Nicole Sabolovic

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.

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.

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.

Monoclonal antibodies to human kidney gamma-glutamyltransferase

Eight hybridoma clones secreting large amounts of monoclonal antibodies against purified human kidney gamma-glutamyltransferase (GGT) were isolated and produced in ascites. None of them inhibits the catalytic activity of GGT. They all bind to the heavy subunit of this dimeric enzyme. Immunoblot analysis showed that these antibodies react with the catalytically active GGT. The monoclonal antibodies also recognize the heavy subunit of the human liver enzyme. This is of interest, as serum GGT is known to originate from the liver. None of the monoclonals reacts with GGTs from rat or pig kidney. After identification of epitopes specificities, the antibodies will be used for the development of immunoassays of GGT especially in human serum.

cis- and trans-Resveratrol Are Glucuronidated in Rat Brain, Olfactory Mucosa and Cultured Astrocytes

Background/Aims: Glucuronidation of cis- and trans-resveratrol (3,5,4’-trihydroxy-trans-stilbene), which is a naturally occurring phytoalexin known to exert a number of beneficial health effects, was investigated in rat brain, cultured astrocytes and olfactory mucosa. Methods: The isomers were incubated with tissue homogenates, microsomes, or rat liver recombinant UDP-glucuronosyltransferases in the presence of UDP-glucuronic acid. The glucuronides were separated by HPLC and quantitated. Astrocytes were exposed to lipopolysaccharide to promote inflammatory conditions. Results: All tissues were able to form resveratrol glucuronides although at a lower extent, when compared to the liver. The reaction was stereo- and regioselective. In brain tissue, trans-resveratrol 3-O-glucuronide was mainly formed, whereas the cis-isomer was glucuronidated at a lower rate on that position. No 4’-O-glucuronide was detected in brain. In olfactory mucosa homogenates, the cis 3-O-glucuronide was mainly formed, whereas the trans-isomer was glucuronidated only on the 3-position. In astrocytes, 3-O-glucuronides of the cis- and trans-resveratrol were only detected. The rat recombinant UGT1A6 and UGT2B1 isoforms were able to glucuronidate cis- and trans-resveratrol. Finally, in inflammatory conditions, trans-resveratrol glucuronidation was enhanced in astrocytes. Conclusion: Brain tissues are effective in the glucuronidation of resveratrol isomers. This metabolism pathway is likely to modulate the concentration of these biologically active substances.

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.

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.

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), β-narphthoflavone, α-napthoflavone, and rifampicin on these drug-metabolizing enzyne 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 β-naphthoflavone treatment in combination with DMSO. This activity was enhanced in Mz-Hep-1 cells more than 83 times by β-naphthoflavone. The enhancement of EROD activity by DMSO and β-naphthoflavone treatment of KYN-2 cells was abolished by α-naphthoflavove treatment. In Mz-Hep-1, only the inducing effect of β-naphthoflavone was abolished by α-naphthoflavone treatment. Rifampicin treatment of KYN-2 cells reversed both the DMSO and β-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 UGT*6 protein detected by immunoblot and supporting this activity were lowered by DMSO treatment and increased by β-naphthoflavone treatment in KYN-2 cells. In Mz-Hep-1 cells, DMSO and β-naphthoflavone had no effect on 1-naphthol glucuronidation activity. DMSO, β-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.

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.

Viral Immortalization and Phenotypic Characterization of a Rat Hepatocyte Cell Line

Viral immortalization (SV40) of adult rat hepatocytes was performed in controlled culture conditions (isolation method, collagen matrix, defined culture medium, short-time culture before virus infecti