O-demethylation Of P-nitroanisole Research Articles

Thiram-induced disturbance of microsomal phospholipid bioformation and phospholipid fatty acid pattern.

In rats, a single oral dose of 30 mg or 1 g thiram per kilogram produced a significant prolongation of the hexobarbital sleeping time or zoxazolamine paralysis time, respectively, a depression of hepatic microsomal O-demethylation of p-nitroanisole to p-nitrophenol, and a decrease in the microsomal cytochrome P-450 content. In addition, incorporation of 14C activity from glucose, glycerol, and palmitic acid into phospholipids (PLs) decreased in most of the components, showing a compensatory increase in only a few of them. Fatty acid (FA) concentrations in phosphatidylcholine (PC) or phosphatidylethanolamine (PE) molecules were increased or decreased from one FA class to the other. This alteration of the FA concentrations in PC and PE, respectively, was virtually exclusively in position 2 of the two PL molecules. The total content of saturated FAs in PC and PE was significantly decreased only after 1 g/kg thiram; this was associated with an increase in total unsaturated FAs. The multiple changes in the bioformation and composition of microsomal membrane PLs suggest a disturbance of the monooxygenase system. It is conceivable that there is an interrelationship between the observed impairment and the inhibitory effect of thiram on the microsomal monoxygenases (MMs).

Aryl ether O-dealkylase activity in the skin of untreated mice in vitro.

1. O-Dealkylation of p-nitroanisole and p-nitrophenetole in 10 000 g supernatant preparations of mouse skin is detectable and quantifiable. The reaction is NADPH-dependent and is mediated by cytochrome P-450. 2. The rate of p-nitroanisole O-demethylation in mouse skin preparations is 2% of that in mouse liver preparations on a mg protein basis, but only 0.23% on a g of tissue basis. 3. For p-nitrophenetole O-deethylation, the cutaneous Vmax is 3.8% of the hepatic Vmax on a mg protein basis. The Km values for the reaction in these two tissues are in a 3:1 ratio, suggesting that there are no marked qualitative differences between cutaneous and hepatic cytochrome P-450.

Inhibition of mixed-function oxidation of p-nitroanisole in perfused rat liver by 2,4-dinitrophenol

The effect of dinitrophenol (52 μ m), an uncoupler of oxidative phosphorylation, on p-nitroanisole O-demethylation in the perfused rat liver was examined. Dinitrophenol inhibited p-nitroanisole metabolism 70% in perfused livers from fasted, phenobarbital-treated rats, and 30% in livers from normal rats, but had no effect on this reaction in isolated microsomes. Rates of p-nitroanisole O-demethylation in livers from fed, phenobarbitaltreated rats were not inhibited by dinitrophenol unless the pentose phosphate shunt was first inhibited by 6-aminonicotinamide pretreatment. Dinitrophenol diminished cellular concentrations of ATP and NADPH 30 and 50%, respectively. Since mixed-function oxidation requires NADPH, these data are consistent with the hypothesis that dinitrophenol interrupts the synthesis and/or transfer of reducing equivalents from the mitochondria into the extramitochondrial space by interfering with energy-dependent NADPH synthesis and substrate shuttle mechanisms. In addition, dinitrophenol diminished conjugation reactions 57 and 89% in all metabolic states studied, most likely because it decreased UDP-glucose levels considerably (40 to 60%).

Non-Michaelian monooxygenase kinetics: studies using competitive inhibitors

Microsomal l~onooxygenase activity has been found frequently to depart from Mic~elis-Menten kinetics in that Lineweaver-Burk plots for the oxidation of a number of xenobiotics have exhibited downwards curvature at high substrate concentrations [l-4]. These results are consistent with catalysis being effected by more than one enzyme, each with a different IS,, value, and this possibility has received some support in the finding that endop~asmic reticulum contains multiple monooxygenases [2,3,5]. Such kinetic behaviour could however be due to a single enzyme possessing multiple interacting sites ]6,7] and it has been proposed that microsom~ monooxygenase activity may be regulated by a ligand concentration-dependent mechanism exhibiting negative cooperativity [8]. Here, we have shown that Michaeiis-Lenten kinetics did not describe the kinetics of O-demethylation of p-nitroanisole by a rat liver microsomal preparation when enzyme activity was measured over a 1300fold range of substrate concentration. In addition, results of studies with 3 competitive inhibitors precluded multiple independent catalytic sites as the source of the observed non-Michaelian kinetics of ether cleavage thereby lending support to the hypothesis that negative cooperativity may be a feature of the control of microsomal monooxygenase activity.

