[Molecular mechanism of mutagenesis induced by aflatoxin B1 using a shuttle vector pSP189/mammalian cell system]

AFB1 is one of the most potent carcinogenic mycotoxins, naturally occurred in foods. We applied a new SV40-based shuttle vector pSP189 and African Green kidney cells (VeroE6 cell line), which constitute shuttle vector/mammalian cell system to detect mutagenesis induced by AFB1 and to study the effects of AFB1 on the mutation site, type and sequence specificity of DNA molecular level through sequence analysis of pSP189 target gene SupF TRNA. The results indicated that through various time treatment of AFB1 and rat liver microsome, the mutants were obtained by transformation of E. Coli MBM 7070 with progeny of pSP189 generated during replication in veroE6 cells, which increased gradually with the lapse of time, and this experiment showed a significant dose-response relationship. Most detected mutants were point mutations evidenced by agarose gel electrophoresis analysis. The results of direct sequencing SupF TRNA of 53 independent mutants showed that most mutations (about 84.9%) were single base substitutions, among which 95.2% of base substitutions happened on the site of G:C base pair, the predominant mutation was G:C-->T:A transversion, accounting for 53.3%, followed by G:C-->A:T transition, accounting for 35.6%. AFB1-induced mutations did not distributed randomly, and had mutation hot spots and sequence specificity, which contained 5 base pair sequence 5'-NNTTC-3'. The mutations on the SupF shuttle vector were consistent with the results from studies of oncogene tumor suppressor genes and DNA adduct.