Expression of a Human Cytochrome P450 in Yeast Permits Analysis of Pathways for Response to and Repair of Aflatoxin-Induced DNA Damage

Abstract

Aflatoxin B₁ (AFB₁) is a human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In humans, AFB₁ is primarily bioactivated by cytochrome P450 1A2 (CYP1A2) and 3A4 to a genotoxic epoxide that forms N⁷-guanine DNA adducts. A series of yeast haploid mutants defective in DNA repair and cell cycle checkpoints were transformed with human CYP1A2 to investigate how these DNA adducts are repaired. Cell survival and mutagenesis following aflatoxin B₁ treatment was assayed in strains defective in nucleotide excision repair (NER) (rad14), postreplication repair (PRR) (rad6, rad18, mms2, and rad5), homologous recombinational repair (HRR) (rad51 and rad54), base excision repair (BER) (apn1 apn2), nonhomologous end-joining (NHEJ) (yku70), mismatch repair (MMR) (pms1), translesion synthesis (TLS) (rev3), and checkpoints (mec1-1, mec1-1 rad53, rad9, and rad17). Together our data suggest the involvement of homologous recombination and nucleotide excision repair, postreplication repair, and checkpoints in the repair and/or tolerance of AFB₁-induced DNA damage in the yeast model. Rev3 appears to mediate AFB₁-induced mutagenesis when error-free pathways are compromised. The results further suggest unique roles for Rad5 and abasic endonuclease-dependent DNA intermediates in regulating AFB₁-induced mutagenicity.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/.

ACKNOWLEDGMENTS

We thank Julian Simon for providing yeast strains; Christian Sengstag for providing hCYP1A2 plasmid; Kerstin Gross-Steinmeyer for advice on MROD assays; Julia Sidorova, Toshio Tsukiyama, James Vary, and Thomas Fazzio for invaluable advice on yeast work; Ed Kelly for early advice in the development of the hCYP1A2-expressing strains; and Shawna Miles and Melisa Dashiell for technical assistance on yeast sporulation and tetrad analysis.

This work was supported in part by R01ES05780 and NIEHS Center grants P30ES07033, U19ES011387, and R01GM41073 and NCI Comprehensive Center grant P30 CA15704.