Effects of diabetes on rabbit kidney and lung CYP2E1 and CYP2B4 expression and drug metabolism and potentiation of carcinogenic activity of N-nitrosodimethylamine in kidney and lung.

There are limited number of studies regarding the influence of diabetes on the regulation of cytochrome P450s and associated drug metabolizing enzyme activities especially in extrahepatic tissues such as kidney. However, there is almost no such study in lung. Alloxan-induced diabetes did not change CYP2B4 expression as measured with immunoblot analysis and associated enzyme, benzphetamine N-demethylase, activity in rabbit kidney and lung. Induction of cytochrome P4502E1 by diabetes was identified by immunochemical detection on Western blots in the lung and kidney microsomes of rabbits. In parallel to CYP2E1 induction, aniline 4-hydroxylase and p-nitrophenol hydroxylase activities were markedly increased in diabetic rabbit lung and kidney. CYP2B4 and CYP2E1 dependent drug metabolism did not show any tissue variation in diabetic rabbit. These findings are in contrast to those of rats, mice and hamster. The results of the present work, in combination with those of the previous work [Arinç, E., Arslan, S., Adali, O., 2005. Differential effects of diabetes on CYP2E1 and CYP2B4 proteins and associated drug metabolizing enzyme activities in rabbit liver. Arch. Toxicol. 79, 427-433], indicate the existence of species-dependent response of CYP-dependent drug metabolizing enzymes to diabetes. A procarcinogen and food contaminant, N-nitrosodimethylamine (NDMA), is converted to its carcinogenic form after it is activated with NDMA N-demethylase. In the current study, a statistically significant increase of liver, kidney and lung NDMA N-demethylase activity associated with CYP2E1 was shown in diabetic rabbit. Thus, it is expected that, the risk of nitrosamine induced carcinogenesis will be greater in liver, kidney and lung of the diabetic subjects.

Effect of phenobarbitone treatment against signal grass (Brachiaria decumbens) toxicity in sheep.

The effect of phenobarbitone against signal grass (Brachiaria decumbens) toxicity was studied in 26 male crossbred sheep. Grazing on signal grass significantly decreased the concentration of cytochrome P-450 and the activity of drug metabolizing enzymes, viz. aminopyrine-N-demethylase, aniline-4-hydroxylase, UDP- glucuronyltransferase and glutathione-S-transferase in liver and kidneys of affected sheep.Oral administration of phenobarbitone (30 mg/kg body weight) for five consecutive days before grazing on B. decumbens pasture, and thereafter, for three consecutive days every two weeks, resulted in significant increases in hepatic and renal activities of drug-metabolizing enzymes. The induction of drug metabolizing activity in sheep grazing on signal grass group was found to be lower than in animals given phenobarbitone alone. Induction by phenobarbitone provided a degree of protection against the toxic effects of B. decumbens as indicated by the delay in the appearance of signs of toxicity. Furthermore, these were much milder compared to those in the sheep not treated with phenobarbitone. The present study suggests that phenobarbitone-type cytochrome P-450 isoenzyme-induction may increase resistance against signal grass (B. decumbens) toxicity in sheep.

Genotoxic and mono-oxygenase system effects of the fungicide maneb.

The in vivo effects of a commercial preparation of maneb on mono-oxygenase activities of hepatic microsomes of basal and induced rats were examined. In vitro experiments with the D7 strain of yeast Saccharomyces cerevisiae were also performed. In both basal and induced rats maneb caused a decrease in cytochrome P-450 content and aniline hydroxylase. Immunoblotting analysis using anti-P-450 IIE1 antibodies confirmed the data obtained for aniline hydroxylase activity. Maneb was toxic in cells of S. cerevisiae. On the basis of in vivo and in vitro experiments it can be concluded that maneb possesses a toxic activity attributable to its main metabolite ethylene thiourea. Immunoblotting analysis indicates that maneb biotransformation influences the IIEI P-450 isoform.

Trimethadione metabolism and microsomal monooxygenases in untreated and phenobarbital-treated rhesus monkeys.

