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Veterans and Agent Orange: Update 2004 (2005)

Chapter: 3 Toxicology

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Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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3
Toxicology

As in Veterans and Agent Orange: Health Effects of Herbicides Used in Vietnam (IOM, 1994; hereafter referred to as VAO), Veterans and Agent Orange: Update 1996 (IOM, 1996; hereafter, Update 1996), Veterans and Agent Orange: Update 1998 (IOM, 1999; hereafter, Update 1998), Veterans and Agent Orange: Update 2000 (IOM, 2001; hereafter, Update 2000), and Veterans and Agent Orange: Update 2002 (IOM, 2003; hereafter, Update 2002), this chapter summarizes recent experimental data that provide the scientific basis for assessment of the biologic plausibility of the effects 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 influenced by dosage (magnitude and frequency of administration); by exposure to other substances, including compounds other than herbicides; by pre-existing health status; by genetic factors; and by the route and rate of the substance’s absorption, distribution, metabolism, and excretion. Attempts to extrapolate from experimental studies to human exposure must therefore carefully consider those variables.

Many chemical compounds were used by the US armed forces in Vietnam. The nature of the substances themselves is discussed in more detail in Chapter 6 of VAO (IOM, 1994). Four herbicides documented 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 (dimenthylarsenic 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 and because

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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considerably more information is available on TCDD than is available for the herbicides. Except as noted, the laboratory studies of those compounds were done with pure formulations of the compounds. The epidemiologic studies discussed in later chapters often track exposures to mixtures.

This chapter begins with a summary of major conclusions presented in past reports. The rest of the chapter consists mostly of overviews and discussions of the relevant experimental studies that have been published since Update 2002 (IOM, 2003) on 2,4-D; 2,4,5-T; picloram; cacodylic acid; and TCDD. Within the update for each substance is a review of the toxicokinetic investigations and a summary of the toxic endpoints and their underlying mechanisms of action. The process of estimating human health risk on the basis of the animal data is then discussed.

HIGHLIGHTS OF PREVIOUS REPORTS

Chapter 4 of VAO and Chapter 3 of Update 1996, Update 1998, Update 2000, and Update 2002 review the results of animal and in vitro studies published through 2002 that investigated the toxicokinetics, mechanisms of action, and disease outcomes of exposure to the herbicides, and the contaminant TCDD, used in Vietnam. The herbicides have not been studied extensively, but in general none of them is considered particularly toxic. High concentrations usually are required to modulate cellular and biochemical processes. In contrast, experimental data reviewed in previous Agent Orange reports led those committees to conclude that TCDD elicits a diverse spectrum of sex-, strain-, age-, and species-specific effects: carcinogenesis, immunotoxicity, reproductive and developmental toxicity, hepatotoxicity, neurotoxicity, chloracne, and loss of body weight. The scientific consensus is that TCDD is not directly genotoxic and that its ability to influence the carcinogenic process is mediated by epigenetic events, such as enzyme induction, cell proliferation, apoptosis, and intracellular communication. Most if not all of TCDD’s effects are mediated through the aryl hydrocarbon receptor (AhR), which interacts with other proteins, binds to DNA, and results in enzyme induction and other biochemical effects.

TOXICITY PROFILE UPDATE OF 2,4-D

Toxicokinetics

Toxicokinetics (also called pharmacokinetics) identifies the routes and rates of uptake, tissue distribution, transformation, and elimination of a toxic substance. Those processes, in part, determine the amount of a particular substance that reaches target organs or cells to influence toxicity. Understanding the toxicokinetics of a compound is an important component for valid reconstruction of exposure.

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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Several studies have examined the pharmacokinetics and metabolism of 2,4-D in animals and humans since the publication of Update 2002. Those data support the previous conclusions that metabolism and elimination of 2,4-D are relatively rapid and that tissue uptake is small.

Van Ravenzwaay et al. (2003) compared the metabolism and elimination of 2,4-D in rats and in dogs to explain dogs’ greater sensitivity. Elimination of 2,4-D from rat plasma was significantly faster than in dogs after oral doses of 5 or 50 milligrams per kilogram (mg/kg) body weight. In the rat, excretion essentially was complete after 24 hours (h) for the low dose and after 48 h for the high dose. For the dog, only about half the dose was eliminated in 5 days. Thus, for an equivalent dosage, the body burden of 2,4-D is significantly higher in dogs, and that finding is consistent with the increased sensitivity of dogs to 2,4-D.

Notably, an interspecies pharmacokinetic analysis by Timchalk (2004) suggested that the dog is not a relevant animal for comparative evaluation of human health risk attributable to 2,4-D exposure. The plasma half-life for 2,4-D in dogs (92–106 h) is substantially longer than in rats (~1 h) or in humans (~12 h) because dogs have less efficient renal clearance mechanisms. The result is a higher body burden in dogs for a substantially longer period than is exhibited by other species. A recent study examining concentrations of 2,4-D and its metabolites in the urine of herbicide applicators was consistent with 2,4-D urinary half-life estimates of 13–40 h for humans (Hines et al., 2003).

Three studies reported that use of sunscreen and chronic consumption of alcohol could significantly increase dermal penetration of 2,4-D. Pont et al. (2004) determined that the total percentages of 2,4-D penetrating excised hairless mouse skin within a diffusion chamber in 24 h ranged from 55% for the no-sunscreen control to 87% in skin treated with sunscreen. All but one of the ingredients tested led to a significant increase in 2,4-D penetration. Penetration enhancement also occurred for human skin (Pont et al., 2004). Brand et al. (2002) observed that of nine sunscreen formulations tested, six led to significant increases in the dermal penetration of 2,4-D in hairless mice. In one case, the penetration was more than twice that of the control. The same laboratory investigated the dermal penetration of 2,4-D through the skin of rats fed either an ethanol-containing or a control diet for 6–8 weeks (Brand et al., 2004). Ethanol consumption more than doubled the rate of 2,4-D penetration. Those studies imply that people who regularly use sunscreens or consume ethanol could be at increased risk for internal exposure to and toxicity from 2,4-D.

Durkin et al. (2004) describe the development of a physiologically based pharmacokinetic (PBPK) model to estimate risk to workers who use backpack pesticide sprayers. There was good correspondence between modeled and observed elimination rates of 2,4-D in rats and humans. Although it might underestimate variability because of a lack of consideration of interindividual differences in the kinetics of 2,4-D, with further refinement, the model could result in

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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more accurate and complete assessments of risk to those who use backpack sprayers and to others who are exposed to 2,4-D.

Chemically reactive metabolites of 2,4-D are believed to mediate the hepatotoxicity of 2,4-D observed in some animal species. Li et al. (2003) determined that 2,4-dichlorophenoxyacetyl-S-acyl-CoA, a metabolite of 2,4-D, binds covalently to human serum albumin and to proteins in rat hepatocytes after in vitro incubation. The authors suggested that the metabolite contributes to induced hepatotoxicity.

Toxic Endpoints and Underlying Mechanisms of Toxic Action

Studies of disease outcomes published since Update 2002 are consistent with the earlier conclusion that 2,4-D is relatively non-toxic and has weak carcinogenic potential. The developing fetus appears to be the most sensitive for several toxic endpoints after maternal exposure. Recent animal studies of disease outcomes of 2,4-D exposure and possible mechanisms are discussed below.

Carcinogenicity and Mechanisms Related to Genotoxic Effects

Previous updates indicated little experimental evidence that 2,4-D produces any carcinogenic activity. No relevant studies on its carcinogenic effects have been published since Update 2002.

Studies reviewed in previous updates indicated that 2,4-D has no genotoxic potential, or that potential is weak, at best. Several more recent reports, however, suggest a weak but positive association between 2,4-D exposure and genotoxic potential in some biologic-model systems. Whereas no effects were observed with pure 2,4-D, in some cases commercial mixtures produced dose-related responses. In others, genotoxicity was observed only when there was evidence of cytotoxicity. Although overall the studies suggest only a weak genotoxicity for 2,4-D, they suggest that the constituents of commercial formulations (like Agent Orange) enhance the toxicity—and, specifically, the genotoxicity—of 2,4-D.

Grabińska-Sota et al. (2002) tested a commercial formulation of 2,4-D in several strains of bacteria. Some genotoxicity was observed, but only at very high concentrations.

The genotoxicity of 2,4-D in fish was evaluated by Ateeq et al. (2002), who assessed the induction of micronuclei and erythrocyte alterations in catfish exposed to 2,4-D at concentrations of as much as 75 parts per million (ppm) in water. The formation of micronuclei and cytotoxicity (vacuolization and echinocyte formation) was concentration dependent. Essentially the same results with similar exposures were observed in a freshwater air-breathing fish, Channa punctatus (Abul Farah et al., 2003). Arias (2003) evaluated the induction of sister chromatid exchange (SCE) and altered cell cycle kinetics in 4-day-old chick embryos

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
×

exposed either to a commercial herbicide preparation containing 37% 2,4-D or to pure 2,4-D. Pure 2,4-D failed to induce a statistically significant change in SCE frequency, and the commercial product produced a change only at the highest dose. It was suggested that genotoxic effects observed with some 2,4-D preparations could be attributable to impurities or adjuvants in the technical-grade products. In these studies, both types of exposure inhibited the mitotic index, but only at the two highest doses (2 and 4 mg/embryo).

Since the last update, two studies have examined the genotoxic effects of 2,4-D exposure in human lymphocytes. Zeljezic and Garaj-Vrhovac (2004) treated cultured lymphocytes with two concentrations (0.4 and 4.0 micrograms per milliliter [µg/mL]) of a commercial formulation of 2,4-D with and without a metabolic activator (rat liver microsomes; S9). The lower concentration of 2,4-D is the acceptable daily intake recommended by the World Health Organization (1–4 picogram/body weight). Both concentrations induced increases in chromatid and chromosome breaks, in the number of micronuclei, and in the number of nuclear buds. Metabolic activation increased the number of chromatid breaks and micronuclei. The investigators also suggested that the genotoxicity could be attributable to compounds other than 2,4-D within the formulation.

A similar study (Holland et al., 2002) examined the effects of exposure to pure and commercial formulations of 2,4-D. Induction of micronuclei was observed in both whole blood and isolated lymphocytes only at a cytotoxic concentration (0.3 milli molar [mM]) of pure or commercial 2,4-D. The replicative index, a measure of cell division kinetics, was decreased at this cytotoxic level, but at a lower concentration (0.005 mM) showed a slight increase which was more pronounced for the commercial formulation. The commercial formulation contained 9.4% pure 2,4-D, which suggests that other ingredients might be responsible for or enhance the effect of 2,4-D on the replicative index. The authors concluded that the genotoxicity of 2,4-D as measured by the micronucleus assay is negligible at environmentally relevant concentrations, but that it might be enhanced in the presence of other chemicals.

Neurotoxicity

Previous updates indicated no evidence that 2,4-D causes effects on the neurologic system in adult animals at doses in the µg/kg/day range. Case reports of acute poisonings of humans exposed to large amounts of 2,4-D formulations (>20 mL) indicated neurologic manifestations of drowsiness, coma, hyperreflectivity, hypertonia, and cerebral edema (Brahmi et al., 2003). One case of ingestion of an unknown quantity of 2,4-D resulted in death. No other relevant studies involving neurotoxicity in adult humans have been published since Update 2002.

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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Reproductive and Developmental Toxicity

Stebbins-Boaz et al. (2004) examined the in vitro sensitivity of amphibian oocytes to 10 mM 2,4-D. Treatment caused depolymerization of perinuclear microtubules and altered cell morphology that was blocked with cotreatment with cytochalasin B, a microfilament inhibitor. 2,4-D treatment also blocked progesterone-induced maturation of the oocytes. Those data indicated that 2,4-D disrupts amphibian oocyte maturation through effects on cytoskeletal organization. Another study (Greenlee et al., 2004) examined the effects of 2,4-D on mouse preimplantation embryo development—a period that corresponds in humans to the first 5–7 days after conception. The 2,4-D exposure of embryos at 10 ng/mL in vitro increased the percentage of cellular apoptosis but had no effect on the development of the embryo to the blastocyst stage or on the mean cell number per embryo. Another group reported that exposure of pregnant mice to herbicide mixture containing low concentrations of 2,4-D (0.01 and 0.1 mg/kg/day), mecoprop (0.004 and 0.04 mg/kg/day), and dicamba (0.0009 and 0.009 mg/kg/day) resulted in significant reductions in implantation sites and live births (Cavieres et al., 2002), but no significant fetotoxicity was observed. Together, those data suggest that the preimplantation embryo might be especially sensitive to 2,4-D. Additional in vivo studies using 2,4-D exclusively are necessary.

Sulik et al. (2002) examined kidney morphology of newborn rats exposed before and after birth to 2,4-D. Dams were exposed to a daily dose of 250 mg/kg (one-third the LD50, the dose that is lethal in 50% of test subjects) in drinking water for 2 months before fertilization and during pregnancy and lactation. Varying degrees of damage to kidney tubules were observed that were more intense after exposure in pregnancy than in the postnatal period. After withdrawal of 2,4-D, the more severe changes observed in the fetus regressed.

Several studies cited in previous updates suggested effects of 2,4-D on the developing brain, and more recent studies present concordant results. Garcia et al. (2001) exposed pregnant rats to 70 mg 2,4-D/kg/day from gestation day 16 through postnatal day 23. Some of the pups were maintained on this exposure until postnatal day 90. Exposure during pregnancy and lactation produced an increased serotonin neuronal area and increased serotonin immunoreactivity in the mesencephalic nuclei. For pups exposed until postnatal day 90, only the serotonin neuronal area from the dorsal raphe nucleus was increased. Changes also were observed in the presence of reactive astrocytes in the mesencephalic nuclei and hippocampus areas; those changes differed with treatment. Those data provide evidence that 2,4-D exposure alters the serotoninergic system during brain development. Another study with the same design also sought to determine whether 2,4-D affected lateralization in the monoamine systems of the basal ganglia and whether there was any correlation with behavioral changes (Bortolozzi et al., 2003). Asymmetrical variations in brain concentrations of dopamine and serotonin were observed that were dependedent on brain region

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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and sex. Some changes appeared to be irreversible, and serotonin changes in left and right striata appeared to correlate with the behavioral alterations (spontaneous circling activity) previously reported.

Ferri et al. (2003) examined the effect of 2,4-D exposure on iron, zinc, and copper concentrations in the brain, serum, and liver of well-nourished and undernourished developing rats. Those metals are known to be essential for normal-brain development. Metal concentrations in tissues were found to be altered only at the highest dose (100 mg/kg) to the dam in well-nourished pups exposed through dams’ milk. A lower dose (70 mg/kg) produced no alterations. However, undernourished pups displayed greater sensitivity to the lower dose of 2,4-D, as indicated by altered metal concentrations and decreased tissue weight. Those data suggest that undernourishment might exacerbate the effects of 2,4-D on developing tissues.

Immunotoxicity

Previous updates indicated that 2,4-D has at most a weak effect on the immune system. Recent publications are consistent with this. One study reported a possible beneficial effect of exposure to low concentrations of 2,4-D (Balagué et al., 2002).

Some studies suggest a relationship between the frequency of errors in antigen receptor gene assembly and an increased risk of lymphoid malignancy. That correlation has been reported for agricultural workers exposed to pesticides (Lipkowitz et al., 1992). Knapp et al. (2003) examined the effects of 2,4-D exposure to mice on the frequency of errors in V(D)J recombination (that is, recombination of the V-gamma and J-beta segments of the T-cell receptor) in thymocytes. At doses of 3–100 mg/kg/day for 4 days, no significant increase in aberrant V(D)J rearrangements was observed. Alterations to bone marrow B-cell populations after exposure of mice to single doses of 50–200 mg/kg were studied by de la Rosa et al. (2003). Decreases in the pre-B and IgM+ B-cell populations were observed 7 days after treatment for the highest dose of 2,4-D.

Escherichia coli is responsible for many urinary tract infections in humans. Weak acids, such as salicylate, that interfere with interaction betweeen bacteria and epithelial cells are sometimes used to treat those infections. Balagué et al. (2002) reported that daily low doses (2.6 and 25 mg/kg) of 2,4-D (also a weak acid) for 20 days significantly decreased or eliminated bacterial cells in mouse bladder and kidneys. A higher dose (70 mg/kg) was not effective after 9 days of treatment. The authors suggested that low exposure to 2,4-D might have a protective effect against urinary tract infections. Several previous investigations (IOM, 2001, 2003) reported kidney toxicity for high 2,4-D concentrations in several animal species. More recent reports detail effects in fish (Gómez et al., 2002).

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
×
Mechanisms Related to Effects on Energy Metabolism or Mitochondrial Function

Several reports cited in previous updates suggested that the toxicity of relatively high concentrations of 2,4-D might be related, at least in part, to its effect on calcium homeostasis and energy metabolism. Those actions might be mediated by a direct action on mitochondria. Some publications suggest it might be attributable to the surfactant in the formulations and not to the 2,4-D itself. However, two recent studies are consistent with the hypothesis that 2,4-D itself causes mitochondrial damage. De Moliner et al. (2002) and Tuschl and Schwab (2003) reported that the exposure of rat cerebellar granule cells and human hepatoma cells, respectively, to 1–16 mM concentrations of 2,4-D elicited cell cycle alterations and apoptotic cell death. Their studies also suggested that the events were caused by a direct effect on mitochondrial membrane potential.

Mechanism Related to Effects on Thyroid Hormones

Effects of 2,4-D on serum concentrations of thyroid hormones, particularly to decreases in thyroxine, were noted in previous updates. Ishihara et al. (2003) examined the effects of industrial and agricultural chemicals on the binding of 3,5,3-L-triiodothronine (T3) to serum thyroid hormone binding proteins (THBPs) and thyroid hormone receptors (TRs). 2,4-D had little or no effect on the binding to THBPs from several species, including humans, or to TRs from chicken or bullfrog.

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 concentrations, 2,4-D is incorporated into cellular membranes to modify membrane structure and integrity.

Duchnowicz and Koter (2003) reported that exposure of isolated erythrocytes to 1 mM 2,4-D resulted in a small but significant increase in hemolysis and in membrane lipid peroxidation. Increased hemolysis was not observed at 0.1 mM (Kleszczyńska et al., 2003). This might be related to a noted concentration-dependent decrease in erythrocyte glutathione (Bukowska, 2003). However, concentrations of 0.5 and 1 mM 2,4-D were found to protect erythrocyte membranes against partial peroxidation induced by ultraviolet irradiation (Bonarska et al., 2003). Özcan Oruç et al. (2004) examined oxidative stress responses in the gills, brains, and kidneys of fish exposed to 2,4-D. Although no significant changes in tissue malondialdehyde, a measure of lipid peroxidation, were observed, there were significant changes in the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GST), and catalase. GST activity in the liver was

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
×

increased at all times from 24–96 h after 2,4-D exposure; SOD activity was increased only at 72 and 96 h (Özcan Oruç and Üner, 2002). It was suggested that tissues adapt to protect cells against oxidative stress elicited by toxins such as 2,4-D. The authors also suggested that increases in tissue SOD and GST activity might serve as good biologic markers of oxidative stress. Orfila et al. (2002) reported that administration of the maximum recommended daily doses of vitamins E and C was ineffective for preventing or altering liver damage produced in rats orally dosed with 200 mg/kg 2,4-D amine daily for 15 days.

TOXICITY PROFILE UPDATE OF 2,4,5-T

No relevant studies on the toxicokinetics of 2,4,5-T or the disease outcomes seen in experimental animals after exposure to 2,4,5-T have been published since Update 2002. Several previous reports indicated that 2,4,5-T has only weak mutagenic potential but that it might alter the profile of enzymes involved in the metabolism of procarcinogens. Previous reports concur that 2,4,5-T is only weakly toxic or carcinogenic.

