Arsenic Carcinogenesis in the Skin

Summary Chronic arsenic poisoning is a world public health issue. Long-term exposure to inorganic arsenic (As) from drinking water has been documented to induce cancers in lung, urinary bladder, kidney, liver and skin in a dose–response relationship. Oxidative stress, chromosomal abnormality and altered growth factors are possible modes of action in arsenic carcinogenesis. Arsenic tends to accumulate in the skin. Skin hyperpigmentation and hyperkeratosis have long been known to be the hallmark signs of chronic As exposure. There are significant associations between these dermatological lesions and risk of skin cancer. The most common arsenic-induced skin cancers are Bowen’s disease (carcinoma in situ), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Arsenic-induced Bowen’s disease (As-BD) is able to transform into invasive BCC and SCC. Individuals with As-BD are considered for more aggressive cancer screening in the lung and urinary bladder. As-BD provides an excellent model for studying the early stages of chemical carcinogenesis in human beings. Arsenic exposure is associated with G2/M cell cycle arrest and DNA aneuploidy in both cultured keratinocytes and As-BD lesions. These cellular abnormalities relate to the p53 dysfunction induced by arsenic. The characteristic clinical figures of arsenic-induced skin cancer are: (i) occurrence on sun-protected areas of the body; (ii) multiple and recrudescent lesions. Both As and UVB are able to induce skin cancer. Arsenic treatment enhances the cytotoxicity, mutagenicity and clastogenicity of UV in mammalian cells. Both As and UVB induce apoptosis in keratinocytes by caspase-9 and caspase-8 signaling, respectively. Combined UVB and As treatments resulted in the antiproliferative and proapoptotic effects by stimulating both caspase pathways in the keratinocytes. UVB irradiation inhibited mutant p53 and ki-67 expression, as well as increased in the number of apoptotic cells in As-BD lesions which resulted in an inhibitory effect on proliferation. As-UVB interaction provides a reasonable explanation for the rare occurrences of arsenical cancer in the sun-exposed skin. The multiple and recurrent skin lesions are associated with cellular immune dysfunction in chronic arsenism. A decrease in peripheral CD4+ cells was noticed in the inhabitants of arsenic exposure areas. There was a decrease in the number of Langerhans cells in As-BD lesion which results in an impaired immune function on the lesional sites. Since CD4+ cells are the target cell affected by As, the interaction between CD4+ cells and epidermal keratinocytes under As affection might be closely linked to the pathogenesis of multiple occurrence of arsenic-induced skin cancer. In this review, we provide and discuss the pathomechanisms of arsenic skin cancer and the relationship to its characteristic figures. Such information is critical for understanding the molecular mechanism for arsenic carcinogenesis in other internal organs.     

BANK regulates BCR-induced calcium mobilization by promoting tyrosine phosphorylation of IP(3) receptor.

B-cell activation mediated through the antigen receptor is dependent on activation of protein tyrosine kinases (PTKs) such as Lyn and Syk and subsequent phosphorylation of various signaling proteins. Here we report on the identification and characterization of the B-cell scaffold protein with ankyrin repeats (BANK), a novel substrate of tyrosine kinases. BANK is expressed in B cells and is tyrosine phosphorylated upon B-cell antigen receptor (BCR) stimulation, which is mediated predominantly by Syk. Overexpres sion of BANK in B cells leads to enhancement of BCR-induced calcium mobilization. We found that both Lyn and inositol 1,4,5-trisphosphate receptor (IP(3)R) associate with the distinct regions of BANK and that BANK promotes Lyn-mediated tyrosine phosphorylation of IP(3)R. Given that IP(3)R channel activity is up-regulated by its tyrosine phosphorylation, BANK appears to be a novel scaffold protein regulating BCR-induced calcium mobilization by connecting PTKs to IP(3)R. Because BANK expression is confined to functional BCR-expressing B cells, BANK-mediated calcium mobilization may be specific to foreign antigen-induced immune responses rather than to signaling required for B-cell development.     

