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8
Variation in Human Sensitivity

There is a marked variation in susceptibility to arsenic-induced toxic effects among mammalian species (see Chapter 4). A variation in susceptibility between human population groups and individuals has also been suggested. Possible factors influencing the susceptibility are genetic polymorphisms (especially in metabolism), life stage at which exposures occur, sex, nutritional status, and concurrent exposures to other agents or environmental factors that influence the toxicity of the chemical.  The problems in understanding human variability in sensitivity to arsenic are compounded by substantial differences in exposure to arsenic among the population groups and individuals. Considerable variation in the quality of analytical data has also made it difficult to compare studies with confidence.

Variability in arsenic metabolism appears to be important in understanding the human response. There is evidence that methylating capacity differs among individuals and population groups. Different capacities would result in variations in tissue concentrations of arsenic. Also, environmental factors, particularly diet, might be important in explaining susceptibility. A diet poor in methionine or protein is likely to decrease the ability to methylate arsenic. Other dietary factors (e.g., selenium and zinc) might play a role in a person's response to arsenic. Those and other factors contributing to variability are discussed in this chapter.

Variation In Arsenic Metabolism

This section discusses variations in the metabolism of inorganic arsenic in humans, especially those variations evidenced by the urinary excretion of



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Page 229 8 Variation in Human Sensitivity There is a marked variation in susceptibility to arsenic-induced toxic effects among mammalian species (see Chapter 4). A variation in susceptibility between human population groups and individuals has also been suggested. Possible factors influencing the susceptibility are genetic polymorphisms (especially in metabolism), life stage at which exposures occur, sex, nutritional status, and concurrent exposures to other agents or environmental factors that influence the toxicity of the chemical.  The problems in understanding human variability in sensitivity to arsenic are compounded by substantial differences in exposure to arsenic among the population groups and individuals. Considerable variation in the quality of analytical data has also made it difficult to compare studies with confidence. Variability in arsenic metabolism appears to be important in understanding the human response. There is evidence that methylating capacity differs among individuals and population groups. Different capacities would result in variations in tissue concentrations of arsenic. Also, environmental factors, particularly diet, might be important in explaining susceptibility. A diet poor in methionine or protein is likely to decrease the ability to methylate arsenic. Other dietary factors (e.g., selenium and zinc) might play a role in a person's response to arsenic. Those and other factors contributing to variability are discussed in this chapter. Variation In Arsenic Metabolism This section discusses variations in the metabolism of inorganic arsenic in humans, especially those variations evidenced by the urinary excretion of

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Page 230 arsenic metabolites. Factors considered are methodological aspects; genetic polymorphism; age, sex, and recreational habits; effects of dose; and individual variation. Methodological Aspects The efficiency of arsenic methylation is often evaluated by the relative distribution  of metabolites  of inorganic  arsenic  (inorganic  arsenic, monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA)) in the urine. However, the relative amounts of arsenic metabolites in the urine might not reflect the actual methylation efficiency in the body (i.e., the fraction of the absorbed dose that is methylated), because the methylated metabolites are excreted in the urine more quickly than inorganic arsenic (Buchet et al. 1981a). Given that inorganic arsenic, especially As(III), is the main form of arsenic interacting with tissues (Buchet et al. 1981a; Vahter and Marafante 1983; Bogdan et al. 1994), a decrease in tissue methylation would result in more arsenic being retained in the body (Marafante and Vahter 1984; Marafante et al. 1985). Ideally, the evaluation of the in vivo methylation of arsenic in humans would be based on the assessment of urinary excretion of MMA and DMA in relationship to the absorbed dose. That assessment is not easily done, because the exact amounts of arsenic inhaled or ingested with drinking water and food, as well as the fraction absorbed, are seldom known. However, a few experimental studies on human volunteers have specified the administered dose and form of arsenic. In a study by Tam et al. (1979), six males ingested 74 As-labeled arsenate (about 0.01 µg of arsenic, greater than 90% As(V), per person), and the excretion of 74 As was followed for 5 days. Results indicate that approximately 58 % of the dose was excreted within that time. In another study, two subjects ingesting 200 µg of As(V) (1 L of bottled water each) excreted about 66% of the dose over 7 days following ingestion (Johnson and Farmer 1991). Taken together, the results of these experimental studies indicate that the proportion of DMA in the urine (relative to U-Asmet) is associated with the urinary excretion of DMA (% of ingested dose), as well as the total urinary excretion of metabolites of inorganic arsenic (U-Asmet), in percentage of the given dose. Similar results were reported in studies of human volunteers exposed to arsenite (Crecelius 1977; Buchet et al. 1981a,b), but the variation was considerable. Thus, on a group basis, a low proportion of DMA in urine (relative to U-Asmet) indicates that the rate of methylation is low and that the overall rate of excretion of arsenic metabolites (in percentage of dose) is low. This would lead to more inorganic arsenic being retained in

