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INVESTIGATION OF DNA REPAIR BY SISTER CHROMATID EXCHANGE (SCE) ANALYSIS AND THE ALKALINE SINGLE CELL GEL ASSAY (SCG) IN MAMMALIAN GO-LYMPHOCYTES AFTER IN VITRO EXPOSURE TO ETHYLENE OXIDE (EO)
Citation:
Kligerman, A D., B. Peng, AND R J. Preston. INVESTIGATION OF DNA REPAIR BY SISTER CHROMATID EXCHANGE (SCE) ANALYSIS AND THE ALKALINE SINGLE CELL GEL ASSAY (SCG) IN MAMMALIAN GO-LYMPHOCYTES AFTER IN VITRO EXPOSURE TO ETHYLENE OXIDE (EO). Presented at International Comet Assay Workshop, Ulm, Germany, July/22-24/2001.
Description:
Investigation ofDNA Repair by Sister Chromatid Exchange (SCE) Analysis and the Alkaline Single Cell Gel Assay (SCG) in Mammalian Go-Lymphocytes after In Vitro Exposure to Ethylene Oxide (EO).
EO is a large volume chemical used primarily as an intermediate in manufacturing and as a fumigant and sterilant. EO is an alkylating agent and mutagen and has been shown in numerous studies to be able to induce chromosome aberrations and SCEs both in vivo and in vitro. EO has been reported to induce long-Iasting lesions that lead to SCEs in peripheral blood lymphocytes many months after exposures have ceased. This has implications for cytogenetic monitoring studies. Our initial studies were designed to investigate the persistence of EO-
induced lesions in vitro in Go-splenic lymphocytes from C57 B16 mice and in human peripheral blood Go-lymphocytes. Splenic lymphocytes and whole human blood were treated with 4 mM EO for 1 h in a 37? C shaker waterbath. The control was treated with a similar volume of 100% methanol. The cells were washed three times with phosphate-buffered saline, and aliquots were either placed in 2 mls of complete medium with mitogen, or mitogen was added at 24, 48, 72, or 96 h after incubation (liquid holding). Just before mitogen addition, a 10 uI aliquot of cells was removed and subjected to the alkaline SCG assay. For both mouse and human cultures, S-bromodeoxyuridine was added 2lh after mitogen addition and the cells were harvested at either 50 h after mitogen addition (mice) or 72 h after mitogen addition (humans). Data obtained from both the SCE and SCG assays were then used to monitor the removal of lesions (DNA repair), and to determine the relationship between the damage shown in the SCG assay compared to the lesions that lead to SCE formation. The results indicate that human lymphocytes show only a small reduction in SCE frequency following 72 h of liquid holding; whereas, mouse splenocytes show a much greater reduction in SCE frequency. The SCG assay shows that DNA damage in both species decreases by about half after about 48 to 72 h of liquid holding. This indicates that the lesions responsible for SCEs, particularly for humans, are not repaired as efficiently as those that are primarily detected by the alkaline SCG assay, and that mouse lymphocytes may be more efficient than human lymphocytes in removing lesions that lead to SCEs.
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