Development of resistance to the mitoinhibitory effects of orotic acid during experimental liver carcinogenesis

Neoplastic cells have long been recognized as having a multiresislantphenotype. However, it is not known whether different properties relatable to resistance were acquired at the initiated ceil stage itself or acquired at various stages during the carcinogenic process. This question was explored using resistance to the mitoinhibitory effects of orotic acid, a liver tumor promoter. Accordingly, male Fischer 344 rats were initiated with diethylnitrosamine (DENA) and later exposed to the resistant hepatocyte (RH) model of liver tumor promotion or to no further treatment. Several weeks later the rats were subjected to 2/3 partial hepatectomy (PH) in the presence of orotic acid at levels which are mitoinhibitory to the normal hepatocytes. Proliferating hepatocytes were labelled with tritiated thymidine. Any focus that had a labelling index higher than the highest labelling index in the surrounding liver was considered as a resistant focus. The results indicated that the majority of the foci generated by DENA alone in the absence of an exogenous treatment are not resistant to the mitoinhibitory effects of orotic acid. In contrast, a large number of foci initiated by DENA and promoted by the RH model are resistant. Further, rats initiated with DENA and exposed to the components of the RH model i.e., 2-acetylaminofluorene (2-AAF) or CCl4 under non-tumor promoting conditions also exhibited foci many of which are resistant to the mitoinhibitory effects of orotic acid. These results are interpreted to indicate that the initiated hepatocyte is not resistant to the mitoinhibitory effects of orotic acid but acquires resistance-upon exposure to either the promoting regimen or to 2-AAF or CCl4. In order to assess the functional significance of this acquired resistance, experiments were designed to determine whether the initiated hepatocytes that acquired resistance to the mitoinhibitory effects of orotic acid can be selectively amplified with 2/3 PH in the presence of levels of orotic acid that are mitoinhibitory to normal hepatocytes. The results indicated that the number, size and the percent area occupied by the foci were significantly higher in initiated rats that were exposed to CCl4 than in those initiated rats not exposed to CCl4, suggesting that induction of resistance by CCl4 in the initiated hepatocytes permits them to be selectively amplified by 2/3 PH in the presence of a mitoinhibitory level of orotic acid. Our results suggest that the initiated hepatocyte is not a resistant phenotype (resistance defined as resistance to the mitoinhibitory effects of erolic acid), but has acquired the ability to express resistance upon exposure to either the promoting regimen (the RH model) or to agents such as 2-AAF and CCl4.