Nitric oxide is involved in arsenite inhibition of pyrimidine dimer excision
D.T. Bau1,2,
J.R. Gurr1 and
K.Y. Jan1,3
1 Institute of Zoology, Academia Sinica, Taipei 11529 and
2 Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 107, Republic of China
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Abstract
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Arsenite is a human carcinogen reported to inhibit DNA repair. The binding of arsenite to functional thiol groups of DNA repair enzymes has in the past been suggested as a possible mechanism for the effect of arsenite on DNA repair. However, recent studies indicate that reactive oxygen species and nitric oxide are involved in arsenite toxicity. This research aims to elucidate the role of these possible mechanisms in the inhibition of UV-induced DNA repair by arsenite. As arsenite inhibits UV-DNA repair in Chinese hamster ovary cells, and this is a commonly used cell line for UV repair experiments, we used these cells to examine the effect of arsenite on the expression of UV-irradiated reporter genes. The T4 UV endonuclease V-incorporated comet assay was used to examine specifically the effect of arsenite on pyrimidine dimer excision. We showed that inhibition of UV-DNA repair by arsenite was suppressed by nitric oxide synthase inhibitors. Arsenite increased nitric oxide production and nitric oxide generators inhibited UV-DNA repair. The involvement of nitric oxide in the inhibition of pyrimidine dimer excision by arsenite was also confirmed in human fibroblasts. Investigation into the effect of oxidant modulators did not give a clear indication that reactive oxygen species are involved in arsenite inhibition of UV-DNA repair. Phenylarsine oxide, a strong thiol-reacting agent, did not inhibit pyrimidine dimer excision and also did not increase nitric oxide production. Our results show conclusively that nitric oxide is involved in the inhibition of pyrimidine dimer excision by arsenite. Reactive oxygen species and the binding of arsenite to functional thiol groups of DNA repair enzymes do not appear to be involved.
Abbreviations: Den V, T4 UV endonuclease V; Fpg, formamidopyrimidine-DNA glycosylase; MTC, S-methyl-L-thiocitrulline; NAME, N-nitro-L-arginine methylester; SIN-1, 3-morpholinosydonimine; Tiron, sodium 4,5-dihydroxybenzene-1,3-disulfonate; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.
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Introduction
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Chronic exposure to arsenic has been related to the etiology of cancers in skin, lung, bladder and kidney (13). Although the clastogenicity of arsenic is well demonstrated, arsenic is generally regarded as a very weak mutagen. It has recently been demonstrated that arsenite is mutagenic in human cells (4) and induces large deletion mutations incompatible with cell survival (5). In addition, arsenite enhances the genotoxicity of several other mutagens (68). The concentration of arsenite required to enhance the genotoxicity of other mutagens is lower than the concentration required for arsenite alone to have a genotoxic effect (9). For this reason the co-genotoxicity of arsenite is potentially hazardous to human health. The mechanism for the co-genotoxicity of arsenite may involve inhibition of DNA repair. Arsenite enhances UV killing in excision-proficient normal human and xeroderma pigmentosum variant cells, but not in excision-defective xeroderma pigmentosum group A cells (10). In nucleotide excision repair, the excision and ligation steps are preferentially inhibited by arsenite (1113).
Inhibition of DNA repair is a major factor in the development of cancer, which may result from mutations in proto-oncogenes and tumor suppressor genes. Reduced DNA repair capacity has been reported for patients with skin (14), colon (15), breast (16) and lung cancer (17). First-degree relatives of patients with cancer also have reduced DNA repair capacity (18). Evidence from molecular epidemiological studies suggests that reduced DNA repair capacity is an important determinant for genetic susceptibility to cancer (19). DNA damage induced by UVC is repaired mainly by nucleotide excision repair, a major pathway for the removal of bulky DNA damage caused by various environmental mutagens. Therefore, arsenite may cause a reduction in the ability of a cell to repair bulky DNA lesions. Since DNA damage is continuously induced by exogenous and endogenous mutagens, inhibition of nucleotide excision repair may also be of general relevance to the carcinogenic potential of arsenic compounds.
