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6
Relationship of Mustard Agent and Lewisite Exposure to Carcinogenesis

There is substantial evidence that some chemical exposures can cause cancer in human tissues. Not all of the mechanisms by which chemicals cause cancer have been delineated, but it is clear that agents that change the genetic memory of the cells—their DNA—are prime candidates for causing cancer. Clearly, not all chemicals are capable of causing cancer, and not all chemicals that cause cancer in experimental animals have been demonstrated to do so in studies of humans. In some cases, a large enough group has not been studied; in others, too little time has elapsed after exposure for the expression of cancer, or the exposure was not at a high enough level to discern the added cancers against the background level of cancer that occurs naturally or due to some other exposure, such as cigarette smoking or occupational exposure to asbestos.

There are several bodies of human data upon which to form a judgment of whether an agent is a carcinogen. The first involves biologic mechanisms: essentially, if a chemical acts in a fashion parallel to a known human carcinogen, it is evidence for a conclusion that the chemical is itself a carcinogen. The second involves evidence of an adverse effect in human cells grown and exposed in the laboratory. The third line of evidence is epidemiologic studies in human populations exposed to the agent in question for some other reason, often because the exposed individuals were involved in its manufacture. Human epidemiologic information about the carcinogenicity of war gas comes from epidemiologic studies of workers exposed in its manufacture, soldiers exposed on the battlefield, and patients exposed to the agents when used for therapeutic purposes—ironically, usually to fight cancer.

In considering the potential carcinogenicity of a chemical, it is



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Veterans at Risk: The Health Effects of Mustard Gas and Lewisite 6 Relationship of Mustard Agent and Lewisite Exposure to Carcinogenesis There is substantial evidence that some chemical exposures can cause cancer in human tissues. Not all of the mechanisms by which chemicals cause cancer have been delineated, but it is clear that agents that change the genetic memory of the cells—their DNA—are prime candidates for causing cancer. Clearly, not all chemicals are capable of causing cancer, and not all chemicals that cause cancer in experimental animals have been demonstrated to do so in studies of humans. In some cases, a large enough group has not been studied; in others, too little time has elapsed after exposure for the expression of cancer, or the exposure was not at a high enough level to discern the added cancers against the background level of cancer that occurs naturally or due to some other exposure, such as cigarette smoking or occupational exposure to asbestos. There are several bodies of human data upon which to form a judgment of whether an agent is a carcinogen. The first involves biologic mechanisms: essentially, if a chemical acts in a fashion parallel to a known human carcinogen, it is evidence for a conclusion that the chemical is itself a carcinogen. The second involves evidence of an adverse effect in human cells grown and exposed in the laboratory. The third line of evidence is epidemiologic studies in human populations exposed to the agent in question for some other reason, often because the exposed individuals were involved in its manufacture. Human epidemiologic information about the carcinogenicity of war gas comes from epidemiologic studies of workers exposed in its manufacture, soldiers exposed on the battlefield, and patients exposed to the agents when used for therapeutic purposes—ironically, usually to fight cancer. In considering the potential carcinogenicity of a chemical, it is

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Veterans at Risk: The Health Effects of Mustard Gas and Lewisite important to acknowledge a dose-response relationship—that is, the more exposure, the more effect. This is important for two reasons:  If a study is small enough, or if the level of exposure in the study is low enough, the study may not detect a chemical-cancer association, even if it is present.  Even if a chemical is a carcinogen at a high level of exposure, it may only rarely cause cancer at low levels of exposure. Two further questions must therefore be addressed in order to make a contribution to the well-being of surviving experimental subjects exposed to chemical war agents. The first is whether the specific agents are likely to be carcinogens. The second is whether, at the level of exposure experienced by these subjects, the added risk that they carry is small or large. ACUTE EFFECTS AND BIOLOGICAL MECHANISMS Sulfur Mustard Sulfur mustard has been produced primarily for its acute toxic effects. Concern has been raised, however, about the long-term health effects of exposure to sulfur mustard in humans. Because the various sulfur mustards are known to be animal carcinogens, much of this concern has centered around their potential carcinogenicity to humans. One part of the process of assessing the carcinogenic risk involves examination of the biologic fate of this compound, its potential genotoxicity, and its ability to induce mutations in living systems. Biologic Fate and Mechanisms of Action After absorption, sulfur mustard undergoes intermolecular cyclization to form an ethylene episulfonium ion intermediate, liberating a free chloride anion. This process is facilitated by heat and by water, a likely explanation for the vulnerability of the warm and moist regions of the body (mucous membranes, eyes, respiratory tract, etc.) to the acute toxic effects of this compound (Somani and Babu, 1989; Ward and Seider, 1984). Cyclization can occur on both ends of the molecule. The cyclic intermediate reacts rapidly with a variety of nucleophiles, according to the affinity of neighboring compounds for the reaction. In pure aqueous media, most sulfur mustard is hydrolyzed to thiodiglycol and hydrochloric acid. Boursnell and colleagues (1946) have shown that 35S-labeled sulfur mustard diffused rapidly throughout the body after intravenous (IV) injection in experiments employing rabbits. Activity was retained chiefly

