Epigenetic Modifications of Dapk and P16 Genes Contribute to Arsenic-induced Skin Lesions and Nondermatological Health Effects

Over 26 million people in West Bengal, India, are exposed to very high levels of arsenic through drinking water, leading to several deleterious endpoints including cancers. To elucidate the role of promoter methylation in arsenic-induced dermato-logical and nondermatological health effects, methylation status of p16 and DAPK genes was determined. A case-control study was conducted involving 72 individuals with arsenic-induced skin lesions (cases) and 50 individuals without skin lesions (controls), having similar arsenic exposure through drinking water. Methylation status was determined by bisulfite conversion of genomic DNA and methylation-specific PCR. Expression of the genes was determined by real-time PCR and Western blot analysis. Associations between the promoter methylation status and nondermatological health effects were determined from epidemiological survey data. Significant hypermethylation was found in the promoters of both DAPK and p16 genes in the cases compared with the controls resulting in downregulation of both the genes in the cases. There was a 3.4-fold decrease in the expression of death-associated protein kinase and 2.2-fold decrease in gene expression of p16 in the cases compared to the controls, the lowest expression being in the cancer tissues. Promoter hyper-methylation of the genes was also associated with higher risk of developing arsenic-induced skin lesions, peripheral neuropa-thy, ocular and respiratory diseases. This study for the first time makes an attempt to correlate epigenetic modifications of the tumor suppressor genes with dermatological and nondermato-logical health outcomes in a population chronically exposed to arsenic. Arsenic is a potent human carcinogen, which affects millions of people in more than 70 countries around the world. At present , more than 137 million people of the world are affected by drinking heavily contaminated ground water, in which arsenic content exceeds much above the maximum permissible limit of 10 µg/l, laid down by World Health Organization (WHO). In India, West Bengal is the worst affected state where more than 26 million people (Chakraborti et al., 2009) are exposed to arsenic by drinking heavily contaminated ground water. Chronic arsenic exposure has resulted in various hazardous health outcomes including noncancerous (raindrop pigmentation and hyperpigmentation), precancerous (palmer, planter, and palmo-planter hyperkeratosis), and cancerous skin lesions (basal cell carcinoma [BCC], squamous cell carcinoma [SCC], and Bowen's diseases [BD]). It also causes cancers of liver, kidney, bladder, and other internal organs (Guha Mazumder, 2001). Although arsenic-induced skin lesions are considered as hallmarks of chronic arsenic toxicity, only 15–20% of the total population show arsenic-induced skin lesions and …

Arsenic is a potent human carcinogen, which affects millions of people in more than 70 countries around the world.At present, more than 137 million people of the world are affected by drinking heavily contaminated ground water, in which arsenic content exceeds much above the maximum permissible limit of 10 µg/l, laid down by World Health Organization (WHO).In India, West Bengal is the worst affected state where more than 26 million people (Chakraborti et al., 2009) are exposed to arsenic by drinking heavily contaminated ground water.Chronic arsenic exposure has resulted in various hazardous health outcomes including noncancerous (raindrop pigmentation and hyperpigmentation), precancerous (palmer, planter, and palmo-planter hyperkeratosis), and cancerous skin lesions (basal cell carcinoma [BCC], squamous cell carcinoma [SCC], and Bowen's diseases [BD]).It also causes cancers of liver, kidney, bladder, and other internal organs (Guha Mazumder, 2001).Although arsenic-induced skin lesions are considered as hallmarks of chronic arsenic toxicity, only 15-20% of the total population show arsenic-induced skin lesions and the rest do not (Banerjee et al., 2011), indicating that genetic variations might play an important role in arsenic susceptibility, toxicity, and carcinogenicity.
DNA methylation is an epigenetic modification of DNA that is tightly regulated in mammalian development and is essential for maintaining the normal functioning of the adult organism (Schaefer et al., 2007).Altered DNA methylation patterns have been associated with multiple human diseases (Robertson, 2005).Global genomic DNA hypomethylation is a hallmark of many types of cancers (Esteller et al., 2001), resulting in illegitimate recombination events and causing transcriptional dysregulation of affected genes (Robertson, 2005).Aberrant CpG methylation of the promoter regions has also been associated with pathogenesis of a number of diseases including cancers (Koyama et al., 2003;Watanabe et al., 2008).
p16 is a tumor suppressor gene that causes growth arrest at G1/S phase by binding to the cyclin-dependent kinase 4-6/ cyclin D complex thereby inhibiting phosphorylation of retinoblastoma protein (pRb) (Serrano et al., 1996).The unphosphorylated pRb specifically binds to the E2F family of transcription factors, thus inhibiting cell cycle progression (Lukas et al., 1996).Death-associated protein kinase (DAPK) is a serine/ threonine protein kinase that contributes to tumor suppression, promotion of apoptosis via Fas ligand-TNF-α, and detachmentinduced cell death (Tyler et al., 2003).
Several hypotheses have been associated with arsenicinduced carcinogenesis including chromosomal abnormalities, oxidative stress, altered DNA repair, p53 gene suppression, gene amplification, transformation, and altered growth factors leading to increased cell proliferation and promotion of carcinogenesis (Hughes, 2002;Kitchin, 2001).In addition to that, it has been hypothesized that altered DNA methylation patterns might contribute to arsenic-induced carcinogenesis.Again, promoter hypermethylation of DAPK and p16 genes with resultant gene inactivation and development of different types of cancers (Lee et al., 2002;Maruyama et al., 2002;Rosas et al., 2001;Tada et al., 2002;Tyler et al., 2003) has been reported previously.So in the present study, our aim was to find out whether DNA methylation patterns in the promoter regions of tumor suppressor p16 and DAPK genes have any role to play in causing arsenic-induced precancerous and cancerous skin lesions.Our results have been compared with a group of individuals exposed to similar levels of arsenic through drinking water but who did not develop any arsenic-induced skin lesions (controls).During our epidemiological survey, we have found that conjunctival irritations of the eyes, peripheral neuropathy, and respiratory distress (nondermatological health effects) have been occurring very frequently in our study population (Banerjee et al., 2011).So, we have also made an attempt to find whether epigenetic modifications of the above-mentioned genes have any association with the non-dermatological health effects of the study population as well.

