Hyperamplification of centrosomes and asynchronous nuclear division induced by N-nitrosodimethylamine in rats

Mechanisms of hepatocyte multinucleation was investigated in rats exposed to N-nitrosodimethylamine (NDMA). By using immunohistochemical reaction to gamma-tubulin it was established that the number of cells containing three and more centrosomes increased in 48 hours after NDMA injection. It was shown that formation of extra-centrosomes in hepatocytes is enhanced by oxidative stress induced by cytochromes P450 superfamily in the course of NDMA metabolism. NDMA administration led to a sharp increase of cytochrome P450 content in the liver, especially in 24 and 48 hours (3.3 and 2.8 times respectively) after NDMA injection. Extensive staining of cytoplasm from centrolobular hepatocytes was indicated by immunohistochemical reaction to cytochrome P4502E1 in 24 and 48 hours after the addition of NDMA. Malone dialdehyde (the derivative of lipid peroxidation) was shown to increase 1.1-2.0 times, whereas catalase activity as antioxidative agent reduced to 1.1-1.3 times in that time. Sunsequently (72-120 hours of NDMA treatment) the number of cells with three or more centrosomes, the intensity of cytoplasmic staining, cytochrome P450 and malone dialdehyde contents in the liver were determined to decrease, whereas catalase activity has increased. After 48 hours of NDMA treatment binucleated hepatocytes with various 3 H-thymidine distribution in nuclei appeared in NDMA-treated cell populations evidencing of asynchronous DNA synthesis. Immunohistochemical reaction against Ki-67 proliferation marker revealed asynchronism of nuclear proliferation activity in binucleated cells spreading not only to S-phase, but also to other phases of cell cycle, and namely G(1),G(2) and M. Thus, main mechanisms of hepatocyte multinucleation under NDMA exposure are accounted for by hyperamplification of centrosomes, asynchronous DNA synthesis and asynchronous mitosis.