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Veterans at Risk: The Health Effects of Mustard Gas and Lewisite in the liver, lungs, and kidneys, with approximately 20 percent of the 35S activity being excreted in 12 hours. In rodents the majority of IV-injected sulfur mustard was excreted in the urine within 72 hours (Davison et al., 1961). The urinary metabolites included thiodiglycol and its conjugate (15 percent), glutamine-bis(b-chloroethylsulfide) conjugates (45 percent), glutamine-bis(b-chloroethylsulfone) conjugates (7 percent), and bis(b-chloroethysulfone) and conjugate (8 percent), with minute amounts of cysteine conjugates. These findings are comparable to subsequent work in rodents after intraperitoneal injection (Roberts and Warwick, 1963). Nucleic Acid and Protein Conjugation The reactive cyclic intermediate, the sulfonium ion, reacts avidly with proteins and nucleic acids, producing alkylation products (see Chapter 5). The alkylation of DNA by sulfur mustards has been studied by many investigators (Ball and Roberts, 1972; Boursnell et al., 1946; Davison et al., 1957, 1961; Gross et al., 1981; Habraken and Ludlum, 1989; Kohn et al., 1965; Lawley and Brookes, 1965; Ludlum et al., 1986; Meier et al., 1984; Papirmeister and Davison, 1964; Papirmeister et al., 1969, 1970, 1984a,b; Price et al., 1968; Roberts et al., 1971; Walker, 1971; Wheeler, 1962). The sulfur mustards can be bifunctional, in that some ion intermediates covalently bind adjacent strands of DNA (a DNA cross-link). This interstrand link has been the subject of much of the study of the genotoxic effect of these agents. DNA cross-links induced by these mustards were shown by Wheeler (1962) to be extremely lethal to cells. Several workers also studied the cell cycle-specific toxicity of this bifunctional agent (Ludlum et al., 1978; Mauro and Elkind, 1968; Roberts et al., 1968, 1986). They have shown that cells in late G 1 phase or S phase of the cell cycle are particularly sensitive to the biologic effects of alkylation. In addition, the repair of DNA lesions induced by sulfur mustards has been studied in many systems, including those employing cells known to be naturally deficient in certain repair enzymes (Ball and Roberts, 1970; Fox and Fox, 1973; Gilbert et al., 1975; Lawley and Brookes, 1968; Murnane and Byfield, 1981; Plant and Roberts, 1971; Reid and Walker, 1966, 1969; Roberts and Kotsaki-Kovatsi, 1986; Roberts et al., 1986; Savage and Breckon, 1981; Walker, 1966; Walker and Reid, 1971; Walker and Smith, 1969). As expected, DNA repair-deficient cells generally are much more sensitive to the DNA cross-linking, and the cells die at significantly lower doses. Recent work has specifically shown that ring nitrogens on DNA are the primary sites of attack. Among the products identified are N-7

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Veterans at Risk: The Health Effects of Mustard Gas and Lewisite alkylguanine and N-3 alkyladenine. Intrastrand and opposite cross-links have been identified at N-7 guanine (Ball and Roberts, 1972; Ludlum et al., 1986; Walker, 1971; Wheeler, 1962). Sulfur mustards also alkylate the 0-6 position of guanine in DNA (Habraken and Ludlum, 1989; Ludlum et al., 1984, 1986). Interestingly, the well-understood DNA repair enzyme O-6-alkylguanine-DNA alkyltransferase does not act to repair these O-6 lesions (Ludlum et al., 1986). Therefore, the 0-6 alkyl product in DNA may be the major mutagenic lesion produced by the sulfur mustards. Cytogenetic and Mutagenic Effects Sulfur mustard induces chromosome aberrations—gross structure breaks visible under light—in a variety of cell systems. In fact, sulfur mustard was the first compound reported to induce chromosome abnormalities in the fruit fly Drosophila melanogaster (Auerbach, 1943). Subsequent work has shown that this type of genetic damage is dose related and that the spectrum of genetic change is similar to that of X-rays, in that it is cell cycle-specific (Nasrat, 1954; Nasrat et al., 1954; Sobels and van Steenis, 1957). Subsequent studies have generalized the data to demonstrate that sulfur mustard induces chromosome aberrations in Vicia faba (fava bean) and marsupial lymphocytes (Popa, 1969; Scott and Bigger, 1972). Further, when cell lines have been studied, cytogenetic (chromosome) sensitivity to sulfur mustard has paralleled that of X-rays (Scott et al., 1974). Interestingly, fishermen exposed to sulfur mustard through netting of leaky barrels of mustard agents dumped at sea after World War II (WWII) have been found to have elevated sister chromatid exchange (SCE) frequencies in their peripheral blood lymphocytes (Wulf et al., 1985). SCEs represent breakage and rejoining of chromosomes. DNA alkylation is well known to be associated, in other circumstances, with induction of SCEs in human cells. Sulfur mustards are also mutagens, inducing heritable alterations in dividing cells. They have been shown to induce mutations in Drosophila (Auerbach, 1951; Auerbach and Robson, 1946, 1947; Auerbach and Sonbati, 1960; Fahmy and Fahmy, 1972; Lee, 1975; Luening, 1951; Sobels, 1962; Sonbati and Auerbach, 1960); in L5178Y mouse lymphoma cells (Capizzi et al., 1973); in the red bread mold Neurospora crassa (Auerbach and Moser, 1950; Jensen et al., 1950; Stevens and Mylroie, 1950); and in the bacteria Salmonella (Ashby et al., 1991). The potency of sulfur mustard in most of these systems is comparable to X-rays. The compound clearly induces somatic mutations in exposed cells in a dose-related fashion. One study has also demonstrated that occupational exposure to sulfur mustard and Lewisite (manufactured in com-

