Major Metabolite Of Inorganic Arsenics Research Articles

Cytotoxic effects of S-(dimethylarsino)-glutathione: A putative intermediate metabolite of inorganic arsenicals

Glutathione (GSH) plays an important role in the metabolism of arsenite and arsenate by generating arsenic-glutathione complexes. Although dimethylarsinic acid (DMA V) is the major metabolite of inorganic arsenicals (iAs) in urine, it is not clear how DMA V is produced from iAs. In the present study we report that S-(dimethylarsino)-glutathione (DMA III(SG)), a putative precursor of dimethylarsinic acid DMA V, was unstable in the culture medium without excess GSH and generated volatile substances which were highly cytotoxic for both rat heart microvascular endothelial cells and HL60, a human leukemia cell line. Cytotoxicity of DMA III(SG) was higher than that of iAs and its LC 50 value was calculated to be 7.8 μM in the endothelial cells. To our surprise DMA III(SG) effectively killed cells in the neighbor wells of the same multi-well dish, indicating that volatile toxic compounds generated from DMA III(SG) in the culture medium. High performance lipid chromatography–inductively coupled plasma mass spectrometry (HPLC–ICPMS) analyses suggested that the freshly generated volatile compounds dissolved into aqueous solution and formed an unstable arsenic compound and the unstable compound was further converted to DMA V. These results suggested that DMA III(SG) exerts its cytotoxicity by generating volatile arsenicals and is implicated in the metabolic conversion of inorganic arsenicals into DMA V, a major final metabolite of inorganic arsenicals in most mammals.

Induction of glutathione S-transferase placental form positive foci in liver and epithelial hyperplasia in urinary bladder, but no tumor development in male Fischer 344 rats treated with monomethylarsonic acid for 104 weeks

The carcinogenicity of monomethylarsonic acid (MMA(V)), a major metabolite of inorganic arsenics in human and experimental animals, was investigated in male Fischer 344 rats. A total of 129 rats at 10 weeks of age were randomly divided into three groups and received drinking water containing MMA(V) at doses of 0 (Control), 50, and 200 ppm ad libitum for 104 weeks. No significant differences were found between the control and the MMA(V)-treated groups regarding clinical signs, mortality, hematological, and serum biochemistry findings. Quantitative analysis of glutathione S-transferase placental form (GST-P) positive foci in liver revealed a significant increase of numbers and areas in the 200 ppm MMA(V)-treated group. In the urinary bladder MMA(V) induced simple hyperplasia and significantly elevated the proliferating cell nuclear antigen (PCNA)-positive index in the urothelium. A variety of tumors developed in rats of all groups, including the controls, but all were histologically similar to those known to occur spontaneously in F344 rats and there were no significant differences among the groups. Thus, it could be concluded that, under the present experimental conditions, MMA(V) induced lesions in the liver and urinary bladder, but did not cause tumor development in male F344 rats even after 2 years exposure.

Promoting effects of monomethylarsonic acid, dimethylarsinic acid and trimethylarsine oxide on induction of rat liver preneoplastic glutathione S-transferase placental form positive foci: a possible reactive oxygen species mechanism.

Dimethylarsinic acid (DMA) is a major metabolite of inorganic arsenicals, which are epidemiologically significant chemicals in relation to liver cancer in mammals. The present study was conducted to determine the promoting effects of organic arsenicals related to DMA [monomethylarsonic acid (MMA) and trimethylarsine oxide (TMAO)] on rat liver carcinogenesis using a liver medium-term bioassay (the Ito test). Male, 10-week-old, F344 rats were given a single i.p. injection of diethylnitrosamine at a dose of 200 mg/kg b.w. as an initiator. Starting 2 weeks thereafter they received 100 ppm of MMA, DMA or TMAO in their drinking water, or no supplement as a control, for 6 weeks. All animals underwent 2/3 partial hepatectomy in week 3 after initiation. Quantification of glutathione S-transferase placental form (GST-P)-positive foci as preneoplastic lesions in liver sections revealed significantly increased numbers and areas in all 3 treated groups compared with controls. Hepatic microsome cytochrome P-450 content was markedly increased with all 3 arsenic treatments. Markedly elevated CYP 2B1 protein levels and CYP 2B1/2 mRNA levels were thus observed in all cases. The potency of promotion was similar for MMA, DMA and TMAO. Since hydroxyradicals were found to be generated in the relatively early phase while methylated arsenicals were metabolized in liver, the resultant oxidative stress might have promoted lesion development.

