Pentoxyresorufin O-depentylation Research Articles

Western Diet Decreases Hepatic Drug Metabolism in Male LDLr-/-ApoB100/100 Mice.

Consumption of a Western diet is an important risk factor for several chronic diseases including nonalcoholic fatty liver disease (NAFLD), but its effect on the xenobiotic metabolizing enzyme activities in the liver has been studied incompletely. In this study, male LDLr-/-ApoB100/100 mice were fed with Western diet (WD) or a standard diet for five months to reveal the effects on drug metabolism such as cytochrome P450 (CYP) oxidation and conjugation activities in the liver. Hepatic steatosis, lobular inflammation, and early fibrosis were observed in WD fed mice, but not in chow diet control mice. When compared to the controls, the WD-fed mice had significantly decreased protein-normalized CYP probe activities of 7-ethoxyresorufinO-deethylation (52%), coumarin 7-hydroxylation (26%), 7-hydroxylation of 3-(3-fluoro-4-hydroxyphenyl)-6-methoxycoumarin (70%), 7-hydroxylation of 3-(4-trifluoromethoxyphenyl)-6-methoxycoumarin (78%), 7-hydroxylation of 3-(3-methoxyphenyl)coumarin (81%), and pentoxyresorufin O-depentylation (66%). Increased activity was seen significantly in sulfonation of 3-(4-methylphenyl)-7-hydroxycoumarin (289%) and cytosol catechol O-methyltranferase (COMT, 148%) in the WD group when compared to the controls. In conclusion, the WD-induced steatosis in male LDLr-/-ApoB100/100 mice was associated with decreased CYP oxidation reactions but had no clear effects on conjugation reactions of glucuronidation, sulfonation, and cytosolic catechol O-methylation. Consequently, the WD may decrease the metabolic elimination of drugs compared to healthier low-fat diets.

Phenethyl isocyanate is not the metabolite of phenethyl isothiocyanate responsible for mechanism-based inhibition of cytochrome P450

Phenethyl isothiocyanate is a chemopreventive phytochemical present in cruciferous vegetables where it exists as the glucosinolate gluconasturtiin. It is a mechanism-based inhibitor of both rat and human cytochrome P450 enzymes. The principal objective of the present study was to ascertain whether phenethyl isocyanate, formed by the cytochrome P450-mediated oxidative desulphuration of phenethyl isothiocyanate, is the metabolite responsible for the mechanism-based inhibition. Phenethyl isothiocyanate, following incubation with Aroclor 1254-induced rat liver microsomes in the presence of NADPH, markedly suppressed the CYP1A-mediated O-deethylation of ethoxyresorufin; extent of inhibition was directly related to the pre-incubation time and was antagonised by reduced glutathione. When human liver microsomes were used, the inhibitory effect of phenethyl isothiocyanate, which was once again related to the pre-incubation time, was even more pronounced. When the ability of phenethyl isothiocyanate and phenethyl isocyanate to directly inhibit the O-deethylation of ethoxyresorufin in rat microsomes was compared, the latter compound was only moderately more effective. In human microsomes, both compounds were equipotent. In phenobarbital-induced lung microsomes, phenethyl isothiocyanate was a direct and potent inhibitor of the O-depentylation of pentoxyresorufin; pre-incubation of the isothiocyanate had no impact. Human precision-cut liver slices were more effective than rat slices in metabolising phenethyl isothiocyanate. Pre-treatment of rats, however, with phenobarbitone significantly enhanced the metabolism of isothiocyanate. It may be inferred from the present studies that: (a) phenethyl isocyanate is not the metabolite of phenethyl isothiocyanate responsible for its mechanism-based inhibition, and (b) CYP2B is an important catalyst of the metabolism of phenethyl isothiocyanate.

