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Our laboratory has shown that arsenite markedly increased the cancer rate caused by solar-simulation ultraviolet radiation (UVR) in the hairless mouse skin model. In the present study, we investigated how arsenite affected DNA photodamage repair and apoptosis after solar-simulation UVR in the mouse keratinocyte cell line 291.03C. The keratinocytes were treated with different concentrations of sodium arsenite (0.0, 2.5, 5.0 microM) for 24 hr and then were immediately irradiated with a single dose of 0.30 kJ/m[2] UVR. At 24 hr after UVR, DNA photoproducts [cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) ] and apoptosis were measured using the enzyme-linked immunosorbent assay and the two-color TUNEL (terminal deoxynucleotide transferase dUTP nick end labeling) assay, respectively. The results showed that arsenite reduced the repair rate of 6-4PPs by about a factor of 2 at 5.0 microM and had no effect at 2.5 microM. UVR-induced apoptosis at 24 hr was decreased by 22.64% at 2.5 microM arsenite and by 61.90% at 5.0 microM arsenite. Arsenite decreased the UVR-induced caspase-3/7 activity in parallel with the inhibition of apoptosis. Colony survival assays of the 291.03C cells demonstrate a median lethal concentration (LC50) of arsenite of 0.9 microM and a median lethal dose (LD50) of UVR of 0.05 kJ/m[2]. If the present results are applicable in vivo, inhibition of UVR-induced apoptosis may contribute to arsenite's enhancement of UVR-induced skin carcinogenesis. [ABSTRACT FROM AUTHOR]

Arsenic is a human carcinogen whose mechanism of action is unknown. Previously, this laboratory demonstrated that arsenite acts as a comutagen by interfering with DNA repair, although a specific DNA repair enzyme sensitive to arsenite has not been identified. A number of stable arsenite-sensitive and arsenite-resistant sublines of Chinese hamster V79 cells have now been isolated. In order to gain understanding of possible targets for arsenite's action, one arsenite-resistant subline, As/R28A, was chosen as a donor for a cDNA expression library. The library from arsenite-induced As/R28A cells was transfected into arsenite-sensitive As/S5 cells, and transfectants were selected for arsenite-resistance. Two cDNAs, asr1 and asr2, which confer arsenite resistance to arsenite-hypersensitive As/S5 cells as well as to wild-type cells, were isolated. asr1 shows almost complete homology with the rat fau gene, a tumor suppressor gene which contains a ubiquitin-like region fused to S30 ribosomal protein. Arsenite was previously shown to inhibit ubiquitin-dependent proteolysis. These results suggest that the tumor suppressor fau gene product or some other aspect of the ubiquitin system may be a target for arsenic toxicity and that disruption of the ubiquitin system may contribute to the genotoxicity and carcinogenicity of arsenite. [ABSTRACT FROM PUBLISHER]

The human enzyme As(III) S-adenosylmethionine methyltransferase (AS3MT) catalyzes arsenic biotransformations and is considered to contribute to arsenic-related diseases. AS3MT is expressed in various tissues and cell types including liver, brain, adrenal gland, and peripheral blood mononuclear cells but not in human keratinocytes, urothelial, or brain microvascular endothelial cells. This indicates that AS3MT expression is regulated in a tissue/cell type-specific manner, but the mechanism of transcriptional regulation of expression of the AS3MT gene is not known. In this study, we define the DNA sequence of the core promoter region of the human AS3MT gene. We identify a GC box in the promoter to which the stress-related transcription factor Sp1 binds, indicating involvement of regulatory elements in AS3MT gene expression., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Antimony (Sb) and its compounds are negative in gene mutation assays in bacteria and cultured mammalian cells but positive in some assays for clastogenicity and/or DNA damage. In order to better understand the modes of action for antimony genotoxicity, we assessed reporter gene activation by antimony and antimony compounds in the new expanded ToxTracker assay. ToxTracker evaluates the activation of biomarkers for different cellular defense mechanisms using a series of green fluorescent protein reporters inserted into mouse embryonic stem cell lines. The assay responds to: 1) DNA damage and inhibition of DNA replication; 2) oxidative stress; 3) unfolded protein response (protein damage); and 4) p53-dependent cellular stress. Sb metal powder, six trivalent (Sb(III)) compounds, and five pentavalent antimony (Sb(V)) compounds were assessed. Sb powder and all six Sb(III) compounds activated oxidative stress ToxTracker reporters at non-toxic doses. Of the five Sb(V) compounds, antimony pentachloride and potassium hexahydroantimonate induced a robust oxidative stress response while sodium antimonate induced only a weak oxidative stress response. At higher concentrations (up to either 75 % toxicity or the highest dissolved concentration tested), Sb powder and all Sb(III) compounds except for antimony trichloride induced the unfolded protein response. Of the five Sb(V) compounds tested, only potassium hexahydroantimonate induced weak activation of the unfolded protein response and was also the only pentavalent compound to yield modest (30 %) cytotoxicity. None of the compounds tested activated the DNA damage/inhibition of DNA replication reporters, nor did they activate the p53-dependent response. All Sb(III) compounds, Sb powder, and three of the five Sb(V) compounds activated the oxidative stress reporters, but there was no activation of reporters associated with DNA damage and repair or p53-dependent cellular stress. The consistent activation of reporters for oxidative stress suggests this mode of action may underlie genotoxicity responses for antimony and its compounds., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Although occupational exposure to antimony and its compounds can produce pulmonary toxicity, human carcinogenic impacts have not been observed. Inhalation studies with respirable antimony trioxide particles administered to rats and mice have, however, induced carcinogenic responses in the lungs and related tissue sites. Genotoxicity studies conducted to elucidate mechanism(s) for tumor induction have produced mixed results. Antimony compounds do not induce gene mutations in bacteria or cultured mammalian cells, but chromosome aberrations and micronuclei have been observed, usually at highly cytotoxic concentrations. Indirect mechanisms of genotoxicity have been proposed to mediate these responses. In vivo genotoxicity tests have generally yielded negative results although several positive studies of marginal quality have been reported. Genotoxic effects may be related to indirect modes of action such as the generation of excessive reactive oxygen species (ROS), altered gene expression or interference with DNA repair processes. Such indirect mechanisms may exhibit dose-response thresholds. For example, interaction of ROS with in vivo antioxidant systems could yield a threshold for genotoxicity (and cancer) only at concentrations above the capacity of antioxidant defense mechanisms to control and/or eliminate damage from ROS., Competing Interests: Declaration of Competing Interest There are no conflicts of interest to declare., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Arsenic methylation, the primary biotransformation in the human body, is catalyzed by the enzyme As(III) S-adenosylmethionine (SAM) methyltransferases (hAS3MT). This process is thought to be protective from acute high-level arsenic exposure. However, with long-term low-level exposure, hAS3MT produces intracellular methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)), which are considerably more toxic than inorganic As(III) and may contribute to arsenic-related diseases. Several single nucleotide polymorphisms (SNPs) in putative regulatory elements of the hAS3MT gene have been shown to be protective. In contrast, three previously identified exonic SNPs (R173W, M287T, and T306I) may be deleterious. The goal of this study was to examine the effect of single amino acid substitutions in hAS3MT on the activity of the enzyme that might explain their contributions to adverse health effects of environmental arsenic. We identified five additional intragenic variants in hAS3MT (H51R, C61W, I136T, W203C, and R251H). We purified the eight polymorphic hAS3MT proteins and characterized their enzymatic properties. Each enzyme had low methylation activity through decreased affinity for substrate, lower overall rates of catalysis, or lower stability. We propose that amino acid substitutions in hAS3MT with decreased catalytic activity lead to detrimental responses to environmental arsenic and may increase the risk of arsenic-related diseases.

