Immunohistochemical detection of CCl 4-induced, mitosis-related DNA synthesis in livers of trout and rat

Abstract

Monoclonal antibodies (MAB) directed against 5-bromo, 2′deoxyuridine (BrdU) were used to detect cells in which carbon tetrachloride-induced DNA synthesis had occurred in livers of rainbow trout ( Salmo gairdneri) and of rats. Trout exposed to CCl 4 (single i.p. injection, 2.5 ml/kg) were injected 45 h later with BrdU (50 mg/kg); 3 h after BrdU administration, trout were sacrificed and livers were processed for immunohistochemistry and histopathology. Anti-BrdU immunohistochemical analysis revealed labeling of only occasional hepatocyte nuclei. On the other hand, in rats given a single i.p. injection of CCl 4 (1.5 ml/kg) and injected 21 h later (3 h prior to sacrifice) with BrdU (50 mg/kg), immunohistochemistry revealed labeling of most hepatocyte nuclei. When control animals (not treated with CCl 4) of both species were administered BrdU and processed for immunohistochemistry, no labeled hepatocyte nuclei were detected. Immunohistochemical studies with cultured human fibroblasts treated with N-methyl- N′-, nitro- N-nitroso guanidine (MNNG) and BrdU demonstrated that replicative DNA synthesis was detected, but unscheduled DNA synthesis (UDS or DNA repair synthesis) was not detected. Therefore, MAB directed against BrdU proved useful in detecting chemically-induced replicative changes in liver cells from both trout and rat. Freeze-dried glycol methacrylate (GMA)-embedded tissue yielded excellent immunostaining and morphology. The immunohistochemical reaction appears particularly applicable to analyzing mitotic response and eliminates production of radioactive waste water when analyzing chemically-induced nuclear changes in aquatic species.