As in Veterans and Agent Orange: Health Effects of Herbicides Used in Vietnam (IOM, 1994; hereafter referred to as VAO), Veterans and Agent Orange: Update 1996 (IOM, 1996; hereafter, Update 1996) and Veterans and Agent Orange: Update 1998 (IOM, 1999; hereafter, Update 1998), this review summarizes the experimental data that serve 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, including exposures to chemicals other than herbicides, 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. Most of the experimental studies of these chemicals, unless otherwise noted, are conducted with pure chemical. This is in contrast to the epidemiologic studies discussed in later chapters in which expo-
sures are often to mixtures of chemicals. Some studies of herbicides are conducted using herbicide mixtures and are noted as such in the text.
This chapter begins with a brief summary of major conclusions derived from the literature reviews in VAO, Update 1996, and Update 1998. This is followed by a summary of toxicological research findings as they relate to human health, and then an overview of the scientific literature published since release of Update 1998, reviewed in detail in this chapter. Note that these more general summaries do not include references to the scientific literature because they are intended to provide background for the nonspecialist.
The “Toxicity Profile Updates” section then provides details of the relevant scientific studies, with references, that have been conducted on 2,4-D,2,4,5-T, picloram, cacodylic acid, and TCDD since Update 1998. The toxicity profile update for TCDD includes a section that discusses the issues involved in estimating potential health risk and factors influencing toxicity. That subsection includes a discussion of the toxic equivalency factor approach to estimating the toxicity of TCDD. It is important, when evaluating the experimental data for all of the compounds, to keep in mind the advantages, disadvantages, and limitations of various types of studies. These considerations are discussed in the final section of the chapter, “Issues in Evaluating the Evidence.”
Highlights of Previous Reports
Chapter 4 of VAO and Chapter 3 of both Update 1996 and Update 1998 review the results of animal and in vitro studies published through 1997 that investigate 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 directly genotoxic and that its ability to influence the carcinogenic process is mediated via epigenetic events such as effects on 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 through 1997 can be found in VAO, Update 1996, and Update 1998.
The distribution of toxicants within the body, or toxicokinetics, can determine the amounts of a particular chemical reaching potential target organs or cells. Earlier data indicate that all four of the herbicides can be absorbed into the body. No data have been published on the toxicokinetics of 2,4-D,2,4,5-T, or picloram since Update 1998. Since Update 1998, some research has been conducted that is relevant to the distribution of cacodylic acid, an organic form of arsenic, in the body. The distribution in the body and excretion out of the body of organic arsenicals were shown to be minimally affected by the dose administered. Data also indicate that some organic forms of arsenic are transferred to the fetus, and it was seen following a human poisoning that organic arsenicals preferentially distribute to organs that are high in lipids.
Studies conducted in veterans of Operation Ranch Hand since Update 1998 have refined estimates of how long it takes for half of the TCDD in the body to be eliminated (i.e., its half-life); the average half-life in humans is 7.6 years. Other studies demonstrate that the distribution of TCDD can be affected by several variables; lipoidal additives in the diet may enhance TCDD excretion, the halflife of TCDD can vary between individuals, and the half-life can vary with dietary modification. Research has also been conducted on how to estimate initial exposure levels using blood measurements of TCDD years after the exposure occurred.
Mechanisms of Toxic Action
There is still little known about the way that herbicides produce toxic effects in animals. Since Update 1998 the ability of 2,4-D to induce mutations has been investigated using a number of assays. Mutations were seen only in one study and there only at very high concentrations of 2,4-D in vivo. 2,4-D did affect the levels of some hormones and cellular components involved in the development and functioning of brain cells. Both 2,4-D and 2,4,5-T inhibited mitochondrial benzoyl coenzyme A (benzoyl-CoA) synthetase and an organic acid transporter. 2,4,5-T also affected Neu tyrosine kinase, a tyrosine kinase receptor that has been shown in other experiments to be correlated with an increased incidence of breast cancer. The relevance of the effects of 2,4,5-T on that enzyme to the toxic effects of 2,4,5-T is unknown. Cacodylic acid can affect microtubule networks at particular points in mitosis. Research on cacodylic acid indicates that it can cause bladder hyperplasia and tumors in rats, lung cancer in mice, and promote skin cancer in mice sensitized by genetic manipulation or exposure to ultraviolet B radiation. One study in mice has demonstrated that it can cause chromosomal abnormalities.
Data published to date are consistent with the hypothesis that TCDD produces most of its biological and toxic effects by binding to a protein that regulates
gene expression, the aryl hydrocarbon receptor (AhR). The binding of TCDD to the AhR triggers a sequence of cellular events that involve interactions with numerous other cellular components. Research in animals that have been engineered not to express the AhR, and in animals with slightly different forms of the receptor, supports a role of the AhR in the toxicity of TCDD. Modulation of genes by AhR may have species-, cell-, and developmental stage-specific patterns, suggesting that the molecular and cellular pathways that lead to any particular toxic event are complex.
Additional research demonstrates that the biochemical and biological outcomes of TCDD exposure can be modulated by numerous other proteins with which the AhR interacts. It is plausible, for example, that the AhR could divert other proteins and transcription factors from other signaling pathways; the disruption of these other pathways could have serious consequences for a number of cell and tissue processes.
With respect to the mechanism underlying the carcinogenic effects of TCDD, it still appears that TCDD does not act directly on the genetic material. Effects on enzymes or hormones could be involved in the carcinogenicity of TCDD.
Recent experiments demonstrated that 2,4-D can cause behavioral effects, muscle weakness, and incoordination in animals, but these effects are seen only at high doses. Reproductive and developmental effects have been seen in animals, but also only at high doses. Furthermore, a precursor of 2,4-D,4-(2,4-dichlorophenoxy)butyric acid (2,4-DB), did not cause an immunotoxic or carcinogenic response in rodents or dogs. Evidence suggests that cacodylic acid can act as a tumor promoter in mice and rats.
Many effects have been observed in animals following exposure to TCDD, and this contaminant is considered more toxic than the pure components of the herbicides used in Vietnam. Sensitivity to TCDD varies among species and strains, but most species studied develop a “wasting syndrome” from acutely toxic doses. This syndrome is characterized by a loss of body weight and fatty tissue. One target of TCDD is the liver, where lethal doses of TCDD cause necrosis, but the effect is dependent on the animal species exposed. Effects on the morphology and function of the liver are seen at lower doses. A recent study demonstrated that TCDD inhibits the ability of the liver to accumulate vitamin A.
TCDD may affect, directly or indirectly, many organs of the endocrine system in a species-specific manner. For example, thyroid hormone levels are altered by treatment of animals with TCDD. Some of the results in different studies of thyroid hormones are contradictory, however, making interpretation of these results difficult.
The adult nervous system has been shown to be sensitive to the effects of TCDD only at high doses. After in utero exposure, however, even these high-
dose effects are not straightforward, with in utero TCDD exposure decreasing performance on certain learning and memory tasks, but improving performance on other tasks.
In animals, one of the most sensitive systems to TCDD toxicity is the immune system. Recent studies have demonstrated that TCDD can alter the levels of immune cells, the measured activity of these cells, and the ability of animals to fight off infection. Effects on the immune system, however, appear to depend on the species, strain, and developmental stage of animal studied.
Reproductive and developmental effects have been seen in animals exposed to TCDD. For example, effects on sperm counts, sperm production, and seminal vesicle weights have been seen in male offspring of rats treated with TCDD during pregnancy. Effects on the female reproductive system have also been seen following developmental exposure to TCDD. In some recent studies, however, the effects on the male and female reproductive system were not accompanied by effects on reproductive outcomes. The mechanism underlying the reproductive effects is not known, but it is possible that they are secondary to effects on reproductive hormones. In recent studies, TCDD did not affect surgically induced endometrial lesions in rats, although effects were seen in earlier studies. Pre- and postnatal exposure of mice to TCDD increased sensitivity to endometrial lesion growth.
TCDD is an extremely potent promoter of neoplasia in laboratory rats. In a recent study, there was an increase in hepatic foci at doses as low as 0.01 ng/kg/ day. This is the lowest dose of TCDD to promote tumors to date. Recent data also suggest that promotion of liver tumors by TCDD in female rats is dependent on continuous exposure to TCDD.
Relevance to Human Health
As indicated above, exposure to 2,3,7,8-TCDD has been associated with both cancer and noncancer end points in animals, and most 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. Animal research indicates that TCDD can cause both cancers and benign tumors, and also enhance the incidence of certain cancers or tumors in the presence of known carcinogens. However, experimental animals differ greatly in their susceptibility to TCDD-induced effects; the sites at which tumors are induced also vary from species to species. Other noncancer health effects vary according to dose and to the animal exposed. Controversy still 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 that health effects caused by Agent Orange occur through chemicals other than TCDD. Al-
though concerns have been raised about nondioxin contaminants of herbicides, far too little is known about the distribution and concentration of these compounds in the formulations used in Vietnam to draw conclusions concerning their impact.
Considerable uncertainty remains about how to apply mechanistic information from non-human studies to an evaluation of the potential health effects in Vietnam veterans of herbicide or dioxin exposure. Therefore, 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. A great deal of research on biological mechanisms has been and continues to be conducted, especially on TCDD. No single mechanism has been established as underlying the toxic effects of TCDD, and with the many different effects seen, more than a single mechanism might exist. It is hoped that as the cellular mechanisms of these compounds are discovered, subsequent VAO updates will have better information on which to base conclusions and to aid in determining the relevance of experimental data to effects in humans.
OVERVIEW OF THE SCIENTIFIC LITERATURE IN UPDATE 2000
Since Update 1998, no data have been published that add to the information available on the toxicokinetics of 2,4-D,2,4,5-T, or picloram. Research has been conducted on the distribution of cacodylic acid, an organic form of arsenic that was used as an herbicide in small quantities in Vietnam. Research in mice demonstrates that the administered dose minimally affects the distribution and excretion of organic arsenicals. In humans it was observed that at least some organic forms of arsenic are transferred to the fetus and that organic arsenicals are distributed more to organs that are high in lipids.
In contrast, a great deal of research conducted since Update 1998 improves the understanding of the processes that affect the distribution of TCDD to different parts of the body. Studies continue to demonstrate that an enzyme, cytochrome P450 1A2 (CYP1A2), plays an important role in the distribution of TCDD. CYP1A2 is expressed at high levels in the liver and binds TCDD. Because of this binding, the levels of TCDD in the liver are more dependent on CYP1A2 levels than on liver lipid content, but this is highly dependent on the concentration of TCDD. Experiments in mice that do not express the Cyp1A2 gene (Cyp1A2 knockout mice) in the liver further demonstrate the importance of CYP1A2 protein in the distribution of TCDD. A greater amount of TCDD is distributed to other organs, and urinary excretion is increased in knockout animals. In addition to CYP1A2 levels, other polyhalogenated aromatic hydrocar-
bons (PHAHs) can affect the toxicokinetics of TCDD; there is decreased retention of TCDD in the presence of other PHAHs.
Studies have been conducted investigating the length of time that TCDD remains in the body and the factors that can influence this. Follow-up examinations in Operation Ranch Hand veterans indicate that TCDD has a mean half-life of 7.6 years and elimination is inversely proportional to bodyfat content, but that age does not have an observable effect on elimination. A study in non-Ranch Hand Vietnam veterans, however, shows that age has a weak effect on the elimination rate of TCDD, and a study in an occupationally exposed cohort also indicates that the elimination rate changes with age, but this may, in part, reflect changes in body composition with age. These studies converge on a consistent estimate of half-life but are inconsistent on the effect of age.
TCDD is also excreted in breast milk, causing both a decrease in maternal TCDD levels and the transfer of TCDD to breast-fed infants. Recent studies show that the volume of breast milk produced can affect the rate at which TCDD is eliminated from the mother. In addition, the concentration of TCDD in breast milk decreases over time with continued breast feeding. Modeling the residue kinetics in infants indicates that the TCDD initially accumulated in infants following exposure from breast feeding is substantially decreased by 2 years of age.
Dietary factors also can affect the absorption and excretion of TCDD. The amount of fat in the diet can greatly affect absorption and excretion. Ingestion of a nonabsorbed dietary fat substitute (olestra) increased the fecal excretion of a very high dose of TCDD.
It is important to know whether the TCDD levels measured in blood are representative of levels in target tissues because TCDD is often measured in blood in human studies. Autopsy studies of human tissues indicate that there is a correlation between the levels of TCDD measured in the blood lipids and the levels measured in adipose, kidney, spleen, liver, and brain tissue, but not in muscle and lung tissue. A study in rodents demonstrates that concentrations of TCDD in the fetal compartment are comparable to the levels in maternal blood.
Mechanisms of Toxic Action
Since Update 1998, the actions of 2,4-D,2,4,5-T, cacodylic acid, and TCDD at the molecular and cellular level have been investigated. These studies enhance our understanding of the actions of these chemicals, particularly TCDD, but the exact mechanisms by which these chemicals are toxic still are not established. No new research has been published that provides data on the mechanisms underlying the toxic effects of picloram.
2,4-D has previously been shown to have low oncogenic potential, with genotoxic effects seen only at high concentrations. Recent evidence is consistent with these earlier data. Only a high concentration of 2,4-D was genotoxic in a wing spot test. There was no evidence of genotoxicity in assays testing for re-
combination; bacterial gene mutation; chromosomal aberrations; forward mutations in the hypoxanthine-guanine phosphoribosyl transferase gene (HGPRT) locus; and induction of DNA damage, repair, and unscheduled synthesis, as well as in tests of the frequency of micronucleated polychromatic erythrocytes in mice.
Research continues to demonstrate effects of 2,4-D on hormone levels and the function of the nervous system. 2,4-D decreased serum thyroxine concentrations, testosterone concentrations in serum and gonads, and serum concentrations of lutenizing hormone, follicle-stimulating hormone, prostaglandin I2, and prostaglandin E2. 2,4-D also inhibited neurite extension in primary cultures of cerebellar granule cells. This effect is accompanied by a reduction in cellular microtubules, disorganization of the Golgi apparatus, and inhibition of ganglioside synthesis. It also inhibits the polymerization of purified tubulin. Although the biological relevance of these affects is not established, it is possible that the effects on hormones and the nervous system are involved in the reproductive and neurological toxicity seen at high doses of 2,4-D.
Both 2,4-D and 2,4,5-T have inhibitory effects on the formation and renal transport of benzylglycine. These compounds inhibit the mitochondrial enzyme benzoyl-CoA synthetase and competitively inhibit an organic acid transporter, inhibiting the secretion of benzoylglycine. 2,4,5-T also activated Neu tyrosine kinase in a cell-free system, stimulated the enzyme in MCF-7 cells, and stimulated foci formation of MCF-7 cells. Although activation of Neu tyrosine kinase has been found to be correlated with an increased incidence of breast cancer in animal models, how these cellular and biochemical effects are related to any toxic end point is unknown.
Most research indicates that cacodylic acid can act as a promoter in the carcinogenic process, and one study has demonstrated that it can cause aneuploidy. It also can disrupt cell growth by affecting the microtubule network. Evidence indicates that it decreases liver glutathione levels, as well as pulmonary and hepatic ornithine decarboxylase levels.
Studies published since Update 1998 are consistent with the hypothesis that TCDD produces its biological and toxic effects by binding to the AhR. For example, recent data indicate that TCDD has only minimal teratogenicity, if any, in AhR knockout mice compared to wild-type mice. Data from knockout mice also suggest that the AhR plays an important, but as yet unknown, developmental and physiological role. Many of the recent data published are consistent also with the notion that cellular processes involving growth, maturation, and differentiation are sensitive to TCDD-induced effects. Findings in animals indicating that reproductive, developmental, and oncogenic end points appear to be sensitive to TCDD are consistent with this notion, and the cellular data provide biologic plausibility for similar end points of toxicity in exposed humans. However, many of the responses to TCDD are tissue- and species-specific and the mechanistic basis for these differences is not completely understood.
The presence of the AhR and ARNT in a variety of tissues from different animal species and strains is well documented. Detailed analysis of variant forms has provided much information associating structure and expression levels with function. Furthermore, experiments in species and strains expressing different forms of the AhR suggest that differences in specific regions of the AhR may be in part responsible for differential sensitivity to TCDD. Evidence continues to indicate that the sequence of the AhR in humans is highly conserved among different individuals.
Research has shown that the association of several proteins with newly synthesized AhR may modulate AhR function. For example, association with 90 kDa heat shock protein (HSP90) is important to maintain the AhR in a conformation that can bind ligand. Recent data are consistent with a mechanism in which HSP90 is released from the ligand-bound AhR following nuclear localization concomitant with ARNT-AhR dimerization. One study, however, demonstrated that dissociation of HSP90 is not required for nuclear translocation of the AhR but is essential for dimerization with ARNT.
Many of the more recent investigations have focused on identifying and characterizing factors that may modulate, by either activation or repression, the ability of the activated AhR-ARNT complex to alter gene expression. In addition, several studies have noted the ability of a variety of AhR ligands to act as receptor antagonists. Studies have investigated the roles of immunophilin proteins, nuclear accessory proteins or coactivators, repression by as yet unidentified cellular factors specific to certain cell types, nuclear factor κB (NF-κB), and histone acetylators and deacetylators.
Investigations into the endogenous ligand for the AhR continue. Although several endogenous compounds which bind to the AhR have been described, it is not yet clear whether any of these have any physiological significance. Naturally occurring ligands for the receptor include resveratrol, curcumin, tryptophan metabolites, galangin, the dietary flavonols quercetin and kaempferol, lipoxin A4, and products of heme metabolism.
Details of the many studies investigating the cellular and molecular effects of TCDD are summarized later in this chapter.
Studies published since Update 1998 are consistent with the previous view that 2,4-D is relatively nontoxic and has weak oncogenic potential. Decreased motor activity, muscle weakness, motor incoordination, decreased weight gain, and serum alterations were seen only at doses greater than 100 mg/kg. Reversible and permanent behavioral alterations have also been seen in rats following treatment with high doses of 2,4-D from gestational day 16 to postnatal day 23. These observations are consistent with previous studies suggesting that 2,4-D could have neurotoxic effects. Exposure to 2,4-D had no effect on lymphocyte blasto-
genesis, immunoglobulin M (IgM) antibody production in response to sheep red blood cells, expression of lymphocyte cell surface markers, or phagocytic function of peritoneal macrophages. Only mild, reversible effects on the skin were observed following 2,4-D treatments. Developing fetuses appear to be the most sensitive to the effects of 4-(2,4–2,4-dichlorophenoxy)butyric acid (2,4-DB), of which 2,4-D is the major metabolite, but even these effects occur at relatively high concentrations. There was no evidence of an oncogenic response in studies of rodents and dogs treated with 2,4-DB.
The ability of 2,4,5-T to produce myelotoxicity was examined using the mouse granulocyte-macrophage (GM) colony-forming unit (CFU) assay and the 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) test for inhibition of proliferation. The concentration that caused a 50 percent inhibition in the assays (i.e., the IC50) was at least 202 µM, indicating a relatively weak potency of 2,4,5-T to produce myelotoxicity. No other studies were found that investigate disease outcomes following exposure to 2,4,5-T.
The pulmonary carcinogenic activity of cacodylic acid (dimethylarsinic acid, DMA) was examined in mice; treated mice developed more pulmonary neoplasms (number per mouse) than untreated mice. Exposure to DMA for 2 years produced bladder hyperplasia and tumors in rats. In other studies, DMA acted as a skin cancer promoter in transgenic mice sensitive to carcinogens and in hairless mice irradiated with ultraviolet B radiation.
There are no recent studies investigating toxic effects following exposure to picloram; one study looking at oxidative functions showed effects of Tordon 75D (a mixture of the triisopropanolamine salts of 2,4-D and picloram) and attributed these effects to the surfactant in the mixture, not picloram.
Many effects have been observed in animals following exposure to TCDD. The classic symptoms of the “wasting syndrome” (i.e., extreme loss of body weight, decreased food consumption with an increase in consumption prior to death, and bloody stool) were observed in female mink treated with TCDD.
Thermoregulatory control is affected by TCDD. A study in rats indicates that the thermoregulatory centers in the hypothalamus are not permanently altered by TCDD.
Neurotoxic effects have been observed after developmental exposure to TCDD, with some learning and memory tasks being affected in rats.
