Metabolism of inorganic arsenic in children with chronic high arsenic exposure in northern Argentina.

This study concerns the metabolism of inorganic arsenic (As) in children in three villages in northern Argentina: San Antonio de los Cobres and Taco Pozo, each with about 200 microg As/l in the drinking water, and Rosario de Lerma, with 0.65 microg As/l. Findings show that the concentrations of As in the blood and urine of the children in the two As-rich villages were on average 9 and 380 microg/l, respectively, the highest ever recorded for children. The concentrations were about 10 and 30 times higher for blood and urine, respectively, than in Rosario de Lerma. Total As in urine was only slightly higher than the sum of metabolites of inorganic As (U-Asmet), i.e., inorganic As, methylarsonic acid (MMA), and dimethylarsinic acid (DMA); this shows that inorganic As was the main form of As ingested. In contrast to previous studies on urinary metabolites of inorganic As in various population groups, the children and women in the present study excreted very little MMA. Thus, there seems to be a polymorphism for the enzymes (methyltransferases) involved in the methylation of As. Interestingly, the children had a significantly higher percentage of inorganic As in urine than the women, about 50% versus 32%. Also, the percentage of inorganic As in the children is considerably higher than in previous studies on children (about 13% in the two studies available) and adults (about 15-25%) in other population groups. This may indicate that children are more sensitive to As-induced toxicity than adults, as the methylated metabolites bind less to tissue constituents than inorganic As. In the children, the percentage inorganic arsenic in urine decreased, and the percentage of DMA increased with increasing U-Asmet, indicating an induction of As methylation with increasing exposure.

ganic As, methylarsonic acid (MMA), and dimethylarsinic acid (DMA); this shows that inorganic As was the main form ofAs ingested. In contrast to previous studies on urinary metabolites of inorganic As in various population groups, the children and women in the present study excreted very little MMA. Thus, there seems to be a polymorphism for the enzymes (methyltransferases) involved in the methylation ofAs. Interestingly, the children had a significantly higher percentage of inorganic As in urine than the women, about 50% versus 32%. Also, the percentage of inorganic As in the children is considerably higher than in previous studies on children (about 13% in the two studies available) and adulu (about 15-25%) in other population groups. This may indicate that children are more sensitive to As-induced toxicity than adults, as the methylated metabolites bind less to tissue contituents thn inorganic As. In the children, the percentage inorganic arsenic in urine decreased, and the percentage of DMA increased with increasing U-As , indicating an induction ofAs methylation with increasing exposure. Key work: age, biotransformation, blood, drinking water, exposure, methylation, urine. Environ Health Perspect 106: 355-359 (1998). [ Online 15 May 1998] htp.//ehpnetl.niehs.'nih.gov/docs/1998/106p355-359conchalabstract.html A large number of people in different parts of the world are exposed to arsenic via drinking water. Such exposure has, in several studies, been associated with an increased risk for skin cancer and various internal cancers (1)(2)(3). Other adverse health effects associated with chronic exposure to As include hyperkeratosis, skin pigmentation, effects on the circulatory system (sometimes leading to gangrene), neurotoxicity, and hepatotoxicity (1,3,4). Although exposure via drinking water implies lifelong exposure beginning in early childhood, there are essentially no studies focused on children. There are only a few observations, particularly of As-induced hyperpigmentation and hyperkeratosis in children (5)(6)(7)(8). As to acute and subacute effects, an incidence of arsenic poisoning due to contaminated formula that gave rise to ingestion of 1.3-3.6 mg As/day in 12,000 infants resulted in hundreds of intoxicated infants and 130 fatalities (4,9). This would indicate that young children are more sensitive to As than adults.
In general, very little is known about factors that influence the toxicity and metabolism of arsenic in humans. Inorganic As is methylated to the more readily excreted and less toxic metabolites methylarsonic acid (MMA) and dimethylarsinic acid (DMA), but there is considerable variation between species and population groups (10,11). Experimental studies have indicated that the methylation of As is influenced by the dose level, mode of administration, form of As administered, and nutritional status of the subject (12)(13)(14). However, the influence of age is essentially unknown.
We have previously reported on elevated exposure to As via drinking water in native women in an Andean village in northern Argentina (11,15,16). In contrast to all previous studies on population groups from Europe, America, and Asia (17), the Andean women were found to excrete very little MMA in the urine, about 2% on average.
The aim of the present study was to investigate the metabolism ofAs in children in the same area, the Salta and Chaco Provinces of northeastern Argentina. We studied two villages with As-rich water (one populated predominately with indigenous people and the other with descendants of the Spanish immigrants) and one area with low water concentrations of As. For evaluation of differences in metabolism between children and adults, some women from the same villages were induded.