Environmental arsenic compounds and their methylated metabolites do not form adducts with DNA, but do cause oxidative DNA damage. Chromosome aberrations are seen at toxic concentrations. Genetic effects that occur at non-toxic concentrations include aneuploidy, comutagenesis (resulting from indirect effects on DNA repair), and delayed mutagenesis (probably secondary to aneuploidy and/or epigenetic effects). Effects of trivalent arsenicals on poly(ADP ribose) polymerase and P53 activation may mediate effects on DNA repair and aneuploidy. A growing literature points to the epigenetic effects of arsenic compounds in cells and in vivo. A review of the current literature on DNA methylation, histone modifications and microRNA effects is presented.

Arsenite is an environmental pollutant. Exposure to inorganic arsenic in drinking water is associated with elevated cancer risk, especially in skin. Arsenite alone does not cause skin cancer in animals, but arsenite can enhance the carcinogenicity of solar UV. Arsenite is not a significant mutagen at non-toxic concentrations, but it enhances the mutagenicity of other carcinogens. The tumor suppressor protein P53 and nuclear enzyme PARP-1 are both key players in DNA damage response. This laboratory demonstrated earlier that in cells treated with arsenite, the P53-dependent increase in p21(WAF1/CIP1) expression, normally a block to cell cycle progression after DNA damage, is deficient. Here we show that although long-term exposure of human keratinocytes (HaCaT) to a nontoxic concentration (0.1 microM) of arsenite decreases the level of global protein poly(ADP-ribosyl)ation, it increases poly(ADP-ribosyl)ation of P53 protein and PARP-1 protein abundance. We also demonstrate that exposure to 0.1 microM arsenite depresses the constitutive expression of p21 mRNA and P21 protein in HaCaT cells. Poly(ADP-ribosyl)ation of P53 is reported to block its activation, DNA binding and its functioning as a transcription factor. Our results suggest that arsenite's interference with activation of P53 via poly(ADP-ribosyl)ation may play a role in the comutagenic and cocarcinogenic effects of arsenite., (2009 Elsevier Inc. All rights reserved.)

Drinking arsenic-contaminated water is associated with increased risk of neoplasias of the skin, lung, bladder and possibly other sites, as well as other diseases. Earlier, we showed that human lymphoblast lines from different normal unexposed donors showed variable sensitivities to the toxic effects of arsenite. In the present study, we used microarray analysis to compare the basal gene expression profiles between two arsenite-resistant (GM02707, GM00893) and two arsenite-sensitive lymphoblast lines (GM00546, GM00607). A number of genes were differentially expressed in arsenite-sensitive and arsenite-resistant cells. Among these, gamma-glutamyltranspeptidase 1 (GGT1) and NF kappa B inhibitor-epsilon (NFKBIE) showed higher expression levels in arsenite-resistant cells. RT-PCR analysis with gene-specific primers confirmed these results. Reduction of GGT1 expression level in arsenite-resistant lymphoblasts with GGT1-specific siRNA resulted in increased cell sensitivity to arsenite. In conclusion, we have demonstrated for the first time that expression levels of GGT1 and possibly NFKBIE might be useful as biomarkers of genetic susceptibility to arsenite. Expression microarrays can thus be exploited for identifying additional biomarkers of susceptibility to arsenite and to other toxicants.

Arsenic in drinking water, a mixture of arsenite and arsenate, is associated with increased skin and other cancers in Asia and Latin America, but not the United States. Arsenite alone in drinking water does not cause skin cancers in experimental animals; therefore, it is not a complete carcinogen in skin. We recently showed that low concentrations of arsenite enhanced the tumorigenicity of solar UV irradiation in hairless mice, suggesting arsenic cocarcinogenesis with sunlight in skin cancer and perhaps with different carcinogenic partners for lung and bladder tumors. Cocarcinogenic mechanisms could include blocking DNA repair, stimulating angiogenesis, altering DNA methylation patterns, dysregulating cell cycle control, induction of aneuploidy and blocking apoptosis. Arsenicals are documented clastogens but not strong mutagens, with weak mutagenic activity reported at highly toxic concentrations of inorganic arsenic. Previously, we showed that arsenite, but not monomethylarsonous acid (MMA[III]), induced delayed mutagenesis in HOS cells. Here, we report new data on the mutagenicity of the trivalent methylated arsenic metabolites MMA(III) and dimethylarsinous acid [DMA(III)] at the gpt locus in Chinese hamster G12 cells. Both methylated arsenicals seemed mutagenic with apparent sublinear dose responses. However, significant mutagenesis occurred only at highly toxic concentrations of MMA(III). Most mutants induced by MMA(III) and DMA(III) exhibited transgene deletions. Some non-deletion mutants exhibited altered DNA methylation. A critical discussion of cell survival leads us to conclude that clastogenesis occurs primarily at highly cytotoxic arsenic concentrations, casting further doubt as to whether a genotoxic mode of action (MOA) for arsenicals is supportable.

The skin cancer enhancing effect of chromium (in male mice) and nickel in UVR-irradiated female Skh1 mice was investigated. The dietary vitamin E and selenomethionine were tested for prevention of chromium-enhanced skin carcinogenesis. The mice were exposed to UVR (1.0 kJ/m(2) 3 x weekly) for 26 weeks either alone, or combined with 2.5 or 5.0 ppm potassium chromate, or with 20, 100 or 500 ppm nickel chloride in drinking water. Vitamin E or selenomethionine was added to the lab chow for 29 weeks beginning 3 weeks before the start of UVR exposure. Both chromium and nickel significantly increased the UVR-induced skin cancer yield in mice. In male Skh1 mice, UVR alone induced 1.9+/-0.4 cancers/mouse, and 2.5 or 5.0 ppm potassium chromate added to drinking water increased the yields to 5.9+/-0.8 and 8.6+/-0.9 cancers/mouse, respectively. In female Skh1 mice, UVR alone induced 1.7+/-0.4 cancers/mouse, and the addition of 20, 100 or 500 ppm nickel chloride increased the yields to 2.8+/-0.9, 5.6+/-0.7 and 4.2+/-1.0 cancers/mouse, respectively. Neither vitamin E nor selenomethionine reduced the cancer yield enhancement by chromium. These results confirm that chromium and nickel, while not good skin carcinogens per se, are enhancers of UVR-induced skin cancers in Skh1 mice. Data also suggest that the enhancement of UVR-induced skin cancers by chromate may not be oxidatively mediated since the antioxidant vitamin E as well as selenomethionine, found to prevent arsenite-enhanced skin carcinogenesis, failed to suppress enhancement by chromate.

