Induction of DNA strand breaks by ethylene oxide in human diploid fibroblasts

Abstract

In vitro exposure of normal human diploid fibroblasts (strain VH-10) to ethylene oxide (EtO) induced DNA strand breaks in the dose range of 2.5-30 mMh of EtO. Alkaline DNA unwinding (ADU), neutral filter elution (NFE), pulsed field gel electrophoresis (PFGE), and the comet assay were used to measure DNA single (SSBs) and double strand breaks (DSBs). Different induction rates of SSBs and DSBs, depending on applied method and also on treatment conditions (cells in monolayer or in suspension were used), were found. A dose-dependent increase of DNA strand breaks was found by the ADU method in the dose range of 2.5-20 mMh of EtO when treatment was performed in monolayer and in suspension. DSBs were detected by NFE only when the cells were treated with EtO in suspension (doses 10-30 mMh). The highest induction rate of DSBs (about 4 DSBs per 100 Mbp per 1 mMh of EtO) was detected in suspension with PFGE applied. We have shown that heat-labile sites are formed by EtO. Presumably, the different DSB levels detected by PFGE and NFE result from the conversion of these sites to DSBs during cell lysis at elevated temperature in the PFGE method. The results of the comet assay confirmed that apoptotic processes are not involved in the formation of DSBs in our experimental conditions (less than 1% of apoptotic cells were observed at all doses studied). Possible mechanisms for the induction of DNA strand breaks by EtO-treatment are discussed. The capacity to repair DSBs in EtO-exposed (5-7.5 mMh) cells was studied, and it was found that a considerable part of the damage (about 50%) could be repaired during 18 hr of incubation.