The contribution of induced cytochrome P450 (P450) isozymes (CMLa; CYP2B, CMLb; CYP2A and CMLc; CYP3A) and related enzymes to trimethadione (TMO) metabolism in phenobarbital-treated rhesus monkey were investigated. The animals received a single dose of TMO (4 mg kg-1) and plasma samples were withdrawn before this administration and again at 0.08, 0.25, 0.5, 1 and 2 h later. Phenobarbital-treatment (20 mg kg-1 day-1 for 3 days; i.p.) significantly increased the plasma dimethadione (DMO)/TMO ratios at 0.08, 0.5, 1 and 2 h one's appropriate controls. Phenobarbital treatment also increased the P450 content (1.7-fold) and activity of aniline p-hydroxylase (1.3-fold), p-nitroanisole O-demethylase (1.8-fold) and benzphetamine N-demethylase (2.3-fold). The content of CMLa, CMLb and CMLc were increased about 12.8, 2.3 and 2.7-fold by phenobarbital pretreatment, respectively. The activity of TMO N-demethylation was inhibited by anti-P450 CMLa and anti-P450 CMLb. However, the anti-P450 CMLc antibody had no effect on this activity in liver microsomes. The results of both in vivo and in vitro studies of the effects of phenobarbital treatment on TMO metabolism indicate that these effects may be attributed to the induction of CMLa. These findings suggest that plasma DMO/TMO ratio in a single blood sampling after TMO administration is very useful for determination the degree of hepatic induction in clinical study.

Effect of 2,4,4'-trichloro-2'-hydroxydiphenyl ether on cytochrome P450 enzymes in the rat liver.

We examined the effect of 2,4,4'-trichloro-2'-hydroxydiphenyl ether (Irgasan DP300) on microsomal cytochrome P450 (P450) enzymes in rat liver. Rats were treated intraperitoneally with Irgasan DP300 daily for 4 days, at doses of 0.2, 0.4 and 0.8 mmol/kg. Among the P450-dependent monooxygenase activities, 7-benzyloxyresorufin O-debenzylase (BROD) and 7-pentoxyresorufin O-depentylase (PROD) in rats, which are associated with CYP2B1, were remarkably induced by all doses of Irgasan DP300. The relative induction to each control activity were from 5.6- to 22.3-fold and 4.9- to 20.2-fold, respectively. Furthermore, immunoblotting showed that CYP2B1/2 protein level in rat liver microsomes was increased from 10.8- to 34.4-fold by Irgasan DP300. In addition, 7-ethoxycoumarin O-deethylase (ECOD) and p-nitrophenol hydroxylase (PNPH) activities were significantly increased by Irgasan DP300 at all doses (from 1.4- to 4.9-fold). Although the activities of other P450-dependent monooxygenases, namely aminopyrine N-demethylase (APND), aniline p-hydroxylase (ANPH), erythromycin N-demethylase (EMND), lauric acid omega-hydroxylase (LAOH) and testosterone 6 beta-hydroxylase (TS6BH) were increased at high doses (> or = 0.4 mmol/kg) of Irgasan DP300, the relative level was lower than those of the CYP2B1-dependent monooxygenases such as BROD and PROD. However, 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD), testosterone 2 alpha-hydroxylase (TS2AH) and testosterone 7 alpha-hydroxylase (TS7AH) activities were not affected by any doses of Irgsan DP300. Immunoblotting showed that CYP3A2/1 and CYP4A1 protein levels were significantly induced from 1.3- to 2.2-fold by Irgasan DP300 (> or = 0.4 mmol/kg), whereas those of CYP1A1/2, CYP2C11/6 and CYP2E1 were not affected by any doses of Irgasan DP300. These results suggest that Irgasan DP300 induces the P450 isoforms of CYP2B subfamily in the rat liver, and that the induced P450 isozymes closely relates to the toxicity of Irgasan DP300 or its chlorinated derivatives.

Influence of long-term ethanol treatment on rat liver biotransformation enzymes.

