Thierry Cresteil

Long-term culture of adult rat hepatocyte spheroids

Hepatocytes from adult rats were cultured on poly-HEMA-coated surface to form spheroids in hormonally defined media as previously shown with newborn rat hepatocytes. Spheroidal aggregates of adult rat hepatocytes were morphologically similar to those of newborn rat hepatocytes and could also form a monolayer of uniform liver parenchyma-like cells when transferred on collagen-coated surfaces even after 2 months of culture. Under these culture conditions, albumin and transferrin secreted in vitro by adult rat hepatocyte spheroids were detectable by immunoprecipitation method at least until 2 months of culture. The production of proteins by hepatocyte spheroids could be regulated in vitro by IL-6: the secretion of alpha 2-macroglobulin was increased and the secretion of albumin was decreased in the presence of this cytokine. In addition, cytochrome P450 IA1 was strongly induced by methylcholanthrene in adult rat hepatocyte spheroids, and the induction remained relatively constant up to 22 days of culture. These cells were also able to metabolize lidocaine to monoethylglycinexylidine when measured up to 14 days of culture, showing the presence of a relatively high level of P450 IIIA2. The UDP-glucuronyltransferase activity, specific for bilirubin conjugation, decreased to 18% of the initial value after 2 weeks of culture. This work showed that adult rat hepatocytes in long-term spheroid culture kept differentiated functions, providing a new model for the in vitro study of hepatocyte functions and complementing that of newborn rat hepatocytes using the same system.

Detection of cytochrome P450 gene expression in human placenta in first trimester of pregnancy.

Human first-trimester placentas were screened for the expression of xenobiotic-metabolizing cytochrome P450 (CYP) genes. mRNAs of CYP1A1, CYP1A2, CYP2C, CYP2D6, CYP2E1, CYP2F1, CYP3A4, CYP3A5, CYP3A7, and CYP4B1 were identified by reverse transcriptase-polymearse chain reaction (RT-PCR) in at least some of the six placental samples studied. CYP2A and CYP2B message were absent in all samples. The level of all of these CYP mRNAs was lower compared to the corresponding levels in liver or lung. the catalytic activity marker (7-ethoxyresorufin O-deethylase) was inducible in the placentas by maternal cigarette smoking. Thus, the regulatory system of placental CYP1A1, mediated by the Ah-receptor, appears to be developed as early as the first trimester of pregnancy. Three immunoreactive bands from placental microsomes were detected by an antihuman CYP3A4 antibody, but no functional activity of CYP3A enzymes could be detected. These results show that placental tissue during the first trimester of pregnancy has the potential of expressing several CYP genes, and forms a basis for subsequent analysis of these forms at the protein and functional level.

High levels of expression of the NAD(P)H : quinone oxidoreductase (NQO1) gene in tumor cells compared to normal cells of the same origin

NAD(P)H:quinone oxidoreductase (NQO1) is a flavoprotein which catalyzes the two-electron reduction of quinones and azo-dyes and thus prevents the formation of free radicals and toxic oxygen metabolites that may be generated by the one-electron reductions catalyzed by cytochrome P450 reductase. Analysis of RNA indicated 20- to 50-fold higher levels of NQO1 gene expression in the liver tumors and in the tissue surrounding the tumors of patients with hepatocarcinoma than in normal individuals. An approximately 50-fold higher level of NQO1 mRNA was also observed in human hepatoblastoma (Hep-G2) cells than in normal liver. By deletion mutagenesis in the human NQO1 gene promoter and subsequent transfection into hepatic and nonhepatic cell lines, a 1.42 kb DNA segment has been identified to contain cis-acting elements responsible for high levels of expression of the NQO1 gene in tumor cells.

