Genome Profiling for Aflatoxin B1 Resistance in Saccharomyces cerevisiae Reveals a Role for the CSM2/SHU Complex in Tolerance of Aflatoxin B1-Associated DNA Damage

Exposure to the mycotoxin aflatoxin B1 (AFB1) strongly correlates with hepatocellular carcinoma (HCC). P450 enzymes convert AFB1 into a highly reactive epoxide that forms unstable 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) DNA adducts, which convert to stable mutagenic AFB1 formamidopyrimidine (FAPY) DNA adducts. In CYP1A2-expressing budding yeast, AFB1 is a weak mutagen but a potent recombinagen. However, few genes have been identified that confer AFB1 resistance. Here, we profiled the yeast genome for AFB1 resistance. We introduced the human CYP1A2 into ∼90% of the diploid deletion library, and pooled samples from CYP1A2-expressing libraries and the original library were exposed to 50 μM AFB1 for 20 hs. By using next generation sequencing (NGS) to count molecular barcodes, we initially identified 86 genes from the CYP1A2-expressing libraries, of which 79 were confirmed to confer AFB1 resistance. While functionally diverse genes, including those that function in proteolysis, actin reorganization, and tRNA modification, were identified, those that function in postreplication DNA repair and encode proteins that bind to DNA damage were over-represented, compared to the yeast genome, at large. DNA metabolism genes also included those functioning in checkpoint recovery and replication fork maintenance, emphasizing the potency of the mycotoxin to trigger replication stress. Among genes involved in postreplication repair, we observed that CSM2, a member of the CSM2(SHU) complex, functioned in AFB1-associated sister chromatid recombination while suppressing AFB1-associated mutations. These studies thus broaden the number of AFB1 resistance genes and have elucidated a mechanism of error-free bypass of AFB1-associated DNA adducts.

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Genome Profiling for Aflatoxin B1 Resistance inSaccharomyces cerevisiaeReveals a Role for the CSM2/SHU Complex in Tolerance of Aflatoxin B1-associated DNA Damage

10.1101/629436 ◽

2019 ◽

Author(s):

Nick St. John ◽

Julian Freedland ◽

Henri Baldino ◽

Frank Doyle ◽

Cinzia Cera ◽

...

Keyword(s):

Dna Damage ◽

Dna Adducts ◽

Aflatoxin B1 ◽

Damage Tolerance ◽

Yeast Genome ◽

Dna Damage Tolerance ◽

Actin Reorganization ◽

Resistant Genes ◽

Functionally Diverse ◽

Pooled Samples

ABSTRACTExposure to the mycotoxin aflatoxin B1 (AFB1) strongly correlates with hepatocellular carcinoma. P450 enzymes convert AFB1into a highly reactive epoxide that forms unstable 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) DNA adducts, which convert to stable mutagenic AFB1formamidopyrimidine (FAPY) DNA adducts. In CYP1A2-expressing budding yeast, AFB1is a weak mutagen but a potent recombinagen. However, few genes have been identified that confer AFB1resistance. Here, we profiled the yeast genome for AFB1resistance. We introduced the human CYP1A2 into ∼90% of the diploid deletion library, and pooled samples from CYP1A2-expressing libraries and the original library were exposed to 50 μM AFB1for 20 hs. By using next generation sequencing to count molecular barcodes, we identified 85 AFB1resistant genes from the CYP1A2-expressing libraries. While functionally diverse genes, including those that function in proteolysis, actin reorganization, and tRNA modification, were identified, those that function in post-replication DNA repair and encode proteins that bind to DNA damage were over-represented, compared to the yeast genome, at large. DNA metabolism genes included those functioning in DNA damage tolerance, checkpoint recovery and replication fork maintenance, emphasizing the potency of the mycotoxin to trigger replication stress. Among genes involved in error-free DNA damage tolerance, we observed thatCSM2, a member of theCSM2(SHU)complex, functioned in AFB1-associated sister chromatid recombination while suppressing AFB1-associated mutations. These studies thus broaden the number of AFB1resistant genes and have elucidated a mechanism of error-free bypass of AFB1-associated DNA adducts.

