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M Christou

Publications and source records attributed to M Christou.

29 records · Page 2Linked to original sources
PubMed1987

Differential expression and function of three closely related phenobarbital-inducible cytochrome P-450 isozymes in untreated rat liver.

The levels of expression of cytochromes P-450b and P-450e (both inducible by phenobarbital (PB) and differing by only 14 of 491 amino acids) in liver microsomes from untreated male rats were separately quantitated by Western blotting with a polyclonal antibody raised against P-450b that is equally effective against P-450e (anti P-450b/e). A protein with mobility identical to P-450e was detected in all microsomal samples. Microsomes from uninduced livers of individual male rats from five different strains exhibited only minor interstrain and interindividual variability in the expression of P-450e (17 +/- 5 pmol P-450e/mg microsomal protein) with the exception of the Brown Norway strain (8.5 +/- 0.5 pmol P-450e/mg). Expression of P-450b varied widely from undetectable levels (less than 2 pmol/mg) in most Sprague-Dawley rats to about 50% of P-450e levels in Fischer and Brown Norway strains. Anti P-450b/e inhibited total metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) by uninduced microsomes, to an extent dependent on rat strain (15-30%), predominantly through inhibition of formation of 12-hydroxymethyl-7-methyl BA (12HOMMBA) (65-85%), the major metabolite of purified P-450e. A specific activity for P-450e-dependent DMBA metabolism was calculated from four sets of microsomes where the P-450b content was either undetectable or very low (0.7-1.0 nmol/nmol P-450e/min-1). Comparable calculated activities were, however, obtained from other untreated rat liver microsomes where P-450b levels were significant. Polymorphism in P-450b was detected but did not affect total P-450b expression or the sensitivity of DMBA metabolism to anti P-450b/e. A fourth band of greater mobility than P-450b (apparent Mr less than 50,000), was also recognized by anti P-450b/e. The intensity of this band did not vary among individual rats or among the different strains and therefore did not correlate with the sensitivity of microsomal DMBA metabolism to anti P-450b/e. A monoclonal antibody (MAb) against P-450b (2-66-3) recognized P-450's b, b2, and e on Western blots but did not react with this higher mobility band. MAb 2-66-3 and two other MAbs produced against P-450b inhibited 12-methylhydroxylation of DMBA by untreated rat liver microsomes to the same extent as anti P-450b/e. Following PB induction, P-450b was induced to about double the level of P-450e in most rat strains examined.(ABSTRACT TRUNCATED AT 400 WORDS)

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1987

Product inhibition of benzo[a]pyrene metabolism in uninduced rat liver microsomes: effect of diol epoxide formation.

Conversion of benzo[a]pyrene (BP) to BP 7,8-dihydrodiol 9,10-oxides (DE) (measured as 7,10/8,9-tetrols) by untreated (UT) rat liver microsomes is over 10 times slower than following 3-methylcholanthrene (MC) induction. Time courses have been subjected to a kinetic analysis analogous to that previously reported for metabolism by MC-induced microsomes (J. Biol. Chem., 259 (1984) 13770-13776). Competition between BP and 7,8-dihydrodiol for P-450 is the major determinant of the rate of DE formation. Glucuronidation of quinones and phenols only increases the isolated BP metabolites including DE by 40%. This indicates far less inhibition by these products than for metabolism in MC-microsomes (4-6-fold). Thus stimulation may result from a decreased quinone-mediated oxidation of metabolites. In the presence of DNA, UT-microsomes metabolize BP to approximately equal amounts of 9-phenol-4,5-oxide (9-PO) and DE/DNA adducts. Addition of uridine diphosphoglucuronic acid (UDPGA) fails to enhance modification of DNA by DE, but formation of the 9-PO adduct is reduced as a result of lower free 9-phenol levels. The kinetic characteristics of BP metabolism by UT-microsomes are highly sensitive to the presence of very small but variable amounts (2-25 pmol/mg) of the very active cytochrome P-450c, which is the predominant form in MC-microsomes. The major effect of elevated levels of P-450c is an 8-fold increase in DE formation at low concentrations of BP due to a lowering of Km (7.9-2.6 microM) and an increase in the regioselectivity for DE formation from 7,8-dihydrodiol (5-15% of total BP metabolites). The formation of DE was directly correlated with the content of P-450c (r = 0.94). The presence of increased levels of P-450c in UT-microsomes is probably due to previous exposure of the animals to environmental inducers and is minimized by controlled housing and feeding.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1987

Induction of mammary cytochromes P-450: an essential first step in the metabolism of 7,12-dimethylbenz[a]anthracene by rat mammary epithelial cells.