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Page 231 the body which was also indicated in a study of arsenic metabolites in the urine of women and children exposed to arsenic via drinking water in northern Argentina (Concha et al. 1998a). The ratio between the arsenic in blood and urine increased significantly with decreasing proportion of DMA in the urine, indicating that more arsenic was bound to blood, probably also tissues, at low methylation efficiency. When evaluating the excretion of arsenic metabolites in urine of human subjects and the factors influencing the concentration of metabolites in tissues and urine, it is essential that the analyses are accurate. As for all analytical data, and especially for those concerning trace elements in body tissues and fluids, the accuracy of the reported data should be verified by appropriate quality-control procedures, the results of which should be documented in the published reports (e.g., Friberg 1988). The situation is perhaps even more problematic for arsenic than for most other common metals in the human environment, because very few standard reference materials (SRMs) with certified concentrations of arsenic in biological media, such as blood and urine, are commercially available. In fact, no SRMs are available for arsenic metabolites in urine.   The need for quality-control procedures was demonstrated in a recent interlaboratory comparison exercise involving seven experienced laboratories determining arsenic metabolites in human urine samples. The variations among the laboratories were 1.3-2.7 µg/L for inorganic arsenic, 1.3-2.7 µg/L for MMA, and 5.8-11.2 µg/L for DMA in a urine sample with low arsenic concentrations; and 3.2-6.0 µg/L for inorganic arsenic, 3.5-5.5 µg/L for MMA, and 14.9-22.3 µg/L for DMA in a medium concentration sample (Crecelius and Yager 1997). Thus, when comparing the results of different studies, the quality-control data have to be considered. Unfortunately, quality-control data are often not reported or adequate. Genetic Polymorphism The average relative distribution of arsenic metabolites in the urine of various population groups seems to be fairly constant, irrespective of the type and extent of exposure. A large number of studies on human subjects exposed to inorganic arsenic occupationally, experimentally, or environmentally have shown that, in general, U-Asmet consists of 10-30% inorganic arsenic, 10-20% MMA, and 60-80% DMA (for a review, see Hopenhayn-Rich et al. 1993; Vahter 1994). However, some populations seem to have a somewhat different distribution of arsenic metabolites in the urine. Recent studies of urinary excretion of arsenic showed an average of 2-4 % MMA in the urine of people (natives and Spanish descendants) exposed to arsenic in drinking water in

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Page 232 northern Argentina (Vahter et al. 1995; Concha et al. 1998a). Studies in San Pedro and Toconao in northern Chile showed that about 5% of the study group (220 individuals) had less than 5 % MMA in their urine (HopenhaynRich et al. 1996a). The percentage of MMA was significantly lower in Atacameños than in subjects of European descent. On the other hand, a study on drinking-water exposure to arsenic in northeastern Taiwan showed an unusually high percentage of MMA in the urine—on average, 27% (Chiou et al. 1997). Whether the reported variations in urinary arsenic metabolites are genetically determined or due to environmental factors remains to be investigated. It should be noted that human polymorphism is reported for other methyltransferases—e.g., histamine N-methyltransferase, nicotinamide N-methyltransferase, thiopurine  S-methyltransferase,  catechol O-methyltransferase, O6-methylguanine-DNA methyltransferase,  and guanidinoacetate methyltransferase (Li et al. 1979; Scott et al. 1988; Aksoy et al. 1996; Krynetski et al. 1996; Scheller et al. 1996; Stockler et al. 1996; Ganesan et al. 1997; Yates et al. 1997). Another example of genetically determined factors possibly influencing arsenic methylation is given in a case report of increased neurotoxicity in a 16-year-old female with 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency and suspected exposure to arsenic from an open bag, kept in the home, containing the pesticide copper acetate arsenite (Brouwer et al. 1992). MTHFR is necessary for the conversion of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the main methyl donor for the remethylation of homocysteine to methionine.  The adolescent had high homocysteine concentrations in plasma and urine, and the resulting deficiency in methyl donors might have lowered the methylation of arsenic. Although all family members were exposed to arsenic, only the 16-year-old with MTHFR deficiency showed signs of arsenic poisoning. Age, Sex, and Recreational Habits In children exposed to background concentrations of arsenic, sex or age had no influence on the arsenic metabolites found in the urine (Buchet et al. 1980; Kalman et al. 1990). However, children in northern Argentina exposed to arsenic at about 200 µg/L in drinking water had a significantly higher percentage of inorganic arsenic and a lower percentage of DMA in the urine, compared with adults (49% vs. 66% DMA; Concha et al. 1998a). That might indicate that children are more sensitive to arsenic than adults. One recent study from Finland indicates that an increase in the proportion of DMA in urine with an increase in age might occur in adults (Kurttio et al. 1998).

