Mutagenesis Of Alternariol On DNA Polymerase Beta In NIH3T3 Cell Line

Objective Alternariol(AOH) is a toxin of mildew isolated from the corn in Lin county,where is the region with a high risk of esophageal carcinoma. Recent evidences showed that AOH may involved in esophageal carcinoma.DNA polymeraseβ(pol β), a member of DNA polymerase family, is primary enzyme involved in chemically induced DNA repair, such as base excision repair(BER). In 2002, Dong et al first reported that pol β not only was highly expressed in tumor tissue but also mutation of the enzyme associated with esophageal carcinoma.In present study, mutagenesis of alternariol on DNA polymeraseβin NIH3T3 cell line were investigated by using of gene analysis.Methods Cultured cells were treated with different concentration of AOH. The concentration used in experiment was 2μmol/L,4μmol/L, 8μmol/L and 16μmol/L respectively. Non treated cells selected as control. After extraction of total RNA from the cells, DNA polpgene was amplified through RT-PCR and then it was inserted into pGEM-T easy vector. To get expression of pol β gene, JM109 Escherichia coli cells were transected with the vector. Analysis of DNA sequence has been done by DNA sequencer. The amount of polβ expression were compared with P-actin. Results The gene sequence from control and 2μmol/L group well matched with GenBank, but two kinds of point mutation were found among every other 3 groups. In those groups that happened to have mutations,a same point mutation(T→C) was shared at 115nt(Tyr-His) while a T→C mutation at 532nt ( Cys-Arg),a T→C mutationat206nt (Ile-Thr) as well as a A→T mutation at 351nt (Glu-Asp) were found in 4μmol/L,8μmol/L and 16μmol/Lgroup respectively . As compared with β-actin, amount of polβgene expression was increased with increasing concentration of AOH. Conclusion The results mentioned above suggested that AOH capable of point mutation in polβ gene. The mechanism underlying cancer induced by AOH might associated with the mutation of polβ gene.