Effect of pyruvate on glutathione s-transferase expression in primary cultures of rat hepatocytes.
Recently, primary rat hepatocyte cultures supplemented with 30 mm pyruvate and various hormones have been proposed as suitable long-term in vitro models for xenobiotic metabolism studies. In this study, the effect of 30 mm pyruvate on the cytosolic phase II biotransformation enzyme glutathione S-transferase (GST, EC 2.5.1.18) has been investigated. Adult male rat hepatocytes were brought into primary culture in serum free Williams' medium E with and without supplementation of 30 mm pyruvate. Total, Mu and Alpha class GST activities as well as GST subunit patterns were determined on the day of the isolation and as a function of culture time. Cell morphology and albumin secretion were also examined. After 7 days of culture, the morphology of pyruvate-treated cultures was still intact and bile canaliculi were clearly visible, whereas control cells without pyruvate had deteriorated and lost their normal morphology. The albumin secretion rate was higher in pyruvate-supplemented than in non-supplemented cultures. Total, Mu and Alpha class GST activities were well maintained in the presence of pyruvate; conversely, without pyruvate, all GST isoenzyme activities were significantly decreased. These findings were confirmed at the protein level. However, when hepatocytes received medium containing pyruvate for 7 days, the Pi class subunit 7, normally not present in adult liver cells, was expressed at a much higher level than was the case in untreated hepatocyte cultures. This study clearly shows that GST activities and GST proteins, as well as cell morphology and albumin secretion, are better maintained in primary monolayer cultures of adult rat hepatocytes supplemented with 30 mm pyruvate than in control cultures. However, it was also found that pyruvate is a very strong inducer of GST Pi. Consequently, the percentage GST subunit pattern of cultured rat hepatocytes is significantly affected by pyruvate supplementation, which may have consequences for in vitro xenobiotic metabolism studies.
Vanhaecke
T
T
Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium.
Vercruysse
A
A
Rogiers
V
V