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Veterans at Risk: The Health Effects of Mustard Gas and Lewisite bination) induces mutations in vivo in human lymphocytes at the hypoxanthine phosphoribosyltransferase (hprt) enzyme gene locus (Yanagida et al., 1988). Summary Mustard agents are mono- and bifunctional DNA-alkylating agents that are extremely cytotoxic at low doses. They alkylate RNA and proteins and produce DNA lesions, which may be repaired only at low doses. Sulfur mustards also alkylate the 0-6 position of guanine; this lesion is likely primarily responsible for the mutagenic consequence of cellular exposure. The sulfur mustards are genotoxic in a wide variety of cells, producing chromosome aberrations and gene mutations in vitro in a dose-related fashion. They also induce SCE and hprt mutations in vivo in the peripheral blood lymphocytes of individuals exposed at low doses. Lewisite There are limited data in the literature on the genetic toxicology of Lewisite. Although data on many types of arsenical compounds have demonstrated significant genotoxic potential, data on Lewisite are incomplete. Biologic Fate and Mechanism of Action Lewisite undergoes a complex hydrolysis involving several reversible reactions. Lewisite oxide (Cl-CH=CH-AsO) is approximately 1 percent soluble in water and 2 percent soluble in a saline solution. It is slightly more soluble at an alkaline pH. At higher pH, Lewisite oxide is cleaved by hydroxyl ions to yield arsenite and acetylene. Lewisite has labile chlorine atoms, trivalent arsenic, and multiple bonds of carbon atoms. It is a very reactive compound. It will undergo rapid nucleophilic substitution by water. In the presence of hydrogen sulfide it forms 2-chlorovinylarsine, an extremely irritating compound. Lewisite also reacts rapidly with mercaptans to form alkylarsine. Lewisite penetrates skin rapidly on contact. It binds avidly to proteins and thiols, and the mechanism of its local and systemic toxicity is likely mediated through this binding. It is concentrated in the thiol-containing tissues throughout the body (e.g., skin and hair). The toxicity of Lewisite is reversed by administration of the dithiol compound BAL, British Anti-Lewisite, or other thiol-containing compounds. The precise chemical nature of any of the genetic lesions (DNA-based lesions) induced by cellular exposure to Lewisite appears to be unknown.

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Veterans at Risk: The Health Effects of Mustard Gas and Lewisite Cytogenetic and Mutagenic Effects Little in vitro genetic toxicology testing appears to have been done on Lewisite. Jostes and colleagues (1989) have completed the most extensive study in Chinese hamster ovary (CHO) cells. They found that Lewisite was cytotoxic after a one-hour exposure to micromolar amounts. Cell survival experiments yielded a D37 of 0.6 µM with an extrapolation number of 2.5. Interestingly, at the dose ranges of 0.125 to 2.0 µM no consistent significant induction of mutations at the hprt gene locus was observed. At doses of 0.25 to 1 µM, Jostes and colleagues noted no significant induction of SCEs in CHO cells, although the dose-response trend was toward a positive response. Lewisite did significantly induce chromosomal aberrations at doses of 0.5 and 0.75 µM, with a definite positive dose-response. Stewart and colleagues (1989) tested the mutagenicity of Lewisite in the Ames Salmonella assay. Four Salmonella strains were tested with and without S9 microsomal activation. This compound (S9) is used in this bioassay system to activate the agent of interest to its biologic intermediates. These intermediates are often the bioactive species and are the compounds of real interest. The strain most sensitive to killing was TA 102. No mutagenic response was observed in any strain with or without S9 activation. No other data evaluating the genotoxicity of Lewisite appear to exist, but there have been studies of the genotoxicity of other arsenicals. Jacobson-Kram and Montalba (1985) have shown that inorganic arsenic induces both chromosome aberrations and SCEs in mammalian cells. Arsenite enhances ultraviolet light (UV) mutagenicity in bacteria (Ross-man, 1981) and viral transformation in mammalian cells (Casto et al., 1979). Arsenite synergistically enhances cis-platinum (a DNA-alkylating agent) and UV-plus-psoralen induced chromosome aberrations (Lee et al., 1986a,b). Using sulfur dioxide and arsenite, Beckman and Nordenson (1986) noted no enhanced induction of SCEs. Recent work has also shown that arsenic will induce gene amplification (an increase in the number of copies of an actively transcribed gene) in mouse cells in culture (Barrett et al., 1989; Lee et al., 1988). Arsenic exposure in vivo also has been reported to induce chromosome aberrations in human lymphocytes (Nordenson et al., 1978). Arsenite reacts avidly with protein sulfhydryl groups. Relatively recent work has shown that arsenite is highly selective in reacting with small, closely spaced dithiol groups in proteins (Joshi and Hughes, 1981; Knowles and Benson, 1983). Dexamethasone binding to glucocorticoid receptors is inhibited by arsenite via a putative mechanism involving the formation of a stable dithioarsenite complex with a single dithiol group within the binding domain of the receptor (Lopez et al., 1990). Arsenite also blocks DNA binding by the receptor, presumably via a similar

