Environmental factors are estimated to be the cause of 19% of cancers. However, human susceptibility to environmental carcinogens is highly variable. Low penetrant alleles may increase the risk for particular environmental-associated cancers. Epidemiological studies, however, are limited by their statistical power and the requirement for large patient numbers. Because many DNA metabolism and housekeeping genes are conserved from yeast to man, high throughput analysis of Saccharomyces cerevisiae (budding yeast) genes that confer resistance to carcinogens have identified human genes that confer resistance to environmental carcinogens. Genomic phenotyping using the ~5,000 yeast single-gene deletion haploid and diploid strains have been highly successful in determining genes that confer resistance to radiation and chemical agents. However, 75% of environmental agents are not carcinogenic per se, but require bioactivation, such as tissue - specific cytochrome P450-mediated metabolic activation. We previously were successful in introducing human CYP1A2 and CYP1A1 into yeast and activating a variety of carcinogens, including aflatoxin B1 (AFB1), benzo[a]pyrene dihydrodiol (BaP-DHD), and heterocyclic amines (HA, food carcinogens).
The aim of this project is to determine which yeast genes are required for resistance to the potent carcinogen, AFB1, using a high throughput systems biology approach. We will introduce plasmids that express human CYP1A2 into the ~5,000 diploid homozygous single-deletion and heterozygous single-deletion strains. In the first aim, we will profile the yeat genome using the diploid single deletion strains for resistance to AFB1. Genes that confer resistance will be identified by high-throughput sensitive assays to measure cell growth and by molecular bar codes using high throughput sequencing or microarrays. Considering that AFB1 is a potent recombinagen, in the second aim, we will identify DNA repair and recombination pathways that confer AFB1 resistance by high throughput sensitivity analysis of double mutants derived from a set of single DNA repair mutants. These studies will thus identify novel genes involved in mediating carcinogen resistance and sensitivity, and provide insights into how recombinational repair processes actively tolerate DNA adducts. The strain collections will be made available to the scientific community and will be a valuable resource for characterizing the genetic susceptibility to environmental agents. The project will be a valuable training tool in systems and computational biology.
Sporadic cancer likely results from a combination of environmental exposure and genetics. Many genetic polymorphisms contribute to cancer risk. However, studies to identify these polymorphisms often lack statistical significance due to small sample sizes. High throughput screening in model organisms will facilitate identifying genes that confer resistance to environmental carcinogens. Since many DNA repair genes are conserved from yeast to mammals, high throughput screening has been successful in identifying genes that confer resistance to multiple DNA damaging agents. Many environmental carcinogens, however, must be metabolically activated by cytochrome P450 enzymes, which are lacking in yeast. We propose to introduce the human P450 gene CYP1A2 into the diploid yeast deletion collection. By high throughput screening we will define a genomic profile for resistance to aflatoxin B1. By computational methods, we will determine DNA repair pathways that confer aflatoxin B1 resistance. This project will give me valuable training in Systems Biology approaches, which will be a powerful method for genomic profiling of resistance to many environmental DNA damaging agents.
|Freedland, Julian; Cera, Cinzia; Fasullo, Michael (2017) CYP1A1 I462V polymorphism is associated with reduced genotoxicity in yeast despite positive association with increased cancer risk. Mutat Res 815:35-43|
|Fasullo, Michael; Freedland, Julian; St John, Nicholas et al. (2017) An in vitro system for measuring genotoxicity mediated by human CYP3A4 in Saccharomyces cerevisiae. Environ Mol Mutagen 58:217-227|
|Fasullo, Michael; Smith, Autumn; Egner, Patricia et al. (2014) Activation of aflatoxin B1 by expression of human CYP1A2 polymorphisms in Saccharomyces cerevisiae. Mutat Res Genet Toxicol Environ Mutagen 761:18-26|