dependent promoters or DNA binding of the receptor. Stable expression of AhR in variant cell clones by retroviral infection fully reconstitutes TCDD responsiveness, including target gene induction and delay of cell cycle progression. These AhR-reconstituted cells, like AhR-

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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containing wild-type cells, show low basal activity of the transiently expressed AhR hybrid. Thus, the increased basal activity in AhR-deficient cells suggests a negative feedback control of AhR activity. In vitro ligand-binding assays are compatible with the idea that the increased basal activity is due to the accumulation of an AhR binding endogenous ligand.

Gradin and colleagues (1995) presented evidence that induction of CYP1A1 and CYP1B1 gene expression by an AhR ligand is repressed by camptothecin, an inhibitor of topoisomerase I. A transiently transfected reporter construct under control of an XRE containing promoter was not affected by the topoisomerase inhibitor and ligand-dependent activation of the AhR to its DNA binding form is not altered by camptothecin. These results imply that topoisomerase I activity is necessary for the primary CYP1A1 induction response, possibly involving dioxin-dependent alterations in the chromatin structure of the CYP1A1 promoter. The inhibitory effect of camptothecin cannot be exerted once the CYP1A1 gene has been activated.

Walsh et al. (1996) observed little or no TCDD-inducible CYP1A1 mRNA or enzyme activity in high-passage cultures of rat skin cells compared to low-passage cultures. Similarly, transfection of a luciferase reporter construct containing -1,317 to +256 base pairs of the 5'-flanking region of the murine CYP1A1 gene was TCDD-inducible in low-but not high-passage cells. Ligand binding and transfection experiments demonstrated the presence of functional AhR complexes in both high-and low-passage cells. Deletion analysis identified a 26-base pair negative regulatory DNA element contained within the upstream regulatory region of the CYP1A1 gene responsible for this effect. Nuclear extracts from both low-and high-passage cells contain a protein that specifically binds to NeRD-containing DNA. Thus, the loss of PAH sensitivity in high-passage rat epidermal cells appears to be due to decreased expression of CYP1A1, and this effect may be mediated by one or more altered NeRD binding factors present in these cells.

Results from a study by Sadar et al. (1996b) suggest that phenobarbital (PB) induction of CYP1A1 in rainbow trout hepatocytes is regulated by cAMP-dependent pathways (PKA), whereas TCDD induction is not dependent on PKA. This conclusion is consistent with the finding that epinephrine, which increases cAMP levels and activates PKA-dependent pathways, was a potent inhibitor of PB induction but had no effect on TCDD induction of CYP1A1 gene expression. Inhibitors of calcium phospholipid-dependent PKC had modest or no effect on PB and TCDD induction of CYP1A1, respectively.

Estimating Potential Health Risk and Factors Influencing Toxicity

Several approaches have been used to estimate the potential health risks associated with TCDD exposures. These include the use of TEFs, quantitative structure-activity relationships, H4IIE-luc cells, toxicity equivalent concentrations (TECs), and body burdens.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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TEF Approach Toxic equivalency factors (TEF) have been used to estimate the potential health risks associated with exposure to TCDD and related chemicals. This approach is described in Update 1996. As discussed there, the TEF approach has been criticized because the relative potency of hydrocarbons may be tissue specific and influenced by interactions occurring among the chemicals present in environmental mixtures. For instance, the total toxicity of a mixture of halogenated aromatic hydrocarbons is not necessarily the sum of the toxicities of individual congeners because individual congeners can compete for the same receptor; therefore, nonadditive behavior may occur. Furthermore, TEFs have not been tested for all effects of dioxin and dioxin-like chemicals, nor have all responses for all chemicals of concern been examined.

The validity of the TEF approach in predicting the toxicity of mixtures was investigated by Pohl and Holler (1995). Minimal risk levels (MRLs) were derived based on the data bases available for chlorinated dibenzo-p-dioxins and chlorinated dibenzofurans. The MRL values were then converted to TCDD toxicity equivalents (TEQs). There was good correlation between intermediate-duration oral MRLs for TCDD and 2,3,4,7,8-pentachlorodibenzo-p-dioxin when expressed in TEQs (7 and 15 pg/kg per day). Although the studies from which these MRLs were derived used different species (guinea pigs and rats, respectively), the toxicity end points (immunological and hepatic for TCDD and hepatic for 2,3,4,7,8-pentachlorodibenzo-p-dioxin) were comparable. Hepatic effects were measured by the same techniques (blood chemistry and histopathology), ensuring similar sensitivity. However, there was a discrepancy between the acute oral MRLs for TCDD and 2,3,4,7,8-pentachlorodibenzo-p-dioxin when they were expressed in TEQs (20 and 500 pg/kg per day, respectively). In this case, not only did the studies used for MRL derivation involve different species (mice and guinea pigs, respectively), but the immunotoxicity end points were measured by techniques with different sensitivity (serum complement activity versus histopathology), making comparisons difficult. The correlations presented support the concept that TEFs are valid only if specific criteria for their derivation are met (e.g., a broad data base of information, consistency across end points, additivity of effects, common mechanism of action).

QSAR Models Quantitative structure-activity relationship (QSAR) models have been used to estimate binding affinity of multiple chemical classes. The predictive nature of these approaches has been largely unsuccessful due to a focus on minimum energy conformations to predict the activity of molecules. Using structural conformations other than those of minimum energy, Mekenyan et al. (1996) developed a model for AhR binding affinity based on halogenated aromatic chemicals known to interact with the receptor. Resultant QSAR models showed good utility across multiple classes of halogenated aromatic compounds.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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H4IIE-luc Cells Using the wild-type cell line H4IIE, Sanderson et al. (1996) developed a method for rapid screening of environmental samples containing Ah-active polyhalogenated aromatic hydrocarbons (PHAHs). The model consists of a recombinant H4IIE rat hepatoma cell line containing a luciferase reporter gene under the control of dioxin-responsive enhancers (H4IIE-luc cell system). The H4IIE-luc cell system was compared to an H4IIE-wt system that expresses AhR-mediated CYP1A induction. Both cell lines exhibited dose-dependent increases in AhR-mediated response on exposure to known agonists. H4IIE-luc cells were three times as sensitive as H4IIE-wt cells to TCDD. PHAHs tested exhibited similar structure-activity relationships in H4IIE-luc as in H4IIE-wt cells. Binary mixtures of TCDD, PCB-126, and PCB-77 showed no departure from additivity in their combined responses when tested. These findings support the use of H4IIE-luc cells as an alternative bioanalytical tool to H4IIE-wt cells for the detection of Ah agonists in environmental samples.

TEC Approach DeVito et al. (1995) investigated the validity of the TEC approach to predicting the toxicity of mixtures. This approach assumes that hydrocarbons act additively and via a common mechanism to cause toxicity. In their studies, eleven TCDD-like congeners and three non-TCDD-like congeners were combined at ratios found in Lake Michigan trout. Signs of toxicity after exposure of newly fertilized eggs to the mixture or to TCDD were indistinguishable. However, dose-response curves for the mixtures were shifted to the right of TCDD dose-response curves. These data suggest that TCDD-like congeners act via a common mechanism to cause toxicity during early trout development, but may not act strictly additively when combined in a mixture of TCDD-and non-TCDD-like congeners at ratios found in Great Lakes fish. Additive effects predicted by these data deviate less than tenfold from the current safety factor approach used in risk assessments, which suggests that this model is a reasonable approach for assessing the risk posed by complex mixtures of PCDDs, PCDFs, and PCBs. In other studies by Birnbaum and DeVito (1995), mice were dosed to reach steady-state conditions, thus precluding bias of TEF values due to pharmacokinetic factors. Slight differences were found in relative potency of some of the congeners when EROD was compared among tissues (liver, lung, and skin), but these differences were less than fivefold. In addition, there appeared to be slight differences for certain congeners with respect to the relative induction potency of CYP1A1 and CYP1A2.

Body Burdens Walker et al. (1996) have suggested that the body burdens of dioxins that produce effects in experimental animals are comparable to body burdens associated with similar effects in humans. In their studies, human body burdens were estimated from lipid-adjusted serum concentrations of dioxins. In the general population, average background concentrations were estimated at 58 ng TEQ/kg serum lipid, corresponding to a body burden of 13 ng TEQ/kg

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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body weight. Populations with known exposure to dioxins have body burdens of 96-7,000 ng TEQ/kg body weight. Results of this investigation indicate that chloracne and induction of CYP1A1, effects clearly associated with dioxin, occur at similar body burdens in humans and animals. Induction of cancer in animals occurs at body burdens of 944-137,000 ng TCDD/kg body weight, whereas noncancer effects in animals occur at body burdens of 10-12, 500 ng/kg. Based on these correlations, investigators concluded that dioxin exposures may result in cancer and noncancer effects at body burdens within one to two orders of magnitude of those in the general population.