The binding of arsenite to protein thiols (20) has been suggested as a mechanism of arsenic toxicity. Since several DNA repair enzymes contain thiol groups in their functional domains, it is possible that arsenite inhibits DNA repair by binding to these functional domains. However, the activity of DNA repair enzymes such as formamidopyrimidine-DNA glycosylase (Fpg), XPA (21), DNA ligase III and poly(ADP-ribosylation) polymerase (22), known to contain thiol groups in zinc finger structures, are not affected by arsenite (22). Moreover, arsenite has been shown to inhibit DNA repair in cells at a much lower concentration than in crude cell extracts (23), or in purified DNA repair enzyme preparations (24). The lack of arsenic-sensitive proteins involved in DNA damage repair suggests that binding of arsenite to the functional thiol groups of a DNA repair protein is not a mechanism for arsenite-mediated DNA repair inhibition.
Recent studies show that reactive oxygen species are involved in arsenite-induced cell signaling and activation of transcription factors (25), DNA strand breakage (26), gene mutation (5), generation of micronuclei (27) and apoptosis (28). Similarly, nitric oxide is involved in arsenite-induced DNA strand breakage (29), poly(ADP-ribosylation) (30) and generation of micronuclei (31). The purpose of this research is to investigate the role of arsenite binding to thiols, reactive oxygen species and nitric oxide in the inhibition of UV-induced DNA repair by arsenite.
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Materials and methods
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Materials
Chinese hamster ovary cells from ATCC (Rockville, MD) and human fibroblasts from Dr Wen Wu-Nan (Department of Biochemistry, National Taiwan University) were grown in McCoy's 5A medium or DMEM medium, supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.03% glutamine. Cells were cultured by incubation at 37°C in a water saturated atmosphere containing 5% CO2. Cell culture chemicals were from Gibco (Grand Island, NY). D-Mannitol, dimethyl sulfoxide, hydrogen peroxide and sodium arsenite were from Merck (Darmstadt, Germany). Catalase, 3-aminotriazole, diethyldithiocarbamate, mercaptosuccinic acid, diphenylene iodonium chloride, 3-morpholinosydonimine (SIN-1), sodium nitrosoprusside, sodium selenite, superoxide dismutase, sodium 4,5-dihydroxybenzene-1,3-disulfonate (Tiron) and uric acid were from Sigma (St Louis, MO). S-Methyl-L-thiocitrulline (MTC) and N-nitro-L-arginine methyl ester (NAME) were from Molecular Probes (Eugene, OR). Phenylarsine oxide and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were from Aldrich (Milwaukee, WI). T4 UV endonuclease V (Den V) was from Epicentre Technologies (Madison, WI). Fpg and proteinase K were from Trevigen (Gaithersburg, MD).
UV irradiation
Cells were washed with phosphate-buffered saline, drained, then exposed to UV radiation at a dose-rate of 0.5 J/m2/s using a Mineralight lamp (Model UVGL-58, Ultraviolet Products) emitting at 254 nm. The fluence rate was checked with an UVX-254 Radiometer (Ultra-violet Product). Plasmids (400 µg in 200 µl TrisEDTA buffer) were irradiated on ice at a dose-rate of 1 J/m2/s.
Plasmids and transfection
Plasmids pGL2 and pCMV-ß-gal containing the reporter genes luciferase and ß-galactosidase, respectively (Promega, Madison, WI) were transformed into competent JM109 bacteria and purified by CsClethidium bromide gradient. Chinese hamster ovary cells were seeded at a density of 1x106 cells per 100 mm dish. After 24 h incubation 1 ml solution containing 6 µg plasmid, 200 µl 200 µg/ml DEAE-dextran and 0.05 M TrisHCl pH 7.8 in serum free McCoy's 5A medium was added to the cells. After 2 h in culture the DNA-containing medium was removed and the cells were washed three times with phosphate-buffered saline. Fresh medium was added and the cultures were incubated a further 24 h to allow reporter gene expression.
