NADPH:cytochrome P-450(c) reductase: biochemical characterization in rat brain and cultured neurons and evolution of activity during development.

NADPH:cytochrome P-450 (c) reductase is a microsomal enzyme which is involved in the cytochrome P-450-dependent biotransformation of many exogenous agents as well as of some endogenous molecules. Using cytochrome c as a substrate, the kinetic parameters of this enzyme were determined in brain microsomes. The comparison of the NADPH:cytochrome P-450 reductase's Vmax values and cytochrome P-450 contents in both fractions, suggests a role of cerebral NADPH:cytochrome P-450 reductase in cytochrome P-450 independent pathways. This is also supported by the different developmental pattern of brain enzyme as compared to the liver enzyme, and by the presence of a relatively high NADPH:cytochrome P-450 reductase activity in immature rat brain and neuronal cultures, while cytochrome P-450 was hardly detectable in these preparations. The enzyme activity was not induced by a phenobarbital chronic treatment neither in the adult brain nor in cultured neurons, suggesting a different regulation of the brain enzyme expression.

Study of the in vitro bioactivation of albendazole in human liver microsomes and hepatoma cell lines.

The metabolism of albendazole (ABZ), a benzimidazole anthelminthic, was studied in either microsomal preparations of human liver biopsies or cultured human hepatoma cell lines. Metabolites were analyzed by HPLC. Our data show that microsomes from human biopsies and two human cell lines, HepG2 and Hep3B, oxidize the drug to the sulfoxide very efficiently, whereas the third cell line tested, SK-HEP-1, does not. Both cytochrome P-450 dependent monooxygenases and flavin-containing monooxygenases appear to be involved in human ABZ metabolism. Using the cell line displaying the highest ABZ-metabolizing activity, HepG2, the cytotoxic and the inducing effects of the parent drug ABZ and of two primary metabolites, the sulfoxide and the sulfone were studied. These three chemicals provoked a rise in mitotic index resulting from cell division blockage at the prophase or at the metaphase (ABZ metabolites) stage, and ABZ was more cytotoxic than its metabolites. With regard to enzyme-inducing effects, our data clearly demonstrate that the sulfoxide and, to a lesser degree, the sulfone are potent inducers of some drug metabolizing enzymes (i.e., cytochrome P-488 dependent monooxygenases and UDP glucuronyltransferase), whereas ABZ fails to increase and even slightly decreases these enzymatic activities. In conclusion, the HepG2 human hepatoma cell line appears to be suitable for the study of many parameters of metabolism and action of ABZ and other structurally related compounds in humans.

Albendazole sulfonation by rat liver cytochrome P-450c.

The metabolism of albendazole (ABZ) was studied in perfused livers from control and ABZ-treated rats (10.6 mg/kg, per os, each day for 10 days). In the perfusion fluid, the concentration of ABZ-sulfoxide (SO-ABZ) remained unchanged in treated, as compared to control animals, whereas ABZ-sulfone (SO2-ABZ) was increased in treated animals. In bile, only SO-ABZ was present. The transformation kinetics of SO-ABZ to SO2-ABZ in microsomes from rats treated with ABZ, 3-methylcholanthrene, Aroclor and isosafrole were biphasic. This suggests that enzyme activity was a consequence of two enzyme systems, one characterized by low affinity and high capacity, the other by high affinity and low capacity, the latter could be induced by 3-methylcholanthrene, ABZ, Aroclor and isosafrole. Cytochrome P-450c was induced potently in vivo by ABZ as proven by increased monooxygenase (7-ethoxyresorufin and 7-ethoxycoumarin-O-deethylase) activities and by Elisa test (a 5-fold increase in hemoprotein concentration was observed). Purified and reconstituted cytochrome P-450c from 3-methylcholanthrene or ABZ-treated rat liver were able to produce SO2-ABZ (2.01 and 1.70 nmol/mg/15 min, respectively, whereas cytochrome P-450b produced 10 times less SO2-ABZ). Immunological assays, as well as activity measurements showed a relationship between cytochrome P-450c-3-methylcholanthrene and cytochrome P-450c-ABZ. We conclude that induction of cytochrome P-450c by ABZ is the probable explanation for the enhanced formation of SO2-ABZ in vivo.

Inducing effect of albendazole on rat liver drug-metabolizing enzymes and metabolite pharmacokinetics.

