Quantitative PCR analysis of diepoxybutane and epihalohydrin damage to nuclear versus mitochondrial DNA.
The bifunctional alkylating agents diepoxybutane (DEB) and epichlorohydrin (ECH) are linked to the elevated incidence of certain cancers among workers in the synthetic polymer industry. Both compounds form interstrand cross-links within duplex DNA, an activity suggested to contribute to their cytotoxicity. To assess the DNA targeting of these compounds in vivo, we assayed for damage within chicken erythro-progenitor cells at three different sites: one within mitochondrial DNA, one within expressed nuclear DNA, and one within unexpressed nuclear DNA. We determined the degree of damage at each site via a quantitative polymerase chain reaction, which compares amplification of control, untreated DNA to that from cells exposed to the agent in question. We found that ECH and the related compound epibromohydrin preferentially target nuclear DNA relative to mitochondrial DNA, whereas DEB reacts similarly with the two genomes. Decreased reactivity of the mitochondrial genome could contribute to the reduced apoptotic potential of ECH relative to DEB. Additionally, formation of lesions by all agents occurred at comparable levels for unexpressed and expressed nuclear loci, suggesting that alkylation is unaffected by the degree of chromatin condensation.
Lariviere
Frederick J
FJ
Department of Chemistry, Washington and Lee University, Lexington, VA 24450, USA.
Newman
Adam G
AG
Watts
Megan L
ML
Bradley
Sharonda Q
SQ
Juskewitch
Justin E
JE
Greenwood
Paul G
PG
Millard
Julie T
JT
D000477:Alkylating Agents / Q000633:toxicity; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects; D002645:Chickens; D003432:Cross-Linking Reagents / Q000633:toxicity; D004247:DNA / Q000187:drug effects / Q000235:genetics; D004249:DNA Damage; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair; D004272:DNA, Mitochondrial / Q000187:drug effects* / Q000235:genetics; D004811:Epichlorohydrin / Q000633:toxicity*; D004852:Epoxy Compounds / Q000633:toxicity*; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D054629:Genome, Mitochondrial / Q000187:drug effects; D016133:Polymerase Chain Reaction
D000477:Alkylating Agents; D003432:Cross-Linking Reagents; D017931:DNA Primers; D004272:DNA, Mitochondrial; D004852:Epoxy Compounds; D004811:Epichlorohydrin; C007366:diepoxybutane; D004247:DNA