Of the many organ systems affected by TCDD, one of the most sensitive is the immune system. Increased parasitic larval burdens occurred in rats following TCDD exposure; there was some indication that age increased the sensitivity of humoral immunity to TCDD exposure. TCDD has been shown to decrease delayed-type hypersensitivity responses, decrease the total percentage of CD4+ cells and the percentage of the CD4+ cells cycling following repeated exposure, and stimulate the production of interleukin-2 (IL-2) and increase the percentage of CD4+ and CD8+ cells in the S and G2M phase of lymphocyte cycling in primed rats. Although there are considerable species and strain differences in immune
responses to TCDD, some evidence indicates that TCDD compromises (suppresses) the immune system of laboratory animals.
Developmental effects on the male reproductive system have been seen following exposure to TCDD. Male offspring of rats gavaged on gestational day 15 with TCDD had significantly decreased body and seminal vesicle weights, and decreased epithelial branching and differentiation in the seminal vesicles. In another study, the number of sperm per cauda epididymis and daily sperm production were decreased, and sperm transit rate was affected at puberty and adulthood in male offspring of female rats treated with TCDD. In the highest-TCDD-exposure group, serum testosterone concentration was decreased at adulthood. In this study, however, reproductive outcomes of those males were not affected. Similarly, female offspring of pregnant female hamsters treated with TCDD on gestational day 15 showed effects on the reproductive system, but reproductive outcomes in female progeny were not reported.
In recent studies TCDD did not affect surgically induced endometrial lesions in rats, although effects were seen in earlier studies. The lesions were increased in mice only with a combination of perinatal and adult exposure to TCDD. Some researchers suggest that TCDD blocks the ability of progesterone to prevent experimental endometriosis, which correlates with its ability to inhibit progesterone-associated transforming growth factor-β2 (TGF-β2) expression and endometrial matrix metalloproteinase suppression.
In utero and lactational exposure of rats to TCDD decreased prostate weight without inhibiting testicular androgen production or decreasing serum androgen concentrations. Additional studies showed that the prostatic epithelial budding process was impaired, suggesting that in utero and lactational TCDD exposure interferes with prostate development by decreasing early epithelial growth, delaying cell differentiation, and producing alterations in epithelial and stromal cell histological arrangement and the spatial distribution of androgen receptor expression.
Data are conflicting as to whether TCDD induces cellular apoptosis. This may be highly dependent on cell type. TCDD failed to induce apoptosis in Fas-deficient and Fas-ligand-defective mice at the lower doses tested, compared to control wild-type mice, suggesting that Fas-Fas ligand interactions may play a role in the TCDD-mediated induction of apoptosis.
TCDD is an extremely potent promoter of neoplasia in laboratory rats. TCDD significantly increased the volume fraction and number of altered hepatic foci at the highest dose. Increases in the number of guanosine 5'-triphosphatase (GTPase) and adenosine 5'-triphosphatase (ATPase) deficient altered hepatic foci per cubic centimeter also occurred at doses as low as 0.01 ng/kg/day. This is the lowest dose of TCDD to promote tumors to date. Recent data also suggest that promotion of liver tumors by TCDD in female rats is dependent on continuous exposure to TCDD.
TOXICITY PROFILE UPDATES
This section updates the toxicity profiles of the five substances discussed in VAO, Update 1996, and Update 1998: (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 1998–2000. Information in this literature update is organized under the following 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
No toxicokinetic studies were identified for the reference period.
Mechanisms of Toxic Action
In Update 1998, several studies supported the view that the mechanism of toxic action of 2,4-D involved disruption of thiol homeostasis. No new studies have been published regarding this aspect.
Several studies, using in vitro and in vivo model systems, published since Update 1998 are consistent with the relatively weak genotoxic potential of 2,4-D. Using the Drosophila melanogaster wing spot test, which assesses somatic mutation and recombination events, a slight, but significant, increase in the frequency of total spots was observed only at the highest concentration (10 mM) of 2,4-D tested. To determine recombinagenic activity, the frequency of Mwh (multiple wing hairs) clones was also evaluated. No increases were observed with any concentration of 2,4-D (Kaya et al., 1999). Another study examined the potential genetic toxicity of 2,4-D and seven of its salts and esters by examining their ability to produce gene mutations in bacteria (Ames test) and their ability to induce DNA damage, repair, and unscheduled DNA synthesis in rat hepatocytes. For all assays, there were no indications of genotoxicity (Charles et al., 1999a). Similarly, no evidence for the genotoxicity of 2,4-D was observed using assays in which the induction of chromosomal aberrations in primary cultures of rat lymphocytes and forward mutations at the HGPRT locus of Chinese hamster ovary cells were examined (Gollapudi et al., 1999). The ability of 2,4-D and its derivatives to induce cytogenetic abnormalities in vivo was also investigated by examining the frequency of micronucleated polychromatic erythrocytes (MN-PCEs) in mice treated with these chemicals by oral gavage. There were no increases in MN-PCE for single doses of 2,4-D up to 400 mg/kg (Charles et al., 1999b).
Notably, all of the above results are consistent with recent data indicating the lack of oncogenic response to 2,4-dichlorophenoxybutyric acid, of which 2,4-D is the major metabolite, following chronic exposure of rats, mice, and dogs (Charles and Leeming, 1998a, b) (see below).
Investigations by Gregus et al. (1999) indicate that both 2,4-D and 2,4,5-T may interfere with the biotransformation and renal elimination of small aromatic carboxylic acids. The effect of these herbicides on glycine conjugation and excretion of benzoic acid was examined. The inhibition of benzoylglycine formation by 2,4-D was demonstrated in the rat following a single intraperitoneal treatment with 110.5 mg/kg. It was found that 2,4-D acted as an inhibitor, but not a substrate, of mitochondrial benzoyl-CoA synthetase. 2,4-D also inhibited the renal secretion of benzoylglycine, apparently by acting as a competitive substrate of the organic acid transporter. 2,4,5-T was approximately equipotent in producing those same effects.
Several studies suggest effects of 2,4-D on hormones and hormone-regulated functions. Prior to Update 1998, investigations reported effects of 2,4-D on serum concentrations of thyroid hormones, particularly, thyroxine. Similarly, Rawlings et al. (1998) observed that two oral doses of 2,4-D of 10 mg/kg/week for 43 days to ewes resulted in significant decreases in serum thyroxine concentrations. No overt signs of toxicity were observed, and there were no significant effects on blood concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone, estradiol, cortisol, or insulin. Treating male rats with 5 and 50 mg/kg/ day for a month, however, led to decreased testosterone levels in serum and gonads. In addition, these dosages produced decreased serum concentrations of LH, FSH, prostaglandin I2, and prostaglandin E2 (Galimov et al., 1998).
A study by Oakes and Pollak (1999) evaluated the toxicity of the components of Tordon 75D (a mixture of the triisopropanolamine salts of 2,4-D and picloram) on the oxidative functions of submitochondrial particles (SMPs). Notably, the concentrations that caused a 50 percent inhibition of oxidative functions (i.e., the effective concentration50, EC50) were in the low micromolar range for 2,4-D and picloram in the presence of other components of Tordon 75D (triisopropanolamine, diethylene glycol monoethyl ether, a silicone defoamer, and a proprietary surfactant). However, in the absence of the other components, the EC50 values for these chemicals were approximately 136 times higher. The results indicate that the toxic effects of Tordon 75D on SMPs and intact rat liver mitochondria were caused mainly, if not solely, by the proprietary surfactant.
Rosso and coworkers (2000) examined the effects of 2,4-D on primary cultures of cerebellar granule cells. A 24-hour exposure to concentrations up to 2 mM produced a dose-dependent inhibition of neurite extension. This inhibition was accompanied by a reduction in the cellular content of microtubules, disorganization of the Golgi apparatus, and inhibition of ganglioside synthesis. They also observed that 2,4-D inhibited the polymerization of purified tubulin in vitro.
Studies published since Update 1998 are consistent with the previous view that 2,4-D is relatively nontoxic and has weak oncogenic potential. Developing fetuses appear to be the most sensitive to the effects of 2,4-D for a number of toxic end points.
Morgulis et al. (1998) studied the acute oral toxicity of 2,4-D in chicks. A number of signs of toxicity were observed, but only at doses higher than 100 mg/ kg. Those effects included decreased motor activity, muscle weakness, motor incoordination, and decreased weight gain. Serum alterations included increased serum uric acid, creatinine and total protein, and serum creatine kinase and alkaline phosphatase activities. These changes were time dependent and reversible at the lower doses. The dose that causes death in 50 percent of the animals (i.e., LD50) was 420 mg/kg. Histopathological postmortem examination indicated vacuolar degeneration of hepatocytes, renal tubular necrosis, and intestinal hemorrhages.
Barile and Cardona (1998) examined 30 different chemicals in cytotoxicity assays using human fetal lung fibroblasts and human skin fibroblasts in culture to evaluate the methods as screens for cytotoxicity and as potential predictors of human toxicity. IC50 values of 2,4-D in these cell lines ranged from 0.87 to 1.3 mg/ml. Evaluation of data for the other chemicals suggests that the experimental IC50 values are as accurate predictors of human toxicity as equivalent blood concentrations derived from rodent LD50 studies.
Bracco and Favre (1999) described an acute fatal case of 2,4-D self-poisoning. A plasma concentration of 2,4-D of 720 µg/kg was measured. Clinical and hemodynamic data indicated the failure of multiple organ systems, supporting an uncoupling of oxidative phosphorylation as a predominant mechanism of 2,4-D toxicity in this case.
Studies by Bortolozzi et al. (1999) examined whether exposure of rats to 2, 4-D during pre- and postnatal development induces behavioral alterations. Pregnant rats were exposed to 70 mg/kg/day from gestational day 16 to postnatal day 23. This exposure produced no overt signs of toxicity, but several types of neurobehavioral alterations were observed. Some of the effects were reversible, others were permanent, and others were seen only after pharmacological challenges. Although the exact mechanism of these effects is unknown, some of the results suggest that alterations in the serotonergic and dopaminergic systems might be involved.
Blakley et al. (1998) examined the effects of a commercial formulation of 2,4-D on immune function in male rats. 2,4-D was administered orally twice per week for 28 days at a dosage of 10 mg/kg. No effect on body weight was observed. Exposure to 2,4-D had no effect on lymphocyte blastogenesis, IgM antibody production in response to sheep red blood cells, expression of lymphocyte cell surface markers, or phagocytic function of peritoneal macrophages.
Kimura et al. (1998) examined the dermatotoxicity of several chemicals with or without ultraviolet (UV) irradiation in hairless descendants of Mexican hairless dogs. Each agent was applied daily for 7 days as a 0.1 percent solution in ethanol:propylene glycol:distilled water (2:1:2 volume per volume [v/v]) to a 3 cm × 3 cm test site at a concentration of 4 µl/cm2. One day after cessation of the 2,4-D treatment only mild histological changes (i.e., slight epidermal thickening) were observed. At 14 days after treatment, no significant lesions were observed that were different from controls at sites treated with 2,4-D with or without UV exposure.
Reproductive or Developmental Toxicity
Charles et al. (1999c) performed developmental toxicity studies in rats and rabbits and a two-generation reproduction toxicity study in rats using 2,4-DB, whose major metabolite is 2,4-D. The maximum tolerated dose (MTD) was exceeded at 125 mg/kg/day as evidenced by maternal body weight loss and resorption of fetuses in several dams. The no-observable-adverse-effect level (NOAEL) for maternal toxicity in rats was 31.25 mg/kg/day based on decreased body weight gain. The NOAEL for embryonic or fetal toxicity was also 31.25 mg/kg/day based on fetal skeletal anomalies. For rabbits, the MTD was exceeded at 60 mg/ kg/day based on decreased body weight gain, abortions in two does, and moribund conditions of two does. The NOAEL for maternal toxicity in rabbits was 30 mg/kg/day based on decreased body weight gain, and the NOAEL for embryonic or fetal toxicity was 60 mg/kg/day. For the two-generation study in rats, maternal performance, fertility rate, and pregnancy were unimpaired at concentrations of 60 and 300 parts per million (ppm). A NOAEL for reproductive toxicity was 1,500 ppm (111.8 mg/kg/day for males and 110.6 mg/kg/day for females), but severe postnatal toxicity was produced at this level. The NOAEL for postnatal toxicity based on litter size, decreased weight gain, and pup mortality was 22.5– 32.6 mg/kg/day in males and 26.4–36.7 mg/kg/day in females. Notably, these NOAEL values are similar to the values obtained for 2,4-D and indicate the relatively low reproductive and developmental toxicity of these chemicals.
An investigation by Charles and Leeming (1998a) evaluated the chronic (2-year) dietary toxicity and oncogenicity of 2,4-DB in rodents. Concentrations of 2,4-DB in food were as high as 1,800 ppm. A NOAEL of 2.48 mg/kg/day for male rats and 3.23 mg/kg/day for female rats was observed based on decreased body weight gain. Minor histopathological lesions were thought to be due to decreased body weight gain. There was no evidence of increased tumors at any dose level. Likewise, no oncogenic effect was observed in an 18-month chronic feeding study in mice. The highest dosages were 128.7 and 100.4 mg/kg/day for 66 weeks in female and male mice, respectively, and 110.6 mg/kg/day for 104 weeks in male rats. A similar 1-year study was conducted with 2,4-DB in dogs (Charles and Leeming, 1998b). The highest dosages for male and female dogs were 12.94 mg/kg/day and 14.16 mg/kg/day for 52 weeks, respectively. Treatment-related findings include reductions in body weight gain and food consumption, and small increases in serum levels of inorganic phosphorus, blood urea nitrogen, creatinine, aspartate aminotransferase, and alanine aminotransferase. Most of these values, except for aspartate aminotransferase, returned to normal in a group allowed a 4-week recovery period following cessation of treatment. Histopathological changes related to treatment were seen predominantly in the liver and kidney. These changes were less marked in animals after the 4-week recovery period. There was no evidence for any immunotoxic or oncogenic response. A NOAEL of 75 ppm 2,4-DB (2.39 and 2.15 mg/kg/day for males and females, respectively) was seen. All three studies were consistent with the generally low toxicity and weak oncogenic potential of 2,4-DB.
Toxicity Profile Update of 2,4,5-T
No toxicokinetic studies of 2,4,5-T were identified for the reference period.
Mechanisms of Toxic Action
Studies summarized in Update 1998 indicate that 2,4,5-T enters cells and disrupts cellular pathways involving acetylcoenzyme A. It was suggested that this might be the mechanism for altered cholinergic transmission and the mechanism for neurotoxicity.
As reported for 2,4-D, 2,4,5-T was found to interfere with both the formation and the renal transport of benzylglycine, the glycine conjugate of benzoic acid, in the rat. Reported data suggest that these effects were due to the inhibition of mitochondrial benzoyl-CoA synthetase by 2,4,5-T and the ability of this chemical to act as a competitive substrate of the renal organic acid transporter. 2,4,5-T was approximately equipotent with 2,4-D for these effects (Gregus et al., 1999).
Activation of the Neu tyro sine kinase has been found to be highly correlated with an increased incidence of breast cancer and may have prognostic value in predicting overall survival and time to breast cancer relapse. Hatakeyama and Matsumura (1999) examined the ability of several organochlorine compounds to activate Neu tyrosine kinase and promote foci formation in human MCF-7 breast tumor cells. 2,4,5-T, at a concentration of 1 nM, was found to significantly activate Neu tyrosine kinase in a cell-free system and stimulate this enzyme at 100 nM in intact MCF-7 cells. A concentration of 100 nM 2,4,5-T also stimulated foci formation of MCF-7 cells to approximately 56 percent the level induced by 100 nM estradiol. Notably, there was a reasonable correlation between the ability of the individual organochlorine compounds to activate Neu tyrosine kinase in the cell-free system and to induce foci formation. The exact mechanism by which 2,4,5-T modulates Neu tyrosine kinase activity is not known. It is also not clear how these effects are related to any toxic end point observed in vivo.
Update 1998 focused on the ability of 2,4,5-T to acutely affect neuronal and muscular function by altering cholinergic transmission. Also, the ability of 2,4,5-T to alter xenobiotic metabolizing enzymes was indicated. No further research has been published in these areas.
Gribaldo et al. (1998) examined several environmental contaminants, including 2,4,5-T, for their ability to produce myelotoxicity using the mouse CFU-GM assay and the MTT test for inhibition of proliferation. The IC50 for the ability of 2,4,5-T to inhibit CFU-GM was >391 µM. For the MTT assay, the IC50 values were 202 and >391 µM for two different cell lines. Those data suggest that 2,4,5-T has a relatively weak potency to produce myelotoxicity.
Toxicity Profile Update of Cacodylic Acid
Cacodylic acid (dimethylarsinic acid, DMA; Chemical Abstracts Service [CAS] registry number 75–60–5) was present (4.7 percent) in an herbicide that was used in Vietnam in defoliation and crop destruction missions. As discussed in VAO, cacodylic acid is a metabolite of inorganic arsenic, but it is very resistant to hydrolysis and no demethylation of cacodylic acid to inorganic arsenic has been observed in any species. Therefore, only studies relevant to organic arsenic, not the entire literature on inorganic arsenic, are reviewed in this toxicity profile.
Arsenic forms reactive metabolites that affect cellular respiration and nearly every organ system in the body. The potency of the arsenical compounds (organic and inorganic), however, depends on absorption rates. Soluble forms are more
readily absorbed than the more insoluble preparations. Methylation of inorganic arsenic to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) is a pathway of inorganic biotransformation. It remains to be determined whether inorganic arsenic, and/or its methylated metabolites or other reactive intermediates formed in the methylation process mediate arsenic toxicity. The primary route of excretion is via the urine.
Hughes et al. (1999) evaluated the differences in inorganic arsenic disposition (methylation) between three strains of mice to determine whether there are strain differences in the methylation of arsenic. There were no overall differences in arsenic disposition between the strains of mice. Hughes and Kenyon (1998) injected mice with radioactive MMA and DMA to determine whether dose affects the excretion and tissue deposition of these two organic arsenicals. It was determined that dose had a minimal effect on excretion and tissue disposition of these compounds. More recently, however, Hughes et al. (2000) found that retention of DMA in the lung increased with increased doses of DMA.
In a study to evaluate transfer of arsenic to the fetus and suckling infant, it was found that the concentration of arsenic in cord blood (9 µg/l) was similar to maternal blood (11 µg/l) (Concha et al., 1998). Essentially all of the arsenic in the blood of both newborns and their mothers was in the form of DMA, an end product of metabolism of inorganic arsenic. Approximately 90 percent of urinary arsenic in both the newborns and their mothers during late gestation was present as DMA. Therefore, the authors suggested that methylation of arsenic is increased during pregnancy and that DMA is the major form of arsenic transferred to the fetus.
Human organs were examined for the distribution of arsenic species following fatal acute intoxication by arsenic trioxide (Benramdane et al., 1999). The liver and kidneys contained the highest concentrations of total arsenic. The total concentration in the blood was up to 350 times less than in organs. Arsenic(III) was the predominant species, while MMA was more concentrated than DMA. MMA and DMA were more prevalent in organs high in lipids compared to other organs.
Mechanisms of Toxic Action
Most evidence indicates that DMA acts in the carcinogenic process as a promoter. A recent study demonstrated that DMA could cause aneuploidy in bone marrow cells following a single intraperitoneal injection in mice.
Kato et al. (1999) investigated the mechanism underlying the suppression of apoptosis by DMA. A heat shock protein (HSP79) was induced by 3-hour exposure to 10 mM DMA. The authors suggest that the induction could mediate DMA’s suppression of apoptosis.
Ochi et al. (1998, 1999) investigated the effects of DMA in Chinese hamster V79 cells. DMA decreased growth and caused morphological changes in these
cells by disrupting the microtubule network during the transition from interphase to mitosis.
Sakurai et al. (1998) showed that methylated arsenicals, including DMA, have lower cytotoxicity in mouse macrophage cells than inorganic arsenicals. Using three different cytotoxicity assays, Petrick et al. (2000) found that DMA has a higher LD50 in Chang human hepatocytes than monomethylarsonic acid, monomethylarsonous acid, and arsenite.
Ahmad et al. (1999) observed decreases in liver glutathione concentration, evidence of hepatic DNA damage, and reduced pulmonary and hepatic ornithine decarboxylase in mice gavaged once with 720 mg DMA/kg. The authors indicate that mice are less responsive to the toxicity of DMA than rats.