Tissue differences in the modulation of rat cytochromes P450 and phase II conjugation systems by dietary doses of phenethyl isothiocyanate

Rats were fed diets supplemented with phenethyl isothiocyanate (PEITC) at 0.06 (low dose, dietary intake level), 0.6 (medium dose) and 6.0 μmole/g (high dose), and xenobiotic-metabolising enzymes were monitored in liver, lung and kidney. At the low dose, inhibition of the hepatic O-dealkylation of ethoxy- and methoxyresorufin was noted, whereas at the high dose increases in the O-depentylation of pentoxyresorufin and O-debenzylation of benzyloxyquinoline were observed, whereas p-nitrophenol hydroxylase was inhibited. Hepatic bioactivation of 2-amino-3-methylimidazo-[4,5- f]quinoline to mutagens was not influenced by the PEITC-treatment. In the lung, at the high dose, ethoxyresorufin dealkylation was elevated and that of pentoxyresorufin suppressed; no significant changes were seen in the kidney. Quinone reductase was markedly elevated at all doses in liver, but the lung enzyme was refractive whereas in the kidney a modest rise was observed at the high dose. Hepatic glutathione S-transferase activity was stimulated by PEITC-treatment, but no effect was evident in the lung or kidney. It is concluded that the effects of PEITC on xenobiotic-metabolising systems are dose- and tissue-dependent, with the liver being the most sensitive and the lung generally resistant. Increased detoxication rather than cytochrome P450 inhibition is the likely mechanism of the chemopreventive activity of PEITC.

Modulation of rat pulmonary carcinogen-metabolising enzyme systems by the isothiocyanates erucin and sulforaphane

The objective of this study was to evaluate the potential of the structurally related aliphatic isothiocyanates erucin and sulforaphane to modulate the pulmonary carcinogen-metabolising enzyme systems in rat lung, a target organ of their chemopreventive activity. Precision-cut rat lung slices were prepared and incubated for 24 h with a range of concentrations of either erucin or sulforaphane, up to 50 μM. Neither compound modulated the O-deethylation of ethoxyresorufin whereas they elevated markedly CYP1A1 and, to a lesser extent, CYP1B1 apoprotein levels. Neither compound influenced the O-depentylation of pentoxyresorufin or CYP2B apoprotein levels, but sulforaphane caused a modest increase in CYP3A2 apoprotein levels. Pulmonary quinone reductase activity, monitored using 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide as substrate, was markedly up-regulated by both compounds and was paralleled by a similar rise in protein levels. Both compounds increased cytosolic glutathione S-transferase activity, measured using 1-chloro-2,4-dinitrobenzene as the accepting substrate; a modest rise was seen in GSTα protein levels, determined immunologically, whereas GSTπ levels were un-affected by the same treatment. Finally, both erucin and sulforaphane increased total glutathione concentration in lung cytosol. It is concluded that these aliphatic isothiocyanates have the potential to antagonise the carcinogenicity of pulmonary carcinogens by stimulating the in situ detoxication of their DNA-binding genotoxic metabolites.

Rat Cytochrome P450 2C11 in Liver Microsomes Involved in Oxidation of Anesthetic Agent Propofol and Deactivated by Prior Treatment with Propofol

Propofol (2,6-diisopropylphenol) is a widely-used anesthetic agent attributable to its rapid biotransformation. Liver microsomal cytochrome P450 (P450) isoforms involved in the biotransformation of propofol in rats and the effects of propofol in vivo on P450 levels in rats were investigated. Of six cDNA-expressed rat P450 isoforms tested, CYP2B1 and CYP2C11 had high catalytic activities from 5 microM and 20 microM propofol concentrations, respectively. Rates of propofol metabolism, at a substrate concentration of 20 microM based on the reported human blood concentration, were decreased by intraperitoneal treatment of propofol with male rats, in contrast to a strong induction by phenobarbital. Single intravenously administered propofol (10 mg/kg) caused the decrease of total P450 and CYP2C contents and activities of testosterone 16alpha-hydroxylation and propofol metabolism in liver microsomes from male rats. The suppressive effects were caused by administered propofol (10 mg/kg) twice every 4 h on CYP2B activities such as testosterone 16beta-hydroxylation or pentoxyresorufin O-depentylation, in addition to the strong suppression of CYP2C function by the single propofol treatment. These results suggest that CYP2C11, presumably deactivated by propofol, has an important role in propofol metabolism in rat liver microsomes. Repeated administration of propofol could markedly decrease the biotransformation of propofol via P450 deactivation.

