Regioselective and stereospecific glucuronidation of trans- and cis-resveratrol in human.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a polyphenol present in wine, which has been reported to have anti-inflammatory, anti-platelet, and anti-carcinogenic effects. The glucuronidation of this compound and that of the cis-isomer also naturally present, has been investigated in human liver microsomes. Both isomers were actively glucuronidated. The reaction led to the formation of two glucuronides (3-O- and 4'-O-glucuronides), whose structure was characterized by LC-MS and proton NMR. Glucuronidation was regio- and stereoselective. It occurred at a faster rate with the cis-isomer and preferred the 3-position on both isomers. In addition, the glucuronidation of resveratrol was tested using several recombinant UDP-glucuronosyltransferase (UGT) isoforms. The reaction was catalyzed by UGT of the family 1A (UGT1A1, 1A6, 1A7, 1A9, 1A10). The bilirubin conjugating UGT1A1 was mainly involved in the 3-O-glucuronidation of trans-resveratrol, whereas the phenol conjugating UGT1A6 activity was restricted to cis-resveratrol. The UGT1A9 and 1A10 were active toward both isomers. The activity supported by UGT2B7 and UGT2B15 was very low and restricted to cis-resveratrol. UGT1A3, 1A4, 2B4, and 2B11 were unable to form resveratrol glucuronides.

Nonsteroidal anti-inflammatory drugs and phenols glucuronidation in Caco-2 cells: identification of the UDP-glucuronosyltransferases UGT1A6, 1A3 and 2B7.

Glucuronidation of phenols (1-naphthol, 4-methylumbelliferone) and nonsteroidal anti-inflammatory drugs (NSAIDs) such as ketoprofen, naproxen and carprofen was investigated in human colon carcinoma Caco-2 cell clones. Glucuronidation of these substances was highly effective in microsomes of the clones PD-7 and TC-7, but much lower in the PF-11 clone. The activity increased up to a maximum after 21 days of culture. RT-PCR experiments indicated that the PD-7 and TC-7 clones expressed the UDP-glucuronosyltransferase (UGT) isoforms UGT1A6, UGT1A3 and UGT2B7, which could account for the glucuronidation of phenols and carboxylic acids observed. Beta-naphthoflavone stimulated by 2-fold the enzyme activity toward 1-naphthol in PD-7 and TC-7 clones, but not in PF-11 cells. This increase was parallel to that of the UGT1A6 mRNA level. Glucuronidation of ketoprofen was also sensitive to the inducing effect of beta-naphthoflavone. Actinomycin D and cycloheximide did not affect the induction of UGT1A6 by beta-naphthoflavone, but suppressed that of ketoprofen UGT. The UGT1A3 mRNA content was enhanced by beta-naphthoflavone; by contrast, that of UGT2B7 was insensitive to the inducer. In conclusion, several UGT isoforms of both families 1 and 2, which glucuronidate phenols and carboxylic NSAIDs, have been identified in Caco-2 cells. They are differently sensitive to beta-naphthoflavone.

Expression and inducibility of UDP-glucuronosyltransferases 1-naphthol in human cultured hepatocytes and hepatocarcinoma cell lines.

The UDP-glucuronosyltransferase (UGTs) isoforms involved in the conjugation of 1-naphthol were characterized in human cultured hepatocytes and in two human hepatocarcinoma cell lines, KYN-2 and Mz-Hep-1 in terms of expression, kinetics and induction by drugs. Their properties were compared to those of UGT1*6 stably expressed in the V79 cell line (V79UGT1*6), which glucuronidates 1-naphthol preferentially. The determination of kinetic constants for glucuronidation of 1-naphthol revealed a two-site model in human hepatocytes, but a one-site model in the two hepatocarcinoma cell lines. Southern blot analysis of RT-PCR products, showed that the UGT1*6 mRNA was expressed in KYN-2, but not in Mz-Hep-1 cells. However, a mRNA encoding a UGT different from UGT1*6 was expressed in Mz-Hep-1 cells. The two inducers, beta-naphthoflavone and rifampicin exerted a differential effect, depending on the cell lines considered. Altogether, the results suggest that, in hepatocytes, two UGT isoforms, which glucuronidate 1-naphthol are expressed and are differentialy regulated by inducers. Both KYN-2 and Mz-Hep-1 cells express one of the two different UGT isoforms found in hepatocytes. The UGT isoform present in KYN-2 cells corresponds to UGT1*6, whereas in Mz-Hep-1 cells the UGT isoform present was different from UGT1*6 and UGT1*7.

Apolipoprotein (apo) E genotype and apoE concentration determine binding of normal very low density lipoproteins to HepG2 cell surface receptors.

