Lack of Effect of Medium Supplementation With Pyruvate and Hormones on Cytochrome P450-mediated Activity of Rat Hepatocytes in Primary Culture

Porntip Wirachwong and Jeffrey R. Fry

The loss of cytochrome P450 (CYP)-dependent activity continues to be a problem in the use of cultured hepatocytes in xenobiotic toxicity studies. It has been reported that the inclusion of pyruvate and various hormones in the culture medium improves the maintenance of various hepatic functions, including that of CYP2C11 mRNA expression. We have studied this further, by investigating the effects of the addition of pyruvate and hormones on various CYP-dependent enzyme activities and on the CYP-dependent toxicity of precocene II in rat hepatocyte cultures. No beneficial effects of this medium supplementation could be demonstrated.
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In Vitro Cytotoxicity of Four Different Buffers for Use in Peritoneal Dialysis

Lena E. Järkelid, Eva Svensson, Reinhold Deppisch and Anders

Various buffers can be used in fluids for peritoneal dialysis (PD). Lactate is the most frequently used buffer, but bicarbonate and pyruvate have been suggested as more biocompatible alternatives. In the past, acetate was used as a buffer in PD fluids, but was abandoned after being linked with sclerosing peritonitis and loss of ultrafiltration. When a new buffer for PD fluids is introduced, one of its most important characteristics is that it must be non-toxic, i.e. that it does not influence fundamental cellular functions. The aim of this study was to investigate the basal cytotoxicity of bicarbonate, acetate, lactate and pyruvate at neutral pH. As target cells, we used cultured mouse fibroblast-like L-929 cells, a well-known cell line approved by the authorities for regulatory use, and primary human mesothelial cells, which are the cells that line the peritoneal cavity and are exposed to the PD fluid in vivo. Pyruvate was more cytotoxic than lactate and bicarbonate, and no significant difference in cytotoxicity was found between lactate and bicarbonate. The human mesothelial cells were more sensitive to exposure to pyruvate than the L-929 fibroblast-like cells, but less sensitive to exposure to pure PD fluids. Thus, we recommend that both types of cell are used for the evaluation of the biocompatibility of PD fluids.
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Pyruvate-induced Long-term Maintenance of Glutathione S-Transferase in Rat Hepatocyte Cultures

Tamara Vanhaecke, André Foriers, Albert Geerts, Elizabeth A. Shephard, Antoine Vercruysse and Vera Rogiers

The addition of pyruvate to the culture medium has been reported to improve the maintenance of P450-dependent enzyme expression in primary rat hepatocyte cultures. In this study, the effects of 30mM pyruvate on cell morphology, albumin secretion and glutathione Stransferase (GST) expression were investigated as a function of the time in culture. The effect of triiodothyronine (T3) exposure on GST expression was also measured in pyruvate-treated cultures. Transmission electron microscopy showed that untreated hepatocytes deteriorated after culture for 7 days, whereas the morphology of the pyruvate-treated cells was similar to that observed in intact liver tissue. The albumin secretion rate was significantly higher in rat hepatocytes exposed to pyruvate than in control cells. In the presence of pyruvate, μ and α class GST activities were well maintained, whereas GST π activity was increased over the entire culture period. HPLC analysis revealed that the complement of GST subunits present in hepatocytes is altered during culture with pyruvate: μ class proteins remained relatively constant, whereas a decrease in the α class content was accompanied by a strong increase in GST subunit P1 (GSTP1). The induction of GSTP1 was confirmed at the mRNA level. In control cultures, π class GST activity was increased, but total, μ, and α class GST activities continuously declined as a function of culture time and became undetectable beyond 7 days in culture. At the protein and mRNA levels, a much smaller increase in GSTP1 was observed than in the pyruvate cultures. When the pyruvate-treated cell cultures were exposed to T3, an inhibitory effect on GST activities and proteins was found. These results indicate that this simple culture model could be useful for studying the expression and regulation of GST.
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