Several recent investigations have focused on the carcinogenic properties of arsenicals in mice and rats. The pulmonary carcinogenic activity of DMA was examined in A/J mice fed 400 ppm DMA for 50 weeks (Hayashi et al., 1998). Treated mice developed more pulmonary neoplasms (number per mouse) than untreated mice. In another study, DMA fed to F344 rats for 2 years produced bladder hyperplasia and tumors at 40 and 100 ppm; more effects were seen in females than in males (Arnold et al., 1999). Using scanning electron microscopy to evaluate urothelial toxicity and hyperplasia after 10 weeks of exposure, it was determined that the urothelial effects of DMA were reversible, that the toxicity is probably not due to urinary solids, and that the toxicity and regeneration are produced in a dose-responsive manner in female rats. In a similar study (M.Wei et al., 1999), urinary bladder tumors were produced in male rats administered 50 or 200 ppm DMA in drinking water for 104 weeks. The authors proposed that DMA may be related to the human carcinogenicity of arsenicals. DMA also promoted urinary bladder neoplasms in α2µ-globulin-deficient rats, the NCI-Black-Reiter rat (Li et al., 1998), and (Lewis x F344) F1 rats (Chen et al., 1999).
Morikawa et al. (2000) demonstrated that DMA can promote skin carcinogenesis. Transgenic mice with ornithine decarboxylase targeted to hair follicle keratinocytes have an increased sensitivity to carcinogens. These mice were initiated with 7, 12-dimethylbenz[a]anthracene and treated topically once with 3.6 mg DMA. Induction of skin tumors was significantly accelerated in mice treated with DMA. Yamanaka et al. (2000) also demonstrated that DMA can be a skin cancer promoter. Hairless mice irradiated with UVB and dosed with DMA (100 ppm in drinking water) had more malignant tumors with severe atypism than irradiated mice not dosed with DMA.
Toxicity Profile Update of Picloram
Since Update 1998, only one study has been published that is relevant to the
toxicity of picloram. Oakes and Pollak (1999) evaluated the toxicity of the components of Tordon 75D (a mixture of the triisopropanolamine salts of 2,4-D and picloram) on the oxidative functions of submitochondrial particles. Details of that study are discussed further in the section on 2,4-D. Results indicate that the toxic effects of Tordon 75D on SMPs and intact rat liver mitochondria were caused mainly, if not solely, by the proprietary surfactant.
Toxicity Profile Update of TCDD
The distribution of planar halogenated aromatic hydrocarbons, including specific congeners of the polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs), has been examined extensively in animal models and to a lesser extent in humans. These chemicals are all hydrophobic and thus tend to be absorbed readily across cell membranes. Properties of the chemicals, properties of the organs and cells, and route of exposure affect the partitioning, absorption, and accumulation of these chemicals. Lipid content is a major factor in the accumulation of TCDD and other PHAHs in different organs and in the body as a whole. Biological processes, especially metabolism, subsequently can affect the distribution and elimination of these chemicals. The concentration of chemical in a given tissue thus depends on the dose as well as the absorption, lipid content, metabolism, and excretion within the organ of concern and in other organs. In addition, binding proteins in some organs may influence the accumulation of TCDD in extrahepatic organs. For example, proteins present in the liver can bind TCDD causing it to accumulate there rather than other organs.
The toxicokinetics of TCDD are discussed in VAO, Update 1996, and Update 1998. These reports indicate that TCDD is distributed to all compartments of the body. Since Update 1998 there have been several findings that improve our understanding of processes affecting this distribution. Earlier, Wang et al. (1997) identified parameters important in modeling the pharmacokinetic (or toxicokinetic) behavior of TCDD. Santostefano et al. (1998) tested the model as related to cytochrome P450 gene expression in rats. The model was further evaluated by Wang et al. (2000) across doses, gender, strain, and species. Those studies and the results of Evans and Anderson (2000) assessing available physiologically-based pharmacokinetic (PBPK) models indicate that PBPK models can validly predict the distribution of TCDD in animals.
DeVito et al. (1998) have addressed further the role of inducible proteins, especially cytochrome P450 1A2, in sequestering TCDD and related PHAHs in liver. CYP1A2 continues to be established as a determining factor in this sequestration. The planar halogenated compounds examined by DeVito et al. (1998), including PCDDs, PCDFs, and PCBs, showed increased concentrations in liver
relative to the concentrations in adipose tissue or skin, a condition not seen with the noncoplanar compounds. Thus, these chemicals do not distribute solely on the basis of lipid solubility. However, an examination of dose-accumulation relationships for TCDD and other dioxin-like compounds in mice showed that TCDD was sequestered in liver to a lesser extent than some other PHAHs. For example, the liver-adipose ratio at a high dose of TCDD was about 3, while for 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) at the same dose, the ratio was about 10. The structure-activity relationships for sequestration are not the same as those determined previously for binding to the AhR (Safe, 1990).
The issue of chemical absorption when the exposure is to mixtures is an important consideration because environmental exposures almost always involve mixtures. In a study in rats, van der Plas et al. (1998) examined possible interactions between dioxin-like and non-dioxin-like compounds and found that combined exposures can alter the toxicokinetics and patterns of elimination of specific compounds. The retention of TCDD is decreased by the presence of other PHAHs. These researchers showed that liver lipid content was positively correlated with retention of non-AhR agonists but that no such correlation was observed with TCDD or TCDF; the authors suggested that the lack of correlation results from CYP1A2 binding of the latter compounds.
The importance of CYP1A2 in influencing the pharmacokinetic behavior of TCDD was emphasized further in rodent studies by Diliberto et al. (1999). The tissue distributions of TCDD, another potent AhR agonist PeCDF, and a PCB congener (PCB 153) that is not bound by CYP1A2, were examined in mice lacking the gene for Cyp1A2 (Cyp1A2 knockout mice). Comparing the distributions in Cyp1A2 knockouts to the distributions in parental strains confirmed that CYP1A2, expressed primarily in the liver, can strongly affect the concentrations of TCDD in other organs. In mice lacking Cyp1A2 expression in the liver, the concentrations of TCDD accumulating in most other organs or tissues examined (lungs, kidneys, spleen, thymus, skin, adipose tissue, and blood) were greater than the concentrations in organs of mice given the same dose that did express hepatic Cyp1A2. Similar results were obtained for 2,3,4,7,8-PeCDF, but not for 2,2′,4,4′,5,5'-hexachlorobiphenyl, which is not an AhR agonist and does not bind to CYP1A2. In Cyp1A2 knockout mice, the ratio of TCDD in liver to TCDD in fat was 0.07, and in the wild type, the liver-fat ratio was 2.49. Notably, the absence of CYP1A2 in knockout mice also was associated with greatly increased urinary excretion of TCDD or PeCDF (Diliberto et al., 1999).
CYP1A2 is expressed in human liver. However, DeVito et al. (1998) comment that although evidence exists for PHAH sequestration in human liver, there are not good data concerning the amounts of the putative human binding protein (CYP1A2) in studies of TCDD distribution in humans. Nevertheless, studies in rodents have implications for understanding the distribution and half-life of TCDD in humans.
Studies of TCDD levels in humans have continued, especially studies of
Vietnam era veterans. In a follow-up study of Ranch Hand veterans, the mean half-life of TCDD was estimated from concentrations in serum collected from 97 of these veterans (Michalek and Tripathi, 1999). Samples were collected in 1982, 1987, 1992, and 1997. The TCDD half-life was estimated as 7.6 years, with a 95 percent confidence interval (95% CI) of 7.0 to 8.2 years. The elimination rate of TCDD decreased significantly with increases in the percentage of body fat, but the rate of elimination did not appear to change with age in this study.
A statistical analysis of serum TCDD levels also was carried out with samples from 1,302 Air Force veterans who were controls for the Agent Orange-exposed group (Michalek et al., 1998). The strength of this study is that it involved the largest single cohort of male Vietnam veterans not known to be exposed to Agent Orange and that all analyses were done in a single laboratory. In this cohort, the mean concentration of TCDD in serum was 4.23±2.53 pg/g (parts per trillion), and the upper bound of the confidence limit was 10.2 pg/g. The statistical analysis supported a weak positive effect of age and a somewhat stronger positive effect of body mass index (BMI, the ratio of weight to square of height) on the levels of TCDD in serum. There was no association between BMI and age. The age effect was suggested to reflect a gradual accumulation of TCDD, which could reflect its long half-life and continual exposure via other sources.
Related to this are estimates of TCDD elimination rates. Van der Molen et al. (1998) employed models to reevaluate the specific elimination rates of TCDD in Vietnam veterans and concluded that TCDD half-life may actually change with age. With the model employed, they predicted half-lives for TCDD in humans ranging from 5.5 years in young adults to 11 years in older adult males. Although this prediction appears to be at variance with the results of Michalek and Tripathi (1999), they emphasize that the mechanism of TCDD elimination in humans is not known, and the model of Van de Molen et al. (1998) does not include hepatic sequestration due to inducible binding to CYP1A2.
Blood and excreta, in addition to milk, are the media in which TCDD residues most often are measured in human subjects. An important concern in toxicology is how the levels of a chemical in blood relate to levels that may occur in other organs. In studies of blood and human tissues taken from eight individuals at autopsy, Iida et al. (1999) found a correlation between the levels of selected chemicals in blood and the levels in some tissues (all expressed as picogram/ gram of tissue lipid). The correlations differed for different congeners of PCDDs and PCBs. TCDD levels in blood were correlated with levels in adipose tissue, kidney, spleen, liver, and brain. However, there was no correlation between blood levels of TCDD and the levels in muscle or lung. Octachlorodibenzo-p-dioxin (OCDD) showed similar correlation between blood and organs except for muscle. PCB 126 showed blood-tissue correlation in fat, kidney, spleen, and liver, while PCB 77, which is rapidly metabolized and does not persist, did not show a good correlation between levels in the blood and any tissue.
In a study of postmortem tissues of 25 humans, Bachour et al. (1998) evalu-
ated the distribution of different PCB congeners in brain, liver, lung, and muscle. They observed that the lower-chlorinated congeners showed a relatively higher degree of accumulation in lung than the higher-chlorinated congeners. Although the distribution to lung may be important in considering the effects of PCBs, this finding may not bear on patterns of distribution of TCDD.
In studies of distribution of TCDD to fetal and embryonic tissues in rodents, Hurst et al. (1998) reported that at selected doses, the amounts of TCDD reaching the fetal compartment were comparable to those measured in maternal blood. A dose of 1.15 µg/kg on gestation day 8 produced fetal body burdens of 18.1 to 39.6 ng/kg, depending on the time of gestation. The authors note that the doses used were in the range of body burdens of 2,3,7,8-TCDD toxicity equivalents (TEQs) estimated to occur in humans exposed through accidents. This suggests that concentrations able to cause effects in developing rodents could potentially accumulate in humans in utero.
Excretion of TCDD in the milk of nursing women has been established as an important route of elimination. Recent studies continue to increase the knowledge of partitioning and elimination in milk. In a study of five women, Schecter et al. (1998a) analyzed PCDDs and PCDFs in predelivery blood, placenta and cord blood, and post-partum blood and milk. They reported a positive relationship between levels in blood, milk, placenta, and cord blood. A further study by Schecter et al. (1998b) showed that total PCDDs, PCDFs, and PCBs in milk and in maternal blood decreased substantially over 2 years in a woman nursing twins. The TCDD levels in milk decreased from 2.7 pg/g milk lipid to <0.4 pg/g in 2.5 years; the TCDD in maternal blood decreased from 2.5 to 1.1 pg/g lipid in just under 2 years. Declines were observed in the other chemicals, and total TEQ in blood declined as well. These results suggest that the volumes of milk expressed may be a factor in the degree of elimination of chemicals from the mother, and that the exposure rate of infants via milk may decline with time.
LaKind et al. (2000) employed a model in an effort to develop reliable estimates of incremental body burden in infants who are exposed to TCDD via nursing. This model considered parameters including chemical absorption, infant growth, TCDD half-life in infants, lipid content of milk, and others. The authors point out that both the progressive depuration from the mother via lactation and the half-life of chemicals in the infant should be considered. They indicate that incremental body burdens of TCDD in infants who are breast-fed initially increase, but the body burdens decrease thereafter as the levels in milk decline over time. They calculate that there is a substantial loss of the initially accumulated TCDD by 2 years and that nursing does not result in a long-term elevation of TCDD body burdens. On the other hand, Patandin et al. (1999) calculated that 12–14 percent of TEQ in adults at age 25 could derive from the TEQ consumed during nursing. However, this is based only on TEQ in breast milk, and does not incorporate a physiologically based model.
Few studies have examined both TCDD intake and elimination in adult
humans. Schlummer et al. (1998) examined the mass balance of PCDDs, PCDFs, PCBs, and hexachlorobenzene (HCB) in seven volunteers, measuring the daily intake by analysis of TCDD in the food consumed and the amount excreted by measuring TCDD in feces. They compared the levels in food and excreta to the levels of TCDD in blood as well. The difference between ingested and excreted TCDD was defined as net absorption. Negative differences were interpreted as net excretion. There were differences in net absorption for different chemicals, including different PCDD, PCDF, and PCB congeners. Interestingly, older subjects showed a greater tendency toward net excretion (greater amounts excreted than ingested) of some compounds than younger subjects. The fat in the diet was found to be a factor affecting absorption and excretion of the compounds, and the authors speculate that distinct processes occur in different parts of the gut, with absorption occurring in the duodenum and jejunum and net excretion back into the gut lumen in the large intestine and particularly the colon.
In a mass balance study of PCDDs and PCDFs in German volunteers, Schrey et al. (1998) measured the dietary intake and fecal excretion of 14 adults, age 24– 64 years. These investigators reported that the measured amounts of TCDD and OCDD excreted, paradoxically, exceeded the amounts consumed in the diet. The intake of TCDD in picograms per day averaged 6.6, while the fecal excretion rate averaged 11.0. The ranges were 3.3–14 pg/day intake and 3.1–20 pg/day excreted. These investigators also reported that the excretion rate of PCDD or PCDF increased with the age of the participants. These authors concluded that there must be missing sources of PCDD for these subjects and that the diet did not reveal total exposure. They also suggested the possibility of formation of PCDDs in vivo, perhaps in the intestine. PCDDs and PCDFs formed in vitro from peroxidase-catalyzed reactions with chlorophenol substrates (Wittsiepe et al., 1999). Such results raise questions about interpretations regarding exposure levels or body burdens based on analysis of excreted residues. On the other hand, this might be explained, in part, if the later dioxin exposure levels were lower than the earlier ones.
Rohde et al. (1999) examined uptake and elimination of TCDD and other PCDDs and PCDFs in men in Germany who had previously accumulated high levels of TCDD in the workplace. Their studies show that concentrations of TCDD in feces had a high positive correlation with blood levels of TCDD (picograms per gram of blood lipid), indicating that fecal excretion contributes to clearance and that fecal PCDD and PCDF may be determined by body burden. The authors also calculate that an amount of TEQ equivalent to that in about 1.7 g of blood lipids is cleared daily through fecal excretion. The results of Rohde et al. (1999) also show that the amounts of TCDD, as well as other congeners, are greater in the excreta than in the food consumed. The excess amount of chemical excreted could be due to the initially high body burdens of these subjects.
Increasing fecal excretion of TCDD may help to increase clearance from the body. Recent studies have reported that ingestion of a nonabsorbed dietary fat
substitute (olestra) can increase the fecal excretion of TCDD. In one study (Geusau et al., 1999), patients with chloracne who had very high blood levels of TCDD were fed olestra, and the rate of intestinal excretion of TCDD was increased up to tenfold, substantially decreasing the half-life, from about 7 years to less than 2 years. In a similar study (Moser and McLachlan, 1999), the excretion of PCDDs, PCDFs, PCBs, and HCB was monitored in three volunteers, first while they were fed a normal diet and then when they were fed a diet supplemented with a fat substitute (olestra). The chemical excretion rates were 1.5 to 11 times greater, depending on the compound, on the supplemented diet. The exact mechanism underlying the increased excretion is not presently understood, although it may simply be due to the increase in the total amount of fat (dietary plus olestra) excreted via the feces (Geusau et al., 1999).
Accurately assessing the risk of TCDD or other PHAH exposures above background levels may require knowledge of the levels accumulated at the time(s) of greatest exposure. Estimating these levels usually requires back-extrapolation from current blood levels in exposed subjects. Heederik et al. (1998) carried out such modeling of TCDD levels in a cohort exposed to TCDD during work at a plant that manufactured chlorophenoxy herbicides. They grouped workers according to several variables, including the category and location of work in the plant during herbicide production, whether they had been employed at the time of an accident in 1963 that caused a release of PCDD, and whether they did or did not have chloracne. Levels of PCDDs, PCDFs, and PCBs were measured in serum drawn in 1993. TCDD levels in serum ranged from 7.6 pg/g serum lipid in nonexposed workers to 105 pg/g in workers exposed to the accident who had chloracne. The back-extrapolation employed a one-compartment first-order model, for which the authors varied assumptions concerning half-life, using three values that have been derived largely from studies of Vietnam veterans: 5.8, 7.1, and 11.3 years. The model assumes that half-lives do not vary between individuals. As expected, the predictions of serum levels at the time of last exposure were strongly dependent on the half-life chosen. This results in differences in levels at which elevated risk occurs, although risk groups did not change. The range in predicted levels might be expected to encompass the real values.
To summarize, studies since Update 1998 have refined estimates of TCDD half-life, suggesting further that the internal distribution of TCDD can be affected by several variables and that excretion rates can be enhanced by lipoidal additives to the diet. There have been refinements in the effort to calculate earlier exposure levels from later measurement of blood levels. The half-life of TCDD can vary between individuals and with dietary modification.
There are a number of controversies surrounding the use of these models. A detailed discussion of the models can be found in EPA’s draft dioxin risk assessment document (U.S. EPA, 2000).
Mechanisms of Toxic Action
Studies published since Update 1998 are consistent with the hypothesis that TCDD produces its biological and toxic effects by binding to a gene regulatory protein, the aryl hydrocarbon receptor. Several earlier studies had suggested that some immunological effects might occur via a non-AhR-mediated mechanism, but now, based on data in AhR-defective animals, these effects are thought to be related to pharmacokinetic factors rather than AhR-related factors. The model indicates that the binding of TCDD to the AhR, dimerization of the AhR with the ARNT protein, binding of this complex to specific DNA sequences (called aryl hydrocarbon-responsive elements [AhREs] or dioxin-responsive elements) present in the 5′-promoter regions of responsive genes, and inappropriate modulation of gene expression represent the initial steps in a series of biochemical, cellular, and tissue changes that result in the toxicity observed. Much of our current understanding of the mechanism of TCDD action is based on analysis of the induction of particular genes, (e.g., cytochrome P4501A1 [CYP1A1]), by TCDD. This hypothesis is supported by numerous studies published evaluating structure-activity relationships of various AhR ligands, the genetics of mutant AhR genes, AhR-deficient mice, and the molecular events contributing to and regulating expression of the AhR and its activity. Although the promoter regions of many genes, including CYP1A1, contain DREs, only a few of these are known to be directly regulated by the AhR-ARNT complex (reviewed by Denison et al., 1998). However, the modulated expression of these genes does not completely explain (at least not as yet) the diversity of toxic effects elicited by TCDD in numerous animal species. By analogy, it is predicted that other genes have inappropriate expression (or repression) directly related to particular toxic events. The findings that many AhR-modulated genes are regulated in a species-, cell-, and developmental stage-specific pattern suggest that molecular and cellular pathways leading to any particular toxic event are complex.
Additional research has shown that the biochemical and biological outcomes of TCDD exposure can be modulated by numerous other proteins with which the AhR interacts. Thus, it is now considered possible that TCDD could modulate gene expression by pathways that do not involve the interaction of the AhR with either ARNT or DREs. In fact, to date there are no data proving that Arnt is required for any toxic effects elicited by TCDD. It is plausible, for example that the AhR (and/or the AhR-ARNT complex) could divert other proteins and transcription factors from other signaling pathways; whose disruption could have serious consequences on a number of cell and tissue processes.