Rat mammary epithelial cells (RMEC) in culture have been shown to activate polycyclic aromatic hydrocarbon (PAH) carcinogens. This study investigates the role of mammary cytochrome P-450 monooxygenases in these metabolic processes. Monooxygenation of 7,12-dimethylbenz[a]anthracene (DMBA) by RMEC in culture exhibited a 6-h lag period before reaching a constant rate. The mechanism for this time-dependent expression of DMBA monooxygenase activity was investigated in lysed cells, where both conjugation and in situ induction of P-450 are prevented. Although metabolism of DMBA by untreated RMEC lysates was undetectable (less than 1 pmol/mg cell protein/h), prior exposure of cultured cells to benz[a]anthracene (BA) induced DMBA metabolism, (approximately 100 pmol/mg cell protein/h). BA pretreatment also eliminated the lag period for metabolism of DMBA by cultured RMEC but did not prevent additional induction of DMBA monoxygenase activity by the substrate. The distribution of monooxygenated DMBA metabolites formed by BA-induced cell lysates was clearly different from that obtained with purified P-450c, the predominant PAH-inducible isozyme in rat liver. For example, the carcinogen precursor DMBA 3,4-dihydrodiol, which is not formed by P-450c, was a clearly detectable product in RMEC. The low epoxide hydratase activity of BA-induced lysate (approximately 400-fold lower compared to that in the liver) limited formation of all DMBA dihydrodiols. The formation of DMBA 3,4-dihydrodiol increased by 5-fold following addition of exogenous purified epoxide hydratase. The DMBA monooxygenase activity of BA-induced RMEC lysates was completely inhibited by alpha-naphthoflavone but was only partially inhibited (50%) by a polyclonal antibody raised against cytochrome P-450c. Anti P-450c completely inhibited formation of some of the metabolites, partially inhibited formation of others and notably stimulated formation of DMBA 3,4-dihydrodiol by 60%. A polyclonal antibody that recognized both rat hepatic P-450a and a group of P-450 isozymes related to P-450h, and which totally inhibited DMBA 3,4-dihydrodiol formation by rat liver microsomes, did not inhibit formation of any DMBA metabolite in RMEC, including DMBA 3,4-dihydrodiol. Western blot analyses of RMEC homogenates demonstrated that BA pretreatment induces P-450c, but not P-450a or any of the P-450h-related isozymes. We conclude that metabolism of DMBA by RMEC depends on induction of P-450c and at least one additional form of cytochrome P-450 which is immunochemically distinct from rat hepatic P-450a and P-450h related isozymes, but is sensitive to alpha-naphthoflavone.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1986

Regulation of rat and bovine adrenal metabolism of polycyclic aromatic hydrocarbons by adrenocorticotropin and 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on polycyclic aromatic hydrocarbon (PAH) metabolism and steroidogenesis in primary cultures of bovine adrenal cortical (BAC) and rat adrenal cortical (RAC) cells have been examined. Remarkably TCDD is an ineffective inducer (15-50%) of PAH metabolism in confluent BAC cells and completely antagonizes a 5-fold induction by benz[alpha]anthracene (BA). In the same concentration range (EC50 5 X 10(-11) M) TCDD suppresses steroidogenesis through an effect on cholesterol metabolism. Adrenocorticotropin (ACTH) and cAMP also suppress PAH metabolism at concentrations which stimulate steroidogenesis (10(-7) M). In RAC cells ACTH potently induces PAH metabolism (7-fold) at a comparable concentration to the stimulation of steroidogenesis. Parallel stimulation of PAH metabolism and steroidogenesis by cAMP suggest that ACTH induction of PAH metabolism is mediated by cAMP. TCDD induces PAH metabolism (2.8-fold, EC50 8 X 10(-11) M) at similar concentrations to the inhibitory effect in BAC cells and this action is additive with ACTH induction. In male rats in vivo TCDD induces adrenal microsomal PAH metabolism (72%) and is more effective in this respect than 3-methylcholanthrene (3MC). Rabbit antibodies against rat liver cytochrome P-450c (the major TCDD-inducible liver form) inhibited the TCDD-induced adrenal metabolism of 7,12-dimethylbenz[alpha]anthracene (DMBA), which also exhibited regioselectivity typical of metabolism by P-450c. Constitutive adrenal microsomal metabolism, which exhibited regioselectivity of DMBA metabolism comparable to the ACTH-sensitive cellular metabolism, was not affected by anti-P-450c. It is concluded that ACTH and TCDD induce distinct forms of cytochrome P-450 in RAC cells and that the latter represents a typical Ah-receptor mediated response. The anomalous effect on PAH metabolism in BAC cells that parallels inhibition of steroidogenesis may derive from repression of a distinct adrenal form of P-450 by the TCDD-Ah-receptor complex.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1986

