Arsenic co-carcinogenesis: inhibition of DNA repair and interaction with zinc finger proteins

Abstract

Arsenic is widely present in the environment and is associated with various population health risks including cancers. Arsenic exposure at environmentally relevant levels enhances the mutagenic effect of other carcinogens such as ultraviolet radiation. Investigation on the molecular mechanisms could inform the prevention and intervention strategies of arsenic carcinogenesis and co-carcinogenesis. Arsenic inhibition of DNA repair has been demonstrated to be an important mechanism, and certain DNA repair proteins have been identified to be extremely sensitive to arsenic exposure. This review will summarize the recent advances in understanding the mechanisms of arsenic carcinogenesis and co-carcinogenesis, including DNA damage induction and ROS generation, particularly how arsenic inhibits DNA repair through an integrated molecular mechanism which includes its interactions with sensitive zinc finger DNA repair proteins.

1. Introduction

Arsenic is ubiquitous in the environment and exposures occur through water, soil, dust, and food. Arsenic is a class I human carcinogen and independently a weak mutagen at environmentally relevant concentrations. Arsenic is the number 1 on the 2019 Substance Priority List of U.S. Agency for Toxic Substances and Disease Registry [https://www.atsdr.cdc.gov/spl/index.html]. The Environmental Protection Agency (EPA) and World Health Organization (WHO) set a maximum contaminant level for arsenic at 10 ppb, which was based on data for arsenic as a single agent exposure and did not consider co-exposures (1). Additionally, recent studies suggest arsenic may affect human health at levels lower than this limit (2,3). There is strong experimental and epidemiological evidence that arsenic in combination with other environmental insults, such as ultraviolet radiation (UVR), increases carcinogenesis at low arsenic concentrations. Thus, the affected population in the U.S. and around the world may be significantly larger than current estimates especially considering many rural populations rely on well water measuring arsenic above the EPA and WHO standard and are at a greater risk for arsenic-associated diseases.

Arsenic is known to generate multiple types of DNA damage including oxidative DNA damage and strand breaks. At lower concentrations, studies show that arsenic functions as a co-carcinogen enhancing the genotoxicity of other DNA damaging agents (4,5). Mechanisms associated with reactive oxygen species (ROS) generation and DNA repair inhibition play important roles in arsenic co-carcinogenicity (6). More importantly, certain zinc finger DNA repair proteins, such as poly (ADP-ribose) polymerase-1 (PARP-1), are sensitive arsenic targets at low and non-cytotoxic concentrations, which indicates the interaction of arsenic with these protein targets may play an important role in its co-carcinogenesis mechanism.

Arsenic exists in various forms, and its metabolic processes consist of multiple organic and inorganic arsenic forms at trivalent and pentavalent states. However, trivalent inorganic arsenic (arsenite, AsIII) is most relevant to environmental exposure from drinking water, soil, and food. This review will focus on the molecular mechanisms that contribute to arsenic carcinogenesis and co-carcinogenesis (highlighting trivalent AsIII) with a particular emphasis on arsenic-mediated DNA damage and repair inhibition and ultimately describe the current research that demonstrates zinc finger proteins as key molecular targets. The significance and impacts of the interaction of arsenic with zinc fingers as an important molecular mechanism of arsenic co-carcinogenesis will be discussed as well as new tools to address research questions related to these mechanisms.

2. Arsenic-induced oxidative stress

Arsenic exposure induces oxidative stress and ROS, which are commonly associated with carcinogenic mechanisms. ROS induce DNA damage and alter DNA repair (7-9), which are main concerns for arsenic exposure and carcinogenesis. Such effects on cellular processes take place in both direct and indirect mechanisms. This section will discuss how arsenic exposure induces oxidative stress, and how arsenic leads to DNA damage and alters DNA repair.

2.1. Mechanisms of oxidative stress induced by arsenic

Oxidative stress is caused by either the induction of ROS or impairment of the antioxidant response system. Arsenic exposure induces oxidative stress through both mechanisms. There are several mechanisms underlying arsenic-induced ROS generation, and their contributions can depend on arsenic concentration and cell type. Studies show arsenic ROS generation occurs in the mitochondria through inhibition of succinic dehydrogenase activity (10). Additionally, at environmentally relevant levels of arsenic, oxidative damage can occur through arsenic-induced activation of NADPH oxidase (NOX) and nitric oxide synthase (NOS) (11-13). NADPH subunits are upregulated by prolonged ROS generation after arsenic exposure increasing its activity (14) and is a main target of arsenic-induced ROS (15). This mechanism is further supported by a study showing arsenic upregulates expression of the p22^phox subunit and increases phosphorylation of the p67^phox subunit while inhibition of NADPH oxidase reduces ROS after arsenic exposure (11). ROS generation also occurs directly through the process of arsenic metabolism within the cell (16,17). Arsenic directly induces the generation of oxygen derived radicals including superoxide anions and hydrogen peroxide (6). Specifically, the arsenic metabolite, dimethylarsine, is shown to interact with molecular oxygen to produce a superoxide anion (18). Arsenic increases the formation of peroxyl radicals, singlet oxygen, and hydroxyl radicals among others via Fenton type reactions (19,20). While treatment with DMSO was shown to inhibit the effects of arsenic-induced ROS by reducing oxygen radicals (21), other types of oxidative stress were not attenuated by this type of treatment. These results indicate arsenic oxidative stress occurs through several different mechanisms.

Antioxidant imbalance through altered mitochondrial function and inhibition of ROS scavengers may also serve as main sources of arsenic oxidative stress. In the cell glutathione serves as an electron donor during arsenic metabolism thereby depleting glutathione leading to antioxidant imbalances (22-24). Arsenic-induced antioxidant imbalances have been demonstrated in other studies, which show alterations to superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase (25-27). In addition to directly affecting antioxidant enzyme function, arsenic may alter synthesis of antioxidants including glutathione (28) and superoxide dismutase (SOD)1 (29). These mechanisms of altered antioxidant response with the concurrent increase in ROS generation likely play important roles in carcinogenic mechanisms of arsenic exposure related to oxidative stress.

2.2. Molecular targets of oxidative damage

Consequences of oxidative stress include damage to major macromolecules in the cell including lipids, proteins, and DNA. Arsenic exposure has been associated with lipid peroxidation in conjunction with ROS generation and DNA damage (30). Arsenic-induced lipid peroxidation was also associated with decreased glutathione (GST) and glutathione peroxidase (GPx) activity likely further contributing to oxidative stress in cells (31). Recent studies show several molecules are capable of reducing arsenic-induced lipid peroxidation including chlorogenic acid and allicin by attenuating ROS and upregulating antioxidant response. Arsenic-induced oxidative damage to lipids may exacerbate oxidative stress in the cell and lead to further perturbations of other components such as DNA. For example, when lipids are oxidized in the cell additional bioactive ROS molecules are generated (19). These bioactive molecules can then perpetuate further lipid damage and target DNA or protein.