Interspecies and Interindividual Differences in Sensitivity Mass mortalities among marine mammal populations in recent years have raised questions about a possible contributory role of contaminants accumulated through the marine food chain. Ross et al. (1996) carried out a 30-month immunotoxicological experiment in which two groups, each containing 11 harbor seals, were fed TCDD-contaminated herring from the Baltic Sea or relatively uncontaminated herring from the Atlantic Ocean. Seals fed Baltic Sea herring accumulated three to four times higher levels of AhR-mediated TCDDs (measured in TEFs) in blubber than their Atlantic counterparts after two years on their respective diets. Blood was sampled 17 times during the course of the experiment for immunological evaluation, during which time the natural cytotoxic activity of peripheral blood mononuclear cells isolated from seals fed Baltic Sea herring declined to a level approximately 25 percent lower than that observed in seals fed Atlantic herring. Since these cells play an important role in the first line of defense against viruses, their observed inactivity in seals feeding on Baltic Sea herring suggests that exposure to contaminants may have an adverse effect on the defense against viral infections in seals inhabiting polluted waters in Europe.

Ema et al. (1994) observed an insertion mutation that results in a stop codon mutation and a truncated AhR. This receptor polymorphism may result in inter-individual differences in responses to dioxin.

Lang et al. (1994) have shown that expression of the AhR in lymphocytes in humans and rodent species is quite different. In mice and rats the AhR is expressed constitutively in lymphocytes, whereas in human peripheral blood lymphocytes, the receptor is not expressed in resting cells and requires culture with mitogens. Differential expression of receptors in target tissues may therefore account for species differences in responsiveness (Lang et al., 1994).

ISSUES IN EVALUATING THE EVIDENCE

A valid surrogate animal model for the study of a human disease must reproduce with some degree of fidelity the manifestations of the disease in humans. Whole-animal studies or animal-based experimental systems continue to be used to study herbicide toxicity because they allow for rigid control of chemi-

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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cal exposures and close monitoring of health outcomes. Because many of the chemical exposures presently associated with certain diseases in humans have been confirmed in experimental studies (Huff, 1993; Huff et al., 1994), data derived from such studies are generally accepted as a valuable guide in the assessment of biological plausibility.

As discussed in this chapter, many of the toxic effects of the herbicides used in Vietnam have been ascribed to 2,3,7,8-tetrachlorodibenzo-p-dioxin, a contaminant of some of the herbicides. This has not, however, simplified the risk assessment process because the toxicologic profile of TCDD is rather complex. In general, there is consensus that most of the toxic effects of TCDD involve interaction with the aryl hydrocarbon receptor, a protein that binds TCDD and other aromatic hydrocarbons with high affinity. The development of AhR knockout mice has helped to establish a definitive association between the AhR and TCDD-mediated toxicity. Formation of an active complex involving the receptor, ligand (the TCDD molecule), and other protein factors is followed by interaction of the activated complex with specific sites on DNA. This interaction results in DNA changes that alter the expression of genes involved in the regulation of cellular processes. In this manner, TCDD and other AhR ligands modulate target cells and presumably exert toxic effects. Attempts to establish correlations between the effects of TCDD in experimental systems and in humans are particularly problematic because species differences in susceptibility to TCDD have been documented. Humans may actually be more resistant than other species to the toxic effects of this chemical (Dickson and Buzik, 1993). Differences in susceptibility involve a toxicokinetic component, since elimination rates in humans may be slower than in rodents. Toxicodynamic interactions are also important because the affinity of TCDD for the AhR is species specific (Lorenzen and Okey, 1991), and responses to occupancy of the receptor vary among different cell types and during different developmental stages.

If TCDD is assumed to be primarily responsible for the harmful effects of herbicides, then toxicity would be predicted to be receptor mediated. Such deductive reasoning, however, has faced considerable challenges, because evidence continues to accumulate that the AhR does not appear to be exclusively responsible for the toxic effects of TCDD, as discussed in Update 1996 and this chapter. Of particular significance is the recognition that a variable region present in the AhR (Dolwick et al., 1993) may account for the multiple forms of AhR that dictate both species-and cell-specific differences in responsiveness to receptor ligands.

Although studies in which transformed human cell lines are employed to study AhR biology minimize the inherent error associated with species extrapolations, caution must be exercised because the extent to which transformation itself influences toxicity outcomes has yet to be fully defined. It is generally accepted that genetic susceptibility plays a key role in determining the adverse effects of environmental chemicals. In the case of TCDD, the drug-metabolizing enzymes induced in humans are different from those induced in rodents, suggesting that

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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the impact of different genetic backgrounds on AhR function is not yet completely understood. This issue is particularly central to the assessment of biologic plausibility, because polymorphisms of the AhR in humans similar to those in laboratory animals would place some individuals at greater risk for the toxic and carcinogenic effects of TCDD. Ultimately, the major challenge in the assessment of biologic plausibility for the toxicity of herbicides and TCDD is not restricted to the understanding of receptor-mediated events. The dose-response relationships that arise from multiple toxicokinetic and toxicodynamic interactions must also be considered. Gene regulation models described to date do not consider the intricacies of the multiprotein interactions between the AhR and other proteins. Thus, future attempts to define the quantitative relationship between receptor occupancy and biologic response to TCDD must consider that multiple biochemical changes may influence the overall cellular response.

REFERENCES

Abbott BD. 1995. Review of the interaction between TCDD and glucocorticoids in embryonic palate . Toxicology 105:365-373.

Abbott BD, Birnbaum LS, Diliberto JJ. 1996. Rapid distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to embryonic tissues in C57BL/6N mice and correlation with palatal uptake in vitro. Toxicology and Applied Pharmacology 141:256-263.

Abbott BD, Probst MR. 1995. Developmental expression of two members of a new class of transcription factors: II. Expression of aryl hydrocarbon receptor nuclear translocator in the C57BL-6N mouse embryo. Developmental Dynamics 204(2):144-155.

Abel J, Li W, Dohr O, Vogel C, Donat S. 1996. Dose-response relationship of cytochrome P4501b1 mRNA induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin in livers of C57BL/6J and DBA/2J mice. Archives of Toxicology 70(8):510-513.

Aozasa O, Ohta S, Mase Y, Miyata H. 1995. Comparative studies on bioaccumulation of PCDDs and PCDFs in C57BL/6 and DBA/2 mice treated with a mixture by oral administration. Chemosphere 30:1819-1828.

Ayotte P, Carrier G, Dewailly E. 1996. Health risk assessment for Inuit newborns exposed to dioxin-like compounds through breast feeding. Chemosphere 32:531-542.


Bacsi SG, Reisz-Porszasz S, Hankinson O. 1995. Orientation of the heterodimeric aryl hydrocarbon (dioxin) receptor complex on its asymmetric DNA recognition sequence. Molecular Pharmacology 47(3):432-438.

Badesha JS, Maliji G, Haks B. 1995. Immunotoxic effects of prolonged dietary exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin. European Journal of Pharmacology 293(4):429-437.

Baker TK, Kwiatkowski AP, Madhukar BV, Klaunig JE. 1995. Inhibition of gap junctional intercellular communication by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat hepatocytes. Carcinogenesis 16(10):2321-2326.

Bank PA, Yao EF, Swanson HI, Tullis K, Denison MS. 1995. DNA binding of the transformed guinea pig hepatic Ah receptor complex: identification and partial characterization of two high-affinity DNA-binding forms. Archives of Biochemistry and Biophysics 317(2):439-448.

Beebe LE, Fornwald LW, Diwan BA, Anver MR, Anderson LM. 1995. Promotion of N-nitrosodiethylamine-initiated hepatocellular tumors and hepatoblastomas by 2,3,7,8-tetrachlorodibenzo-p-dioxin or Aroclor 1254 in C57BL/6, DBA/2, and B6D2F1 mice. Cancer Research 55:4875-4880.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
×

Bergesse JR, Balegro HF. 1995. 2,4-Dichlorophenoxyacetic acid influx is mediated by an active transport system in Chinese hamster ovary cells. Toxicology Letters 81:167-173.

Berkers JA, Hassing I, Spenkelink B, Brouwer A, Blaauboer BJ. 1995. Interactive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and retinoids on proliferation and differentiation in cultured human keratinocytes: quantification of cross-linked envelope formation. Archives of Toxicology 69:368-378.

Bhattacharyya KK, Brake PB, Eltom SE, Otto SA, Jefcoate CR. 1995. Identification of a rat adrenal cytochrome P450 active in polycyclic hydrocarbon metabolism as rat CYP1B1. Demonstration of a unique tissue-specific pattern of hormonal and aryl hydrocarbon receptor-linked regulation. Journal of Biological Chemistry 270(19): 11595-11602.

Birnbaum LS, DeVito MJ. 1995. Use of toxic equivalency factors for risk assessment for dioxins and related compounds. Toxicology 105:391-401.