Onset of xenobiotic metabolism in children: Toxicological implications

The level of expression of cytochromes P450 shows a wide interindividual variability, depending on the age and tissue investigated. Several lines of evidence indicate that the human foetal liver is an active site for the biotransformation of drugs, chemicals and hydrophobic endogenous molecules. Besides this high degree of maturity, many studies have shown a discrepancy in the onset of activities and suggested that cytochrome P450 isoforms developed independently. Thus, many cytochromes P450 are absent or barely detectable in the foetal liver and develop postnatally. The postnatal evolution of P450 was explored in a liver bank constituted with samples collected from neonates aged less than 24 h to 10 years. Three major groups of cytochrome P450 could be described: a first group of cytochromes P450 expressed in the foetal liver includes the CYP3A7 and 4A1, mostly active on endogenous substrates; a second group (termed early neonatal P450) includes CYP2D6 and 2E1. They surged within hours after birth although proteins could not be detected in foetal samples. A third group of P450s (neonatal P450) develops later. CYP3A4 and CYP2Cs rose during the first weeks after parturition and CYP1A2 was the last isoform to be expressed in the human liver. Among phase II enzymes, epoxide hydrolase and glutathione S-transferase pi are very active in the foetal liver, whereas glutathione S-transferases mu and alpha and UDP-glucuronosyltransferases develop within 3 months after birth. These data clearly emphasize the delayed maturation of certain biotransformation pathways in the human liver during the perinatal period and constitute a scientific basis for improving safety during chemical exposure in children.

Evidence for phosgene formation during liver microsomal oxidation of chloroform

Abstract When aerobically incubated with liver microsomes and NADPH, chloroform produces a stable adduct with cysteineas a nucleophilic trapping agent. The adduct was identified by thin layer chromatography, gas-liquid chromatography and combined gas chromatography-mass spectrometry as the reaction product of cysteine with phosgene.

The role of cytochrome P450 3A (CYP3A) isoform(s) in oxidative metabolism of testosterone and benzphetamine in human adult and fetal liver

Testosterone metabolism was studied in human adult and fetal liver microsomes. In fetal livers 6 beta-hydroxylase (6 beta OH) activity (1-2% of adult activity) and 2 alpha-hydroxylase (2 alpha OH) activity (about 40% of adult activity) were present. Also some fetal livers produced two unknown metabolites. Androstenedione was formed in all fetal livers studied (10-20% of adult activity). Testosterone hydroxylations at 6 beta-, 2 beta-, 15 alpha- and 15 beta-positions were associated with CYP3A isoform(s) in adult liver, because they were strongly inhibited by midazolam, a known substrate for CYP3A4 and by anti-CYP3A4 antibody. Fetal liver activities were consistently inhibited less than the activities in adult livers. The formation of androstenedione was not affected by these inhibitors in fetal or adult liver microsomes. Benzphetamine N-demethylase activity in the fetal livers was about 40% of adult activity. Anti-CYP3A4 antibody had no effect on that activity in fetal or in adult liver microsomes, whereas a monoclonal antibody 1-68-11 (generated against rat CYP2C11) slightly inhibited benzphetamine N-demethylase activity in adult liver. This study indicates that human fetal and adult liver are dissimilar in their testosterone metabolism pattern. The formation of androstenedione from testosterone in fetal liver may have a physiological role. Testosterone hydroxylases are less inhibited by anti-CYP3A4 antibody, midazolam and progesterone in fetal than in adult liver.

Evidence for the presence of distinct flavin-containing monooxygenases in human tissues

