Benzyloxyresorufin O-dealkylase Research Articles

Selective induction of hepatic cytochrome P450 2B activity by leelamine in vivo, as a potent novel inducer.

Cytochrome P450 (CYP) is an important enzyme that can act on xenobiotic substances such as toxic chemicals or drugs. Phenobarbital (PB) has been widely used to induce CYP2B activity to investigate the drug-drug interaction of CYP2B substrate drugs. Leelamine is a diterpene compound, and is the current focus of efforts to develop a treatment for diabetes. In this study, we identified the selective and potent inductive effect of leelamine on CYP2B at doses of 5, 10, or 20mg/kg in male ICR mice for 1 or 3days. In liver, the activity of CYP2B significantly increased 3.6-fold after treatment with leelamine, compared to vehicle-treated group. Activities of benzyloxyresorufin O-dealkylase and pentoxyresorufin O-dealkylase significantly increased 6.3- and 5.3-fold, respectively, with a single treatment of 20mg/kg leelamine for 1day. Furthermore, immunoblot analysis showed that significantly and dose-dependently increased CYP2B10 protein levels in liver. However, PCR results showed that there were no significant changes in the CAR and CYP2B mRNA levels after leelamine treatment. Accordingly, we suggest that leelamine is a novel substitute of PB for the selective induction of CYP2B activity in vivo.

Bimodal action of miroestrol and deoxymiroestrol, phytoestrogens from Pueraria candollei var. mirifica, on hepatic CYP2B9 and CYP1A2 expressions and antilipid peroxidation in mice

Miroestrol and deoxymiroestrol are phytoestrogens isolated from Pueraria candollei var. mirifica. The influence of miroestrol and dexoymirosestrol on hepatic cytochrome P450 (P450) enzymes and antioxidative activity in brain was examined in C57BL/6 mice compared with that of a synthetic female sex hormone estradiol. We hypothesized that miroestrol and deoxymiroestrol would induce CYP2B9 expression, whereas CYP1A2 expression would be suppressed compared with estradiol. Miroestrol and deoxymiroestrol treatment significantly increased uterus weight and volume. In addition, both of these phytoestrogens induced the expression of CYP2B9 and suppressed the expression of CYP1A2, as expected. Hepatic P450 activities correspondingly showed that both compounds increased benzyloxyresorufin O-dealkylase activity, whereas methoxyresorufin O-dealkylase activity was reduced. These observations suggested that miroestrol and deoxymiroestrol might affect hepatic P450 enzymes, including the CYP2B9 and CYP1A2 P450 isoforms. Assessment of lipid peroxidation demonstrated that miroestrol and deoxymiroestrol markedly decreased levels of malondialdehyde formation in the mouse brain. This is the first report suggesting miroestrol and deoxymiroestrol as potential alternative medicines to estradiol because of their distinctive ability to regulate mouse hepatic P450 expression and their beneficial antioxidative activities.