ANTITUMOR DRUG ELLIPTICINE INHIBITS THE ACTIVITIES OF RAT HEPATIC CYTOCHROMES P450

Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine also forms the cytochrome P450 (CYP)-mediated covalent DNA adducts. Here, we study the effect of ellipticine on CYP enzymes in rat hepatic microsomes, studying its binding to the enzymes and its potential to inhibit the CYP activities measured with their selective substrates. Although ellipticine was reported to be a selective and strong inhibitor of CYP1A1/2, we found that its inhibitory potential is non-specific. Ellipticine is the most potent inhibitor for CYP3A-dependent 6beta-hydroxylation of progesterone, followed by CYP1A1/2-dependent ethoxyresorufin O-deethylation and CYP2B-mediated pentoxyresorufin O-depentylation. Lower inhibition was detected for 1'-hydroxylation of bufurarol, 21-hydroxylation of progesterone and 6-hydroxylation of chlorzoxazone catalyzed by CYP2D, CYP2C and CYP2E1, respectively. Ellipticine binds to several CYPs of rat hepatic microsomes. The binding titration of ellipticine typically give reverse type I spectrum with CYPs in rat hepatic microsomes. The results indicate that inhibition of CYPs by ellipticine cannot be explained only by its differential potency to bind to individual CYPs.

Black tea intake modulates the excretion of urinary mutagens in rats exposed to 6-aminochrysene: induction of cytochromes P450 by 6-aminochrysene in the rat

Rats were exposed to black tea (2.5% w/v) as their sole drinking liquid for either 1 day (short-term) or 1 month (long-term), while controls received water. After exposure, all animals received a single oral dose of 6-aminochrysene and urine was collected for 72 h. Urinary mutagenicity was determined in the Ames test using an activation system comprising hepatic cytosol from Aroclor 1254-induced rats and utilizing the Salmonella typhimurium O-acetylase overexpressing bacterial strain YG1024. Both tea treatments suppressed the urinary excretion of indirect acting mutagens; no direct acting mutagenic activity was detectable. Furthermore, both tea treatments induced hepatic CYP1A2 activity, as exemplified by the O-demethylation of methoxyresorufin, when compared with the corresponding controls; similarly, an increase in CYP1A2 apoprotein levels was observed. The O-depentylation of pentoxyresorufin was also induced by the long-term tea treatment only, but the effect was less pronounced. No significant changes were seen in glutathione S-transferase and glucuronosyl transferase activities. When rats were exposed to caffeine at a dose level corresponding to that in black tea, a marked decrease was observed in the urinary excretion of indirect acting mutagens following a single oral dose of 6-aminochrysene. It is concluded that even after short-term exposure, black tea enhances the metabolism of 6-aminochrysene and that this is probably related to the up-regulation of hepatic CYP1A2 by the caffeine present in black tea. Finally, 6-aminochrysene was a potent inducer of CYP1A1, as assessed by the O-deethylation of ethoxyresorufin and immunoblot analysis. The same treatment modestly increased glutathione S-transferase activity when assessed using 1-chloro-2,4-dinitrobenzene as the accepting substrate.

Cyclosporine A and Tacrolimus: In vitro investigations on the differential interactions with the cytochrome P450 system in rat and human liver

Species differences in the interactions of cyclosporine A (CSA) and tacrolimus (TAC) with the cytochrome P450 (CYP) system in male rat and human liver were investigated in vitro by assessing effects on a series of model reactions for different CYP isoforms. CSA and TAC concentration dependently inhibited ethoxyresorufin O-deethylation, ethoxycoumarin O-deethylation and pentoxyresorufin O-depentylation and 7alpha- and 17-testosterone hydroxylation (TH) activities in both species. In rat liver no effect of CSA was seen on ethylmorphine N-demethylation and 2alpha- and 6beta-TH activities, but an inhibition due to TAC. Both CSA and TAC, however, distinctly decreased ethylmorphine N-demethylation and 2beta- and 6beta-TH activities in human liver. The same results were seen with 14alpha- and 15beta-TH activities. 2alpha-, 16alpha- and 16beta-TH activities were only inhibited in human liver with TAC, whereas only in this case 6alpha-TH activity was left unaffected. p-Nitrophenol hydroxylase activity was not influenced by either substance in both species. Thus, CSA mainly interacts in rat with the CYP isoforms 1A, 2A and 2B and in man with the CYP subtypes 1A, 2A, 2B, 2C and 3A. TAC seems to interfere predominantly in rat with the CYP isoforms 2A, 2B, 2C and 3A and in man with the CYP subtypes 1A, 2B, 2C and 3A. In summary, our results point to distinct species differences in the interactions with the CYP system with both substances, and although from literature CSA and TAC are known to be metabolized mainly by CYP 3A, according to our findings in rat liver CSA seems not to interact with this CYP subtype.