Materials and Methods
Study area and investigated population. One of the areas investigated was San Antonio de los Cobres (hereafter called S. A. Cobres), a village of about 5,000 inhabitants, mainly indigenous (18). The village is located at an altitude of 3,800 m above sea level in the western part of the Salta Province, in the arid Puna region of the middle Andes. In this area, the volcanic bedrock has a high content of As. There is no industrial source of As pollution in the area. The local economy is based on a rudimentary agriculture (potatoes and other vegetables) and breeding of llamas, goats, and sheep. The basic diet consists of meat, maize, rice, and vegetables. According to the statistics of the local Primary Health Care Committee (work area 29, Los Andes, Gerardo Vogler, Head), about 47% of the population is below 14 years of age. About 12% of the children 2-5 years of age suffer from malnutrition. The mortality rate is 52/1,000 for children under 1 year of age. No signs of chronic As intoxication, such as palmo-plantar hyperkeratosis, pigmentation changes, or skin cancer, have been observed by the local physician.
Taco Pozo is located on the plains of the Chaco Province, east of Salta. The village has 6,600 inhabitants, mainly "criollos" (descendants of Spanish immigrants). The As in the drinking water is of geological origin (19). There are no mines in the region, and the most important industries are related to cotton production and to the breeding of cattle, goats, and pigs. According to the local physician (Luis A. Veron, personal communication), signs of palmo-plantar hyperkeratosis are quite common in the area, also in children, but no statistics are available. In the present study, only one 13year-old boy and one 43-year-old woman showed palmo-hyperkeratosis.
For comparison, a group of children in Rosario de Lerma, belonging to the Salta Province, was studied. The village is located at an altitude of 1,000 m above sea level, has 18,000 inhabitants, mainly indigenous, and is known to have a low concentration ofAs in the drinking water.
The study comprised 24 children and 15 women in S. A. Cobres, 12 children and 12 women in Taco Pozo, and 21 children and 12 women in Rosario de Lerma. The participants were recruited by the physicians at the local hospitals and via the local radio or television. For investigation of As exposure and metabolism, samples of the local drinking water, food, urine, and blood were collected in each village. All the children and women were interviewed about their water consumption and dietary habits. Children below 10 years of age were interviewed with the help of their mothers.
Sampling and chemic. Drinking water from the publi bution systems in each village in 100-ml acid-washed polyeth In S. A. Cobres, the sampling urine was carried out at the ho, 1994. Because some of the u were lost, the sampling was November 1994. In Rosario d sampling was carried out at the in November 1994. In Taco ] were collected in the homes o and children in September 199' ples were collected from tl (cubital) using Venoject tubes ( Terumo, Leuven, Belgium) EDTA (K2) as an anticoagular samples were collected in acid-) containers and transferred to 100-ml acidc water distriwashed polyethylene bottles. The pH of the was collected urine and the presence of glucose and pro-Lylene bottles. tein were tested using N-Combur-Test of blood and (Boeringer Mannheim, GmbH, Germany). spital in April Concentrated HCI (1 ml to 100 ml urine) irine samples was added to prevent bacterial growth. All repeated in samples were frozen directly and kept at le Lerma, the -20°C until they were transported (on wet local hospital ice) to Sweden for analysis. Pozo, samples The concentrations of total As in drinkf the women ing water, food, blood, and urine were deter-5. Blood sam-mined using hydride generation atomic he arm vein absorption spectrophotometry (HG-AAS) (VP-100SDK; following dry ashing (20)(21)(22). The As con-, containing centrations in urine were adjusted for variait. Spot urine tion in density, determined by a Hand washed plastic refractometer (Atago, Japan). Because the concentration of total As in blood and urine lht) collected at may be highly influenced by dietary As of seafood origin, mainly arsenobetaine, we also Abbreviations: B-As, total arsenic in blood; U-Asm.t, sum of arsenic metabolites in urine; Inorg As, inorganic As; MMA, monomethylarsonic acid; DMA, dimethylarsinic acid.
&Adjusted to a density of 1.019 g/mI (average of all urine samples). bNo urinary arsenic speciation.
