Induction and inhibition of cytochrome P450-dependent monooxygenases of rats by fungicide bitertanol

The effects of fungicide bitertanol on cytochrome P450-dependent monooxygenases were studied using rats treated intraperitoneally with the N-substituted triazole for 4 days. Treatment with 10, 25, and 100 mg/kg bitertanol produced 2-, 4-, and 14-fold increases of 7-ethoxyresorufin O-deethylation activity in liver microsomes, respectively. Immunoblot analysis of microsomal proteins revealed that 25 mg/kg bitertanol increased CYP1A1 protein in the liver, kidney, and lung by 10-, 13-, and 17-fold, respectively. Bitertanol produced smaller increases of CYP2B and CYP3A catalytic activity and protein than that of CYP1A1 in liver. RT-PCR analysis of total RNA indicated that bitertanol-induced CYP1A1, CYP2B, and CYP3A mRNA. Additions of 0.01–100 μM bitertanol to liver microsomes from rats treated with 25 mg/kg bitertanol or 3-methylcholanthrene inhibited microsomal 7-ethoxyresorufin O-deethylation activity (IC₅₀ = 0.8 or 0.9 μM). Bitertanol at 100 mg/kg increased liver UDP-glucuronosyltransferase and glutathione S-transferase activities by 2-fold. Bitertanol at 25 mg/kg produced a minor increase in metabolic activation of benzo[a]pyrene by liver S-9 fraction in the Ames mutagenicity test while the increase was blocked by addition of 100 μM bitertanol. These findings show that bitertanol is an inducer of CYP1A1, CYP2B, and CYP3A in vivo and an inhibitor of CYP1A catalytic activity in vitro.     

Induction of cytochrome P450s by rutaecarpine and metabolism of rutaecarpine by cytochrome P450s

Rutaecarpine is an alkaloid originally isolated from the unripe fruit of Evodia rutaecarpa. Recently, rutaecarpine has been characterized to have an anti-inflammatory activity through cyclooxygenase-2 inhibition. In the present studies, the effects of rutaecarpine on liver cytochrome P450 s (P450s) and P450 s involved in the metabolism of rutaecarpine were studied in vivo and in vitro, respectively, because the data are crucial in the early development of rutaecarpine as a new drug candidate. Oral administration to male ICR mice of rutaecarpine for 3 consecutive days induced liver P450 1A-, 2B- and 2E1-selective monooxygenase activities. The induction of P450 1A and 2B by rutaecarpine was confirmed by Western immunoblotting. When rutaecarpine was incubated with rat liver microsomes in the presence of an NADPH-generating system, five metabolites were detected by UV and mass spectral analyses. The 3-methylcholanthrene- and phenobarbital-induced microsomes greatly increased the formation of metabolites. Our present results suggest that rutaecarpine might induce P450 1A and 2B in mice, and that P450 1A and 2B might predominantly metabolize rutaecarpine in rat liver microsomes.     

Effects of chronic exposure to cadmium on renal cytochrome P450-dependent monooxygenase system in rats