A review of animal models for the study of arsenic carcinogenesis

As inorganic arsenic is a proven human carcinogen, significant effort has been made in recent decades in an attempt to understand arsenic carcinogenesis using animal models, including rodents (rats and mice) and larger mammals such as beagles and monkeys. Transgenic animals were also used to test the carcinogenic effect of arsenicals, but until recently all models had failed to mimic satisfactorily the actual mechanism of arsenic carcinogenicity. However, within the past decade successful animal models have been developed using the most common strains of mice or rats. Thus dimethylarsinic acid (DMA), an organic arsenic compound which is the major metabolite of inorganic arsenicals in mammals, has been proven to be tumorigenic in such animals. Reports of successful cancer induction in animals by inorganic arsenic (arsenite and arsenate) have been rare, and most carcinogenetic studies have used organic arsenicals such as DMA combined with other tumor initiators. Although such experiments used high concentrations of arsenicals for the promotion of tumors, animal models using doses of arsenicals species closed to the exposure level of humans in endemic areas are obviously the most significant. Almost all researchers have used drinking water or food as the pathway for the development of animal model test systems in order to mimic chronic arsenic poisoning in humans; such pathways seem more likely to achieve desirable results.

Lack of promoting effect due to oral administration of dimethylarsinic acid on rat lung carcinogenesis initiated with N-bis(2-hydroxypropyl)nitrosamine

Dimethylarsinic acid (DMA), a major metabolite of inorganic arsenics, and arsenic exposure is associated with tumor development in a wide variety of human tissues. In the present study, we examined whether DMA has tumor-promoting activity on rat lung carcinogenesis initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). Male, 8-week-old, F344 rats were treated with DHPN at a concentration of 0.1% in drinking water for 1 week, and starting 1 week thereafter, DMA was administered at concentrations of 0, 100, 200 or 400 ppm in the drinking water for 30 weeks. Induction of epithelial lesions, classified as alveolar epithelial hyperplasia, adenoma, and adenocarcinoma was evident in the lungs of DHPN-initiated animals, but no significant differences were found between DMA-treated groups and control groups. Furthermore, no significant differences in 5-bromo-2′-deoxyuridine labeling indices, as a marker of cell proliferation were observed among the groups. An additional group treated with DMA at concentrations of 200 ppm alone, without prior DHPN initiation was found to develop no epithelial lesions in the lung. There was no significant gain in 8-hydroxydeoxyguanosine formation, as a marker of oxidative stress, in the lungs of rats treated with DMA in their drinking water. In conclusion, oral-administered DMA does not exert promoting effects on rat lung carcinogenesis initiated with DHPN.

Promotion of skin carcinogenesis by dimethylarsinic acid in keratin (K6)/ODC transgenic mice.

Dimethylarsinic acid (DMA) is a major metabolite of inorganic arsenicals in mammals, and arsenic exposure is associated with tumor development in a wide variety of human tissues, particularly the skin. Transgenic mice with ornithine decarboxylase (ODC) targeted to hair follicle keratinocytes are much more sensitive than littermate controls to carcinogens. In this study we investigated the promoting effect of DMA on skin carcinogenesis in such K6/ODC transgenic mice. The back skin of female C57BL/6J K6/ODC transgenic mice, 10 to 14 weeks old, was initiated with topical application of 7,12‐dimethylbenz[α]anthracene (DMBA) at a dose of 50 μg or acetone alone on day 1 of the experiment, followed by treatment with 3.6 mg of DMA, 5 μg of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) or neutral vehicle (control) twice a week for 18 weeks. Mice were killed 1 week after the end of the treatment. Induction of skin tumors was significantly accelerated in the DMA‐treated group, as well as in the TPA‐treated group, indicating that DMA has a promoting effect on skin tumorigenesis in K6/ODC transgenic mice.

Promotion of NCI-Black-Reiter male rat bladder carcinogenesis by dimethylarsinic acid an organic arsenic compound

Dimethylarsinic acid (DMAA) is a major metabolite of inorganic arsenicals in mammals. In the present study, we investigated its promoting effects on urinary bladder carcinogenesis in NCI-Black-Reiter (NBR) rats, which lack α2u-globulin synthesizing ability. Male 9–14-week-old NBR rats were treated sequentially with 0.05% N-butyl- N-(4-hydroxybutyl)-nitrosamine (BBN) for 4 weeks and then given 100 ppm DMAA in their drinking water (group 1) for 32 weeks. Induction of preneoplastic lesions (papillary or nodular hyperplasia) in this DMAA-treated group was significantly increased as compared to the carcinogen alone control group ( P<0.01). The development of carcinomas was also enhanced and a significant increase in the 5-bromo-2′-deoxyuridine (BrdU) labeling index of the urinary bladder epithelial cells was observed for the DMAA treatment group. These results indicate that DMAA has promoting effects on urinary bladder carcinogenesis even in NBR rats, so its effects are not dependent on the presence of α2u-globulin.

Promotion of rat hepatocarcinogenesis by dimethylarsinic acid: association with elevated ornithine decarboxylase activity and formation of 8-hydroxydeoxyguanosine in the liver.