The influence of rats' long-term ethanol consumption on liver enzymes that could be involved in the biotransformation of benzo(a)pyrene [B(a)P] has been studied. Male and female Wistar rats received an increasing amount of ethanol in their drinking water up to 15% (w/v) in three weeks. The ethanol content was kept at a concentration of 15% for another three weeks. One group of rats also received B(a)P in the last week of the ethanol treatment. Livers were isolated, and microsomal and cytosolic fractions were prepared. In every enzyme measurement sex differences were observed. Long-term ethanol consumption induced P450, especially aniline 4-hydroxylase (P4502E1). However, testosterone 6 beta-hydroxylase (P4503A2 and P4502C13) in males and testosterone 12 beta-hydroxylase in females were decreased. The phase 2 enzymes glutathione S-transferase (subunit 1) and epoxide hydrolase were also decreased in their activity. Our results support the hypothesis that the effect of long-term ethanol consumption on B(a)P biotransformation as found in in vivo and in vitro studies, consisting of lowered formation of phenolic and diolic metabolites, is the result of a decrease of constitutive P450 isoenzymes.

Translational activation of ethanol-inducible cytochrome P450 (CYP2E1) by isoniazid.

The molecular mechanism of ethanol-inducible cytochrome P450(CYP2E1) induction by isoniazid was studied and compared to that of pyridine, an inducer of CYP2E1. Aniline hydroxylase and immunoreactive CYP2E1 protein were significantly induced by isoniazid without or with only slight activation of other cytochromes P450. In contrast, pyridine increased the activities of a broad range of P450s. The effects of two structural analogs of isoniazid, isonicotinamide and isonicotinic acid were also tested and found to have a markedly decreased ability to induce CYP2E1. The induction of CYP2E1 by isoniazid was not accompanied by an increased level of CYP2E1 mRNA, and was completely blocked by pretreatment with cycloheximide or sodium fluoride, inhibitors of mRNA translation. These data thus suggest that CYP2E1 induction by isoniazid is due to activation of CYP2E1 mRNA translation and that the hydrazide group on the pyridine ring of isoniazid is important both in the selective induction of CYP2E1 and for magnitude of effect.

Elevation of serum triacylglycerol concentration in association with hepatic microsomal enzyme induction after treatment with phenylbutazone and diclofenac sodium in rats.

1. The relationship between serum triacylglycerol concentration and hepatic microsomal enzyme activity was examined in rats. 2. Two groups of rats were injected with diclofenac sodium at doses of 2.5 and 5 mg day-1 kg-1. A third group was injected with phenylbutazone at a dose of 20 mg day-1 kg-1. The treatment was continued for 15 days and the rats were killed 24 h after the last dose. 3. In all drug-treated rats, the serum triacylglycerol concentration and the hepatic microsomal activities of aminopyrine N-demethylase and aniline hydroxylase were significantly increased as compared with the corresponding values in control rats. The correlations between the serum triacylglycerol concentrations and the activities of the two enzymes, as indices of the hepatic microsomal activity, were highly significant. 4. These results indicate that the possibility of hypertriglyceridaemia as an adverse effect of the induction of the hepatic microsomal enzymes after the administration of phenylbutazone and diclofenac sodium should be considered.

Induction of cytochrome P-450 by dimethyl sulfoxide in primary cultures of adult rat hepatocytes.

Treatment of hepatocyte cultures with dimethyl sulfoxide (DMSO) induced P-450IIE1-specific aniline 4-hydroxylase activity and P-450IA1-specific ethoxyresorufin O-deethylase activity at a concentration of 0.1% (v/v). The P-450IIB-specific pentoxyresorufin O-deethylase activity was induced only at the 2% (v/v) level. Dot blot analysis of the total cellular RNA and cycloheximide treatment of the culture suggested that induction of ethoxyresorufin O-deethylase activity by DMSO may be due to the increase of de novo synthesis of the P-450IA1 protein, not to accumulation of mRNA in the hepatocyte culture.

Pre-translational induction of pentoxyresorufin O-depentylase by pyridine.

