Failure to discriminate initiation from promotion of liver tumors in a long-term study with the phenobarbital-type inducer alpha-hexachlorocyclohexane and the role of sustained stimulation of hepatic growth and monooxygenases.

alpha-Hexachlorocyclohexane (alpha-HCH) was administered p.o. to female Wistar rats for periods of up to 33 months; doses were 20 mg/kg/day, 200 mg/kg every second week, or 420 mg/kg every third week. Increases of liver size, DNA, RNA, and protein (by 50 to 100%) and of drug-metabolizing enzyme activities (up to 300%) observed previously after single doses of alpha-HCH were found to persist after approximately one-third, 1, and 2 years of treatment. At 1 and 2 years, DNA synthesis was measured by [3H]thymidine uptake and was no higher than in controls. All changes regressed upon withdrawal of alpha-HCH after 1 year of treatment. These findings provide no evidence to suggest a protracted development of toxicity or of growth autonomy in the majority of liver cells. Foci of altered cells, neoplastic nodules, and in 2 animals hepatocellular carcinoma were detected histologically in the livers of 24 of 34 treated rats. In livers of 10 of 22 untreated control rats, foci of altered cells developed "spontaneously" between 12 and 34.5 months. If neoplastic lesions were induced by a single dose of diethylnitrosamine, 75 or 150 mg/kg, subsequent treatment with alpha-HCH led to the appearance of hepatocellular carcinoma with 7 months. Altogether, hepatocellular carcinoma within 7 months. Altogether, hepatocellular carcinomas were found in 18 of 21 rats treated with both agents but in only 3 of 26 animals treated with diethylnitrosamine alone. The results show that determination of tumor numbers alone in a long-term animal experiment does not allow one to decide whether alpha-HCH (and similar "xenobiotic inducers") is an initiating carcinogen or merely promotes tumorigenesis from "spontaneous" lesions. Our findings support the latter possibility by the failure to detect evidence suggesting initiating potential of alpha-HCH, by the enhanced mitotic response to alpha-HCH, by the enhanced mitotic response to alpha-HCH in foci of altered cells as reported elsewhere, and by the observation of a permanent stimulatory action on liver growth during prolonged exposure to alpha-HCH.