Arsenicals are epidemiologicaUy significant chemicals in relation to induction of liver cancer in man. In the present study, we investigated the dose‐dependent promotion potential of dimethylarsinic acid (DMAA), a major metabolite of inorganic arsenicals in mammals, in a rat liver carcinogenesis model. In experiment 1, glutathione‐S‐transferase placental form (GST‐P)‐positive foci, putative preneoplas‐tic lesions, were employed as endpoints of a liver medium‐term bioassay for carcinogens (Ito test). Starting 2 weeks after initiation with diethylnitrosamine, male F344 rats were treated with 0, 25, 50 or 100 ppm of DMAA in the drinking water for 6 weeks. All animals underwent two‐thirds partial hepatectomy at week 3 after initiation. Examination of liver sections after termination at 8 weeks revealed dose‐dependent increases in the numbers and areas of GST‐P‐positive foci in DMAA‐treated rats as compared with controls. In experiment 2, ornithine decarboxylase activity, which is a biomarker of cell proliferation, was found to be significantly increased in the livers of rats treated with DMAA. In experiment 3, formation of 8‐hydroxydeoxyguanosine, which is a marker of oxygen radical‐mediated DNA damage, was significantly increased after administration of DMAA. These results indicate that DMAA has the potential to promote rat liver carcinogenesis, possibly via a mechanism involving stimulation of cell proliferation and DNA damage caused by oxygen radicals

Exposure to dimethylarsinic acid, a main metabolite of inorganic arsenics, strongly promotes tumorigenesis initiated by 4-nitroquinoline 1-oxide in the lungs of mice.

The effect of dimethylarsenics on the pulmonary tumorigenesis initiated by 4-nitroquinoline 1-oxide (4NQO) in mice was examined. The exposure of mice to dimethylarsinic acid (DMAA), a major metabolite of inorganic arsenics in mammals, resulted in not only promotion but also progression of the tumorigenic process in the lungs of mice administered 4NQO. Furthermore, dimethylarsenics influenced the differentiation process in lung tumorigenesis by 4NQO. These results may pave the way for the elucidation of lung carcinogenesis caused by arsenics.

Promoting effects of dimethylarsinic acid on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder carcinogenesis in rats.

Arsenicals are epidemiologically significant chemicals in relation to induction of urinary bladder cancer in man. In the present study, we investigated the dose-dependent promotion potential of dimethylarsinic acid (DMA), a major metabolite of inorganic arsenicals in mammals, for rat urinary bladder carcinogenesis. In experiment 1, 6-week-old male F344 rats were treated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 4 weeks and then given one of several concentrations of DMA in their drinking water (groups 1-6: 0, 2, 10, 25, 50 and 100 p.p.m.) for 32 weeks. The development of preneoplastic lesions and tumors (papillomas and carcinomas) in the urinary bladder was enhanced by treatment with DMA in a dose-dependent manner. A significant increase in multiplicity of tumors (papillomas and carcinomas) was observed even at a low concentration of DMA (10 p.p.m.). On the other hand, no preneoplastic lesions and tumors were observed in the rats treated with DMA alone. In experiment 2, different concentrations of DMA (groups 1-4: 0, 10, 25 and 100 p.p.m.) in drinking water were administered to the rats for 8 weeks without prior initiation by BBN. A significant increase in the 5-bromo-2'-deoxyuridine labeling index and alteration of the surfaces of the urinary bladder epithelial cells, as revealed by scanning electron microscopy, provided evidence of a dose-dependent increase in cell proliferation due to the DMA treatment. These results suggest that DMA has the potential to promote rat urinary bladder carcinogenesis and one of the mechanisms involved is its stimulation of cell proliferation in the urinary bladder epithelium.

DNA-Strand Breaks Induced by Dimethylarsinic Acid, a Metabolite of Inorganic Arsenics, Are Strongly Enhanced by Superoxide Anion Radicals.

We previously reported that dimethylarsinic acid (DMAA), a major metabolite of inorganic arsenics, induced DNA single-strand breaks (ssb) both in vivo and in cultured alveolar type II (L-132) cells in vitro, possibly via the production of dimethylarsenic peroxyl radicals. Here, the interaction of superoxide anion radicals (O2-) in the induction of ssb in L-132 cells was investigated using paraquat, an O2(-)-producing agent. A significant enhancement of ssb formation was observed in the DMAA-exposed cells when coexposed to paraquat. This enhancement occurred even when post-exposed to DMAA after washing, suggesting that the DMAA exposure caused some modification of DNA such as DNA-adducts, which was recognized by active oxygens to form ssb. An experiment with UV-irradiation, which was likely to induce ssb at the modified region, supported the possibility of DNA modification by DMAA exposure. An ESR study indicated that O2- produced by paraquat in DMAA-exposed cells was more consumed than in non-exposed cells, assumingly through the reaction with the dimethylarsenic-modified region of DNA. The species of active oxygens were estimated by using diethyldithiocarbamate, aminotriazole, diethylmaleate, hydrogen peroxide (H2O2), gamma-irradiation and ethanol. O2- but neither H2O2 nor hydroxyl radicals was very likely to contribute to the ssb-enhancing action of paraquat.

