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--> systems allows us to reasonably determine whether these effects can occur in humans. Toxicokinetics During the past three years new information was published on the distribution of 2,4-D and 2,4,5-T in the body. There is also new information on the metabolism of cacodylic acid. Both 2,4-D and 2,4,5-T remain in the body for a short period of time—less than two weeks. 2,4-D enters liver cells and binds to protein and lipid molecules; the consequence of this is unknown. Both 2,4-D and 2,4,5-T enter the brain. Cacodylic acid is metabolized to a more toxic and reactive form called the dimethylarsenic radical. TCDD, unlike the herbicides, stays in the body for a long time. It is removed from the body as it metabolizes to less toxic forms that are more easily excreted than TCDD itself. During the past three years, models have been further developed that provide insight into the complex ways in which TCDD is distributed in the body. Mechanism of Action Little is known about the way in which the herbicides produce toxic effects in animals. Tests with 2,4-D and cacodylic acid indicate that these substances are toxic to the body's cells and genetic material. The recent discovery that inorganic arsenicals are metabolized in mammals to dimethylarsenic acid suggests that the toxic effects of 2,4-D may be similar to that produced by cacodylic acid, which is dimethylarsenic acid. To date, the consensus is that TCDD is not toxic to the body's genetic material and that its ability to cause cancer in animals is due to other events. It may cause cancer by affecting the body's enzymes, the way cells reproduce, and the rate at which cells die. New tests suggest that TCDD causes chloracne by affecting the development of one of the layers of skin: the epidermis. New tests also indicate that wasting syndrome results from TCDD's preventing glucose uptake by fat cells and by cells in the brain and pancreas. Most of the tests published during the past three years have examined the molecular mechanisms of TCDD toxicity. The tests confirm earlier findings that the toxic effects of TCDD are caused by the binding of TCDD to a protein called the aryl hydrocarbon (Ah) receptor. The binding of TCDD to this receptor triggers other effects that result in a toxic sequelae. However, some tests also suggest that other events, in addition to the binding of TCDD to the protein receptor, are involved.
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--> Disease Outcomes and Mechanisms of Toxicity Tests with 2,4-D, 2,4,5-T, cacodylic acid, and picloram indicate that these substances are not very toxic. High concentrations are needed to produce toxic effects in laboratory animals and nonhuman systems. No new information has been published to indicate that these herbicides cause cancer in animals. Nerve damage has been reported in rats given high doses of 2,4-D in food and teratogenic effects have been seen in sea urchin eggs exposed to 2,4,5-T during early development. Cacodylic acid has been shown to produce lesions in the kidneys of rats. TCDD has been reported to produce a number of toxic effects in several different animal species. The toxic effects include carcinogenicity, immunotoxicity, reproductive/developmental toxicity, hepatotoxicity, neurotoxicity, chloracne, and loss of body weight. Not all these effects are seen in all species. Some are seen in males and not females, and vice versa; some are seen in young animals only. TCDD does cause cancer in animals. Recent tests show that young animals are particularly sensitive to TCDD. The reproductive capability of newborn males exposed to TCDD is adversely affected. Recent studies also show that TCDD can damage animals' nerves, enlarge their livers, and impair the ability of their hearts to contract. Tests using nonhuman systems suggest that TCDD can also affect the cells in the kidney, but these effects have not been shown in live animals. Although TCDD has been shown to cause adverse effects on the immune response in nonhuman systems, the immune response in live animals does not appear to be affected. Summary Of VAO Multiple chemicals were used for various purposes in Vietnam. Four herbicides documented in military records were of particular concern and were extensively addressed in Chapter 4 of VAO (IOM, 1994). These included 2,4-dichlorophenoxyacetic acid (2,4-D); 2,4,5-trichlorophenoxyacetic acid (2,4,5-T); picloram; and cacodylic acid. In addition, the toxicologic properties of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin), a contaminant of 2,4,5-T, were described. Complete toxicity profiles for each of the five substances were presented. The chapter focused to a large extent on the toxicological effects of TCDD, because considerably more information was available on TCDD than on the herbicides. As stated in Chapter 4 of VAO (IOM, 1994), the primary purpose of reviewing the animal studies on the five substances was to contribute to an understanding of the biologic plausibility of the associations observed in epidemiologic studies that are relevant to herbicide exposure in Vietnam. In examining the individual toxicity profiles of the chemicals, it was recognized that differences in chemical levels, frequency of administration, single or combined exposures, preexisting health status, genetic factors, and routes of exposure significantly influence