TOXICITY PROFILE UPDATE OF CACODYLIC ACID

Toxicokinetics

Cacodylic acid was present at 4.7% in a herbicide used for defoliation in Vietnam. Cacodylic acid is DMA, which also is a metabolic product of exposure to inorganic arsenic. Methylation of inorganic arsenic generally has been considered a detoxification process: it produces less acutely toxic methylated species (monomethyl arsenic (MMA) and DMA), and it increases excretion of arsenic. More recently, however, some of the methylated metabolic intermediates have been thought to be more toxic than is the parent compound. The methylation pathway of inorganic arsenic results in the formation of pentavalent DMA (DMAv) and trivalent DMA (DMAIII) (IOM, 2003). DMAv appears to be less toxic than DMAIII (IOM, 2003); about 80% of DMA is excreted unchanged and more rapidly than is inorganic arsenic (reviewed in Duzkale et al., 2003). DMAIII in fingernails and DMAv in fingernails and hair can serve as biologic markers of arsenic exposure (Mandal et al., 2003).

Endpoints and Underlying Mechanisms of Toxic Action

Since Update 2002, the only new literature concerning the toxic activity of cacodylic acid addressed genotoxicity, a major mechanism of carcinogenesis. In addition to being produced as an herbicide, cacodylic acid, or DMA, is a metabolic product of organic arsenic exposure in humans. The committee considered the relevance of data on inorganic arsenic to DMA. Although inorganic arsenic is

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
×

a human carcinogen, there is no evidence that direct exposure to its metabolite, 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 effects observed from exposure to inorganic arsenic also occur from 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). 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). 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, 2003).

Since Update 2002, further studies have investigated the potential carcinogenicity of DMA itself. In a 2-year bioassay, F344 rats were administered drinking water that contained 0, 12.5, 50, or 200 ppm DMA for 104 weeks (Wei et al., 2002). Between weeks 97 and 104, urinary bladder tumors were observed in 8 of 31 rats treated with 50 ppm DMA and in 12 of 31 rats administered 200 ppm DMA. No urinary bladder neoplasms were observed in the groups given 0 or 12.5 ppm DMA. The urinary bladder tumors had a low rate of H-ras mutations, but no alterations of the p53, K-ras, or B-catenin genes were reported. In another study, F344 rats were exposed to 100 ppm DMA for 2 weeks. DMA produced cytotoxicity and regenerative hyperplasia of the urothelium of the urinary bladder (Cohen et al., 2002).

Salim et al. (2003) administered DMA in drinking water (0, 50, or 200 ppm for 18 months) to P53 heterozygous (+/-) knockout mice and wild-type (+/+) C57BL/6J mice. Treatment resulted in a significant increase in total numbers of tumors (at the 50- and 200-ppm doses) in the wild-type mice and significant earlier induction of tumors in more organs and tissues of both the p53 +/- knockout (50 and 200 ppm) and the wild-type mice (50 and 200 ppm). The lack of organ specificity or mutations in the residual allele or in wild-type alleles in both genotypes suggests that DMA is a non-genotoxic carcinogen.

In an initiation–promotion experiment, carcinogenesis in F344 rats was initiated with a single injection (200 mg/kg) of diethylnitrosamine and promoted with 100 ppm DMA (Nishikawa et al., 2002). DMA treatment resulted in a significant increase in numbers and areas of GST-P positive foci (preneoplastic lesions) in the liver.

Duzkale et al. (2003) found that DMA exerted differential antiproliferation and cytotoxic activity against leukemia and multiple myeloma cells, but not against normal peripheral blood progenitor cells, and induced apoptosis in the malignant cells. However, the concentrations of DMA necessary to achieve those effects were 500–1,000 times those required when arsenic trioxide was used. Other researchers have observed increased apoptosis in cell cultures exposed to DMA and noted that DMA requires intracellular reduced glutathione to induce apoptosis (Sakurai, 2003; Sakurai T et al., 2002).

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
×

DNA strand breaks, a form of oxidative damage, were generated by DMA in cultured human cells and in isolated bacterial DNA (Schwerdtle et al., 2003). DMA also was cytotoxic to cultured Chinese hamster V79 cells and caused chromosome structural aberrations (Ochi et al., 2003). Nesnow et al. (2002) showed that the DNA-damaging activity of DMA is an indirect genotoxic effect mediated by reactive oxygen species formed concomitantly with the oxidation of DMAIII to DMAv. Induction of the tumor suppressor protein p53, another indicator of DNA damage, was produced by DMA in cultured human cells in a dose- and time-dependent manner (Filippova and Duerksen-Hughes, 2003). DMA also has induced chromosome aberrations in Chinese hamster ovary cells and in the SCE assay (Kochhar et al., 2003). However, DMA cultured with primary rat astroglia cells did not produce changes in cell viability or cause DNA damage at micromolar concentrations; treatment of the astroglia cells with inorganic arsenicals resulted in decreased cell viability and increased DNA damage (Jin et al., 2004).

DMA was a clastogen in human lymphocytes and a mutagen at the Tk+/-locus in mouse lymphoma cells (Kligerman et al., 2003). Those authors did not consider DMA to be a gene mutagen.

Coexposure of human liver ferritin and DMA resulted in more DNA damage (Plasmid pBR322) than did exposure to DMA alone (Ahmad et al., 2002). The authors proposed that iron-dependent DNA damage could be a mechanism of action of human arsenic carcinogenesis.

Cacodylic acid, at doses on the order of 200 ppm, has been shown to act as a tumor promoter in the kidneys and urinary bladders of laboratory animals (IOM, 2003).

TOXICITY PROFILE UPDATE OF PICLORAM

The compounds 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D) are components of Agent White, an herbicide formulation used in Vietnam. Studies reviewed in previous updates and in VAO reported fairly rapid elimination of picloram and suggest that some carcinogenic and neurologic effects can be attributed to exposure, although only at extremely high doses. Some cellular abnormalities in liver and inconsistent developmental effects also have been reported, and there is some evidence that picloram causes male-mediated birth defects including persistent histologic effects in testes, in animals (IOM, 2003). However, blood concentrations of either agent associated with a dose that is high enough to elicit such effects were not likely to occur in occupational exposure to Agent White.

No relevant studies of picloram have been published since the preparation of Update 2002.

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
×

TOXICITY PROFILE UPDATE OF TCDD

Toxicokinetics

The terms “toxicokinetics” and “pharmacokinetics” refer to processes and rates involved in the movement of a toxic chemical or drug into, within, and from an animal’s body. Thus, toxicokinetics encompasses the routes and rates of uptake, tissue distribution, transformation, and elimination of a toxic substance from the body. Those processes can determine the amounts of a particular substance that will reach specific target organs or cells, thereby influencing toxicity in those organs or cells. It is important to determine the concentrations of TCDD and other polychlorodibenzo-p-dioxin (PCDD) congeners in different organs in the process of reaching conclusions about toxic effects in target organs.

The distribution of TCDD and other chlorodibenzo-p-dioxin congeners has been examined extensively in experimental animal models over the past 25 years. Other planar halogenated aromatic hydrocarbon (PHAH) compounds are thought to act by similar mechanisms, especially the polychlorinated dibenzofurans and non-ortho-polychlorinated biphenyls, and they also have been examined extensively. 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 as related to accidental or background exposure.

As discussed in numerous papers reviewed in previous reports, those compounds are hydrophobic and tend to be absorbed readily across cell membranes. TCDD is distributed to all compartments of the body, although the amounts differ from organ to organ. Properties of the compounds, properties of the organs and cells, and the route of exposure affect the partitioning, absorption, and accumulation of the chemicals. Lipid content affects the accumulation of TCDD and other PHAHs in different organs and in the body as a whole. Biologic processes, especially metabolism, subsequently can affect their distribution and elimination. The concentration in a given organ or tissue thus depends on dose, absorption, lipid content, and metabolism in the organ of concern. Adipose tissue–serum partition coefficients derived from concentration ratios can indicate the degree of accumulation in fatty tissues, and the amount of fat in a particular organ can determine partitioning into that organ. Interindividual differences in absorption, distribution, and elimination can lead to a range in the organ–serum partition coefficients within a population. Whole-blood or serum concentrations of TCDD can also fluctuate with differences in physiologic state and metabolic processes that can affect the mobilization of lipids and possibly the mobilization of compounds from lipid stores. Moreover, the processes in one organ can influence distribution to others. Thus, binding proteins in some organs, such as cytochrome P450 1A2 (CYP1A2) in liver, can influence accumulation in extrahepatic organs.

Modeling the toxicokinetics or pharmacokinetics of TCDD has several objec-

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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tives: to estimate organ distribution from 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. Pharmacokinetic or toxicokinetic parameters can be similar or differ substantially among species, depending on similarities or differences in anatomy, physiology, and metabolic biochemistry—particularly for processes involved in foreign chemical metabolism—which vary greatly. PBPK models apply knowledge of mechanisms, including organ size and blood flow, binding proteins in blood, xenobiotic metabolism, and excretion and clearance rates to the process of predicting concentrations and kinetic behavior of a compound, given a particular exposure. Allometric models rely largely on relationships between body size and lipid content and the kinetic parameters of elimination. PBPK models can offer more information about tissue distribution, but it still is possible to predict elimination of TCDD with knowledge of fat content and body mass index (BMI). In this way, toxicokinetic modeling and exposure assessment are linked.

Animal Studies

There have been several additions to the literature since the last report (IOM, 2003) that detail the processes that affect distribution of TCDD. Studies in rodent models have continued to support the value of PBPK models for predicting the disposition of TCDD. Kim et al. (2002) used a mechanistic PBPK model to estimate body burden after exposure of female rats to TCDD, as compared with a kinetic model that applies effective dose for a specific known molecular effect. That study showed that the use of a simpler kinetic approach to body burden gave results similar to those based on a PBPK model for estimating body burden, with a defined exposure regimen.

Efforts have continued to identify dietary or other approaches that can enhance the elimination of dioxins, decreasing their uptake and half-life. The goals of such studies are to decrease absorption of dioxins from contaminated food in the digestive tract and to stimulate the excretion and elimination of dioxins from highly contaminated individuals. Studies over the past several years have attempted to enhance elimination of TCDD, for example with activated charcoal, crude dietary fiber, Olestra, and seaweed (reviewed in previous updates). Elimination of TCDD residues is generally in the feces, which can include ingested matter that is not absorbed, and in residues excreted from the body in bile. Aozasa et al. (2003) fed mice soluble or insoluble dietary fiber—the latter with or without several porphyrin (chlorophyllin) derivatives differing in the metal complexed with the porphyrin—after the mice had been exposed to the dioxin congener 1,2,3,4,7,8-HxCDD. Mice were given an oral dose of 10 µg of the HxCDD congener per kilogram of body weight. Three days later, the mice were fed diets that contained various amounts of fiber. Results showed that a diet

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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with 10% insoluble fiber promoted dioxin excretion by about 60% over a soluble fiber diet, and that with Cu-porphyrin added, the stimulation was 144%. A similar objective was approached in a study with rats, using nori, a traditional constituent of Japanese diets that is prepared from red algae, to affect TCDD uptake and elimination (Morita and Tobiishi, 2002). The experiment included administration of nori simultaneously with a mixture of polychlorinated dibenzofurans (PCDF) and PCDD congeners, including TCDD, and administration of nori 7 days after administration of the PCDD–PCDF. The two approaches should indicate effects on absorption and elimination from the body, respectively. The results with a 10% nori diet showed a 5-fold increase in fecal excretion of TCDD when nori and TCDD were given together, indicating reduced uptake of TCDD. When the 10% nori diet began 7 days after TCDD, there was a 2.4-fold increase in TCDD excretion and a slight but significant decrease in TCDD body burden, indicating an effect on elimination. Similar results were obtained for total toxic equivalents (TEQ), with a greater amount of dioxins and dibenzofurans eliminated with the nori diet in both experimental approaches.

Such dietary approaches are not yet sufficiently effective to reliably accomplish a therapeutic enhancement of elimination for TCDD. Sakurai K et al. (2002) carried out a study in guinea pigs to establish the tissue distribution of TCDD given at different doses and for different periods. The objective was to establish a baseline in the guinea pig for eventual use of this species as a model to test the ability of dietary additions to enhance elimination of TCDD. The TCDD concentrations achieved in the guinea pig showed tissue distribution (adipose/liver ratios) similar to that in humans, suggesting that the guinea pig could be a useful model for low-dose studies. The TCDD concentrations were higher in adipose tissue than they were in liver, suggesting that CYP1A2 might not be as effective at sequestering TCDD in guinea pigs as it is in rats or mice, or that the dose was too low to induce CYP1A2. The longer half-life of TCDD in guinea pigs (94 days) than in rats (20 days), as reported by Olson (1986), may involve a slower rate of excretion of metabolites or parent compound at low doses and a higher body fat content in the guinea pig rather than a difference in their rates of metabolism.

The major source of exposure to dioxins is the diet. Cavret et al. (2003) used an in vitro model (Caco-2 cells derived from a human colon carcinoma) and an in vivo model system (pig fitted with vascular catheters, allowing sampling of portal and brachiocephalic blood) to define the factors that influence uptake of TCDD and other compounds (phenanthrene and benzo[a]pyrene) in the intestine. The studies showed proportional absorption of the compounds in the cells and in the in vivo model, suggesting that the companion systems might be useful in defining conditions and factors involved in accumulation of TCDD.

Emond et al. (2004) refined a PBPK model for pregnant rats, to predict TCDD concentrations in both maternal and fetal tissues, throughout gestation. The model was simplified by incorporating only those tissue compartments known to significantly influence the distribution of TCDD. This simplified model

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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gave similar fits to the data as the full model, a result of the fact that liver and fat compartments variably represent 80–95% of the total body burden of TCDD. The validated rodent model could be useful in assessing fetal human exposures.

Human Studies

Since Update 2002, few studies have addressed toxicokinetic modeling of TCDD in humans, although those under way continue to support the validity of PBPK modeling to estimate internal dose from amount ingested. Bois (2003) used a PBPK model to assess the effect that ingestion of TCDD at concentrations above background would have on internal concentration. The model predicted that ingestion of 100 picograms (pg) of TCDD twice a week for 3 weeks would increase the internal (blood) concentrations by less than 1% relative to the background TCDD exposure in Europe.

In a study in Japan, Maruyama et al. (2002a) determined tissue–blood partition coefficients for use in a PBPK model for humans, measuring the concentrations of 17 PCDD and PCDF congeners in various tissues (liver, kidney, gut, viscera, skin, muscle, fat, bile) taken at autopsy from 8 men and women who had not been accidentally exposed to dioxins. The partition coefficients were used in a model previously described by Lawrence and Gobas (1997), and the model was used to estimate the concentrations in two other sets of people for whom PCDD data were available. There was good agreement between the concentrations estimated by the model and the actual concentrations measured in the tissues. The estimated concentration in adipose tissue of the samples from Japan was 4.1 pg/g lipid, which falls between the concentrations reported earlier for a group of Massachusetts Vietnam veterans (6.9 pg/g) (Schecter et al., 1990) and those reported in a population in Munich, Germany (2.6 pg/g) (Thoma et al., 1990).

Maruyama et al. (2002b) explored individual variation in human TCDD concentration using the PBPK model. As discussed in previous Updates, individual variation arises from differences in biologic variables, such as body weight, sex, and BMI, as well as from differences in metabolism rates and exposure. TCDD concentrations in Japanese adults vary greatly, ranging from 0.0025 to 0.025 pg/g lipid in blood, and from 0.036 to 1.11 pg/g lipid in liver. From their analysis, Maruyama et al. (2002b) concluded that differences in concentration of TCDD in the diet affect TCDD accumulation more than do any of the possible differences in half-life of the chemical. The levels of various PCDDs in multiple organs of the Japanese individuals analyzed in the study by Maruyama et al. (2002a) also showed that the concentrations of some congeners were greater in liver than in adipose tissue, which could be an indication of sequestration by CYP1A2.

Since Update 2002, new studies have reported on TCDD and other PHAHs in a limited number of human tissues, focusing on blood. Several reported on TCDD, PHAH, and TEQ in human milk as well.

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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Petreas et al. (2004) examined the differences between concentrations of persistent lipophilic chemicals, including PCDDs, in abdominal adipose tissue and in breast tissue that came from surgical specimens. The objective was to determine whether concentrations in one kind of tissue could be used to predict concentrations in the other. The study provided further information on individual variation in the content of the compounds. Adipose tissue is generally thought to be suitable for estimating the steady state of highly lipophilic contaminants. The average concentration of TCDD alone in abdominal–breast pairs was 3.9 pg/g lipid, with a range of 0.3–9.5 pg/g. In general, the concentrations in one tissue were highly correlated with those in the other. In this study, the correlation was weakest for TCDD, and there was a suggestion of preferential deposition of TCDD in abdominal adipose rather than in breast tissue, although in other studies TCDD is often evenly distributed in different fat compartments (IOM, 2003).

Kim et al. (2001) examined current blood concentrations in Korean veterans of the Vietnam conflict for exposure assessment related to health outcomes. Blood concentrations were measured in pooled samples from each of 4 groups defined on the basis of service in Vietnam and from a control group of veterans who did not serve in Vietnam. TCDD analyses were performed at the US Centers for Disease Control and Prevention. Concentrations averaged 0.3 pg/g serum lipid in the non-Vietnam group and 0.63–0.84 pg/g in the 4 exposed groups. The authors suggest a trend related to conditions of service in Vietnam, but they acknowledge that it is nearly impossible to use those numbers to determine what exposure had occurred 25 years earlier. The TCDD concentrations in the presumably exposed groups were all substantially lower than were concentrations in control groups from the Ranch Hand or other studies.

Concentrations of contaminants in workers at a hazardous-waste incinerator, a source of dioxins, were studied by Agramunt et al. (2003) to evaluate the worker exposure to metals and organic chemicals that emanate from the incinerator, which began operation in 1999. The study compared incinerator operators (4 composite samples), laboratory workers (1 composite), and administration workers (1 composite). Total PCDDs and PCDFs did not differ for the groups. Moreover, TCDD concentrations declined from an average of 4.3 pg/g blood lipid in 1999 to an average of 0.9 pg/g in 2003. The total PCDD and PCDF concentrations followed a similar pattern.

Dwernychuk and colleagues (2002) examined concentrations of TCDD in environmental matrices and in Vietnamese residents from 4 sites in Vietnam that had been exposed at different rates during the Vietnam conflict. The TCDD concentrations in soil, fish fat, and duck fat were highest in a village in the Aluoi Valley on the site of a former US base that, among the sites considered in this study, had been used the longest by US special forces. Samples of blood and milk were obtained from village residents in 1999. Blood samples from each site were pooled by sex and age (younger or older than 25 years). Values in the 4 pooled-blood samples from the most contaminated location were 31 and 41 pg/g lipid in

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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males and 14 and 16 pg/g in females. Concentrations were lower in samples from the other sites and were below the limits of detection in those from the least-contaminated site. Milk samples from 4 women in each location had average values that fell in the same ranking, although the individual variation was as high as 4-fold within a group. Total TEQ followed the pattern, though TCDD values differed more between sites than did the TEQ. The results suggest that contamination that occurred decades earlier still contributes to elevated exposure of the populations in those areas, presumably via contamination of fish and fowl that are used for food.

