Controlled Extension of the Tumor Promotion Period Enhances Progression of GSTP-Positive Hepatocellular Lesions in a Rat Model of Hepatocarcinogenesis.

Abstract

Histochemical approaches are critical for detecting early, clinically silent stages of hepatocarcinogenesis; however, the widely used diethylnitrosamine/2-acetylaminofluorene initiation-promotion (IP) model is time-constrained, limiting evaluation of lesion progression beyond the preneoplastic stage. This study established and validated an extended IP protocol to enhance histochemical detection and stage-resolved analysis of preneoplastic liver lesions while assessing the effects of intermittent fasting (IF) during promotion. Male Wistar rats underwent either the standard IP protocol or an extended version (IPx), in which promotion was prolonged by 3 weeks. IF was implemented during promotion in both models. Preneoplastic lesions were identified by immunohistochemical detection of rat placental glutathione S-transferase P (rGST-P), complemented by histological and molecular analyses. Promotion extension markedly increased the liver area occupied by rGST-P-positive altered hepatic foci and induced pronounced histoarchitectural alterations, improving assessment of advanced preneoplastic stages without progression to overt carcinoma. IF reduced lesion burden and partially preserved liver architecture, particularly in the extended model, and was associated with changes in markers of apoptosis, proliferation, inflammation, and metabolic regulation. These findings validate promotion extension as a methodological refinement of the IP model, enabling detailed analysis of advanced preneoplastic stages and effective evaluation of interventions that modulate early hepatocarcinogenic progression.