Akira Hasegawa

Induction of DNA damage by dimethylarsine, a metabolite of inorganic arsenics, is for the major part likely due to its peroxyl radical

To reveal the mechanisms of previously reported lung-specific DNA strand scissions in murine after oral administration of dimethylarsinic acid (DMAA), a main metabolite of inorganic arsenics in mammals, the ultimate substance causing DNA lesion was investigated using dimethylarsine which was a further metabolite of DMAA. The alkaline elution assay using 3H-labeled DNA showed that a major portion of the strand breaks was not suppressed by SOD and catalase, suggesting an ultimate substance other than active oxygen participated in the DNA damage. By ESR analysis, a radical estimated to be (CH3)2AsOO. was detected as a reaction product of dimethylarsine and molecular oxygen. This peroxyl radical, rather than active oxygen, was assumed to play a major role in DNA damage.

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.

Dimethylated arsenics induce DNA strand breaks in lung via the production of active oxygen in mice

In order to study the genotoxicity of arsenics, we focused our attention on dimethylarsinic acid (DMAA) which was a main metabolite of inorganic arsenics in mammals. ICR mice were orally administered DMAA-Na (1500mg/kg). DNA single-strand breaks occurred specifically in lung at 12h after administration. An in vitro experiment indicated that the breaks were not caused directly by DMAA but by dimethylarsine, a further metabolite of DMAA. Furthermore, the dimethylarsine-induced breaks were diminished by the addition of SOD and catalase, suggesting that active oxygen produced by dimethylarsine was involved in the induction of DNA damage.

Metabolic methylation is a possible genotoxicity-enhancing process of inorganic arsenics

To elucidate if the metabolic methylation participates in the induction of inorganic arsenic-responsible genetic damage, arsenite (ARS) and its methylated metabolites, methanearsonic acid (MMAA) and dimethylarsinic acid (DMAA), were comparatively assayed for the induction of DNA damage by determining DNA repair synthesis using polymerization inhibitors such as aphidicolin (aph) and hydroxyurea (HU). When human alveolar epithelial type II (L-132) cells in culture were exposed to either one of these three arsenic compounds, DNA single-strand breaks resulting from the inhibition of repair polymerization were remarkably produced by exposure to DMAA at 5 to 100 microM, while not by that to ARS and MMAA even at 100 microM. Furthermore, a bromodeoxyuridine (BrdrU)-photolysis assay indicated that the induction of DNA repair synthesis was observed only in the case of exposure to DMAA. When L-132 cells were exposed to 100 microM MMAA in the presence of 10 mM S-adenosyl-L-methionine (SAM), which is a well-known methyl-group donor in metabolic methylation of arsenics, DNA repair synthesis was induced along with an increase in the amount of dimethylarsenic in the cells. These results indicate that metabolic methylation of inorganic arsenics to dimethylarsenics is predominantly involved in the induction of DNA damage.

The role of orally administered dimethylarsinic acid, a main metabolite of inorganic arsenics, in the promotion and progression of UVB-induced skin tumorigenesis in hairless mice

The effect of dimethylarsinic acid (DMA) on skin tumorigenesis by UVB irradiation was examined. Hairless mice (Hos: HR-1) irradiated with UVB at a dose of 2 kJ/m(2) twice weekly, were fed with drinking water containing 1000 ppm DMA, a main metabolite of inorganic arsenics, produced more skin tumors than DMA-untreated mice. Histopathological examination revealed that the mouse malignant tumors with severe atypism appeared only in the treatment group of UVB plus 1000 ppm DMA. These positive results point out the importance of dimethylated metabolites of inorganic arsenic in the process of skin carcinogenesis.

DNA strand breaks in mammalian tissues induced by methylarsenics.

DNA damage induced by administration of dimethylarsinic acid (DMAA) to rats and mice was investigated. At 12 h after administration of DMAA, DNA single-strand breaks were induced markedly in lung. The majority of dimethylarsine, one of the main metabolites, in the expired air was excreted within 6–18 h after administration of DMAA to rats. In vitro experiments using nuclei isolated from lung of mice indicated that DNA strand breaks were caused by dimethylarsine. Furthermore, the strand breaks after exposure to dimethylarsine were reduced in the presence of catalase and/or superoxide dismutase. These results strongly suggest that the strand breaks are induced not by dimethylarsine itself but by active oxygen, e.g., O2− and ·OH, produced both by dimethylarsine and molecular oxygen. When DNA was exposed to dimethylarsine, thiobarbituric acid (TBA)-reactive intermediates andcis-thymine glycol were produced. Dimethylarsine appears to induce DNA damage by the mechanism similar to the damage produced by ionizing radiation.

