Characterization of global transcriptional responses and DNA repair following aflatoxin B(1) treatment in Saccharomyces cerevisiae.

摘要

Aflatoxin B1 (AFB1) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, it is bioactivated by cytochrome P450 (CYP450) enzymes, primarily hCYP1A2, to a genotoxic epoxide that forms N7-guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB 1, the YHE2 strain of S. cerevisiae engineered to express hCYP1A2, was exposed to dose and time-dependent AFB1 treatment resulting in minimal cytolethality, but substantial genotoxicity. Flow cytometric analysis revealed dose and time dependence of S-phase delay under the same treatments. Replicate cDNA microarray analyses were performed for each experimental point. The significance analysis of expression array data demonstrated the complexity of the transcriptional profile in response to AFB1 exposure. Among the findings, environmental stress response genes (Gasch, Spellman et al. 2000) were underrepresented in the study. The DNA damage signature detected in the study overlapped with that reported in a previous study with other DNA-damaging agents (Gasch, Huang et al. 2001), suggesting activation of DNA damage response pathway by common DNA structure. A rapid and coordinated repression was also shown in the histone genes as well as a large proportion of M phase and daughter-specific transcripts, suggesting a checkpoint-dependent transcriptional pathway that targets these genes for repression. A series of yeast haploid mutants defective in DNA repair and cell cycle checkpoints were transformed with hCYP1A2 to investigate how bulky AFB-DNA adducts are repaired. The stability of hCYP1A2 expression in these mutants was characterized by Western blots and Methoxyresorufin O-demethylase (MROD) enzymatic activity. Mutant clones with hCYP1A2 activity comparable to the wild-type strain were treated with various doses of AFB1. Colony-forming ability and the L-Canavanine resistance forward mutation assay were employed to measure cell survival and mutation, respectively. The data suggested the involvement of nucleotide excision repair, postreplication repair and checkpoints in the repair/tolerance of AFB1-induced DNA damage in the yeast model. Furthermore, Rad51-mediated homologous recombination repair is found to play a key role in the error-free repair of AFB1-induced DNA lesions, and there exist unique roles for checkpoints and AP endonucleases-dependent DNA intermediates in mediating AFB1-induced mutagenicity.