Detection of DNA single strand breaks induced by carcinogens in chinese hamster ovary cells cocultured with rat hepatcytes

Abstract

A sensitive hepatocyte-mediated mutagenesis at premutagenic step has been evaluated in Chinese hamster ovary (CHO) cells with the detection of single strand breaks (SSB) induced by procarcinogens. SSB was detected by alkaline elution technique. Rat primary hepatocytes were prepared from adult rats using the in situ collagenase perfusion technique. Benzo(a)pyrene did not induce any SSB in CHO cells cultured alone. But when the CHO cells were cocultivated with rat primary hepatocytes, a significant SSB was induced at $5\times10^{-5}$ or $10^{-4}$M. Aflatoxin Bl induced SSB to a small extent in CHO cell cultured alone, but in the cocultivation with hepatocyte SSB was greatly increased at $10^{-6},\;5\times10^{-6}$, and $10^{-5}$M. The elution of DNA are 4.4, 8.5 and 14.4\% for $10^{-6},\;5\times10^{-6}$, and $10^{-5}$M of AF-B$_1$ in 6 hr exposure, respectively, and 10.5, 14.1 and 15.4\% in 12 hr exposure, respectively. In general, SSB induced by procarcinogens was dosedependent and exposure time dependent in this system. When the proteinase-K was added in cell lysis, the elutions of DNA were increased more 9 to 15\% than those of samples not using the enzymes.