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Previous laboratory studies have shown that exposures to inorganic As (iAs) disrupt insulin production or glucose metabolism in cellular and animal models. Epidemiological evidence has also linked chronic human exposures to iAs to an increased risk of diabetes mellitus, a metabolic disease characterized by impaired glucose tolerance and insulin resistance. We have recently shown that arsenite and its methylated metabolites inhibit insulin-stimulated glucose uptake in cultured adipocytes by disrupting insulin-activated signal transduction pathway and preventing insulin-dependent translocation of GLUT4 transporters to the plasma membrane. Here, we present results of follow-up studies using male C57BL/6 mice chronically exposed to arsenite (1 to 50 ppm As) or to its metabolite methylarsonite (0.1 to 5 ppm As) in drinking water for 8 weeks. Results of these studies show that only the exposure to arsenite at the highest level of 50 ppm As produces symptoms attributable to impaired glucose tolerance. Notably, tissue concentrations of iAs and its methylated metabolites in pancreas and in major glucose metabolizing tissues in mice in this exposure group were comparable to the concentrations of total As reported in livers of Bangladeshi residents exposed to much lower concentrations of iAs in drinking water. These results suggest that because mice clear iAs and its metabolites more rapidly than humans, much higher exposure levels may be needed in mouse studies to produce the diabetogenic effects of iAs commonly found in human populations exposed to iAs from environmental sources.
Inorganic As (iAs) is one of the most potent environmental carcinogens . However, chronic exposures to iAs have also been associated with various non-cancerous diseases, including diabetes mellitus. Increased risks of developing or dying of diabetes mellitus have been reported in populations exposed to iAs in drinking water and among workers exposed to iAs in occupational settings (reviewed in ). The most recent evidence linking iAs exposure to diabetes has been provided by Coronado-Gonzâlez and associates  who examined 200 diabetes mellitus cases and 200 community controls in Coahuila State (Mexico) where residents are exposed to iAs in drinking water (20 to 400 ppb). This study utilized appropriate clinical criteria to diagnose diabetes; exposure to iAs was characterized by measurements of total As concentrations in urine. These investigators found a dose-response relationship between the risk of diabetes and the level of total As in urine (μg As/g creatinine). The adjusted odds ratios (OR) were as follows: 1 for As < 63.5; 2.16 (95%CI 1.23-3.79) for 63.5 ≤ As> 104, and 2.84 (95%CI 1.64-4.92) for As> 104.
Diabetes mellitus is a complex metabolic disease characterized by an impaired production of insulin by pancreas (type-1 diabetes) or by an insufficient utilization of glucose due to resistance of the liver or/and peripheral tissues to insulin signal (type-2 diabetes). Numerous laboratory studies have demonstrated that iAs and some organic As compounds suppress insulin production by pancreatic?-cells, and modulate glucose uptake by various cells, including adipocytes and myocytes (reviewed [2,4]).
Other studies have shown that exposures to iAs produce either hyper- or hypoglycemia in laboratory animals, depending on the exposure conditions and animal species . However, because of differences in the exposure level, As species, and animal or cellular models, previous laboratory studies provide only a limited insight into the mechanisms of the diabetogenic effects of iAs exposure in humans.
Research in our laboratory has focused mainly on effects of iAs and its metabolites on the insulin-activated signal transduction pathway that regulates the insulin-dependent glucose uptake in peripheral tissues. We found that trivalent arsenicals, arsenite (iAsIII), methylarsonite (MAsIII) and dimethylarsinite (DMAsIII), inhibit insulin-stimulated glucose uptake by cultured murine 3T3-L1 adipocytes at concentrations that do not affect cell viability: 5-100 μM iASIII, 0.5-5 μM MAsIII, and 5-10μM DMAsIII . Examination of individual steps in the insulin-activated signal transduction pathway showed that iAsIII (50 μM) and MAsIII (2 μM) inhibited the phosphorylation of protein kinase-B (PKB/Akt) by phosphoinositide-dependent protein kinase (PDK)-l and 2 (Figure 1), thus preventing the insulin-dependent translocation of GLUT4 transporters from the perinuclear compartment to the plasma membrane (5). In contrast, DMAsIII inhibited GLUT4 translocation by interfering with signaling steps downstream from PKB/Akt. Our findings contrasted sharply those of some of the previous studies that showed high-cytotoxic concentrations of iAsIII or phenylarsine oxide stimulated insulin independent glucose uptake through activation/phosphorylation of the p38 mitogen activated protein kinase (MAPK) (reviewed in ). In our study, the subtoxic concentrations of iAsIII (up to 50 μM) and MAsIII (up to 2 μM) that inhibited insulin-dependent glucose uptake by adipocytes induced phosphorylation of two other MAPKs, ERK 1 and 2, but did not affect phosphorylation of p38 or JNK (Figure 2). Taken together, these data suggest that although exposures to toxic concentrations of iAs may induce glucose uptake by activating stress signaling pathways, exposures to subtoxic concentrations inhibit insulin-activated glucose uptake without inducing stress in a manner consistent with the diabetogenic effects of chronic exposure to iAs.
