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4 Information Related to Biologic Plausibility The committee reviewed all relevant experimental studies of 2,4-dichlo- rophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), pi- cloram, cacodylic acid, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) that have been published since Update 2006 (IOM, 2007) and has incorporated the findings, when it was appropriate, into this chapter or into the biologic-plausibil- ity sections of Chapters 6â9 when they are of consequence for particular health outcomes. For each substance, this chapter includes a review of toxicokinetic properties, a brief summary of the toxic outcomes investigated in animal experi- ments, and a discussion of underlying mechanisms of action as illuminated by in vitro studies. To achieve the goals of this chapter more effectively, the current committee has slightly modified the presentation of toxicologic information used by previous Veterans and Agent Orange (VAO) committees. The toxicology chapter of each earlier update presented information about each of the several hundred potentially relevant toxicologic articles published in the preceding 2 years. In contrast with the committeeâs responsibility to evaluate each potentially relevant epidemiologic study of the chemicals of interest published in the preceding 2 years, its charge with respect to the toxicologic literature is to distill experimental toxicologic findings to judge whether it is biologically plausible to attribute adverse health outcomes reported in epidemiologic investigations to the chemicals. The current committee recognized that for most readers of the VAO series the implications of most toxicologic results reported are not immediately obvious. Therefore, start- ing with this update, the committee will focus on integrating and interpreting the toxicologic evidence rather than delineating the entire body of new experimental findings. 65
66 VETERANS AND AGENT ORANGE: UPDATE 2008 Establishment of biologic plausibility through laboratory studies strengthens the evidence of a causeâeffect relationship between herbicide exposure and health effects reported in epidemiologic studies and thus supports the existence of the less stringent relationship of association, which is the target of this committeeâs charge. Experimental studies of laboratory animals or cultured cells allow obser- vation of effects of herbicide exposure under highly controlled conditions that are difficult or impossible to control in epidemiologic studies. Such conditions include frequency and magnitude of exposure, exposure to other chemicals, pre- existing health conditions, and genetic differences between people, all of which can be controlled in a laboratory animal study. Once a chemical contacts the body, it begins to interact through the processes of absorption, distribution, metabolism, and excretion. Those four biologic pro- cesses characterize the disposition of a foreign substance that enters the organ- ism. Their combination determines the concentration of the compound in the body and how long each organ is exposed to it and thus influences its toxic or pharmacologic activity. Absorption is the entry of the substance into the organism, normally by uptake into the bloodstream via mucous surfaces, such as the intestinal walls of the digestive tract during ingestion. Low solubility, chemical instability in the stomach, and inability to permeate the intestinal wall can all reduce the extent to which a substance is absorbed after being ingested. The solubility of a chemical in fat and its hydrophobicity influence the pathways by which it is metabolized (structurally transformed) and whether it persists in the body or is excreted. Absorption is a critical determinant of the chemicalâs bioavailability, that is, the fraction of it that reaches the systemic circulation. Other routes of absorption ex- perienced by free-ranging humans are inhalation (entry via the airways) and der- mal exposure (entry via the skin). Animal studies may involve additional routes of exposure that are not ordinarily encountered by humans, such as intravenous or intraperitoneal injection, in which the chemical is injected into the bloodstream or abdominal cavity, respectively. Distribution refers to the travel of a substance from the site of entry to the tissues and organs where they will have their ultimate effect or be sequestered. Distribution takes place most commonly via the bloodstream. The term metabo- lism is used to describe the breaking down that all substances begin to experience as soon as they enter the body. Metabolism of most foreign substances takes place in the liver by the action of oxidative enzymes collectively termed cyto- chrome P450. As metabolism occurs, the initial (parent) chemical is converted to new chemicals called metabolites. When metabolites are pharmacologically or toxicologically inert, metabolism deactivates the administered dose of the parent chemical reducing its effects on the body. Sometimes metabolism may activate the compound to a metabolite more potent or more toxic than the parent compound. Excretion, also referred to as elimination, is the removal of substances or
INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY 67 their metabolites from the body, most commonly in urine or feces. Excretion is often incomplete, and incomplete excretion results in the accumulation of foreign substances that can adversely affect function. The routes and rates of absorption, distribution, metabolism, and excretion of a toxic substance collectively are termed toxicokinetics (or pharmacokinetics). Those processes determine the amount of a particular substance or metabolite that reaches specific organs or cells and that persists in the body. Understanding the toxicokinetics of a chemical is important for valid reconstruction of exposure of humans and for assessing the risk of effects of a chemical. The principles involved in toxicokinetics are similar among chemicals, although the degree to which different processes influence the distribution depends on the structure and other inherent properties of the chemicals. Thus, the lipophilicity or hydrophobicity of a chemical and its structure influence the pathways by which it is metabolized and whether it persists in the body or is excreted. The degree to which different toxicokinetic processes influence the toxic potential of a chemical depends on metabolic pathways, which often differ among species. For that reason, attempts at extrapolation from experimental animal studies to human exposures must be done with extreme care. Many chemicals were used by the US armed forces in Vietnam. The nature of the substances themselves was discussed in more detail in Chapter 6 of the original VAO report (IOM, 1994). Four herbicides documented in military records were of particular concern and are examined here: 2,4-D, 2,4,5-T, 4-amino-3,5,6- trichloropicolinic acid (picloram), and cacodylic acid (dimethyl arsenic acid, DMA). This chapter also examines 2,3,7,8-tetrachlorodibenzo-p-dioxin (referred to in this report as TCDD to represent a single, and the most toxic, congener of the tetrachlorodibenzo-p-dioxins [tetraCDDs], also commonly referred to as dioxin), a contaminant of 2,4,5-T, because its potential toxicity is of concern; considerably more information is available on TCDD than on the herbicides. Other contaminants present in 2,4-D and 2,4,5-T are of less concern. Except as noted, the laboratory studies of the chemicals of concern used pure compounds or formulations; the epidemiologic studies discussed in later chapters often tracked exposures to mixtures. TCDD Chemistry TCDD is a polychlorinated dibenzo-p-dioxin that has a triple-ring structure consisting of two benzene rings connected by an oxygenated ring (Figure 4-1); chlorine atoms are attached at the 2, 3, 7, and 8 positions of the benzene rings. The chemical properties of TCDD include a molecular weight of 322, a melting point of 305â306Â°C, a boiling point of 445.5Â°C, and a log octanolâwater parti- tion coefficient of 6.8 (NTP substance profile). It is virtually insoluble in water