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Veterans at Risk: The Health Effects of Mustard Gas and Lewisite mechanism within the DNA-binding domain of the relevant protein (Simons et al., 1990). Recent work of Wiencke and Yager (1992) hypothesized that arsenite might interact with DNA repair proteins that are known to contain so-called zinc fingers. These zinc fingers, which contain closely spaced dithiols, are likely important in gene regulation. Proteins that contain zinc fingers include the UVRA protein (Husain et al., 1986), polyadenosine diphosphoribose polymerase (Cherney et al., 1987; Uchida et al., 1987), the RAD-18 protein (Jones et al., 1988), and the XPAC protein (Tanaka et al., 1990). All of these are proteins that are thought to be central to DNA repair of genetic lesions. Wiencke and Yager showed that arsenite alone induced both SCEs and chromosome aberrations. However, when the DNA cross-linking agent diepoxybutane (DEB) was added to human lymphocyte culture in the presence of arsenite, the induction of chromosome aberrations was synergistically enhanced. The induction of SCEs was only additively increased. Interestingly, the synergistic enhancement of chromosome aberrations was most pronounced in individual lymphocytes previously known to be relatively more sensitive to the clastogenic action of DEB (Wiencke et al., 1991). Wiencke and Yager concluded that the specific co-clastogenic effects of arsenite were mediated by its interference with DNA repair activities. All of this work may indicate that arsenicals could interact with DNA-alkylating agents when they are given concomitantly. Although there is no direct evidence that the genetic effects of sulfur mustard exposure are enhanced by concomitant Lewisite exposure, it remains a possibility. Summary In contrast to mustard agents, the genetic toxicology of Lewisite has been poorly studied. Its hydrolysis has been examined and arsenite is one significant product likely produced in man after exposure. Lewisite itself clearly induces chromosome aberration in one type of cellular assay. It appears not to be mutagenic in Salmonella. Arsenicals in general and arsenite have been shown to be clastogenic and to induce SCE in human and other mammalian cells. Arsenite synergistically enhances the clastogenic action of alkylating agents, perhaps through binding to DNA repair proteins. EVIDENCE OF LONG-TERM HEALTH EFFECTS Animal Studies This section reviews the results of the published experimental carcinogenesis studies of sulfur mustard and nitrogen mustard. The latter

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Veterans at Risk: The Health Effects of Mustard Gas and Lewisite are included because there is a dearth of experiments on sulfur mustard. Further, the similarity of action of nitrogen mustard to sulfur mustard provides information that is useful in assessing the types and sites of malignancy that may occur from exposure to sulfur mustard. In what follows, in accordance with historical usage, we use the term sulfur mustard, although the terms HS, HD, or H were often used in past experimental literature. HD and HS refer to distilled sulfur mustard (approximately 96 percent pure), while H refers to an impure preparation known as Levinstein mustard. Experiments have been conducted with two nitrogen mustards, HN2 [methylbis(b-chloroethyl)amine or the hydrochloride] and HN3 [tris(b-chloroethyl)amine]. By far the most data are available for HN2, which is a common chemotherapeutic agent, usually in combination with other chemicals (see Medical and Therapeutic Exposure section of this chapter). The designations HN2 and HN3 are used as appropriate. Early Studies in Mice and Rats One of the earliest reports of the carcinogenic effect of the nitrogen mustards is an interim report describing preliminary results on the intravenous, intraperitoneal, and subcutaneous injections of HN2 and HN3 into Swiss mice and albino rats (Griffin et al., 1950). At the time of the report, tumors had developed in 15 to 20 percent of treated animals, whereas no tumors had appeared in controls. The tumors included fibrosarcomas, lymphosarcomas, and adenocarcinomas (no sites or numbers were given), and administration of a single dose proved as effective in inducing tumors as multiple injections. Another early study on the carcinogenicity of mustard compounds was conducted at the Chester Beatty Research Institute in London (Boyland and Horning, 1949). Aqueous solutions of HN2 or HN3 were administered by subcutaneous injection weekly for 50 weeks to two groups of 20 stock mice each. The weekly dose of each substance was 1 mg/kg body weight. This dose was toxic and led to a high early mortality; only 10 animals administered HN2, and 4 animals administered HN3, survived 150 days. These 14 survived from 284 to 580 days. At autopsy, among the 10 injected with HN2, there were 3 lung carcinomas, 1 lung adenoma, and 1 with ''early bronchogenic tumors." One animal had a liver lymphosarcoma and another had a uterine fibromyoma. Three were free of tumors. Among the 4 administered HN3, there were 2 lung carcinomas and 1 lung adenoma. One of the animals with carcinoma also had a spindle-celled sarcoma at the site of injection. Substantial other non- or premalignant pathology was described in all of the treated animals. Of 40 untreated mice sacrificed between 14 and 18 months of age, 6 had lung adenomas and 2