MATERIAlS AND METhODS
Study site and participants.A total of 122 individuals from highly arsenic-affected Murshidabad district of West Bengal were recruited for this study.The study population was divided into 72 individuals with arsenicinduced skin lesions (cases) and 50 individuals with no skin lesions (controls), both groups having similar arsenic exposure through drinking water.The arsenic content in their drinking water was much above the permissible limits laid down by WHO (10 µg/l).The study subjects were matched with respect to age, sex, and socioeconomic status.Individuals ranging from 18 to 60 years of age with at least 10 years of arsenic exposure were selected as arsenic-exposed study participants.Occupationally, majority of the study participants were farmers and household workers.The exposed individuals were chosen on the basis of the arsenic content in urine samples (for both skin lesion and no skin lesion groups) and arsenic-induced skin lesions because skin lesions are hallmarks of chronic arsenic toxicity (for skin lesion group).An interview was performed based on a structured questionnaire that elicited information about demographic factors, lifestyle, occupation, diet, smoking, medical, and residential histories (Ghosh et al., 2007).An expert dermatologist with 15 years of experience identified the characteristic arsenic-induced skin lesions and helped in the recruitment of exposed study participants.He also confirmed that the control group had no arsenic-induced skin lesions.Then specialists in the fields of ophthalmology, neurology, and respiratory diseases examined each participant to diagnose nondermatological health effects in them.Some of the individuals with cancerous skin lesions were requested to attend a surgical camp, where the surgeons collected the cancerous skin tissues and peripheral unaffected tissues.Fifteen cancer tissue samples and peripheral tissues from the individuals with skin lesions were used in the present study.Cancerous tissues consisted of BD, SCC, and BCC.Urine, water, and blood samples were collected only from those subjects who provided informed consent to participate in the study.This study was conducted in accord with the Helsinki II Declaration and approved by the Institutional Ethics Committee of CSIR-Indian Institute of Chemical Biology.
Arsenic estimation in water and urine samples.Study participants were provided with acid-washed (nitric acid-water [1:1]) polypropylene bottles for collection of drinking water (Das et al., 1995).First morning voids (approximately 100 ml) were collected in precoded polypropylene bottles for arsenic estimation as these give the best measure of the recent arsenic exposure (Buchet et al., 1981).Immediately after collection, the samples were stored in salt-ice mixture and brought to the laboratory where they were kept at −20°C, until estimation of arsenic was carried out.The urine samples were filtered and diluted with deionized water as required and were then quantified for arsenic using a mixture of trivalent, pentavalent arsenic, monomethyl arsenic acid, and dimethyl arsenic acid as the standard.Concentration of arsenic in the samples was determined from the standard curve obtained.Freezedried urine standard (certified value: 0.137 ± 0.011 mg/l; NIES CRM No. 18) from the National Institute of Standards and Technology was used to calibrate the instrument for estimation of arsenic in urine.Arsenic contained in water samples was reduced to trivalent forms of arsenic by adding 1 ml each of 1% potassium iodide (Merk, India), 1% ascorbic acid (Rankem, India), and concentrated hydrochloric acid (Merk, India) to 7 ml of diluted (1 ml of original sample + 6 ml of deionized water) water sample.The samples were then mixed well and allowed to stand at room temperature for 45 min for complete reduction to trivalent form and were then analyzed in the atomic absorption spectrometer (AAS) instrument.Water samples were then quantified for arsenic using trivalent arsenic solution as the standard.Standard reference material for water (Soft drinking water UK-ERMCA022a, Middlesex, UK) was used to calibrate the instrument and as standard reference.Arsenic measurement was done employing the Perkin-Elmer Model-Analyst 700 (Boston, MA) spectrometer equipped with a Hewlett-Packard (Houston, TX) Vectra computer with GEM software (Perkin-Elmer EDL System-2) and arsenic lamp (lamp current 380 mA).