Assay of luciferase and ß-galactosidase activity
Cells harvested with a rubber policeman, were frozen at 70°C and lysed by three freezethaw cycles. Cell lysates were centrifuged at 12 000 r.p.m. for 10 mins and supernatants were transferred to a new tube. Protein content of the supernatant was determined using the Bio-Rad Protein Assay kit. Luciferase activity in cell lysates prepared from cells transfected with pGL2 was determined using the Dual-light kit from Tropix (Bedford, MA). Cell lysates containing 30 µg protein were added to a cuvette and the volume was increased to 10 µl with distilled water. Then 25 µl buffer A (Dual-light kit) was added to each cuvette which were placed in a LUMAC Biocounter M2500-luminometer. The relative light unit obtained with luciferase was measured for 10 s starting within 2 s after the addition of 100 µl buffer B (Dual-light kit). ß-Gal activity was determined 1 h after measurement of luciferase activity. ß-Gal activity was initiated by the addition of 100 µl Accelerator-II (Dual-light kit). The relative light unit of ß-gal was measured for 10 s following a 2 s delay.
Colony forming ability
Procedures for measuring colony forming ability have been previously described (7).
Single-cell alkaline electrophoresis (Comet assay)
The standard comet assay without enzyme digestion has been described previously (29). In addition to the standard comet assay, Den V, Fpg and proteinase K were used to digest UV-, arsenite- and UV plus arsenite-induced comets. After cell lysis the slides were washed with distilled water, then incubated with reaction buffer at 37°C for 30 min. Reaction buffers were 50 mM TrisHCl, 5 mM EDTA pH 7.5 for Den V digestion, 10 mM TrisHCl, 1 mM EDTA pH 7.5, 50 mM NaCl for Fpg digestion and phosphate-buffered saline for proteinase K digestion. Following incubation in reaction buffer, 2 U Den V, 2 U Fpg or 0.01 µg proteinase K in 20 µl reaction buffer was added to each slide. A coverslip was added and slides were placed in a sealed moist box for incubation at 37°C for 2 h. Den V digestion was performed twice. Slides were prepared for alkaline denaturation and electrophoresis as described in the standard comet assay.
Nitrite determination
Nitric oxide can react with molecular oxygen and water to form the stable oxidized products, nitrite and nitrate. The detection of nitrite released in cell culture medium was used to indicate nitric oxide generation. The fluorometric assay of nitrite by using 2,3-diaminonaphthalene was described previously (29).
Statistics
Data are expressed as means ± SE throughout the paper. All experiments were performed independently at least three times. Statistical analyses were performed with one-way ANOVA, except when the effects of UV plus arsenite were considered. In this case the effect of UV plus arsenite and the expected additive value of UV alone and arsenite alone were compared by Student's t-test. P values <0.05 were considered to be statistically significant.
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Results
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Arsenite inhibited UV-DNA repair
When arsenite was added to cell cultures after transfection with UV-irradiated pGL2, a greater decrease in luciferase activity was observed in comparison to cultures in which no arsenite was added (Figure 1A
). The decrease in the luciferase activity in cultures transfected with UV-irradiated reporter plasmid followed by the addition of arsenite was significantly greater than the expected additive inhibitory effect of UV-irradiated plasmid alone and arsenite treatment of host cells alone (Figure 1A and B
). This suggests that UV and arsenite act synergistically to decrease reporter gene activity. Supporting results were obtained using the ß-galactosidase reporter gene (Figure 1C and D
). A 2 h DEAE-dextran pre-treatment decreased the colony forming efficiency of Chinese hamster ovary cells to 87.8 ± 3.4%. However, without this pre-treatment a 24 h treatment using up to 8 µM arsenite was not cytotoxic. A 2 h DEAE-dextran treatment plus a 24 h treatment with 4 µM arsenite decreased the colony forming efficiency to 76.8 ± 3.5%. Therefore, the decrease in reporter gene expression and UV-DNA repair in arsenite-treated cells was not the result of cell death.

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Fig. 1. Effects of treatment with arsenite on the enzyme activity of UV-irradiated and unirradiated reporter gene. (A) Chinese hamster ovary cells after transfection with pGL2 irradiated with 0, 1000 or 2000 J/m2 UV were incubated without ( , UV), or with 4 µM sodium arsenite ( , UV-As). (B) Cells after transfection with pGL2 unirradiated ( , As), or irradiated with UV 1000 J/m2 ( , UV-As), were incubated with 04 µM sodium arsenite. (C) and (D) Plasmid pCMV-ß-gal was used instead of pGL2. The chemiluminescence intensities of sham-, pGL2- and pCMV-ß-gal-transfected cells were 4.5 ± 0.6, 4376 ± 254 and 15 458 ± 672 relative light units, respectively. (* indicates P < 0.05 by comparing UV or arsenite treatment alone with UV plus arsenite treatment.)
