Arsenite induces oxidative DNA adducts and DNA-protein cross-links in mammalian cells.

Arsenic is generally recognized as a nonmutagenic carcinogen because sodium arsenite induces DNA damage only at very high concentrations. In this study we demonstrate that arsenite concentrations above 0.25 microM induce DNA strand breaks in both human leukemia cells and Chinese hamster ovary cells. Therefore, DNA damage may be involved in arsenic-induced carcinogenesis. Formamidopyrimidine-DNA glycosylase and proteinase K greatly increased DNA strand breaks in arsenite-treated cells, providing evidence that a large portion of arsenite-induced DNA strand breaks come from excision of oxidative DNA adducts and DNA-protein cross-links. Because DNA strand breaks appear only temporarily during excision repair, the level of detectable DNA strand breaks will be low at any given time point. For this reason many previous studies have only detected low levels of DNA strand breaks. We also show that catalase, and inhibitors of calcium, nitric oxide synthase, superoxide dismutase, and myeloperoxidase, could modulate arsenite-induced DNA damage. We conclude that arsenite induces DNA adducts through calcium-mediated production of peroxynitrite, hypochlorous acid, and hydroxyl radicals.

Nitric oxide is involved in arsenite inhibition of pyrimidine dimer excision.

Arsenite is a human carcinogen reported to inhibit DNA repair. The binding of arsenite to functional thiol groups of DNA repair enzymes has in the past been suggested as a possible mechanism for the effect of arsenite on DNA repair. However, recent studies indicate that reactive oxygen species and nitric oxide are involved in arsenite toxicity. This research aims to elucidate the role of these possible mechanisms in the inhibition of UV-induced DNA repair by arsenite. As arsenite inhibits UV-DNA repair in Chinese hamster ovary cells, and this is a commonly used cell line for UV repair experiments, we used these cells to examine the effect of arsenite on the expression of UV-irradiated reporter genes. The T4 UV endonuclease V-incorporated comet assay was used to examine specifically the effect of arsenite on pyrimidine dimer excision. We showed that inhibition of UV-DNA repair by arsenite was suppressed by nitric oxide synthase inhibitors. Arsenite increased nitric oxide production and nitric oxide generators inhibited UV-DNA repair. The involvement of nitric oxide in the inhibition of pyrimidine dimer excision by arsenite was also confirmed in human fibroblasts. Investigation into the effect of oxidant modulators did not give a clear indication that reactive oxygen species are involved in arsenite inhibition of UV-DNA repair. Phenylarsine oxide, a strong thiol-reacting agent, did not inhibit pyrimidine dimer excision and also did not increase nitric oxide production. Our results show conclusively that nitric oxide is involved in the inhibition of pyrimidine dimer excision by arsenite. Reactive oxygen species and the binding of arsenite to functional thiol groups of DNA repair enzymes do not appear to be involved.

NADH oxidase activation is involved in arsenite-induced oxidative DNA damage in human vascular smooth muscle cells.

Arsenic is atherogenic, carcinogenic, and genotoxic. Because atherosclerotic plaque has been considered a benign smooth muscle cell tumor, we have studied the effects of arsenite on DNA integrity of human vascular smooth muscle cells. By using single-cell alkaline electrophoresis, apparent DNA strand breaks were detected in a 4-hour treatment with arsenite at a concentration above 1 micromol/L. DNA strand breaks of arsenite-treated cells were increased by Escherichia coli formamidopyrimidine-DNA glycosylase and decreased by diphenylene iodinium, superoxide dismutase, catalase, pyruvate, DMSO, or D-mannitol. Extract from arsenite-treated cells showed increased capacity for producing superoxide when NADH was included in the reaction mixture; however, addition of arsenite to extract from untreated cells did not increase superoxide production. The superoxide-producing ability of arsenite-treated cells was also suppressed by diphenylene iodinium, 4,5-dihydroxy-1, 2-benzenedisulfonic acid disodium salt (Tiron), or superoxide dismutase. Superoxide production and DNA strand breaks in arsenite-treated cells were also suppressed by transfecting antisense oligonucleotides of p22phox, an essential component of NADH oxidase. Treatment with arsenite also increased the mRNA level of p22phox. These results suggest that arsenite activates NADH oxidase to produce superoxide, which then causes oxidative DNA damage. The result that arsenite at low concentrations increases oxidant levels and causes oxidative DNA damage in vascular smooth muscle cells may be important in arsenic-induced atherosclerosis.

