CONJUGATION REACTIONS IN HUMAN LIVER AND HUMAN LIVER CELLS IN CULTURE

Abstract

Glucuronidation and sulphate conjugation are quantitatively the major conjugation reactions in drug metabolism. The adult human liver is capable of carrying out both forms of conjugation. This study showed that the cultured foetal liver cells from second trimester aborted foetuses are capable of sulphate conjugation. Low levels of glucuronidation could also be detected. Cultured foetal liver cells have the full complement of enzymes required for sulphation. PAPS generation involving the enzymes ATP sulphurylase and APS kinase was measured by a radiometric assay. The synthesis of PAPS was linear for 15 mins with a pH optimum at 8.0. The Km for inorganic sulphate was 0.24mM. Phenolsulphotransferase (PST) activity and the overall sulphation of N-acetyldopamine (NADA) was measured using extracts of cultured foetal liver cells. Both were radiometric assays employing PAP35s and sodium 35sulphate respectively. The PST activity and the overall sulphate conjugation reactions could be demonstrated with three different substrates: NADA, dopamine and 1-naphthol. Kinetic studies were carried out with NADA. The apparent Km of NADA was 38uM as determined by both assays. For the overall sulphation, the apparent Km for inorganic sulphate was O.11mM, and that for PAPS as determined by the PST reaction was 2.57uM. Two pH optima of 6.7 and 8.6 were observed for both assays. An anti-rat liver paracetamol PST antibody was used to screen a rat liver lambda Zap II cDNA library. A 1.10kb cDNA was subsequently isolated. A fast and sensitive procedure employing high performance liquid chromatography with fluorometric detection (HPLC-fluorometry) was developed to study UDP-glucuronosyltransferase (UDPGT) activity. The formation of harmol glucuronide was linear for 20mins with adult liver microsomes. Two apparent Kms were observed for harmol (3.15uM and 38.3uM) and for UDPGA (158uM and 473uM), These data indicate the presence of a high and a low affinity form of UDPGT in the adult liver, Extracts of cultured foetal liver cell were capable of forming harmol glucuronide. However, only the low affinity form was detected. The apparent Kms were 51.4UM for harmol and 396uM for UDPGA. The UDPGT activity in the foetal liver cells was induced by treating the cultures with phenobarbital. A more dramatic increase (> 4 folds) was observed when HgC12 was used. HgC12 also induced the expression of the high-affinity UDPGT form in the cells. These changes brought about by HgC12 were observed using harmol and 1-naphthol as acceptors for glucuronic acid. Administration of HgC12 to young adult mice also produced significant increase in renal UOPGT activity. Intact, cultured cells were capable of sulphating NADA and forming harmol glucuronide. Despite an initial decline in the activities of both reactions, a relatively constant level could be maintained in confluent cultures, thus enabling detailed kinetic studies to be carried out. A comparison of the activities of both conjugating reactions in the cells with those of the adult liver showed that sulphate conjugation is much more developed than glucuronidation.