Cellular changes that occur within the liver before and during liver fibrosis have been studied. At various times (0-90 days) after γ irradiation (0-25 Gy) of the whole liver, hepatocytes and liver nonparenchymal cells were isolated by enzymatic dissociation of the liver and differential centrifugation. Differential cell counts were done to quantify the yield of hepatocytes and nonparenchymal cells. Hepatocyte function in vitro was assayed by the uptake of rose bengal and compared with the clearance of the dye in vivo. Intracellular alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were used to estimate the effect of radiation on the phenotypic expression of hepatocytes. Gene expression of transforming growth factor β1 (TGF-β1), a cytokine which has been found to play a role in hepatic fibrosis, was measured by <tex-math>${}^{32}{\rm P}\text{-}{\rm cDNA}\colon {\rm mRNA}$</tex-math> hybridization of poly( A+) mRNA isolated from purified populations of hepatocytes and nonparenchymal cells. Morphologically, hemorrhages were evident in perfused livers 28 days after 25 Gy whole-liver irradiation. This injury was preceded by a significant increase in liver nonparenchymal cells at 2 weeks, the earliest time after irradiation at which measurements were made. Significant increases in liver nonparenchymal cells occurred at doses greater than 10 Gy and persisted throughout the follow-up period after irradiation. The lowest dose studied (5 Gy) inhibited both normal growth of the liver and the associated increase in the number of hepatocytes. Doses greater than 10 Gy resulted in a dose-dependent decline in liver mass and hepatocytes. Viable hepatocytes isolated from irradiated dysfunctional livers, with respect to clearance of rose bengal, were functionally normal in vitro. A dose-dependent decline in the ALT and AST contents of liver cell suspensions and homogenates was observed but the relative decline in ALT was greater, resulting in lower ALT/AST ratios. This decline in the ALT/AST ratios occurred at doses (<15 Gy) which caused no loss of hepatocytes, suggesting possible conversion of ALT-rich periportal hepatocytes to ALT-poor hepatocytes due to changes in the microenvironment and/or aging of the cell population. TGF-β1 mRNA was detected mainly in nonparenchymal cells, and radiation preferentially enhanced the TGF-β1 gene expression in these cells. These data suggest that radiation-induced alterations in liver nonparenchymal cell populations may be responsible for microvascular fibrosis, which results in a cascade of pathological events which lead to hepatocyte loss.

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