The aim of this study was to evaluate the effects of chronic exposure to cadmium (Cd) on the renal cytochrome P450-dependent monooxygenase system. For this purpose, male Wistar rats were intoxicated with Cd administered in drinking water at a concentration of 5 or 50 mg Cd/l for 6, 12 and 24 weeks. Concentrations of cytochrome P450 and cytochrome b₅ as well as activities of NADPH-cytochrome P450 reductase and NADH-cytochrome b₅ reductase were determined in the kidney microsomal fraction. Protein content of CYP1A1, CYP2E1 and CYP3A1 cytochrome P450 isoforms was evaluated as well. In the rats exposed to 5 mg Cd/l, the concentration of cytochrome P450 decreased (by 41%) after 24 weeks of the experiment. The activity of NADPH-cytochrome P450 reductase decreased (by 24%) after 6 and 12 weeks, whereas after 24 weeks it remained unchanged, compared with the control group. Moreover, a decrease in the concentration of cytochrome b₅ (by 25, 15 and 26% at 6, 12 and 24 weeks, respectively) and the activity of its NADH reductase (by 26 and 31% at 6 and 24 weeks, respectively) was noted in these animals. At the exposure to 50 mg Cd/l, the concentrations of cytochrome P450 and cytochrome b₅ and the activities of their corresponding reductases were decreased at each time-point. Western blot analysis revealed that all isoforms of cytochrome P450 studied were affected by Cd and the effect was dependent on the level and the duration of exposure. The results of this study indicate that chronic exposure to Cd in a dose- and time-dependent manner affects the kidney cytochrome P450-dependent monooxygenase system by decreasing the concentrations of cytochrome P450 and cytochrome b₅ and inhibiting the activities of their corresponding reductases. The effect of Cd on the cytochrome P450 content is associated with its ability to stimulate or inhibit of various P450 isoforms. A very important finding of this study is that Cd affects the kidney cytochrome P450-dependent monooxygenase system at relatively low exposure and low kidney Cd accumulation (2.40±0.15 g/g). As the experimental model used reflects human exposure to Cd, we conclude that Cd can affect the kidney cytochrome P450-dependent monooxygenase system in environmentally exposed humans. Previously we have reported disorders in the system in the liver of rats at the same levels of exposure as in this study. Thus, we hypothesize that the metabolism and detoxification of many substances, including xenobiotics, may be seriously affected in Cd-exposed subjects.Part of this work was presented at the EUROTOX 2001, Istanbul, Turkey, 2001     

Oxidation of ranitidine by isozymes of flavin-containing monooxygenase and cytochrome P450

Rat and human liver microsomes oxidized ranitidine to its N-oxide (66-76%) and S-oxide (13-18%) and desmethylranitidine (12-16%). N- and S-oxidations of ranitidine were inhibited by metimazole [flavin-containing monooxygenase (FMO) inhibitor] to 96-97% and 71-85%, respectively, and desmethylation of ranitidine was inhibited by SKF525A [cytochrome P450 (CYP) inhibitor] by 71-95%. Recombinant FMO isozymes like FMO1, FMO2, FMO3 and FMO5 produced 39, 79, 2180 and 4 ranitinine N-oxide and 45, 0, 580 and 280 ranitinine S-oxide pmol x min(-1) x nmol(-1) FMO, respectively. Desmethyranitinine was not produced by recombinant FMOs. Production of desmethylranitidine by rat and human liver microsomes was inhibited by tranylcypromine, a-naphthoflavon and quinidine, which are known to inhibit CYP2C19, 1A2 and 2D6, repectively. FMO3, the major form in adult liver, produced both ranitidine N- and S-oxides at a 4 to 1 ratio. FMO1, expressed primarily in human kidney, was 55- and 13-fold less efficient than the hepatic FMO3 in producing ranitidine N- and S-oxides, respectively. FMO2 and FMO5, although expressed slightly in human liver, kidney and lung, were not efficient producers of ranitidine N- and S-oxides. Thus, urinary contents of ranitidine N-oxide can be used as the in vivo probe to determine the hepatic FMO3 activity.     

Induction of hepatic cytochrome P-450c-dependent monooxygenase activities by dantrolene in rat

The effect of dantrolene sodium, a skeletal muscle relaxant, on drug metabolizing enzymes has been investigated after treatment of rats with a dose of 200 mg/kg for five days. We observed an induction of cytochrome P-450c and epoxide hydrolase in immunoassays and activities. An enhancement of the UDP-glucuronosyltransferase (GT1) activity was observed. We also reported a decrease of both liver cytochrome P-450 content and microsomal cytochrome P-450b dependent N-demethylation activities. On the other hand, the binding of dantrolene on microsomal cytochrome P-450 produced a type I difference spectrum, these data were obtained with liver microsomal cytochrome P-450c induced by 3-methylcholanthrene.     

