Cytochrome P-450- and flavin-containing monooxygenase-catalyzed formation of the carcinogen N-hydroxy-2-aminofluorene and its covalent binding to nuclear DNA.

The metabolic N-oxidation of the carcinogen 2-aminofluorene was examined in vitro using fortified hepatic microsomes from a variety of species. Rat, dog, human, and pig liver microsomes catalyzed the formation of N-hydroxy-2-aminofluorene (N-OH-AF) from AF at rates of 1.6, 1.0, 1.2, and 3.5 nmol/min/mg protein, respectively. The involvement of both cytochrome P-450 and the flavin-containing monooxygenase was demonstrated with hepatic microsomes and with purified enzymes by using specific enzyme inhibitors. 2-[(2,4-Dichloro-6-phenyl)phenoxy]ethylamine, a potent cytochrome P-450 inhibitor, decreased microsomal N-OH-AF formation by 96, 83, 70, and 46% in the rat, dog, human, and pig, respectively; and further addition of methimazole, a high-affinity flavin-containing monooxygenase substrate, abolished the residual N-hydroxylating activity. Using the purified porcine flavin-containing monooxygenase, metabolic formation of N-OH-AF occurred at a rate of 4.9 nmol/min/nmol flavin adenine nucleotide and was insensitive to 2-[(2,4-dichloro-6-phenyl)phenoxy]ethylamine inhibitor. In addition, purified rat liver cytochrome P-450 (isolated from 5,6-naphthoflavone-induced animals) N-hydroxylated AF (1.1 nmol/min/nmol P-450) and was completely inhibited by 2-[(2,4-dichloro-6-phenyl)-phenoxy]ethylamine, but the reaction was insensitive for methimazole. To determine whether or not the metabolic formation of N-OH-AF could lead directly to covalently bound adduct(s) with DNA under these incubation conditions (30 min, pH 7.5), the binding of synthetic and metabolically formed [3H]-N-OH-AF to added calf thymus DNA and to DNA in isolated rat liver nuclei was investigated. In all cases, the amount of DNA-bound carcinogen accounted for 0.08 to 0.15% of the N-OH-AF present in the incubation mixtures. These data, when compared to the levels of AF bound to hepatic nuclear DNA reported in vivo, suggest that the nonenzymatic reaction of N-OH-AF with nuclear DNA may be sufficient to account for a substantial portion of the observed in vivo binding of this carcinogen.

Flavin-containing monooxygenases: enzymes adapted for multisubstrate specificity.

Unlike all other oxidases, microsomal flavin-containing monooxygenases (FMO) discriminate between essential and foreign compounds by excluding the former rather than selectively binding the latter. As Daniel Ziegler describes here, xenobiotics that readily cross cell membranes can enter the catalytic cavity, whereas charged groups on essential metabolites that prevent their passive diffusion out of the cell also block their access to FMO. FMO appears to be ideally adapted to catalyse the detoxification of structurally diverse soft nucleophiles (e.g. alkaloids with basic side-chains and organic sulfur xenobiotics) so abundant in food derived from plants.

Studies on substrate specificity of the hog liver flavin-containing monooxygenase. Anionic organic sulfur compounds.

The influence of anionic groups on interaction of nucleophilic sulfur compounds with the purified hog liver flavin-containing monooxygenase was evaluated from kinetic constants obtained with various dithiobenzoates, thiolbenzoates, and thiolalkylcarboxylic acids. All compounds tested bearing a single negative charge localized on sulfur were excellent substrates but derivatives with a carboxylic acid group one or two carbons removed from the heteroatom exhibited low or no substrate activity. The effect of a carboxylic acid group more distal from sulfur appeared to depend on steric factors that are not well defined. For instance, none of the carboxylic acids (C2-C8) bearing a single thiol on the terminal carbon were oxygenated at detectable rates, whereas dihydrolipoic acid appeared to be a substrate although the concentration required for half-maximal activity was quite high (approximately 2 mM). Lipoic acid was a much better substrate (Km = 0.12 mM), and kinetic constants obtained with lipoic acid analogues suggest that position of the negative charge relative to the dithiolane ring is critical, since increasing the length of the side chain increased the Km. None of the alicyclic disulfides or sulfides containing one or more carboxylic acid groups showed detectable substrate activity. However, the more lipophilic sulfur-containing fatty acids inhibited the enzyme which may mask their potential substrate activity.

Use of thiocarbamides as selective substrate probes for isoforms of flavin-containing monooxygenases.

