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Evaluation of Metabolic Activation of 7,12-Dimethylbenz(a)anthracene in Vitro by Aroclor 1254-induced Rat Liver S-9 Fraction¹

Chemical Carcinogenesis Program, Frederick Cancer Research Center, Frederick, Maryland 21701

Short-term assays for detection of chemical carcinogens frequently rely on an Aroclor 1254-induced rat liver S-9 fraction for metabolic activation of test compounds. The ability of this in vitro system to reproduce the activation occurring in target tissue was investigated by examining the DNA adducts produced when the polycyclic aromatic hydrocarbon carcinogen, 7,12-dimethylbenz(a)anthracene (DMBA), was incubated with the S-9 fraction and calf thymus DNA. Analyses by Sephadex LH-20 column chromatography of hydrocarbon-deoxyribonucleoside adducts obtained after enzymic digestion of the [³H]DMBA-modified DNA revealed that the products of binding of DMBA to DNA in the presence of the S-9 fraction vary with the relative concentration of DMBA to S-9 fraction. Further analyses of these adducts by high-pressure liquid chromatography in the presence of the diol-epoxide-DNA adduct (isolated from mouse embryo cells exposed to [¹⁴C]DMBA) and chemically synthesized ultraviolet-absorbing markers of DMBA 5,6-oxide-deoxyribonucleoside adducts showed that, at high DMBA-S-9 ratios, DMBA 5,6-oxide-deoxyribonucleoside adducts were prominent among the products while, at low DMBA-S-9 ratios, the products included the diol-epoxide-DNA adduct found in target tissue. However, this adduct was always accompanied by other adducts not found in intact cellular systems. Inclusion of a metabolic inhibitor (1,1,1-trichloropropylene oxide) in the Salmonella mutagenicity assay demonstrated that high levels of revertants can be obtained from rat liver S-9 fraction-activated DMBA under conditions which should prohibit formation of the diol-epoxide. These results suggest that Aroclor 1254-induced rat liver S-9 fraction does not exactly reproduce the metabolic activation of this particular carcinogen in vivo and therefore should not be assumed to do this for other carcinogens.

¹ This work was supported by Contract N01-CO-75380 with the National Cancer Institute, NIH, Bethesda, Md. 20205.

² To whom requests for reprints should be addressed, at Chemical Carcinogenesis Program, Frederick Cancer Research Center, P. O. Box B, Frederick, Md. 21701.

Received 9/17/79. Accepted 11/30/79.

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				C. Bigger, J. Tomaszewski, A Dipple, and R. Lake Limitations of metabolic activation systems used with in vitro tests for carcinogens Science, July 25, 1980; 209(4455): 503 - 505. [Abstract] [PDF]