Fibrates and their newly synthesized glycinate or glycinate-methylester derivatives: Comparison of the interactions with liver cytochrome P450 dependent monooxygenase- and oxidase-functions in vitro

Different fibrates (bezafibrate, ciprofibrate, clofibrate, fenofibrate, gemfibrozil) were investigated in comparison with their newly synthesized glycinate and glycinate-methylester derivatives. Interactions with the cytochrome P450 (CYP) system were studied by assessing binding to CYP and effects on CYP mediated monooxygenase functions in rat liver 9000 g supernatants, as measured by six model reactions for different CYP isoforms (ethoxyresorufin O-deethylation, ethoxycoumarin O-deethylation, pentoxyresorufin O-depentylation, p-nitrophenol-hydroxylation, ethylmorphine N-demethylation, lauric acid 11- or 12-hydroxylation). Possible prooxidant or antioxidant properties were investigated by the stimulated lipid peroxidation, hydrogen peroxide production, and lucigenin and luminol amplified chemiluminescence using rat liver microsomes. Additionally, the influence on luminol amplified rat whole blood chemiluminescence was examined. All substances tested displayed binding to CYP. Effects on the monooxygenase model reactions were in general more distinct with the glycinates than with the parent compounds and most pronounced with the glycinate-methylester derivatives. The slightest effects on all model reactions were seen with clofibrate and its derivatives. On the whole, low antioxidative rather than prooxidative effects were observed. In general and with most model reactions, the antioxidative capacity of the glycinate and glycinate-methylester derivatives slightly exceeded that of the respective parent compounds. Summarizing the results it can be concluded that with respect to possible interactions with the CYP system in vivo and thus with the biotransformation of other concomitantly administered compounds no advantages of the glycinate or glycinate methylester derivatives over their parent fibrates are to be expected. Only the antioxidative capacity of the derivatives was somewhat higher than that of the parent substances, though most probably only of minor therapeutical relevance.

Expression and inducibility of cytochrome P450 isoforms in 1-year-old intrasplenic liver cell transplants in rats

Syngenic fetal liver tissue suspensions were transplanted into the spleens of 60- to 90-day-old male Fischer 344 inbred rats. Transplant recipients were compared with age-matched control rats. One year after surgery, the animals were treated orally with beta-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or the respective solvents 24 or 48 h before being killed. Expression of cytochrome P450 (P450) isoforms in spleens and orthotopic livers was assessed by immunohistochemistry and P450-dependent monooxygenase functions by the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), pentoxyresorufin O-depentylation (PROD) and ethylmorphine N-demethylation (EMND). Spleens of control animals displayed almost no expression of P450 isoforms and P450-mediated monooxygenase functions. Similar to liver, in the transplanted hepatocytes no P450 1A1 but distinct P450 2B1 and 3A2 expression was observed. Furthermore, the transplant-containing spleens displayed significant EROD, ECOD, PROD and EMND activities. Similar to normal liver, BNF treatment enhanced P450 1A1 and 2B1, PB induced P450 2B1 and 3A2, and DEX induced P450 3A2 expression in the transplanted hepatocytes. Correspondingly, in the transplant-containing spleens EROD, ECOD and PROD activities were significantly enhanced following BNF treatment, EROD, ECOD, PROD and EMND activities after PB administration, and EMND activity by DEX treatment. These results demonstrate that hepatocytes originating from fetal liver tissue suspensions can survive in the spleen at least for 1 year. They have differentiated into adult hepatocytes and even 1 year after transplantation express different P450 isoforms which are inducible by BNF, PB and DEX, corresponding to normal adult liver.

Expression and inducibility of cytochrome P450 isoforms in 1-year-old intrasplenic liver cell transplants in rats

