Number of Simultaneously Expressed Enzyme Alterations Correlates with Progression of N‐Ethyl‐N‐hydroxyethylnitrosamine‐induced Hepatocarcinogenesis in Rats

Preneoplastic and neoplastic liver cell lesions, induced by EHEN (N‐ethyl‐N‐hydroxyethylnitrosamine) in rats, were investigated to establish the numbers of simultaneously expressed altered enzyme phenotypes within the lesion cells. The lesions were divided into 5 classes on the basis of altered expression in one or more of the following 5 enzymes: glutathione S‐transferase placental form, glucose‐6‐phosphate dehydrogenase, glucose‐6‐phosphatase, adenosine triphosphatase, and γ‐glutamyl transpeptidase. Class 1 lesions contained cells expressing one altered enzyme. Similarly, class 2, 3, 4 and 5 lesions had cells simultaneously expressing 2, 3, 4, and 5 enzyme alterations, respectively. Four histopathological categories of lesions, ACF (altered cell foci) (274 lesions), HN (hyperplastic nodules) (47 lesions), HCC (hepatocellular carcinomas) (99 lesions) and THC (transplanted hepatocellular carcinomas) (5 lesions) were studied. Proliferation potential was assessed in terms of 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation. The distribution profiles of classes 1 to 5 showed a clear reciprocal change from low class (1 to 2 enzymes) predominance in ACF to high class (4 to 5 enzymes) predominance in HN. Increase of BrdU labeling indices was clearly correlated with progression from HN to HCC. Only a small population of class 5 ACF showed a high BrdU labeling index, indicating particular potential for further development. Thus, the stages of EHEN‐induced neoplasia were found to be characterized by gradual increase in the number of altered enzyme phenotypes, with acquisition of proliferative potential being associated with further progression towards malignant conversion.