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The comet assay was used to confirm the above results. Cells analyzed immediately after irradiation with 10 J/m2 UV revealed very little DNA strand breakage using the standard comet assay. However, the incorporation of Den V digestion following cell lysis significantly increased the level of DNA strand breakage (Figure 2A
). When UV-irradiated cells were allowed to recover for 24 h in normal medium, the amount of DNA strand breakage decreased to a very low level, even with Den V digestion (Figure 2B
). This is probably due to the induction of DNA repair mechanisms. When UV-irradiated cells were incubated for 24 h in medium containing 2 and 4 µM arsenite, an increase in DNA strand breakage was observed (Figure 2C
). Since a 24 h treatment with arsenite did not induce DNA strand breakage with or without Den V digestion (Figure 2C
), the increase in DNA strand breakage in cells treated with UV plus arsenite suggests that arsenite inhibits the excision of UV-induced Den V-digestible adducts. However, it is also possible that arsenite acts synergistically with UV to induce more DNA adducts. To test this possibility, we compared the amount of DNA strand breaks in cells treated with either UV alone, arsenite alone or UV plus arsenite. This was done using the standard comet assay incorporating digestion with Den V, Fpg and proteinase K. Fpg catalyzes the excision of oxidized bases such as formamidopyrimidine and 8-oxoguanine (32) while proteinase K releases DNA strand breaks from DNAprotein cross-links (33). The amount of DNA strand breaks revealed by Den V digestion in cells treated with UV plus arsenite was almost equal to the expected additive value of treatment with UV and arsenite alone (Figure 2D
). The Den V-digestible adducts from treatment with UV plus arsenite must come from UV treatment because arsenite did not induce Den V-digestible adducts. The amount of DNA strand breaks revealed by Fpg or proteinase K digestion in cells treated with UV plus arsenite was significantly less than that of the expected additive value of treatment with UV and arsenite alone. This is probably because UV and arsenite induce Fpg- and proteinase K-digestible adducts redundantly. Thus, the results do not support the notion that UV and arsenite act synergistically to induce more DNA adducts.

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Fig. 2. Effect of arsenite on the excision of pyrimidine dimers in Chinese hamster ovary cells. (A) Exponentially growing cells were irradiated with UV and harvested immediately for comet assay without ( ) or with Den V digestion (). * indicates P < 0.05 by comparing without versus with Den V digestion. (B) UV-irradiated cells were harvested after a 24 h incubation in normal medium. After lysis, slides were incubated with ( ) or without Den V ( ) (* indicates P < 0.05 by comparing without versus with Den V digestion). (C) Cells treated with various concentrations of arsenite for 24 h ( , As), or irradiated with UV 10 J/m2 and harvested immediately (, UVR0), or treated with UV plus arsenite ( , UV-As). After lysis, slides were digested with Den V (+ indicates P < 0.05 by comparing experiments without and with arsenite). (D) Cells were untreated (Unt) or irradiated with UV 10 J/m2 and harvested immediately (UV10), or treated with 4 µM sodium arsenite for 4 h (As4), or treated with UV plus arsenite (UV10-As4). Cells were then subjected to the comet assay without enzyme digestion (no enz) or further digested with Den V, Fpg or proteinase K (PK). The expected additive values, shown in shaded bars, were calculated from the values of UV alone plus arsenite alone minus untreated (*, + and #, indicate P < 0.05 by comparing without versus with enzyme digestion, without versus with UV or arsenite treatment, and the expected additive value versus the observed value, respectively).
