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3 Toxicology The purpose of this chapter is to summarize recent experimental data that provide the scientiï¬c basis of assessment of the biologic plausibility of the ef- fects of herbicide exposure as reported in epidemiologic studies. Establishment of biologic plausibility through laboratory studies strengthens the evidence of the effects of herbicide exposure that are believed to occur in humans. Toxic effects are inï¬uenced by dosage (magnitude and frequency of administration); by exposure to other substances, including compounds other than herbicides; by pre-existing health status; and by genetic factors. Those variables are difï¬cult to control in epidemiologic studies of humans exposed to herbicides. Experimental studies using laboratory animals or cultured cells allow observation of effects of herbicide exposure under conditions that control for such variables. The routes and rates of uptake, tissue distribution, metabolism, and elimina- tion of a toxic substance collectively are termed toxicokinetics (also pharmaco- kinetics). Those processes determine the amount and persistence of a particular chemical or metabolite that reaches speciï¬c organs or cells in the body. Under- standing the toxicokinetics of a compound is important for valid reconstruction of exposure in humans and for assessing the risk of effects of a particular toxicant. The principles involved in toxicokinetics are similar among chemicals, although the degree to which different processes may inï¬uence the distribution depends on the structure and other inherent properties of the chemicals. Thus, properties such as the lipophilicity or hydrophobicity of a chemical inï¬uence the pathways by which it is metabolized (structurally transformed) and whether it persists in the body or is excreted. Chemical structure and pathways of metabolism also determine the reactivity and toxic potential of a chemical. Those properties differ among the chemicals of concern in this report and may differ among species as 47
48 VETERANS AND AGENT ORANGE: UPDATE 2006 well. Attempts to extrapolate from experimental studies to human exposure must therefore consider them carefully. Many chemical compounds were used by the US armed forces in Viet- nam. The nature of the substances themselves is discussed in more detail in Chapter 6 of Veterans and Agent Orange: Health Effects of Herbicides Used in Vietnam, hereafter referred to as VAO (IOM, 1994). Four herbicides docu- mented in military records were of particular concern and are examined here: 2,4- dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-amino-3,5,6-trichloropicolinic acid (picloram), and cacodylic acid (dimethyl- arsinic acid, DMA). This chapter also focuses on 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD, or dioxin), a contaminant of 2,4,5-T, because its potential toxicity is of concern; considerably more information is available on TCDD than on the herbicides. Other contaminants present in 2,4-D and 2,4,5-T are of less concern. Except as noted, the laboratory studies of the chemicals of concern were done with pure compounds or formulations; the epidemiologic studies discussed in later chapters often track exposures to mixtures. This chapter begins with a discussion of major conclusions presented in reports by predecessors of the current committee: VAO (IOM, 1994); Veterans and Agent Orange: Update 1996, hereafter referred to as Update 1996 (IOM, 1996); Veterans and Agent Orange: Update 1998, or Update 1998 (IOM, 1999); Veterans and Agent Orange: Update 2000, or Update 2000 (IOM, 2001); Veter- ans and Agent Orange: Update 2002, or Update 2002 (IOM, 2003); and Veterans and Agent Orange: Update 2004, or Update 2004 (IOM, 2005). The rest of the chapter consists mostly of overviews and discussions of the relevant experimen- tal studies that have been published since Update 2004 (IOM, 2005) on 2,4-D, 2,4,5-T, picloram, cacodylic acid, and TCDD. The update for each substance includes a review of the toxicokinetic investigations and a summary of the toxic endpoints and their underlying mechanisms of action. HIGHLIGHTS OF PREVIOUS REPORTS Prior reports have reviewed the results of animal and in vitro studies pub- lished through 2004 that investigated the toxicokinetics, mechanisms of action, and disease outcomes of exposure to the herbicides used in Vietnam and TCDD, the contaminant of Agent Orange. The herbicides have not been studied exten- sively, but in general none of them is considered highly toxic. High concentra- tions usually are required to alter cellular and biochemical processes. In contrast, experimental data reviewed in previous reports led to the conclusion that TCDD elicits a spectrum of toxic effects that vary with exposure level and the age, sex, and species of the animals studied. Carcinogenicity, immunotoxicity, reproduc- tive and developmental toxicity, hepatotoxicity, and neurotoxicity have been observed in several species. The scientiï¬c consensus is that TCDD is not directly genotoxic and that its ability to inï¬uence the carcinogenic process is mediated
TOXICOLOGY 49 by epigenetic events, such as enzyme induction, cell proliferation, apoptosis, and intracellular communication. Most, if not all, of the biochemical and toxic effects of TCDD are mediated by the aryl hydrocarbon receptor (AhR), a cellular protein that functions as a regulator of gene transcription. Studies to understand the role of AhR in normal physiology are important for understanding the risks associated with human exposure to TCDD. UPDATED TOXICITY PROFILE OF 2,4-D The herbicide 2,4-D is a synthetic chemical that mimics speciï¬c plant- growth regulators, the auxins, and interferes with their function. According to previous reports, 2,4-D is considered moderately toxic, eliciting a number of adverse effects in animal studies, including carcinogenesis, immunotoxicity, teratogenesis, endocrine disruption, renal toxicity, and hepatotoxicity. The stud- ies reported in the last 2 years continue to indicate that the toxicity of 2,4-D can involve effects on several processes. Thus, studies reviewed in Update 2006 and previous updates indicate that 2,4-Dâs effects include membrane disruption, uncoupling of oxidative phosphorylation, lipid peroxidation, altered redox status, and chromosomal abnormalities. 2,4-D can be a peroxisome proliferator and can induce cytotoxic effects, including apoptosis. Those effects occur at very high doses, and the mechanisms of 2,4-D action are not understood. 2,4-D does cause changes in gene expression, so newer studies that identify pathway changes by using gene-expression proï¬ling may provide insights into the mechanisms of 2,4-D toxicity. Toxicokinetics 2,4-D is moderately hydrophobic and is rapidly excreted as the unchanged parent compound. The half-life in animals varies from hours to weeks and in humans from less than a day to several days, depending on the route of exposure. After oral administration, most of the dose typically will be excreted within 48 hours. Since the publication of Update 2004, several studies have examined the toxicocokinetics and metabolism of 2,4-D in animals and humans. Their re- sults support the previous conclusions that metabolism of 2,4-D to its primary metabolite, 2,4-dichlorophenol (2,4-DCP), is rapid and that the elimination of 2,4-D and 2,4-DCP is relatively rapid, thus limiting the amount that is taken up into tissues. In a study in rats, Aydin et al. (2005) examined the accumulation of 2,4-D in kidney; the kidney is a target organ in subchronic studies of 2,4-D toxicity in rodents. Male and female rats were given different doses of 2,4-D in drinking water and food for 30 days, and kidney tissue was analyzed for residues of 2,4- D and 2,4-DCP. Both compounds were found in kidney with all dose regimens,
50 VETERANS AND AGENT ORANGE: UPDATE 2006 and the amounts of metabolite exceeded the parent compound at all doses. Also in rodents, Sturtz et al. (2006) examined the distribution of 2,4-D to milk of dams after parturition. The rats were fed diets supplemented with 2,4-D at doses equivalent to 15, 25, 50, and 70 mg/kg per day for 16 days. There was a dose- dependent increase in 2,4-D content in the damsâ serum and milk and in serum collected from the pups. Analysis of the lipid composition of the milk found a dose-dependent reduction in polyunsaturated fatty acids. Two papers documented the effects of sunscreen ingredients on dermal up- take of 2,4-D, which is of concern because sunscreens may be used in agricultural or other occupational settings where exposure to herbicides might occur. The ï¬rst study (Pont et al., 2003) used hairless mouse skin explants as the model. Six com- binations of sunscreen active ingredients were applied and then 2,4-D, and mate- rial that passed the dermal barrier was collected and measured at various times over 24 hours. All six formulations enhanced the penetration of 2,4-D in through the skin. Among the active ingredients, N,N-diethyl-m-toluamide (DEET) was most effective in enhancing penetration; octocrylene was the only ingredient found to antagonize 2,4-D uptake. In a related study using the same mouse skin model, the same group (Brand et al., 2003) examined the effects of the physical blockers titanium oxide (TiO2) and zinc oxide (ZnO), which are UVA absorbers, on the uptake of 2,4-D in the presence of commercially formulated sunscreens or in an alternative carrier, phenyl trimethicone. Five of nine sunscreens tested alone increased the transdermal absorption of 2,4-D. However, ZnO impeded uptake of 2,4-D, and TiO2 had no effect when phenyl trimethicone was the solvent. Thus, inert ingredients can modify the enhancing effect of active sunscreen agents. The study also included washing the skin as a variable; washing between applications resulted in the same penetration effect of a second application, and repeating the application without washing resulted in greater penetration of the second dose. Underlying Mechanisms of Toxic Action Studies of effects of 2,4-D published since Update 2004 are consistent with the earlier conclusion that 2,4-D is not acutely toxic and has only weak carcino- genic potential. Recent animal studies of disease outcomes after 2,4-D exposure and possible mechanisms are discussed below. Genotoxic Effects and Mechanisms Related to Carcinogenicity Carcinogenicity may occur as a result of mutations (genotoxicity) or epigen- etic effects involving promotion of tumor-cell proliferation and tumor growth. Studies reviewed in previous updates indicated that 2,4-D has weak genotoxic po- tential. A recent report supports the suggestion of a weak but positive association between 2,4-D exposure and genotoxic potential. Gonzalez et al. (2005) exam- ined deoxyribonucleic acid (DNA) damage and cytogenetic endpoints in Chinese
TOXICOLOGY 51 hamster ovary (CHO) cells exposed to 2,4-D and to a formulation containing 2,4-D dimethylamine salt (2,4-DMA), a derivative that is used in Argentina. The chemicals were applied to cells in culture at 0, 2, 6, or 10 g/mL of media. DNA strand breaks increased in a dose-dependent manner with a doubling at the high- est dose. Mitotic indexes were decreased only at the higher doses. Comet assay showed damage by both 2,4-D and 2,4-DMA. The mechanism of the effects is not known, but it is speculated that the chemicals stimulate production of reactive oxygen species. Overall, the studies suggest only weak genotoxicity of 2,4-D. In a study of 2,4-D effects on human prostatic-cancer cells, Kim et al. (2005) examined the androgenic action of 2,4-D and of 2,4-DCP as synergists of dihy- drotestosterone (DHT) on activation of the androgen receptor (AR). Two human prostatic-cancer cell lines were exposed to the chemicals at various doses with or without the AR agonist DHT. Among endpoints examined was induction of an AR-dependent reporter gene (luciferase). Neither 2,4-D nor 2,4-DCP alone increased reporter-gene expression, but they both enhanced the agonist-mediated activation of the reporter gene by DHT. Further analysis suggested that the effect might involve facilitation of translocation of the DHT-bound AR to the nucleus. Neurotoxicity Update 2004 cited case reports of acute poisonings of humans exposed to large amounts of 2,4-D formulations, indicating neurologic manifestations of drowsiness, coma, hyperreï¬ectivity, hypertonia, and cerebral edema (Brahmi et al., 2003). No relevant studies involving neurotoxicity in adult humans have been published since Update 2004. However, a variety of studies address neuro- logic systems in animal models, and several studies support effects of 2,4-D on the developing brain in animal models. Studies concerning neurologic effects dur- ing development are detailed in the section on developmental toxicity below. Some studies have suggested that 2,4-D has effects on brain processes (dopamine metabolism and action) and structures (such as the nigrostriatus) im- plicated in movement disorders, such as Parkinsonâs disease. A study by Thiffault et al. (2001) addressed the hypothesis that 2,4-D causes damage to dopaminergic terminals and contributes to nigrostriatal degeneration. Male C57BL/6 mice 7â8 weeks old were given a single subcutaneous injection of 100 or 200 mg/kg 2,4-D methyl ester. After 7 days, dopamine and dopamine metabolites (3,4- dihydroxyphenylacetic acid and homovanillic acid) were measured in the stria- tum. Neither dose produced any change in the concentrations of dopamine. The only statistically signiï¬cant change was a slight (about 15 percent) decrease in 3,4-dihydroxyphenylacetic acid at the highest dose. Challenge with 2,4-D 7 days after a 15-mg/kg dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a known dopaminergic toxicant, did not potentiate the effects of MPTP. The results do not support a link between acute exposure to 2,4-D and nigrostriatal injury in the mouse model.
52 VETERANS AND AGENT ORANGE: UPDATE 2006 Zafeiridou et al. (2006) used nerve preparations from a nonmammalian ver- tebrate model, the frog Rana ridibunda, to address effects of 2,4-D on peripheral nerve function. The study involved isolated sciatic nerves exposed to 2,4-D in a specialized chamber that allowed the action potential to be measured as an indica- tion of proper physiologic functioning of the nerves. The effective concentration 50 percent (EC50) of 2,4-D for neurotoxicity in this preparation was determined to be 3.8 mM. Inasmuch as 2,4-D is a weak acid, pH is thought to affect its toxicity. Changing the pH in the nerve media from a physiologic pH of 7.2 to 3.3 enhanced the toxicity and reduced the EC50 to 0.24 mM. The results suggest an effect of 2,4-D on the peripheral nervous system, but the mechanisms are unknown, and the signiï¬cance of the non-physiologic-pH result is not clear. Reproductive and Developmental Toxicity Previous studies have indicated that 2,4-D is embryotoxic in vivo and that the fetus may be more sensitive than adults to its toxic effects. Since Update 2004, studies continue to indicate that 2,4-D has effects in developing animals, includ- ing multiple effects on the developing central nervous system. Sameshima et al. (2004) examined the effects of 2,4-D on rat embryos maintained in culture to determine whether toxicity involved direct effects on the embryo. Embryos of Wistar rats were isolated on day 9.5 of gestation and were exposed to 2,4-D in culture media at 50, 100, 200, or 500 g/mL. Several indexes of growth (number of somites, crown-rump length, head length, and yolk sac diameter) and structure (such as eye development, ear development, neural tube defects, and forelimb development) were examined. Growth retardation and morphologic defects were progressively more severe at higher doses, and all endpoints were signiï¬cantly affected at the highest dose. The results indicate that 2,4-D can directly affect the embryo in vitro, but the mechanisms for the effects are not apparent. Bortolozzi et al. (2004) examined the effects of 2,4-D on the ontogeny of dopaminergic D2 receptors in rat pups that had been exposed to 2,4-D in utero, during lactation, and after weaning. Eight female Wistar rats were exposed to 2,4-D in the diet at 70 mg/kg per day from gestation day 16 to postpartum day 23, while four control female rats were fed vehicle in the diet. After weaning, half the litters in the 2,4-D group were fed the control diet until day 90, and the others were fed the 2,4-D diet. A speciï¬c binding assay for D2 receptors was used to measure D2 receptor concentrations in striatum, prefrontal cortex, hippocam- pus, and cerebellum at various times up to postnatal day 90. There were modest statistically signiï¬cant increases in D2 receptors in all four regions of brains of pups that had been fed the 2,4-D diet after weaning. However, the effect was not evident in pups that were exposed in utero and then fed a control diet after weaning. The results suggest that the effect induced in utero or during lactation was reversible on cessation of exposure.
TOXICOLOGY 53 A study by the same group (Garcia et al., 2004) examined the effects of 2,4-D on tyrosine hydroxylase activity in midbrain areas (the substantia nigra and the ventral tegmental area) in neonatal rats exposed to 2,4-D only through lactation. After birth of litters, female mice were treated with 2,4-D by intraperitoneal injec- tion at 70 or 100 mg/kg per day for 16 days; control female mice received injec- tions of carrier. Pups were nursed in one of the three groups. On postnatal day 25, cellular expression of tyrosine hydroxylase (TH) and serotonin transporters (5-HTT) was determined with immunohistochemical staining and image analysis. In both brain regions, there were reductions in TH and 5-HTT in substantia nigra of both dose groups. In the ventral tegmental area, there were reductions in both in the high-dose group and of 5-HTT in the low-dose group. A similar study (Garcia et al., 2006) examined concentrations of dopamine -hydroxylase, the enzyme that synthesizes noradrenaline from dopamine, in the locus coeruleus of neonatal rats that had been exposed to 2,4-D by lactation. As in the previous study, 9-day-old primaparous female Wistar rats were exposed by intraperitoneal injection to 2,4-D at 70 or 100 mg/kg per day for 14 days (post- natal days 9â22), and control female rats received carrier. On day 22, brains were examined for dopamine hydroxylase with immunohistochemistry. Pups exposed to either dose of 2,4-D showed a pronounced and signiï¬cant (p 0.001) reduc- tion (of 73â75 percent) in dopamine hydroxylase staining in the locus coeruleus. Taken together the Garcia studies indicate that the neonatal rat brain can be inï¬uenced by lactational exposure to 2,4-D. Chemicals that interact with estrogen receptors may cause reproductive problems or could affect estrogen-dependent cancers, including breast cancer. Lemaire et al. (2006) addressed possible interaction of 2,4-D with human estro- gen receptors ER and ER . Cell lines stably transfected with human ER or ER , with a luciferase reporter construct, were used to assess the interactions of 49 chemicals, including 2,4-D. 2,4-D showed no agonist activity and no antago- nist activity with either ER or ER . Immunotoxicity Previous updates concluded that 2,4-D has at most a weak effect on the im- mune system. Recent publications are consistent with that conclusion. The ability of 2,4-D to cause thymic involution was studied in C57BL/6 mice exposed to 2,4-D and another herbicide, propanil, or to mixtures of the two (de la Rosa et al., 2005). Mice 6â8 weeks old were treated by injection of either propanil or 2,4-D at 50, 100, 150, or 200 mg/kg of body weight or with 1:1 combinations of the stated doses of each chemical. Two days after treatment, thymus weight was measured, and CD4 CD8 thymocyte populations were mea- sured by ï¬ow cytometry. Thymic atrophy was observed only in the highest-dose groups, that is, with the combination of chemicals both at 150 mg/kg and with each chemical alone at 200 mg/kg. A decrease in CD4+ CD8+ and CD4â CD8+
54 VETERANS AND AGENT ORANGE: UPDATE 2006 cells occurred at the next-lower dose. A role for glucocorticoids was assessed by determining whether thymic effects could be reversed by treatment with a gluco- corticoid receptor antagonist (RU486) 2 hours before and 12 hours after dosing. The effects of the herbicide on the thymus appeared at least partly to require the glucocorticoid receptor. A study of 2,4-D effects on antibody responses was carried out with a design similar to that used above by the same group (Salazar et al., 2005). C57BL/6 mice were exposed to 2,4-D at 150 mg/kg, to propanil, or to mixtures of the two by intraperitoneal injection within an hour of receiving an injection of heat-killed Streptococcus pneumoniae. Responses to two speciï¬c antigens were determined: a T-cell-independent type 2 antigen (phosphorylcholine) and a T-cell-dependent antigen (pneumococcal surface protein). At 3, 5, 7, 10, and 14 days after treat- ment, spleen and bone marrow cells were obtained, cell populations were ana- lyzed with ï¬ow cytometry, and antibody-secreting cells and antibody production were determined with immunoassay. The number of phosphorylcholine antigen- speciï¬c bone marrow antibody-secreting B cells was decreased by 2,4-D. How- ever, the number of antibody-secreting B cells in spleen was not changed. The response to pneumococcal surface-protein antigen A was not affected. The results suggest a modest inhibitory effect of 2,4-D on the ability to mount a humoral immune response. Mechanisms Related to Effects on Energy Metabolism or Mitochondrial Function Several reports cited in previous updates suggested that the toxicity of rela- tively high concentrations of 2,4-D might be related, at least in part, to its effect on calcium homeostasis and energy metabolism. Those effects might be mediated by a direct action on mitochondria. It is generally recognized that the toxicity of 2,4-D is not understood at the molecular level. There may be multiple avenues of toxicity, and uncovering the pathways that are affected by 2,4-D could lead to hypotheses about the mechanisms. A study by Argese et al. (2005) examined 15 phenolic and phenoxy herbi- cides, including 2,4-D, for toxic effects on mitochondrial function and the depen- dence of EC50 on structural and physicochemical properties of the chemicals. The assay used submitochondrial particles prepared from beef-heart mitochondria, and effects on NADH generation by the particles in vitro were assessed. An EC50 for the effect was determined as the concentration of herbicide that gave 50 percent inhibition of NADH generation. The EC50 for 2,4-D was 32 M. The investigators concluded that the compounds were acting by a nonspeciï¬c mode of action at the membrane. Effects of 2,4-D on metabolic enzymes of the glycolytic, citric acid, and pentose phosphate pathways involved in generating NADH and NADPH were examined by Yilmaz and Yuksel (2005). Enzyme activities were measured in