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Dietary Resveratrol Alleviates AFB1-Induced Ileum Damage in Ducks via the Nrf2 and NF-κB/NLRP3 Signaling Pathways and CYP1A1/2 Expressions

Agriculture ◽

10.3390/agriculture12010054 ◽

2022 ◽

Vol 12 (1) ◽

pp. 54

Author(s):

Hao Yang ◽

Yingjie Wang ◽

Chunting Yu ◽

Yihan Jiao ◽

Ruoshi Zhang ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Signaling Pathways ◽

Dna Adducts ◽

Aflatoxin B1 ◽

Mrna Level ◽

Basal Diet ◽

Protective Effects ◽

Expression Of Genes ◽

And Oxidative Stress

The aim of this study was to explore the mechanism underlying the protective effects of resveratrol against Aflatoxin B1-induced ileum injury in ducks. A corn–soybean meal-basal diet and two test diets (500 mg/kg resveratrol +0.2 mg Aflatoxin B1/kg, 0.2 mg AFB1/kg) were used in a 10-wk design trial (n = 15 ducks/group). These results showed that the toxicity of Aflatoxin B1 significantly reduced the antioxidant capacity of duck ileum and induced inflammation, oxidative stress, mitochondrial dysfunction and DNA damage in ducks. The expression of genes, including CYP1A2, CYP2A6, and CYP3A4, at the mRNA level was significantly upregulated (p < 0.05) by AFB1. The level of Nrf2 was suppressed (p < 0.05) and the mRNA and protein level of NF-κB was activated (p < 0.05) in the AFB1 group. However, supplementation with 500 mg/kg dietary resveratrol in Aflatoxin B1-induced ducks significantly ameliorated these alterations and decreased the mRNA expression of CYP1A1 and CYP1A2 (p < 0.05) and the production of AFB1-DNA adducts (p < 0.05). The results proved that resveratrol alleviated ileum injury induced by AFB1, decreased the production of AFB1-DNA adducts by downregulating the expression of CYP1A1 and CYP1A2, and reduced DNA damage and oxidative stress via the Nrf2/ Keap1 and NF-κB/NLRP3 signaling pathways.

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Identification of New Markers of Alcohol-Derived DNA Damage in Humans

Biomolecules ◽

10.3390/biom11030366 ◽

2021 ◽

Vol 11 (3) ◽

pp. 366

Author(s):

Valeria Guidolin ◽

Erik S. Carlson ◽

Andrea Carrà ◽

Peter W. Villalta ◽

Laura A. Maertens ◽

...

Keyword(s):

Dna Damage ◽

Dna Adducts ◽

Neutral Loss ◽

Alcohol Exposure ◽

Dose Dependence ◽

Accurate Mass ◽

Constant Neutral Loss ◽

Covalent Modifications ◽

Underlying Mechanisms

Alcohol consumption is a risk factor for the development of several cancers, including those of the head and neck and the esophagus. The underlying mechanisms of alcohol-induced carcinogenesis remain unclear; however, at these sites, alcohol-derived acetaldehyde seems to play a major role. By reacting with DNA, acetaldehyde generates covalent modifications (adducts) that can lead to mutations. Previous studies have shown a dose dependence between levels of a major acetaldehyde-derived DNA adduct and alcohol exposure in oral-cell DNA. The goal of this study was to optimize a mass spectrometry (MS)-based DNA adductomic approach to screen for all acetaldehyde-derived DNA adducts to more comprehensively characterize the genotoxic effects of acetaldehyde in humans. A high-resolution/-accurate-mass data-dependent constant-neutral-loss-MS3 methodology was developed to profile acetaldehyde-DNA adducts in purified DNA. This resulted in the identification of 22 DNA adducts. In addition to the expected N2-ethyldeoxyguanosine (after NaBH3CN reduction), two previously unreported adducts showed prominent signals in the mass spectra. MSn fragmentation spectra and accurate mass were used to hypothesize the structure of the two new adducts, which were then identified as N6-ethyldeoxyadenosine and N4-ethyldeoxycytidine by comparison with synthesized standards. These adducts were quantified in DNA isolated from oral cells collected from volunteers exposed to alcohol, revealing a significant increase after the exposure. In addition, 17 of the adducts identified in vitro were detected in these samples confirming our ability to more comprehensively characterize the DNA damage deriving from alcohol exposures.

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