Evaluation of the genotoxicity of Orthosiphon stamineus aqueous extract

Orthosiphon stamineus, Benth, also known as Misai Kucing in Malaysia and Java tea in Indonesia, is traditionally used in Southeastern Asia to treat kidney dysfunctions, diabetes, gout and several other illnesses. Recent studies of Orthosiphon stamineus pharmacological profile have revealed antioxidant properties and other potentially useful biological activities thereby lending some scientific support to its use in folk medicine. So far the genotoxicity of Orthosiphon stamineus extracts has not been evaluated. In this study the genotoxic potential of Orthosiphon stamineus aqueous extract was investigated by the Salmonella/microsome mutation assay and the mouse bone marrow micronucleus test. Chemical composition of Orthosiphon stamineus aqueous extract was analyzed by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD). The Salmonella/microsome assay (TA97a, TA98, TA100 and TA1535; plate incorporation method) was performed in the presence or in the absence of extrinsic metabolic activation (S9 mixture). In the mouse micronucleus assay, Orthosiphon stamineus extract was administered by gavage (0, 500, 2000 and 4000 mg/kg body weight/day for 3 days) to male and female Swiss Webster mice (N=6 per dose per sex) and bone marrow cells were harvested 24 h after the last dose. Ethoxy-resorufin-O-dealkylase (EROD) and benzyloxy-resorufin-O-dealkylase (BROD) activities were determined in mouse liver microsomes. The chemical analysis revealed that the Orthosiphon stamineus extract contained flavonoids (sinensetin, eupatorin), caffeic acid, and rosmarinic acid (44.00±1.879 μg/mg), the latter seemed to be one of its major constituent. Tested at doses up to 5000 μg/plate, the Orthosiphon stamineus extract was not toxic to Salmonella tester strains and did not increase the number of revertant colonies over the background incidence. In the mouse bone marrow assay, the extract did not alter the polychromatic:normochromatic erythrocytes (PCE:NCE) ratio, nor did it increase the incidence of micronucleated polychromatic erythrocytes (MNPEs). No overt toxicity and no change of CYP1A (EROD) and 2B9/10 (BROD) activities were noted. Based on the aforementioned findings, it is concluded that the use of Orthosiphon stamineus in the traditional medicine poses no genotoxic risk.

Hepatic CYP1A Induction by Chlorinated Dioxins and Related Compounds in the Endangered Black-Footed Albatross from the North Pacific

The present study assesses effects of dioxins and related compounds (DRCs) including polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and dioxin-like polychlorinated biphenyls (DL-PCBs) on cytochrome P450 1A (CYP1A) expression level in liver of black-footed albatrosses (Phoebastria nigripes) collected from the North Pacific. Total 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-T(4)CDD) toxic equivalents (TEQs) derived from toxic equivalency factor for birds proposed by World Health Organization were in the range of 2100 to 10 000 pg/g lipid wt (120-570 pg/g wet wt). Simultaneously, microsomal alkoxyresorufin O-dealkylase (AROD) activities, including methoxy-, ethoxy-, pentoxy-, and benzyloxy-resorufin O-dealkylase activities were also measured in the same specimens. Total TEQs and TEQ (on wet wt basis) from some individual DRC congeners had significant positive correlations with AROD activities, suggesting induction of CYP1A by DRCs. Congeners like 2,3,7,8-T(4)CDD and most of the DL-PCBs that showed no significant positive correlations between the concentrations and AROD activities, exhibited significant negative correlations between AROD activities and the concentration ratio of the congener to a recalcitrant CB169, suggesting preferential metabolism of these congeners by induced CYP1A. As far as we know, this is the first direct evidence revealing that hepatic CYP1A level is elevated with the accumulation of DRCs in the wild black-footed albatross population. The present study gives more robust estimate of impacts of DRCs on CYP1A induction in this rare pelagic species than indexes like hazard quotient and TEQ-threshold comparison that have been so far carried out.

Impact of Pueraria candollei Root Cultures on Cytochrome P450 2B9 Enzyme and Lipid Peroxidation in Mice

Pueraria candollei (Leguminosae) has long been used as a traditional medicine for rejuvenation, and the major constituents in the tuberous root are isoflavonoids. The effects of the root cultures of P. candollei (r-PC) on cytochrome P450 enzymes in mouse liver and antioxidant activity in mouse brain including gross anatomy were examined. r-PC significantly enlarged the uterus length while no change in weight was noted. The total P450 content was not significantly altered by either estradiol benzoate (ES) or r-PC. Benzyloxyresorufin O-dealkylase activity was significantly increased by r-PC, comparable to the increase induced by ES. Correspondingly, r-PC demonstrated a tendency to upregulate CYP2B9 mRNA expression. Furthermore, assessment of lipid peroxidation in mouse brain using the thiobarbituric acid assay demonstrated that r-PC remarkably decreased the level of malondealdehyde formation. These observations suggest r-PC as a potential alternative to ES with the extrabenefit of its antioxidant activity.