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Page 233 In people exposed to inorganic arsenic via the drinking water in northern Chile, sex, ethnicity, length of exposure, and smoking, but not age, seemed to have a small but significant influence on the relative amounts of urinary arsenic metabolites (Hopenhayn-Rich et al. 1996a). Multiple linear regression (p = 0.01) indicated that women had about 3 % more DMA and less MMA in the urine than men. Smoking 10 cigarettes a day resulted in an increase of a few points in the percentage of MMA and a corresponding decrease in the percentage of DMA. Also, one study of human exposure to arsenic via drinking water (up to 600 µg/L) in northeastern Taiwan indicates that women had a somewhat higher percentage of DMA and lower percentage of MMA in the urine than men (Hsu et al. 1997). However, the concentrations of arsenic in the urine were not given. In another study from northeastern Taiwan (where U-Asmet  were found to be 173 µg/L), no sex difference was found in the relative amount of the various urinary metabolites of arsenic (Chiou et al. 1997). Neither was sex a determining factor for the relative distribution of urinary arsenic metabolites in the recent study from Finland (Kurttio et al. 1998). The observed higher relative amount of DMA in urine in women compared with men in some of the above-mentioned studies might be related to hormonal effects. Recently, pregnant women in the third trimester were reported to have more than 90 % DMA in plasma and urine, a percentage that was significantly higher than that in nonpregnant women (Concha et al. 1998b). Differences in birth rate result in differences in the number of pregnant women in study populations. Effect of Dose As mentioned in Chapter 5, experimental animal studies have shown a decreased methylation at higher doses of arsenic, as well as an inhibition of the second step in the methylation of arsenic in rat-liver cells incubated in vitro with high concentrations of arsenite. In a study using four human volunteers who ingested a daily dose of 125, 250, 500, or 1,000 µg of arsenic as arsenite (one subject per dose) for 5 consecutive days (Buchet et al. 198lb), the excretion of DMA tended to level off at the highest dose (1,000 µg per day). That response has been interpreted by some as saturation of methylation and has provoked considerable discussion in the literature (EPA 1988; Carlson-Lynch et al. 1994; Beck et al. 1995). However, only one individual was tested per dose, and the urinary excretion of arsenic metabolites is known to vary in individuals; therefore, the results should be interpreted with great caution.