Pentoxyresorufin O-depentylase activity, mainly associated with phenobarbital-inducible cytochrome P450IIB1 (designated CYP2B1), was increased after a single treatment of pyridine (250 mg/kg, i.p.), and further increased by repeated treatments for 5 days. The catalytic activity and immunoreactive protein of CYP2B recognized by polyclonal antibodies were significantly induced by a relatively high dose of pyridine (250 mg/kg, i.p.) while ethanol-inducible cytochrome P450IIE1 (CYP2E1) could be induced by a low dosage (25 mg/kg, i.p.). Unlike CYP2E1 induction without changing its mRNA level, the induction of CYP2B by pyridine was accompanied by an elevation of its mRNA, indicating a pre-translational activation of this enzyme. These results indicate that pyridine induces various isozymes of cytochromes P450 by different induction mechanisms.

Ethanol-inducible microsomal aniline hydroxylase activity and cytochrome P-450 isozymes in adult hen liver.

1. Cytochrome P-450 was induced in adult hen liver by administering 15% ethanol in drinking water and compared with other inducers such as phenobarbital and beta-naphthoflavone. 2. Aniline was the only substrate whose turnover was induced by ethanol treatment when measured in the presence of 100 microM alpha-naphthoflavone. 3. The inhibitor alpha-naphthoflavone differentiated aniline and p-nitrophenol hydroxylase activities, while p-hydroxyphenyl imidazole and SKF differentiated p-nitrophenol hydroxylase activity between ethanol- and beta-naphthoflavone-induced microsomes. 4. Ethanol treatment also slightly induced some P-450 isozymes related to phenobarbital and beta-naphthoflavone inducers.

Unimpaired induction of drug-metabolizing enzymes in hepatocytes isolated from rats with micronodular cirrhosis.

To test further the competence of the cirrhotic liver to metabolize xenobiotics, hepatocytes were isolated from control and CCl4-induced cirrhotic male or female rats. Histologically micronodular cirrhosis was present in all CCl4-treated rats, while control rats had normal livers. Portal perfusion pressure and intrahepatic collagen content were also significantly increased by CCl4 administration. In male rats, no significant differences in levels of circulating transaminases nor in alkaline phosphatase was observed between cirrhotic and control rats, while CCl4-treated females had slightly higher than normal serum transaminase levels at the time of the studies. Hepatocytic cytochrome P-450 and basal xenobiotic biotransformation were unaffected by micronodular cirrhosis in both genders; calculation of the aminopyrine and 7-ethoxycoumarin intrinsic clearances (Cli) revealed, however, a slightly decreased transformation potential in hepatocytes obtained from cirrhotic females, a phenomenon not observed in cirrhotic male rats. It is speculated that the observed reduction in Cli may have been independent of cirrhosis per se, owing to the perduring cytotoxic effect of CCl4 as evidenced by the higher than normal level of transaminases in female rats. Finally, male rats were subjected to in vivo administration of phenobarbital or 3-methylcholanthrene; both compounds led to significant induction of the mixed-function oxidase system, which was similar in magnitude and in selectivity in control and cirrhotic rats as illustrated by calculation of the Michaelis-Menten kinetic parameters for aniline p-hydroxylation, aminopyrine-N-demethylation, 7-ethoxycoumarin-O-deethylation, and p-nitrophenol UDP-glucuronyl transferase. We conclude that in well-established but compensated and hepatolysis-free micronodular cirrhosis, hepatocytes are fully able to transform xenobiotics and to respond normally and selectively to inducers of drug metabolism.

Pharmacokinetics of 4-acetylaminophenylacetic acid. 2nd communication: tissue accumulation and enzyme induction in rats after repeated administration, and placental and milk transfer after single dosing.

The absorption, distribution, metabolism and excretion of 14C-labeled 4-acetylaminophenylacetic acid (MS-932) were studied in male rats after administration of an oral dose of 10 mg/kg once a day for 21 days. Comparison with the single dosing showed no marked alterations in absorption, distribution, metabolism and excretion. There were no significant differences in the activities of hepatic aniline hydroxylase and aminopyrine N-demethylase between the MS-932 treated group (10 mg/kg for 8 days) and the 0.5% aqueous sodium carboxymethyl cellulose control group (p greater than 0.05). Placental transfer of radioactivity was studied after single oral administration of 10 mg/kg of 14C-MS-932 to pregnant rats on the 12-13th and 19-20th days of gestation. Radioactivity concentrations were highest in the maternal plasma and lowest in the amniotic fluid and fetus for both middle and late pregnancies. The concentrations in the amniotic fluid and fetus decreased more slowly than did the concentration in the maternal plasma. Excretion of radioactivity to milk was studied after single oral administration of 10 mg/kg of 14C-MS-932 to lactating rats on the 10th day after parturition. Radioactivity concentrations in the rat milk were maximal at 1 h after dosing and were lower than in the maternal plasma at all the sampling times.