TOXICOLOGY 55 liver of offspring of females that had received injections of 2,4-D at 3.38 mg/kg of body weight (0.01 of the LD30), or ethanol or saline. The study was repeated in three generations of mice and the offspring were exposed only in utero and through lactation. Increases in malate dehydrogenase were noted, but the effects were modest and highly variable. The authors also examined chromosomal struc- ture in bone marrow cells; no abnormalities were observed. Mechanism Related to Effects on Thyroid Hormones Effects of 2,4-D on serum concentrations of thyroid hormones, particularly decreases in thyroxine, were noted in previous updates. No new studies related to possible involvement of the thyroid were identiï¬ed. Mechanisms Related to Effects of Cell Stress Responses Several investigations examined the ability of 2,4-D to promote or inhibit oxidative damage to cell membranes. Together, they suggest that at high concen- trations 2,4-D is incorporated into cellular membranes and modiï¬es membrane structure and integrity. A number of prior studies implicated oxidative stress in the mechanisms of 2,4-D toxicity. Oxidative stress was addressed further by Celik et al. (2006) in a study of serum enzymes, antioxidant defenses, and lipid peroxidation in various tissues of rats exposed to 2,4-D. Male (Sprague-Dawley) rats 4 months old (weight, 150â200 g) were given 2,4-D (of unspeciï¬ed source) in drinking water ad li- bitum for 25 days. The doses amounted to an intake of about 1.5 or 3 mg/kg per day. After 25 days, serum, red blood cells, and other tissues were obtained for assay. Serum alanine aminotransferase, lactate dehydrogenase, and creatine phosphokinase were signiï¬cantly increased by both doses of 2,4,D, but aspartate aminotransferase was not affected. The lipid peroxidation end product malondial- dehyde was signiï¬cantly increased in liver, kidney, and heart but not in red cells or brain. Glutathione was signiï¬cantly depleted in kidney and brain at both doses, and other organs variably. Other antioxidant enzymes showed varied responses in the different organs. Together, the data implicate oxidative stress as a factor in the toxicity of 2,4-D. Gene-Expression Proï¬ling The ï¬rst expression-proï¬ling study of 2,4-D effects was that of Bharadwaj et al. (2005), who examined global gene expression in human hepatoma cells (HepG2 cells) exposed to 2,4-D at low concentrations. HepG2 cells (at four to 10 passages) were continuously cultured in a commercial formulation of 2,4-D at concentrations 0.1 nM to 4 mM, a range spanning the full spectrum of toxic and environmental concentrations. RNA isolated from control and treated cells
56 VETERANS AND AGENT ORANGE: UPDATE 2006 was prepared and hybridized to Human 1.7k-Expressed Sequence Tag microar- rays. At least 87 genes showed signiï¬cant changes (two-fold threshold), which were evenly divided between those showing upregulation and those showing downregulation. Changes began to appear at a 2,4-D concentration of 1.0 nM. The affected genes included genes involved in cell-cycle control, stress response, immune function, and DNA repair. Bharadwaj et al. (2005) concluded that âthe cellular response to 2,4-D is complexâ and ostensibly associated with altered expression of many genes. UPDATED TOXICITY PROFILE OF 2,4,5-T Commercial production of 2,4,5-T resulted in the formation of TCDD as a contaminant. This section summarizes the toxicity of 2,4,5-T itself. TCDD toxic- ity is summarized later in this chapter. The herbicide 2,4,5-T is an auxin mimic. It is similar to 2,4-D in its mode of action. VAO and the updates concur that 2,4,5-T is only weakly toxic or carci- nogenic. Updates have indicated that 2,4,5-T has only weak mutagenic potential but that it might alter the proï¬le of enzymes involved in the metabolism of pro- carcinogens. Earlier reports indicated that 2,4,5-T could interfere with the forma- tion of the neurotransmitter acetylcholine, which could be involved in effects on growth and the nervous system. Earlier studies also indicated membrane disrup- tion and possibly oxidative stress as effects of 2,4,5-T. A few studies relevant to mechanisms of toxicity of 2,4,5-T have been published since Update 2004; most of them included assessment of 2,4-D as well as 2,4,5-T, and those studies are discussed in the sections on each of these herbicides. Toxicokinetics 2,4,5-T is moderately hydrophobic and, like 2,4-D, is generally rapidly ex- creted, largely as the unchanged parent compound although some is conjugated to amino acids. The half-life in animals varies from hours to weeks and in humans from less than a day to several days, depending on the route of exposure. No relevant studies on the toxicokinetics of 2,4,5-T in experimental animals or humans after exposure to 2,4,5-T were identiï¬ed in the search for Update 2006. Mechanisms of Toxic Action Neurotoxicity The study by Zafeiridou et al. (2006), described in the section on 2,4-D tox- icity, also addressed the effects of 2,4,5-T on peripheral nerve function. Isolated sciatic nerve preparations from a frog model (Rana ridibunda) were exposed to 2,4,5-T in a specialized chamber that allowed the action potential to be measured
TOXICOLOGY 57 as an indication of nerve vitality. Adding 2,4,5-T to this preparation at various concentrations resulted in a dose-dependent decrease in the time to reduce the amplitude of the action potential relative to saline. An EC50 of 0.9 mM was de- termined for 2,4,5-T. Because 2,4,5-T also is a weak acid, changing the pH in the nerve media from physiologic (7.2) to 3.3 reduced the EC50 to 0.2 mM. The results suggest an effect of 2,4,5-T on the peripheral nervous system, but the mechanisms of the effect are unknown. Cell Stress Responses One report describing cellular effects of 2,4,5-T and its metabolite 2,4,5- trichlorophenol (2,4,5-TCP) on human erythrocytes has been published (Bukowska, 2004a). The study examined the effects of 2,4,5-T and 2,4,5-TCP on indexes of oxidative stress in human red blood cells (RBCs). Exposure of RBCs to the chemicals was in buffered saline, in which RBCs constituted 5 percent of the volume. Superoxide dismutase, catalase, reduced and total glutathione, glutathione reductase, and adenylate energy charge were measured in the RBCs. Modest decreases in superoxide dismutase and catalase were observed in cells ex- posed to 2,4,5-TCP at 250 ppm but not in those exposed to 2,4,5-T. The decrease in catalase activity was pronounced at a dose of 1,000 ppm. (In comparison, at that dose, 2,4-D and its metabolite 2,4-DCP did not affect RBC catalase activity.) Both 2,4,5-T and 2,4,5-TCP elicited modest decreases in the content of reduced glutathione but did not affect total glutathione. The two compounds also caused changes in RBC structure that suggested effects on membrane integrity. Energy Metabolism or Mitochondrial Function Argese et al. (2005) used SMPs prepared from beef heart to assay effects of 2,4,5-T on NADH generation as a measure of toxicity. Effects on mitochondrial respiratory functions were considered in relation to structural and physicochemi- cal properties of 2,4,5-T and other chemicals in the study. The EC50 of 2,4,5-T for inhibition of NADH generation determined with this assay was 21 M. Com- paring effects of a number of chemicals in relation to their chemical properties, the investigators concluded that 2,4,5-T had a nonspeciï¬c mode of action at the membrane. Reproductive and Developmental Toxicity Chemicals that interact with estrogen receptors can impair reproduction. Lemaire et al. (2006) addressed possible interaction of 2,4,5-T with estrogen re- ceptors. Cell lines stably transfected with human ER or ER , with a luciferase reporter construct, were used to assess the interaction of 49 chemicals, including 2,4,5-T, with the receptors. 2,4,5-T did not show any agonist activity with either ER or ER . However, 2,4,5-T showed antiestrogenic activity in the ER cells. A 2,4,5-T concentration of 10 M inhibited the response induced by E2, an ovarian
58 VETERANS AND AGENT ORANGE: UPDATE 2006 steroid hormone, by 20 percent. Whole-cell competition binding assays showed that 2,4,5-T could inhibit the binding of E2 to the ER . UPDATED TOXICITY PROFILE OF CACODYLIC ACID Cacodylic acid, or DMA, and its sodium salt constituted about 30 percent of Agent Blue, one of the mixtures used for defoliation in Vietnam. DMA is a metabolic product of exposure to inorganic arsenic. Methylation of inorganic arsenic generally has been considered a detoxiï¬cation process, producing less acutely toxic methylated speciesâmonomethyl arsonic acid and DMAâand increasing excretion. More recently, however, some of the methylated metabolic intermediates have been thought to be more toxic than the parent compound. The methylation pathway of inorganic arsenic results in the formation of pentavalent DMA (DMAV) and trivalent DMA (DMAIII) (IOM, 2005). The committee con- sidered the relevance of data on inorganic arsenic to DMA. Although inorganic arsenic is a human carcinogen, there is no evidence that direct exposure to DMA produces cancer in humans. DMA also is not demethylated to inorganic arsenic. It has not been established, nor can it be inferred, that the observed effects of exposure to inorganic arsenic are also caused by exposure to DMA. Therefore, the literature on inorganic arsenic is not considered in this report. The reader is referred to Arsenic in Drinking Water (NRC, 1999a) and Arsenic in Drinking Water: 2001 Update (NRC, 2001). Toxicokinetics DMAV appears to be less toxic than DMAIII, perhaps in part because of its rapid excretion (IOM, 2005). Recent studies conï¬rm that DMAV administered intravenously to rats is excreted unchanged and rapidly in the urine with none detected in the bile and less than 4 percent distributing into tissues after 12 hours (Cui et al., 2004; Suzuki et al., 2004). However, about 10 percent binds to hemoglobin in RBCs (Suzuki et al., 2004), and rat hemoglobin exhibits a bind- ing afï¬nity to DMAV 10 times higher than human hemoglobin; this may account for its accumulation in rat blood (Lu et al., 2004). Chronic exposure of normal rat hepatocytes to DMAV resulted in reduced uptake over time and in acquired cytotoxic tolerance (Kojima et al., 2006); the tolerance was mediated by induc- tion of glutathione-S-transferase activity and of multiple-drug-resistant protein expression. Endpoints and Underlying Mechanisms of Toxic Action Neurotoxicity In one report related to the effects of DMA on neuronal ion channels, Kruger et al. (2006) found that DMAIII and DMAV signiï¬cantly attenuated ion currents
TOXICOLOGY 59 through N-methyl-D-aspartate (NMDA) receptor ion channels, whereas only DMAV inhibited ion currents through -amino-3-hydroxy-5-methylisoxazole-4- propionic acid receptors. The data suggest that those methylated forms of arsenic may have neurotoxic potential. Immunotoxicity In a preliminary study, a low concentration of DMAV (10â7 M) increased proliferation of human peripheral blood monocytes after their stimulation with phytohemoagglutinin, whereas only a high concentration (10â4 M) inhibited release of interferon- ; this suggested that immune modulatory effects of DMAV are concentration-speciï¬c (Di Giampaolo et al., 2004). Genotoxicity and Carcinogenicity Since Update 2004, most of the new literature concerning the toxic activ- ity of DMA has addressed genotoxicity, a major mechanism of carcinogenesis. Cancer has been induced in the urinary bladder, kidneys, liver, thyroid glands, and lungs of laboratory animals exposed to high concentrations of DMA (IOM, 2003, 2005). DMA might act through induction of oxidative damage or damage to DNA, and exposure results in necrosis of the urinary bladder epithelium followed by regenerative hyperplasia (IOM, 2005). In a new study, DMAIII was considerably more potent than DMAV in induc- ing DNA damage in CHO cells (Dopp et al., 2004), and this was associated with a 10 percent uptake of DMAIII into the cells compared with 0.03 percent for DMAV. Additional study showed that DMAV is poorly membrane-permeable, but when forced into cells by electroporation it can induce DNA damage (Dopp et al., 2005). Furthermore, DMAV induced protein-DNA adducts in lung ï¬broblast cells (MRC-5) (Mouron et al., 2005) and transformation loci in 3T3 ï¬broblasts after post-treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) (Tsuchiya et al., 2005). However, DMAV was devoid of promotion activity in 3T3 ï¬broblasts when cells were pretreated with either 3-methylcholanthrene or sodium arsenite. DMAV, but not inorganic arsenic species, exhibited genotoxicity in Dro- sophila as assessed by the somatic mutation and recombination test (Rizki et al., 2006). Drosophila lacks the ability to methylate arsenic, so these data suggest that arsenic biomethylation is a key determinant of arsenic genotoxicity. Both DMAIII and DMAV have been shown to induce DNA damage by in- creasing oxidative stress. Chronic exposure of ddY mice to DMAV at 400 ppm in drinking water increased staining for 4-hydroxy-2-nonenal adducts, which are indicative of oxidative stress, and for 8-oxo-2 -deoxyguanosine (8-oxodG), reac- tive oxygen species-induced DNA damage, in Clara cells of the lung (An et al., 2005). Gomez et al. (2005) demonstrated that DMAIII induced a dose-related increase in DNA damage and oxidative stress in Jurkat cells.
60 VETERANS AND AGENT ORANGE: UPDATE 2006 Two separate studies investigated the degree to which oxidative stress may mediate DMAV cytotoxicity. In one study, an antioxidant (N-acetylcysteine, vita- min C, or melatonin) and DMA at 100 ppm DMAV were coadministered to F344 rats for 10 weeks (Wei et al., 2005). N-Acetylcysteine inhibited DMAV-induced proliferation of the urinary bladder epithelium, whereas neither vitamin C nor melatonin had an effect; this suggested that oxidative stress may mediate the cytotoxic process in the urothelium. In a second study, metallothionein wild- type and null mice were exposed to a single oral dose of DMAV at 0, 188, 375, or 750 mg/kg (Jia et al., 2004). DMAV induced a dose-dependent increase in metallothionein in the liver of wild-type mice but metallothionein was undetect- able and uninducible in the null mice. At 24 hours after exposure, DMAV induced dose-dependent DNA adducts, DNA strand breaks, and pulmonary and bladder apoptosis in both genotypes, but the incidence of damage was signiï¬cantly higher in the null mice. Those results suggest that metallothionein may play a protective role against DMAV-induced DNA damage. In a recent study, gene-expression proï¬ling of bladder urothelium after chronic exposure to DMAV in the drinking water showed signiï¬cant increases in genes that regulate apoptosis, the cell cycle, and oxidative stress (Sen et al., 2005). Furthermore, doses that were nontoxic, according to a lack of histologic and ultrastructural changes, could be distinguished from toxic doses on the basis of the expression of a subset of genes involved in control of cell signaling and the stress response, such as thioredoxin, E-cadherin, and heat shock 70 kDa. Since Update 2004, further studies have investigated the carcinogenicity of DMA. Xie et al. (2004) administered DMAV at 1,000 ppm in drinking water to v-Ha-ras transgenic mice for 17 weeks and after 4 weeks of treatment applied TPA to the skin twice a week. The results were an initial 10 percent body-weight loss, a cumulative mortality of 20 percent, hepatic arsenic accumulation, he- patocellular degeneration and foci of inï¬ammation without evidence of liver tumors, and hepatic DNA hypomethylation. Hepatic gene-expression proï¬ling showed that DMAV exposure induced changes consistent with oxidative stress, including induction of heme oxygenase, NAD(P)H:quinone oxidoreductase, and glutathione-S-transferase. Mizoi et al. (2005) found that chronic administration of DMAV at 400 ppm to mice after their initiation with 4-nitroquinolone 1-oxide (4NQO) signiï¬cantly increased the number of lung tumors and the percentage of mice with lung tu- mors. DMAV also signiï¬cantly increased pulmonary 8-oxodG adducts regardless of whether the mice had been treated with 4NQO. Hairless mice treated with DMAIII on the skin after initiation with dimethylbenz[a]anthracene exhibited a signiï¬cant increase in epidermal 8-oxodG adducts and skin tumors. In a 2-year bioassay, F344 rats were exposed to DMAV at 0, 2, 10, 40, or 100 ppm in drinking water, and C57BL/6 mice were exposed at 0, 8, 40, 200, or 500 ppm (Arnold et al., 2006). The rats developed epithelial carcinomas and papil- lomas in the urinary bladder and nonneoplastic changes in the kidney. In contrast, the mice failed to develop any tumors but exhibited glomerular nephropathy,
TOXICOLOGY 61 nephrocalcinosis, and vacuolation of the urinary epithelium. The murine no- observed-effect level (NOEL) based on non-neoplastic changes was 40 ppm in male mice and 8 ppm in female mice; the rat NOEL based on neoplastic and nonneoplastic changes was 10 ppm in both sexes. UPDATED TOXICITY PROFILE OF PICLORAM Picloram (4-amino-3,5,6-trichloropyridine-2-carboxylic acid or 4-amino- 3,5,6-trichloropicolinic acid) was used with 2,4-D in the herbicide formulation Agent White, which was sprayed in Vietnam. Picloram also is commonly used in Australia in a formulation under the trade name Tordon 75DÂ®. Tordon 75D contains several chemicals, including 2,4-D, picloram, a surfactant diethylene- glycolmonoethyl ether, and a silicone defoamer. A number of studies on picloram actually used such mixtures as Tordon or other mixtures of 2,4-D and picloram that are similar to Agent White. The initial VAO committee reviewed studies on the toxicokinetics, carci- nogenicity, genotoxicity, acute toxicity, chronic systemic toxicity, reproductive and developmental toxicity, and immunotoxicity of picloram. Studies in animals showed rapid absorption through the gastrointestinal tract and rapid elimination of picloram in urine as the unaltered parent compound. Previously reviewed carcinogenicity assays reported ï¬nding thyroid or liver tumors in rodents treated with picloram, but the results were largely negative. An Environmental Protection Agency review panel concluded that the one report of a signiï¬cant increase in liver tumors was attributable to hexachlorobenzene contaminant of the picloram test agent. Some cellular abnormalities in liver and inconsistent developmental effects were reviewed in VAO and updates. Update 2002 assessed possible ef- fects of picloram in male Sprague-Dawley rats exposed to commercial-grade Tordon 75D. Effects included atrophy and histologic changes in testes (Oakes et al., 2002). The animals had been exposed to various doses, some very high, over various periods. The authors did not determine which of the chemicals in the Tordon 75D formulation caused the testicular damage but cited other studies that suggested that picloram was not responsible; in the long-term carcinogenesis studies in Fischer 344 rats conducted by Stott et al. (1990), administration of daily doses of picloram up to 200 mg/kg for 2 years resulted in no evidence of testicular atrophy. Oakes and Pollack (1999) also examined effects of Tordon 75D on mitochondrial function and reported that effects probably could be attributed to the surfactant in the formulation rather than to picloram. Update of the Scientiï¬c Literature No relevant studies of picloram have been published since the preparation of Update 2004. Two earlier studies that were not reviewed, however, do merit mention. Nolan et al. (1984) examined the toxicokinetics of picloram in six healthy
62 VETERANS AND AGENT ORANGE: UPDATE 2006 male human volunteers, who were given single oral doses of 0.5 or 5.0 mg/kg and a dermal dose of 2.0 mg/kg. Picloram was rapidly absorbed in the gavage study and rapidly excreted as unchanged compound in the urine. More than 75 percent of the dose was excreted within 6 hours, and the remainder was excreted with an average half-life of 27 hours. On the basis of the quantity of picloram excreted in urine in the skin study, the authors noted that only 0.2 percent of the picloram applied to the skin was absorbed. Thus, because of its rapid excretion, picloram has a low potential to accumulate in humans. The other study reported on effects of picloram on induction of drug- metabolizing enzymes in the liver (Reidy et al., 1987). Male and female Sprague- Dawley rats were treated with a variety of doses up to 200 mg/kg for 7 or 14 days. Modest increases (by a factor of 5â8) in activity of microsomal ethoxyresoruï¬n- O-deethylase activity (which is associated with the cytochrome P450 enzymes that are under control of the AhR) were observed but only at the highest doses of picloram. In contrast, 3-methylcholanthrene, a known inducer of CYP1A, induced the activity by a factor of 150. In general, then, the literature on picloram toxicity continues to be sparse. Studies in humans and animals indicate that picloram is rapidly eliminated as the parent compound. Studies in animals have indicated that picloram is sparingly toxic at very high doses. UPDATED TOXICITY PROFILE OF TCDD Toxicokinetics Unlike the herbicides described earlier in this chapter, which tend to be rapidly excreted from the body and do not accumulate to any substantial degree in tissues, TCDD is poorly metabolized and highly lipid-soluble, so it can ac- cumulate in the body and distribute among tissues. The toxicokinetics of TCDD are therefore important in predicting the amounts that will reach speciï¬c target organs or cells and thereby contribute to toxicity in thems. The more complex models, known as physiologically based pharmacokinetic (PBPK) models, are powerful tools that divide the body into compartments that represent organs and integrate the exposure dose with organ mass, blood ï¬ow, metabolism, and lipid content to predict the movement of toxicants into and out of each organ. The distribution of TCDD and other chlorodibenzo-p-dioxin congeners has been examined extensively in experimental-animal toxicokinetic models over the last 25 years. In animal models, it is possible to control exposure and thus to test the validity of PBPK or other models. In humans, the utility of such models is determined by examining TCDD tissue and blood concentrations in relation to occupational or environmental exposure. The models show that TCDD is distributed to all compartments of the body although the amounts differ from organ to organ, and lipid content has a substantial effect on the net accumulation
TOXICOLOGY 63 of TCDD in different organs and in the body as a whole. Whole-blood or serum concentrations of TCDD can also ï¬uctuate with differences in physiologic states and metabolic processes, which can affect the mobilization of lipids and possibly of compounds stored in them. Moreover, processes in one organ can inï¬uence distribution to others. For example, binding proteins in the liver, such as hepatic cytochrome P450 1A2 (CYP1A2) in liver, can inï¬uence accumulation in other organs. Modeling the toxicokinetics of TCDD has several objectives: to estimate organ distribution on the basis of concentrations measured in surrogate tissue, such as blood; to determine organism concentrations from diet or other external sources of exposure; and to back-extrapolate from current tissue concentrations to those at the time of original exposure. The most recent animal and human studies modeling the toxicokinetics of TCDD are reviewed below. Animal Studies Several additions to the literature since the last report (IOM, 2005) detail the processes that affect distribution of TCDD. TCDD is known to distribute to and accumulate in adipose. Irigaray et al. (2005) compared the kinetics of uptake and release of TCDD in isolated porcine adipocytes with those of the free fatty acid palmitic acid. Their study revealed that under conditions of lipogenesis the uptake kinetics of palmitic acid and TCDD were similar; however, under conditions of lipolysis, palmitic acid was released signiï¬cantly faster than TCDD. Thus, they concluded that the storage rate of TCDD in adipose is greater than the release rate and results in net accumulation at steady state and that increases in serum TCDD could be likely in the presence of radical weight loss. In addition to the distribution to adipose, studies continue to investigate the distribution of TCDD to the liver and the role of hepatic sequestration by CYP1A2 in TCDD pharmacokinetics and toxicity. Iwata et al. (2004) showed a preferential deposition of TCDD-like dioxins and biphenyls into the liver, rather than adipose, of Baikal seals as exposure increased; this showed that concentration-dependent hepatic sequestration of these congeners is common in mammals. The hepatic sequestration is believed to result from direct binding of TCDD to TCDD-induced CYP1A2 (Poland et al., 1989). Dragin et al. (2006) demonstrated that distribution of TCDD is highest in adipose, mammary gland, and serum of pregnant mice lacking CYP1A2 expression compared with wildtype mice that show the highest TCDD concentrations in liver. Furthermore, fetuses from CYP1A2 null dams ex- hibit higher sensitivity to teratogenicity and lethality than fetuses of wildtype dams at the same maternal TCDD dose. Notably, knock-in of human CYP1A1 and 1A2 genes into the CYP1A2 null mouse reduces the TCDD teratogenic sensitivity of the fetuses back to the wildtype levels. Thus, the study revealed that maternal hepatic CYP1A2 sequesters TCDD and protects against fetal teratogenicity, and human CYP1A2 provides the same degree of protection.