68 VETERANS AND AGENT ORANGE: UPDATE 2008 Cl O Cl Cl O Cl 2,3,7,8-tetrachlorodibenzo-p-dioxin FIGURE 4-1â Chemical structure of TCDD. Figure 4-1.eps (19.3 ng/L), but is soluble in organic solvents, such as benzene and acetone. It has been suggested (EPA 2004 Draft Document) that volatilization of dioxin from water may be an important mechanism of transfer from the aqueous to the atmospheric phase. Absorption, Distribution, Metabolism, and Elimination The absorption, distribution, metabolism, and elimination of TCDD have been extensively studied in a number of animal models in the last 25 years. Given the plethora of data, this section only highlights and summarizes key find- ings. A more exhaustive review may be found at http://www.epa.gov/ncea/pdfs/ dioxin/nas-review. TCDD is absorbed into the body rapidly but is eliminated slowly. Because of the slow elimination, the concentration of TCDD in lipid or blood is thought to be in dynamic equilibrium with that in other tissue compartments and is thus considered to be reasonable for use in estimating total body burdens. Exposure of humans to TCDD is thought to occur primarily via the mouth, skin, and lungs. In laboratory animals, oral administration of TCDD has been shown to result in absorption of 50â93% of the administered dose (Nolan et al., 1979; Rose et al., 1976). Similarly, a study performed in a 42-year-old man found that 87% of the oral dose was absorbed. Dermal absorption appears to be dose-dependent, with lower absorption occurring at higher doses (Banks and Birnbaum, 1991). Studies performed in humans indicate that human skin may be more resistant to absorp- tion (Weber, 1991). After ingestion and gastrointestinal absorption, TCDD associates primarily with the lipoprotein fraction of the blood and later partitions into the cellular membranes and tissues (Henderson and Patterson, 1988). TCDD is distributed to all compartments of the body; the amounts differ from organ to organ, but most studies indicate that the primary disposition of TCDD is in the liver and adipose tissues. For example, in a human volunteer, it was found that at 135 days after ingestion, 90% of TCDD was in fat (Poiger and Schlatter, 1986); in the rhesus monkey, TCDD is very persistent in adipose tissue (Bowman et al., 1989). The
INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY 69 disposition and elimination of TCDD depend on the tissue examined, the time that has elapsed since exposure, total exposure, and other factors. For example, the concentration of cytochrome P450 1A2 (CYP1A2) (Poland et al., 1989) in the liver is increased by TCDD. Direct binding of TCDD to the CYP1A2 is thought to result in sequestration of TCDD in the liver and to inhibit its distribution to other tissues. The importance of CYP1A2 concentrations for the toxic actions of TCDD has also been shown in studies performed in laboratory animals in which maternal hepatic CYP1A2 was found to sequester TCDD and protect the fetus against TCDD-induced teratogenesis (Dragin et al., 2006). In addition, distribu- tion of TCDD is age-dependent, as shown by studies in which young animals displayed the highest concentration of TCDD in the liver and older animals the highest concentrations in kidney, skin, and muscle (Pegram et al., 1995). Finally, the elimination rate of TCDD, in particular after low exposures, depends heavily on the amount of adipose tissue mass (Aylward et al., 2005; Emond et al., 2005, 2006). In laboratory animals and humans, metabolism of TCDD occurs slowly. It is eliminated primarily in feces as both the parent compound and its more polar metabolites. However, elimination appears to be dose-dependent; at low doses, about 35% of the administered dose of TCDD was detected in the feces; at higher doses, about 46% was observed (Diliberto et al., 2001). The dose-dependent occurrence of TCDD metabolites in the feces is thought to be due to increased expression of metabolizing enzymes at higher doses. A measure of elimination is half-life, which is defined as the time required for the plasma concentration or the amount of a chemical in the body to be reduced by one-half. The half-life of TCDD in humans varies with body mass index, age, sex, and concentration and has been found to vary from 0.4 to over 10 years (Table 4-1). In light of the variables discussed above and the effect of differences in physiologic states and metabolic processes, which can affect the mobilization of lipids and possibly of compounds stored in them, complex models known as physiologically based pharmacokinetic models have been developed to integrate exposure dose with organ mass, blood flow, metabolism, and lipid content to predict the movement of toxicants into and out of each organ. A number of recent modeling studies have been performed in an effort to understand the relevance of animal experimental studies to exposures that occur in human populations (Aylward et al., 2005a,b; Emond et al., 2005). Toxicity Profile The administration of TCDD to laboratory animals affects many tissues and organs.â The effects of TCDD in laboratory animals have been observed in a number of species (rats, mice, guinea pigs, hamsters, monkeys, cows, and rabbits) after the administration of a variety of doses and after periods that represent acute (less than 24 hr), subchronic (more than 1 day up to 3 months), and chronic (more
70 VETERANS AND AGENT ORANGE: UPDATE 2008 TABLE 4-1â Estimates of TCDD Half-Life in Humans and Animals Confidence Reference Half-Lifea Interval Comment Human studies: Leung et al., 2006 0.4 year Breast-fed infants, 0â1 year after exposure Kumagai and Koda, 2005 1.1â2.3 years Adult male, incinerator workers, 0â1.3 years after exposure Aylward et al., 2005a < 3 years Calculated for exposures > 10,000 pg/g of serum lipid > 10 years Calculated for exposures < 50 pg/g of 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, 0â3 years after exposure 2.9 yearsb Adult female, severe exposure, 0â3 years after exposure Michalek et al., 2002 0.34 yearb Adult males, Seveso cohort, 0â3 months after exposure 6.9 years Adult males, Seveso cohort, 3â16 years after exposure 9.8 years Adult females, Seveso cohort, 3â16 years after exposure 7.5 years Adult males, Ranch Hands, 9â33 years after exposure Needham et al., 1994 7.8 years 7.2â9.7 Adults, Seveso cohort years Pirkle et al., 1989 7.1 years 5.8â9.6 Adult males, Ranch Hands, 9â23 years years after exposure Animal studies: Neubert et al., 1990 73.7 days 60.9â93.8 Monkeys, marmoset, single injection days 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 and Tuomisto, 21.9 days Rats, male Han/Wistar, resistant 1990 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 Â± 3.0 days Rats, male Sprague-Dawley a Half-livesof 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 contami- nated persons consistent with nonlinear elimination predicted by physiologically based pharmacoki- netic modeling (for example, 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.
INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY 71 than 3 months) exposures. Some differences are observed in the different species, particularly with respect to their degree of sensitivity, but in general the effects observed are qualitatively similar. Relatively high exposures of TCDD affect a variety of organs and result in organ dysfunction and death. The specific organ dysfunction that constitutes the lethal event, however, is not known. A character- istic of TCDD exposure is a wasting syndrome with loss of adipose and muscle tissues and severe weight loss. In most rodents, exposure to TCDD affects the liver, as indicated by hepatic enlargement, the presence of hepatic lesions, and impaired hepatic function. The thymus is also sensitive. Finally, in both humans and nonhuman primates, TCDD exposure results in chloracne and associated dermatologic changes. As will be discussed in more detail in Chapters 6â9, stud- ies performed in animal models have indicated that exposure to TCDD adversely affects the heart, the skin, and the immune, endocrine, and reproductive systems, and increases the incidence of cancers of the liver, skin, thyroid, adrenal cortex, hard palate, nasal turbinates, tongue, and respiratory and lymphatic systems (Huff et al., 1994). When TCDD has been administered to pregnant animals, such birth defects as cleft palate, malformations of the reproductive organs of the male and female progeny, and abnormalities in the cardiovascular system have been observed. The administration of TCDD to laboratory animals and cultured cells affects enzymes, hormones, and receptors.â In addition to adversely affecting the ability of specific organs to fulfill their normal physiologic roles, TCDD has been found to alter the function and expression of essential proteins. Some of the proteins are enzymes, specialized proteins that increase the rates of chemical reactions and aid in the bodyâs ability to convert chemicals into different molecules. The metabolism of foreign chemicals often changes their biologic properties and in some cases increases the bodyâs ability to eliminate them in urine. The enzymes that are most affected by TCDD are ones that act on or metabolize xenobiotics and hormones. Xenobiotics are chemicals that are not expected to be present in the body, and hormones are made by the body and serve as chemical messengers that transport a signal from one cell to another. Among the enzymes affected by TCDD, the best studied is CYP1A1, which metabolizes xenobiotics. In labora- tory animals, exposure to TCDD commonly results in an increase in the CYP1A1 present in most tissues; CYP1A1 therefore is often used as a marker of TCDD exposure. Other enzymes that are affected by TCDD are ones that metabolize hormones such as thyroid hormones, retinoic acid, testosterone, estrogens, and adrenal ste- roids. Those hormones transmit their signals by interacting with specific proteins called receptors and in this manner initiate a chain of events in many tissues of the body. For example, binding of the primary female sex hormone, estrogen, to the estrogen receptor promotes the formation of breasts and the thickening of the endometrium and regulates the menstrual cycle. Exposure to TCDD can increase