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Page 234 In an analysis of data available in studies published up to that time, no major differences were observed in the average relative distribution of urinary metabolites of inorganic arsenic between people exposed to arsenic occupationally, people exposed to high doses of arsenic experimentally or via drinking water, and people exposed to much less arsenic in the general environment (Hopenhayn-Rich et al. 1993). A number of studies indicate that the methylation of arsenic is fairly insensitive to high doses that are in the range of exposure occuring via drinking water. The average distribution of arsenic metabolites in urine was 10-30% inorganic arsenic, 10-20% MMA, and 60-80% DMA in most studies. A study of people in Nevada with high concentrations of arsenic in their well water (up to 1,300 µg/L) showed that a group of 18 subjects with an average U-Asmet of 750 µg/L had about 22% MMA and 58% DMA in the urine (Warner et al. 1994). Similar data were reported for a control group of 18 subjects whose drinking water contained arsenic at less than 10 µg/L, but the speciation data were close to detection limit and not as reliable as those for the exposed group. A few reports have shown that as the exposure to arsenic via drinking water increased, the percentage of urinary DMA (in relationship to total arsenic metabolites in the urine) decreased and that of MMA increased slightly (Hopenhayn-Rich et al. 1996a; Del Razo et al. 1997; Hsu et al. 1997). Although the changes were statistically significant, they were small. For example, speciation of arsenic in urine from people exposed to arsenic via drinking water in northern Chile indicated that a 500-µg/L increase in total UAsmet corresponded to a 2% increase in urinary MMA and a 3 % decrease in DMA (Hopenhayn-Rich et al. 1996a). A temporary (2 months) change in the source of water, involving a decrease in arsenic concentration from 600 to 45 µg/L, resulted in a small decrease in the average proportion of inorganic arsenic (about 3 %) and in the ratio of MMA to DMA in the urine, but those changes were not related to the magnitude of the decrease in concentration of total urinary metabolites (Hopenhayn-Rich et al. 1996b). A further support for arsenic methylation being relatively insensitive to the drinking-water concentration is a case report of a man who developed neuropathy after 4 months of daily consumption of well water containing 25,000 µg/L of arsenic (Kosnett and Becker 1988). Urine sampling before the DMSA chelation therapy was initiated showed U-Asmet at 5,500 µg/L, out of which 72% was in the form of MMA and DMA (about 36% of each) and 26% inorganic arsenic. Thus, the observed effects of the arsenic dose on the methylation efficiency seems to be small and mainly affecting the ratio of MMA to DMA. As available data indicate that the rate of excretion of MMA is similar to or slightly higher than that of DMA-about 75 % in 4 days (Buchet et al. 1981a)

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Page 235 for MMA and DMA compared with about 50 % for inorganic arsenic (Tam et al. 1979; Buchet et al. 198 1a)—a small change in the MMA-to-DMA ratio is unlikely to have any toxicological significance. However, the formation of reactive As(III) intermediates cannot be excluded (Cullen et al. 1989a,b; Styblo et al. 1997), although they have not been detected in vivo. Furthermore, the increased excretion of MMA at higher doses can indicate that the second methylation step is inhibited by inorganic As(III) in the tissues; that would indicate that As(III) is increasing in the tissues (Thompson 1993). There are also a few studies indicating an increase in the percentage of DMA in urine at higher exposure concentrations. In children exposed to arsenic via drinking water in northern Argentina, the percentage of urinary DMA increased with increasing U-Asmet concentration (about a 30% increase in DMA with a 400-µg/L increase in U-Asmet) (Concha et al. 1998a). That response was not observed in adults, who had a significantly higher proportion of DMA in the urine than the children. However, in a study of adults in northeastern Taiwan, the percentage of urinary DMA increased and that of MMA decreased with increasing arsenic concentrations in the drinking water (three groups of people drinking water with 0-50, 51-300, and > 300 µg/L, respectively; Chiou et al. 1997). Interindividual Variation When studies of arsenic metabolites in human urine are scrutinized, a substantial interindividual variation in the relative amounts of the metabolites is obvious, although group averages seem to be fairly consistent between studies. Some studies on the distribution of arsenic metabolites in the urine of people exposed to arsenic via drinking water are presented in Table 8-1. Obviously, few studies are published, and several of them include few subjects. Thus, although the variation can be assumed to be an integrated result of the various factors influencing the methylation of arsenic (e.g., genetic, physiological, nutrition, recreational, and analytical), little is known about the relative importance of the various factors. In fact, even the day-today variation in the urinary arsenic-metabolite pattern in one individual is unknown. Nutritional Status This section discusses the modulation of the responses to arsenic toxicity on the basis of the nutritional status of the individual. As mentioned in

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Page 236 Table 8-1 Interindividual Variation in the Relative Amounts (Median or Mean Values and Total Ranges) of Arsenic Metabolites (Inorganic Arsenic, MMA, and DMA) in the Urine of Individuals Exposed to Inorganic Arsenic via Drinking Water Drinking-Water Exposure Cases, U-Asmet , Urinary Arsenic Metabolites, %   to Arsenic No. µg/L Inorganic Arsenic MMA DMA Reference Argentina, 200 µg/L, women             S.A. de los Cobres 11 256 25 2.1 74 Vahter et al. 1995     109-405 6.5-42 0.6-8.3 54-93   Taco Pozo 12 386 39 2.2 58 Concha et al. 1998a     90-606 18-52 1.1-3.5 46-80   Argentina, 200 µg/L, children             S.A. de los Cobres 22 323 49 3.6 47 Concha et al. 1998a     125-578 21-76 0.9-12 22-69   Taco Pozo 12 440 42 3.4 54 Concha et al. 1998a     337-621 26-54 1.3-7.9 44-68   Chile, 600 µg/L, adults             San Pedro 122 482 18 15 67 Hopenhayn-Rich et al.     61-1,893 5.6-39 1.7-31 42-93 1996a Chile, 15 µg/L, adults             Toconao 98 49 14 9.7 76 Hopenhayn-Rich et al.     6-267 3.6-31 3.1-24 51-92 1996a China, <1 to >300 µg/L, adults             Taiwan 115 173 12 27 61 Chiouet al. 1997     ± 19 (SE) ± 1.0 (SE) ± 1.2 (SE) ± 1.4 (SE)   (Table continued on next page)