Inorganic arsenic (arsenite and arsenate) in drinking water has been associated with skin cancers and increased incidence of cardiovascular diseases. Additionally, studies have demonstrated the pro-angiogenic effect of arsenite and its potential promotion of tumor angiogenesis and tumor progression. Furthermore, recent reports demonstrated reversal of skin co-carcinogenesis by an organoselenium compound. The present study was undertaken to determine the effect and mechanism on angiogenesis of arsenite at low level and its potential reversal by various selenium-derived compounds. The pro-angiogenesis effects and mechanisms of sodium arsenite were determined using the chick chorioallantoic membrane (CAM) model over 3 days and compared with standard pro-angiogenesis factors, such as basic fibroblast growth factor (b-FGF). Additionally, the potential effect of various selenium-derived compounds--such as dimethyl selenone, diphenyl selenone, sodium selenite or Se-methyl selenocysteine--in reversing the pro-angiogenesis effect of arsenite or b-FGF was also determined in the CAM model. The pro-angiogenesis effect of arsenite or b-FGF was significantly (P < 0.01) blocked by dimethyl selenone, diphenyl selenone, sodium selenite or Se-methyl selenocysteine. The pro-angiogenesis effect of either sodium arsenite at 33 nM or b-FGF was blocked (P < 0.01) by the extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation inhibitor, PD 98059. Additionally, the pro-angiogenic effect of arsenic or b-FGF was blocked as well (P < 0.01) by the alphavbeta3 antagonist, XT199. These data suggest that the pro-angiogenesis effect of arsenic is initiated at the plasma membrane integrin alphavbeta3, involves activation of the ERK1/2 pathway and is effectively reversed by various selenium-derived compounds.

Arsenic-induced carcinogenesis is a worldwide problem for which there is currently limited means for control. Recently, we showed that arsenite in drinking water greatly potentiates solar ultraviolet radiation (UVR) induced skin cancer in mice, at concentrations as low as 1.25 mg/l. In this study, we examined the protective efficacy of vitamin E and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) against tumors induced by UVR and UVR + arsenite. Hairless mice were exposed to UVR alone (1.0 kJ/m(2) x 3 times weekly) or UVR + sodium arsenite (5 mg/l in drinking water) and fed lab chow supplemented or not with vitamin E (RRR-alpha-tocopheryl acetate, 62.5 IU/kg diet) or p-XSC (10 mg/kg) for 26 weeks. The tumor yield for mice receiving UVR alone was 3.6 tumors/mouse and the addition of arsenite to the drinking water increased the yield to 7.0 tumors/mouse (P < 0.005). Vitamin E and p-XSC reduced the tumor yield in mice given UVR + arsenite by 2.1-fold (P < 0.001) and 2-fold (P < 0.002), respectively. Vitamin E, but not p-XSC, reduced the tumor yield induced by UVR alone by 30% (P < 0.05). No significant difference in tumor types or grade of malignancy was observed in mice treated with or without chemopreventives. Immunostaining of mouse skin for 8-oxo-2'-deoxyguanosine (8-oxo-dG) revealed a significant reduction of 8-oxo-dG formation in mice treated with vitamin E or p-XSC compared with those treated with UVR + arsenite. These results show that vitamin E and p-XSC protect strongly against arsenite-induced enhancement of UVR carcinogenesis.

Recent evidence suggests that inflammatory cytokines and growth factors contribute to arsenite (As)-induced human carcinogenesis. We investigated the expression of inflammatory cytokine mRNAs during the transformation process induced by chronic As exposure in non-tumorigenic human osteogenic sarcoma (N-HOS) cells using gene arrays, and results were confirmed by RT-PCR and protein arrays. Caffeic acid phenethyl ester (CAPE), a naturally occurring immunomodulating agent, was used to evaluate the role of inflammatory factors in the process of As-mediated N-HOS cell transformation and in As-transformed HOS (AsT-HOS) cells. We found that an 8-week continuous exposure of N-HOS to 0.3 microM arsenite resulted in HOS cell transformation. That exposure also caused substantial decreases in inflammatory cytokine mRNAs, such as interleukin (IL) IL-1alpha, IL-2, IL-8, IL-18, MCP-1, TGF-beta2, and TNF-alpha, while it increased c-jun mRNA in a time-dependent manner. Co-incubation of N-HOS with As and CAPE (0.5-2.5 microM) prevented As-mediated declines in cytokine mRNAs in the co-treated cells, as well as their transformation to anchorage independence, while it caused decreases in c-jun mRNA. CAPE (up to 10 microM) had no effect on growth of N-HOS cells. However, CAPE (1-10 microM) treatment of AsT-HOS cells inhibited cell growth, induced cell cycle G2/M arrest, and triggered apoptosis, accompanied by changes in cytokine gene expression, as well as decreases in cyclin B1 and cdc2 abundance. Resveratrol (RV) and (-)(.) epigallocatechin gallate (EGCG), preventive agents present in grapes and green tea, respectively, induced similar changes in AsT-HOS cell growth but required much higher doses than CAPE to cause 50% growth arrest (<2.5 microM CAPE versus 25 microM RV or 50 microM EGCG). Overall, our findings suggest that inflammatory cytokines play an important role in the suppressive effects of CAPE on As-induced cell transformation and in the selective cytotoxicity of CAPE to As-transformed HOS cells.

Arsenite is a toxicant and environmental pollutant associated with multisite neoplasias and other health effects. The wide range of doses used and the claims that some high doses are "not toxic" in some assays have confounded studies on its mechanism of action. The purpose of this study is to determine whether the treatment time and particularly the duration between treatment and assay are important factors in assessing arsenite toxicity. We compared three commonly used assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red (NR), and clonal survival, using human osteogenic sarcoma (HOS) cell line U-2OS. Results from the assays were well correlated only when the factor of time was taken into account. In both the MTT and NR assays, exposure to arsenite for 24 h induced much less toxicity than exposure for 48 or 72 h, which gave similar results. In contrast, results in clonal survival assays showed only a small difference between 24-h exposure and longer exposure times. Arsenite demonstrated delayed cytotoxicity, killing the cells even after its removal from the medium in NR assay. Apoptosis was assessed by TUNEL staining and caspase-3 activation. After treatment for 24 h with 0.1 and 1 microM arsenite, no apoptosis was seen. However, after an additional 24 h in arsenite-free medium, a small amount of apoptosis could be detected, and much more apoptosis was seen after 48 h. In contrast, 10 microM arsenite triggered rapid necrosis and failed to activate caspase 3 or cause TUNEL staining. We also confirmed previous reports that exposure to low concentrations of arsenite caused transient stimulation of cell growth. Our finding of delayed toxicity by arsenite suggests that to avoid underestimation of toxicity, the duration between treatment and assay should be taken into account in choosing appropriate doses for arsenite as well as for other toxicants that may show similar delayed toxicity. The NR and MTT assays should be performed only after an interval of at least 48 h after a 24-h exposure to arsenite.

