Reduction and glucuronidation of naftazone by human and rat liver microsomes.

Reduction and glucuronidation of the vasoprotectant drug, naftazone, by human and rat liver microsomes and by recombinant UDP-glucuronosyltransferases (UGT) stably expressed in V79 cells were studied. The oxo group was first reduced in the presence of NADPH or NADH, and was subsequently readily glucuronidated on the phenolic moiety leading to a 1 beta-O-glucuronide, as revealed from MS and by proton and 13C-NMR. Glucuronide extracted from the urine of rats treated with the drug presented the same structure. In all enzyme systems tested, NADH was the most efficient electron donor, when compared with NADPH. The reaction was strongly inhibited by quercitrin, a specific inhibitor of carbonyl reductase. Attempts to isolate the reduced intermediate were unsuccessful because of its marked instability. In humans, a large interindividual variation for the formation of glucuronide was observed with microsomes of seven different liver samples (3.98 +/- 3.22 nmol/min.mg). In rat, glucuronidation of reduced naftazone was strongly induced (12-fold) by 3-methylcholanthrene and, to a lesser extent (2.6-fold) by phenobarbital, but was not affected by clofibrate. In addition, liver microsomes from Gunn rats, which present a genetic defect in bilirubin and phenol UGTs could not form glucuronide of reduced naftazone. The drug, after addition of NADH, was a substrate of the human liver recombinant UGT1*6 that presents a strict specificity toward planar phenolic substances, but not that of UGT2B4 and UGT2B1 expressed in V79 fibroblasts. The reducing step by the endogenous V79 cellular reductase was rate-limiting. In conclusion, the powerful inducing effect exerted by 3-methylcholanthrene, the lack of glucuronidation in the Gunn rat and the ability of UGT1*6 encoded by the UGT1 gene to glucuronidate reduced naftazone suggest that, in humans and in the rat, the compound is metabolized by a UGT isoform (UGT1*6 and the rat orthologous form) belonging to family 1, with a restricted specificity toward the drug.

Oral administration of [3H]navelbine in patients: comparative pharmacokinetics using radioactive and radioimmunologic determination methods.

[3H]Navelbine (NVB) was administered orally to two patients. Drug levels in biological fluids were monitored by radioimmunoassay (RIA) and direct radioactivity (RA) determinations. NVB absorption was rapid: maximum plasma concentrations appeared in the first 2 h after oral administrations. Pharmacokinetic parameters estimated from RIA data were in complete accordance with those obtained from i.v. injections. Bioavailability (i.v./po) estimated from RIA and RA data averaged 40.6 and 93.0%, respectively. NVB urine excretion was low. Fecal excretion remained its main elimination route. Moreover, large differences were observed in area under NVB plasma concentration-time curve (AUC) values obtained by the two methods, implying intense drug bio-transformations.

Circadian synchronization of liver regeneration in adult rats: the role played by adrenal hormones.

The role played by the adrenal hormones in the regulation of liver proliferation in adult rats was investigated under various experimental conditions. In untreated control groups, cell growth was very low and endogenous corticosterone levels showed a clearly-defined circadian rhythm with a peak in the evening. Adrenalectomy depressed the level of endogenous corticosterone immediately and the growth rate of the liver increased significantly. We were able to prevent this effect by repeated injections of corticosterone at physiological doses. After a 1/3 hepatectomy and a sham-operation, the corticosterone blood level maintained its normal circadian pattern with the exception of a transient increase during the first two post-operative hours. After a hepatectomy of this kind, a negative correlation was found to exist between the adrenal hormone level and the waves of DNA synthesis; the subsequent mitoses appeared in two successive circadian waves of decreasing amplitude, a maximum value being reached in the morning. In rats submitted to a 1/3 hepatectomy and an adrenalectomy simultaneously, the endogenous corticosterone level fell significantly after a post-operative peak. The regenerating pattern was completely different from that induced by 1/3 hepatectomy alone. The rise in the labelling index began earlier and rose to significantly higher values; it was then followed by a single large mitotic wave without any circadian rhythm. These results favour the hypothesis that adrenal hormones have a significant effect on the negative control of liver regeneration. Circadian changes in the corticosterone level were responsible for the nycthemeral pattern observed in the regenerating liver after a partial hepatectomy. The results show a marked inhibition of the G1-S transition, particularly in the evening, when the endogenous corticosterone concentration was at its highest. Also discussed is the relationship between corticoids and 'chalones', which synergetically inhibit the passage from G0 into the cell cycle.