Alkoxyresorufin O-dealkylase assay using a rat hepatocyte spheroid microarray

Hepatocyte multicellular aggregates (spheroids), which maintain high expression of liver functions, have been advocated as a useful culture technique for various cell-based assays. In this study, we investigated the drug metabolic function of a hepatocyte spheroid microarray (HSM) chip, which contained an array of 672 spheroids of primary rat hepatocytes within a 100-mm(2) region in the center of a poly(methylmethacrylate) plate (24 x 24 mm) and used an alkoxyresorufin (ethoxy-, methoxy-, pentoxy- and benzyloxyresorufin) O-dealkylase assay system. Ethoxyresorufin O-dealkylase (EROD) activity of the HSM chip initiated by 3-methylcholanthrene (3-MC), an inducer of cytochrome P450 enzymes, was 5- to 10-fold higher than that of monolayer hepatocytes, with activity being maintained for at least 2 weeks. We also demonstrated that 3-MC induced EROD, methoxyresorufin O-dealkylase (MROD) and benzyloxyresorufin O-dealkylase (BROD) activities in the HSM chip, while sodium phenobarbital (P450 inducer) induced pentoxyresorufin O-dealkylase (PROD), BROD, EROD and MROD activities. Induction of these activities was confirmed by increased gene expression of the related P450 enzymes. These results showed that the HSM chip had a good response to P450 inducers and that function was maintained for long periods of time. The HSM chip therefore may be a promising cellular platform for drug metabolic assays using hepatocytes.

Differential inhibition of hepatic microsomal alkoxyresorufin O-dealkylation activities by tetrachlorobiphenyls

Polychlorinated biphenyls (PCBs) elicit a spectrum of biochemical and toxic effects in exposed animals. In the present study, we assessed the effect of PCB structure, using four symmetrically-substituted PCBs, on cytochrome P450 (CYP)-mediated methoxy-, ethoxy- and benzyloxyresorufin O-dealkylase (MROD, EROD and BROD, respectively) activities. We found that 2,2′,4,4′-tetrachlorobiphenyl (PCB 47), 2,2′,5,5′-tetrachlorobiphenyl (PCB 52), 2,2′,6,6′-tetrachlorobiphenyl (PCB 54) and 3,3′,4,4′-tetrachlorobiphenyl (PCB 77) inhibited alkoxyresorufin O-dealkylase activities in hepatic microsomes from 3-methylcholanthrene (MC) or phenobarbital (PB)-treated rats. Measurement of the in vitro inhibitory potencies of the tetrachlorobiphenyls revealed that MROD, EROD and BROD activities were differentially inhibited and the degree of inhibition was determined by the chlorination pattern of the PCB. PCB 77 was more potent than PCB 47 or PCB 52 at inhibiting MROD and EROD activities in hepatic microsomes from MC-treated rats, while no inhibition of either activity was observed with PCB 54. In contrast, BROD activity measured in hepatic microsomes from PB-treated rats was inhibited by PCB 47, PCB 52 and PCB 54 but not by PCB 77. The mode of inhibition for each activity was also evaluated statistically. Inhibition of the alkoxyresorufin O-dealkylase activities could not be discerned in hepatic microsomes from corn oil-treated rats because the activities were inherently too low. No evidence for mechanism-based inhibition of MROD, EROD or BROD activities or an effect via CYP reductase was found. The results demonstrate that relatively coplanar PCBs such as PCB 77 preferentially inhibit EROD and MROD activities, whereas noncoplanar PCBs such as PCB 54 preferentially inhibit BROD activity.