Polycomb (PcG) Proteins, BMI1 and SUZ12, Regulate Arsenic-induced Cell Transformation

Inorganic arsenic is a well-documented human carcinogen associated with cancers of the skin, lung, liver, and bladder. However, the underlying mechanisms explaining the tumorigenic role of arsenic are not well understood. The present study explored a potential mechanism of cell transformation induced by arsenic exposure. Exposure to a low dose (0.5 μm) of arsenic trioxide (As(2)O(3)) caused transformation of BALB/c 3T3 cells. In addition, in a xenograft mouse model, tumor growth of the arsenic-induced transformed cells was dramatically increased. In arsenic-induced transformed cells, polycomb group (PcG) proteins, including BMI1 and SUZ12, were activated resulting in enhanced histone H3K27 tri-methylation levels. On the other hand, tumor suppressor p16(INK4a) and p19(ARF) mRNA and protein expression were dramatically suppressed. Introduction of small hairpin (sh) RNA-BMI1 or -SUZ12 into BALB/c 3T3 cells resulted in suppression of arsenic-induced transformation. Histone H3K27 tri-methylation returned to normal in BMI1- or SUZ12-knockdown BALB/c 3T3 cells compared with BMI1- or SUZ12-wildtype cells after arsenic exposure. As a consequence, the expression of p16(INK4a) and p19(ARF) was recovered in arsenic-treated BMI1- or SUZ12-knockdown cells. Thus, arsenic-induced cell transformation was blocked by inhibition of PcG function. Taken together, these results strongly suggest that the polycomb proteins, BMI1 and SUZ12 are required for cell transformation induced by organic arsenic exposure.     

Computational analysis of calcium signaling and membrane electrophysiology in cerebellar Purkinje neurons associated with ataxia

ABSTRACT: BACKGROUND: Mutations in the smooth endoplasmic reticulum (sER) calcium channel Inositol Trisphosphate Receptor type 1 (IP3R1) in humans with the motor function coordination disorders Spinocerebellar Ataxia Types 15 and 16 (SCA15/16) and in a corresponding mouse model, the IP3R1delta18/delta18 mice, lead to reduced IP3R1 levels. We posit that increasing IP3R1 sensitivity to IP3 in ataxias with reduced IP3R1 could restore normal calcium response. On the other hand, in mouse models of the human polyglutamine (polyQ) ataxias, SCA2, and SCA3, the primary finding appears to be hyperactive IP3R1-mediated calcium release. It has been suggested that the polyQ SCA1 mice may also show hyperactive IP3R1. Yet, SCA1 mice show downregulated gene expression of IP3R1, Homer, metabotropic glutamate receptor (mGluR), smooth endoplasmic reticulum Ca-ATP-ase (SERCA), calbindin, parvalbumin, and other calcium signaling proteins. RESULTS: We create a computational model of pathological alterations in calcium signaling in cerebellar Purkinje neurons to investigate several forms of spinocerebellar ataxia associated with changes in the abundance, sensitivity, or activity of the calcium channel IP3R1. We find that increasing IP3R1 sensitivity to IP3 in computational models of SCA15/16 can restore normal calcium response if IP3R1 abundance is not too low. The studied range in IP3R1 levels reflects variability found in human and mouse ataxic models. Further, the required fold increases in sensitivity are within experimental ranges from experiments that use IP3R1 phosphorylation status to adjust its sensitivity to IP3. Results from our simulations of polyglutamine SCAs suggest that downregulation of some calcium signaling proteins may be partially compensatory. However, the downregulation of calcium buffer proteins observed in the SCA1 mice may contribute to pathology. Finally, our model suggests that the calcium-activated voltage-gated potassium channels may provide an important link between calcium metabolism and membrane potential in Purkinje cell function. CONCLUSION: Thus, we have established an initial platform for computational evaluation and prediction of ataxia pathophysiology. Specifically, the model has been used to investigate SCA15/16, SCA1, SCA2, and SCA3. Results suggest that experimental studies treating mouse models of any of these ataxias with appropriately chosen peptides resembling the C-terminal of IP3R1 could adjust receptor sensitivity, and thereby modulate calcium release and normalize IP3 response. In addition, the model supports the hypothesis of IP3R1 supersensitivity in SCA1.     

