Collagen gel sandwich and immobilization cultures of rat hepatocytes: Problems encountered in expressing glutathione S-transferase activities

Abstract

Collagen gel sandwich and immobilization cultures of rat hepatocytes are two recently developed organotypical culture models. Basic information with respect to the maintenance of xenobiotic biotransformation pathways and the expression of key enzyme activities, however, is lacking, making their use in pharmaco-toxicological studies rather speculative. The expression of the glutathione S-tranferase (GST; EC 2.5.1.18) activity, a key phase II enzyme, has been chosen to study the various problems that may arise in expressing the results of cytosolic enzyme activities when rat hepatocytes are cultured using both new culture models. Collagen gel matrix easily entraps culture medium proteins. These interfere with the cytosolic protein content, a parameter versus which cytosolic enzyme activities, including GSTs, are usually expressed. The following solutions are proposed: expression of the cytosolic enzyme activity results versus either (i) microsomal proteins, these are not contaminated by medium proteins, or versus (ii) cytosolic proteins after a complete collagenase digestion (0.05% collagenase type I of Sigma, 45 min, 37 °C) of the collagen matrix. Expression of enzyme activities versus cellular DNA appears to be unacceptable since unreliable results were obtained due to entrapped DNA in the collagen matrix. Once it was known how to express cytosolic enzyme activity, the maintenance of GST activities was investigated in both culture models using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) as substrates for total and Mu class GSTs, respectively. Two culture media were compared, control medium (DMEM) with and without supplementation of l-proline (final concentration 60 μg/ml). In both culture models, after an initial decrease, total GST activities increased significantly up to values higher than those observed for freshly isolated cells. The Mu class GST activities were maintained constant for 7 days and increased thereafter. l-Proline supplementation of the culture medium prevented the initial decline in total and Mu class GST activities in both culture configurations but did not seem to be of crucial importance in the maintenance of GST activities in both culture models.