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Nitric oxide but not reactive oxygen species was involved in arsenite inhibition of UV-DNA repair
As nitric oxide and reactive oxygen species mediate arsenite toxicity, the involvement of these molecules in arsenite inhibition of UV-DNA repair was investigated. We reasoned that if these molecules were involved then interference of nitric oxide and reactive oxygen species production would suppress the inhibitory effect of arsenite. Results indicate that arsenite inhibition of UV-irradiated pGL2 expression was not affected by diphenyleneiodonium chloride (110 µM), an inhibitor of superoxide; Tiron (0.054 mM), a scavenger for oxidase; superoxide dismutase (18 µg/ml), a superoxide scavenger; diethyldithiocarbamate (0.252 mM), an inhibitor of superoxide dismutase; catalase (100800 U/ml), a hydrogen peroxide scavenger; 3-aminotriazole (1040 mM), an inhibitor of catalase; sodium selenite (540 µM), a glutathione peroxidase activator; mercaptosuccinic acid (0.252 mM), an inhibitor of glutathione peroxidase; D-mannitol (0.10.5 mM), a hydroxyl radical scavenger; uric acid (0.054.0 mM), a scavenger for both hydroxyl radical and peroxynitrite (data not shown). However, both Trolox (Figure 3A
), a scavenger for hydroxyl radical and peroxynitrite, and dimethyl sulfoxide (data not shown), a hydroxyl radical scavenger, suppressed arsenite inhibition of UV-irradiated pGL2 expression. Overall, these results suggest that reactive oxygen species are not involved in the inhibition of UV-DNA repair by arsenite. On the other hand, nitric oxide synthase inhibitors, NAME (Figure 3A
) and MTC (data not shown) significantly suppressed arsenite inhibition of UV-irradiated pGL2 expression, but had no effect on arsenite inhibition of unirradiated pGL2 expression.
The comet assay was also used to investigate the involvement of nitric oxide and reactive oxygen species in the inhibition of UV-DNA repair by arsenite. Results show that while post UV incubation with arsenite resulted in the accumulation of Den V-digestible adducts, the inclusion of NAME, MTC, Trolox or uric acid during arsenite treatment significantly reduced the Den V-digestible adducts induced by UV plus arsenite (Table I
). Diphenyleneiodonium chloride, dimethyl sulfoxide and D-manitol also suppressed arsenite inhibition of UV-DNA repair. However, Tiron, catalase, mercaptosuccinic acid and 3-aminotriazole had no effect. Thus, the comet experiments show that nitric oxide is involved in arsenite inhibition of UV-DNA repair but the involvement of reactive oxygen species remains ambiguous.
We reasoned that if arsenite inhibition of UV-DNA repair is mediated by nitric oxide and not by reactive oxygen species, then nitric oxide generators will act in a similar way to arsenite while oxidants will act differently. This was confirmed by the observation that sodium nitrosoprusside, a nitric oxide donor, and SIN-1, a peroxynitrite generator, decreased the expression of UV-irradiated pGL2 more severely than unirradiated pGL2 (Figure 4A and B
). Conversely, H2O2 decreased the expression of UV-irradiated and unirradiated pGL2 with the same potency (Figure 4C
). The comet assay also showed that sodium nitrosoprusside and SIN-1 inhibited the excision of UV-induced Den V-digestible adducts (Figure 4D
).
Binding to thiols was not involved in arsenite inhibition of UV-DNA repair
As thiol groups are important functional groups in DNA repair enzymes, phenylarsine oxide, a strong thiol-reacting agent, was used to test if arsenite inhibits UV-DNA repair by reacting with thiol groups. Results show that in contrast to arsenite, phenylarsine oxide inhibited the expression of unirradiated and UV-irradiated pGL2 with similar potency (Figure 5A
). The IC50 of phenylarsine oxide to inhibit cell growth and pGL2 gene expression were estimated to be 5 and 0.4 µM, respectively (data not shown). Therefore, the reduction of luciferase activity by phenylarsine oxide was not due to cell death. Unlike arsenite, NAME (Figure 5B
) and MTC (data not shown) did not suppress the inhibitory effect of phenylarsine oxide, and phenylarsine oxide did not inhibit the excision of UV-induced Den V-digestible adducts (Figure 5C
, D and E).