Cdc2/cyclin B1 interacts with and modulates inositol 1,4,5-trisphosphate receptor (type 1) functions

The resistance of inositol 1,4,5-trisphosphate receptor (IP3R)-deficient cells to multiple forms of apoptosis demonstrates the importance of IP3-gated calcium (Ca2+) release to cellular apoptosis. However, the specific upstream biochemical events leading to IP3-gated Ca2+ release during apoptosis induction are not known. We have shown previously that the cyclin-dependent kinase 1/cyclin B (cdk1/CyB or cdc2/CyB) complex phosphorylates IP3R1 in vitro and in vivo at Ser421 and Thr799. In this study, we show that: 1) the cdc2/CyB complex directly interacts with IP3R1 through Arg391, Arg441, and Arg871; 2) IP3R1 phosphorylation at Thr799 by the cdc2/CyB complex increases IP3 binding; and 3) cdc2/CyB phosphorylation increases IP3-gated Ca2+ release. Taken together, these results demonstrate that cdc2/CyB phosphorylation positively regulates IP3-gated Ca2+ signaling. In addition, identification of a CyB docking site(s) on IP3R1 demonstrates, for the first time, a direct interaction between a cell cycle component and an intracellular calcium release channel. Blocking this phosphorylation event with a specific peptide inhibitor(s) may constitute a new therapy for the treatment of several human immune disorders.     

Inositol 1,4,5-trisphosphate receptor type 1 phosphorylation and regulation by extracellular signal-regulated kinase

Type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) is a widely expressed intracellular calcium-release channel found in many cell types. The operation of IP(3)R1 is regulated through phosphorylation by multiple protein kinases. Extracellular signal-regulated kinase (ERK) has been found involved in calcium signaling in distinct cell types, but the underlying mechanisms remain unclear. Here, we present evidence that ERK1/2 and IP(3)R1 bind together through an ERK binding motif in mouse cerebellum in vivo as well as in vitro. ERK-phosphorylating serines (Ser 436) was identified in mouse IP(3)R1 and Ser 436 phosphorylation had a suppressive effect on IP(3) binding to the recombinant N-terminal 604-amino acid residues (N604). Moreover, phosphorylation of Ser 436 in R(224-604) evidently enhance its interaction with the N-terminal "suppressor" region (N223). At last, our data showed that Ser 436 phosphorylation in IP(3)R1 decreased Ca(2+) releasing through IP(3)R1 channels.     

Protein kinase A and two phosphatases are components of the inositol 1,4,5-trisphosphate receptor macromolecular signaling complex

The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed intracellular calcium (Ca(2+)) release channel on the endoplasmic reticulum. IP3Rs play key roles in controlling Ca(2+) signals that activate numerous cellular functions including T cell activation, neurotransmitter release, oocyte fertilization and apoptosis. There are three forms of IP3R, all of which are ligand-gated channels activated by the second messenger inositol 1,4,5-trisphosphate. Channel function is modulated via cross-talk with other signaling pathways including those mediated by kinases and phosphatases. In particular IP3Rs are known to be regulated by cAMP-dependent protein kinase (PKA) phosphorylation. In the present study we show that PKA and the protein phosphatases PP1 and PP2A are components of the IP3R1 macromolecular signaling complex. PKA phosphorylation of IP3R1 increases channel activity in planar lipid bilayers. These studies indicate that regulation of IP3R1 function via PKA phosphorylation involves components of a macromolecular signaling complex.     

Cdc42 regulates arsenic-induced NADPH oxidase activation and cell migration through actin filament reorganization

Although arsenic is a human carcinogen, the molecular mechanisms of its action remain to be understood. The present study reports that exposure to arsenic induced actin filament reorganization, resulting in lamellipodia and filopodia structures through the activation of Cdc42 in SVEC4-10 endothelial cells. It was also found that arsenic induced the formation of the superoxide anion (O2*) in SVEC4-10 cells. Immunoprecipitation and Western blotting analysis demonstrated that arsenic stimulation induced serine phosphorylation of p47phox, a key component of NADPH oxidase, indicating that arsenic induces O2* formation through NADPH oxidase activation. Inhibition of arsenic-induced actin filament reorganization by either overexpression of a dominant negative Cdc42 or pretreatment of an actin filament stabilizing regent, jasplakinolide, abrogated arsenic-induced NADPH oxidase activation, showing that the activation of NADPH oxidase was regulated by Cdc42-mediated actin filament reorganization. This study also showed that overexpression of a dominant negative Rac1 was sufficient to abolish arsenic-induced O2*- production, implying that Rac1 activities are required for Cdc42-mediated NADPH oxidase activation in response to arsenic stimulation. Furthermore, arsenic stimulation induced cell migration, which can be inhibited by the inactivation of either Cdc42 or NADPH oxidase. Taken together, the results indicate that arsenic is able to activate NADPH oxidase through Cdc42-mediated actin filament reorganization, leading to the induction of an increase in cell migration in SVEC4-10 endothelial cells.     

Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B-cell receptor activation.

Bruton's tyrosine kinase (Btk) is essential for B-lineage development and represents an emerging family of non-receptor tyrosine kinases implicated in signal transduction events initiated by a range of cell surface receptors. Increased dosage of Btk in normal B cells resulted in a striking enhancement of extracellular calcium influx following B-cell antigen receptor (BCR) cross-linking. Ectopic expression of Btk, or related Btk/Tec family kinases, restored deficient extracellular Ca2+ influx in a series of novel Btk-deficient human B-cell lines. Btk and phospholipase Cgamma (PLCgamma) co-expression resulted in tyrosine phosphorylation of PLCgamma and required the same Btk domains as those for Btk-dependent calcium influx. Receptor-dependent Btk activation led to enhanced peak inositol trisphosphate (IP3) generation and depletion of thapsigargin (Tg)-sensitive intracellular calcium stores. These results suggest that Btk maintains increased intracellular calcium levels by controlling a Tg-sensitive, IP3-gated calcium store(s) that regulates store-operated calcium entry. Overexpression of dominant-negative Syk dramatically reduced the initial phase calcium response, demonstrating that Btk/Tec and Syk family kinases may exert distinct effects on calcium signaling. Finally, co-cross-linking of the BCR and the inhibitory receptor, FcgammaRIIb1, completely abrogated Btk-dependent IP3 production and calcium store depletion. Together, these data demonstrate that Btk functions at a critical crossroads in the events controlling calcium signaling by regulating peak IP3 levels and calcium store depletion.     

Serum levels of the extracellular domain of the epidermal growth factor receptor in individuals exposed to arsenic in drinking water in Bangladesh

Epidermal growth factor receptor-dependent mechanisms have been implicated in growth signal transduction pathways that contribute to cancer development, including dermal carcinogenesis. Detection of the extracellular domain of the epidermal growth factor receptor (EGFR ECD) in serum has been suggested as a potential biomarker for monitoring this effect in vivo. Arsenic is a known human carcinogen, producing skin and other malignancies in populations exposed through their drinking water. One such exposed population, which we have been studying for a number of years, is in Bangladesh. The purpose of this study was to examine the EGFR ECD as a potential biomarker of arsenic exposure and/or effect in this population. Levels of the EGFR ECD were determined by enzyme-linked immunosorbent assay in the serum samples from 574 individuals with a range of arsenic exposures from drinking water in the Araihazar area of Bangladesh. In multiple regression analysis, serum EGFR ECD was found to be positively associated with three different measures of arsenic exposure (well water arsenic, urinary arsenic and a cumulative arsenic index) at statistically significant levels (p≤0.034), and this association was strongest among the individuals with arsenic-induced skin lesions (p ≤ 0.002). When the study subjects were stratified in tertiles of serum EGFR ECD levels, the risk of skin lesions increased progressively for each increase in all three arsenic measures (also stratified in tertiles) and this increasing risk became more pronounced among subjects within the highest tertile of EGFR ECD levels. These results suggest that serum EGFR ECD levels may be a potential biomarker of effect of arsenic exposure and may indicate those exposed individuals at greatest risk for the development of arsenic-induced skin lesions.     

Regulation of the type 1 inositol 1,4,5-trisphosphate receptor by phosphorylation at tyrosine 353

The inositol 1,4,5-trisphosphate receptor (IP3R) plays an essential role in Ca2+ signaling during lymphocyte activation. Engagement of the T cell or B cell receptor by antigen initiates a signal transduction cascade that leads to tyrosine phosphorylation of IP3R by Src family nonreceptor protein tyrosine kinases, including Fyn. However, the effect of tyrosine phosphorylation on the IP3R and subsequent Ca2+ release is poorly understood. We have identified tyrosine 353 (Tyr353) in the IP3-binding domain of type 1 IP3R (IP3R1) as a phosphorylation site for Fyn both in vitro and in vivo. We have developed a phosphoepitope-specific antibody and shown that IP3R1-Y353 becomes phosphorylated during T cell and B cell activation. Furthermore, tyrosine phosphorylation of IP3R1 increased IP3 binding at low IP3 concentrations (<10 nm). Using wild-type IP3R1 or an IP3R1-Y353F mutant that cannot be tyrosine phosphorylated at Tyr353 or expressed in IP3R-deficient DT40 B cells, we demonstrated that tyrosine phosphorylation of Tyr353 permits prolonged intracellular Ca2+ release during B cell activation. Taken together, these data suggest that one function of tyrosine phosphorylation of IP3R1-Y353 is to enhance Ca2+ signaling in lymphocytes by increasing the sensitivity of IP3R1 to activation by low levels of IP3.     

