Metabolism and disposition of arsenic species from controlled dosing with dimethylarsinic acid (DMAV) in adult female CD-1 mice. V. Toxicokinetic studies following oral and intravenous administration.

Arsenic species contaminate food and water, with typical dietary intake below 1 µg/kg bw/d. Exposure to arsenic in heavily contaminated drinking water is associated with human diseases, including cardiovascular and respiratory disorders, diabetes, and cancer. Dietary intake assessments show that rice and seafood are the primary contributors to intake of both inorganic arsenic and dimethylarsinic acid (DMAV) and at similar magnitudes. DMAV plays a central role in the toxicology of arsenic because enzymatic methylation of arsenite produces DMAV as the predominant metabolite, which may promote urinary clearance but also generates reactive intermediates, predominantly DMAIII, that bind extensively to cellular thiols. Both inorganic arsenic and DMAV are carcinogenic in chronically exposed rodents. This study measured pentavalent and trivalent arsenic species in blood and tissues after oral and intravenous administration of DMAV (50 µg As/kg bw). DMAV underwent extensive first-pass metabolism in the intestine and liver, exclusively by reduction to DMAIII, which bound extensively to blood and tissues. The results confirm a role for methylation-independent reductive metabolism in producing fluxes of DMAIII that presumably underlie arsenic toxicity and indicate the need to include all dietary intake of inorganic arsenic and DMAV in risk assessments.

The distribution in tissues and urine of arsenic metabolites after subchronic exposure to dimethylarsinic acid (DMAV) in rats.

Dimethylarsinic acid (DMA(V)) acted as cancer promoter promoted urinary bladder, liver, and lung carcinogenesis in rats. Understanding of the distribution of arsenicals in critical sites will aid to define the action of DMA(V)-induced toxicity and carcinogenicity. The present experiment was conducted to compare the accumulated levels of arsenicals in the liver, kidney, and bladder of both male and female rats after subchronic exposure to DMA(V). After exposure to DMA(V) in drinking water for 10 weeks, urinary DMA concentrations of 100 and 200 ppm DMA(V)-treated rats increased significantly compared with those of the control rats. Smaller amount of trimethylarsinic acid (TMA) was detected in urine, but not in liver, kidney, and bladder muscle. In the liver and kidney, the levels of DMA in DMA(V)-treated rats significantly increased compared with those of the control group, but there was no difference between 100 and 200 ppm DMA(V)-treated rats. DMA did not accumulate in bladder muscle. There was no difference for DMA concentrations between male and female rats. Our results suggest that the accumulation of DMA in the liver and kidney was saturated above 100 ppm DMA(V) treatment concentration, and DMA(V) was a little partly metabolized to TMA, and TMA was rapidly excreted into urine.

Time course of urothelial changes in rats and mice orally administered arsenite.

Inorganic arsenic (arsenite and arsenate) at high exposures is a known human carcinogen, inducing tumors of the urinary bladder, skin, and lungs. In two experiments, we examined the urothelial proliferative effects of treatment with 173 ppm sodium arsenite (100 ppm arsenic) in the drinking water for 6 and 24 hr, and 3, 7, and 14 days in female F344 rats and 43.3 ppm sodium arsenite (25 ppm arsenic) in female C57BL/6 wild-type and arsenic (+3 oxidation state) methyltransferase knockout (As3mt KO) mice that are unable to methylate arsenicals. In the rat and both mouse genotypes, scanning electron microscopy showed cytotoxic urothelial changes as early as 6 hr after the start of arsenic exposure. The severity of As(III)-induced cytotoxic urothelial changes increased over time in the rat and in the As3mt KO mouse. Light microscopy showed an increase in urothelial hyperplasia in the rat. No significant increases in bromodeoxyuridine-labeling index were observed. The data support the hypothesis that the sequence of events in the mode of action for urothelial effects of orally administered inorganic arsenic in the rat and mouse involves superficial cytotoxicity with consequent regenerative increased cell proliferation similar to the findings associated with the administration of dimethylarsinic acid (DMA(V)) in rats.

Effect of topical dimethylarsinic acid on the expression of apoptosis-related proteins in mouse skin.

