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

Abstract

Aflatoxin B 1 (AFB 1 ) is a human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In humans, AFB 1 is primarily bioactivated by cytochrome P450 1A2 (CYP1A2) and 3A4 to a genotoxic epoxide that forms N 7 -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 B 1 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 AFB 1 -induced DNA damage in the yeast model. Rev3 appears to mediate AFB 1 -induced mutagenesis when error-free pathways are compromised. The results further suggest unique roles for Rad5 and abasic endonuclease-dependent DNA intermediates in regulating AFB 1 -induced mutagenicity.Mutagenic substances, including many environmental contaminants, are structurally diverse and range from simple compounds such as methylating agents to bulky DNA damaging agents (57). Differences in DNA repair capacity may play an important role in determination of individual susceptibility to endogenous and exogenous mutagens (88). DNA repair and mutagenesis have been less well studied for bulky mutagens, partly because the majority of them require efficient bioactivation by biotransformation enzymes that are absent in many in vitro study models (35).A yeast model has been established in this study to delineate the mechanisms involved in the elimination and/or tolerance of DNA damage produced by a bulky mutagen, aflatoxin B 1 (AFB 1 ). A natural toxin produced by the common fungal mold Aspergillus flavus (21), AFB 1 is one of the most potent mutagens ever characterized in eukaryotes (12,31,62). It requires biotransformation by cytochrome P450 (CYP450)-dependent monooxygenases to the reactive, yet highly unstable AFB 1 -8,9-exo-epoxide (AFBO) to become toxic and carcinogenic (21). AFBO can react with the N 7 guanine residue of DNA to form an unstable trans-8,9-dihydro-(N 7 -guanyl)-9-hydroxy-aflatoxin B 1 adduct (AFB 1 -N 7 -Gua) that is thought to either be removed hydrolytically to form an abasic (AP) site or to undergo a hydrolytic reaction that opens the guanine imidazole ring, forming a stable and persistent formamidopyrimidine (AFB 1 -FAPY) derivative (61,93). Studies have shown that human CYP1A2 (hCYP1A2) is the high-affinity CYP450 enzyme active at the low AFB 1 concentrations typically encountered in dietary exposures (26). Consequences of AFB 1 -induced DNA lesions include chromoso...

Participation of DNA Polymerase ζ in Replication of Undamaged DNA in Saccharomyces cerevisiae

“…The Rad14 protein is involved in damage recognition during NER (Guzder et al 1993). The deletion of the RAD14 gene results in a weak, but clearly measurable, spontaneous mutator phenotype ( Figure 1A, Table S1), consistent with previous reports (Bertrand et al 1998;Harfe and JinksRobertson 2000;Scheller et al 2000;Yu et al 2003;Guo et al 2005). This indicates that the rad14 strain indeed accrues potentially mutagenic DNA damage.…”

Gene expression profile of MAP kinase PTC1 mutant exposed to Aflatoxin B1: dysfunctions of gene expression in glucose utilization and sphingolipid metabolism

“…The exception was the RNR3 gene, which codes for ribonuclotide diphosphate reductase, in which the expression was induced by AFB 1 . In contrast, the DNA repair protein-coding genes RAD2, 4,6,7,14,16,26,28,51,54, and 59 were induced by AFB 1 . Based on expression levels, glucose metabolism-related genes essential for stress response or cell integrity were detected (Table 3).…”

“…In many cases, genotoxicity is noted though acute poisoning that originated in the cytotoxicity is reported well. Specifically, AFB 1 is converted into exo-or endo-type 8,9-epoxide in the intracellular space of host organisms, and exo-type epoxide binds to DNA [4]. The exo-type epoxide more easily binds to DNA compared to the endo-type, and the intensity of binding is correlated to toxicity level [5] [6].…”

Enhanced production of human Cytochrome P450 2C9 by Escherichia coli BL21(DE3)pLysS through the novel use of grey relational analysis and Plackett–Burman design

“…Thus, efforts in recent years have realized the expression of some CYP isoforms in bacteria (Mast et al 2004;Kagawa et al 2004), yeast (Guo et al 2005;Rodriguez-Antona et al 2005), and cultured mammalian cells (Mezentsev et al 2005). Among the heterologous expression systems, E. coli expression systems have advantages compared with other expression systems in terms of the low cost to maintain them, the ease of use, and the high yield of CYP with a relatively short period of incubation.…”