In vivo genotoxicity evaluation of dimethylarsinic acid in Muta™Mouse

Dimethylarsinic acid (DMA) induces DNA damage in the lung by formation of various peroxyl radical species. The present study was conducted to evaluate whether arsenite or its metabolite, DMA, could initiate carcinogenesis via mutagenic DNA lesions in vivo that can be attributed to oxidative damage. A transgenic mouse model, MutaMouse, was used in this study and mutations in the lacZ transgene and in the endogenous cII gene were assessed. When DMA was intraperitoneally injected into MutaMice at a dose of 10.6 mg/kg per day for 5 consecutive days, it caused only a weak increase in the mutant frequency (MF) of the lacZ gene in the lung, which was at most 1.3-fold higher than in the untreated control animals. DMA did not appreciably raise the MF in the bladder or bone marrow. Further analysis of the cII gene in the lung, the organ in which DMA induced the DNA damage, revealed only a marginal increase in the MF. Following DMA administration, no change in the cII mutation spectra was observed, except for a slight increase in the G:C to T:A transversion. Administration of arsenic trioxide (arsenite) at a dose of 7.6 mg/kg per day did not result in any increase in the MF of the lacZ gene in the lung, kidney, bone marrow, or bladder. Micronucleus formation was also evaluated in peripheral blood reticulocytes (RETs). The assay for micronuclei gave marginally positive results with arsenite, but not with DMA. These results suggest that the mutagenicity of DMA and arsenite might be too low to be detected in the MutaMouse.

Oxidative DNA damage following exposure to dimethylarsinous iodide: the formation of cis-thymine glycol.

The purpose of the present study was to elucidate the genotoxic mechanism of trivalent dimethylated arsenic, particularly the induction mechanism of oxidative stress in nuclear bases. Cis-thymine glycol was used as a biomarker of DNA oxidation damage. The treatment of thymine with dimethylarsinous iodide (DMI), a model compound of dimethylarsinous acid, induced the formation of cis-thymine glycol. This oxidative damage was induced via the production of dimethylated arsenic peroxide, but not via the production of superoxide anion or hydrogen peroxide. Trivalent dimethylated arsenic may thus play an important role in arsenic carcinogenesis through the induction of oxidative base damage.

Active arsenic species produced by GSH-dependent reduction of dimethylarsinic acid cause micronuclei formation in peripheral reticulocytes of mice.

Dimethylarsine and trivalent dimethylated arsenic, metabolites of inorganic arsenics, have received considerable attention in current research because of their biological activities. We attempted to determine the appearance of micronucleated reticulocytes (MNRETs) in mouse peripheral blood following intraperitoneal administration of dimethylarsinous iodide (DMI) and trimethylarsine (TMA), model compounds of trivalent dimethylated arsenic and dimethylarsine, respectively. A significant increase in the number of MNRETs was observed with TMA, but not with DMI. Furthermore, MNRETs only appeared with 10.6 mg/kg of dimethylarsinic acid (DMA) following its co-injection with reduced glutathione (GSH). These results suggest that micronucleus formation may need further metabolic reduction of trivalent dimethylated arsenic, i.e. the production of dimethylarsine, by an excess amount of GSH. Meanwhile, the increase in MNRETs by administration of arsenite at 7.6 mg/kg, an equivalent dose to DMA as As, was remarkably diminished by co-administration with GSH. These results indicate that GSH plays an important role in the genotoxic process of arsenics, particularly by dimethylated arsenic.

Accentuation by pertussis toxin of the 5-hydroxytryptamine-induced potentiation of ATP-evoked responses in rat pheochromocytoma cells

We previously demonstrated that 5-hydroxytryptamine (5-HT) enhances the cationic current activated by extracellular ATP in rat pheochromocytoma PC12 cells. We report here that pertussis toxin (PTX) modulates this 5-HT-dependent enhancement in these cells. 5-HT potentiated ATP-evoked intracellular Ca2+ concentration ([Ca]i) rise and dopamine release over a concentration range from 1 to 100 microM. When cells were pre-treated with PTX, this potentiation was accentuated. Pretreatment with PTX also accentuated the 5-HT-dependent enhancement of the ATP-activated current. These results suggest that the enhancement by 5-HT of the ATP-evoked responses is negatively regulated by a mechanism mediated through PTX-sensitive GTP-binding protein.