Alkyltransferase-mediated toxicity of bis-electrophiles in mammalian cells

Aley G. Kalapila, Anthony Pegg

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The primary function of O6-alkylguanine-DNA alkyltransferase (AGT) is to maintain genomic integrity in the face of damage by both endogenous and exogenous alkylating agents. However, paradoxically, bacterial and mammalian AGTs have been shown to increase cytotoxicity and mutagenicity of dihaloalkanes and other bis-electrophiles when expressed in bacterial cells. We have extended these studies to mammalian cells using CHO cells that lack AGT expression and CHO cells stably transfected with a plasmid that expresses human AGT. The cytotoxicity of 1,2-dibromoethane, dibromomethane and epibromohydrin was significantly increased by the presence of AGT but cytotoxicity of butadiene diepoxide was not affected. Mutations caused by these agents were assessed using hypoxanthine-guanine phosphoribosyltransferase (HPRT) as a reporter gene. There was a small (c. 2-3-fold) but statistically significant AGT-mediated increase in mutations caused by 1,2-dibromoethane, dibromomethane and epibromohydrin. Analysis of the mutation spectrum induced by 1,2-dibromoethane showed that the presence of AGT also altered the types of mutations with an increase in total base substitution mutants due to a rise in transversions at both G:C and A:T sites. AGT expression also led to mutations arising from the transcribed strand, which were not seen in cells lacking AGT. Although the frequency of deletion mutations was decreased by AGT expression, the formation of large deletions (≥3 exons) was increased. This work demonstrates that interaction of AGT with some bis-electrophiles can cause mutagenicity and diminished cell survival in mammalian cells. It is consistent with the hypothesis that DNA-AGT cross-links, which have been characterized in experiments with purified AGT protein and such bis-electrophiles, can be formed in mammalian cells.

Original languageEnglish (US)
Pages (from-to)35-42
Number of pages8
JournalMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume684
Issue number1-2
DOIs
StatePublished - Feb 3 2010

Fingerprint

Alkyl and Aryl Transferases
Ethylene Dibromide
Mutation
CHO Cells
Hypoxanthine Phosphoribosyltransferase
Sequence Deletion
Alkylating Agents
Reporter Genes
Exons
Spectrum Analysis
Cell Survival
Plasmids
DNA
Proteins

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics
  • Health, Toxicology and Mutagenesis

Cite this

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title = "Alkyltransferase-mediated toxicity of bis-electrophiles in mammalian cells",
abstract = "The primary function of O6-alkylguanine-DNA alkyltransferase (AGT) is to maintain genomic integrity in the face of damage by both endogenous and exogenous alkylating agents. However, paradoxically, bacterial and mammalian AGTs have been shown to increase cytotoxicity and mutagenicity of dihaloalkanes and other bis-electrophiles when expressed in bacterial cells. We have extended these studies to mammalian cells using CHO cells that lack AGT expression and CHO cells stably transfected with a plasmid that expresses human AGT. The cytotoxicity of 1,2-dibromoethane, dibromomethane and epibromohydrin was significantly increased by the presence of AGT but cytotoxicity of butadiene diepoxide was not affected. Mutations caused by these agents were assessed using hypoxanthine-guanine phosphoribosyltransferase (HPRT) as a reporter gene. There was a small (c. 2-3-fold) but statistically significant AGT-mediated increase in mutations caused by 1,2-dibromoethane, dibromomethane and epibromohydrin. Analysis of the mutation spectrum induced by 1,2-dibromoethane showed that the presence of AGT also altered the types of mutations with an increase in total base substitution mutants due to a rise in transversions at both G:C and A:T sites. AGT expression also led to mutations arising from the transcribed strand, which were not seen in cells lacking AGT. Although the frequency of deletion mutations was decreased by AGT expression, the formation of large deletions (≥3 exons) was increased. This work demonstrates that interaction of AGT with some bis-electrophiles can cause mutagenicity and diminished cell survival in mammalian cells. It is consistent with the hypothesis that DNA-AGT cross-links, which have been characterized in experiments with purified AGT protein and such bis-electrophiles, can be formed in mammalian cells.",
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Alkyltransferase-mediated toxicity of bis-electrophiles in mammalian cells. / Kalapila, Aley G.; Pegg, Anthony.

In: Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 684, No. 1-2, 03.02.2010, p. 35-42.

Research output: Contribution to journalArticle

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