72 VETERANS AND AGENT ORANGE: UPDATE 2008 the metabolism of estrogen, and this leads to a decrease in the amount of estrogen available for binding and activating the estrogen receptor. The ultimate effect of TCDD is an interference with all the bodily functions that are regulated by estro- gens. Similarly, the actions of TCDD on the adrenal steroids can adversely affect their ability to regulate glucose tolerance, insulin sensitivity, lipid metabolism, obesity, vascular function, and cardiac remodeling. In addition to changing the amount of hormone present, TCDD has been found to interfere with the ability of receptors to fulfill their role in transmitting hormone signals. Animal models have shown that exposure to TCDD can increase the amounts of enzymes in the body and interfere with the ability of hormones to activate their specific hormone re- ceptors. Those actions of TCDD on enzymes and hormone receptors are thought to underlie, in part, observed developmental and reproductive effects and cancers that are hormone-responsive. TCDD alters the paths of cellular differentiation.â Research performed primarily in cultured cells has shown that TCDD can affect the ability of cells to undergo such processes as proliferation, differentiation, and apoptosis. During the pro- liferative process, cells grow and divide. When cells are differentiating, they are undergoing a change from less specialized to more specialized. Cellular dif- ferentiation is essential for an organism to mature from a fetal to an adult state. In the adult, proper differentiation is required for normal functions of the body, for example, in maintaining a normally responsive immune system. Processes of controlled cell death, such as apoptosis, are similarly important during develop- ment of the fetus and are necessary for normal physiologic functions in the adult. Apoptosis is a way for the body to eliminate damaged or unnecessary cells. The ability of a cell to undergo proliferation, differentiation, and apoptosis is tightly controlled by an intricate network of signaling molecules that allows the body to maintain the appropriate size and number of all the specialized cells that form the fabric of complex tissues and organs. Disruption of that network that alters the delicate balance of cell fate can have severe consequences, including impair- ment of the function of the organ because of the absence of specialized cells. Alternatively, the presence of an excess of some kinds of cells can result in the formation and development of tumors. Thus, the ability of TCDD to disrupt the normal course of a specific cell to proliferate, differentiate, or undergo apoptosis is thought to underlie (at least in part) its adverse effects on the immune system and the developing fetus and its ability to promote the formation of certain cancers. Definition of Dioxin-like Compounds and TEF and TEQ Terminology Many compounds have dioxin-like properties: they have similar chemical structure, have similar physiochemical properties, and cause a common battery of toxic responses. Because of their hydrophobic nature and resistance to me-
INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY 73 tabolism, these chemicals persist and bioaccumulate in fatty tissues of animals and humans. Several hundred chemicalsâsuch as the polychlorinated dibenzo- p-dioxins, polychlorinated dibenzofurans, polybrominated dibenzo-p-dioxins, polybrominated dibenzofurans, and polychlorinated biphenylsâare described as dioxin-like compounds (DLCs), although only a few of them are thought to display dioxin-like toxicity. For most purposes, only 17 polychlorinated dibenzo- p-dioxins and polychlorinated dibenxofurans and a few of the coplanar poly- chlorinated biphenyls that are often encountered in environmental samples are recognized as being true DLCs. In the context of risk assessment, these polychlo- rinated dibenzo-p-dioxins, polychlorinated dibenxofurans, and polychlorinated biphenyls are commonly found as complex mixtures when detected in environ- mental media and biologic tissues or when measured as environmental releases from specific sources. That complicates the human health risk assessment that may be associated with exposures to varied mixtures of DLCs. To address the problem, the concept of toxic equivalency has been elaborated by the scientific community, and the toxic equivalency factor (TEF) has been developed and in- troduced to facilitate risk assessment of exposure to those chemical mixtures. On the most basic level, TEFs compare the potential toxicity of each DLC found in a mixture with the toxicity of TCDD, the most toxic member of the group. The procedure involves assigning individual TEFs to the DLCs with consideration of chemical structure, persistence, and resistance to metabolism. TEF ascribe spe- cific order-of-magnitude toxicity to each DLC relative to that of TCDD, which is assigned a TEF of 1.0. The DLCs have TEFs ranging from 0.00001 to 1.0. When several compounds are present in a mixture, the toxicity of the mixture is estimated by multiplying the TEF of each DLC in the mixture by its mass concentration and summing the products to yield the TCDD toxicity equivalent quotient (TEQ) of the mixture. Mechanism of Action TCDD binds and activates the aryl hydrocarbon receptor (AHR). The AHR is a member of a family of basic-helix-loop-helix (bHLH) transcription factors, that is, one of many proteins in the cell that controls the transfer (or transcription) of genetic information from DNA to RNA. bHLH proteins are characterized by the presence of a string of basic amino acid residues followed by two alpha helices joined by a loop. Generally, the larger of the two helices participates in binding to DNA in a specific sequence motif; the specificity is determined largely by the amino acid sequence of the helix. bHLH transcription factors are dimeric, form- ing functional heterodimers with other members of the family. By mechanisms that are poorly understood, binding of the heterodimeric complex to DNA recruits the transcriptional machinery needed to activate gene expression and results in a large increase in the rate of synthesis of mRNA molecules for the genes regulated by the complex and ultimately in a large increase in the corresponding protein.
74 VETERANS AND AGENT ORANGE: UPDATE 2008 The best known AHR target is the expression of a mixed-function oxidase en- zyme that was termed aryl hydrocarbon hydroxylase (AHH) in the 1960s and is now better known as the CYP1A1 enzyme. Expression of this protein is an acute outcome of AHR activation and may not faithfully represent the consequences of chronic exposure to AHR ligands. In its inactive state, the AHR is found in the cytosol of the cell, where it is protected from proteolytic degradation by several chaperones and cochaperones. As a receptor, the AHR is a protein capable of receiving and forming a com- plex with specific substances, termed ligands, which confer on it the ability to perform a biologic function. In the case of the AHR, the function is to induce the transcription of specific target genes. Hence, the AHR belongs to a class of ligand-activated transcription factors. If the ligand is a chemical, such as TCDD, the AHR dissociates from the chaperones and translocates into the nucleus of the cell, where it forms a heterodimer with another bHLH protein, the AHR nuclear translocator (ARNT). This heterodimer binds to its cognate DNA motifs and recruits the macromolecular complexes needed to initiate gene transcription. AHR Functional Domains The AHR contains several regions, or domains, that perform distinct func- tions. The receptor is a member of the Per-Arnt-Sim (PAS) bHLH subfamily (Burbach et al., 1992; Fukunaga et al., 1995). The bHLH motif is found in the amino terminus of the protein and is common to all transcription factors in this subfamily (Jones, 2004). The members of the bHLH family also have several highly conserved domains with functionally distinctive biochemical roles. One of the domains is the basic region, described earlier, which is involved in the binding of AHR/ARNT complexes to DNA. Another domain is the HLH region, which facilitates the stable interaction between AHR and ARNT. A third domain is termed the PAS domain and consists of a stretch of 200â350 amino acids with high sequence relatedness to protein domains that were originally found in the Drosophila melanogaster genes period (Per) and single minded (Sim) and in the AHRâs dimerization partner ARNT; hence the name PAS. The AHR contains two PAS domains, PAS-A and PAS-B (Ema et al., 1992). The PAS domains support secondary interactions with other PAS-domainâcontaining proteins, with the chaperones and cochaperones, and with many other transcription factors, coacti- vators and corepressors. The ligand-binding site of AHR is in the PAS-B domain (Coumailleau et al., 1995) and contains several conserved residues critical for ligand binding (Goryo et al., 2007). A fourth important domain in the carboxyl terminus of the protein, is rich in glutamine and is involved in coregulator recruit- ment and transactivation (Kumar et al., 2001).
INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY 75 AHR Ligands From an environmental point of view, there are two classes of AHR ligandsâ synthetic and naturally occurringâthat total more than 400 known ligands. Many of the first ligands to be discovered were synthetic polycyclic aromatic hydrocar- bons (PAHs), such as 3-methylcholanthrene, benzo[a]pyrene (B[a]P), benzanthra- cene, and naphthoflavone. The biologic consequences of experimental exposure of mice to those chemicals led to the prediction of a receptor-dependent mecha- nism long before the existence of the AHR was directly demonstrated. Compari- son of the effects of 3-methylcholanthrene treatment in two inbred mouse strains revealed a major difference in PAH responsiveness. Hepatic CYP1A1 enzyme increased more than 6-fold after 3-methylcholanthrene treatment in C57BL/6 mice but not in DB/2 mice. Appropriate genetic crosses between responsive C57BL/6 mice but in non-responsive DB/2 mice indicated that responsiveness in these prototype strains was inherited as a simple autosomal dominant trait. The genetic locus defined in the crosses was termed the aromatic hydrocarbon responsiveness (Ahr) locus (Nebert et al., 1982). Molecular biologic studies dur- ing the next decade showed that responsive and nonresponsive mice had equally functional CYP1A1 enzymes and that the Ahr locus encoded a regulatory gene responsible for induction of the Cyp1a1 gene. The members of the polyhalo- genated aromatic hydrocarbonsâsuch as the dibenzodioxins, dibenzofurans, and polychlorinated and polybrominated biphenylsâwere recognized as AHR ligands much later, after the discovery by Poland and co-workers that dioxin was a potent inducer of hepatic AHH in the rat. At that time, it was found that high concentrations of TCDD could induce AHH activity in the nonresponsive DB/2 mouse to levels as high as those in the responsive C57BL/6 mouse. The difference between responsive and nonresponsive strains was in sensitivity to the inducer: DB/2 mice required 18 times more TCDD than C57BL/6 mice for 50% of the maximal response. Later, a receptor protein for TCDD, 3-methylcholanthrene and other PAHs, was identified, characterized in the hepatic cytosol of C57BL/6 mice, and termed Ah receptor (Poland et al., 1976). The available evidence indicated that the protein was the product of the Ahr locus, which was localized to mouse chromosome 12 and human chromosome 7, and later cloned (Burbach et al., 1992; Ema et al., 1992). Recent work has focused on naturally occurring compounds in hopes of identifying an endogenous ligand (Denison and Nagy, 2003). Several such natu- rally occurring compounds have been identified as AHR ligands, including the tryptophan derivatives indigo and indirubin (Adachhi et al., 2001), the tetrapyr- roles bilirubin (Sinal and Bend, 1997), the arachidonic acid metabolites lipoxin A4 and prostaglandin G (Seidel et al., 2001), modified low-density lipoprotein (McMillan and Bradfield, 2007), several dietary carotenoids (Denison and Negy, 2003; Probst et al., 1993), and cAMP (Oesch-Bartlomowicz et al., 2005). One assumption made in the search for an endogenous ligand is that the ligand will
76 VETERANS AND AGENT ORANGE: UPDATE 2008 be a receptor agonist, that is, that it will activate the receptor. However, recent work has shown that may not be the case inasmuch as one such natural ligand, 7-ketocholesterol, competitively inhibits AHR-dependent signal transduction (Savouret et al., 2001). AHR Signaling Pathway In the absence of bound ligand, the inactive AHR is retained in the cyto- plasm of the cell in a complex consisting of two molecules of the heat shock protein hsp90, one molecule of prostaglandin E synthase 3 (p23) (Kazlauskas et al., 1999), and one molecule of the immunophilin-like protein hepatitis B virus X-associated protein 2 (XAP2) (Petrulis et al., 2003), previously identified as AHR-interacting protein (Ma and Whitlock, 1997) and AHR-activated 9 (Carver and Bradfield, 1997). The hsp90 dimerâp23 complex plays multiple roles in the protection of the AHR from proteolysis, maintaining it in a conformation that makes the receptor accessible to ligand binding at the same time that it prevents the premature binding of ARNT (Carver and Bradfield, 1994; Pongratz et al., 1992; Whitelaw et al., 1993). XAP2 interacts with the carboxyl terminus of hsp90 and with the AHR nuclear-localization signal (NLS), a short amino acid domain in the bHLH region that targets the receptor for interaction with nuclear-transport proteins. Binding of XAP2 blocks such interaction, preventing the inappropriate trafficking of the receptor into the nucleus (Petrulis et al., 2003). Binding of ligand induces the release of XAP2 and the exposure of the NLS and leads to the binding of nuclear-import proteins and translocation of the cytosolic complex into the nucleus (Davarinos and Pollenz, 1999; Song and Pollenz, 2002). Once in the nucleus, chaperones and cochaperones dissociate from the AHR, exposing the two PAS domains and allowing the binding of ARNT (Hoffman et al., 1991; Probst et al., 1993). The activated AHR/ARNT heterodimeric complex is then capable of directly or indirectly interacting with DNA by binding to recognition sequences in the regulatory region of responsive genes (Dolwick et al., 1993; Probst et al., 1993). The canonical DNA recognition motif of the AHR/ARNT complex is re- ferred to as the AHR-responsive element (AHRE, also referred to as the dioxin- responsive element [DRE] or the xenobiotic-responsive element [XRE]). This element is found in the promoter region of AHR-responsive genes and contains the core sequence 5â²-GCGTG-3â² (Shen and Whitlock, 1992), which is part of a more extensive consensus binding sequence, 5â²-T/GNGCGTGA/CG/CA-3â² (Lusska et al., 1993; Yao and Denison, 1992). The AHR/ARNT complex binds to the AHRE core sequence in such a manner that ARNT binds to the 5â²-GTG-3â² and AHR binds to 5â²-TC/TGC-3â² (Bacsi et al., 1995; Swanson et al., 1995). A second type of element, termed AHRE-II, 5â²-CATG(N6)C[T/A]TG-3â², has recently been shown to be capable of acting indirectly with the AHR/ARNT complex (Boutros et al., 2004; Sogawa et al., 2004). The end result of the process is the recruitment
INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY 77 of the transcriptional machinery associated with RNA polymerase II and the initiation of differential changes in the expression of the genes bearing the AHR/ ARNT recognition motif. Many of the genes code for proteins responsible for detoxification reactions directed at the elimination of the ligand. Recent research suggests that posttranslational modifications in histone proteins may modify the response (Hestermann and Brown, 2003; Schnekenburger et al., 2007). AHR Physiology The vertebrate AHR is presumed to have evolved from its counterpart in invertebrates, in which it serves a ligand-independent role in normal develop- ment processes. The ancestral function of the AHR appears to be the regulation of specific aspects of embryonic development, it having acquired the ability to bind xenobiotic compounds only during vertebrate evolution (Hahn, 2001). The invertebrate AHR also functions as a transcription factor and binds to the same dimerization partner (ARNT) and DNA response elements as the vertebrate pro- tein, but it does not respond to any of the environmental ligands recognized by the vertebrate receptor. Instead, it regulates diverse developmental processes that are independent of exogenous ligand exposure, such as neuronal differentiation dur- ing worm development in Caenorhabditis elegans (Huang et al., 2004; Qin and Powell-Coffman, 2004) or normal morphogenesis of legs, antennae, and bristles in Drosophila melanogaster (Adachi-Yamada et al., 2005). In developing verte- brates, the AHR seems to play a role in cellular proliferation and differentiation and, in keeping with this role in invertebrates, also possesses a developmental role in craniofacial, renal, and cardiovascular morphogenesis (Birnbaum et al., 1989; Fernandez-Salguero et al., 1997; Lahvis et al., 2005). The clearest adaptive physiologic response of AHR activation is the induc- tion of xenobiotic-metabolizing enzymes involved in detoxification of toxic li- gands. Evidence of that response, which was described above, was first observed from the induction of Cyp1a1, resulting from exposure to PAHs or TCDD, and directly related to activation of the AHR signaling pathway (Israel and Whitlock, 1983, 1984). Because of the presence of AHRE motif in their gene promoters, other metabolizing genes were tested and found to be induced by AHR ligands, and this led to the identification of a so-called AHR gene battery of phase I and phase II detoxification genes that code for the drug-metabolizing enzymes CYP1A1, CYP1A2, CYP1B1, NQO1, ALHD3A1, UGT1A2, and GSTA1 (Ne- bert et al., 2000). Presumably, vertebrates have evolved those enzymes to detect a wide array of foreign, potentially toxic chemicals, represented in the wide variety of substrates that AHR is able to bind whose biotransformation and elimination it is able to facilitate. A potential complication of the adaptive responses elicited by AHR activa- tion is the induction of a toxic response. Toxicity may result from the adaptive response itself if the induction of metabolizing enzymes results in the production