DNA damage in arsenite- and cadmium-treated bovine aortic endothelial cells.

Reactive oxygen species have been shown to be involved in the mutagenicity, clastogenicity, and apoptosis of mammalian cells treated with arsenic or cadmium. As these endpoints require several hours of cellular processing, it is not clear that reactive oxygen species damage DNA directly or interfere with DNA replication and repair. Using single-cell alkaline electrophoresis, we have detected DNA strand breaks (DSBs) in bovine aortic endothelial cells by a 4-h treatment with sodium arsenite (As) and cadmium chloride (Cd) in sublethal concentrations. As-induced DSBs could be decreased by nitric oxide (NO) synthase inhibitors, superoxide scavengers, and peroxynitrite scavengers and could be increased by superoxide generators and NO generators. Treatment with As also increased nitrite production. These results suggest that As-increased NO may react with O2*- to produce peroxynitrite and cause DNA damage. The results showing that Cd increased cellular H2O2 levels and that Cd-induced DSBs could be modulated by various oxidant modulators suggest that Cd may induce DSBs via O2*-, H2O2, and *OH. Nevertheless, the DSBs in both As- and Cd-treated cells seem to come from the excision of oxidized bases such as formamidopyrimidine and 8-oxoguanine, as the Escherichia coli enzyme formamidopyrimidine-DNA glycosylase (Fpg) increased DSBs in cells treated with As, 3-morpholinosydnonimine (a peroxynitrite-generating agent), Cd, or H2O2.

Dithiothreitol enhances arsenic trioxide-induced apoptosis in NB4 cells.

Recently, arsenic trioxide (As2O3) was reported to induce clinical remission in patients with acute promyelocytic leukemia. Modulation of protein phosphorylation by binding to the vicinal thiols has been suggested as a possible mechanism. We found that phenylarsine oxide, a strong vicinal thiol-binding agent, neither induced nuclear fragmentation or DNA laddering nor increased caspase activity in NB4 cells; however, As2O3 and a weak thiol-binding agent, dimethylarsinic acid, did increase activity. Dithiothreitol (DTT) effectively suppressed the phenylarsine oxide-inhibited cellular reductive capacity, but unexpectedly, enhanced As2O3-induced apoptosis in NB4 cells. As2O3-induced and As2O3-plus-DTT-induced apoptosis in NB4 cells was modulated by oxidant modifiers, but not by nitric oxide synthase inhibitors. These results demonstrate that DTT, a dithiol agent and known antidote for trivalent inorganic arsenic, enhances the toxicity of As2O3, thereby opening a new research direction for the mechanisms of arsenic toxicity and perhaps also helping in the development of new therapeutic strategies for treating leukemias.

Calcium-dependent nitric oxide production is involved in arsenite-induced micronuclei.

Arsenic, a human carcinogen is known to induce sister-chromatid exchanges, chromosome aberrations and micronuclei (MN), but its mechanisms remain unknown. Recently, independent studies have suggested that intracellular calcium and reactive oxygen species are involved in arsenite-induced MN, and nitric oxide (NO) is involved in arsenite-induced poly(ADP-ribosylation). The aim of this research is to investigate the involvement of these molecules in arsenite-induced MN. The intracellular oxidant level and calcium level were monitored with a flow cytometer by using dichlorofluorescein diacetate and fluo3-AM, respectively. The NO production was estimated from the nitrite in cell culture medium with a spectrophotometer by using diaminonaphthalene. The results show that a 4-h treatment with arsenite above 5 microM, caused a dose-dependent increase of oxidant, NO, as well as intracellular calcium level. The arsenite-increased intracellular oxidant level was inhibited by NO synthase inhibitors, S-methyl-l-thiocitrulline and Nomega-nitro-l-arginine methyl ester and calcium chelators, ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 2-[(2-bis-[carboxymethyl]-amino-5-methylphenoxy)-methyl]-6-methoxy-8- bis[carboxy-methyl]aminoquinoline, but not by catalase inhibitor, 3-aminotriazole. The arsenite-increased NO could also be suppressed by NO synthase inhibitors and calcium chelator. However, the arsenite-increased intracellular calcium level was inhibited by calcium chelators, but not by NO synthase inhibitors. A 4-h treatment with arsenite above 10 microM, also induced MN dose-dependently. The arsenite-increased MN could be reduced by NO synthase inhibitors, calcium chelators, as well as superoxide dismutase and uric acid. These results suggest the involvement of peroxynitrite in arsenite-induced MN. We surmise that the disturbance of NO production may cause cardio/peripheral vascular disorders, and the peroxynitrite-mediated DNA damages may cause genetic instability and, hence, cancers in arsenic-exposed humans.

