Rapid DNA degradation in primary rat hepatocytes treated with diverse cytotoxic chemicals: analysis by pulsed field gel electrophoresis and implications for alkaline elution assays.
The use of genetic toxicology tests for hazard identification is complicated by the fact that some in vitro tests using cultured mammalian cells are subject to potential artifacts that can make it difficult to distinguish between direct, chemically-induced genotoxicity, and DNA damage that occurs secondary to chemically-induced cytotoxicity (e.g., mediated by endogenous nucleases). Recently, we demonstrated that cytotoxicity-induced DNA double strand breaks (dsb) can produce artifacts in the in vitro alkaline elution/rat hepatocyte assay [Elia et al., 1993]. To explore this further, we used pulsed field gel/DNA dsb assays to characterize the relationship between chemically-induced cytotoxicity and the degradation of genomic DNA to high molecular weight fragments. Two sets of compounds were tested: 17 cytotoxic agents judged to be neither genotoxic nor carcinogenic, and 10 known genotoxic carcinogens. We found a close correlation between chemically-induced cytotoxicity and the rapid degradation of DNA to high molecular weight, double-stranded fragments. In contrast, the classic genotoxic chemicals tested generally did not trigger DNA dsb fragmentation at doses that were genotoxic but not immediately cytotoxic. These data indicate that pulsed field gel/DNA dsb assays can be used with in vitro genetic toxicology assays to help distinguish between genotoxic and cytotoxic mechanisms of DNA damage.
Department of Genetic and Cellular Toxicology, Merck Research Laboratories, West Point, Pennsylvania.
M C
MC
Elia
R D
RD
Storer
T W
TW
McKelvey
A R
AR
Kraynak
J E
JE
Barnum
L S
LS
Harmon
J G
JG
DeLuca
W W
WW
Nichols
D000818:Animals; D015197:Carcinogenicity Tests; D002478:Cells, Cultured; D005591:Chemical Fractionation; D003603:Cytotoxins / Q000633:toxicity*; D004247:DNA / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D016521:Electrophoresis, Gel, Pulsed-Field; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D009154:Mutation / Q000187:drug effects / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley
D003603:Cytotoxins; D004247:DNA