Induction of liver microsomal cytochrome P-450 isozymes by 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide.

1. 1-(2-Chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide (RP 52028), an antagonist of peripheral type benzodiazepine binding sites, a potential anticonvulsant, has been shown to have an inducing effect on drug-metabolizing enzymes. 2. RP 52028 was administered orally at 20-800 mg/kg for 1 week, and enzymic activities were determined using a panel of substrates. Western blot analyses were performed using several specific polyclonal and monoclonal antibodies directed against cytochrome P-450 isozymes. 3. RP 52028 appears to be an inducer of cytochrome P-450 II B1 (P-450b) and related enzymic activities; i.e. benzphetamine, ethylmorphine and aminopyrine demethylation.

Identification of the molecular structure of the phenolic primary metabolite of dimetindene in animals and man.

The metabolic pathway of dimetindene (dimetindene maleate, Fenistil), an antiallergic drug commercially available for more than 20 years, has remained unknown. By means of HPLC, GC-MS and MS-MS, dimetindene was found to be hydroxylated on the indene moiety of the molecule both in vitro with hepatic microsomes of several species and in vivo in man. Cochromatographic analysis (HPLC and GC-MS) of the primary metabolites produced in vitro and in vivo demonstrated their similarities but revealed differences from the chemically synthesized 5-hydroxy-dimetindene. Using large volumes of in vitro incubations of rat microsomes with dimetindene and cofactors, 1.15 mg of pure primary metabolite were first produced and then extensively purified. The study of the NMR proton in one and two dimensions (COSY) clearly demonstrated that this metabolite is hydroxylated on the C6 of the indene moiety as compared to the C5 for the synthetic product.

Pharmacokinetics of a new anticancer drug, navelbine, in patients. Comparative study of radioimmunologic and radioactive determination methods.

A study was designed to investigate the fate of navelbine (NVB) and its excretion routes in two cancer patients treated with tritiated NVB (30 mg/m2) by i.v. bolus injection. Plasma and red blood cell concentrations and urine and stool elimination were monitored over long periods of time. NVB plasma and urine concentrations were measured by both radioimmunoassay (RIA) and direct radioactive (RA) determination. Samples were also analyzed by high-performance liquid chromatography to evaluate the importance of NVB metabolism. Whereas the major excretion route for NVB was the stool (from 34% to 58.4% of the total dose given over 21 days), urinary excretion was low (about 21% within the same time period), corresponding mainly to that of unchanged drug. Thus, a good correlation was found between RIA and RA determinations in urine. In contrast, plasma area under the curve (AUC) values obtained after RA and RIA analysis differed markedly (AUC RIA/AUC RA = 0.23-0.31), demonstrating that a significant proportion of the plasma-circulating drug was biotransformed, mainly during the last elimination phase. This could have important pharmacological and toxicologic implications in clinical practice.

Differences in the metabolism of epicainide in rats and man.

1. Epicainide is a new potent antiarrhythmic agent, metabolized differently in rats and man. 2. In rats, the aromatic part of the molecule (the two phenyl rings linked to the quaternary carbon atom) undergoes metabolic attack by the mono-oxygenases. The hydroxy and methoxy-hydroxy metabolites are predominant and are excreted in urine and bile both as free and conjugated forms. 3. In contrast, the aromatic moiety of epicainide remains unchanged in man. It is the ethylpyrrolidine ring which is mainly attacked, resulting in the expected N-deethylation, subsequent oxidation of the pyrrolidine moiety, and reduction or hydrolysis of the amide function. 4. A preliminary kinetic analysis of epicainide in man reveals a linear open 2-compartment model. The radioactivity recoveries confirm the absence of any accumulation of the drug in the organism.