Arsenite stimulates poly(ADP-ribosylation) by generation of nitric oxide.

Recent studies indicate that arsenic may generate reactive oxygen species to exert its toxicity. Because reactive oxygen species are known to induce poly(ADP-ribosylation), which is implicated in DNA repair, signal transduction, and apoptosis, we have investigated the effect of arsenite on poly(ADP-ribosylation). The results showed that arsenite treatment induced poly(ADP-ribosylation), NAD depletion, DNA strand breaks, and micronuclei in CHO-K1 cells. Increase of nitrite level, a stable product of nitric oxide, was also detected in medium of arsenite-treated cultures. S-methyl-L-thiocitrulline and N omega-nitro-L-arginine methyl ester, inhibitors of nitric oxide synthase, could suppress the arsenite-induced NAD depletion, DNA strand breaks, and micronuclei, whereas 3-aminobenzamide, an inhibitor of poly (ADP-ribose) polymerase, could enhance micronucleus production and NAD depletion in arsenite-treated cells. These results suggest that arsenite treatment may generate nitric oxide to damage DNA and which then stimulate poly(ADP-ribosylation). Because arsenite also induced DNA strand breaks and NAD depletion in bovine aortic endothelial cells, and these could also be suppressed by S-methyl-L-thiocitrulline, the induction of nitric oxide may be important to the etiology of arsenic-induced vascular disorders in humans.

Arsenite retards DNA break rejoining by inhibiting DNA ligation.

Arsenic has been shown to inhibit methyl methane-sulphonate (MMS)-induced DNA repair but the exact mechanism remains controversial. The purpose of this investigation is to examine which step of DNA repair is most sensitive to arsenite (As) and how As inhibits it. The results from single-cell alkaline electrophoresis, showing post-treatment with As increased DNA strand breaks in MMS-treated cells, suggest that that the excision step seems to be less sensitive to As than later steps. To test this hypothesis, hydroxyurea (Hu) plus cytosine-beta-D-arabinofuranoside (AraC) were used to block DNA polymerization, allowing the DNA strand breaks to accumulate. These experiments indicated that As had weak inhibitory effects on DNA strand break accumulation. However, As inhibited the rejoining of those DNA strand breaks which could be rejoined within 4 h after release from blockage by Hu plus AraC. To further elucidate this mechanism, a cell extract was used to compare the relative sensitivity of the various steps in DNA repair to As. The potency of the As inhibitory effect as deduced from concentration-response curves were: ligation of poly(rA).oligo(dT) > ligation of poly(dA).oligo(dT) approximately DNA polymerization > or = DNA repair synthesis > excision. As is known to inhibit the activity of pyruvate dehydrogenase by interacting with vicinal dithiol groups. Dithiothreitol could effectively remove As inhibition of both the ligation of poly(rA).oligo(dT) and the activity of pyruvate dehydrogenase but had no obvious effect on As inhibition of poly(dA).oligo(dT) ligation. Since DNA ligase III contains vicinal dithiol groups, we postulate that As may inhibit DNA break rejoining by interacting with the vicinal dithiols to inactivate DNA ligation in MMS-treated cells.

Glutathione peroxidase and catalase modulate the genotoxicity of arsenite.

