Harald Gschaidmeier

Paracetamol glucuronidation by recombinant rat and human phenol UDP-glucuronosyltransferases

Stably expressed human and rat phenol UDP-glucuronosyltransferases (UGTs) of the UGT1 complex (HlugP1, HlugP4 and 3-methylcholanthrene-inducible rat UGT1A1, the latter considered to be an orthologous enzyme to HlugP1) have been used to investigate the role of UGTs in paracetamol glucuronidation. Kinetic analysis of recombinant UGTs was compared to that of total UGT activities in liver microsomes. Paracetamol was found to be an overlapping substrate of several UGTs. It shows higher affinity for HlugP1 and rat UGT1A1 (apparent Km values of 2 and 3 mM, respectively) than for HlugP4 (Km = 50 mM) and other UGTs present in liver microsomes (Km values of > 12 mM). Glucuronidation of paracetamol with HlugP1 contrasts with that of 6-hydroxychrysene and of 4-methylumbelliferone, which are conjugated with higher affinity by HlugP4 than by HlugP1. Due to the wide tissue distribution of rat UGT1A1, paracetamol glucuronidation was also investigated in extrahepatic rat and human tissues. Paracetamol UGT activity was present and inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat kidney, lung and spleen. It was also detected in human kidney. A selective cDNA probe for exon 1 of HlugP1 cross-reacted with mRNA from both human liver and kidney. The results demonstrate that paracetamol is conjugated by HlugP1 and its rat orthologue UGT1A1 with higher affinity than by HlugP4 and other UGTs.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-mediated membrane translocation of c-Src protein kinase in liver WB-F344 cells.

Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental contaminant and the most potent agonist of the aryl hydrocarbon receptor (AhR). Persistent activation of the AhR has been shown to be responsible for most TCDD-mediated toxic responses, including liver tumour promotion. However, the mechanisms responsible for these complex toxic reactions are still unknown. TCDD (1 nM) has previously been shown to reduce DNA synthesis of primary hepatocyte cultures and cell contact inhibition of confluent WB-F344 cells. The latter model was used to study early effects of TCDD on protein tyrosine kinase c-Src in confluent WB-F344 cells. It was found that TCDD decreased cytosolic c-Src (protein and tyrosine kinase activity) after 20–60 min, and increased c-Src in the membrane fraction. Membrane translocation of c-Src occurred in the presence of 100 μM cycloheximide and was observed after treatment with 1 nM TCDD or 50 nM 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin. Under these conditions epidermal growth factor (EGF) receptor tyrosine phosphorylation was also studied. As expected, its phosphorylation was low in confluent cells but was significantly enhanced by TCDD treatment. Pretreatment of WB-F344 cells for 1 h with 1 μM geldanamycin, which disrupts cytosolic heat shock protein Hsp90 complexes with AhR and Src, abolished TCDD-mediated Src translocation and TCDD-mediated reduction of cell contact inhibition. The WB-F344 cell model appears to be very useful to study TCDD effects on protein tyrosine kinases and of signaling pathways responsible for modulation of the cell cycle by TCDD.

FUNCTIONS AND TRANSCRIPTIONAL REGULATION OF PAH-INDUCIBLE HUMAN UDP-GLUCURONOSYL-TRANSFERASES

Functions and regulation of selected human UDP-glucuronosyltransferases (UGT1A1, UGT1A4, UGT1A6, UGT1A9, UGT2B7, UGT2B15) are summarized. Evidence for at least two PAH-inducible UGTs (UGT1A6 and UGT1A9) is presented, which, however, are also constitutively expressed in a tissue- and cell-specific manner. These isoforms have recently been characterized to conjugate planar and bulky phenols, respectively. Using a selective RT-PCR method, UGT1A6 expression was detected in a variety of tissues (liver, kidney, lung, intestine, and pharyngeal mucosa). PAH-inducible UGTs may cooperate in the metabolism of phenolic metabolites of benzo(a)pyrene. Studies with stably expressed isoforms suggest that UGT1A9 is responsible for the formation of benzo(a)pyrene-3.6-diphenol diglucuronide, the major biliary metabolite of benzo(a)pyrene.