78 VETERANS AND AGENT ORANGE: UPDATE 2008 of toxic metabolites. For example, the PAH B[a]P, an AHR ligand, induces its own metabolism and detoxification by the AHR-dependent signaling mechanism described earlier but paradoxically becomes bioactivated to a toxic metabolite in several tissues by metabolism that depends on CYP1A1 and CYP1B1 enzymatic activity (Harrigan et al., 2004). A second potential source of AHR-mediated tox- icity may be aberrant changes in global gene expression beyond those observed in the AHR gene battery. The global changes in gene expression may lead to deleterious changes in cellular processes and physiology. Microarray analysis has proved invaluable in understanding and characterizing that response (Martinez et al., 2002; Puga et al., 2000, 2004; Vezina et al., 2004). It is clear that the AHR is an essential component of the toxicity of dioxin and of DLCs. Homozygous deletion of the AHR in mice leads to a phenotype that is resistant to the toxic effects of TCDD and to the carcinogenic effects of B[a]P (Fernandez-Salguero et al., 1996; Lahvis and Bradfield, 1998; Schmidt et al., 1996). AHR knockout mice, however, have other phenotypic effects, including reduced liver size and hepatic fibrosis and cardiovascular abnormalities. Hence, it is likely that dioxin has effects due to gratuitous deregulation of endogenous AHR functions, unrelated to the intrinsic toxicity of some of its ligands. Carcinogenic Classification of TCDD The US Environmental Protection Agency (EPA) and the International Agency for Research on Cancer (IARC), a branch of the World Health Organiza- tion, have defined criteria to classify the potential carcinogenicity of chemicals on the basis of the weight of scientific evidence from animal, human, epidemiologic, mechanistic, and mode-of-action studies. EPA classified TCDD as a âprobable human carcinogenâ in 1985 and as âcarcinogenic to humansâ in a 2003 reassess- ment. In 1998, the IARC panel of experts concluded that the weight of scientific evidence supported the classification of dioxin as a class I carcinogen, that is, as âcarcinogenic to humans.â Four years later, the US National Toxicology Program upgraded its classification to âknown to be a human carcinogen.â In 2006, a panel of experts convened by the National Research Council to evaluate the EPA reas- sessment concluded that TCDD was âlikely to be carcinogenic to humans.â That designation reflected the revised EPA Guidelines for Carcinogen Risk Assessment made public in 2005. Other Toxic Health Outcomes of Dioxin There is an extensive body of evidence from experimental studies in animal model systems that TCDD, other dioxins, and several DLCs are immunotoxic. Although the available evidence on humans is scant, mechanistic considerations support the notion that chemical alterations to immune function would cause adverse health outcomes because of the critical role that the immune system
INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY 79 plays in general protection, fighting off infection and eliminating cancer cells at early stages. Because of those considerations, these compounds are potential immunotoxicants. Similarly, reproduction and embryonic development clearly are targets of TCDD, other dioxins, and DLCs; it is found consistently that the adverse effects are more prevalent during fetal development than in the adult. However, data on those effects in humans are practically nonexistent. TCDD, other dioxins, and DLCs are also recognized as potentially capable of causing birth defects, reproductive disorders, immunotoxicity, and chloracne. Human and animal studies have revealed other potential health outcomes includ- ing cardiovascular disease, hepatic disease, thyroid dysfunction, lipid disorders, neurotoxicity, and metabolic disorders, such as diabetes. A number of effects of TCDD exposure in vitro appear to be independent of AHR-mediated mechanisms. The salient ones are induction of transforming growth factor-Î± and other genes involved in extracellular matrix deposition in cells from mice with homozygous ablation of the Ahr gene (Guo et al., 2004); mobilization of calcium from intracellular sources and imported from the culture medium (Puga et al., 1995); and related to calcium mobilization, the induction of mitochondria oxidative stress (Senft et al., 2002). Calcium mobilization by TCDD may have an important effect on signal-transduction mechanisms that control gene expression, inasmuch as several proto-oncogenes, such as c-fos, are activated by calcium changes. Recent Findings from Mechanistic Studies That May Be Relevant to Health Outcomes Several reports published since Update 2006 (IOM, 2007) have contributed to the understanding of how TCDD exposure may have contributed to particular health outcomes, as follows: â¢ Insight into the role of the AHR repressor protein in mediating cell- typeâspecific responsiveness to TCDD (Evans et al., 2008; Haarmann- Stemmann et al., 2007). â¢ Identification of genes that may be regulated by TCDD through activation of AHR that are involved in cholesterol biosynthesis, lipogenesis, and glu- cose metabolism (Sato et al., 2008; Zarzour et al., 2008); development of thymocytes (the KLF2 regulon) (McMillian et al., 2007); glucose uptake (the glucose transporter) (Tonack et al., 2007); and metabolism of the ste- roid hydroxysteroid 17-beta dehydrogenase; plus a novel gene (Hectd2) with poorly defined function (Hayes et al., 2007). â¢ Examination of the effect of TCDD on signaling pathways, such as that of protein kinase C (Lee et al., 2007) and integrin signaling (Liu and
80 VETERANS AND AGENT ORANGE: UPDATE 2008 Jefcoate, 2006) that may underlie TCDDâs adverse effects on neurons in cell culture and cell adhesion, respectively. â¢ Elucidation of crosstalk between the AHR and receptors involved in regulation of cellular responses to stress (Burchiel et al., 2008; Dvorak et al., 2008; Sonneveld et al., 2007) and estrogen (Boverhof et al., 2008; Marquez-Bravo and Gierthy, 2008; Tanaka et al., 2007). â¢ Progress pertaining to TCDDâs effects on oxidative stress and DNA dam- age (Lin et al., 2007; Lu et al., 2007) and events in the mitochondria (Biswas et al., 2008; Shertzer et al., 2006). â¢ Study of TCDDâs effects on determination of cell-fate. Mechanisms by which TCDD induces G1 arrest in hepatic cells (Mitchell et al., 2006; Weiss et al., 2008) and decreases viability of endometrial endothelial cells (Bredhult et al., 2007), insulin-secreting beta cells (Piaggi et al., 2007), peripheral T cells (Singh et al., 2008), and neuronal cells (Bredhult et al., 2007) were identified. The findings may be relevant to cancer, reproduc- tive health, diabetes, immune function, and neurotoxicity. Insights were also gained into TCDDâs effects on maturation and differentiation of adipocytes (Arsenescu et al., 2008), granule neuron precursors (Collins et al., 2008), dendritic cells (Lee et al., 2007), and how TCDD induces the differentiation of regulatory T cells (Funatake et al., 2008; Gill et al., 2008; Hollingshead et al., 2008; Kimura et al., 2008; Quintana et al., 2008; Veldhoen et al., 2008; Vogel et al., 2008). â¢ Increased evidence that the TCDDâAHR pathway may impinge on the cytokine-inflammatory response (Chiaro et al., 2008; Dong and Matsumura, 2008; Hollingshead et al., 2008; Ito et al., 2008; Li et al., 2007; Vogel et al., 2007a,b). Those publications collectively have added to what was already strong sup- port of an association of TCDD exposure with adverse health effects in laboratory animals and, by extension, in humans. Summary on Biologic Plausibility of TCDD Inducing Adverse Effects in Humans Mechanistic studies in vitro and in laboratory animals have characterized the biochemical pathways and types of biologic events that contribute to adverse effects of exposure to TCDD. For example, much evidence indicates that TCDD acting via the AHR in partnership with ARNT alters gene expression. Receptor binding may result in release of other cytoplasmic proteins that alter the expres- sion or activity of other cell-regulatory proteins. Mechanistic studies also indicate that many other cellular component proteins contribute to the gene-regulatoring effect and that the response to TCDD exposure involves a complex interplay between genetic and environmental factors. Comparative data from animal and
INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY 81 human cells in vitro and from tissues suggest a strong qualitative similarity among species in response to TCDD, and this further supports the applicability to humans of the generalized model of initial events in response to dioxin ex- posure. Biochemical and biologic responses to TCDD exposure are considered adaptive or simply reflective of exposure and not adverse in themselves if they take place within the normal homeostatic parameters of an organism. However, they may exceed physiologic parameters or constitute an early event in a pathway leading to damage to sensitive members of the population. In the latter case, the response is toxic and would be expected to cause an adverse health effect. From a mechanistic standpoint, adverse effects identified in vitro are expected to occur in all the cells of an organism and to all the organisms that express these proteins. That generalization sets the ground rules for the concept of biologic plausibil- ity, which relies on extrapolation from laboratory tests to human risks, and on the precautionary principle, which bases decision-making on precaution if the precise nature or magnitude of the potential damage that a substance may cause in humans is uncertain. When deemed useful, the findings from individual publications are presented in the biologic-plausibility sections associated with specific health outcomes. PHENOXY HERBICIDES: 2,4-D AND 2,4,5-T Chemistry 2,4-D (Chemical Abstracts Service [CAS] No. 94-75-7) is an odorless and, when pure, white crystalline powder (Figure 4-2); it may appear yellow when phenolic impurities are present. The melting point of 2,4-D is 138Â°C, and the free acid is corrosive to metals. It is soluble in water and in a variety of organic sol- vents (such as acetone, alcohols, ketones, ether, and toluene). 2,4,5-T (CAS No. 93-76-5) is an odorless, white to light-tan solid with a melting point of 158Â°C. FIGURE 4-2â Structure of selected phenoxy herbicides. Figure 4-2.eps