The Effect of Aspartame on Rat Brain Xenobiotic-Metabolizing Enzymes

This study demonstrates that chronic aspartame (ASP) consumption leads to an increase of phase I metabolizing enzymes (cytochrome P450 (CYP)) in rat brain. Wistar rats were treated by gavage with ASP at daily doses of 75 and 125 mg/kg body weight for 30 days. Cerebrum and cerebellum were used to obtain microsomal fractions to analyse activity and protein levels of seven cytochrome P450 enzymes. Increases in activity were consistently found with the 75 mg/kg dose both in cerebrum and cerebellum for all seven enzymes, although not at the same levels: CYP 2E1-associated 4-nitrophenol hydroxylase (4-NPH) activity was increased 1.5-fold in cerebrum and 25-fold in cerebellum; likewise, CYP2B1-associated penthoxyresorufin O-dealkylase (PROD) activity increased 2.9- and 1.7-fold respectively, CYP2B2-associated benzyloxyresorufin O-dealkylase (BROD) 4.5- and 1.1-fold, CYP3A-associated erythromycin N-demethylase (END) 1.4- and 3.3-fold, CYP1A1-associated ethoxyresorufin O-deethylase (EROD) 5.5- and 2.8-fold, and CYP1A2-associated methoxyresorufin O-demethylase (MROD) 3.7- and 1.3-fold. Furthermore, the pattern of induction of CYP immunoreactive proteins by ASP paralleled that of 4-NHP-, PROD-, BROD-, END-, EROD- and MROD-related activities only in the cerebellum. Conversely, no differences in CYP concentration and activity were detected in hepatic microsomes of treated animals with respect to the controls, suggesting a brain-specific response to ASP treatment.

Selective Enhancement of Cytochrome P-450 Activity in Rat Hepatocytes by in Vitro Heat Shock

We investigated the effect of heat shock on cytochrome P-450 activity in rat hepatocytes and report a significant, selective, and time-dependent enhancement of cytochrome P-450 activity in heatshocked hepatocytes. Stable long-term cultures of rat hepatocytes were heat shocked (42.5 degrees C) for 1 to 3 h and allowed to recover at 37 degrees C. Cytochrome P-450-dependent ethoxyresorufin O-dealkylase (EROD) and benzyloxyresorufin O-dealkylase (BROD) activities were measured up to 48 h after heat shock treatment. In general, the optimal heat shock exposure time was between 2 and 3 h. BROD activity (induced by sodium phenobarbital) increased approximately 6-fold in hepatocytes heat shocked for 3 h in comparison with hepatocytes maintained at 37 degrees C. EROD activity (induced by 3-methylcholanthrene) increased 2-fold on exposure to heat shock for 2 h. The expression of inducible heat shock proteins Hsp70 and Hsp32 was verified by Western immunoblot analyses. In the absence of the appropriate inducer, heat shock treatment did not enhance cytochrome P-450 activity. Furthermore, enhanced P-450 enzyme activity was delayed for heat-shocked hepatocytes. It is hypothesized that heat shock treatment attenuates the negative effects triggered by the addition of the toxic inducers and possibly stabilizes the levels of cytochrome P-450 proteins. These results suggest that heat shock treatment may be used to enhance the functionality of hepatocytes, specifically, in bioartificial liver assist devices.

Characterization of enzymes responsible for biotransformation of the new antileukotrienic drug quinlukast in rat liver microsomes and in primary cultures of rat hepatocytes.

The promising new drug quinlukast, 4-(4-(quinoline-2'-yl-methoxy)phenylsulphanyl)benzoic acid (VUFB 19363), is under investigation for its anti-inflammatory and anti-asthmatic effects. The main metabolite of quinlukast identified in incubations of rat microsomal fraction, and in primary culture of rat hepatocytes, is quinlukast sulfoxide (M2). Also, several other metabolites of quinlukast were found: two dihydrodiol derivatives (M3, M5) and quinlukast sulfone (M4). This study was conducted to characterize the enzymes involved in quinlukast biotransformation in rat in-vitro. Primary cultures of rat hepatocytes were treated with inducers of different cytochrome P450s (CYPs) for 48 h. Quinlukast (100 microM) was incubated for 24 h in a primary culture of induced or control hepatocytes. The effects of CYP inhibitors, ketoconazole, methylpyrazole, metyrapone and alpha-naphthoflavone (2, 10, 50 microM), on quinlukast metabolism were tested in induced and control hepatocytes. Significant induction of M2 (6 times), M5 (twice) and M3 (by 50%) formation by dexamethasone and strong concentration-dependent inhibition by ketoconazole indicated that CYP3A participates in formation of these metabolites. CYP1A catalyses formation of metabolite M3 mainly, as beta-naphthoflavone induced (10 times) production of M3 and a strong inhibitory effect of alpha-naphthoflavone on its formation was observed. A significant inhibitory effect of quinlukast (2, 10, 50 microM) on ethoxyresorufin, methoxyresorufin and benzyloxyresorufin O-dealkylase activity was observed as well.