The X-ray hypersensitive Chinese hamster ovary (CHO) cells, xrs-5, are also more sensitive to sodium arsenite in terms of cell growth and micronucleus induction than CHO-K1 cells. Since reactive oxygen species are suggested to be involved in arsenic toxicity, we have measured antioxidant mechanisms in xrs-5 as well as CHO-K1 cells. There were no apparent differences in the activities of superoxide dismutase, glutathione S-transferase, glutathione reductase, and the levels of glutathione between xrs-5 and CHO-K1 cells. However, the activities of glutathione peroxidase and catalase were 5.4- and 5.8-fold lower, respectively, in xrs-5 cells. The addition of catalase or glutathione peroxidase to cultures reduced the arsenite-induced micronuclei in xrs-5 cells. Whereas, simultaneous treatment with mercaptosuccinate, an inhibitor of glutathione peroxidase, and 3-aminotriazole, an inhibitor of catalase, synergistically increased the arsenite-induced micronuclei. These results suggest that both catalase and glutathione peroxidase are involved in defense against arsenite genotoxicity. The xrs-6 cells, another line of x-ray hypersensitive CHO cells, which had 1.6-fold higher catalase activity and 2.5-fold higher glutathione peroxidase activity than xrs-5 cells, were also more sensitive than CHO-K1 cells but were less sensitive than xrs-5 cells to cell growth inhibition of arsenite. Moreover, a 1.6-fold increase of glutathione peroxidase activity by selenite adaptation effectively removed the arsenite-induced micronuclei in CHO-K1 cells. These results suggest that glutathione peroxidase is more important than catalase in defending against arsenite toxicity. Our results also suggest that increasing the intracellular antioxidant level may have preventive or therapeutic effects in arsenic poisoning.

Cadmium inhibits DNA strand break rejoining in methyl methanesulfonate-treated CHO-K1 cells.

The cogenotoxicity of Cd has been recognized. This effect may stem from Cd inhibition of DNA repair. We studied the effects of Cd on DNA repair of methyl methanesulfonate (MMS)-damaged Chinese hamster ovary cells (CHO-K1) by single-cell alkaline electrophoresis. The results indicate that in the presence of Cd, DNA strand breaks accumulated in MMS-treated cells. Using hydroxyurea (Hu) plus cytosine-beta-D-arabinofuranoside (AraC) to block DNA polymerization, DNA strand breaks accumulated and Cd had little inhibitory effects on these accumulations. However, Cd inhibited the rejoining of these DNA strand breaks, which could be rejoined 6 hr after release from Hu plus AraC blockage. These results indicate that the potency of Cd inhibition of DNA repair replication and/or ligation may be greater than the inhibition of DNA adduct excision. To further elucidate this mechanism, we used an in vitro cell-free assay system to analyze the Cd effects on DNA repair synthesis, DNA polymerization, and DNA ligation. We have shown a dose-dependent inhibition of these three activities by Cd in CHO-K1 cell extract. The IC50s of Cd were 55, 26, and 10 microM, respectively. Moreover, Cd inhibition of DNA ligation in cell extract could be recovered partially by thiol compounds such as glutathione, beta-mercaptoethanol, dithiothreitol, and metallothionein. Since both in vivo and in vitro studies demonstrated that Cd was more effectively involved in interfering with the DNA ligation step and that thiol agents could partially remove Cd inhibition of DNA ligation, we speculate that part of the Cd inhibition of DNA repair may be through binding of Cd to the proteins participating in DNA ligation.

Reactive oxygen species are involved in nickel inhibition of DNA repair.

Nickel has been shown to inhibit DNA repair in a way that may play a role in its toxicity. Since nickel treatment increases cellular reactive oxygen species (ROS), we have investigated the involvement of ROS in nickel inhibition of DNA repair. Inhibition of glutathione synthesis or catalase activity increased the enhancing effect of nickel on the cytotoxicity of ultraviolet (UV) light. Inhibition of catalase and glutathione peroxidase activities also enhanced the retardation effect of nickel on the rejoining of DNA strand breaks accumulated by hydroxyurea plus cytosine-beta-D-arabinofuranoside in UV-irradiated cells. Since DNA polymerization and ligation are involved in the DNA-break rejoining, we have investigated the effect of ROS on these two steps in an extract of Chinese hamster ovary cells. Nickel inhibition of the incorporation of (3H)dTTP into the DNase I-activated calf thymus DNA was stronger than the ligation of poly(dA) x oligo(dT), whereas H2O2 was more potent in inhibiting DNA ligation than DNA polymerization. Nickel, in the presence of H2O2, exhibited a synergistic inhibition on both DNA polymerization and ligation and caused protein fragmentation. In addition, glutathione could completely recover the inhibition by nickel or H2O2 alone but only partially recover the inhibition by nickel plus H2O2. Therefore, nickel may bind to DNA-repair enzymes and generate oxygen-free radicals to cause protein degradation in situ. This irreversible damage to the proteins involved in DNA repair, replication, recombination, and transcription could be important for the toxic effects of nickel.