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Veterans at Risk: The Health Effects of Mustard Gas and Lewisite hepatomas. The number of tumors at any site and the number of lung tumors in animals administered HN2 were significantly greater than expected at the 95 percent confidence level. Boyland and Horning noted a particular excess of lung tumors and lymphosarcomas compared to the number expected. Heston's Studies in Mice From 1949 through 1953, W. E. Heston and his colleagues conducted a series of studies in which sulfur mustard and nitrogen mustard were administered to strain A mice and the occurrence of pulmonary tumors was studied (Heston, 1949, 1950, 1953a,b; Heston et al., 1953). Strain A mice are highly inbred and have an extremely high genetic susceptibility for development of pulmonary adenomas; the incidence of spontaneous pulmonary tumors is about 50 percent in animals 12 months of age and 90 percent at 18 months of age. Thus, in several of Heston's experiments all of the animals developed adenomas, and comparisons can be made only between the total number of tumor nodules found in different exposure groups. The administered amounts of nitrogen and sulfur mustards were highly toxic, and a significant number of early deaths of animals occurred. The results of the first two studies (Heston, 1949, 1950) are summarized in Table 6-1. In Experiment I, 100 percent of the experimental animals that survived acute mortality had tumors at sacrifice (13 to 16 weeks), compared to 13 percent of controls (the remaining 8 experimental and 8 control mice were followed longer, but no data were provided on their outcome). The mean number of lung nodules in the experimental group was 3.48, compared with 0.13 in the control group. The 9 animals receiving 4 injections had 5.11 nodules on average, the 13 receiving 3 injections had 2.62 nodules, and the lone animal with 2 injections had 2 nodules. The administered doses of nitrogen mustard clearly increased the number of nodules and number of strain A mice affected. In Experiment II, all experimental animals received four injections and follow-up was continued for 10 months, rather than 3 or 4. The full dosing scheme led to several animals dying prior to completion of the experiment, but the results are in agreement with those of Experiment I, with a greater number of nodules being associated with the longer follow-up period. In Experiment III, a single larger injection of HN2 resulted in higher short-term mortality, but the incidence of tumor nodules in animals that survived 10 months was similar to the group that received the full dose in four injections. Experiments IV and V followed a similar methodology to examine the effects of intravenous injection of sulfur mustard. Experiment IV saw a

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Veterans at Risk: The Health Effects of Mustard Gas and Lewisite TABLE 6-1 Pulmonary Tumors in Strain A  Mice Injected Intravenously with Nitrogen Mustard and Sulfur Mustard Experiment Follow-up Duration No. of Mice % with Nodules Mean No. of Nodules Nitrogen mustard Experiment I: two to four intravenous injections of 1 mg/kg HN2 (14 received 4 injections; 22 received 3; and one, 2) at 2-day intervals 13-16 wks 29 100 3.48 Control 13-16 wks 30 13 0.17 Experiment II: Four intravenous injections of 1 mg/kg HN2 at 2-day intervals 10 mos 20 100 9.60 Control 10 mos 32 63 0.81 Experiment III: One intravenous injection of 4 mg/kg HN2 10 mos 9 100 7.56 Control 10 mos 31 58 0.94 Sulfur mustard Experiment IV: Four intravenous injections of 0.65 mg/kg at 2-day intervals 16 wks 15 93 2.60 Control 16 wks 28 61 0.93 Experiment V: Four intravenous injections of 0.65 mg/kg at 2-day intervals 16 wks 47 69 1.09 Control 16 wks 46 13 0.13   SOURCE: Heston, 1949, 1950. high early mortality: 9 males and 4 females of 30 animals injected died shortly after completion of the injections. The results for the animals surviving 16 weeks showed somewhat fewer mice with lung nodules and a lower mean number of nodules compared to Experiment I with HN2. Further, an unusually high number of controls developed adenomas for which no cause could be identified. When Experiment IV was repeated as Experiment V, by using a lower concentration of sulfur mustard, fewer mice developed lung nodules (68 vs. 93 percent) and the mean number of lung nodules was substantially less (1.09 vs. 2.60). Obviously, there is uncertainty about the actual dose to the animals in the sulfur mustard experiments, particularly in Experiment IV, where the administered dose may have exceeded 1 mg/kg body weight. A later study by Heston and colleagues (1953) compared the separate and combined effects of intravenous HN2 and X-irradiation (Table 6-2). With only two injections of 1 mg/kg HN2, some animals were tumor free