Collection of blood samples and isolation of peripheral blood mononuclear cells.
Blood was drawn by vein-puncture method, collected in EDTA-vacutainer tubes, and immediately put on ice.Blood samples were brought to the laboratory within 2 h after collection and the subsequent works were done.Peripheral blood mononuclear cells were obtained from EDTA-blood by density gradient centrifugation using Histopaq (Himedia, India).Cell viability was determined by trypan blue dye exclusion and was always greater than 98% (Banerjee et al., 2008).
Isolation of genomic DNA.Genomic DNA was isolated from fresh whole blood using salting-out method (Johns and Paulus-Thomas, 1989) with sodium perchlorate followed by isopropanol precipitation.The isolated DNA was dissolved in 100-200 μl of Tris-EDTA acid buffer (pH 8.0).DNA was isolated from 15 cancerous tissue samples and unaffected peripheral tissues from the patients with BD, BCC, and SCC using DNeasy Blood and Tissue Kit (Qiagen GmbH, Germany), following manufacturers' instructions.Optical density (OD) was measured in a spectrophotometer (OD 260/280 ) and run in 1% agarose gel.

Bisulfite conversion of DNA.
Methylation conversion of genomic DNA has been done using the EpiTect Bisulfite Kit (Qiagen GmbH, Germany) following manufacturer's instructions.Briefly, the procedure consisted of bisulfite-mediated conversion of unmethylated cytosine residues in genomic DNA to uracil and conservation of methylated cytosines as cytosines.This converted DNA was used for methylation-specific PCR (MSP), based on specially designed methylation-specific primers.

Methylation-specific PCR.
A highly specific and sensitive MSP was carried out to study the promoter methylation status in DAPK and p16 genes as previously described by Rosas et al. (2001).Briefly, the principle is as follows: Bisulfite modification of DNA converted unmethylated cytosine to uracil, whereas the methylated cytosine remains unaltered.The modified DNA was then subjected to PCR amplification using methylation-and unmethylation-specific primers to discriminate between methylated and unmethylated DNA, taking advantage of sequence difference that results from bisulfite modification.To amplify the chosen regions, PCR was performed in a 20-μl reaction volume using Hotstar PCR master mix (Qiagen GmbH, Germany) containing 2 μl of bisulfite-treated DNA.Primers for determination of methylated or unmethylated DAPK and p16 alleles have been described elsewhere (Jabłonowski et al., 2011;Rosas et al., 2001;Tyler et al., 2003).PCR conditions for p16 were as follows: initial denaturation and hotstart at 95°C for 15 min, then 40 cycles consisting of 30 s at 95°C, 30 s at 60°C for unmethylated or 65°C for methylated reaction, and 1 min at 72°C.All the primers were bought from Xcleris Labs Pvt.Ltd (Ahmedabad, India).Cycling was performed in Eppendorf Mastercycler (Hamburg, Germany) as follows for DAPK: initial denaturation and hotstart at 95°C for 15 min, then 40 cycles consisting of 30 s at 95°C, 30 s at 60°C for unmethylated or 65°C for methylated reaction, and 1 min at 72°C.All PCR products were analyzed by 8% acrylamide gel electrophoresis, stained with ethidium bromide, and photographed under ultraviolet light.

