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Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae
Published on: April 5, 2015
An in vitro system for measuring genotoxicity mediated by human CYP3A4 in Saccharomyces cerevisiae
Michael Fasullo1,2, Julian Freedland1, Nicholas St John1
1College of Nanoscale Sciences and Engineering, State University of New York Polytechnic Institute, Albany, New York.
Abstract:
P450 activity is required to metabolically activate many chemical carcinogens, rendering them highly genotoxic. CYP3A4 is the most abundantly expressed P450 enzyme in the liver, accounting for most drug metabolism and constituting 50% of all hepatic P450 activity. CYP3A4 is also expressed in extrahepatic tissues, including the intestine. However, the role of CYP3A4 in activating chemical carcinogens into potent genotoxins is unclear. To facilitate efforts to determine whether CYP3A4, per se, can activate carcinogens into potent genotoxins, we expressed human CYP3A4 in the DNA-repair mutant (rad4 rad51) strain of budding yeast Saccharomyces cerevisiae and tested the novel, recombinant yeast strain for ability to report CYP3A4-mediated genotoxicity of a well-known genotoxin, aflatoxin B1 (AFB1 ). Yeast microsomes containing human CYP3A4, but not those that do not contain CYP3A4, were active in hydroxylation of diclofenac, a known CYP3A4 substrate drug, a result confirming CYP3A4 activity in the recombinant yeast strain. In cells exposed to AFB1 , the expression of CYP3A4 supported DNA adduct formation, chromosome rearrangements, cell death, and expression of the large subunit of ribonucleotide reductase, Rnr3, a marker of DNA damage. Expression of CYP3A4 also conferred sensitivity in rad4 rad51 mutants exposed to colon carcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). These data confirm the ability of human CYP3A4 to mediate the genotoxicity of AFB1 , and illustrate the usefulness of the CYP3A4-expressing, DNA-repair mutant yeast strain for screening other chemical compounds that are CYP3A4 substrates, for potential genotoxicity. Environ. Mol. Mutagen. 58:217-227, 2017. © 2017 Wiley Periodicals, Inc.
Insights
This study demonstrates that the human CYP3A4 enzyme activates carcinogens like aflatoxin B1, leading to DNA damage and cell death. A novel yeast strain expressing CYP3A4 can be used to screen for genotoxicity of other compounds.
Area of Science:
- Biochemistry and Molecular Biology
- Toxicology and Environmental Health
Background:
- Cytochrome P450 (P450) enzymes, particularly CYP3A4, are crucial for metabolizing xenobiotics, including chemical carcinogens.
- CYP3A4 is the most abundant P450 in the liver and also present in extrahepatic tissues, but its role in carcinogen activation remains unclear.
- Understanding CYP3A4's role in genotoxicity is vital for assessing the risks associated with various chemical exposures.
Purpose of the Study:
- To investigate whether CYP3A4 can directly activate chemical carcinogens into genotoxic agents.
- To develop and validate a recombinant yeast strain for reporting CYP3A4-mediated genotoxicity.
- To assess the genotoxic potential of aflatoxin B1 (AFB1) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) using the developed yeast model.
Main Methods:
- Human CYP3A4 was expressed in a DNA-repair deficient (rad4 rad51) Saccharomyces cerevisiae strain.
- CYP3A4 activity was confirmed by the hydroxylation of diclofenac, a known CYP3A4 substrate.
- The recombinant yeast strain's response to AFB1 and MeIQx exposure was evaluated by measuring DNA adducts, chromosome rearrangements, cell death, and Rnr3 expression.
Main Results:
- Yeast microsomes expressing CYP3A4 showed significant activity in hydroxylating diclofenac.
- Exposure to AFB1 in CYP3A4-expressing yeast resulted in increased DNA adducts, chromosome aberrations, cell death, and Rnr3 expression, indicating genotoxicity.
- CYP3A4 expression conferred sensitivity to MeIQx in the DNA-repair mutant yeast, further supporting its role in genotoxicity.
Conclusions:
- Human CYP3A4 is capable of mediating the genotoxicity of chemical carcinogens like AFB1.
- The developed CYP3A4-expressing, DNA-repair mutant yeast strain is a useful tool for screening potential genotoxicity of CYP3A4 substrates.
- This model facilitates research into the specific contribution of CYP3A4 to chemical carcinogen activation and associated health risks.