DNA is considered a major target of oxidative damage often implicated in carcinogenic mechanisms, and arsenic-induced oxidative DNA damage has been well studied (32-34). As a result of increased oxidative stress, arsenic can induce several types of DNA lesions, including strand breaks. One mechanism of arsenic-induced oxidative DNA damage is through NADH oxidase activation leading to increased superoxide production (35). Additionally, arsenic exposure can produce hydroxy radicals that react with DNA nucleobases producing DNA lesions including 8-Hydroxy-2'-deoxyguanosine (8-OHdG), 5-hydroxycytosine, and 5-hydroxyuracil (36). More detail on arsenic-induced DNA damage will be discussed in section 3.1. While arsenic can induce DNA damage through direct oxidative stress, oxidative damage or alterations to DNA repair proteins can lead to altered DNA repair and therefore additional or unrepaired DNA damage.

Oxidative stress is also associated with protein modifications after arsenic exposure. Direct damage to proteins by arsenic-induced oxidative damage has been demonstrated by an increase in carbonyl residues, which are used as a biomarker for arsenic oxidative protein damage and a decrease in protein thiols (27,37,38). A recent study also demonstrated a significant association of oxidized low-density lipoprotein with arsenic intake from drinking water in a cohort from the Navajo Nation (39). Several studies have investigated how arsenic-induced oxidative stress alters post-translational modifications of proteins (40). Specifically, studies show certain posttranslational modifications to proteins are sensitive to redox states (41,42), which may be altered by arsenic-induced oxidative stress. Tu et al., 2018 (43) found arsenic significantly alters histone modifications associated with the transformation of bronchial epithelial cells, and this finding is supported by other studies (44-46). Specifically, arsenic has been found to alter phosphorylation status of proteins leading to changes in signaling pathways and protein function (23,47-49). Other studies have examined changes in posttranslational modifications of cysteines on proteins by arsenic associated with altered DNA repair protein function (50,51). Arsenic oxidative stress leads to modification of cysteine residues, especially those containing zinc finger proteins, which are likely targets of arsenic-induced ROS (52-54). Details on modification of cysteine in zinc fingers and DNA repair proteins is further discussed in Section 5. Arsenic is also thought to affect protein tyrosine phosphorylation due to its effects on redox-sensitive cysteines (55-57).

2.3. Signaling pathways in oxidative stress

Arsenic-induced oxidative stress affects signaling pathways for cell cycle, apoptosis, and gene transcription related to DNA damage and repair. All these pathways have the potential to promote carcinogenic mechanisms and studies show arsenic-induced ROS affect signaling in many of these pathways specifically related to DNA damage (58,59). For example, although arsenic induces DNA damage, studies show arsenic allows cell cycle checkpoint bypass resulting in unregulated cell proliferation and perpetuation of DNA damage (60-62). In addition to altering cell cycle control, arsenic has also been shown to promote proliferation and survival through ERK, EGFR and MAPK pathways while simultaneously inhibiting cell death pathways, including apoptosis, although the cells are under increased oxidative stress (63-65). EGFR may also be activated without EGF binding due to transient inactivation of protein tyrosine phosphatases (PTPs). Because PTPs have preserved cysteine residues sensitive to ROS, and EGFR transactivation is negatively regulated by PTPs, arsenic-induced oxidative stress could inhibit PTPs, thus activating downstream tyrosine kinases (66). Interestingly, a recent study demonstrated melatonin was able to overcome arsenic-induced oxidative stress associated with upregulation of pro-inflammatory pathways as well as DNA damage (67). All these mechanisms promote survival of arsenic exposed cells despite DNA damage and increased oxidative stress potentially promoting tumorigenesis.

In addition to affecting proteins in signaling pathways arsenic may also affect transcription of genes in those pathways resulting in altered function. For example, arsenic activates the E2F family of transcription factors affecting cell cycle effects contributing to uncontrolled cell proliferation (68). Similarly, arsenic affects expression of proteins involved in apoptosis, for example, increasing Bax (pro-apoptotic) and decreasing Bcl2 (antiapoptotic), and these effects occurred despite persistently increased DNA damage (69). The combination of transcriptional effects with direct alteration of proteins in these pathways contributes to arsenic carcinogenic mechanisms and tumor progression.

3. DNA damage induced by arsenic exposure

The effects of arsenic on DNA damage and repair underly the carcinogenic and co-carcinogenic mechanisms of arsenic. Unrepaired or incorrectly repaired DNA damage leads to various types of mutations across the genome, which increases the risk of developing cancer. Arsenic exposure can not only cause DNA damage, but also inhibit DNA repair. When co-exposed with other DNA damaging agents, DNA repair inhibition by arsenic leads to the retention of DNA damage and enhanced mutagenesis (4). Arsenic-induced DNA damage is predominantly mediated through oxidative dependent mechanisms, but other mechanisms also play roles which will be discussed in this section.

3.1. Arsenic induces DNA damage and alters DNA methylation

Chromosome instability

Common features of genome instability associated with cancer development include changes to chromosome structure or copy number (70-72). Several in vitro, in vivo, and ex vivo studies have identified chromosome aberrations, copy number alterations, and micronuclei in association with arsenic exposure (73-75). A study on primary human lymphocytes discovered sub-micromolar AsIII treatment resulted in a concentration-dependent increase in abnormal metaphase cells. The most prevalent chromosomal aberrations found in the AsIII exposed lymphocytes were chromatid breaks and aneuploidy (76). Aneuploidy is a common characteristic of cancer cells arising from aberrant cell division. A study assessing lung squamous cell carcinoma tumors found an association between arsenic exposure and DNA copy number alterations. Arsenic induced DNA losses in several chromosomes and a gain at chromosome 19q13.33, which is known to contain oncogenes (77). Colognato et al. found arsenic-induced aneuploidy led to the development of micronuclei in arsenic exposed human peripheral lymphocytes (78). Micronuclei are associated with DNA damage and cancer and have been found to promote tumorigenesis (70,79). An epidemiology study of a population in Southern Assam, India, linked arsenic exposure through drinking water with enhanced cytogenetic damage. More specifically, cytome assay analysis of buccal epithelial cells revealed arsenic exposure increased the percentage of micronucleus, nuclear bud, binucleated, and pyknotic cells correlating with greater levels of DNA damage in lymphocytes measured via comet assay (80). These findings indicate that arsenic can induce genome instability leading to the development of a variety of changes to chromosome structure and copy number, all of which can promote tumorigenesis.

Oxidative DNA damage

There is a vast amount of literature demonstrating the capability of arsenic to generate ROS leading to oxidative stress and DNA damage. As discussed in section 2, oxidative damage via arsenic can occur through various mechanisms including mitochondrial dysfunction, antioxidant imbalance, and the activation of NADPH oxidase and nitric oxide synthase (11,81,82). Arsenic-induced ROS and reactive nitrogen species (RNS) attack DNA bases producing lesions such as 8-OHdG and 8-nitroguanine, respectively. Indeed, 8-OHdG has widely been used to measure oxidative DNA damage after arsenic exposure (81,83-87). A study on human in utero arsenic exposure showed a significant increase in both 8-OHdG and 8-nitroguanine DNA lesions in arsenic exposed newborns. The study also revealed a significant increase in DNA strand breaks associated with in utero arsenic exposure which could arise from unrepaired damage (87).

