Different Properties of Microsomal UDP‐Glucuronyltransferase in Buffalo Rat Liver and a Clonal Strain of Rat Hepatoma Cells Derived from the Same Rat Strain

Abstract

Abstruct:Optimal conditions for the synthesis of o-aminophenol and p-nitrophenol glucuronides by a clonal strain of rat hepatoma cells (MHIC1) in culture were established. Properties of glucuronyltransferase (UDP-glucuronate glucuronyltransferase (acceptor unspecific), E. C. 2.4.1.17) in homogenates of cultured hepatoma cells, subcutaneous tumours derived from these cells in rats, as well as livers of Buffalo rats were studied. In rat liver 83-90 % and 92-95 % of the glucuronyltransferase activity in homogenates to p-nitrophenol and o-aminophenol respectively were latent, the latency being most pronounced in male animals. With homogenates of cultured hepatoma cells, on the other hand, digitonin activated 1.3 and 1.8-fold only with p-nitrophenol and o-aminophenol as acceptors; other potential activators (Triton X-l 00, UDP-N-acetylglucosamine and diethylnitrosamine) were either without effect or inhibited the enzyme. A I .5-2-fold higher degree of activation of glucuronyltransferase was found in homogenates of hepatoma tumours derived from the same cells injected subcutaneously into Buffalo rats. The specific activity of fully activated glucuronyltransferase in homogenates of cultured hepatoma cells was, however, 2-6.5-fold higher than that of the fully activated rat liver enzyme. The rate of p-nitrophenol glucuronide synthesis by cultures of hepatoma cells increased up to a concentration of 0.10 mM of the aglycone in the growth medium. At concentrations of 0.3 mM and higher, a peculiar lag period was seen before any glucui-onide appeared in the medium. This phenomenon was not seen with o-aminophenol as acceptor and not in broken cell preparations with either substrate. The maximal rate of o-aminophenol glucuronidation in cultures of the hepatoma cells corresponded to that found for homogenates with 0.254.50 mM UDPglucuronate added to the incubation mixture. This value is in good agreement with the presumed intracellular levels of UDP-glucuronate in the liver cell in vivo.