We investigated the effect of the topical application of dimethylarsinic acid (DMA) on skin thickness and the expression of several apoptosis-related proteins in skin. After administration of DMA during pregnancy, skin thickness and skin expression of Bcl-2, Bcl-3, Bad, Bid, and caspases-3, -6, -8, -9, and -12 were examined in dams and their offspring. DMA treatment caused significant increases in skin thickness (p < 0.05) and the expression of Bcl-2, Bad, and capase-12 in the skin of dams at the mRNA and protein levels (p < 0.01). However, maternal exposure to DMA did not significantly alter the expression of the studied apoptosis-related factors in the skin of the offspring. These findings indicate that DMA may induce skin apoptosis, in part, by modulating the expression of Bcl-2, Bad, and caspase-12 in maternal skin. Additionally, our results suggest that maternal exposure to DAM during pregnancy may not induce apoptosis in the skin of the offspring.

Dimethylarsinic acid in drinking water changed the morphology of urinary bladder but not the expression of DNA repair genes of bladder transitional epithelium in F344 rats.

Inorganic arsenic increases urinary bladder transitional cell carcinoma in humans. In F344 rats, dimethylarsinic acid (DMA[V]) increases transitional cell carcinoma. Arsenic-induced inhibition of DNA repair has been reported in cultured cell lines and in lymphocytes of arsenic-exposed humans, but it has not been studied in urinary bladder. Should inhibition of DNA damage repair in transitional epithelium occur, it may contribute to carcinogenesis or cocarcinogenesis. We investigated morphology and expression of DNA repair genes in F344 rat transitional cells following up to 100 ppm DMA(V) in drinking water for four weeks. Mitochondria were very sensitive to DMA(V), and swollen mitochondria appeared to be the main source of vacuoles in the transitional epithelium. Real-time reverse transcriptase polymerase chain reaction (Real-Time RT PCR) showed the mRNA levels of tested DNA repair genes, ataxia telangectasia mutant (ATM), X-ray repair cross-complementing group 1 (XRCC1), excision repair cross-complementing group 3/xeroderma pigmentosum B (ERCC3/XPB), and DNA polymerase beta (Polbeta), were not altered by DMA(V). These data suggested that either DMA(V) does not affect DNA repair in the bladder or DMA(V) affects DNA repair without affecting baseline mRNA levels of repair genes. The possibility remains that DMA(V) may lower damage-induced increases in repair gene expression or cause post-translational modification of repair enzymes.

Effects of co-administration of antioxidants and arsenicals on the rat urinary bladder epithelium.

Oxidative stress has been increasingly recognized as a possible mechanism in the toxicity and carcinogenicity of various chemicals, including arsenic. Therefore, treatment with antioxidants may afford a protective effect against arsenic-induced cytotoxicity and carcinogenesis. Dimethylarsinic acid (DMAV) has been shown to be a bladder carcinogen in rats when administered at high doses (100 ppm) in the diet or in the drinking water. The main purpose of the present study was to evaluate the effects of co-administration of antioxidants with arsenicals on the rat urinary bladder epithelium in vitro and in vivo. In a previous experiment, treatment with 1000 ppm melatonin for two weeks did not inhibit cell proliferation induced in the rat urothelium by 100 ppm DMAV. In the current study, we examined the effects of five antioxidants that act via different mechanisms, on the in vitro cytotoxicity of various arsenicals, for the purpose of determining which antioxidants might have protective effects against arsenic-induced cytotoxicity. The antioxidants that inhibited cytotoxicity in vitro were then studied also in vivo. Melatonin showed slight inhibition of the cytotoxicity of arsenite, but had no effect on the other arsenicals. N-acetylcysteine (NAC) inhibited the cytotoxicity of monomethylarsonous acid (MMAIII), DMAV, dimethylarsinous acid (DMAIII), and trimethylarsine oxide (TMAO). Vitamin C inhibited cytotoxicity induced by arsenate, arsenite, MMAIII) and DMAIII. Tiron and Trolox had no effect on the cytotoxicity of any arsenical. The in vitro inhibitory effects of NAC and vitamin C on DMAV and on DMAIII, suggested that these antioxidants might afford preventive effects on DMAV-induced bladder cytotoxicity and carcinogenesis in rats. To test this hypothesis, a 10-week rat bioassay was conducted. Melatonin was also included to clarify the results of the previous two-week experiment. The sodium salt of vitamin C (Na-Asc), but not melatonin or NAC, inhibited the proliferative effects of DMAV on the bladder epithelium in rats. These results suggest that oxidative stress is at least in part involved in DMAV-induced rat bladder toxicity and proliferation, and therefore, vitamin C may afford inhibitory effects in DMAV-induced bladder carcinogenesis in rats. Microarray analysis of DMAV-responsive genes revealed that DMAV did not have a consistent modifying effect on gene expression in the rat bladder epithelium, suggesting that proteins and/or lipids may be the targets of damage by DMAV-induced oxidative stress.