Arsenic-induced oxidative damage to DNA can be enhanced with co-exposure to a DNA damaging agent. For example, 10 μM AsIII treatment of HaCaT cells is required for a significant increase in 8-OHdG lesions, whereas the addition of UVR leads to enhanced oxidative damage with only 2 μM AsIII (81,88). Additionally, a recent epidemiology study found chronic low levels of arsenic in drinking water induced ROS correlated with DNA damage (by comet assay) in airway cells and 8-OHdG DNA damage in plasma (84). Further evidence of ROS-induced DNA damage after arsenic exposure is illustrated by reduction in DNA damage with the addition of free radical scavenging enzymes such as superoxide dismutase and catalase and treatments upregulating antioxidant responses (53,89-91). These findings exemplify arsenic as a co-carcinogen.

DNA Methylation

DNA methylation influences gene expression and is mediated through DNA methyltransferases (DNMTs) that catalyze the addition of a methyl group to cytosine. CpG island methylation within promoter regions is generally associated with decreased gene transcription. DNA methylation patterns are often altered in cancer cells supporting gene-specific promoter hypermethylation and global genome hypomethylation. These alterations can lead to the downregulation of tumor suppressor genes, such as p53 and p16, and the activation of transposable elements and proto-oncogenes promoting carcinogenesis (92,93). p53 and p16 are DNA damage response factors capable of initiating cell cycle arrest in the presence of DNA damage. These mechanisms are essential for preventing the propagation of genetically damaged cells that could lead to the development of cancer. Blood samples from individuals chronically exposed to arsenic in West Bengal, India, revealed a dose-dependent increase in hypermethylation of tumor suppressor genes p53 and p16. However, a small subgroup of individuals exposed to high levels of arsenic displayed hypomethylation of p53, which is a common duality of arsenic on DNA methylation (94,95).

Various studies have demonstrated arsenic induced global genome hypomethylation, which may occur as a result of arsenic metabolism. The biotransformation of arsenic can deplete S-Adenosyl methionine levels which is also required for DNA methylation via DNMTs. This can lead to genomic hypomethylation, especially in the context of nutritional deficiencies that limit the synthesis and reutilization of S-Adenosyl methionine (73,93,94,96). In addition, several studies demonstrate the ability of AsIII to reduce both transcript and protein levels of DNMT1, DNMT3A, and DNMT3B in HaCaT and lung epithelial cells, potentially leading to global hypomethylation (97-99). Gestational arsenic exposure increases global DNA hypomethylation in the sperm of C3H mice, particularly at the retrotransposon LINEs and LTRs. This arsenic mediated effect can lead to an increase in retrotransposon activity which is known to induce various types of cancer (100,101). Several epidemiology studies have also shown hypomethylation of transposable elements with arsenic exposure (73). As mentioned previously, hypomethylation of proto-oncogenes has been reported with arsenic exposure. Proto-oncogene c-Myc and c-Ha-ras hypomethylation and resulting increase in expression was demonstrated in arsenic treated Syrian hamster embryo cells (102). c-Myc upregulation is heavily associated with cancer contributing to the development of over 40% of tumors (103). Activating mutations within H-ras are commonly found within tumors promoting cancer cell proliferation and survival (104). Altogether, alterations in DNA methylation patterns by arsenic can lead to gene expression changes that promote carcinogenesis.

Mitochondrial DNA

Mitochondrial DNA (mtDNA) encodes for products involved in cellular respiration and protein synthesis and is vulnerable to oxidative damage. Mitochondria have limited DNA repair capabilities compared to DNA in the nucleus and mtDNA lacks the protective features provided by histones and nucleosome assembly. ROS generated through the electron transport chain is a common cause of oxidative damage to mtDNA and the development of somatic mutations. Mutations within mtDNA can further disrupt oxidative phosphorylation resulting in heightened ROS production, rate of DNA mutations, and risk for cancer induction (105). Arsenic is known to disrupt the electron transport chain which can enhance the levels of mitochondrial ROS leading to direct mtDNA damage. A concentration-dependent increase in oxidative damage to mtDNA was observed in arsenic exposed human keratinocytes, which was partially rescued with antioxidant treatment. Additionally, elevated levels of mtDNA oxidative damage and mutations were found in lesional skin tissue of individuals with arsenic-induced Bowen’s disease compared to perilesional tissue (106). Another study showed arsenic trioxide (ATO) treatment of human acute promyelocytic leukemia cells resulted in an increase in mtDNA mutation spots corresponding with an increase in cellular apoptosis. The types of mutations that arose from ATO exposure included transitions, transversions, and codon insertions and deletions (107).

In addition to mutations, alterations in mtDNA copy number are also linked to cancer development (108,109). The D-loop region of mtDNA regulates replication and transcription and D-loop hypomethylation can lead to an increase in mtDNA copy number (110). An epidemiology study on a population in West Bengal, India, demonstrated an increase in D-loop hypomethylation in blood samples of individuals exposed to arsenic through drinking water. These findings corresponded with an arsenic-induced increase in mtDNA copy number (111). Alternatively, studies have also found an arsenic-induced increase in mtDNA deletions and decrease in mtDNA copy number, which can also support carcinogenesis (112,113). Altogether, these findings demonstrate the genotoxicity of arsenic not only affects nuclear DNA, but also alters the integrity of mtDNA, which is known to be associated with cancer development.

Somatic Mutations

Arsenic is known to induce mutations, which may be in part due to ROS generation (21). A study utilizing the mouse lymphoma TK assay revealed a significantly increase in mutation frequency with exposure to at least 10 μM arsenic. This effect was observed with various arsenic species including AsIII, ATO, monomethylarsonic acid, and dimethylarsinic acid (114). Even at low concentrations, AsIII treatment of normal human neonatal epidermal keratinocytes (HEKn) showed a concentration-dependent increase in HPRT mutations from 0.1 to 1 μM AsIII (91). The role of arsenic-enhanced mutations in carcinogenic mechanisms is a promising trajectory for arsenic research. There is little information on specific site mutations induced by arsenic, and a majority of the literature on this topic is focused on the effect of arsenic in cases of pre-existing mutations (115,116). To date, studies have focused on site mutations in specific genes limiting the ability to determine the mechanisms leading to these events. To better understand arsenic co-carcinogenesis, whole-genome or whole-exome sequencing can be used to determine mutational processes by analyzing mutational signatures.

The whole genome mutational profile can be viewed as a record bearing all the mutational signatures of mutational processes (such as failed DNA repair, etc.) caused by environmental exposures. There is a computational framework for deciphering individual mutational signatures from whole-genome sequencing data. Thus, mutational signatures are emerging as a powerful tool for identifying environmental exposures from the cancer genome and for understanding their role in cancer development. The field has made great strides in recent years, yet few papers have investigated environmental mutagens and only one has looked at arsenic, using mutational signature analysis (117-119). Mutational processing analyses should be of great help in the demonstration of the molecular mechanisms of arsenic throughout the genome, including co-carcinogenesis, deciphering the contribution of oxidative DNA damage, and the inhibition of DNA repair.

3.2. Arsenic synergy with DNA damaging agents

Ultraviolet radiation (UVR)

Arsenic exposure increases the risk of developing skin lesions and several types of skin cancer including squamous cell carcinoma in situ, invasive squamous cell carcinoma, and basal cell carcinoma (120,121). In the United States, nonmelanoma skin cancer is the most prevalent malignancy and is associated with increased cost, morbidity, and mortality. Over 3 million people in the United States were treated for nonmelanoma skin cancer in 2012, a substantial increase from 2006 (122). Approximately 90% of nonmelanoma skin cancers are caused by exposure to UVR (123). In 2004, Burns et al. discovered environmentally relevant levels of arsenic greatly enhanced the carcinogenicity of UVR leading to increased cancer yield in mice (124). An epidemiology study by Chen et al. also revealed this association in humans (125). The link between consumption of arsenic contaminated drinking water and the development of skin cancer is apparent even at concentrations below the EPA Maximum Contaminant Level of 10 μg/L (126).

UVR exposure produces various types of DNA damage depending on wavelength. UVA can generate ROS leading to DNA single strand breaks and oxidized DNA bases, whereas UVB predominantly contributes to the generation of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4 PPs) (127). Various repair pathways are activated to remove damage caused by UVR, such as single strand break repair and base excision repair (BER) for remediating DNA single strand breaks and oxidized DNA bases, respectively. For bulkier DNA lesions such as CPDs and 6-4 PPs, recognition and repair are facilitated by nucleotide excision repair (NER) (128). Exposure to low levels of arsenic can lead to the inhibition of BER and NER, thus reducing the repair capacity for UVR generated DNA lesions (6). Studies on UVR exposed HEKn cells revealed a significant increase in the levels of 8-OHdG, CPDs, DNA strand break marker pH2AX, and HPRT mutations with only 1 μM AsIII treatment (86,91). Levels of UVR induced CPDs, 6-4 PPs, and pH2AX were also significantly increased in SHK-1 mice treated with 5 mg/L of AsIII in their drinking water for 28 days (91). Evidence suggests arsenic generated ROS and RNS play a role in the synergistic effects of arsenic and UVR through the inhibition of PARP-1 (11,51,129-131). In addition, many studies have found arsenic can regulate the expression of various NER and BER proteins impacting the ability of cells to repair UVR generated DNA lesions (6,132). Altogether, the synergy between arsenic and UVR provides a clear example of how arsenic can function as a co-carcinogen.

Polycyclic aromatic hydrocarbons (PAHs)

Exposure to polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (BaP), are linked with the development of skin, lung, and bladder cancers and are derived from both natural and anthropogenic sources (133). For example, cigarette smoke contains BaP, along with other carcinogens as discussed below (134). Exposure to BaP can also occur from emissions from fossil fuel combustion, release of hazardous waste, and ultimately through drinking water. These sources concurrently serve as common sources of arsenic, and therefore, result in BaP-arsenic co-exposures. Reactive metabolites of BaP, such as benzo(a)pyrene diolepoxide (BPDE), generate helix distorting DNA lesions at concentrations as low as 10 nM (135). The dominant DNA adduct formed with exposure to BaP is BPDE-N²-dG, which unrepaired can lead to G to T transversions in DNA (136). These transversions are often found within the p53 gene of lung cancer patients and are associated with PAH exposure through cigarette smoking (137).

Studies have demonstrated the ability of arsenic to promote BaP-induced DNA damage. For example, the addition of AsIII to BaP exposed mouse hepatoma cells enhanced the number of BaP-DNA lesions by as much as 18-fold. Given alone, 0.5 μM BaP and 2.5 μM AsIII did not induce HPRT mutations, but together increased the mutation frequency by 8-fold (138). Another study found Sprague–Dawley rats treated with BaP and AsIII displayed an increase in the retention of DNA adducts compared to BaP alone, suggesting the inhibition of DNA repair (136). More recent studies by Burchiel et al. have demonstrated a synergy between arsenic and PAHs in the suppression of immune cells (139,140). AsIII promoted the genotoxicity of BaP metabolites in mouse thymus cells. Further investigation revealed mechanisms underlying the observed effect may include PARP inhibition and increased BaP metabolism (140). Altogether, studies have provided evidence for the synergism of arsenic and BaP leading to increased DNA damage, mutagenesis, and altered immune functions.

Tobacco

Cigarette smoke contains an assortment of carcinogens including PAHs, aromatic amines, and nitrosamines, and can generate ROS leading to oxidative damage to DNA (134). Arsenic has been shown to increase genotoxicity and lung cancer risk in cigarette smokers (80,141,142). In vivo studies have demonstrated an increase in oxidative DNA damage with co-exposure to arsenic and cigarette smoke. For example, the addition of aerosolized arsenic compounds to Syrian golden hamsters exposed to cigarette smoke led to a 5-fold increase in 8-oxo-2’-deoxyguanosine, predominantly in the nuclei of airway epithelium and sub-adjacent interstitial cells (143). Another study revealed an AsIII-induced increase in plasma 8-OHdG levels within cigarette smoke exposed mice (144). An epidemiology study of tobacco chewers in Southern Assam, India, revealed a significant increase in cytogenetic damage when co-exposed to arsenic. More specifically, arsenic enhanced the percentage of buccal epithelial cells containing micronuclei and nuclear buds in tobacco chewers signifying chromosomal damage (80). Altogether, these findings demonstrate the ability of arsenic to enhance the genotoxicity of tobacco products resulting in genomic instability and carcinogenesis.

Ionizing radiation (IR)

Radiotherapies utilizing X-ray or gamma radiation generate DNA double-strand breaks, which can be lethal or mutagenic if not repaired correctly thus making an effective treatment against cancer (145). Though a number of cancers can be treated with radiation alone, some require co-therapy to improve treatment outcomes (146). ATO has been shown to significantly increase IR-induced apoptosis of endothelial HDMEC and tumor OSCC-3 cells, while protecting osteoblasts. This arsenic effect was also demonstrated in vivo, where the addition of ATO to IR treated mice inhibited tumor growth, angiogenesis, and metastasis, as well as protected against radiation-induced bone loss (147). A study on IR exposed glioblastoma multiforme cells found treatment with ATO upregulated Bax and caspase-3 and downregulated Bcl-2 leading to cellular apoptosis (148). Arsenic exposure has been shown to inhibit DNA double-strand break repair (DSBR) leading to the retention of DNA damage which can result in the activation of p53 and the induction of apoptosis (86,149-154). These findings may support the usefulness of arsenic and IR as a cancer co-therapy.