Institute of Standards and Technology (NIST), Gaithersburg, MD] and the Standard Reference Material 2670 (toxic metals in freeze-dried urine) were analyzed for total As (certified concentrations 82 ± 1.2 pg/l and 480 ± 100 pg/l, respectively) together with the collected water and urine samples. The obtained concentrations were 87 ± 6.2 pg/l (mean ± SD; n = 97) and 520 pg/I ± 50 (n = 92). The QC sample used in the determination of arsenic metabolites in urine consisted of a human urine sample spiked with 55.2 pg/l of arsenate, MMA, and DMA in the ratio 20:20:60. The total concentration of the As metabolites was set to 60 pg/l, based on several previous analyses at our laboratory and a reference laboratory (Analytica AB). When analyzed together with the collected urine samples, the average concentration of the sum of the As metabolites was 63 ± 6.2 pg/l (n = 43). In addition, 10 urine samples of children with MMA concentrations below 3% were spiked with known concentrations of MMA and DMA. The average recovery was 88% (range 72-117%) for MMA and 108% (96-126%) for DMA. Participation in an interlaboratory comparison for As metabolites in human urine samples (27) showed good agreement between our results and the average values of the six participating laboratories (11). The food samples were analyzed together with the standard reference materials bovine liver 1577, and rice flour, with certified concentrations of 0.055 ± 0.005 pg/g dry weight (dw) and 0.41 ± 0.005 pg/g dw, respectively. The obtained results were 0.054 ± 0.005 pg/g (n = 4) for bovine liver 1577 and 0.47 ± 0.01 pg/g (n = 3) for rice flour. We also spiked, in duplicate, five food samples with 17 ng As/g (SD 0.27) using Standard Reference Water (1643c, NIST). The average recovery was 96% (range 90-102%). Statistical analyses were performed using the Mann Whitney Rank Sum Test (comparison of groups) and Spearman Rank Order Correlation Test. Cobres and Taco Pozo and less than 1 pg/i in Rosario de Lerma (Table 1), whereas the concentration of selenium was less than 2 pg/1. In the As-rich villages, the food contained high As concentrations (Table 1), especially soup, which was consumed twice a day by about 70% of the investigated individuals in S. A. Cobres and 30% of those in Taco Pozo. Also maize porridge, which was the most common food in Taco Pozo, contained high concentrations ofAs.
The concentrations of total As in blood (B-As) were about as high in the children as in the women in both S. A. Cobres and Taco Pozo, but these concentrations were about 10 times higher than in Rosario de Lerma ( Table 2). The B-As was not influenced by sex or age. The selenium concentrations in blood plasma of three children and four women from S. A. Cobres were 51-63 pg/l and 67-88 pg/l, respectively.
The concentrations of the sum of U-Asmet in children and women from the Asrich areas were more than 30 times higher than in the control area (Table 2) and were about as high in the children as in the women. With the exception of the women in Taco Pozo, the concentrations of total As in urine were only slightly higher (median of children and women in S. A. Cobres 327 and 307 pg/l and Taco Pozo 457 and 464 pg/l, respectively) than those of the metabolites of inorganic As, indicating that the main exposure was to inorganic As. Probably, the women in Taco Pozo were exposed to more organic As compounds via food than inorganic As. U-Asmet was not influenced by sex or age. There was a significant positive correlation between the concentrations of B-As and U-Asmet in the As-rich areas (r = 0.42; p = 0.002; Fig. 1).
As shown in Table 2, most of the children and women had only low levels of MMA in the urine. However, children had significantly higher (p<0.0001) inorganic As in the urine than the women. This difference was most apparent in S. A. Cobres, where six of the children had over 60% inorganic As. Among the children, there was no significant association between age and the percentage of inorganic As, MMA, or DMA in the urine. There was also no difference between girls and boys, with the exception of a higher percentage of MMA in boys (mean 6.0%) as compared to girls (mean 3.1%) in S. A. Cobres (p = 0.023). As shown in Figure 2, there was a significant negative association between the percentage of inorganic As and U-Asmet (r = -0.54; p =0.001) in children and a corresponding positive association between the percentage of DMA in urine and U-Asmet (r = 0.57; p<0.0001). A similar association was not seen in the women.
To evaluate the effect of methylation (measured as the percentage of DMA in urine) on the retention and excretion of U-Asmet, we calculated the ratio B-As/U-Asmet for all individuals except the women in Taco Pozo who were found to have higher total U-As than U-Asmet, which would indicate exposure to other arsenic compounds than inorganic As, possibly via food (23). Obviously, such an exposure to other As compounds would influence also B-As, which we did not speciate. Figure 3, shows a significant decrease of the ratio BAs/U-Asmet with increasing percentage of DMA in the urine (r = -0.48; p = 0.001).

Discussion
The concentrations of As in the drinking water collected at different sampling sites in S. A. Cobres and Taco Pozo were about 20 times higher than the provisional World Health Organization (WHO) drinking water guideline of 10 pg As/I (28). There were also high As concentrations in the major foods, all of which were prepared using the local water. The intake of inorganic As via drinking water and food caused highly elevated concentrations in the blood and urine of the investigated children and women-about 10 and 30 times, respectively, the concentrations observed in the control area, Rosario de Lerma, in which the As concentration in the drinking water was below 1 pg/l. The concentrations of B-As (average 9 pg/l) and U-Asmet (average 380 pg/I) are the highest ever reported for children.