Biokinetics and subchronic toxic effects of oral arsenite, arsenate, monomethylarsonic acid, and dimethylarsinic acid in v-Ha-ras transgenic (Tg.AC) mice.

Previous research demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment increased the number of skin papillomas in v-Ha-ras transgenic (Tg.AC) mice that had received sodium arsenite [(As(III)] in drinking water, indicating that this model is useful for studying the toxic effects of arsenic in vivo. Because the liver is a known target of arsenic, we examined the pathophysiologic and molecular effects of inorganic and organic arsenical exposure on Tg.AC mouse liver in this study. Tg.AC mice were provided drinking water containing As(III), sodium arsenate [As(V)], monomethylarsonic acid [(MMA(V)], and 1,000 ppm dimethylarsinic acid [DMA(V)] at dosages of 150, 200, 1,500, or 1,000 ppm as arsenic, respectively, for 17 weeks. Control mice received unaltered water. Four weeks after initiation of arsenic treatment, TPA at a dose of 1.25 microg/200 microL acetone was applied twice a week for 2 weeks to the shaved dorsal skin of all mice, including the controls not receiving arsenic. In some cases arsenic exposure reduced body weight gain and caused mortality (including moribundity). Arsenical exposure resulted in a dose-dependent accumulation of arsenic in the liver that was unexpectedly independent of chemical species and produced hepatic global DNA hypomethylation. cDNA microarray and reverse transcriptase-polymerase chain reaction analysis revealed that all arsenicals altered the expression of numerous genes associated with toxicity and cancer. However, organic arsenicals [MMA(V) and DMA(V)] induced a pattern of gene expression dissimilar to that of inorganic arsenicals. In summary, subchronic exposure of Tg.AC mice to inorganic or organic arsenicals resulted in toxic manifestations, hepatic arsenic accumulation, global DNA hypomethylation, and numerous gene expression changes. These effects may play a role in arsenic-induced hepatotoxicity and carcinogenesis and may be of particular toxicologic relevance.

Urinary sulfur-containing metabolite produced by intestinal bacteria following oral administration of dimethylarsinic acid to rats.

Our long-term oral administration of dimethylarsinic acid (DMAV) in rats revealed that three unidentified metabolites, M-1, M-2, and M-3, were detected in urine and feces. DMAV and trimethylarsine oxide (TMAO) were converted to M-2 and M-3 and M-1 by Escherichia coli strain A3-6 isolated from the ceca of DMAV-administered rats, respectively. In this study, we report on the mechanism of production and the chemical properties of these unknown metabolites. To investigate the pattern of conversion of DMAV or TMAO by A3-6 in the presence of cysteine (Cys), arsenic metabolites of DMAV or TMAO in medium after incubation with A3-6 and Cys were analyzed by liquid chromatography with inductively coupled plasma mass spectrometry (LC-ICP-MS). DMAV was reduced to dimethylarsinous acid (DMAIII) to form M-2 in the presence of Cys and A3-6, and M-2 was further converted to M-3. TMAO was rapidly converted to M-1 by A3-6. The cytotoxicity of the unidentified metabolites was investigated. M-2 was more cytotoxic than DMAV, M-1, and M-3 in V79 cells. The cytotoxicity of M-2 in HL-60 cells was decreased by the addition of superoxide dismutase, suggesting that the cytotoxicity of M-2 might be due to the production of reactive oxygen species. In addition, we examined the chemical properties of M-2 by LC-ICP-MS and LC-MS. M-2 was oxidized to DMAV by hydrogen peroxide, suggesting that M-2 may be a reduced form of DMAV. M-2 was consistent with the reactant of DMAV with metabisulfite-thiosulfate reagent but not DMAIII by analyses of LC-ICP-MS and LC-MS. The molecular weight of M-2 was 154, and M-2 was a sulfur-containing metabolite.

Urothelial cytotoxicity and regeneration induced by dimethylarsinic acid in rats.