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--> toxicity outcomes. Thus, any attempt to extrapolate from experimental studies to human exposure must carefully consider such variables before conclusions are made. The remainder of this section summarizes what appeared in VAO on what was known about the chemistry, toxicokinetics, disease outcomes, and mechanisms of toxicity of the five substances. Chemistry 2,4-D and 2,4,5-T are called chlorophenoxy acids and are made up of carbon, hydrogen, oxygen, and chlorine. They both dissolve in water and are very similar in structure to a natural plant hormone called auxin. As a result of this similarity, 2,4-D and 2,4,5-T can mimic the action of auxin in some plants, and this activity is thought to account for herbicidal activity. TCDD forms as a by-product during the manufacture of 2,4,5-T and also contains carbon, hydrogen, oxygen, and chlorine. TCDD dissolves easily in fats and oils, but not in water, and is persistent in the environment. The primary source of TCDD in the environment is combustion and industrial processes. The primary source of human exposure is through food. Cacodylic acid contains carbon, hydrogen, oxygen, and arsenic and was called Agent Blue. Picloram contains carbon, hydrogen, oxygen, chlorine, and nitrogen. The combination of cacodylic acid and 2,4-D was known as Agent White. Both compounds dissolve in water. Toxicokinetics TCDD is ingested by animals through contaminated food. More than 50 percent is absorbed into the body through the gastrointestinal tract. Most of the TCDD breathed in the air is thought to be absorbed through the lungs, but this route of exposure is not well studied. In contrast, TCDD is not absorbed well through the skin. The same pattern of absorption applies for 2,4-D and 2,4,5-T, and probably for picloram and cacodylic acid. TCDD is distributed primarily to the liver and to body fat. The amount of time that TCDD remains in the liver or fat is different for different species. 2,4-D and 2,4,5-T are distributed widely in the body; the distribution patterns of picloram and cacodylic acid are less well understood. Some cacodylic acid that is absorbed is bound to red blood cells. Although cacodylic acid binds readily to red blood cells in rats, it does not bind to human red blood cells. TCDD is metabolized by enzymes in the liver to form derivatives that can dissolve in water and thus are more easily eliminated from the body than TCDD itself, which does not dissolve in water. Water-soluble derivatives of TCDD are thought to be much less toxic to animals than TCDD itself. 2,4-D, 2,4,5-T, and cacodylic acid are not metabolized to any significant extent in the body.
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--> Mice and rats eliminate TCDD from the body in both urine and feces, whereas all other species studied eliminate TCDD primarily through feces. Elimination from the body is slow; in humans it may take seven to ten years or more for half of the body burden of TCDD to be removed. 2,4-D, 2,4,5-T, picloram, and cacodylic acid are eliminated rapidly from humans, mostly in the urine. Cacodylic acid that is bound to red blood cells is eliminated as the cells die naturally. Disease Outcomes and Mechanisms of Toxicity In this section, we summarize studies that investigated the toxic effects of TCDD and the herbicides. If known, the mechanism of toxicity is also explained. Carcinogenicity: TCDD The ability of TCDD to cause cancer in animals has been studied using rats, mice, and hamsters exposed to TCDD for one to two years. The results of these studies were summarized in detail in VAO. In these studies, TCDD was fed to animals, applied to their skin, injected under their skin, or injected into their abdominal cavities. Increased tumor rates have been reported to occur at several different sites in the body. In studies in which liver cancer occurred, other toxic changes in the liver also occurred. Other organs in which increased cancer rates were observed in animals exposed to TCDD include the thyroid and adrenal glands, the skin, and the lungs. Organs in which decreased cancer rates were observed in animals exposed to TCDD include the uterus, the pancreas, and the pituitary, mammary, and adrenal glands. In addition to increasing cancer rates in animals by itself, TCDD can increase tumor formation by other chemicals. For example, when a single dose of a known carcinogen is applied to the skin of mice and that dose is followed by multiple doses of TCDD over a period of several months, more skin tumors are seen than would be expected from the single dose of carcinogen alone. Similar results are obtained in rat livers when a single dose of a liver carcinogen is followed by multiple doses of TCDD. In rats, liver tumor formation associated with TCDD exposure is dependent on the presence of ovaries; in other words, only female rats that have not had their ovaries removed can develop liver tumors when they are exposed to TCDD. This observation indicates that complex hormonal interactions are likely to be involved in TCDD-induced carcinogenesis. Mechanism of Toxicity TCDD has a wide range of effects on growth regulation, hormone systems, and other factors associated with the regulation of activities in normal cells. These actions of TCDD may affect tumor formation. Understanding