Effects of dibromoacetonitrile on rats following 13-week drinking water exposure

The subchronic toxicity of dibromoacetonitrile (DBAN), a disinfection by-product in drinking water, was studied in the rat. Male (180±18 g) and female (152±9 g) Sprague–Dawley rats (10 animals per group) were fed DBAN in organic-free distilled water at concentrations of 0.1, 1, 10 and 100 ppm for 13 weeks. Control rats received organic-free distilled water only. Water intakes in the highest dose males and females were reduced by 25 and 32% as compared to the controls, respectively ( P<0.05), with no significant reductions in food consumption and body weight gain. The organ to body weight ratio was significantly increased in the highest-dose males and females for kidneys but not for the brain, liver, spleen, thymus and testicles. In the males, decreases were detected in serum uric acid levels at 1 and 100 ppm, and in urinary uric acid at 10 and 100 ppm. Decreased serum protein was detected in the highest-dose males and decreased serum LDH was found in the highest-dose females. Both the white blood cell and lymphocyte counts were significantly elevated in the highest-dose females. A significant increase in hepatic catalase activity was observed only in males starting at 1 ppm, and increased palmitoyl-CoA oxidase (PCO) activity was found in males and females of the highest dose group. In the males, decreased thiobarbituric acid reactive substance (TBARS) level was detected in the liver at 1.0 and 100 ppm groups, while increased TBARS was found in the serum at 100 ppm DBAN. No treatment-related changes were detected in the activities of hepatic benzyloxyresorufin O-dealkylase (BROD), pentoxyresorufin O-dealkylase (PROD) and ethoxresorufin O-deethylase (EROD), and in hepatic UDP-glucuronosyltransferase (UDPGT) and glutathione S-transferases (GST). Although DBAN is a potent inhibitor of hepatic aldehyde dehydrogenase (ALDH) and GST in vitro, there was no evidence of suppression of these enzymes in the treated animals. Mild histological changes were detected in animals receiving the highest dose, consisting of collapsed angularity, increased epithelial height in the thyroid of both sexes, and cytoplasmic vacuolation and nuclear vesiculation in the thyroid of females, increased myeloid to erythroid ratio in the bone marrow of both sexes, and cytoplasmic inclusions in the proximal tubules of male kidneys. In summary, treatment effects occurred predominantly at 100 ppm and included in both sexes: increased kidney weights, histological changes in the thyroid and bone marrow, and increased peroxisomal enzyme activities; and in males: decreased serum and urinary uric acid levels, and indication of oxidative stress. The no-observed-adverse-effect level (NOAEL) was therefore judged to be 10 ppm, equivalent to 1.11 and 1.21 mg/kg/day in the males and females, respectively.

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.

Biochemical effects of chloral hydrate on male rats following 7-day drinking water exposure.