Blakley BR. 1997. Effect of roundup and Tordon 202C herbicides on antibody production in mice. Veterinary and Human Toxicology 39(4):204-206.

Blakley, PM, Kim ES, Firneisz GD. 1989. Effects of paternal subacture exposure to Tordon 202C on fetal growth and development in CD-1 mice . Teratology 39(3):237-241.

Brodie AE, Azarenko VA, Hu CY. 1996. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibition of fat cell differentiation. Toxicology Letters. 1996 84:55-59.

Brodie AE, Azarenko VA, Hu CY. 1997. Inhibition of increases of transcription factor mRNAs during differentiation of primary rat adipocytes by in vivo 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment. Toxicology Letters 90:91-95.

Brown D J, Van Beneden R J, Clark GC. 1995. Identification of two binding proteins for halogenated aromatic hydrocarbons in the hard-shell clam, Mercenaria mercenaria. Archives of Biochemistry and Biophysics 319(1):217-224.

Burleson GR, Lebrec H, Yang YG, Ibanes JD, Pennington KN, Birnbaum LS. 1996. Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on influenza virus host resistance in mice. Fundamental and Applied Toxicology 29(1):40-47.

Cantrell SM, Lutz LH, Tillitt DE, Hannink M. 1996. Embryotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): the embryonic vasculature is a physiological target for TCDD-induced DNA damage and apoptotic cell death in Medaka (Orizias latipes). Toxicology and Applied Pharmacology 141:23-34.

Carrier G, Brunet RC, Brodeur J. 1995a. Modeling of the toxicokinetics of polychlorinated dibenzo-p-dioxins and dibenzofurans in mammalians, including humans. I. Nonlinear distribution of PCDD/PCDF body burden between liver and adipose tissues. Toxicology and Applied Pharmacology 131:253-266.

Carrier G, Brunet RC, Brodeur J. 1995b. Modeling of the toxicokinetics of polychlorinated dibenzo-p-dioxins and dibenzofurans in mammalians, including humans. II. Kinetics of absorption and disposition of PCDDs/PCDFs. Toxicology and Applied Pharmacology 131:267-276.

Celander M, Weisbrod R, Stegeman JJ. 1997. Glucocorticoid potentiation of cytochrome P4501A1 induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin in porcine and human endothelial cells in culture. Biochemical and Biophysical Research Communications 232:749-753.

Chaffin CL, Peterson RE, Hutz RJ. 1996. In utero and lactational exposure of female Holtzman rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin: modulation of the estrogen signal. Biology of Reproduction 55:62-67.

Charles JM, Bond DM, Jeffries TK, et al. 1996a. Chronic dietary toxicity/oncogenicity studies on 2,4-dichlorophenoxyacetic acid in rodents. Fundamental and Applied Toxicology 33:166-172.

Charles JM, Cunny HC, Wilson RD, Bus JS. 1996b. Comparative subchronic studies on 2,4-dichlorophenoxyacetic acid, amine, and ester in rats. Fundamental and Applied Toxicology 33:161-165.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
×

Charles JM, Dalgard DW, Cunny HC, Wilson RD, Bus JS. 1996c. Comparative subchronic and chronic dietary toxicity studies on 2,4-dichlorophenoxyacetic acid, amine, and ester in the dog. Fundamental and Applied Toxicology 29:78-85.

Chen I, Safe S. Bjeldanes L. 1996. Indole-3-carbinol and diindolylmethane as aryl hydrocarbon (Ah) receptor agonists and antagonists in T47D human breast cancer cells. Biochemical Pharmacology 51 (8): 1069-1076.

Chen YH, Riby J, Srivastava P, Bartholomew J, Denison M, Bjeldanes L. 1995. Regulation of CYP1A1 by indolo[3,2-b]carbazole in murine hepatoma cells. Journal of Biological Chemistry 270(38): 22548-22555.

Chen YH, Tukey RH. 1996. Protein kinase C modulates regulation of the CYP1A1 gene by the arylhydrocarbon receptor. Journal of Biological Chemistry 271(42):26261-26266.

Cheung YL, Lewis DF, Ridd TI, Gray TJ. Ioannides C. 1997. Diaminonaphthalenes and related aminocompounds: mutagenicity, CYP1A induction and interaction with the Ah receptor. Toxicology 118(2-3):115-127.

Christou M, Savas U, Schroeder S, Shen X, Thompson T, Gould MN, Jefcoate CR. 1995. Cytochromes CYP1A1 and CYP1B1 in the rat mammary gland: cell-specific expression and regulation by polycyclic aromatic hydrocarbons and hormones. Molecular and Cellular Endocrinology 115(1):41-50.

Couture LA, Harris MW, Birnbaum LS. 1990a. Characterization of the peak period of sensitivity for the induction of hydronephrosis in C57B1/6N mice following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fundamental and Applied Toxicology 15:142-150.

Couture LA, Abbott BD, Birnbaum LS. 1990b. A critical review of the developmental toxicity and teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin: recent advances toward understanding the mechanism. Teratology 42:619-627.

Corton JC, Moreno ES, Hovis SM, Leonard LS, Gaido KW, Joyce MM, Kennett SB. 1996. Identification of a cell-specific transcription activation domain within the human Ah receptor nuclear translocator. Toxicology and Applied Pharmacology 139(2):272-280.

Coumailleau P, Poellinger L, Gustafsson JA, Whitelaw ML. 1995. Definition of a minimal domain of the dioxin receptor that is associated with hsp90 and maintains wild type ligand binding affinity and specificity. Journal of Biological Chemistry 270(42):25291-25300.

Cummings AM, Metcalf JL. Birnbaum L. 1996. Promotion of endometriosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats and mice: time-dose dependence and species comparison. Toxicology and Applied Pharmacology 138:131-139.

de Heer C, Schuurman HJ, Liem AK, Penninks AH, Vos JG, van Loveren H. 1995a. Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the human thymus after implantation in SCID mice. Toxicology and Applied Pharmacology 134(2):296-304.

de Heer C, van Driesten G, Schuurman HJ, Rozing J, van Loveren H. 1995b. No evidence for emergence of autoreactive V beta 6+ T cells in Mls-1a mice following exposure to a thymotoxic dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicology 103(3):195-203.

De Krey GK, Kerkvliet NI. 1995. Suppression of cytotoxic T lymphocyte activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin occurs in vivo, but not in vitro, and is independent of corticoster-one elevation. Toxicology 97(1-3):105-112.

DeVito MJ, Birnbaum LS. 1995. The importance of pharmacokinetics in determining the relative potency of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran. Fundamental and Applied Toxicology 24:145-148.

DeVito MJ, Birnbaum LS, Farland WH, Gasiewicz TA. 1995. Comparisons of estimated human body burdens of dioxinlike chemicals and TCDD body burdens in experimentally exposed animals. Environmental Health Perspectives 103:820-831.

Dickson LC, Buzik SC. 1993. Health risks of ''dioxins": a review of environmental and toxicological considerations. Veterinary and Human Toxicology 35(1):68-77.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
×

Diliberto JJ, Jackson JA, Birnbaum LS. 1996. Comparison of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) disposition following pulmonary, oral, dermal, and parenteral exposures to rats. Toxicology and Applied Pharmacology 138:158-168.

Dohr O, Sinning R, Vogel C, Munzel P, Abel J. 1997. Effect of transforming growth factor-betal on expression of aryl hydrocarbon receptor and genes of Ah gene battery: clues for independent down-regulation in A549 cells. Molecular Pharmacology 51 (5):703-710.

Dohr O, Vogel C, Abel J. 1995. Different response of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-sensitive genes in human breast cancer MCF-7 and MDA-MB 231 cells. Archives of Biochemistry and Biophysics 321 (2):405-412.

Dolwick KM, Swanson HI, Bradfield CA. 1993. In vitro analysis of Ah receptor domains involved in ligand-activated DNA recognition. Proceedings of the National Academy of Sciences (USA) 90:8566-8570.

Duffard R, Garcia G, Rosso S, et al. 1996. Central nervous system myelin deficit in rats exposed to 2,4-dichlorophenoxyacetic acid throughout lactation. Neurotoxicology and Teratology 18:691-696.

Dunn RT II, Ruh TS, Burroughs LK, Ruh MF. 1996. Purification and characterization of an Ah receptor binding factor in chromatin. Biochemical Pharmacology 51(4):437-445.

Ema M, Ohe N, Suzuki M, Mimura J, Sogawa K, Ikawa S, Fujii-Kuriyama Y. 1994. Dioxin binding activities of polymorphic forms of mouse and human arylhydrocarbon receptors. Journal of Biological Chemistry 269(44):27337-27343.

Enan E, Matsumura F. 1995a. Evidence for a second pathway in the action mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Significance of Ah-receptor mediated activation of protein kinase under cell-free conditions. Biochemical Pharmacology 49(2):249-261.