Studies summarized below report on the structural and functional aspects of the AhR and ARNT, processes regulating DNA binding and transcriptional events, biochemical and biological processes associated with AhR activation, interactions of the AhR with several cellular signaling processes, evidence for different mechanisms of toxicity, and interactions with other chemicals. Many of
these data are consistent with the notion that cellular processes involving growth, maturation, and differentiation are most sensitive to TCDD-induced modulation as mediated by the AhR. The findings in animals indicating that reproductive, developmental, and oncogenic end points appear to be most sensitive to this chemical are consistent with that notion. These data support a biologic plausibility for similar end points of toxicity that may occur in exposed humans. However, it is clear that many of these responses are tissue and species specific. The mechanistic basis for these differences is not completely understood.
It is important to consider exposure levels when discussing animal data, because experimental studies are often conducted at much higher levels than humans are exposed to, even in intense occupational settings. A discussion of the level of TCDD in the herbicides used in Vietnam and the possible levels of exposure of U.S. military personnel is found in Update 1998 and in Chapter 5 of this report.
Structural and Functional Aspects of AhR and ARNT
Several publications have documented the presence of the AhR and ARNT in a variety of tissues from different animal species and strains. Detailed analysis of variant forms has provided much information associating structure and expression levels with function. Jana et al. (1998) observed a good correlation between the steady-state level of AhR expression and the relative inducibility of CYP1 A1 in eight different rat strains. Analysis of ARNT demonstrated the presence of a wild-type form and an alternatively spliced variant in all strains. The expressed levels of both ARNT forms were equal in five strains, but the presence of the wild-type form predominated in the other three strains. Data on the tissue-specific expression of both forms suggested that their relative expression could contribute to differential induction of CYP1A1.
The Han/Wistar (H/W) rat strain has been shown to be at least a thousandfold less sensitive to the acute lethal effects of TCDD than most other rat strains. However, the H/W strain appears to be just as sensitive to other biological effects of TCDD, including CYP1A1 induction. Genetic analysis has shown that two genes, the Ahr gene and an additional unknown gene, contribute to this resistance. However, the Ahrhw allele appears to be the major determinant (Tuomisto et al., 1999). It was found that the apparent mass of the AhR from the H/W rats (~98 kDa) is lower than that of the receptor of other rat strains (~106 kDa) (Pohjanvirta et al., 1999). This difference is due to a deletion-insertion-type change at the 3′-end of exon 10 in the receptor cDNA resulting from a single point mutation at the first nucleotide at intron 10 and subsequent altered mRNA splicing (Pohjanvirta et al., 1998). The mutation leads to a loss of 38 to 43 amino acids within the transactivation domain of the AhR. Although the functional consequences of this loss remain to be characterized, the data suggest that this region may provide differential selectivity in the responses elicited by TCDD.
Korkalainen et al. (2000) recently reported the sequence of the AhR contained in the hamster. Hamsters are highly resistant to the acute toxicity of TCDD. The amino terminal was found to be highly conserved with other species. However, the glutamine-rich C-terminal region known to be involved in transactivation is substantially expanded. The functional significance is not known, but the authors hypothesized that it may account for the selective responsiveness of this species.
Several different forms of the AhR have been identified in fish. Two forms of the AhR were characterized in rainbow trout (Abnet et al., 1999). These forms demonstrated significant sequence homology with AhRs cloned from mammalian species, especially in the basic helix-loop-helix (bHLH) and the PAS domain. The glutamine-rich sequence found in the transactivation domain of mammalian AhR, however, was absent. Both forms bound TCDD with high affinity but demonstrated tissue-specific differences in expression and distinct preferences for different enhancer DNA sequences. The data suggest that the two AhR forms may regulate different genes and contribute to tissue-specific responses to AhR agonists. Similarly, two divergent AhR forms have been characterized in the teleost Fundulus heteroclitus (Karchner et al., 1999). Although both possess bHLH and PAS domains related to mammalian AhR, the two forms were found to be highly divergent in other domains. In particular, the transactivation domain in one form lacks the glutamine-rich region. Furthermore the two genes displayed different tissue-specific patterns of expression. The authors suggested that one form represents a novel subfamily of bHLH-PAS proteins within the vertebrate AhR family. Notably, the glutamine-rich region found in the transactivation domain of mammalian AhR was also found to be lacking in zebrafish AhR (Tanguay et al., 1999).
The hepatic binding affinity of the AhR for TCDD was compared between the beagle dog and cynomolgus monkey. The AhR from both species possessed a low binding affinity for TCDD (Kd values of 17.1 and 16.5 nM, respectively) similar to that previously reported for humans (Sandoz et al., 1999). Wanner et al. (1999) also demonstrated high conservation of the Ahr gene sequence in humans by an analysis for polymorphisms in 14 chemical workers involved in a trichlorophenol accident. Only one amino acid-exchanging polymorphism was identified (arginine-lysine at codon 554); this polymorphism did not appear to influence susceptibility to induced chloracne. Human AhR and ARNT were found to be transcriptionally active in response to TCDD when they were expressed in yeast; yeast possess no natural counterpart to the AhR pathway (Miller, 1999). Different AhR agonists displayed additive responses within this system. The author indicated that this may provide a useful model for further study and characterization of AhR agonists and AhR-dependent signal transduction pathways. It should be pointed out that although only one AhR sequence polymorphism has been identified and this has no apparent influence on the susceptibility to induced chloracne, other data suggest some heterogeneity of AhR concentrations and characteristics
in the human population (Perdew and Hollenback, 1995; Roberts et al., 1990, 1991). In addition, because as indicated above there are a number of factors and pathways regulating AhR activity, it is possible that although the AhR in humans might have lower affinity for TCDD than other species, differences in other regulatory factors might actually increase the relative responsiveness under certain conditions. At present, there are no clear data on the molecular properties of the AhR to conclude whether humans would be more or less susceptible to TCDD as compared to other species.
Research has shown that the association of newly synthesized AhR with a 90 kDa heat shock protein is important to maintain the AhR in a conformation that can bind ligand. Much of this work has been performed using AhR isolated from mice and rats. Recent investigations using AhR from the guinea pig and rabbit suggest some species differences in ligand binding-initiated stability of the AhR. Using AhR in which HSP90 dissociation was induced by increased salt content, these studies indicated that, at least for some species, HSP90 is not absolutely required to maintain the AhR in a ligand binding conformation (Phelan et al., 1998). Association with HSP90 is also thought to regulate nuclear uptake by blocking an N-terminal nuclear localization sequence (see below). However, it is not clear whether ligand binding-elicited dissociation of HSP90 occurs prior to or following nuclear localization. Additional work has shown the unliganded and unchaperoned AhR to be extremely labile in cells (Lees and Whitelaw, 1999). These data are consistent with a mechanism in which HSP90 is released from ligand-bound AhR following nuclear localization and concomitant with ARNT dimerization. Further studies by Heid et al. (2000) demonstrated that dissociation of HSP90 is not required for nuclear translocation of the AhR but is essential for dimerization with ARNT.
Hahn (1998) recently compared the AhR signal transduction pathway in mammalian and nonmammalian species. Together the information suggests that the AhR is an ancient protein and that its development stemmed from functions not related to regulation of CYP1A genes and divergent from the ability of the mammalian AhR to bind TCDD and related xenobiotics.
A number of publications have demonstrated that mammalian AhR and ARNT are expressed in a tissue-specific manner. Furthermore, a number of factors including developmental and differentiation stage, presence and activation of other transcription factors, and prior exposure to AhR ligands have been found to have a significant influence on the relative expression of the AhR. Garrison and Denison (2000) characterized the 5′-flanking region of the mouse Ahr gene. Putative binding sites for several transcription factors, such as stimulatory protein 1 (Sp1), were identified.
TCDD exposure elicits developmental alterations in many species and induces cleft palate in the mouse embryo. Abbott et al. (1999a) examined the expression of AhR, ARNT, and CYP1A1 mRNAs in embryonic craniofacial tissues during their development. CYP1A1 mRNA was found to be expressed in
developing craniofacial tissue and was highly induced by TCDD exposure. AhR and ARNT mRNAs were upregulated during early palatogenesis. ARNT mRNA expression was about five- to sixfold lower than that of AhR. Note that these data might support a hypothesis that TCDD may act by activating the AhR and diverting ARNT away from other normal functions of this protein. This study also observed a decreased expression of ARNT mRNA following TCDD treatment. Abbott et al. (1999b) also compared AhR and ARNT expression in cultured human embryonic palate with mouse palate. Palate from human embryos expressed approximately 350 times less AhR mRNA and 135 times less Arnt mRNA than the mouse palate. Within the 18 different human palatal tissues examined, there was a strong correlation between TCDD-induced CYP1A1 mRNA and AhR content. However, it was previously observed that human palates required about 200 times more TCDD to produce the same changes in cellular differentiation and proliferation and affect changes in growth factor (i.e., epidermal growth factor [EGF] and transforming growth factor) compared to mouse palatal tissue exposed under the same conditions in vitro (Abbott et al., 1998, 1999b). Together these data suggest that the embryonic human palate may not be as susceptible to altered growth and differentiation effects as the mouse palate and that the relative expression of the AhR and ARNT in these tissues may influence their responsiveness.
In the male rat, in utero and lactational TCDD exposure impairs development of the accessory sex organs, including the prostate, and alters sexual behavior (see below). Sommer et al. (1999) examined the effects of stage of development and TCDD treatment on AhR protein, ARNT protein, and mRNA concentrations in rat prostate. AhR protein in developing ventral and dorsolateral prostate decreased with age between postnatal day (pnd) 1 and 21. A similar decrease in ARNT protein was seen in the dorsolateral prostate. These changes were associated with decreases in AhR and ARNT mRNA. Exposure of the adult male rat to TCDD decreased AhR but not ARNT protein in prostate tissue, vas deferens, and epididymis. In utero and lactational exposure to TCDD reduced prostate AhR protein levels on pnd 7 and delayed the developmental decrease in AhR protein. These data indicate that prostatic AhR and ARNT levels are regulated during development in the rat. AhR and ARNT were also found to be expressed in normal human fetal prostate, with the most intense reactivity found in smooth muscle cells followed by epithelial cells and fibrocytes (Kashani et al., 1998). Decreased AhR expression was observed in benign hyperplastic tissue, but increased expression was frequently observed in carcinomatous prostate tissue from adult subjects.
Exposure of experimental animals to TCDD during pregnancy has been shown to cause in utero death and a variety of developmental abnormalities. Studies by Robles et al. (2000) examined AhR expression in mouse ovary. AhR protein was present at relatively high concentrations in oocytes and granulosa cells of follicles at all stages of development. Other studies detected AhR mRNA,
ARNT mRNA, and AhR protein in preimplantation rabbit embryos (Tscheudschilsuren et al., 1999a) and during early gestation in the rabbit uterus (Tscheudschilsuren et al., 1999b). In contrast, very low levels of AhR mRNA were found in the preimplantation uterus. In addition, Kuchenhoff et al. (1999) demonstrated cycle- and age-dependent AhR expression in the human endometrium, with maximal receptor levels observed near the time of ovulation. Together these data suggest that AhR and ARNT have some functional role in fetomaternal interactions during early gestation. Additional data obtained using Ahr null-allele animals are consistent with this hypothesis (see below).
TCDD exposure has been shown to elicit a wide variety of immune system alterations in all experimental animals examined. Stromal cells in bone marrow play an essential role in the support and direction of hematopoiesis, and it is possible that TCDD may exert its effects through alterations in stromal cell support capacity. Lavin et al. (1998) demonstrated the presence and functionality of AhR and ARNT in three mouse bone marrow stromal cell lines and in primary bone marrow cell cultures. These data demonstrate that the molecular machinery necessary for mediating TCDD-dependent alterations is present and functional in bone marrow stromal cells.
It has been demonstrated previously by many investigators that nuclear uptake of ligand-bound AhR is an essential event leading to transcriptional alterations of TCDD-responsive genes. A recent study by Ikuta et al. (2000) suggests that the shuttling of the AhR from nucleus to cytosol is also essential for the inducible expression of the CYP1A1 gene. Although the exact mechanisms responsible for these events are not clear, an additional study by Ikuta et al. (1998) demonstrates that the AhR contains both nuclear localization and export signals in the N-terminal region. The minimum AhR nuclear localization signal was contained in residues 13–39 of the human AhR, while the minimal nuclear export signal was contained in residues 55–75.
Several investigations utilizing whole animals and cells in culture demonstrated that TCDD treatment elicits a sustained depletion of AhR protein, without an effect on AhR mRNA, in a variety of tissues (Giannone et al., 1998; Pollenz et al., 1998; Roman et al., 1998a). Additional research has shown this induced degradation to involve TCDD-elicited nuclear uptake of the ligand-bound receptor, subsequent nuclear export and ubiquination, and proteasomal degradation (Davarinos and Pollenz, 1999; Roberts and Whitelaw, 1999; Ma and Baldwin, 2000). These data suggest a novel mechanism for the regulatory control of activated AhR and cellular AhR content. Notably, Shimba et al. (1998) reported that AhR protein levels decreased with ongoing adipose differentiation in 3T3-L1 cells and that cellular responsiveness to TCDD was concomitantly decreased. Whether the regulation of AhR levels in the latter case is related to TCDD-elicited degradation via proteasomes has yet to be determined. Nevertheless, these data indicate that normal cellular processes regulate AhR protein and the responsiveness of its signal transduction pathway.
As indicated in Update 1998, the resistance of Ahr null-allele (knockout) mice to the enzyme-inductive and toxic effects of even very high doses of TCDD further corroborates the role of this protein in these responses. Recent data from Peter et al. (1999) demonstrating only minimal teratogenicity of TCDD in knockout mice compared to wild-type mice are consistent with this. Previous data from investigations with Ahr null-allele mice also suggested that the AhR plays an important, but as yet unknown, developmental and physiological role. Data published by Abbott et al. (1999c) demonstrated a number of adverse reproductive outcomes in Ahr null-allele mice. Null-allele females had difficulty maintaining the conceptus, surviving pregnancy and lactation, and rearing pups to weaning. Null-allele pups demonstrated poor survival during lactation and after weaning. However, across different genotypes, the sex ratios and genotypic frequencies were comparable. A recent study by Robles et al. (2000) examined the ovarian germ cell dynamics in Ahr null-allele and wild-type mice. An analysis of serial ovarian sections revealed a twofold greater number of primordial follicles in Ahr null-allele animals at day 4 postpartum. Additional investigations suggested that this may result from a decrease in the death rate of the developing germ cell line because AhR deficiency attenuated oocyte apoptosis in fetal ovaries cultured in vitro. It was hypothesized that the AhR may have some role in regulating the size of the oocyte reserve by affecting germ cell death. These data are consistent with those published by Benedict et al. (2000), who provide evidence to suggest that the AhR may be involved in regulation of the number of antral follicles in postnatal life. Because antral follicles are necessary for the secretion of several reproductive hormones, it is possible that these results could explain the reproductive abnormalities observed in Ahr null-allele animals by Abbott et al. (1999c). Recently, data reported by Thurmond et al. (2000) in chimeric Ahr+/+ and Ahr–/– animals also suggested that the AhR may have some role in B-lymphocyte maturation processes. Finally, Lahvis et al. (2000) recently observed alterations in vascular development in AhR-deficient mice. It should be noted that although the knockout mice generated by several laboratories have shown many similar characteristics, distinct phenotypes have also been observed (Lahvis and Bradfield, 1998). Whether these differences occur because of local environmental effects or different genetic backgrounds has yet to be determined.
DNA Binding and Transcriptional Interference
As indicated above, the ability of TCDD to directly modulate gene expression depends on: (1) its ability to activate the AhR to a form that can dimerize with ARNT; (2) the ability of this complex to bind to specific DNA sequences located in the regulatory regions of certain genes; (3) and the alteration of transcriptional events regulating the expression of those genes. DNA binding and transactivation domains have been identified in both the AhR and ARNT. However, it is clear that the ability of this activated complex to induce particular genes
and alter cell morphology and/or function is very tissue and species specific. It seems likely that the relative presence of tissue-specific factors may play a large role in regulating these responses to AhR ligands. Many of the more recent investigations have focused on identifying and characterizing factors that may modulate, by either activation or repression, the ability of the activated AhR-ARNT complex to alter gene expression. In addition, several studies have noted the ability of a variety of AhR ligands to act as receptor antagonists.
The AhR has been shown to associate with several proteins. One of these is known to be a member of the immunophilin family. The presence of this protein has been shown to enhance the transcriptional activity of the AhR-ARNT complex in several different cellular systems (Carver et al., 1998; Meyer et al., 1998). The exact function of this protein is not clear, but some evidence suggests that although it is not required for the interaction of HSP90 with the AhR, it increases the level of active AhR by regulating the rate of AhR turnover (Meyer and Perdew, 1999; La Pres et al., 2000). In contrast, a 23 kDa protein has been shown to associate with the ligand-binding form of the AhR and is thought to play a role in stabilizing the complex containing HSP90 (Kazlauskas et al., 1999).
Nuclear accessory proteins, or coactivators, have been shown to act as bridging factors between enhancer-binding proteins, such as the estrogen receptor, and the basal transcription complex. For example, RIP 140 has been found to interact with several nuclear receptors to enhance transcriptional activity. Similarly, Kumar et al. (1999) demonstrated that in several cell lines the presence of RIP140 enhanced TCDD-mediated, AhR-dependent transcriptional activity. Mapping of the interactions sites indicated that the AhR recruits RIP 140 via the glutamine-rich region within the transactivation domain. Nguyen et al. (1999) also found that the coactivator ERAP140 immunoprecipitates with the AhR-ARNT complex, enhances AhR-ARNT binding to DREs, and increases AhR-mediated gene expression.
Several publications have suggested the repression of AhR-mediated signal transduction by cellular factors. Gradin et al. (1999) identified the presence of a factor in human fibroblasts that is able to repress the activity of the AhR. Within these cells, genes controlled by the activated AhR-ARNT complex have been shown to be nonresponsive to TCDD. The represser was shown to act by binding with ARNT to form an inactive complex able to bind to the DRE. Using mouse hepatoma cells, Eltom et al. (1999) identified the presence of an ARNT-like protein that may act as a selective suppressor of CYP1B1 gene expression. The presence of this factor may account for the selective expression of CYP1A1 over CYP1B1 in some cell types with and without TCDD exposure. Studies by Mimura et al. (1999) identified and characterized an AhR represser (AhRR) in mice that acts by competing with the AhR for dimerization with ARNT. The gene for this represser was activated by the TCDD-AhR-ARNT complex. The encoded sequence exhibited a great degree of similarity to the AhR. However, the PAS-A domain is variable and the PAS-B sequence, which functions in ligand binding
and interaction with HSP90, is missing. This is thought to represent a novel regulatory mechanism of AhR function.
The mouse AhR has been shown to physically associate with the pleiotropic transcription factor NF-κB, and this interaction appears to produce a mutual functional repression of their actions (Tian et al., 1999). These authors suggest that the ability of the TCDD-activated AhR to interact with NF-κB and divert it from other actions may provide a possible mechanistic explanation for some of the toxic responses elicited by TCDD. Similarly, AhR and ARNT proteins were found to coimmunoprecipitate with SMRT (silencing mediator for retinoic acid and thyroid hormone receptor), and this interaction was found to block both AhR-ARNT DRE binding and AhR-mediated gene expression (Nguyen et al., 1999). The actual mechanism by which these interactions occur and their relative importance in the gene- and tissue-specific responses elicited by TCDD are unknown.
Histone acetylation and deacetylation have been demonstrated to be highly specific regulatory processes that control the activation and repression of some genes. Several coactivators and repressers possess histone acetylase and deacetylase activities. Garrison et al. (2000) reported that the treatment of cells in culture with histone deacetylase inhibitors increased the constitutive activity of an AhR-dependent DRE-driven gene reporter construct. Cotransfection with a protein having histone acetylase activity decreased the activity of this promoter. These studies provide evidence that histone acetylation-deacetylation may regulate AhR-responsive genes.
As indicated above, several lines of evidence suggest a normal functional role of the AhR. However, an endogenous ligand that has clear functionality under physiological conditions has not been identified. Previous reports have indicated stimulation of AhR-dependent processes in the absence of added ligand. A recent report by Chang and Puga (1998) found that CYP1A1-deficient mouse hepatoma c37 cells possess transcriptionally active AhR-ARNT complexes in the absence of exogenous ligands. A similar finding was observed following treatment of Hepa-1 cells with an inhibitor of CYP1A1 activity. Likewise the expression of a DRE-responsive reporter gene in CV-1 cells leads to high levels of reporter gene expression. These data suggest that a CYP1A1 substrate that accumulates in cells lacking this enzyme activity is an endogenous ligand for the AhR. This ligand has yet to be identified.