Inorganic arsenic (arsenite and arsenate) in drinking water has been associated with skin cancers in several countries such as Taiwan, Chile, Argentina, Bangladesh, and Mexico. This association has not been established in the United States. In addition, inorganic arsenic alone in drinking water does not cause skin cancers in animals. We recently showed that concentrations as low as 1.25 mg/l sodium arsenite were able to enhance the tumorigenicity of solar UV irradiation in mice. The tumors were almost all squamous cell carcinomas (SCCs). These data suggest that arsenic in drinking water may need a carcinogenic partner, such as sunlight, in the induction of skin cancers. Arsenite may enhance tumorigenicity via effects on DNA repair and DNA damage-induced cell cycle effects, leading to genomic instability. Others have found that dimethlyarsinic acid (DMA), a metabolite of arsenite, can induce bladder cancers at high concentrations in drinking water. In those experiments, skin cancers were not produced. Taken together, these data suggest that arsenite (or possibly an earlier metabolite), and not DMA, is responsible for the skin cancers, but a second genotoxic agent may be a requirement. The differences between the US and the other arsenic-exposed populations with regard to skin cancers might be explained by the lower levels of arsenic in the US, less sun exposure, better nutrition, or perhaps genetic susceptibility differences.

The present study was designed to establish the form of the dose-response relationship for dietary sodium arsenite as a co-carcinogen with ultraviolet radiation (UVR) in a mouse skin model. Hairless mice (strain Skh1) were fed sodium arsenite continuously in drinking water starting at 21 days of age at concentrations of 0.0, 1.25, 2.5, 5.0, and 10 mg/L. At 42 days of age, solar spectrum UVR exposures were applied three times weekly to the dorsal skin at 1.0 kJ/m2 per exposure until the experiment ended at 182 days. Untreated mice and mice fed only arsenite developed no tumors. In the remaining groups a total of 322 locally invasive squamous carcinomas occurred. The carcinoma yield in mice exposed only to UVR was 2.4 +/- 0.5 cancers/mouse at 182 days. Dietary arsenite markedly enhanced the UVR-induced cancer yield in a pattern consistent with linearity up to a peak of 11.1 +/- 1.0 cancers/mouse at 5.0 mg/L arsenite, representing a peak enhancement ratio of 4.63 +/- 1.05. A decline occurred to 6.8 +/- 0.8 cancers/mouse at 10.0 mg/L arsenite. New cancer rates exhibited a consistent-with-linear dependence on time beginning after initial cancer-free intervals ranging between 88 and 95 days. Epidermal hyperplasia was elevated by arsenite alone and UVR alone and was greater than additive for the combined exposures as were growth rates of the cancers. These results demonstrate the usefulness of a new animal model for studying the carcinogenic action of dietary arsenite on skin exposed to UVR and should contribute to understanding how to make use of animal data for assessment of human cancer risks in tissues exposed to mixtures of carcinogens and cancer-enhancing agents.

Arsenic is a common environmental contaminant of our air, water and food, but not every individual who drinks arsenic-contaminated water shows clinical signs of toxicity. Large inter-individual variations are also found in arsenite-induced aneuploidy, chromosome aberrations and sister chromatid exchanges in peripheral blood lymphocytes from different human donors. Lymphoblasts are virally immortalized lymphocytes that retain most of the properties of lymphocytes. Individual lymphoblast cell lines retained their arsenite sensitivity after cryopreservation and subsequent revival. We measured the accumulation of 73[As]-arsenite into lymphoblast lines derived from 11 normal individuals. Arsenite accumulation rate varied 6.3 fold between the slowest and the fastest subjects. Assays in 14 lymphoblast lines showed variability to the toxic effects of arsenite, as measured by growth inhibition. Lymphoblast lines also vary with regard to their growth rates, but there is no relationship between growth rate and arsenite sensitivity. Surprisingly, we also found no correlation between arsenite accumulation rate and cellular sensitivity to growth inhibition, suggesting that the arsenite accumulation rate may not be the main determinant of cellular sensitivity to arsenic. We were also unable to detect evidence for a human homolog for the yeast arsenite efflux gene ACR3, using RT-PCR.

Epidemiological evidence shows an association between inorganic arsenic in drinking water and increased risk of skin, lung and bladder cancers. The lack of animal models has hindered mechanistic studies of arsenic carcinogenesis in the past, but some promising new models for these cancers are now available. The various forms of arsenic to which humans are exposed, either directly or via metabolism of inorganic arsenic to various methylated forms, further complicate the issue of mechanism, since these compounds can have different effects, both genotoxic and non-genotoxic. This review will try to integrate all of these issues, with a strong bias toward effects that are produced by environmentally relevant arsenic concentrations.

Arsenite is the most likely carcinogenic form of arsenic in the environment. Previously, expression cloning for cDNAs whose overexpression confers arsenite-resistance in Chinese hamster V79 cells identified two genes: fau and a novel gene, asr2. The fau gene encodes a ubiquitin-like protein (here called FUBI) fused to the ribosomal S30 protein. Since the expression of the fox sequence (antisense to fau) increased the tumorigenicity of a mouse sarcoma virus, it was proposed that fau might be a tumor suppressor gene. We intended to test its ability to block arsenite-induced transformation of human osteogenic sarcoma (HOS) cells to anchorage-independence. Instead, we found that overexpressing fau itself was able to transform HOS cells. When the two domains were expressed separately, only FUBI was transforming and only the S30 domain conferred arsenite resistance. An incidental finding was the transforming activity of the selectable marker, hyg. FUBI belongs to the ubiquitin-like protein group that is capable of forming conjugates to other proteins, although none have so far been identified. Alternatively, FUBI may act as a substitute or inhibitor of ubiquitin, to which it is most closely related, or to close ubiquitin-like relatives UCRP, FAT10, and/or Nedd8.

Arsenite is a human multisite carcinogen, but its mechanism of action is not known. We recently found that extremely low concentrations (

Although epidemiologic evidence shows an association between inorganic arsenic in drinking water and increased risk of skin, lung, and bladder cancers, no animal model for arsenic carcinogenesis has been successful. This lack has hindered mechanistic studies of arsenic carcinogenesis. Previously, we and others found that low concentrations (< or =5 microm) of arsenite (the likely environmental carcinogen), which are not mutagenic, can enhance the mutagenicity of other agents, including ultraviolet radiation (UVR) and alkylating agents. This enhancing effect appears to result from inhibition of DNA repair by arsenite, but not via inhibition of DNA repair enzymes. Rather, low concentrations of arsenite disrupt p53 function and upregulate cyclin D1. Failure to find an animal model for arsenic carcinogenesis might be because arsenite is not a carcinogen per se but acts as an enhancing agent (cocarcinogen) with a genotoxic partner. We tested this hypothesis with solar UVR in hairless but immunocompetent Skh1 mice. Mice were given 10 mg/L sodium arsenite in drinking water (or not) and irradiated with 1.7 KJ/m(2) solar UVR 3 times weekly. As expected, no tumors appeared in any organs in control mice or in mice given arsenite alone. After 26 weeks irradiated mice given arsenite had a 2.4-fold increase in skin tumor yield compared with mice given UVR alone. The tumors were mostly squamous cell carcinomas, and those occurring in mice given UVR plus arsenite were much larger and more invasive. These results are consistent with the hypothesis that arsenic acts as a cocarcinogen with a second (genotoxic) agent by inhibiting DNA repair and/or enhancing positive growth signaling. Skin cancers in populations drinking water containing arsenic may be caused by the enhancement by arsenic compounds of carcinogenesis induced by UVR (or other environmental agents). It is possible that lung and bladder cancers associated with arsenic in drinking water may also require a carcinogenic partner.