Abstract Syngenic fetal liver tissue suspensions were transplanted into the spleens of 60- to 90-day-old male Fischer 344 inbred rats. Transplant recipients were compared with agematched control rats. One year after surgery, the animals were treated orally with β-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or the respective solvents 24 or 48 h before being killed. Expression of cytochrome P450 (P450) isoforms in spleens and orthotopic livers was assessed by immunohistochemistry and P450-dependent monooxygenase functions by the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), pentoxyresorufin O-depentylation (PROD) and ethylmorphine N-demethylation (EMND). Spleens of control animals displayed almost no expression of P450 isoforms and P450-mediated monooxygenase functions. Similar to liver, in the transplanted hepatocytes no P450 1A1 but distinct P450 2B1 and 3A2 expression was observed. Furthermore, the transplantcontaining spleens displayed significant EROD, ECOD, PROD and EMND activities. Similar to normal liver, BNF treatment enhanced P450 1A1 and 2B1, PB induced P450 2B1 and 3A2, and DEX induced P450 3A2 expression in the transplanted hepatocytes. Correspondingly, in the transplant-containing spleens EROD, ECOD and PROD activities were significantly enhanced following BNF treatment, EROD, ECOD, PROD and EMND activities after PB administration, and EMND activity by DEX treatment. These results demonstrate that hepatocytes originating from fetal liver tissue suspensions can survive in the spleen at least for 1 year. They have differentiated into adult hepatocytes and even 1 year after transplantation express different P450 isoforms which are inducible by BNF, PB and DEX, corresponding to normal adult liver.

Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of different cytotoxins on cytochrome P450 mediated monooxygenase functions and on oxidative state

Syngenic fetal liver tissue suspensions were transplanted into the spleens of adult male Fisher 344 inbred rats. Four months after surgery, transplant recipients and age matched control rats were treated with different cytotoxins (allyl alcohol [AAL], bromobenzene [BBZ], carbon tetrachloride [CCl4], or thioacetamide [TAA]) or the respective solvents 24 or 48 hours before sacrifice. Effects of the cytotoxins on P450 mediated monooxygenase functions in liver and spleen 9,000 g supernatants were assessed by measuring the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), pentoxyresorufin O-depentylation (PROD), and ethylmorphine N-demethylation (EMND). Additionally, the influence on the oxidative state was investigated by assessing the liver and spleen tissue content of lipid peroxidation (LPO) products and of reduced and oxidized glutathione (GSH;GSSG). The livers of both solvent treated transplant recipients and control rats displayed regular EROD, ECOD, PROD and EMND activities. After AAL treatment EROD and EMND activities within the livers were not affected, but ECOD and PROD activities were increased. BBZ administration caused a decrease in EROD and EMND activities, ECOD activity remained unaffected, and PROD activity was even increased. CCl4 and TAA administration caused a strong reduction in the activity of all four model reactions. Spleens of control rats displayed almost no P450 mediated monooxygenase functions, independent whether the rats had been treated with the cytotoxins or not. In the transplant containing spleens, however, significant EROD and ECOD, but hardly any PROD or EMND activities were seen. After AAL administration EROD activity was not affected in the transplant containing spleens, but ECOD activity was increased. BBZ treatment led to a decrease in EROD and an elevation in ECOD activity. CCl4 and TAA strongly reduced the activity of both of these model reactions. The tissue content of LPO products within livers and transplant containing spleens was significantly increased after BBZ and CCl4 treatment. An elevation in LPO products was also seen in the spleens of the control rats due to CCl4 administration. Tissue GSH and GSSG content in both livers and transplant containing spleens were strongly reduced after BBZ treatment. After CCl4 administration only a significant decrease in liver GSSG contents was seen. TAA treatment caused a reduction in the GSH and GSSG content in the spleens of both transplant recipients and control rats, but not in the livers. From these results it can be concluded, that the effects of cytotoxins like AAL, BBZ, CCl4 or TAA on P450 dependent monooxygenase functions and on oxidative state are exerted in the ectopic intrasplenic liver cell transplants in a similar way as in normal orthotopic liver.

Induction of cytochrome P450 2B1-mRNA and pentoxyresorufin O-depentylation after exposure of precision-cut rat liver slices to phenobarbital

Precision-cut rat liver slices were prepared from male Wistar rats with a Krumdieck slicer and cultured in William's medium E for up to 24 h. In untreated control slices, CYP2B1-mRNA concentration, which was quantified by competitive RT-PCR, did not decrease during this time. After exposure of the slices to 100 μM phenobarbital, CYP2B1-mRNA increased by about 10- or 60-fold after 6 or 24 h, respectively. The extent of this in vitro induction was similar to that after in vivo administration of 60 mg/kg phenobarbital. Pentoxyresorufin O-depentylation (PROD) was also inducible in vitro after 24 h, but to a lesser extent than the corresponding CYP-mRNA. Precision-cut liver slices proved to be a simple and reliable in vitro system for the sensitive detection of an induction by phenobarbital.