Differential induction of peroxygenase-dependent microsomal aniline hydroxylase by chronic ethanol ingestion.

The liver microsomal-mediated hydroxylation of aniline, which is selectively induced by chronic (EtOH) ingestion, has been studied as a function of NADPH plus dioxygen (O2)- or hydroperoxide-dependent reactions. Consistent with the well-documented induction of aniline hydroxylase following chronic ethanol -ingestion, the results showed selectivity towards aniline hydroxylase by the NADPH plus O2- and tert-butyl hydroperoxide (t-BuOOH)-dependent reactions with microsomes from EtOH-fed rats. On the other hand, the cumene hydroperoxide (CumOOH)-dependent aniline hydroxylase activity was not discriminated between microsomes from EtOH- and pair-fed rats. In parallel experiments with positive controls, CumOOH did show selectivity for phenobarbital (PB)-induced microsomal aniline hydroxylase compared to chow-fed rats. The Kcat/KM values, which indicate the efficiency of enzyme catalysis, for NADPH plus O2-, t-BuOOH, and CumOOH-dependent aniline hydroxylase from EtOH-fed rats were 102, 37, and 5 and from pair-fed rats were 68, 4, and 4 (nmol p-aminophenol/min/nmol cytochrome P-450)/mM aniline, respectively. The relative Kcat/KM ratio for EtOH-fed to that of pair-fed microsomal aniline hydroxylase from NADPH plus O2-, t-BuOOH-, and CumOOH-dependent reactions were 1.5, 7.4, and 1.2, respectively. The present preliminary studies indicate that the catalytic efficiency of EtOH-induced aniline hydroxylase is significantly greater for the t-BuOOH-dependent reaction.

Expression of cytochrome P-450 isozymes in the liver of hypophysectomized rats. Evidence for different regulation mechanisms concerning P450IIB and P450IIIA subfamilies.

1. Monooxygenase activities have been examined in rat liver to determine the effects of castration and hypophysectomy on cytochrome P-450 species. In adult males, hypophysectomy caused a decrease of total P-450 concentration, aniline hydroxylase, benzopyrene hydroxylase, benzphetamine demethylase, testosterone hydroxylase and imipramine hydroxylase and demethylase activities. The treatment of hypophysectomized animals with human growth hormone or testosterone did not restore the full activity. 2. When probed with antibodies, microsomes from hypophysectomized males and females exhibited an intense reaction with a polyclonal anti-(phenobarbital-induced P-450) which was not observed with a monoclonal antibody of anti-(phenobarbital-induced P-450). 3. These microsomal preparations also reacted with an antibody raised against a developmentally regulated P-450. No sex difference could be detected with this antibody. Furthermore, administration of human growth hormone to hypophysectomized males prevented this immunoreaction. 4. Total RNA has been prepared from the same liver; when probed with cDNAs, no changes occurred in the content in P-450 b/e, PB 24 (a constitutive member of the phenobarbital subfamily) and phenobarbital-inducible mRNA for UDP-glucuronosyltransferase. 5. In contrast, P-450 mRNA induced by pregnenolone 16 alpha-carbonitrile was modulated by hormonal manipulations: lower in females and castrated males than in intact males, increased in both sexes after hypophysectomy. Treatment of hypophysectomized males with human growth hormone abolished this rise in pregnenolone-16 alpha-carbonitrile-induced P-450 mRNA accumulation. Data collected in this study support the assumption that hypophysectomy acts differently on the regulation of various P-450 isozymes and that this regulation clearly does not involve the phenobarbital subfamily of P-450s.