The oxidation of thiourea, phenylthiourea, 1,3-diphenylthiourea, 1,3-bis-(3,4-dichlorophenyl)-2-thiourea and 1,1-dibenzyl-3-phenyl-2-thiourea was measured in reactions catalyzed by purified pig liver flavin-containing monooxygenase (FMO-1) and by microsomal fractions isolated from pig, guinea pig, chicken, rat and rabbit tissues. The reactions, followed by measuring substrate-dependent thiocholine oxidation [Guo and Ziegler, Anal Biochem 198: 143-148, 1991], were carried out in the presence of 2 mM 1-benzylimidazole to minimize potential interference from reactions other than those catalyzed by isoforms of the flavin-containing monooxygenase (FMO). While at saturating substrate concentrations the Vmax for purified FMO-1 catalyzed oxidation of all five thiocarbamides was essentially constant, velocities for the microsomal catalyzed reactions varied not only with tissue and species but also with the van der Waals' surface area of the thiocarbamide. Rat liver, rat kidney and rabbit liver microsomes failed to catalyze detectable oxidation of thiocarbamides larger than 1,3-diphenylthiourea and lung microsomes from a female rabbit only accepted substrates smaller than 1,3-diphenylthiourea. On the other hand, liver microsomes from chickens, pigs and guinea pigs catalyzed the oxidation of larger thiocarbamides, but the rates decreased with increasing substrate size and chicken liver microsomes showed no detectable activity with the largest thiocarbamide tested. To define more precisely the parameters affecting thiocarbamide substrate specificity of microsomal preparations, activities present in detergent extracts of guinea pig liver microsomes were separated into three distinct fractions. The substrate specificities of these partially purified fractions were different and consistent with the difference observed with microsomal catalyzed reactions. This strongly suggests that thiocarbamides that differ in size may be useful probes for measuring the number of activities of FMO isoforms in crude tissue preparations.

Studies on the biliary efflux of GSH from rat liver due to the metabolism of aminopyrine.

The biliary efflux of GSH and GSSG due to aminopyrine was studied using perfused rat livers. The infusion of 0.8 mM aminopyrine led to a rapid rise in the amount of GSH released into the bile with only a small increase in the amount of GSSG released; caval GSH + GSSG efflux was unaffected. N-Benzylimidazole, an inhibitor of cytochrome P-450, completely blocked the response while phenobarbital pretreatment of the rats doubled the rate of GSH efflux. H2O2 and selenium-containing glutathione peroxidase were not involved since livers from selenium-deficient rats perfused with aminopyrine released GSH at the same rate as control livers. Aminopyrine injected i.p. into conscious rats also stimulated biliary GSH efflux to the same extent as with perfused livers. Biliary release of GSH in the perfused livers could be duplicated by infusing formaldehyde. It is proposed that formaldehyde produced during the N-demethylation of aminopyrine by cytochrome P-450 combines reversibly with GSH to form S-hydroxymethylglutathione which is oxidized by formaldehyde dehydrogenase to S-formylglutathione. Formaldehyde formed in excess of its capacity to be metabolized enzymatically is released into the bile as S-hydroxymethylglutathione which then dissociates to its initial reactants.

Nitroglycerin and isosorbide dinitrate stimulation of glutathione disulfide efflux from perfused rat liver.

Nitroglycerin (GTN) and isosorbide dinitrate (ISD) are metabolized by glutathione S-transferase to nitrite with production of GSSG from GSH. Infusion of organic nitrates into perfused rat liver led to efflux of GSSG in the bile and nitrite in the perfusate. Biliary GSSG increased more rapidly than did nitrite release as GTN infusion rate was increased, indicating that GSSG reducing capacity was being exceeded. Rapid GTN-induced oxidation of GSH may be the mechanism of tissue GSH depletion by GTN and other alkylnitrates. Such depletion of glutathione may reduce nitrite production from organic nitrates and underlie tolerance to these drugs.

Multisubstrate flavin-containing monooxygenases: applications of mechanism to specificity.

Kinetic studies on mechanism of the flavin-containing monooxygenase (FMO1) from pig liver microsomes are described in detail with special emphasis on the interpretation of constants derived from the rate equation. The evidence reviewed indicates that oxidation of xenobiotic substrates by the 4a-hydroperoxyflavin form of the enzyme is a second order reaction not saturable by substrate. Under steady-state conditions decomposition of the hydroxyflavin (an intermediate form of the enzyme that does not require enzyme-substrate or enzyme-product equilibrium complexes) is rate limiting. The lack of detectable equilibrium binding is also consistent with rate constants defining Km deduced from steady-state measurements. A model consistent with all evidence currently available indicates that at saturating concentrations of xenobiotic substrates that catalytic site on the enzyme is unoccupied most of the time. This property may explain why non-substrate analogs of xenobiotic substrates do not inhibit FMO activity. Rate constants for the oxidation of xenobiotics by the enzyme-bound and synthetic 4a-hydroperoxyflavin indicate that while enzyme protein accelerates the reaction with xenobiotics bearing nitrogen, it has only marginal effects on the oxidation of substrates bearing sulfur. Differences in the nucleophilicity of compounds bearing these heteroatoms may be primarily responsible but other, as yet undefined, factors may also contribute. In addition, analysis of rate constants affected by protonated lipophilic amines indicates that these allosteric effectors apparently modify enzyme structure so as to affect two or more rate constants and, depending on the nature and concentration of the xenobiotic substrate, protonated amines can either stimulate or inhibit catalytic activity.

Cost-effectiveness of clozapine therapy for severe psychosis.

