Analysis of cellular responses to aflatoxin B1 in yeast expressing human cytochrome P450 1A2 using cDNA microarrays

Yingying Guo, Linda L. Breeden, Wenhong Fan, Lue Ping Zhao, David L. Eaton, Helmut Zarbl

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Aflatoxin B1 (AFB1) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, AFB 1 is bioactivated by cytochrome P450 (CYP450) enzymes, primarily CYP1A2, to the genotoxic epoxide that forms N7-guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB 1, a strain of Saccharomyces cerevisiae engineered to express human CYP1A2 was exposed to doses of AFB1 that resulted in minimal lethality, but substantial genotoxicity. Flow cytometric analysis demonstrated a dose and time dependent S phase delay under the same treatment conditions, indicating a checkpoint response to DNA damage. Replicate cDNA microarray analyses of AFB1 treated cells showed that about 200 genes were significantly affected by the exposure. The genes activated by AFB 1-treatment included RAD51, DUN1 and other members of the DNA damage response signature reported in a previous study with methylmethane sulfonate and ionizing radiation [A.P. Gasch, M. Huang, S. Metzner, D. Botstein, S.J. Elledge, P.O. Brown, Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p, Mol. Biol. Cell 12 (2001) 2987-3003]. However, unlike previous studies using highly cytotoxic doses, environmental stress response genes [A.P. Gasch, P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, P.O. Brown, Genomic expression programs in the response of yeast cells to environmental changes, Mol. Biol. Cell 11 (2000) 4241-4257] were largely unaffected by our dosing regimen. About half of the transcripts affected are also known to be cell cycle regulated. The most strongly repressed transcripts were those encoding the histone genes and a group of genes that are cell cycle regulated and peak in M phase and early G1. These include most of the known daughter-specific genes. The rapid and coordinated repression of histones and M/G1-specific transcripts cannot be explained by cell cycle arrest, and suggested that there are additional signaling pathways that directly repress these genes in cells under genotoxic stress.

Original languageEnglish (US)
Pages (from-to)121-142
Number of pages22
JournalMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume593
Issue number1-2
DOIs
StatePublished - Jan 29 2006
Externally publishedYes

Fingerprint

Cytochrome P-450 CYP1A2
Aflatoxin B1
Oligonucleotide Array Sequence Analysis
Yeasts
Genes
DNA Damage
Histones
Cytochrome P-450 Enzyme System
Methyl Methanesulfonate
cdc Genes
Aspergillus flavus
DNA Adducts
Epoxy Compounds
Guanine
Microarray Analysis
Cell Cycle Checkpoints
Ionizing Radiation
S Phase
Cell Division
Saccharomyces cerevisiae

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics
  • Health, Toxicology and Mutagenesis

Keywords

  • Aflatoxin B
  • DNA damage response
  • Daughter-specific genes
  • Histone genes
  • Yeast cDNA microarrays
  • Yeast transcriptional response

Cite this

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title = "Analysis of cellular responses to aflatoxin B1 in yeast expressing human cytochrome P450 1A2 using cDNA microarrays",
abstract = "Aflatoxin B1 (AFB1) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, AFB 1 is bioactivated by cytochrome P450 (CYP450) enzymes, primarily CYP1A2, to the genotoxic epoxide that forms N7-guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB 1, a strain of Saccharomyces cerevisiae engineered to express human CYP1A2 was exposed to doses of AFB1 that resulted in minimal lethality, but substantial genotoxicity. Flow cytometric analysis demonstrated a dose and time dependent S phase delay under the same treatment conditions, indicating a checkpoint response to DNA damage. Replicate cDNA microarray analyses of AFB1 treated cells showed that about 200 genes were significantly affected by the exposure. The genes activated by AFB 1-treatment included RAD51, DUN1 and other members of the DNA damage response signature reported in a previous study with methylmethane sulfonate and ionizing radiation [A.P. Gasch, M. Huang, S. Metzner, D. Botstein, S.J. Elledge, P.O. Brown, Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p, Mol. Biol. Cell 12 (2001) 2987-3003]. However, unlike previous studies using highly cytotoxic doses, environmental stress response genes [A.P. Gasch, P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, P.O. Brown, Genomic expression programs in the response of yeast cells to environmental changes, Mol. Biol. Cell 11 (2000) 4241-4257] were largely unaffected by our dosing regimen. About half of the transcripts affected are also known to be cell cycle regulated. The most strongly repressed transcripts were those encoding the histone genes and a group of genes that are cell cycle regulated and peak in M phase and early G1. These include most of the known daughter-specific genes. The rapid and coordinated repression of histones and M/G1-specific transcripts cannot be explained by cell cycle arrest, and suggested that there are additional signaling pathways that directly repress these genes in cells under genotoxic stress.",
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Analysis of cellular responses to aflatoxin B1 in yeast expressing human cytochrome P450 1A2 using cDNA microarrays. / Guo, Yingying; Breeden, Linda L.; Fan, Wenhong; Zhao, Lue Ping; Eaton, David L.; Zarbl, Helmut.

In: Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 593, No. 1-2, 29.01.2006, p. 121-142.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Analysis of cellular responses to aflatoxin B1 in yeast expressing human cytochrome P450 1A2 using cDNA microarrays

AU - Guo, Yingying

AU - Breeden, Linda L.

AU - Fan, Wenhong

AU - Zhao, Lue Ping

AU - Eaton, David L.

AU - Zarbl, Helmut

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AB - Aflatoxin B1 (AFB1) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, AFB 1 is bioactivated by cytochrome P450 (CYP450) enzymes, primarily CYP1A2, to the genotoxic epoxide that forms N7-guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB 1, a strain of Saccharomyces cerevisiae engineered to express human CYP1A2 was exposed to doses of AFB1 that resulted in minimal lethality, but substantial genotoxicity. Flow cytometric analysis demonstrated a dose and time dependent S phase delay under the same treatment conditions, indicating a checkpoint response to DNA damage. Replicate cDNA microarray analyses of AFB1 treated cells showed that about 200 genes were significantly affected by the exposure. The genes activated by AFB 1-treatment included RAD51, DUN1 and other members of the DNA damage response signature reported in a previous study with methylmethane sulfonate and ionizing radiation [A.P. Gasch, M. Huang, S. Metzner, D. Botstein, S.J. Elledge, P.O. Brown, Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p, Mol. Biol. Cell 12 (2001) 2987-3003]. However, unlike previous studies using highly cytotoxic doses, environmental stress response genes [A.P. Gasch, P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, P.O. Brown, Genomic expression programs in the response of yeast cells to environmental changes, Mol. Biol. Cell 11 (2000) 4241-4257] were largely unaffected by our dosing regimen. About half of the transcripts affected are also known to be cell cycle regulated. The most strongly repressed transcripts were those encoding the histone genes and a group of genes that are cell cycle regulated and peak in M phase and early G1. These include most of the known daughter-specific genes. The rapid and coordinated repression of histones and M/G1-specific transcripts cannot be explained by cell cycle arrest, and suggested that there are additional signaling pathways that directly repress these genes in cells under genotoxic stress.

KW - Aflatoxin B

KW - DNA damage response

KW - Daughter-specific genes

KW - Histone genes

KW - Yeast cDNA microarrays

KW - Yeast transcriptional response

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