Ah receptor-controlled transcriptional regulation and function of rat and human UDP-glucuronosyltransferase isoforms

Transcriptional regulation and function of rat and human PAH-inducible UDP-glucuronosyltransferase (UGT) isoforms have been studied. 1. At least two PAH-inducible UGT isoforms are expressed in a variety of tissues, the rat isoforms UGT1A6 and UGT1A7, and the human isoforms UGT1A6 and UGT1A9. 2. For the rat and human UGT1A6 isoforms two modes of tissue- and cell-specific regulation were found, PAH-inducible and constitutive expression. 3. Transient transfection studies, using human UGT1A6/CAT fusion constructs and colon carcinoma Caco-2 cells, revealed that PAH induction of human UGT1A6 is mediated by the Ah receptor. 4. Cell-expressed UGT isoforms were used to study their function in PAH metabolism. Rat UGT1A7 and human UGT1A9 appear to be more efficient than the corresponding UGT1A6 isoforms in catalyzing glucuronide formation of PAH phenols and diphenols. Several isoforms may act together in the formation of benzo(a)pyrene-3.6-diol diglucuronide, the major glucuronide found in rat bile. The results suggest complex modes of transcriptional regulation of PAH-inducible UGTs. They also suggest a major role of these UGT isoforms in detoxication of PAHs.

Formation of mono- and diglucuronides and other glycosides of benzo(a)pyrene-3,6-quinol by V79 cell-expressed human phenol UDP-glucuronosyltransferases of the UGT1 gene complex

Glucuronidation of quinols of polycyclic aromatic hydrocarbons (PAHs) represents an important detoxication pathway preventing toxic quinone/quinol redox cycles. Therefore, mono- and diglucuronide formation of benzo(a)pyrene-3,6-quinol was investigated and compared to that of structurally related 3,6-dihydroxychrysene and simple phenols (1-naphthol and 4-methylumbelliferone) using V79 cell-expressed human UGT1.6 (= P1) and human UGT1.7 (= P4). Properties of human UGT1.6 were compared to those of the rat ortholog. Cofactors related to UDP-glucuronic acid such as UDP-galacturonic acid and UDP-glucose were also studied. It was found that rat and human UGT1.6 and human UGT1.7 catalyse monoglucuronide formation of planar PAH quinols. Diglucuronide formation was only detectable with human UGT1.7. The UGT isozymes studied also formed galacturonides and, although only to a minor extent, glucosides. Rat UGT1.6 (but not the human ortholog) catalysed digalacturonide formation of benzo(a)pyrene-3,6-quinol; the in vivo significance of galacturonide formation remains to be established. The results suggest that planar PAH phenols and quinols are conjugated more efficiently by human UGT1.7 than by UGT1.6, which preferentially conjugates simple planar phenols.

Radiation inactivation analysis of microsomal UDP-glucuronosyltransferases catalysing mono-and diglucuronide formation of 3,6-dihydroxybenzo(a)pyrene and 3,6-dihydroxychrysene

Indirect evidence has suggested that multiple subunits of microsomal UDP-glucuronosyltransferases (UGTs) are involved in diglucuronide formation of diphenols of polycyclic aromatic hydrocarbons (Bock et al., Mol Pharmacol 42: 613-618, 1992). To substantiate this suggestion functional target sizes of UGTs catalysing these reactions were determined in microsomes in situ by radiation inactivation analysis. Target sizes of UGTs catalysing the glucuronidation of 1-naphthol and 6-hydroxychrysene were found to be 91 +/- 29 and 120 +/- 27 kDa, respectively. However, target sizes for mono- and diglucuronide formation of 3,6-dihydroxybenzo(a)pyrene were 118 +/- 33 and 218 +/- 24 kDa, respectively. Similarly, using 3,6-dihydroxychrysene as substrate target sizes of 109 +/- 21 and 101 +/- 23 kDa were found for 6-O-monoglucuronide and 3-O-monoglucuronide formation and a target size of 192 +/- 34 kDa observed for diglucuronide formation. Based on subunit molecular masses of 50-60 kDa for UGTs, these results suggest that UGTs involved in monoglucuronide formation of phenols may function as dimers. In contrast, UGTs involved in diglucuronide formation of diphenols of polycyclic aromatic hydrocarbons may function as tetramers in microsomes in situ.

