Apoptosis in cultured rat hepatocytes: The effects of tumour necrosis factor α and interferon γ

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Abstract
We investigated the cytotoxic effects of tumour necrosis factor α (TNF α) and interferon γ(IFN γ) on rat hepatocytes in culture. Under phase contrast microscopy, we found a small number of dying hepatocytes in control cultures, each having been transformed into a cluster of small spheres. Under transmission electron microscopy, these cells showed the characteristics of apoptosis. TNF α and a combination of TNF α and IFN γ exerted a cytotoxic effect, whereas IFN γ showed no significant cytotoxicity when assessed by neutral red assay and by measuring LDH activity in culture medium. Under phase contrast microscopy, the number of apoptotic cells increased with the addition of either TNF α or IFN γ, and markedly with the addition of both. DNA extracted from apoptotic cells cultured with TNF α and IFN γ was fragmented, and a set of bands of the ‘200 bp ladder’, which is characteristic of the DNA of apoptotic cells, was observed in agarose gel electrophoresis. These findings indicate that cultured hepatocytes die from apoptosis. TNF α killed cultured rat hepatocytes by increasing apoptosis, and this effect was potentiated by the addition of INF γ, which by itself was also weakly cytotoxic.