In another study in Southeast Asia, Schecter et al. (2003) examined chlorinated dioxin, dibenzofuran, and biphenyl concentrations in blood and milk from residents of Laos. TCDD in blood was detectable in 3 of 6 individuals and in 1 of 4 pooled samples. Detected values ranged from 1.2 to 4 pg/g blood lipid. Those values are similar to the concentrations detected in blood from residents of unsprayed areas of Northern Vietnam, measured at 1.2–2.3 pg/g blood lipid. The report also notes that the samples from Laos are comparable to those in 2 pooled-blood samples from Dallas, Texas (1 and 3.8 pg/g lipid), and to the mean blood concentrations of TCDD from 13 persons in Munich (2.4 pg/g lipid). TCDD in milk from 3 of the Laotian women ranged from 0.06 to 0.35 pg/g, as compared with the average of 1.6 pg/g in milk from 69 individual samples taken in Germany.

Eskenazi et al. (2004) recently analyzed archived samples from individuals from Seveso, Italy, collected soon after the accident. The analyses confirmed that the highest concentrations of TCDD were in people residing in the most highly contaminated areas, but although the average values for blood levels of TCDD in zone A residents are higher than in zone B, the levels in some zone B individuals were higher than those in some individuals from zone A. Age at first exposure was the other strong predictor of TCDD serum concentration. It is noteworthy that the actual concentrations of TCDD measured in the immediate postexposure samples by Eskenazi et al. (2004) were similar to the estimates that Landi et al. (1998) had obtained by extrapolating back from levels measured in blood samples collected in 1996, 20 years after the accident. Eskenazi et al. (2004) also reported greater than anticipated total TEQ in blood samples archived in 1976 from women from zone non-ABR (unexposed individuals) and found that substantial TEQ (about 80% of the total TEQ) was due to compounds other than TCDD. It is likely that compounds other than TCDD contributed to the overall TEQ in women from exposed areas also. These findings are consistent with the results from more contemporary Seveso samples reported by Weiss et al. (2003) and discussed below. This suggests that inclusion of compounds additional to TCDD contributing to TEQ could be important in health assessments and might in fact confound the determination of the effects of TCDD alone.

Weiss et al. (2003) reported on measurement of dioxin, dibenzofuran, and polychlorinated biphenyl (PCB) congeners in milk samples from residents near

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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Seveso (zones R and B), as compared with milk from women in Milan, Italy, and from a rural area that was unaffected by the Seveso accident. Samples were collected 25 years after the heaviest exposure at Seveso. Samples collected from 12 women at each site in the first week postpartum and 3 months after delivery were pooled separately. TCDD concentrations were elevated in the samples from Seveso—4.45 pg/g lipid in the first-week sample from Seveso as compared with 1.63 and 1.58 pg/g in the other locations. However, TEQ from PCDD, PCDF, and PCB congeners were similar in all areas.

Elevated TCDD concentrations in breast milk have been observed by numerous investigators over the years. The occurrence of TCDD in breast milk is important because it indicates a flux of chemical through the organ, which could have implications for effects in breast tissue. Breast milk also is the dominant source of TCDD contamination in infants. Analyses show that breast-fed infants accumulate substantially more TCDD than do bottle-fed infants. Several variables influence the concentration of TCDD and other contaminants in breast milk, including the time from delivery that milk is obtained, the number of children who have been nursed, and the variation in milk composition (for example see NRC, 1999b). Lorber and Phillips (2002) note the importance of including such information in pharmacokinetic models for estimating infant exposures. They suggest that models enhanced by adding information on metabolism of specific congeners as a function of age and of the initial exposure could estimate the persistence of elevated concentrations as children grow.

Analysis of dioxins in tissues of infants who died suddenly in Westphalia, Germany (Bajanowski et al., 2002) gave results consistent with several of the observations mentioned above. The cases were divided into two groups: one of 15 children who died in 1991–1992; a second of 12 children who died in 1996–1997. The results showed similar TCDD concentrations in adipose and liver tissue, although several PCDD and PCDF congeners were more concentrated in liver, which would reflect binding by CYP1A2, as in the Japanese study by Maruyama et al. (2002a). There was a consistent difference between breast-fed and non-breast-fed infants. TCDD concentrations in adipose tissue were 2.5 and 0.6 pg/g lipid, respectively, in breast-fed and bottle-fed infants. The data also showed that duration of breast-feeding was directly proportional to dioxin concentration. Total TEQ was 22 pg/g fat in the 1991–1992 group and about 6 pg/g in the tissue from the child who died in 1996–1997. This is attributed to a general decrease of about 50% from 1989 to 1998 in background levels of PCDDs and PCDFs in human milk.

The trend of decreasing levels is supported by studies of Aylward and Hays (2002), who examined human TCDD body burdens in studies over nearly 30 years in the United States, Canada, Germany, and France. The analysis identified temporal trends in human blood levels, with a nearly 10-fold decrease in levels from the early 1972 to 2000, from nearly 20 pg/g blood lipid to just over 2 pg/g blood lipid. Using half-life estimates that are average for humans (about 7.5 years)

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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and first-order elimination kinetics, they conclude that the decreases must result from decreased intake levels in the diet. The study’s best estimate of daily TCDD absorption was 0.04 pg TCDD/kg. The authors pointed out that, despite limitations, the conclusion of a reduction in intake over this time is strong.

The dose of a compound such as TCDD to an infant through milk could constitute an important proportion of lifetime exposure. Aylward et al. (2003) reviewed several studies to examine the extent to which concentrations in milk corresponded to blood concentrations in the same people. The objective was to determine whether blood concentrations might be used to estimate general exposure for infants. Their analyses of the somewhat limited data suggest that the concentrations of TCDD and other compounds human milk generally reflect those in blood.

Metabolism and Half-Life Studies

It is generally agreed that the toxicity of TCDD is related in part to the fact that it is so persistent in the body. But recent estimates of TCDD half-life in humans have been reported to range much more widely than once thought. The half-life of TCDD in humans varies with BMI, age, sex, and, most substantially, with initial exposure. Miniero et al. (2001) reviewed data on the half-life of TCDD and how it was correlated with body weight. Michalek and Tripathi (1999) observed that the elimination rate of TCDD in Ranch Hand personnel was related to body fat. Although the major determinants of TCDD half-life are thought to be lipophilicity, metabolism, and sequestration in the liver, the half-life seems to be correlated empirically with body weight in mammals. However, elimination also appears to occur biphasically, with initial exposure determining the initial rate of elimination. Some studies addressed in Update 2002 are mentioned here, given their significance to determination of half-lives of TCDD.

As reviewed in Update 2002, the elimination of TCDD from highly exposed individuals was examined by Geusau and coworkers (2002), who studied 2 patients who had extremely high concentrations of TCDD (144,000 pg/g blood fat in one and 26,000 pg/g blood fat in the other). Overall TCDD half-lives of 1.5 and 2.9 years (Table 3-1) were reported for the more and less severely contaminated individuals, respectively. Those values are considerably shorter than are the commonly reported values, which range from 6.9 to 9.8 years (Table 3-1). The implication that the rate of elimination is greater for more highly contaminated persons was supported by the analysis of TCDD toxicokinetics in adults from Seveso (Michalek et al., 2002). Serum samples obtained within days after exposure, which provided a measure of the initial dose accumulated by the individuals, gave a mean half-life of only 0.34 years in the Seveso males over the first 3 months after exposure. The mean half-life during the period from 3 years to just over 16 years after exposure was 6.9 years. By comparison, the mean half-life in Ranch Hand personnel, at 9–33 years after exposure, was 7.5 years—only slightly

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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TABLE 3-1 Estimates of TCDD Half-Life in Humans and Animals

Reference

Half-lifea

Confidence Interval

Comment

Human Studies

Aylward et al., 2004

<3 years

 

Calculated for exposures >10, 000 pg/g serum lipid

 

>10 years

Calculated for exposures <50 pg/g serum lipid

Flesch-Janys et al., 1996

7.2 years

Adult males, Boehringer cohort

Geusau et al., 2002

1.5 yearsb

Adult female, severe exposure 0–3 years PE

 

2.9 yearsb

Adult female, severe exposure 0–3 years PE

Michalek et al., 2002

0.34 yearsb

Adult males, Seveso cohort, 0–3 months PE

 

6.9 years

Adult males, Seveso cohort, 3–16 years PE

9.8 years

Adult females, Seveso cohort, 3–16 years PE

7.5 years

Adult males, Ranch Hands 9–33 years PE

Needham et al., 1994

7.8 years

7.2–9.7 years

Adults, Seveso cohort

Pirkle et al., 1989

7.1 years

5.8–9.6 years

Adult males, Ranch Hands 9–23 years PE

Animal Studies

Neubert et al., 1990

73.7 days

60.9–93.8 days

Monkeys, Marmoset, single injection

DeVito and Birnbaum, 1995

15 days

 

Mice, female B6C3F1

Gasiewicz et al., 1983

11.0 daysc

Mice, C5BL/6J

 

24.4 daysc

Mice, DBA/2J

 

12.6 daysc

Mice, B6D2F1/J

Koshakji et al., 1984

20 days

Mice, male ICR/Ha Swiss

Hurst et al., 1998

8 days

Rats, Long-Evans, excretion from liver

Pohjanvirta et al., 1990

21.9 days

Rats, male Han/Wistar resistant strain

Viluksela et al., 1996

20.2 days

Rats, Long-Evans TurkuAB strain

 

28.9 daysd

Rats, Long-Evans Charles River strain

Weber et al., 1993

16.3 ± 3.0 days

Rats, male Sprague-Dawley

a Half-lives of TCDD in humans based on measurement of TCDD in serum samples.

b Shorter half-lives measured in humans during first months after exposure or in severely contaminated persons consistent with nonlinear elimination predicted by PBPK modeling (e.g., by Carrier et al., 1995). 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.

ABBREVIATION: PE, postexposure.

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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different from the rate in Seveso males. The pattern of elimination in the Seveso cohort is consistent with a two-compartment model that should be considered in toxicokinetic models of TCDD accumulation and in attempts to extrapolate original exposures in other contaminated populations.

Aylward et al. (2004) reexamined the toxicokinetic modeling to estimate exposure history of adults from several groups (Seveso, the National Institute for Occupational Safety and Health [NIOSH] cohort, and the highly exposed Austrian women). They optimized the fit of the model to the data by varying the elimination rates, based on evidence that there are concentration-dependent elimination rates. The modeling indicates substantial variability in elimination rates between individuals, as well as confirming a faster elimination in males than females and in younger than older individuals. The half-lives for TCDD in males, resulting from the data and models, range from less than 3 years at serum levels greater than 10,000 pg/g lipid to over 10 years at serum lipid levels less than 50 pg/g lipid. Employing varying elimination rates may provide more reliable ranges for estimating exposure concentrations by back-extrapolation for individuals.

Once accumulated in a person, internal exposure to a compound such as TCDD, which has a half-life of years, would continue over time, as it is slowly eliminated from the body. Time-dependent cumulative dose estimates or “area under the curve” dose estimates are based on the concentrations measured in blood and the time over which those concentrations were present in the individual. In a study of the NIOSH cohort, Salvan et al. (2001) used a PBPK model to calculate cumulative dose as related to all cancer risk. In the case of the Ranch Hands, it was suggested that the elimination rate (the area under the curve) should be tested for a relationship to the incidence of diabetes in the Ranch Hand cohort (Michalek et al., 2003). Differences in elimination rates have been observed among Ranch Hands, and those variations would relate to differences in cumulative dose; that is, to an accurate calculation of the area under the curve for total internal exposure. The analysis did not show a relationship between elimination rate and diabetes. In their analysis of elimination rates, Michalek et al. (2003) also noted that the data on elimination rates based on blood samples collected years after the exposure could not be extended to earlier periods.

The issue of cumulative internal dose over time also concerns potential effects of background exposure. In many parts of the world, background exposure to TCDD and related chemicals that contribute to total TEQ continues. If the area under the curve were to be used as the exposure metric related to outcomes, then background exposure would be important, either alone or as added to an accidental exposure. There is no consensus about whether the area under the curve over time, the actual tissue concentration at any given time, or some other expression is the most valid dose metric for any particular health outcome. For cancer, however, it is quite clear that total area under the curve, as was attempted by Salvan et al. (2001), is not optimal.

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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It is generally thought that the elimination rate of TCDD is related to the rate of metabolism of the compound. Thus, the shorter half-life in rodents than in humans could be related to a lower percent body fat, but could also involve more rapid metabolism of TCDD in rodents. However, there are few measurements of the TCDD metabolism rate by human enzymes. Inouye et al. (2002) examined 12 human cytochrome P450 forms (expressed in yeast) for their ability to catalyze the metabolism of PCDDs. The expressed CYP1A1 showed the highest rates of activity with a series of mono-, di-, and trichlorinated dibenzo[p]dioxins. However, the rates of TCDD metabolism were below the limits of detection in this study. There are virtually no measurements of TCDD metabolism in vivo. Recent studies have addressed how the structure of rat cytochrome P450 1A1 is related to the mechanism and rate of dioxin metabolism by that enzyme. Shinkyo et al. (2003) used structural models of rat CYP1A1 to suggest parts of the enzyme molecule that might govern the rate at which the enzyme metabolizes TCDD. Subsequently, they used genetic engineering to modify the rat enzyme and reported that altering the structure in accord with the model results did increase the rate of TCDD metabolism by the enzyme.

In humans, there are individual differences in the rate of TCDD elimination. It is not known whether there are human CYP1A variants that oxidize TCDD at different rates or whether that could influence the disposition or elimination of TCDD. It should be noted, however, that the rates of TCDD metabolism in humans still are slow enough that the influence on accumulation patterns could be minimal. In any case, the major determinants of the half-life of TCDD in all mammals examined, including humans, are the percent body fat at background exposures and hepatic sequestration at high doses.

Summary

Studies that model the disposition and effects of TCDD in rodents continue to be refined and to support the development and use of PBPK models to estimate congener-specific concentrations in human tissues. It will be important to continue to refine PBPK models for evaluating tissue distribution in humans.

The information on TCDD toxicokinetics is expanding, and new studies tend to support the conclusions of earlier work. PBPK models could predict distribution and elimination rates, but allometric models also appear to explain elimination rates, including differences between individuals. The data show that BMI and body fat content are important determinants of TCDD half-life, particularly at low exposure. It is worth emphasizing again that 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. Regardless, biphasic elimination continues to confound back-extrapolation to initial exposure for persons who might have experienced high exposures years before blood or other tissue samples were

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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obtained for analysis. Efforts to enhance the rate of absorption of TCDD from the diet and to enhance elimination by inclusion of fiber or other dietary supplements continue to show promise. Back-extrapolation is still an important issue to be addressed. Accurately assessing risk of TCDD or other PHAH exposure above background could require information about the amounts accumulated at the times of greatest exposure. To make such estimates usually requires researchers to work backwards from current blood concentrations in exposed subjects. Although differences in dioxin concentrations in blood serum obtained years or decades after external exposure could relate to differences in that exposure, individual differences in elimination rates could be substantial.

Toxic Endpoints and Underlying Mechanisms of Toxic Action

Studies published since Update 2002 are consistent with the hypothesis that TCDD produces its biologic and toxic effects by binding to a gene regulatory protein, the AhR, which can modulate gene expression through several mechanisms. Research 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 specific DNA sequences (often called Ah-responsive elements, AhREs; dioxin-responsive elements, DREs; or xenobiotic-responsive elements, XREs) present in the 5′-promoter regions of particular genes lead to the inappropriate modulation of gene expression. Those molecular changes are the initial steps in a series of biochemical, cellular, and tissue changes that result in the toxicity observed. That hypothesis is supported by numerous studies that have evaluated structure–activity relationships of various compounds that bind to the AhR, the genetics of mutant genes that express the AhR, AhR-deficient mice, and the molecular events that contribute to and regulate AhR expression and its activity. Many studies published since Update 2002 are consistent with this mechanism of AhR action. However, newer studies also indicate that the TCDD-bound AhR modulates genes and cellular signaling pathways by mechanisms that do not involve direct binding to DNA but that occur by its interaction with other cellular proteins. The exact relationships among the mechanisms, the modulated expression of known genes, and the diversity of toxic effects elicited by TCDD in humans and numerous animal species have yet to be uncovered.

The finding that many AhR-regulated genes are modulated in a species-, cell-, and developmental-stage-specific pattern suggests that the molecular and cellular pathways that lead to a particular toxic event are complex. Much of the data are consistent with the notion that the cellular processes that involve growth, maturation, and differentiation are most sensitive to TCDD-induced modulation as mediated by the AhR. The findings in animals indicate that the reproductive, developmental, and oncogenic endpoints are sensitive to TCDD. The data support the biologic plausibility of similar endpoints in exposed humans. Many of

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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the responses, however, are tissue and species specific. As such, the appearance of some toxic endpoint in one or even several animal species exposed to TCDD does not necessarily indicate the same endpoint will occur in exposed humans, or vice versa. The exact mechanisms responsible for the differences are not known.

The conclusions indicated above are similar to those in Update 2002. Since that update, 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 2002 that was identified by the committee, closer attention is given only to studies that add substantial new information, particularly as it might be relevant to the exposure of Vietnam veterans. As discussed in Update 2002, it is important to consider exposure and species sensitivity when discussing animal data and their relevance to humans.

Structural and Functional Aspects of the AhR

AhR Gene and Protein Published data cited in previous updates are consistent with the conservation of AhR structure and function among species. Nevertheless, specific differences in amino acids or amino acid sequences within domains of the AhR protein are associated with altered AhR function and with responsiveness to TCDD and other ligands. Some differences might account for species–species differences in sensitivity to TCDD. Hahn et al. (2004) identified 25 single nucleotide polymorphisms in the AhR gene of a population of Atlantic killifish that has evolved genetic resistance to TCDD and related chemicals. However, none of the encoded proteins from the identified alleles differed functionally when expressed in cultured cells. Backlund and Ingelman-Sundberg (2004) reported that a tyrosine at the 320 position of the rat AhR is critical in its responsiveness to several AhR ligands, but not to TCDD. Tyrosine 9 in the mouse AhR was found to be essential for recognition of DREs (Minsavage et al., 2003).

Bunger et al. (2003) reported that mice carrying a mutation in the nuclear localization sequence of the AhR were resistant to TCDD-induced hepatomegaly, thymic involution, and cleft palate formation. The data suggest that most, if not all, TCDD-induced toxicity requires nuclear localization of the TCDD-bound AhR. Two publications by Simanainen et al. (2003, 2004a) reported that genetic differences in the C-terminal transactivation domain of the rat AhR modify some, but not all, of the TCDD-induced toxic responses in these animals. The authors postulate that there are at least two different AhR-mediated signaling pathways that lead to different toxic endpoints. Furthermore, the related resistance of the rat strains carrying the genetic variances in the AhR develops differently as they age (Simanainen et al., 2004b). The concepts are consistent with the tissue-specific sensitivity to TCDD observed in vertebrate species. Two forms of the AhR, AhR1 and AhR2, are expressed in the zebrafish. Consistent with the finding that

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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the AhR2 but not the AhR1 binds TCDD, Prasch et al. (2003a) reported that the AhR2 mediates the developmental toxicity of TCDD in zebrafish.