This paper describes our recent studies that examined effects of chronic exposures to iAsIII and MAsIII in drinking water on fasting blood glucose levels and glucose tolerance in laboratory mice.
iAsIII, sodium salt (99% pure), was purchased from Sigma-Aldrich (St. Louis, MO). MAsIII (methylarsine oxide) was synthesized by Dr. William Cullen (UBC, Vancouver). Sodium borohydride was from EM Science (Gibbstown, NJ) and ultrapure phosphoric acid from J.T. Baker (Phillipsburg, NJ). Sodium arsenate (96%, Sigma), disodium monomethylarsonate (98%, Chem Service, West Chester, PA), dimethylarsinic acid (98%, Strem Chemicals, Inc., Newburyport, MA) and trimethylarsine oxide (gift from Dr. Cullen) were used as standards for speciation analysis of As. All other chemicals were the highest grade available.
Four-week-old male weanling C57BL/6 (B6) mice (The Jackson Laboratory, Bar Harbor, ME, USA) were housed five per cage with free access to food (Lab Diet 5058, Nutrition International, Brentwood, MO) and drinking water. All mice were housed in the University of North Carolina Animal Facility with 12-h light/dark cycle, 22 ± 1°C and humidity of 50 ± 10%. Two independent trials were carried out during which mice were exposed for 8 weeks to iAsIII or MAsIII in drinking water as follows:
Trial 1: Five groups of mice (5 animals each), including controls drinking deionized water (DIW) and mice drinking DIW with iAsIII (1 or 10 ppm As) or MAsIII (0.1 or 1 ppm As).
Trial 2: Five groups of mice (5 animals each), including controls drinking DIW and mice drinking DIW with iAsIII (25 or 50 ppm As) or MAsIII (2.5 or 5 ppm As).
Water containing iAsIII and MAsIII was freshly prepared every 3-4 and every 2 days, respectively. Water consumption in all exposure groups was monitored weekly and body weights every two weeks.
Mice were fasted 5 hours before administration of the IPGTT. D-glucose (Sigma) was dissolved in phosphate buffered saline and administered to mice via i.p. injection (2 g/kg). Samples of whole blood (2-3 μl each) were collected from a tail clip bleed immediately before (the fasting blood) and 15, 30, 60, 90, and 120 min after glucose injection. Blood glucose levels were measured using a Freestyle Glucose Monitoring System (Abbott Laboratories, Abbott Park, IL). After IPGTT, mice were sacrificed and liver, pancreas, quadriceps and adipose tissues were collected for analysis of As species.
Dissected tissues were snap frozen in liquid N2 and stored at -70° C. Prior to analysis, tissues were digested overnight in 2 M ultrapure phosphoric acid (90°C) . The digestion converts all trivalent arsenicals to pentavalency. Pentavalent arsenicals in digestates were determined by a cryotrap-coupled hydride generation-atomic absorption spectrometry (HG-AAS), using a Perkin-Elmer model 5100 atomic absorption spectrometer (PerkinElmer, Norwalk, CT, USA) as previously described . This method routinely resolves arsines generated from arsenate (iAsV), methylarsonate (MAsV), dimethylarsinate (DMAsV) and trimethylarsine oxide (TMAsVO). Five concentrations (0.05, 0.25, 0.5, 1 and 2.5 ng/ml) of each of these arsenicals were used to prepare calibration curves. Arsines generated from tissue samples were identified by spiking with standards. The concentration of total speciated As for each tissue sample was calculated as the sum of concentrations of iAsV, MAsV, DMAsV, and TMAsVO. The total As was analyzed in microwave digested tissues by graphite furnace (GF)-AAS, using a Perkin-Elmer model 5100 atomic absorption spectrometer . Recovery of As during HG-AAS analysis was calculated as the concentration of total speciated As (determined by HG-AAS) divided by the concentration of total As (determined by GF-AAS). HG-AAS was also used for analysis of As species in the laboratory diet.
Data collected in this study were evaluated by analysis of variance with Tukey multiple comparison posttest using a GraphPad Instat statistical software package (GraphPad Software, San Diego, CA). Differences among means with p < 0.05 were considered statistically significant.
Average daily As intake from drinking water per mouse (Figure 3) was calculated based on water consumption. Control mice and mice in most exposure groups consumed an average of 4 to 5 ml of water per day. However, mice exposed to iAs at the 25 and 50 ppm As levels consumed in average only 3.8 ml and 2.5 ml per day, respectively. The laboratory diet used in Trial 1 contained 47 to 66 ppb of total As; the total As levels in diet used in Trial 2 ranged from 20 to 29 ppb. iAs represented 70 to 80% of the total As in the diets. Over the 8-week period, mice in all treatment groups gained about 7 g of weight with exception of mice exposed to iAs at 50 ppm As level which gained in average only 5.3 g (data not shown). However, this difference was not statistically significant. No significant differences were noted in liver weights between experimental groups (data not shown). No overt signs of pathology were observed in tissues of As-treated mice.