Albendazole (ABZ), methyl (5-(propylthio)-1H-benzimidazol-2-yl)carbamate, is a broad spectrum anthelmintic drug. S-oxidation to the sulfoxide (SO-ABZ) and the sulfone (SO2-ABZ) are the first steps of its bioconversion. SO-ABZ is pharmacologically active and embryotoxic in rats. In the present study, rat liver microsomal drug-metabolizing enzymes were assayed after 10 days oral administration with 40 mumol ABZ/kg per day. The activities of 4-nitroanisole O-demethylase, benzo[a]pyrene hydroxylase, 7-ethoxycoumarin O-deethylase, and 7-ethoxyresorufin O-deethylase increased 6-, 7-, 8-, and 30-fold, respectively. By immunoblotting an increase in cytochrome P-448 was observed. UDP-glucuronosyltransferase (GT) type 1 activities (1-naphthol, 7-hydroxycoumarin, 4-nitrophenol, and 4-methylumbelliferone) were significantly higher than in control microsomes (3- to 4-fold), while GT type 2 activities and bilirubin-GT remained unchanged. Microsomal epoxide hydrolase (benzo[a]pyrene oxide) increased 2-fold. Microsomal gamma-glutamyltransferase activity was unchanged. The in vivo SO-ABZ plasma level was decreased when the SO2-ABZ plasma level was increased. In vitro sulfoxidation and sulfonation were, however, unchanged. Although a range of imidazole derivatives, including benzimidazole itself, were commonly reported as inhibitors of monooxygenase activities, ABZ behaved as an inducer of cytochrome P-448, GT1, and epoxide hydrolase.

Properties of human hepatic UDP-glucuronosyltransferases. Relationship to other inducible enzymes in patients with cholestasis.

Glucuronidation of 4-nitrophenol, nopol (a monoterpenoid alcohol) and bilirubin, which in the rat, are catalyzed by three different enzymes, has been examined in liver biopsies from patients with various liver diseases, in particular cholestasis. These different activities were not correlated, which strongly suggests that at least three independently regulated forms of UDP-glucuronosyltransferases were present in the microsomes. Non ionic detergents (Triton X100, Emulgen 911) and deoxycholate produced similar activation (more than 2-fold) of the glucuronidation of 4-nitrophenol. Amphipathic substances, such as CHAPS (3-[3-cholamidopropyl-dimethylammonio]-1-propane sulfonate), and lysophosphatidylcholines maximally increased this UDP-glucuronosyltransferase activity, the most potent being oleoyl lysophosphatidylcholine (4-fold increase). Discriminant analysis of the data revealed no correlation between the three different UDP-glucuronosyltransferase activities and the age or sex of the patients. A good correlation was found on multidimensional analysis between form 1 of the enzyme (4-nitrophenol glucuronidation) and, in decreasing order of magnitude, epoxide hydrolase (measured with benzo(a)pyrene-4,5-oxide as substrate), cytochrome P-450, 7-ethoxycoumarin deethylase, aspartate aminotransferase and gamma-glutamyltransferase (r = 0.89); and between Form 3 of the enzyme (bilirubin glucuronidation) and NADPH cytochrome c reductase, alkaline phosphatase, (r = 0.81). These relationships may reflect the differential variation in enzymatic activities in various hepato-biliary diseases.

Comparison of cytochrome P-450 content and activities in liver microsomes of seven animal species, including man.

The cytochrome P-450 content found in human livers obtained post mortem was between 0.21-0.42 nmol/mg protein. The kinetic parameters of the mono-oxygenase activities--Km and Vmax--were determined in liver microsomes for N-demethylation (aminopyrine, benzphetamine, ethylmorphine), O-demethylation (4-nitroanisole), O-deethylation (7-ethoxycoumarin) and hydroxylation (benzo[a]pyrene), in an attempt to establish an inter-species comparison between man and the six animal species studied. The four substrates studied (aminopyrine, benzphetamine, ethylmorphine, benzo[a]pyrene) were shown to be less active in humans than the male rat, which is the most commonly used model. However, other animal species, such as the female Sprague-Dawley rat and the pig, are much more similar to man. From a procedural point of view, the optimal substrate concentrations vary from one experimental species to another. Due to the apparent Km observed, for example, the activities of the guinea-pig require a higher substrate concentration.