Enan E, Matsumura F. 1995b. Regulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) of the DNA binding activity of transcriptional factors via nuclear protein phosphorylation in guinea pig adipose tissue. Biochemical Pharmacology 50:1199-1206.

Enan E, Matsumura F. 1996. Identification of c-Src as the integral component of the cytosolic Ah receptor complex, transducing the signal of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through the protein phosphorylation pathway. Biochemical Pharmacology 52:1599-1612.

Enan E, Lasley B, Stewart D, Overstreet J, Vandevoort CA. 1996a. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) modulates function of human luteinizing granulosa cells via cAMP signaling and early reduction of glucose transporting activity. Reproductive Toxicology 10:191-198.

Enan E, Moran F, VandeVoort CA, Stewart DR, Overstreet JW, Lasley BL. 1996b. Mechanism of toxic action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in cultured human luteinized granulosa cells. Reproductive Toxicology 10:497-508.

Enan E, Overstreet JW, Matsumura F, VandeVoort CA, Lasley BL. 1996c. Gender differences in the mechanism of dioxin toxicity in rodents and in nonhuman primates. Reproductive Toxicology 10:401-411.


Fan F, Rozman KK. 1995. Short-and long-term biochemical effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in female Long-Evans rats. Toxicology Letters 75:209-216.

Fan F, Pinson DM, Rozman KK. 1995. Immunomodulatory effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin tested by the popliteal lymph node assay. Toxicologic Pathology 23(4):513-517.

Fan F, Wierda D, Rozman KK. 1996. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on humoral and cell-mediated immunity in Sprague-Dawley rats. Toxicology 106(1-3):221-228.

Fan F, Yan B, Wood G, Viluksela M, Rozman KK. 1997. Cytokines (IL-lbeta and TNFalpha) in relation to biochemical and immunological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rats. Toxicology 116(1-3):9-16.

Fernandez-Salguero P, Pineau T, Hilbert DM, McPhail T, Lee SS, Kimura S, Nebert DW, Rudikoff S, Ward JM, Gonzalez FJ. 1995. Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor [see comments]. Science 268(5211):722-726.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Fiorella PD, Olson JR, Napoli JL. 1995. 2,3,7,8-tetrachlorodibenzo-p-dioxin induces diverse retinoic acid metabolites in multiple tissues of the Sprague-Dawley rat. Toxicology and Applied Pharmacology 134:222-228.

Fitzgerald CT, Fernandez-Salguero P, Gonzalez FJ, Nebert DW, Puga A. 1996. Differential regulation of mouse Ah receptor gene expression in cell lines of different tissue origins. Archives of Biochemistry and Biophysics 333(1):170-178.

Fukunaga BN, Probst MR, Reisz-Porszasz S, Hankinson O. 1995. Identification of functional domains of the aryl hydrocarbon receptor. Journal of Biological Chemistry 270(49):29270-29278.

Gaido KW, Maness SC. 1995. Post-transcriptional stabilization of urokinase plasminogen activator mRNA by 2,3,7,8-tetrachlorodibenzo-p-dioxin in a human keratinocyte cell line. Toxicology and Applied Pharmacology 133(1):34-42.

Gasiewicz TA, Kende AS, Rucci G, Whitney B, Willey JJ. 1996. Analysis of structural requirements for Ah receptor antagonist activity: ellipticines, flavones, and related compounds . Biochemical Pharmacology 52(11):1787-1803.

Gassmann M, Kvietikova I, Rolfs A, Wenger RH. 1997. Oxygen-and dioxin-regulated gene expression in mouse hepatoma cells. Kidney International 51(2):567-574.

Gebremichael A, Tullis K, Denison MS, Cheek JM, Pinkerton KE. 1996. Ah-receptor-dependent modulation of gene expression by aged and diluted sidestream cigarette smoke. Toxicology and Applied Pharmacology 141(1):76-83.

Germolec DR, Adams NH, Luster MI. 1995. Comparative assessment of metabolic enzyme levels in macrophage populations of the F344 rat. Biochemical Pharmacology 50(9):1495-1504.

Germolec DR, Henry EC, Maronpot R, Foley JF, Adams NH, Gasiewicz TA, Luster MI. 1996. Induction of CYP1A1 and ALDH-3 in lymphoid tissues from Fisher 344 rats exposed to 2,3,7,8-tetrachlorodibenzodioxin (TCDD). Toxicology and Applied Pharmacology 137(1):57-66.

Gilday D, Gannon M, Yutzey K, Bader D, Rifkind AB. 1996. Molecular cloning and expression of two novel avian cytochrome P450 1A enzymes induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Journal of Biological Chemistry 271(51):33054-33059.

Gohl G, Lehmkoster T, Munzel PA, Schrenk D, Viebahn R, Bock KW. 1996. TCDD-inducible plasminogen activator inhibitor type 2 (PAI-2) in human hepatocytes, HepG2 and monocytic U937 cells. Carcinogenesis 17(3):443-449.

Golub MS, Jacobson SW. 1995. Workshop on perinatal exposure to dioxin-like compounds. IV. Neurobehavioral effects. Environmental Health Perspectives 103 (Suppl) 2:151-155.

Gonzalez FJ, Fernandez-Salguero P, Lee SS, Pineau T, Ward JM. 1995. Xenobiotic receptor knockout mice. Toxicology Letters 82:83117-83121.

Gradin K, McGuire J, Wenger RH, Kvietikova I, Whitelaw ML, Toftgard R, Tora L, Gassmann M, Poellinger L. 1996. Functional interference between hypoxia and dioxin signal transduction pathways: competition for recruitment of the Arnt transcription factor. Molecular and Cellular Biology 16(10):5221-5231.

Gradin K, Toftgard R, Berghard A. 1995. Differential effects of a topoisomerase I inhibitor on dioxin inducibility and high-level expression of the cytochrome P450IA1 gene. Molecular Pharmacology 48(4):610-615.

Gray LE Jr, Kelce WR, Monosson E, Ostby JS, Birnbaum LS. 1995. Exposure to TCDD during development permanently alters reproductive function in male Long-Evans rats and hamsters: reduced ejaculated and epididymal sperm numbers and sex accessory gland weights in offspring with normal androgenic status. Toxicology and Applied Pharmacology 131:108-118.

Gray LE Jr, Ostby JS. 1995. In utero 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters reproductive morphology and function in female rat offspring. Toxicology and Applied Pharmacology 133:285-294.

Guiney PD, Smolowitz RM, Peterson RE, Stegeman JJ. 1997. Correlation of 2,3,7,8-tetrachlorodibenzo-p-dioxin induction of cytochrome P4501A in vascular endothelium with toxicity in early life stages of lake trout. Toxicology and Applied Pharmacology 143(2):256-273.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Hahn ME, Chandran K. 1996. Uroporphyrin accumulation associated with cytochrome P4501A induction in fish hepatoma cells exposed to aryl hydrocarbon receptor agonists, including 2,3,7,8-tetrachlorodibenzo-p-dioxin and planar chlorobiphenyls. Archives of Biochemistry and Biophysics 329(2):163-174.

Hahn ME, Karchner SI. 1995. Evolutionary conservation of the vertebrate Ah (dioxin) receptor: amplification and sequencing of the PAS domain of a teleost Ah receptor cDNA . Biochemical Journal 310(Pt 2):383-387.

Hakkola J, Pasanen M, Pelkonen O, Hukkanen J, Evisalmi S, Anttila S, Rane A, Mantyla M, Purkunen R, Saarikoski S, Tooming M, Raunio H. 1997. Expression of CYP1B1 in human adult and fetal tissues and differential inducibility of CYP1B1 and CYP1A1 by Ah receptor ligands in human placenta and cultured cells. Carcinogenesis 18(2):391-397.

Hanberg A, Kling L, Hakansson H. 1996. Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the hepatic stellate cell population in the rat. Chemosphere 32:1225-1233.

Hanneman WH, Legare ME, Barhoumi R, Burghardt RC, Safe S, Tiffany-Castiglioni E. 1996. Stimulation of calcium uptake in cultured rat hippocampal neurons by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicology 112:19-28.

Harper N, Steinberg M, Thomsen J, Safe S. 1995. Halogenated aromatic hydrocarbon-induced suppression of the plaque-forming cell response in B6C3F1 splenocytes cultured with allogenic mouse serum: Ah receptor structure activity relationships. Toxicology 99(3):199-206.

Hassoun EA, Bagchi D, Stohs SJ. 1995. Evidence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced tissue damage in fetal and placental tissues and changes in amniotic fluid lipid metabolites of pregnant CF1 mice. Toxicology Letters 76:245-250.

Hassoun EA, Stohs SJ. 1996. Comparative teratological studies on TCDD, endrin and lindane in C57B/6J and DBA/2J mice. Comparative Biochemistry and Physiology: C. Pharmacology, Toxicology, and Endocrinology 113(3)393-398.

