Microsomal hydroxylase induction in liver cell culture by phenobarbital, polycyclic hydrocarbons, and p,p'-DDT.

Aryl hydrocarbon hydroxylase induction by phenobarbital, polycyclic hydrocarbons, and the insecticide, 2.2-bis(p-chlorophenyl)-1,1,1-trichloroethane (p,p'-DDT), occurs in rat fetal liver cell cultures. The maximum net rate at which the hydroxylase activity accumulates is about the same when phenobarbital, 3-methlcholanthrene, or benz[a]anthracene is in the growth medium at optimum concentrations. An additive effect is obtained when either phenobarbital or p.p'-DDT is present with a polycyclic hydrocarbon in the growth medium, but not when the cells are treated with phenobarbital plus p.p'-DDT or with the combination of two polycyclic hydrocarbons.

Genetic regulation of hepatic steroid 16 alpha-hydroxylase activities in inbred strains of mice.

Steroid 16 alpha-hydroxylase activities and properties were studied in C57Bl/6J, 129/J, AKR/R, DBA/2J, C3H/I, and BALB/c mouse liver using four different substrates. The highest enzymatic activities were measured in the female mice, with the exception of the 129/J females. As in the rat liver, the sexual differentiation of the steroid 16 alpha-hydroxylation observed in adult male and female mice took place at puberty. In the adult mouse liver, two steroid 16 alpha-hydroxylase activities (forms I and II) could be differentiated on the basis of their relative affinities for the various steroid substrates and their relative proportions in male and female mouse livers. In the immature mouse liver, no sexual differences could be detected, and the mice of both sexes presented phenotypes identical to those of the adult female. The adult 129/J females appeared genetically deficient with respect to the form I of the steroid 16 alpha-hydroxylase and presented a phenotype identical to that of the adult male mice of the various strains tested. Differences in hydroxylase activities between the C57Bl/6J and 129/J strains were investigated using standard genetic breeding protocols. Steroid 16 alpha-hydroxylase seemed to be inherited additively in the liver of the female mice obtained by crossing the C57Bl/6J male and the 129/J female or the 129/J male and the C57Bl/6J female. In the male mice, regardless of genotype, the observed phenotype was always identical to the two male parental types. Both hormonal and genetic regulations were responsible for the different phenotypes occurring in adult male and female C57Bl/6J and 129/J mouse livers.

Monoclonal antibodies against human liver cytochrome P-450.

Monoclonal hybridomas which produce antibodies against human liver microsomal cytochrome P-450 were developed. Three similar hybridomas produced antibodies which recognized an epitope specific to a family of human P-450 isozymes (P-450(5)). This epitope was also present on cytochrome P-450 PCN-E (pregnenolone-16 alpha-carbonitrile induced) from rat liver microsomes, but this isozyme differed from the human P-450(5) by its molecular weight. These antibodies enabled us to quantify cytochrome P-450(5) in human liver microsomes and to demonstrate an important quantitative polymorphism in the human liver monooxygenase system.

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.

Effects of gastroplasty on body weight and related biological abnormalities in morbid obesity.

Obesity is a prevalent metabolic disorder associated with high morbidity and mortality rates. Medical treatment rarely succeeds, and bariatric surgery has been proposed as an alternative therapy. The purpose of this non-controlled retrospective study was to evaluate time-course changes in body weight in severely obese patients who underwent vertical ring gastroplasty or adjustable silicone gastric banding, and to assess the prevalence and potential reversibility of several of the biological abnormalities associated with morbid obesity. From an initial cohort comprising 658 patients, regular body weight measurements and biological data were obtained in 505 patients [419 females, 86 males; age 36 +/- 11 years; body mass index 42.7 +/- 6.9 kg/m2; (mean +/- SD)] with a mean follow-up of 26 +/- 14 months. Mean weight loss was 32 +/- 16 kg. Most weight reduction occurred within the first 6 months, followed by near-stabilisation or even slight weight regain. Most biological parameters were obtained before surgery and after at least 6 months of follow-up. The high prevalence and severity of metabolic disturbances associated with the insulin resistance syndrome (hyperglycaemia, hyperinsulinaemia, decreased HDL cholesterol, hypertriglyceridaemia, elevated fibrinogen levels and hyperuricaemia) before gastroplasty were significantly decreased after weight loss. No major biological deficiencies were observed following gastroplasty, except low iron serum levels. It is concluded that marked weight loss associated with gastroplasty involved a remarkable reduction in the prevalence and severity of several biological abnormalities classically considered as cardiovascular risk factors.