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--> how TCDD influences tumor formation in laboratory animals may help us understand whether TCDD affects tumor formation in humans. It has been shown that TCDD binds to a protein in animal and human cells called the Ah receptor. It is thus possible that TCDD, together with the Ah receptor, can interact with sites on DNA and alter the information obtained from DNA in such a way that a normal liver cell is transformed into a cancerous liver cell. Direct proof of this possibility has not been obtained. Carcinogenicity: Herbicides Several studies of the carcinogenicity of 2,4-D, 2,4,5-T, picloram, and cacodylic acid have been performed in laboratory animals. In general, they produced negative results, although some were not performed using rigorous criteria for the study of cancer in animals, and some produced equivocal results that could be interpreted as either positive or negative. These studies and their results were summarized in detail in VAO. 2,4-D was administered to rats, mice, and dogs in their food, by injecting it under their skin, or by placing it directly into their stomachs. All the results were negative, except for one study that found an increased rate of brain tumors in male rats, but not female rats, receiving the highest dose. These tumors also occurred in the control group and thus may have occurred spontaneously and not as a result of 2,4-D exposure. 2,4,5-T has been administered to rats and mice in their food, in their drinking water, by injecting it under their skin, or by placing it directly into their stomachs. Picloram has been tested in rats and mice in their food. Results of all of these studies were uniformly negative, with the exception of one study using picloram in which liver tumors appeared, but were attributed to the presence of a contaminant, hexachlorobenzene. Cacodylic acid has been tested in a very limited study in mice both in their food and by placing it directly into their stomachs. Immunotoxicity: TCDD TCDD was shown to have a number of effects on the immune systems of laboratory animals. Studies in mice, rats, guinea pigs, and monkeys indicated that TCDD suppresses the function of certain components of the immune system in a dose-related manner; that is, as the dose of TCDD increases, its ability to suppress immune function increases. TCDD suppressed the function of cells of the immune system, such as lymphocytes (cell-mediated immune response), as well as the generation of antibodies by B cells (humoral immune response). Increased susceptibility to infectious disease has been reported following TCDD administration. In addition, TCDD increased the number of tumors that formed in mice following injection of tumor cells.
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--> The effects of TCDD on the immune system appear to vary among species. Mechanism of Toxicity It is likely that the Ah receptor plays a role in some types of immunotoxicity. Some studies indicate that an animal's hormonal status may contribute to its sensitivity to immunotoxicity. The fact that TCDD induces such a wide variety of effects in animals suggests that it is likely to have some effect in humans as well. Immunotoxicity: Herbicides The potential immunotoxicity of the herbicides used in Vietnam has been studied to only a very limited extent. Effects on the immune system of mice were reported for 2,4-D administered at doses that were high enough to produce clinical toxicity, but these effects did not occur at low doses. The potential for picloram to act as a contact sensitizer (i.e., to produce an allergic response on the skin) was tested, but other aspects of immunotoxicology were not examined. Reproductive and Developmental Toxicity: TCDD TCDD was reported to have a number of effects on the reproductive and developmental functions of laboratory animals. For example, administration of TCDD to male rats, mice, guinea pigs, marmosets, monkeys, and chickens can elicit reproductive toxicity by affecting testicular function, decreasing fertility, and decreasing the rate of sperm production. TCDD has also been found to decrease the levels of hormones such as testosterone in rats. The reproductive systems of adult male laboratory animals are considered to be relatively insensitive to