Cisplatin

Cisplatin is a DNA crosslinking agent used in the treatment of several different types of cancers including carcinomas, germ cell tumors, lymphomas, and sarcomas. Cisplatin crosslinks purine bases within DNA, activating a number of DNA repair pathways (155). NER, mismatch repair (MMR), homologous recombination, translesion synthesis, and Fanconi anemia all play a role in the repair of cisplatin generated DNA lesions (156). The disadvantages of cisplatin as a chemotherapy are the development of cisplatin resistant cancer cells and the various side effects of the treatment (155). Combination therapies can often be more effective at lower doses decreasing potential side effects and increasing cytotoxicity towards drug resistant cancer cells (157). The synergistic nature of arsenic and cisplatin has sparked many investigations into the co-therapeutic potential of arsenic in the treatment of cancer (158). The addition of ATO to cisplatin treated human ovarian cancer cells resulted in a concentration-dependent decrease in proliferation and increase in apoptosis. The synergistic effect of arsenic and cisplatin treatment was evident in both cisplatin sensitive and resistant ovarian cancer cell lines, demonstrating the usefulness of arsenic in the treatment of ovarian cancer (159). A number of mechanisms are involved in cisplatin resistance, including upregulation of DNA repair pathways. The addition of AsIII to cisplatin treated mice increased platinum accumulation in tumors and prevented the upregulation of NER factor XPC, suggesting AsIII may sensitize cancer cells to cisplatin through its ability to suppress NER and increase platinum uptake in tumors (160).

4. Arsenic regulation of DNA repair

At environmental relevant concentrations, arsenic acts mainly as a co-carcinogen enhancing the carcinogenic effects of other carcinogens. One of the most important mechanisms of arsenic co-carcinogenesis is the inhibition of DNA repair. Epidemiology studies show arsenic-exposed populations have reduced DNA repair capacity (161). While not fully understood, arsenic orchestrates a multitude of different effects on cellular function that decrease DNA repair and promote the carcinogenicity of DNA damaging agents. Indeed, evidence of arsenic mediated changes in the levels and recruitment of NER, BER, DSBR, and MMR proteins have been reported (97,151,161-172). In addition, arsenic can alter the activity of DNA repair proteins via direct binding to critical zinc finger domains, further discussed in section 5 (6). Retained DNA damage from arsenic inhibition of DNA repair can result in the generation of DNA double-strand breaks when encountered by the DNA replication fork (173,174). Arsenic is known to induce DNA strand breaks, particularly in conjunction with a DNA damaging agent (6,129,173). DNA double-strand breaks are a common cause of chromosomal rearrangements and can also occur through improper DNA repair. Studies have shown alterations in DSBR by arsenic favor error prone pathways, such as non-homologous-end joining, and are associated with structural changes to chromosomes such as chromosomal translocations (149-152). Here we summarize and review recent advances in arsenic inhibition of DNA repair, showing the effects at all levels, from transcription to post-translation.

4.1. Transcriptional regulation

Canonical pathways of protein expression begin with changes in mRNA at the transcriptional level. Several studies have demonstrated arsenic-induced alterations in transcript levels of DNA repair genes, particularly those involved in NER and BER (165,166). Mechanistically, arsenic may alter the expression of DNA repair genes by affecting DNA methylation and histone modifications. Importantly, arsenic-induced changes in gene expression can depend on various conditions. A study conducted by Osmond et al. investigated AsIII regulation of BER gene transcript levels in mice. Transcript levels of BER genes APE1, LIG1, OGG1, PARP1, and POLB were all significantly reduced in 24-week-old mice treated with 2 ppm AsIII for 2 weeks compared to untreated mice. However, acute 24hr AsIII treatment increased BER genes APE1, LIG3, and POLB. These findings along with the significant differences between transcript levels in 24-week-old versus 1-week-old mice, highlights the complexity of arsenic and the potential reasoning behind the diverse and sometimes conflicting results seen in arsenic research (170). Many studies have demonstrated arsenic may impact DNA repair directly at the transcriptional level, though much of the research has been conducted using arsenic levels significantly higher than environmentally relevant. Detailed mechanisms, such as DNA methylation and histone modifications, will be discussed in this section.

DNA Methylation

As mentioned in section 3.1, arsenic can downregulate DNA damage response genes and upregulate proto-oncogenes and transposons by altering DNA methylation patterns. Additionally, arsenic is associated with the downregulation of DNA repair genes via promoter hypermethylation. BRCA1 is an essential DNA repair protein involved in homologous recombination, an error free DSBR pathway, and promoter hypermethylation is commonly linked with sporadic breast tumors and estrogen receptor negativity (175). AsIII treatment led to BRCA1 hypermethylation and decreased BRCA1 and estrogen receptor alpha expression in both MCF7 cells and MCF7 cell mammary tumor xenografts (151). These findings may demonstrate a mechanism by which arsenic promotes cancer through the inhibition of an error free DSBR pathway. Indeed, a study utilizing DSBR reporter assays showed arsenic exposure influences pathway choice favoring more error prone repair mechanisms, such as non-homologous-end joining, which are associated with chromosomal aberrations (149,150).

Arsenic-induced changes in DNA methylation are also linked with the downregulation of MMR genes. AsIII treatment of HaCaT cells has been shown to increase promoter hypermethylation of the MMR gene MSH2 resulting in decreased expression (97). In addition, a study evaluating the impact of arsenic on individuals within West Bengal, India, revealed significant promoter hypermethylation of both MLH1 and MSH2 genes and a corresponding downregulation in gene transcript levels (163). MMR is an important pathway in the recognition and remediation of DNA replication and recombination generated base mismatches, insertions, and deletions (176). Recombination events can occur from unrepaired DNA lesions, particularly when encountered by the replication fork (174). Arsenic exposure leads to the retention of unrepaired DNA lesions which may increase replication stress and DNA strand breaks, thus increasing the requirement of MMR (6,174,176). Therefore, the loss of MMR in the context of arsenic exposure may have a great effect on genome stability.

Histone Modifications

Chromatin structure is tightly regulated by the methylation of histones, such as di-methylation of histone H3 on lysine 9 (H3K9me2), which is most often found within heterochromatin and is associated with gene silencing (177). Recently, Ding et al. discovered an AsIII induced increase in H3K9me2 levels within promoter regions of BER genes MPG, XRCC1, and PARP1, which corresponded with a decrease in transcript and protein levels of each gene (164). Partial rescue of protein levels was achieved with methyltransferase inhibitor treatments, indicating the role of histone methylation in the mechanism of action of arsenic (164). BER is an essential pathway for the removal of oxidized DNA bases, particularly in the context of arsenic exposure which stimulates the production of ROS (178). Without this protective mechanism, mutations may arise and promote tumorigenesis (179).