Average U-Asmet of about 50-200 pg/I have been reported in a few previous studies on children living close to copper smelters in the United States and Mexico (29,30). In other studies of children living in the vicinity of As-emitting industries, such as coal-fired power plants, coal mines, cadmium refineries, and copper and lead smelters, the average U-Asmet values were below 20 pg As/I (31)(32)(33)(34)(35)(36). In children without known exposure to As, the average U-Asmet were 5-10 pg/l (30,35).
The children in the present study, especially those in S. A. Cobres, had a considerably higher percentage of inorganic As (about 47%) and a lower percentage of DMA (50%) in urine than the women, but both had similar low percentages of MMA. There are only two previous reports on the speciation of As metabolites in the urine of children. Buchet et al. (32) reported average values of 12% inorganic As, 28% MMA, and 60% DMA in children in Belgium (n = 14), and Kalman et al. (31) reported average values of 13% inorganic As, 16% MMA, and 71% DMA in children in the United States (n = 158). However, in both these studies, the U-Asmet was low (<20 pg/l), only slightly higher than the U-Asmet in children from Rosario de Lerma (on average 10 pg/l), which was too low for reliable speciation with our methods. In the present study, only two children in S. A. Cobres had a pattern of As metabolites in the urine similar to the pattern reported by Buchet et al. (32) and Kalman et al. (31). The low percentage of MMA in children and women was similar to that observed in our previous studies on As exposure in native Andean women (11,16).
All other published reports on urinary As metabolites in human subjects (including populations in Europe, America, and Asia exposed to inorganic As in the general environment or occupationally) consistently show average values of 10-30% inorganic As, 10-20% MMA, and 60-80% DMA (17). However, a few studies of factory workers exposed to As (37,38) and subjects exposed to As in the drinking water (39) have reported low urinary percentages of MMA in a few individuals. Thus, there is a considerable interindividual variation in As methylation. Further research is needed to elucidate the reasons for such variations. The results indicate a genetic polymorphism for As methyltransferase. This enzyme is not fully characterized, but has been isolated from tissues of several mammalian species (40,41). It should also be noted that adequate quality control has not always been reported for U-Asmet' A recently published interlaboratory comparison (experienced laboratories only) ofAs species in spiked human urine samples showed extensive variation in the results, especially at low concentrations (27). Thus, some of the reported variation in metabolite pattern may be due to analytical variation.
More important, from a toxicological point of view, is the finding of very high concentrations of inorganic As in the urine of children in the present study, indicating a low general methylation efficiency. In total, 27% of the children in S. A. Cobres had more than 60% inorganic As and less than 40% DMA in the urine. This may indicate that the children are more sensitive to As-induced toxicity, as inorganic As is the main form interacting with tissue constituents (14). Further studies on the metabolism and effects ofAs in children are urgently needed. Unfortunately, there are no statistics available on As-related health effects in the areas studied in the present project. However, according to the local physicians, signs ofpalmo-plantar hyperkeratosis are common in Taco Pozo, but not in S.A. Cobres. Experimental studies have indicated that selenium may protect against As intoxication (42). However, the selenium concentrations in water were low, and the concentrations in plasma were similar to those reported for other countries (43).
Another interesting finding in the present study was the increasing percentage of urinary DMA, and the corresponding decreasing percentage of inorganic As, with increasing U-Asmet in the children, but not in the women. However, our previous studies showed a higher percentage of DMA and a lower percentage of inorganic As in the urine of the women in S. A. Cobres, as compared to women with fairly low exposure in other Andean villages (5-15 pg/I in drinking water) (11). This may indicate an induction of the methylation of inorganic As to DMA. In contrast, previous studies on adult subjects have shown a decrease in the percentage of DMA, and a corresponding increase in the percentage of MMA, with increasing U~Asmet (39). Our results could also be interpreted as a higher U-Asmet excretion in individuals with a higher methylating capacity, as experimental studies have shown that DMA is more rapidly excreted in the urine than is inorganic As (14). To evaluate if that was the case in the present study, we calculated the B-As/U-Asmet ratio assuming that B-As reflects the retention of As and U-Asmet reflects the excretion. As the B-As/U-Asmet ratio decreased significantly with the increasing percentage of DMA in the urine, which was used as a measure of the methylating capacity, it seems dear that the individuals with the most efficient methylation also had the lowest retention ofAs. However, the higher percentage of DMA observed in children with about 600 pg/l U-Asmet, compared to those with 200 pg/l U-Asmet (60% vs. 44%), could not explain all the differences in U~Asmet (400 pg/l), why the results still indicate an induction of As methylation with increasing exposure level. Further, B-As/U-Asmet also decreased with the increasing percentage of DMA in the women in S. A. Cobres, for whom there was an apparent increase in percentage of DMA with increasing U-Asmet' Obviously, further studies on factors influencing As methylation and excretion in humans are needed.