Induction of DNA-protein crosslinks by dichloromethane in a V79 cell line transfected with the murine glutathione-S-transferase theta 1 gene.
Dichloromethane (DCM) is considered a probable human carcinogen. Laboratory studies have shown an increased incidence of lung and liver cancer in mice but not in rats or hamsters. Despite the correlation between metabolism of DCM by the glutathione-S-transferase (GST) pathway and the occurrence of tumors in different species, the mechanism of tumor induction by DCM metabolites produced through the GST pathway remains unclear. In this study a V79 cell line stably transfected with the murine GST theta 1 gene (mGSTT1) was compared to the parent cell line (MZ) to determine how the construct affects DCM metabolism and the sensitivity of the cell line to DNA damage and cytotoxicity. V79 cells were treated with DCM (2.5-10mM) or formaldehyde (150-600muM) for 2h. Also, formaldehyde produced by V79 cytosol metabolism of DCM was measured spectrophotometrically. DNA damage and DNA-protein crosslinks were measured by the standard and proteinase K-modified alkaline single cell gel electrophoresis (SCG) assays. Cytotoxicity was assessed by trypan blue stain exclusion, the Live/Dead((R)) cell viability/cytotoxicity kit for animal cells, and the neutral red assay. After DCM treatment a significant concentration-dependent increase in tail moment in the V79 MZ cells was observed compared to a significant concentration-dependent decrease in tail moment in the V79 mGSTT1 cells. Post-incubation with proteinase K significantly increased DNA migrations in DCM-treated V79 mGSTT1 cells. DCM formed significantly higher levels of formaldehyde in the cytosol of the V79 mGSTT1 cells than in the cytosol of the V79 MZ cells. Results using the cytotoxicity assays were comparable using the trypan blue and Live/Dead((R)) assays, neither showing a difference in response between the two cell lines when exposed to either formaldehyde or DCM. These results indicate that V79 mGSTT1 can metabolize DCM to a genotoxic and cytotoxic metabolite, which is likely formaldehyde. This is the first time that the magnitude of the GSTT1 effect can be observed in mammalian cells without confounding caused by using cells with different genetic backgrounds.
Hu
Yunping
Y
Environmental Carcinogenesis Division, B-143-06, NHEERL, US EPA, Research Triangle Park, NC 27711, USA.
Kabler
Sandra L
SL
Tennant
Alan H
AH
Townsend
Alan J
AJ
Kligerman
Andrew D
AD
D000818:Animals; D002273:Carcinogens / Q000378:metabolism* / Q000633:toxicity; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D020552:Comet Assay; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000187:drug effects; D005557:Formaldehyde / Q000378:metabolism / Q000633:toxicity; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D008752:Methylene Chloride / Q000378:metabolism* / Q000633:toxicity; D012680:Sensitivity and Specificity; D013997:Time Factors; D014162:Transfection
D002273:Carcinogens; D003432:Cross-Linking Reagents; D018736:DNA Adducts; D005557:Formaldehyde; D008752:Methylene Chloride; C413545:glutathione S-transferase T1; D005982:Glutathione Transferase