Hayes HM, Tarone RE, Cantor KP, Jessen CR, McCurnin DM, Richardson RC. 1991. Case-control study of canine malignant lymphoma: positive association with dog owner's use of 2,4-dichlorophenoxyacetic acid herbicides. Journal of the National Cancer Institute 83(17):1226-1231.

Hayes HM, Tarone RE, Cantor KP. 1995. On the association between canine malignant lymphoma and opportunity for exposure to 2,4-dichlorophenoxyacetic acid. Environmental Research 70: 119-125.

Henry EC, Kent TA, Gasiewicz T. 1997a. DNA binding and transcriptional enhancement by purified TCDD cntdot Ah receptor complex. Archives of Biochemistry and Biophysics 339(2):305-314.

Henry TR, Spitsbergen JM, Hornung MW, Abnet CC, Peterson RE. 1997b. Early life stage toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish (Danio rerio). Toxicology and Applied Pharmacology 142:56-68.

Henshel DS, Martin JW, Norstrom R, Whitehead P, Steeves JD, Cheng KM. 1995. Morphometric abnormalities in brains of great blue heron hatchlings exposed in the wild to PCDDs. Environmental Health Perspectives 103 (Suppl 4):61-66.

Hoffer A, Chang CY, Puga A. 1996. Dioxin induces transcription of fos and jun genes by Ah receptor-dependent and-independent pathways. Toxicology and Applied Pharmacology 141(1): 238-247.

Hoffman EC, Reyes H, Chu FF, Sander F, Conley LH, Brooks BA, Hankinson O. 1991. Cloning of a factor required for activity of the Ah (dioxin) receptor, Science 252(5008):954-958.

Hogenesch JB, Chan WK, Jackiw VH, Brown RC, Gu YZ, Pray-Grant M, Perdew GH, Bradfield CA. 1997. Characterization of a subset of the basic-helix-loop-helix-PAS superfamily that interacts with components of the dioxin signaling pathway. Journal of Biological Chemistry 272(13):8581-8593.

Hossain A, Kikuchi H, Ikawa S, Sagami I, Watanabe M. 1995a. Identification of a 120-kDa protein associated with aromatic hydrocarbon receptor nuclear translocator. Biochemical and Biophysical Research Communications 212(1):144-150.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Hossain A, Kikuchi H, Ikawa S, Sagami I, Watanabe M. 1995b. Identification of cellular protein that can interact specifically with the basic helix-loop-helix domain of the aromatic hydrocarbon receptor. Biochemical and Biophysical Research Communications 215(1):405-411.

Huang Y, Harper PA, Okey AB. 1995. Aromatic hydrocarbon receptor in cultured fetal cells from C57BL/6J and DBA/2J mice: similarity in molecular mass to receptors in adult livers. Canadian Journal of Physiology and Pharmacology 73(1):18-26.

Huff J. 1993. Chemicals and cancer in humans: first evidence in experimental animals. Environmental Health Perspectives 100:201-210.

Huff J, Lucier G, Tritscher A. 1994. Carcinogenicity of TCDD: experimental, mechanistic, and epidemiologic evidence. Annual Review of Pharmacology and Toxicology 34:343-372.

Hughes MF, Mitchell CT, Edwards BC, Rahman MS. 1995. In vitro percutaneous absorption of dimethylarsinic acid in mice. Journal of Toxicology and Environmental Health 45:279-290.

Huisman M, Koopman-Esseboom C, Fidler V, et al. 1995a. Perinatal exposure to polychlorinated biphenyls and dioxins and its effect on neonatal neurological development. Early Human Development 41:111-127.

Huisman M, Koopman-Esseboom C, Lanting CI, et al. 1995b. Neurological condition in 18-month-old children perinatally exposed to polychlorinated biphenyls and dioxins. Early Human Development 43:165-176.

Hushka DR, Greenlee WF. 1995. 2,3,7,8-tetrachlorodibenzo-p-dioxin inhibits DNA synthesis in rat primary hepatocytes. Mutation Research 333:89-99

Ilian MA, Sparrow BR, Ryu BW, Selivonchick DP, Schaup HW. 1996. Expression of hepatic pyruvate carboxylase mRNA in C57BL/6J Ah(b/b) and congenic Ah(d/d) mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Journal of Biochemical Toxicology 11:51-56.


Janz DM, Bellward GD. 1996a. In ovo 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure in three avian species. 1. Effects on thyroid hormones and growth during the perinatal period. Toxicology and Applied Pharmacology 139:281-291.

Janz DM, Bellward GD. 1996b. In ovo 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure in three avian species. 2. Effects on estrogen receptor and plasma sex steroid hormones during the perinatal period. Toxicology and Applied Pharmacology 139:292-300.

Jorgensen ECB, Autrnp H. 1995. Effect of a negative regulatory element (NRE) on the human CYP1A1 gene expression in breast carcinoma MCF-7 and hepatoma HepG2 cells. FEBS Letters 365(2-3):101-107.

Jorgensen ECB, Autrup H. 1996. Autoregulation of human CYP1A1 gene promoter activity in HepG2 and MCF-7 cells. Carcinogenesis 17(3):435-441.


Kale PG, Petty BT Jr, Walker S, Ford JB, Dehkordi N, Tarasia S, Tasie BO, Kale R, Sohni YR. 1995. Mutagenicity testing of nine herbicides and pesticides currently used in agriculture. Environmental and Molecular Mutagenesis 25(2):148-153.

Karras JG, Morris DL, Matulka RA, Kramer CM, Holsapple MP. 1996. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) elevates basal B-cell intracellular calcium concentration and suppresses surface Ig-but not CD40-induced antibody secretion. Toxicology and Applied Pharmacology 137(2):275-284.

Kerkvliet NI. 1995. Immunological effects of chlorinated dibenzo-p -dioxins. Environmental Health Perspectives 103 (Suppl 9):47-53.

Kerkvliet NI, Baecher-Steppan L, Shepherd DM, Oughton JA, Vorderstrasse BA, DeKrey GK. 1996. Inhibition of TC-1 cytokine production, effector cytotoxic T lymphocyte development and alloantibody production by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Journal of Immunology 157(6):2310-2319.

Kharat I, Saatcioglu F. 1996. Antiestrogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin are mediated by direct transcriptional interference with the liganded estrogen receptor. Cross-talk between aryl hydrocarbon-and estrogen-mediated signaling. Journal of Biological Chemistry 271(18):10533-10537.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Kim CS, Binienda Z, Sandberg JA. 1996. Construction of a physiologically based pharmacokinetic model for 2,4-dichlorophenoxyacetic acid dosimetry in the developing rabbit brain. Toxicology and Applied Pharmacology 136:250-259.

Kleman M, Gustafsson JA. 1996. Interactions of procarcinogenic heterocyclic amines and indolocarbazoles with the dioxin receptor. Biological Chemistry 377(11):741-762.

Ko HP, Okino ST, Ma Q, Whitlock JP Jr. 1996. Dioxin-induced CYP1A1 transcription in vivo: the aromatic hydrocarbon receptor mediates transactivation, enhancer-promoter communication, and changes in chromatin structure. Molecular and Cellular Biology 16(1):430-436

Kohn MC, Lucier GW, Clark GC, Sewall C, Tritscher AM, Portier CJ. 1993. A mechanistic model of effects of dioxin on gene expression in the rat liver. Toxicology and Applied Pharmacology 120(1):138-154.

Kohn MC, Sewall CH, Lucier GW, Portier CJ. 1996. A mechanistic model of effects of dioxin on thyroid hormones in the rat. Toxicology and Applied Pharmacology 136:29-48.

Kraemer SA, Arthur KA, Denison MS, Smith WL, DeWitt DL. 1996. Regulation of prostaglandin endoperoxide H synthase-2 expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Archives of Biochemistry and Biophysics 330(2):319-328.

Kremer J, Lai ZW, Esser C. 1995. Evidence for the promotion of positive selection of thymocytes by Ah receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin. European Journal of Pharmacology 293(4):413-427.

Krishnan V, Porter W, Santostefano M, Wang X, Safe S. 1995. Molecular mechanism of inhibition of estrogen-induced cathepsin D gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells. Molecular and Cellular Biology 15(12):6710-6719.

Lamb JC, Moore JA, Marks TA. 1980. Evaluation of 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity in C57BL/6 mice. Reproduction and Fertility in Treated Male Mice and Evaluation of Congenital Malformations in Their Offspring. National Toxicology Program.

Lang DS, Becker S, Clark GC, Devlin RB, Koren HS. 1994. Lack of direct immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on human peripheral blood lymphocyte subsets in vitro. Archives of Toxicology 68(5):296-302.

Lawrence BP, Leid M, Kerkvliet NI. 1996. Distribution and behavior of the Ah receptor in murine T lymphocytes. Toxicology and Applied Pharmacology 138(2):275-284.