The amount of the AhR protein could influence a tissue’s relative responsiveness to TCDD. Several publications have reported on factors that control the expression and stability of AhR protein in cells. Racky et al. (2004) identified 7 polymorphisms of the human AhR promoter region, although they did not appear to alter AhR gene expression in lymphocytes. However, the analysis also demonstrated the involvement of Sp1 protein in AhR gene regulation. Exposure of cells and animals to TCDD has been shown to stimulate pathways that mediate the degradation of the AhR protein, leading to a subsequent decrease in AhR-mediated gene alterations. Additional data have improved our understanding of those pathways (Ma and Baldwin, 2002; Pollenz, 2002; Santiago-Josefat and Fernandez-Salguero, 2003; Song and Pollenz, 2003; Wentworth et al., 2004). Notably, TCDD plasma concentrations in people exposed 20 years earlier in Seveso were associated with a reduction in AhR expression in lymphocytes. Plasma TCDD concentrations showed a negative association with induced cytochrome P450 (CYP)1A1-mediated enzyme activity after exposure of lymphocytes from those persons to TCDD in vitro (Baccarelli et al., 2004; Landi et al., 2003). Those studies might suggest that long-term exposure of humans to TCDD perturbs AhR gene regulation. Song and Pollenz (2002) reported that treatment of cells with geldanamycin, which binds to 90 kilodalton heat shock protein (hsp90) and disrupts the conformation of the hsp90–AhR complex, resulted in a substantial degradation of the AhR and to decreased cellular responsiveness to TCDD. Spink et al. (2003) reported that a continued presence of estrogen was required to maintain high concentrations and activity of AhR protein in MCF-7 breast cancer cells. Although hypophysectomy significantly lowered AhR expression in rat liver, no change in induction of CYP1A1 by AhR ligands was observed (Timsit et al., 2002). Hestermann et al. (2002) reported that serum withdrawal from cultured teleost hepatoma cells led to decreased AhR expression and to lowered inducibility of CYP1A1. AhR gene silencing using small inhibitory RNA decreased the responsiveness of cells to TCDD (Abdelrahim et al., 2003).

One of the many difficulties of extrapolating from experimental data in animals is the fact that, although AhRs in humans and animals are homologous with a high percentage of identity, there may be functional differences. At least one form of human AhR binds and responds differently to TCDD and related xenobiotics than animal counterparts. Moriguchi et al. (2003) generated a mouse possessing the human AhR (hAhR) in place of the mouse AhR. Mice homozygous for the hAhR gene exhibited weaker induction of target genes such as cyp1a1 and cyp1a2 than did wild-type mice. After maternal exposure to TCDD, mouse fetuses homozygous for hAhR developed hydronephrosis but not cleft palate; wild-type mice developed both. Although some of those differences might reflect the previous findings indicating that the human AhR has lower affinity for TCDD than does the AhR in other species (IOM, 2003), it is apparent from the investigations

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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of Moriguchi et al. (2003) that the relative sensitivity could depend not only on the type of AhR present, but also on the dose and the endpoint examined. This could also vary for different human AhR allele products.


Interaction of the AhR with Other Proteins As indicated in Update 2002, the function and regulation of the AhR depends on the presence of several other intracellular proteins. Additional studies have been published on the interactions of the AhR with Arnt (Chapman-Smith et al., 2004; Korkalainen et al., 2003), a protein called X-associated protein 2 (XAP2; also called ARA9 and AhR-interacting protein, or AIP) (Berg and Pongratz, 2002; Lees and Whitelaw, 2002; Lees et al., 2003; Petrulis et al., 2003; Ramadoss et al., 2004), hsp90 (Cox and Miller, 2003, 2004), p23 (Cox and Miller, 2004; Shetty et al., 2003), the export protein CRM-1 (Berg and Pongratz, 2002), and the nuclear import protein importin (Petrulis et al., 2003). Those proteins function in stabilizing the AhR in cells and in regulating the intracellular localization of the AhR. Ikuta et al. (2004a) observed that changes in cell density regulate the intracellular localization and transcriptional activity of the AhR. This could be related to altered interaction of the AhR with the above proteins, as mediated by the phosphorylation of the AhR at particular amino acid residues (Ikuta et al., 2004a,b).

Ramadoss et al. (2004) noted that the hAhR is biochemically different from the mouse AhR in its ability to interact with factors that regulate cytoplasmic–nuclear localization. Interactions of the AhR with TRAP/DRIP/ARC/Mediator protein complex (Wang S et al., 2004), transcription elongation factor (Tian et al., 2003), histone deacetylase (Wei et al., 2004), and ubiquitin-like protein Nedd8 (Antenos et al., 2002) also modulate, through a variety of mechanisms, the ability of the AhR to regulate genes.

Updates 2000 and 2002 noted the identification of an AhR repressor (AhRR) protein that inhibits AhR function by competing with the AhR for dimerization with Arnt. Additional data on this protein are consistent with previous information. Tsuchiya et al. (2003a) used a variety of human cell lines to show that several polycyclic aromatic hydrocarbons, including TCDD, induced the expression of the AhRR gene, but in a compound- and cell-specific manner. Yamamoto et al. (2004) observed that in human tissues the expression of AhRR was extremely high in testis. It was very high in lung, ovary, spleen, and pancreas from adults but low in the same tissues from fetuses. They also observed variable expression of AhRR in mononuclear cells from various sources. The authors suggest that this might account, at least in part, for differences in sensitivity of human tissues to TCDD and related compounds. Korkalainen et al. (2004), however, determined that the relative expression and inducibility of AhRR in several rat strains did not account for strain differences in sensitivity to TCDD.

Several investigations demonstrated AhR-mediated alteration of the estrogen receptor (ER) signaling pathway, and vice versa, that could occur through a variety of mechanisms, including inhibition of ER protein expression, enhanced

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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metabolism of estrogens, induction of inhibitory factors, binding of the AhR to inhibitory DREs, competition for common nuclear coregulatory proteins, and proteosome-dependent degradation of the AhR and ER (Reen et al., 2002; Safe and Wormke, 2003). Ohtake et al. (2003) reported that the AhR–Arnt complex can associate directly with ER-α and ER-β, which could lead to the recruitment of the ERs to estrogen-responsive promoters and to estrogenic effects. Those data are particularly relevant because TCDD exposure in animals is known to affect estrogen-dependent responses in several tissues, especially those involved in reproduction.


Chemicals Other Than TCDD That Affect AhR Function As indicated in previous updates, data on the ability of various dioxin-like chemicals to bind to the AhR and cause toxicity show that the AhR can mediate the toxicity of those substances; newer information supports that conclusion. Since Update 2002, several new assays and reagents have been developed to detect AhR ligands and to determine how they modulate AhR function (Behnisch et al., 2002; Fukuda et al., 2004; Han et al., 2004; Ramadoss and Perdew, 2004; Sun et al., 2004; Swanson et al., 2002). Several compounds alter AhR function by binding directly to the AhR. The indole derivatives indirubin and indigo (Adachi et al., 2004; Guengerich et al., 2004; Knockaert et al., 2004; Sugihara et al., 2004), several pesticides (Long et al., 2003), and some dietary flavonoids (Gouedard et al., 2004; Zhang et al., 2003) appear to act as AhR agonists. Other compounds, such as other naturally occurring flavonoids (Zhang et al., 2003); synthetic flavonoids (Palermo et al., 2003; Roblin et al., 2004; Zhou and Gasiewicz, 2003); the drug salicylamide (MacDonald et al., 2004); and omeprazole, 2-mercapto-5-methoxybenzimidazole, and promaquine (Backlund and Ingelman-Sundberg, 2004) also bind to the AhR, but they act as AhR antagonists. Notably, several compounds used for their ability to inhibit specific kinases also act either as AhR agonists (Andrieux et al., 2004) or as antagonists (Joiakim et al., 2003; Shibazaki et al., 2004). Their ability to produce agonist or antagonist activity appears to depend on their relative affinity for the AhR and on properties related to their intrinsic efficacy, that is, on their ability to produce a response once bound to the AhR. The latter could be related to ligand structure (Beger et al., 2002) and to the relative ability of a chemical to elicit a particular conformational change in AhR structure (Henry and Gasiewicz, 2003). Phenylthiourea is a weak activator of the AhR, but it can inhibit TCDD-induced responses under some conditions (Wang W-D et al., 2004). The relative agonist or antagonist activity of a compound also is species and gene specific (Gouedard et al., 2004; Zhou and Gasiewicz, 2003; Zhou et al., 2003).

Recent reports indicate the presence of AhR ligands in airborne particulate organic matter (Arrieta et al., 2003), in fish exposed to effluents from a bleached-pulp and paper mill (Hewitt et al., 2003), and in plant food extracts (Amakura et al., 2004).

Other chemicals, such as o,p′-DDT (Jeong and Kim, 2002), estradiol, and

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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dexamethasone (Lai et al., 2004), appear to alter TCDD- and AhR-dependent gene transcription by mechanisms that are not related to their ability to bind the AhR. Pearce et al. (2004) reported that 6-methyl-1,3,8-trichlorodibenzofuran, a weak AhR agonist and partial antagonist, also is a partial ER agonist.

It is postulated that TCDD is toxic by mimicking an endogenous ligand for the AhR and activating the receptor at inappropriate times or for inappropriately long periods. The actual physiologic ligand for the AhR, if any, is not known. Roblin et al. (2004) determined that a variant rat hepatoma cell line contains some agent that acts as an AhR agonist. Song et al. (2002) identified a compound, 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester, from porcine lung that binds to the AhR from several vertebrates, including human, and activates it to a transcriptionally active form. It is not clear whether this is truly a physiologically relevant ligand for the AhR. Previous investigations (IOM, 2001) noted that metabolites of tryptophan can act as AhR agonists. A study by Bittenger et al. (2003) reported that aspartate aminotransferase could convert tryptophan into indole-3-pyruvate, which then spontaneously reacts in aqueous solution to form several compounds that act as AhR agonists.


AhR-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 altered intracellular signaling pathways. Several genes that are modulated by TCDD and by dioxin-like compounds in a variety of biologic systems, including human cells, are listed in Table 3-2, which includes citations to papers published since Update 2002. Genes whose mRNA or protein concentrations have been altered are included, but enzymes or proteins whose biologic activities are altered by some other mechanism are not. Several genes are regulated by direct interaction of the AhR–Arnt complex with DREs in the promoter regions. Other genes are suspected, but not yet proven to be induced by this mechanism. The expression of several genes is inhibited by the ability of the AhR to bind to DREs near the DNA-binding sites for other transcription factors, such as the ER. The expression of other genes appears to be altered by posttranscriptional mechanisms. Table 3-2 also lists genes that are modulated in a variety of cells and tissues in several species, including humans, although the mechanisms of alteration are not understood. It is likely that the induction or repression of many of those genes is secondary to the ability of the AhR–Arnt complex to act directly on other genes. The size of the latter category emphasizes the ability of the ligand-bound AhR to initiate a cascade of molecular and biochemical events that eventually lead to cell and tissue alterations. That they are tissue-, species-, and developmental-stage-specific events also emphasizes the complex nature of the biochemical events that lead to particular toxic response (Wilson, 2004; Wood et al., 2002).

Since Update 2002, several investigations have examined the molecular mechanisms underlying the specificity of the responses elicited by TCDD and

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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TABLE 3-2 Genes and Proteins Known to Be Modulated by TCDD and/or Dioxin-like Chemicals

Reference

Genes

Genes and Proteins Directly Regulated by AhR

Thomsen et al., 2004

hairy and enhancer of Split homolog-1 (HES-1)

Son and Rozman, 2002

plasminogen activator inhibitor-1

Baba et al., 2001

aryl hydrocarbon receptor repressor (AhRR)

Porter et al., 2001

heat shock protein 27 (inhibition)

Jeon and Esser, 2000

interleukin-2

Gao et al., 1998

ecto-ATPase

Gillesby et al., 1997

pS2 (inhibition)

Kraemer et al., 1996

cyclooxygenase-2

Krishnan et al., 1995

cathepsin D (inhibition); Sp1 (inhibition)

Gaido and Maness, 1994

plasminogen activator inhibitor-2

Lamb et al., 1994

UDP glucuronosyltransferase1

Sutter et al., 1994

CYP1B1

Pimental et al., 1993

glutathione-S-transferase Ya

Takimoto et al., 1992

aldehyde dehydrogenase 4

Favreau and Pickett, 1991

NAD(P)H-menadione oxidoreductase 1

Tukey and Nebert, 1984

CYP1A2

Poland and Knutson, 1982

CYP1A1

Genes Suspected to Be Directly Regulated by the AhR

Gouedard et al., 2004

paraoxonase

Santiago-Josephat et al., 2004

TGF-β binding protein 1 (inhibition)

Niermann et al., 2003

T cadherin (inhibition)

Huang et al., 2002

proopiomelanocortin (ACTH precursor)

Ma, 2002

poly(ADP-ribose) polymerase

Matikainen et al., 2002

Bax

Park and Rho, 2002

Cu–Zn superoxide dismutase

Rivera et al., 2002

CYP2S1

Zhao et al., 2002

RANTES

Ogi et al., 2001

polκ

Ohbayashi et al., 2001

DIF-3

Sugawara et al., 2001

steroidogenic acute regulatory protein

Kim et al., 2000

c-myc

Lai et al., 1996

transforming growth factor-β (TGF-β); interleukin-6; interferon-γ

Masten and Shiverick, 1995

BSAP

Genes and Proteins Modulated by Posttranscriptional Mechanisms

Henley et al., 2004a

interleukin-1β

Dong et al., 1997

MHC Q1

Gaido et al., 1992

TGF-α; urokinase plasminogen activator

Puga et al., 1992

c-fos; c-jun

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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Reference

Genes

Other Genes Reported, Since Update 2002, to Be Altered by AhR Ligand Exposure

Adachi et al., 2004

ADP ribosylation factor 4; basic transcription factor 2 34-kDa subunit; cadherin 2; CDC-like kinase; complement component 5; cyclin-dependent kinase inhibitor 1A; cyclin-dependent kinse 1; CYP19A1; DNA mismatch repair protein; early growth response protein; 110-kDa heat-shock protein; heat shock factor-binding protein 1; heat shock protein 60-kDa protein; insulin-like growth factor-binding protein 10; insulin-like growth factor binding protein 1; insulin-like growth factor II; integrin β; interleukin 1 receptor type 1; 45-kDa interleukin enhancer-binding factor 2; NEDD5 protein homolog; Niemann-Pick C disease protein; retinoblastoma-binding protein 3; Rab geranylgeranyl transferase β subunit; RNA polymerase II elongation factor SIII p15 subunit; Sec61-γ; sex-determining region Y box-containing gene 9; short/ branched chain-specific acyl-CoA dehydrogenase; solute carrier family 2 member 2; T-complex protein 1 τ and δ subunits; thyroid receptor-interacting protein 15; topoisomerase I and II α; transcription factor HTF4; translation initiation factor 4E 25-kDa subunit

Fisher et al., 2004

Bcl-2 family genes bik, bid, Hrk, bok/mtd, mcl-1, bcl-x, and bcl-w; IAP family genes X-linked IAP, NAIP1, and NAIP5; Myd88; p21; p53; RIP; TNFR family genes OX40, Fas, CD30, Ltβ-R, and TNFR1; TNF family genes LIGHT, OX40L, and Bar-like; TRAF2

Johnson et al., 2004

actin α; Ahr; alcohol dehydrogenase 1, complex; angiopoietin-like 4; angiotensinogen; brain derived neurotrophic factor; cadherin 16; calbindin-28k; carbonic anhydrase 3; carboxylesterase 3; Cd44 antigen; coagulation factor II; cytokine receptor-like factor 1; epiregulin; fibroblast growth factor 7; fibroblast growth factor receptor 4; follistatin; forkhead box a2 and f2; Fos-like antigen 1; glutamyl aminopeptidase; Gro1 oncogene; high mobility group at-hook 2; α-2-hs-glycoprotein; hydroxysteroid 11-β dehydrogenase 2; insulin-like growth factor 2; insulin-like growth factor binding proteins 3, 5, and 6; integrin α 3, α 6, and β 4; IL-6; interferon activated gene 202a; lymphocyte antigen 6 complex, loci e, A and H; lysyl oxidase; matrix metalloproteinase 3 and 9; mitogen regulated protein proliferin 3; NADH dehydrogenase 1; osteopontin; p21; peripherin; phospholipase a2 group VII; proliferin 2; Ras-related protein; rennin 1 structural; retinol binding protein 4, plasma; RNA binding motif, single stranded interacting protein 1; secreted phosphoprotein 1; small proline-rich proteins 2b, 2c and 2f; spleen tyrosine kinase; squalene epoxidase; stratifin; thrombomodulin; TNF receptor family member 1b; tumor-associated calcium signal transducer 2

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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Reference

Genes

Karyala et al., 2004

ADP-ribosylation-like factor 6 interacting protein 5; calcium binding protein A11; CCAAT/enhancer-binding protein; esterase 10; immediate early response 3; nicotinic acetylcholine receptor subunit α 6; nuclear factor erythroid derived 2, like 2; prenylated SNARE protein; RIKEN-CDNA FLJ13933 FIS, clone Y79AA1000782; RIKEN-phosphogluconate dehydrogenase inhibitor; S100 calcium-binding protein A4; vanin 1; Vomeronasal organ family 2, receptor, 11; distal-less homeobox 5

Moennikes et al., 2004 (constitutively active AhR)

DEAD/H box polypeptide 3; DnaJ (hsp40) homolog, subfamily B, member 1; fatty acid binding protein 2 (intestinal); heat shock 70 kDa protein 5; heat shock protein 1α, hsp90; heat shock protein 105; hepatic nuclear factor 4 (HNF4); HIV-tat interactive protein 2; homocysteine-inducible, ER stress-inducible, ubiquitin-like domain member 1, Herp; C-type lectin-like receptor 2; lectin (galactose binding, soluble 1); malic enzyme; mannoside acetylglucosaminyltransferase 2; phosphoribosyl pyrophosphate amidotransferase; pleckstrin homology domain containing (family B number 1); Ras homolog gene family member E; ribosomal protein L12; S-100 calcium binding protein A10 (calpactin); signal transducer and activator of transcription 2; solute carrier protein 21 (organic anion transporter, member 10); TNFα-induced adipose-related protein; ubiquitin-specific protease 2; vaccinia related kinase 2; zinc finger protein 191

Murphy et al., 2004

matrix metalloproteinase-1

Vogel et al., 2004

CCAAT/enhancer-binding protein

Hoegberg et al., 2003

lecithin:retinol acyltransferase

Son et al., 2003

CK8 polypeptide; glutathione peroxidase; Ig λ-1 chain C region; Ig λ-2 chain C region

Bhathena et al., 2002

CYP2C11

Bruno et al., 2002

albumin; ATP synthetase β subunit; calreticulin precursor; cytochrome B5; CYP2D4; 25DX; endoplasmic reticulum protein ERP29 precursor; ferritin light chain; 78 kDa glucose-regulated protein precursor; glutamate dehydrogenase; glyceraldehydes-3-phosphate dehydrogenase; heat shock protein 72; 3-α-hydroxysteroid dehydrogenase; IκB kinase 2; 150 kDa iodothyronine 5′ monodeiodinase; isocitrate dehydrogenase; oxygen-regulated protein; peroxiredoxin IV; prohibitin; protein disulfide isomerase ER60 precursor

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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Reference