Selective interactions of cytochromes P-450 with the hydroxymethyl derivatives of 7,12-dimethylbenz[a]anthracene.

Competition between a hydroxylated metabolite and the parent polycyclic aromatic hydrocarbon (PAH) for metabolism at cytochromes P-450 may result in the generation of hydroxylated dihydrodiol epoxides. The effectiveness of the competition between 7-hydroxymethyl-12-methylbenz[a]anthracene (7HOMMBA) or 12-hydroxymethyl-7-methylbenz[a]anthracene (12HOMMBA) and 7,12-dimethylbenz[a]anthracene (DMBA) is highly dependent on the form(s) of cytochrome P-450 in the microsomes. The inhibitory effects of exogenously added 7HOMMBA or 12HOMMBA on DMBA metabolism were 30- to 50-fold greater in 3-methylcholanthrene (MC)-induced rat liver microsomes (Ki = 0.4 microM) compared to either uninduced or phenobarbital (PB)-induced liver microsomes (Ki = 14 and 11 microM, respectively). Similarly, product inhibition of total DMBA metabolism by metabolites generated in situ was significant only in MC-induced liver microsomes (Ki' = 2.5 microM). Metabolism of 7HOMMBA in these microsomes was strongly restricted by an unusual substrate inhibition derived from the inhibitory binding of a second molecule of 7HOMMBA. This same phenomenon was observed with reconstituted cytochrome P-450c but not with PB-induced or uninduced microsomes. Complex formation by binding of DMBA, 7HOMMBA, and 12HOMMBA to purified P-450c reconstituted in phospholipid micelles was determined by optical spectroscopy and fluorescence quenching. Binding affinities of both the 7HOMMBA and 12HOMMBA (Kd = 95 and 110 nM, respectively), were 2.5-fold higher compared to that of DMBA (Kd = 265 nM). These results provide a first demonstration that hydroxylation of a PAH can lead to preferential metabolism through an increased affinity for cytochrome P-450.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1986

Modulation of carcinogenesis by dietary factors.

The purpose of this report is to present recent data on two modulating factors of carcinogenesis that are found in Western-type diets: a beef-derived mutagenesis modulator that has been shown to inhibit the initiation of epidermal carcinogenesis in mice, and the possible role of net energy rather than dietary fat per se in the enhancement of rat mammary carcinogenesis.

Animals↗
PubMed1986

Metabolic N-hydroxylation of substituted acetophenone imines. I. Evidence for formation of isomeric oximes.

A series of chemically stable substituted acetophenone imines and their potential N-hydroxylated metabolites (i.e., oximes) have been synthesized and characterized by spectroscopic methods. The enzymic N-hydroxylation of acetophenone imines in vitro has been demonstrated as a general metabolic pathway in several mammalian species including the guinea-pig. The oxime metabolites were formed as mixtures of two geometric isomers, Z (syn-phenyl) and E (anti-phenyl), wherein the phenyl and hydroxyl group are cis and trans to each other respectively. The E (anti-phenyl) isomer was the quantitatively predominant isomeric form metabolically produced by all species studied. The relative proportions of the E and Z isomers in metabolic mixtures were found to be species dependent.

Acetophenones↗
PubMed1984

The role of secondary metabolism in the metabolic activation of 7,12-dimethylbenz[a]anthracene by rat liver microsomes.