Inorganic arsenic is a known human carcinogen of the skin and respiratory tract. Epidemiologic evidence indicates that it is also carcinogenic to the urinary bladder and other internal organs. Lack of an animal model has limited progress on understanding the mechanism of arsenic carcinogenesis. It was recently reported that high doses of an organic arsenical, dimethylarsinic acid (DMA), increased urinary bladder tumors in rats when administered in the diet or in the drinking water for 2 years, with the female being more sensitive than the male. We previously showed that high doses of DMA (40 or 100 ppm of the diet) fed for 10 weeks increased urothelial cell proliferation in the rat. Treatment with DMA also increased renal calcification and increased urinary calcium concentration. In 2 experiments, we examined the urothelial proliferative effects of treatment with 100 ppm DMA in the diet in female F344 rats for 2 and 10 weeks and for 6 and 24 h, and 3, 7, and 14 days. Cytotoxic changes in the urothelium were evident by SEM as early as 6 h after treatment was begun. Foci of cellular necrosis were detected after 3 days of treatment, followed by widespread necrosis of the urothelium after 7 days of treatment. The bromodeoxyuridine (BrdU) labeling index was not increased until after 7 days of treatment, suggesting that administration of DMA results in cytotoxicity with necrosis, followed by regenerative hyperplasia of the bladder epithelium. Although the rat provides an animal model to study the urothelial effects of DMA, the relevance of this finding to inorganic arsenic carcinogenesis in humans must be extrapolated cautiously, due to the high doses of DMA necessary to produce these changes in the rat and the differences in metabolism of arsenicals in rodents, especially rats, compared to humans.

Dose-dependent effects on tissue distribution and metabolism of dimethylarsinic acid in the mouse after intravenous administration.

Most mammals methylate inorganic arsenic to dimethylarsinic acid (DMA). This organic arsenical causes organ-specific toxicity and is a multi-organ tumor promoter. The objective of this study was to examine whether dose could affect the distribution and metabolism of DMA. Female B6C3F1 mice (3-4/time point) were administered 1.11 or 111 mg/kg of DMA (1 microCi of [14C] or unlabeled) intravenously and killed serially (5-480 min). Blood was separated into plasma and red blood cell fractions and liver, kidney and lung were removed, weighed and homogenized. Tissue samples were oxidized and analyzed for DMA-derived radioactivity. Blood and several organs of the non-radioactive DMA-treated animals were digested in acid and analyzed by hydride generation atomic absorption spectrophotometry for DMA and metabolites. Concentration-time profiles showed a biexponential decrease of DMA-derived radioactivity in all tissues examined. Kidney had the highest concentration (1-20% dose/gm) of radioactivity of all tissues up to 60 min post-administration. Concentration of radioactivity was greater in plasma than red blood cells at 5 and 15 min and then was similar for the remaining time points. A dose-dependent effect on the concentration of radioactivity was observed in the lung. The retention of radioactivity in the lung was altered compared with liver and kidney, with a much longer t1/2beta and a disproportionate increase in area under the curve with increased dose. No methylated or demethylated products of DMA were detected in blood or any organ up to 8 h post-exposure. The dose-dependent distribution of DMA in the lung may have a role in the toxic effects DMA elicits in this organ.

Effects of dietary dimethylarsinic acid on the urine and urothelium of rats.

Dimethylarsinic acid (DMA), fed to rats for 2 years, produced bladder hyperplasia and tumors at doses of 40 and 100 p.p.m., more in females than males. No urothelial proliferation was seen in mice. Our objectives were to investigate the mode of action of bladder tumor formation, evaluate the dose-response and the role of diet and to determine if the urothelial effects were reversible. The study included groups of female F344 rats fed DMA in Purina 5002 diet at doses of 0, 2, 10, 40 or 100 p.p.m. for 10 weeks; two groups of females fed DMA (0 and 100 p.p.m.) in Altromin 1321 for 10 weeks; two groups of males fed DMA (0 and 100 p.p.m.) in Purina 5002 for 10 weeks; a female high-dose recovery group (100 p.p.m. in Purina 5002 diet for 10 weeks followed by control diet for 10 weeks); and two female groups (0 and 100 p.p.m.) in Purina diet for 20 weeks. Urothelial toxicity and hyperplasia were detected by light and scanning electron microscopy (SEM), and the bromodeoxyuridine labeling index was increased in the female 40 and 100 p.p.m. groups. The effects were less in males, but were similar in females fed DMA in Altromin 1321. SEM detected no abnormal urinary solids related to treatment in any group. Urinary calcium was increased in the females fed 40 and 100 p.p.m. in Purina diet, despite overall urinary dilution. Calcification was increased in kidneys of female rats fed Purina diet. The urothelial effects of DMA were reversible. The findings support a non-DNA reactive mechanism for DMA rat bladder carcinogenicity related to urothelial toxicity and regeneration. The toxicity is probably not due to urinary solids. The toxicity and regeneration are produced in a dose-responsive manner in female rats, are greater in female than in male rats, and are reversible.