Ursodeoxycholic acid prevents hepatic cytochrome P450 isozyme reduction in rats with deoxycholic acid-induced liver injury

Background/Aims: Hydrophobic bile acids, such as deoxycholic acid produce cholestatic liver injury. Ursodeoxycholic acid has been shown to be useful in the treatment of cholestatic liver disease. Methods: In this study, we investigated the effects of deoxycholic acid or ursodeoxycholic acid (1% of diet, for 14 days) and their combination (1% each) on expression of hepatic cytochrome P450 isozymes, their related enzyme activities and mRNA level in rats. Results: Adding 1% deoxycholic acid to chow caused a marked increase in serum total bilirubin (47-fold) and total bile acid (8-fold) concentrations and in alkaline phosphatase (2.5-fold, p<0.01) and alanine aminotransferase activities (23.5-fold, p<0.01). Adding the same dose of ursodeoxycholic acid along with the deoxycholic acid mitigated both the rise in serum total bilirubin and bile acid concentrations and that in alkaline phosphatase and alanine aminotransferase activities, although the use of ursodeoxycholic acid alone did not affect any of the above. Feeding 1% deoxycholic acid caused a decrease (48% of control) in total cytochrome P450 content in hepatic microsomes. Addition of 1% ursodeoxycholic acid along with the 1% deoxycholic acid completely prevented the decrease in total cytochrome P450 content. Feeding ursodeoxycholic acid alone did not affect the total cytochrome P450 content. The expression of cytochrome P450 2B1, 2E1, 3A2, 2C6, 2C11 and 4A1 proteins in hepatic microsomes was decreased by deoxycholic acid (44, 51, 23, 59, 30 and 74% of control, respectively). Likewise, the activities of cytochrome P450 2B1 (pentoxyresorufin O-depentylation), 2E1 (aniline p-hydroxylation) and 3A2 (testosterone 6β-hydroxylation) isozymes and the 3A2 mRNA levels in liver were decreased by deoxycholic acid. Addition of 1% ursodeoxycholic acid to 1% deoxycholic acid also prevented the decrease in these cytochrome P450 proteins, related enzyme activities and mRNA levels in liver. Conclusions: These results indicate that, in rats with deoxycholic acid-induced liver injury, ursodeoxycholic acid prevents the decrease in hepatic cytochrome P450 isozymes and suggest that ursodeoxycholic acid is useful for the treatment of liver injury in terms of aiding the normalization of the hepatic drug-metabolizing system.

Ciprofibrate — racemate and enantiomers: Effects of a four-week treatment on male inbred Fischer rats. A biochemical and morphological study