Lee IJ, Jeong KS, Roberts BJ, Kallarakal AT, Fernandez-Salguero P, Gonzalez FJ, Song BJ. 1996. Transcriptional induction of the cytochrome p4501al gene by a thiazolium compound, yh439 . Molecular Pharmacology 49(6):980-988.

Lesca P, Peryt B, Larrieu G, Alvinerie M, Galtier P, Daujat M, Maurel P, Hoogenboom L. 1995. Evidence for the ligand-independent activation of the ah receptor. Biochemical and Biophysical Research Communications 209(2):474-482.

Li SY, Dougherty JJ. 1997. Inhibitors of serine/threonine-specific protein phosphatases stimulate transcription by the Ah receptor/Arnt dimer by affecting a step subsequent to XRE binding. Archives of Biochemistry and Biophysics 340(1):73-82.

Li W, Donat S, Dohr O, Unfried K, Abel J. 1994. Ah receptor in different tissues of C57BL/6J and DBA/2J mice: use of competitive polymerase chain reaction to measure Ah-receptor mRNA expression. Archives of Biochemistry and Biophysics 315(2):279-284.

Li X, Johnson DC, Rozman KK. 1997. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases release of luteinizing hormone and follicle-stimulating hormone from the pituitary of immature female rats in vivo and in vitro. Toxicology and Applied Pharmacology 142:264-269.

Li X, Rozman KK. 1995. Subchronic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and their reversibility in male Sprague-Dawley rats. Toxicology 97:133-140.

Lindebro MC, Poellinger L, Whitelaw ML. 1995. Protein-protein interaction via PAS domains: role of the PAS domain in positive and negative regulation of the bHLH/PAS dioxin receptor-Arnt transcription factor complex. EMBO Journal 14(14):3528-35239.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Liu H, Safe S. 1996. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on insulin-induced responses in MCF-7 human breast cancer cells. Toxicology and Applied Pharmacology 138(2): 242-250.

Liu PC, Matsumura F. 1995. Differential effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the "adipose-type" and "brain-type" glucose transporters in mice. Molecular Pharmacology 47:65-73.

Lorenzen A, Kennedy SW, Bastien LJ, Hahn ME. 1997. Halogenated aromatic hydrocarbon-mediated porphyrin accumulation and induction of cytochrome P4501A in chicken embryo hepatocytes. Biochemical Pharmacology 53:373-384.

Lorenzen A, Okey AB. 1991. Detection and characterization of Ah receptor in tissue and cells from human tonsils. Toxicology and Applied Pharmacology 107:203-214.

Lu YF, Santostefano M, Cunningham BD, Threadgill MD, Safe S. 1996a. Substituted flavones as aryl hydrocarbon (Ah) receptor agonists and antagonists. Biochemical Pharmacology 51(8): 1077-1087.

Lu YF, Sun G, Wang X, Safe S. 1996b. Inhibition of prolactin receptor gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 human breast cancer cells. Archives of Biochemistry and Biophysics 332(1):35-40.

Luebke RW, Copeland CB, Andrews DL. 1995. Host resistance to Trichinella spiralis infection in rats exposed to 2,3,7,8-tetrachlorodibenzo-p -dioxin (TCDD). Fundamental and Applied Toxicology 24(2):285-289.

Luebke RW, Copeland CB, Diliberto JJ, Akubue PI, Andrews DL, Riddle MM, Williams WC, Birnbaum LS. 1994. Assessment of host resistance to Trichinella spiralis in mice following preinfection exposure to 2,3,7,8-TCDD. Toxicology and Applied Pharmacology 125(1):7-16.

Ma Q, Whitlock JP Jr. 1996. The aromatic hydrocarbon receptor modulates the Hepa 1c1c7 cell cycle and differentiated state independently of dioxin. Molecular and Cellular Biology 16(5):2144-2150

Ma X, Stoffregen DA, Wheelock GD, Rininger JA, Babish JG. 1997. Discordant hepatic expression of the cell division control enzyme p34cdc2 kinase, proliferating cell nuclear antigen, p53 tumor suppressor protein, and p21Waf1 cyclin-dependent kinase inhibitory protein after WY14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid) dosing to rats. Molecular Pharmacology 51:69-78.

Mahon MJ, Gasiewicz TA. 1995. Ah receptor phosphorylation: localization of phosphorylation sites to the C-terminal half of the protein. Archives of Biochemistry and Biophysics 318(1): 166-174.

Masten SA, Shiverick KT. 1995. The Ah receptor recognizes DNA binding sites for the B cell transcription factor, BSAP: a possible mechanism for dioxin-mediated alteration of CD19 gene expression in human B lymphocytes. Biochemical and Biophysical Research Communications 212(1):27-34.

Masten SA, Shiverick KT. 1996. Characterization of the aryl hydrocarbon receptor complex in human B lymphocytes: evidence for a distinct nuclear DNA-binding form. Archives of Biochemistry and Biophysics 336(2):297-308.

McGrath LF, Cooper KR, Georgopoulos P, Gallo MA. 1995. Alternative models for low dose-response analysis of biochemical and immunological endpoints for tetrachlorodibenzo-p-dioxin. Regulatory Toxicology and Pharmacology 21:382-396.

McGuire J, Whitelaw ML, Pongratz I, Gustafsson JA, Poellinger L. 1994. A cellular factor stimulates ligand-dependent release of hsp90 from the basic helix-loop-helix dioxin receptor. Molecular and Cellular Biology 14(4):2438-2446.

Mekenyan OG, Veith GD, Call DJ, Ankley GT. 1996. A QSAR evaluation of Ah receptor binding of halogenated aromatic xenobiotics. Environmental Health Perspectives 104(12):1302-1310.

Merchant M, Safe S. 1995. In vitro inhibition of 2,3,7,8 -tetrachlorodibenzo-p-dioxin-induced activity by alpha-naphthoflavone and 6-methyl-1,3,8-trichlorodibenzofuran using an aryl hydrocarbon (Ah)-responsive construct. Biochemical Pharmacology 50(5):663-668.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Michalek JE, Caudill SP, Tripathi RC. 1997. Pharmacokinetics of TCDD in veterans of Operation Ranch Hand: 10-year follow-up. Erratum. Journal of Toxicology and Environmental Health 52(6):557-558.

Michalek JE, Pirkle JL, Caudill SP, Tripathi RC, Patterson DG Jr, Needham LL. 1996a. Pharmacokinetics of TCDD in veterans of Operation Ranch Hand: 10-year follow-up. Journal of Toxicology and Environmental Health 47:209-220.

Michalek JE, Tripathi RC, Kulkarni PM, Pirkle JL. 1996b. The reliability of the serum dioxin measurement in veterans of Operation Ranch Hand. Journal of Exposure Analysis and Environmental Epidemiology 6:327-338.

Miranda S, Vollrath V, Wielandt AM, Loyola G, Bronfman M, Chianale J. 1997. Overexpression of mdr2 gene by peroxisome proliferators in the mouse liver. Journal of Hepatology 26(6): 1331-1339.

Moos AB, Kerkvliet NI. 1995. Inhibition of tumor necrosis factor activity fails to restore 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced suppression of the antibody response to sheep red blood cells. Toxicology Letters 81(2-3):175-181.

Mufti NA, Bleckwenn NA, Babish JG, Shuler ML. 1995. Possible involvement of the Ah receptor in the induction of cytochrome P-450IA1 under conditions of hydrodynamic shear in microcarrier-attached hepatoma cell lines. Biochemical and Biophysical Research Communications 208(1):144-152.

Mýnzel P, Bock-Hennig B, Schieback S, Gschaidmeier H, Beck-Gschaidmeier S, Bock KW. 1996. Growth modulation of hepatocytes and rat liver epithelial cells (WB-F344) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Carcinogenesis 17(2):197-202.

Neubert R, Maskow L, Delgado I, Helge H, Neubert D. 1995. Chlorinated dibenzo-p-dioxins and dibenzofurans and the human immune system. 2. In vitro proliferation of lymphocytes from workers with quantified moderately-increased body burdens. Life Sciences 56(6): 421-436.

Nodland KI, Wormke M, Safe S. 1997. Inhibition of estrogen-induced activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the MCF-7 human breast cancer and other cell lines transfected with vitellogenin A2 gene promoter constructs. Archives of Biochemistry and Biophysics 338(1):67-72.


Ochi T, Nakajima F, Sakurai T, Kaise T, Oya-Ohta Y. 1996. Dimethylarsinic acid causes apoptosis in HL-60 cells via interaction with glutathione. Archives of Toxicology 70:815-821.

Okino ST, Whitlock JP Jr. 1995. Dioxin induces localized, graded changes in chromatin structure: implications for Cyp1A1 gene transcription. Molecular and Cellular Biology 15(7):3714-3721.

Oliveira GH, Palermo-Neto J. 1995. Toxicology of 2,4-dichlorophenoxyacetic acid (2,4-D) and its determination in serum and brain tissue using gas chromatography-electron-capture detection. Journal of Analytical Toxicology 19:251-255.