Magnesium reduces nickel inhibition of DNA polymerization.

The activities of DNA polymerization and DNA ligation in extract of Chinese hamster ovary cells were both stimulated by MgCl2. DNA polymerization was stimulated by MgCl2 above 0.25 mM, whereas, MgCl2 above 2 mM was required to stimulate DNA ligation. The activity of DNA polymerization maintained a plateau at MgCl2 1-12 mM, whereas DNA ligation reached a maximal activity at MgCl2 6 mM and decreased thereafter. NiCl2 0.1-0.2 mM also had a stimulatory effect on DNA polymerization, but was much less potent than MgCl2. However, nickel ion (Ni2+) had no detectable stimulating effect on the activity of DNA ligation. In the presence of MgCl2, the activities of DNA polymerization and DNA ligation decreased with increasing concentration of NiCl2. Ni2+ inhibition of DNA polymerization was reduced by increasing the concentration of MgCl2, but increasing the concentration of MgCl2 did not reduce Ni2+ inhibition of DNA ligation. Preincubating cell extract with MgCl2 decreased the Ni2+ inhibition of DNA polymerization but not DNA ligation. These results suggest that Ni2+ may compete with magnesium ion (Mg2+) to reduce DNA polymerization, but this mechanism seems not applicable to Ni2+ inhibition of DNA ligation.

Arsenite induces apoptosis in Chinese hamster ovary cells by generation of reactive oxygen species.

Arsenic, a human carcinogen, possesses a serious environmental threat but the mechanism of its toxicity remains unclear. Knowledge of how arsenic induces cell death and how cells escape the death path may help to understand arsenic carcinogenesis. We have investigated the nature of sodium arsenite-induced cell death in Chinese hamster ovary K1 cells. Following phosphate-citric acid buffer extraction, apoptotic cells with lower DNA content than the G1 cells were detected by flow cytometry. Immediately after 4 h of 40 microM arsenite treatment, no appreciable fraction of cells with sub-G1 DNA content was detected; however, the sub-G1 cell fraction increased with postarsenite incubation time, and detectable increase started at 8 h of incubation, whereas the intracellular peroxide level as measured by the fluorescent intensity of 2',7'-dichlorofluorescein increased immediately following a 4-h arsenite treatment. Simultaneous treatment with arsenite plus antioxidant (N-acetyl-cysteine, Trolox, and Tempo); copper ion chelator (neocuproine); protein kinase inhibitor (H-7) or protein synthesis inhibitor (cycloheximide) reduced the fraction of sub-G1 cell and internucleosomal DNA degradation. Trolox, neocuproine, or cycloheximide given after arsenite treatment also effectively reduced apoptosis. These results lead to a working hypothesis that arsenite-induced apoptosis in CHO-K1 cells is triggered by the generation of hydrogen peroxide, followed by a copper-mediated Fenton reaction that catalyzes the production of hydroxyl radicals, which selectively activates protein kinase through de novo synthesis of macromolecules.

Glutathione can rescue the inhibitory effects of nickel on DNA ligation and repair synthesis.

The purpose of this investigation was to explore the reason why nickel chloride enhances the cytotoxicity and genotoxicity of ultraviolet (UV) light, but not that of methyl methanesulfonate (MMS) in Chinese hamster ovary cells. The cellular glutathione content was increased by treatment with MMS or nickel, but not with UV. Post-treatment with nickel synergistically raised the cellular glutathione content in MMS-treated cells; this phenomenon was not observed in UV-irradiated cells. Preventing cellular glutathione induction by buthionine sulfoximine increased the cytotoxicity, the frequency of sister chromatid exchange and prolonged the cell cycle in cells treated with nickel or MMS plus nickel. Pretreatment with N-acetylcysteine, a glutathione precursor, increased the clonogenic survival of cells treated with UV plus nickel. In vitro assays indicated that nickel could inhibit oligonucleotide ligation and the repair synthesis of UV- or MMS-treated plasmids and glutathione could relieve nickel inhibition. These results suggest that the enhancement by nickel of UV cytotoxicity and genotoxicity may be due to its inhibition of DNA repair, whereas treating cells with MMS plus nickel increased cellular glutathione levels, which may help in neutralizing the toxicity of nickel. The results also suggest that the activity of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione biosynthesis, may be increased by treatment with MMS, nickel and more so with MMS plus nickel.