There have been several reports of naturally occurring high-affinity ligands for the receptor. Many of these exist in plants that are consumed by humans. These include resveratrol (Ciolino et al., 1998a; Casper et al., 1999; Ciolino and Yeh, 1999), curcumin (Ciolino et al., 1998b), tryptophan metabolites (Heath-Pagliuso et al., 1998; Y.D.Wei et al., 1999), galangin (Ciolino and Yeh, 1998; Quadri et al., 2000), the dietary flavonols quercetin and kaempferol (Ciolino et al., 1999), lipoxin A4 (Schaldach et al., 1999), and products of heme metabolism (Sinal and Bend, 1997). Several of these, such as the tryptophan metabolites,
lipoxin A4, appear primarily to have AhR agonist activity. Galangin, quercetin, kaempferol, and curcumin have concentration-dependent agonist and antagonist activity, whereas resveratrol appears to act mainly as an AhR antagonist. Under certain conditions, several of these compounds have been shown to have some anticancer activity, but a role of the AhR in that activity has not yet been demonstrated. Although there are good data available on the structure-activity relationships for AhR agonists, the structural requirements and mechanisms of AhR antagonists are poorly understood. A study by Henry et al. (1999) examined certain flavone derivatives for their ability to produce AhR antagonist activity in mouse hepatoma cells. Overall, the data indicate that particular substituent groups, such as a 3′-methoxy group or one possessing terminal atoms with high electron density that can form hydrogen bonds or interact electrostatically with receptor amino acids, promote antagonist activity. Additional studies indicate that these chemicals act as antagonists by binding the same site on the AhR as TCDD but fail to elicit a conformational change resulting in nuclear translocation, dissociation of HSP90, and ARNT dimerization. Recently, Ashida et al. (2000a) suggested that dietary levels of certain flavones and flavanols may act as AhR antagonists.
As indicated above, the AhR and ARNT have been localized to a variety of different tissues at various stages of development. This information, although useful, does not indicate when and where the AhR-ARNT complex might be activated in vivo by various agonists. This information could be used to determine tissues that are sensitive to TCDD and to explore the physiological function of the AhR. Willey et al. (1998) developed a transgenic mouse model that can be used to indicate the temporal and spatial context of transcriptionally active AhR following agonist exposure in vivo. Transgenic mice containing a TCDD-responsive lacZ reporter gene construct demonstrated TCDD-inducible β-galactosidase activity in several tissues following adult and in utero exposure. These tissues included palate, liver, lung, genital tubercle, paws, tooth buds, intestine, brain, and developing ear. These data indicate that TCDD can initiate altered gene expression in these tissues, thereby identifying potential targets of toxicity.
Biological Consequences of Activation
Many toxic effects of TCDD have been described in experimental animals and exposed human populations. The exact mechanisms responsible for most, if not all, of these species- and tissue-specific effects are not known. Although there is information about particular genes that may be altered under certain conditions, the relationships between altered gene expression and more complex biological responses such as altered growth, differentiation, developmental effects, and neoplasia are not understood. A major obstacle to clarifying these issues has been the lack of understanding of the actual direct cell targets and the develop-
mental stage of greatest sensitivity. Since Update 1998, several investigations have been conducted to identify both cellular and gene targets for TCDD.
In animals lethally intoxicated with TCDD, a wasting syndrome characterized by decreased food intake and body weight loss is most often observed. The biochemical pathways responsible for this syndrome are not understood, but reduced gluconeogenesis has been thought to play a role in the process. Work by Viluksela et al. (1999) demonstrated a dose-dependent decrease in hepatic phosphoenolpyruvate carboxykinase (PEPCK) activity, the rate-limiting enzyme of gluconeogenesis, following the exposure of TCDD-resistant H/W rats to TCDD. This decrease was not observed in TCDD-sensitive Long-Evans (L-E) rats. However, TCDD treatment attenuated the increase in PEPCK activity observed in pair-fed controls. Although the change in PEPCK activity alone could not explain the difference in sensitivity between the two rat strains, the gluconeogenic response following TCDD treatment appeared to be altered. Unkila et al. (1998) also compared these two rat strains for the effects of TCDD on tryptophan homeostasis. In L-E rats, TCDD treatment produced an elevation in brain tryptophan and plasma free tryptophan concentrations and a decrease in hepatic tryptophan pyrrolase. Much smaller or no effects were seen in the H/W animals. Although there appeared to be some correlation between the relative sensitivity of these strains to TCDD-induced body weight loss and alterations in tryptophan metabolism, the analysis of other dioxin congeners did not demonstrate the same relationships. CAAT-enhancer binding proteins (C-EBP) have been suggested to play a role in the coordination of energy homeostasis. Liu et al. (1998) examined the effect of TCDD treatment (100 µg/kg) on C-EBPβ and C-EBPβ expression and function in mice. There was a time- and dose-dependent decrease in the amount of C-EBPβ mRNA in adipose tissue and liver and an increase in C-EBPβ mRNA. These changes were also reflected in the protein-C-EBP recognition element complexes formed using hepatic nuclear extracts from control and TCDD-treated mice. Induced changes in the expression of these transcription factors may have a role in the mechanism by which TCDD treatment causes a wasting syndrome in animals.
Gao et al. (1998) reported the induction of an ecto-ATPase in mouse hepatoma cells that is AhR dependent and is regulated at the transcriptional level. Ecto-ATPases influence several physiological processes including the metabolism of nucleic acids. However, their exact role in induced toxicity is not understood.
Liver lesions have been reported in several animal species following exposure to TCDD, and this chemical is a potent tumor promoter in the liver of male and female mice, as well as female rats. Two recent studies evaluated the relative contributions of different cell types to the hepatic lesions observed in rats. Using a chimeric Ahr null-allele model, Thurmond et al. (1999) found that hematopoietic cells contribute to the hepatic lesions induced by TCDD. More specifically,
the presence of the AhR in hepatic parenchyma alone was sufficient for TCDD-elicited hepatic necrosis. An inflammatory response is secondary to this damage but depends on the presence of the AhR in immune cells. Riebniger and Schrenk (1998) examined the expression of TGF-β1 in fat-storing cells isolated from rat liver. TGF-β1 is an inhibitor of hepatocellular proliferation but is synthesized predominantly in nonparenchymal hepatic cells. Exposure of these cells to TCDD either in vivo or in vitro had no effect on TGF-β1 gene expression. In addition, no effect on CYP1A1 expression was observed in these cells. This was likely due to the absence of a detectable amount of the AhR. Increased portal fibrosis and small liver size have been reported in Ahr null-allele mice. Zaher et al. (1998) examined the relationship between TGF-β expression and apoptosis in this mouse line. Livers from Ahr null-allele animals had increased expression of TGF-β1 and TGF-β3 proteins and increased numbers of hepatocytes undergoing apoptosis compared to wild-type animals. These parameters were especially increased in the portal areas. Primary hepatocytes from null-allele animals exhibited increased numbers of cells in apoptosis and increased secretion of TGF-β3 into the media. Conditioned media from null-allele cells stimulated apoptosis in hepatocytes from wild-type mice. These data suggest that abnormal liver morphology in Ahr null-allele animals may be mediated by abnormal levels of TGF-β and increased apoptosis of hepatocytes. The AhR may have a normal function in regulating these processes. Puga et al. (2000a) observe that exposure of human hepatoma HepG2 cells to 10 nM TCDD resulted in the altered expression of 310 known genes; 30 of these were upregulated and 78 were downregulated. The significance of this information to the specific toxic responses in the liver is not clear.
Several reports on exposed animal and human populations suggest that halogenated aromatic hydrocarbons, such as TCDD, may alter cognitive function and learning, especially when exposure occurs during critical periods of brain development. Hong et al. (1998) examined the effect of TCDD on evoked excitatory postsynaptic potentials (EPSPs) using hippocampal slices from adolescent and adult male rats. The hippocampus has been shown to have a role in learning and memory. A TCDD concentration of 100 nM was shown to decrease EPSPs in ventral slices but not in slices from the middle third of the hippocampus. The calcium channel blocker nifedipine blocked the inhibition of EPSPs by TCDD but not by 2, 2', 5, 5'-tetrachlorobiphenyl. These data suggest that the effect of TCDD is mediated by L-type calcium channels and is congener specific.
Newborn infants are often susceptible to vitamin K deficiency, which may lead to hemorrhagic disorders. Bouwman et al. (1999) investigated whether TCDD exposure might affect vitamin K-dependent blood coagulation. Single oral doses up to 10 µg/kg to adult female and male rats caused a dose-dependent reduction in vitamin K-dependent coagulation factor VII. This was negatively correlated with an induction of CYP1 A1 activity. The greatest reduction achieved was 44 percent in female rats. No effect on male rats was observed. Sex-dependent differences in vitamin K-dependent carboxylase and vitamin K 2, 3-epoxide
reductase were also observed. The carboxylase was greatly induced in female rats. Further studies in newborns are needed.
TCDD has been shown to induce the expression of various cytokines in a variety of cell types. Lang et al. (1998) examined the expression of various cytokine genes in human airway epithelial cells, alveolar macrophages, and peripheral blood monocytes and lymphocytes. CYP1A1 activity and mRNA were increased in a concentration-dependent manner in brochoepithelial cells and blood lymphocytes, with epithelial cells being the most inducible (EC50 of 30 pM). TCDD treatment did not affect the expression or production of IL-6 or IL-8 in these cells.
Perinatal exposure to TCDD has been shown to alter thermoregulatory control in hamsters and rats. Gordon and Miller (1998) examined behavioral thermoregulation, ability to develop fever, and thermoregulatory stability in rats exposed perinatally at gestational day (gd) 15 to 1 µg TCDD/kg. Although the data confirmed that TCDD induced nocturnal hypothermia in males up to 11 months of age, the hypothalamic thermoregulatory centers did not appear to be permanently altered. Accentuated fever in TCDD-treated animals in response to lipopolysaccharide was also observed. These data suggest that there are functional alterations in the hypothalamic-pituitary-thyroid axis. Whether these findings relate to previously reported alterations in blood thyroid hormone levels in response to TCDD is unknown. Using primary rat anterior pituitary cells in culture, Bestervelt et al. (1998) assessed whether TCDD exposure directly affected pituitary function. Maximal effects of TCDD on adrenocorticotropic hormone (ACTH) secretion occurred at media concentrations between 10–11 and 10–15 M after 24-hour exposure. Alterations in secretion were observed as early as 6 hours and persisted up to 10 days. Decreased pituitary responsiveness to corticotropin-releasing hormone and arginine-8-vasopressin was also observed. These data support the hypothesis that TCDD might directly interfere with anterior pituitary gland function, and this might occur at very low cellular concentrations.
It has been known for some time that TCDD alters the expression of several types of cytochrome P450 isozymes. Although the effect of these changes on the metabolism of drugs and xenobiotics has been widely documented, the relationship of these alterations to the complex biological and toxic effects of TCDD is not understood. Walker et al. (1999) investigated the dose-dependent expression of CYP1A1, CYP1A2, and CYP1B1 in the livers of female rats exposed for 30 weeks to doses of TCDD up to 125 ng/kg/day. There was a dose-dependent induction of all three isozymes by TCDD. However, at the highest levels of exposure, the protein levels of CYP1A1 and CYP1A2 induced were between forty- and a hundredfold higher than those of CYP1B1. The authors suggested that if CYP1B1 is involved in TCDD-induced hepatocarcinogenesis, its function is unique and does not overlap with that of CYP1A1 or CYP1A2. Mahajan and Rifkind (1999) observed differential induction of CYP1A4 and CYP1A5 in a variety of tissues from the chick embryo following TCDD exposure. The data suggest that the ability of
TCDD to induce CYP1A isozymes is very tissue and species specific because of differences in the function of these isozymes and that these differences may contribute to differential sensitivity of animal species to TCDD.
AhR Signaling Interactions
Since Update 1998, additional evidence has been published indicating that the AhR affects and is affected by other signal transduction pathways. Furthermore, it seems likely that the ability of the TCDD-activated AhR to interact with these pathways results in the altered regulation of processes involving tissue development, growth, and differentiation that is observed following exposure of animals to TCDD. There is also additional evidence that the AhR may have some role in the normal regulation of the cell cycle. The newest data on AhR interactions with growth and differentiation processes, redox signaling, kinase activities, and hormone receptor signaling are discussed below.
Growth and Differentiation Signaling
TCDD has been demonstrated to be a potent tumor promoter in several types of initiation-promoter models. Its ability to induce cell proliferation and altered differentiation is believed to be an important factor in the mechanism of TCDD-induced carcinogenesis. A study by Walker et al. (1998) investigated the time course and reversibility of cell proliferation in control and diethylnitrosamine-initiated female rats exposed biweekly to an average daily dose of 125 ng TCDD/ kg/day for up to 60 weeks. Cell proliferation, as determined by the incorporation of 5-bromo-2′-deoxyuridine into DNA, was not increased until after 30 weeks of exposure. In contrast, CYP1 A1 activity was increased at all times examined after 14 weeks. Even 16 weeks after the cessation of TCDD treatment, cell proliferation remained significantly elevated over controls. Proliferation was not increased, however, 30 weeks after withdrawal of treatment. Notably, dosimetric analysis suggested that the rat liver tissue burden of TCDD was predictive of CYP1A1 expression but not of cell proliferation. The measurement of total dose over time was not predictive of CYP1 A1 expression or cell proliferation. Therefore, tissue burden correlated with a simple reversible response (i.e., CYP1A1 activity), but not with a more complex response.
The regulation of and balance between cellular differentiation, proliferation and cell death (apoptosis) are essential components of a tissue’s ability to succumb to or escape chemical-induced developmental abnormalities or carcinogenesis. The proto-oncogene c-myc is an important factor in apoptosis regulation and has been shown to be increased in human and rodent liver neoplasia. A study by Christensen et al. (1999) examined whether apoptosis is altered in hepatocytes that overexpress c-myc and whether TCDD alters that apoptosis. Concentrations of TCDD up to 1 nM did not affect c-myc-induced apoptosis in hepatocytes.
Reiners and Clift (1999) examined the relationship between AhR content of mouse hepatoma cells and susceptibility to ceramide-induced apoptosis. Ceramide exposure caused a concentration-dependent inhibition of cell proliferation and induction of apoptosis. In variant cells that possess approximately 10 percent of the AhR content of wild-type cells, ceramide also arrested growth but did not induce apoptosis. Furthermore, modulation of the AhR content confirmed the relationship between AhR content and susceptibility to ceramide-induced apoptosis. This relationship appears to be relatively specific for ceramide-affected pathways because AhR content did affect apoptosis induced by other agents. Ceramide, however, was able to induce apoptosis in a cell line possessing AhR but lacking ARNT, suggesting that the AhR modulates ceramide-induced apoptosis by a mechanism that does not require ARNT protein and exogenous AhR ligands.
Dioxin-like chemicals have profound effects on the immune system of experimental animals. The direct cell targets that mediate these responses in vivo have not been unequivocally identified, in part because of the multicellular nature of the immune system, conflicting data from in vivo and in vitro studies, and the possibility that immune responses may be mediated indirectly through an action on other tissue systems (e.g., endocrine effects). Several studies have examined whether TCDD-induced changes in the immune system are caused by altered differentiation and maturation processes that lead to cellular arrest, proliferation, or apoptosis. Based on previous publications indicating that TCDD induces thymic atrophy through its ability to elicit apoptosis in thymocytes, Kamath et al. (1998) examined phenotypic changes in thymocytes from animals treated with 50 µg TCDD/kg and compared these changes to cells undergoing spontaneous apoptosis in vitro. Similar phenotypic changes were observed in both populations. Therefore, phenotypic alteration in the density of thymocyte surface molecules might be a biomarker of apoptosis. Such a biomarker would be useful because detection of apoptosis in vivo is often difficult. Pryputniewicz et al. (1998) examined whether resting or activated T cells are more susceptible to TCDD exposure. T cells isolated from the popliteal lymph nodes of mice treated with 50 µg TCDD/kg had a decreased proliferative response to anti-CD3 antibody compared to cells from control animals. Freshly isolated cells from TCDD-treated animals did not show increased apoptosis. However, activated cells that were cultured for 24 hours alone or with anti-CD3 antibody demonstrated increased apoptosis compared to resting lymph node T cells. These data suggest that TCDD elicits differential effects on activated and resting T cells. This same group of researchers also determined that Fas-deficient and Fas ligand-defective mice were resistant to TCDD-induced thymic atrophy and apoptosis. TCDD treatment resulted in increased FasL protein but not Fas mRNA (Kamath et al., 1999). Fas ligand is a cytokine belonging to the tumor necrosis factor family, and it appears to be involved in controlling pathways that lead to activation of caspases and subsequent cell death. The data from Kamath et al. (1999) suggest that Fas
ligand may play an important role in TCDD-mediated immune effects through its ability to control apoptotic events. A study by Hart et al. (1999) demonstrated an increased number of apoptotic cells in the lymphoid regions of the spleen and pronephros from fish (Tilapia) treated with 5 µg/kg/day TCDD for 5 days. No significant effect was observed at a dosage of 1 µg/kg/day TCDD. Finally, another study found that TCDD concentrations of 1–40 nM induced apoptosis in two cultured human leukemic lymphoblastic T cell lines. However, two other AhR ligands, 2, 3, 7, 8-tetrachlorodibenzofuran and (β-naphthoflavone failed to elicit apoptosis even at concentrations up to 20 µM. Cell death was not observed in a cell line containing a dominant-negative mutant of c-Jun N-termal kinase (JNK). Those data suggest that the AhR may not be involved in TCDD-induced apoptosis and that the INK signal transduction pathway may play a role.
In contrast to the studies cited above, other investigations in mice indicate that apoptosis is not involved in TCDD-induced thymic atrophy. Overexpression of the antiapoptotic oncogene Bcl-2 in the thymus did not prevent atrophy induced by 30 µg TCDD/kg. Although distinct phenotypic changes in thymocytes were observed, no signs of apoptosis were detected using several different methods (Staples et al., 1998a). However, apoptosis was detected in animals treated with dexamethasone, and the overexpression of Bcl-2 prevented that apoptosis. In another publication, Staples et al. (1998b) demonstrated that thymic alterations induced by TCDD were strictly dependent on the presence of the AhR in hematopoietic cells. These studies were performed using chimeric animals with either TCDD-responsive (Ahr+/+) stromal components and TCDD-unresponsive (Ahr–/–) hematopoietic components, or the reverse. Thymic atrophy was not observed in animals with Ahr+/+ stroma and Ahr–/– hematopoietic cells, or in animals with Ahr–/– in both components. However, the same degree of atrophy was observed in animals with Ahr–/– stroma and Ahr+/+ hematopoietic cells as in animals Ahr+/+ in both components. These results indicate that the targets for TCDD-induced atrophy are in the hematopoietic compartment. Nohara et al. (2000) examined phenotypic changes in thymus and lymph nodes following treatment of rats with 1 or 2 µg TCDD/kg. Similar phenotypic changes in thymocytes were observed as previously reported, with the exception that a reduction in CD4+CD8+ cells did not occur. However, the phenotype of lymph node cells indicated that the number of recent thymic emigrants was decreased. Based on this observation, the authors suggest that TCDD might result in immunosuppression, in part, through its ability to alter differentiation of T cells and cause a subsequent reduction in the peripheral T-cell repertoire. Prell et al. (2000) report a hyporesponsive cytotoxic T-cell response in TCDD-treated mice because of a disruption of precursor activation. This was found not to be caused by insufficient IL-2 or apoptotic deletion CD8+ T cells. On the other hand, in mice exposed to TCDD and infected with influenza virus, the number of CD8+ mediastinal lymph node cells was reduced, these cells failed to develop cytolytic activity, and the production of IL-2 and interferon-γ (IFN-γ) was suppressed. Exposure to TCDD
also decreased the production of virus-specific antibodies, the recruitment of CD8+ cells to the lung, and the percentage of cells bearing a cytotoxic T-lymphocyte (CTL) phenotype (Warren et al., 2000). The cytolytic activity of lung lavage cells from TCDD- and vehicle-treated mice, however, was equivalent in this study.