Human epidermal blister: a convenient tissue for toxicological and genetic studies of benzo(a)pyrene metabolism.

Benzo(a)pyrene (BP) metabolism has been studied in epidermal blisters maintained in a culture medium for 24 h and 48 h. The viability of the cells has been assayed by [3H]proline incorporation into proteins and by [14C]BP metabolism into unconjugated metabolites. A screen of BP metabolism in 19 individuals shows a great variation of basal epidermal activity. Induction of BP metabolism by the application of coal tar 24 h before the epidermal blister sampling, resulted in two- to eight-fold increase in BP metabolism. This induction is not increased when the coal tar application is repeated.

Benzo[a]pyrene metabolism in mouse liver. Association of both 7,8-epoxidation and covalent binding of a metabolite of the 7,8-diol with the Ah locus.

The 7,8-epoxidation of benzo[a]pyrene, and the 9,10-epoxidation of benzo[a]-pyrene trans-7,8-dihydrodiol coupled with covalent binding of the highly reactive diol-epoxide, are two key P-450-mediated reactions believed to be important in cancer initiation, mutagenesis and teratogenesis. New assays for these two reactions were developed with mouse liver microsomes. These two activities have apparent Km values (approximately 6 microM) similar to that of aryl hydrocarbon hydroxylase activity. Twenty-six individual 3-methylcholanthrene-treated Ahb/Ahd and Ahd/Ahd progeny of the (C57BL/6N)(DBA/2N) F1 X DBA/2N backcross were studied. Both of the newly described activities appear to represent P-450 protein(s) that are responsible for aryl hydrocarbon hydroxylase activity and that are coordinately controlled by the Ahb allele.

Differences in the biochemical properties of aldrin epoxidase, a cytochrome P-450-dependent monooxygenase, in various tissues.

Aldrin epoxidase, a cytochrome P-450-dependent monooxygenase, was studied in the lung and kidney of male rats. The sensitivity of the liver enzyme activity to different chemicals in vitro was influenced by the treatment of the animals with phenobarbital or methylcholanthrene. These results confirm that more than one form of cytochrome P-450 supports aldrin epoxidase in the liver. The lung and kidney aldrin epoxidase activity was not modified by the administration of chemical inducers to the rats. In vitro, the lung and kidney aldrin epoxidase activities were activated by tetrahydrofurane and progesterone, respectively. The results obtained from the lung and kidney indicate that one single species of cytochrome P-450, associated with aldrin epoxidase, exists in these organs, but it may be a different type, or regulated in a different manner in these tissues.

[Clometacin--1. Metabolism and bioavailability of clometacin and its metabolites in the rat]. / Travaux sur la clométacine--1re partie. Métabolisme et biodisponibilité de la clométacine et de ses métabolites chez le rat.

The metabolism and bioavailability of 14C clometacin was studied in the rat. The drug is rapidly excreted in the urine and in the feces. It is not detected in any tissues of the treated animals 48 hours after administration. The metabolism of clometacin is very limited under the experimental conditions used in our study. More than 85% of the drug is excreted in an unchanged from. One metabolite, representing 4 to 10% of the excreted radioactivity, was found in the urine and identified as 1-(2-methyl-3-p-chloro phenylcarbinol-6-hydroxy) indol acetic acid. This metabolite was not detected in the feces, nor in the different tissues 4 or 48 hours after i.v. or oral administration of the drug.

[Clometacin--2. Metabolism and bioavailability in the monkey and in man]. / Travaux sur la clométacine--2e partie. Métabolisme et biodisponibilité chez le singe et chez l'homme.

Contrarily to the rat (see part I), clometacin is not metabolized in the monkey and in man. Urinary elimination is greater in the monkey (80 to 90% of the administered drug) than in humans (14 to 33%). Urinary excretion is rapid, i.e. more than 80% within the 24 first hours. Retention of the drug by any organ was not detected in the monkey.