4.2. Post-translational regulation

Degradation

Arsenic regulates proteasomal-mediated degradation of proteins through various means (166,180-185). For example, ATO is used in the treatment of acute promyelocytic leukemia due to its ability to bind PML-RARα oncoprotein and stimulate ROS leading to the formation of PML-RARα multimers and targeted degradation (186). Arsenic binding also contributes to the degradation of TIP60 involved in DSBR (187). The mechanism and consequences of arsenic binding to proteins is further discussed in section 5. Another mechanism by which AsIII influences protein degradation is through the upregulation of E3 ubiquitin ligases. A study on p53 mutant expressing HaCaT and MIA PaCa-2 cell lines revealed ATO transcriptionally increased the levels of an E3 ubiquitin ligase, Pirh2, which in turn increased the polyubiquitination and degradation of mutant p53 (185). XPC is an integral factor in global genome NER and can bind to a variety of DNA lesions such as those caused by exposure to UVR, chemotherapy drugs, and tobacco smoke (134,188). High concentrations of AsIII decreased XPC protein levels in human lung fibroblasts and primary mouse keratinocytes. XPC expression in human lung fibroblasts was partially rescued with the addition of a proteasome inhibitor, MG-132, suggesting levels of XPC were regulated via AsIII induced proteasomal degradation (166). In addition to specific targets, global levels of polyubiquitinated proteins were increased in ATO treated mouse cardiomyocytes. These findings corresponded with a concentration-dependent increase in proteasome activity with ATO exposure, which was found to play a protective role against arsenic mediated cell death (184). In addition to transcriptional regulation, these findings demonstrate AsIII can alter the levels of DNA damage response and repair genes via protein degradation.

Recruitment

Protein phosphorylation is a post-translational modification which can induce changes in protein activity, stability, binding, and cellular localization. As mentioned in section 2.3 arsenic is known to regulate several different signaling pathways via mechanisms such as generating ROS, binding to sulfhydryl groups, and altering gene expression. EGFR signaling mediates DNA repair pathways such as MMR (189). The activation of EGFR leads to the phosphorylation of the DNA sliding clamp, proliferating cell nuclear antigen (PCNA), which in turn inhibits the binding and activation of MMR factors by PCNA (190). PCNA is a critical processivity factor that facilitates DNA replication and replication-coupled DNA repair pathways by acting as a scaffold for the recruitment of proteins (191). HeLa cells treated with 5, 10, and 15 μM AsIII resulted in a concentration-dependent increase in EGFR levels in which corresponded with an increase in the phosphorylation of PCNA. Whole cell extracts from AsIII treated HeLa cells had reduced MMR activity which was partially restored by the addition of exogenous PCNA (190). These findings demonstrate a mechanism by which arsenic alters the activity of MMR through PCNA phosphorylation (189,190).

As mentioned previously, Bhattacharjee et al. demonstrated hypermethylation of MMR genes in individuals chronically exposed to arsenic in West Bengal, India. In addition to this finding, the authors discovered another mechanism by which arsenic inhibits the activity of MMR, alterations in histone methylation. The study found that arsenic exposure was associated with the reduction in tri-methylation of histone H3 on lysine 36 (H3K36me3), particularly in the subpopulation with skin lesions (163). H3K36me3 is required for the recruitment of MMR machinery to chromatin, and thus, its function. Loss of H3K36me3 is linked with microsatellite instability and enhanced spontaneous mutation frequency, as seen in cells deficient in MMR (192). Alterations in histone acetylation and ubiquitination have also been demonstrated with arsenic exposure. AsIII can bind and inhibit histone modifiers such as TIP60 histone acetyltransferase and RNF20-RNF40 histone E3 ubiquitin ligase resulting in a decrease in acetylation of lysine 16 on histone H4 and monoubiquitination of lysine 120 on histone H2B respectively. These histone modifications are important in the recruitment of DSBR factors by relaxing chromatin around DNA double-strand breaks (6,152,187). Altogether, the different mechanisms in arsenic mediated regulation of DNA repair proteins illustrates the multifaceted effects of arsenic on cell function and genome stability.

5. Arsenic interrupting zinc finger protein targets: molecular mechanisms.

The DNA repair system is highly sensitive to arsenic exposure. Even at low, non-cytotoxic concentrations, arsenic exposure inhibits DNA repair activity coinciding with inhibition of specific zinc finger DNA repair proteins that are sensitive to arsenic exposure. In this section, we will summarize and review recent advances on arsenic inhibition of zinc finger DNA repair proteins. In addition, we will discuss how this mechanism works in concert with oxidative stress mechanisms, creating a cohesive picture of arsenic co-carcinogenesis.

5.1. Zinc finger proteins: key role of the zinc ion.

Zinc finger proteins form a large family that function in various physiological and pathological processes (193,194). Zinc fingers are small, folded motifs thermodynamically held together by the coordination of a zinc ion with a combination of four cysteine and/or histidine residues (195,196). Zinc plays an important role in the structure and function of zinc fingers, as well as protecting the sensitive thiol groups from oxidation (52,197). Zinc finger proteins can be categorized by the number of cysteine and histidine residues they contain. For example, the “classical” zinc finger consists of 2 cysteine and 2 histidine residues, designated a C2H2 conformation (194). Other non-classical zinc finger proteins have different combinations of cysteine and histidine residues, such as C3H1 and C4 (193,195). Zinc finger proteins can also have more complicated structures, such as RING, PHD, and LIM types (198-202), however, these complex structures are in reality combinations of C3H1 or C4 zinc fingers (193,195,200,203). Over 80% of zinc finger proteins are found in the C2H2 conformation, while a minority are categorized as the C3H1 and C4 conformations (193).

Over five percent of human proteins contain zinc finger domains that perform various functions including binding to DNA, RNA, lipids, proteins, and are involved in post translational modifications (204). Recognizing and binding to other macro-molecules are mediated by zinc finger motifs (194,195,200,205). Removing the zinc ion from zinc fingers results in conformational changes and loss of function. As a result, arsenic displacement of zinc ion, through direct or indirect mechanisms, could severely disrupt zinc finger protein function, which will be discussed in following sections.

5.2. Zinc finger DNA repair proteins as an arsenic target

PARP-1 has two C3H1 zinc fingers in its DNA binding domain and is an essential DNA repair protein that is recognized as a highly sensitive target of arsenic exposure (6). Early studies show low levels of arsenic not only inhibit PARP-1 expression, but also its function, setting the stage for future investigation into how arsenic affects zinc finger proteins (206,207). PARP-1 binds to DNA strand breaks via its zinc fingers in the DNA-binding domain, stimulating poly ADP-ribosylation, recruitment of DNA repair factors, and the initiation of repair pathways such as BER, NER, single-strand break repair, and DSBR (208,209). These pathways are required for the repair of UVR generated DNA damage and therefore, PARP-1 is an essential protein for maintaining genome stability in the context of UVR exposure (128). Indeed, knockdown of PARP-1 significantly increases the retention of UVR-induced 8-OHdG and DNA strand breaks in HaCaT cells (50,129).