Isolation of RNA.
RNA was isolated by TRIzol reagent (Invitrogen) from peripheral blood mononuclear cells and tissue samples according to manufacturer's instructions.The RNA was dissolved in RNAse-free DEPC water (diethyl pyrocarbonate) and concentration was checked by measuring OD 260/280 in a spectrometer.

Complementary DNA conversion.
RNA was converted to complementary DNA (cDNA) using RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas Life Sciences) following manufacturers' instructions.Briefly, 2 µg RNA (after DNase I digestion [Takara]) was converted using random hexamer primers, reaction buffer, RNAse inhibitors, dNTP mix, and reverse transcriptase provided in the kit.Thermal cycling parameters were as follows: 60 min at 42°C, incubation for 5 min at 25°C followed by 60 min at 42°C, and termination by heating at 70°C.The cDNA was stored at −20°C till real-time PCR was done.

Gene expression analysis.
Quantitative real-time PCR was carried out on a Stratagene Mx3000P quantitative PCR system (Stratagene, La Jolla, CA).Real-time PCR was done using MESA GREEN qPCR Master MIX Plus SYBR assay I Low Rox Kit (Eurogentec) following manufacturers' instructions.The primers used were p16F: 5′-AAGGTCCCTCAGACATCCC-3′, p16R: 5′-TGGACATTTACGGTAGTGGG-3′, 18sF: 5′-CGGACAGGATTGACAGAT TGATAGC-3′, and 18sR: 5′-TGCCAGAGTCTCGTTCGTTATCG-3′.The primers used for DAPK were DAPKF: 5′-GGAGGATAGTTGGATTGAGTTAAT GTT-3′ and DAPKR: 5′-CAAATCCCTCCCAAACACCAA-3′.All reactions were performed in triplicate in a 20 µl total volume containing a 10 µl of the Mastermix provided in the kit, 1 µl of cDNA, and the sense and antisense primers at a final concentration of 100nM.Reactions were carried out in a Stratagene Mx3000P quantitative PCR system (Strategene) for 1 cycle at 94°C for 5 min; 40 cycles at 94°C for 30 s, 59°C for 30 s, and 72°C for 30 s; 1 cycle at 94°C for 1 min; and 1 cycle at 55°C for 30 s. Fluorescence values of Mesa Green dye, representing the amount of product amplified at that point in the reaction, were recorded in real time at both the annealing step and the extension step of each cycle.The cycle threshold (C t ), defined as the point at which the fluorescence signal was statistically significant above background, was calculated for each amplicon in each experimental sample by use of MxPro QPCR software.Gene expression was quantified by the comparative ΔΔCT method.The transcript level of each specific gene was normalized to 18s rRNA amplification as previously described (Hong et al., 2010).Postamplification melting curve analyses were performed to assure product specificity.

Isolation of protein.
Protein isolation from blood and tissue samples was done using TRIzol reagent (Invitrogen), according to manufacturer's instructions.The protein concentration was determined using Bradford assay, and 150 µg proteins were loaded for Western blot analysis.
Western blot analysis.Western blot analysis was done in the cases and controls as previously described (Banerjee et al., 2008).Aliquots of equal amounts of proteins were subjected to 12% SDS-PAGE.Thereafter, proteins were electrophoretically transferred to nitrocellulose membrane (Millipore) and nonspecific sites were blocked with 5% bovine serum albumin (Sigma-Aldrich, St Louis, MO) in 20mM Tris-buffered saline (pH 7.5) containing 1% Tween-20 and reacted with a primary monoclonal antibody (Calbiochem), anti-p16, anti-DAPK, or β-actin (1:1000) for 4 h at room temperature.After washing with Tris-buffered saline containing 0.1% Tween-20, the membrane was then incubated with alkaline phosphatase-conjugated goat anti-rabbit secondary or goat anti-mouse secondary antibodies (1:2000) as required.The protein bands were visualized using nitro-blue tetrazolium and 5-bromo-4-chloro-3'-indolyphosphate.Equal loading of the Western blots was determined with the loading control β-actin.Both bands from each blot were pooled to get one of the triplicate measurements.Image J was used for analysis of the band intensity of the blots.