Ciprofibrates (racemate and both enantiomers, Raccip, R- and Scip) were administered orally in doses of 1 and 10 mg/kg once daily over 28 days to male inbred Fischer 344 rats, age 90-110 days at the beginning of the experiment. Body mass gain was observed in all groups. The 1 mg groups showed almost no difference to the control group. The 10 mg groups exhibited less body mass gain, most pronounced in the Scip group. Liver masses were increased in a dose dependent manner up to more than 200%, only the 10 mg Scip group was not significantly different from the 1 mg group which exhibited an increase in liver weight to about 175%. Also the kidney weights increased to 130%, whereas thymus and spleen weights were decreased in the high dose groups. Liver microsomal cytochromes P450 (P450) concentrations were not altered in the 1 mg groups and distinctly lowered in the 10 mg groups. Ethoxyresorufin and ethoxycoumarin O-deethylations were lowered in all experimental groups in a dose dependent manner, after administration of the high doses down to 30% of the control levels or less. Pentoxyresorufin O-depentylation, however, was increased in all 1 mg groups. In the high dose groups it was not altered. Ethylmorphine N-demethylation was decreased after administration of the high doses by about 50%, but only Scip decreased this reaction also after administration of the low dose. NADPH/Fe2+-stimulated microsomal luminol and lucigenin amplified chemiluminescence was increased, whereas hydrogen peroxide formation was depressed even by the low doses to 50% of the normal values, to about 25% by the high doses. Microsomal lipid peroxidation, however, was only slightly or not influenced. Glutathion concentrations (in the reduced and the oxidized form) were increased in a dose dependent manner by about 20 to 30%, the concentration of lipid peroxides was not significantly influenced. Thus, the effects of the enantiomers were not different and were similar to those of the racemate. In serum, cholesterol and triglycerides were only moderately lowered. Albumin concentrations were significantly enhanced in all groups, total proteins after 1 mg/kg Raccip only. Serum bilirubins were not altered, and among the indicator enzymes for liver damage only ALAT, alkaline phosphatase and the dehydrogenases were increased, in no case higher than twofold. Histologically distinct effects were seen after administration of both doses, more pronounced after 10 mg/kg, but with no differences between the enantiomers and Raccip: marked hypertrophy of the hepatocytes, reduced staining of the nuclei, strongly acidophilic granulated cytoplama, no basophilia of the cell bodies, loss of glycogen. These changes were most pronounced around the central veins. Hepatocyte apoptoses also were observed. By immunohistochemistry an increased staining was seen for all P450 isoforms tested (1A1, 2B1, 2E1, 3A2 and 4A1), predominantly perivenously and most pronounced after administration of the high doses without differences between Rcip, Scip or Raccip (preliminary results). By electron microscopy a moderate proliferation of peroxisomes after treatment with 1 mg/kg Cips with a ratio between mitochondria and peroxisomes of about 1:1 (controls: 10:1) was observed, and the peroxisomes were a more heterogeneous population. The relative portions of glycogen and both forms of the ER decreased. Treatment with 10 mg/kg Rcip, Scip or Raccip led to a strong increase in the number of peroxisomes, in some hepatocytes the ratio between mitochondria and peroxisomes was 1:3 with an increased heterogeneity among the peroxisomes evidenced by a broad range of electron densities. Most peroxisomes lacked a nucleoid. Thus, the biochemical effects differed only slightly and the morphological effects of the enantiomers were not different and were similar to those of the racemate.

Effects of the agrochemicals butachlor, pretilachlor and isoprothiolane on rat liver xenobioticmetabolizing enzymes

1. The herbicides butachlor (2-chloro-2′,6′,diethyl-N-[buthoxymethyl] acetanilide) and pretilachlor (2-chloro-2′,6′-diethyl-N-[2-propoxyethyl] acetanilide) are widely used in Asia, South America, Europe and Africa. Isoprothiolane (diisopropyl-1,3-dithiolan-2-ylidenemalonate) is used as a fungicide and an insecticide in rice paddies. We administered these agrochemicals to the male rat and examined their effects on cytochrome P450 (P450), glutathione S-transferase (GST), UDP-glucuronosyltransferase (UDPGT), and NAD(P)H-quinone oxidoreductase 1 (NQO1)-related metabolism in the liver. 2. Administration of isoprothiolane, butachlor or pretilachlor to rat induced hepatic P4502B subfamily-dependent enzyme activities (pentoxyresorufin O-depentylation and testosterone 16 β-hydroxylation) up to 271-413% of control, which coincided with the increase in expression levels of the P4502B apoprotein. 3. Activities of GST toward 1-chloro-2,4-nitrobenzene and 3,4-dichloronitrobenzene were slightly induced (127-133% of control) in the liver of the rat treated with these pesticides. On the other hand, marked elevations of UDPGT activities toward p-nitrophenol (164-281% of control) were observed. NQO1-related metabolism (menadione reductase activity) was also induced (123-176% of control) in the liver of rat treated with these agrochemicals. 4. These results indicate that some of the agrochemicals currently in use are capable of inducing phase I and II xenobiotic-metabolizing enzyme activities in an isozyme selective manner. The induction of these activities may disrupt normal physiologic functions related to these enzymes in exposed animals.

Fractionation of green tea extracts: correlation of antimutagenic effect with flavanol content