TCDD, because high doses are required to elicit effects. Potential developmental toxicity following exposure of male animals to TCDD has not been studied. Studies in female animals are limited but demonstrate reduced fertility, decreased ability to remain pregnant throughout gestation, decreased litter size, increased fetal death, impaired ovary function, decreased levels of hormones such as estradiol and progesterone, and increased rates of fetal abnormalities. Most of these effects may have occurred as a result of TCDD's general toxicity to the pregnant animal, however, and not as a result of a TCDD-specific mechanism that acted directly on the reproductive system. Mechanism of Toxicity Little information is available on the cellular and molecular mechanisms of action that mediate TCDD's reproductive and developmental effects in laboratory animals. Evidence from mice indicates that the Ah receptor may play a role: mice with Ah receptors that have a relatively high affinity for TCDD respond to lower doses than do mice with a relatively low affinity. Other, as-yet-unidentified, factors also play a role, however, and it is
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--> possible that these effects occur only secondarily to TCDD-induced general toxicity. Extrapolating these results to humans is not straightforward, because the many factors that determine susceptibility to reproductive and developmental effects vary among species. Reproductive and Developmental Toxicity: Herbicides Several studies evaluated the reproductive and developmental toxicity of herbicides in laboratory animals. Results indicated that 2,4-D does not affect male or female fertility and does not produce fetal abnormalities, but when pregnant rats or mice are exposed it does reduce the rate of growth of offspring and increase their rate of mortality. Very high doses were required to elicit these effects. 2,4,5-T was toxic to fetuses when administered to pregnant rats, mice, and hamsters. Cacodylic acid is toxic to rat, mouse, and hamster fetuses at high doses that are also toxic to the pregnant mother. Limited data suggested that picloram may produce fetal abnormalities in rabbits at doses that are also toxic to the pregnant animals. Investigations of the developmental toxicity of the herbicides suggested that they can be toxic to developing animals, but high doses are required. Other Toxicities: TCDD TCDD has been reported to elicit several other kinds of toxicity in laboratory animals. Effects of TCDD on the liver include increasing the rate at which liver cells multiply, increasing the rate of other cell death, increasing fat levels in liver cells, decreasing bile flow, and increasing the levels of protein and of substances that are precursors to heme synthesis. TCDD also increases the levels of certain enzymes in the liver, but this effect is not considered toxic. Mice and rats are susceptible to TCDD-induced liver toxicity, but guinea pigs and hamsters are not. It is possible that liver toxicity is associated with susceptibility to liver cancer. Other toxic effects of TCDD that have been reported in laboratory animals include reduced blood glucose levels and starvation, increased rates at which cells in the gastrointestinal tract multiply, and changes in skin cells. Other Toxicities: Herbicides The herbicides used in Vietnam were reported to elicit adverse effects in a number of organs in laboratory animals. The liver is a target organ for toxicity induced by 2,4-D, 2,4,5-T, and picloram, with changes reportedly similar to those induced by TCDD. Some kidney toxicity was reported in animals exposed to 2,4-D and cacodylic acid. Exposure to 2,4-D has also been associated with effects on blood, such as reduced levels of heme and red blood cells.
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--> Literature Update In this section, the results of experimental studies published during the period 1992-95 are reviewed. The section begins with an overview of the toxicology data on dioxin and the four herbicides. This is followed by updates of the toxicological profiles reviewed in VAO. These include 2,4-dichlorophenoxyacetic acid (2,4-D); 2,4,5-trichlorophenoxyacetic acid (2,4,5-T); picloram; and cacodylic acid; as well as the contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). As in VAO, this review is intended to summarize the experimental data that provide the scientific basis for the assessment of biologic plausibility of the health outcomes reported in epidemiologic studies. Efforts to establish the biologic plausibility of effects due to herbicide exposure in the laboratory strengthen the evidence for the effects of the herbicides suspected to occur in humans. Overview This chapter reviews the results of animal studies published during the past three years that investigated the toxicokinetics, mechanism of action, and disease outcomes of dioxin and the four herbicides employed in Vietnam. These data were used to provide the scientific basis for the assessment