Pressure treatment with chromium, copper, and arsenic (CCA) is the most prevalent method for protecting wood used in outdoor construction projects. Although these metals are tightly bound to the wood fibers and are not released under most conditions of use, we examined the bioavailability of metals in CCA pressure-treated wood dust in vitro. Cytotoxicity and metallothionein (MT) mRNA expression were examined in V79 Chinese hamster lung fibroblast cells incubated with respirable-size wood dust generated by sanding CCA-treated and untreated (control) Southern yellow pine. In colony survival studies, increased cytotoxicity (p < 0.05) occurred in V79 cells treated with CCA wood dust (351 +/- 77 microg/ml, mean +/- SE) compared with control wood dust (883 +/- 91 microg/ml). Increased cytotoxicity with CCA wood dust also occurred in an arsenic resistant subline of V79 cells, thus suggesting that arsenic was not responsible for the increased cytotoxicity. Metallothionein mRNA was significantly increased after 48 h of treatment with CCA wood dust compared with control wood dust. Incubation of CCA wood dust in cell culture media resulted in the transfer of copper, but not chromium or arsenic, into the media. Moreover, the treatment of cells with this filtered extract resulted in significantly increased metallothionein mRNA, suggesting that bioavailable copper is responsible for inducing metallothionein mRNA in V79 cells. Thus, these bioassays suggest that metals become bioavailable during in vitro culture of phagocytic V79 cells with CCA wood dust.

Identifying genes upregulated in lead-resistant cells should give insight into lead toxicity and cellular protective mechanisms and may also result in identification of proteins that may be useful as biomarkers. Glial cells are thought to protect neurons against heavy metals. Rat glioma C6 cells share many properties of normal glial cells. To identify and analyze genes upregulated in a lead-resistant variant, PbR11, suppression subtractive hybridization (SSH) between mRNAs of wild-type and PbR11 cells was performed. Sequencing and database searches identified three genes, thrombospondin-1, heparin sulfate 6-sulfotransferase, and neuropilin-1, which play important roles in angiogenesis and axon growth during development. Two genes, HSP90 and UBA3, are involved in the ubiquitin-proteosome system. One gene was identified as that of a rat endogenous retrovirus and another, 2C9, is a transcript expressed in fos-transformed cells. PbR11 also overexpresses c-fos. Expression of these genes and effects of short-term lead exposure (24 h, up to 600 microM) on their expression in C6 cells was examined. The rat endogenous retrovirus and 2C9 are expressed only in PbR11 cells, and show no expression, either constitutive or lead-induced, in wild-type C6 cells. HSP90 is expressed at low level constitutively in C6 cells, but can be induced in a dose-dependent manner by lead. In contrast, thrombospondin-1 is repressed in a dose-dependent manner by lead. The other genes (HS6ST, neuropilin, and UBA3) show low constitutive expression and are neither upregulated nor downregulated by exposure to lead. We suggest that neuropilin-1, heparin sulfate 6-sulfotransferase, and thrombospondin-1 may be important targets for lead-induced developmental neurotoxicity.

Although epidemiological evidence shows an association between arsenic in drinking water and increased risk of skin, lung, and bladder cancers, arsenic compounds are not animal carcinogens. The lack of animal models has hindered mechanistic studies of arsenic carcinogenesis. Previously, this laboratory found that low concentrations of arsenite (the likely environmental carcinogen) which are not mutagenic can enhance the mutagenicity of other agents, including ultraviolet radiation (UVR). This enhancing effect appears to result from inhibition of DNA repair by arsenite. Recently we found that low concentrations of arsenite disrupted p53 function and upregulated cyclin D1. These results suggest that the failure to find an animal model for arsenic carcinogenesis is because arsenite is not a carcinogen per se, but rather acts as an enhancing agent (cocarcinogen) with a genotoxic partner. We tested this hypothesis with solar UVR as carcinogenic stimulus in hairless Skh1 mice. Mice given 10 mg/l sodium arsenite in drinking water for 26 weeks had a 2.4-fold increase in yield of tumors after 1.7 KJ/m(2) UVR three times weekly compared with mice given UVR alone. No tumors appeared in mice given arsenite alone. The tumors were mostly squamous cell carcinomas, and those occurring in mice given UVR plus arsenite appeared earlier and were much larger and more invasive than in mice given UVR alone. These results are consistent with the hypothesis that arsenic acts as a cocarcinogen with a second (genotoxic) agent by inhibiting DNA repair and/or enhancing positive growth signaling., (Copyright 2001 Academic Press.)

Cells lacking mismatch repair (MMR) exhibit elevated levels of spontaneous mutagenesis. Evidence exists that MMR is involved in repair of some DNA lesions besides mismatches. If some oxidative DNA lesions are substrates for MMR, then the excess mutagenesis in MMR(-) cells might be blocked by dietary antioxidants. Effects of the dietary antioxidants ascorbate, alpha-tocopherol, (-)-epigallocatechin gallate (EGCG) and lycopene on spontaneous mutagenesis were studied using mismatch repair-deficient (hMLH1(-)) human colon carcinoma HCT116 cells and HCT116/ch3 cells, in which normal human chromosome 3 has been added to restore mismatch repair. HCT116 cells have a 22-fold higher spontaneous mutation rate compared with HCT116/ch3 cells. HCT116 cells cultured in 1% fetal bovine serum (FBS) have twice the spontaneous mutation rate of those cultured in 10% FBS, most likely due to reduction in serum antioxidants in the low serum medium. As expected, alpha-tocopherol (50 microM) and ascorbate (284 microM) reduced spontaneous mutagenesis in HCT116 cells growing in 1% serum more dramatically than in cells cultured in 10% serum. The strongest antimutagenic compound was lycopene (5 microM), which reduced spontaneous mutagenesis equally (about 70%) in HCT116 cells growing in 10 and 1% FBS and in HCT116/ch3 cells. Since lycopene was equally antimutagenic in cells growing in low and high serum, it may have another antimutagenic mechanism in addition to its antioxidant effect. Surprisingly, EGCG (10 microM) was toxic to cells growing in low serum. It also reduced spontaneous mutagenesis equally (nearly 40%) in HCT116 and HCT116/ch3 cells. The large proportion of spontaneous mutagenesis that can be blocked by antioxidants in mismatch repair-deficient cells support the hypothesis that a major cause of their excess mutagenesis is endogenous oxidants. Blocking spontaneous mutagenesis, perhaps with a cocktail of antioxidants, should reduce the risk of cancer in people with a genetic defect in mismatch repair as well as other individuals.