Ascorbic acid effect on methylcholanthrene-induced transformation in C3H10T1/2 clone 8 cells.

Ascorbic acid (50 micrograms/ml), added to the culture medium on a biweekly basis, suppresses the methylcholanthrene-induced transformation of C3H10T1/2 cells.

Synthesis of 16 alpha-3H androgen and estrogen substrates for 16 alpha-hydroxylase.

The synthesis of 16 alpha-3H androgens and estrogens is described. 1-(3H)-Acetic acid in the presence of zinc dust reacts with 16 alpha-bromo-17-ketosteroids to produce 16 alpha-3H-17-ketosteroids. This chemical reaction was used to prepare 16 alpha-3H-dehydroepiandrosterone (I) and 16 alpha-3H-estrone acetate (XI) from 16 alpha-bromo-dehydroepiandrosterone (X) and from 16 alpha-bromo-estrone acetate (XII), respectively. Using appropriate microbiological techniques, it was possible to convert these radiolabelled substrates into 16 alpha-3H-androstenedione (II) and 16 alpha-3H-estradiol-17 beta (VII). 16 alpha-3H-Estrone (VI) was obtained by the chemical hydrolysis of 16 alpha-3H-estrone acetate. The label distribution as determined by microbiological 16 alpha-hydroxylations indicated a specific labelling of 77% for androgens and 65% for estrogens in the 16 alpha position. These substrates can be used for measuring the 16 alpha hydroxylase activity, an important step in the biosynthesis of estriol (VIII) and estetrol (IX).

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.

Competition between benzo[a]pyrene and various steroids for cytochrome P-450-dependent rat liver monooxygenases.

Cytochrome P-450-dependent monooxygenases are able to oxidize a large variety of endogenous and exogenous substrates. This paper describes the in vitro interaction between benzopyrene and steroids at the level of two rat liver monooxygenases: steroid-16 alpha-hydroxylase and aryl hydrocarbon hydroxylase (AHH). The results obtained suggest the following conclusions: (1) Steroid-16 alpha-hydroxylase is partially supported by a specific cytochrome P-450 form which is not inhibited in vitro by exogenous substrates. Steroid-16 alpha-hydroxylase is completely independent from cytochrome P1-450 (or P-448), as it is insensitive, in vitro, to alpha-naphthoflavone; (2) AHH is supported by two cytochrome P-450 forms: a specific form which is inducible by methylcholanthrene and inhibited in vitro by alpha-naphthoflavone, but is insensitive to metyrapone and steroids; and another less specific form which is inhibited by metyrapone and steroids in vitro.

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.

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.

Mutational specificity of benzo[a]pyrene diolepoxide in monkey cells.

Benzo[a]pyrene diolepoxide (BPDE) is thought to be the major mutagenic and carcinogenic intermediate in benzo[a]pyrene metabolism in mammalian cells. In order to test the mutagenic specificity of this compound in mammalian cells, we have used the pZ189 shuttle vector system to identify and analyze point mutations induced when DNA treated in vitro with BPDE is replicated in monkey cells. We find that point mutations occur almost exclusively at G.C base pairs; G.C----T.A and G.C----C.G transversions and single base pair deletions occur most frequently. This pattern is consistent with the known preferential covalent binding of BPDE to G residues.

Biochemical aspects of chemical carcinogenesis.

This article briefly outlines three major biochemical problems in relation to the presence of chemical carcinogens in mammalian cells: (a) the enzymatic systems responsible for the chemical carcinogen metabolism; (b) the nature of the reactive metabolites and (c) the cellular target(s). References and examples are only given for the field of the polycyclic aromatic hydrocarbons.

[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.

[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.