Novel arachidonate metabolites generated by cytochrome P450-dependent mono-oxygenases

Cytochrome P450-dependent arachidonic acid (AA) metabolism by medullary thick ascending limb of the loop of Henle (mTALH) cells, corneal epithelium and other transporting epithelia, such as those of the intestines, generate metabolites which affect Na(+)-K(+)-ATPase activity, vasomotion and, thereby, organ function. Further, these novel AA metabolites contribute to the control of blood pressure in the SHR through participation in the local control of blood flow and in the regulation of extracellular fluid volume.     

Herb-drug interactions with Danshen (Salvia miltiorrhiza): a review on the role of cytochrome P450 enzymes

Danshen, the dried root and rhizome of Salvia miltiorrhiza Bunge, is a widely used medicinal plant for the treatment of cardiovascular diseases in China and a complementary medicine in the West. Danshen is indexed in the 2010 Chinese Pharmacopoeia, with more than 35 formulations and concoctions containing Danshen water-extracts, ethanolic extracts or their combination, which are rich in phenolic acids and different levels of tanshinones. There are rare reports on the adverse effects of Danshen preparations. It is, however, well-known that Danshen leads the anticoagulation failure of warfarin. The Danshen-warfarin interaction may be mediated via both pharmacodynamic and pharmacokinetic mechanisms. This review does not summarize recent progress, but the effects of Danshen and its active ingredients on the interactions of cytochrome P450 (CYP450) and drug transporters, as well as the analysis of ingredients, and the metabolism and pharmacokinetics that are related to these interactions. Tanshinones play significant roles in the inhibition and induction of several CYP450 isozymes. It can be concluded that precautions should be taken when using Danshen preparations rich in tanshinones for CYP-related herb-drug interactions.     

Inhibition of the hepatic cytochrome P-450-dependent monooxygenase system by rubratoxin B in male mice

The in vivo effects of rubratoxin B on hepatic mixed-function oxidase enzymes in male mice were examined. Animals were exposed to a single ip dose of 0.25, 0.5, 1.0, or 1.5 mg rubratoxin B/kg or daily doses of 0.5 mg/kg rubratoxin B for 14 days. Rubratoxin B (1 mg/kg) markedly inhibited the hepatic cytochrome P-450-dependent monooxygenase system when measured 1, 16, 48, 72, and 96 hr after a single exposure. At 48 hr, dose levels as low as 0.25 mg rubratoxin B/kg inhibited pentobarbital hydroxylase and ethylmorphine demethylase. These same enzymes, along with cytochrome P-450 content and NADPH-dependent dehydrogenase, continued to be depressed to about the same degree when animals were exposed to rubratoxin B for 14 days. NADPH-cytochrome c reductase was not affected by rubratoxin B on single or multiple exposure. These studies demonstrated that a single exposure to rubratoxin B caused a depression in activity of the cytochrome P-450 dependent monooxygenase enzyme system and that after maximum inhibition by a single dose at 48 hr, the trend was for the enzymes to return to control values.     

Circadian changes of cytochrome P-450-dependent monooxygenase system in the rat liver.

Circadian changes in cytochrome P-450 and cytochrome b5 content and activity of NADPH-cytochrome P-450 and NADH-cytochrome b5 reductases have been studied in rat liver microsomes in season autumn. The obtained results indicate, that cytochrome P-450 in 6-month-old animals shows 12 h rhythm, but in older ones 24 h rhythm. NADPH-cytochrome P-450 reductase activity shows 24 h rhythm in oldest animals only. Cytochrome b5 and its reductase has 24 h rhythm in all examined groups of rats.     

Induction of hepatic and extrahepatic cytochrome P-450 and monooxygenase activities by N-substituted imidazoles

1. Seven N-substituted imidazoles, with abilities to induce rat hepatic cytochrome P-450 from 1.5- to 4-fold after 3 days of treatment (75 mg/kg daily), were investigated for their concurrent inductive effect in kidney, intestine and lung. 2. The ability of a compound to induce cytochrome P-450 in the liver did not correlate with the ability to induce in extrahepatic tissues, the highest magnitude hepatic inducer (clotrimazole) having little inductive effect in other organs. 3. Induction of cytochrome P-450 concentration was greater in kidney and intestine than in lung but, with the exception of the two imidazoles bearing either a benzyl or a 2-naphthylmethyl substituent, the degree of induction in the extrahepatic organs did not approach that seen in liver. 4. Different monooxygenase activities were preferentially induced by the individual N-substituted imidazoles in a single tissue, and activities induced by a compound in one tissue were not uniformly induced by that compound in other tissues. Induction of activities did not always correlate with an increase in cytochrome P-450 concentration.     