Arsenite, the most likely environmental carcinogenic form of arsenic, is not significantly mutagenic at non-toxic concentrations, but is able to enhance the mutagenicity of other agents. Evidence suggests that this comutagenic effect of arsenite is due to inhibition of DNA repair, but no specific repair enzyme has been found to be sensitive to low (<1 microM) concentrations of arsenite. To determine whether arsenite affects signaling which might alter DNA repair, this study assesses the effect of arsenite on p53-related signal transduction pathways after ionizing radiation. Long-term (14 day) low dose (0.1 microM) arsenite caused a modest increase in p53 expression in WI38 normal human fibroblasts, while only toxic (50 microM) concentrations increased p53 levels after short-term (18 h) exposure. When cells were irradiated (6 Gy), p53 and p21 protein concentrations were increased after 4h, as expected. Both long-term, low dose and short-term, high dose exposure to arsenite greatly suppressed the radiation-induced increase in p21 abundance. In addition, long-term, low dose (but not short-term, high dose) exposure to arsenite resulted in increased expression of cyclin D1. These results show that in cells treated with arsenite, p53-dependent increase in p21 expression, normally a block to cell cycle progression after DNA damage, is deficient. At the same time, low (non-toxic) exposure to arsenite enhances positive growth signaling. We suggest that the absence of normal p53 functioning, along with increased positive growth signaling in the presence of DNA damage may result in defective DNA repair and account for the comutagenic effects of arsenite.

When cells are exposed to toxicants, changes in gene expression ensue. To date, there is little information on gene expression changes induced by metals in mammalian cells. The basic methods for identifying altered gene expression of both a temporary and a permanent nature are outlined, with examples drawn mostly from what is known about metal-induced changes in gene expression. The application of this information in the development of new biomarkers of exposure and effect, in identifying individuals with altered susceptibility to metal compounds, and in the choice of genes for microarrays is discussed., (Copyright 2000 Wiley-Liss, Inc.)

Glial cells are thought to protect neurons from heavy-metal toxicity. To gain a better understanding of mechanisms of protection against lead compounds, a number of lead-resistant C6 rat glioma cell sublines have been isolated. After 8 mo of growth in the absence of lead nitrate, three sublines still maintain their lead-resistant phenotype. None of the lead-resistant sublines are cross-resistant to Cd(II) or Ni(II), but all are cross-resistant (in varying degrees) to Hg(II), As(III), Sb(III), and Sn(II), and one is resistant to trimethyl tin. No inducible lead resistance is seen in any glioma line. One subline has been used to create cell-cell hybrids with wild-type cells. The hybrids exhibit dominance of the lead-resistant phenotype. To identify and analyze altered gene expression at the mRNA level in the lead-resistant sublines, the differential display technique was used. Numerous differences are seen between amplified fragments from wild-type and lead-resistant cells. Candidate clones are now being analyzed to confirm the differential expression and to isolate cDNAs that confer lead resistance.

Little is known about endogenous processes causing spontaneous mutagenesis in mammalian cells. To study this problem, a mathematical model and method developed previously in our laboratory was used to measure the spontaneous mutation rate in mammalian cells at the transgenic gpt locus in Chinese hamster G12 cells. We found that spontaneous mutagenesis increased when cells were cultured in low (<0.25%) serum. These cells also contained higher oxidant levels, measured by dichloroflourescein (DCF) fluorescence, suggesting that the elevated spontaneous mutagenesis resulted from endogenous oxidants which are normally quenched by serum antioxidants. This was found to be the case. Spontaneous mutagenesis was significantly reduced in serum-depleted as well as control cells when catalase (100 ng/ml) or the antioxidants ascorbate (50 microg/ml) or mannitol (100-500 microg/ml) were added to the medium. Overexpression of metallothionein in these cells also suppressed spontaneous mutagenesis and mutagenesis induced by oxygen radical-generating compounds. Cells expressing metallothionein antisense RNA become mutators. Taken together, these results suggest that the major cause of spontaneous mutagenesis in mammalian cells is endogenously-generated oxidative DNA damage which can be blocked by metallothionein or by dietary antioxidants carried by the blood supply., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

Chinese hamster V79 cells and their arsenite-resistant variants were found to have an arsenite- and antimonite-inducible tolerance mechanism which protects against the subsequent cytotoxic effects of arsenate, arsenate and antimonite. Inducible tolerance requires de novo mRNA and protein synthesis, and is independent of the heat shock response. In contrast, we report that the arsenite hypersensitive variant line As/S27D lacks the inducible tolerance response. Numerous attempts were made to detect an inducible tolerance response to arsenite in a variety of human cells. An assay based on Neutral red uptake was used in order to study inducible tolerance in cells with poor clonability. Neither normal diploid cells nor human tumor cells of different origins were found to elicit an inducible tolerance response to arsenite. This finding may help to explain why rodents do not develop tumors after exposure to arsenite, while humans do. In addition, all human cell lines tested were much more sensitive to arsenite compared to Chinese hamster cells. Human keratinocytes were especially sensitive. In general, human cells resemble arsenic hypersensitive Chinese hamster As/R27D cells, which have lost a protective mechanism found in wild-type Chinese hamster cells.

The functions of metallothioneins (MTs) have been debated for at least a decade. Because it seems unlikely that they evolved only to protect cells against exogenous heavy metals, it has been suggested that MTs have roles in scavenging reactive intermediates, controlling zinc and copper homeostasis, and controlling transfer of zinc to transcription factors and other proteins. Previously, we demonstrated that Chinese hamster G12 cells which overexpress MT have greatly reduced spontaneous mutation rates, suggesting that MT evolved to prevent spontaneous mutagenesis induced by free nuclear zinc ions. We have now isolated G12 transfectants which express antisense RNA to MT. Immunofluorescent staining reveals MT protein in both the nucleus and the cytoplasm in parental cells. A clone expressing high levels of antisense RNA (AMT30) shows reduced basal and induced levels of MT protein. AMT30 cells are hypersensitive to cadmium, zinc, copper and mercury chlorides as well as to menadione. Glutathione levels in AMT30 and G12 cells do not differ. AMT30 cells are spontaneous mutators, showing a spontaneous mutation rate 5-10 times that of G12 cells or G12 cells transfected with vector alone. Only transfectants which show a high level of MT antisense expression (i.e., AMT30) had greatly elevated spontaneous mutation rates. These results support our hypothesis that a major role of MT is to act as an endogenous antimutagen probably via scavenging of reactive intermediates in the nucleus. AMT30 cells should be useful in delineating the sources of spontaneous mutagenesis.

When estimating a spontaneous mutation rate from either a single culture (C=1) or from the C parallel cultures (C>1) of a fluctuation experiment, the use of a large initial population size N0 to seed each culture will permit a gaussian approximation for the probability distribution of the number M of mutants at the time when the culture(s) has (have) grown to size N=N02g, i.e., experienced g doublings. Using this gaussian approximation we find that the maximum likelihood estimate mu of the expected number mu of mutants present in a culture in generation g is (exactly) (equation: see text) where r = 2g / g and M 2 is the average of the squares of the C mutant counts. The maximum likelihood estimate p of the unknown mutation rate p is p = 2 mu / gN assuming an 'ideal' experiment and that there were no mutants in the initial population. A well-behaved maximum likelihood estimate is known to be efficient in large samples and we illustrate by Monte Carlo simulation that indeed p is better (has smaller mean squared error) than our previous (Rossman et al., 1995) estimator (equation: see text) (M is the average mutant count) provided N0 is of the order 1/p or larger. This advantage exists even without a fluctuation experiment, i.e., for C = 1.