Arsenite increased nitric oxide production
Nitric oxide generation was measured in cell cultures to ascertain whether arsenite inhibits UV-DNA repair via the generation of nitric oxide. Results show that treatment with arsenite increased nitric oxide production; however, treatment with phenylarsine oxide or UV has no effect (Figure 6A
D). NAME, MTC, Trolox and dimethyl sulfoxide, which all suppressed arsenite inhibition of UV-DNA repair, also decreased nitric oxide production in cells treated with UV plus arsenite. Consistent with DNA repair data, nitric oxide production in cells treated with UV plus arsenite was decreased by Trolox and dimethyl sulfoxide, but not by Tiron, superoxide dismutase, catalase or 3-aminotriazole.
Nitric oxide is also involved in arsenite inhibition of UV-DNA repair in human fibroblasts
As human fibroblasts are widely used in nucleotide excision repair research, these cells were used to confirm the involvement of nitric oxide in the inhibition of pyrimidine excision by arsenite. A 6 h post-UV treatment with 14 µM arsenite was found to inhibit the excision of pyrimidine dimers in human fibroblasts (Figure 7A
C) as well as Chinese hamster ovary cells. The nitric oxide synthase inhibitors, NAME and MTC, again decreased the inhibitory effect of arsenite (Figure 7D
). Arsenite treatment also increased nitrite production in human fibroblasts (data not shown).
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Discussion
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Since arsenite did not induce Den V-digestible adducts, the results in Figures 2, 5C, 5D, 7C and 7D

clearly demonstrate that arsenite inhibits excision of UV-induced Den V-digestible adducts. This is consistent with previous reports that arsenite inhibits UV-induced DNA adduct excision in Chinese hamster ovary cells (11,12) and human fibroblasts (13). Arsenite has been shown to inhibit both transcription-coupled repair and global genome repair (13). While the Den V-incorporated comet assay reflects global genome repair, the host cell-mediated DNA repair assay requires the expression of a reporter gene and, hence, may reflect transcription-coupled repair. However, in human cells no clear evidence of transcription-coupled repair was detected for expression of the reporter gene (34). It is tempting to speculate that arsenite may inhibit proteins common for both global genome repair and transcription-coupled repair. Moreover, since Den V specifically cleaves cyclobutane pyrimidine dimers, the Den V-incorporated comet assay seems to reflect only damage recognition and incision steps, not steps thereafter. This discussion leads to a suggestion that arsenite may inhibit the nucleotide excision repair steps participated by proteins such as XPA, XPF and XPG.
The notion that nitric oxide is involved in arsenite inhibition of UV-DNA repair was supported by the following observations: (i) arsenite treatment increased nitric oxide production; (ii) nitric oxide synthase inhibitors suppressed arsenite-induced nitric oxide production; (iii) nitric oxide synthase inhibitors suppressed arsenite inhibition of UV-irradiated pGL2 expression; (iv) nitric oxide synthase inhibitors also suppressed arsenite inhibition of the excision of Den V-digestible adducts; (v) like arsenite, nitric oxide donors suppressed the expression of UV-irradiated reporter plasmids more severely than that of unirradiated plasmids; (vi) nitric oxide donors inhibited the excision of Den V-digestible adducts; (vii) treatment with phenylarsine oxide did not increase nitric oxide production and phenylarsine oxide did not inhibit the excision of Den V-digestible adducts. Our conclusion is consistent with a recent report that inflammatory cytokines inhibit oxidative DNA repair in cholangiocarcinoma cells by a nitric oxide-dependent mechanism (35). Nitric oxide may interact with thiol groups of proteins to produce S-nitrosoproteins (36). It has been suggested that S-nitrosylation of proteins could mediate signaling functions similar to those of protein phosphorylation (37). Arsenite has been shown to initiate gene transcription by altering signal transduction molecules (38,39); however, much higher concentrations of arsenite were used in those studies. Therefore the involvement of signal molecules in arsenite toxicology remains to be determined. Alternatively, nitric oxide may react with superoxide to produce peroxynitrite, which can titrate critical tyrosine residues of proteins involved in DNA repair. Exogenous nitric oxide and peroxynitrite donors have been shown to inhibit DNA ligase (40), formamidopyrimidine-DNA glycosylase (41) and O6-methylguanine-DNA methyltransferase (42). Further experiments are needed to demonstrate that the amount of nitric oxide induced by arsenite is able to interfere with the normal function of proteins involved in DNA repair.