Much evidence indicates that TCDD exposure during development results in altered tissue growth and differentiation processes. Several studies demonstrate that in utero and lactational exposure of male rats to TCDD impairs prostate development. Pregnant rats were treated with 1 µg TCDD/kg at gd 15, and male pups were examined between gd 20 and pnd 32. At gd 20 an impairment of the prostatic epithelial budding process was observed. Cell proliferation of the ventral prostate was decreased on pnd 1 but was not altered at later periods. There were delays in the differentiation of the prostate periductal smooth muscle cells and luminal epithelial cells. In addition, there were alterations in the arrangement of cell types in the ventral prostate characterized by hyperplastic epithelium, increased density or continuous layer of basal epithelial cells, and thickened periductal smooth muscle sheaths (Roman et al., 1998b). These effects may be related to an observed transient downregulation in the expression of androgenregulated prostatic mRNAs used as markers of differentiated ductal epithelium. Increased expression of CYP1A1 mRNA, however, was also seen (Roman and Peterson, 1998). Hamm et al. (2000) demonstrated that treatment of rats with 1 µg TCDD/kg at gd 15 resulted in decreased seminal vesicle weights in offspring 8–11 months of age. Decreased seminal vesicle epithelial branching and differentiation were also observed.
Developmental effects on female offspring of in utero exposure to TCDD have been observed in several other studies. A single oral dose of 2 µg TCDD/kg to pregnant hamsters at gd 11.5 altered reproductive tissue and function in female offspring (Wolf et al., 1999). Body weights were reduced, vaginal opening was delayed, and vaginal estrous cycles were altered. In addition, there was increased incidence of external urogenital malformations characterized by clefting of the phallus, which has also been observed in the rat. More recently, Dienhart et al. (2000) reported that a single dose of 1 µg TCDD/kg at gd 15 to pregnant rats results in abnormal vaginal development in female fetuses by inhibiting regression of the Wolffian ducts, increasing the size of the interductal mesenchyme, and preventing fusion of the Mullerian ducts. Cummings et al. (1999) report that exposure of mice at gd 8 to TCDD (3 µg/kg), supplemented by adult exposure, resulted in increased sensitivity to the promotion of surgically-induced endometrial lesion growth; no effects were observed in the rat. It is not clear if this growth is related to previous reports of increased incidence of endometriosis in exposed monkeys. A recent publication by Yang et al. (2000), however, indicates that TCDD exposure of cynomolgus monkeys exerted a bimodal effect on survival and growth of endometrial implants. This study is of particular interest because the dose levels used (1–25 ng/kg, 5 days per week for 12 months)
resulted in tissue dioxin levels representative of dioxin concentrations in human adipose tissue. Exposure of pregnant rats to 1 µg TCDD/kg at gd 15 reduced the number of ovarian antral and preantral follicles of certain size classes in female offspring examined at pnd 21–22 (Heimler et al., 1998). The loss of these follicles did not appear to be due to apoptotic events.
Two studies suggest that TCDD and the AhR may have effects on the developing mammary gland. Brown et al. (1998) investigated whether prenatal exposure to TCDD predisposes rats to mammary cancer. The exposure of pregnant rats to 1 µg TCDD/kg at gd 15 resulted in significantly more terminal end buds and fewer lobules in 50-day-old female offspring. There were also increased numbers of 7, 12-dimethylbenz[a]anthracene (DMBA) induced mammary adenocarcinomas in rats prenatally treated with TCDD. The alterations in mammary gland differentiation were correlated with increased susceptibility to mammary cancer from prenatal TCDD exposure. Hushka et al. (1998) examined the actions of 2,3,7,8-tetrachlorodibenzofuran (TCDF) and AhR presence on AhR signal transduction pathways in the developing mouse mammary gland. In untreated 6-to 8-week-old Ahr null-allele littermates, there were reductions of terminal end buds and increased numbers of blunt-ended terminal ducts. Treatment of mammary glands in vitro with TCDF suppressed lobule development and DNA synthesis. These data suggest that normal mammary gland development may be dependent on the presence of the AhR and that exposure to TCDF during this period may alter normal AhR function to suppress development.
Previous studies suggest that the presence of dioxin-like compounds in mother’s milk may affect the mineralization of children’s permanent first molars. Developmental dental defects have also been reported in monkeys and rats exposed to TCDD, and in humans exposed in utero following the accidental contamination of cooking oil with PCBs and dibenzofurans in Taiwan (Rogan et al., 1988). To examine the role of EGF in the actions of TCDD on tooth development, Partanen et al. (1998) cultured embryonic teeth from mice with TCDD in the absence or presence of EGF. Exposure to 0.5–1.0 µM TCDD caused depolarization of ondontoblasts and ameloblasts and improper mineralization and deposition of enamel matrix. The presence of EGF attenuated many of the effects of TCDD. These results suggest that interference of TCDD with mouse molar development in vitro involves EGF receptor (EGFR) signaling. Studies by Willey et al. (1998) suggest the presence of transcriptionally active AhR in developing mouse tooth buds exposed to TCDD in vivo.
Hurst et al. (2000) examined the embryonic concentrations of TCDD that were associated with developmental defects. Pregnant rats received a single oral dose of radiolabeled TCDD on gd 15, and fetal concentrations were measured at gd 16 and 21. In an animal that was administered 0.2 µg TCDD/kg, at gd 16 there were 13.2 pg TCDD/g present in an individual fetus. This concentration has been previously associated with delayed puberty, decreased epididymal sperm counts in male pups, and malformations in the external genitalia of females.
Several groups have investigated the signal transduction pathways that are altered by TCDD exposure and that may be responsible for altered tissue development and cellular differentiation. Kolluri et al. (1999) demonstrated that TCDD-induced activation of the AhR induces the p27Kip1 cyclin/cdk inhibitor by altering Kip1 transcription in hepatoma cells and in cultured fetal thymus glands. Hepatoma cells expressing Kip1 antisense RNA are resistant to TCDD-induced suppression of proliferation and delay of cell cycle through the G1 phase. In addition, Kip1-deficient thymic glands are less sensitive to the inhibition of proliferation by TCDD. Because Kip1 appears to act as a tumor suppressor gene, at present it seems unlikely that the effects of TCDD on this gene could underlie some of TCDD’s carcinogenicity and tumor-promoting activity.
It has been shown that TCDD induces a G1 cell cycle arrest in rat 5L hepatoma cells via the AhR (Ma and Whitlock, 1996). In addition, retinoblastoma (RB) protein is known to control cell cycle progression through G1, and to promote cell differentiation (Goodrich and Lee, 1992; Richon et al., 1992; Sehy et al., 1992). Two publications have implicated an interaction of the AhR with the RB protein in the ability of TCDD to mediate cell cycle arrest via the AhR. Ge and Elferink (1998) demonstrated that the RB protein and the AhR interact directly, that the interaction occurs through two distinct regions in the AhR, and that the RB preferentially associates with ligand-bound AhR. Puga et al. (2000b) also verified this interaction using both the yeast two-hybrid system and human breast carcinoma MCF-7 cells. They also demonstrated that the AhR acts in synergy with the RB to repress E2 promoter binding factor (E2F)-dependent transcription and induce cell cycle arrest. The AhR has also been shown to interact with p300, an important coactivator in the regulation of the cell cycle (Tohkin et al., 2000).
Arachidonic acid and its metabolites modulate cell growth and differentiation and may be involved in the multistep process of carcinogenesis (Tsunamoto et al., 1987; Gaillard et al., 1989). Work by Lee et al. (1998) examining the profile of CYP1A isozymes in different tissues from mice and chicks treated with TCDD suggest that species-specific differences in the catalytic activities of these isozymes and subsequent differences in arachidonic acid metabolism may contribute to species differences in sensitivity to TCDD. Using inhibitors of cyclooxygenase (COX) and lipoxygenase activities, Wolfle et al. (2000) examined the potential role of arachidonic acid metabolism in the promotion of malignant transformation of mouse fibroblasts by TCDD. The promoting effects of 1.5 pM TCDD were blocked by cotreatment with COX inhibitors. However, selective inhibition of COX-2 did not abolish the effect of TCDD. Long-term treatment with TCDD induced both COX-1 and COX-2 mRNA and protein, and this resulted in the accumulation of PGE2 and 6-keto-PGF1α. The data suggest that the stimulation of arachidonic acid metabolism by COX induction may be a critical event in the promotional actions of TCDD in mouse fibroblasts. In contrast, exposure of mice to a single dose of 15 µg TCDD/kg did not alter COX mRNA or
protein in the spleen (Lawrence and Kerkvliet, 1998). Furthermore, several inhibitors blocked arachidonic acid metabolism but did not affect either TCDD-induced suppression of the cytotoxic T-lymphocyte response to tumor cells or antibody formation in response to sheep red blood cells.
Several previous reports indicated that TCDD modulates the expression of prostaglandin endoperoxide H synthase 2 (PGHS-2) in different cell lines. A study by Vogel et al. (1998) further examined whether this activity is altered in TCDD-exposed mice and whether any alteration was mediated by the AhR. A single dose of 10 µg TCDD/kg to C57BL/6J mice did not alter PGHS-1 in any tissue examined. However, TCDD treatment increased PGHS-2 mRNA in the lung and spleen, but not in the liver and kidney. A nearly hundredfold higher dose of TCDD was necessary to produce the same response in lungs of DB A/2J mice, which possess a defective AhR that has a lower affinity for TCDD. These studies indicate that TCDD modulates PGHS-2 activity in selected tissues by a mechanism involving the AhR.
A study by J.H.Yang et al. (1999) examined the relationships between the malignant transformation of human epidermal keratinocyte cells in culture by TCDD and the expression of several growth regulatory factors. Concentrations of TCDD up to 100 nM altered the expression of TGF-β1, plasminogen activator inhibitor-2 (PAI-2), and TNF-α in the transformed cells compared to the parental cells. There was a concentration-dependent increase in PAI-2 mRNA in the parental cells but not in the transformed cells. Although there were significant differences in TGF-β1 and TNF-α mRNA expression between the transformed and parental cells, TCDD exposure did not produce a change in the expression of these growth factors. There was also a concentration-dependent increase in expression of IL-β1 and PAI-2 mRNA in immortalized human endometrial stromal cells following TCDD treatment (Yang, 1999). Expression of PAI-2 mRNA appeared to be altered by TCDD at the posttranscriptional level.
Abbott et al. (1998) examined the expression of several growth factors in nine separate samples of human embryonic palate placed in culture following their exposure to 10 nM TCDD. AhR protein levels were increased in five individuals. This increase was accompanied by increased expression of TGF-β, EFG, glucocorticoid receptor (GR), and/or TGF-β2. EGF was increased in tissues where there was either no change in or decreased expression of the AhR. TGF-β expression was increased in six of nine samples. The effect of TCDD on the expression of TGF-β2 was dependent on the age of the tissue; younger tissue demonstrated decreased expression, while older tissue had increased expression. The authors suggest that differences in the patterns of expression between human and mouse palates exposed to TCDD, as well as genetic variability, may provide a partial explanation for the relative insensitivity of human palatal cells to TCDD in culture.
Enan et al. (1998a) demonstrated, in cynomolgus macaques that had an increased incidence of endocervical squamous metaplasia approximately 1 year
after a single doses of 2 or 4 µg TCDD/kg, that the expression of several growth factors was altered in endocervical cells from these monkeys. EGFR binding activity, Cdk4 protein levels, and DNA binding activity of AP-1 were significantly decreased, whereas H-ras, p53, wafl/p21, and Cdc2p34 protein levels increased. Significant alterations were also observed in c-Src kinase, protein tyrosine kinase, and casein kinase II (see below).
The mutational activation of the K-ras oncogene often occurs in human and mouse lung adenocarcinomas. TCDD treatment (1.6 µg/kg) increased the expression of K-ras p21 in the lungs of several strains of mice and altered the membrane-cytosol ratio of this protein (Ramakrishna and Anderson, 1998). Those effects were strain dependent and also depended on the Ahr genotype expressed. Treatment of immortalized alveolar type 2 E10 cells with 10 nM TCDD increased K-ras p21 levels in the membrane fraction approximately fourfold. It is not known how these data are related to proposed mechanisms underlying human lung cancer.
Urokinase plasminogen activator (uPA) is one of the genes shown to be altered by TCDD exposure. This protein is a serine protease that is involved in matrix turnover and growth of tumor cells. Unlike many other genes that are affected by TCDD at the transcriptional level, uPA is upregulated by TCDD due to mRNA stabilization. The mechanism of this regulation is still poorly understood. Shimba et al. (2000) demonstrated that a liver cytoplasmic protein of approximately 50 kDa recognizes the 3′-untranslated region of uPA mRNA in a TCDD-dependent manner. Additional work suggests that the effect is mediated by a protein phosphorylation cascade, not by de novo protein synthesis. These data indicate that TCDD alters uPA expression by a mechanism that apparently does not alter gene expression at the transcriptional level, suggesting that the binding of the AhR-ARNT complex to DREs is not involved in the process.
As discussed in Update 1998, exposure to TCDD might affect cellular redox (oxidation-reduction) status and associated signaling pathways through several mechanisms. Since Update 1998, more evidence has accumulated suggesting that cross-talk occurs between the AhR and hypoxia inducible factor (HIF) signaling pathways. Chan et al. (1999) investigated whether competition for ARNT or other cellular factors in rat hepatoma cells is involved in the cross-talk between the signaling pathways, because some HIFs complex with ARNT to bind with hypoxia responsive enhancers (HREs). In transfection assays, hypoxia inhibited DRE-driven transcription (i.e., induction of CYP1A1). AhR agonists inhibited activation of the HRE and induction of erythropoietin (EPO), a gene that regulates adaptation to low oxygen. Using endogenous loci activation of hypoxia pathways still inhibited CYP1A1 upregulation; however, AhR activation enhanced the induction of EPO. Because of this result, the investigators looked at the
promoter region of EPO and discovered that it contains DREs. Therefore, EPO appears to be an AhR-regulated gene. In addition, they found that the DREs present in the EPO promoter region can compensate for the inhibitory effects that TCDD has on HRE-mediated transcription. These are novel mechanisms for multiple levels of reciprocal cross-talk. Studies by Kim and Sheen (2000) also demonstrated that hypoxia inhibits TCDD-induced CYP1A1 expression in hepatoma cells.
TCDD is known to induce a number of enzyme systems, through the AhR, that are involved in either the generation or the metabolism of oxidative intermediates. An imbalance of these intermediates could have profound effects on the cellular signaling pathways that regulate growth control and differentiation. In mice, treatment with 5 µg TCDD/kg three times daily produced an oxidative stress response characterized by increased hepatic oxidized glutathione levels and increased urinary levels of 8-hydroxydeoxyguanosine, a product of DNA base oxidation and excision repair (Shertzer et al., 1998). The latter persisted up to 8 weeks following treatment. In a recent study by Slezak et al. (2000) oxidative stress was characterized in several tissues following oral subchronic (0.15–150 ng/kg; 5 days/week for 13 week) or acute (0.001–100 µg/kg) exposure to TCDD. Acute treatment with doses of 10 and 100 µg/kg resulted in increases in hepatic superoxide anion production and lipid peroxidation. These were also increased at a subchronic dose of 150 ng/kg/day with a liver TCDD concentration of only 12 ng/kg. Subchronic doses as low as 0.15 ng/kg/day decreased glutathione concentrations in lung and kidney, but this was not observed at higher doses of TCDD.
Hassoun et al. (1998) examined the ability of subchronic exposure to TCDD to produce oxidative stress in the brains of mice. Exposure to 0.45 to 150 ng/kg/ day for 5 days per week for 13 weeks resulted in a dose-dependent increase in the production of superoxide anion and lipid peroxidation in brain tissues. Similarly, Bagchi et al. (2000) noted dose-dependent increases in mouse brain and liver lipid peroxidation, DNA fragmentation, and production of superoxide anion. Notably, these effects of TCDD were exacerbated in mice deficient in the tumor suppressor p53. Machala et al. (1998) found that TCDD induced the activity of microsomal glutathione S-transferase (GST) in trout liver. Using TCDD and a variety of other chemicals, these investigators determined that induction of microsomal GST might be an early biochemical indicator of oxidative stress. Several previous investigations suggest that reactive oxygen species (ROS) might be generated by the increased presence of several of the cytochrome P450 isozymes known to be induced by TCDD and related halogenated aromatic hydrocarbons. More recently Schlezinger et al. (1999) demonstrated, using tissue microsomes from humans and in vitro expressed enzymes of other species, that the slowly metabolized AhR agonist 3,3',4,4'-tetrachlorobiphenyl stimulates the release of ROS by an uncoupling of the CYP1A1 enzyme reaction.
Investigations by Smith et al. (1998) indicate that iron potentiates the hepatic porphyria and toxicity of TCDD in AhR-responsive mouse strains. The adminis-
tration of iron prior to a single dose of 75 µg TCDD/kg increased the formation of hydroxylated and peroxylated uroporphyrin derivatives and the induction of GST compared to animals treated with vehicle or TCDD alone. Iron overload, however, resulted in a reduction of TCDD-induced CYP1A activities. These data suggest that iron may potentiate an oxidative process elicited by TCDD.
Research continues to support the hypothesis that TCDD modulates cellular function in part through an alteration of kinase activities. How these changes relate to specific end points of TCDD toxicity, however, has yet to be determined. Evidence also suggests that phosphorylation-dephosphorylation processes regulate the activity of the AhR-ARNT complex, but where the phosphorylation sites are located or how they regulate AhR function has yet to be determined.
Ashida et al. (2000b) studied the effects of TCDD treatment on nuclear protein kinases and phosphatases that affect the transcription factors c-myc and AP-1 in guinea pig liver. A single TCDD treatment of 1 µg/kg resulted in an increase in nuclear protein tyrosine kinase within 1 day after treatment. The activity had returned to near control levels by day 40. There was a reduction in casein kinase II (CKII) activity, however, at all times studied up to 40 days. A similar effect on CKII activity was also seen in this study following TCDD treatment of liver explants in culture. DNA binding of AP-1 was increased, but a biphasic effect on protein binding to the c-myc response element occurred, with an initial increase in binding followed by a suppression of binding. Additional studies demonstrated that changes in phosphorylation were responsible for TCDD-induced changes in DNA binding of these transcription factors and that CKII played an important role in transducing the actions of TCDD.
The p53 protein is a key mediator of apoptosis after genotoxic stress, and inhibition of apoptosis of preneoplastic cells is thought to be a major mechanism of action of tumor promoters. The activity of p53 is controlled primarily at the posttranslational level by hyperphosphorylation which decreases the ability of p53 to activate transcription (see review by Colman et al., 2000). Exposure of primary rat hepatocytes to TCDD at concentrations up to 10 nM resulted in a concentration-dependent suppression of apoptosis induced by UV light and an increase in p53 phosphorylation (Worner and Schrenk, 1998). This effect of TCDD on p53 was also observed in hepatocyte extracts in the same study. Treatment of the extracts with anti-src antibodies almost completely eliminated the effect of TCDD on p53 phosphorylation, suggesting a key role of c-src in these effects of TCDD. It is of interest that recent investigations suggest that low levels of p53 are associated with the resistance of certain mouse strains to TCDD toxicity and that this resistance is additive to that associated with the Ahdd locus, which encodes a defective AhR protein (A.L.Yang et al., 1999).
The results of several studies suggest a role of c-src kinase in the toxicity
induced by TCDD. Some of these investigations further suggest that c-src might directly interact with the AhR. Enan et al. (1998b) report that treatment of guinea pigs with 1 µg TCDD/kg increases src kinase activity in adipose tissue from males but not females and that the increase is associated with an increased phosphorylation of cytosolic protein that occurs in males but not females. This and other publications by the same group also report that treatment of guinea pigs with the tyrosine kinase inhibitors geldanamycin and quercetin and src deficiency in mice partially protect against some effects of TCDD-induced toxicity, including the excess fatty deposits and mottled appearance of liver (Matsumura et al., 1997; Enan et al., 1998c; Dunlap et al., 1999). Kohle et al. (1999) examined the effects of TCDD on c-src activity and membrane translocation and cell contact inhibition in WB-F344 cells. Exposure to 1 nM TCDD decreased cytosolic c-src and increased c-src in the plasma membrane. EGFR tyrosine phosphorylation was also enhanced by TCDD treatment. Pretreatment of cells with geldanamycin, which also disrupts HSP90 protein complexes, abolished the TCDD-induced translocation of src to the membrane and the TCDD-mediated reduction of cell contact inhibition. The increase in membrane src did not depend on de novo protein synthesis. These data suggest that the membrane translocation of c-src and altered cell contact inhibition are mediated by activation of the cytosolic AhR-HSP90 complex.