Several Chinese hamster V79 cell line variants resistant to arsenite and one arsenite-hypersensitive variant have been isolated. The basis for the variation in arsenite sensitivity was studied by transport experiments using radiolabeled arsenite. Two arsenite-resistant variants (As/R7 and As/R27) exhibited decreased accumulation of arsenite, and the hypersensitive variant (As/S5) exhibited increased arsenite accumulation compared with the parental line. Cells depleted of endogenous energy reserves were loaded with radiolabeled arsenite, and the rate of arsenic efflux was measured. Arsenite-resistant variants exhibited an increased rate of efflux, while the hypersensitive variant exhibited a decreased efflux rate. Efflux was decreased in cells incubated with the protonophore carbonyl cyanide m-chlorophenylhydrazine, demonstrating its energy dependence. Two inhibitors of glutathione S-transferase also decreased arsenite efflux, suggesting the involvement of an arsenite-glutathione complex. However, separation of the products of extrusion and the intracellular arsenic species by paper chromatography followed by autoradiography failed to show the appearance of an arsenite-glutathione complex in either case. Rather, all label in the product of the transport reaction appeared to be arsenite whether cells were loaded with arsenate or arsenite, indicating first that intracellular reduction of As(V) to As(III) had occurred and second that the arsenite was transported as an unconjugated species. All intracellular label was associated with high-molecular-weight material, possibly protein. Our results demonstrate the existence of an energy-dependent arsenical efflux pump in mammalian cells and show that arsenic is extruded as arsenite.

Spontaneous mutagenesis is thought to play a crucial role in spontaneous carcinogenesis. We recently described a new mathematical model for estimation of the spontaneous mutation rate (mutation/gene/generations) based on the assumption that mutations are fixed in the S-phase of the cell cycle. With this definition, the spontaneous mutation rate should be independent of the growth rate. In the present study, we tested this hypothesis, using cell line G12, a transgenic Chinese hamster V79 derivative, which contains a single copy of the Escherichia coli gpt gene as a target for mutagenesis. The growth rate was modulated by varying the serum concentration or the seeding density, or by addition of suramin, transforming growth factor beta, or dichlorobenzimidazole riboside to the medium. Significant increases in the spontaneous mutation rate occurred when cell proliferation was blocked by serum deprivation. Density-dependent inhibition of growth and inhibition of growth by suramin, transforming growth factor beta, or dichlorobenzimidazole riboside did not result in significant increases in spontaneous mutation rates. The level of oxidants in cells cultivated in the presence of low concentrations of serum was higher compared to control cells, suggesting that the increases in the spontaneous mutation rates under low serum conditions may be partly a result of oxidative stress due to a lack of serum antioxidants. This was shown to be the case, because spontaneous mutation rates were significantly reduced in serum-depleted cells when antioxidants were added to the medium. We suggest that during carcinogenesis, when tumors are in a prevascularized state, the spontaneous mutation rate may be elevated, and this process may contribute to the genetic instability of the tumor cells.

Certain mathematical artifacts which had been appended by others to Luria and Delbrück's [Genetics 28: 491-511, 1943] model of spontaneous mutagenesis in bacterial populations have added confusion to the modeling and measurement of spontaneous mutation rates. Additional confusion arises when models which had been tuned for experiments with bacterial cultures grown from a small inoculum are adapted for use with mammalian cell cultures grown from a large initial population. As one consequence, biologists still tend to grow the large number of parallel cultures required by the fluctuation test in order to avoid large errors due to the high variability in the number of mutants in a growing culture. By avoiding models with infinite mean values and certain mathematical approximations that lead to conceptual and practical difficulties, the large variance of the number of mutants can be avoided (and the precision of the estimated mutation rate controlled) through the use of sufficiently large initial cell populations. A direct consequence is that simpler experiments with fewer cultures may suffice. In this paper, after a discussion of the confusions, we extend our previous approach [Rossman et al.: Mutat Res 328:21-30, 1995] by giving improved formulas for the standard error of the estimated mutation rate. The improvement results from using a more inclusive model based on consideration of the variability due to both the biological phenomenon of the growing culture (growth and mutation) and the protocols used for selection (sampling and plating efficiency). Also included is the situation where the initial cell population is not assumed to be free of mutants but the initial mutant fraction is measured instead. These standard error formulas are useful in planning experiments that yield mutation rate estimates with planned precision and for comparing and testing hypotheses about mutation rates in two or more populations which are grown under different conditions.

The study of spontaneous mutation rates in mammalian cells has been hampered by the lack of an alternative to the cumbersome Luria and Delbrück fluctuation test. A brief review of mathematical treatments of spontaneous mutagenesis, along with some of the limitations of the fluctuation test, is presented. A new experimental method and a simple mathematical model for deriving the spontaneous mutation rate are described. Data from the transgenic Chinese hamster G12 cell line growing at two different rates is analyzed according to this model. The results support the concept that, at least for growing cells, the spontaneous mutation rate is independent of the growth rate, and the mutant fraction increases in a linear fashion with the number of generations.

G12, a transgenic Chinese hamster V79 cell derivative which contains a single copy of the Escherichia coli gpt gene as a target for mutagenesis, has little constitutive metallothionein (MT) expression. It was transfected with a vector containing the mouse MT-I gene, and MT-I-overproducing lines were isolated. MT-I transfectants had lower spontaneous mutation frequencies compared with the G12 parental cell line. Mutagenesis by alkylating agents was unchanged. MT expression in G12 and MT transfectants could be modulated by exposure to Zn(II) or Cd(II). The spontaneous mutation frequencies in Zn(II)- and Cd(II)-treated cells was inversely related to MT expression. In G12 cells grown in concentrations of Zn(II) up to 12 microM, a significant dose-dependent increase in spontaneous mutagenesis was observed. At higher (but subtoxic) concentrations in which endogenous MT was induced, a dramatic decrease in spontaneous mutagenesis was observed. In contrast, MT-I transfectants exhibited much lower spontaneous mutagenesis after growth in all concentrations of Zn(II). These data demonstrate a possible role for MT in modulating spontaneous mutagenesis and point to a role for Zn(II) in contributing to spontaneous mutagenesis. Because there is variability in human MT expression, low MT expression might be a risk factor for cancer.

Both V79 and As/R28A cells (an arsenite-resistant Chinese hamster V79 cell variant) show increased resistance to toxic concentrations of arsenite after pretreatment with a nontoxic concentration. The induced tolerance can be completely inhibited by actinomycin D or cycloheximide. Pretreatment with a nontoxic heat shock (45 degrees C, 10 min) resulted in a clear increased thermotolerance in both cell lines but failed to induce arsenite tolerance in either cell line. Pretreatment with arsenite induced a thermotolerance in V79 cells but not in As/R28A cells. These results are consistent with a model whereby the signal for induction of arsenite tolerance involves binding of arsenite to a protein effector which is amplified in the As/R28A line, thereby preventing action of arsenite in the regulation of heat shock factor which induces the heat shock response.

