Biology subjects

Fasullo, M.

Publications and source records attributed to Fasullo, M..

2 recordsLinked to original sources

bioRxiv2022

High-Throughput Screening of the Saccharomyces cerevisiae Genome for 2-Amino-3-Methylimidazo Quinoline Resistance Identifies Colon Cancer-Associated Genes

Heterocyclic aromatic amines (HAAs) are potent carcinogenic agents found in charred meats and cigarette smoke. However, few eukaryotic resistance genes have been identified. We used Saccharomyces cerevisiae (budding yeast) to identify genes that confer resistance to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). CYP1A2 and NAT2 activate IQ to become a mutagenic nitrenium compound. We introduced an expression vector that contains human CYP1A2 and NAT2 genes into selected mutant strains and the diploid yeast deletion collection. The deletion libraries expressing CYP1A2 and NAT2 or no human genes were exposed to either 400 or 800 M IQ for five or ten generations. DNA barcodes were sequenced using the Illumina HiSeq 2500 platform and statistical significance was determined for exactly matched barcodes. Four screens for IQ resistance in the "humanized" collection identified 1160 unique ORFs, of which 337 were validated or duplicated in at least two screens. Two screens of the original yeast library identified 101 genes that overlap with the 337 previously identified. Selected genes were validated by growth curves, competitive growth assays, or trypan blue assays. Prominent among both sets are ribosomal protein genes, while nitrogen metabolism, cell wall synthesis, and phosphatase genes were identified among the "humanized" library. Protein complexes identified included the casein kinase 2 (CK2) and histone chaperone (HIR) complex. DNA repair genes included NTG1, RAD18, RAD9, PSY2 and UBC13. Polymorphisms in human NTHL1, the NTG1 ortholog, and RAD18 are risk factors for colon cancer. These studies thus provoke questions of whether genetic risk factors for colon cancer confer more HAA-associated toxicity.

genetics↗

bioRxiv2021

CYP1B1 converts procarcinogens into genotoxins in Saccharomyces cerevisiae

CYP1B1 activates many chemical carcinogens into potent genotoxins, and allelic variants are risk factors in lung, breast, and prostate cancer. However, genetic instability phenotypes incurred by CYP1B1-activated metabolites have been investigated for only few compounds. In this study, we expressed human CYP1B1 in yeast strains that measure DNA damage-associated toxicity and frequencies of chromosomal translocations and mutations. DNA damage-associated toxicity was measured in a rad4 rad51 strain, defective in both DNA excision and recombinational repair. Frequencies of chromosomal translocations were measured in diploid yeast strains containing two his3 fragments, and mutation frequencies were measured by selecting for canavanine resistance (CanR) in haploid strains. These strains were exposed to benzo[a]pyrene dihydrodiol (BaP-DHD), aflatoxin B1 (AFB1), and the heterocyclic aromatic amines, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). We observed that AFB1, BaP-DHD, IQ, and MeIQx conferred toxicity in the DNA repair mutant expressing CYP1B1. Translocation frequencies increased eight-fold and three-fold after exposure to 50 M AFB1 and 33 M BaP-DHD respectively. Only a two-fold increase in mutation frequency was observed after exposure to 50 M AFB1. However, a robust DNA damage response was observed after AFB1 exposure, as measured by the induction of the small subunit of ribonucleotide reductase, Rnr3. While CYP1B1-mediated activation of BaP-DHD and heterocyclic aromatic amines was expected, strong activation of AFB1 was not. These studies demonstrate that CYP1B1-mediated activation of carcinogens does not only activate compounds to become mutagens but also can convert compounds to become potent recombinagens.

pharmacology and toxicology↗