64 VETERANS AND AGENT ORANGE: UPDATE 2006 Previous data from experimental animal models show that as TCDD ex- posure increases, its apparent half-life decreases; this suggests dose-dependent elimination (Diliberto et al., 2001; Michalek et al., 2002). In a recent study, Emond et al. (2006) investigated the inï¬uence of cytochrome P450 1A2 induction and adipose tissue mass on the dose-dependent elimination of TCDD by using a rat PBPK model. When an inducible-elimination component was included in the PBPK model on the basis of dose-dependent induction of hepatic CYP1A2, the TCDD half-life of elimination varied signiï¬cantly with dose, with high doses resulting in much faster elimination. In addition, Emond et al. (2006) found that increasing adipose tissue mass slows the elimination of TCDD at low doses, but the relationship between adipose tissue mass and TCDD elimination reaches a maximal level at higher doses as CYP1A2 is induced. Thus, the authors suggest that at low TCDD exposures the diffusion from adipose is the rate-limiting step in TCDD elimination, whereas at higher TCDD exposures metabolic elimination is the rate-limiting step. Human Studies Efforts have continued to identify dietary or other approaches that can en- hance the elimination of dioxins, decreasing their uptake and half-life, and previ- ous studies over the last several years have attempted to enhance elimination of TCDD, for example, with activated charcoal, crude dietary ï¬ber, Olestra, and sea- weed (reviewed in previous updates). TCDD residues are generally eliminated in the feces, which can include ingested matter that is not absorbed, and in residues excreted from the body in bile. In a pilot study with nine subjects, Sakurai et al. (2004) showed that treatment of hyperlipoproteinemia patients with the anion- exchange resin colestimide for 6 months decreases blood TCDD by 20 percent without signiï¬cantly reducing any of the lipid fractions. Clearly, randomized placebo-controlled studies are needed to conï¬rm those results. Since Update 2004, two new human PBPK models have been developed in an effort to incorporate the increasing evidence that TCDD elimination is dose- dependent. The models challenge the current dogma of using a ï¬rst-order elimi- nation model and assuming an 8.7-year half-life for TCDD to back-extrapolate peak human exposures from current serum concentrations. In humans, when initial exposure is very high (over 1,000 ppt), TCDD-inducible elimination results in estimated elimination half-life of 1â3.6 years. Thus, the new human PBPK models incorporate a concentration-dependent TCDD-elimination component, which is assumed to be a function of hepatic CYP1A2 induction in one model (Emond et al., 2005) and of hepatic TCDD concentration in the other (Aylward et al., 2005a,b). Aylward et al. (2005a) modiï¬ed a previously published toxicokinetic model (Carrier et al., 1995a,b) to include a concentration-dependent distribution of TCDD to liver and adipose and tissue-speciï¬c rates of elimination from these
TOXICOLOGY 65 two compartments. To optimize the model, serial measurements of serum TCDD concentrations from 39 people were used with initial serum lipid TCDD con- centrations of 130â144,000 ppt, including 36 adults exposed during the Seveso accident and three poisoned in Vienna, Austria. The model predicts that the ap- parent half-life of TCDD is less than 3 years when serum TCDD concentrations are greater than 10,000 ppt and over 10 years when serum TCDD concentrations are less than 50 ppt. The model results also indicate that men eliminate TCDD faster than women and young people faster than older people. Application of the model to serum sampling data from the National Institute for Occupational Safety and Health (NIOSH) cohort indicates that previous esti- mates of peak serum TCDD concentrations, based on ï¬rst-order elimination, have been underestimated and the underestimation may be by as much as a factor of 10 for the most highly exposed subcohorts. Aylward and colleagues (2005b) then compared a concentration- and age-dependent elimination model (CADM) with a ï¬rst-order elimination model with an 8.7-year half-life to predict the cumulative serum TCDD concentration. The CADM resulted in a signiï¬cantly better ï¬t to the measured serum lipid data than the ï¬rst-order elimination model. Furthermore, use of the CADM to back-extrapolate cumulative serum TCDD exposure predicts signiï¬cantly higher cumulative-exposure estimates than the ï¬rst-order elimina- tion model. Thus, Aylward et al. (2005b) conclude that the underestimation of occupational exposures by use of a ï¬rst-order elimination model may result in an overestimation of TCDDâs carcinogenic potency. Cheng et al. (2006) applied the same CADM to estimate the cancer risk as- sociated with occupational TCDD exposure in the NIOSH cohort. They found that the model predicted cumulative serum TCDD concentrations 4â5 times higher than those obtained with the ï¬rst-order elimination model and an 8.7-year ï¬xed-half-life model. Further use of the PBPK model failed to ï¬nd a signiï¬cant relation between untransformed TCDD exposure and cancer response using any lag period, but did identify a positive association between logarithmically trans- formed TCDD exposure and cancer mortality when a lag period of 1â15 years was applied and when the individuals with the most extreme exposures (i.e., highest 5 percent) were excluded from the analysis. Several explanations could account for the strengthened association between cancer risk and TCDD exposure for a sustained period, including the experimental evidence that TCDD acts as a potent tumor promoter, which requires a sustained period to accelerate tumor growth. Using a similar approach, Emond et al. (2005) extrapolated a previously published rodent pharmacokinetic model of TCDD (Emond et al., 2004), which incorporates dose-dependent elimination as a function of hepatic CYP1A2 induc- tion, to human exposures. The model was optimized by using serial serum TCDD concentrations from 20 Ranch Hand veterans, data from a single human volunteer exposed to a high dose of TCDD (Poiger and Schlatter, 1986), and data from two women poisoned with TCDD (Geusau et al., 2002). The model shows good cor-
66 VETERANS AND AGENT ORANGE: UPDATE 2006 relations with measured serum TCDD concentrations for both the Ranch Hand cohort and the two highly exposed women. Application of the model to serum sampling data from another group of Ranch Hand veterans to back-extrapolate peak serum TCDD concentrations at the time of military discharge indicates that previous estimates, based on ï¬rst-order elimination, are much too low and may result in exposure misclassiï¬cation of people in epidemiology studies. It should be noted that there was substantial inter-individual variability in the TCDD kinet- ics of the data sets used to estimate the parameters in the PBPK models developed by Aylward et al. (2005) and Emond et al. (2005). This inter-individual variability will add to the uncertainty in quantitative dose-response assessment for cohorts relying on back-extrapolated exposures. In summary, the new concentration-dependent PBPK models, which predict that TCDD elimination is considerably faster after high exposure, suggest that (1) previous back-extrapolation of peak serum TCDD concentrations may be signiï¬- cantly underestimated and result in a potential for exposure misclassiï¬cations and (2) peak human exposures may be more similar to doses used in animal toxicity studies than previously estimated. Metabolism and Half-Life Studies It is generally agreed that the toxicity of TCDD is related in part to its per- sistence in the body; however, a wide range of elimination half-life estimates in humans have been reported (Table 3-1). Recent studies suggest that TCDD half- life in humans is concentration-dependentâthat rapid elimination follows hepatic sequestration at high doses and much slower elimination follows redistribution of TCDD to adipose at lower body burdens. Thus, the half-life of TCDD in humans varies with body-mass index (BMI), age, sex, and TCDD concentration (reviewed in IOM, 2005). The relevant papers were reviewed in Update 2004 and are discussed above in the context of new PBPK models, and they will not be reviewed again here. Rather, papers that have been published since Update 2004 will be discussed. One and 16 months after occupational exposure of incinerator workers in Japan to TCDD-like polychlorinated dibenzodioxins and polychlorinated diben- zofurans, Kumagai and Koda (2005) measured blood lipid concentrations. Blood samples were also collected from unexposed control subjects. The mean TCDD- equivalent concentration was 49.1 and 29.4 ppt on a lipid basis 1 and 16 months, respectively, after exposure, whereas control subjects averaged 18 ppt. With a one-compartment model, the half-life estimates of those TCDD-like chemicals were 1.1â2.3 years. Leung et al. (2006) measured TCDD intake and blood lipid concentrations in two infants exposed via breast milk and then estimated their TCDD-elimination half-life. Blood lipid concentrations of TCDD increased by factors of 1.7 and 1.9 in the two infants from birth to about 1 year of age. The average elimination
TOXICOLOGY 67 TABLE 3-1 Estimates of TCDD Half-Life in Humans and Animals Conï¬dence Reference Half-Lifea Interval Comment Human Studies Leung et al., 2006 0.4 years Breast-fed infants, 0â1 year PE Aylward et al., 2005a 3 years Calculated for exposures 10,000 pg/g serum lipid 10 years Calculated for exposures 50 pg/g serum lipid Kumagai and Koda, 2005 1.1â2.3 years Adult men, incinerator workers, 0â1.3 years PE Geusau et al., 2002 1.5 yearsb Adult women, severe exposure 0â3 years PE 2.9 yearsb Adult women, severe exposure 0â3 years PE Michalek et al., 2002 0.34 yearsb Adult men, Seveso cohort, 0â3 months PE 6.9 years Adult men, Seveso cohort, 3â16 years PE 9.8 years Adult women, Seveso cohort, 3â16 years PE 7.5 years Adult men, Ranch Hands 9â33 years PE Flesch-Janys et al., 1996 7.2 years Adult men, Boehringer cohort Needham et al., 1994 7.8 years 7.2â9.7 years Adults, Seveso cohort Pirkle et al., 1989 7.1 years 5.8â9.6 years Adult men, Ranch Hands 9â23 years PE Animal Studies Hurst et al., 1998 8 days Rats, Long-Evans, excretion from liver Viluksela et al., 1996 20.2 days Rats, Long-Evans TurkuAB strain 28.9 daysd Rats, Long-Evans Charles River strain DeVito and Birnbaum, 1995 15 days Mice, female B6C3F1 Weber et al., 1993 16.3 3.0 days Rats, male Sprague-Dawley Neubert et al., 1990 73.7 days 60.9â93.8 days Monkeys, Marmoset, single injection Pohjanvirta et al., 1990 21.9 days Rats, male Han/Wistar resistant strain Koshakji et al., 1984 20 days Mice, male ICR/Ha Swiss Gasiewicz et al., 1983 11.0 daysc Mice, C5BL/6J 24.4 daysc Mice, DBA/2J 12.6 daysc Mice, B6D2F1/J ABBREVIATION: PE, postexposure. a Half-lives of TCDD in humans based on measurement of TCDD in serum samples. b Shorter half-lives measured in humans during ï¬rst months after exposure or in severely contaminated persons consistent with nonlinear elimination predicted by PBPK modeling (e.g., by Carrier et al., 1995b). Greater half-life in females attributed to greater body-mass index. c Total cumulative excretion of 3H-TCDD-derived radioactivity. d Attributed to differences in dilution due to different growth rates.
68 VETERANS AND AGENT ORANGE: UPDATE 2006 half-life estimate of TCDD from the infants was 0.4 year, considerably lower than the roughly 7.6 years in adult humans. Summary Studies that model the disposition and effects of TCDD in rodents continue to be reï¬ned and to support the development and use of PBPK models to estimate congener-speciï¬c concentrations in human tissues. It will be important to con- tinue to reï¬ne PBPK models for evaluating tissue distribution in humans. The information on TCDD toxicokinetics is expanding, and new studies are beginning to challege the previous paradigm of a one-compartment, ï¬rst- order elimination model to back-extrapolate to earlier exposure estimates. The data show that BMI and body fat content are important determinants of TCDD half-life, particularly after low exposure. However, persons who accumulate high concentrations of TCDD show an initial phase of elimination that is rapid with half-lives that are much shorter than average. The mechanism underlying the rapid phase of elimination is not known but appears to be related to hepatic CYP1A2 induction that sequesters TCDD in liver and prevents it from distribut- ing; prevention of distribution results in rapid elimination from the liver into the bile. Thus, biphasic elimination continues to confound back-extrapolation to ini- tial exposure in persons who might have experienced high exposures years before blood or other tissue samples were obtained for analysis. Estimates from biphasic elimination models suggest that signiï¬cant individual variation in elimination can occur and that the use of the ï¬rst-order models may lead to misclassiï¬cation of people into the wrong exposure groups on the basis of back-extrapolation to earlier exposure concentrations. The implications for exposure misclassiï¬cation are discussed in more detail in Chapter 5. Evidence presented in the new studies suggests that a multicompartment model is appropropriate. However, it remains unclear which type of model should be used for dose reconstruction. As the biphasic models are validated and opti- mized further, continued assessment of the potential for misclassiï¬cation with the ï¬rst-order elimination models is highly warranted for epidemiologic studies that rely on back-extrapolated estimates of initial exposure concentrations. Efforts to enhance the rate of absorption of dietary TCDD and to enhance elimination by inclusion of ï¬ber or other dietary supplements continue to show promise. Underlying Mechanisms of Toxic Action Studies published since Update 2004 are consistent with the hypothesis that TCDD produces its biologic and toxic effects by binding to a gene regulatory pro- tein, the AhR, which can modulate gene expression through several mechanisms that will be discussed in greater detail in the following sections. The hypothesis
TOXICOLOGY 69 that TCDD toxicity requires the AhR is supported by numerous studies that have evaluated structureâactivity relationships of various compounds that bind to the AhR, the genetics of mutant genes that express the AhR, AhR-deï¬cient mice, and the molecular events that contribute to and regulate AhR expression and its activity. As our understanding of the mechanisms involved in TCDD-induced toxic- ity and carcinogenicity unfolds, it is clear that our attention must also include an appreciation of the physiologic role of the AhR. The AhR signaling pathway may be viewed as regulating multiple physiologic processes that may not be mutu- ally exclusive, including the adaptive pathway that mediates the metabolism of xenobiotics, such as the polycyclic aromatic hydrocarbons; a toxic pathway that mediates the deleterious effects of xenobiotic exposure; and pathways that regu- late developmental and adult cardiovascular homeostasis and adult fertility. The ï¬nding that many AhR-regulated genes are modulated in a species-, cell-, and developmental-stage-speciï¬c pattern suggests that the molecular and cellular pathways that lead to a particular toxic event are complex. Many of the data are consistent with the notion that the cellular processes most sensitive to TCDD-induced modulation as mediated by the AhR are those involving growth, maturation, and differentiation. The ï¬ndings in animals indicate that the repro- ductive, developmental, and oncogenic endpoints are sensitive to TCDD. The data support the biologic plausibility of similar endpoints in exposed humans. Many of the responses, however, are tissue- and species-speciï¬c. Hence, the ap- pearance of some toxic endpoint in one or even several animal species exposed to TCDD does not necessarily indicate that the same endpoint will occur in exposed humans, or vice versa. The mechanisms responsible for the differences are not known exactly but will be considered in greater detail in the sections below. The conclusions indicated above are similar to those in Update 2004. Since those updates, many cellular and molecular interactions of the AhR have been reported. However, in many cases, it is not clear how they might be related to a particular toxic endpoint. Therefore, although the text below cites related work published since Update 2004 that was identiï¬ed by the committee, closer atten- tion is given only to studies that add substantial information, particularly as it might be relevant to the exposure of Vietnam veterans. As discussed in Update 2004, it is important to consider exposure and species sensitivity in discussing animal data and their relevance to humans. Likewise, it is important to note that many of the effects that are reported in animals are seen at dose levels of greater than 1 to 10 Î¼g/kg, far higher than currently measured and most back-extrapolated estimates of veteransâ exposure levels. TCDD-Mediated Alterations of Gene Expression Much of our current understanding of the mechanism of TCDD action is based on analysis of the induction of particular genes and analysis of altered
70 VETERANS AND AGENT ORANGE: UPDATE 2006 intracellular signaling pathways. The genes modulated by TCDD exposure are involved in numerous biologic processes, as described in Update 2004. As shown in Tables 3-2 and 3-3, a number of recently reported genomic and proteomic stud- ies lend insight into the gene pathways that are altered by TCDD in many cell types and tissues. Examples of pathways or processes (with some speciï¬c genes involved indicated in square brackets) that are altered by TCDD exposure are: metabolism of xenobiotics, carbohydrates, glucose, fatty acids, and cholesterol and transport of small molecules (Yueh et al., 2005); cell-cycle control [cyclin E1, cyclin G2]; oncogenesis and proliferation [myc] (Yang et al., 2005); EGFR ligands (Martinez et al., 2004); apoptosis or programmed cell death [Bax]; matrix remodeling [matrix metalloproteinase-1 and -9, plasminogen-activator inhibitor- 2]; inï¬ammation [interleukin 1 , TNF ]; angiogenesis [VEGF, endothelin]; and altered immune function [TNF -induced protein 2). It is presumably through alterations of those basic processes that TCDD exerts its carcinogenic and toxic effects. Like many ligand-activated transcription factors, the AhR alters gene ex- pression via mechanisms that can be classiï¬ed as genomic mechanisms that require direct binding of the AhR/ARNT heterodimer to DNA (âgenomic/direct DNA bindingâ), genomic mechanisms that do not require direct DNA bind- ing (âgenomic/non-DNA bindingâ), or nongenomic mechanisms that occur at the post-transcriptional level and include modiï¬cation of translation and signal transduction. A large body of literature indicates that the binding of TCDD to the AhR, the dimerization of the AhR with a nuclear protein (AhR nuclear-transport protein, or ARNT), and the interaction of that complex with speciï¬c DNA se- quences (often called Ah-responsive elements, or AhREs; dioxin-responsive ele- ments, or DREs; or xenobiotic-responsive elements, or XREs) present in the 5 -promoter regions of particular genes lead to the inappropriate modulation of gene expression. Genes that are directly regulated by binding of the AhR/ARNT heterodimer to DREs (the âgenomic/direct DNA binding mechanismâ) include CYP1A1, CYP1A2, and CYP1B1, which are upregulated after TCDD exposure, as described in Update 2004. The recent ï¬nding that the AhR/ARNT heterodimer upregulates NF-E2 p45-related factor (NRF2; Miao et al., 2005), which is an im- portant regulator of phase II metabolizing enzymes, presents an additional means by which TCDD can alter xenobiotic metabolism via the âgenomic/direct DNA binding mechanism.â Also included in the mechanism are genes that are down- regulated by TCDD, such as pS2 (Gillesby et al., 1997). Given that the mecha- nism requires the appropriate recruitment of coactivators (Hankinson, 2005) and possibly corepressors, the relative cellular concentration of the coregulators may strongly inï¬uence TCDDâs actions on gene expression and may be an underlying contributor to at least some of TCDDâs tissue- and cell-speciï¬c effects. In the âgenomic/non-DNA-bindingâ mechanism of TCDDâs actions, gene transcription would be altered in a manner that does not require direct binding of the AhR/ARNT heterodimer to DNA. This mechanism includes the TCDD modu-
TABLE 3-2 Overview of TCDD-Induced Changes in Gene Expression as Identiï¬ed by Microarray Analysis Biological Model Conditions Outcomes Major Pathways Affected Example References Three-day-old 0.5 or 5 nM TCDD-induced Metabolic processes Handley- zebraï¬sh TCDD, expression pericardial Xenobiotic metabolism CYP1A1 Goldstone in heart analyzed edema/circulatory Fatty acid metabolism Acyl-coenzyme A dehydrogenase et al., 2005 after 24 hr impairment Steroid synthesis Sarcomeric components Myosin heavy chain Mitrochondrial energy transfer NADH dehydrogenase Ribosomal machinery Mitochondrial 12S rRNA C57BL/6N 1.5, 3, and 6 g/kg TCDD- Metabolic processes Thackaberry pregnant mice, TCDD, expression induced cardiac Xenobiotic metabolism CYP1A1 et al., 2005a GD 12.5 in hearts at GD tetratogenicity, Carbohydrate metabolism Fbp1 17.5 impaired Organic transporters Abcc3 cardioproliferation Cell cycle (G1/S) Cyclin E1 Extracellular matrix MMP13 C57BL/6 20 g/kg TCDD, TCDD-induced Metabolic processes Fujita et al., pregnant mice, expression in brain decrease in Xenobiotic metabolism CYP1A1 2006 ED 7 at ED 12 neocortical Organogenesis Mab21L2 neurogenesis (Mitsuhashi et al., 2003) 71 continued
TABLE 3-2 Continued 72 Biological Model Conditions Outcomes Major Pathways Affected Example References 6â8 wk female 150 and 1500 Gene expression Metabolic processes Puga et al., C57BL/6J ng/kg TCDD, consistently altered Xenobiotic metabolism CYP1B1 2004 mice expression in aorta in both systems Glycolysis Glyoxalase after 40 weeks by TCDD was Insulin signaling Igfbp2 OR emphasized Cell cycle Cyclin G2 Signal transduction/mitogenesis Slp65 Vascular 5 nM TCDD, Membrane channels Scn5a smooth muscle expression after Growth arrest/DNA damage GADD45g cells 8 hr Transcription factors Jmy Immune function Ly4911 Platelet function Clec2 10-wk female 1 and 10 g/kg Metabolic processes Watanabe C57BL/6J TCDD / E2, Xenobiotic CYP1A1 et al., 2005 ovariectomized expression in Fatty acid metabolism Fabp1 uterus after 6 hr Cell structure Connexin 26 Proliferation PCNA Immune function Immunoglobulin heavy chains 28 PND female 30 g/kg TCDD No TCDD-induced TCDD Only Boverhof C57BL/6 / E2 (100 effect on E2 Metabolic processes et al., 2006 mice, ovari- g/kg), expression induced uterine Xenobiotic metabolism CYP1A1 ectomized in uterus after wet weight, Protein/small molecular transport Kpna6 2â72 hr luminal epithelial Amino acid metabolism Asns cell height or Fatty acid transport/metabolism Lrp2 stromal thickness