So far, it has been demonstrated that arsenite increases nitric oxide production in Chinese hamster ovary cells (30), C3H 10T 1/2 cells (43), human umbilical vein endothelial cells (unpublished data) and in bovine aorta endothelial cells (29). In contrast, arsenite is reported not to increase nitric oxide production in rat aortic smooth muscle cells (44), hepatocytes (45) or in human liver cells (46). Moreover, arsenite also inhibits inducible nitric oxide synthase gene expression in cytokine-stimulated human liver cells (46), rat pulmonary artery smooth muscle cells (47) and in hepatocytes (45). Thus the effect of arsenite on nitric oxide production seems to be cell-type specific. It is tempting to ask: Does arsenite inhibit UV-DNA repair in those cells in which arsenite can not increase or even inhibit nitric oxide production?
Inhibition of UV-DNA repair by arsenite was found not to be affected by oxidant modulators such as Tiron, superoxide dismutase, catalase, 3-aminotriazole and mercaptosuccinic acid using both the host cell-mediated DNA repair assay and the comet assay. However, oxidant modulators, such as uric acid, D-mannitol and diphenyleneiodonium chloride, reduced arsenite inhibition of UV-DNA repair but had no apparent effect on arsenite inhibition of UV-irradiated pGL2 expression. Therefore, the present results did not give a clear indication that oxidants are involved in arsenite inhibition of UV-DNA repair. The differential results may be due to the Den V-incorporated comet assay being more specific for the effect of arsenite on pyrimidine dimer excision, whereas the host cell-mediated DNA repair assay may include the effect of arsenite on all DNA repair steps including transcription, translation, protein modification and enzyme activity.
As mentioned in the Introduction, although reaction with thiol is generally assumed to be the major mechanism for arsenite executing its biological activities, it seems unlikely that arsenite inhibits DNA repair by interacting with the thiol groups of repair enzyme proteins. The possibility that arsenite interacts with thiol groups of other proteins in the DNA repair process is not supported by the following observations: (i) while phenylarsine oxide inhibited gene expression of UV-irradiated and unirradiated pGL2 with equal potency, arsenite inhibited the former more severely than the latter; (ii) while nitric oxide synthase inhibitors substantially suppressed arsenite inhibition of UV-irradiated pGL2 gene expression, they had no suppressive activity on that of phenylarsine oxide; (iii) arsenite inhibited the excision of UV-induced Den V-digestible adducts, but phenylarsine oxide did not; (iv) arsenite increased nitric oxide production but phenylarsine oxide did not. These results, in opposition to the thiol-reacting hypothesis, together with the suggestion of nitric oxide involvement, indicate the necessity for re-examination of the involvement of thiol reacting activity in arsenic toxicity.
It is well recognized that hamster cells repair UV-induced DNA damage less efficiently than human cells. Rodent cells are proficient in the removal of 64 pyrimidinepyrimidone photoproducts from the overall genome, but the cyclobutane pyrimidine dimers are removed only from the transcribing strand of active genes (48,49). However, the results in Figures 2B and 5C
indicate that Chinese hamster ovary cells repair UV-induced Den V-digestible adducts efficiently. More experiments are needed to investigate this discrepancy. It is also known that genetic damage induced by UV is primarily in the form of cyclobutane pyrimidine dimers (6590%) and 64 pyrimidinepyrimidone photoproducts (1035%) (50,51). The results in Figure 2D
indicate that in addition to Den V-digestible adducts, UV also induced a substantial amount of Fpg- and proteinase K-digestible adducts. The biological significance of UV-induced oxidative DNA adducts and DNAprotein cross-links is largely unknown.
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Notes
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3 To whom correspondence should be addressed at: Institute of Zoology, Academia Sinica, Taipei 11529, Republic of China Email: ZOJKY{at}sinica.edu.tw 
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Acknowledgments
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We thank Mr Tyler Rainsbury for English editorial service. This work was supported by grant NSC 89-2320B001-031, ROC.
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Received September 8, 2000;
revised December 4, 2000;
accepted January 10, 2001.