Catalytic activities and substrate specificity of flavin-containing monooxygenase were examined in human tissues. During incubation with imipramine, human hepatic microsomes efficiently carried out cytochrome P450-dependent reactions but not the formation of N-oxide, while in kidney imipramine N-oxide was the only metabolite formed during in vitro incubation. The production of imipramine N-oxide was essentially due to flavin-containing monooxygenase as shown by thermal inactivation. In contrast, thiobenzamide and dimethylaniline were actively transformed by both human liver and kidney flavin-containing monooxygenase. Neither the modification of pH nor the solubilization of microsomal membranes increased imipramine N-oxidation in human liver. Kinetic analysis indicated a poor affinity (about 7 mM) of human liver microsomes for imipramine versus 0.3 mM in kidney. Immunological studies were undertaken to support enzymatic data. Antibodies raised against rat liver flavin-monooxygenase reacted strongly with human kidney microsomes but extremely weakly with liver microsomes. The relative amount of immunochemically determined protein correlated well with imipramine N-oxidation activity. A dose-dependent inhibition of imipramine N-oxidation by anti-flavin-monooxygenase antibodies was observed in human kidney, as well as in rat kidney and liver. Taken together, the results can be interpreted by the possible existence in human tissues of distinct flavin-containing monooxygenases exhibiting a partial overlapping substrate specificity. The protein involved in imipramine N-oxidation is missing from human liver but actively carries out the reaction in kidney, while another protein catalyzes the oxidation of thiobenzamide and dimethylaniline in both tissues.

Immunological and enzymatic comparison of hepatic cytochrome P-450 fractions from phenobarbital-, 3-methylcholanthrene-, β-naphtoflavone- and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats

A comparison of the cytochrome P-450 forms induced in rat liver microsomes by phenobarbital on the one hand, and 3-methylcholanthrene, beta-naphthoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin on the other hand, was performed using specific antibodies: anti-P-450 B2 PB IG (against the phenobarbital-induced cytochrome P-450) and anti-P-450 B2 BNF IG (against the beta-naphthoflavone-induced cytochrome P-450). On DEAE-cellulose chromatography, four cytochrome P-450 fractions were separated, called P-450 A (non-adsorbed), P-450 Ba, P-450 Bb and P-450 Bc, from control, phenobarbital-, 3-methylcholanthrene, beta-naphthoflavone- and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats. Cytochrome P-450 A fractions appeared to be unmodified by the inducers, whereas the specifically induced cytochrome P-450 forms were always recovered in Bb fractions. The P-450 Bb fractions induced by 3-methylcholanthrene, beta-naphthoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibited common antigenic determinants, comparable catalytic activities (benzphetamine N-demethylase, benzo[a]pyrene hydroxylase) and similar mol. wts. Moreover, the inhibition patterns by the two antibodies of benzphetamine N-demethylase and benzo[a]pyrene hydroxylase activities catalysed by 3-methylcholanthrene, beta-naphthoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin microsomes or by the corresponding P-450 Bb fractions in a reconstituted system were quite identical. By these different criteria, beta-naphthoflavone, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin seem to induce a common cytochrome P-450 species in rat liver.

Specific evidence that schizandrins induce a phenobarbital-like cytochrome P-450 form separated from rat liver.

Abstract Cytochromes P-450 present in liver microsomes from rats treated with Schizandrin B, Schizandrol B and DDB were separated on DEAE cellulose column and enzymatically and immunologically compared to the corresponding fractions of cytochrome P-450 obtained from untreated or phenobarbital-treated animals. A cytochrome P-450 species showing the same chormatographic behaviour on DEAE cellulose, which possesses a common antigenic site with phenobarbital-induced cytochrome P-450 and exhibits a high benzphetamine demethylase activity is induced by schizandrins, whereas the other cytochromes P-450 are not affected. Thus, the biosynthesis of a phenobarbital-like cytochrome P-450 was promoted by a class of molecules struturally different of phenobarbital.

Enzymatic and immunological evidences that phenobarbital induces cytochrome P 450 in fetal and neonatal rat liver

Abstract Some characteristics of the liver monooxygenase system were investigated in near-term, newborn and adult rats. When cytochromes P 450 were separated by chromatography on DEAE cellulose, the fraction eluted by NaCl was not significantly increased by transplacental phenobarbital treatment as it is in adult treated animals, but exhibited reconstituted enzyme activities and immunological characteristics qualitatively similar to those of phenobarbital-treated adults. This inductive effect was more acute in 5-d-old newborns and finally in adults. Thus, the mechanism responsible for the induction of cytochrome P 450 by phenobarbital is present but not very active in rat fetuses, and exhibits a rapid development after birth.