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Veterans at Risk: The Health Effects of Mustard Gas and Lewisite TABLE 6-2 Pulmonary Tumors in Strain A Mice Injected Intravenously with Nitrogen Mustard and/or Exposed to X-Radiation Experiment Follow-up Duration No. of Mice % with Nodules Mean No. of Nodules Control mice 11 mos 64 39 0.7 900 R of X-radiation 11 mos 55 24 0.6 Two injections of 1 mg/kg bw of nitrogen mustard 11 mos 59 93 4.2 900 R of X-radiation plus 2 injections of 1 mg/kg bw of nitrogen musard 11 mos 55 38 1.1   SOURCE: Heston et al., 1953. after 11 months of follow-up. Those receiving the radiation had fewer tumors than controls; those receiving both radiation and HN2 had a tumor frequency similar to controls, but higher than those receiving radiation. The carcinogenicity of sulfur mustard and HN2 was further studied by Heston (1953a), using a variety of subcutaneous injections of sulfur mustard and HN2 in strain A, C3H, or C3Hf mice. (C3Hf female mice are less susceptible to mammary tumors than C3H mice, but C3Hf male mice are more likely to develop hepatomas.) The results, summarized in Table 6-3 for C3H and C3Hf mice and Table 6-4 for strain A mice, show that tumors, including sarcomas, were commonly found at the site of injection: among C3H  and C3Hf mice, 7 of 87 injected with sulfur mustard and 12 of 73 injected with HN2 had injection site tumors. No tumors appear at the site of injection of 16 mice administered olive oil, nor did any tumors occur in noninjected controls in the region chosen for injection. Of the tumors at sites other than the site of injection, only pulmonary nodules from HN2 injections were significantly different from control animals: 34 of 73 injected animals developed pulmonary tumors, compared with 14 of 74 control animals. For all C3H and C3Hf animals, tumors appeared in 8.4 percent injected with sulfur mustard, compared with 15.8 percent of controls. In the strain A mice only 1 tumor was observed in the injection site and there were no differences between the control and experimental group in number of tumors in other sites (Table 6-4). Heston conducted another experiment in which strain A mice were exposed to sulfur mustard by inhalation. It is likely that the concentration of sulfur mustard in the experimental chamber reached relatively high levels during the 15-minute exposure: of a total of 80 mice exposed, 13 died in the 4 months prior to follow-up. Control and experimental mice were sacrificed periodically from 4 to 11 months after exposure. At

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Veterans at Risk: The Health Effects of Mustard Gas and Lewisite served in other studies adds weight to the evidence of the carcinogenicity of sulfur mustard. Battlefield Exposure Jackson and Adams (1973) conducted a case control study to explore the causes of aggressive and disfiguring cases of basal cell carcinoma: 33 cases of "horrifying" basal cell carcinoma were compared with 435 other cases of basal carcinoma. Of interest to this discussion, 2 of the 33 cases had histories of mustard vapor burns. One case, reported in some detail, developed a basal cell carcinoma in the scar on his neck left by sulfur mustard in World War I (WWI). This study does not provide an estimate of the risk to veterans with mustard agent burns of subsequently developing dermatologic cancers. The study nevertheless reveals a possible etiologic association between mustard agent burns and aggressive basal cell carcinoma. Case and Lea (1955) studied a group of 1,267 men who were receiving pensions for the effects of mustard agent poisoning resulting from WWI combat exposures. The mortality experience of this group was compared with expected mortality for males in England and Wales and with the mortality experience of 1,421 other WWI pensioners disabled by bronchitis and another 1,114 pensioners with limb amputations, but without bronchitis, who had not been exposed to sulfur mustard. The results showed that both the cohort exposed to sulfur mustard and the cohort with bronchitis, but not the amputees, had significant excesses of mortality for "all causes" and for "cancer of the lung and pleura." The authors did not feel that these results support the hypothesis that sulfur mustard acts as a direct carcinogen. The major limitation of this study was the lack of information on cigarette smoking. Pension records showed that 81 percent of the exposed cohort were disabled by bronchitis or other pulmonary conditions, but there is no way to determine if their bronchitis is mustard-associated, smoking-associated, or both. It is possible that members of this cohort, like U.S. veterans who were exposed to sulfur mustard (Norman, 1975), smoked less than their peers. Thus, this study provided weak evidence that mustard exposure is not associated with an excess of cancer of the lung. Beebe (1960) conducted a similar study of U.S. veterans of WWI that was designed to compare the mortality and morbidity of three groups: (1) soldiers hospitalized for exposure to mustard vapor; (2) soldiers with pneumonia; and (3) soldiers requiring amputation. Unlike the British study, all members of the gas cohort had to have had medical evidence of eye or skin burns from mustard agents. Smoking data were also available on a sample of all three cohorts and documented a deficit of smoking in the gas cohort. The sulfur mustard cohort had 1.3 percent