82 VETERANS AND AGENT ORANGE: UPDATE 2008 2,4,5-T is noncorrosive and is soluble in alcohol and water. It reacts with organic and inorganic bases to form salts and with alcohols to form esters. Uses of 2,4-D and 2,4,5-T 2,4-D has been used commercially in the United States since World War II to control the growth of broadleaf plants and weeds on range lands, lawns, golf courses, forests, roadways, parks, and agricultural land and remains today a widely used herbicide approved for use by the European Union and the US EPA. Formulations include 2,4-D amine and alkali salts and esters, which are mobile in soil and easily absorbed through the leaves and roots of many plants. Like 2,4-D, 2,4,5-T was developed and marketed as a herbicide during World War II. How- ever, the registration for 2,4,5-T was canceled by EPA in 1978 when it became clear that it was contaminated with TCDD during the manufacturing process. The herbicidal properties of 2,4-D and 2,4,5-T are related to their ability to mimic the plant growth hormone indole acetic acid. They are selective herbicides in that they affect the growth of only broadleaf dicots (which include most weeds) and do not affect monocots, such as wheat, corn, and rice. Absorption, Distribution, Metabolism, and Elimination Several studies have examined the absorption, distribution, metabolism, and excretion of 2,4-D and 2,4,5-T in animals and humans. Data on both compounds are consistent among species and support the conclusion that absorption of oral or inhaled doses is rapid and complete. Absorption through the skin is much lower but may be increased with the use of sunscreens or alcohol (Brand et al., 2002; Pont et al., 2004). After absorption, 2,4-D and 2,4,5-T are distributed widely in the body but are eliminated quickly, predominantly in unmetabolized form in urine (Sauerhoff et al., 1977). Neither 2,4-D nor 2,4,5-T is metabolized to a great extent in the body although 2,4,5-trichlorophenol and 2,4-dichlorophenol have been identified as trace metabolites in urine. The half-life in humans after single doses of 2,4-D or 2,4,5-T has been estimated to be about 18â23 hours (Gehring et al., 1973; Kohli et al., 1974; Sauerhoff et al., 1977; WHO, 1984). Results of a recent study that examined concentrations of 2,4-D and its metabolites in the urine of herbicide applicators was consistent with 2,4-D urinary half-life esti- mates of 13â40 hours in humans (Hines et al., 2003). Toxicity Profile The toxicity data base on 2,4-D is extensive (http://www.epa.gov/ttn/atw/ hlthef/di-oxyac.html: accessed January 21, 2009), whereas the data available on the toxicity of purified 2,4,5-T, independent of its contamination by TCDD, are sparse. TCDD is much more toxic than 2,4,5-T, and much of the toxicity at-
INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY 83 tributed to 2,4,5-T in early studies was later shown to be caused by the TCDD contaminant. The following summary therefore focuses on 2,4-D toxicity, and information on pure 2,4,5-T is added when it is available. After a single oral dose, 2,4-D is considered to produce moderate acute toxic- ity with an LD50 (dose lethal to 50% of exposed animals) of 375 mg/kg in rats, 370 mg/kg in mice, and from less than 320 to 1,000 mg/kg in guinea pigs. Rats and rabbits have dermal LD50s of 1,500 mg/kg and 1,400 mg/kg, respectively. 2,4,5-T itself also produces moderate acute toxicity, with oral LD50s of 389 mg/kg in mice and 500 mg/kg in rats. Death from acute poisoning with 2,4-D or 2,4,5-T has been attributed to the ability of the chemicals to uncouple oxidative phosphorylation, a vital process used by almost all cells in the body as the pri- mary means of generating energy. After exposure to high doses, death can occur rapidly from multiple organ failure. Studies in rats, cats, and dogs indicate that the central nervous system is the principal target organ for acute 2,4-D toxicity in mammals and suggest that the primary site of action is the cerebral cortex or the reticular formation (Arnold et al., 1991; DÃ©si et al., 1962a,b). Neurotoxicity in humans is the predominant effect of acute inhalation and oral exposure to 2,4-D; symptoms include stiffness of arms and legs, incoordination, lethargy, anorexia, stupor, and coma. 2,4-D is also an irritant of the gastrointestinal tract, causing nausea, vomiting, and diarrhea. Chronic exposure to 2,4-D at relatively high concentrations has been shown to produce a variety of toxic effects, including hepatic and renal toxicity, neuro- toxicity, and hematologic changes. A no-observed-effect level (NOEL) of 2,4-D of 1 mg/kg was identified for renal toxicity in rats (Hazleton Laboratories Amer- ica, 1987). The reproductive toxicity of 2,4-D is limited to reduced survival and decreased growth rates of offspring of mothers fed high doses during pregnancy and was associated with maternal toxicity. However, even at high exposures, 2,4- D did not affect fertility and did not produce teratogenic effects in the offspring. The purity of 2,4,5-T has been shown to influence its reproductive toxicity; TCDD contamination increases its fetotoxic effects and induces teratogenic ef- fects. Immunotoxicity of 2,4-D has been reported in a small number of studies. At high doses that produced clinical toxicity, suppression of the antibody response was observed, whereas other measures of immune function were normal. The immunotoxicity of 2,4,5-T has not been evaluated in laboratory animals. Carcinogenicity The carcinogenicity of 2,4-D or 2,4,5-T has been studied in rats, mice, and dogs after exposure in their food, direct placement in their stomachs, or expo- sure of their skin. All the studies had negative results except one that found an increased incidence of brain tumors in male rats, but not female rats, that received the highest dose of 2,4-D. The occurrence of malignant lymphoma in dogs kept as pets was reported to be higher when owners reported that they used 2,4-D on their
84 VETERANS AND AGENT ORANGE: UPDATE 2008 lawns than when they did not (Hayes et al., 1991, 1995), but detailed reanalysis did not confirm this finding (Kaneene and Miller, 1999). A controlled study using dogs exposed to 2,4-D in the laboratory had negative results. Timchalk (2004) suggested that dogs are not relevant for comparative evaluation of human health risk attributable to 2,4-D exposure, because they excrete 2,4-D less efficiently than rats or humans. 2,4-D is not metabolized to reactive intermediates capable of interacting with DNA, and the evidence supports the conclusion that 2,4-D is not a carcinogen. CACODYLIC ACID Chemistry Arsenic (As) is a naturally occurring element that exists in trivalent form (As+3 or AsIII) and pentavalent form (As+5 or AsV). The AsIII in sodium arsenite is generally considered to be the most toxicâsee Figure 4-3 for chemical struc- FIGURE 4-3â Structure of selected arsenic-containing compounds. Figure 4-3.eps
INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY 85 tures of selected arsenic-containing compounds. Arsenic is commonly present in drinking-water sources associated with volcanic soils and can reach high concentrations (over 50 ppb). Numerous human health effects have been attrib- uted to drinking-water exposure, particularly bladder, skin, and lung cancers and vascular diseases. Arsenic exists in both inorganic and organic (methylated) forms and is read- ily metabolized in humans and other species. Inorganic arsenic can be converted to organic forms, but organic forms cannot be converted into inorganic forms (Cohen et al., 2006). Cacodylic acid has a valence of +5 and is commonly referred to as dimethylarsinic acid (DMAV). Cacodylic acid, disodium methanearsonate, and monosodium methanearsonate are herbicides that EPA-approved for use in the United States, where they are occasionally applied on golf courses and large open spaces. Cacodylic acid was the form of arsenic used in Agent Blue; DMAV made up about 30% of Agent Blue, one of the mixtures used for defoliation in Vietnam. Agent Blue was chemically and toxicologically unrelated to Agent Or- ange, which consisted of phenoxy herbicides contaminated with DLCs. As shown in Figure 4-4, DMA and monomethyl arsonic acid (MMA) are metabolic products of exposure to inorganic arsenic. Methylation of inorganic arsenic used to be considered a detoxification process associated with increased excretion (Vahter and Concha, 2001). More recently, however, some of the methylated metabolic intermediates have been thought to be more toxic than the parent compound. The MMAIII iAsV Limited MMAV cellular uptake DMAV DMAs 60â80% of human urinary excretion iAsIII iAsIII DMAIII Extensive TMA cellular uptake TMAO TMAO MMAs None found in 10â20% of human human urine, urinary excretion 5â10% of rat urinary excretion FIGURE 4-4â General pathways of arsenic metabolism after exposure to inorganic arsenic (iAs). SOURCE: Adapted with permission from Cohen et al., 2006. Figure 4-4.eps