Genes

Martinez et al., 2002

activin receptor type II B; acyl-coenzyme A oxidase; aminoaclase 1; B-cell lymphoma protein 3; basic transcription element binding protein 1; bone morphogenic protein; β-catenin; Cdc42; CDK-2 associated protein; cellular retinoic acid binding protein 1; collagen IV α 3 chain; collagen VI α 3; cyclin-dependent kinase 4 inhibitor C; cyclin-dependent kinase inhibitor 2B isoform; CYP27A1; discoidin receptor tyrosine kinase; E2F dimerization partner 2; early growth response 1; EGF-containing fibulin-like extracellular matrix protein; ephrin A1, isoform a; epidermal growth factor receptor substrate 15; epithelial-cadherin; fibroblast growth factor; fibronectin receptor β subunit; Fos-related protein; GABA A receptor; GATA binding protein 1; glucocorticoid receptor; GTPase activating protein; homospermidine synthase; hsp 70 kDa protein insulin-like growth factor 1 receptor; GABA A receptor ε subunit; 25 kDa GTP binding protein; l hsp 70 kDa 2; hyaluronidase 1; insulin induced protein 1; interferon-induced protein 56 and p78; interferon γ receptor 1; interferon regulatory factor 4; IL-6 receptor β; IL-8; Kruppel-like factor 5; lamanin B2 chain and α 3b chain; leukemia inhibitor factor; low density liproprotein receptor-related protein; macrophage inflammatory protein 1-β; MAP kinase-activated protein kinase 2; MAP kinase phosphatase-1; matrix metalloproteinase 1 and 9; mesoderm specific transcript isoform; mitotic arrest defective protein; multifunctional DNA repair enzyme; neurotrophic tyrosine kinase; NFκB p100/p49 subunits; nuclear receptor coactivator 2; ornithine cyclodeaminase; 8-oxo-dGTPase; p53; p53-binding protein Mdm4; peripheral benzodiazepine receptor; polyamine oxidase; protein kinase C α; protein kinase C-like 2; protein tyrosine phosphatase type 1; pyruvate dehydrogenase kinase; replication licensing factor; retinoic acid receptor β; RNA polymerase II; S100 calcium binding protein; serine/threonine kinase 4; serine/threonine specific protein phosphatase; serum/glucocorticoid regulated kinase; STAT1; thioltransferase; thioredoxin reductase; thrombin receptor; thrombomodulin; thymosin β 10; tissue inhibitor of metalloproteinase-3; translation initiation factor 3 and 4H; transmembrane 4 superfamily member; tumor-associated calcium signal transducer 4; tyrosine-protein kinase receptor; ubiquitin-like interferon, α-inducible protein; vasoactive intestinal polypeptide receptor; VEGF; vitronectin; WAP four-disulfide core domain 2, isoform 1 precursor; zinc finger protein 42

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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Reference

Genes

Zeytun et al., 2002

angiogenin; Bad; bcl-w (Bcl2-like 2); casper; caspses 1, 3, 7, 8, 11, and 14; CRADD; cyclin-dependent kinase inhibitor p21 Waf1; DAXX (Fas-binding protein); DR5 (TRAIL death-inducing receptor); Fas ligand; IAP 1 and 2 (inhibitor of apoptosis proteins 1 and 2); fibroblast growth factor; G-CSF; GADD45 (DNA-damage inducible transcript 1); HGF (hepatocyte growth factor); ILs 3, 4, 5, 6, 7, 9, 10, 12α, 15 and 18; mdm2; NFκb1; NF-κB inducing kinase; p53 responsive protein; PDGFα; retinoblastoma supsceptibility protein; RIP (cell death protein); thrombospondin 3; TNFβ; TRAF2 (TNF receptor associated factor 2); (TRAF3 (death adaptor molecule); TRAF6 (CD40 associated factor); Trail (TNF-related apoptosis inducing ligand); TRIP (TRAF-interacting protein); tumor necrosis factor I and II receptors; VEGF-B, C, D and I

mediated by the AhR. The relative expression of the AhR and AhRR could influence those mechanism, as could the relative presence or activity of other molecules, such as Sp1 (Tsuchiya et al., 2003b), the retinoid receptor (Soprano and Soprano, 2003), antioxidants (Münzel et al., 2003), histone acetylases and DNA methylases (Nakajima et al., 2003), and nuclear factor erythroid 2-related factor 2 (Ma et al., 2004). Nickel and other hypoxia-mimicking agents (Davidson et al., 2003) could influence AhR activity at specific gene sites and in specific cells or tissues.

There also have been several reports that TCDD exposure to cells alters the activity of enzymes such as cAMP/protein kinase A (Vogel et al., 2004), mitogen-activated protein kinases (Kwon et al., 2003; Tan et al., 2002), and protein kinase C (Williams et al., 2004) by mechanisms that might not depend directly on the ability of the AhR–Arnt complex to bind to specific DNA sites. It has been postulated that the chronic activation of those and other kinase pathways is significant in the toxic actions of TCDD (Matsumura, 2003). The ability of TCDD to elicit formation of the nuclear AhR complex also could activate proteasomal degradation of the ER (Wormke et al., 2003). That mechanism, in addition to the ability of the AhR–Arnt complex to bind to inhibitory response elements in estrogen-regulated genes, could be responsible for the observation that AhR ligands suppress estrogen-induced responses in several estrogen-responsive tissues. Caruso et al. (2004) also reported that the AhR could affect the trafficking or processing of cellular proteases found in endosomes and lysosomes. TCDD might cause many of its effects by altering the expression of genes involved in matrix remodeling. Murphy et al. (2004) reported that TCDD in-

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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duces the expression of matrix metalloproteinase-1 and that the AhR-signaling pathways interact with the retinoic-acid-signaling pathways for the regulation of the expression. Several other investigators reported TCDD-induced expression of matrix metalloproteinases in experimental systems (Table 3-2).

As noted in Update 2002, TCDD and other AhR ligands alter cell proliferation and differentiation in a variety of model systems. This is believed to be an important factor in the mechanism of TCDD-induced carcinogenesis and toxic effects in many tissues, especially in reproductive tissues and in developing fetuses. Several studies have been published that identify the ability of TCDD, via the AhR, to disrupt the cell cycle (Abdelrahim et al., 2003; Hestermann et al., 2002; Levine-Fridman et al., 2004; Puga et al., 2002; Shimba et al., 2002), possibly by mechanisms that are related to the ability of the AhR to form complexes directly with cellular proteins, including retinoblastoma protein, that are critical in cell cycle regulation. The AhR also could regulate genes for cell cycle regulatory proteins (Table 3-2).

Data on several individual genes are discussed below in the context of particular tissue systems or toxic endpoints that could be affected by TCDD. However, this should not be interpreted to indicate that the ability of TCDD to modulate the expression of a particular gene is limited to that tissue. Although the effects of TCDD are high tissue and cell specific, genes or biochemical pathways modulated in one tissue are often found to be modulated in several other tissues.

Mechanisms Related to Particular Toxic Endpoints

An accumulation of studies in experimental animals indicates that TCDD affects a variety of tissues, and the type of effect observed is often tissue specific. Effects 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 TCDD’s 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.

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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Lethality and Wasting Syndrome As indicated above and in Update 2002, there is some variation among species in susceptibility to the lethal effects of TCDD that is attributable in part to differences of primary amino acid sequences and expression of the AhR protein. Exposure of most animal species to relatively high doses of TCDD elicits a wasting 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 identified. 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 research has focused on the ability of TCDD to perturb hypothalamus function. Cheng et al. (2003a) reported that the treatment of rats with TCDD caused a down-regulation of nitric oxide synthase and NADPH–diaphorase activity in the hypothalamus. They suggested that decreased production of nitric oxide results in the reported increased activity of the enkephalinergic system that modulates food intake (Cheng et al., 2003b). The expression of several hypothalamic neuropeptides—neuropeptide Y, proopiomelanocortin, cocaine- and amphetamine-regulated transcript, and melanin-concentrating hormone—were increased in TCDD-treated rats that displayed a decrease in body weight gain (Fetissov et al., 2004). The investigators also reported that the AhRR was present in the nuclei of neurons found in the hypothalamus, and they suggested that the AhR has a physiologic role in regulating food intake. Yang et al. (2004) reported that TCDD treatment elicited an induction of Sim1 in a neuronal cell line as well as in mouse kidney and hypothalamus. Sim1 is essential for the differentiation of the paraventricular nucleus of the hypothalamus, and the authors suggested that Sim1 mediates the effect of TCDD on feeding behavior in laboratory animals. Uno et al. (2004) reported that mice that lack expression of a functional cyp1a1 gene (cyp1a1 -/- mice) were protected against TCDD-elicited lethality and wasting syndrome. Although it is known that this cyp1a1 enzyme generates oxidants and metabolites of endogenous and exogenous compounds, how those materials operate in the induced wasting syndrome is not clear.


Effects on Skin and Adipose Tissue Skin lesions, including chloracne, often are reported for 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 an increased expression of IL-1β in human keratinocytes by a posttranscriptional mechanism (Table 3-2). 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); that process is AhR dependent.

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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Several groups have examined the mechanism because it could help explain how TCDD acts in various tissues. Fibroblasts stimulated by a hormone mixture undergo a cascade of molecular events to initiate adipocyte differentiation. Those events include regulation of c-myc, fos, and jun and then up-regulation of CCAAT-enhancer-binding proteins and of the peroxisome proliferators-activated receptor (PPAR) γ. Hanlon et al. (2003) and Cimafranca et al. (2004) determined that AhR- and ERK-dependent pathways function synergistically to mediate TCDD-induced suppression of PPARγ and subsequent inhibition of adipocyte differentiation. Vogel and Matsumura (2003) reported that the inhibitory effect of TCDD was absent in fibroblasts from c-Src-deficient mice, suggesting that c-Src kinase mediates the antiadipogenic action of TCDD. Notably, the AhR protein is depleted during adipose tissue differentiation, resulting in the loss of responsiveness to TCDD and related xenobiotics. Shimba et al. (2003) determined that the Ahr gene is regulated during adipogenesis at the transcriptional level by an unidentified trans-acting factor that was higher in preadipocytes than it is in adipocytes. Down-regulation of this factor during adipose differentiation could result in suppression of Ahr gene transcription.

Several investigations cited in previous updates noted that TCDD exposure alters plasma and tissue lipid content in animals. Stanton et al. (2002) reported increased plasma concentrations of total triacylglycerides and specific fatty acids in immature male chickens exposed to TCDD but no increase in free fatty acid concentrations. Treatment with TCDD antagonized estrogen-induced increases in fatty acids. The data suggested that TCDD treatment increased plasma lipids through a mechanism other than increased adipose tissue mobilization. Chen et al. (2002) examined the lipid content of pregnant female rats and fetuses exposed to mixtures of dioxin-like compounds. The lipid content of placenta, liver, and serum from treated dams was lower than it was in the control group at gestational days 16 and 21 and at postnatal day 4. The lipid content of the offspring was not affected until after birth, when they were exposed by lactation, at which time the lipid content in treated mice was higher than it was in controls.


Effects on Bone and Teeth Previous studies have suggested that defects in children’s tooth development 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). Tooth development in rats and mink appears to be a target of TCDD toxicity that is at least partially a result of coexpression of the AhR and Arnt during early tooth development (IOM, 2003). TCDD can arrest molar tooth development in rats after in utero and lactational exposure. Recent studies in rats have identified the “critical window” of in utero exposure that effects development of the molars (Miettinen et al., 2002) and incisors (Kiukkonen et al., 2002), as well as impairment of tooth development in tissue culture (Partanen et al., 2004). Pregnant female dioxin-sensitive line C rats were exposed to a single oral dose of TCDD at 1 µg/kg on gestation days 11, 13,

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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or 19 and on postnatal days 0, 2, and 4 (Miettinen et al., 2002). Pups were killed at 40 days of age. The offspring exposed in utero and via lactation lacked third molars; the greatest effect was exhibited in pups exposed on gestation day 11. Postnatal exposure to TCDD did not affect third-molar development, and none of the treatments affected development of the first and second molars. TCDD accelerated the eruption of the lower incisors and retarded the eruption of the third molars. That effect was most pronounced if exposure had occured during early morphogenesis and from tooth initiation to the early-bud stage, after which sensitivity decreased substantially.

In a study in which mouse embryonic molar tooth explants were exposed to TCDD at 1 µM in tissue culture, morphogenesis of the first molar teeth was not inhibited, but the development of the second molars was arrested when they were explanted before the early-bud stage (Partonen et al., 2004). Later exposure led to smaller tooth size and altered cuspal morphology. The results of in vitro studies also revealed that TCDD enhanced apoptosis of dental epithelial cells predetermined to undergo apoptosis during normal development. The authors concluded that TCDD can arrest tooth development in vitro if exposure starts at the initiation stage and that TCDD interferes with tooth development by stimulating apoptosis in cells of the dental epithelium.

Hans/Wistar (TCDD-resistant) and Long-Evans (TCDD-sensitive) female rats were administered subcutaneous total doses of TCDD at 0.17, 1.7, 17, or 170 (H/W rats only) µg/kg (Kiukkonen et al., 2002). The treatments began when the rats were 10 weeks old and continued for 20 weeks. The exposures covered two life cycles of the incisor. At the high doses (17 and 170 µg/kg), there were color defects and pulpal perforation of the lower incisors and arrest of dentin formation of the incisor teeth. The authors concluded that there was a dose-dependent effect in the mesenchymal and, to a lesser extent, the epithelial elements of the forming tooth.

No relevant studies on bone have been published since Update 2002. In general, possible effects of TCDD on bone have not been thoroughly investigated.


Cardiovascular Toxicity TCDD can affect the developing cardiovascular system, but there is little evidence that the cardiovascular system is a major target of TCDD toxicity in adult animals (IOM, 2003). It has been proposed, however, that exposure to dioxin increases the incidence of ischemic heart disease by exacerbating its severity (Dalton et al., 2001).

Male marmosets (Callithrix jacchus) treated with a single subcutaneous dose of TCDD at 1, 10, or 100 ng/kg TCDD showed no overt signs of toxicity by 2 or 4 weeks after treatment, and there was no difference in heart weight, compared with control monkeys. Histochemistry (Riecke et al., 2002) revealed an increase in collagen (picrosirius red stain) that presented in different patterns in the intracellular matrix of the myocardium; the changes were associated with activation of TGFβ1, which the authors suggested was a mediator.

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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Cardiomyopathy and chronic active arteritis increased in a dose-related manner in Harlan Sprague-Dawley rats gavaged 5 days per week for 2 years with TCDD at 0, 3, 10, 22, 46, or 100 ng/kg/day (Jokinen et al., 2003; NTP, 2004). The severity of the cardiomyopathy was minimal on average, and the chronic active arteritis occurred mainly in the mesentery and pancreas at the 46 and 100 ng/kg doses.

Treatment of female rats with the dioxin-like 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126) for 12 weeks affected several cardiovascular risk factors, including heart weight, serum cholesterol, and blood pressure (Lind et al., 2004). Niermann et al. (2003) reported that TCDD exposure of rat vascular smooth muscle cells repressed, through an AhR-dependent mechanism, the expression of T-cadherin, an adhesion molecule that is highly expressed in vascular tissues and cells (Table 3-2).


Pulmonary Toxicity This and previous updates report evidence suggestive of an association between herbicide exposure in Vietnam and respiratory cancer (see Carcinogenesis below). Although several published reports have suggested an association between TCDD exposure and chronic obstructive pulmonary disease, this committee found insufficient evidence to support a relationship between herbicide exposure and respiratory disorders that are not considered cancer. This is, in part, based on the findings that the pulmonary system of animals is somewhat resilient to the toxic effects of TCDD. Low doses (1–10 µg/kg) of TCDD that result in toxicity to other organ systems do not appear to damage the lungs of animals. No data have been reported since Update 2002 to elucidate the toxic effects of TCDD on the respiratory system. However, increased lung weight and bronchiolar metaplasia of the alveolar epithelium has been observed in Harlan female Sprague-Dawley rats gavaged 5 days per week for 2 years with TCDD at 0, 3, 10, 22, 46, or 100 ng/kg/day (NTP, 2004).

Martinez et al. (2002) examined the effects of TCDD exposure on global gene expression profiles in human lung cell lines. Althered gene responses included xenobiotic metabolizing genes, genes known to be involved in cell cycle, and genes involved in cell-signaling pathways and that mediate cellular communication (Table 3-2). Some differences were observed between malignant and non-malignant cells. The authors concluded that TCDD exposure can modify signaling pathways associated with pulmonary disease.


Hepatotoxicity The liver is a primary target of TCDD and related compounds in many animals, but the severity of effects varies considerably among species. The liver and its cells often are used to study the effects of TCDD on biochemical pathways that could be responsible for toxic endpoints. In a 2-year NTP study (2004), rats administered TCDD at a dose of at least 10 ng/kg (5 days per week for 104 weeks) exhibited several non-neoplastic lesions in the liver. The lesions included hepatocyte hypertrophy, multinucleated hepatocytes, inflammation, pig-

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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mentation, diffuse fatty change, necrosis, bile duct hyperplasia, bile duct cyst, nodular hyperplasia, portal fibrosis, and cholangiofibrosis.

In the process of crossbreeding TCDD-sensitive and TCDD-resistant rat strains, Niittynen et al. (2003) observed a new type of TCDD-induced toxicity in the livers of those animals. The toxicity was characterized by the accumulation of bile pigments, made up primarily of biliverdin and related compounds, progressive sinusoidal distension, and hepatic peliosis with membrane-bound cysts. Patterson et al. (2003) reported that TCDD could potentiate hepatic apoptosis during non-lethal hepatic endotoxemia by an unknown mechanism.

AhR-null allele mice develop liver fibrosis characterized by increased hepatic retinoid concentrations, tissue transglutaminase type II activity, TGFβ overexpression, and accumulation of collagen and by reduced expression of PPARγ. Andreola et al. (2004) reported that the effects were reversed when the mice were fed a diet deficient in vitamin A. Previous updates have reported that TCDD exposure in laboratory animals significantly disrupts the homeostasis of vitamin A. Several recent reports are consistent with the ability of the AhR-signaling pathway to interact with the retinoid receptor pathways by different mechanisms. Schmidt et al. (2003) reported that TCDD exposure in rats results in the significant elevation of concentrations of tissue all-trans-retinoic acid and in decreased concentrations of several other retinoid acid metabolites. A review by Soprano and Soprono (2003) pointed out that several retinoids, in addition to affecting the RXR–RAR signaling pathway, also can activate the AhR pathway.

Several research groups have suggested that induction of cellular oxidative stress is a mechanism by which TCDD could elicit damage via the AhR to lead to many of the toxic endpoints observed, including liver injury. Since Update 2002, several groups have examined the mechanism. Senft et al. (2002) noted that the production of hydrogen peroxide (H2O2) by mitochondria from AhR-null-allele mice was one-fifth that found in wild-type mice. TCDD treatment also caused an increase in succinate-stimulated mitochondrial H2O2 production in wild-type, but not in AhR-null-allele, animals. Their data suggested that constitutive and TCDD-induced mitochondrial reactive oxygen production are associated with a function of the AhR. Shertzer et al. (2004a) reported that, although TCDD treatment decreased microsomal production of H2O2, the addition of other halogenated aromatic hydrocarbons to TCDD-induced microsomes in vitro actually stimulated the release of H2O2 production. The authors postulated that the pathway contributes to oxidative stress response and to toxicity of those compounds. CYP1A2, an AhR-regulated protein (Table 3-2), was found to protect against reactive oxygen production in mouse liver microsomes (Shertzer et al., 2004b). Hilscherova et al. (2003) reported that when chick eggs were exposed before incubation there was significant effects on the indicators of oxidative stress in liver, but not in the brain, of the hatchling chicks. The treatment increased susceptibility to lipid peroxidation and oxidative DNA damage that was only partially mitigated by administration of vitamins E and A. Mice treated with chitosan

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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oligosaccharide II were protected against TCDD-induced lipid peroxidation, inhibition of glutathione peroxidase, and glutathione S-transferase activities and to losses in body weight and in liver weight (Shon et al., 2002). Similar chitooligosaccharides were found to scavenge superoxide and hydroxyl radicals in vitro; to inhibit the growth of cancer cells; and to increase the activity of macrophages, T cells, and natural killer cells in tumor-bearing mice.