Induction of cytochrome P450 enzymes by albendazole treatment in the rat

The anthelmintic drug albendazole (ABZ), methyl(5-(propylthio)-1H-benzimidazol-2-yl)carbamate, is a benzimidazole highly efficient in the treatment of neurocysticercosis. The effects of ABZ treatment (i.p. and p.o. administration) on the expression of several cytochrome P450 (CYP) enzymes were evaluated in rat liver in order to characterize the spectrum of altered CYP enzymes involved in the metabolism of environmental mutagens and carcinogens, after drug intake. Intraperitoneal administration of ABZ (50 mg/kg body weight/day/three days in corn oil) to rats, caused an induction of hepatic activities of CYP1A1-associated ethoxyresorufin O-deethylase (EROD) 65 fold, CYP1A2-associated methoxyresorufin O-demethylase (MROD) 6 fold, CYP2B1-associated penthoxyresorufin O-dealkylase (PROD) 4 fold, CYP2B2-associated benzyloxyresorufin O-dealkylase (BROD) 14 fold, as well as a partial reduction of CYP2E1-associated 4-nitrophenol hydroxylase (4-NPH) activity. CYP3A-associated erythromycin N-demethylase (END) activity was not modified under the same treatment conditions. Western blot analysis was conducted to explore if the increased catalytic activity was a result of an increased protein content; only CYP1A1/2 showed a visible increase in protein concentration after ABZ inoculation, therefore, the increased PROD and BROD activities could be attributed to the induction of CYP1A1/2. Results with the two main metabolites of ABZ (15 mg/kg body weight/day/three days, i.p.) indicated that ABZ sulfoxide (ABZSO) but not ABZ sulfone (ABZSO(2)) displayed the same pattern of CYP induction than ABZ. Oral administration of ABZ at the human therapeutic dose of 20 mg/kg body weight/day/three days, produced an increase in CYP1A1/2 protein content 24 h after the first intake. The protein level remained high during the treatment, and up to 72 h after the last administration; basal protein levels were almost recovered 48 h later.     

Propranolol as an inhibitor of some cytochrome P450-dependent monooxygenase activities in native and induced rat liver microsomes

The in vitro effect of propranolol (10(-3) M and 10(-4) M), a nonselective and extensively metabolized beta-adrenergic blocking agent, on rat liver drug metabolism in native and induced (with phenobarbital and beta-naphthoflavone [beta-NF]) microsomes was studied. The type of inhibition and the inhibitory constants of some cytochrome P450-dependent microsomal enzyme reactions (hexobarbital oxidation [HBO], ethylmorphine-N-demethylation [EMND], aniline hydroxylation [AH], ethoxycoumarin-O-deethylation [ECOD], ethoxyresorufin-O-dealkylation [EROD] and penthoxyresorufin-O-dealkylation [PROD]) were estimated. The results showed that propranolol competitively inhibited AH activity in native microsomes. The type of inhibition was changed from competitive to noncompetitive in all other enzyme activities studied. This inhibition was more pronounced after phenobarbital induction in PROD (Ki = 0.11 +/- 0.01 mM), ECOD (Ki = 0.40 +/- 0.09 mM) and EMND (Ki = 0.59 +/- 0.1 mM), and after beta-NF induction in AH (Ki = 0.28 +/- 0.05 mM) and in HBO (Ki = 0.35 +/- 0.1 mM) in native microsomes. It was assumed that the noncompetitive type of inhibition is due to the covalent binding of reactive metabolites derived from propranolol to hepatic microsomal proteins. The competitive type of inhibition of AH suggested a common P450 isoenzyme in the metabolism of propranolol and aniline. Thus, in this study, propranolol has been found to be not only a selective inhibitor of CYP2D6 isoenzyme-dependent reactions, but also a nonspecific inhibitor of other cytochrome P450 isoenzymes.     

