Published in Cancer Detection and Prevention 1999; 23(5):485-495.

Nonlinearity of Nuclear Enlargement in Hepatocytes Induced by the Carcinogen N’-Nitrosomorpholine in Ova

Christiane Wiemann, D.V.M.,a Harald Enzmann, M.D.,b Eckehard Löser, Ph.D.,b and Gerhard Schlüter, Ph.D.b

aDepartment of Toxicology, BASF AG, Ludwigshafen, Germany, and bInstitute of Toxicology, Bayer AG, Wuppertal, Germany

Address all correspondence and reprint requests to: Address correspondence and reprint requests to: Chrsistiane Wiemann, D.V.M., Department of Toxicology, BASF AG, 67056 Ludwigshafen, Germany.

Abstract. In this study we investigated the effects of different doses of the carcinogenic nitrosamine N’- nitrosomorpholine (NNM) on the occurrence of enlarged nuclei in embryonic turkey liver in order to evaluate whether this parameter might represent a quantitative indicator of chemically induced hepatocarcinogenesis. Therefore fertile embryo turkey eggs were injected with NNM over a dose range of 125 µg 8 mg/egg at the first day of incubation. After incubation for 24 days, the embryonic livers were removed and processed for histologic evaluation. The induction of hepatocytes with enlarged nuclei (nuclear profiles > 35 µm2) was quantitated morphometrically in hematoxylin and eosin (H&E)-stained sections. The NNM treatment increased both the number of enlarged hepatocyte nuclei and the areas of the individual profiles of the enlarged nuclei in a dose-dependent manner. Exposure to 500 µg-8 mg NNM/egg resulted in a statistically significant increase in the number of hepatocytes with enlarged nuclei. The lower doses of 250 µg and 125 µg NNM/egg showed a similar albeit not significant trend. Signs for cytotoxic effects on the hepatocytes, such as necrosis or enhanced cytoplasmic vacuolization, were observed in tissue samples of embryos exposed to 4 or 8 mg NNM, but not after treatment with lower doses. The dose-effect curve for the induction of the nuclear enlargement was nonlinear, with a moderate slope for lower dose levels of 125-500 µg/egg and a steep slope for higher dose levels of 1-8 mg. Findings in rodents indicate a pathogenic link between the occurrence of enlarged nuclei and hepatocarcinogenesis. Based on the results with NNM, it is suggested that the in ovo model may represent a rapid, convenient, and inexpensive experimental approach for dose effect investigations on chemically induced hepatocarcinogenesis.

KEY WORDS: carcinogenesis, cell nucleus, dose response, morphometry, nonanimal, short-term test, toxicology.