Abraham et al. (2002) tested the ability of the caffeine test for the measurement of CYP1A2 induction in human subjects with variable exposure to TCDD. CYP1A2 was induced at least 10 times in 2 subjects who had been highly exposed (initial blood fat concentrations of TCDD at 144,000 and 26,000 ppt, corresponding to doses of TCDD at 25 and 6 µg/kg). However, the test was not able to show induced CYP1A2 activity in a person who had moderate TCDD exposure (initial blood fat TCDD concentration of 856 ppt). The researchers concluded that direct quantification of TCDD was more specific and sensitive for determining exposure. The body burdens of TCDD at 25 and 6 µg/kg observed in 2 of the subjects are among the highest ever recorded in humans and are at least 100-fold higher than body burdens in most, if not all, Vietnam veterans. However, those concentrations are comparable to the doses used in most studies with experimental animals.


Pancreatic and Gastrointestinal Tract Effects An NTP study by Nyska et al. (2004) evaluated the effects in the pancreas of chronic exposure of female rats to TCDD (2 years; 3–100 ng/kg/day). Several dose-related non-neoplastic changes were observed, including cytoplasmic vacuolation, chronic inflammation, atrophy, and arteritis. Low incidences of pancreatic acinar adenoma and carcinoma also were observed. The data suggest that the pancreatic acini are target tissues for TCDD.


Neurotoxicity Few studies have examined the possibility of nervous system damage in adult animals exposed to TCDD. For those studies that have been performed, the developing brain appears to be more sensitive (see Developmental Toxicity) than does the brain in the juvenile or adult animal. Some studies cited in Update 2002 suggest that the nervous system may also be affected in adult animals, albeit at higher doses. More recent studies are consistent with this (see Lethality and the Wasting Syndrome).

Several investigators have suggested that TCDD-induced oxidative stress elicits damage in the nervous system. Some of those studies were cited in Update 2002. Hassoun et al. (2003) observed that subchronic exposure of rats to TCDD (10–46 ng/kg/day for 13 weeks) caused dose-related increases in the production of superoxide anion and lipid peroxidation in the cerebral cortex and hippocampus. The changes were associated with decreases in superoxide dismutase. Lee et al. (2002) also reported that alteration of cellular redox balance could mediate a TCDD-induced inhibition of proliferation of human neuronal cells. In that case,

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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however, TCDD treatment of isolated SK–N–SH neuroblastoma cells suppressed the basal generation of reactive oxygen species, caused an inhibition of lipid peroxidation, and increased the glutathione concentration. Increases in superoxide dismutase and catalase were reported, but decreases in glutathione peroxidase and glutathione reductase also were observed. Disruption of the blood–brain barrier by TCDD through oxidative stress could compromise neuronal homeostasis and potentiate neurotoxicity. Filbrandt et al. (2004) reported that the AhR was present and functionally active in cerebral vascular endothelial cells and astrocytes.

Previous investigations reported neuropathy and changes in synaptic transmission in the brain of TCDD-exposed rats (IOM, 1996, 1999, 2001). Cho et al. (2002) investigated the possibility that TCDD affects the expression of synaptic proteins in E18 rat cortical cells. After 4 days of exposure, cell viability was significantly reduced and there were decreased numbers of secondary or higher order dendritic processes. NMDA receptor subunits were found to be up-regulated, but there was a down-regulation of several synaptic organizing proteins (PSD-95, Densin-180, septin6 homologue) and a synapse-enriched enzyme (αCaM kinase II). The authors suggested that the altered expression of synaptic proteins is responsible for the altered synaptic transmission and neuropathy observed after TCDD exposure.

Studies cited in previous updates indicate that TCDD affects the homeostasis of vitamin A. Huang et al. (2003) used cellular retinal-binding protein (CRBP-1) knockout mice to determine whether the effect of TCDD on gene expression in the brain and pituitary might be modulated by the retinoid system. In general, the relative induction of CYP1A1 and the AhR was lower in the brains of CRBP-1 knockout animals. However, the basal expression of the AhRR was higher in the pituitary from knockout mice.


Immunotoxicity The immune system of laboratory rodents is highly sensitive to the toxic effects of TCDD, and the immune suppression observed after TCDD exposure is mediated through the AhR. Many cell types make up the immune system, and most have been shown to express the AhR. Developing an understanding of the specific cells that are altered by TCDD and how they contribute to alterations in immune function induced by TCDD is of great interest to the research community. Understanding how TCDD affects the immune system in rodents enhances the ability to extrapolate the experimental results to assessment of human risks. Since Update 2002, several papers have addressed the mechanisms of TCDD’s effects on the immune system.

Involution of the thymus is a hallmark of TCDD exposure. The mechanism of action of TCDD on the thymus is open to question—some data support indirect effects on the thymocytes via the thymic epithelial or dendritic cells, others support direct effects on thymocytes. Two recent studies, one using differential expression of the AhR (Laiosa et al., 2003) and one using differential ARNT

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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expression (Tomita et al., 2003), showed that the AhR–ARNT-signaling component that leads to thymic involution is required in the thymocytes but not in the dendritic cells or in other thymic stromal cells. Laiosa et al. (2003) also showed that TCDD blocked the ability of early thymocytes to divide, perhaps because they are incapable of homeostatic repopulation of the thymus. TCDD also induced the expression of adseverin (a protein that could influence cell differentiation) in thymocytes but not thymic epithelial cells, whereas CYP1A1 was induced in both types of cells (Svensson et al., 2002). A direct effect of TCDD on the function of thymic epithelial cells was supported by Riecke et al. (2003), who reported that exposure to low concentrations of TCDD promotes the terminal differentiation of thymic epithelial cells in vitro and alters their expression of several adhesion molecules. ARNT2, another protein that dimerizes with the AhR, is not involved in thymic atrophy (Laiosa et al., 2002). Those new results favor the hypothesis that thymic involution observed after TCDD exposure results from direct AhR-mediated toxicity to the developing thymocytes that could be exacerbated by additional effects on thymic epithelial cells.

Mature lymphocytes in the secondary lymphoid organs also are affected by TCDD. Recent studies using wild-type or AhR-knockout cells show that the AhR must be expressed in CD4+ and CD8+ T cells for TCDD to suppress the generation of a cytotoxic T lymphocyte response (Kerkvliet et al., 2002). Induction of CYP1A1 in purified T cells exposed to TCDD in vitro validates the presence of a functional AhR (Doi et al., 2003). Camacho et al. (2002) suggested that activation of the Fas gene by TCDD induces apoptosis of activated T cells, leading to immune suppression. Kwon et al. (2003) used human Jurkat T cells to show that the mitogen-activated protein kinase signaling pathway was activated by TCDD, including activation of caspase 3, a mediator of apoptosis. Gene array analysis has identified other genes altered in the thymus or spleen of TCDD-treated mice (Park et al., 2001; Zeytun et al., 2002), but their functional significance to TCDD’s immunotoxicity has not been determined. Low concentrations of TCDD also were shown to directly alter the activation of dendritic cells derived from bone marrow cell culture (Ruby et al., 2002). Dentritic cells are required for presenting antigens to T cells to initiate an immune response.

Several new projects attempted to explain how TCDD enhances mortality of mice infected with influenza virus. Previous studies had shown that the virus was cleared from the lungs of TCDD-treated mice even though the T cell and antibody-mediated immune responses to the virus were suppressed. Vorderstrasse et al. (2003) reported that virus-specific IgA concentrations were significantly higher in TCDD-treated mice and that all other classes of antibodies were reduced. There also was an increase in the number of neutrophils in the lungs of virus-infected, TCDD-treated mice; natural killer cell numbers and inflammatory cytokine concentrations (TNFα, IL-1, IFNα/β) were not changed (Neff-LaFord et al., 2003). Damage to the lung caused by the increase in neutrophils was postulated to explain the toxicity of TCDD in influenza-infected mice. Increased

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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numbers of neutrophils in mouse spleen and blood with a growing tumor allograft also were reported by Choi et al. (2003). The neutrophils from TCDD-treated mice were defective at killing tumor cells but were enhanced in respiratory burst capability, which could lead to tissue damage. However, those results contrast with a recent in vitro study in which TCDD suppressed the oxidative burst of human neutrophils (Abrahams et al., 2003). Kim HJ et al. (2003) reported that repeated dosing of rats with TCDD resulted in higher numbers of CD11b+ cells (macrophages or neutrophils) in the spleen and in an increase in serum IL-6.

An alternative hypothesis for mortality in TCDD-treated mice infected with influenza is based on mitochondrial toxicity, similar to that seen in Reye’s syndrome in humans (Luebke et al., 2002). However, no effect of TCDD on serum NH3 or on glucose concentrations, two biologic markers of Reye’s syndrome, was observed. Rather, enhanced pulmonary inflammation, consisting of increased macrophage and neutrophil numbers, was reported; there were no effects on the concentrations of TNFα, MIP-1α, MIP-2, overall protein, or LDH in the lung. Those results confirm and extend the findings of Neff-LaFord et al. (2003).

B cells also appear to be direct targets of TCDD; they express high concentrations of CYP1A1 after exposure to TCDD (Doi et al., 2003). Several genes could be regulated by TCDD in B cells, including p27kip1, a regulator of cell survival and differentiation (Crawford et al., 2003); AP-1, a transcription factor that influences B cell function (Suh et al., 2002); and Pax5, a repressor of B cell differentiation (Yoo et al., 2004). Recent studies have validated earlier work that showed that PCB congeners that are chlorinated in the di-ortho positions inhibit

TCDD-induced suppression of B cell functions; the non-ortho-substituted PCB (PCB77) produced additive effects on B cells with TCDD (Suh et al., 2003). TCDD has long been known to suppress primary antibody responses, primarily IgM production. Two new studies address the effect of TCDD on antibody class switching to IgG production, an important aspect of the immune response that is crucial for vaccination effectiveness. Ito et al. (2002) showed that TCDD suppressed the production of key cytokines (IL-2, IL-4, IL-5, IL-6) produced by T cells that are important for B cell switching. IL-5 was particularly sensitive to suppression by TCDD because of its effect on T cells rather than antigen-presenting cells. The proliferation of germinal center B cells, which is essential for the production of high-affinity antibodies, also was suppressed in TCDD-treated mice; and TCDD did not enhance B cell apoptosis (Inouye et al., 2003).

A novel effect of AhR signaling to deplete peritoneal B1 cells was described in mice that express a constitutively active AhR (Andersson et al., 2003). B1 cells are thought to operate in the innate response to infection by viruses and bacteria. The ability of TCDD to activate the AhR to cause a similar depletion of peritoneal B1 cells has not been investigated.

TCDD increases the number of hematopoietic progenitor cells in the bone marrow. Sakai et al. (2003) verified and validated that finding, and they reported that stem cells from TCDD-treated mice (40 µg/kg) were incapable of reconstitut-

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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ing the bone marrow of irradiated recipient mice. Those effects were not linked to changes in cell proliferation or cell death, and they were not seen in AhR-deficient mice.

Perinatal exposure of rats to low doses of TCDD was found to suppress the contact hypersensitivity response to dinitrofluorobenzene as measured in 6-month-old female offspring (Walker et al., 2004).

Rheumatoid arthritis is a chronic inflammatory condition that affects joints. It is characterized by the proliferation of cells called synoviocytes and by the production of proinflammatory cytokines and chemokines. Tamaki et al. (2004) reported that several AhR agonists, including TCDD, could increase the mRNA for IL-1 β in a human-fibroblast-like synoviocyte line, and that it occurred via the AhR. AhR agonists are contained in cigarette smoke, so the authors posit that might provide the basis for a link between cigarette smoking and rheumatoid arthritis.


Carcinogenesis TCDD has been demonstrated to be a carcinogenic agent and potent tumor promoter in several model systems. A 2-year bioassay in female rats revealed carcinogenicity that was evidenced by increased incidence of cholangiocarcinoma and hepatocellular adenoma of the liver, by cystic keratinizing epithelioma of the lung, and by gingival squamous cell carcinoma of the oral mucosa (NTP, 2004).

The ability of TCDD to induce cell proliferation and to alter differentiation is believed to be an important factor in its mechanism of carcinogenesis. Ray and Swanson (2003, 2004) investigated whether the tumor-promoting activity of TCDD results from its ability to alter proliferation, differentiation, or senescence of normal human epidermal keratinocytes. TCDD accelerated differentiation, as demonstrated by increased expression of the differentiation markers involucrin and filaggrin. TCDD also increased proliferation, as indicated by an increased production of NADH–NADPH and changes in cell cycle. Dioxin exposure attenuated senescence and repressed expression of the tumor suppressor proteins p53 and p16INK-4a. The ability of TCDD to immortalize human keratinocytes was indicated by those authors to be a novel mechanism by which the compound could lead to malignancy. Parfett (2003) examined the ability of TCDD and other tumor promoters to induce the expression of proliferin (PLF), a glycoprotein suggested to influence the regulation of cellular proliferation and differentiation, in C3H/10T/1/2 cells. TCDD induced the expression of basal- and serum-induced PLF mRNA. The authors suggested that the induction of PLF in that cell line could be used as a short-term marker for chemical agents with promotional activity.

Several of the enzymes induced by TCDD, including CYP1A1, CYP1A2, and CYP1B1, are responsible for the metabolic activation of many promutagens, so activation of the AhR is considered important for the carcinogenic activity of many compounds. Lin P et al. (2003) reported an association between increased CYP1B1 and AhR expression in human non-small-cell lung cancer tissues.

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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CYP1B1 expression was detected in 3 of 19 normal lungs, but in 42 of 89 non-small-cell lung cancers. A similar study found that CYP1B1 mRNA concentrations were elevated in peripheral leukocytes of lung cancer patients (Wu et al., 2004). Both studies controlled for age, smoking status, and sex. Previous reports indicate the development of lung cancer in female rats but not male rats exposed to TCDD. Lin et al. (2004) reported that cotreatment with 17 β-estradiol (E2) and TCDD significantly enhanced the toxicity observed in human bronchial epithelial cells, and that TCDD cotreatment increased the production of E2 metabolites. The authors suggested that the metabolites enhance the effects on cells.

Chronic bioassays have shown TCDD exposure to increase the incidence of hepatic tumors in female rats but not in male rats. Moennikes et al. (2004) used a transgenic mouse line for expression of a constitutively active AhR to investigate the role of that protein in hepatocarcinogenesis. The presence of the constitutively active AhR dramatically increased the incidence of N-nitrosodiethylamine-initiated tumors. Whereas only 1 tumor was observed in the 15 wild-type mice, 19 tumors were present in the 19 transgenic mice. Notably, and as indicated in Update 2002, increased stomach tumors were observed in the transgenic mice not treated with N-nitrosodiethylamine (Andersson et al., 2002). Chen et al. (2003) confirmed that AhR activation and induction of CYP1A1 was required for benzo[a]pyrene-7,8-dihydrodiol-induced apoptosis in human HepG2 cells. That activity also was linked to increased mitogen-activated protein kinase activity that was AhR dependent. However, Schrenk et al. (2004) reported that TCDD inhibited apoptosis in rat hepatocytes initiated by ultraviolet irradiation. The suppression of apoptosis by TCDD coincided with increases in concentration and hyperphosphorylation of the tumor suppressor protein p53.

Non-genotoxic carcinogens (such as TCDD) are thought to induce tumor formation by altering the balance between cell proliferation, differentiation, and death. Mally and Chipman (2002) examined the effect of TCDD treatment (2.5–250 ng/kg for 2 days/week for 4 weeks) on gap junction formation in relation to proliferation and apoptosis. Dose-dependent reductions in gap junction formation were observed in rat liver but not in rat thyroid or kidney. Alterations in gap junctions did not correlate with induction of cell proliferation. However, Dietrich et al. (2002) reported that TCDD treatment of confluent WB-F344 rat liver epithelial cells induced a release from contact inhibition and a 2-fold increase in cell number in the presence of serum. This did not occur when TCDD was added to exponentially growing or to subconfluent, serum-deprived cells. The contact-inhibited cells also demonstrated loss of G1 arrest and increases in cyclin D2 and cyclin Q protein. The cdk2–cdc2-specific inhibitor olomoucine abolished the TCDD response. The same research group reported that TCDD exposure to those cells caused a down-regulation of γ-catenin protein and mRNA (Dietrich et al., 2003). Notably, catenin, a cell membrane protein, also is considered a tumor suppressor. Chramostova et al. (2004) reported that strong or moderate AhR

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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ligands, including TCDD, increased WB-F344 cell numbers that corresponded to an increased percentage of cells in S phase.

Several studies have implicated the β-catenin-signaling pathway in prostate cancer. The cytoplasmic accumulation of free β-catenin leads to increased binding to and activation of particular transcription factors that control genes involved in proliferation and differentiation. Accumulation of intracellular β-catenin occurs in a variety of cancers. Chesire et al. (2004) reported that the overexpression of a mutant hyperactive form of β-catenin leads to increased AhR in human prostate cancer cells. A possible role of this increase in the development of prostate cancer has yet to be determined.

Investigations by Lamartiniere (2002) indicated that if TCDD is given to pregnant rats, the offspring are more susceptible to dimethylbenz[a]anthracene-induced mammary cancer as adults. Offspring exposed prenatally to TCDD had mammary glands with more terminal end bud structures and fewer lobules. The terminal end buds are considered more undifferentiated and more susceptible to for tumor initiation; the lobules are more mature and are the least susceptible structures. Thus, the timing of exposure to TCDD could be extremely important for carcinogenesis. Van Duursen et al. (2003) reported that exposure to TCDD and several dioxin-like compounds decreased the ratio of 4-hydroxyestrogens to 2-hydroxyestrogens but increased the concentrations of potentially carcinogenic estrogen metabolites in human mammary epithelial cell lines. A high estrogen 4-/2-hydroxylation ratio has been identified as a likely indicator of the presence of mammary tissue neoplasms. The authors suggested that the value of this ratio as a prognostic marker for cancer risk should be further examined. Spink et al. (2003) reported that the presence of estrogen was required to maintain high AhR expression in those cells and inducibility of the enzymes responsible for the altered metabolism of estrogen.

Kim AH et al. (2003) used a differential-dose regimen to examine the possible use of area under the curve as a dose metric for tumor promotional responses in female rats exposed to TCDD. The volume fraction of GST-positive foci in livers was higher in the TCDD group given a high peak dose during the first week of treatment than it was in the group that received the same average daily dose for the duration of the experiment. The authors interpret those findings to indicate that the peak magnitude of TCDD in the liver rather than the area under the curve is more important in the tumor-promoting ability of TCDD.


Effects on the Testis Many effects of TCDD in male rodents have been reported previously, including decreases in the size of the accessory sex organs and daily sperm production. Both AhR and Arnt are expressed in rat and human testis, and the data suggest that AhR in that tissue is regulated by follicle-stimulating hormone (FSH) (Schultz et al., 2003). The latter result suggests a possible action by the AhR in controlling spermatogenesis. The expression of the AhRR also is very high in the human testis (Yamamoto et al., 2004), suggesting at least the

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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possibility that the human testis might be relatively resistant to the effects of TCDD.

Other investigations suggest that TCDD causes tissue damage by induction of oxidative stress. Latchoumycandane and Mathur (2002) observed that subchronic treatment of rats with TCDD (1–100 ng/kg/day for 45 days) resulted in a significant decrease in the weights of the testis, epididymis, seminal vesicles, and ventral prostate. There also was a substantial decline in daily sperm production. Testicular activities of superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase were decreased, and there were increased tissue concentrations of H2O2 and lipid peroxidation. Acute-exposure studies produced a similar effect of TCDD-induced oxidative stress on epididymal sperm (Latchoumycandane et al., 2003). A report by Kwon et al. (2004) noted a protective effect for dietary ursodeoxycholic acid (UDCA), a biliary component in bears, on the acute effects of TCDD on testes in mice. TCDD induced decreased testicular weight, and decreases in serum-luteinizing hormone and FSH were abrogated in animals also receiving UDCA. The mechanism of the protective effect is unknown. Fukuzawa et al. (2004) reported that TCDD treatment at relatively high doses (20–100 µg/kg) to wild-type, but not AhR-null-allele mice, caused a significant reduction in testicular leutinizing hormone receptor and cytochrome P450 side chain cleavage protein and mRNA, as well as decreased testosterone synthesis.