Hypoxia and cold: Influence on cellular oxygen consuming systems in guinea pig liver

Traditionally any biochemical changes found in animals exposed to high altitude have been interpreted solely in relation to hypobaric hypoxia. The present work has been carried out to study the influence of cold and hypoxia in guinea pig native to high altitude. The three major oxygen consuming systems of the liver were measured using cytochrome oxidase as a mitochondrial marker, catalase as a peroxisomal marker, and the o-demethylation of p-nitroanisole and the hydroxilation of hexobarbital as markers for microsomal activity. Serum levels of thyroid hormones (T 3 and T 4) were also determined. Evidence is presented showing that cold produces a dramatic increase of liver catalase and cytochrome oxidase activities, and of serum T 3 and T 4. Interestingly, the increase in thyroid hormones did not precede the increase of the two enzyme activities in the liver of guinea pig exposed to 4°C. On the other hand, it was found that hypoxia appears to have no significant influence upon any of the three major oxygen consuming systems of the liver.

NADH- and NADPH-dependent reconstituted p-nitroanisole O-demethylation system containing cytochrome P-450 with high affinity for cytochrome b5.

A reconstituted system containing a form of cytochrome P-450, cytochrome b5, NADPH-cytochrome P-450 reductase, and NADH-cytochrome b5 reductase, all purified from rabbit liver microsomes, could catalyze O-demethylation of p-nitroanisole in the presence of both NADPH and NADH. Omission of either cytochrome P-450 or cytochrome b5 from the system led to complete loss of the activity. The reconstituted activity was sensitive to carbon monoxide, metyrapone, phenyl isocyanide, and cyanide, indicating that the cytochrome P-450 used is cyanide-sensitive and is involved in the catalytic process. The maximal demethylase activity was attained when the system contained cytochrome P-450 and cytochrome b5 at a 1 : 1 molar ratio. Trypsin digestion of cytochrome b5 abolished the capacity of this cytochrome to reconstitute the demethylase activity. These results suggest that O-demethylation of p-nitroanisole by this particular form of cytochrome P-450 absolutely requires the intact form of cytochrome b5 and that the second electron needed for the demethylation may be donated to the cytochrome P-450 only by way of cytochrome b5.

Stimulation of p-nitroanisole O-demethylation in perfused livers by xylitol and sorbitol

Xylitol and sorbitol, two sugar alcohols which readily enter into pathways of hepatic carbohydrate metabolism, stimulated p-nitroanisole O-demethylation in perfused livers from fasted, but not fed, phenobarbital-treated rats. The increase in mixed-function oxidation correlated well with the production of NADH from the metabolism of xylitol and sorbitol (half-maximal stimulation for both processes was observed with concentrations between 0.1 and 0.2 mM). p-Nitroanisole metabolism by isolated hepatic microsomes was unaffected by the addition of xylitol and sorbitol; however, when NADH was added to microsomes, or was generated from sorbitol, sorbitol dehydrogenase and NAD +, a synergistic increase in p-nitroanisole metabolism occurred. Ethanol (0.2 mM), which does not enter into pathways of carbohydrate metabolism, also caused an increase in the pyridine nucleotide redox state and stimululated p-nitroanisole O-demethylation in livers from fasted rats. In addition, sorbitol and xylitol stimulated p-nitrophenol conjugation in livers from fasted, phenobarbital-treated animals, probably by supplying substrate for increased UDP-glucuronic acid synthesis. The data indicate that sugars which influence the pyridine nucleotide redox state alter rates of mixed-function oxidation and conjugation in whole cells.

Pesticides as inducers of hepatic drug-metabolizing enzymes—I. Mixed function oxidase activity

1. 1. Sixteen pesticides, one carcinogen (3-methylcholanthrene), one cotton defoliant, two drugs (phenobarbital and chloral hydrate) and two insecticide synergists (piperonyl butoxide and sesamex) were administered to adult male mice by different routes in order to evaluate the inductive effects of pesticides on hepatic drug metabolizing enzymes. 2. 2. Of the experimental insecticides screened, TH-6042 (a substituted pyrazoline) showed the highest level of induction of cytochrome P-450 (>3-fold) and also significant increases in O-demethylation of p-nitroanisole and N-demethylation of aminopyrine. 3. 3. Maximum induction was generally achieved when compounds were administered orally for each of 3 days.