Requirement of inositol 1,4,5-trisphosphate receptors for tumor-mediated lymphocyte apoptosis

Tumor cells strategically down-regulate Fas receptor expression to evade immune attack and up-regulate expression of Fas ligand to promote apoptosis of infiltrating T lymphocytes. Many pathways leading to apoptotic cell death require calcium release from inositol 1,4,5-trisphosphate receptors (IP3Rs). Here, we show that Fas-dependent killing of Jurkat T lymphoma cells by SW620 colon cancer cells requires calcium release from IP3R. General suppression of IP3R signaling significantly reduced SW620-mediated Jurkat cell apoptosis. Significantly, a specific inhibitor of apoptotic calcium release from IP3R strongly blocked lymphocyte apoptosis. Thus, selective pharmacological targeting of apoptotic calcium release from IP3R may enhance tumor cell immunogenicity.     

Nitric oxide donor, V-PROLI/NO, provides protection against arsenical induced toxicity in rat liver cells: requirement for Cyp1a1

Arsenic is a cancer chemotherapeutic but hepatotoxicity can be a limiting side effect. O²-vinyl 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (V-PROLI/NO) is a nitric oxide (NO) donor prodrug and metabolized by liver cytochromes P450 (CYP450) to release NO. The effects of V-PROLI/NO pretreatment on the toxicity of arsenic (as NaAsO₂) were studied in a rat liver cell line (TRL 1215). The cells acted upon the prodrug to release NO, as assessed by nitrite levels, in a time-dependent fashion to maximal levels of 8-fold above basal levels. Pretreatment with V-PROLI/NO markedly reduced arsenic cytolethality which was directly related to the level of NO produced by V-PROLI/NO treatment. Cyp1a1 expression was directly related to the level of NO production and to reduced arsenic cytotoxicity. V-PROLI/NO pretreatment markedly reduced arsenic-induced apoptosis and suppressed phosphorylation of JNK1/2. V-PROLI/NO pretreatment facilitated additional increases in arsenic-induced metallothionein, a metal-binding protein important in arsenic tolerance. Thus, V-PROLI/NO protects against arsenic toxicity in rat liver cells, reducing cytolethality, apoptosis and dysregulation of MAPKs, through generation of NO formed after metabolism by liver cell enzymes, possibly including Cyp1a1. CYP450 required for NO production from V-PROLI/NO treatment in the rat and human appears to differ as we have previously studied the ability of V-PROLI/NO to prevent arsenic toxicity in human liver cells where it reduced toxicity apparently through a CYP2E1-mediated metabolic mechanism. None-the-less, it appears that both rat and human liver cells act upon V-PROLI/NO via a CYP450-related mechanism to produce NO and subsequently reduce arsenic toxicity.     

ERK binds, phosphorylates InsP3 type 1 receptor and regulates intracellular calcium dynamics in DT40 cells

Modulation on the duration of intracellular Ca(2+) transients is essential for B-cell activation. We have previously shown that extracellular-signal-regulated kinase (ERK) can phosphorylate inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) at serine 436 and regulate its calcium channel activity. Here we investigate the potential physiological interaction between ERK and IP(3)R1 using chicken DT40 B-cell line in which different mutants are expressed. The interaction between ERK and IP(3)R1 is confirmed by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) assays. This constitutive interaction is independent of either ERK kinase activation or IP(3)R1 phosphorylation status. Back phosphorylation analysis further shows that type 1 IP(3)R (IP(3)R1) is phosphorylated by ERK in anti-IgM-activated DT40 cells. Finally, our data show that the phosphorylation of Ser 436 in the IP(3)-binding domain of IP(3)R1 leads to less Ca(2+) release from endoplasmic reticulum (ER) microsomes and accelerates the declining of calcium increase in DT40 cells in response to anti-IgM stimulation.     