Ontogenetic variation in rat liver, lung and kidney monooxygenase induction by low doses of benzo(A)pyrene and cigarette-smoke condensate.

The specific lung-AHH induction, which we previously observed after the inhalation of cigarette smoke, is not due to the route followed by the inhaled smoke, for the same phenomenon occurs after i.p. injection of either cigarette smoke condensate (CSC) or benzo(a)pyrene in low doses. In this respect lung AHH behaves completely differently from the liver and kidney enzyme, in which organs, basal AHH activity (which is low in the foetus) increases rapidly after birth to reach the adult level 2 months later, and is only inducible by CSC and low doses of BP in unweaned rats. In the lung, the basal AHH activity (low in the foetus) increases abruptly at birth, peaks in 5-day-old rats and then decreases slightly. Contrary to enzyme activity in other tissues, lung AHH cannot be induced in unweaned young animals. The enzyme subsequently becomes sensitive to inducing agents and is highly inducible in 90-day-old rats. Similar behaviour occurs in 2 other enzymes linked to cytochrome P1450: ethoxycoumarin deethylase and ethoxyresorufin deethylase. The results could be related to the particular susceptibility of the lung to develop cancer after the inhalation of cigarette smoke.

Multiplicity of cytochrome P-450 in primary fetal hepatocytes in culture.

Primary fetal rat hepatocytes in culture display different monooxygenase activities which can be induced by several chemical inducers. Until now, these hepatocytes were believed to produce only one single cytochrome P-450 species, namely the cytochrome P1-450 (or P-448). It now seems possible to induce other cytochrome P-450 species in these hepatocytes, providing that they receive an appropriate hormonal treatment. We have examined the effect of dexamethasone on the cytochrome P-450 type supporting different enzymic activities (aryl hydrocarbon hydroxylase, ethoxycoumarin deethylase, aldrin monooxygenase). Our results show that the presence of dexamethasone in the culture medium produces qualitative and quantitative changes in the monooxygenase-supporting cytochrome(s) P-450. For low dexamethasone concentrations (between 1 nM and 0.1 microM) a cytochrome P-450 is formed displaying biochemical and biophysical properties similar to those induced by phenobarbital in the adult rat liver. At higher concentrations (above 1 microM), similar qualitative changes are observed; but a quantitative phenomenon occurs, the (cytochrome P-450)-dependent enzymic activities being also induced. Dexamethasone also has a synergistic effect on the induction of enzymic activity by the mixture of phenobarbital plus benzanthracene. The various biochemical changes induced by dexamethasone in the fetal cell cultures parallel those observed in vivo during the perinatal period of life. The cell culture system may thus constitute an interesting model for studying the ontogenic development of liver monooxygenases.

Significant variation in mouse-skin aryl hydrocarbon hydroxylase inducibility as a function of the hair growth cycle.

An easy, rapid and improved technique for homogenizing whole skin is described. This technique consists of reducing skin to a powder in liquid N2 by using a metallic mortar, and homogenizing the powder in a Potter-Elvehjem tube. Using this homogenizing method, we have shown that skin AHH activity in C57BL/6K and C3H/Ico mice can be induced by i.p. injected or topically applied methylcholanthrene during a defined period of the hair growth cycle, i.e. between the 8th and 14th days after depilation (Stage 6 of the anagen period). In each experimental model, there is an optimal methylcholanthrene concentration which yields a maximum induction. Topical methylcholanthrene is also responsible for a smaller aryl hydrocarbon hydroxylase (AHH) induction when the chemical is applied the same day that the club hairs are plucked. On the other hand, skin AHH activity is never induced by methylcholanthrene in DBA/2J mice, a genetically non-responsive strain. No clear-cut segregation of skin AHH inducibility levels is found among the offspring from the back-cross between (C57BL/6J X DBA/2J)F1 and non-inducible DBA/2J mice.

