Induction of DNA Strand Breaks in Cultured Rat Embryo Cells by Crocidolite Asbestos as Assessed by Nick Translation1

Asbestos, a proven carcinogen, is reported to have no genotoxic effects. We hypothesized, however, in light of its clastogenic effects that one mechanism by which asbestos induces cell transformation and tumorigenesis involves the induction of DNA strand scission. Cultured rat embryo cells were exposed to low concentrations of International Union Against Cancer crocidolite and examined at intervals ranging from 2 to 48 h. The induction of DNA strand breaks was examined using the technique of nick translation followed by autoradiography or scintillation counting. Our results indicate that cells exposed to crocidolite have a higher incidence of DNA breaks and that this effect becomes apparent within 2-6 hours of exposure. Ball-milled crocidolite as well as riebeckite have a significantly lower effect while glass fibers induce a more pro nounced DNA strand damage. These observations support the role fiber length plays in carcinogenesis and suggest that the classification of asbestos as a nongenotoxic carcinogen be reconsidered. UICC' crocidolite fibers using the technique of nick translation (26). Nick translation was initially developed to study the replication of DNA in vitro (27) and has recently been used to investigate the availability for transcription of the DNA in chromosomes (28-30) as well as in cultured cells and tissue sections (31, 32). Exogenous DNA polymerase I binds at DNA sites containing free 3'-hydroxy termini and, due to its polym erase and exonuclease activities, synthesizes a new DNA strand while digesting the nicked DNA chain. Thus, the amount of synthesis that occurs reflects the presence of strand breaks and the degree of damage sustained by DNA. This technique ap pears to be sufficiently sensitive to detect chemical and UVinduced DNA damage (33) and to differentiate between active and inactive chromatin (28, 34). It has been used to document heat-induced DNA damage at earlier times then was possible with alkaline elution (35).