Urinary bladder carcinogenicity of dimethylarsinic acid in male F344 rats.

The present study was conducted to determine the carcinogenicity of dimethylarsinic acid (DMA) administered to male F344 rats in a 2 year bioassay. A total of 144 rats (10 weeks old at the start) were divided into four groups of 36 rats each. Groups 1-4 received DMA (purity 100%) at concentrations of 200, 50, 12.5 and 0 p.p.m. in the drinking water, respectively, for 104 weeks. From weeks 97 to 104, urinary bladder tumors were observed in 12 of 31, eight of 31 and none of 33 in groups 1-3, respectively. No bladder tumors were observed in group 4. The present study demonstrated that long-term p. o. administration of DMA induced urinary bladder carcinomas in male F344 rats. Therefore, the results indicate that DMA is carcinogenic for the rat urinary bladder, which may be related to the human carcinogenicity of arsenicals.

Dose-dependent effects on the disposition of monomethylarsonic acid and dimethylarsinic acid in the mouse after intravenous administration.

The organic arsenicals monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) are the primary metabolites of inorganic arsenic, a known human carcinogen. The objective of this study was to examine if dose would affect the excretion and terminal tissue disposition of MMA and DMA in the mouse. 14C-MMA (4.84 and 484 mumol/kg) and -DMA (8.04 and 804 mumol/kg) were administered to female mice via the tail vein. The mice were placed in metabolism cages for collection of urine (1, 2, 4, 8, 12, and 24 h) and feces (24 h). The animals were then sacrificed at 24 h and tissues were removed and analyzed for radioactivity. The urine was also analyzed for parent compound and metabolites. Urinary excretion of MMA- and DMA-derived radioactivity predominated over fecal excretion. Dose did not affect the overall urinary excretion of both compounds. However, fecal excretion was significantly lower in the low-dose MMA-treated animals as opposed to in the high-dose group, whereas in the high-dose DMA-treated group excretion was lower than in the low-dose DMA group. The retention of radioactivity was low (< 2% of dose) and the distribution pattern similar for both compounds, with carcass > liver > kidney > lung. The concentration of radioactivity (% dose/g tissue) was greater in kidney than in liver, lung, and blood for both compounds. The distribution and concentration of MMA-derived radioactivity was significantly greater in the liver and lung of the high-dose group. The MMA-treated animals excreted predominantly MMA in urine and lower amounts of DMA (< 10% of the dose). The percentage excreted as DMA was significantly higher in the low-dose MMA group. In the urine of DMA-treated animals, an unstable metabolite and the parent compound were detected. Overall, it appears the dose of organic arsenical administered has a minimal effect on its excretion and terminal tissue disposition in the mouse. The rapid elimination and low retention of MMA and DMA explain in part their low acute toxicity.

Cellular response to oxidative damage in lung induced by the administration of dimethylarsinic acid, a major metabolite of inorganic arsenics, in mice.

Oral administration of dimethylarsinic acid (DMAA), a major metabolite of inorganic arsenics, induces DNA damage in the mouse and rat lung due to both active oxygens and dimethylarsenic peroxyl radical produced in the metabolism of DMAA. Our paper describes the cellular response to DMAA in the mouse lung. In male ICR mice given a single po dose (1500 mg/kg) of DMAA-Na, the activities of mitochondrial superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase significantly increased at 6 hr or longer after dosing, while cytosolic superoxide dismutase and catalase did not. With regard to cellular sulfhydryls after DMAA dosing, levels of reduced glutathione and nonprotein sulfhydryl decreased, while mixed disulfides significantly increased. Further, NADPH markedly decreased at 6-9 hr after DMAA dosing. These cellular variations suggest that the mouse pulmonary cell produced active oxygens, i.e., superoxide anion radical, hydrogen peroxide, and subsequent radicals in the metabolism of DMAA and that these and also the dimethylarsenic peroxyl radical were responsible for pulmonary DNA damage.