Rates Of P-nitroanisole O-demethylation Research Articles

Selective reduction of hepatic cytochrome P450 content in patients with intrahepatic cholestasis: A mechanism for impairment of microsomal drug oxidation

Alteration of the components of the mixed-function oxidase system, including cytochrome P450, cytochrome b5, cytochrome P450 reductase, and cytochrome b5 reductase, was examined in liver microsomes prepared from needle biopsy samples of 12 patients with intrahepatic cholestasis. The rate of p-nitroanisole O-demethylation in the microsomes was also measured as the activity of cytochrome P450-dependent drug oxidation. The cytochrome P450 content (0.29 ± 0.05 nmol/mg microsomal protein) in the patients was significantly lower than that in the 11 control subjects (0.41 ± 0.09). The rate of p-nitroanisole O-demethylation was also significantly lower in the patients. However, the cytochrome b5 content and the activities of the reduced forms of nicotinamide adenine dinucleotide phosphate- and nicotinamide adenine dinucleotidecytochrome c reductases did not differ between the two groups. Thus, selective reduction of cytochrome P450 seems to play a major role in the impairment of microsomal drug oxidation during intraheptic cholestasis. Moreover, the reduction of cytochrome P450 was correlated with the serum concentrations of total bilirubin and bile acid but not with the serum glutamic oxaloacetic transaminase value. These observations suggest that the reduction of cytochrome P450 might be related to the severity of cholestasis.

Effect of aging on the metabolism of p-nitroanisole and p-nitrophenol in isolated rat hepatocytes

The effects of aging on the monooxygenation of p-nitroanisole (PNA) and conjugation of p-nitrophenol (PNP) were determined in hepatocytes isolated from young (2–4 months) and old (20–24 months) male Fischer 344 rats. The rate of PNA O-demethylation was significantly decreased (41%) in aged rats. PNP formed from the monooxygenation of PNA was predominantly conjugated by suIfation. The decrease in monooxygenation of PNA was accompanied by a 57% decrease in the rate of PNP-sulfate production. No change in the glucuronidation of PNP or level of unconjugated PNP was observed during aging when PNA was used as substrate. With PNP as substrate, glucuronidation was the major conjugative pathway. An age-related decrease (70%) in the suIfation capacity was observed at both 100 and 200 μM PNP, but no change in the glucuronidation of PNP was seen in aged rats. Therefore, the overall conjugation of PNP was decreased by 30% with age. Microsomal glucuronyltransferase activity toward PNP was unaffected by aging, and microsomal cytochrome P-450 levels were reduced 34% in aged rats. This study demonstrates age-related decreases in PNA monooxygenation and PNP suIfation in isolated rat hepatocytes, while PNP glucuronidation capacity is unaffected during aging.

Effect of aging on oxidative metabolism and conjugation in perfused livers of an inbred strain of mice: A pilot study

A study was carried out to examine the possibility that altered mitochondrial function with aging might cause changes in hepatic rates of oxygen uptake and β-oxidation of fatty acids. Livers of 2–4 month and 20–25 month old CBA mice were perfused, and rates of respiration and ketogenesis were measured. Livers of old mice had significantly decreased rates of oxygen utilization (190 ± 16 vs 151 ± 9 μmol/gm/hr, p.05) and significantly lower rates of ketogenesis as indicated by rates of acetoacetate and β-hydroxybutyrate production (84 ± 8 vs 63 ± 7, p <.05). To determine whether NADPH supply, which is largely derived from β-oxidation of fatty acids in the fasted state, might be rate limiting for mixed-function oxidation in aged mice, rates of p-nitroanisole O-demethylation were measured in livers of young and old mice. Age had no effect on mixed-function oxidation of p-nitroanisole. Rates of glucuronidation of p-nitrophenol were also not affected by age; however, rates of sulfation of p-nitrophenol were significantly higher in livers of old (7.2 ±.6) than in young (3.5 ± 0.8) mice. This pilot study raises the possibility that the disposition of ingested fatty acids and xenobiotic compounds requiring sulfation may be significantly altered with aging. Moreover, it demonstrates that the perfused mouse livers can be used as a model in aging studies.

P-Nitroanisole O-demethylation in perfused hamster liver : High rates of pentose cycle-independent mixed-function oxidation

Rates of p-nitroanisole O-demethylation in perfused livers from Syrian golden hamsters were three to four times greater than comparable rates measured in preparations from Sprague-Dawley rats. Hamsters also had greater microsomal p-nitroanisole O-demethylase activity and cytochrome P-450 contents than rats. In general, phenobarbital caused similar increases in these properties in both species. Fasting of hamsters for 24 hr increased p-nitroanisole O-demethylase activity in microsomes but did not affect rates in perfused livers. Rates were also unaffected in the perfused liver by pretreatment with 6-aminonicotinamide, an inhibitor of the pentose phosphate shunt. Hamster livers had low activities of pentose cycle enzymes but high activities of malic enzyme and isocitrate dehydrogenase compared to rats. In hamster livers, maximal rates of p-nitroanisole O-demethylation were not maintained but declined steadily over 40 min with prolonged p-nitroanisole infusion. The decreased rates of mixedfunction oxidation in the non-recirculating perfusion system could not be explained by diminished tissue viability or degradation of cytochrome P-450 but were likely due to a decline in the formation of reduced cofactor. Hepatic concentrations of α-ketoglutarate and malate increased during ρ-nitroanisole infusion. Furthermore, rates of p-nitroanisole O-demethylation were inhibited by ethanol and aminooxyacetate, agents which inhibit the generation and/or movement of mitochondrial reducing equivalents into the cytosol. The infusion of pyruvate stimulated p-nitroanisole O-demethylation in perfused livers from fasted hamsters. This effect was maximal with 0.1 mM pyruvate, did not require gluconeogenesis, and was insensitive to 6-aminonicotinamide treatment. Thus, stimulation of p-nitroanisole metabolism by pyruvate in hamster livers is likely related to the mitochondrial oxidation of pyruvate, rather than to increased NADPH generation via the pentose phosphate cycle. These data indicate that mitochondrial sources of NADPH supply reducing equivalents for mixed-function oxidation in hamster liver.

Genetic regulation of NADPH supply in perfused mouse liver. Role of the Ah locus during induction by 3-methylcholanthrene.

Rates of p-nitroanisole O-demethylation in perfused livers from control DBA/2J and C57BL/6J, as well as from 3-methylcholanthrene-treated DBA/2J mice, reached maximal values of 15-17 mumol/g/h after 10 min of p-nitroanisole (0.2 mM) infusion. During the subsequent 30 min of p-nitroanisole infusion these rates declined steadily to 50% of the maximal rate. Infusion of 20 mM glucose completely reversed this decline in rate. Phenobarbital treatment of C57BL/6J and DBA/2J mice increased rates of p-nitroanisole O-demethylation about 2.5-fold to 44.4 +/- 3.4 and 35.1 +/- 3.4 mumol/g/h, respectively, which also declined during subsequent perfusion. In contrast, the kinetics of p-nitroanisole O-demethylation in perfused livers from 3-methylcholanthrene- and beta-naphthoflavone-treated C57BL/6J were distinctly different. These livers maintained high maximal rates of p-nitroanisole O-demethylation (41.1 +/- 4.7 mumol/g/h) throughout the 40 min of p-nitroanisole infusion. 3-Methylcholanthrene-treated (C57BL/6J)(DBA/2J)F1 perfused livers also maintained high rates of monooxygenation throughout the perfusion. All livers from the 3-methylcholanthrene-treated, Ah locus-responsive F1 X DBA/2J backcross group maintained high rates of p-nitroanisole O-demethylation throughout perfusion; however, 3-methylcholanthrene-treated, Ah locus-nonresponsive perfused livers resembled livers from untreated mice. The NADP+/NADPH ratio in livers from 3-methylcholanthrene-treated C57BL/6J but not DBA/2J mice was significantly higher than controls in the absence of p-nitroanisole. Addition of p-nitroanisole increased this ratio in both groups; however, higher values were observed in livers from C57BL/6J than DBA/2J mice. These data provide genetic evidence that the ability of mouse liver to sustain high rates of monooxygenation following 3-methylcholanthrene treatment is an autosomal dominant trait corresponding with the Ah locus and most likely is due to enhanced NADPH turnover.

Evaluation of a role of acetaldehyde in the mechanism of inhibition of p-nitroanisole O-demethylation in isolated hepatocytes by ethanol

The rate of p-nitroanisole O-demethylation is markedly inhibited by ethanol. To evaluate a role of acetaldehyde in the inhibition by ethanol, a comparison was made of the effects of ethanol and acetaldehyde on the metabolism of p-nitroanisole by isolated liver cells. No effect on the metabolism of p-nitroanisole was found at low concentrations of acetaldehyde (<0.5 m m), whereas inhibition occurred at high concentrations (1 m m). In fact, acetaldehyde was not any more inhibitory than crotonaldehyde, which is a poor substrate for the low- K m mitochondrial aldehyde dehydrogenase. Cyanamide, an inhibitor of acetaldehyde oxidation, did not prevent the inhibition by ethanol. Crotonol, an alcohol that does not change the mitochondrial redox state, in contrast to ethanol, proved to be a more effective inhibitor of the metabolism of p-nitroanisole than ethanol. Greater sensitivity to crotonol was also found in isolated microsomes and may reflect hydrophobic effects by crotonol, relative to ethanol. These results suggest that although high levels of acetaldehyde can be inhibitory, physiological levels of acetaldehyde did not affect the metabolism of p-nitroanisole. It is unlikely that acetaldehyde itself plays a major role in the mechanism by which ethanol inhibits the metabolism of p-nitroanisole. The inhibition of p-nitroanisole O-demethylation by ethanol was prevented by pyruvate or fructose, but not by xylitol, sorbitol, or lactate. All these substrates by themselves stimulated metabolism of p-nitroanisole. Pyruvate and glyceraldehyde (which arises from the metabolism of fructose) can oxidize cytosolic NADH. These results suggest that the generation of cytosolic NADH from the oxidation of ethanol, the subsequent requirement for substrate shuttles to transfer NADH into the mitochondria, and redox inhibition of the citric acid cycle, interfere with the transport of NADPH out of the mitochondria, and consequently with drug metabolism.

Absence of enhanced detoxication of permethrin in pyrethroid-resistant house flies

The mechanisms of resistance to pyrethroids were studied in a permethrin-selected (147-R) strain of the house fly, Musca domestica L. Approximately 12-fold synergism was obtained with a mixture of (1 R)- trans-permethrin:piperonyl butoxide (1:5) so that the resistance decreased from 97-fold to 22-fold. Tests with the esterase inhibitor S,S,S-tributyl phosphorotrithioate produced very little synergism in either the resistant (R) strain (1.6-fold) or the susceptible (S) strain (1.9-fold). An investigation of the microsomal components revealed that compared to the S strain, the R strain demonstrated twice as much cytochrome P-450 and cytochrome b 5 and double the rate of NADPH-cytochrome c reductase activity. In addition, the rate of p-nitroanisole O-demethylation was found to be six times greater in the R strain. An in vivo accumulation study showed that the R strain displayed a decreased rate of penetration of trans-[ 14C]permethrin. When treated at equitoxic doses the R strain was found to tolerate 50-fold more internal permethrin than the S strain. An in vitro metabolism study indicated that there was no difference between strains in the overall rate of metabolism of trans-[ 14C]permethrin. The evidence obtained supports the conclusion that several resistance factors are involved but that decreased sensitivity of the nervous system to the action of pyrethroids is the principal mechanism of resistance in the 147-R strain.

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%).

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.

The state of NADPH/NADP + during mixed-function oxidation of hexobarbital in hemoglobin-free perfused rat liver

The effect of dinitrophenol (52 μm), 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%).