Mechanism of inhibition of mixed-function oxidation by ethanol.

p-Nitroanisole (PNA) O-demethylation in perfused rat livers was inhibited by low concentrations of ethanol (Ki = 1 mM) and acetaldehyde (Ki = 0.5 mM). Ethanol and acetaldehyde also inhibited microsomal PNA metabolism, but only at much higher concentrations (Ki = 130 mM and 5 mM, respectively). Moreover, the dose-dependent inhibition of PNA metabolism in the perfused liver correlated strongly with changes in surface fluorescence of pyridine nucleotides and flavoproteins resulting from the metabolism of ethanol and acetaldehyde. t-Butanol (20 mM) did not cause pyridine nucleotide or flavoprotein reduction, nor did it inhibit PNA metabolism. When ethanol metabolism was blocked with 4-methylpyrazole, the inhibition of PNA metabolism by ethanol was diminished.

Regulation of p-nitroanisole O-demethylation in perfused rat liver. Adenine nucleotide inhibition of NADP+-dependent dehydrogenases and NADPH-cytochrome c reductase.

Perfusion of rat livers with 10 mM-fructose or pretreatment of the rat with 6-aminonicotinamide (70 mg/kg) 6 h before perfusion decreased intracellular ATP concentrations and increased the rate of p-nitroanisole O-demethylation. This increase was accompanied by a decrease in the free [NADP+]/[NADPH] ratio calculated from concentrations of substrates assumed to be in near-equilibrium with isocitrate dehydrogenase. After pretreatment with 6-aminonicotinamide the [NADP+]/[NADPH] ratio also declined. Reduction of NADP+ during mixed-function oxidation may be explained by inhibition of of one or more NADPH-generating enzymes. Glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase and "malic" enzyme, partially purified from livers of phenobarbital-treated rats, were inhibited by ATP and ADP. Inhibitor constants of ATP for the four dehydrogenases varied considerably, ranging from 9 micrometer for "malic" enzyme to 1.85 mM for glucose 6-phosphate dehydrogenase. NADPH-cytochrome c reductase was also inhibited by ATP (Ki 2.8 mM) and by ADP (Ki 0.9 mM), but not by AMP. Concentrations of ATP and ADP that inhibited glucose 6-phosphate dehydrogenase and the reductase were comparable with concentrations in the intact liver. Thus agents that lower intracellular ATP may accelerate rates of mixed-function oxidation by a concerted mechanism involving deinhibition of NADPH-cytochrome c reductase and one or more NADPH-generating enzymes.

The obligatory requirement of cytochrome [formula omitted]5 in the [formula omitted]-nitroanisole O-demethylation reaction catalyzed by cytochrome P-450 with a high affinity for cytochrome [formula omitted

The role of cytochrome b 5 in the p -nitroanisole O-demethylation was studied with a reconstituted system containing a unique cytochrome P-450, isolated from rabbit liver microsomes as a species with a high affinity for cytochrome b 5. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome b 5 reductase, and Triton X-100 in addition to cytochrome b 5. The omission of cytochrome b 5 from the complete system entirely abolished the activity. These results clearly show that cytochrome b 5 is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome b 5 in such a way that the second electron is transferred from cytochrome b 5 and thus exhibits the demethylase activity.

Relationship between alveolar PO2 and the rate of p-nitroanisole O-demethylation by the cytochrome P-450 pathway in isolated rabbit lungs.