Several gpt+ transgenic cell lines were derived from hprt V79 cells to study mutagenesis mechanisms in mammalian cells. The G12 cell line was previously shown to be hypermutable by X-rays and UV at the gpt locus compared to the endogenous hprt gene of the parental V79 cells (Klein and Rossman, 1990), and is now shown to be highly mutable by the clastogenic anti-tumor agent bleomycin sulfate. A second transgenic cell line G10, which has a different gpt insertion site, was studied in comparison with G12. Both G12 and G10 cell lines carry the stable gpt locus at a single integration site in the Chinese hamster genome, and neither spontaneously deletes the integrated gpt sequence at a high frequency. Although spontaneous mutation to 6-thioguanine resistance in G10 cells is 3-4 times higher than in G12 cells, the cell lines differ to a much greater extent when mutated by clastogens. In comparison to G12 cells, the gpt locus in G10 cells is up to 13 times more sensitive to bleomycin mutagenesis and 5 times more responsive to X-ray mutagenesis. In contrast, there is much less difference in UV-induced mutagenesis and no differences in mutagenesis induced by alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The dose-dependent decrease in survival of the transgenic cells is the same for all mutagens tested, and does not differ from that of V79 cells. Neither transgenic cell line is generally hypermutable, since mutagenesis at an endogenous gene, Na+K+/ATPase, is similar to that of the parental V79 cell line. Although both cell lines can be induced to delete the transgene following clastogen exposure, deletions are not the only recovered mutations, and the cell lines can also be used to study mutations within the PCR recoverable gpt gene. The utility of these transgenic cells to investigate genome position effects related to mammalian mutagenesis mechanisms is discussed.

Nickel is an established human and animal carcinogen, but efforts to demonstrate its mutagenicity in a number of cell types have not been successful. In this report we describe the mutational response to nickel compounds in the G12 cell line, an hprt deficient V79 cell line containing a single copy of the E. coli gpt gene. This cell line has a low spontaneous background, making it suitable for assessment of mutagenic responses to environmental contaminants. When G12 cells were treated with insoluble particles of crystalline nickel sulfide < 5 microns in diameter, a strong, dose-dependent mutagenic response was observed up to 80 times the spontaneous background. Of 48 mutant gpt(-) clones isolated that were induced by insoluble nickel, all were capable of DNA amplification of the gpt sequences by polymerase chain reaction (PCR). The ability to produce full-length PCR products is an indication that large deletions of gene sequences have not occurred. When G12 cells were treated with soluble nickel sulfate, the mutational response was not significantly increased over the spontaneous background. This difference in mutagenic response reflects a large difference in the mutagenic potential of soluble and insoluble nickel compounds, which reflects the carcinogenic potential of these forms of nickel.

A number of Chinese hamster V79 cell sublines that are arsenite resistant or arsenite sensitive were isolated. After more than 6 months of growth in the absence of arsenite, these sublines still maintain their arsenite-resistant or -sensitive phenotypes. At least some arsenite-resistant cell lines are also cross-resistant to sodium arsenate and potassium antimonyl tartrate. Wild-type or arsenite-resistant sublines show a further increase in resistance to toxic concentrations of arsenite after pretreatment with a nontoxic concentration. Pretreatment of cells with a nontoxic dose of arsenite also increased their survival to toxic doses of antimonite. Likewise, pretreatment with antimonite increased survival to arsenite as well as to antimonite. The inducible arsenite resistance increases with pretreatment time, reaching a plateau after 8 hr of pretreatment. Fusion of arsenite-resistant cells with arsenite-sensitive cells demonstrated a clear dominance of arsenite resistance. These results suggest that mammalian cells contain an arsenite/antimonite pump whose activity may be modulated by prior exposure to arsenite or antimonite.

We have previously reported that lead(II) is weakly mutagenic to Chinese hamster V79 cells. A transgenic cell line G12 containing a single copy of the E. coli gpt gene was developed in this laboratory from Chinese hamster V79 cells. The gpt locus in the G12 cells is more mutable by radiation and oxidative agents compared with the endogenous hprt locus of wild-type V79 cells. We have investigated the mutagenicity of two lead compounds at the gpt locus in G12 cells. Only at a toxic dose is lead acetate significantly mutagenic to G12 cells. Lead nitrate is not significantly mutagenic at any dose. Although both compounds are water-soluble, lead acetate, but not lead nitrate, forms a fine white insoluble precipitate upon addition to growth medium. A nick translation assay on cells treated with lead compounds and then permeabilized indicated that lead nitrate and, to a greater extent, lead acetate causes the appearance of nicks in chromosomal DNA. Lead ions in the presence of hydrogen peroxide, but not alone, introduced nicks into supercoiled plasmid DNA in vitro, suggesting that lead ions can partake in a Fenton reaction and thereby damage DNA. At lower nonmutagenic concentrations, lead acetate enhances the mutagenicity of MNNG and ultraviolet light. DNA damage by ultraviolet light is not enhanced by lead ions in vitro. Our data support the concept that non-toxic concentrations of lead(II) can inhibit DNA repair. Thus, at biologically relevant doses, lead(II) could act as a comutagen and possibly a cocarcinogen, but is not likely to act as an initiating genotoxic carcinogen.

Previous evidence that cadmium(II) causes gene mutations in bacteria or mammalian cells was weak. However, alterations in protocol have recently led to better evidence for its mutagenicity, especially in bacteria. Mutagenicity results may be confounded by tolerance mechanisms. Exposure of DNA in vitro to Cd2+ or to Cd2+ hydrogen peroxide does not result in strand breaks or alkali-labile sites. The fact that bacterial and mammalian cells appear to sustain some type of repairable DNA damage after exposure to Cd2+ suggests that the damage must be caused in an indirect manner. Recently, the ability of cadmium-metallothionein complex to cause DNA strand breaks has been described. Cd2+ also induces a "pro-oxidant state" by causing a depletion of cellular glutathione. This finding is consistent with the role of Cd2+ as a clastogen and may explain its weak mutagenicity at loci which cannot detect complex mutations. Cd2+ can also inhibit DNA repair, and can therefore act synergistically with certain mutagens and, presumably, carcinogens.

Gene amplification contributes to carcinogenesis by enhancing proto-oncogene activity and causing chromosomal instability. The ease of detecting amplified tumor-virus sequences has encouraged use of this system as a surrogate for studying the molecular events involved in endogenous gene amplification. We report here a new system for studying carcinogen-induced amplification of both endogenous and viral sequences in the SV40-transformed human keratinocyte line AG06. Treatment with carcinogens induced a transient dose-dependent amplification of the integrated SV40 sequences. The amplified sequences appeared in the extrachromosomal fraction. Treatment of these cells with carcinogens prior to methotrexate (MTX) selection increased the frequency of MTX-resistant colonies, 67% of which exhibited dihydrofolate reductase (dhfr) amplification. The abilities of five carcinogens with different DNA-damaging activities (the DNA-damaging agents N-methyl-N-nitro-N-nitrosoguanidine, mitomycin C (MMC), ultraviolet light C, and X-rays and the non-DNA-damaging agent arsenite) to induce SV40 and dhfr amplification at concentrations that result in 50% clonal survival were compared. All four DNA-damaging carcinogens (as well as growth arrest) were able to elicit some SV40 amplification, but responses varied markedly, from 1.8-fold for X-rays to sevenfold to eightfold for MMC. There was no correlation between the ability to elicit the two amplification responses. Arsenite, which did not induce SV40 amplification, was the best inducer of MTX resistance. These results point to different controls involved in the induction of viral and dhfr amplification. The signal for amplification of viral genes may be triggered by DNA damage and growth arrest, whereas amplification of dhfr, and perhaps other endogenous sequences, seems to be triggered by other signals as well.