Induction of the hepatic cytochrome P-450 dependent monooxygenase system by cis- and trans-5,10-dihydrogen mirex

The hepatic induction of cytochrome P-450-dependent monooxygenase components by cis- and trans-5,10-dihydrogen mirex was studied in male and female laboratory rats. There were sex-dependent differences between the two isomeric derivatives of mirex. The cis-isomer significantly increased aniline hydroxylase activity in the female, but not in the male. In contrast, aminopyrine N-demethylase was significantly increased by the cis-isomer in both sexes. The trans-isomer increased the hydroxylase and N-demethylase activities in both sexes. The cis-isomer induced NADPH-cytochrome c reductase in the female, and the trans-isomer did not. Both isomers induced hepatic reductase activity in the male rat. No sex-dependent differences in the hepatic induction of cytochrome P-450 were observed for either isomer.     

Induction and inhibition of cytochrome P450 and drug-metabolizing enzymes by climbazole

To determine the effect of climbazole on hepatic microsomal cytochrome P450 (P450) and drug-metabolizing enzymes, four different P450 isoforms (CYP2B1, 3A2, 2E1, and 2C12) were examined in female Long-Evans rats. Treatment of rats with climbazole resulted in the induction of P450 content. Climbazole both induced and inhibited aminopyrine N-demethylase activity, but not erythromycin N-demethylase activity. Uridine 5'-phosphate (UDP)-glucuronosyl transferase and glutathione S-transferase activities were also increased with climbazole treatment. Immunoblot analyses revealed that climbazole induces CYP2B1 and CYP3A2 at the lower dose examined, but it failed to increase CYP2B1 at the higher dose. Northern blot analysis revealed that climbazole markedly increases P450 2B1 mRNA. These results indicate that climbazole induces and inhibits P450-dependent drug-metabolizing enzymes in vivo and may have the dose-differential effect on CYP2B1 in rat liver.     

Induction and suppression of renal and hepatic cytochrome P450-dependent monooxygenases by acute and chronic streptozotocin diabetes in hamsters

 The acute and chronic effects of streptozotocin diabetes on kidney and liver microsomal monooxygenases were studied using hamsters 2 days and 6 weeks following treatment with the diabetogen, respectively. Acute diabetes increased aniline hydroxylation and N-nitrosodimethylamine demethylation, decreased pentoxyresorufin O-dealkylation, without affecting benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation in kidney and liver microsomes. The effects of chronic diabetes on the microsomal monooxygenases were similar to the effects of acute diabetes, except that the chronic diabetic condition markedly decreased benzo(a)pyrene and 7-ethoxycoumarin oxidations in kidney microsomes. Total cytochrome P450 content and NADPH-cytochrome P450 reductase activity in kidney and liver microsomes of the diabetic hamsters were similar to the controls. Gel electrophoresis of microsomes from control and streptozoptocin treated hamster tissues revealed that diabetes enhanced the intensity of protein band(s) in the P450 molecular weight region. Immunoblotting of microsomal proteins showed that acute and chronic streptozotocin diabetes induced proteins immunorelated to P450s 2E1 and 1A in kidney and liver. In marked contrast, the acute and chronic diabetic conditions decreased the level of a P450 2B-immunorelated protein(s) in kidney and liver. The present study demonstrates that acute and chronic streptozotocin diabetes has the ability to induce P450 2E1 and 1A and suppress P450 2B in hamster kidney and liver and that the hamster monooxygenase responds to diabetes differently from the rat enzyme. Received: 27 March 1995 / Accepted: 4 July 1995     

Distribution and induction of cytochrome P-450 and two cytochrome P-450-dependent monooxygenase activities in rat liver parenchymal cell subpopulations separated by centrifugal elutriation