Identification of nondermatological health effects in the study population.
Identification of the nondermatological health effects like conjunctival irritations of the eyes, peripheral neuropathy, and respiratory problems was done by expert physicians in the relevant field by the methods as described previously (Banerjee et al., 2011).

Statistical analyses.
Data are expressed as mean ± SD.Mann-Whitney test was done to test for significant differences in age, arsenic content in urine and water samples, and expression levels of DAPK and p16 between the two study groups.Chi-square test was employed to compare the distribution of gender, tobacco usage, and methylation status between the groups.Odds ratio (OR), 95% confidence intervals, and two-tailed p values were calculated for assessing the risk of the development of skin lesions and health effects associated with methylation status of the two genes.Microsoft Excel and Graph Pad Instat3 (San Diego, CA) software were used for the purpose.

Demographic Characteristics of the Study Participants
Seventy-two arsenic-exposed individuals with precancerous (palmer and planter hyperkeratosis) and cancerous skin lesions and 50 exposed individuals without skin lesions were recruited for this study.BD, SCC, and BCC were found in 19, 9, and 2 individuals, respectively.From the individuals with skin lesions, 15 cancerous tissue sections and peripheral unaffected tissues were randomly selected for this study.Rest of the exposed individuals with skin lesions had raindrop pigmentation and palmer or planter hyperkeratosis or both.Hyperkeratosis of skin is considered as a precursor of arsenic-induced skin cancer (Ahsan et al., 2003), as skin cancers often appear at the sites of existing hyperkeratosis (National Research Council, 1999).Figure 1 shows typical arsenic-induced noncancerous, precancerous, and cancerous skin lesions in the study participants.Descriptive characteristics of the study subjects are summarized in Table 1.The average age of controls and cases were 40.20 and 42.56 years, respectively.Majority of the male individuals are farmers and females are housewives in both the categories.There is no significant difference in the age or sex distribution patterns, tobacco usage status, and arsenic content in urine or water between the two groups.

Determination of Methylation Status in the Study Subjects
Representative gel pictures of MSP are shown in Figure 2. Results (Table 2) show significant hypermethylation (p < .001) in both DAPK and p16 gene promoters in the individuals with skin lesions compared with the individuals without skin lesions.It also shows that individuals with DAPK and p16 hypermethylation had higher risk of developing skin lesions as is predicted from the OR values.

Gene Expression Study by Real-time PCR
Gene expression data are shown in Figure 3. DAPK at the mRNA levels was about 3.4-fold times downregulated in the individuals with skin lesions compared with the individuals with no skin lesions.Again, p16 at the mRNA level was about 2.2-fold   downregulated in the individuals with skin lesions compared with the individuals with no skin lesions.Interestingly, in the cancerous tissues of the patients, DAPK and p16 were about eight-and sevenfold downregulated compared with the no skin lesion controls, respectively.The expression was about 2-and 1.7-fold lower in the cancerous tissues compared with the peripheral unaffected tissues for DAPK and p16, respectively (data not shown in the figure).

Western Blot Analysis
The results of Western blot analysis are shown in Figure 4 where we find that there is downregulation of both p16 and DAPK proteins in cases compared with the controls.Both DAPK and p16 proteins were downregulated in the order: exposed individuals with no skin lesions > exposed individuals with skin lesions ~ peripheral unaffected tissues > cancer tissues.Equal loading of the proteins was determined from the loading control β-actin.

Association of Promoter Methylation Status With Nondermatological Health Effects
The results of association of promoter hypermethylation status with nondermatological health effects are shown in  Tables 3 and 4. Individuals with DAPK promoter hypermethylation had higher risk of developing conjunctival irritation of the eyes (OR: 2.46 [1.17-5.20]),peripheral neuropathy (OR: 2.43 [1.66-5.12]),and respiratory problems (OR: 3.07 [1.20-7.84])compared with the individuals who do not have such hypermethylation.Similarly, individuals with p16 promoter hypermethylation had higher risk of developing peripheral neuropathy .81])compared with the individuals lacking such hypermethylation.Neither eye nor respiratory problems showed any association with p16 promoter hypermethylation.