Olnes MJ, Verma M, Kurl RN. 1996. 2,3,7,8-tetrachlorodibenzo-p-dioxin modulates expression of the prostaglandin G/H synthase-2 gene in rat thymocytes. Journal of Pharmacology and Experimental Therapeutics 279(3):1566-1573.

Ou X, Ramos KS. 1995. Regulation of cytochrome P4501A1 gene expression in vascular smooth muscle cells through aryl hydrocarbon receptor-mediated signal transduction requires a protein synthesis inhibitor. Archives of Biochemistry and Biophysics 316(1):116-122.


Palmeira CM, Moreno AJ, Madeira VM. 1995a. Effects of paraquat, dinoseb and 2,4-D on intracellular calcium and on vasopressin-induced calcium mobilization in isolated hepatocytes. Archives of Toxicology 69:460-466.

Palmeira CM, Moreno AJ, Madeira VM. 1995b. Thiols metabolism is altered by the herbicides paraquat, dinoseb and 2,4-D: a study in isolated hepatocytes. Toxicology Letters 81:115-123.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Park JY, Shigenaga MK, Ames BN. 1996. Induction of cytochrome P4501A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin or indolo (3,2-b) carbazole is associated with oxidative DNA damage. Proceedings of the National Academy of Sciences of the United States of America 93(6): 2322-2327.

Paulino CA, Guerra JL, Oliveira GH, Palermo-Neto J. 1996. Acute, subchronic and chronic 2,4-dichlorophenoxyacetic acid (2,4-D) intoxication in rats. Veterinary and Human Toxicology 38:348-352.

Paulino CA, Palermo-Neto J. 1995. Effects of acute 2.4-dichlorophenoxyacetic acid on cattle serum components and enzyme activities. Veterinary and Human Toxicology 37:329-332.

Peters JM, Wiley LM. 1995a. Murine preimplantation embryos express aryl hydrocarbon receptor nuclear translocator (Arnt) mRNA. Teratology 51(3):193.

Peters JM, Wiley LM. 1995b. Evidence that murine preimplantation embryos express aryl hydrocarbon receptor. Toxicology and Applied Pharmacology 134(2):214-221.

Phillips M, Enan E, Liu PC, Matsumura F. 1995. Inhibition of 3T3-L1 adipose differentiation by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Journal of Cell Science 108(Pt 1):395-402.

Pohl H. Holler J. 1995. Halogenated aromatic hydrocarbons and toxicity equivalency factors (TEFs) from the public health assessment perspective. Chemosphere 31(1):2547-2559.

Pollenz RS, Sullivan HR. Holmes J, Necela B, Peterson RE. 1996. Isolation and expression of cDNAs from rainbow trout (Oncorhynchus mykiss) that encode two novel basic helix-loop-Helix/PER-ARNT-SIM (bHLH/PAS) proteins with distinct functions in the presence of the aryl hydrocarbon receptor. Evidence for alternative mRNA splicing and dominant negative activity in the bHLH/PAS family. Journal of Biological Chemistry 271(48):30886-30896.

Prell RA, Kerkvliet NI. 1997. Involvement of altered B7 expression in dioxin immunotoxicity: B7 transfection restores the CTL but not the autoantibody response to the P815 mastocytoma. Journal of Immunology 158(6):2695-2703.

Prell RA, Oughton JA, Kerkviet NI. 1995. Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on anti-CD3-induced changes in T-cell subsets and cytokine production. International Journal of Immunopharmacology 17(11):951-961.

Rhile MJ, Nagarkatti M, Nagarkatti PS. 1996. Role of Fas apoptosis and MHC genes in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity of T cells. Toxicology 110(1-3):153-167.

Rier SE, Martin DC, Bowman RE, Dmowski WP, Becker JL. 1993. Endometriosis in rhesus monkeys (Macaca mulatta) following chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin [see comments]. Fundamental and Applied Toxicology 21(4):433-441.

Rin K, Kawaguchi K, Yamanaka K, Tezuka M, Oku N, Okada S. 1995. DNA-strand breaks induced by dimethylarsinic acid, a metabolite of inorganic arsenics, are strongly enhanced by superoxide anion radicals. Biological and Pharmaceutical Bulletin 18:45-48.

Roman BL, Sommer RJ, Shinomiya K, Peterson RE. 1995. In utero and lactational exposure of the male rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin: impaired prostate growth and development without inhibited androgen production. Toxicology and Applied Pharmacology 134:241-250.

Ross PS, De Swart RL, Timmerman HH, Reijnders PJH, Vos JG, Van Loveren H, Osterhaus ADME. 1996. Suppression of natural killer cell activity in harbour seals (Phoca vitulina) fed Baltic Sea herring. Aquatic Toxicology 34(1):71-84.

Rowlands JC, McEwan IJ, Gustafsson JA. 1996. Trans-activation by the human aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator proteins: direct interactions with basal transcription factors. Molecular Pharmacology 50(3):538-548.


Sadar MD, Ash R, Sundqvist J, Olsson PE, Andersson TB. 1996a. Phenobarbital induction of CYPIA1 gene expression in a primary culture of rainbow trout hepatocytes. Journal of Biological Chemistry 271(30):17635-17643.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Sadar MD, Blomstrand F, Andersson TB. 1996b. Phenobarbital induction of cytochrome P4501A1 is regulated by cAMP-dependent protein kinase-mediated signaling pathways in rainbow trout hepatocytes . Biochemical and Biophysical Research Communications 225(2):455-461.

Sadar MD, Westlind A, Blomstrand F, Andersson TB. 1996c. Induction of CYP1A1 by GABA receptor ligands. Biochemical and Biophysical Research Communications 229(1):231-237.

Sandberg JA, Duhart HM, Lipe G, Binienda Z, Slikker W Jr, Kim CS. 1996. Distribution of 2,4-dichlorophenoxyacetic acid (2,4-D) in maternal and fetal rabbits. Journal of Toxicology and Environmental Health 49:497-509.

Sanderson JT, Aarts JM, Brouwer A, Froese KL, Denison MS, Giesy JP. 1996. Comparison of Ah receptor-mediated luciferase and ethoxyresorufin-O-deethylase induction in H4IIE cells: implications for their use as bioanalytical tools for the detection of polyhalogenated aromatic hydrocarbons. Toxicology and Applied Pharmacology 137(2):316-325.

Santostefano M, Safe S. 1996. Characterization of the molecular and structural properties of the transformed and nuclear aryl hydrocarbon (Ah) receptor complexes by proteolytic digestion. Chemico-Biological Interactions 100(3):221-240.

Sastry BV, Janson VE, Clark CP, Owens LK. 1997. Cellular toxicity of 2,4,5-trichlorophenoxyacetic acid: formation of 2,4,5-trichlorophenoxyacetylcholine. Cellular and Molecular Biology 43(4):549-557.

Schmidt JV, Bradfield CA. 1996. Ah receptor signaling pathways. Annual Review of Cell and Developmental Biology 12:55-89.

Schmidt JV, Su, GH, Reddy JK, Simon MC, Bradfield CA. 1996. Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development. Proceedings of the National Academy of Sciences of the United States of America 93(13):6731-6736.

Schuetz EG, Schuetz JD, Thompson MT, Fisher RA, Madariage JR, Strom SC. 1995. Phenotypic variability in induction of P-glycoprotein mRNA by aromatic hydrocarbons in primary human hepatocytes. Molecular Carcinogenesis 12(2):61-65.

Selmin O, Lucier GW, Clark GC, et al. 1996. Isolation and characterization of a novel gene induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver. Carcinogenesis 17:2609-2615.

Sewall CH, Clark GC, Lucier GW. 1995a. TCDD reduces rat hepatic epidermal growth factor receptor: comparison of binding, immunodetection, and autophosphorylation . Toxicology and Applied Pharmacology 132:263-272.

Sewall CH. Flagler N, Vanden Heuvel JP, et al. 1995b. Alterations in thyroid function in female Sprague-Dawley rats following chronic treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicology and Applied Pharmacology 132:237-244.

Smialowicz RJ, Williams WC, Riddle MM. 1996. Comparison of the T cell-independent antibody response of mice and rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fundamental and Applied Toxicology 32(2):293-297.

Sommer R J, Ippolito DL, Peterson RE. 1996. In utero and lactational exposure of the male Holtzman rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin: decreased epididymal and ejaculated sperm numbers without alterations in sperm transit rate. Toxicology and Applied Pharmacology 140:146-153.

Stahl BU. 1995. 2,3,7,8-tetrachlorodibenzo-p-dioxin blocks the physiological regulation of hepatic phosphoenolpyruvate carboxykinase activity in primary rat hepatocytes. Toxicology 103:45-52.