Liver parenchymal cells from the periportal and centrilobular zones differ in their morphological, biochemical and functional characteristics. In an effort to obtain fractions enriched in either periportal or centrilobular cells, isolated rat liver parenchymal cells were separated into five subpopulations by centrifugal elutriation. The mean diameters of the cells present in fractions I–V were 19.6, 21.1, 21.8, 22.7 and 23.5 m, respectively. The content of cytochrome P-450 as well as benzphetamine N-demethylase and 7-ethoxyresorufin O-deethylase activities were higher in the larger parenchymal cells than in the smaller ones. After administration of phenobarbital the content of cytochrome P-450 was approximately two-fold greater in the cells present in fractions 3–5, when compared to the same subpopulations isolated from untreated rats; the activity of benzphetamine N-demethylase was enhanced to a similar extent in all five fractions. 3-Methylcholanthrene treatment resulted in a significant increase of cytochrome P-450 content and 7-ethoxyresorufin O-deethylase activity in all five fractions: both parameters were slightly higher in fractions 4 and 5 than in fractions 1 and 2. In conclusion, the elutriated liver parenchymal cells seem to preserve the biochemical heterogeneity observed in the intact liver; the potential enrichment of periportal and centrilobular cells in the different fractions by centrifugal elutriation is discussed.     

Species variation in the response of the cytochrome P-450-dependent monooxygenase system to inducers and inhibitors

1. In the safety evaluation of drugs and other chemicals it is important to evaluate their possible inducing and inhibitory effects on the enzymes of drug metabolism. 2. While many similarities exist between species in their response to inducers and inhibitors, there are also important differences. Possible mechanisms of such variation are considered, with particular reference to the cytochrome P-450 system. 3. Differences in inhibition may be due to differences in inhibitory site of the enzyme involved, which is not always the active site of the enzyme, in competing pathways or in the pharmacokinetics of the inhibitor. 4. Differences in induction could be due to differences in the nature of the induction mechanism, in the isoenzyme induced, in tissue- or age-dependent regulation, in competing pathways for the substrate or its products, or in the pharmacokinetics of the inducing agent. 5. Examples of each of these possible differences are considered, often from our own work on the P450 IA subfamily, and results in animals are compared with those in humans, where possible. 6. At present, the differences between species in their response to inducers and inhibitors make extrapolation to humans from the results of animal studies difficult, so that ultimately such effects should be studied in the species of interest, humans.     

Nitric oxide inhibits renal cytochrome P450-dependent epoxygenases in the rat

1. Nitric oxide (NO), or peroxynitrite, is known to inhibit haemoproteins, including cytochrome P450 mono-oxygenases. The present study explores the functional correlates of the inhibition by NO of renal epoxygenase on the vascular responses to arachidonic acid (AA) in the perfused kidney. 2. Control kidneys produce measurable amounts of epoxyeicosatrienoic acids (epoxides), which were increased from 0.6 +/- 0.2 to 1.8 +/- 0.9 ng/min (P < 0.05) following the addition of AA 5 micro g. Sodium nitroprusside (SNP; 100 micro mol/L), an NO donor, blunted the basal and AA-stimulated efflux of epoxides. 3. Sodium nitroprusside at 10 and 100 micro mol/L inhibited renal microsomal conversion of [14C]-AA to epoxides and its hydration products dihydroxyeicosatrienoic acid (diols). Microsomes harvested from rats 3 h after treatment with Escherichia coli endotoxin (lipopolysaccharide; LPS) also inhibited renal epoxygenase activity (81 +/- 8%; P < 0.05). 4. In the phenylephrine-preconstricted and indomethacin (2.8 micro mol/L)-treated kidney, AA at 5, 10 and 25 micro g elicited vasodilation that was blunted by miconazole (2 micro mol/L), 80 mmol/L KCl, tetraethylammonium (10 mmol/L), a K+ channel blocker, or SNP (100 micro mol/L). 5. Vasodilation induced by AA, but not 5,6-epoxide, was reduced in rats treated with LPS, an effect that was abolished by Nomega-nitro-l-arginine (100 mg/kg in drinking water for 10 days). 6. These data suggest that NO inhibits renal epoxygenase activity and inhibits epoxide-mediated AA-induced vasodilation in the rat kidney.