The clinical relevance of apoE concentration in lipoprotein fractions should be evaluated. We investigated the impact of the common apolipoprotein (apo) E polymorphism in conjunction with very low density lipoprotein (VLDL) apoE concentration on the receptor binding properties of VLDL preparations from 17 normolipidemic subjects of the HepG2 cell surface receptors. All six apoE genotypes were studied. When apoE genotype alone was considered, two subgroups could be distinguished: VLDL without apoE isoform E2 (VLDL-3/3, VLDL-3/4, and VLDL-4/4) showed significantly higher affinity than VLDL with apoE2 (VLDL-4/2, VLDL-3/2, and VLDL-2/2). Once we adjusted for VLDL apoE content, we observed that VLDL affinity to HepG2 cell surface receptors decreased, according to apoE genotype, in the following order: VLDL-4/4 (100%) > VLDL-3/4 (93%) > VLDL-3/3 (82%) > VLDL-4/2 (53%) > VLDL-3/2 (36%) > VLDL-2/2 (30%). Moreover, we found that VLDL apoE concentration could modify isoform-specific binding. An analysis in 47 subjects showed that the concentration of total VLDL protein and the VLDL apoE concentration varied considerably. The variation of VLDL apoE was independent of apoE genotype and corresponding serum apoE levels. We conclude that, in addition to apoE genotype, apoE content of VLDL is an important determinant of the receptor binding properties of VLDL.

Inducibility of ethoxyresorufin deethylase and UDP-glucuronosyltransferase activities in two human hepatocarcinoma cell lines KYN-2 and Mz-Hep-1.

Two human hepatoma cell lines, KYN-2 and Mz-Hep-1 were characterized in terms of glucuronidation capacity and inducibility of cytochrome P4501A1/1A2 and several UDP-glucuronosyltransferases (UGTs). Cytochrome P4501A1/1A2 activity was measured using 7-ethoxyresorufin and that of UGTs with 16 different substrates. The effects of dimethyl sulfoxide (DMSO), 3-naphthoflavone, alpha-naphthoflavone, and rifampicin on these drug-metabolizing enzyme activities were studied. DMSO treatment increased in a dose-dependent manner the ethoxyresorufin O-deethylase (EROD) activity in KYN-2 cells, while an opposite effect was observed in Mz-Hep-1 cells. In KYN-2 cells, EROD was more responsive toward beta-naphthoflavone treatment in combination with DMSO. This activity was enhanced in Mz-Hep-1 cells more than 83 times by beta-naphthoflavone. The enhancement of EROD activity by DMSO and beta-naphthoflavone treatments of KYN-2 cells was abolished by alpha-naphthoflavone treatment. In Mz-Hep-1, only the inducing effect of beta-naphthoflavone was abolished by alpha-naphthoflavone treatment. Rifampicin treatment of KYN-2 cells reversed both the DMSO and beta-naphthoflavone effects on the EROD activity. Glucuronidation of steroids, bile acids, fatty acids and drugs was effective in KYN-2 and Mz-Hep-1 cells. Both 1-naphthol glucuronidation and the level of UGT1*6 protein detected by immunoblot and supporting this activity were lowered by DMSO treatment and increased by beta-naphthoflavone treatment in KYN-2 cells. In Mz-Hep-1 cells, DMSO and beta-naphthoflavone had no effect on 1-naphthol glucuronidation activity. DMSO, beta-naphthoflavone and rifampicin also affected the glucuronidation of various substrates supported by different UGT isoforms. These results indicate that KYN-2 and Mz-Hep-1 cells can be used as new in vitro models for the studies of drug metabolism and the regulation of the corresponding enzymes.

Glucuronidation in the Caco-2 human intestinal cell line: induction of UDP-glucuronosyltransferase 1*6.

The ability of the differentiated human intestinal cell line, Caco-2, to glucuronidate various endobiotic and xenobiotic molecules was investigated. Glucuronidation of hydroxylated or carboxylic acid compounds such as 1-naphthol, thymol, androsterone, estriol, hyodeoxycholic acid, lithocholic acid, chloramphenicol, paracetamol and morphine could be determined in microsomal fractions of Caco-2 cells. The activity toward 1-naphthol was the highest glucuronidation activity measured in Caco-2 cells. This activity was specifically increased four-fold upon addition of beta-naphthoflavone into culture medium but not by rifampicine or clofibrate and was related to a biosynthesis of UDP-glucuronosyltransferase 1*6 (UGT1*6). alpha-Naphthoflavone did not affect the inducing property of beta-naphthoflavone. 7-Ethoxyresorufin-O-dealkylation activity, supported by cytochrome P4501A1, was induced more than 1000-times in Caco-2 cells by beta-naphthoflavone treatment, and this effect was partially abolished by alpha-naphthoflavone treatment. The results suggest that several isoforms, including UGT1*6, are expressed in Caco-2 cells.

Human cell lines in pharmacotoxicology. An introduction to a panel discussion.