Effects on the Prostate Prostate cells and prostate cancer cell lines are responsive to TCDD in terms of induction of a variety of genes, including those involved in drug metabolism. Some polycyclic aromatic hydrocarbons exhibited antiandrogenic effects in human prostate carcinoma (LNCaP) cells as determined by the analysis of the 5α-dihydrotestosterone-stimulated induction of prostate-specific antigen (PSA) (Kizu et al., 2003). TCDD was not examined in those studies. Nevertheless, the effects were found to depend on the AhR and were likely mediated through the noted elevation of c-fos and c-jun expression. Those proteins are known to inhibit binding, through formation of the AP-1 complex, of the androgen receptor (AR) to the androgen-responsive element of target genes, such as PSA. Studies by Morrow et al. (2004) demonstrated that TCDD inhibits growth of LNCaP cells and hormone-induced up-regulation of AR protein. However, the effects appeared to depend on the promoter region of the particular gene, suggesting that although there might be cross-regulation between the AR- and AhR-signaling pathways, the interactions are complex.


Effects on the Ovary and Female Reproductive Tissue The ovaries of experimental animals provide targets for the action of TCDD. Abnormal follicle development and decreased numbers of ova have been observed. There also is a widening of the mesenchyme that separates the Mullerian ducts while the zone of unfused ducts is increased, and TCDD exposure delays vaginal opening and

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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causes persistent vaginal threads (IOM, 2003). The AhR and Arnt are expressed in rabbit ovary within the steroid-secreting interstitial cells, in follicular and granulose cells, and in lutein cells (Hasan and Fischer, 2003) as well as in oocytes and surrounding cumulus cells (Pocar et al., 2004). Those cells are responsive to TCDD in terms of AhR-mediated altered transcription. Dasmahapatra et al. (2002) reported that TCDD induced CYP1A1 and CYP1B1 mRNA in rat ovary, but that it was highly dependent on the phase of the estrous cycle. The authors suggested that this could be important for the metabolism of estrogens. Pocar et al. (2004) reported that there is constitutive expression of CYP1A1 in immature oocytes, but not in cumulus cells, and that a significant increase in CYP1A1 occurs in both cell types after maturation. This expression of CYP1A1 was found to be AhR dependent, suggesting that the AhR could have some physiologic role in oocyte maturation. Estradiol was found to enhance, but estriol to inhibit, TCDD-induced expression of CYP1A1 in a mouse ovarian cancer cell line (Son et al., 2002).

TCDD alters processes involved in ovarian steroid synthesis. Moran et al. (2003a,b) noted that treatment with TCDD of human luteinized granulose cells resulted in an approximate 50% decrease in the expression of cytochrome P450 17α-hydroxylase/17,20-lyase, concomitant with a 65% decrease in 17,20-lyase activity. The decreases were proportional to the observed inhibition of estradiol secretion by the cells. The data are consistent with those from a study by Petroff and Mizinga (2003). Diminished serum progesterone and estradiol concentrations after the exposure of rats to TCDD were attributed to altered steroid synthesis and release rather than to an alteration in pharmacokinetics. Mizuyachi et al. (2002) also reported that TCDD treatment blocked ovulation in immature rats primed with chorionic gonadotropin to stimulate ovulation. An analysis of the ovaries indicated increased concentrations of CYP1A1 and Arnt, plasminogen activator inhibitor types 1 and 2, urokinase plasminogen activator, and tissue plasminogen activator 24 h after treatment. TCDD inhibited the expression of cyclooxygenase-2 (COX-2). That effect was thought to be critical because a reduction in COX-2 expression is associated with ovulation failure. Minegishi et al. (2003) reported that treatment of rat granulose cells with TCDD resulted in a significant decrease in the FSH-induced expression of luteinizing hormone receptor. Luteinizing hormone acts on granulose cells to stimulate steroidogenesis, luteinization, and ovulation. Williams et al. (2004) indicated that TCDD exposure induced PCKδ protein expression and phosphotransferase activity in mouse ovarian cancer cells. Benzo[a]pyrene, but not TCDD, was found to cell adhesion proteins in human uterine cells (McGarry et al., 2002).


Effects on the Uterus TCDD exposure has been reported to decrease uterine weight in rodents, to alter endometrial structure in rodents, and to increase the incidence of endometriosis in rhesus monkeys (IOM, 2003). Takemoto et al. (2004) demonstrated that TCDD treatment blocks an estrogen-induced increase in mouse uterine weight. However, they also observed that this effect did not

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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occur in either Ahr -/- or cyp1b1 -/- animals, suggesting that AhR-dependent-increased metabolism of estrogen could be involved in this antiestrogenic response to TCDD.

TCDD exposure increased the prevalence and severity of endometriosis in non-human primates. Some concern exists about the possibility that TCDD also increases the prevalence of endometriosis in humans. Rier and Foster (2002), who reviewed the evidence, concluded that the data are consistent with a hypothesis that TCDD and related compounds promote endometriosis by stimulating chronic inflammation that leads to an alteration of the estrogen- and progesterone-dependent processes that normally limit the development of endometriosis. TCDD has been shown to modulate the expression of many genes involved in regulating inflammatory processes (Table 3-2). Nevertheless, the exact mechanisms are not known. Zhao et al. (2002) reported that functional AhR is present in human endometrial stromal cells and that TCDD induces the expression of RANTES, a chemokine shown to regulate inflammation. The authors suggested that could be a mechanism for relating TCDD exposure and endometriosis. However, in a review of the literature on endometriosis in humans and non-human primates, Guo (2004) concluded that there is insufficient evidence to support the hypothesis that dioxin exposure leads to the development of endometriosis.


Effects on the Mammary Gland TCDD exposure results in a reduction of primary branches, decreased epithelial elongation, and fewer alveolar buds and lateral branches in the mammary glands. Consistent with this, Vorderstrasse et al. (2004) reported that AhR over-activation during pregnancy could disrupt mammary gland differentiation and lactation. Exposure of pregnant mice to TCDD (5 µg/kg) on days 0, 7, and 14 of pregnancy resulted in severe defects in hormone-stimulated breast development, including stunted growth, decreased branching, and poor formation of lobular alveolar structures. The impaired differentiation resulted in the decreased expression of whey protein in the gland, and all pups born to the dams died within 24 hours. The exact mechanism of this effect has not been determined. However, because the effects preceded hormone-induced events, it was proposed that altered hormone concentrations were unlikely to have been a mechanism of impaired mammary development.

Human breast cancer cells have been useful in investigations of the mechanisms of AhR signaling and of the effects of TCDD on hormone-induced responses, especially responses to estrogen. Previous updates reported that TCDD blocks many estrogen-induced responses in human breast cancer cells. Davis et al. (2003) reported that the AhR antagonist 3′-methoxy-4′-nitroflavone blocked the ability of TCDD to inhibit apoptosis induced in mammary epithelial cells by epidermal growth factor withdrawal. A concentration-dependent analysis suggested that TCDD inhibited apoptosis by several mechanisms, including an effect on the expression of transforming growth factor-α.

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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Endocrine and Other Effects As indicated in previous updates, TCDD and related compounds affect the thyroid and thyroid hormones in several animal species. Possible mechanisms include the displacement of hormones from serum transport proteins, the alteration of deiodinase activity, and the increasing thyroid hormone catabolism via glucuronidation. Viluksela et al. (2004) recently reported that alterations in tissue 5′-deiodinases I and II in rats after TCDD treatment were secondary to elicited decreases in serum thyroxine (T4) concentrations, and that altered deiodinase activities did not significantly influence the circulating levels of T4 and triiodothryonine (T3). Yamada-Okabe et al. (2004) reported that many genes found to be altered by T3 in HeLa cells overexpressing the thyroid hormone receptor were further modulated in cells exposed to both T3 and TCDD. The results indicated that TCDD augments the cellular responses to T3 by hyper-activating thyroid-hormone-mediated gene expression. The National Toxicology Program (2004) reported that female rats exposed chronically (2 years) to TCDD demonstrated altered thyroid follicles characterized by decreased luminal size and increased height of the follicular epithelial cells. Tani et al. (2004) characterized that effect as the result of a reversible hypertrophic response of the thyroid follicular cell.

Huang et al. (2002) reported that TCDD induced several genes in the pituitary after in vivo and in vitro exposure. Most notably, an increased expression of the adrenocorticotrophic hormone precursor proopiomelanocortin (POMC) gene was observed in TCDD-treated mice and in a pituitary cell line. This could be an AhR-responsive gene; DRE sequences were found in the promoter region. The data suggest that the pituitary gland is a direct target for TCDD and that, in particular, the up-regulation of POMC expression is significant in many endocrine alterations induced by TCDD. Petroff et al. (2003) observed that TCDD treatment in immature female rats stimulates a premature gonadotropin release by interacting with an estradiol- and phenobarbital-sensitive neural signal but not by acting directly on the gonadotrophin-releasing-hormone releasing neurons in the hypothalamus.

In the National Toxicology Program study (NTP, 2004), female Harlan Sprague-Dawley rats were administered TCDD at 3, 10, 22, 46, or 100 ng/kg body weight by gavage 5 days a week for 104 weeks. Exposure to TCDD increased the incidence of non-neoplastic lesions in the liver, lungs, oral mucosa, pancreas, thymus, adrenal cortex, heart, clitoral gland, kidney, forestomach, and thyroid glands. In the pancreas, the incidence of chronic active inflammation, acinar atrophy, and arterial chronic active inflammation was increased in the 100 ng/kg group at 2 years. At 2 years, there also was cortical atrophy of the adrenal cortex in the 100 ng/kg group; cortical hyperplasia was observed in groups administered 10 ng/kg or more. Thymic atrophy was observed at 2 years in groups administered 22 ng/kg or more, and follicular cell hypertrophy was noted in some exposure groups. Serum-free T4 concentrations were generally lower and serum T3 concentrations were higher, as were the thyroid-stimulating hormone concen-

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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trations in a dose–response fashion. Gingival squamous hyperplasia was observed in the oral mucosa, as was squamous hyperplasia of the forestomach. A minimal to mild nephropathy was generally observed, as was an infrequent moderate-to-marked nephropathy.


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 this. All the following are studies in which effects of TCDD on the developing embryo or fetus were investigated after maternal exposure to TCDD. There have been no studies since Update 2002 in which the effect of TCDD on the fetus was investigated after paternal exposure.

Work by Ishimura et al. (2002) suggested that the increased fetal death observed in rats after exposure to TCDD is attributable to placental hypoxia. They compared the content of placental proteins from TCDD-treated rats and animals subjected to hypoxic conditions. Increased glyceraldehyde 3-phosphate dehydrogenase, a marker protein of hypoxia, in both sets of placentas at gestational day 20 suggested that the TCDD-exposed placentas were in a hypoxic state at the end of the pregnancy.

Since Update 2002, there have been several reports detailing effects of TCDD on the developing cardiovascular system. Some investigations were stimulated by the finding that the AhR appears to influence the development of normal vascular architecture. Vasquez et al. (2003) demonstrated that cardiac hypertrophy is a consequence of AhR inactivation in mice. In their investigations, AhR-knockout animals were not found to be hypertensive, but they developed cardiomyopathy and diminished cardiac output. They did not exhibit the molecular markers characteristic of cardiac hypertrophy. The data suggested that increased cardiomyocyte size is a consequence of the absence of the AhR. Thackaberry et al. (2003) also observed that AhR-null embryos develop cardiac enlargement, and that the phenotype depends in part on the maternal genotype. However, markers of cardiac hypertrophy, β-myosin heavy chain and atrial natriuretic factor, were increased in AhR-null embryos. Thackaberry et al. (2003) also reported that the AhR is required for normal insulin regulation in pregnant and older mice and for cardiac development in embryonic mice. Pregnant and non-pregnant females at particular ages had significantly decreased fasting plasma insulin concentrations and a reduced ability to respond to exogenous insulin. Lund et al. (2003) reported that cardiac hypertrophy in AhR-null mice is correlated with elevated angiotensin II, endothelin-I, and mean arterial blood pressure and that all of these were reduced by treatment with an angiotensin-converting enzyme inhibitor. Additional investigations by Guo et al. (2004) indicate that the expression of TGF-β-regulated genes is deregulated in aortic smooth muscle cells from AhR-knockout mice. The AhR has been shown to mediate resolution and maturation of the fetal vascular structure, in particular closure of the fetal liver

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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vascular structure known as the ductus venosus. Work by Walisser et al. (2004) demonstrated that Arnt also is essential to AhR developmental signaling that mediates those processes in vascular development. Bunger et al. (2003) reported that liver development in mice carrying a mutation in the nuclear localization sequence of the AhR was identical to that observed in AhR-null-allele mice, indicating that a role of the AhR in vascular development requires nuclear localization of that protein.

Chick embryos are susceptible to cardiac effects after TCDD exposure. Data from work by Ivnitski-Steele and Walker (2003) indicate that TCDD inhibits early coronary vascular outgrowth in chick embryos by a mechanism that depends on vascular endothelial growth factor (VEGF). Treatment of embryos with exogenous VEGF rescued them from TCDD-induced inhibition of coronary vasculogenesis. In addition, hearts from TCDD-treated embryos exhibited a significant reduction in VEGF mRNA and in protein. Kanzawa et al. (2004) noted that TCDD induced a differential expression of the CYP1A family genes in chick embryo heart and liver.

The cardiovascular system, in particular the vascular endothelium of the developing embryo, also has been identified as a primary target of TCDD toxicity in fish. Studies cited in Update 2002 concluded that circulatory failure and oxidative stress in vascular endothelial cells are primary events that mediate the toxicity. Several recent studies using zebrafish and rainbow trout are consistent with this (Bello et al., 2004; Carvalho et al., 2004; Dong et al., 2004). Dong et al. (2004) provided further evidence that an effect of TCDD on vascular endothelium leads to local circulation failure and apoptosis in zebrafish dorsal midbrain. Zodrow and Tanguay (2003) reported that TCDD inhibits caudal fin regeneration that could occur by a down-regulation of genes that are important in vascularization. Further studies indicated that the pathology observed could not account alone for the inhibitory effects of TCDD on fin regeneration (Zodrow et al., 2004). Bello et al. (2004) reported that TCDD significantly inhibits growth of the common cardinal vein in zebrafish and that is dependent on AhR2. Hill et al. (2004) reported that the TCDD-induced edema seen in zebrafish results from an increased permeability to water across the surface of the developing embryo. Prasch et al. (2003b) observed that hypoxia decreases responses to TCDD in zebrafish embryos, thus suggesting interactions between the signaling pathways for HIF-α (which regulates VEGF) and the AhR. Investigations by Teraoka et al. (2003) suggested that induction of CYP1A1 is required for circulation failure and edema induced by TCDD toxicity in zebrafish.

Several of the developmental defects observed in animals exposed to TCDD can be explained, at least in part, by the failure of the vascular system to develop normally. TCDD induces myocardial defects during chick embryogenesis. Ivnitski-Steele et al. (2004) reported that those effects could be attributable to the ability of TCDD to disrupt oxygen gradients necessary for normal coronary vascular development and to alter expression of HIF-1a and VEGF-A. Prasch et

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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al. (2003b) likewise observed an interaction between the AhR- and hypoxia-signaling pathways in developing zebrafish. Hypoxia decreased TCDD induction of CYP1A mRNA and decreased the potency of TCDD in causing edema.

Studies by Blankenship et al. (2003) tested the hypothesis that oxidative stress, the generation of radical oxygen species, and induction of CYP1A1 could promote TCDD-induced abnormalities and embryo lethality in chickens. However, under the conditions of the study, cotreatments with inhibitors, antioxidants, and radical scavengers failed to significantly alter the outcomes induced by TCDD.

Abbott et al. (2003) examined the potential roles of epidermal growth factor (EGF) and TGF-α in developmental toxicity—specifically cleft palate and hydronephrosis—elicited by TCDD by the use of EGF, TGF-α, and double-knockout mice. The EGF (-/-) mice were less responsive to induced cleft palate, but more sensitive for the induction of hydronephrosis; the TGF-α (-/-) and double-knockout animals demonstrated sensitivity that was no different from that of the wild-type mice. The data demonstrated that the EGF receptor pathway is important in developmental responses to TCDD but that the relative mechanism appears to be specific to the endpoint. Miettinen et al. (2004) recently reported that EGF receptor deficiency in mice did not alter the ability of TCDD to produce cleft palate and hydronephrosis or change the time to eye opening. The data suggest that although EGF could influence those endpoints mediated by TCDD, signaling through the EGF receptor is not absolutely required. Davis et al. (2002) reported the localization in mouse chromosome 11 of a quantitative trait locus that affects the ability of TCDD to induce alterations in the mandible. The exact genes responsible for this have not been identified. Notably, work by Falahatpisheh and Ramos (2003), who used metanephric cultures from AhR (-/-) and AhR (+/+) mice, suggested that the AhR is involved in normal kidney development. That effect appears to involve regulation of the Wilms’s tumor suppressor gene, which is important in mesenchymal–epithelial transition and differentiation during nephrogenesis, and it is consistent with the noted ability of TCDD to disrupt the process.

Previous updates cited several reports indicating that development of the male reproductive system is exceptionally sensitive to in utero and lactational TCDD exposure. Those effects have included alterations in sperm production, increased numbers of abnormal sperm, decreased prostatic weight and growth and seminal vesicle growth, and decreased urogenital–glans penis length (IOM, 2003). Nevertheless, the TCDD-exposed males were able to impregnate females to produce viable fetuses (IOM, 2003). Simanainen et al. (2004a) reported that genetic differences in the C-terminal transactivation domain of the rat AhR modified sensitivity to some TCDD-induced male reproductive effects. TCDD-altered growth of the reproductive organs was not affected by the allelic differences; sperm numbers appeared to be affected differentially. Impaired prostate growth has been shown consistently. Ko et al. (2002) determined that the effect of TCDD

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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on the prostate was lobe specific. The inhibition of ventral prostate development was characterized by the complete absence of branching morphogenesis. The impaired development of the dorsal, lateral, and anterior prostate is associated with inhibition of processes involved in duct formation. The inhibition of prostate bud formation in the ventral and dorsolateral prostate was not the result of an insufficient amount of 5α-dihydrotestosterone (Lin T-M et al., 2003) or to the impairment of the androgen-signaling pathway (Ko et al., 2004a). Abbott et al. (2003) used EGF- and TGF-α–knockout animals, to demonstrate that both growth factors are important in the formation of prostatic buds and the ability of TCDD to inhibit bud formation in a region-specific manner. An additional study (Ko et al., 2004b) demonstrated that the changes that occur in the urogenital sinus epithelium that are responsible for the inhibited prostate budding are secondary to those initiated in the mesenchyme.

TCDD harms the reproductive systems of immature and adult female animals. Salisbury and Marcinkiewicz (2002) reported that in utero and lactational exposure to TCDD disrupted estrous cycles and inhibited ovulation rates in female offspring. Decreased rates of ovulation occurred in the presence of exogenous gonadotrophins, suggesting that TCDD directly affects the ovaries.

