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Immunohistochemical Detection of Activated Caspases in Apoptotic Hepatocytes in Rat Liver

Institute of Pharmacology and Toxicology, Department of Toxicology, University of Tübingen, Wilhelmstr. 56, 72074 Tübingen, Germany

Albrecht Buchmann

Institute of Pharmacology and Toxicology, Department of Toxicology, University of Tübingen, Wilhelmstr. 56, 72074 Tübingen, Germany

Wilfried Bursch

Institut für Tumorbiologie-Krebsforschung, Borschkegasse 9a, A-1090 Vienna, Austria

Rolf Schulte-Hermann

Institut für Tumorbiologie-Krebsforschung, Borschkegasse 9a, A-1090 Vienna, Austria

Michael Schwarz

Institute of Pharmacology and Toxicology, Department of Toxicology, University of Tübingen, Wilhelmstr. 56, 72074 Tübingen, Germany, michael.schwarz@ uni-tuebingen.de

In our study we tested the utility of antibodies that specifically recognize the cleaved large (active) subunits of caspase-3 and caspase-9 for immunohistochemical detection of apoptotic hepatocytes in rat liver sections using archival material from cyproterone acetate (CPA)-treated and control rats. CPA blocks apoptosis of hepatocytes and discontinuation of CPA treatment results in a syncronized wave of hepatocyte apoptosis. By comparing liver sections from CPA-treated and control rats with high and low rates of apoptosis we observed a close correlation between the occurrence of cleaved caspase-positive apoptotic figures and H&E-stained apoptotic bodies when evaluated in parallel sections. Caspase-stained figures were either immuno-positive apoptotic bodies or pre-apoptotic hepatocytes showing cytoplasmic and/or nuclear caspase-staining with otherwise normal cellular appearance. In extension of these observations we developed a double-immunohistochemical staining procedure which enables the detection of caspase-3-positive apoptotic hepatocytes within glutathione-S-transferase-P (GST-P)-positive preneoplastic liver foci. By use of this technique, inhibition of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin as detected by counting of H&E-stained apoptotic bodies was found to correlate with a strong reduction of cleaved caspase-positive hepatocytes in GST-P-positive preneoplastic foci. In summary, this study demonstrates that cleaved caspase-positive apoptotic hepatocytes could be reliably identified and quantified both in normal and neoplastically transformed liver tissue.

Key Words: Apoptosis • caspase-3 • caspase-9 • immunohistochemistry • liver • hepatocarcinogenesis

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