Mono- and diglucuronide formation from benzo[a]pyrene and chrysene diphenols by AHH-1 cell-expressed UDP-glucuronosyltransferase UGT1A7

Polycyclic aromatic hydrocarbon (PAH)-type compounds induce at least two rat UDP-glucuronosyltransferase isoforms, UGT1A6 and UGT1A7. Among the glucuronidation reactions of PAH metabolites studied, mono- and diglucuronide formation of benzo[a]pyrene and chrysene-3,6-diphenol showed the highest induction factors in rat liver microsomes. Availability of AHH-1 cells stably expressing UGT1A7 allowed us to study whether this PAH-inducible isoform could catalyze benzo[a]pyrene and chrysene-3,6-diphenol glucuronidation. It was found that UGT1A7 indeed catalyzed mono- and diglucuronide formation of both benzo[a]pyrene and chrysene 3,6-diphenols. V79 cell-expressed rat UGT1A6 also catalyzed these reactions, except for chrysene diphenol diglucronide formation (Bock et al., Mol Pharmacol 42: 613-618, 1992). Enzyme kinetic studies of the glucuronidation of 6-hydroxychrysene (used as a stable PAH phenol) indicated that UGT1A7 conjugated this compound with a lower apparent Km value (0.1 microM) than UGT1A6 (10 microM). The results suggest that the two PAH-inducible UGTs may cooperate in conjugating PAH metabolites, but that UGT1A7 is more efficient.

Comparative bioavailability of the microemulsion formulation of cyclosporine (Neoral) with a generic dispersion formulation (Cicloral) in young healthy male volunteers.

The aim of this study was to compare the bioavailability of cyclosporine (CyA) from the generic dispersion formulation Cicloral (CIC) with the microemulsion formulation Neoral (NEO) and the original Sandimmune (SIM) capsules after single doses of 100, 300, or 600 mg of drug, respectively. The study was performed according to an open 3-period cross-over design with 12 young healthy male volunteers for each dosage. The concentrations of CyA and its main metabolites were determined by high performance liquid chromatography in whole blood and urine up to 48 hours postdosing. Peak concentrations and area under the time-concentration curve were greater for the NEO and CIC formulations compared with SIM, and the mean bioavailability of CIC was significantly (P<0.05) lower compared with NEO. The bioavailability of SIM compared with NEO was 54% to 71%, in agreement with previous results. Bioequivalence was not demonstrated between CIC (test) and NEO (reference) as the 90% confidence intervals were outside the 80% to 125% guidelines based on log-transformed AUCs, and were 75.2% to 87.7% at 100 mg, 79.2% to 91.8% at 300 mg, and 76.6% to 94.5% at 600 mg doses. The respective values for Cmax were 78.9% to 94.6%, 80.7% to 95.0%, and 71.4% to 84.1%. A good correlation was demonstrated between the urinary recovery of CyA and the AUC4. Therefore, the urinary recovery of CyA may be helpful as a surrogate parameter for the systemic exposure of patients to CyA. Whereas the relative amount of hydroxylated metabolites (AM1, AM9, AM1c) was similar for all formulations and doses, the urinary recovery of the N-demethylated metabolite AM4N decreased with increasing dose indicating saturable metabolism. No relationship could be demonstrated between CYP3A activity using dextromethorphan as a probe for the metabolic clearance of CyA.