DNA Hypermethylation of Promoter of Gene p53 and p16 in Arsenic-Exposed People with and without Malignancy

Sarmishtha Chanda 1 Uma B. Dasgupta 1 Debendranath GuhaMazumder 0 Mausumi Gupta 1 Utpal Chaudhuri 1 Sarbari Lahiri 0 Subhankar Das 0 Nilima Ghosh 0 Debdutta Chatterjee 0 0 Institute of Post Graduate Medical Education and Research (IPGME & R) , Kolkata- 700020 , India 1 Department of Biophysics, Molecular Biology and Genetics, University of Calcutta , 92, APC Road, Kolkata- 700009 , India Chronic arsenic exposure is known to produce arsenicosis and cancer. To ascertain whether perturbation of methylation plays a role in such carcinogenesis, the degree of methylation of p53 and p16 gene in DNA obtained from blood samples of people chronically exposed to arsenic and skin cancer subjects was studied. Methylation-specific restriction endonuclease digestion followed by polymerase chain reaction (PCR) of gene p53 and bisulfite treatment followed by methylation-sensitive PCR of gene p16 have been carried out to analyze the methylation status of the samples studied. Significant DNA hypermethylation of promoter region of p53 gene was observed in DNA of arsenic-exposed people compared to control subjects. This hypermethylation showed a dose-response relationship. Further, hypermethylation of p53 gene was also observed in arsenic-induced skin cancer patients compared to subjects having skin cancer unrelated to arsenic, though not at significant level. However, a small subgroup of cases showed hypomethylation with high arsenic exposure. Significant hypermethylation of gene p16 was also observed in cases of arsenicosis exposed to high level of arsenic. In man, arsenic has the ability to alter DNA methylation patterns in gene p53 and p16, which are important in carcinogenesis. - Environmental exposure to inorganic arsenic is a major health hazard in many countries. West Bengal in India is one of the most affected areas in the world, as large number of people drink arsenic-contaminated subsoil water. Health effects due to chronic arsenic toxicity include various skin manifestations like pigmentation, keratosis, and systemic diseases like lung and liver disease, neuropathy, vascular abnormalities, and skin, bladder, and lung cancer (GuhaMazumder, 2001; GuhaMazumder et al., 1998; National Research Council, 1999). From epidemiological study in West Bengal, a clear dose-response re The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration. 1 To whom correspondence should be addressed. E-mail: . lationship between the prevalence of skin lesions and level of arsenic in water was observed (GuhaMazumder et al., 1998). Though the elevated risk of cancer associated with chronic arsenic exposure is well established (IARC, 2004; Waalkes, 1995), the mechanism of carcinogenesis is not clear. Arsenic is a poor mutagen (Jacobson and Moltalbano, 1985; Lee et al., 1985; Rossman et al., 1980) and does not induce significant point mutation. However, its effect on DNA repair, sister chromatid exchange, aneuploidy, and micronuclei development has been documented (IARC, 2004). After entering the body, inorganic arsenic (As) is methylated to monomethyl arsonic acid (MMA) and dimethylarsinic acid (DMA) and excreted through urine. This biotransformation is effected by the methyltransferase enzyme (Cyt19), using Sadenosylmethionine (SAM) as the methyl group donor, in presence of endogenous reductase (Lin et al., 2002; Hayakawa et al., 2005). Omega class of GST enzymes are involved in the reductase activity, and MMA(V) reductase is the same as GST O1-1 (Zakharyan et al., 2001). Arsenic methylation is presumed to occur at high level in liver, which is a suspected target organ for arsenic-induced carcinogenesis (Donohue and Abernathy, 2001; Goering et al., 1999; Vahter, 1990). However, expression of Cyt19 is higher in some other tissues like kidney or testis. It has been hypothesized that alteration of DNA methylation is involved in arsenic-induced carcinogenesis. This mechanism has been proposed because the SAM/methyltransferase pathway for biotransformation of arsenic overlaps with the DNA methylation pathway, in which donation of methyl groups from SAM to cytosine produces 5-methylcytosine in DNA. It is known that the regulation of expression of many genes is controlled by the extent of methylation of cytosine, often in long stretches of cytosine-rich sequences known as CpG islands, especially in the promoter regions. Various workers have tested the hypothesis of altered methylation on DNA extracted from cultured cell lines exposed to different doses of arsenic compounds. Mass and Wang (1997) demonstrated hypermethylation in the CpG island of p53 tumor suppressor gene in arsenic-exposed murine cell lines. Subsequently, both hyper- and hypomethylation of DNA have been demonstrated by Zhong and Mass (2001) using methyl-sensitive PCR on human lung and kidney cell lines. We report the results of a study on a cross-section of arsenicexposed population of West Bengal, India. Blood samples were collected from cases showing evidence of arsenicosis and arsenic-related cancer. Two tumor suppressor genes, p53 and p16, have been chosen for this study. p53 gene, known as guardian of the genome protects the genome against insults and induces growth arrest at G1 and apoptosis at G2. Gene p16 has also been included in this study, as it also induces growth arrest at G1/S in damaged cells by binding to CDK 4/6, thus working on the Retinoblastoma-1 genemediated pathway (Lewin, 2004). In one group of cases, promoter region of p53 has been studied using the methyl sensitive restriction enzyme HpaII. In another group, promoter region of p16 has been studied by bisulfite modification of the DNA followed by methyl-sensitive PCR analysis. Hypermethylation of promoter region of both the genes were observed in people suffering from arsenicosis. Also, promoter region of p53 gene was found to be hypermethylated in individuals with arsenic-induced skin cancer compared to those with skin cancer unrelated to arsenic exposure. MATERIALS AND METHODS Subjects. Subjects of this study were taken from the arsenic clinic of Institute of Post Graduate Medical Education and Research, Kolkata, India. The arsenic clinic is a tertiary referral center. All the cases recruited were referred cases from South 24 Parganas, one of the worst affected districts of West Bengal. Epidemiological studies were being carried out in this district since 1995 (GuhaMazumder et al., 1998; Haque et al., 2003). Arsenic exposure data of all these people were therefore known to the investigators. Criteria for diagnosis of arsenicosis were based on parameters described earlier (GuhaMazumder, 2001). Briefly these are as follows. (1) History of taking arsenic contaminated water (>50 lg/l) for more than 6 months. (2) Presence of characteristic skin manifestation of chronic arsenic toxicity: d HyperpigmentationIt may be diffuse, spotty or blotchy, scattered all over the bo (...truncated)