The present study was undertaken to evaluate the role of individual flavanols in the antimutagenic potential of green tea. Aqueous extracts of green tea were fractionated into four fractions, each of which was fully defined with respect to its content of (-)-epigallocatechin gallate, (-)-epicatechin gallate, (-)-epigallocatechin, (-)-epicatechin and gallic acid. The ability of each fraction to antagonize the mutagenicity of four model mutagens, namely N-nitrosopyrrolidine, benzo(a)pyrene, 2-aminoanthracene and Glu-P-1 (2-amino-6-methyldipyrido[1,2-a: 3,2-d]imidazole), was investigated in the Ames test. No correlation could be established between any of the flavanols and antimutagenic potential. Similarly, no correlation was evident between the flavanol content of each fraction and its ability to inhibit CYP1A, as exemplified by the O-dealkylations of methoxy- and ethoxy-resorufin. Furthermore, no relationship could be established between CYP2B activity, as exemplified by the O-depentylation of pentoxyresorufin and the antimutagenic potential of green tea. Using a modified Ames test procedure, the ability of each tea fraction to scavenge the metabolically generated reactive intermediates of the model mutagens was investigated, this being an additional mechanism of the antimutagenic potential of green tea. Generally, fractions with high flavanol content were more effective scavengers. It is concluded that the contribution of flavanols to the antimutagenic activity of green tea is, at best, limited. ©1997 SCI

Xenobiotic Metabolism by Rat Thymus: Induction of Thymic P450 Enzymes

We investigated the drug-metabolizing capacity of the thymus, and compared it with that of the liver. An attempt was also made to characterize the activity of different thymic P450 enzymes. Post-mitochondrial and microsomal fractions of thymus were prepared from female rats. The microsomal enzyme inducers were pregnenolone-16α-carbonitrile (PCN), phenobarbital and acetone. The aromatic hydroxylation of zoxazolamine and p-nitrophenol, N-demethylation of ethylmorphine and erythromycin, and O-depentylation of pentoxyresorufin were assayed. It was found that thymic tissue could affect drug biotransformations. PCN, phenobarbital and acetone induced metabolism of both erythromycin and p-nitrophenol in liver and thymus. However, liver resorufin depentylase was induced only by phenobarbital; no activity was detected in thymus. The thymus can metabolize xenobiotics, although to a lesser extent than the liver. Classic microsomal inducers increased the drug-metabolizing activity of the thymus, and CYP3A1 and CYP2E1 activity was identified.

Interaction of chlormezanone with rat liver microsomes and its degradation.

Chlormezanone binds to oxidized cytochrome P450 in rat liver microsomes with a binding curve according to type I like hexobarbital but less pronounced and with a general shift to the left. Ethylmorphine N-demethylation, ethoxycoumarin and ethoxyresorufin O-deethylation are inhibited by chlormezanone in mM concentrations only whereas pentoxyresorufin O-depentylation is inhibited by about 50% in microM concentrations. Luminol and lucigenin amplified chemiluminescence indicating the formation of reactive oxygen species was not influenced in concentration ranges between mM and microM, whereas NADPH/Fe stimulated lipid peroxidation showed a tendency of inhibition. But scavenger activity could not be demonstrated: the zymosan stimulated chemiluminescence of whole blood was not influenced significantly. The degradation process of chlormezanone was elucidated. The first step involves ring opening by chemical hydrolysis with subsequent formation of an unstable acylhalfaminal which is the source of 4-chlorobenzaldehyde. This aldehyde undergoes enzymatically controlled oxidation to 4-chlorobenzoic acid which is the parent compound of following phase II reactions. The second degradation product is 2-carboxyethane-sulfinic-acid-N-methylamide, which is hydrolyzed very quickly. Neither oxidation of the sulfinic acid or its N-methylamide derivative could be observed nor N-demethylation of chlormezanone.

QUANTITATIVE PREDICTION OF PHENOBARBITAL INDUCTION OF CYP2B IN VIVO FROM CULTURED HEPATOCYTES STUDIES

In vitro-in vivo scaling works well for metabolic enzyme inhibition, but is less successful with enzyme induction. In this paper, we are attempt to establish the prediction method for enzyme induction in vivo from cultured hepatocytes studies. Phenobarbital (PB) was used as a model inducer for CYP2B and the enzyme activities were assessed by pentoxyresorufin O-depentylation (PROD). In vivo induction was determined in liver microsomes isolated from rats after administration of PB. In vitro induction was assessed in cultured rat hepatocytes treated with PB. To compare in vitro induction with in vivo induction, plasma unbound concentration of PB has been used as a method of approximating unbound hepatocellular PB concentration in rats. Maximum induction (6 fold) and EC50 values (15-20 μM) after single administration of PB were well agreed between in vitro and in vivo studies. Similar results were obtained in repeated administration of PB. In conclusion, although there still remain some problems for quantitative prediction of the enzyme induction, we showed the possibility and effectiveness of the prediction of inducibility in vivo from cultured hepatocytes.