EE TCDD Metabolic processes Amino acid metabolism Asns Small molecule transport Slc 25a5 Proliferation PCNA Signal transduction Inpp5a Protease function Serpinh1 Microtubule structure Dctn2 Transcription factors Rcor3 Rat ovarian 100 pM TCDD, FSH induced Metabolic processes Miyamato, granuloa cells expression after differentiation Xenobiotic metabolism CYP1B1 2004 24 hr Cell cycle Cyclin G Cell structure proteins -actin Cell adhesion Filamin A Ovarian differentiation LH receptor Steroidogenesis P450 scc C3H 10 T1/2 10 nM TCDD IDMB-induced IDMB-induced change inhibited Hanlon cells (mouse treatment during differentiation by TCDD et al., 2005 embryonic adipogenesis, into adipocytes, Metabolic processes ï¬broblasts) expression after cooperative Oxidation-linked Metabolism Cytosolic epoxide hydrolase 24 hr enhancement with Triglyceride synthesis ADRP the addition of Cholesterol regulation Phospholipid transferase protein EGF Cell structure Extracellular matrix Procollagen VI Proteoglycans Osteoglycin Protease function Plasminogen activator inhibitor 1 Secreted proteins Adiponectin Transcription factors C/EBP Plasma membrane receptors Anion carrier protein 4 Signal transduction ERK3 Cytokines Clustein 73 continued
TABLE 3-2 Continued 74 Biological Model Conditions Outcomes Major Pathways Affected Example References C3H 10 T1/2 10 nM TCDD TCDD-regulated genes under Trasande cells (mouse treatment 48 hr four conditions (quiescent, EGF, et al., 2006 embryonic prior to 24 hr IDMB, EGF IDMB) induced ï¬broblasts) treatment with changes in genes not altered by EGF and IDMB IDMB alone Metabolic processes Xenobiotic metabolism CYP1B1 Glucose-linked energy metabolism Phosphoglycerate kinase 1 Iron metabolism Coproporphyrinogen oxidase Cytokines Interleukin receptor type 1 Oxidative stress response Superoxidase dismutase 3 Growth factor signaling Glypican 1 HPL1A 0.1â10 nM TCDD, Metabolic processes Toyoshiba lung airway expression after Xenobiotic metabolism CYP1A1 et al., 2004 epithelial cells 24 hr Fatty acid -oxidation Human peroxisomal acyl-CoA oxidase Retinoic acid transport Cellular retinoic acid binding protein 1 Differentiation Retinoic acid receptor
30 PND female 30 g/kg TCDD, liver weight at Metabolic processes Boverhof C57BL/6 mice, expression in liver 24, 72, and 168 hr, Xenobiotic metabolism CYP1A1 et al., 2005 ovariectomized after 2â168 hr apoptosis, immune Fatty acid uptake/Metabolism Apolipoprotein A-1 cell accumulation Glucogenogenesis Gpd2 or at 168 hr Oncogenesis Myc Apoptosis Hip1 0.001â300 g/kg liver weight at Immune function Cd44 TCDD, expression 100 and 300 g/kg, in liver after 24 hr mild to moderate cytoplasmic vacuolization (0.001â.1 g/kg) (lipid accumulation) alanine aminotransferase at 24 and 168 hr serum cholesterol (72 and 168 hr) FFA at 24, 72, and 168 hr triglycerides at 24, 72, and 168 hr 75 continued
TABLE 3-2 Continued 76 Biological Model Conditions Outcomes Major Pathways Affected Example References 250â300 g 0.4 or 40 g/kg body weight (40 Metabolic processes Fletcher male Sprague- TCDD, expression g/kg, 7 day) Xenobiotic metabolism CYP1A1 et al., 2005a Dawley in liver after 6 hr, serum Lipid metabolism Acaa1 outbred CD 24 hr, and 7 days cholesterol (24 hr Carbohydrate metabolism Mel rats and 7 day) Insulin signaling Igfbp1 serum triglycerides Nitrogen metabolism Glu (40 g/kg, 24 hr 40 g/kg, 7 day) Retinoid metabolism CES3 serum glucose Steroid metabolism Srd5a1 (40 g/kg, 7 day) Protein/small molecule transport Slc17a1 total protein, Cell cycle Ccnd1 globulin (40 g/kg, Cell signaling Tgfb1i4 7 day) Immune response Fkb4 hemoglobin (40 Cell structure MPL3 g/kg, 6hr, 24 hr, and 7 day) alanine aminotransferase (0.4 and 40 g/kg, 7 day) 24 hr and 7 d, evidence of centrilobular hypertrophy (40 g/kg)
Female Harlan 1â100 ng/kg/day Liver hypertrophy, Metabolic processes Vezina SD rats TCDD, expression multinucleated Xenobiotic metabolism CYP1A1 et al., 2004 in liver after 13 hepatocytes and Oxidative stress Cytochrome c oxidase subunit VIII-H weeks diffuse fatty Cell adhesion C-Cam 4 change in 100 ng Protease function Serpin 7A dose (NTP, 2004a) Immune response Ig non-productively rearranged lamda-chain Cell structure CAP2 Signal transduction iNO synthatase Cell signaling EGF 10-wk male 1,000 g/kg AHR effects independent of Tijet et al., Ahr / or TCDD, expression TCDD 2006 wild-type Ahr in liver after 19 hr Metabolic processes / C57BL/6J Xenobiotic metabolism CYP1A2 mice Steroid metabolism CYP17a1 Glucose metabolism Pck1 Protein/small molecule transport Slc16a5 Protease function Serpina 12 Retinoic acid signaling Retinal binding protein 1 Cell structure Tuba 8 AHR-Dependent effects of TCDD Metabolic processes Xenobiotic metabolism CYP1A1 Immune response TNF 19 Apoptosis Ti-PARP Protease function Serpina 7 Cell structure Tuba 8 77 continued
TABLE 3-2 Continued 78 Biological Model Conditions Outcomes Major Pathways Affected Example References 30 PND female 30 g/kg TCDD, Same as those in TCDD induced both in vivo and Dere et al., C57BL/6 mice, expression in liver Boverhof et al. in vitro 2006 ovariectomized after 2â168 hr (2005) Metabolic processes or Xenobiotic metabolism CYP1A1 0.001â300 g/kg Fatty acid synthesis Ptgs 1 TCDD, expression Small molecule transport Slc20a1 in liver after 24 hr Growth arrest/DNA damage GADD45b Cell cycle Cda5 Mitochondrial function Mrp137 Development Cfdp 1 Immune response Irf1 , Tnfaip2 Cell structure Col1a1 TCDD repressed both in vivo and in vitro Metabolic processes Gyk DNA modiï¬cation Dnmt 1 Proliferation Tk1 TCDD induced in vivo and repressed in vitro CO2 hydration Carbonic anhydrase 2 Fatty acid signaling Fabp5 Growth arrest/DNA damage GADD45gip1 Oncogenesis Myc TCDD repressed in vivo and induced in vitro Small molecule transport Slco Ib2 Immune function Btg2
TCDD-elicited gene expression responses unique to C57BL/6 hepatic tissue Metabolic processes Xenobiotic metabolism Gsta2 Fatty acid metabolism Elovl5 Glucose metabolism Gpd2 CO2 hydration Car3 Growth arrest Gas1 Development Notch1 Immune response Cd3d Mouse 10 nM TCDD, hepatoma cells expression after (Hepa1c1c7 1â48 hr wild-type and or c4 ARNT- 0.001 nMâ100 deï¬cient) nM, expression after 168 hr Mouse Basal conditions Differences in Relative to wild-type Fong et al., hepatoma cells, morphology and Cell structure/cytoskeleton 2005 Hepa1c1c7 proliferation C1 Anxa7 (c1, CYP1A1 (c1 WT c4,c12) Dncl2a deï¬cient; C4 Mapt c4, ARNT Kif20a deï¬cient; c12, C12 Vim AHR deï¬cient) Epb4.115 Vcl 79 continued
TABLE 3-2 Continued 80 Biological Model Conditions Outcomes Major Pathways Affected Example References Bioenergetics C1 Nadufv C4 Cox6b Ndufs2 C12 Cox8a Proliferation C1 Banf1 Lyar tgfb1i4 C4 Degs Oaz1 C12 Arg2 Cdkn1a Ranbp1 Human 50 nM TCDD, Metabolic processes CYP1A1 Kim et al., hepatoma cell expression after Proliferation JUN 2006 lines, Hep3B 1â4 hr Signal transduction SOS1 and HepG2 Development TBX3 Long-Evans 100 g/kg TCDD, TCDD modulated AHRE-II genes and Sprague expression in liver Metabolic processes Dawley rats after 3 and 19 hr Xenobiotic metabolism CYP2b19 Cell signaling Tgf 3 Proteosome function Proteasome type5 Circadian rhythm Period 2 Ribosomal proteins Rib L 29
TOXICOLOGY 81 TABLE 3-3 Overview of TCDD-Induced Changes in Gene Expression as Identiï¬ed by Proteomics Model Conditions Proteins Differentially Expressed by TCDD Exposure Reference 5-wk, 1â50 g/kg Apoliprotein A-IV Jiang et al., male TCDD, -1-Macroglobulin 2005 Sprague- expression in Acidic ribosomal protein P0 Dawley liver after 7 Bal-647 rats days Endoplasmic reticulum protein 29 Proteasome subunit type 3 -1-macroglobulin or Ba1-647-newly expressed MAWD binding protein-newly expressed 0.01â2.5 g/ -Aminolevulinate dehydratase kg TCDD, MAWD binding protein expression in Phosphatidylethanolamine binding proteins; liver after 4 hippocampal cholinergic neurostimulating peptide weeks Transthyretin precursor; prealbumin Histidine triad nucleotide binding protein Phosphoglycerate mutase type B subunit 10-wk, 100 g/kg Protein and mRNA expression altered by TCDD in Pastorelli male TCDD, sensitive (Long-Evans) rats et al., 2006 Long- expression in Aldehyde dehydrogenase family 3, member A1 Evans liver after 5 Apolipoprotein A-1 and Hans days Programmed cell death protein 8 /Wistar Argininosuccinate synthetase rats Carbonic anhydrase 3 MAWD-binding protein Paraoxonase 3 Selenium binding protein 2 Sulfotransferase family 1A, member 1 Transferrin G. gallus 20 ng Liver (p < 0.05) Bruggeman eggs TCDD/egg, Fibrinogen gamma chain precursor et al., 2006 (ED0) expression Hypothetical protein: high homology with NADH in liver and ubituinone oxidoreductase, 42 kDa subunit ovary in Ovary (p < 0.05) 1-day-old 60 kDa heat shock protein chicks Regucalcin continued
82 VETERANS AND AGENT ORANGE: UPDATE 2006 TABLE 3-3 Continued Model Conditions Proteins Differentially Expressed by TCDD Exposure Reference 59 wk ( 100 ng/kg Liver Oberemm 12 wk) TCDD, Voltage-dependent anion channel protein 1 et al., 2005 marmosets expression Transferrin in liver and Lamin A thymus after 4 Heat shock 70 kDa protein 1, Chain A weeks Hemoglobin beta chain Eukaryotic translation initiation factor 4H Thymidine phosphorylase precursor Delta 3,5-delta2,4-dienoyl-CoA isomerase Fructose-bisphosphate aldolase B Dihydrolipoamide succinyltransferase Thymus NADH-ubiquinone oxidoreductase 51 kDa subunit 60 kDa heat shock protein Isocitrate dehydrogenase subunit alpha Glycerol-3-phosphoate dehydrogenase Dihydrolipoamide dehydrogenase Isocitrate dehydrogenase subunit alpha Citrate synthase Mitochondrial innter membrane protein Adseverin Pyruvate dehydrogenase subunit beta Mitochondrial aldehyde dehydrogenase Motor protein Heat shock cognate 71 kDa protein Protein disulï¬de isomerase A3 Cytoplasmic actin 1 Thiopurine S-methyltransferase Ca-dependent protease Vimentin Rat 5L 1 nM TCDD, Histones Sarioglu hepatoma expression Histone H1 strongly et al., 2006 cells after 8 hr Structural proteins Vimentin Molecular chaperones GRP78 Oxidant defense Superoxide dismutase [Mn], mitochondrial precursor Ribosomal proteins 60 S ribosomal protein L7a Elongation factors Eukaryotic translation initiation factor 4B Nucleolus biogenesis Nucleophosmin (B23) Glycolysis Mitochondrial hexokinase I Xenobiotic metabolism Aldehyde dehydrogenase
TOXICOLOGY 83 lation of interactions between the AhR and other nuclear transcription factors, such as the estrogen receptor (ER)-alpha (Beishlag and Perdew, 2005; Mathews et al., 2005), NK (Tian et al., 2002), and Rb (Puga et al., 2002). Recent inves- tigations into the toxicologic signiï¬cance of the interaction between the AhR and Rb have revealed that the corepression elicited by it facilitates the TCDD-induced growth-arrest response (Huang and Elferink, 2005; Marlowe et al., 2004). When AhR is activated by TCDD, it binds to ARNT; this could decrease the ARNT available for complexing with other PAS proteins (e.g., hypoxia-inducible fac- tors such as HIF1 ) that employ ARNT as a dimer partner and could likewise affect formation of ARNT/ARNT homodimers, thereby affecting the regulation of additional genes. As noted in Update 2004, the AhR cross-talks with the ER signaling path- way via a number of mechanisms that include its direct interaction with ER . Recent data have expanded our understanding of this mechanism by revealing that both the AhR and ER are recruited to the CYP1A1 promoter (Beischlag and Perdew, 2005; Matthews et al., 2005), and this indicates that ER is a co- regulator of the AhR. It is not clear whether ER may act as a coactivator or a corepressor. Furthermore, recruitment of the liganded AhR to the breast-cancer gene BRCA-1 appears to be important in the ability of estrogen to upregulate BRCA-1 transcription (Hockings et al., 2006). Although the relevance of those events to human health is unclear, they may prove important in sex- or age- dependent (premenopausal vs postmenopausal) effects of TCDD. Additional transcription factors that have been recently reported to inter- act with the AhR include SMRT (Widerak et al., 2006) and C/EBP (Liu and Matsumura, 2006), which may play a role in TCDDâs ability to interfere with reti- noic acid receptor signaling and glucose transport, respectively. An additional âgenomic/non-DNA-binding mechanismâ would include TCDDâs alteration in the methylation status of genes that is thought to result in gene silencing. Recent studies have shown that in some experimental conditions TCDD suppresses the expression p16Ink4a and p53, tumor-suppressor genes that regulate the onset of cellular senescence, via a mechanism that appears to involve DNA methylation (Ray and Swanson, 2004). Similarly, when an approach that involved exposure of mouse embryos to TCDD before exposure was used, it was reported that TCDD increased methylation of two growth-related imprinted genes, H19 and Igf2, in the developing fetus (Wu et al., 2004). Progress in understanding the ânongenomic mechanismsâ by which TCDD exerts its biologic effects has been restricted largely to its effect on signal trans- duction. TCDD has been reported to activate the p38-mitogen-activated protein kinase (MAPK) (Park et al., 2005; Weiss et al., 2005) and c-Src kinase (Mazina et al., 2004) pathways. TCDDâs activation of at least MAPK appears to occur in an AhR-dependent manner (Weiss et al., 2005).
84 VETERANS AND AGENT ORANGE: UPDATE 2006 Factors That May Contribute to the Tissue-, Species-, and Developmental-Stage-Speciï¬c Actions of TCDD As indicated in previous updates, factors that underlie the tissue-, species-, and developmental-stage-speciï¬c actions of TCDD include its variations in expression levels of the AhR expression, variations in the expression levels of proteins that regulate its activity, and variations in AhR amino acid composition, phosphorylation status, and cellular localization. High expression of the AhR is presumed to correspond to higher responsiveness to the effects of TCDD, but this has not been conclusively demonstrated. As described in Update 2004, expression of the AhR represents a balance between synthesis (regulated at the transcrip- tional and translational levels) and degradation (mediated by agonist-dependent and agonist-independent mechanisms). Recent progress in our understanding of the factors involved in regulation of AhR synthesis includes the discovery that IL-4 can induce AhR synthesis at the transcriptional level in B cells (Tanaka et al., 2005). Additional progress in understanding regulation of AhR expression has come from studies in which the investigators questioned whether the expression of the AhR varies during the progression of diseases and between speciï¬c cell types. With respect to disease state, the best examined is the progression of can- cer. For example, as mentioned previously, the expression of either the AhR or its target genes, CYP1B1 and CYP2S1, is upregulated in tumor tissues isolated from human pancreatic, lung, prostatic, ovarian, and colon cancers compared with that detected in normal tissues (Abdelrahim et al., 2003; Downie et al., 2005; Koliopanos et al., 2002; Kumarakulasingham et al., 2005; Tokizane et al., 2005). More recently, it has been shown that expression of the AhR is lower in endometrial cancer than in the normal endometrium (Michalak et al., 2005) and in hematologic malignancies, such as acute lymphoblastic leukemia due to hyper- methylation of the AhR promoter (Mulero-Navarro et al., 2006). Expression of both the AhR and its dimerization partner, ARNT, has been reported to be lower in pancreatic islets isolated from subjects with type 2 diabetes than in patients without this disease (Gunton et al., 2005). The observations that in some cases the expression of the AhR is cell-type- speciï¬c imply that a speciï¬c population of cells in an organ may elicit the pri- mary effects of TCDD. In the lung, AhR expression has recently been shown to be higher in bronchiolar Clara cells than in the progenitor human small-airway epithelial cells (Chang et al., 2006). In the liver, the hepatocytes appear to be the âprimary responderâ to the toxic effects of TCDD (Walisser et al., 2005). And the ï¬nding that both the cerebral endothelial cells and astrocytes express a functional AhR signaling pathway implies that components of the bloodâbrain barrier are also targets of TCDD toxicity (Filbrandt et al., 2004). Also, agonist-induced degradation of the AhR is thought to play a major role in limiting its signaling capacity. Recent progress reveals that agonist-induced degradation of the AhR involves multiple mechanisms and appears to depend
TOXICOLOGY 85 on the absence or presence of a functional transcriptional activation domain and binding of the AhR to DNA (Pollenz et al., 2005). Inï¬uence of Other Proteins on the Ability of TCDD to Activate the AhR As mentioned in Update 2004, the ability of TCDD to activate the AhR is inï¬uenced by a number of interacting proteins that have various functions. XAP2 mediates cellular localization and stability of the AhR and appears to be capable of inducing a conformational change in the AhR that excludes p23 from the AhR complex, retains the AhR in the cytoplasm, and hence represses AhR transcriptional activity (Hollingshead et al., 2004). An additional chaperone pro- tein, CyP40, has recently been found to assist in formation of the AhR/ARNT heterodimer (Shetty et al., 2004). The AhR repressor (AhRR) associates with the AhR and negatively regulates its function. Although high expression levels of AhRR have been shown in cultured cells to inhibit AhR signaling (Nishihashi et al., 2006), an expected negative correlation between its tissue-dependent ex- pression and AhR agonist activity was not observed, so expression of the AhRR may not dictate a tissueâs responsiveness to TCDD (Bernshausen et al., 2006). Additional proteins that are critical for appropriate AhR function are transcrip- tional coactivators. Recently discovered AhR transcriptional coactivators of the AhR are coiled-coil coactivator (Kim and Stallcup, 2004), GRIP1-associated co- activator 63 (Chen et al., 2006), the Epstein-barr virus-encoded EBNA-3 protein (Kashuba et al., 2006), and thyroid-hormone receptor/retinoblastoma-interacting protein 230 (Beishlag et al., 2004). Effect of Phosphorylation on AhR Activity In addition to variations in protein expression, phosphorylation status can dictate a cellâs responsiveness to the effects of TCDD. Recent progress has shown that the AhR-signaling pathway is regulated by c-scr (Backlund and Ingelman-Sundberg, 2005), the mitogen-activated protein kinases (Chen et al., 2005; Shibazaki et al., 2004; Tan et al., 2004), and protein kinase C (Kawajiri and Ikuto, 2004; Kayano et al., 2004; Machemer and Tukey, 2005; Minsavage et al., 2004). Those kinases not only regulate transcriptional activity of the AhR but participate in targeting the AhR for degradation. The phosphorylation status of the AhR chaperone HSP90 also plays a role in the ability of the AhR to form a cytosolic complex capable of binding TCDD (Ogiso et al., 2004). Effect of Pharmacologic Agents, Dietary Factors, and Environmental Exposures on the AhR and TCDDâs Adverse Effects It is predicted that coexposure to agents that act as AhR agonists or AhR antagonists that may be encountered pharmacologically, via the diet, and in the environment may modulate a personâs response to TCDD. For example, although
86 VETERANS AND AGENT ORANGE: UPDATE 2006 omeprazole, an antiulcer drug, is an AhR agonist, its metabolite that is generated by CYP3A4 acts as a potent AhR antagonist (Gerbal-Chaloin et al., 2006). New data indicate that activators of PPAR (for example WY-146430) may modulate the ability of the AhR to upregulate its target genes, albeit via mechanisms that do not require their direct binding to the AhR (Fallone et al., 2005; Shaban et al., 2005). As discussed in Update 2004, a number of dietary agents may act as AhR agonists or antagonists. Several investigators have proposed that diets rich in particular micronutrients can play an important role in protecting people from the adverse effects of xenobiotics, such as TCDD. Of particular interest are the ï¬avonoids, catechins, and theaï¬avins found in fruits, vegetables, and black and green tea (Chen et al., 2004; Kim et al., 2004; Fukuda et al., 2004a,b, 2005; Ramadass et al., 2003). The observations that those compounds at relatively low concentrations act as AhR antagonists, which inhibit the ability of TCDD to induce formation of the AhR/ARNT complex and thus induction of CYP1A1, indicate that their ingestion via dietary means may be sufï¬cient to confer a pro- tective effect. A number of environmental agents have also been found to alter the ability of TCDD to activate AhR signaling. For example, the heavy metals Hg2+, Pb2+, and Cu2+ have been shown to decrease TCDD-mediated induction of CYP1A1 activity via a mechanism that appears to involve enhanced heme degradation (Korashy and El-Kadi, 2004, 2005). Furthermore, the heavy metals As 3+, Cd2+, and Cr6+ have been reported to mediate oxidative stress, downregulate CYP1A1 at posttranscriptional levels, and potentiate Nqo1 and GstYa at the transcriptional level (Elbekai and El-Kadi, 2005). Examination of the effects of other halogena- tated aromatic compounds on the ability of TCDD to induce the AhR-signaling pathway revealed that the coplanar polychlorinated biphenyls (PCBs) 77 and 126 that act as AhR agonists exhibit additive behavior with TCDD (Chen and Bunce, 2004). The nonplanar PCB 153, in contrast, exhibits antagonist activity and com- petitively inhibits the actions of TCDD. The environmental estrogen bisphenol A was reported to inhibit the expression of the AhR. It has also been postulated that TCDD exerts its toxicity by mimicking an endogenous ligand for the AhR and activating the receptor at inappropriate times or for inappropriately long periods. The physiologic ligand for the AhR has not yet been identiï¬ed, but endogenous AhR agonists reported thus far and de- scribed in Update 2004 include 2-(1 H-indole-3 -carbonyl)-thiazole-4-carboxylic acid methyl ester, metabolites of tryptophan, and lipoxin A4. Recently, it has been shown that although 2-(1 H-indole-3 -carbonyl)-thiazole-4-carboxylic acid methyl ester can act as a potent AhR agonist both in vitro and in vivo, it failed to incur adverse effects typically associated with TCDD exposure, that is, cleft- palate formation, hydronephrosis, and thymic atrophy (Henry et al., 2006). Simi- larly, studies performed in vivo support the idea that lipoxin A4, which mediates a number of inï¬ammatory events, is an AhR agonist (Machado et al., 2006).
