Jacques Van Cantfort

Radioactive assay for aryl hydrocarbon hydroxylase. Improved method and biological importance

By addition of two volumes of a 1M aqueous KOH/dimethylsulfoxide (1585; v/v) mixture to the enzymatic incubation medium, it is possible to selectively extract the unmetabolized benzo(a)pyrene in hexane. Therefore, the radio-activity remaining in the water phase corresponds to all the in vitro synthesized metabolites. This isotopic method is very sensitive (2 × 10−11 moles) and is almost insensitive to the room lighting. The aryl hydrocarbon hydroxylase activities found with this method are 2,3 and 10 times higher in the liver, lung and kidney respectively compared to those obtained with the fluorimetric method.

Cholesterol 7 α‐Hydroxylase

70% of the microsome-bound cholesterol is directly accessible to cholesterol 7alpha-hydroxylase in an assay in vitro. After 5 min of incubation this endogenous cholesterol makes a single pool with the exogenously added substrate and modifies its specific radioactivity. Thus, an accurate estimation of the enzymic activity should take the participation of endogenous cholesterol into account. Cholesterol 7alpha-hydroxylase activity is enhanced in vitro by thiol-containing substances like mercaptoethanol, dithiothreitol, or cysteamine. On the contrary, the enzymic activity is inhibited by heavy cations (Hg2+, Pb2+, Cu2+, Zn2+), or --SH-blocking agents (mersalic acid p-chloro-mercuribenzoic acid). Several steroids are potent inhibitors (Ki less than Km) of the enzyme, among them pregnenolone and its acetate derivative and the cholesterol closely related 7-oxocholesterol and 7-dehydrocholesterol. The cholesterol esters are neither substrates nor inhibitors of cholesterol 7alpha-hydroxylase. Only a high concentration (1 mM) of biliary acids or of their glyco or tauro derivatives inhibits cholesterol 7alpha-hydroxylase. The quantitatively less important lithocholic acid and deoxycholic acid are the strongest inhibitors; the most common cholic acid does not affect the enzymic activity even at 1 mM.

Genetic and Hormonal Regulation of Steroid Hydroxylases and Drug Metabolizing Enzymes in Rat Liver

Two microsomal steroid hydroxylase activities (cholesterol-7α-hydroxylase and progesterone-16α-hydroxylase) were measured in the livers of Sprague-Dawley and Wistar rats and compared to three other monooxygenase activities (aryl hydrocarbon hydroxylase, p-nitro-anisole-O-demethylase and aminopyrine-N-demethylase). Cholesterol-7α-hydroxylase behaves in a very unique manner. It is the only one of the studied enzymes to be more active in the female than in the male, it is very poorly induced by phenobarbital and methylcholanthrene, but responds quickly to the administration of glucocorticoids. In fact, the cholesterol-7α-hydroxylase activity presents a very pronounced circadian rhythm which is under the control of the hypothalamo-adrenal axis. Marked differences are also found in the response of the various enzymatic activities to the administration of inducers as well as in their relative activities in untreated male and female animals.ZusammenfassungZwei mikrosomale Steroidhydroxylase-Aktivitäten (Cholesterin-7α-hydroxylase und Progesteron-16α-hydroxylase) wurden in Lebern von Sprague-Dawley und Wistar-Ratten gemessen und mit drei anderen Monooxygenase-Aktivitäten (Aryl hydrocarbon hydroxylase, p-Nitro-anisol-O-demethylase und Aminopyrin-N-demethylase) verglichen.Cholesterin-7α-hydroxylase weist ein besonderes Verhalten auf. Es ist das einzige der untersuchten Enzyme, welches bei weiblichen Tieren stärker aktiv ist als bei männlichen Tieren, das durch Phenobarbital und Methylcholanthren nur sehr wenig induziert wird, aber sehr schnell auf die Verabreichung von Glucocorticoiden reagiert. Die Cholesterin-7α-hydroxylase Aktivität weist einen stark ausgeprägten circadianen Rhythmus auf, welcher ja durch die Hypothalamoadrenale-Achse kontrolliert wird.Ausgeprägte Unterschiede werden auch in der Reaktion der verschiedenen enzymatischen Aktivitäten auf die Verabreichung von Induktoren festgestellt, wie auch in ihren relativen Aktivitäten bei unbehandelten männlichen und weiblichen Tieren.

RELATION BETWEEN THE CIRCADIAN RHYTHMS OF MITOTIC RATE AND CHOLESTEROL‐7α‐HYDROXYLASE ACTIVITY IN THE REGENERATING LIVER

The evolution of mitoses and cholesterol‐7α‐hydroxylase activity were studied in parallel in the regenerating liver after a partial hepatectomy performed at two different hours of the nycthemeral period, i.e. 10 a.m. and 8 p.m.

The in-vitro formation and the strong inhibitory action of 7-keto-cholesterol on cholesterol-7α-hydroxylase activity

Abstract 7-keto-cholesterol is a non-physiological compound usually formed in vitro during the incubation of cholesterol. Experimental data indicates that this substance does not derive from 7α-hydroxycholesterol. Therefore, its formation should not be taken into account in the measurement of cholesterol-7α-hydroxylase activity. However, 7-keto-cholesterol strongly inhibits the activity of this enzyme.

Determination of papaverine in blood samples by gas-liquid chromatography and mass fragmentography

The measurement of papaverine in blood samples by using either a glass capillary column with a flame-ionization detector or a packed column with mass fragmentographic detection is described. The two methods permit the determination of the normal range of concentrations of papaverine in blood (2-500 ng/ml). Owing to its high specificity, mass fragmentography is greatly superior to capillary chromatography, which is sometimes subject to interferences by solvent impurities.

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.

Influence of a chronic administration of diethylnitrosamine on the relation between specific tissular and division functions in the rat liver

Abstract The evolution of the division and specific tissular functions, in the liver, which are mutually exclusive of young and partially hepatectomised adult rats, was studied during hepatic carcinogenesis induced by the chronic administration of diethylnitrosamine. The results show that one of the phenomena implicated in this chemical carcinogenesis is the deregulation of the normal equilibrium between the specific tissular and the division functions. In effect, during the preneoplasic stages, these two functions are progressively disrupted; they lose their normal exclusive relationship and their specific nychtermeral rhythms. These abnormalities are observed in the unoperated as well as partially hepatectomised rats.

The influence of five monooxygenase inducers on liver cytosol estradiol receptor levels in the ovariectomized adult rat

Abstract The intraperitoneal injection of the well known monooxygenase inducers (phenobarbital, β-naphtoflavone, pregnenolone-16α-carbonitrile, benzo(a)-pyrene, methylcholanthrene) elicits a net decrease in the specific binding of estradiol to its cytosol receptor in female rat livers. Amongst the five chemicals tested, only phenobarbital did not exhibit such a phenomenon, but caused a slight increase. This observation was neither due to a competitive inhibition by these compounds, nor to an enhanced metabolism of [ 3 H]-estradiol. Moreover, when this effect was produced by polycyclic hydrocarbons, it was inversely correlated to the activity of aryl hydrocarbon hydroxylase, induced by these same chemicals.