Induction of lung-specific DNA damage by metabolically methylated arsenics via the production of free radicals.

To clarify the genotoxicity of inorganic arsenics, we focused on the genotoxic effect of metabolically methylated arsenics in mammals. Oral administration to mice of dimethylarsinic acid (DMAA), a major metabolite of inorganic arsenics, induced lung-specific DNA damage, i.e., DNA single-strand breaks and the clumping of heterochromatin. The lung-specific strand breaks were not caused by DMAA itself, but by dimethylarsine, a further metabolite of DMAA. An in vitro experiment indicated that DNA single-strand breaks by dimethylarsine were suppressed by the presence of superoxide dismutase and catalase, suggesting that the strand breaks were induced via the production of free-radical species including active oxygens. Dimethylarsenic peroxyl radical [(CH3)2AsOO.] and superoxide anion radical produced from the reaction between molecular oxygen and dimethylarsine were detected by electron-spin resonance analysis using a spin-trapping agent and the cytochrome-c method, respectively. Of these two radicals, the dimethylarsenic peroxyl radical rather than the superoxide anion radical is assumed to play the dominant role in causing the DNA damage, at least for DNA single-strand breaks.

DNA damage induced in cultured human alveolar (L-132) cells by exposure to dimethylarsinic acid.

Gene damage in cultured human alveolar (L-132) cells induced by exposure to dimethylarsinic acid (DMAA), a major metabolite of inorganic arsenics in mammals, was studied. DNA single-strand breaks and DNA-protein cross-links were induced by the treatment of L-132 cells with 10 mM DMAA. These kinds of damage appeared at 8 hr after start of exposure to DMAA. As regards DNA-protein cross-links, the DNA was found to bind not only to core histone proteins but also linker histone (H1) and nonhistone proteins. Furthermore, the cross-links were formed by the binding to serine or threonine residues of H1 or nonhistone proteins through phosphate moieties of the DNA. The induction of the alkali-labile sites in DNA in DMAA-treated L-132 cells was observed prior to that of DNA single-strand breaks and DNA-protein cross-links. As one of the alkali-labile sites in DNA, we estimated apurinic/apyrimidinic (AP) sites in DNA. The present study suggests that the DNA single-strand breaks and DNA-protein cross-links induced by the treatment of L-132 cells with DMAA occurred via the formation of AP sites in the DNA and that the DNA-protein cross-links were produced by a Schiff-base reaction between amino groups of nuclear proteins and aldehyde groups of AP sites in the DNA and the DNA single-strand breaks, by a beta-elimination reaction on AP sites in the DNA.

Gene Damage Induced in Human Alveolar Type II (L-132) Cells by Exposure to Dimethylarsinic Acid

To analyze the mechanisms of arsenic-induced gene damage, found previously in lungs of mice and rats orally administered dimethylarsinic acid (DMAA), a major metabolite of inorganic arsenics, we employed an in vitro system using human alveolar type II (L-132) cells. The exposure to 10mM DMAA for 10 hr caused significant single-strand breaks in DNA of the cells. At an earlier period of the exposure, the replicative DNA synthesis was markedly suppressed, and the chain length of the nascent DNA was shorter than that of the control, suggesting that the template DNA received some modification other than strand breaks. The modification, being repairable, was sensitive to ultraviolet irradiation to cause strand breaks. These results suggest the possibility of DNA-adduct formation at the early period of DMAA exposure in L- 132 cells.

Preferential increase of heterochromatin in venular endothelium of lung in mice after administration of dimethylarsinic acid, a major metabolite of inorganic arsenics.

Dimethylarsinic acid (DMAA) administration induced the preferential increase of the heterochromatic area forming the inside of the interphase nucleus. A histopathological study of the lung and liver in mice after DMAA administration was carried out by transmission electron microscopy. Ultrastructural alterations in the endothelial nuclei of the alveolar wall were observed 12-48 h after administration. Heterochromatin tended to collect in a dense, compact mass lining the inner walls of the nucleus. A significant increase in heterochromatin induced by DMAA administration was observed by morphometric analysis. However, no substantial differences appeared in the sinusoidal endothelium of the liver. This study suggests that the much greater induction of morphological alterations, such as increased heterochromatin, in lung endothelial nuclei than in the liver might explain the high risk of lung cancer by arsenics, and that there may be a close relationship between heterochromatin alteration and DNA damages.

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.