nitrophenols and pentoxyresorufin

Little is known about the effects of the cardiovascular drug verapamil (VRP) on metabolic processes in fish. Most calcium channel blockers including VRP are metabolized by cytochrome P450 (CYP450) enzymes. In this study we investigated the in vivo effect of VRP on some CYP450-mediated reactions in juvenile rainbow trout (Oncorhynchus mykiss). Fish were exposed to sublethal concentrations of VRP (0.5, 27 and 270 μg l(-1)) for 0, 21, and 42 day. The following CYP450-mediated reactions were studied in hepatic microsomes: O-dealkylation of ethoxyresorufin, methoxyresorufin, and pentoxyresorufin, hydroxylation of coumarin, tolbutamide, and p-nitrophenol, and O-debenzylation of 7-benzyloxy-4-trifluoromethylcoumarin. The amounts of products of these reactions did not differ among fish exposed to different levels of VRP and control fish. This suggests that the levels of VPR used did not alter catalytic activity of the selected CYP450 enzymes. In conclusion, none of the investigated CYP450-mediated reactions has potential as a biomarker to monitor VRP contamination of the aquatic environment.

Topics: Animals; Biomarkers; Calcium Channel Blockers; Coumarins; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Liver; Microsomes, Liver; Nitrophenols; Oncorhynchus mykiss; Oxazines; Verapamil; Water Pollutants, Chemical

Despite their substantially lower levels relative to hepatic tissue, pulmonary cytochrome P-450 (CYP) monooxygenases play an important role in the metabolic activation of substrates that cause lung injury. The target- and species-selective toxicity of a number of pulmonary toxicants has been attributed to the presence and distribution of activating enzymes with high kcat in target airways of susceptible species. However, experimental demonstration of these concepts and quantitative assessment of the contribution of individual CYP isoforms is lacking. This study was undertaken to characterize the catalytic activities of CYP2F2 with naphthalene, a murine Clara cell toxicant, as well as with other xenobiotics that either undergo metabolic activation to cytotoxic intermediates or that function as "isoform-selective" substrates. Recombinant CYP2F2 was produced using the baculovirus expression vector system in Spodoptera frugiperda and Trichoplusia ni cells, accounting up to approximately 20% of the total cellular protein. Incubations containing naphthalene, recombinant CYP2F2, NADPH-cytochrome P-450 oxidoreductase, and NADPH-regenerating system metabolized naphthalene with a high degree of stereoselectivity to 1R, 2S-naphthalene oxide (66:1 enantiomeric ratio). The Km and kcat values, along with the specificity constant, for naphthalene metabolism by recombinant CYP2F2 were 3 microM, 104 min-1, and 5.8 x 10(5) M-1 s-1, respectively. Recombinant CYP2F2 also metabolized ethoxyresorufin, pentoxyresorufin, p-nitrophenol, and 1-nitronaphthalene at easily detectable levels. The results from this work suggest that CYP2F2 1) plays a key role in the species- and cell-selective toxicity of naphthalene and 2) efficiently metabolizes a number of other substrates, including the lung toxicant 1-nitronaphthalene.

Topics: Animals; Baculoviridae; Biotransformation; Blotting, Western; Cytochrome P-450 CYP1A1; Cytochrome P-450 Enzyme System; Electrophoresis, Polyacrylamide Gel; Kinetics; Moths; Naphthalenes; Nitrophenols; Oxazines; Recombinant Proteins; Spodoptera; Stereoisomerism; Xenobiotics

We examined which cytochrome P-450 (P-450) species other than CYP1A participates in the oxidative metabolism of theophylline (TP) in mouse hepatic microsomes. Among the three metabolic pathways of TP, only 8-hydroxylation was selectively enhanced by acetone, a potent inducer of CYP2E. We assumed that two P-450 populations with different metabolic ability were involved in this metabolic process, and kinetic analyses revealed that the enhancement was due to the induction of a high-capacity P-450 population. The 8-hydroxylation at a substrate concentration, where most of the total activity was attributed to the catalysis of the high-capacity phase, was markedly impaired by CYP2E inhibitors such as 4-methylpyrazole and aminoacetonitrile, whereas the N-demethylations were little affected by these agents. The activity of TP 8-hydroxylation was significantly correlated with that of p-nitrophenol hydroxylation, a probe for CYP2E, in untreated microsomes. The activities of these oxidative reactions were modified to a similar degree by known enzyme inhibitors with a range of inhibitory potencies and affinity for P-450 isoforms. On the other hand, a relationship between TP N-demethylations and p-nitrophenol hydroxylation was not apparent, but there was a behavioral similarity between the two types of N-demethylations. The results indicated that TP 8-hydroxylation, which accounts for a large portion of TP oxidations, involves CYP2E, and that its N-demethylations are mediated by a common or closely similar P-450 species distinct from CYP2E.