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Page 237 (Table continued from previous  page)   Cases, U-Asmet , Urinary Arsenic Metabolites, %   Drinking-Water Exposure to Arsenic No. µg/L Inorganic Arsenic MMA DMA Reference Mexico, 415 µg/L, adults             Santa Ana 35 544 31 11 54 Del Razo et al. 1997     429-689 28-34 9.5-13 50-58   United States, 300-400 µg/L, adults             California 10 161 24 18 55 Hopenhayn-Rich et al.     66-299 8-44 13-27 38-77 1993 Abbreviations: U-Asmet, urinary arsenic metabolites; SE, standard error.

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Page 238 Chapter 5, experimental studies have shown that rabbits fed diets with low amounts of methionine, choline, or proteins had a marked decrease in the urinary excretion of DMA and an increased tissue retention of arsenic (Vahter and Marafante 1987). In addition, the subcellular distribution of arsenic was altered. Whether that is also true for humans is not known. In areas with severe arsenic-related health effects due to ingestion of drinking water with high arsenic concentrations (i.e., southwestern Taiwan and the Antofagasta region in northern Chile), the inhabitants were reported to have a low socioeconomic level and a poor nutritional status (Borgono et al. 1977; Tseng 1977; Zaldivar and Guillier 1977; Hsueh et al. 1995). Several studies have suggested that poor nutrition might increase the health effects of arsenic. In addition to other studies noted in this section, people in and around West Bengal who were below 80% of the standard body weight for their age and sex had a 1.6-fold increase (2.1 in females, 1.5 in males) in the prevalence of keratoses, suggesting that malnutrition might increase susceptibility (Guha Mazumder et al. 1998). Poor nutritional status might indicate an increased susceptibility to arsenic toxicity, leading to reduced methylation of arsenic and therefore increased tissue retention of arsenic. However, the percentage of methylated arsenic metabolites in the urine seems to vary only to a small degree across populations (see Table 8-1 and Hopenhayn-Rich et al. 1993). No information is available on how responses to arsenic toxicity are modulated by the nutritional status of individuals. For example, there is disagreement among investigators concerning the nutritional status of arsenic-exposed subjects with blackfoot disease, mainly because of the lack of proper studies (Yang and Blackwell 1961; Engel and Receveur 1993; Hsueh et al. 1995). The remainder of this section discusses (1) the various nutritional factors that influence the toxicity of arsenic (i.e., methyl group donors, selenium, and zinc) and (2) what is known about the nutritional status of the Taiwanese populations on which EPA (1988) based its estimate of the carcinogenic potency of arsenic. Methyl Group Donors Sound but sparse data demonstrate that S-adenosyl methionine (SAM) is the main methyl donor in the methylation of inorganic arsenite. Challenger (1945) suggested that arsenic methylation involved reduction and oxidative methylation by the addition of a carbonium ion to arsenic in the + 3 oxidation state. In vivo studies on rabbits given periodate-oxidized adenosine (PAD), a potent inhibitor of S-adenosylhomocysteine hydrolase, showed that SAM is