of biologic plausibility of the health outcomes in epidemiological studies. In examining the individual toxicity profiles of the chemicals in question, readers must consider that differences in chemical levels, frequency of administration, predetermined health status, genetic factors, and routes of exposure influence toxicity outcomes. The toxicity of the herbicides used in Vietnam remains poorly studied. In general, 2,4-D, 2,4,5-T, cacodylic acid, and picloram have not been identified as particularly toxic substances, since high concentrations are often required to modulate cellular and biochemical processes. However, some studies reported during the period 1992-95 have observed impairment of motor function in rats administered high single oral doses of 2,4-D. The ability of 2,4,5-T to interfere with calcium homeostasis in vitro was documented and linked to the teratogenic effects of 2,4,5-T on the early development of sea urchin eggs. Cacodylic acid was reported to induce renal lesions in rats. No studies were published pertaining to the toxicity of picloram. The half-life of 2,4-D and 2,4,5-T is relatively short and does not appear to extend beyond two weeks. 2,4-D binds covalently to hepatic proteins and lipids, but the molecular basis of this interaction and its biologic consequences are unknown. Evidence was presented suggesting that both 2,4-D and 2,4,5-T are capable of gaining access to the central nervous system and that the uptake process is energy-dependent. A series of studies indicates that high concentrations of cacodylic acid result in the formation of a toxic intermediate, the dimethylarsenic radical. TCDD elicits a diverse spectrum of sex-, strain-, age-, and species-specific
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--> effects, including carcinogenicity, immunotoxicity, reproductive/developmental toxicity, hepatotoxicity, neurotoxicity, chloracne, and loss of body weight. To date, the consensus is that TCDD is not genotoxic and that its ability to influence the carcinogenic process is mediated via epigenetic events such as enzyme induction, cell proliferation, apoptosis, and intracellular communication. Recent studies on the effects of TCDD and related substances on the immune system amplify earlier findings and suggest that these compounds affect primarily the T-cell arm of the immune response. The effects may result from decreased functions of the thymus gland. Direct effects of TCDD on T cells in vitro, however, have not been demonstrated, suggesting that the action of TCDD may be indirect. A number of recent studies suggest that cytokines may play an important role in regulating the immune response. It should be emphasized that very little change to the overall immune competence of the intact animal has been reported. In contrast, a number of animal studies of the reproductive and developmental toxicity of TCDD suggest that developing animals may be particularly sensitive to the effects of TCDD. Specifically, male reproductive function has been reported to be altered following perinatal exposure to TCDD. In addition, experimental studies in rats of the effects of TCDD on the peripheral nervous system suggest that a single low dose of TCDD can cause a toxic polyneuropathy. Other studies published during the reference period provide evidence that hepatotoxicity of TCDD involves AhR-dependent mechanisms. Specifically, there is evidence that the AhR receptor plays a role in the co-mitogenic action of TCDD with epidermal growth factor and in the induction of liver enzymes involved in the metabolism of xenobiotics. Acute exposures to TCDD have been correlated with effects on intermediary metabolism and hepatomegaly. The myocardium has been shown to be a target of TCDD toxicity; impairment of a cAMP-modulated contraction has been implicated. TCDD has been reported to decrease an acidic type I Keratin involved in epidermal development, leading to keratinocyte hyperproliferation and skin irritations such as chloracne. Recent evidence suggests that the inhibition of glucose transport in adipose tissue, pancreas, and brain may be one of the major contributing factors to the wasting syndrome. Finally, in vitro studies have identified glomerular mesangial cells as sensitive cellular targets. These findings are consistent with epidemiologic reports that aromatic hydrocarbons result in glomerulonephritis. By far, the majority of the studies identified during the reference period focused on the elucidation of the molecular mechanism of TCDD toxicity. The evidence further supports the concept that the toxic effects of TCDD involve AhR-dependent mechanisms. A better appreciation of the complexity of TCDD effects in target cells has led to the development of refined, physiologically based pharmacokinetic models. These models take into account intracellular diffusion, receptor and protein binding, and liver induction to establish the fractional distribution of the total body burden as a function of the overall body concentration.
