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Arsenic (As) is a human carcinogen. Our prior work showed that chronic (>18 weeks) low level (500 nM) arsenite (As3+) exposure induced malignant transformation in a rat liver epithelial cell line (TRL 1215). In these cells, metallothionein (MT) is hyper-expressible, a trait often linked to metal tolerance. Thus, this study examined whether the adverse effects of arsenicals and other metals were altered in these chronic arsenite-exposed (CAsE) cells. CAsE cells, which had been continuously exposed to 500 nM arsenite for 18 to 20 weeks, and control cells, were exposed to As3+, arsenate (As5+), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), antimony (Sb3+), cadmium (Cd2+), cisplatin (cis-Pt), and nickel (Ni2+) for 24 h and cell viability was determined by metabolic integrity. The lethal concentration for 50% of exposed cells (LC50) for As3+ was 140 microM in CAsE cells as compared to 26 microM in control cells, a 5.4-fold increase in tolerance. CAsE cells were also very tolerant to the acute toxic effects of As5+ (LC50 > 4000 microM) compared to control (LC50 = 180 microM). The LC50 for DMA was 4.4-fold higher in CAsE cells than in control cells, but the LC50 for MMA was unchanged. There was a modest cross-tolerance to Sb3+, Cd2+, and cis-Pt in CAsE cells (LC50 1.5-2.0-fold higher) as compared to control. CAsE cells were very tolerant to Ni2+ (LC50 > 8-fold higher). Culturing CAsE cells in As(3+)-free medium for 5 weeks did not alter As3+ tolerance, implicating an irreversible phenotypic change. Cellular accumulation of As was 87% less in CAsE cells than control and the accumulated As was more readily eliminated. Although accumulating much less As, a greater portion was converted to DMA in CAsE cells. Altered glutathione (GSH) levels were not linked with As tolerance. A maximal induction of MT by Zn produced only a 2.5-fold increase in tolerance to As3+ in control cells. Cell lines derived from MT normal mice (MT+/+) were only slightly more resistant (1.6-fold) to As3+ than cells from MT null mice (MT-/-). These results show that CAsE cells acquire tolerance to As3+, As5+, and DMA. It appears that this self-tolerance is based primarily on reduced cellular disposition of the metalloid and is not accounted for by changes in GSH or MT.

Inorganic arsenic is considered a high-priority hazard, particularly because of its potential to be a human carcinogen. In exposed human populations, arsenic is associated with tumors of the lung, skin, bladder, and liver. While it is known to be a human carcinogen, carcinogenesis in laboratory animals by this metalloid has never been convincingly demonstrated. Therefore, no animal models exist for studying molecular mechanisms of arsenic carcinogenesis. The apparent human sensitivity, combined with our incomplete understanding about mechanisms of carcinogenic action, create important public health concerns and challenges in risk assessment, which could be met by understanding the role of metabolism in arsenic toxicity and carcinogenesis. This symposium summary covers three critical major areas involving arsenic metabolism: its biodiversity, the role of arsenic metabolism in molecular mechanisms of carcinogenesis, and the impact of arsenic metabolism on human risk assessment. In mammals, arsenic is metabolized to mono- and dimethylated species by methyltransferase enzymes in reactions that require S-adenosyl-methionine (SAM) as the methyl donating cofactor. A remarkable species diversity in arsenic methyltransferase activity may account for the wide variability in sensitivity of humans and animals to arsenic toxicity. Arsenic interferes with DNA methyltransferases, resulting in inactivation of tumor suppressor genes through DNA hypermethylation. Other studies suggest that arsenic-induced malignant transformation is linked to DNA hypomethylation subsequent to depletion of SAM, which results in aberrant gene activation, including oncogenes. Urinary profiles of arsenic metabolites may be a valuable tool for assessing human susceptibility to arsenic carcinogenesis. While controversial, the idea that unique arsenic metabolic properties may explain the apparent non-linear threshold response for arsenic carcinogenesis in humans. In order to address these outstanding issues, further efforts are required to identify an appropriate animal model to elucidate carcinogenic mechanisms of action, and to define dose-response relationships.

The aim of this paper is to report on the presence of chemical interferences in the quantitation of methylated arsenic species in human urine when using a method based on selective volatile arsine species generation, chromatographic separation, and hydride generation atomic absorption spectrometry (HGAAS) detection. An abnormal profile of methylated arsenic species characterized by the absence of the peak corresponding to dimethylarsinic acid (DMA) was observed in urine from some individuals exposed to arsenic via drinking water and living in rural communities of northwestern Argentina. The absence of this peak persisted even after the addition of known amounts of DMA to the samples. However, the DMA peak appeared after urine digestion with hydrochloric acid (2M). Samples showing interferences were provided by individuals who had mate consumption and coca-leaf chewing habits. Because the relative proportions of methylated arsenic species present in urine have been used to evaluate the efficiency of the methylation process, interferences in the formation or detection of methylarsines may cause underestimation of As exposure and also lead to erroneous conclusions about relative biomethylation efficiencies. Therefore, we recommend that urine samples should be digested with 2M HCl before performing speciation analysis using HGAA techniques. Further studies on the impact of this type of interferences on other arsenic speciation methods are also required.