Topics: Acetone; Aminoacetonitrile; Aminopyrine; Animals; Cytochrome P-450 CYP2E1; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Fomepizole; Hydroxylation; Male; Mice; Mice, Inbred Strains; Microsomes, Liver; Nitrophenols; Oxazines; Phenobarbital; Pregnenolone Carbonitrile; Pyrazoles; Theophylline

The dealkylations of 7-ethoxy- and 7-pentoxyresorufin,p-nitrophenol hydroxylation, and regio- and stereoselective hydroxylation of testosterone were measured to study the stability and inducibility of cytochrome P450 activities in cultured human hepatocytes. The results showed that human hepatocytes in primary culture retain the ability to increase specific cytochrome P450 activities upon incubation with inducers. 3-Methylcholanthrene produced a strong increase (6- to 21-fold over control) in 7-ethoxyresorufin O-deethylase activity and a small enhancement (1.5- to 2.5-fold) of the p-nitrophenol hydroxylation rate. Incubation of cells with phenobarbital resulted in moderate increases in 7-pentoxyresorufin O-depentylation (1.5- to 2-fold) and in testosterone hydroxylation at 16 alpha (1.5- to 4.5-fold) and 16 beta (1.3- to 4-fold) positions. Ethanol specifically increased p-nitrophenol hydroxylase activity (1.5- to 3.5-fold) and reduced 15 beta- and 6 beta-hydroxylations of testosterone. Treatment of hepatocytes with dexamethasone produced an increase of almost all the activities studied, with 6 beta- (2- to 3-fold) and 16 beta-hydroxytestosterone (1.4- to 2.4-fold) formation showing the greatest enhancement. Clofibric acid exposure resulted in 1.5- to 3-fold increases in 7-pentoxyresorufin O-depentylase and in testosterone 6 beta- and 2 beta-hydroxylase activities. Isosafrol selectively increased 7-ethoxyresorufin O-deethylase activity (2- to 3-fold), and it moderately reduced the other activities studied.

Topics: Adult; Aged; Cells, Cultured; Clofibric Acid; Cytochrome P-450 Enzyme System; Dexamethasone; Enzyme Induction; Ethanol; Female; Humans; Isoenzymes; Liver; Male; Methylcholanthrene; Middle Aged; Nitrophenols; Oxazines; Oxidation-Reduction; Phenobarbital; Safrole; Substrate Specificity; Testosterone

Cytochrome P-450 (P-450) 2E1 is under transcriptional and post-transcriptional control. Well-defined time courses were carried out to compare the effect of pyrazole and 4-methylpyrazole on catalytic activities, apo-P-450 2E1 levels and mRNA levels to evaluate whether induction of P-450 2E1 is preceded by altered mRNA levels. Two days of treatment with pyrazole or three days of treatment with 4-methylpyrazole resulted in significant induction of P-450 2E1, as assessed by Western blots and by oxidation of dimethylnitrosamine or p-nitrophenol. No changes in mRNA levels were detected with either inducer. Within 2 h of the second treatment with pyrazole, maximal induction of P-450 2E1 was observed, however, a 8-12 h time-dependent period was required after the third treatment with 4-methylpyrazole for maximal induction. Irrespective of the time period, increased catalytic activity and P-450 2E1 appears to reflect a post-transcriptional mechanism. A single treatment with 4-methylpyrazole increased P-450 2B1/B2 levels and oxidation of pentoxyresorufin about 2- to 3-fold. No change in mRNA levels for 2B1/B2 was observed. Although significant, the induction of 2B1/B2 by 4-methylpyrazole is more than an order of magnitude less than that by phenobarbital. Pyrazole did not induce 2B1/B2. It appears that, similar to acetone and ethanol, 4-methylpyrazole may increase several P-450 isozymes, whereas pyrazole is more specific for induction of P-450 2E1.

Topics: Animals; Base Sequence; Cytochrome P-450 CYP2E1; Cytochrome P-450 Enzyme System; Dimethylnitrosamine; Enzyme Induction; Fomepizole; Immunoblotting; Male; Molecular Sequence Data; Nitrophenols; Oligonucleotide Probes; Oxazines; Oxidoreductases, N-Demethylating; Pyrazoles; Rats; Rats, Inbred Strains; RNA, Messenger; Time Factors