The biochemical and toxicological effects of chloral hydrate were investigated. Four groups (n = 7 per group) of male Sprague-Dawley rats (161-170 g) were administered chloral hydrate in drinking water at concentrations of 20, 200 or 2000 ppm for 7 days. The control group received phosphate-buffered water only. There were no treatment-related changes in the body weight gains, relative weights of major organs or haematological parameters. Trichloroacetic acid was significantly (P < 0.05) elevated in the serum of high-dose animals (7.75 +/- 5.14 mg dl(-1), mean +/- SD). In the high-dose animals there was a 36% increase in protein level in the liver homogenates but not in the corresponding 9000 g supernatants. Concurrently, there was a threefold increase in the activity of the hepatic peroxisomal enzyme palmitoyl CoA oxidase (PCO). A prominent change was the dose-related suppression in hepatic aldehyde dehydrogenase (ALDH) activity observed in all treatment groups, with the decrease ranging from 15% at 20 ppm to 68% at 2000 ppm. There were no significant decreases in the activity of hepatic enzymes ethoxyresorufin O-deethylase (EROD), benzyloxyresorufin O-dealkylase (BROD) and UDP-glucuronosyl-transferase (UDPGT). In the high-dose group there was a 30% increase in hepatic glutathione-S transferase (GST) activity, accompanied by a 13% increase in glutathione (GSH). Significant effects on lipids were observed in the liver of the high-dose animals, with a 15% decrease in hepatic cholesterol and triglyceride levels. There were no treatment-related changes in serum chemistry parameters, including cholesterol and triglyceride levels. Although in vitro assays showed chloral hydrate to be an inhibitor of serum pseudocholinesterase activity, with a 50% inhibition concentration (ic(50)( of approximately 0.7 mM at 5 mM butyrylthiocholine, no decrease in serum pseudocholinesterase activity was found in the treated animals. It was concluded that the liver is the target organ for chloral hydrate, with suppression of ALDH as the most sensitive endpoint followed by alteration in the GSH level and GST activity. Changes observed in the high-dose animals, such as increased peroxisomal PCO activity in the liver and perturbation of lipid homeostasis in the liver and blood, were likely to be associated with trichloracetic acid, the major metabolite of chloral hydrate.

Xenobiotic metabolism by cultured primary porcine hepatocytes.

Considering the large yield of viable cells comparable to human liver, primary porcine hepatocytes offer a valuable resource for constructing a bioartificial liver device. In this study, the ability of cultured primary porcine hepatocytes to detoxify xenobiotics has been examined using various known substrates of cytochrome P450 isoenzymes and UDP-glucuronosyltransferases. Present investigation demonstrated the stability of the isoenzymes responsible for the metabolism of diazepam in native state and stabilization of other isoenzymes, as judged by ethoxycoumarin o-dealkylase (ECOD), ethoxyresorufin o-dealkylase (EROD), benzyloxyresorufin o-dealkylase (BROD), and pentoxyresorufin o-dealkylase (PROD) activities following induction in culture environment, for a period of 8 days. Resorufin O-dealkylase activities were found to be the most unstable and deteriorated within first 5 days in culture. These activities were restored following induction with 3-methylcholanthrene (3-MC) or sodium phenobarbital (PB) to 20-fold of 1 activity for EROD, and 60 and 174% of day 1 activity for PROD and BROD on day 8, respectively. Metabolism of methoxyresorufin was most strikingly increased following induction with 3-MC to approximately 60-fold of day 1 activity, on day 8. UDP-glucuronosyltransferase-dependent glucuronidation of phenol red, however, stayed intact during the course of our study without induction. Our study indicated that porcine hepatocytes in vitro maintain many important liver-specific functions including detoxification (steady state and inducibility).

Dose-response effects of lycopene on selected drug-metabolizing and antioxidant enzymes in the rat