AsIII treatment of HEKn cells results in a concentration-dependent decrease in PARP-1 zinc content, signifying AsIII mediated displacement of zinc from the PARP-1 zinc fingers (86). The mechanism of zinc displacement from PARP-1 was revealed by MALDI-TOF-MS analysis demonstrating AsIII binding to the PARP-1 zinc finger peptide, even in the presence of excess zinc (50,210). AsIII was shown to reduce chromatin bound PARP-1 and UVR stimulated poly ADP-ribosylation in HEKn cells providing evidence for AsIII mediated inhibition of the zinc finger containing DNA binding domain of PARP-1 (86,211). PARP-1 inhibition by AsIII may also be a mechanism underlying the enhancement of UVR-induced 6-4 PPs, CPDs, 8-OHdG, pH2AX, and gene mutations in HEKn cells exposed to AsIII (91). AsIII treatment of SHK-1 mice also led to an increase in UVR-induced 6-4 PPs, CPDs, and pH2AX. Furthermore, zinc supplementation reduced arsenic enhancement of DNA damage both in vitro and in vivo (91). These findings support a mechanism of AsIII induced zinc loss from PARP-1 leading to DNA-binding domain inhibition and the retention of DNA damage with co-exposure to UVR.

PARP-1 is not a unique DNA repair protein target for arsenic. XPA is an essential NER factor that binds to DNA lesions via its C4 zinc finger domain and acts as a scaffold to mediate repair (212). AsIII treatment of both HaCaT and HEKn cells leads to a significant reduction in XPA zinc content (210,211). The loss of zinc in XPA corresponds with increased cysteine oxidation and decreased chromatin recruitment of XPA in HEKn cells exposed to AsIII (52,211). The inhibition of XPA by arsenic may contribute to the retention of UVR induced 6-4 PPs, CPDs, and DNA strand breaks in cells (91,212). In addition, XPA inhibition by arsenic could hinder the removal of bulky DNA lesions caused by cisplatin and BaP, thus supporting the observed synergistic effects described in section 3.2 (134,188).

Several studies by Wang et al. demonstrate arsenic targeting RING finger containing DNA repair proteins. The RING finger is distinct from the zinc finger domains of XPA and PARP-1 in that it is composed of two interdigitated zinc-binding sites. Upon DNA damage, the RNF20-RNF40 E3 ubiquitin ligase complex is responsible for the monoubiquitination of histone H2B which leads to chromatin relaxation and facilitates DSBR factor recruitment (152). Both RNF20 and RNF40 RING fingers contain C4 and CHC2 zinc binding sites (213). MALDI-TOF-MS and UV absorption spectra analyses of RNF20 and RNF40 RING finger peptides revealed direct AsIII binding resulting in conformational changes of the zinc finger domain. A decrease in the monoubiquitination of H2B was observed in several different cell lines after 24 h exposure to 5 μM AsIII. In addition, 5 μM AsIII treatment of HeLa cells displayed decreased recruitment of DSBR factors BRCA1 and RAD51 to laser-induced DNA double-strand break sites (152). These findings are consistent with the observation that arsenic inhibits the repair of IR induced DNA double strand breaks (214). Another study revealed AsIII binding to the RING finger containing DNA repair protein FANCL. FANCD2 monoubiquitination by the E3 ubiquitin ligase, FANCL, is a critical step during DNA interstrand crosslink repair. This repair pathway is necessary for the removal of interstrand crosslinks generated by DNA damaging agents such as cisplatin and endogenous metabolites (215). AsIII exposure of HeLa cells resulted in decreased FANCD2 monoubiquitination and chromatin recruitment (216). Altogether, these findings illustrate a mechanism of arsenic co-carcinogenicity via the inhibition of DNA repair zinc finger domains.

5.3. Arsenic directly and selectively binds to zinc fingers.

Arsenic can react with the sulfur of thiol group in cysteine to form As-S bond. Therefore, proteins with free cysteine residues are potential arsenic targets. However, arsenic does not show significant binding to free cysteine or thiol-containing small molecules in vivo. Protein studies indicate low reactivity of arsenic to proteins with single cysteine or two cysteine residues (217). Kinetics experiments show arsenic binding to one or two cysteine residues have a higher dissociation rate constant than three or four cysteine residues (218,219). It was demonstrated that the half-life of the AsIII-trithiol peptide complex is two orders of magnitude greater than that of the corresponding AsIII-dithiol peptide complex. This dramatic difference in chemical kinetics implies that trivalent arsenite may be capable of interacting with single or two cysteine residues, but a more stable interaction of arsenite with a C3 or C4 zinc finger protein may be necessary to sustain biological impact. Furthermore, trivalent arsenic appears to have a spatial requirement for binding with three cysteines simultaneously. Reactivity of arsenite to cysteine requires at least 3 cysteines to be present with a strict spatial requirement of 3 cysteine thiols oriented towards the same center within the radius of the arsenic atom. Cysteine in zinc finger motifs naturally fulfill this spatial requirement, where all cysteine thiols point to and bind with the zinc ion in the center.

The differential reactivity of arsenic to different numbers of cysteine residues in zinc finger motif suggests arsenic binding selectivity. Trivalent arsenic selectively binds to C3H1 and C4 zinc fingers, but not C2H2 zinc fingers, as demonstrated by mass spectrometry and site direct mutation studies (210,220). When inorganic arsenite is metabolized to become monomethylarsonous acid (MMAIII), the methyl group will occupy one of the three binding sites. As expected, MMAIII not only binds with C3H1 and C4 zinc fingers, but also C2H2 (223). Even more interestingly, two MMAIII were found on the same C4 zinc finger motif. These findings provide consistent evidence that arsenic binding selectivity is determined chemically by its valence state.

Since a majority of zinc finger proteins in biological systems are of C2H2 configuration, selective binding of trivalent arsenic with the minority C3H1 and C4zinc finger proteins may make them particularly targeted. For example, PARP-1 can be inhibited by AsIII at concentrations as low as 0.1 μM in cells (50), leading to zinc release and DNA binding activity inhibition. GATA-1, a transcription factor regulating red blood cell differentiation, can be inhibited by arsenic exposure starting from the concentration of 0.1 μm in cellular and animal models (222). This selective binding may explain why arsenic shows a co-carcinogenesis effect at environmentally relevant concentrations. Moreover, C4 configuration zinc fingers may be more sensitive to arsenic exposure compared to the C3H1 configuration due to higher number of available cysteine residues (211).

A kinetic and thermodynamic study revealed that zinc and arsenic have relatively similar binding affinities towards zinc finger peptides, suggesting that zinc finger occupancy in a cellular system may be strongly influenced by the relative concentrations of each metal (223). A slight kinetic advantage for arsenic over zinc may lead to significant arsenic binding to newly synthesized zinc finger proteins, especially during the process of significant induction of protein expression, such as during a DNA damage response with significant PARP-1 induction. Similarly, this may occur in red blood cell differentiation when a large amount of GATA-1 is induced. Thus, under these circumstances, certain zinc finger protein targets may be highly sensitive to arsenic exposure.

Because of their similar binding affinities towards zinc finger motifs, arsenic competing with zinc ions in zinc fingers is key to a zinc chemo-prevention strategy, which involves supplementing zinc to rescue the effect of arsenic-inhibited DNA repair proteins (91). Therefore, zinc supplement could be an effective strategy attenuating zinc finger inhibition in various arsenic toxicity scenarios.