DISCuSSION
Chronic arsenic exposure has been associated with various deleterious endpoints including cancers.Although a number of mechanisms have been thought to contribute to arsenicinduced carcinogenesis, but none of the mechanisms have been clearly understood.DNA methylation has been hypothesized to be an important contributor in number of cancers.So in the present study, we wanted to find out whether promoter methylation patterns of two tumor suppressor genes p16 and DAPK have any role to play in inducing arsenic-induced precancerous and cancerous skin lesions in the chronically exposed population of West Bengal.We have also investigated the effect of aberrant promoter methylation patterns on commonly observed nondermatological health effects in these study subjects.
Aberrant DNA methylation patterns of CpG islands by DNA methylases have been shown to be associated with gene inactivation and play vital roles in the development of cancers.Hypoand hypermethylation of genes could mediate carcinogenesis through upregulation of oncogene expression or downregulation of tumor suppressor gene expression, respectively (Chanda et al., 2006;Chen et al., 2007).In this context, it is worthwhile to discuss that there might be an association between arsenicinduced toxicity and DNA methylation because arsenic metabolism and DNA methylation both utilize the same methyl donor, S-adenosyl methionine (SAM).Arsenic metabolism is characterized by methylation of arsenic as SAM is the unique methyl group donor in each conversion step of biomethylation resulting in the production of monomethylarsonic acid and dimethylarsinic acid.The same SAM is utilized in DNA methylation process to convert cytosine to 5-methyl cytosine catalyzed by DNA methyl transferase.Thus, long-term exposure to arsenic may lead to SAM insufficiency and global DNA hypomethylation (Goering et al., 1999;Sciandrello et al., 2004).In contrast to this, it has been found that, in a cross-sectional study, exposure to arsenic contaminated water was associated with a global DNA hypermethylation (Majumdar et al., 2010) although the participants in the highest estimated exposure group had methylation levels  that were comparable with those in the two lowest groups.In another study by Pilsner et al. (2007), there was a positive association between urinary arsenic and DNA hypermethylation in 294 arsenic-exposed individuals.It has also been found that arsenic interferes with DNA methyl transferases resulting in inactivation of tumor suppressor genes through DNA hypermethylation (Goering et al., 1999).This contrasting behavior in methylation patterns may be due to the induction of cytosine methyltransferase, which leads to the hypermethylation of certain genes as a consequence (Chanda et al., 2006), the mechanism of which is not clearly understood till now.Thus, mechanism of DNA hypermethylation after arsenic exposure remains unclear.
Hypermethylation of promoters of both p16 and DAPK genes has previously been associated with a number of tumor types and cancers as mentioned above.In our study, we have found hypermethylation in the promoter regions of both p16 and DAPK genes, resulting in downregulation of p16 and DAPK in the arsenic-exposed people with arsenic-induced precancerous and cancerous skin lesions, compared with the individuals without skin lesions and the lowest expression being in the cancerous tissues.Also, the expression of these proteins is lower in the cancerous tissues compared with unaffected tissue mass of the same individual.Thus, our results indicate a reciprocal association between downregulation of p16 and DAPK with arsenicinduced skin lesions.Because p16 and DAPK are normally associated with cell cycle arrest at G1/S phase and gamma interferon-induced programmed cell death, respectively (Tyler et al., 2003), their epigenetic silencing would render the cells immortal, leading to carcinogenesis.This might also throw some light on the fact that out of 26 million people exposed to arsenic, only 15-20% show arsenic-induced skin lesions and not the rest.Till date, it was thought that only genetic variations like polymorphisms might contribute to the phenotypic difference between the two groups (Banerjee et al., 2011, Kundu et al., 2011), but our results indicate that epigenetic silencing of these particular genes could also contribute to the phenotypic expression of the skin lesions in the cases and not in the controls.Thus, in addition to the genetic variations, the epigenetic modifications might act as biomarkers in identifying arsenic susceptibility; larger cohort-based studies might establish the fact.However, it may  Notes.CI, confidence interval.Bold indicates that p16 hypermethyaltion is associated with higher risk of developing peripheral neuropathy only.
be speculated that if methylation status is somehow disrupted in them in the future, then the individuals without skin lesions might develop cancerous lesions as well.Future research might throw some light in this respect.Our results are in accordance with several previous findings in human populations chronically exposed to arsenic (Chanda et al., 2006;Chen et al., 2007) and also in in vitro study (Chai et al., 2007) with arsenic salts.However, all these studies did not throw any light on the effect of promoter hypermethylation on their respective gene expression.Here, we report for the first time that hypermethylation in the p16 and DAPK gene promoters leads to downregulation of the two genes, and p16 and DAPK genes are important contenders in contributing to arsenic-induced precancerous and cancerous skin lesions.
Here, we have also found the association of the commonly occurring nondermatological health effects with promoter methylation status of the study subjects.Hypermethylation in the DAPK gene was associated with increased risk of conjunctival irritations of the eyes, peripheral neuropathy, and respiratory diseases, whereas hypermethylation of p16 genes was associated with the risk of developing peripheral neuropathy only.Results indicate that promoter methylation in DAPK and p16 genes is a good indicator for risk assessment of dermatological and nondermatological health effects in the arsenic-exposed population.Previously, hypermethylation in CpG sites of DAPK has been strongly correlated with ocular adnexal MALT lymphomagenesis in South Korea (Choung et al., 2012) and RB1/p16 INK4a gene pair displayed aberrant methylated alleles in 15% of cases in type 2-associated schwannomas (Gonzalez-Gomez et al., 2003).Again, hypermethylation of both DAPK and p16 genes was found in patients with lung cancers when compared with the patients with nonmalignant pulmonary diseases (Fujiwara et al., 2005).Because DAPK is known to have anti-inflammatory effects (Nakav et al., 2012), the decreased production of DAPK results in the inflammation of the skin, eyes, myelin sheath of the nerves, and respiratory tracts and contributes to the precancerous and cancerous skin lesions, conjunctival irritations, peripheral neuropathy, and respiratory distress in the cases of our study population.The effect of epigenetic silencing of p16 is not so pronounced (even if p16 is known to have anti-inflammatory effect; Murakami et al., 2012) in predicting the risk of eye and respiratory problems in our study population.This may be due to the fact that the diseases mentioned are complex diseases and are influenced by a number of gene products and their associations with different disease outcomes might be as a result of interactions with other genes and environmental factors.So it might be said that epigenetic modifications of the particular genes may be one of the contributors and not the sole contributor in contributing to the disease states in the arsenic-exposed people and might act as epigenetic biomarkers in predicting the risk of arsenic-induced dermatological and nondermatological health consequences.At this point, it is worthwhile to mention that because cancer progression is linked to inflammation (Coussens et al., 2002), the epigenetic silencing of DAPK and p16 genes provides an explanation for the formation of precancerous and cancerous lesions in the skin of cases only and not in the controls.This is also confirmed by the fact that the epigenetic silencing is highest in the cancer tissues where inflammatory conditions are very much pronounced.

