Peroxisome proliferators (PPs)-induced DNA hypomethylation has been proposed as a mechanism of their toxicity, including carcinogenic action. The effect of di-butyl phthalate (DBP), a known peroxisome proliferators, on the methylation level of the c-myc promoter region in rat liver was studied. Changes in the methylation status of the c-myc gene were correlated with changes in DNA synthesis, DNA methyltransferase (DNMTs) activity and liver weight. Male Wistar rats received DBP in one, three or fourteen daily oral doses of 1800 mg/kg body weight (b.w.) x day(-1) (this dose is close to the dose that increases the numbers of peroxisomes in male Wistar rats). We have demonstrated that DBP decreased the methylation of the c-myc gene. Cytosine hypomethylation in the analyzed CpG sites of the c-myc gene promoter occurred during the whole period of study, although after 14 doses of DBP the difference from control was only on the borderline of significance (p = 0.066). An increase in DNA synthesis was only observed after 24 hours of treatment with DBP, and it preceded liver growth. We hypothesize that DBP-induced demethylation of the c-myc gene was an active mechanism, not associated with DNMTs activity and DNA replication.
We investigated the relationship between dibutyl phthalate (DBP)-induced hypomethylation of the c-Myc promoter region (as evident in our early study) and the expression of c-Myc and DNMT1 genes (at messenger RNA (mRNA) and protein level) in the rat liver. Male Wistar rats received DBP in 1, 3, or 14 daily doses of 1800 mg kg(-1) body weight. Levels of DNMT1, c-Myc mRNA, and proteins were detected using real-time polymerase chain reaction and Western blot analysis, respectively. Our findings indicate that DBP caused an increase in mRNA levels of c-Myc at all time points. The results showed that protein levels of c-Myc in rat liver also increased significantly by DBP treatment, which were more pronounced at last time point (after 14 doses). Furthermore, overexpression of DNMT1gene have been found after one dose of DBP, which was confirmed at the protein level by Western blot analysis. Reduced levels of DNMT1mRNA and proteins (3 and 14 doses) were coordinated with depletion DNA synthesis (reported previously). Based on our previous results and those presented here, the following conclusion could be drawn: (1) DBP exerted biological activity through epigenetic modulation of c-Myc gene expression; (2) it seems possible that DBP-induced active demethylation of c-Myc gene through mechanism(s) linked to generation of reactive oxygen species by activated c-Myc; and (3) control of DNA replication was not directly dependent on c-Myc transcriptional activity and we attribute this finding to DNMT1gene expression which was tightly coordinated with DNA synthesis.
The aim of the study was to investigate the early effect of acute dichlorodiphenyltrichloroethane (DDT) exposure on the methylation status of the promoter region of two tumor suppressor genes: p53 and p16(INK4a) (p16) in rat liver. We analyzed their transcript and protein expression profiles concurrently with the examination of transcriptional and protein expression levels of DNA (cytosine-5)-methyltransferase 1 (Dnmt1). Male Wistar rats were treated with a single dose of DDT (57 mg kg(-1) of body weight) and the methylation status of p53 and p16 genes was examined after 24 h using methylation-sensitive restriction analysis-MSRA. The obtained results indicate that DDT induced alternations in methylation of the promoter region in both p53 and p16 genes. In all the tested samples, the promoter CpG islands of p53 (-261, -179, and -450) were methylated within 100% as compared to control samples (0%). The methylation status of the p16 promoter (-11 and +77) was also altered due to exposure to DDT. Methylated cytosines were detectable in 75% of the tested DNA samples. The Real-time PCR and western blot analyses showed a decrease in mRNA and protein levels of p53, respectively, which was related to the increase in DNA synthesis. These relationships were also observed for mRNA and protein expressions of p16, although to a slighter extent. We also showed that hypermethylation in the promoter region of both tumor suppressor genes was consistent with an increased Dnmt1 mRNA level, and this relationship was further confirmed at the protein level of DNMT1. Concluding, our data suggests that epigenetically mediated changes in gene expression may play an important role in the mechanism of DDT toxicity, including carcinogenic action.
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