Stegeman JJ, Hahn ME, Weisbrod R, Woodin BR, Joy JS, Najibi S, Cohen RA. 1995. Induction of cytochrome P4501A1 by aryl hydrocarbon receptor agonists in porcine aorta endothelial cells in culture and cytochrome P4501A1 activity in intact cells. Molecular Pharmacology 47(2):296-306.

Swanson HI, Chan WK, Bradfield CA. 1995. DNA binding specificities and pairing rules of the Ah receptor, ARNT, and SIM proteins. Journal of Biological Chemistry 270(44):26292-26302.

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Tritscher AM. Seacat AM, Yager JD, et al. 1996. Increased oxidative DNA damage in livers of 2,3,7,8-tetrachlorodibenzo-p-dioxin treated intact but not ovariectomized rats. Cancer Letters 98:219-225.

Tuomisto J, Andrzejewski W, Unkila M, et al. 1995. Modulation of TCDD-induced wasting syndrome by portocaval anastomosis and vagotomy in Long-Evans and Han/Wistar rats. European Journal of Pharmacology 292:277-285.


Unkila M, Pohjanvirta R, Tuomisto J. 1995a. Biochemical effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds on the central nervous system. International Journal of Biochemistry and Cell Biology 27:443-455.

Unkila M, Ruotsalainen M, Pohjanvirta R, et al. 1995b. Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on tryptophan and glucose homeostasis in the most TCDD-susceptible and the most TCDD-resistant species, guinea pigs and hamsters. Archives of Toxicology 69:677-683.


Van Birgelen AP, Van der Kolk J, Fase KM, et al. 1995. Subchronic dose-response study of 2.3,7,8-tetrachlorodibenzo-p-dioxin in female Sprague-Dawley rats. Toxicology and Applied Pharmacology 132:1-13.

Vasiliou V, Kozak CA, Lindahl R, Nebert DW. 1996. Mouse microsomal class 3 aldehyde dehydro-genase: AHD3 cDNA sequence, inducibility by dioxin and clofibrate, and genetic mapping. DNA and Cell Biology 15(3):235-245.

Viluksela M, Stahl BU, Rozman KK. 1995. Tissue-specific effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the activity of phosphoenolpyruvate carboxykinase (PEPCK) in rats. Toxicology and Applied Pharmacology 135:308-315.

Voskoboinik I, Ooi SG, Drew R, Ahokas JT. 1997. Peroxisome proliferators increase the formation of BPDE-DNA adducts in isolated rat hepatocytes. Toxicology 122(1-2):81-91.


Walker MK, Cook PM, Butterworth BC, Zabel EW, Peterson RE. 1996. Potency of a complex mixture of polychlorinated dibenzo-p-dioxin, dibenzofuran, and biphenyl congeners compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin in causing fish early life stage mortality. Fundamental and Applied Toxicology 30:178-186.

Walker MK, Pollenz RS, Smith SM. 1997. Expression of the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator during chick cardiogenesis is consistent with 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced heart defects . Toxicology and Applied Pharmacology 143(2):407-419.

Walsh AA, Tullis K, Rice RH, Denison MS. 1996. Identification of a novel cis-acting negative regulatory element affecting expression of the CYP1A1 gene in rat epidermal cells. Journal of Biological Chemistry 271(37):22746-22753.

Wang X, Thomsen JS, Santostefano M, Rosengren R, Safe S, Perdew GH. 1995. Comparative properties of the nuclear aryl hydrocarbon (Ah) receptor complex from several human cell lines. European Journal of Pharmacology 293(3):191-205.

Wanibuchi H, Yamamoto S, Chen H, et al. 1996. Promoting effects of dimethylarsinic acid on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder carcinogenesis in rats. Carcinogenesis 17:2435-2439.

Wanner R, Brommer S, Czarnetzki BM, Rosenbach T. 1995. The differentiation-related upregulation of aryl hydrocarbon receptor transcript levels is suppressed by retinoic acid. Biochemical and Biophysical Research Communications 209(2):706-711.

Wanner R, Panteleyev A, Henz BM, Rosenbach T. 1996. Retinoic acid affects the expression rate of the differentiation-related genes aryl hydrocarbon receptor, ARNT and keratin 4 in proliferative keratinocytes only . Biochimica et Biophysica Acta 1317(2):105-111.

Wamgard L, Bager Y, Kato Y, Kenne K, Ahlborg UG. 1996. Mechanistical studies of the inhibition of intercellular communication by organochlorine compounds. Archives of Toxicology Supplement 18:149-159.

Suggested Citation:"3 Toxicology." Institute of Medicine. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: The National Academies Press. doi: 10.17226/6415.
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Watson MA, Devereux TR, Malarkey DE, Anderson MW, Maronpot RR. 1995. H-ras oncogene mutation spectra in B6C3F1 and C57BL/6 mouse liver tumors provide evidence for TCDD promotion of spontaneous and vinyl carbamate-initiated liver cells. Carcinogenesis 16(8): 1705-1710.

Weber LW, Lebofsky M, Stahl BU, Smith S, Rozman KK. 1995. Correlation between toxicity and effects on intermediary metabolism in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated male C57BL/ 6J and DBA/2J mice. Toxicology and Applied Pharmacology 131:155-162.

Weiss C, Kolluri SK, Kiefer F, Gottlicher M. 1996. Complementation of Ah receptor deficiency in hepatoma cells: negative feedback regulation and cell cycle control by the Ah receptor. Experimental Cell Research 226(1 ):154-163.

Weston WM, Nugent P, Greene RM. 1995. Inhibition of retinoic-acid-induced gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin . Biochemical and Biophysical Research Communications 207(2):690-694.

White TE, Rucci G, Liu Z, Gasiewicz TA. 1995. Weanling female Sprague-Dawley rats are not sensitive to the antiestrogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicology and Applied Pharmacology 133(2):313-320.

Whitelaw ML, McGuire J, Picard D, Gustafsson JA, Poellinger L. 1995. Heat shock protein hsp90 regulates dioxin receptor function in vivo. Proceedings of the National Academy of Sciences of the United States of America 92(10):4437-4441.

Wilker C, Johnson L, Safe S. 1996. Effects of developmental exposure to indole-3-carbinol or 2,3,7,8-tetrachlorodibenzo-p-dioxin on reproductive potential of male rat offspring. Toxicology and Applied Pharmacology 141(1):68-75.

Wolfle D, Marquardt H. 1996. Antioxidants inhibit the enhancement of malignant cell transformation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Carcinogenesis 17:1273-1278.

Xiao GH, Pinaire JA, Rodrigues AD, Prough RA. 1995. Regulation of the Ah gene battery via Ah receptor-dependent and independent processes in cultured adult rat hepatocytes. Drug Metabolism and Disposition 23(6):642-650.


Yamaguchi Y, Kuo MT. 1995. Functional analysis of aryl hydrocarbon receptor nuclear translocator interactions with aryl hydrocarbon receptor in the yeast two-hybrid system. Biochemical Pharmacology 50(8):1295-1302.

Yamamoto S, Wanibuchi H, Hori T, Yano Y, Matsui-Yuasa I, Otani S, Chen H. Yoshida K, Kuroda K, Endo G, Fukushima S. 1997. Possible carcinogenic potential of dimethylarsinic acid as assessed in rat in vivo models: a review. Mutation Research 386(3):353-361.

Yao Y, Hoffer A, Chang CY, Puga A. 1995. Dioxin activates HIV-1 gene expression by an oxidative stress pathway requiring a functional cytochrome P450 CYP1A1 enzyme. Environmental Health Perspectives 103(4):366-371.


Zhao W, Ramos KS. 1995. Inhibition of DNA synthesis in primary cultures of adult rat hepatocytes by benzo[a]pyrene and related aromatic hydrocarbons: role of Ah receptor-dependent events. Toxicology 99(3):179-189.

Zorn NE, Russell DH, Buckley AR, Sauro MD. 1995. Alterations in splenocyte protein kinase C (PKC) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin in vivo. Toxicology Letters 78:93-100.

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Third in a series of six congressionally mandated studies occurring biennially, this book is an updated review and evaluation of the available scientific evidence regarding the statistical association between exposure to herbicides used in Vietnam and various adverse health outcomes suspected to be linked with such exposures. As part of the review, the committee convened a workshop at which issues surrounding the reanalysis and the combination of existing data on the health effects of herbicide and dioxin exposure were addressed.

This book builds upon the information developed by the IOM committees responsible for the 1994 original report, Veterans and Agent Orange, and Veterans and Agent Orange: Update 1996, but will focus on scientific studies and other information developed since the release of these reports. The two previous volumes have noted that sufficient evidence exists to link soft tissue sarcoma, non-Hodgkin's lymphoma, Hodgkin's disease, and chloracne with exposure. The books also noted that there is "limited or suggestive" evidence to show an association with exposure and a neurological disorder in veterans and with the congenital birth defect spina bifida in veterans' children. This volume will be critically important to both policymakers and physicians in the federal government, Vietnam veterans and their families, veterans organizations, researchers, and health professionals.

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