Inhibitory effect of zinc-protoporphyrin on the induction of heme oxygenase and the associated decrease in cytochrome P-450 content in rats.

Pretreatment of rats with zinc-protoporphyrin, which has shown to be a potent competitive inhibitor of heme oxygenase, resulted in the inhibition of bromobenzene-mediated induction of heme oxygenase and decreases of the cytochrome P-450 content, aminopyrine demethylase and aniline hydroxylase activities. Such an inhibitory effect of zinc-protoporphyrin on the induction of heme oxygenase and concomitant decreases of drug-metabolizing enzymes occurred in a dose-dependent manner with complete inhibition of these effects at a dose of 40 mumol/kg. The effects of zinc-protoporphyrin were also observed in thioacetamide- and BCG-treated rats and ascitic tumor AH 66-bearing rats. Likewise, a decrease of cytochrome b5 content observed under these experimental conditions was also restored significantly by zinc-protoporphyrin. These results strongly suggest that the induction of heme oxygenase is a primarily important early event which consequently leads to the decrease in cytochrome P-450 content and associated enzyme activities.

Cytochrome P450 induction by phenobarbital (PB) is inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA): evidence that protein kinase C regulates induction.

The hepatic microsomal monooxygenase system was studied in hypophysectomized male rats exposed for 24 or 48 h to PB and/or TPA, an activator of kinase C. TPA attenuated basal and PB-induced levels of P450, aniline hydroxylase (ANH), ethylmorphine demethylase (EDM) and cytochrome c reductase. Hence, PB may effect induction via the inhibition of kinase C. Supporting this is spectral evidence that PB and TPA do not bind and the fact that TPA did not decrease P450 when co-incubated with O2 and NADPH. Hemin failed to increase P450 levels previously depressed by TPA indicating that TPA acts by lowering apocytochrome levels. This is consistent with its attenuation of PB-effected increases in hepatic RNA. TPA effects were associated with increased hepatic RNA and were blocked by puromycin.

Biochemical and immunochemical evidence for the induction of an ethanol-inducible cytochrome P-450 isozyme in male Syrian golden hamsters.

The effects of ethanol and of phenobarbital pretreatment on hamster microsomal metabolism of aniline and p-nitrophenol have been investigated. Hydroxylation of both compounds was increased over 2-fold by ethanol pretreatment, whereas phenobarbital pretreatment had little effect on either activity. Ethanol pretreatment had no effect on the specific content of total cytochrome P-450, while phenobarbital pretreatment increased the specific content 1.6-fold. Comparison of the specific activities for aniline hydroxylation and p-nitrophenol hydroxylation of individual microsomal samples from control, ethanol-pretreated and phenobarbital-pretreated animals showed a high degree of correlation (r2 = 0.98) consistent with the involvement of the same site for catalysis of these two compounds. Antibody to rabbit ethanol-inducible cytochrome P-450 (isozyme 3a) inhibited over 80% of the aniline (high affinity) and p-nitrophenol hydroxylase activities of microsomes from ethanol-treated hamsters. A comparison of the antibody-inhibitable rates for both hydroxylase activities with microsomes from untreated, ethanol- or phenobarbital-pretreated hamsters suggested that an isozyme homologous to rabbit isozyme 3a (hamster cytochrome P-450alc) was induced in hamsters about 3.5-fold by ethanol and was unaffected by phenobarbital. The induction of hamster cytochrome P-450alc was confirmed by immunoblot analysis of hamster microsomes. A single protein with a molecular weight of approximately 54,000 was recognized by antibody to the rabbit isozyme. Quantification of the immunoblots demonstrated that the hamster isozyme was increased about 3-fold, in good agreement with the induction determined by a comparison of the antibody-inhibitable rates. The results indicated that, although there was no change in the total spectrally observable cytochrome P-450, there was a marked change in the distribution of the isozymes of cytochrome P-450, with an increase in the alcohol-inducible form after 28-day ethanol consumption by chow-fed hamsters. This isozyme can be readily monitored by either high-affinity aniline or p-nitrophenol hydroxylation or by Western immunoblot analysis and appears to be the ethanol-inducible form of cytochrome P-450 in hamsters.