Food borne bacterial models for detection of benzo[a]pyrene-DNA adducts formation using RAPD-PCR

Abstract

Random amplified polymorphic DNA (RAPD) PCR is a feasible method to evaluate genotoxin-induced DNA damage and mutations. In this study, Lactobacillus plantarum ATCC 14917T, Enterococcus faecium DSMZ 20477T, Escherichia coli PQ37 and Saccharomyces cerevisiae S441 were screened for DNA genetic alterations by DNA fingerprinting using M13 and LA1 primers after treatment with three compounds forming covalent adducts with DNA [benzo[a]pyrenediol epoxide (BPDE), methyl methanesulfonate and 1,2,3,4-diepoxybutane (DEB)]. M13 RAPD fingerprinting revealed that the total number of bands decreased in all treated DNA compared to control samples and generally the lost bands were characterized by high molecular weight. Some extra bands were detected for L. plantarum and E. faecium, while in E. coli and S. cerevisiae DNAs BPDE and DEB treatments did not result in new extra bands. Besides qualitatively analysis, cluster analysis based on Unweighted Pair-Group Method with Average algorithm was performed to compare DNA fingerprints before and after treatments. This analysis confirmed the absence of significant differences between negative controls and treated DNA in S. cerevisiae and E. coli however the disappearance of some bands can be detected. The data indicate that this approach can be used for DNA damage detection and mutations induced by genotoxic compounds and highlighted the possible use of L. plantarum and E. faecium M13 based fingerprinting as reference for hazard identification in risk assessment.