Investigations by El-Sabeawy et al. (1998) indicate that exposure of rats to TCDD during pubertal development affects kinase activities and EGFR binding in the testis, as well as sperm motility. Doses of TCDD up to 10 µg/kg were administered to 21-day-old male rats, and tissues were examined up to 90 days of age. Treatment with 10 µg/kg caused testicular atrophy, a decrease in the diameter of the seminiferous tubules, and an absence of spermatogonia cells. Doses as low as 1 µg/kg decreased testicular sperm numbers, affected sperm motility, and affected acrosome reactions. Decreases in EGFR were found in rat testicular tissue from 34 to 90 days. TCDD significantly increased c-src kinase activity but decreased the activities of protein tyrosine kinase, mitogen-activated kinase, and protein kinase C. Pretreatment with genedanamycin blocked the testicular atrophy observed with the 10 µg/kg dose of TCDD. These data suggest the involvement of src kinase and EGFR in TCDD-induced effects on testicular development.
Several lines of evidence indicate that phosphorylation of the AhR and/or ARNT contributes to both the DNA binding and the transactivation functions of the complex. The actual sites of phosphorylation on these proteins and their role in regulating the action of these transcription factors are unknown. Several reports by Long and coworkers (Long et al., 1998, 1999; Long and Perdew, 1999) demonstrate that protein kinase C activity is required for AhR-mediated trans activation function. This activity, however, does not affect AhR or ARNT protein levels, nuclear localization, or AhR-ARNT dimerization. Additional work indicates that the action of protein kinase C is not dependent on the transactivation
domains present in the AhR or ARNT, further suggesting that coactivators recruited by these domains are not affected by protein kinase C activity.
Hormone Receptor Signaling
During the past 2 years, considerable research has continued to focus on the interactions among hormone receptor signaling pathways that might play a role in TCDD toxicity, especially on interactions between the estrogen receptor and the AhR. Several groups have investigated the mechanisms by which these pathways might interact. Proposed mechanisms include altered hormone metabolism, effects on receptor expression, competition for accessory transcription factors, and binding to overlapping response elements in responsive genes.
As discussed in Update 1998, TCDD is a more potent hepatocarcinogen in female rats than in male rats or ovariectomized female rats, suggesting a role of ovarian hormones in the mechanism of TCDD-induced hepatocarcinogenesis. Wyde et al. (2000) studied the effects of cotreatment with TCDD and 17β-estradiol in diethylnitrosamine (DEN) initiated ovariectomized rats. Rats were initiated with DEN and treated with TCDD at 100 ng/kg/day for 20 or 30 weeks with and without 17β-estradiol treatment using constant-release pellets. Hepatotoxicity was observed in the TCDD-treated groups, but no excess hepatotoxicity was associated with 17β-estradiol supplementation in the ovariectomized animals. Notably, liver TCDD concentrations were similar between intact rats and ovariectomized rats supplemented with 17β-estradiol but were higher in ovariectomized rats not supplemented with 17β-estradiol. A study by Petroff et al. (2000) investigated whether estrogens increased the toxic effects of TCDD on growth and ovarian function in the rat. Immature rats were treated with estradiol followed by a single dose of 10 µg TCDD/kg. Follicular development was induced with equine chorionic gonadotropin followed by an ovulator dose of human chorionic gonadotropin. TCDD treatment inhibited ovulation; this inhibition was potentiated by estradiol treatment in hypophysectomized but not intact animals. Only hypophysectomized rats exposed to both TCDD and estradiol showed weight loss. Inhibition of ovulation by TCDD was alleviated by estradiol treatment into the ovarian bursa. This treatment, however, increased the body weight loss elicited by TCDD. These data demonstrate the complex interactions between TCDD, estrogen, and other hormonal systems in intact animals and suggest that the interactions occur systemically and in the ovary. Gao et al. (2000) demonstrated that several AhR agonists blocked ovulation in gonadotropin-primed immature female rats.
Chaffin et al. (2000) investigated the regulation of AhR and ARNT mRNAs in liver and ovarian tissues during the rat estrous cycle. Hepatic AhR mRNA was increased on the morning of proestrus and decreased dramatically by the evening of proestrus. Hepatic ARNT mRNA decreased between diestrus and the morning of proestrus and between the evening of proestrus and the morning of estrus.
Ovarian AhR mRNA did not change between diestrus and proestrus but decreased on the evening of proestrus. Changes in ovarian ARNT mRNA were similar to those seen in the liver. Treatment with estradiol or an estradiol antagonist did not affect AhR mRNA in the liver. This study suggests that changes in the reproductive cycle also regulate the expression of both AhR and ARNT, although estradiol levels may not play a major role in those changes.
Enan et al. (1998d) investigated the effect of TCDD treatment on the ability of estradiol to alter kinase activities in adipose tissues of immature and mature female rats. In mature females, estradiol treatment (15 µg/kg) increased tyrosine kinase (TK) and protein kinase A (PKA) activities and decreased mitogen-activated protein 2 kinase (MAP2K) activity. TCDD treatment at doses of 10–115 µg/kg blocked the stimulatory effect of estradiol on TK and PKA activities. In immature females, estradiol treatment decreased TK and PKA activities, and TCDD exposure potentiated these effects. TCDD treatment also decreased binding of radiolabeled estradiol to estrogen receptors (ERs) in both mature and immature females and blocked the stimulatory effect of estradiol on ER binding activity. Geldanamycin treatment abolished most of the effects of TCDD. These data indicate the ability of TCDD to disrupt estrogen-dependent signal transduction pathways in female rats.
Several other studies have demonstrated the ability of TCDD to disrupt estrogen signaling processes in cultured cells. Smeets et al. (1999) demonstrated that several AhR agonists, including TCDD, suppress estradiol-induced secretion of the yolk protein vitellogenin in carp hepatocytes. This was not correlated with the induction of CYP1A1 activity by TCDD, since induction of this enzyme occurred at lower concentrations of TCDD. Exposure of human endometrial cells to TCDD reduced the ER level by 40 percent and reduced ER-mediated transcription by 50 percent (Ricci et al., 1999a).
Estrogen treatment has also been shown to affect the AhR-mediated signal transduction pathway. Sarkar et al. (2000) demonstrated that estradiol (5 µg/kg) enhances the induction by TCDD (0.3 µg/kg) of CYP1A1 in female rat livers. The increase in nuclear AhR content resulting from TCDD treatment, however, was not altered by estradiol treatment, suggesting that estrogen affects the induction of the CYP1A1 gene after formation of the active AhR-ARNT complex. In contrast, estradiol treatment decreased CYP1A1 expression (constitutive and TCDD induced) in cultured human endometrial cells (Ricci et al., 1999b). The authors suggested that the activated pathways were competing for available transcription factors. Studies by Schuur et al. (1998) demonstrated that thyroid status did not affect the induction of CYP1 A1 activity by TCDD.
Using GST-pulldown assays and immunoprecipitation, Klinge et al. (2000) demonstrated that the AhR directly interacts with ERα, COUP-TF (an orphan nuclear receptor expressed in estrogen target tissues), and ERRα1 (an estrogenrelated receptor) in vitro. Agonist-bound AhR showed a stronger interaction than antagonist-bound AhR. These receptors do not appear to interact with ARNT.
COUP-TF1 was found to bind the DRE in vitro, and overexpression of this protein in MCF-7 breast cancer cells inhibited TCDD-induced reporter gene activity. TCDD treatment inhibited estradiol-activated reporter genes, but COUP-TF did not block the antiestrogenic effect of TCDD. COUP-TF may regulate AhR-dependent responses by direct protein-protein interaction and by DRE binding competition.
Data from Jana et al. (1999a) suggest that ERα acts as a positive modulator in the regulation of AhR-responsive genes. The relationship between ERα activity and TCDD responsiveness was examined using human uterine endometrial carcinoma cells. RL95–2 cells were highly responsive to TCDD, but KLE cells were not, despite the fact that KLE cells express higher levels of the AhR. Nuclear translocation of ERα, however, was shown to be defective in KLE cells. Transient expression of ERα in KLE cells restored the cells’ responsiveness to estradiol and TCDD. Caruso et al. (1999) investigated the role of HSP90 in mediating cross-talk between ER and AhR-regulated pathways. Overexpression of HSP90 in human breast cancer cells inhibited TCDD responsiveness, but had no effect on estrogen responsiveness. Expression of an ER deletion mutant that does not bind DNA or the ligand binding domain of the AhR increased basal and TCDD-inducible CYP1A1 expression. Notably, HSP90 mainly localized to the cytoplasm in ER-positive cell lines, whereas in ER-negative cells, HSP90 was equally distributed between cytosol and nucleus. These data suggest that cellular ER content may regulate AhR responsiveness by a mechanism that involves HSP90.
Tian et al. (1998) demonstrated that TCDD causes tissue-specific downregulation of ER mRNA. Treatment of mice with 5 µg TCDD/kg resulted in decreased ER mRNA in the ovaries, uterus, liver, and lungs. The most pronounced changes occurred in the ovaries and uterus. No changes were observed in the kidney and brain. These data suggest that the ovary and uterus may be the most sensitive organs to the antiestrogenic effects of TCDD.
TCDD treatment (10 nM) of MCF-7 cells blocked estradiol induced c-fos protooncogene mRNA levels (Duan et al., 1999). This inhibitory response was not observed in the presence of an AhR antagonist and in variant Ah-nonresponsive cells, indicating that the AhR was required for the effect of TCDD. Further studies indicated that the activated AhR complex bound to a DRE sequence that overlapped with an estradiol-responsive site rich in guanine and cytosine. The data suggest that TCDD acts by activating the AhR, which then binds to an inhibitory DRE complex, thus blocking ER/Sp1 DNA complex formation and subsequent activation of the c-fos gene. Additional work by Klinge et al. (1999) suggests that the activated AhR-ARNT complex inhibits estrogen action by inhibiting ER binding to imperfect estrogen responsive elements (ERE) sites that are adjacent to or overlap DRE sites.
Jana et al. (1999b) demonstrated an interaction between the AhR and testosterone signal transduction pathways. Normal and testosterone-stimulated growth
of LNCaP prostate cancer cells were inhibited by 10–100 nM TCDD, and testosterone treatment inhibited the induction of CYP1A1 mRNA by TCDD in a concentration-dependent manner. TCDD inhibited testosterone-dependent transcriptional activity and testosterone-regulated prostate-specific antigen expression. Treatment of porcine preovulatory follicles with 10 nM TCDD decreased the number of proliferating cells, reduced testosterone secretion, and increased estradiol secretion (Grochowalski et al., 2000). The exposure of isolated porcine luteal cells to 10 nM TCDD also resulted in decreased progesterone secretion by these cells (Gregoraszczuk et al., 2000).
Exposure to TCDD has been shown to produce hyperkeratosis of the skin in humans and several other species. The characteristics of this response resemble those of vitamin A deficiency. Krig and Rice (2000) reported that TCDD suppressed retinoid-induced transglutaminase mRNA in a human squamous carcinoma cell line.
Research has identified other groups of chemicals that may either interact directly with the AhR or affect AhR function indirectly. When exposure to such chemicals and TCDD occurs, the toxicological consequences of TCDD exposure can be modified. Likewise, exposure to TCDD and AhR ligands may modify the toxicity of other chemicals.
Loeffler and Peterson (1999) investigated the effects of in utero and lactational exposure of rats to mixtures of TCDD and p,p’-dichlorodiphenyldichloroethylene (DDE). Rats were treated with 0.25 µg TCDD/kg on gd 15 and 100 mg/ kg DDE on gd 14–18, or with 0.25 µg TCDD/kg on gd 15 and 100 mg/kg DDE on gd 14–18. Male offspring were examined on pnd 21 through 63. Treatment with TCDD or DDE alone decreased prostate weights in a time-dependent manner. Coexposure appeared to potentiate these effects at pnd 21 but not at later periods. Individual exposures reduced epididymal sperm numbers, but cotreatment did not further reduce sperm numbers. Roman et al. (1998b) demonstrated that TCDD treatment produced a hyperplastic and disorganized pattern of androgen receptor staining, and Loeffler and Peterson (1999) found that DDE treatment alone decreased epithelial androgen receptor staining in ventral prostate. Androgen receptor staining following cotreatment exhibited a pattern that was characteristic of the effects of the individual compounds.
Several studies have examined the effect of exposure to both PCBs and dioxins. Wolfle (1998) studied the interactions between these compounds in an in vitro promotion assay that measures malignant transformation of carcinogeninitiated mouse fibroblasts. PCB 126 (a coplanar AhR agonist) (1 and 0.3 pM) and TCDD (0.15 pM) produced an additive effect in this assay system, but PCB 153 (a diortho-substituted compound) (3 or 30 nM) antagonized the TCDD (1.5 pM) mediated promotion. Smialowicz et al. (1997) examined the effect of cotreat-
ment with 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) and TCDD on the antibody plaque-forming cell (PFC) response to sheep red blood cells in B6C3F1 mice. While exposure to TCDD alone resulted in a dose-related suppression of the PFC response, exposure to PCB153 alone resulted in an enhanced response at 358 mg/kg. Combined exposure did not change the response relative to PCB treatment alone. However, the PFC response was enhanced at combined exposures with low concentrations of TCDD, but suppressed with combined exposures with a higher concentration of TCDD. The data suggested that PCB 153 acts as a functional antagonist rather than an AhR antagonist, but the effect may be concentration dependent. Some of these interactions with PCBs and their implications are discussed further in the section on toxic equivalency factors (TEFs).
Studies by Kim et al. (1999) suggest that compounds present in ginseng extracts may protect against some end points of TCDD-induced toxicity. Treatment of guinea pigs with a single dose of 1 µg TCDD/kg produced decreased body weight gain and decreased testicular weight (there was no effect on testes weight, however, when the data were expressed as percentage of body weight). Light and electron microscopic examination of the testes showed smaller tubules and maturation arrest of spermatogonia, dissolution of germinal epithelium, and disruption of tight junctions between Sertoli cells. Cotreatment with extracts from Panax ginseng protected against the effects of TCDD on testes weight and the morphological alterations.
Humans are exposed to both cigarette smoke and diesel exhaust. Publications by Meek and Finch (1999) and Meek (1998) indicate that both mainstream cigarette smoke and diesel exhaust particles contain chemicals that bind to the AhR and ER and regulate AhR- and ER-responsive genes. Additional work by Dertinger et al. (1998) indicated that cigarette smoke contains chemicals that transform the AhR to an active transcription factor. These investigators also demonstrated that the genotoxic effect of cigarette smoke condensate, as determined by reticulocyte micronuclei formation, was potentiated in mouse hepatoma cells pretreated with 1 nM TCDD. This effect was attenuated in hepatoma cells that contained approximately tenfold lower levels of AhR. Furthermore, while the cigarette smoke condensate increased the incidence of micronucleated reticulocytes in Ahr+/+ mice, no increase was observed in Ahr null-allele mice. The data suggest that AhR-regulated enzyme induction plays an important role in mediating the genotoxicity of chemicals found in cigarette smoke. A study by Chang et al. (1999) examined the differential response of human lung adenocarcinoma cell lines to benzo[a]pyrene and demonstrated that a variation in AhR-mediated CYP1A1 induction contributes to the differential susceptibility of different cell lines to benzo[a]pyrene metabolite-DNA adduct formation. The above data are consistent with recent work by Shimizu et al. (2000) showing that Ahr-positive mice develop tumors in response to benzo-[a]pyrene treatment, but Ahr null-allele animals do not develop tumors. The latter study provides direct evidence that AhR-regulated signaling pathways are involved in carcinogenesis induced by certain chemicals. Together these data are consistent with the proposed hypothesis that exposure to TCDD or other AhR agonists, some
of which are found in tobacco smoke, results in the induction of cytochrome P450 isozymes that are further responsible for the metabolic activation of polycyclic aromatic hydrocarbons in smoke or other pro-carcinogens to genotoxic intermediates. However, the exact quantitative relationships between the level of enzyme induction, metabolic activation of pro-carcinogens, and increased tumor incidence have yet to be determined.
Lethality There is considerable variation in species susceptibility to the lethal effects of TCDD. Sensitivity among strains of rats can vary as much as one thousandfold. The LD50 values between species range from 0.6 µg/kg for guinea pig to more than 5 mg/kg for hamster (U.S. EPA, 1985). Most species, however, commonly develop a wasting disease that follows exposure to acute toxic doses of TCDD, which is characterized by loss of body weight and fatty tissue. The mechanism responsible for this syndrome remains unclear.
A study conducted to assess the toxicity of dietary TCDD in female mink identified “LD50S” of approximately 0.264 and 0.047 µg TCDD/kg body weight per day for 28 and 125 days of exposure, respectively (Hochstein et al., 1998). In this study, mink were fed diets supplemented with 0.001, 0.01, 0.1, 1.0, 10, or 100 parts per billion (ppb) TCDD for up to 125 days. Mortality occurred in the 1, 10, and 100 ppb groups, and these mink displayed the classic symptoms of the wasting syndrome (i.e., extreme loss of body weight, decreased food consumption with an increase in consumption prior to death, and bloody stool). The mink remained content and alert until they became moribund. This phenomenon has been widely accepted as a manifestation of TCDD toxicity.
Dermal Toxicity There have not been any studies the past 2 years demonstrating significant health effects of TCDD on the skin of animals. Although chloracne is a hallmark of TCDD toxicity in humans, the skin of most animals is not as sensitive to the condition.
Cardiovascular Toxicity Very little attention has been given recently to the cardiovascular effects of TCDD in animals. Although prior to Update 1998 there were reports of developmental defects in the cardiovascular system of TCDD-treated animals suggesting that the cells lining the blood vessels can be a target of TCDD toxicity, to date there is little information available regarding the potential for TCDD to act as a cardiovascular toxicant following postnatal exposure (acute or chronic). Therefore, the cardiovascular system does not appear to be a primary target organ of TCDD toxicity in animals.
Renal Toxicity The kidney does not appear to be a primary target organ of TCDD toxicity in animals. Significant lesions are not reported in the kidney, even
at doses of TCDD that induce major effects on other organs. A recent report indicates, however, that halogenated aromatic hydrocarbons can interfere with the mitochondrial function and glutathione homeostasis of renal tissue (Parrish et al., 1998).
Hepatotoxicity As discussed in Update 1998, the liver is a primary target organ of halogenated aromatic hydrocarbons. The severity of damage varies considerably between species. Alterations in the liver are usually associated with the Ah locus. Hepatomegaly occurs at sublethal doses and is a result of hyperplasia and hypertrophy of hepatocytes. Lethal doses of TCDD result in necrosis of hepatocytes. Alterations in liver morphology are accompanied by impaired hepatobiliary function, including increased microsomal monooxygenase activity, liver enzyme leakage, impaired plasma membrane function, porphyria, hyperlipidemia, hyperbilirubinemia, hyperproteinemia, and increased regenerative DNA synthesis. TCDD can also inhibit DNA synthesis of liver cells, decrease receptors in liver cell membranes, and inhibit liver enzymes. It has been recently confirmed that TCDD can also inhibit normal hepatic accumulation of dietary vitamin A (Kelley et al., 1998).
Endocrine Effects TCDD affects the levels of thyroid hormone. Those effects appear to be species dependent and may reflect both the dose and duration of exposure. For example, in one study discussed there, serum total thyroxine (T4) levels were dose dependently decreased, whereas serum total triiodothyronine (T3) levels were unaffected by TCDD when female rats were killed 4 days after dosing. However, at 90 days, postdosing, serum T3 and T4 were both dose dependently elevated (Fan and Rozman, 1995). In other studies, when rats and mice were exposed to multiple doses of TCDD, T3 (in mice) and T4 (in mice and rats) were decreased dose dependently. Follicular hyperplasia and hypertrophy of the thyroid gland occurred in rats gavaged biweekly for 30 weeks with 0.1–125 ng/ kg/day of TCDD. Thyroid-stimulating hormone (TSH) was elevated in these animals, and there was increased excretion of T3 glucuronide (Sewall et al., 1995). These contrasting results confuse direct interpretation of the effects of TCDD on the production and activity of thyroid hormones.
Other endocrine effects of TCDD focus on reproduction, and these effects are discussed below in “Reproductive and Developmental Toxicity.”