Several reports of studies in animals and exposed humans suggest that perinatal exposure to TCDD or to dioxin-like compounds can impair brain development. Rats exposed in utero to TCDD at 1 µg/kg maternal body weight showed deficits in spatial discrimination–reversal learning (RL) tasks but showed an increase in task spatial learning and memory (Update 2002). Prenatal exposure of primates to TCDD facilitated some spatial tasks and impaired visual RL tasks (IOM, 2003). Other research with rats exposed in utero to TCDD has resulted in reports of dose-dependent reductions in the number of revolutions on running wheels, in lever response rates, and in accuracy in lever chambers (IOM, 2003). GABA neurons in the brain are targets of TCDD; virtually all GABA neurons expressed the AhR gene (IOM, 2003). It has been proposed that GABAergic neurons in the brain are targets of TCDD that mediate developmental effects via affecting GAD67 gene expression in the preoptic area of the brain that controls reproductive functions (Hays et al., 2002).

Zareba et al. (2002) used male and female Sprague-Dawley rats to determine the effects of TCDD exposure in utero on brain cortical dominance. The rats were exposed to TCDD at 0, 20, 60, or 180 ng/kg gestational day 18 (Zareba et al., 2002). In TCDD-treated males, several brain areas reversed from right hemispheric dominance to left hemispheric dominance; in the females, brain dominance moved from left (normal) toward right hemispheric dominance. Motor activity was not affected in the TCDD-treated offspring. In agreement, Kakeyama et al. (2003) determined that perinatal exposure of rats significantly altered sexual behavior of male offspring. In normal animals, mating behavior induces c-fos mRNA in the preoptic area and brain-derived neurotrophic factor (BDNF) mRNA

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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in the frontal cortex. The investigators determined that TCDD exposure decreased the up-regulation of BDNF, but that it had no effect on c-fos expression. Another study examined the effects of gestational and lactational exposure to TCDD on spatial and visual discrimination/RL in Sprague-Dawley rats using two-lever operant testing chambers (Widholm et al., 2003). The mothers were administered TCDD orally at 0.1 µg/kg on gestational days 10–16. TCDD-exposed rats made more errors in spatial RL when they were beginning to learn new reinforcement contingencies, but no overall differences in the number of errors committed were noted between the TCDD-exposed and the control rats. TCDD-induced visual RL effects in males resulted in a reduction in errors on original learning; in females, there was a reduction in errors on the second reversal. The authors concluded that alterations in cognitive function after early exposure to TCDD are subtle and, under some conditions, that learning is facilitated rather than impaired. Ishizuka et al. (2003) reported that low-dose TCDD treatment of pregnant rats on gestational day 15 altered the sex-dependent expression of hepatic CYP2C11. Together those data are consistent with the hypothesis that perinatal exposure to TCDD changes the sexual differentiation of the neonatal brain in male rats.

Yamaguchi et al. (2003) reported that low (0.01–1 pg/mL) exposure to TCDD in vitro disrupted glial differentiation in the presence of low toluene exposure by up-regulating the synthesis of glial fibrillary acidic protein at the translational level. Hill et al. (2003) reported that exposure of zebrafish embryos to TCDD caused a 30% reduction in the total number of neurons in the 168-h brain. It was linked to decreased expression of the developmentally regulated genes neurogenin and sonic hedgehog. Publications from two laboratories indicate the importance of the AhR for neuronal development in Caenorhabditis elegans, although the AhR in this species apparently lacks the ability to bind to TCDD (Huang et al., 2004; Qin and Powell-Coffman, 2004).

Embryos from cynomologus macaques treated with a single dose of TCDD (4 µg/kg) on gestational days 15 or 20 were examined to determine morphology of the developing neural tube (Moran et al., 2004). Maternal blood was analyzed for fatty acid concentrations. The TCDD-treated embryos exhibited increased cell death and intracellular spaces in the neural tube. Significant decreases were observed in the n-3 (40–60%) and n-6 (47–57%) essential fatty acids in the treated pregnancies. The authors suggest that because neural tube development depends, in part on n-3 and n-6 fatty acids, it is possible that the effect resulted in the observed defects in early brain development. Stanton et al. (2003) examined the relationship between TCDD-elicited alterations of serum fatty acid concentrations and alterations in brain symmetry in neonatal chickens. Altered fatty acid concentrations correlated with hemispheric differences in dorsal width and angle and in dorsal–tectal length, suggesting that altered fatty acid concentrations are associated with altered brain morphology induced during development by TCDD.

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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SUMMARY OF TOXICITY PROFILES

This section synthesizes the experimental data on 2,4-D, 2,4,5-T, picloram, cacodylic acid, and TCDD reviewed here and in previous VAO reports, with a focus on recent data.

2,4-D

Most studies of 2,4-D have reported it to be relatively non-toxic; health effects are exhibited in animals only at high doses. Tissue uptake of 2,4-D is poor and metabolism fairly rapid, which could partially explain its low toxicity.

Earlier studies demonstrated that high doses of 2,4-D can cause behavioral effects, muscle weakness, and coordination problems in animals. A recent study indicated effects on the nervous system in humans, but at very high levels of exposure. Since Update 2002, there were no additional relevant studies to examine the effects of 2,4-D on the adult nervous system. The reproductive and developmental effects of 2,4-D also have been examined recently. Some studies suggest that oocytes and the preimplantation embryo may be especially sensitive to 2,4-D. Several studies cited in previous updates had suggested effects of 2,4-D on developing brain and recent studies are consistent with this. Elicited changes in brain neurotransmitter content appeared to correlate with behavioral alterations, and some of those changes appeared to be irreversible. Other studies suggested that undernourishment enhances the effects of 2,4-D on development. Recent studies are consistent with a weak effect on the immune system. Carcinogenicity tests of 2,4-D have generally been negative, and it is either non-genotoxic or only weakly mutagenic in the many assays used. However, recent investigations suggest that chemicals in commercial formulations (like Agent Orange) may enhance the genotoxicity of 2,4-D. Previous studies have suggested that 2,4-D might affect thyroid hormones (more specifically serum thyroxine). A recent study found 2,4-D had no effect on the ability of T3 to bind to serum thyroid hormone binding proteins.

Mechanistic studies have been conducted for 2,4-D that show a number of effects on cells or biochemical measures, including effects on some hormones, on cellular components involved in the development and functioning of brain cells, and on some enzymes and transporters. Effects on calcium metabolism and energy metabolism, possibly through direct effects on mitochondrial function, also have been reported for 2,4-D treatment, as have effects on stress proteins. The relationship of any of those effects to any disease outcomes in animals or humans, however, is unknown.

Taken together, the experimental data reviewed here and in previous reports indicate that pure 2,4-D is relatively non-toxic, but that it causes neuro-developmental effects after neonatal exposure (at 100 mg/kg body weight per day). The herbicide 2,4-D, however, was often contaminated with dioxins other than 2,3,7,8-TCDD.

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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2,4,5-T

Although not a great deal of research has been conducted recently on 2,4,5-T, the available data indicate that 2,4,5-T itself is relatively non-toxic. No relevant studies on the disease outcomes in experimental animals after exposure to 2,4,5-T have been published since Update 2002. Previous studies indicate that 2,4,5-T is absorbed into the body after oral exposure; absorption after dermal exposure is much slower. No recent toxicokinetic studies have been conducted. Studies of the reproductive effects of 2,4,5-T have demonstrated that it can be fetotoxic in rodents at doses greater than 20 mg/kg body weight per day on days 6–15 of pregnancy, retarding growth and causing increased embryolethality and cleft palate. No such effects were seen in rabbits, sheep, or monkeys, and evidence suggests that TCDD contamination of the 2,4,5-T might underlie the reproductive effects seen in rodents. The carcinogenicity of 2,4,5-T also has been investigated; no indications of carcinogenicity were seen. Studies of 2,4,5-T show it to have weak genotoxic potential. Little is known regarding the cellular effects of 2,4,5-T, but it does alter cellular metabolism (for example, on the acetylcoenzyme A system), affect cholinergic transmission and the tyrosine kinase receptor, and disrupt apoptosis. As in the case of 2,4-D, the relevance of those effects to human diseases is not known, and the data consistently indicate that 2,4,5-T is relatively non-toxic.

Cacodylic Acid

Cacodylic acid, or DMA, is a metabolite of inorganic arsenic. Because the relevance of studies of inorganic-arsenic exposure for evaluating effects of exposure to cacodylic acid has not been established and cannot be inferred (Chapter 2), the literature on inorganic arsenic is not considered in this report. Methylation of inorganic arsenic to DMA was long thought to be a detoxification pathway. However, the trivalent methylated forms of arsenic, DMAIII and MMAIII, have been shown to cause toxic effects; after acute exposure MMAIII is about 4 times more toxic than is inorganic arsenic, and DMAIII’s toxicity is similar to that of arsenicIII (NRC, 2001). Urinary excretion of DMA appears to be species dependent; rapid excretion occurs in many animals. Rats, however, accumulate DMA in red blood cells and tissues.

Few animal studies are available on the non-cancer health effects of cacodylic acid, but previous reports indicate that high, maternally toxic doses are fetotoxic and teratogenic in rats and mice. There is evidence that DMA can promote skin tumorigenesis in animals that is initiated chemically or with ultraviolet radiation. Evidence of cacodylic acid’s pulmonary and bladder carcinogenic activity has been presented for mice and rats, respectively. In other studies, however, cacodylic acid did not promote kidney tumors or lung tumors in nitrosamine-initiated rats. In recent studies, high exposure levels of DMA have also been

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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shown to induce urinary bladder tumors in rats, and increase the total number of tumors present in both wild-type and p53 +/- mice.

A primary mechanism of the acute toxicity of arsenic is interference of cellular respiration, but the mechanisms underlying the effects of cacodylic acid are not well understood. Some data indicate that cacodylic acid (DMA) acts through induction of oxidative damage or damage to DNA. Recent studies demonstrate that DMA is a potent inducer of apoptosis (or programmed cell death) and this is consistent with previous studies on cacodylic acid.

Picloram

Few studies have examined the toxicity of picloram, but those done indicate that it is relatively non-toxic. No relevant studies of picloram have been published since Update 2002. Two of three carcinogenicity studies reviewed in VAO indicate that picloram is not carcinogenic; a third was positive for liver tumors, but on review of the data, an Environmental Protection Agency committee concluded that the tumors had resulted from hexachlorobenzene (HCB) contamination. The VAO committee did note, however, that because the study was carried out with technical picloram, the compound used in Vietnam most likely contained similar amounts of HCB. Although the data on reproductive effects are not extensive, no effects have been seen that are considered treatment related. Notably, a study of the male-mediated reproductive toxicity of Tordon 75D® (a commercial mixture of 2,4-D and picloram) reported no effects on fetal survival or malformations. Another commercial mixture of 2,4-D and picloram, Tordon 202C®, had immunotoxic effects, reducing antibody production in mice in response to sheep red cell inoculation at concentrations only marginally above those expected to be encountered after recommended application of the herbicide. The immunotoxic effects observed with this commercial mixture may be due to their contamination with dioxin-like compounds. Once again, however, the relevance of the few effects seen to human health outcomes is not known; taken together, the data indicate that picloram is relatively non-toxic.

TCDD

In contrast with the effects of the herbicides themselves, the effects of TCDD, a contaminant of 2,4,5-T, have been studied extensively. TCDD is hydrophobic and therefore is absorbed well across membranes, distributes to all compartments of the body, and partitions with lipids. Data also indicate that TCDD is transferred across the placenta to the fetus and that it is transferred to neonates through lactation. The enzyme cytochrome P450 1A2 (CYP1A2) is important in the distribution of TCDD. Studies of TCDD in Ranch Hand Vietnam veterans indicate that it has a mean half-life of 7.6 years. Recent clinical studies of two women

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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exposed to very high amounts of TCDD, however, revealed an elimination half-life of 1.5 and 2.9 years in the more and less exposed women, respectively, indicating that the half-life depends on body burden. Recent data from Seveso also indicate that the half-life is shorter in the first 3 months after exposure than it is from 3 to 16 years after exposure. This, however, makes back-extrapolation from current to original levels in exposed individuals tenuous at best, especially because individual differences in elimination rates could be substantial. Those data on half-life are consistent with a two-compartment toxicokinetic model for TCDD, but a more complex PBPK model would provide a more satisfactory fit. Those findings emphasize the difficulties of attempts to extrapolate back to original exposures of exposed individuals, especially Vietnam veterans. Olestra somewhat increased the excretion of TCDD in the two heavily exposed patients, and this is consistent with earlier studies that indicate that the diet can affect the toxicokinetics of TCDD. A study in rats demonstrated that dietary seaweed can increase TCDD excretion. Other studies indicted that the consumption of insoluble dietary fiber or nori, a Japanese dietary item prepared from red algae, can increase the elimination of TCDD, but only slightly. The apparent correlation between TCDD half-life and body weight may actually be best explained in terms of body composition: the greater the portion of body composition represented by adipose tissue, the longer the half-life. TCDD concentrations are often measured in blood, and autopsy studies indicate that blood concentrations correlate with tissue concentrations. Studies also have been conducted to validate PBPK models to estimate the distribution and tissue concentrations of TCDD. Such models appear to be useful for toxicokinetic predictions.

Many effects have been observed in animals after exposure to TCDD, and TCDD is considered more toxic than were the active ingredients of the herbicides used in Vietnam. Sensitivity to the lethal effects of TCDD varies among species and strains, but after acutely toxic doses most species studied develop a wasting syndrome that is characterized by a loss of body weight and fatty tissue. Several recent reports suggest that this may be due to direct effects of TCDD on the hypothalamus. One target of TCDD is the liver, in which lethal doses of TCDD cause necrosis, although the effect depends on the species. Effects on the structure and function of the liver are also seen at lower doses. Several recent studies demonstrated that TCDD significantly disrupts the homeostasis of vitamin A in the liver. TCDD could affect, directly or indirectly, many organs of the endocrine system in a species-specific manner. Thyroid hormone concentrations have been shown to be affected, although some study results are contradictory and species specific, making interpretation of those data and a determination of their relevance to humans difficult.

The adult nervous system has been shown to be sensitive to TCDD only at high doses. After in utero exposure, however, the developing brain appears to be more sensitive. In utero TCDD exposure decreases performance in some learning

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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and memory tasks but improves performance in others. Several studies reported that low-dose in utero exposure altered learning and memory, hearing, sexual behavior, and the sex-dependent expression of a hepatic protein in male offspring.

The immune systems of animals are particularly sensitive to TCDD. Recent studies have demonstrated that TCDD can alter the number of immune cells, the measured activity of the cells, and the ability of animals to fight off infection. Effects on the immune system, however, appear to depend on the species, strain, and developmental stage of the animal studied.

Previous studies indicated that TCDD exposure increases the prevalence and severity of endometriosis in non-human primates. However, the data in rodent models show mixed results. Several recently published reviews of the literature are not in agreement as to whether there is evidence to support the hypothesis that dioxin exposure may lead to the development of endometriosis. Reproductive and developmental effects have been seen in animals exposed to TCDD; TCDD exposure affects sperm count, sperm production, and seminal vesicle weights in male offspring and affects the reproductive systems of female offspring. In some recent studies, however, the reproductive-system effects were not accompanied by effects on reproductive outcomes. Effects on the developing cardiovascular system also have been seen in several animal species after TCDD exposure. However, there is little evidence that the cardiovascular system is a target of TCDD in adult animals. TCDD has been shown to affect the thyroid and thyroid homones in several animal species. Possible mechanisms include the displacement of hormones from serum transport proteins, altering deiodinase activity, and increasing thyroid hormone catabolism. The relevance of these data to exposed veterans is not clear.

TCDD is carcinogenic and an extremely potent promoter of neoplasia in laboratory rats. Liver cancers have been seen consistently after TCDD treatment, and increases in skin cancer, lung cancer, and cancers of the thyroid and adrenal glands have been seen in some studies. A recently completed 2-year bioassay in female rats indicated increased incidences of cholangiocarcinoma and hepatocellular adenoma of the liver, cystic keratinizing epithelioma of the lung, and gingival squamous cell carcinoma of the oral mucosa. A decrease in cancers of the uterus; the pancreas; and the pituitary, mammary, and adrenal glands also has been seen previously. Most of those tumors decreased only at the high dose and the decrease was associated with decreases in body weight gain. The decrease in mammary tumors was seen in one study. A previous study showed an increase in hepatic foci in rats at TCDD doses as low as 0.01 ng/kg body weight per day—the lowest dose of TCDD known to promote tumors. In addition, promotion of liver tumors by TCDD in female rats depends on continuous exposure. In a recent study, TCDD was found to immortalize keratinocytes, and this was suggested to be a possible mechanism by which this chemical may lead to malignancy.

Data published since Update 2002 are consistent with the hypothesis that TCDD produces most or all of its effects by binding to a protein that regulates

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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gene expression, the AhR. The binding of TCDD to the AhR and interaction of the complex with other proteins is followed by its binding to DNA, which triggers cellular events that include the induction of numerous proteins. Research on animals that have been engineered not to express the AhR and on animals with slightly different forms of the AhR provides evidence that the AhR is a necessary mediator for the toxicity of TCDD. Modulation of genes by the AhR appears to have species-, cell-, and developmental-stage-specific patterns, which suggest that the molecular and cellular pathways that lead to any particular toxic event are complex.

Additional research has demonstrated that the outcomes of TCDD exposure can be modulated by numerous other proteins with which the AhR interacts. It is plausible, therefore, that the AhR could divert proteins and transcription factors from other signaling pathways; the disruption of the other pathways could have serious consequences for cellular and tissue processes.

Despite the large amount of research on the cellular effects of TCDD, details of the mechanisms that underlie its effects have not been elucidated. Possible mechanisms discussed in this chapter include effects on protein kinase expression, effects on vitamin stores, effects on cellular differentiation and the cell cycle, and oxidative stress. Although the mechanisms underlying the carcinogenic effects of TCDD remain unknown, available data indicate that TCDD does not act directly on the genetic material; most genotoxic assays have negative results. Effects on enzymes or hormones could be involved in the carcinogenicity of TCDD.

RELEVANCE TO HUMAN HEALTH

Exposure to TCDD has been associated with cancer and non-cancer endpoints in animals, and most, if not all, TCDD effects are mediated through the AhR. Although structural differences in the AhR have been identified, it operates similarly in animals and humans, and a connection between TCDD exposure and human health effects is, in general, considered biologically plausible. Animal research indicates that exposure to TCDD can cause cancers and benign tumors, and that it can increase the incidence of some cancers or tumors in the presence of known carcinogens. However, experimental animals differ greatly in susceptibility to TCDD-induced effects, and the sites at which tumors are induced vary from species to species. Non-cancer health effects also vary according to dose, time, and species. Whether the effects of TCDD and other exposures are threshold dependent—that is, whether some exposures are too low to induce any effect—is an open question. The relationship between mechanism and the shape of the dose–response curve—linear or non-linear—is complex, not well understood, and could be different for different endpoints.

Little information is available on the biologic plausibility of causation of health effects by Agent Orange through chemicals other than TCDD. Although

Suggested Citation:"3 Toxicology." Institute of Medicine. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. doi: 10.17226/11242.
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concerns have been raised about non-dioxin contaminants of herbicides, far too little is known about their distribution and concentration in the formulations used in Vietnam to permit conclusions concerning their impact.

Considerable uncertainty remains about how to apply mechanistic information from non-human studies to an evaluation of the potential health effects of herbicide or dioxin exposure in Vietnam veterans. Although the data specific to humans is inadequate to demonstrate strong relationships between exposure and disease conditions or pathologies, the growing and abundant evidence from experimental studies of laboratory animals and wildlife strongly suggests that similar adverse effects are likely in human populations—the issues of sensitivity and dose–response perhaps being paramount in species differences. It is hoped that, as the cellular mechanisms of those compounds are discovered, future VAO updates will have better information on which to base conclusions, including better information on the relevance of experimental data to effects in humans.

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