Delay of the excision of UV light-induced DNA adducts is involved in the coclastogenicity of UV light plus arsenite.

Chromatid exchanges and chromatid breaks were synergistically increased by a 2-h post-treatment with arsenite (VS treatment) but not with arabinofuranosyl cytosine (VA treatment) of UV-irradiated late-G1 Chinese hamster ovary cells. In order to determine the mechanism of this UV-arsenite coclastogenicity, we have compared the effects of arsenite and arabinofuranosyl cytosine on the generation of DNA strand breaks in UV-irradiated cells by alkaline elution and alkaline sucrose sedimentation. Only very small numbers of DNA breaks were detected immediately after VS treatment, however the breaks in parental strands increased as the cells reached mitosis in drug-free medium, whereas a large number of breaks were detected immediately after VA treatment but the breaks decreased thereafter. By labelling the newly synthesized DNA, we have also shown that the VS-treated cells had more breaks in daughter strands than the VA-treated cells at the time of reaching mitosis. The effect of a 2-h post-treatment with arsenite on the excision of UV-induced DNA adducts was further investigated by using the exponentially growing cells. The results confirmed that very low amount of breaks was detectable immediately after VS treatment, however the amount of breaks increased upon the removal of arsenite. Therefore, the breaks in the daughter strands of VS-treated cells may come from DNA replication using templates containing unexcised adducts, or using broken templates. It is conceivable that gaps in the overlapping regions of parental and daughter strands may result in chromatid breaks and that misreplication, because of unexcised adducts or gaps in the parental strands, may result in chromatid exchanges.

Differential effects of luminol, nickel, and arsenite on the rejoining of ultraviolet light and alkylation-induced DNA breaks.

When Chinese hamster ovary cells were treated with ultraviolet (UV) light or methyl methanesulfonate (MMS), a large number of DNA strand breaks could be detected by alkaline elution. These strand breaks gradually disappeared if the treated cells were allowed to recover in a drug-free medium. The presence of nickel or arsenite during the recovery incubation retarded the disappearance of UV-induced strand breaks, whereas the disappearance of MMS-induced strand breaks was retarded by the presence of arsenite or of luminol, a new inhibitor for poly(ADP-ribose) synthetase. Luminol, however, had no apparent effect on the repair of UV-induced DNA strand breaks, and nickel had no effect on the repair of MMS-induced DNA strand breaks. When UV- or MMS-treated cells were incubated in cytosine arabinofuranoside (AraC) plus hydroxyurea (HU), a large amount of low molecular weight DNA was detected by alkaline sucrose sedimentation. The molecular weight of these DNAs increased if the cells were further incubated in a drug-free medium. This rejoining of breaks in cells pretreated with UV plus AraC and HU was inhibited by nickel and by arsenite, but not by luminol. The rejoining of breaks in cells pretreated with MMS plus AraC and HU was inhibited by luminol and by arsenite, but not by nickel. These results suggest that different enzymes may be used in DNA resynthesis and/or ligation during the repairing of UV- and MMS-induced DNA strand breaks, and that nickel, luminol, and arsenite may have differential inhibitory effects on these enzymes.

Induction of chromatid breaks and tetraploidy in Chinese hamster ovary cells by treatment with sodium arsenite during the G2 phase.

Treatment of Chinese hamster ovary (CHO) cells with sodium arsenite during the G2 phase induced poorly condensed chromosomes and chromatid breaks. The induction of chromatid breaks was confirmed by the appearance of micronucleated cells after arsenite-treated G2 cells were allowed to re-enter interphase. When the duration of the G2 phase was artificially divided into 4 periods, more chromatid breaks were induced by treatment with arsenite during the very early G2 phase (or G2/S boundary). In addition to the induction of chromatid breaks, arsenite treatment also remarkably retarded the re-entry of mitotic cells into interphase. By replating and incubating arsenite-treated G2 cells in drug-free medium, we subsequently observed the appearance of a population of cells whose DNA content was between 4C and 8C, and metaphase cells with near-tetraploid chromosome numbers in the next mitotic division.