Our objective was to evaluate the activities of some enzymes of the heme biosynthesis pathway and their relationship with the profile of urinary porphyrin excretion in individuals exposed chronically to arsenic (As) via drinking water in Region Lagunera, Mexico. We selected 17 individuals from each village studied: Benito Juarez, which has current exposure to 0.3 mg As/l; Santa Ana, where individuals have been exposed for more than 35 years to 0.4 mg As/l, but due to changes in the water supply (in 1992) exposure was reduced to its current level (0.1 mg As/l), and Nazareno, with 0.014 mg As/l. Average arsenic concentrations in urine were 2058, 398, and 88 microg As/g creatinine, respectively. The more evident alterations in heme metabolism observed in the highly exposed individuals were: (1) small but significant increases in porphobilinogen deaminase (PBG-D) and uroporphyrinogen decarboxylase (URO-D) activities in peripheral blood erythrocytes; (2) increases in the urinary excretion of total porphyrins, mainly due to coproporphyrin III (COPROIII) and uroporphyrin III (UROIII); and (3) increases in the COPRO/URO and COPROIII/COPROI ratios. No significant changes were observed in uroporphyrinogen III synthetase (UROIII-S) activity. The direct relationships between enzyme activities and urinary porphyrins, suggest that the increased porphyrin excretion was related to PBG-D, whereas the increased URO-D activity would enhance coproporphyrin synthesis and excretion at the expense of uroporphyrin. None of the human studies available have reported the marked porphyric response and enzyme inhibition observed in rodents. In conclusion, chronic As exposure alters human heme metabolism; however the severity of the effects appears to depend on characteristics of exposure not yet fully characterized.

The cytogenetic effects of arsenic exposure were studied among rural populations that live in the same geographical area and have similar socioeconomic status, but different degree of exposure to inorganic arsenic (As) via drinking water. A group of inhabitants of Santa Ana (408.17 micrograms/l of As in drinking water) were considered the exposed individuals and a group of inhabitants of Nazareno (29.88 micrograms/l) were considered as controls. Blood and urine samples were obtained from volunteers. Past and current exposure, health, and nutritional status as well as the presence of arsenic skin lesions were ascertained in study participants through questionnaires and physical examination. The frequencies and types of chromosomal aberrations in first-division metaphases were studied in whole blood lymphocyte cultures while the presence of micronuclei (MN) was studied in exfoliated epithelial cells obtained from the oral mucosa and from urine samples. Total arsenic (TAs) content, and the relative proportions of inorganic arsenic (IAs), and the metabolites monomethylarsonic (MMA) and dimethylarsinic (DMA) acid were determined in urine samples. Exposed individuals showed a significant increase in the frequency of chromatid and isochromatid deletions in lymphocytes and of MN in oral and urinary epithelial cells. Males were more affected than females, and a higher number of micronucleated oral cells were found among those individuals with skin lesions. The type of cytogenetic damage observed gives evidence of arsenic as a clastogenic/aneugenic carcinogen.

Relationships between alterations in the profile of urinary arsenic (As) species and the presence of cutaneous signs of arsenicism were studied in Region Lagunera, Mexico. The use of urinary concentrations of putative substrates and products of the As metabolism pathway, as indicators of metabolic efficiency is also discussed. Arsenic was determined by hydride generation atomic absorption spectrophotometry and separation of As species was performed by ion exchange chromatography. The exposed group had an average of 0.408 mg As/l of total As (TAs) in their drinking water, whereas "control' individuals had 0.031 mg/l. Urinary concentrations of arsenic species and TAs were 20 to 95 times higher in the exposed group. Significant increases in the relative proportions of inorganic arsenic (Asi) and monomethylarsonic acid (MMA), accompanied by decreases of dimethylarsinic acid (DMA) were also found in exposed individuals. Therefore, significant decreases in the value of the MMA/Asi, DMA/MMA and DMA/ Asi ratios were observed, suggesting a decreased As methylating ability. Exposed individuals bearing cutaneous signs had a significantly longer time of exposure, higher urinary concentrations and proportions of MMA and MMA/Asi values, and significantly lower DMA/ MMA than exposed individuals without cutaneous signs. Further research is needed to identify better parameters for assessing the efficiency of As metabolism in chronically exposed populations and to confirm the potential relationship between metabolic alterations and overt signs of As toxicity.