86 VETERANS AND AGENT ORANGE: UPDATE 2008 methylation pathway of inorganic arsenic results in the formation of pentavalent DMA (DMAV) and trivalent DMA (DMAIII). The committee contemplated the relevance to DMA of data on exposure to inorganic arsenic. Although inorganic arsenic is a human carcinogen, there is no evidence that direct exposure to DMA produces cancer in humans. DMA also is not demethylated to inorganic arsenic (Vahter et al., 1984). It has not been established, nor can it be inferred, that the observed effects of exposure to inorganic arsenic are also caused by exposure to DMA. Therefore, the literature on inorganic arsenic is not considered in this report. The reader is referred to Arsenic in Drinking Water (NRC, 1999a) and Arsenic in Drinking Water: 2001 Update (NRC, 2001). Toxicokinetics The metabolism and disposition of DMAV has recently been reviewed (Cohen et al., 2006). In general, DMAV is rapidly excreted mostly unchanged in the urine of most animal species after systemic exposure. However, rats are unique in that a small percentage (10%) of DMAV binds to hemoglobin in red blood cells and leads to a longer half-life in blood (Cui et al., 2004; Suzuki et al., 2004). Rat he- moglobin is unique in its binding of DMAV in that its binding affinity to DMAV is 10 times higher than that of human hemoglobin (Lu et al., 2004). Chronic expo- sure of normal rat hepatocytes to DMAV resulted in reduced uptake over time and in acquired cytotoxic tolerance (Kojima et al., 2006); the tolerance was mediated by induction of glutathione-S-transferase activity and of multiple-drugâresistant protein expression. Adair et al. (2007) recently examined the tissue distribution of DMA in rats after dietary exposures for 14 days; they found that it was extensively metabolized to trimethylated forms that may play a role in toxicity. Recently, a physiologically based pharmacokinetic model for intravenous and ingested DMAV has been developed based on mouse data (Evans et al., 2008). Similar models have been developed for humans on the basis of exposure to inor- ganic arsenic (El-Masri and Kenyon, 2008), but these models have limited utility in considering the toxicity of DMAV exposures that are relevant to veterans. Toxicity Profile This section discusses the toxicity associated with organic forms of arsenic, most notably DMAV because this is the active ingredient in Agent Blue. The toxicity of inorganic arsenic is not considered relevant to veteran exposures to Agent Blue. Neurotoxicity Kruger et al. (2006) found that DMAIII and DMAV significantly attenu- ated neuronal ion currents through N-methyl-D-aspartate receptor ion channels
INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY 87 whereas only DMAV inhibited ion currents through Î±-amino-3-hydroxy-5- methylisoxazole-4-propionic acid receptors. The data suggest that those methyl- ated forms of arsenic may have neurotoxic potential. Immunotoxicity Previous studies have shown that a low concentration of DMAV (10â7 M) could increase proliferation of human peripheral blood monocytes after their stim- ulation with phytohemagglutinin whereas it took a high concentration (10 â4 M) to inhibit release of interferon-Î³; this suggested that immunomodulatory effects of DMAV are concentration-specific (Di Giampaolo et al., 2004). Genotoxicity and Carcinogenicity Cancer has been induced in the urinary bladder, kidneys, liver, thyroid glands, and lungs of laboratory animals exposed to high concentrations of DMA (Wei et al., 2002). Exposure to DMAV resulted in necrosis of the urinary bladder epithelium followed by regenerative hyperplasia (Cohen et al., 2002). DMAIII was considerably more potent than DMAV in inducing DNA dam- age in Chinese hamster ovary cells (Dopp et al., 2004), and this was associated with a 10% uptake of DMAIII into the cells compared with 0.03% uptake of DMAV. An additional study showed that DMAV is poorly membrane-permeable, but when forced into cells by electroporation it can induce DNA damage (Dopp et al., 2005). Furthermore, DMAV induced protein-DNA adducts in lung fi- broblast cells (MRC-5) (Mouron et al., 2005) and transformation loci in 3T3 fibroblasts following subsequent treatment with the tumor promoter 12-O- tetradecanoylphorbol-13-acetate (TPA) (Tsuchiya et al., 2005). However, DMAV was devoid of promotion activity in 3T3 fibroblasts when cells were pretreated with 3-methylcholanthrene or sodium arsenite. DMAV, but not inorganic arsenic species, exhibited genotoxicity in Drosophila as assessed with the somatic mutation and recombination test (Rizki et al., 2006). Drosophila lacks the ability to methylate arsenic, so the data suggest that arsenic biomethylation is a key determinant of arsenic genotoxicity. DMAIII and DMAV have been shown to induce DNA damage by increasing oxidative stress. Chronic exposure of ddY mice to DMAV at 400 ppm in drinking water increased staining for 4-hydroxy-2-nonenal adducts, which are indicative of oxidative stress, and for 8-oxo-2â²-deoxyguanosine (8-oxodG), reactive oxygen- speciesâinduced DNA damage, in Clara cells of the lung (An et al., 2005). Gomez et al. (2005) demonstrated that DMAIII induced a dose-related increase in DNA damage and oxidative stress in Jurkat cells. Two studies investigated the degree to which oxidative stress may mediate DMAV cytotoxicity. In one study, an antioxidant (N-acetylcysteine, vitamin C, or melatonin) and DMAV at 100 ppm were coadministered to F344 rats for 10 weeks
88 VETERANS AND AGENT ORANGE: UPDATE 2008 (Wei et al., 2005). N-Acetylcysteine inhibited DMAV-induced proliferation of the urinary bladder epithelium whereas neither vitamin C nor melatonin had an ef- fect; this suggested that oxidative stress may mediate the cytotoxic process in the urothelium. In the second, metallothionein wild-type and null mice were exposed to a single oral dose of DMAV at 0, 188, 375, or 750 mg/kg (Jia et al., 2004). DMAV induced a dose-dependent increase in metallothionein in the livers of wild-type mice, but metallothionein was undetectable and uninducible in the null mice. At 24 hours after exposure, DMAV induced dose-dependent DNA adducts, DNA strand breaks, and pulmonary and bladder apoptosis in both genotypes, but the incidence of damage was significantly higher in the null mice. Those results suggest that metallothionein may play a protective role against DMAV-induced DNA damage. Gene-expression profiling of bladder urothelium after chronic exposure to DMAV in drinking water showed significant increases in genes that regulate apop- tosis, the cell cycle, and oxidative stress (Sen et al., 2005). Furthermore, doses that were nontoxic, according to a lack of histologic and ultrastructural changes, could be distinguished from toxic doses on the basis of the expression of a subset of genes involved in control of cell signaling and the stress response, such as thioredoxin E-cadherin. Xie et al. (2004) administered DMAV at 1,000 ppm in drinking water to v-Ha-ras transgenic mice for 17 weeks and after 4 weeks of treatment applied TPA to the skin twice a week. The results were an initial 10% body-weight loss, a cumulative mortality of 20%, hepatic arsenic accumulation, hepatocellular degeneration and foci of inflammation without evidence of hepatic tumors, and hepatic DNA hypomethylation. Hepatic gene-expression profiling showed that DMAV exposure induced changes consistent with oxidative stress, including induction of heme oxygenase, NAD(P)H:quinone oxidoreductase, and glutathione-S-transferase. Mizoi et al. (2005) found that chronic administration of DMAV at 400 ppm to mice after their initiation with 4-nitroquinolone 1-oxide (4NQO) significantly increased the number of lung tumors and the percentage of mice that had lung tumors. DMAV also significantly increased pulmonary 8-oxodG adducts regard- less of whether the mice had been treated with 4NQO. Hairless mice treated with DMAIII on the skin after initiation with dimethylbenz[a]anthracene exhibited a significant increase in epidermal 8-oxodG adducts and skin tumors. In a 2-year bioassay, F344 rats were exposed to DMAV at 0, 2, 10, 40, or 100 ppm in drinking water, and C57BL/6 mice were exposed at 0, 8, 40, 200, or 500 ppm (Arnold et al., 2006). The rats developed epithelial carcinomas and papillomas in the urinary bladder and nonneoplastic changes in the kidneys. The mice failed to develop any tumors but exhibited glomerular nephropathy, nephrocalcinosis, and vacuolation of the urinary epithelium. The murine NOEL based on nonneoplastic changes was 40 ppm in males and 8 ppm in females; the rat NOEL based on neoplastic and nonneoplastic changes was 10 ppm in both sexes. In a recent study, Cohen et al. (2007) exposed F344 rats to DMAV at
INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY 89 2â100 ppm in the diet for 2 years and found an increase in bladder tumors; they postulated that trimethylated forms of arsenic may be responsible for bladder cancer in rats. Similar findings have not been reported in other species. In light of the significant differences in metabolism of arsenic by different species and the lack of supportive data in humans, it cannot be concluded that DMAV leads to an increase in cancer risk in humans. Mechanisms Oxidative stress is a common theme that runs through the literature on the mechanisms of action of arsenic, particularly with regard to cancer in animals, although some studies have suggested that methylated arsenicals (MMAIII and DMAIII) can induce mutations in mammalian cells at concentrations below those required to produce oxidative stress after in vitro exposures (Klein et al., 2008). Recent studies have shown that mice deficient in DNA-repair enzymes associated with oxidative stress are highly susceptible to formation of tumors, particularly lung tumors, induced by DMAV (Kinoshita et al., 2007). The chemical reaction of arsenicals with thiol groups in sensitive target tissues, such as red blood cells and kidneys, may also be a mechanism of action of organic arsenicals (Naranmandura and Suzuki, 2008). The variation in the susceptibility of various animal species to tumor for- mation caused by inorganic and organic arsenic is thought to depend heavily on differences in metabolism and distribution. Thus, genetic differences may play an important role. Numerous investigators are examining potential human susceptibility factors and gene polymorphisms that may increase a personâs risk of cancer and other diseases induced by arsenicals. Several such studies were reported during the update period covered by this update (Hernandez et al., 2008; Huang SK et al., 2008; Huang YK et al., 2008; McCarty et al., 2007; Meza et al., 2007; Steinmaus et al., 2007). However, the studies are in early stages, and it is not possible to identify polymorphisms that may contribute to a personâs suscep- tibility to DMA-induced cancer or tissue injury. PICLORAM Chemistry Picloram (4-amino-3,5,6-trichloropyridine-2-carboxylic acid or 4-amino- 3,5,6-trichloropicolinic acid; see chemical structure in Figure 4-5) was used with 2,4-D in the herbicide formulation Agent White, which was sprayed in Vietnam. Picloram is also used commonly in Australia in a formulation with the trade name Tordon 75DÂ®. Tordon 75D contains several chemicals, including 2,4-D, picloram, a surfactant diethyleneglycolmonoethyl ether, and a silicone defoamer. A number
90 VETERANS AND AGENT ORANGE: UPDATE 2008 Picloram [1918-02-1] CI NH 2 HO CI O N CI FIGURE 4-5â Structure of picloram. Figure 4-5.eps of studies of picloram used such mixtures as Tordon or other mixtures of 2,4-D and picloram that are similar to Agent White. Toxicokinetics The original VAO committee reviewed studies of the toxicokinetics of pi- cloram. Studies of animals showed rapid absorption through the gastrointestinal tract and rapid elimination of picloram as the unaltered parent compound in urine. Nolan et al. (1984) examined the toxicokinetics of picloram in six healthy male volunteers who were given single oral doses of 0.5 or 5.0 mg/kg and a dermal dose of 2.0 mg/kg. Picloram was rapidly absorbed in the gavage study and rapidly excreted as unchanged compound in urine. More than 75% of the dose was excreted within 6 hours, and the remainder with an average half-life of 27 hours. On the basis of the quantity of picloram excreted in urine in the skin study, the authors noted that only 0.2% of the picloram applied to the skin was absorbed. Because of its rapid excretion, picloram has low potential to accumu- late in humans. In general, the literature on picloram toxicity continues to be sparse. Studies of humans and animals indicate that picloram is rapidly eliminated as the parent compound. Studies of animals have indicated that picloram is sparingly toxic at high doses. Toxicity Profile The original VAO committee reviewed studies of the carcinogenicity, geno- toxicity, acute toxicity, chronic systemic toxicity, reproductive and developmental toxicity, and immunotoxicity of picloram. In general, there is limited evidence for cancer in some rodent models but not in other species (NCI, 1978). In those studies, there was some concern that contaminants in the picloram (in particular, hexachlorobenzene) might be responsible for the carcinogenicity. Therefore, picloram has not been established as a chemical carcinogen. There is also no evidence that picloram is a genotoxic agent on the basis
INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY 91 of studies conducted by EPA (1988c). Picloram is considered a mild irritant; erythema is seen in rabbits only at high doses. The available information on the acute toxicity of picloram is also paltry. Some neurologic effectsâincluding hyperactivity, ataxia, and tremorsâwere reported in pregnant rats exposed to picloram at 750 or 1,000 mg/kg (Thompson et al., 1972). Chronic Systemic Toxicity Several studies have reported various effects of technical-grade picloram on the livers of rats. In the carcinogenicity bioassay conducted by Stott and col- leagues (1990) described above, treatment-related hepatomegaly, hepatocellular swelling, and altered tinctorial properties in the central regions of the liver lobules were noted in the groups exposed at 60 and 200 mg/kg per day. In addition, males and females exposed at the high dose had higher liver weights than controls. The NOEL was 20 mg/kg per day, and the lowest effect level was 60 mg/kg per day for histologic changes in centrilobular hepatocellular tissues. According to EPA, hexachlorobenzene (at 197 ppm) was probably not responsible for the hepatic ef- fects (EPA, 1988c). Gorzinski and colleagues (1987) also reported a dose-related increase in liver weights, hepatocellular hypertrophy, and changes in centrilobular tinctorial properties in male and female F344 rats exposed to picloram at 150 mg/kg per day and higher in the diet for 13 weeks. In a 90-day study, cloudy swelling in the liver cells and bile duct epithelium occurred in male and female F344 rats given 0.3% or 1% technical picloram in the diet (EPA, 1988c). Hepatic effects have also been reported in dogs exposed to picloram: increased liver weights were reported in beagles that received 35 mg/kg per day or more in the diet for 6 months (EPA, 1988c). No other effects of chronic exposure to picloram have been reported. Reproductive and Developmental Toxicity The reproductive toxicity of picloram was evaluated in a two-generation study; however, too few animals were evaluated, and no toxicity was detected at the highest dose tested, 150 mg/kg per day (EPA, 1988c). Some developmental toxicity was produced in rabbits exposed to picloram by gavage at 400 mg/kg per day on days 6â18 of gestation. Fetal abnormalities included single-litter inci- dences of forelimb flexure, fused ribs, hypoplastic tail, and omphalocele (John- Greene et al., 1985). Some maternal toxicity was observed at that dose, however, and EPA concluded on the basis of the low-litter incidence of the findings, that the malformations were not treatment-related (EPA, 1988c). No teratogenic effects were produced in the offspring of rats given picloram by gavage at up to 1,000 mg/kg per day on days 6â15 of gestation, although the occurrence of bilateral accessory ribs was significantly increased (Thompson et al., 1972).
92 VETERANS AND AGENT ORANGE: UPDATE 2008 Immunotoxicity Studies of the potential immunotoxicity of picloram included dermal sen- sitization and rodent immunoassays. In one study, 53 volunteers received nine 24-hour applications of 0.5 mL of a 2% potassium picloram solution on the skin of both upper arms. Each volunteer received challenge doses 17â24 days later. The formulation of picloram (its potassium salt) was not a skin sensitizer or an irritant (EPA, 1988c). In a similar study, a 5% solution of picloram (M-2439, Tordon 101 formulation) produced slight dermal irritation and a sensitization response in 6 of the 69 volunteers exposed. When the individual components of M-2439âpicloram, triisopropanolamine (TIPA) salt, and 2,4-D TIPA saltâwere tested separately, no sensitization reaction occurred (EPA, 1988c). Tordon K+, but not technical-grade picloram, was also found to be a skin sensitizer in guinea pigs (EPA, 1988c). CD1 mice exposed to Tordon 202C (94% 2,4-D and 6% piclo- ram) had no consistent adverse effects on antibody responses (Blakley, 1997). Mechanisms There are no well-characterized mechanisms of toxicity known for picloram, and therefore they are not discussed here. REFERENCES Adachi J, Mori Y, Matsui S, Takigami H, Fujino J, Kitagawa H, Miller C III, Kato T, Saeki K, Matsuda T. 2001. Indirubin and indigo are potent aryl hydrocarbon receptor ligands present in human urine. Journal of Biological Chemistry 276:31475â31478. Adachi-Yamada T, Harumoto T, Sakurai K, Ueda R, Saigo K, OâConnor MB, Nakato H. 2005. Wing- to-leg homeosis by spineless causes apoptosis regulated by fish-lips, a novel leucine-rich repeat transmembrane protein. Molecular & Cellular Biology 25(8):3140â3150. Adair BM, Moore T, Conklin SD, Creed JT, Wolf DC, Thomas DJ. 2007. Tissue distribution and uri- nary excretion of dimethylated arsenic and its metabolites in dimethylarsinic acid- or arsenate- treated rats. Toxicology and Applied Pharmacology 222(2):235â242. An Y, Kato K, Nakano M, Otsu H, Okada S, Yamanaka K. 2005. Specific induction of oxidative stress in terminal bronchiolar Clara cells during dimethylarsenic-induced lung tumor promoting process in mice. Cancer Letters 230(1):57â64. Arnold EK, Beasley VR, Parker AJ, Stedelin JR. 1991. 2,4-D toxicosis II: A pilot study of clinical pathologic and electroencephalographic effects and residues of 2,4-D in orally dosed dogs. Veterinary and Human Toxicology 33:446â449. Arnold LL, Eldan M, Nyska A, van Gemert M, Cohen SM. 2006. Dimethylarsinic acid: Results of chronic toxicity/oncogenicity studies in F344 rats and in B6C3F1 mice. Toxicology 223(1-2): 82â100. â Throughout the report the same alphabetic indicator following year of publication is used con- sistently for the same article when there were multiple citations by the same first author in a given year. The convention of assigning the alphabetic indicator in order of citation in a given chapter is not followed.
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