Arsenite enhances DNA double-strand breaks and cell killing of methyl methanesulfonate-treated cells by inhibiting the excision of alkali-labile sites.

Analysis of DNA strand breaks by alkaline elution indicates that DNA repair of Chinese hamster ovary cells treated with methyl methanesulfonate (MMS) was inhibited by sodium arsenite. Comparing the profiles of a 36-min elution with buffer pH 12.1 and a 12-h elution with buffer pH 12.4 revealed that alkali-labile sites were increased more than frank breaks in the combined treatment with MMS plus arsenite. Enhancement of alkali-labile sites was detected with low doses of MMS and arsenite, whereas enhancement of frank breaks required higher doses of MMS and arsenite. Double-strand breaks were detected after incubating the MMS-treated cells in an arsenite-containing medium for 18 or 12 h but not less than 6 h. No double-strand breaks were detected when MMS-damaged cells were posttreated with arsenite for 3 h; however, double-strand breaks were detected after further incubating these cells in arsenite-free medium for 18 h. Thus, inhibition of arsenite on the excision of methylated bases may have accumulated a large number of alkali-labile sites in the parental strands, and DNA replication may then generate breaks in the non-methylated daughter strands. Double-strand breaks may result from overlapping gaps between the parental and daughter strands and/or postreplication repair. These double-strand breaks may then result in the synergistic cell death as observed with posttreatment of MMS-damaged cells with arsenite for 1 or 3 h.

Factors affecting zero background frequency of sister-chromatid exchange in mosquito cells.

A background level of sister-chromatid exchange (SCE) is found in mammalian cells. However, in the somatic cells of Drosophila melanogaster, there was no spontaneous SCE if a minimum concentration of bromodeoxyuridine (BrdU) was used. In this communication we report that in a mosquito (Aedes albopictus) cell line (C6/36), some cells also have no background SCE (0-SCE). Subclones of high (44%) or low (12%) frequency of 0-SCE cells were obtained, but none of them contained 100% 0-SCE cells. Increasing frequency of SCE/cell concomitant with decreasing frequency of 0-SCE cells was observed by raising the BrdU concentration in the culture medium during the first cell cycle, culturing cells at lower density and depleting reduced glutathione (GSH) with buthionine sulfoximine. Since these mosquito cells contain much higher level of GSH than Chinese hamster ovary (CHO) cells, and feeding cells with BrdU increased GSH in mosquito cells but decreased GSH in CHO cells, we speculate that GSH may play a role in the low or non-existent background SCE and in the high resistance to many DNA damaging agents of mosquito cells.

Glutathione as a cellular defence against arsenite toxicity in cultured Chinese hamster ovary cells.

The cytotoxic effect of arsenite seems to be inversely related to the intracellular glutathione (GSH) content, and GSH seems to facilitate the metabolism of arsenic in cell. Arsenite is also known to induce chromosome aberration, to enhance the cytotoxicity and clastogenicity of ultraviolet (UV) light, and to inhibit UV-induced DNA repair. We have investigated whether these toxic effects of arsenite and the cellular arsenic content are also modulated by the intracellular GSH. A 2-h pretreatment of the cultured ovary (CHO) cells with GSH reduced the clastogenicity and cytotoxicity of arsenite. The enhancing effects of arsenite on chromosome aberrations and cell destruction induced by UV were also reduced by a 2-h pretreatment with GSH. The inhibitory effect of arsenite on the strand-break rejoining during UV-induced DNA repair was reduced by GSH pretreatment and was enhanced by pretreatment with buthionine sulfoximine, which is known to deplete the cellular GSH. The cellular arsenic content was reduced by GSH pretreatment and increased by buthionine sulfoximine pretreatment. GSH given before or simultaneously with arsenite, effectively reduced the clastogenicity and coclastogenicity of arsenite. GSH given after treatment with arsenite decreased the cellular arsenic content, and increased the cell survival, but did not reduce the clastogenicity or the coclastogenicity of arsenite.