TOXICOLOGY 87 Impact of Genetic Polymorphisms on Responses to TCDD As noted in Update 2004, the species-speciï¬c effects observed with respect to TCDDâs toxic responses appear to be due largely to differences in the amino acid composition of the AhR. As recently reviewed by Conner and Aylward (2006), the data have consistently demonstrated that humans have lower respon- siveness than many laboratory animals. Results of a recent study (Ramadoss and Perdew, 2004) that examined the ligand-binding capabilities of the human AhR compared with that obtained from the mAhRd and mAhRb-1 are consistent with that statement and show that the mAhRd and mAhRb-1 have 2 and 10 times the relative binding afï¬nity of the human AhR, respectively. That the species-speciï¬c effects of TCDD toxicity are due largely to differences in amino acid composition was also recently conï¬rmed in a study of avian species, which display one of the most dramatic differences in sensitivity to TCDD (Karchner et al., 2006). It was reported that the tern AhR has lower activity than the chicken AhR because of two amino acids, Val325 and Ala 381. An additional approach is being used to understand the genetic basis of susceptibility to TCDDâs toxic effects in a study of a population of killiï¬sh that are dioxin-resistant; recently, it was reported that although two alleles were identiï¬ed (AHR1*1 and AHR1*3), they did not con- tribute to functional differences in AhR action (Hahn et al., 2004). Given that polymorphisms of the AhR have been found in rats, mice, and birds that signiï¬cantly alter the ability of TCDD to activate the AhR-signaling pathway, it has been proposed that similar differences may exist in the human population. Only eight polymorphisms have been identiï¬ed in the human popu- lation (Harper et al., 2002). Most human AhR polymorphisms are restricted to exon 10, which encodes the C-terminal region of the AhR known as the transcrip- tional activation domain, in particular, at codons 517, 554, and 570. However, the polymorphisms that have been discovered thus far in the human AhR have not yet been shown to change their ability to confer responsiveness to TCDD substantially (Okey et al., 2005). And although differences in the composition of the C-terminal region of the AhR had previously been thought to result in only subtle differences in AhR function, this idea is now challenged by a recent report that the C-terminal region of the AhR plays a role in determining its cellular lo- calization and hence its ability to respond to the presence of TCDD (Ramadoss and Perdew, 2005). Several human population studies designed to identify an association between the AhR, ARNT, or AhRR and human disease states have recently been reported. Study of cleft-palate formation resulted in the association of nonsyndromic oral clefts and a polymorphic form of ARNT (IVS12-19T/G) (Kayano et al., 2004). With respect to reproduction, it has been found that micropenis and male infer- tility are associated with the Pro185Ala polymorphism of AhRR (Soneda et al., 2005; Watanabe et al., 2004). The same polymorphism has been found to be asso- ciated with susceptibility to and severity of endometriosis (Tsuchiya et al., 2005).
88 VETERANS AND AGENT ORANGE: UPDATE 2006 Finally, a recent report (Long et al., 2006) indicates that the AhR polymorphism Lys554Arg may confer increased susceptibility to breast cancer. Effects Related to Particular Toxic Endpoints and Health Outcomes Accumulated studies in experimental animals indicate that TCDD affects a variety of tissues, and the types of effect observed are often tissue-speciï¬c. Ef- fects are most often dose-dependent; that is, some toxic endpoints appear to be more sensitive to low exposures, and others occur only at high concentrations. Toxic effects also have been found to depend on the species examined and often on the age and sex of the animal. There is no reason to suspect that humans would be different in that respect. Findings in animals suggest that reproductive, developmental, and oncogenic endpoints are the most sensitive to TCDD, and this is consistent with the notion that growth, maturation, and differentiation are the most sensitive cellular processes. The data support the biologic plausibility of similar toxic endpoints in humans. Although the exact biologic mechanisms of those endpoints and the observed differences are not yet understood, recent data show the possibility that at least some of the effects are mediated by TCDDâs ability, through the AhR, to modulate cell-cycle control, signaling pathways that lead to cell death or inappropriate cell activation, hormones and growth factors and the responses to them, or the biochemical pathways that lead to oxidative stress. Those mechanisms are implicated in many of the toxic endpoints discussed below. Lethality and Wasting Syndrome As indicated above and in Update 2004, there is some variation among spe- cies in susceptibility to the lethal effects of TCDD that is attributable in part to differences in primary amino acid sequences and expression of the AhR protein. Exposure of most animal species to relatively high doses of TCDD elicits a wast- ing syndrome characterized by decreased food consumption and loss of body weight. The biochemical pathways affected by TCDD that lead to the wasting syndrome have not been identiï¬ed. Several groups posit that TCDD, via the AhR, alters a body-weight set point. The hypothalamus contains neuroendocrine cells that regulate several physiologic processes, including energy balance. Recent re- search that has focused on the ability of TCDD to perturb hypothalamic function has found that the effects of TCDD differed from those of leptin and indicated that TCDD affected primarily orexigenic factors (LindÃ©n et al., 2005). Several studies focused on the ability of different agents to block the TCDD- induced wasting syndrome. Treatment with curcumin (Ishida et al., 2004) protected against the loss of body-weight gain but failed to alter a classic AhR-mediated event, induction of hepatic ethoxyresoruï¬n-O-deethylase activity; TCDDâs ef- fect on body-weight gain might not require activation of the AhR. Cotreatment
TOXICOLOGY 89 with the antioxidants vitamins A and E protected against TCDDâs effect on body weightâan indication that TCDD may induce the wasting syndrome by increas- ing oxidative stress (Alsharif and Hassoun, 2004). Effects on Skin and Adipose Tissue Skin lesions, including chloracne, are often reported in animals and humans after exposure to TCDD and related compounds. Chloracne is characterized by altered proliferation and differentiation of epidermal cells. TCDD affects the temporal expression of protein markers of keratinocyte terminal differentiation during murine skin morphogenesis (Loertscher et al., 2002). Henley et al. (2004a) reported that TCDD exposure induced increased expression of IL-1 in human keratinocytes by a posttranscriptional mechanism; the investigators also reported that ERK and JNK MAP kinase pathways are necessary for this to occur (Henley et al., 2004b). As indicated in previous updates, TCDD inhibits the differentiation of some preadipocyte cell lines to adipocytes (fat cells); the process is AhR-dependent. Several groups have examined the mechanism because it could help to explain how TCDD acts in various tissues. Fibroblasts stimulated by a hormone mix- ture undergo a cascade of molecular events to initiate adipocyte differentiation, including regulation of c-myc, fos, and jun and then upregulation of CCAAT- enhancer-binding proteins and of the peroxisome proliferators-activated receptor . A recent microarray analysis has revealed that the ability of TCDD to inhibit adipocyte differentiation appears to involve a synergistic interaction between the AhR and the growth factors EGF and FGF that results in changes in cell adhe- sion (Hanlon et al., 2005). Several investigations cited in previous updates noted that TCDD exposure alters plasma and tissue lipid content in animals. A micro- array study performed with liver mRNA isolated from male rats treated with a relatively high dose of TCDD supports the idea that TCDD alters cell adhesion and demonstrated that in the liver TCDD exposure results in major deregulation of cholesterol metabolism and bile acid synthesis and transport (Fletcher et al., 2005a). Effects on Bone and Teeth Previous studies have suggested that defects in childrenâs tooth develop- ment may be associated with environmental exposure to dioxins and dioxin-like chemicals (Alaluusua et al., 2002; Funatsu et al., 1971; Lind et al., 1999, 2000a,b; Rogan et al., 1988). In a recent follow-up study of dental aberrations in subjects exposed to dioxin in Seveso, Italy (Alaluusua et al., 2004), developmental enamel defects were not signiï¬cantly increased in individuals that had serum TCDD less than 226 ng/kg at the time of accident, compared to unexposed individu- als. However, the percentage of individuals with developmental enamel defects
90 VETERANS AND AGENT ORANGE: UPDATE 2006 was signiï¬cantly increased for those that had serum TCDD between 238â592 ng/kg (45 percent) or 700â26,000 ng/kg (60 percent) at the time of the accident, compared to unexposed individuals (26 percent). Furthermore, 93 percent of the subjects with developmental enamel defects had been under 5 years old at the time of the Seveso accident. A number of recent animal studies conï¬rm the effects of developmental TCDD exposure on tooth development. Offspring of female mink exposed in utero and via lactation to dioxin, dioxin-like chemicals, and non-dioxin-like chemicals (including non-dioxin-like PCBs and polybrominated diphenyl ethers) exhibited mandibular and maxillary squamous epithelial cell proliferation, which could contribute to tooth loss (Bursian et al., 2006). The incidence of tooth ab- normalities was signiï¬cantly increased in surviving offspring of rhesus monkeys exposed in utero and via lactation to TCDD (Yasuda et al., 2005); the abnormali- ties occurred at maternal body burdens maintained at 300 g/kg and included precocious eruption, dysplasia, incomplete calciï¬cation, and missing teeth; no effects were observed at maternal body burdens of 30 g/kg. When rat offspring exposed in utero and via lactation to a single maternal TCDD dose of 0.03, 0.1, 0.3, or 1.0 g/kg were challenged with a sugar-rich diet and exposure to Strepto- coccus mutans at the age of 11 weeks (Miettinen et al., 2006), TCDD exposure increased the number of caries lesions in the enamel at the lowest maternal dose and in the dentin at the two highest maternal doses; changes in mineral composi- tion were not related to the increased caries incidence. TCDD also can interfere with enamel maturation when exposure occurs only postnatally. A single oral dose of TCDD at 50 or 1,000 g/kg given to lactating Han/Wistar dams 1 day after delivery induced retention of enamel matrix and retarded dentin mineralization in pups on postnatal day 22 (Gao et al., 2004). The changes were associated with decreased AhR and CP1A1 immunoreactivity in ameloblasts and odontoblasts. Only two relevant studies of TCDDâs effects on bone have been published since Update 2004. Ilvesaro et al. (2005) found that rat bone cell cultures ex- pressed mRNA for both the AhR and ARNT and that osteoclasts expressed much higher levels of the AhR than osteoblastic mononuclear cells. However, treatment of rat osteoclasts with 10 nM TCDD failed to alter osteoclast activity. In the sec- ond study, three strains of rats, which varied in their sensitivity to TCDD because of a mutated AhR, were exposed to TCDD at 0, 0.1, 0.3, or 1.0 g/kg at different times during gestation or lactation, and offspring were analyzed 5â6 or 52 weeks after birth for bone mineral density and geometry (Miettinen et al., 2005). Only the most sensitive line of rats exhibited signiï¬cant decreases in tibial and femoral mineral density and breaking force and decreases in femoral cross-sectional area, length, and endosteal and periosteal circumference. Earlier exposure induced more severe defects, but gestational exposure alone was not sufï¬cient. Notably, most of the bone defects returned to normal limits 52 weeks after birth.
TOXICOLOGY 91 Cardiovascular Toxicity It is well established that TCDD can affect the developing cardiovascular system, and there is growing evidence from a variety of experimental models that the cardiovascular system may also be a target of TCDD toxicity in adult animals. The evidence is derived from studies of the AhR-null mice, of constitutively active-AhR mice, of TCDDâs effects on vascular smooth muscle and endothelial cell cultures, and of chronic TCDD exposure of rodents. A role of the AhR in cardiovascular homeostasis in adults has been demon- strated by a number of previously published studies. Brieï¬y, the studies showed that AhR-null mice develop age-progressive cardiac hypertrophy and ï¬brosis (Fernandez-Salguero et al., 1997; Thackaberry et al., 2003; Vasquez et al., 2003), which was preceded by increases in blood pressure, plasma angiotensin II, and plasma endothelin-1 (Lund et al., 2003). Blockade of angiotensin II synthesis ameliorated the hypertension and cardiac hypertrophy (Lund et al., 2003), but more recent studies demonstrated that blockade of endothelin-1 signaling was signiï¬cantly more effective, normalizing blood pressure and cardiac hypertrophy to AhR-wildtype values (Lund et al., 2006). In a recent study, Lund et al. (2005) showed that high endothelin-1 mediated increases in reactive oxygen species (ROSs) that were associated with cardiac hypertrophy via increased NAD(P)H oxidase activity and superoxide anion generation. Those studies suggest that the AhR may play a protective role in the adult cardiovascular system and that genetic deletion abolishes that beneï¬t. Constitutive activation of the AhR also results in disruption of cardiovascular homeostasis. Brunnberg et al. (2006) have shown that male mice expressing a constitutively active AhR develop an age- progressive cardiac hypertrophy associated with greater expression of CYP1A1 in coronary capillary endothelial cells than in AhR-wildtype mice. Three recent cell-culture studies demonstrate that sustained activation of the AhR by TCDD or the AhR agonist 3-methylcholanthrene disrupts vascular- cell gene expression, proliferation, and function. Exposure of human umbilical vein endothelial cells to either 3-methylcholanthrene or TCDD inhibited basal proliferation, and exposure to TCDD also inhibited proliferation stimulated by vascular endothelial growth factor (Ivnitski-Steele and Walker, 2003; Juan et al., 2006). Both AhR agonists also inhibited angiogenesis as assessed in vitro. In a third study, global gene-expression proï¬ling after TCDD exposure of mouse aorta in vivo or cultured vascular smooth muscle cells in vitro showed that more than 4,500 genes differed in expression between the two models but that a subset of 35 genes showed similar responses to dioxin exposure (Puga et al., 2004). Commonly responding genes included phase I and phase II metabolic enzymes, signal-transduction kinases and phosphatases, and regulators of DNA repair and the cell cycle.