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--> The association of TCDD with the cytosolic AhR was shown to require a second protein, known as ARNT, for DNA binding capability and transcriptional activation of target genes. Comparison of the murine and human AhR have demonstrated that the carboxy terminal region involved in transactivation is hypervariable, a feature which may account in part for species differences in TCDD responsiveness. During the referenced period, expression of multiple forms of the AhR was documented suggesting differences in cellular responsiveness to TCDD, and the multiplicity of biological effects may be related to such differences. Evidence has also begun to accumulate suggesting that events in addition to receptor binding also influence the biological response to TCDD. Specifically, there is evidence for ligand-independent activation of the AhR evidence for transcriptional independent responses, and data to support the review that the transcriptional regulation of the AhR-responsive genes is dictated by combinatorial interactions among proteins. It is now also clear that AhR-related signaling influences, and is itself influenced by, other signal transduction mechanisms at low concentrations. Signaling interactions explaining the toxic effects of TCDD may involve growth factors, free radicals, the interaction of TCDD with the estrogen transduction pathway, and protein kinases. The normal cellular functions of the AhR and its role in cellular homeostasis remain undefined. Dioxin-independent activation of the AhR has now been demonstrated, suggesting that unidentified endogenous ligands are operational in the absence of TCDD. The exciting development of AhR-deficient ''knock-out" mice by homologous recombination in embryonic stem cells will likely provide the tools to define the roles of the AhR in mammalian species (Fernandez-Salguero et al., 1995). Toxic equivalency factors (TEFs) have been used to estimate the potential health risks associated with exposure to TCDD and complex mixtures containing structurally similar chemicals. The approach assumes linearity of the toxic response for TCDD and is based on a receptor-mediated mechanism of action. The TEF approach has come under increasing scrutiny because it disregards potentially significant kinetic interactions, tissue-specific effects, and interactions among chemicals present in complex mixtures. These factors, in addition to dietary factors and interspecies and interindividual differences in sensitivity, may influence TCDD toxicity. Update Of Toxicity Profiles In this section, we update the toxicological profiles on the five substances discussed in VAO: 2,4-dichlorophenoxyacetic acid (2,4-D); 2,4,5-trichlorophenoxyacetic acid (2,4,5-T); picloram; cacodylic acid; and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin). Each update begins with a summary, which is followed by a review of the experimental studies published during the period
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--> 1992-95. This information is organized under the headings of toxicokinetics, mechanism of action, and disease outcomes and mechanisms of toxicity. Toxicity Profile Update of 2,4-D Summary Studies published during the period 1992-95 provide evidence that 2,4-D binds covalently to hepatic proteins and lipids; the molecular basis of this interaction and its biologic consequences are unknown. 2,4-D has also been shown to accumulate in the brain. This process is mediated through an active anion transport system. 2,4-D is not considered to be particularly toxic because high concentrations are often required to modulate cellular and biochemical processes. Impairment of motor functions has been reported in rats orally administered a single high oral dose of 2,4-D. Toxicokinetics Measurable amounts of 2,4-D can be detected in the blood and urine of dogs several days after exposure to contaminated lawns under natural conditions (Reynolds et al., 1994). Among 44 dogs potentially exposed to 2,4-D-treated lawns for an average of 10.9 days, 33 dogs (75 percent) had urine concentrations of 2,4-D greater than or equal to 10 µg/l, and 17 dogs (39 percent) had urine concentrations of ≥; 50 µg/l. Among 15 dogs with no known exposure to 2,4-D-treated lawns in the previous 42 days, 4 (27 percent) had 2,4-D in urine, 1 at a concentration of ≥; 50 µg/l. The highest mean concentration of 2,4-D in urine (21.3 mg/l) was found in dogs sampled within two days after application of the herbicide. The hepatocellular distribution of 2,4-D was examined by Evangelista et al. (1993). The herbicide decreases total lipids, especially phospholipids, both in total liver and in microsomes. 2,4-D crosses the liver plasma membrane and can be detected in all subcellular fractions. 2,4-D binds covalently to hepatic proteins and lipids, with protein binding being ten-fold higher than lipid. The mechanism of and implications for covalent binding of 2,4-D remain to be defined. 2,4-D has been demonstrated to accumulate in the brain without damaging the blood–brain barrier. This accumulation is related to the biochemical properties of 2,4-D, which is a very strong acid and is partially soluble in water. Recent experiments have demonstrated that the mechanism that mediates the accumulation of 2,4-D in the brain is the saturation of an active organic anion transport system in the choroid plexus (Kim et al., 1988; Kim et al., 1994). The brain depends on the active transport of organic anions by the choroid plexus to keep potentially toxic anions, including foreign chemicals, as 2,4-D and endogenous neurotransmitter metabolites, at low concentrations in the central nervous system.