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Page 239 an important methyl donor in the formation of MMA and DMA (Marafante and Vahter 1984; Marafante et al. 1985). The use of SAM as a methyl donor was confirmed by Buchet and Lauwerys (1985) and Styblo and Thomas (1997) using in vitro enzyme assays of crude rat-liver cytosol, and by Zakharyan et al. (1995) using a 2,100-fold purified rabbit-liver arsenite and MMA methyltransferases preparation. Because the major source of SAM is the essential amino acid methionine, methylation of inorganic arsenite by SAM is dependent on the nutritional intake of methionine, cysteine, related vitamins, and cofactors. The importance of such nutritional factors for arsenic metabolism was clearly shown by Vahter and Marafante (1987) in a study showing that low amounts of methionine, choline, or protein in the diet decreased the methylation of inorganic arsenite in the rabbit. Cyanocobalamin (vitamin B12) and its coenzymes, 5'-deoxycobalamin and methylcobalamin, have also been implicated (Buchet and Lauwerys 1985), but the involvement of vitamin B12 or its coenzymes is far from clear since in vitro methylation of arsenite in the presence of methylcobalamin can occur in the absence of any enzymes (Buchet and Lauwerys 1985). In the case report described earlier of a 16-year-old female with a MTHFR deficiency and a history of exposure to the pesticide copper acetate arsenite, Brouwer et al. (1992) suggested that she had arsenic-related neurotoxicity, which was not seen in the rest of her family, presumably because of the MTHFR inborn error of metabolism. MTHFR is a metabolic source of methyl groups. These observations obviously show that nutritional factors affecting one-carbon and methyl metabolism must be taken into account in any survey of the nutritional status of persons chronically exposed to inorganic arsenic. Although the Brouwer et al. (1992) report is based on only one subject, the importance of the study should not be minimized. Selenium A strong interaction between arsenic and selenium was first observed in 1938 (Moxon 1938) and has since been confirmed in many animal experiments. Arsenites and arsenates, whether applied through the diet or by injection, reduce the toxicity of most selenium compounds. Organic arsenic compounds are less active than inorganic forms in modifying selenium toxicity; arsenic sulfides have no effect. In contrast to the protection afforded by arsenic against excessive exposure to most selenium compounds, arsenic increases the toxicity of the metabolic products dimethylselenide and the trimethylselenonium  ion through an unknown mechanism.  With that

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Page 240 exception, the nature of the arsenic-selenium interaction in animals is well known. While reducing the elimination of dimethylselenide in exhaled air, sodium arsenite (1 mg/kg) strongly increases the concentration and excretion of selenium in bile at the expense of hepatic concentrations. The magnitude of those changes is thought to explain the reduction of toxicity and the formation of a selenoarsenide compound in the liver, proposed as one mechanism responsible for the increased biliary excretion (Levander 1977).  That hypothesis is supported by the observation that injected sodium selenite (0.5 mg/kg) increases the biliary excretion of injected arsenite and reduces the hepatic arsenic concentration. Although additional pathways of detoxification might exist, the proposed selenoarsenide compound would explain the well-established bidirectional nature of this interaction: arsenic and selenium reduce each other's toxicity. Enhanced biliary excretion is the common mechanism; however, biliary excretion differs among species and the extent to which that difference applies to humans is unknown. The health relevance of those interactions has been observed only under laboratory conditions, where selenium reduced the teratogenic, clastogenic, and cytotoxic effects of arsenic, just as arsenic reduced the toxic effects of selenium (ATSDR 1993). Whether such health effects also occur under practical conditions is much less clear. Those interactions could offer some potentially beneficial applications to reduce the toxicity of excessive environmental exposure to arsenic or selenium by raising the exposure to the respective antagonist. A number of arsenicals, approved as feed additives, were tested in South Dakota where the high selenium concentrations in forage proved to be toxic to cattle and sheep, but their use was found to be impractical (Levander 1977). No such plans have been considered for human populations. There is no evidence of risks to animal and human populations from a toxic synergism between arsenic and methylated forms of selenium, although the possibility has been discussed (Kenyon et al. 1997). Finally, there is no evidence from animal experiments that arsenic, superimposed on a marginal selenium status, would induce selenium deficiency (Levander 1977). The reports discussed here lead to the conclusion that, despite convincing evidence for a strong arsenic-selenium interaction in experimental animals, there is as yet no direct evidence for its health effect in humans. Such a health effect, however, resulting from the lack of adequate selenium to counteract arsenic excesses would be consistent with the situation in the blackfoot-disease areas of Taiwan. Selenium status there should be considered a moderator of arsenic toxicity and taken into account when the Taiwanese data are applied to populations with adequate selenium intakes.