The cost-effectiveness of using the atypical antipsychotic medication clozapine for severe psychosis was examined in a rural public-sector community mental health setting in Virginia. Based on a sample of 20 patients, use of clozapine resulted in estimated cost savings of between $3,000 and $9,000 per patient per year, including the costs of dropouts from treatment. Savings were mainly due to a decline in hospitalization from 47.7+/-59.8 days per patient in the year before clozapine treatment to 4.6+/-11.3 days in the year after. Although this study had methodological limitations, the results suggest that clozapine may be cost-effective in this setting.

Flavoenzymes inhibited by indomethacin.

The effect of indomethacin on the activity of five different flavoenzymes, three dehydrogenases and six hydrosases, was determined. Indomethacin at concentration 1.0 mM inhibited the activity, in decreasing order of sensitivity, of the following flavoenzymes: D-amino acid oxidase (pig kidney), flavin-containing monooxygenases (pig liver microsomal), cyclohexanone monooxygenase (Acinetobacter), NADPH-quinone reductase (pig liver), and glutathione reductase (yeast), but it had no effect on the activity of glucose oxidase (Aspergillus) or liver microsomal NADPH-cytochrome P-450 reductase. Indomethacin was competitive with D-alanine for the D-amino acid oxidase (Ki=30 microM) and with NADPH for all other flavoenzymes sensitive to this compound (Kis 170-500 microM). While indomethacin also inhibited two of the three NAD(P)+-dependent dehydrogenases tested, the Kis were relatively high (<1, 500 microM), and of the six different hydrolases tested only one, liver microsomal esterase, was inhibited by indomethacin (Ki=600 microM). Indomethacin also inhibited aminopyrine demethylation catalyzed by the liver microsomal P-450 monooxygenase (Ki=1,000 microM). Although the exact mechanism for the inhibition of functionally different flavoenzymes sensitive to indomethacin is not known, the inhibition is probably not due to the detergent properties of this drug.

Effects of dietary protein-energy interrelationships on Holstein steer performance and ruminal bacterial fermentation in continuous culture.

In vivo and in vitro 3 x 2 factorial experiments were conducted concurrently to evaluate the incorporation of 0, 15, or 30% sugar beet pulp (SBP) as an energy source in diets fed to growing Holstein steers with either soybean meal (SBM) or alcohol-treated, defatted soybean flakes (ATSBF) as primary supplemental protein sources. Three groups of 42 Holstein steers each were fed six different diets from 54 kg initial BW to 320 kg in three experimental periods. There were no overall SBP level x protein source interactions (P greater than .05). Beet pulp level tended to decrease ADG (linear, P = .05) and increase feed/gain (linear, P less than .05) and DMI (quadratic, P less than .05). Each grower diet was used in a substrate for ruminal microbial metabolism in six dual-flow, continuous-culture fermenters. Organic matter and carbohydrate digestion were similar (P greater than .05) among diets. Increasing dietary levels of SBP caused a concomitant increase (P less than .05) in acetate and decrease (P less than .05) in butyrate and isobutyrate concentrations. Beet pulp level x protein source interactions (P less than .05) were observed for CP degradation, ammonia and nonammonia N, and dietary N flow. Crude protein degradation was higher (P less than .05) for the 0% SBP with SBM diet (81.3%) than for the 30% SBP with ATSBF diet (64.4%). Efficiency of bacterial synthesis was similar (P greater than .05) among diets. Results indicated that SBP is an effective dietary energy source for high-energy grower diets at 15 or 30% of the DM but may cause a decrease in some performance traits. There were no nutritional benefits of using ATSBF vs SBM as the supplemental N source.

Evaluation of various nitrogen supplements in starter diets for growing Holstein steers and their effects on ruminal bacterial fermentation in continuous culture.

Concurrent in vivo and in vitro studies were conducted to evaluate urea (U), soybean meal (SBM), ground soybeans (RAW), extruded soybeans (ES) or extruded soybeans plus urea (ES + U) as primary supplemental N sources in starter diets for Holstein steers. Three groups of 48 Holstein steers each were fed five different starter diets to 181 kg BW in three experimental periods over 2 yr. Average daily gains were similar (P greater than .05) for steers fed ES + U (1.12 kg), ES (1.08 kg) and SBM (1.09 kg) but lower (P less than .05) for those fed U (1.00 kg) or RAW (.97 kg) diets. Feed/gain was similar (P greater than .05) for ES-fed steers vs those fed other diets except U. From 181 to 477 kg, all steers were fed the same diet. Steers fed the RAW starter diet had the lowest (P less than .05) ADG for the entire period. The starter diets were used as substrates for ruminal microbial metabolism in eight dual-flow continuous culture fermenters. True OM digestion was higher and NDF and ADF digestion was lower (P less than .05) for the ES + U diet than for the ES diet. Dietary protein degradation was lowest (P less than .05) for the ES diet (64.4%). Total bacterial N flow was higher (P less than .05) with the ES + U, SBM and U diets than with the ES diet. Lysine flow was higher (P less than .05) for the ES + U diet than for all other diets except ES. Results of these experiments indicate that ES as a protected ruminal escape N source with or without added urea did not improve steer performance above that obtained from SBM in starter diets.