Comparison of aryl hydrocarbon hydroxylase and epoxide hydratase. Induction in primary fetal rat liver cell culture.

The activity of aryl hydrocarbon hydroxylase (AHH) and/or epoxide hydratase (EH) is induced in primary fetal rat liver cell culture by benz-[alpha]anthracene (BA), phenobarbital (PB), cigarette smoke condensate (CSC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and trans-stilbene oxide (TSO). The response of the two enzymes to the different chemicals varies as follows: (a) AHH is induced by lower concentrations of BA, PB and CSC than those required to significantly induce EH; (b) AHH is selectively induced by TCDD and by low BA concentrations; (c) the kinetics of AHH induction by BA, PB and CSC is faster than that of EH; (d) TSO is a selective inducer of EH. As described earlier for AHH, RNA and protein synthesis and the continuous presence of the inducer are required in the early phases of EH induction. Later when the EH activity has reached a plateau, intact RNA and protein synthesis is not necessary to maintain the enzyme at its optimal value. The removal of the inducer determines a decay of the EH activity, allowing the estimation of a biological tau 1/2 of about 72 h. TSO prevents the AHH induction by PB, but not that mediated by BA and CSC. Added together with PB, BA, CSC or PB plus BA, TSO induces the EH activity in a more than additive manner. This effect is only seen after 6 days of continuous treatment. These results indicate that in this tissue culture model, the mechanism of AHH and EH induction can clearly be dissociated.

Dose-response relationship of rat aryl hydrocarbon hydroxylase and epoxide hydratase induction.

This paper summarizes our recent results supporting the hypothesis that different regulation mechanisms are involved in the control of AHH and EH activity and that the AHH induction in the extrahepatic tissues might also be affected by liver specific inducers. In the rat, lung and kidney AHH is highly sensitive to the inducers present in cigarette smoke and cigarette smoke condensate, the EH activity not being affected by the same agents. Phenobarbital is also able to potentiate the inducing action of low doses of benzo(a)pyrene on the lung AHH activity. In primary rat liver cells in culture, AHH and EH can be selectively induced. Low doses of benz(a)anthracene preferentially enhance the AHH activity while trans-stilbene oxide and various antioxidants modify only the EH activity. Phenobarbital, which also induces the AHH activity in cell culture, produces a more than additive effect when added to the culture medium in a mixture with benz(a)anthracene. Trans-stilbene oxide prevents the AHH induction by phenobarbital and not by benz(a)anthracene. Our results suggest that, in addition to its own induction capacity, phenobarbital is also able to potentiate the action of chemicals belonging to a different class of inducers.

Benzo[a]pyrene metabolism in rat fetal hepatocytes culture. Improved methodology and effect of substrate concentration.

The different characteristics of benzo[a[pyrene (BP) metabolism in primary fetal rat liver cell culture have been investigated. We have determined the extent of the in vivo [3H]BP metabolism by measuring all of the metabolites retained in the cell and excreted into the culture medium. The extent of the conjugation as well as the nature of the conjugates was established and the pattern of these metabolites analyzed by high performance liquid chromatography (HPLC). The fetal hepatocytes very actively metabolize BP and readily excrete in the culture medium all the produced metabolites in the form of sulfate and glucuronide conjugates. The relative proportion of those compounds varies as a function of the substrate concentration added to the cell culture, the higher the BP concentration, the more glucuronide conjugates. The HPLC analysis of the metabolites shows that BP-1,6-quinone and -3,6-quinone are the major excreted products, indicating the probable existence of an active 6 hydroxylation reaction in the fetal hepatocytes. On the other hand, the pattern of the different metabolites is influenced by the BP concentration. At low BP doses (0.8 microM), the relative amount of polar metabolites is twice as high and that of primary phenols twice as low, when compared to those produced by cells treated with 80 microM BP. The AHH activity drastically modifies the overall rate of the BP metabolism but does not affect the qualitative pattern of the excreted metabolites. The overall metabolism of [3H]BP by the cell culture can easily be estimated by measuring the release of the tritiated water from the substrate into the culture medium.