92 VETERANS AND AGENT ORANGE: UPDATE 2006 Pulmonary Toxicity This and previous updates report evidence suggestive of an association between herbicide exposure in Vietnam and respiratory cancer (see âCarcino- genicity,â below). Several published reports have suggested an association be- tween TCDD exposure and chronic obstructive pulmonary disease, but the present committee found insufï¬cient evidence to support a relationship between herbicide exposure and respiratory disorders that are not considered cancer, not- ing the lack of toxicity to the pulmonary system in laboratory animals exposed acutely to low doses (1â10 g/kg) of TCDD that result in toxicity to other organ systems. Recent studies have shown that when mice exposed to TCDD are challenged with a respiratory virus (inï¬uenza), they experience a higher mortality. The in- crease in mortality did not reï¬ect TCDD suppression of the antiviral adaptive immune response, as initially suspected, but was associated with an enhanced inï¬ux of neutrophils into the lung (IOM, 2005). Neutrophils produce several toxic substances (for the purpose of killing pathogens), so it is possible that excess neutrophils in the lung produce excess collateral damage and pathologic changes that result in increased mortality. Testing that hypothesis, Teske et al. (2005) showed that prevention of the neutrophilia (by using an antineutrophil antibody) provided partial protection of TCDD-treated mice from inï¬uenza-induced death. Furthermore, the increased mortality and neutrophilia were AhR-dependent and not observed in TCDD- treated AhR-knockout mice. However, TCDD exposure did not alter the concen- trations of common lung neutrophil chemoattractantsâmacrophage inï¬ammatory protein 1 (MIP-1 ), MIP-2, keratinocyte chemoattractant, lipopolysaccharide- induced CXC chemokine, interleukin 6, and complement split product C5aâthe expression of adhesion molecules by neutrophils (CD11a, CD11b, CD49d, CD31, CD38), or the normal neutrophil apoptotic process. Likewise, concentrations of ROSs and myeloperoxidase activity were normal in neutrophils from TCDD- treated mice. In the absence of evidence of TCDD effects at the level of neutro- phil recruitment or function, several indicators of lung damage were assessed (Bohn et al., 2005). However, TCDD exposure did not alter the concentrations of lactate dehydrogenase (a marker of lung cell damage) or enhance edema (mea- sured by proteins in bronchoalveolar-lavage ï¬uid and wet-to-dry weight ratios). Lung concentrations of Clara cell secretory protein, an inï¬ammatory mediator produced by lung-associated Clara cells, were also not altered by TCDD. Despite the absence of functional changes, TCDD induced CYP1A1 expression in the Clara cells of the lung and in lung endothelial cells and Type II pneumocytes, indicating the ability of TCDD to affect the lung directly via AhR activation (Bohn et al., 2005). CYP1A1 and CYP1B1 were also induced in the lungs of rats given a single high dose (5 g/kg) of TCDD 4 hours before termination (Harrigan et al., 2006),
TOXICOLOGY 93 and treatment of human broncoalveolar H358 cells with 10 nM TCDD in vitro induced the expression of enzymes as well (Jiang et al., 2005). In a unique design, Esser et al. (2005) investigated the possible long-term effects of TCDD on the health of aged mice that had been given a single dose of TCDD (2.5 or 25 g/kg) when they were young. Histologic examination of several tissues revealed no differences except in the lungs, where TCDD was as- sociated with an increased incidence of activated bronchus-associated lymphoid tissue; the signiï¬cance of this ï¬nding remains to be determined. In terms of chronic-exposure studies, treatment of rats with low doses of TCDD for 2 years, but not 1 year, resulted in increased lung weight and bronchiolar metaplasia of the alveolar epithelium in female rats (IOM, 2003, 2005). Recent studies show that CYP1A1 and CYP1B1 are strongly induced in Clara cells after treatment of rat lung slices in vitro with TCDD (Chang et al., 2006). In human lung tissue, AhR and CYP1A1 are expressed mainly in bronchiolar epithelial cells (Lin et al., 2003). Using primary cultures of human small-airway epithelial (SAE) cells that differentiate into Clara cells in vitro, Chang et al. (2006) showed mRNA and protein levels of CYP1A1, CYP1B1, and the AhR are increased in SAE cells cultured with TCDD. The authors suggest that Clara cells may be the most sensitive lung-cell type responsive to TCDD. Previous toxicogenomic studies identiï¬ed EGR1 as a potential novel target for TCDD and other AhR agonists in human lung epithelial cells (IOM, 2005). Follow-up studies have now shown that TCDD (via the AhR) does not act as a transcriptional promoter but rather appears to increase the half-life of the mRNA via a post-transcriptional mechanism that leads to higher EGR1 protein con- centrations in treated cells (Martinez et al., 2004). EGR1 is of interest because it functions as a transcription factor to regulate the expression of several genes involved in cell growth, apoptosis, and differentiation and may play a role in tumor development. Hepatotoxicity Several studies addressed mechanisms of TCDD-induced hepatotoxicity. Chang et al. (2005) examined AhR expression, AhR activation (CYP 1A2 induc- tion), and hepatocellular pathology together, in young male mice, and correlated these factors in the same animal and in the same cells in centrilobular location, providing direct evidence on the relationship between AhR activation and hepatic toxicity. Boverhof et al. (2005) sought to further characterize TCDD toxicity by com- prehensive analysis of temporal and dose-response changes in hepatic tissues, using a microarray approach. Liver from immature ovariectomized C57BL/6 mice that had been treated with 30 Î¼g/kg of TCDD or vehicle were sacriï¬ced after times up to 168 h or were exposed by gavage to doses up to 300 Î¼g/kg of TCDD and sacriï¬ced after 24 h. There were 443 and 315 features that exhibited
94 VETERANS AND AGENT ORANGE: UPDATE 2006 a signiï¬cant change at one or more doses or time points, respectively. Gene ex- pression changes were associated with physiological processes such as oxidative stress and metabolism, differentiation, apoptosis, gluconeogenesis, and fatty acid uptake and metabolism. Histopathology and clinical chemistry showed pheno- types supporting a mechanism for TCDD-mediated fatty liver, one that would involve mobilization of peripheral fat and inappropriate increases in hepatic uptake of fatty acids. Vezina et al. (2004) employed DNA microarrays to seek unique gene expres- sion patterns in liver, associated with subchronic exposure to TCDD and related chemicals. Female Harlan Sprague-Dawley rats were exposed to toxicologi- cally equivalent doses of four different compounds, based on the TEFs of each chemical: TCDD (100 ng/kg/day), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF; 200 ng/kg/day), 3,3â,4,4â,5-pentachlorobiphenyl (PCB126; 1,000 ng/kg/day), or 2,2â,4,4â,5,5â-hexachlorobiphenyl (PCB153; 1,000 microg/kg/day). Global gene expression proï¬les assessed with Affymetrix GeneChips were compared by prin- cipal components analysis. TCDD, PeCDF, and PCB126 produced very similar expression proï¬les that were distinct from the PCB153. Such ï¬ndings may help to uncover some fundamental features of dioxin toxicity. An expression proï¬ling study to identify further genes involved in hepa- totoxicity and hepatocarcinogenesis by TCDD was carried out using C57BL/6 (AhR / , wild-type) and B6.129-J (AhR / , knockout) mice (Yoon et al., 2006). Mice were injected intraperitoneal (ip) with TCDD at 100 g/kg. Relative liver weight was signiï¬cantly increased after 72 hr after TCDD in AhR / mice, but without apparent histopathological change. The liver was analyzed for gene ex- pression proï¬les 72 hr later. As compared with AhR / mice, the expression of 51 genes ( 3-fold) was changed in AhR / mice; 28 genes were induced, while 23 genes were repressed. Most of the genes were associated with chemotaxis, inï¬ammation, carcinogenesis, acute-phase response, immune responses, cell metabolism, cell proliferation, signal transduction, and tumor suppression. The study suggests that the analysis of genes in liver of AhR / and AhR / mice may help clarify the mechanisms of AhR-mediated hepatotoxicity and hepato- carcinogenesis by TCDD. Oxidative stress is induced by TCDD, but the mechanism is not understood. Shen et al. (2005) examined oxidative stress in mouse liver after one dose of TCDD (5 /kg body weight). Mitochondrial succinate-dependent production of superoxide and H2O2 in doubled at 7â28 days, then subsided by day 56, at the time that levels of cytosolic and mitochondrial GSH and GSSG increased. Comparing AhR( / ) knockout and wild-type mice, showed that TCDD-induced thiol changes in both cytosol and mitochondria were dependent on the AhR. The TCDD-stimulated increase in production of reactive oxygen paralleled a four-fold increase in formamidopyrimidine DNA N-glycosylase (FPG)-sensitive cleavage sites in mitochondrial DNA, compared with nuclear DNA. TCDD-dependent oxi-
TOXICOLOGY 95 dative stress in mitochondria and mitochondrial DNA damage appear to involve the mitochondrial thiol reduction state. A couple of studies assessed TCDD effects on retinoid metabolism. Fletcher et al. (2005b) examined the effects of long-term low-dose TCDD exposure on retinoid, thyroid hormone, and vitamin D homeostasis in Long-Evans and Han/ Wistar rats, given a tumor initiation regimen. These results showed that TCDD disrupts both retinoid storage and metabolism of retinoic acid and retinoic acid metabolites in liver, kidney, and plasma from doses as low as 1 ng/kg body weight (bw)/day. Furthermore, 9-cis-4-oxo-13,14-dihydro-retinoic acid (RA) was identi- ï¬ed as a novel and sensitive indicator of TCDD exposure, in a resistant and sensi- tive rat strain, thereby extending the database of low-dose TCDD effects. Fletcher et al. (2005a), in a separate study, sought to identify novel genes and pathways possibly associated with TCDD-induced hepatotoxicity. Male Sprague-Dawley rats were given single low or high doses of TCDD and gene expression analyzed by microarray. In addition to altering expression of phase I and phase II metabo- lizing enzymes, 0.4 Î¼g/kg bw TCDD also altered the expression of Gadd45a and Cyclin D1, genes indicative of cellular stress or DNA damage and associated with cell-cycle control. At the high dose, widespread changes occurred with genes encoding cellular signaling proteins, cellular adhesion, cytoskeletal and mem- brane transport proteins, as well as transcripts coding for lipid, carbohydrate and nitrogen metabolism, and some genes involved in cholesterol metabolism. Yang et al. (2005a) investigated the effects of vitamin A on TCDD effects on liver of mice. Mice were given a single oral dose of 40 g TCDD/kg bw with or without the continuous administration of vitamin A (2500 IU/kg bw/day) or were given daily an oral dose of 0.1 Î¼g TCDD/kg bw with or without vitamin A. Mice were sampled at various days. TCDD caused liver damage and increased liver weights, and the liver damage was less severe in mice receiving TCDD vitamin A. EROD activities, CYP1A1 expression, and AhR mRNA expression in vitamin A TCDD-treated mice were lower than those in TCDD-treated mice; supplementation of vitamin might attenuate the liver damage caused by TCDD. In a companion study, Yang et al. (2005b) found that TCDD affected the metabolism of vitamin A as well. TCDD signiï¬cantly decreased the hepatic all-trans-retinol level and increased the hepatic all-trans-RA content, increased the mRNA and enzymatic activities of retinal oxidase. Pancreatic and Gastrointestinal Tract Effects Novelli et al. (2005) examined TCDD effects on secretory function of iso- lated pancreatic islets from rat. At 24 hr after TCDD (1 /kg bw, ip), rats showed no signiï¬cant differences in levels of plasma glucose, insulin, triglycerides, and leptin, but plasma-free fatty acids increased signiï¬cantly compared to untreated rats. Insulin content of islets isolated from TCDD-treated rats was signiï¬cantly decreased although DNA and protein content were unchanged. Incubation of
96 VETERANS AND AGENT ORANGE: UPDATE 2006 islets with varied concentrations of glucose resulted in signiï¬cant impairment of glucose-stimulated insulin secretion in islets isolated from TCDD-treated rats. A signiï¬cant reduction of [3H]-2-deoxy-glucose uptake was seen in pancreatic tissue of TCDD-treated rats, while no signiï¬cant reduction in GLUT-2 protein levels was detected in islets from TCDD-treated rats. The results suggest that low-dose TCDD could rapidly induce signiï¬cant alterations of the pancreatic endocrine function in the rat. Previous 2-year studies of TCDD on female Harlan Sprague-Dawley rats revealed acinar-cell vacuolation, atrophy, inï¬ammation, and arteritis, as well as a rare occurrence of pancreatic acinar-cell adenomas and carcinomas. Yoshizawa et al. (2005a) sought to identify mechanisms involved in early stages of acinar- cell lesions. Pancreas from animals treated for 14 and 31 weeks with 100 ng TCDD/kg bw or corn oil vehicle were examined for acinar-cell kinetics and proliferating cell nuclear antigen, CYP1A1, AhR cholecystokinin-A receptor (CCK-A receptor; CCKAR), duodenal cholecystokinin 8 (CCK), and amylase localization. Increased apoptotic activity in acinar cells occurred in 14- and 31- week-treated animals, with an increase in proliferative activity in the latter. Also in the latter, in the vacuolated acinar cells, CYP1A1 was overexpressed, and statistically signiï¬cant decreases in expressions of AhR, CCKAR, and amylase occurred. Changes in the expression of the various genes may be related to acinar- cell lesions. TCDD effect on the intestine is not well understood. Ishida et al. (2005) examined the effect of dioxin on the pathology and function of the intestine in AhR-sensitive and -less-sensitive mice, after oral administration of TCDD (100 g/kg). C57BL/6J mice showed changes in villous structure and nuclear/cyto- plasm ratio in the epithelial cells of the intestine. In an oral glucose tolerance test, the serum glucose level was signiï¬cantly increased in the C57BL/6J mouse but not in the DBA/2J mouse. The expression of intestinal mRNAs coding sodium- glucose co-transporter 1 (SGLT1) and glucose transporter type 2 were increased only in C57BL/6J mice. The intestinal activity of sucrase and lactase also was signiï¬cantly increased in C57BL/6J mice by TCDD. Neurotoxicity Several of the studies referred to in the preceding section concerning devel- opmental effects of TCDD in fact deal with effects in developing brain (e.g., Chang et al., 2005; Mitsui et al., 2006) A number of other papers concern possible mechanisms by which chemicals including chemicals of interest might cause effects on neurological and cognitive function. For example, Parkinsonâs disease (PD) is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra and movement defects. Oxidative stress has been implicated in the pathogenesis of PD based on its role in the cascade of biochemical changes that lead to DA neuronal death. Thiruchelvam et al. (2005) analyzed the role of
TOXICOLOGY 97 oxidative stress as a mechanism of the DA neurotoxicity in mice, showing pos- sible protection by overexpression of superoxide dismutase or glutathione per- oxidase. Choi et al. (2006) also showed that defense against reactive oxygen can protect against DA neuron damage in a mouse model. Although not concerning the chemicals of interest, such studies continue to support suggestions that the level of reactive oxygen species could alter the functions of speciï¬c signaling cascades and may be involved in neurodegeneration. This is potentially relevant to chemicals of concern, as TCDD has been reported to elicit oxidative stress in some organs (e.g., Shen et al., 2005). TCDD is thought to produce neurobehavioral abnormalities associated with both cognitive and locomotor systems, yet the regional and cellular targets in- volved in developmental neurotoxicity are largely unknown. Williamson et al. (2005) assessed whether developing cerebellar granule neuroblasts may be direct targets for TCDD. AhR and ARNT proteins were present in mouse cerebellum from birth throughout postnatal development. AhR protein levels peaked between postnatal days 3â10, a critical period for neuroblast growth and maturation. Both AhR and ARNT were expressed in cerebellar granule neuroblast cultures, and TCCD elicited time-dependent and concentration-dependent increases in CYP1A1 and 1B1 expression. TCDD treatment also reduced thymidine incorpo- ration and granule neuroblast survival in a concentration-dependent manner. The study suggests that granule neuroblasts are direct targets for developmental AhR- mediated TCDD neurotoxicity and that TCDD exposure may disrupt granule cell neurogenesis. Kim and Yang (2005) also addressed possible molecular mechanism and intracellular targets by which TCDD could lead to neurodevelopmental and neurobehavioral deï¬cit. They analyzed TCDD-induced neurotoxic effects in the granule cells from the cerebellum, where certain cognitive abilities and motor function command are executed. TCDD induced a dose-dependent increase of total protein kinase C activity that was AhR-dependent and N-methyl-D-aspartate receptor (NMDAR)-independent, a PKC isozyme-speciï¬c pattern of induction. Increase of the ROS formation was also observed in the cells treated with TCDD in a dose-dependent and an AhR-dependent manner. TCDD also increased intra- cellular free calcium [Ca2 ], which is associated with ROS formation and PKC activation in the cerebellar granule cells. It is suggested that TCDD activates the NMDAR, which may induce a sustained increase of [Ca2 ] in neurons followed by the ROS formation. In an effort to develop other model systems, Ton et al. (2006) examined several parameters of neurotoxicity during development in zebraï¬sh exposed to 7 well-characterized compounds. Embryos were exposed by immersion from 6 hrs postfertilization (hpf). Dying cells in the brain were assessed by acridine orange staining (likely to be apoptotic). Motor neurons were assessed by antiacetylated tubulin staining and catecholaminergic neurons were visualized by antityrosine hydroxylase staining. TCDD was primarily teratogenic and not speciï¬cally neu-
98 VETERANS AND AGENT ORANGE: UPDATE 2006 rotoxic, while 2,4-D showed neurotoxicity. The results showed a correlation with mammalian data and suggest that zebraï¬sh is a predictive animal model for neurotoxicity screening. Lensu et al. (2006) explored possible brain areas that could be involved in mechanisms by which TCDD causes wasting syndrome (a dramatic loss of body weight over 2â5 weeks). The studies used Long-Evans (Turku/AB) (L-E) and Han/Wistar (Kuopio) rats, which show 1,000-fold differences in TCDD sensitiv- ity. Rats were examined for brain areas that might be activated by a single dose of TCDD (50 g/kg) given 24 hr prior to sampling. Leptin (1.3 mg/kg, ip) was used as a reference compound, as its neural pathway for decreasing food intake is fairly well known. Serial sections of brain were stained with antibody against c-Fos, and selected areas in the hypothalamus were examined. TCDD alone did not elicit any major alterations in c-Fos protein levels in the hypothalamic nuclei at 24 hr after administration, in either rat strain, while leptin increased the num- ber of c-Fos-immunopositive cells in the hypothalamic ventromedial and arcuate nuclei. The ï¬ndings are not suggestive of a primary role for the hypothalamus in the wasting syndrome, however in a related study (LindÃ©n et al., 2005), TCDD mainly affected expression of orexigenic but not anorexigenic factor mRNAs, and there were temporal differences in response found between the rat strains. Immunotoxicity The immune system has been recognized as a sensitive target for the toxicity of TCDD for many years (see previous updates). Immunotoxicity is manifested as increased susceptibility to infectious disease and increased tumor development in laboratory animal studies. Many cell types make up the immune system, and most of the cells have been shown to express the AhR, which is required for initiating the toxicity of TCDD. Identifying the speciï¬c cells that are altered by TCDD and how they contribute to TCDD-induced alterations in immune function is of great interest to the research community. Understanding how TCDD affects the immune system in rodents increases the ability to extrapolate experimental results to assessment of human risks. Since Update 2004, several papers have addressed the mechanisms of TCDDâs effects on the immune system. A hallmark of TCDDâs immunotoxicity is involution of the thymus, the gland that serves as the site of T-cell development. The mechanism of action of TCDD on the thymus includes both direct AhR-mediated changes in developing thymo- cytes and indirect effects on thymocytes via the AhR-expressing thymic epithelial and dendritic cells. Studies published since Update 2004 have investigated the effects of TCDD exposure on the thymus. Nottebrock et al. (2006) studied the mechanisms underlying thymic involution in marmosets treated with a low dose of TCDD. They reported a dose-dependent increase in expression of several extra- cellular matrix proteins, TGF- 1, and integrins CD49d and CD29. Camacho et al. (2005) reported that activation of the AhR by TCDD in murine thymic stromal
TOXICOLOGY 99 cells induced the expression of Fas ligand (FasL), a death-receptor ligand that induces apoptosis when it binds to cells that express Fas. The increase in FasL expression occurred through AhR-dependent activation of the transcriptional regu- lator NFkB. When TCDD-treated thymic stromal cells were incubated with thymic T cells, the T cells underwent apoptosis, which is consistent with the process of thymic atrophy seen in TCDD-treated mice. In other studies, Ji et al. (2005) reported that down-modulation of the Fas gene by an herbal extract of Artemisia iwayomogii protected thymocytes from TCDD-induced apoptosis in vitro. The main function of the thymus is T-cell selection, a two-step process that has the overarching goal of allowing T cells that recognize foreign material to survive while T cells that react to self-antigens are deleted through apoptosis. Self-reactive T cells that escape thymic selection have the potential to trigger autoimmune responses that lead to autoimmune disease. Using an HY-T cell receptor transgenic mouse model and an acute dose of TCDD (50 g/kg), Fisher et al. (2005a) reported that exposure of mice to TCDD altered the process of T- cell selection in the thymus and produced an increase in self-reactive T cells in the periphery of male mice. The ï¬ndings contrast with the lack of effect of TCDD on T-cell selection in a different model system (de Heer et al., 1995). Other studies reported that exposure to TCDD promoted the premature emigration of double-negative thymocytes from the thymus to the peripheral lymphoid tissues (Temchura et al., 2005). The effect was associated with increased expression of calgranulin B, a calcium-binding protein involved in cell migration. Prema- ture emigration of the double-negative precursor population could contribute to TCDD-induced thymic atrophy. The ability of mature lymphocytes in the secondary lymphoid organs to respond to antigenic challenge is also affected by TCDD. Many prior studies showed profound and dose-dependent suppression of primary immune responses in TCDD-treated animals. Recent studies show that suppression of the antibody response by TCDD in mice is related to suppression of IL-5 production by T helper cells (Inouye et al., 2005). TCDD was also shown to alter the expression of several genes in CD4+ T cells and B cells after immunization of mice with OVA (Nagai et al., 2005). Downregulation of genes involved in GTP-binding protein-linked signaling was most notable in CD4+ T cells. Funatake et al. (2004) used OVA-speciï¬c transgenic T cells to track the effects of TCDD on the early response of CD4+ T cells to antigen stimulation. As in previous studies, the early proliferative response of the T cells to antigen stimulation was not altered by TCDD, but their numbers declined signiï¬cantly on day 4. The decline was preceded by a decreased expression of CD62L and CD11a but no change in CD49d. By day 4, proliferation of the cells had ceased in TCDD-treated mice, and by day 5, the frequency of apoptotic cells was increased. Fas-FasL signaling was excluded as the mechanism of increased T-cell death. However, the expres- sion of several other genes associated with cell survival or death was increased in the T cells by TCDD exposure. Many of the upregulated genes belong to the
100 VETERANS AND AGENT ORANGE: UPDATE 2006 TNF/TNFR superfamilies, including 4-1BB, CD30, trance, and trail. P53 was also increased in T cells exposed to TCDD. The results suggest that a complex interplay of survival and death signals exists in the T cells and that AhR activa- tion by TCDD alters the delicate balance and leads to changes in T-cell survival. A novel explanation of altered T-cell numbers in TCDD-treated mice is the induction of T regulatory cells. Funatake et al. (2005) showed that treatment of mice with TCDD during T-cell activation generated a population of T cells that expressed high levels of CD25 and suppressed the proliferation of naÃ¯ve T cells in vitro. Those changes depended on AhR expression in the T cells. There is extensive evidence that suppression of immune function by TCDD depends on signaling through the AhR. Recently, mice expressing a constitutively active AhR in T cells were genetically engineered (Nohara et al., 2005). They showed CYP1A1 expression in the thymus and spleen, thymic atrophy, and sup- pressed T-cell and B-cell numbers in the spleen after immunization. The effects were similar in scope but not in magnitude to those seen after AhR activation by TCDD. In similar studies, Ito et al. (2004) used transient transfection to study the effects of a constitutively active AhR in human Jurkat T cells in vitro. Their results suggest that AhR activation causes apoptosis and cell-cycle arrest through changes in AhR-regulated gene expression. In addition, Ndebele et al. (2004) showed that treatment with TCDD suppressed the production of IL-2 by Jurkat cells; suppression was associated with decreased NFkB. NFkB signaling in B cells is also affected by TCDD exposure. Recent studies indicate that the dioxin- response element overlaps with an NFkB-response element in the enhancer re- gion of the gene for immunoglobulin heavy chain. Interference with enhancer activity may explain the suppression of antibody production in TCDD-treated B cells. Suppression of B-cell responses by TCDD may also be induced by AhR- dependent induction of the gene for suppressor of cytokine signaling 2 in B cells treated with TCDD (Boverhof et al., 2004). The AhR is expressed in a variety of cells in most species, including humans. However, the ability of the AhR to bind TCDD and activate gene expression can vary with the species. The human AhR has been considered to have less afï¬nity for binding TCDD than the AhR in many other species. This has been inter- preted to mean that humans are among those species having lower sensitivity to toxic effects of TCDD. Nohara et al. (2006) recently reported results of a study comparing TCDD induction of lymphocytes from humans with the responses in lymphocytes from two species with high-afï¬nity AhR (SD rats and C57BL/6 mice) and a species with a low-afï¬nity AhR (DBA/2 mice). The EC 50 for induc- tion of CYP1A1 mRNA (at the time of peak response) in human lymphocytes was similar to that in the DBA/2 mice and about 10-fold lower than in lymphocytes from the SD rats or the C57BL/6 mice. However, the levels of CYP1A1 mRNA induced at maximally effective doses were greater in the human lymphocytes than in the other species. The authors suggest that some unknown mechanisms modulate the extent of CYP1A1 gene expression in lymphocytes.