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Page 241 Zinc The clinical relevance of the interaction of arsenic and zinc is more tenuous. Injected parenterally, zinc protects mice against acute arsenic toxicity by way of an unknown mechanism (Kreppel et al. 1994), not related to the induction of metallothionein. Long-term protection by dietary zinc, however, has not been demonstrated.  Lin and Yang (1988) measured unusually low zinc concentrations in blood and urine of blackfoot-disease patients in Taiwan. Engel and Receveur (1993) estimated the nutritional adequacy of the diet of the Taiwanese population in the blackfoot-disease endemic area and believed that only zinc might be present in inadequate amounts. However, the calculated zinc intake in the blackfoot-disease endemic area of 9 mg per day is above the recent FAO-IAEA-WHO (1996) recommendation of 5.7 and 4.0 mg per day (population minimal requirement for moderate dietary availability for males and females, respectively, 18-60 years of age and older). Early reports of beneficial effects of zinc treatment in patients with peripheral vascular diseases (Henzel et al. 1969, 1971) have not been followed up. Nutritional Status of Populations in Taiwan As early as 1961, Yang and Blackwell reported that blackfoot-disease patients had poor nutritional status. Their report appeared before arsenic was implicated as a possible causative agent for blackfoot disease. The study pointed out that the mean protein of the diet made up only 9% of the caloric intake. The mean methionine intake was 1.2 g per day. Blackwell was a member of a highly respected U.S. naval research group who remained in Taiwan for at least 2 years to study blackfoot disease. In a brief critique of the Yang and Blackwell (1961) study, Engel and Receveur (1993) using the food consumption data of Yang and Blackwell made estimations and stated that ''intake of protein and essential amino acids were adequate, and fat intake accounted for only 5.3% of the energy intake. . . .  Our results indicate an inadequate zinc intake at 58%  of the recommended daily allowance (NRC  1989), which is based on the maintenance of existing zinc status in healthy young adults on a mixed U.S. diet."  Engel and Receveur (1993) in their letter speculated that "zinc deficiency may behave synergistically with arsenic in carcinogenesis and atherosclerosis. " The most recent recommendation for dietary zinc intake was established in 1996 (FAO-IAEA-WHO). On the basis of the recent data, the

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Page 242 dietary zinc intake reported in the Yang and Blackwell (1961) study appears to be adequate. The malnutrition of persons with blackfoot disease is further supported by Hsueh et al. (1995), who studied 1,571 subjects from the blackfoot-disease endemic area in Taiwan and pointed out that "undernourishment" related to high consumption of dried sweet potato, as indicated from questionnaire data, was associated with the increased prevalence of skin cancer induced by arsenic. Again one must emphasize that these are epidemiological data derived from questionnaires and are not measurements of sweet potato consumption or chemical analysis of sweet potato composition. There is no question that the nutritional status of persons chronically exposed to arsenic is crucial to understanding the signs and symptoms of arsenic toxicity. Animal studies have shown clearly that methionine, the factors involved in its metabolism, and selenium influence the metabolism and toxicity of arsenate and arsenite. A recent series of studies using biochemical biomarkers and clinical observations indicate a very low selenium status of the population in some areas of Taiwan. A study of selenium, manganese, cobalt, chromium, and zinc concentrations in the urine of 40 patients with blackfoot disease and 40 healthy controls reported concentrations of selenium between 3 and 4 µg/L, regardless of health status (Pan et al. 1996a). That range compares with 3.3 µg/L (26 subjects) in the population of the Keshan area of China, where selenium-responsive cardiomyopathy is endemic (Oster 1992). Both concentrations represent the lowest values in a worldwide comparison; other values are 26 µg/L (173 subjects), 60 µg/L (167 subjects), and 125 µg/L (10 subjects) in populations in China, the United States, and Canada, respectively (Oster 1992). Urinary excretion accounts for approximately half of the dietary selenium over a wide range of intakes (FAO-IAEA-WHO 1996); thus, assuming a daily urine output of 2 L, the data suggest an average intake of 12-16 µg per day. That selenium intake compares with an average daily intake of 11 µg in the areas of China with endemic Keshan disease and with average daily intakes of 116 µg, 60-159 µg, and 98-224 µg in other areas of China, the United States, and Canada, respectively (Oster 1992). The recommended dietary allowance for selenium in the United States is 55 and 70 µg per day (NRC 1989), the lower limit of the WHO safe ranges for population mean intake are 30 and 40 µg per day for females and males, respectively (FAO-IAEA-WHO 1996). Consistent with the urinary data, the serum selenium concentrations in areas of Taiwan also ranked lowest in a worldwide comparison. Thirty-two patients with idiopathic dilated cardiomyopathy from central Taiwan presented an average of 27.6 µg/L compared with 42.7 µg/L in 31 age-matched normal volunteers, a significant difference (Chou et al. 1998). The patients' values