5.4. Arsenic indirectly disrupts zinc fingers through ROS/RNS.

Arsenic induces oxidative and nitrosative stress, affecting related enzymes and protein targets such as HO-1 (20). Specifically, zinc finger proteins containing redox sensitive cysteine residues are molecular targets of ROS induced by arsenic. The number of cysteine residues in certain proteins is a factor of the likelihood of oxidation by ROS. Thus, C4 zinc finger proteins are more likely to be oxidized by ROS than C3H1 and C2H2. Oxidative modification on cysteine residues in a zinc finger motif leads to zinc release, conformational change, and ultimately functional loss of certain zinc finger protein targets (51,52). Zinc finger protein targets such as PARP-1 are highly sensitive to arsenic-induced ROS. For example, PARP-1 can be inhibited by AsIII as low as 0.1 μM, while the effect can be rescued by antioxidants (51).

There are different types of oxidative modifications on cysteine residues; some are reversible (i.e. −SOH and −SO₂H) while others are irreversible (i.e. −SO₃H and −SS−). Arsenic-induced ROS leads to both types of modifications (52), suggesting that some oxidative changes induced by arsenic might be reversed by antioxidants or zinc supplement, but in certain conditions the damage could be permanent. Arsenic also induces nitric oxide production at low and environmentally relevant concentrations (130,224). Nitrosative stress can lead to nitrostative modification of zinc finger proteins such as PARP-1 (225). Furthermore, ROS and RNS mechanisms may occur at the same time and synergistically. For example, superoxide and nitric oxide produce a peroxynitrite, which inhibits PARP-1 activity (53). Therefore, arsenic-generated ROS/RNS not only can directly cause DNA damage, but also inhibit DNA repair through oxidative or nitrosative modifications to key DNA repair zinc finger proteins.

5.5. The interplay between direct and indirect mechanisms.

Zinc binding protects cysteine residues within a zinc finger motif from being oxidized easily. Under increased oxidative stress, free cysteine should be oxidized prior to zinc-protected cysteine on zinc finger motifs (208). Taking PARP-1 as an example: its regulatory domain responsible for enzyme activity contains 2 free cysteines, while the cysteines in its DNA binding domain are all located in zinc fingers which are bound to zinc ions (208). Therefore, when ROS levels increase, PARP-1 DNA binding activity should be less sensitive to oxidative damage than the enzyme activity. However, while the two free cysteines in the regulatory domain remain unchanged, arsenic is able to cause oxidation of the cysteines on PARP-1 zinc finger and inhibit PARP-1 activity at low and non-cytotoxic concentrations (52). This result suggests that arsenic displacement of zinc from the zinc finger configurations eliminates zinc protection of the zinc finger cysteine residues, thus rendering these cysteines vulnerable to attack by AsIII-generated ROS.

Under oxidative stress, arsenic binding to zinc fingers is unstable. When arsenic replaces zinc from zinc finger motifs, thiol groups on the cysteines becomes vulnerable to oxidative modification, making arsenic binding to zinc fingers a transient process (52). Arsenic presence in zinc finger proteins increases at the beginning of exposure, but then decreases quickly while oxidative modifications on zinc finger proteins persist and continue to increase (52). These results indicate that cysteine oxidation further disrupts AsIII binding to the zinc finger motif, thereby releasing AsIII to interact with another target protein. Because the released AsIII would be free to interact with another target protein and then repeat this cycle, it effectively functions in a catalyst-like manner for oxidation of targeted proteins. The bind-and-release model could explain why a very low concentration of AsIII (e.g. submicromolar) is capable of causing significant inhibition of PARP-1 activity in cells. Since a significant portion of arsenic-induced oxidative damage to zinc fingers is likely permanent, it explains why adding antioxidants only partially reverses the impact of arsenic exposure (226).

Arsenic selectively binds to specific zinc finger proteins, making these proteins vulnerable to oxidative damage. The interplay and the unified mechanisms of arsenic binding (direct mechanism) and oxidation (indirect mechanism) of zinc fingers by arsenic explains the high sensitivity of certain zinc finger proteins to arsenic exposure, and more importantly, sheds light on prevention and intervention strategies by utilizing zinc supplements and antioxidants.

6. Summary and future perspectives

Arsenic carcinogenesis and co-carcinogenesis present a significant impact to human health at environmentally relevant concentrations. Research on the molecular mechanisms of arsenic co-carcinogenesis should be of great help in prevention and intervention of the impact of arsenic exposure. In addition to the many other effects of arsenic exposure such as DNA damage, epigenetic alterations, and gene regulation, inhibition of DNA repair plays a central role in arsenic co-carcinogenesis. Here we summarized research on the advances in molecular mechanisms of arsenic inhibiting zinc finger DNA repair proteins. Recent work revealed a clear mechanism of selective binding of arsenic to zinc finger proteins, as well as the interplay of binding and ROS. The overall concept of the molecular mechanism is summarized in Fig. 1. This illustrated mechanism not only demonstrates how arsenic inhibits DNA repair but is also meant to inspire future research on arsenic interacting with zinc fingers as a key molecular mechanism involved in the co-carcinogenicity of other environmental toxicants (222) or to be applied to pharmaceutical applications (227).

Figure 1.

Schematic illustration of arsenic co-carcinogenesis

Meanwhile, there are still many important gaps in our understanding of the mechanisms of arsenic carcinogenesis and co-carcinogenesis. One of the major gaps of knowledge is how arsenic is involved in mutagenic processes that lead to cancers. For example, while there are many types of environmental mutagens, why does arsenic exposure and co-exposure lead to cancers at such low concentrations? Also, UVR exposure results in certain mutational processes leading to unique mutational patterns in the genome. Whether arsenic co-exposure alters such patterns, and how such potential changes lead to strong co-carcinogenesis effects is still unclear. In the future, the state-of-art computational genomic approaches may enable these questions to be answered.

Abbreviations:

6-4 PPs

pyrimidine (6-4) pyrimidone photoproducts

8-OHdG

8-Hydroxy-2'-deoxyguanosine

AsIII

arsenite

ATO

arsenic trioxide

BaP

benzo(a)pyrene

BER

base excision repair

BPDE

benzo[a]pyrene diolepoxide

CPD

cyclobutane pyrimidine dimer

DNMT

DNA methyltransferase

DSBR

double strand break repair

EGFR

epidermal growth factor receptor

EPA

Environmental Protection Agency

GPx

glutathione peroxidase

GST

glutathione

H3K36me3

tri-methylation of histone H3 on lysine 36

H3K9me2

di-methylation of histone H3 on lysine 9

HEKn

human epidermal keratinocytes

IR

ionizing radiation

MMAIII

monomethylarsonous acid

MMR

mismatch repair

mtDNA

mitochondrial DNA

NER

nucleotide excision repair

NOS

nitric oxide synthase

NOX

NADPH oxidase

PAHs

polycyclic aromatic hydrocarbons

PARP-1

poly(ADP-ribose)polymerase-1

PCNA

proliferating cell nuclear antigen

PTPs

protein tyrosine phosphatases

RNS

reactive nitrogen species

ROS

reactive oxygen species

SOD

superoxide dismutase

−SOH

sulfenic acid

−SS−

disulfide bond

UVR

ultraviolet radiation

WHO

World Health Organization