The effect of induction by either phenobarbital (PB) or 3-methylcholanthrene (MC) on the kinetics of both primary and secondary metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) have been studied. PB and MC induction stimulate the initial rate of total DMBA metabolism 4- and 8-fold, respectively. Individual metabolites exhibited distinct time courses dependent on the inducer. With both induced and uninduced microsomes, formation of DMBA 5,6-dihydrodiol exhibited a 2-5 min time lag before reaching a maximum rate while, in contrast, formation of 7-hydroxymethyl-12-methylbenz[a]anthracene (7HOMMBA) declined substantially from linearity at a very early point in the reaction. This was most apparent after 2 min and total levels decreased after 5 min. Regioselectivity for both DMBA and 7HOMMBA were measured based on the initial rates of mono-oxygenation derived from these kinetics. In each set of microsomes, prior 7-hydroxylation redirected metabolism towards DMBA 3,4-dihydrodiol formation and away from 5,6-dihydrodiol. Large differences were noted for the effectiveness and regioselectivity of secondary metabolism via 7HOMMBA. MC-induced liver microsomes exhibited a stronger preference for secondary metabolism of 7HOMMBA than PB-induced and control microsomes. In all cases, 7HOMMBA dihydrodiols, formed from DMBA, appeared in the same ratio as when formed directly from 7HOMMBA, suggesting formation from 7HOMMBA rather than from 7-hydroxylation of primary dihydrodiols. The extent of secondary metabolism of primary products was calculated as the difference between the calculated formation (determined from the appropriate regioselectivity and DMBA disappearance) and the quantitated level at a particular reaction time. However, for MC-induced microsomes, quantitation of 7HOMMBA mono-oxygenated metabolites accounted for less than 50% of the calculated 7HOMMBA secondary metabolism. This discrepancy results from selective further oxidation of 7HOMMBA phenols. For control and PB-induced liver microsomes, quantitation of identifiable 7HOMMBA mono-oxygenated metabolites agreed well with calculated secondary 7HOMMBA metabolism. Based on the results presented here, factors that favor hydroxylation of PAH prior to bay-region diol-epoxide formation are discussed.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1984

Binding and metabolism of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene by seven purified forms of cytochrome P-450.

Seven different forms of cytochrome P-450 have been purified from rat liver microsomes. The major 3-methylcholanthrene (MC) inducible cytochrome P-450 (form c) exhibits the greatest activity toward both benzo[a]pyrene (BP) (58 min-1) and 7,12-dimethylbenz[a]anthracene (DMBA) (29 min-1) and forms substantially high spin, high affinity complexes (Kd = 10 nM) with both hydrocarbons. Cytochrome P-450d, a minor MC-inducible form, has far lower activity for metabolism of both polycyclic aromatic hydrocarbons (PAH), yet also forms high affinity complexes (Kd approximately 100 nM) with both PAH, retaining the full high spin state of the free cytochrome. Although two phenobarbital (PB)-induced forms (P-450's b and e) differ by only 13 amino acids, they exhibit significant differences in metabolism of PAH and in complex formation. Whereas P-450b is only active in metabolism of DMBA (9.8 min-1 versus 1.9 min-1 for BP), P-450e has low activity for both substrates (3.3 and 1.2 min-1). Nevertheless, P-450e forms a high affinity complex (Kd approximately 100 nM) with both PAH that enhances the proportion of the high spin state (from 30% to 70%). Failure to displace n-octylamine (NOA) suggests binding that is removed from the heme. P-450b remains low spin in the presence of PAH and NOA is again not displaced. In addition, the two forms can be distinguished by their regioselectivities for both PAH. P-450's a, h, and pregnenolone-16 alpha-carbonitrile (PCN) exhibit little activity toward BP or DMBA, but P-450 PCN does form a low spin complex with BP (not DMBA). Regioselectivity in metabolism of DMBA by PB-induced microsomes does not agree with that of the major constituent forms. Only the minor, less active purified forms (e and a) mediate substantial 12-hydroxylation and 3,4-epoxidation of DMBA. Thus, additional factors in microsomal reactions must contribute to these differences.

9,10-Dimethyl-1,2-benzanthracene↗
PubMedDate not supplied

Primary aliphatic amine metabolism: the formation of a stable imine metabolite.

The possible role of hydroxylamine and imine intermediates in the metabolism of amphetamine and related primary amines is discussed. The inherent instability of imines, has hitherto precluded the recognition of these compounds as metabolites of primary amines. During studies on the metabolism of o-methylbenzhydrylamine using washed hepatic microsomes from a number of mammalian species, the formation of o-methylbenzophenoneimine as a metabolite was observed. This metabolite was identified by preparative thin layer chromatography followed by gas-chromatography combined with mass spectrometry.

Amphetamines↗