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Page 243 are only slightly higher than the 21-µg/L value in the blood of 173 subjects from the Keshan disease area of China; healthy control values are 95  µg/L (111 subjects), 109.9 µg/L (1,025 subjects), and 143.9 µg/L (268 subjects) in populations in China, the United States, and Canada, respectively (Oster 1992). Two additional studies of blackfoot-disease patients and healthy controls from  Taiwan reported slightly higher selenium  concentrations but still indicated a low selenium status. Wang et al. (1993) in a whole-blood study reported that patient values were 46.7 µg/L (113 subjects) and control values were 62.7 µg/L (49 subjects). In a serum study by Pan et al. (1996b), patient values were 51.2 µg/L (40 subjects) and control values were 56.5 µg/L (40 subjects). Another report of very high selenium concentrations in the plasma of 36 stroke patients (214 µg/L) and 21 controls (230  µg/L) in Taiwan (Chang et al. 1998) cannot be evaluated because the analyses were not performed by the authors, and no data on analytical methods and quality control were provided. The sporadic occurrence of idiopathic dilated cardiomyopathy in central Taiwan (Chou et al. 1998), if related to the cardiomyopathy of the Keshan area of China, might be taken as additional, suggestive evidence of marginal selenium deficiency in the population. Summary and Conclusions Variability in response to arsenic might have its origin in one or a number of intrinsic or extrinsic factors, many of which affect the body's ability to methylate and eliminate arsenic. Other factors, such as nutrition, life stage, pre-existing health conditions, or recreational habits, might play a role in the response to arsenic but have not been studied extensively. It is plausible but not proved that poor diet substantially exacerbates the toxicity of arsenic. Much more work is needed to draw any definitive conclusions about the role of specific dietary components in the manifestations of arsenic toxicity. Important factors to consider in evaluating diet in this context are methionine, cysteine, vitamin B12, and folic acid, as well as essential trace elements, such as selenium and zinc. On the basis of its review of the data on the variations in human sensitivity to arsenic exposure, the subcommittee concludes the following: ·      Studies on human volunteers show that a low proportion of DMA in urine is associated with a low rate of methylation of ingested inorganic arsenic and a low rate of excretion of arsenic metabolites. ·      A few studies indicate a slight decrease in the percentage of DMA and

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Page 244 a corresponding increase in the percentage of MMA with increasing exposure concentrations. However, a few studies indicate an increase in the percentage of DMA with increasing exposure concentrations, particularly in children. At this time, there is no clear relationship between the dose of arsenic ingested and the relative amounts of arsenic metabolites in the urine. ·      There is substantial variation among individuals, and among some populations, in the fractions of methylated forms of arsenic in urine. Factors that can contribute to variation are age, sex, nutrition, and genetic polymorphism. Although such factors appear to influence the kinetics and extent of biomethylation, the implications of that for chronic toxicity, including carcinogenesis are uncertain. ·      The influence of nutritional factors on arsenic metabolism and toxicity is not clear. ·      A wider margin of safety might be needed when conducting risk assessments of arsenic because of variations in metabolism and sensitivity among individuals or groups. Recommendations Factors influencing the susceptibility to or expression of arsenic-associated cancer and noncancer effects need to be better characterized. Particular attention should be given to the study of the extent and reasons for interindividual and intraindividual variation in arsenic metabolism, tissue accumulation, and excretion (including total and relative amounts of urinary arsenic metabolites) under various exposure scenarios. Gene products responsible for metabolism, diet, and other environmental factors that might influence the susceptibility or expression of arsenic-associated toxicity need to be better characterized in comparative studies involving populations and individuals with different susceptibilities and suitable animal models. Studies of the potential differences in arsenic methylation efficiency between young children and adults need to be validated and considered in the risk assessment of arsenic. Future studies of arsenic metabolism and toxicity should include quality-control data on the method used to analyze for arsenic species in the urine, because assurance is needed that the variation is not due to the analytical methods or procedures used. References Aksoy, S., R. Raftogianis, and R. Weinshilboum. 1996. Human histamine

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