Arsenic species were analyzed in pancreas, the insulin producing tissue, and in the major glucose metabolizing tissues, including liver, muscle (quadriceps) and adipose tissue (Figure 4). In mice exposed to iAsIII and to high concentrations of MAsIII (2.5 and 5 ppm As) the levels of total speciated As in these tissues generally correlated with the concentration of iAsIII in drinking water and with the estimated As intake. No such correlation was found for mice exposed to low concentrations of MAsIII. Concentrations of total speciated As in adipose tissues and quadriceps were lower in mice drinking water with low concentrations of MAsIII (0.1 and/or 1 ppm As) compared to control mice, suggesting that exposure to MAsIII mobilized As species that originated from the diet and were retained in these tissues. In addition, the tissue concentrations of As species significantly differed between the control mice in Trial 1 and 2. These differences may reflect differences in the content and speciation of As in diets used in both trials, as well as variations in diet composition. DMAsV and MAsV represented the major fraction of As in most tissues, particularly in mice exposed to the highest levels of iAsIII or MAsIII. Because of a limited access to GF-AAS, the total As contents were analyzed only in livers and quadriceps from mice exposed to 50 ppm As as iAsIII. The comparison of data provided by GF-AAS and HG-AAS showed that the average recovery of As during HG-AAS analysis was 110% for skeletal muscle and 105% for the liver.
Glucose tolerance in mice in all exposure groups was assessed by IPGTT. Figure 5 shows IPGTT profiles for mice in the highest iAsIII and MAsIII exposure groups and in the corresponding control groups. No significant differences in fasting blood glucose levels were found between the control mice and mice exposed to iAsIII or MAsIII. All groups exhibited the characteristic rapid rise in blood glucose within 15-30 min of glucose challenge, followed by a gradual decrease in blood glucose level. With exception of mice exposed to 50 ppm As as iAsIII, no significant differences were found in the IPGTT profiles among the experimental groups. The average 15-, 30-and 60-min blood glucose concentrations were significantly higher in mice from the 50 ppm As group as compared to the corresponding control values (Figure 5A).
Our results show that exposure to iAsIII in drinking water can induce impaired glucose tolerance in B6 mice. However, the concentration of iAs in drinking water needed to produce this effect (50 ppm) is order of magnitude higher than iAs concentrations that increase risk of diabetes in humans. For example, in Coahuila (Mexico) exposures to 63.6 to 104 ppb As in drinking water have been shown to significantly increase OR (2.16) for developing diabetes (3). In arseniasis-endemic areas of Bangladesh, the concentrations of As in drinking water reach up to 3.4 ppm . Samples of liver from Bangladeshi residents who developed hepatomegaly as a result of drinking water with 0.22 to 2 ppm As contained up to 6,000 μg As/kg dry weight  (i.e., 1,200 μg As/kg of intact liver). In this study, a similar average concentration of total speciated As (1,165 μg As/kg) was found in livers of mice drinking water with 50 ppm As as iAsIII. For comparison, livers of mice exposed 1 ppm As as iAsIII contained on average only 11 μg to As/kg. These data are consistent with the higher rate of iAs metabolism and clearance in mice compared to humans [11,12]. Thus, significantly higher exposure levels are needed to produce in mice the symptoms of chronic iAs toxicity (including impaired glucose tolerance) described for humans.
The concentrations of total speciated As in tissues of mice that developed impaired glucose tolerance after drinking water with iAsIII (50 ppm As) ranged from 20 to 1,165 μg/kg (i.e., ~0.27 to 15.5 μM). These concentrations are in a good agreement with the concentrations of trivalent arsenicals that inhibited the insulin-stimulated glucose uptake by cultured 3T3-L1 adipocytes: ≥ 0.5 μM MAsIII and ≥ 5 μM iAsIII or DMAsIII . MAsIII was more potent than either iAsIII or DMAsIII as an inhibitor of the insulin-stimulated glucose uptake and insulin signaling in adipocytes. In contrast, exposures to MAsIII in drinking water (up 5 ppm As) had no effects on glucose tolerance in B6 mice (Figure 5). However, concentrations of total speciated As in tissues of these mice were much lower that those found in tissues of mice exposed to 50 ppm As as iAsIII (Figure 4) which developed impaired glucose tolerance. Thus, it is reasonable to hypothesize that the diabetogenic effects of iAs exposure are associated with accumulation of critical amounts of As metabolites, possibly MAsIII, in tissues that regulate insulin production and/or in the major glucose metabolizing tissues.
This work has been supported by a US EPA Cooperative Agreement 282952201 and a Clinical Nutrition Research Center Grant DK 56350 from NIH. This manuscript has been reviewed in accordance with the policy of the National Health and Environmental Effects Research Laboratory, U.S. EPA, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.