The administration of lycopene to female rats at doses ranging from 0.001 to 0.1 g/kg b.w. per day for 2 weeks was found to alter the drug-metabolizing capacity and antioxidant status of the exposed animals. An investigation of four cytochrome P450-dependent enzymes revealed that benzyloxyresorufin O-dealkylase activity in the liver was significantly induced in a dose-dependent fashion at all lycopene doses investigated. Likewise, ethoxyresorufin O-dealkylase activity was induced, although only at the two highest lycopene concentrations tested. An investigation of selected phase 2 detoxification enzymes provided evidence that lycopene was capable of inducing hepatic quinone reductase, approximately two-fold, at doses between 0.001 and 0.05 g/kg b.w. per day, whereas no effect was observed at the remaining doses tested. Glutathione transferase, using the two substrates, 2,4-dichloronitrobenzene and 1-chloro-2,4-dinitrobenzene, was significantly induced at the 0.1 g/kg b.w. per day dose, whereas no effect was observed at the remaining lycopene doses. Analysis of the antioxidant status of the blood compartment revealed that three out of four antioxidant enzymes were affected by lycopene treatment. The activity of superoxide dismutase was thus significantly induced at lycopene doses of 0.005 and 0.05 g/kg b.w, whereas glutathione reductase and glutathione peroxidase was only induced at the 0.005 g/kg b.w. per day dose. For all antioxidant enzymes investigated, the activities seemed to return to the control level after exerting peak induction at doses between 0.005 and 0.05 g/kg b.w. per day. The explanation for this remains unknown. The plasma concentration of lycopene at dietary levels of 0.001, 0.005, 0.05 and 0.1 g/kg b.w. per day was estimated to be 16, 32, 71 and 67 nM, which is barely within the lower range of the mean human plasma concentration of lycopene, which ranges from 70–1790 nM. Oxidative stress induced by the heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo [4,5– b]pyridine (PhIP), and investigated by analyzing for malondialdehyde in plasma, was not found to be affected by prior lycopene exposure. The level of PhIP–DNA adducts in the liver or colon was likewise not affected by lycopene at any dose. Overall, the present study provides evidence that lycopene administered in the diet of young female rats exerts minor modifying effects toward antioxidant and drug-metabolizing enzymes involved in the protection against oxidative stress and cancer. The fact that these enzymatic activities are induced at all of these very low plasma levels, could be taken to suggest that modulation of antioxidant and drug-metabolizing enzymes may indeed be relevant to humans, which in general exhibit a plasma lycopene level several fold above the effective levels observed in this study.

Induction of Hepatic CYP2B Is a More Sensitive Indicator of Exposure to Aroclor 1260 than CYP1A in Male Rats

To evaluate the effect of exposure to an environmentally relevant polychlorinated biphenyl mixture, adult male rats were treated with Aroclor 1260 for 7 days and levels of several cytochrome P450 (CYP) enzymes were measured in liver microsomes prepared 3 days after the last dose. Treatment with Aroclor 1260 at dosages ranging from 0.5 to 50 mg/kg/day had no effect on body weight, but liver weight was increased significantly in rats treated with the two highest dosages. Of the monooxygenase activities examined, benzyloxyresorufin O-dealkylase and testosterone 16β-hydroxylase activities were increased to the greatest extent with maximal induction of both activities reached at 5 mg/kg/day. Densitometric quantitation of blots probed with antibody against CYP2B revealed that CYP2B1 and CYP2B2 protein levels were increased approximately 55-fold and 16-fold, respectively, after treatment with Aroclor 1260 at 5 mg/kg/day. Ethoxyresorufin O-deethylase activity and CYP1A1 protein levels displayed linear dose-dependent increases, but the hepatic CYP1A1 content did not exceed 10% that of CYP2B1 at all dosages of Aroclor 1260. Microsomal CYP3A- and CYP2A1-mediated enzyme activities and protein levels were also increased by treatment with Aroclor 1260 but to a lesser extent, whereas CYP2C11-mediated enzyme activities and protein levels were reduced. A separate time-course study showed that induction of CYP2B, but not of CYP1A, enzymes persisted for at least 48 days after treatment with Aroclor 1260 at 10 mg/kg/day. In summary, the results indicate that induction of CYP2B enzymes is a more sensitive biomarker of exposure to Aroclor 1260 than CYP1A.

Ornithine alpha-ketoglutarate counteracts the decrease of liver cytochrome P-450 content in burned rats.