The main purpose of this study was to assess environmental contamination by arsenic and cadmium in a smelter community (San Luis Potosí City, México) and its possible contribution to an increased body burden of these elements in children. Arsenic and cadmium were found in the environment (air, soil, and household dust, and tap water) as well as in the urine and hair from children. The study was undertaken in three zones: Morales, an urban area close to the smelter complex; Graciano, an urban area 7 km away from the complex; and Mexquitic, a small rural town 25 km away. The environmental study showed that Morales is the most contaminated of the zones studied. The range of arsenic levels in soil (117-1396 ppm), dust (515-2625 ppm), and air (0.13-1.45 micrograms/m3) in the exposed area (Morales) was higher than those in the control areas. Cadmium concentrations were also higher in Morales. Estimates of the arsenic ingestion rate in Morales (1.0-19.8 micrograms/kg/day) were equal to or higher than the reference dose of 1 microgram/kg/day calculated by the Environmental Protection Agency. The range of arsenic levels in urine (69-594 micrograms/g creatinine) and hair (1.4-57.3 micrograms/g) and that of cadmium in hair (0.25-3.5 micrograms/g) indicated that environmental exposure has resulted in an increased body burden of these elements in children, suggesting that children living in Morales are at high risk of suffering adverse health effects if exposure continues.

Lymphocyte cultures from individuals exposed to high levels of hydroarsenicism showed a slower cell cycle kinetics than cultures from low-exposed individuals. Since this difference in proliferation could be due to chronic arsenic exposure, the in vitro effects of inorganic arsenic in human whole blood lymphocyte cultures were investigated. When lymphocytes were exposed to concentrations of arsenite and arsenate similar to those found in the blood of exposed subjects (10(-7), 10(-8) and 10(-9) M) during the last 24 h before harvesting, a dose-related inhibition of proliferation was observed. Cultures were also treated with 10(-9) M of arsenite and arsenate for 2, 6 and 24 h at the beginning of the cultures in the presence or absence of phytohemagglutinin (PHA). Inhibition of stimulation and proliferation was directly related to the length of treatment. The results show that, at the concentrations tested, arsenite and arsenate impair lymphocyte stimulation and proliferation and confirm the fact that chronic arsenic exposure can affect the proliferation of whole blood lymphocytes.

The acute administration of sodium arsenite (AsIII) to rats resulted in a biphasic alteration of the hepatic cytosolic "free" heme pool. The first stage was an increase in the cytosolic "free" heme without significant effects on the content of cytochrome P-450 or on bilirubin excretion. The second stage consisted of a continuous fall of the cytosolic "free" heme and of the content of cytochrome P-450. These changes were concurrent with an eight-fold increase in heme oxygenase activity and associated with marked elevations in the biliary excretion of bilirubin. The bile was collected from chronically cannulated rats to avoid artifacts related to anesthesia or post anesthetic effects. The rapid increase in biliary excretion of labeled heme degradation products indicated an increased breakdown of newly synthesized heme. Immunoelectrophoresis of bile proteins showed an altered pattern of bile protein excretion. The increased biliary haptoglobin suggested some hemolysis, while the reduction in the free immunoglobulin A (IgA) secretory component showed an AsIII-related decreased protein transport across hepatocytes to bile. Further research is required to assess the direct role of an increased heme degradation in the genesis of the hepatotoxic effects of AsIII.

Acute arsenic (As) administration produced in rat liver a decrease in the heme saturation of tryptophan pyrrolase (TP), accompanied by dose-related increases in 5-aminolevulinate synthetase (ALAS) and heme oxygenase (HO) activities, along with a corresponding decrease in cytochrome P-450 (P-450) concentration. The relationship between heme synthesis and degradation was altered as a result of As treatment. The magnitude of these effects was related to the oxidation state of arsenic, sodium arsenite (AsIII) being more potent than sodium arsenate (AsV). These results support the contention that the heme saturation of TP is sensitive to treatments that modify liver heme concentration. The increase in HO activity produced by As appears to be mediated by a mechanism largely or entirely independent of heme. The main effects of continuous exposure to AsIII were an initial decrease in the heme saturation of TP, which remained constant during the period of treatment, and an initial increase in ALAS activity, which after ten days of exposure dropped somewhat but remained above control values. No significant effects on HO or P-450 concentration were observed. These results were interpreted as indicative that a new balance between heme synthesis and degradation had been reached and that an adaptive response to the subchronic effects of AsIII was taking place.