In Vitro - In Vivo Correlation of Gene Expression Alterations Induced by Liver Carcinogens

G.      Guenther; J.      G. Hengstler; A.      Oberemm; A.      Lampen; U.      Gundert-Remy; H.      Mielke; P.      Godoy; R.      Reif; J.      Sisnaiske; T.      Heise; A.      Ghallab; J.      Rahnenführer; B.      Hellwig; H.      J. Ahr; H.      Ellinger-Ziegelbauer; D.      Storm; M.      Schug

Abstract

Although cultivated hepatocytes are widely used in the studies of drug metabolism, their application in toxicogenomics is considered as problematic, because previous studies have reported only little overlap between chemically induced gene expression alterations in liver in vivo and in cultivated hepatocytes. Here, we identified 22 genes that were altered in livers of rats after oral administration of the liver carcinogens aflatoxin B1 (AB1), 2-nitrofluorene (2-NF), methapyrilene (MP) or piperonyl-butoxide (PBO). The functions of the 22 genes have been classified into two groups. Genes related to stress response, DNA repair or metabolism and genes associated with cell proliferation, respectively. Next, rat hepatocyte sandwich cultures were exposed to AB1, 2-NF, MP or PBO for 24h and expression of the above mentioned genes was determined by RT-qPCR. Significant correlations between the degree of gene expression alterations in vivo and in vitro were obtained for the stress, DNA repair and metabolism associated genes at concentrations covering a range from cytotoxic concentrations to non-toxic/in vivo relevant concentrations. In contrast to the stress associated genes, no significant in vivo/in vitro correlation was obtained for the genes associated with cell proliferation. To understand the reason of this discrepancy, we compared replacement proliferation in vivo and in vitro. While hepatocytes in vivo, killed after administration of hepatotoxic compounds, are rapidly replaced by proliferating surviving cells, in vitro no replacement proliferation as evidenced by BrdU incorporation was observed after washing out hepatotoxic concentrations of MP. In conclusion, there is a good correlation between gene expression alterations induced by liver carcinogens in vivo and in cultivated hepatocytes. However, it should be considered that cultivated primary hepatocytes do not show replacement proliferation explaining the in vivo/in vitro discrepancy concerning proliferation associated genes.

Keywords: Hepatocytes, in vitro, sandwich culture, toxicogenomics, carcinogens, genotoxic, non-genotoxic, stress response genes, proliferation