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Veterans at Risk: The Health Effects of Mustard Gas and Lewisite die of lung cancer compared with 1.0 percent of the wounded control, which while elevated was not significant. Compared with the mortality patterns of the general population, however, the gas cohort had 39 observed deaths from respiratory cancer compared with 26.6 expected. This statistically significant excess is even more impressive if one considers that the gas cohort had a deficit of smokers. Significant lung cancer excess was not observed for the pneumonia or the wounded cohorts. Beebe concluded that, since there was no substantial difference in lung cancer mortality between sociologically similar groups of soldiers, yet there was a substantial difference with the general population, this study is "quite suggestive" but "unproved." Norman (1975) followed Beebe's cohorts for an additional 10 years. The mortality ratios for the mustard-exposed, pneumonia, and wounded populations were 0.99, 0.93, and 0.92, respectively, which may reflect an excess in that soldiers must pass a screening examination before induction. The mortality ratio of the gas-exposed cohort compared to wounded controls was 1.3, with 95 percent confidence intervals extending from 0.9 to 1.9. With "a more sensitive test," this difference in lung cancer deaths in the mustard-exposed cohort was even higher. A further case control analysis, for which little methodological detail was provided, indicated that mustard exposure added little excess risk compared with cigarette smoking. The authors recognized that this comparison might not accurately reflect the cumulative amount of cigarette smoking in each group; indeed, they provided data that smokers who were gassed quit substantially earlier than those in the other two groups. This study provides some evidence supporting the conclusion that a battlefield exposure associated with hospitalization is later associated with an excess mortality from lung cancer. This study also demonstrates the degree of information that would be essential to disentangle unequivocally the effect of mustard exposure from cigarette smoking. Medical Therapeutic Exposure In the early 1970s, reports began appearing of acute nonlymphocytic leukemia (ANL) in patients with multiple myeloma and lymphoma who had been treated with nitrogen mustards (Kyle et al., 1970; Rosner and Grunwald, 1975). Initially, it was thought that this occurrence might represent a consequence of the late natural history of these diseases. However, ANL was also found to occur in other types of cancer patients who had been treated with nitrogen mustards and other alkylating agents (Einhorn, 1978; Fisher et al., 1985; Greene et al., 1982). In particular, patients with ovarian cancer treated with melphalan and chlorambucil developed this complication. The reason why the initial

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Veterans at Risk: The Health Effects of Mustard Gas and Lewisite observations of this complication were in multiple myeloma, lymphoma, and ovarian cancer patients is undoubtedly that these were the first patients to receive prolonged therapy with nitrogen mustards who survived long enough to develop this complication. As experience with cancer chemotherapy has increased, it has become obvious that this complication occurs in approximately 3 to 5 percent of all patients treated with therapeutic courses of nitrogen mustards and other alkylating agents (Tucker et al., 1988), and in some groups of patients treated with prolonged, intensive courses, this rate has been as high as 30 percent (Einhorn, 1978). The acute leukemia that is seen after alkylating agent therapy is very malignant and responds poorly to conventional therapy. The peak time of onset of this leukemia has been reported to be between three and nine years after the original treatment (Blayney et al., 1987). As larger numbers of cancer patients have been treated with alkylating agents, the evidence has become very strong that there is also an increase in the rate of solid tumors in these patients (Tucker et al., 1988). In one study of patients who had been treated for Hodgkin's disease, the patients' 10-year actuarial risk of ANL was estimated to be 5.9 percent, for lymphoma 3.5 percent, and for solid tumors 5.8 percent (Koletsky et al., 1986). Similar risks have been described in other studies, including children treated for cancer. Therapeutic nitrogen mustards are administered systemically (except for a small experience with topical application to the skin, see Chapter 9) and are given repeatedly for periods of weeks to months. They are less reactive and have a different systemic pharmacology than sulfur mustard (Colvin and Chabner, 1990). Therefore, it is difficult to make quantitative extrapolations to the carcinogenicity of sulfur mustard and to which tumors sulfur mustard would be expected to produce. For example, acute leukemia has not been reported as a late consequence of sulfur mustard exposure. This might well be because those patients with sufficient skin or inhalation exposure to deliver a leukemic dose to the bone marrow would have succumbed to pulmonary and other complications. However, because sulfur mustards and nitrogen mustards have similar effects on DNA, the clinical experience with nitrogen mustards supports the evidence that sulfur mustard is carcinogenic in man. SUMMARY Gaps in the Literature There are three major gaps in the epidemiologic literature on occupational exposure. The first gap concerns the limited cohorts of workers that have been studied, Japanese and British. Other countries (including the United States) manufactured war gases; hence other cohorts of

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Veterans at Risk: The Health Effects of Mustard Gas and Lewisite workers could be identified and studied, which would contribute additional information. The second gap concerns the limited information available on exposure levels. Historical records vary considerably in their detail. Exposure measurements, if any, were usually done to determine levels for particularly troublesome parts of the manufacturing process or were collected for specific areas of the plant, which limits their value in characterizing particular jobs. The third gap concerns assessment of risk. The available epidemiologic studies focus on determining whether exposure is associated with a increased rate of malignancy in the exposed workers. This endeavor is complicated, and its conclusions—whether or not war gases are carcinogenic—are not the most appropriate questions to be addressed by this committee. Even if one concludes that the studies demonstrate a relationship between exposure and human cancer, the question remains: What would be the likely rate of cancer in the chamber and field test volunteers, or those who worked with these agents, exposed at levels different from the battlefield or occupational situations in Japan or Great Britain? This question requires a quantitative risk assessment, which cannot be developed from the data available in the literature. Conclusions Mustard agents are well known to be monofunctional and bifunctional DNA alkylating agents. They are extremely cytotoxic at low doses. They alkylate RNA and proteins and produce DNA lesions, which may be repaired only at low doses. The sulfur mustards also alkylate the 0-6 position of guanine. DNA alkylation is likely primarily responsible for the mutagenic consequence of cellular exposure. The sulfur mustards induce a wide variety of genetic lesions in many types of mammalian cells in vitro in a dose-related fashion. They also induce genetic damage in vivo in peripheral blood lymphocytes at low doses. In contrast to mustard agents, the genetic toxicology of Lewisite has been poorly studied. Lewisite induces chromosome aberration in one type of cellular assay. It appears not to be mutagenic in Salmonella. Sulfur mustard produces a variety of cancers through different exposure routes. It produced skin malignancies in chamber exposure in rats. Intravenous injection produced a significant increase in pulmonary tumors in highly susceptible strain A mice. Subcutaneous injections produced sarcomas and other tumors at the injection site in C3H, C3Hf, and strain A mice, but did not produce an increase of tumors at other sites. Nitrogen mustard, particularly HN2, was more widely tested and

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Veterans at Risk: The Health Effects of Mustard Gas and Lewisite found to be a carcinogen. It produced pulmonary tumors from both intravenous and intraperitoneal injections in strain A mice. Subcutaneous exposures produced injection site tumors and pulmonary tumors in selected strains of mice. Its carcinogenic potency appeared to be similar to sulfur mustard, and it was one of the most potent carcinogens amongst the alkylating agents tested in the strain A bioassay program. Evidence indicates that occupational exposure to sulfur mustard is associated with respiratory tract cancer. The battlefield experience is somewhat more equivocal, although the lung cancer excess is suggestive of an association. Evidence from therapeutic use of nitrogen mustard clearly indicates a causal association with skin cancer (see Chapter 9) and leukemia. An excess of skin cancer or leukemia was not evident in the occupational or battlefield studies. The weight of the evidence—cellular, epidemiological, and toxicologic—indicates a causal association between sulfur mustard exposure and the occurrence of excess respiratory cancer, and skin cancer, and possibly leukemia. Inadequate exposure information limits accurate estimation of the cancer excesses that may be expected. The evidence is insufficient to indicate a causal relationship for Lewisite carcinogenesis. Based on the foregoing, the committee concludes that human subjects of the WWII chamber tests are probably at increased risk of respiratory tract and skin cancer. This conclusion is based upon estimates of exposure to sulfur mustard that occurred among the subjects of the chamber tests (see Chapter 3), which approximated the battlefield exposure of surviving WWI soldiers. Studies of WWI gassing victims demonstrate a suggestive excess of cancer of the respiratory tract. Limitations on information on exposure of study subjects and WWI gassing victims limit the precision of this risk projection. REFERENCES Abell CW, Falk HL, Shimkin MB. 1965. Uracil mustard: a potent inducer of lung tumors in mice. Science 147:1443-1445. Ashby J, Tinwell H, Callander RD, Clare N. 1991. Genetic activity of the human carcinogen sulfur mustard toward Salmonella and the mouse bone marrow. Mutation Research 257:307-312. Auerbach C. 1943. Chemically induced mutations as rearrangements. Drosophila Information Service 17:48-50. Auerbach C. 1951. The effect of oxygen concentration on the mutagenic action of mustard gas. Kurze Mitteilungen 15:341-342. Auerbach C, Moser H. 1950. Production of mutations by monochloro-mustards. Nature 166:1019-1020. Auerbach C, Robson JM. 1946. Chemical production of mutations. Nature 157:302. Auerbach C, Robson JM. 1947. Production of mutations by chemical substances. Proceedings of the Royal Society of Edinburgh. Section B: Biology 62:271-283.

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