TOXICOLOGY 101 Inï¬ammation Cells of the immune system contribute to inï¬ammation. That is advantageous in ï¬ghting infections. However, if inï¬ammation is inappropriately enhanced or prolonged, tissue damage can result. In several human diseases (such as rheuma- toid arthritis and coronary arterial disease), prolonged inï¬ammatory responses appear to contribute to tissue damage and disease severity. Past studies have shown that TCDD can act on macrophages and increase inï¬ammatory mediator produc- tion. Park et al. (2005) now show that TCDD activates ERK and p38 MAPKs in a macrophage cell line (RAW 264.7 cells). Using a different macrophage cell line (U937), Vogel et al. (2005) showed that TCDD increases the expression of a number of genes related to inï¬ammation, including cyclo-oxygenase-2, TNF , IL-8, and C-reactive protein. Those effects could underlie the enhanced inï¬am- matory response reported in several early studies of TCDD toxicity. Carcinogenicity and Contributing Molecular Mechanisms TCDD has been demonstrated to be a carcinogenic agent and potent tumor promoter in several model systems. As mentioned in Update 2004, the carci- nogenic actions of TCDD have been revealed in a 2-year bioassay in female rats. It was reported that TCDD treatment increased the incidence of cholangio- carcinoma and hepatocellular adenoma of the liver, cystic keratinizing epithe- lioma of the lung, and gingival squamous-cell carcinoma of the oral mucosa (NTP, 2004). Observations reported in a follow-up study support the idea that formation of gingival squamous-cell carcinoma induced by TCDD is preceded by gingival squamous hyperplasia that is associated with dysplasia (Yoshizawa et al., 2005b). Further evidence that TCDD exposure may contribute to the formation of squamous-cell carcinoma is provided by a study performed by Wyde et al. (2004), who used a genetically initiated mouse tumorigenesis model (TgAC transgenic mice). They reported that exposure to TCDD via either gavage or dermal ad- ministration signiï¬cantly increased formation of papillomas and squamous-cell carcinomas in the skin. With respect to the role that TCDD may play in hepatocarcinogenesis in mice, a follow-up of the NTP study (NTP, 2004) revealed that during the course of TCDD treatment, proliferation of the hepatocytes, biliary epithelium, and oval cells was increased in a time- and dose-dependent manner (Hailey et al., 2005). In considering the mechanisms by which TCDD may exert these effects, the authors concluded that the evidence was not sufï¬cient to support the idea that the TCDD-induced response was regenerative in the liver and suggested that at this stage the response should be classiï¬ed as a âproliferative response.â A role of the AhR in eliciting TCDDâs tumor-promoting effects in the liver is supported by the recent ï¬nding that in mice bearing the constitutively active form of the AhR, tumor formation initiated by the hepatocarcinogen N-nitrosodiethylamine
102 VETERANS AND AGENT ORANGE: UPDATE 2006 was signiï¬cantly greater than that in wild-type mice (Moennikes et al., 2004). Histopathologic examination of the spontaneous stomach tumors formed in the mice that express the constitutively active form of the AhR revealed that although the tumors penetrated into the layers of the muscle and exhibited metaplasia, they did not metastasize and were not dysplastic (Andersson et al., 2005). Hahn and Weinberg (2002) have proposed that the development of human cancers requires only six changes: resistance to growth inhibition, evasion of apoptosis, immortalization, independence from mitogenic stimulation, angiogen- esis, and metastasis and invasion. Those changes are thought to be due to inherited and acquired alterations in DNA that include such genetic events as DNA muta- tions and chromosomal aberrations, such epigenetic events as DNA methylation that silences gene expression, and DNA damage that follows exposure to such agents as ROSs. Although it is apparent that TCDD can participate in genotoxic events by inducing such enzymes as CYP1A1, CYP1A2, and CYP1B1âwhich are responsible for the metabolic activation of many promutagens, increasing oxidative stress and increasing DNA methylation of tumor-suppressor genes (Ray and Swanson, 2004; Wu et al., 2004)âa large body of evidence indicates that TCDDâs carcinogenic actions occur primarily by nongenotoxic means that would facilitate a damaged cellâs progression through Weinbergâs six requisite changes (Table 3-4). TABLE 3-4 Impact of TCDD on the Six Requisite Changes Proposed by Hahn and Weinberga to Be Required for the Development of Human Cancers TCDD-Regulated Gene Proposed Set of Acquired Products That May Mediate Capabilities Actions of TCDD Its Actions Resistance to growth Increases p27Kip1, E2F inhibition Decreases ER, AR Evasion of apoptosis Increases Decreases Immortalization Increases p16Ink4a, p53 Telomerase activity Independence from Increases EGFR ligands mitogenic stimulation Kinases Angiogenesis Inhibits HIF, VEGF Metastasis and ? TGF , MMPs invasion Increases aAdapted from Hahn and Weinberg (2002).
TOXICOLOGY 103 TCDD Exposure and DNA Damage At the mechanistic level, TCDD is con- sidered to be a tumor promoter that lacks genotoxic actions, that is, direct inter- action with DNA. However, some evidence supports the idea that TCDD may participate in genotoxic effects through its induction of cytochrome P450s that increase formation of ROSs, increase oxidative DNA damage, and increase the frequency of chromosomal abnormalities. For example, in mice given a single dose of TCDD by injection, increases in superoxide and H2O2 and a decrease in GSH relative to GSSG (classic indicators of an oxidative-stress response) were observed in the liver (Shen et al., 2005). Given that the changes in thiol concen- trations were observed in the wild-type mice, but not in mice that lack the AhR, the TCDD response appears to be AhR-dependent. Furthermore, the TCDD- inducible oxidative stress response appears to involve localization of CYPs nor- mally present in the endoplasmic reticulum of the mitochondria (Genter et al., 2004). Similar increases in ROSs and oxidative DNA damage have been reported when primary rat hepatocytes were used (Knerr et al., 2006). Changes in lipid peroxidation, hemoglobin oxidation, and catalase and glutathione peroxidase activity (measures of oxidative stress) in human RBCs are also consistent with the idea that TCDD induces oxidative stress (Bukowska, 2004b). In fact, it has been proposed that the TCDD-induced increase in intracellular oxygen-species formation may underlie its ability to increase the cytotoxic actions of mitomycin C used in chemotherapeutic treatment for solid tumors (Collier et al., 2006). In contrast, it has been reported that in a CHO cell line, TCDD failed to increase oxidative stress (Chan et al., 2004a) but increased the frequency of DNA double- strand break repair (Chan et al., 2004b) and homologous recombination (Chan et al., 2004a). In vivo evidence that TCDD exposure may lead to chromosomal abnormali- ties was found in studies of sheep ï¬ocks that reside in the provinces of Naples and Caserta, Italy (Perucatti et al., 2006). Animals with high levels of dioxins ex- hibited a higher rate of chromosomal abnormalities (aneuploidy, gaps, chromatid breaks, chromosomal breaks, and fragments) than control sheep raised away from the control area. A follow-up study of two ï¬ocks that had higher concentrations of dioxin (milk mass of human World Health Organization TCDD equivalent, 50.65 and 39.51 pg/g of fat in the two exposed herds) similarly reported higher rates of increases in chromosomal abnormalities and sister-chromatid exchange in the exposed group than in the control animals (Perucatti et al., 2006). Finally, in a study of two human patients inadvertently exposed to TCDD (TCDD blood concentrations, 85,600 and 17,700 pg/g of blood lipids 4 months after intoxication was diagnosed), the presence of genotoxic events was evaluated by measuring micronuclei, sister-chromatid exchange, and DNA strand breaks (comet assay tail factor) (Valic et al., 2004). High concentrations of micronuclei (16.0 and 21.8 per 500 binucleated cells compared with the laboratory baseline of 3.7 0.6 per 500) and comet assay tail factor (33.5 and 4.6 percent compared with the laboratory baseline of 3.6 percent 0.5 percent) were observed, but
104 VETERANS AND AGENT ORANGE: UPDATE 2006 sister-chromatid exhange values remained within the normal range. The values of micronuclei and comet assay tail factor were transient and after 13 months were reported to be nearly normal. Resistance to Growth Inhibition As discussed in Update 2004, TCDD has been found to alter cell-cycle progression. In most cases, activation of the AhR by TCDD results in antiproliferative effects that are thought to be mediated by an induction of cell-cycle arrest (Huang and Elferink, 2005), or by inhibition of either insulin-induced (Park et al., 2004), estrogen-induced (Oenga et al., 2004), or androgen-induced (Barnes-Ellerbe et al., 2004) proliferation. However, the effect of TCDD on cell proliferation may be cell-dependent or dose-dependent inasmuch as TCDD has also been reported to increase proliferation of nontumori- genic human breast luminal epithelial cells (Ahn et al., 2005). Alternatively, the effect of TCDD on cell proliferation may depend on the stage of their conï¬uence in the culture dish. For example, recent data (VondrÃ¡Ë ek et al., 2005) are consis- c tent with those reported previously (Hoelper et al., 2004; Milstone and Lavigne, 1984; Ray and Swanson, 2003) in demonstrating that TCDD induces proliferation in contact-inhibited cells. Effect of TCDD on Apoptosis As reported in Update 2004 (IOM, 2005), TCDD has been shown to exert antiapoptotic effects. The effect of TCDD on apoptosis appears to depend on whether apoptosis is induced by the intrinsic or extrinsic pathways. For example, although TCDD has been shown to inhibit apoptosis induced by either the genotoxin diethylnitrosoamine (Paajarvi et al., 2005) or ultraviolet radiation (Park and Matsumura, 2006)âthe instrinsic apop- totic pathwayâthe presence of a functional AhR promotes apoptosis stimulated by FasLâthe extrinsic pathway (Park et al., 2005). Effect of TCDD on Immortalization The immortalization process involves primarily inactivation of the Rb/p16Ink4a pathway, inactivation of the p53 path- way, and activation of telomerase. As reported in Update 2004 (IOM, 2005), TCDD has been shown to extend the life span of normal human keratinocytes by a mechanism that appears to involve inhibition of senescence and silencing of p53 and p16Ink4a (Ray and Swanson, 2003). A recent report indicates that TCDD may also affect the immortalization process favorably by inducing telomerase activity (Sarkar et al., 2006). Effect of TCDD on Promoting Independence from Mitogenic Stimula- tion Mitogens, such as ligands of the epidermal growth factor, exert their growth-stimulating effects by initiating signal-transduction cascades that ulti- mately result in activation of the immediate early gene family. As mentioned previously, TCDD has been shown to increase the activity of a number of kinases: c-scr (Backlund and Ingelman-Sundberg, 2005), the MAPKs (Chen et al., 2005;
TOXICOLOGY 105 Shibazaki et al., 2004; Tan et al., 2004), and protein kinase C (Kayano et al., 2004; Machemer and Tukey, 2005; Minsavage et al., 2004; Puebla-Osorio et al., 2004). Additional means by which TCDD can contribute to activation of this pathway include upregulation of the expression of the mitogen epiregulin (Patel et al., 2006) and the transcription factor EGR1 (Martinez et al., 2004). Effect of TCDD on Angiogenesis TCDD has been shown to inhibit angiogen- esis in diverse models, such as during ï¬n regeneration in the zebraï¬sh (Zodrow and Tanguay, 2003; Zodrow et al., 2004) and during embryonic development in the chick embryo (Ivnitksi-Steele and Walker, 2003), and this inhibition is as- sociated with downregulation of vascular endothelial growth factor (VEGF) and other genes involved in vascularization. The degree to which CYP1A1 induction or oxidative stress in the vascular endothelium contributes to inhibition of angio- genesis remains unclear. Recent studies further demonstrate that TCDD or other AhR agonists disrupt neovascularization in various models, including suppres- sion of vascular remodeling in the placenta during late gestation (Ishimura et al., 2006), reduction of responsiveness of endothelial cells to angiogenic stimuli in the chick embryo (Ivnitski-Steele et al., 2005), and inhibition of endothelial cell proliferation and tube formation in vitro after VEGF stimulation (Ivnitski-Steele and Walker, 2005; Juan et al., 2006). The degree to which TCDD inhibition of angiogenesis affects its carcinogenic potential remains to be elucidated. Effect of TCDD on Tumor Invasion and Metastasis The ï¬nal stage of the cancer process requires alterations in cellâcell contact and cellâmatrix adhesion (a transition from the epithelial to mesenchymal cellular structure), an increase in cell migration, and invasion into neighboring tissue and metastasis (Christofori, 2006). Gene pathways that play important roles include those of transforming growth factor (TGF ), matrix metalloproteinases, and osteopontin. A role of the AhR in regulating the migration process is supported by recent ï¬ndings that lack of the AhR leads to impaired migration of xenotransplanted tumors (Mulero- Navarro et al., 2005). Lack of the AhR also results in overexpression of a negative regulator of TGF , latent TGF binding protein-1 (Corchero et al., 2004), and a decrease in TGF activity (Gomez-Duran et al., 2006). Furthermore, TCDD has been shown to increase the invasiveness of cultured melanoma cells (Villano et al., 2006) and the expression of matrix metalloproteinases (Haque et al., 2005; Murphy et al., 2004; Villano et al., 2006) in several cultured cell lines. Finally, in mice that express a constitutively active form of the AhR, downregulation of os- teopontin that correlated with the development of stomach tumors was observed (Kuznetsov et al., 2005). Taken together, those ï¬ndings indicate that activation of the AhR by TCDD can facilitate ï¬ve of the six steps required for the develop- ment of human cancers.
106 VETERANS AND AGENT ORANGE: UPDATE 2006 Effects on the Male Reproductive Organs Effects on the Testis Many effects of TCDD in male rodents have been re- ported previously, including decreases in the size of the accessory sex organs and daily sperm production. Both the AhR and ARNT are expressed in rat and human testis, and studies suggest that TCDD causes tissue damage by induction of oxidative stress (IOM, 2005). In a recent study, Khorram et al. (2004) demon- strated that human sperm express abundant AhR and ARNT mRNA; other studies have identiï¬ed gene targets of TCDD in the testis that may mediate toxicity. Yamano et al. (2005) identiï¬ed the novel spermatogenesis-related factor-2, which is expressed primarily in the spermatocyte and which was signiï¬cantly decreased in the rat testis after TCDD exposure of neonatal rat pups. Kuroda et al. (2005) found that the mouse homologue of the Drosophila wapl (wings apart-like) gene was expressed exclusively in murine testis and that its expression was suppressed by TCDD exposure. Lai et al. (2005a) showed that exposure of primary Sertoli cells in culture to TCDD signiï¬cantly induced P450 aromatase and sertolin mRNA, increased estra- diol secretion, and suppressed MÃ¼llerian-inhibiting substance and testin mRNA expression, although these changes required TCDD doses 100â1,000 times those needed to induce CYP1A1. Lai et al. (2005b) also showed that exposure of primary Sertoli cells to TCDD signiï¬cantly reduced P450 side-chain cleavage expression, progesterone secretion, and hCG-stimulated testosterone secretion. TCDD exposure of the medaka demonstrated signiï¬cant histologic changes in the testis, including disorganized spermatogenesis, Leydig cell swelling, and Sertoli cell vacuolation (Volz et al., 2005). Those changes were not associated with CYP1A1 induction but were associated with signiï¬cant changes in gene ontology pathways, including proteolysis, signal transduction, metabolism, cell proliferation, and cell motility. Two studies investigated the effects of TCDD on spermatogenesis and erec- tile function in vivo. Simanainen et al. (2004a) exposed three rat lines, which vary in their sensitivity to TCDD, to a single dose of TCDD at 0, 30, 300, or 3,000 g/kg and analyzed effects on male reproductive organs 17 days later. The highest dose of TCDD reduced serum testosterone equally in all three rat lines, but the reduction in spermatogenesis was smaller in two more resistant lines. In a second study, TCDD also inhibited spermatogenesis in rabbits treated with 1.0 g/kg, and this was associated with reduced contractile and relaxation responses in smooth muscle isolated from erectile tissue (Moon et al., 2004). One study also demonstrated that TCDD-induced effects on sperm require the AhR and are mediated by ROSs (Fisher et al., 2005b). Exposure of C57BL/6 male mice to TCDD at 0, 0.1, 1.0, 10, or 50 g/kg induced a dose-dependent loss of mitochondrial membrane potential in AhR-wild-type mice but not in AhR-null mice. TCDD increased sperm concentrations of ROSs, and treatment with an antioxidant prevented the increase in ROSs and the loss of mitochondrial membrane potential.
TOXICOLOGY 107 Effects on the Prostate Prostate cells and prostatic-cancer cell lines are re- sponsive to TCDD in induction of various genes, including those involved in drug metabolism. Simanainen et al. (2004b) used different rat lines (TCDD-resistant Hans/Wistar and TCDD-sensitive Long Evans) and showed that TCDD treat- ment resulted in a signiï¬cant decrease in the weight of prostate lobes; however, the effect did not appear to be line-speciï¬c. In contrast, the TCDD reduction in sperm does appear to be line-speciï¬c and not fully related to the effects of TCDD on serum testosterone (Simanainen et al., 2004a). TCDD effects appear to occur through actions on the urogenital sinus (Lin et al., 2004). In utero and lactational exposure to TCDD appears to retard the aging process in the prostate (Fritz et al., 2005). As mentioned in Update 2004, exposure of human prostatic-cancer cell line 9LnCaP to TCDD inhibits androgen-dependent growth. Further probing of the mechanisms that underlie that effect indicate that TCDD alters cell-cycle regula- tory proteins by blocking androgen-induced hyperphosphorylation of retinoblas- toma protein, reducing cyclin D, and inducing p21 expression (Barnes-Ellerbe et al., 2004). Effects on the Female Reproductive Organs The ovaries of experimental animals provide targets for the action of TCDD. The ovary expresses both the AhR and ARNT and is responsive to TCDD- inducible CYP1A1 and 1B1 expression, which depends on the phase of the estrous cycle (IOM, 2005). Earlier studies have established that TCDD alters ovarian steroidogenesis, reducing ovarian expression of luteinizing and follicle- stimulating hormone receptors, reducing circulating progesterone and estradiol, and decreasing fertility (IOM, 2005). Bussman and BaraÃ±ao (2006) characterized the regulation of AhR expression in rat granulosa cells by the endogenous hormones follicle-stimulating hormone and estradiol and by the exogenous AhR ligand -naphthaï¬avone. Both follicle- stimulating hormone and estradiol reduced AhR protein and mRNA expression in a time-dependent manner, and -naphthaï¬avone induced a rapid decrease in AhR protein via proteasomal degradation but also induced a delayed increase in AhR mRNA after prolonged exposure. Two new studies further elucidate TCDD-induced changes in gene expression and function in the ovary. Exposure of rat granulosa cells to 100 pM TCDD for 24 hours signiï¬cantly suppressed expression of genes that are essential to ovarian function, including luteinizing-hormone receptor and P450 side-chain cleavage, the latter required for ovarian steroidogenesis (Miyamoto, 2004). Hombach- Klonisch et al. (2006) determined that exposure of oviduct epithelial cells to 0, 0.1, 1.0, or 10 nM TCDD downregulated the amount of ER in the nucleus and suppressed ER-dependent signaling in a dose-dependent manner. Three recent studies have shown that TCDD reduces fertility and repro- ductive success. Chronic dietary exposure of female zebraï¬sh to TCDD for 20
108 VETERANS AND AGENT ORANGE: UPDATE 2006 days decreased serum estradiol and serum vitellogenin, and these changes were associated with a decreased number of follicles and increased number of atretic follicles, which probably accounted for more than a 50 percent decrease in egg production (Heiden et al., 2006). Li et al. (2006) showed that TCDD accumulated in the uterus of pregnant mice to the same degree as in the liver, and this expo- sure during early pregnancy reduced circulating progesterone and the numbers of implanted embryos and implantation sites. Franczak et al. (2006) reported that a single TCDD exposure of female rats before puberty delayed the onset of puberty and induced premature reproductive senescence; life-long TCDD expo- sure resulted in a loss of cyclicity and accelerated the transition to reproductive senescence. Previous studies had shown that TCDD-induced placental dysfunction, including placental hypoxia, contributes to reduction in reproductive success (Ishimura et al., 2002). A recent study expands on that observation. TCDD- induced fetal death was associated with delayed disappearance of glycogen cells and the presence of cysts at the placental junctional zone when pregnant rats were exposed on gestation day (GD)15 (Kawakami et al., 2006). Pregnant Holtzman rats were signiï¬cantly more susceptible to those effects than Sprague-Dawley rats, but the sensitivity was not a result of sequence differences in the AhR or of inducibility of placental CYP1A1. Ishimura et al. (2006) showed that a single TCDD dose to pregnant Holtzman rats on GD15 suppressed vascular remodeling in the labyrinth zone of the placenta during late gestation, which was associated with decreased expression of Tie2, a gene associated with vascular remodeling. The lack of remodeling resulted in constriction of fetal capillaries in the placenta. Global gene-expression proï¬ling of the placentas of TCDD-exposed pregnant Holtzman rats demonstrated that TCDD strongly induced glucose transporters, interferon-inducible genes, and antiangiogenic cytokines (Mizutani et al., 2004). Effects on the Uterus Update 2004 (IOM, 2005) reported that TCDD decreased uterine weight, altered endometrial structure, and blocked estrogen-mediated endometrial proliferation and hypertrophy via an AhR-dependent mechanism in rodents and increased the incidence of endometriosis in rhesus monkeys. There have been some additional studies regarding TCDDâs effects on the uterus and the development of endometriosis. Mueller et al. (2005) showed that glycodelin, a glycoprotein with contraceptive and immunosuppressive effects, was a direct AhR-mediated target of TCDD in human endometrial endothelial cells and that TCDD exposure signiï¬cantly increased gene transcription and protein secre- tion. In another study, uterine gene expression was examined in ovariectomized C57BL/6 mice that were treated with TCDD, ethynylestradiol (EE), or both (Boverhof et al., 2006). Of the 281 genes regulated by TCDD, 228 were also regulated by EE, but TCDD-mediated responses temporally lagged behind EE responses. An ER antagonist blocked the estrogen-like gene expression induced
TOXICOLOGY 109 by TCDD, and this suggested that the responses are ER-mediated. Kitajima et al. (2004) demonstrated that TCDD blocked an estrogen-induced increase in pro- liferation of a pre-existing endometriotic lesion and signiï¬cantly increased the expression of both the estrogen receptor and the AhR in mice. In an earlier review of the literature on endometriosis in humans and non- human primates, Guo (2004) concluded that there was insufï¬cient evidence to support the hypothesis that dioxin exposure leads to the development of endo- metriosis. One recent study provides some new correlative evidence of a link between TCDD and endometriosis. Igarashi et al. (2005) showed that TCDD exposure of normal human endometrial stromal cells signiï¬cantly reduced the ratio of the expression of progesterone receptor B (PR-B) to that of progesterone receptor A (PR-A). TCDD also blocked the ability of progesterone to suppress matrix metalloproteinase (MMP) expression, speciï¬cally MMP-3 and MMP-7. Both the reduced PR-B:PR-A ratio and the resistance to progesterone-mediated MMP suppression are observed in endometrial tissue from women who have endometriosis. Effects on the Mammary Gland As discussed in Update 2004, TCDD ex- posure disrupts mammary gland differentiation and lactation, reduces formation of primary and lateral branches of the mammary glands, decreases epithelial elongation, and decreases the number of alveolar buds. The mechanism of those effects has not been determined. However, because the effects preceded hormone- induced events, it has been proposed that altered hormone concentrations were unlikely to have been a mechanism of impaired mammary development. More recent investigations into the mechanisms that regulate branching morphogenesis have revealed that expression of the AhR, ARNT, and CYP1A1 and CYP1B1 is associated with the extracellular matrix interactions that promote cellâcell and cellâextracellular matrix adhesion (Larsen et al., 2004). Human breast-cancer cells have been useful in investigations of the mecha- nisms of AhR signaling and of the effects of TCDD on hormone-induced re- sponses, especially responses to estrogen. Previous updates reported that TCDD blocks many estrogen-induced responses in human breast-cancer cells. Recent data have demonstrated that TCDD-activated AhR inhibits the ability of the ER to upregulate the trifunctional carbamoylphosphate synthetase/aspartate trans- carbamylatransferase/dihydroorotase (CAD) gene that mediates some of the effects of estrogen on cell-cycle progression (Khan et al., 2006). Additional work has shown that ER and the TCDD-activated AhR directly interact and thereby modulate the ability of the AhR to upregulate CYP1A1 (Beishlag and Perdew, 2005; Matthews et al., 2005). TCDD-activated AhR was also shown to inhibit the ability of estrogen to upregulate ER and (Kietz et al., 2004). Finally, TCDD-activated AhR was shown to inhibit the ability of estrogen to upregulate tumor-suppressor gene BRACA-1 (Hockings et al., 2006). TCDD has previously been shown to alter growth, differentiation, and apop-
110 VETERANS AND AGENT ORANGE: UPDATE 2006 tosis of cultured breast epithelial cells. Along those lines, Park et al. (2004) have shown that the ability of TCDD to inhibit growth and differentiation of MCF10A (mammary epithelial) cells appears to involve TCDD inhibition of insulin signaling and c-Src kinase and ERK activation (Park et al., 2004). With respect to TCDD effects on apoptosis in those cells, more recent data have shown that the antiapoptotic actions of TCDD also involve c-Src/ERK signaling (Park and Matsumura, 2006). Most previous studies have reported that TCDD exerts antiproliferative effects on breast epithelial cells, but it has been by using cells with stem-cell and luminal characteristics that the effects of TCDD followed a U-shaped curve: with 1 nM TCDD, cell proliferation increased, but cell prolif- eration decreased at higher concentrations of TCDD. TCDD was also shown to increase the ability of the cells to grow in soft agarâan indicator of anchorage- independent growth (Ahn et al., 2005). Kanno et al. (2006) have shown that over- expression of the AhR target gene, AhRR, inhibits the growth of breast-cancer cells; this may be an additional mechanism by which TCDD activation of the AhR inhibits proliferation of breast-cancer cells (Kanno et al., 2006). Finally, the AhR target genes CYP1A1 and CYP1B1 can metabolize - estradiol to metabolites, some of which can act as tumor initiators. Recent data indicate that those reactions may be important in increasing formation of ROSs and oxidative damage in breast-cancer cells (Chen et al., 2004). Endocrine and Other Effects TCDD and related compounds affect the thyroid and thyroid hormones in several animal species (IOM, 2003, 2005) and affect other endocrine organs, such as the pituitary and adrenal gland (IOM, 2005). Previously proposed mechanisms of altering thyroid hormone function include the displacement of hormones from serum transport proteins, the alteration of deiodinase activity, and an increase in thyroid hormone catabolism via glucuronidation (IOM, 2005). A recent study showed that TCDD signiï¬cantly alters expression of enzymes necessary for thyroid hormone biosynthesis and release (Pocar et al., 2006). TCDD exposure of primary porcine thymocytes signiï¬cantly downregulated mRNA expression of the sodium iodide symporter, which is required for accumulation of iodide in the thyroid, and cathepsin B, which is required for proteolysis of thyroglobulin and release of thyroid hormones from the thyroid gland. Nishimura et al. (2005a) demonstrated that lactational but not in utero ex- posure was responsible for a TCDD-induced decrease in serum thyroxin and increase in thyroid-stimulating hormone in offspring. That disruption of thyroid hormone homeostasis required expression of the AhR; TCDD exposure of AhR- null pups had no effect on thyroid hormone levels (Nichimura et al., 2005b). Elango et al. (2006) showed that TCDD exerted estrogenic effects on the rainbow trout pituitary by stimulating a concentration-dependent increase in growth hormone and prolactin mRNA expression.
