title: Immunohistochemical detection of activated caspases in apoptotic hepatocytes in rat liver.
abstract: 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.
authors.affiliation: Institute of Pharmacology and Toxicology, Department of Toxicology, University of Tübingen, Wilhelmstr. 56, 72074 Tübingen, Germany.
authors.forename: Veit-Simon
authors.initials: VS
authors.lastname: Eckle
authors.forename: Albrecht
authors.initials: A
authors.lastname: Buchmann
authors.forename: Wilfried
authors.initials: W
authors.lastname: Bursch
authors.forename: Rolf
authors.initials: R
authors.lastname: Schulte-Hermann
authors.forename: Michael
authors.initials: M
authors.lastname: Schwarz
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000032:analysis / Q000096:biosynthesis*; D002452:Cell Count; D017373:Cyproterone Acetate / Q000633:toxicity; D005260:Female; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D007150:Immunohistochemistry / Q000379:methods*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D011230:Precancerous Conditions / Q000201:enzymology; D051381:Rats; D017208:Rats, Wistar
chemical_list: D017373:Cyproterone Acetate; C505410:Casp3 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases