The effects of cobalt chloride, SKF-525A, and N-(3,5-dichlorophenyl)succinimide on in vivo hepatic mixed function oxidase activity as determined by single-sample plasma clearances.

1. Four drugs--antipyrine, theophylline, quinidine, and ethosuximide--were used as probes of in vivo hepatic mixed function oxidase (MFO) activity. Functional MFO activity was evaluated by estimating probe clearances subsequent to pretreatment of rats with either cobalt chloride, SKF-525A, or N-(3,5-dichlorophenyl) succiminide (NDPS). 2. Clearances of each probe were estimated from single plasma concentration measurements. Each pretreatment altered the clearances of this panel of probes in a different way. NDPS pretreatment increased theophylline clearance while slowing quinidine and ethosuximide clearances. SKF-525A slowed all probe clearances except for ethosuximide. Cobalt chloride slowed all probe clearances except for theophylline. 3. The use of multiple probes as substrates for the hepatic cytochrome P-450 system can provide some insight into the functional consequences of xenobiotic exposures on that system. Moreover, xenobiotic-induced functional changes on hepatic MFO when assessed in vivo appear to be modest relative to changes in in vitro activity or hepatic cytochrome P-450 content. This minimally invasive multiprobe method may be useful for assessing xenobiotic influences on human hepatic MFO in vivo.     

Inhibition of hepatic mixed function oxidase activity by propyl gallate

Propyl gallate was found to inhibit microsomal benzpyrene hydroxylase activity and demethylase activity with ethylmorphine, aminopyrine or benzphetamine as a substrate. The extent of inhibition with different substrates varied with the age and diet of the animals. The benzpyrene hydroxylase activity of the microsomes of the 3-methylcholanthrene-treated rats was shown to be less susceptible to propyl gallate inhibition. Propyl gallate does not inhibit the NADPH-dependent reduction of cytochrome P-450; therefore, the site of inhibition is not on NADPH-cytochrome c reductase as suggested previously. Propyl gallate interacts with cytochrome P-450 to produce a positive absorption peak around 420 nm, and it may also interfere with the binding of a type I substrate, benzphetamine. It inhibits ethylmorphine demethylation by a noncompetitive mechanism and aminopyrine demethylation by a mixed mechanism. The mode of propyl gallate inhibition and the implications of these observations are discussed.     

Components of the heme biosynthetic pathway and mixed function oxidase activity in human fetal tissues

Components of the enzyme systems involved in heme biosynthesis and mixed function oxidase activity were examined in tissues from human fetuses between the ages of 10.8 and 17.5 weeks, aborted by hysterotomy. The activity of δ-aminolevulinic acid synthetase (ALAS) was highest in liver, and successively lower in adrenal, placenta, kidney and lung. The mean level of ALAS activity in fetal liver [61.9 ± 13.4(S.E.)nmoles ALA/g/hr] was three times higher than the level reported in human adult liver. ALAS activity was directly correlated with the concentrations of porphyrins in liver, lung and placenta (r = 0.96). Protoporphyrin predominated in liver, while coproporphyrin predominated in lung and kidney. Ferrochelatase measured in livers from two fetuses (47.5 and 60.2 nmoles heme/g/hr) was sufficient to account for complete conversion to heme of the ALA produced by ALAS. As with other species, ALAS and ferrochelatase were found mainly in the mitochondrial subfraction of cells. Aryl hydrocarbon hydroxylation (AHH) could be measured in liver, adrenal, lung, kidney, intestine and placenta, but aminopyrine demethylation could not. In the adrenal, the concentration of cytochrome P-450 (1.92 nmoles/mg of microsomal protein) and mean AHH activity [258.2 ± 36.6 (S.E.) pmoles 3-OH benzo(a)pyrene/mg of protein/hr] were both four times higher than in the liver. In the fetal liver, the mean concentration of P-450 was within the range reported for human adult liver, but the activity of AHH [55.1 ± 17.3 (S.E.) pmoles/mg of protein/hr] was only 2 per cent of the activity reported for the adult. Cytochrome P-450 could not be detected in kidney or lung, though low levels of AHH were found (7.1 and 7.2 pmoles/mg of protein/hr). The presence of higher levels of ALAS and lower levels of cytochrome P-450 and AHH in the fetal liver than in the adrenal indicates that the activity of the heme biosynthetic pathway is not the main determinant of hemoprotein concentration and the extent of mixed function oxidase activity in human fetal tissues. The data also suggest that limitations in heme and hemoprotein synthesis may contribute to the very low mixed function oxidase activity in human fetal lung and kidney, but that other factors must account for the disparity in hepatic mixed function oxidase activity between the human fetus and adult.     

Perinatal development of hepatic microsomal mixed function oxidase activity in swine

Hepatic microsomal cytochrome P-450, cytochrome b₅, NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase levels were measured in fetal (107-days gestation), newborn and 1-, 2-, 3-, 4- and 6-week-old swine. Cytochrome P-450 levels and NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase activities increased in near parallel with ethylmorphine demethylase (V_max) activity between the first and the sixth postnatal week. The activities or levels of all parameters measured appeared to plateau between the fourth and sixth week post-partum. The only qualitative change observed after 1 week of age was a slight increase in the K_m for ethylmorphine demethylation. NADPH-cytochrome c reductase activity of fetal liver was relatively high, being approximately 40 per cent of the values attained at 6 weeks of age. This was in contrast to very low levels of NADPH-cytochrome P-450 reductase activity and cytochrome P-450 content of fetal liver. Clearly the activity of the flavoprotein NADPH-cytochrome c reductase does not limit the rate of reduction of cytochrome P-450 in the microsomal fraction of fetal liver. The possibility that cytochrome P-450 exists in a different form, or ratio of forms, in fetal liver could not be ascertained from carbon monoxide (CO) or ethylisocyanide (EtCN) difference spectra of fetal microsomal preparations. However, the dithionite difference CO spectra of cytochrome P-450 did not change with age.     

Effect of storage on microsomal mixed function oxidase activity in mouse liver

The stability of mixed function oxidase components from hepatic microsomes of mice was studied. Microsomal pellets were overlaid with isotonic, buffered KCl and frozen at ?20° for up to 20 days. Ethyl morphine N-demethylase and aniline hydroxylase were the least stable, showing significant decreases in activity after 3 and 5 days of storage respectively. NADPH cytochrome c reductase and cytochrome P-450 were both stable for at least 10 days. Electron micrographs of resuspended pellets after storage showed an increasing tendency toward aggregation of microsomal vesicles as the length of storage increased.     

Dietary ascorbic acid and hepatic mixed function oxidase activity in the guinea pig

Studies were carried out to characterize the response of hepatic mixed function oxidase (MFO) activity to chronic ascorbic acid deficiency and excessive ascorbic acid intake in the guinea pig. When guinea pigs were fed excessive ascorbic acid, there was a small increase in hepatic cytochrome P-450 which was unaccompanied by any alteration in drug-metabolizing enzyme activity. Similarly, induction of MFO activity by phenobarbital was not modified by excessive ascorbic acid administration. Chronic ascorbic acid deficiency resulted in depressed metabolism of aniline, aminopyrine, ethoxycoumarin and benzphetamine, but not of ethylmorphine, in comparison with animals fed diets containing control and/or excessive amounts of ascorbic acid. In contrast to the metabolism of all drugs studied, the 7 alpha-hydroxylation of cholesterol was depressed by both inadequate and excessive vitamin C intake, demonstrating the unique sensitivity of cholesterol 7 alpha-hydroxylase to dietary ascorbate.     

Histamine inhibition of mixed function oxidase activity in rat and human liver microsomes and in the isolated perfused rat liver

The imidazole ring is a common structural feature of some xenobiotics that inhibit cytochrome P-450-catalysed reactions. Histamine is a 4-substituted imidazole and a preliminary study has shown it to be an inhibitor of rat liver microsomal drug oxidation. This work has now been extended. Histamine appears to be a competitive inhibitor of the alpha-hydroxylation (HM) (Ki = 164 microM; IC50 at 20 microM = 308 microM) and O-demethylation (ODM) (Ki = 243 microns; IC50 at 20 microM = 400 microM) of metoprolol in rat liver microsomes. Of the metabolites of histamine only N-acetylhistamine showed comparable inhibitory potency to that of the parent compound. Histamine impaired the disappearance of lignocaine when incubated with rat liver microsomes. This was accompanied by a corresponding inhibition of 3-hydroxy-lignocaine appearance. Histamine produced a type II spectral interaction with rat liver microsomes (lambda max = 432 nm, lambda min = 408 nm; Ks = 0.11 mM). When histamine was incubated alone with rat liver microsomes no loss of substrate was observed. The oxidation of metoprolol by human liver microsomes was impaired by histamine (IC50 values for ODM appearance at 25 microM: liver HL1 greater than 10, HL3 = 3.8 and HL4 = 3.7 mM). In comparison, cimetidine had an IC50 value of 1.5 mM using microsomes from liver HL3. Addition of histamine impaired the elimination of metoprolol by the isolated perfused rat liver in a dose-dependent manner (P less than 0.001, one-way analysis of variance). These data demonstrate that histamine can enter hepatocytes, interact with cytochrome P-450 and inhibit some drug oxidation reactions. The physiological relevance of inhibition of drug metabolism by histamine remains to be determined.     

Specificity of medicarpin and related isoflavonoids in inhibition of rat hepatic mixed function oxidase activity

The cytochromes P-450 of the mixed function oxidase system metabolize a wide variety of endogenous compounds to either nontoxic products or toxic metabolites. A number of natural products, such as flavonoids, influence this metabolism. Exposure to these compounds may therefore be a factor in animal and human responsiveness to cytochrome P-450 substrates. We have examined the effect of the pterocarpan medicarpin on the cytochrome P-450-dependent aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin deethylase (ECD) activities of rat liver microsomes. Medicarpin and maackiain and two of their biosynthetic precursors inhibit the constitutive and phenobarbital (PB)-induced types of AHH, but have little effect on the 3-methylcholanthrene (MC)-induced type of AHH. This is in contrast to the effect of the commonly used cytochrome P-450 inhibitor 7,8-benzoflavone, which inhibits the hepatic AHH of MC-treated rats and has no effects on the AHH of control or PB-treated rats. However, medicarpin inhibited the constitutive as well as the PB- and MC-induced ECD. The specific modulatory effect as well as its relative availability suggests the utility of medicarpin as a probe for different forms of cytochrome P-450 in animal tissues.     

Effect of cimetidine or ranitidine pretreatment on hepatic mixed function oxidase activity in the rat

This study compared the effect of single equimolar oral doses of cimetidine (100 mg/kg) or ranitidine (139 mg/kg) on rat hepatic mixed function oxidases. Cimetidine significantly (p less than 0.05) increased hexobarbital sleeping time and prolonged aminopyrine and theophylline elimination. In contrast, ranitidine did not significantly affect hexobarbital sleeping time and theophylline elimination but significantly (p less than 0.025) increased aminopyrine elimination. Aminopyrine N-demethylase activity in vitro was significantly (p less than 0.05) inhibited by cimetidine pretreatment but significantly (p less than 0.025) increased by ranitidine pretreatment. The direct addition of cimetidine or SKF 525A to the 10,000g supernatant fraction from controlled liver homogenates decreased aminopyrine N-demethylase activity, whereas the direct addition of ranitidine tended to increase aminopyrine N-demethylase activity. A significant correlation (r = 0.65, p less than or equal to 0.005) was observed between hexobarbital sleeping time in vivo and aminopyrine N-demethylase activity in vitro in the same rat. The results of this study showed that cimetidine inhibited mixed function oxidases, whereas ranitidine had no effect or tended to stimulate mixed function oxidases.     

Hydralazine as a selective probe inactivator of aldehyde oxidase in human hepatocytes: estimation of the contribution of aldehyde oxidase to metabolic clearance

Aldehyde oxidase (AO) metabolism could lead to significant underestimation of clearance in prediction of human pharmacokinetics as well as unanticipated exposure to AO-generated metabolites, if not accounted for early in drug research. We report a method using cryopreserved human hepatocytes and the time-dependent AO inhibitor hydralazine (K(I) = 83 ± 27 μM, k(inact) = 0.063 ± 0.007 min(-1)), which estimates the contribution of AO metabolism relative to total hepatic clearance. Using zaleplon as a probe substrate and simultaneously monitoring the AO-catalyzed formation of oxozaleplon and the CYP3A-catalyzed formation of desethyzaleplon in the presence of a range of hydralazine concentrations, it was determined that >90% inhibition of the AO activity with minimal effect on the CYP3A activity could be achieved with 25 to 50 μM hydralazine. This method was used to estimate the fraction metabolized due to AO [f(m(AO))] for six compounds with clearance attributed to AO along with four other drugs not metabolized by AO. The f(m(AO)) values for the AO substrates ranged between 0.49 and 0.83. Differences in estimated f(m(AO)) between two batches of pooled human hepatocytes suggest that sensitivity to hydralazine varies slightly with hepatocyte preparations. Substrates with a CYP2D6 contribution to clearance were affected by hydralazine to a minor extent, because of weak inhibition of this enzyme. Overall, these findings demonstrate that hydralazine, at a concentration of 25 to 50 μM, can be used in human hepatocyte incubations to estimate the contribution of AO to the hepatic clearance of drugs and other compounds.     

Phenol sulfotransferase 1A1 activity in human liver: kinetic properties, interindividual variation and re-evaluation of the suitability of 4-nitrophenol as a probe substrate

Sulfation is an important metabolic pathway in humans for xenobiotics, hormones and neurotransmitters, and is catalysed by the cytosolic sulfotransferase (SULT) enzymes. Phenol SULTs, especially SULT1A1, are particularly important in xenobiotic and drug metabolism because of their broad substrate specificity and extensive tissue distribution. A common variant SULT1A1 allozyme (SULT1A1*2) exists in the population, and is less stable than the wild-type SULT1A1*1. 4-Nitrophenol is widely used as a substrate for quantifying SULT1A1 activity. However, our kinetic experiments suggest that 4-nitrophenol is not an ideal substrate when determining SULT1A1 activity in human liver. Assays with a bank of 68 human liver cytosols revealed three distinct kinetic profiles for 4-nitrophenol sulfation in the population: linear, biphasic and inhibition. Sulfation of 4-nitrophenol by purified, recombinant SULT1A1*1 and SULT1A1*2 shows marked substrate inhibition, with inhibition at 4-nitrophenol concentrations greater than 4 and 10 microM, respectively. Furthermore, sulfation of 4-nitrophenol by purified recombinant SULT1B1 was significant at concentrations of 4-nitrophenol less than 10 microM. Western blots showed that the SULT1A1 levels in liver are highly variable between liver samples and that no correlation was observed between SULT1A1 activity and protein level in liver cytosols. However, a correlation between SULT1A1 activity and protein level was observed in human placental cytosols, where SULT1B1 is not expressed. We believe that in human liver other SULT isoforms (particularly SULT1B1) contribute to the sulfation of 4-nitrophenol. Therefore, 4-nitrophenol is not an ideal substrate with which to quantitate SULT1A1 activity in human liver tissue.     

Induction of mixed function oxidase activity in man by rifapentine (MDL 473), a long-acting rifamycin derivative.

The effects of rifapentine (MDL 473) administration on hepatic mixed function oxidase activity in man have been investigated in six healthy volunteers. Administration of rifapentine (600 mg 48 h-1) for 10 days resulted in a significant reduction in antipyrine half-life (from 13.2 +/- 1.0 h to 7.7 +/- 0.4 h) and a corresponding increase in its total body clearance (from 41.8 +/- 5.5 ml min-1 to 67.4 +/- 5.6 ml min-1). Twelve days after stopping rifapentine administration, these values had largely returned to base-line. 24-Hour excretion of 6 beta-hydroxycortisol was significantly increased, by approximately three-fold, following administration of rifapentine for 10 days. Again, 12 days after stopping drug administration, 6 beta-hydroxycortisol excretion had returned to pretreatment values. Clearance of antipyrine to its three oxidative metabolites was increased by rifapentine administration, although the increase for 3-hydroxymethylantipyrine was not significant. The greatest increase (+140%) was observed for norantipyrine. Twelve days after the last dose of rifapentine, all values had returned to control levels. It is concluded that, like rifampicin, rifapentine is a potent inducer of mixed function oxidase activity in man and that the possibility of clinically significant drug interactions should be anticipated in the therapeutic use of this compound.