Neurotoxicity Although some early studies indicated that TCDD can be neurotoxic (see Update 1998), many acute toxicity studies have been conducted with TCDD without noted signs of neurotoxicity. Intravenous injections of 8 µg/kg, resulting in total brain concentrations of 356 ppb TCDD, were not associated with systemic toxicity or neurotoxicity in rats (Stahl and Rozman, 1990). Rats administered 1,000 µg/kg TCDD intraperitoneally had significant decreases in body weight but no significant neurological impairment (Sirkka et al., 1992). The
nervous system does not appear to be a sensitive target organ for TCDD toxicity, at least in adult animals.
Prenatal exposure of L-E rats to 1 µg TCDD/kg (per os) on gd 15 reduced the nocturnal core temperature in male offspring (Gordon and Miller, 1998). At some ages, however, the diurnal core temperature of TCDD-treated rats was elevated. No effect on behavioral thermoregulation was seen. The ability to develop a fever following administration of a lipopolysaccharide (LPS) endotoxin was also increased in the TCDD group. Although prenatal exposure to TCDD led to dysfunction in temperature control, the authors concluded that the normal behavioral regulation of core temperature suggests that hypothalamic thermoregulatory centers are not permanently altered.
Seo et al. (1999) gavaged pregnant Sprague-Dawley rats with 0 and 1 µg/kg/ day of TCDD on gestational days 10–16. Male and female offspring were assessed beginning on day 80 for three spatial learning and memory tasks (radial arm maze [RAM], Morris water maze [MWM], and spatial discrimination-reversal learning [RL]), as well as a nonspatial learning task (visual RL). Both male and female TCDD-exposed rats showed a deficit in learning on the visual RL task, but male rats exposed during gestation and lactation showed a facilitation of task-specific spatial learning and memory. These data concurred with data from monkeys showing that perinatal exposure to TCDD facilitated certain spatial tasks but impaired visual RL tasks.
Immunotoxicity Of the many organs and systems affected by TCDD, one of the most sensitive is the immune system. Many immunotoxicological studies involving TCDD have been conducted over the past two decades in mice. It has been demonstrated by many investigators that the consequences of TCDD exposure include suppression of the antibody plaque-forming cell (PFC) response to T-cell-dependent antigens, inhibition of T-cell helper function, and inhibition of cytotoxic T-lymphocyte activity. In addition, in general, TCDD suppresses the production of cytokines (IL-1, IL-2, IFN-γ, and TNF); prevents maturation of thymocytes to mature T cells; inhibits B-lymphocyte differentiation; skews thymocyte subsets toward CD4-CD8+ cells; and increases the expression of CYP1A1 in selected immune cells. Most of these effects appear to be mediated by specific binding to the AhR. TCDD does not appear to alter many macrophage activities such as phagocytosis, tumor cytolysis and cytostasis, or antigen presentation, but it may stimulate macrophage-generated inflammatory cytokines and reactive oxygen species (IOM, 1994, 1999).
Since Update 1998, a study has been conducted to determine whether TCDD affects the resistance of mice and rats to parasitic infection with comparison of effects between exposures in the young and aged (Luebke et al., 1999). Mice and rats were gavaged with a single dose of 1, 10, or 30 µg TCDD/kg for 7 days before being infested with Trichinella spiralis (Ts). Eleven days later, young
controls eliminated a greater proportion of the original parasite burden from the intestine than did aged control animals; this TCDD effect was not seen in aged rodents. Rats in the experiment were also evaluated for larval burdens. Increased larval burdens occurred in young rats at 30 µg/kg and in aged rats at 10 and 30 µg/ kg doses. In addition, parasite-specific splenocyte and lymph node cell proliferation was suppressed by TCDD in young mice. The authors concluded that age-related immunosuppression did not exacerbate TCDD-induced suppression of T-cell-mediated expulsion of adult parasites and, in fact, provided some degree of protection. However, a lower dose of TCDD in aged rats suppressed humoral and cellular responses that limited the burden of encysted larvae, suggesting that age increases the sensitivity of humoral immunity to TCDD exposure.
The effects of TCDD on cytotoxic T lymphocytes in the lungs of mice were evaluated by Warren et al. (2000). The number of CD8+ mediastinal lymph node (MLN) cells was reduced by 60 percent in mice exposed to a single dose of TCDD (10 µg/kg) and infected with influenza virus compared to vehicle-treated mice. The cytolytic activity of lavaged cells from lung in both TCDD- and vehicle-treated mice, however, was equivalent, and interferon levels in the lungs of TCDD-treated mice were tenfold higher than those in control mice. The authors indicated that the link between these effects remains unclear.
Since arachidonic acid metabolites are potent immunoregulatory molecules, Lawrence and Kerkvliet (1998) examined the effects of TCDD on the production of AA metabolites. Mice (C57BL/6) exposed to 15 µg/kg TCDD (intrapentoneally) had a 2-fold increase in the release of AA from spleen cell membranes, a 1.4-fold enhancement of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) production in the spleen, and 3-fold higher levels of PGE2 in the peritoneal cavity during the immune response to allogenic p815 tumor cells. Metabolic inhibitors did not affect TCDD-induced suppression of the cytotoxic T-lymphocyte response and antibody formation. TCDD, however, did not alter the message or protein levels of COX-1, COX-2, or IL-1. The investigators, therefore, concluded that AA metabolites most likely do not mediate TCDD immunotoxicity.
It has been suggested that the proliferation and/or differentiation of hematopoietic stem cells is affected by TCDD, contributing to a reduced capacity of bone marrow to generate pro-T lymphocytes (Murante and Gasiewicz, 2000). In addition, data from a recent study suggest that TCDD exerts its effect on those cells entering and/or within the mature B-lymphocyte subpopulation and that effects noted in the earlier stages of B-lymphocyte maturation are a compensatory response to the effect on the mature cells (Thurmond and Gasiewiez, 2000).
TCDD also appears to interfere with normal physiological cell death (apoptosis) and to induce apoptosis in most laboratory mice (Kamath et al., 1998, 1999). However, TCDD failed to induce apoptosis in Fas-deficient and Fas ligand-defective mice at a lower dose than in control wild-type mice. These studies suggest that Fas-Fas ligand interactions may play a role in the TCDD-mediated
induction of apoptosis. However, other investigations suggested that TCDD exposure does not induce apoptosis in T cells (Staples et al., 1998a). These effects may be highly dependent on both dose and specific maturation stage.
TCDD suppresses the response of cytotoxic-T-lymphocytes (CTL) to allogeneic tumor cells in mice; this suppression is accompanied by a decrease in expression of CD86 and suppression of IL-2 and IFN-γ production. In a series of experiments to determine the role of IL-2, IFN-γ, and CD8+ cells in this process, data indicated that TCDD induces an early defect in CTL precursor activation and that the defect is not due to insufficient IL-2 production or deletion of CD8+ cells (Prell et al., 2000). The authors suggest that ligands of the AhR may disrupt CTL precursor activation.
The immune system of laboratory rodents appears to be highly sensitive to in utero exposure to TCDD. Rats (F344) exposed on gd 14 to TCDD (3 µg/kg) had significantly decreased delayed-type hypersensitivity responses up to 4 months (female) and 19 months (male) of age (Gehrs and Smialowicz, 1999). The lowest maternal dose of TCDD that produced DTH suppression in offspring at 14 months of age was 0.1 µg TCDD/kg for males and 0.3 µg TCDD/kg for females. Cell phenotype analysis was performed on thymus and lymph node suspensions, but no correlations were established between altered phenotypes and suppressed DTH responses. The authors concluded that suppression of the DTH response following perinatal TCDD exposure is persistent through late adulthood, occurs at a low dose to the dam (0.1 µg/kg), and is most pronounced in male offspring.
Many investigators have evaluated immune effects following exposure to a single dose of TCDD. Huang and Koller (1999) compared the effects of single and repeated dosing of TCDD on splenic T-cell subpopulations in L-E rats. They demonstrated that repeated dosing of TCDD decreased the total percentage of CD4+ cells and the percentage of the CD4+ cells cycling 9 days, postexposure, while an analogous single dose of TCDD failed to affect the CD4+ cell subpopulation. The authors suggest that the disease pattern of TCDD toxicity could differ between single and equivalent multiple (cumulative) exposures. This is consistent with results indicating that the immunotoxic effects of TCDD are associated with the length and repetitive nature of exposure.
In a study to determine if TCDD alters (suppresses) the activation events that follow exposure to a superantigen, it was concluded that TCDD further stimulated the production of IL-2, as well as increased the percentage of CD4+ and CD8+ cells in the S and G2M phase of lymphocyte cycling in rats primed with a superantigen (Huang and Koller, 1998). In this investigation, IL-6, TNF, IL-2R, IL-1, and T-cell receptor (TCR) expression were basically unaffected by TCDD exposure. Although reduced body weight gain and histopathology confirmed that a single 25 µg/kg oral dose of TCDD caused morbidity but not mortality in L-E rats, the effect on the immune system was one of stimulation or no effect in an activated (TCDD+superantigen) versus nonactivated (TCDD) immune system, respectively.
These studies collectively demonstrate that there are considerable species differences between rats and mice, as well as strain differences, in their immune responses following exposure to TCDD. Nevertheless, TCDD appears to compromise (suppresses) the immune system of laboratory animals and the developmental period appears to be extremely sensitive to the immunosuppressive effects of TCDD in multiple species.
Reproductive and Developmental Toxicity As discussed in Update 1998, low doses of TCDD can affect reproductive development and fertility of progeny. Recently, considerable interest has been focused on “endocrine disrupters,” that is, chemicals in the environment that affect the endocrine system and its target organs, especially the endocrine-associated developmental effects of TCDD. In one study (Faqi et al., 1998), the male offspring of female rats treated with TCDD 2 weeks prior to mating, and throughout mating, pregnancy, and lactation were evaluated for developmental effects. The number of sperm per cauda epididymis was decreased in the TCDD groups at puberty and adulthood. Daily sperm production was permanently decreased, as was the sperm transit rate. TCDD-exposed groups showed an increased number of abnormal sperm at adulthood. In the highest TCDD exposure group, serum testosterone concentration was decreased at adulthood. The authors concluded that sperm parameters were more susceptible than other end points investigated. Nevertheless, all TCDD-exposed males were able to impregnate unexposed female rats that yielded viable fetuses. In addition, mating, pregnancy, and fertility indices were not affected, and the number of implantations, resorption rates, number of viable and dead fetuses, fetal weights, and sex ratios were similar among groups. Female offspring from this study (Faqi and Chahoud, 1998) had a delay in vaginal opening (1 day) and reduced uterine weight. The authors regarded these effects as antiestrogenic. Since “endocrine-related” effects occurred only at the highest dose of TCDD in the female, it was concluded that male offspring are more susceptible to TCDD than female progeny when exposure occurs throughout pregnancy and lactation. Additional studies have shown that when pregnant female rats were administered a single oral dose of TCDD (10 µg/kg) on gd 15, TCDD interfered with vaginal development in female fetuses by impairing regression of the Wolffian ducts, increasing the size of interductal mesenchyme, and preventing fusion of the Mullerian ducts (Dienhart et al., 2000).
When pregnant female hamsters were dosed orally with 2 µg TCDD/kg body weight on gd 15, material viability, body weight, fertility, and F1 litter size did not differ between treated and control groups (Wolf et al., 1999). In the offspring, body weight was permanently reduced by about 30 percent, vaginal opening was delayed, and the vaginal estrous cycle was altered by TCDD, but the regular 4-day estrous cycle in female progeny was not disrupted. In this study, 20 percent of the female offspring did not become pregnant, 38 percent of pregnant F1 females from the TCDD group died near term, and the number of implants and
live births was reduced by TCDD treatment. Effects in the progeny occurred at a dosage nearly four orders of magnitude below the toxic level for adult hamsters.
To evaluate the effects of TCDD on endometriosis, rats and mice were treated on gd 8 with 1 and 3 µg TCDD/kg, respectively (Cummings et al., 1999). Female offspring were reared to adulthood, and endometriosis was surgically induced. All animals were treated with 0, 3, or 10 µg TCDD/kg 3 weeks prior to surgery; at the time of surgery; and 3, 6, and 9 weeks after surgery. When the animals were killed 12 weeks after surgery, TCDD was seen not to affect the surgically induced endometrial lesions in the rats, although effects were seen in rats in an earlier study (Cummings et al., 1996). In mice, the lesions were increased only with a combination of perinatal and adult exposure to TCDD. A similar protocol in rhesus monkeys revealed that TCDD facilitated the survival of endometrial implants but had no effect on circulating gonadal steroid levels or on the menstrual cycle (Yang et al., 2000). Other researchers (Bruner-Tran et al., 1999) suggest that TCDD blocks the ability of progesterone to prevent experimental endometriosis, which correlates with its ability to inhibit progesterone-associated TGF-β2 expression and endometrial matrix metalloproteinase suppression.
In utero and lactational exposure of Holtzman rats to TCDD decreased prostate weight without inhibiting testicular androgen production or decreasing serum androgen concentrations (Roman et al., 1998a). Additional studies (Roman et al., 1998b) showed that the prostatic epithelial budding process was impaired, suggesting that in utero and lactational TCDD exposure interferes with prostate development by decreasing early epithelial growth, delaying cytodifferentiation, and altering epithelial and stromal cell histological arrangement and the spatial distribution of androgen receptor expression.
Pregnant L-E rats were gavaged on gd 15 with 1.0 µg/kg TCDD, and their male offspring were necropsied at intervals up to 120 days postnatally to observe the effects of TCDD on seminal vesicles (Hamm et al., 2000). TCDD treatment significantly decreased body and seminal vesicle weights in male rats 49–120 days postnatally. Epithelial branching and differentiation were decreased in the seminal vesicles of TCDD-treated rats. It was concluded that TCDD decreases seminal vesicle growth by impairing development of the epithelium.
During the past few years, reproductive and developmental TCDD research has focused on exposure of pregnant females. As discussed in previous reports, the reproductive systems of adult male laboratory animals are considered to be relatively insensitive to TCDD because high doses are required to elicit effects.
Carcinogenicity TCDD is considered to be a carcinogen in animals and humans. TCDD is not directly genotoxic and acts as a promoter in multistage models of carcinogens. The carcinogenic activity of TCDD depends on the presence of the AhR, and involves multiple pathways in regulating cell proliferation and differentiation; the multiple site specificity suggests multiple mechanisms of action. Two possible mechanisms of tumor promotion by TCDD may involve
oxygen radicals and interference of gap junction intercellular communication. TCDD is a known hepatocarcinogen in rats and mice (IOM, 1999).
In a two-stage model of hepatocarcinogenesis, TCDD significantly increased the volume fraction and number of altered hepatic foci at the highest dose (10 ng/kg/day) (Teeguarden et al., 1999). Increases in the number of GTPase- and ATPase-deficient altered hepatic foci per cubic centimeter occurred at doses as low as 0.01 ng/kg/day. This is the lowest dose of TCDD to promote tumors to date and indicates that TCDD is an extremely potent promoter of neoplasia in laboratory rats.
When female Sprague-Dawley rats were administered 1.75 µg/kg TCDD biweekly continuously for 60 weeks via oral gavage, an increase (nonsignificant) in liver tumors occurred in these rats compared to corn oil controls (Walker et al., 2000). The incidence of hepatocellular adenomas and carcinomas, however, was significantly decreased in rats treated with 1.75 µg/kg TCDD for 30 weeks followed by no TCDD treatment for an additional 30 weeks, compared to the TCDD continuously treated and vehicle control groups. Although there may be several other explanations, such as differences in total dosage delivered, the authors interpreted these data to suggest the possibility that the promotion of liver tumors by TCDD in female rats is dependent upon continuous exposure to TCDD.
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 toxic equivalency factors, quantitative structure-activity relationship (QSAR) models, in vitro screening methods, and toxicity equivalent concentrations (TECs).
TEF Approach In recent years the TEF methodology for comparing the relative toxicity of dioxin-like chemicals has been used by several government agencies around the world. Although it is considered one of the best approaches for assessing the relative risk of complex mixtures of these contaminants, it is an interim approach and there are several inherent uncertainties with this procedure.
TEFs are determined by inspection of the available congener-specific biological and biochemical data and assignment of an “order-of-magnitude” estimate of relative toxicity when compared to 2,3,7,8-TCDD. TEF values are by no means precise; scientific judgment and expert opinion, based on all the available data, form the basis of these values. The actual scientific data upon which these values are based may vary considerably, often by several orders of magnitude depending on the different biological end points chosen for a particular dioxin-like chemical. Therefore, considerable uncertainty exists about the use of these values, and it is often difficult to quantify that uncertainty. Although the recent World Health Organization TEF values (van den Berg et al., 1998) are most often cited and generally accepted, the TEF values used can differ slightly depending
on the state, country, and particular health organization, as well as the classification schemes accepted by an agency. Nevertheless, most agencies in the United States, including the U.S. Environmental Protection Agency, support the basic approach as a “reasonable estimate” of relative toxicity. Furthermore, numerous countries and several international organizations have adopted this approach although, again, the accepted values may differ.
The basic TEF concept is based on the premise that the toxic and biologic responses of all of these chemicals are mediated through the AhR. Although all of the data to date support this concept, the set of data for each particular chemical considered to be “dioxin-like” is incomplete. One possible limitation of the approach is that it does not consider synergistic or antagonistic interactions among these chemicals. In addition, this approach does not consider possible actions or interactions of these chemicals that are not mediated by the AhR. Indeed, little research has been done in this area.
For a chemical mixture such as PCBs, another limitation of the TEF methodology is that the risk from non-dioxin-like chemicals (i.e., noncoplanar PCBs) is not evaluated.
Furthermore, the actual kinetics and metabolism for each dioxin-like chemical differ considerably. Data are often available only on tissue concentrations at any given time and not necessarily on the original exposure of the organism. Sometimes tissue concentrations are not available. Extrapolation to a meaningful dose may add considerable uncertainty to calculation of the 2,3,7,8-TCDD toxicity equivalent (TEQ) to which an individual may have been exposed.
QSAR Models As discussed in Update 1998, quantitative structure-activity relationship (QSAR) models have been used to estimate the 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. Some QSAR models have shown good utility across multiple classes of halogenated aromatic compounds.
In Vitro Screening Methods As discussed in Update 1998, techniques such as the use of H411E-luc cells have been developed for the detection of AhR agonists in environmental samples. Since Update 1998, other methods have been proposed for detecting TCDD and assaying TEQs. Li et al. (1999) concluded that enzyme immunoassay could be used as a rapid and sensitive screening tool in many circumstances. Bovee et al. (1998) concluded that a chemical-activated luciferase gene expression bioassay is promising for detecting dioxins in milk. Smeets et al. (1999) demonstrat ed that vitellogenin secretion from primary carp hepatocytes can indicate the presence of compounds with estrogenic or antiestrogenic activity, including TCDD.
ISSUES IN EVALUATING THE EVIDENCE
For an animal model to be valid in the study of a human disease, the model must reproduce with some consistency the manifestations of the disease in humans. Whole-animal studies or animal-based experimental systems are used to study herbicide toxicity because they allow for rigid control of chemical 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 AhR, 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. Furthermore, it is possible that TCDD, via the AhR, may modulate signal transduction pathways by mechanisms not involving the interaction of the AhR with DNA. Regardless of the specific mechanism, TCDD and other AhR ligands modulate target cells and presumably exert toxic effects.
Establishing a correlation between the effects of TCDD in experimental systems and in humans, however, is 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), however, there are data suggesting that for certain end points humans may be at least as sensitive as some experimental animals (DeVito et al., 1995). Differences in susceptibility involve a toxicokinetic component, since elimination rates in humans are slower than in rodents (Ahlborg and Hanberg, 1992). Toxicodynamic interactions are also important because the affinity of TCDD for the AhR is species and strain specific (Lorenzen and Okey, 1991) and responses to occupancy of the receptor vary among different cell types and during different developmental stages. The drug-metabolizing enzymes induced in humans are different from those induced in rodents (Neubert, 1992), suggesting that the impact of different genetic backgrounds on AhR function is not yet completely understood. It is generally accepted that genetic susceptibility plays a key role in determining the adverse effects of environmental chemicals.
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 however, the challenge in the assessment of the biologic plausibility of the toxicity of herbicides and TCDD is not restricted to understanding 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 many interactions between the AhR and other proteins. 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.
Although studying AhR biology in transformed human cell lines minimizes 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.
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