A critical role for IkappaB kinase beta in metallothionein-1 expression and protection against arsenic toxicity

Arsenic is a widespread environmental toxic agent that has been shown to cause diverse tissue and cell damage and at the same time to be an effective anti-cancer therapeutic agent. The objective of this study is to explore the signaling mechanisms involved in arsenic toxicity. We show that the IkappaB kinase beta (IKKbeta) plays a crucial role in protecting cells from arsenic toxicity. Ikkbeta(-)(/)(-) mouse 3T3 fibroblasts have decreased expression of antioxidant genes, such as metallothionein 1 (Mt1). In contrast to wild type and IKKbeta-reconstituted Ikkbeta(-)(/)(-) cells, IKKbeta-null cells display a marked increase in arsenic-induced reactive oxygen species (ROS) accumulation, which leads to activation of the MKK4-c-Jun NH(2)-terminal kinase (JNK) pathway, c-Jun phosphorylation, and apoptosis. Pretreatment with the antioxidant N-acetylcysteine (NAC) and expression of MT1 in the Ikkbeta(-)(/)(-) cells prevented JNK activation; moreover, NAC pretreatment, MT1 expression, MKK4 ablation, and JNK inhibition all protected cells from death induced by arsenic. Our data show that two signaling pathways appear to be important for modulating arsenic toxicity. First, the IKK-NF-kappaB pathway is crucial for maintaining cellular metallothionein-1 levels to counteract ROS accumulation, and second, when this pathway fails, excessive ROS leads to activation of the MKK4-JNK pathway, resulting in apoptosis.     

Arsenic Induces Thioredoxin 1 and Apoptosis in Human Liver HHL-5 Cells

To further characterize the mechanisms underlying liver toxicity induced by arsenic, we examined in this study the effect of arsenic on thioredoxin (Trx) and the apoptotic signaling pathways in human liver HHL-5 cells. The cells were treated with 0, 2, 5, and 10 μM of sodium arsenite for 24 h, and the changes of Trx1 and thioredoxin reductase (TrxR1) as well as intracellular ROS and apoptosis were examined. A concentration-dependent increase in mRNA and protein levels of Trx1 and TrxR1 was observed in arsenic-treated cells. Intracellular ROS levels and apoptosis were also significantly increased in a concentration-dependent manner. In line with this, protein levels of Bax and cytochrome C were increased and Bcl-2 was decreased by arsenic treatments. Increases in caspase 3 activity were observed. These results indicate that Trx is involved in arsenic-induced liver cell injury, probably through the apoptotic signaling pathway. However, further studies are needed to elucidate on these findings.     

Inorganic arsenic compounds cause oxidative damage to DNA and protein by inducing ROS and RNS generation in human keratinocytes

Arsenic is a naturally occurring element that is present in food, soil, and water. Inorganic arsenic can accumulate in human skin and is associated with increased risk of skin cancer. Oxidative stress due to arsenic exposure is proposed as one potential mode of carcinogenic action. The purpose of this study is to investigate the specific reactive oxygen and nitrogen species that are responsible for the arsenic-induced oxidative damage to DNA and protein. Our results demonstrated that exposure of human keratinocytes to trivalent arsenite caused the generation of 8-hydroxyl-2′-deoxyguanine (8-OHdG) and 3-nitrotyrosine (3-NT) in a concentration- and time-dependent manner. Pentavalent arsenate had similar effects, but to a significantly less extent. The observed oxidative damage can be suppressed by pre-treating cells with specific antioxidants. Furthermore, we found that pre-treating cells with Nω-nitro-l-arginine methyl ester (l-NAME), an inhibitor of nitric oxide synthase (NOS), or with 5,10,15,20-tetrakis (N-methyl-4′-pyridyl) porphinato iron (III) chloride (FeTMPyP), a decomposition catalyst of peroxynitrite, suppressed the generation of both 8-OHdG and 3-NT, which indicated that peroxynitrite, a product of the reaction of nitric oxide and superoxide, played an important role in arsenic-induced oxidative damage to both DNA and protein. These findings highlight the involvement of peroxynitrite in the molecular mechanism underlying arsenic-induced human skin carcinogenesis.