Regulation of arsenic methylation: identification of the transcriptional region of the human AS3MT gene.

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.

Antimony and its compounds: Health impacts related to pulmonary toxicity, cancer, and genotoxicity.

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.

Nonsynonymous Polymorphisms in the Human AS3MT Arsenic Methylation Gene: Implications for Arsenic Toxicity.

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.

Mode of action assessment of the genotoxic properties of antimony and its compounds evaluated in the ToxTracker assay.

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.

Arsenite induced poly(ADP-ribosyl)ation of tumor suppressor P53 in human skin keratinocytes as a possible mechanism for carcinogenesis associated with arsenic exposure.

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.

fau and its ubiquitin-like domain (FUBI) transforms human osteogenic sarcoma (HOS) cells to anchorage-independence.

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-induced alterations of DNA photodamage repair and apoptosis after solar-simulation UVR in mouse keratinocytes in vitro.

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/m2 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/m2. If the present results are applicable in vivo, inhibition of UVR-induced apoptosis may contribute to arsenite's enhancement of UVR-induced skin carcinogenesis.

Caffeic acid phenethyl ester (CAPE) prevents transformation of human cells by arsenite (As) and suppresses growth of As-transformed cells.

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 cocarcinogenesis: an animal model derived from genetic toxicology studies.

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.

Arsenic-induced enhancement of ultraviolet radiation carcinogenesis in mouse skin: a dose-response study.

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.

In vitro bioavailability of heavy metals in pressure-treated wood dust.

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.

Arsenite induces delayed mutagenesis and transformation in human osteosarcoma cells at extremely low concentrations.

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

Mechanism of arsenic carcinogenesis: an integrated approach.

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.

Genetic and epigenetic effects of environmental arsenicals.

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.

Gene expression levels in normal human lymphoblasts with variable sensitivities to arsenite: identification of GGT1 and NFKBIE expression levels as possible biomarkers of susceptibility.

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.

Further evidence against a direct genotoxic mode of action for arsenic-induced cancer.

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.

Pro-angiogenesis action of arsenic and its reversal by selenium-derived compounds.

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.

Dietary chromium and nickel enhance UV-carcinogenesis in skin of hairless mice.

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.

Vitamin E and organoselenium prevent the cocarcinogenic activity of arsenite with solar UVR in mouse skin.

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.