TOXICOLOGY 111 Developmental Toxicity Extensive data from studies in animal experiments suggest that developing tissues are highly sensitive to the toxic effects of TCDD, as mediated by the AhR, and that tissue growth and differentiation processes are affected. Recent publications are consistent with that. All the following are studies in which effects of TCDD on the developing embryo or fetus were investigated after maternal exposure to TCDD; they include an array of animal models and a variety of ex- perimental endpoints. Since Update 2004, no studies in which the effect of TCDD on the fetus was investigated after paternal exposure have been published. Elimination of TCDD from the Embryo It is notable that previous studies have shown that several frog species are relatively insensitive to the developmen- tal toxicity of TCDD (Jung and Walker, 1997). Philips et al. (2006) investigated whether the insensitivity might result from rapid elimination of the toxicant. They found that although 1-month-old tadpoles exhibited fast elimination of TCDD, frog embryos exhibited very little elimination of TCDD; mechanisms other than TCDD elimination might account for the insensitivity of frog species to TCDD- induced developmental toxicity. Effects on Cardiovascular Structure, Function, and Gene Expression Since Update 2004, several reports have detailed the role of AhR signaling and the effects of TCDD on the developing cardiovascular system. Developmentally, the AhR plays a normal role in vascular remodeling and, in particular, is required for normal closure of the hepatic ductus venosus (Lahvis et al., 2000). A recent study in which the AhR was genetically deleted only from hepatocytes or only from endothelial cells demonstrated that AhR expression in endothelial cells was necessary for developmental closure of the ductus venosus (Walisser et al., 2005). That study illustrates the requirement for endothelial-cell AhR signaling during development. Not surprisingly, the vascular endothelium of the developing embryo also has been identiï¬ed as a primary target of TCDD toxicity, and studies cited in Update 2002 and Update 2004 (IOM, 2003, 2005) conclude that circu- latory failure, oxidative stress in vascular endothelial cells, and abnormal angio- genesis are primary events that mediate the toxicity. Further study has shown that reduced angiogenesis in the chick embryo after TCDD exposure is associated with reduced secretion of VEGF and reduced responsiveness of endothelial cells to angiogenic stimuli (Ivnitski-Stele et al., 2005). Garrick et al. (2005) showed that endothelial cells isolated from differ- ent microvascular beds of the eel exhibited signiï¬cantly different sensitivity to TCDD-induced cytochrome P450 1A activity. Cardiac endothelial cells exhibited the highest induction response, renal endothelium was signiï¬cantly lower, and rete mirabile endothelium was virtually unresponsive. A number of new studies have investigated the effects of TCDD exposure
112 VETERANS AND AGENT ORANGE: UPDATE 2006 on the morphogenetic development of the heart. Exposure of zebraï¬sh embryos to TCDD immediately after fertilization reduced heart size and cardiac myocyte cell number and induced cardiac structural malformations and abnormal looping (Antkiewicz et al., 2005). The structural changes were associated with reduced ventricular contraction that progressed to ventricular standstill. The observed cardiotoxicity occurred within 72 hours of fertilization and before circulatory failure, suggesting that the heart is a direct target of TCDD. Similar effects were described in zebraï¬sh larvae exposed to TCDD 3 days after fertilization (Carney et al., 2006); this exposure regimen decreased perfusion of intersegmental vessels, cardiac stroke volume and output, and cardiac ejection fraction within 8â12 hours of exposure. The changes were later associated with pericardial edema, altered cardiac looping, and decreased myocyte number by 24 hours after exposure. Two additional studies using antisense morpholinos showed that zebraï¬sh ARNT1 is required for the developmental cardiovascular toxicity of TCDD (Prasch et al., 2006) but that induction of CYP1A is not required (Carney et al., 2004). The latter ï¬nding contrasts with an earlier study by Teraoka et al. (2003) that showed that knocking down CYP1A expression by antisense morpholinos protected against TCDD-induced developmental toxicity. The reasons for the discrepancy between the two studies remain to be elucidated but may be related to differences in the timing of assessment of the speciï¬c toxic endpoints. Developmental exposure of chick embryos and murine fetuses to TCDD has shown effects on cardiac chronotropic responses. Electrocardiographic (ECG) recordings in chick embryos in ovo after TCDD exposure showed an increased incidence of arrhythmias, normal basal heart rate, and decreased responsiveness to -adrenergic-stimulated tachycardia (Sommer et al., 2005). Additional data suggest that those changes resulted from alterations in signal transduction up- stream of adenylyl cyclase. In contrast, ECGs of mouse pups after in utero and lactational TCDD exposure demonstrated signiï¬cant reductions in basal heart rate but normal chronotropic responses to -adrenergic stimulation (Thackaberry et al., 2005b). Three studies have conducted global gene-expression proï¬ling of the devel- oping heart after TCDD exposure in an attempt to identify changes that mediate cardiotoxicity. In 3-day-old zebraï¬sh larvae, signiï¬cant alterations in expression of cardiac sarcomere and mitochondrial energy-transfer components after em- bryonic exposure to TCDD were identiï¬ed, including induction of cardiomyosin light chains 1 and 2, myosin heavy chain, and cytochromes b and c (Handley- Goldstone et al., 2005). Those changes are consistent with the dilated cardiomy- opathy phenotype observed in piscine and avian embryos. A similar study exposed 3-day-old zebraï¬sh larvae to TCDD for 1 hour and found signiï¬cant induction of xenobiotic metabolism genes within 1 hour and signiï¬cant repression of genes that regulate cell division and proliferation after 12 hours, corresponding to the onset of TCDD-induced cardiac toxicity (Carney et al., 2006). In murine fetuses on GD 17.5, 3 days after TCDD exposure, signiï¬cant alterations were identiï¬ed
TOXICOLOGY 113 in expression of cell-cycle and extracellular-matrix genes, including induction of two matrix metalloproteinases and downregulation of cyclins associated with the progression from G1 to S phase (Thackaberry et al., 2005a). Those changes are consistent with reduced myocyte proliferation, thinner ventricle walls, and low heart size observed in piscine, avian, and murine fetuses (Antkiewicz et al., 2005; Ivnitski et al., 2001; Thackaberry et al., 2005b; Walker and Catron, 2000). Effects on Male Reproductive Structure, Function, and Gene Expres- sion Previous updates cited several reports that indicated that development of the male reproductive system is exceptionally sensitive to in utero and lactational TCDD exposure. Those effects have included impaired development of the pros- tate that is lobe-speciï¬c, with the ventral prostate exhibiting absence of branching morphogenesis and the dorsal, lateral, and anterior prostate exhibiting inhibi- tion of duct formation (IOM, 2005). More recent work has shown that TCDD inhibition of murine ventral prostate development after in utero and lactational exposure persisted into senescence as determined at the ages of 100 and 510 days (Fritz et al., 2005). Castration of TCDD-exposed mice at senescence resulted in a more signiï¬cant reduction in prostate weight and androgen-dependent gene expression than castration of control mice; TCDD inhibits the normal aging of the prostate when the organ becomes increasingly androgen-independent with age. In another study, in utero and lactational TCDD exposure of rats reduced ventral prostate weight without affecting the weight of other male reproductive organs or reducing sperm number. However, when the TCDD-exposed male offspring were mated to unexposed females, the percentage of male pups in F2 generation (38/100, 38 percent) was signiï¬cantly lower than female pups (62/100, 62 percent; p 0.05), compared to the sex ratio produced from unexposed male ones (male pups, 83/159, 52 percent; female pups, 76/159, 48 percent) (Ikeda et al., 2005a). Yonemoto et al. (2005) related the maternal dose that signiï¬cantly reduces ventral prostate weight postnatally to the maternal body burden and fetal concentration of TCDD 1 day after dosing; they found that a maternal dose of 800 ng/kg on GD15 resulted in a maternal body burden of 290 pg/g and a fetal concentration of 52 pg/g on GD16. Lin et al. (2004) demonstrated that the urogenital sinus (UGS), from which the prostatic epithelial bud is derived, is a direct target of TCDD requiring the AhR. TCDD exposure of the UGS in organ culture prevented prostatic epithelial buds from forming in the UGS isolated from AhR-wild-type mice but not in the UGS from AhR-null mice. Effects on Fetal and Postnatal Steroidogenesis The developmental toxicity of TCDD also includes alterations in fetal and postnatal steroidogenesis. Pregnant Wistar rats were administered a single oral dose at 0, 0.01, 0.1, or 1.0 g/kg TCDD on GD15, and expression of steroidogenetic enzymes and steroid recep- tors was analyzed in the fetal testis on GD20 (Mutoh et al., 2006). Only the
114 VETERANS AND AGENT ORANGE: UPDATE 2006 highest dose of TCDD reduced mRNA expression of steroid acute regulatory protein (StAR); 3 -hydroxysteroid dehydrogenase; ER ; androgen receptor; cytochromes P450 CYP11A1, CYP17, and CYP11B1; and SF-1, a transcription factor that positively regulates StAR, while there was no effect of the lower doses. In addition, expression of circulating luteinizing hormone (LH) was reduced in the fetal serum following exposure of pregnant dams to 1.0 g/kg TCDD. Ad- ministration of chorionic gonadotropin on GD17 prevented the reduction in fetal testis StAR mRNA induced by maternal exposure to 1.0 g/kg TCDD, while 100 nM TCDD had no effect on StAR mRNA expression when the fetal testis was exposed in organ culture. These data suggest that TCDD may impair fetal steroidogenesis by targeting pituitary gonadotropins. In another study, pregnant Sprague-Dawley rats were given a single oral dose of 0, 0.04, 0.2, or 1.0 g/kg TCDD on GD13, and steroidogenic outcomes were evaluated in male and female pups postnatally (Haavisto et al., 2006; Myllymaki et al., 2005). In male offspring, plasma testosterone was increased on postnatal day (PND)10 at the highest TCDD dose, but unchanged on PND14 at all TCDD doses. Additionally, neither LH nor follicle-stimulating hormone concentrations were affected in male offspring at any dose of TCDD. In contrast, a maternal dose of 1.0 g/kg TCDD reduced plasma estradiol in female pups on PND14 and PND16, but increased plasma LH and follicle-stimulating hormone at PND14 without affecting plasma progesterone. Ovarian expression of StAR and cytochrome P450 aromatase mRNA was reduced on PND14, but only by the 0.2 g/kg TCDD, while P450 aromatase activity was reduced in isolated ovarian follicles at the 1.0 g/kg TCDD dose. Effects on Avian Reproductive Development TCDD affects the reproductive systems of birds after in ovo exposure. Bruggeman et al. (2006) showed that ex- posure of fertile chicken eggs to 20 ng of TCDD before incubation signiï¬cantly increased expression of 60-kDa heat-shock protein and decreased expression of regucalcin in the hatchling ovary. That exposure also reduced hatchability and body-weight gain of hatchlings and resulted in the retention of the right oviduct in laying hens, which usually regresses during late embryonic development (Bruggemen et al., 2005). However, there were no effects on circulating steroid hormones or laying performance. Effects on Brain Sexual Differentiation, Structure, and Gene Expression on Neurobehavior Several reports of studies in animals and exposed humans suggest that perinatal exposure to TCDD or to dioxin-like compounds can im- pair brain development and induce neurobehavioral deï¬cits. Update 2004 (IOM, 2005) reported that perinatal TCDD exposure decreased neuron number and re- duced or reversed sexual dimorphic brain development, and these changes were associated with altered sexual behavior and learning. Recent studies conï¬rm and extend those observations. Nayyar et al. (2003) found that offspring of F-344 rats
TOXICOLOGY 115 that were exposed to TCDD at 0 or 700 ng/kg on GD15 exhibited a signiï¬cant decrease in the hippocampal expression of the N-methyl-D-aspartate receptor subtype 1 mRNA and protein. Chang et al. (2005) exposed pregnant Sprague- Dawley rats to a single dose of TCDD at 0 or 2 g/kg on GD15 and analyzed ex- pression of Bcl-2 gene family members in the brain of offspring on PND0 and at the age of 4 months. On PND0, expression of the anti-apoptotic gene Bcl-xL was increased in the cerebral cortex and decreased in the cerebellum of male rats; this pattern was reversed in female rats. At 4 months, expression of the antiapoptotic gene Bcl-2 was increased in the cerebellum of both male and female rats but was increased in the cerebral cortex only of female rats. The variable patterns of expression of these genes indicate that early exposure of dioxin could effect the development of certain brain regions with gender differences. Hojo et al. (2006) administered TCDD to pregnant Sprague-Dawley rats at 0 or 180 ng/kg on GD8 and found that it altered cortical-cell size distribution only in male rats, increasing the number of small cells and decreasing the number of large cells. The perinatal exposure also induced a reversal of hemisphere lateral- ization in cell number in both male and female rats. Altered sexual differentiation of the brain was also described in offspring of pregnant Holtzman rats given TCDD at 0, 200, or 800 ng/kg on GD15 (Ikeda et al., 2005b). The sex ratio of brain aromatase activity (male:female) was sig- niï¬cantly decreased on PND2, and the volume of the sexual dimorphic nucleus was signiï¬cantly reduced in male rats on PND98. Those changes were associated with demasculinized behavior in male rats as reï¬ected in increased consumption of a saccharin solution. Altered brain development after perinatal TCDD exposure was associated with other behavioral changes later in life. Rainbow trout swim-up fry that had been exposed to TCDD as newly fertilized eggs exhibited decreased densities of retinal ganglion and corresponding deï¬cits in visual and motor function that resulted in decreased prey-capture rate (Carvalho and Tillitt, 2004). Exposure of pregnant Wistar rats to TCDD at 0 or 1 g/kg on GD15 altered response to contextual fear conditioning in adult male offspring (Mitsui et al., 2006); this response was associated with a decreased percentage of neurons in the hippocam- pus that express phosphorylated cyclic AMP response-element-binding protein, a transcription factor associated with learning and memory. Negishi et al. (2006) evaluated the social behavior of surviving offspring of rhesus monkeys that had been exposed in utero and via lactation to TCDD at such a dosage that maternal body burdens were maintained at 300 g/kg. Perinatal TCDD exposure had no effect on learning or interest in or hostility to an observer but signiï¬cantly increased visual exploration and mutual proximity during a ï¬rst-encounter test. To begin to elucidate changes in fetal gene expression that may account for alterations in fetal brain development, Fujita et al. (2006) exposed pregnant C57BL/6 mice to TCDD at 20 g/kg on GD7 and analyzed global gene expres-
116 VETERANS AND AGENT ORANGE: UPDATE 2006 sion in the fetal brains on GD12. TCDD exposure signiï¬cantly altered the ex- pression of 40 genes, including induction of xenobiotic metabolizing enzymes CYP1A1 and 1B1 and suppression of transcription factors associated with human morphogenetic malformations. Effects on Craniofacial Development A number of studies have reported de- tailed morphology and mechanisms of TCDD-induced jaw, palatal, and subman- dibular gland defects. Yamada et al. (2006) treated pregnant A/J, C57BL/6, and ICR mice with a single dose of TCDD at 0, 10, 20, or 40 g/kg and assessed the timing and incidence of cleft palate and lip. The potency of TCDD-induced cleft palate was similar in C57BL/6 and ICR mice and occurred at a high incidence in the absence of fetal mortality. In contrast, the doseâresponse relationships for cleft-palate incidence and fetal mortality were similar in A/J mice. TCDD induced the highest incidence of cleft palate when administered in the period GD11.5â12.5 but failed to induce cleft lip. Branching morphogenesis and cleft formation of the salivary gland was signiï¬cantly impaired in cultured murine GD13 submandibular gland exposed to 0.1, 1.0, or 2 M TCDD (Kiukkonen et al., 2006). Neither epidermal growth factor nor ï¬bronectin could rescue this abnormal morphogenesis. Teraoka et al. (2006) found that zebraï¬sh embryos exposed to TCDD at 0, 0.3, 0.5, or 1.0 ppb 24 hours after fertilization exhibited reduced expression of sonic hedgehog a and b mRNA in the upper and lower jaw; expression was res- cued by antisense morpholinos against AhR2. Cell proliferation in the developing jaw was also signiï¬cantly reduced by TCDD. Cleft palate induced by TCDD in fetal C57BL/6 mice in utero or in palatal shelves in organ culture was characterized by a reduced number of ï¬lopodial extensions and increased cellularity at the medial epithelial edge resembling the phenotype observed in transforming growth factor 3-null (Tgf 3-null) mice (Thomae et al., 2005). Addition of Tgf 3 to a palate culture prevented the TCDD- induced reduction in ï¬lopodial outgrowth, the increased cellularity, and the palatal clefting; thus, the Tgf 3 pathway may be involved in TCDD-induced teratogenesis. Thomae et al. (2006) demonstrated that the sensitivity to TCDD- induced cleft palate in mice was signiï¬cantly inï¬uenced by a locus on chromo- some 3 and modestly inï¬uenced by a locus on chromosome 12. Effects on Thymus Differentiation and Function The effects of perinatal TCDD exposure on the differentiation of the thymus and postnatal function of the immune system have been reported in two recent studies. Besteman et al. (2005) reported that when pregnant C57BL/6 mice were exposed to TCDD at 0, 5, or 10 g/kg on GD 14 or 16, the fetal thymuses on GD 18 exhibited loss of distinction between the cortical and medullary regions, decreased thymocyte viability, and a dose-related increase in thymocyte early apoptosis. Vorderstrasse et al. (2006) studied the effects of perinatal TCDD exposure of C57BL/6 mice on immune
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