Various types of cells lines are used in pharmacotoxicology. Established cell lines are easily available, with few ethical restrictions. Some specific properties are preserved, although they have kept the phenotype of the original tissue, which is frequently a tumor phenotype. They are usually more resistant to toxic compounds than freshly isolated cells. Some drug-metabolizing enzymes are expressed and regulated in these cells. Immortalized cell lines are also of interest in toxicology. They are mainly examined for their potential in mutagenicity testing. These cells and numerous others of animal or human origin can be transfected with cDNA coding for human enzymes. They are used for determination of the individual enzyme involved in a particular metabolic pathway, or, when multiple transfections are successfully achieved, for mutagenicity testing. Regulation studies are also possible in such cells after transfection of DNA elements regulating gene transcription.

Gamma-glutamyl transpeptidase activity in the epididymis during fetal and postnatal development in rats.

The histochemical and biochemical distributions of gamma-glutamyl transpeptidase (gamma-GT) were investigated in the epididymis of rats during fetal and postnatal development. In the epididymal homogenates, gamma-GT activity was detected on the fifth day after birth. A sharp increase was observed after 30 days of life in the caput homogenates. Moderate levels of the enzyme were found in the cauda epididymis. Gamma-GT is histochemically detected from the 15th day of gestation in Wolffian ducts and in 17- to 18-day-old fetuses in newly differentiated epididymal tubules. Enzyme activity, was associated with the plasma membranes (apical, lateral, and basal), was preponderant on the apical part of the epithelial cells. During the first 15 days of the postnatal life, the histochemical reaction intensities were identical from the caput to the cauda epididymidis. From the 18th day onwards, enzyme activity decreased in the corpus and in the cauda, while gamma-GT increased in the caput epididymidis, and a strong activity was found on the apical surface of epithelial cells. Weak or moderate gamma-GT activity of spermatozoa in the caput tubules, increasing steadily from caput to cauda epididymidis, suggests that gamma-GT may be related to the functional maturation of spermatozoa.

Expression and regulation of drug metabolizing enzymes in an immortalized rat hepatocyte cell line.

A hepatic cell line has been immortalized after simian vacuolating virus 40 infection of adult rat hepatocytes maintained in defined culture conditions. This cell line, designated SVHep B4, expressed nuclear large T antigen, exhibited an extended lifespan (50 subcultures) and had a hepatocyte-like morphology. Expression and regulation of drug metabolizing enzymes were studied in long-term cultures of SVHep B4 cells. Significant activities of phase I and phase II enzymes were detected. gamma-Glutamyltransferase, a marker often increased in neoplastic and dedifferentiated hepatocytes, showed a low activity whereas the hepatospecific enzyme tyrosine aminotransferase was expressed at levels similar to those in liver. Responsiveness of drug metabolizing enzymes to inducers was investigated with phenobarbital, dexamethasone and methylcholanthrene. IIB and IA subfamilies of cytochrome P450 were increased, respectively, by phenobarbital (170%) and methylcholanthrene (500%). Glucuronidation of 1-naphthol was increased by phenobarbital (140%) and 3-methylcholanthrene (160%). Phenobarbital, methylcholanthrene and dexamethasone were found to increase significantly gamma-glutamyltransferase while tyrosine aminotransferase activity was enhanced by dexamethasone. Stable expression and inducibility of drug metabolizing enzymes in long-term cultures of the SVHep B4 cell line demonstrate that immortalization of adult hepatocytes represents a promising tool for drug biotransformation studies in vitro.

Influence of the isolation method on the stability of differentiated phenotype in cultured rat hepatocytes.

Primary cultures of adult rat hepatocytes were established using two different isolation procedures: a two-step collagenase perfusion and a method using ethylenediaminetetraacetate (EDTA) as the dissociating agent. Both techniques provided good yields of hepatocytes with comparable viability. The evolution of hepato-specific protein levels and several drug-metabolizing enzyme activities were followed for 8 days in cultured hepatocytes obtained by both methods. EDTA-isolated hepatocytes maintained a low gamma glutamyltransferase (GGT) activity, whereas collagenase-treated cells acquired a high GGT level. Transferrin secretion and tyrosine aminotransferase (TAT), alanine aminotransferase (ALT), and microsomal epoxide hydrolase (mEH) activities were stable in both EDTA- and collagenase-isolated hepatocytes, whereas albumin secretion, aspartate amino transferase (AST) activity, total cytochromes P-450 content, IA1 and IIB1 P-450 isoenzymes, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) levels, and bilirubin glucuronidation decreased faster in collagenase-treated cells. The most important difference observed was the maintainance of the mixed-function oxidase system in EDTA-isolated hepatocytes. These results emphasize the critical role of isolation technique in stabilization of differentiated hepatocytes in primary culture.