CONCluSION
In conclusion, we might say that DNA hypermethylation in promoter of the tumor suppressor DAPK and p16 genes are responsible for their epigenetic silencing, which might lead to several dermatological and nondermatological health effects in the chronically arsenic-exposed population of West Bengal.Hence, reversal of DNA methylation at these sites may be a potential therapeutic strategy as this reversal may restore expression of these transcriptionally regulated genes.

FIg. 2 .
FIg. 2. Representative gel pictures showing methylation patterns in the study subjects.L: ladder, B: blank lane, and U and M: bands of PCR using unmethylated (U) or methylated (M) MSP primer sets for p16 and DAPK genes.

FIg. 3 .
FIg. 3. Comparison of gene expression in the study groups.Figure (A) shows fold change in gene expression of DAPK in different study groups and figure (B) shows fold change in gene expression of p16 in different study groups.Wosl = without skin lesions; Wsl = with skin lesions; Can tissue = cancer tissue.

FIg. 4 .
FIg. 4. Comparison of protein levels in the study groups.Figure (A) shows representative Western blots.Relative intensity of each band after normalization with the intensity of β-actin in a blot (below each Western blot) was measured.Figure (B) shows fold change in protein expression levels as measured by Image J software.*p < .001for Mann-Whitney test when compared with the no skin lesion group.**p < .001for one-way ANOVA with Tukey-Kramer post test when compared with the no skin lesion group.Each experiment has been repeated thrice.
FIg. 4. Comparison of protein levels in the study groups.Figure (A) shows representative Western blots.Relative intensity of each band after normalization with the intensity of β-actin in a blot (below each Western blot) was measured.Figure (B) shows fold change in protein expression levels as measured by Image J software.*p < .001for Mann-Whitney test when compared with the no skin lesion group.**p < .001for one-way ANOVA with Tukey-Kramer post test when compared with the no skin lesion group.Each experiment has been repeated thrice.

TABlE 3 Association of Aberrant Promoter Methylation in DAPK With Nondermatological health Effects
Notes.CI, confidence interval.Bold indicates that DAPK hypermethylation is associated with higher risk of developing all three non-dermatological health effects.