The relationship between alveolar PO2 and the rate of O-demethylation of p-nitroanisole, a model substrate for cytochrome P-450 -linked mixed-function oxidation, was evaluated in the isolated rabbit lung perfused with Krebs-Ringer bicarbonate buffer. The appearance of the product, p-nitrophenol, in the pulmonary perfusate was measured spectrophotometrically, The PO2 of the ventilating gas was varied with an accurate gas mixing pump and measured with an electrochemical O2 analyzer. In control lungs ventilated with 5% CO2 in air, the rate of p-nitrophenol production was approximately equal to 3.1 +/- 0.04 (mean +/- SE; n = 9) mumol/h per g dry wt. p-Nitrophenol production was unaltered when O2 in the ventilating gas was decreased to 1%, but it was depressed reversibly when alveolar O2 WAS 0.1% OR LESS AND WAS ABOLISHED DURING VENTILATION WITH 0.005% O2. The rate of the reaction was inhibited by 50% when alveolar PO2 was 0.3 mm Hg representing and intracellular [O2] OF approximately equal to muM. In the presence of metyrapone (0.1--1 mM), an inhibitor of cytochrome P-450-dependent reactions, p-nitrophenol production was 0.07--0.17 mumol/h per g dry wt. Ventilation of lungs with varying CO concentration in 20% O2 resulted in 50% inhibition of p-nitrophenol production when CO concentration was 10% (CO/O2 = 0.5). These results indicated that O-demethylation of p-nitroanisole by the lung is a cytochrome P-450-dependent reaction and that its rate is not affected until alveolar PO2 is less than 1 mm Hg.

O-demethylation of p-nitroanisole by mixed function oxidase system of the rainbow trout ( Salmo gairdneri) liver

1. 1. O-demethylation of p-ntiroanisole has been characterized in the 10,000 g supernatant of the rainbow trout liver. 2. 2. The reaction was inhibited by CO indicating the involvement of the microsomal cytochrome P-450. 3. 3. The PCBs induced O-demethylating activity was markedly inhibited by α-napthoflavone, while the uninduced reaction was poorly affected. SKF 525A and testosterone strongly inhibited both the induced and the uninduced reaction. 4. 4. The pretreatment with PCBs increased the apparent V max value 6–7 times, while the apparent K m value was not changed.

Sensitive radioassay of microsomal O-demethylation of 14CH 3O- or C 3H 3O- p-Nitroanisole for comparative studies

1. Radioassay of p-nitroanisole O-demethylation in liver homogenates and/or microsomes is described. Disappearance of as little as five nanomoles substrate can be precisely determined. 2. O-Demethylation rates from radioassays and from spectroassays of formaldehyde correspond, but both are greater than apparent rates of p-nitrophenol production measured by spectrophotometry. 3. Comparative studies were performed using homogenates and microsomes from vertebrates (9 species) and invertebrates (4 species). Results indicated species differences, enzyme induction and characteristic tissue levels of O-demethylation activity. 4. Cost/time considerations favor the radioassay over usual spectrophotometric procedures for comparative studies involving large numbers of samples.

Liver microsomal oxidations in baboon (Papio papio)

1. The activity of baboon (Papio papio) hepatic microsomes was evaluated using several model substrates. 2. As with other species studied in this laboratory, baboon microsomal enzyme activities were in the decreasing order: aldrin epoxidation greater than p-nitroanisole O-demethylation greater dihydroisodrin hydroxylation greater than benzo(a)pyrene oxidations. 3. Substrate, time, protein and cofactor requirements were examined. 4. Activities of stored frozen tissue and microsomal pellets remained similar up to seven days. 5. In the six animals studied (three male, three female), no differences in hepatic microsomal activity due to sex were apparent.

THE INVOLVEMENT OF CYTOCHROME P-448 AND P-450 IN NADH-DEPENDENT O-DEMETHYLATION OF p-NITROANISOLE IN RAT LIVER MICROSOMES

These studies have shown that addition of p-nitroanisole to a reaction mixture containing rat liver microsomes resulted in an increase the reoxidation rate of NADH- reduced cytochrome b5. Fortification of rat liver microsomes with partially purified cytochrome b5 produces an increase in both NADPH-dependent and NADH-dependent p-nitroanisole O-demethylation activity. Antiserum to cytochrome P-450 isolated from phenobarbital-treated rat liver microsomes inhibited the NADH-dependent O-deme-thylation activity as well as the NADPH-dependent O-demethylation activity seen in rat liver microsomes. Addition of either purified cytochrome P-450 or cytochrome P-448 to an incubation mixture containing phenobarbital-treated rat liver microsomes enhanced the NADH-dependent p-nitroanisole O-demethylation activity. These results suggest that NADH-dependent and, in part, NADPH-dependent O-demethyl- ations are catalyzed by cytochrome P-448 and cytochrome P-450 receiving electrons from cytochrome b5.

Alteration of hepatic detoxication enzyme activity by dietary arsenic trioxide

The effects of dietary arsenic trioxide (As 2O 3) on liver detoxication enzymes were studied. Enzyme activity was monitored by measuring hexobarbitone sleeping time, oxidative cleavage of O-ethyl- O-p-nitrophenyl phenylphosphonothioate (EPN) and O-demethylation of p-nitroanisole, in rats fed diets containing 0, 100, 500, 1000, 2000 or 5000 ppm As 2O 3 for 15 days. Hexobarbitone sleeping time was decreased in rats given 100 ppm As 2O 3 or more; the other enzyme responses were stimulated considerably in rats given 1000 ppm As 2O 3 and most of the rats given 2000 or 5000 ppm died. The capacity of the enzyme system to be stimulated by dietary sodium phenobarbitone was not impaired by As 2O 3.

Effects of clophen A50, 4-, 2,5,2′,5′- tetra- and 2,4,5,2′,4′,5′-hexachlorobiphenyl on the mixed-function oxidase system of rainbow trout ( salmo gairdnerii rich.) liver

1. Clophen A50 and 2,4,5,2′,4′,5′-hexachlorobiphenyl enhanced the O-demethylation of p-nitroanisole to p-nitrophenol and the content of cyt P-450 in the rainbow trout liver. Dose dependence in relation to time of the induction by Clophen A50 is described. 2. Over a 14-day period a single intraperitoneal injection of Clophen A50, 4-, 2,5,2′,5′-tetra- or 2,4,5,2′,4′,5′-hexachlorobiphenyl did not increase the NADPH-cyt c reductase activity or the microsomal protein and phospholipid contents. 3. The qualitative pattern of induction by PCBs and chlorobiphenyls of the mixed function oxidase system of rainbow trout liver are discussed in relation to the mammalian aryl hydrocarbon hydroxylase system.

In vitro interactions of di-, tetra- and hexa-chlorobiphenyl with rabbit liver monooxygenase

2,4,6,2',4',6'-Hexa,- 2,5,2',5'-tetra- and 2,2'-dichloro-biphenyl (HCB, TCB and DCB) caused a type-I-binding spectrum with liver microsomes of phenobarbital treated rabbits. They had a high affinity to cytochrome P-450, which increased with decreasing chlorine content of the compounds. In parallel, the O-demethylation of p-nitroanisole was most effectively inhibited by DCB, to a smaller extent by TCB and HCB. Inhibition was competitive. While DCB was rapidly metabolized, metabolism of TCB was slow. No metabolites have been detected of HCB. Although HCB was not metabolized it stimulated the microsomal NADPH- and oxygen-consumption and also the reduction of cytochrome P-450. The stoichiometry of the NADPH- and O 2-consumption of 2:1 in the presence of HCB is compatible with the notion that NADPH is oxidized to H 2O, i.e. HCB may act as uncoupler of electron transport from monooxygenation.

Hepatic hydroxylation and glucuronidation in the rat after subacute pyrazole treatment

Male Wistar rats were given pyrazole subcutaneously at a dose of 100 mg/ kg daily, for 4 days, and the in vitro activities of drug metabolizing enzymes and the concentrations of copper, zinc and magnesium were measured in the liver. During the course of treatment, the animals failed to thrive at the rate of the controls. Although protein content of microsomes was unchanged, the phospholipid content increased. Pyrazole administration significantly decreased the activity of NADPH-cytochrome c reductase, while it did not affect the O-demethylation of p-nitroanisole and the microsomal content of cytochrome P-450. The activities of UDPglucose dehydrogenase, UDPglucuronosyltransferase and l-gulonate dehydrogenase were markedly enhanced. On the contrary, pyrazole administration produced a decrease in the activities of UDPglucuronic acid pyrophosphatase, β-glucuronidase and d-glucuronolactone dehydrogenase. The pentobarbital sleeping-time was prolonged after pyrazole treatment for 4 days but the animals showed a decreased spontaneous locomotor activity already on the third day of treatment. This suggests that a depression of the central nervous system was present, which was due to pyrazole itself. The total hepatic content of copper was significantly higher in pyrazole-treated animals than in the control group. The concentrations of zinc and magnesium were increased in the hepatic cytosolic fraction. These results indicate that pyrazole affects the enzymes involved both in hydroxylation and in glucuronidation reactions. In view of the unspecificity of the compound in affecting various enzyme systems, its use in studies on ethanol metabolism in vivo does not seem warranted.