The effect of ornithine alpha-ketoglutarate (OKG) on cytochrome P-450 enzyme activities was studied in a well-defined model of injury (burn followed by fasting then subsequent hypocaloric diet) administered to young rats for 3 d. Hepatic microsomes were prepared by ultracentrifugation and levels of cytochromes P-450 were determined spectrophotometrically. The activities of ethoxy-resorufin-O-deethylase (EROD), benzyloxy-resorufin-O-dealkylase (BROD), and erythromycin demethylase were measured as markers of P-450 1A, 2A, and 3A isotypes respectively. The level of total hepatic microsomal proteins (8 mg/mL) remained constant. The level of cytochrome P-450 (1.14+/-0.08 nmol/mg microsomal proteins) was decreased by a hypocaloric diet (23%, P = 0.003) and burn further enhanced this phenomenon (15%, P = 0.03). Both healthy and burned rats receiving OKG showed the same level of cytochrome P-450 as the rats fed ad libitum. OKG supplementation counteracted the enhancement (40%) of EROD activity induced by hypocaloric diet but did not influence BROD and erythromycin demethylase activities. OKG sustained cytochrome P-450 levels in rats fed a hypocaloric diet, even after burning. These findings indicate that OKG may favor drug metabolism in this injured population.

Glucose consumption by rats decreases cytochrome P450 enzyme activity by altering hepatic lipids

Although glucose is a ubiquitous nutrient, increased consumption of glucose decreases the metabolism of numerous drugs in humans and animals. To understand the mechanisms involved that cause decreased drug metabolism in rats that consume glucose in their water, enzyme activity and expression as well as determining the contribution of the lipids toward decreasing in vitro metabolic activity were investigated. Enzyme assays of hepatic CYP1A2, 2C6, 2C11 and 3A2 showed significant decreases in activity from glucose-treated rats compared to control. While immunodetection of CYP1A1, 2B1/2, 2C11, and 3A1/2 showed no significant difference in protein expression. Hepatic fatty acid synthase activity increased in the glucose-treated rats compared to controls. Studies with glucose-treated microsomal lipids reconstituted with microsomal proteins from control rats caused a significant decrease in benzyloxyresorufin O-dealkylase activity. The results presented here support the hypothesis that the activities of cytochrome P450 proteins are altered by modulating their catalytic activity as a result of the lipid environment rather than changing the level of expression of the individual enzymes.

Cytochrome P450 (P450) isoenzyme specific dealkylation of alkoxyresorufins in rat brain microsomes.

Characterization of xenobiotic metabolizing cytochrome P450s (P450s) was carried out in rat brain microsomes using the specific substrates, 7-pentoxy- and 7-ethoxyresorufin (PR and ER), metabolized in the liver by P450 2B1/2B2 and 1A1/1A2 respectively and 7-benzyloxyresorufin (BR), a substrate for both the isoenzymes. Brain microsomes catalysed the O-dealkylation of PR, BR and ER in the presence of NADPH. The ability to dealkylate alkoxyresorufins varied in different regions of the brain. Microsomes from the olfactory lobes exhibited maximum pentoxyresorufin-O-dealkylase (PROD), benzyloxyresorufin-O-dealkylase (BROD) and ethoxyresorufin-O-dealkylase (EROD) activities. The dealkylation was found to be inducer selective. While pretreatment with phenobarbital (PB; 80 mg/kg; i.p. x 5 days) resulted in significant induction in PROD (3-4 fold) and BROD (4-5 fold) activities, 3-methylcholanthrene (MC; 30 mg/kg; i.p. x 5 days) had no effect on the activity of PROD and only a slight effect on that of BROD (1.4 fold). MC pretreatment significantly induced the activity of EROD (3 fold) while PB had no effect on it. Kinetic studies have shown that this increase in the activities following pretreatment with P450 inducers was associated with a significant increase in the velocity of the reaction (Vmax) of O-dealkylation. In vitro studies using organic inhibitors and antibodies have further provided evidence that the O-dealkylation of alkoxyresorufins is isoenzyme specific. While in vitro addition of alpha-naphthoflavone (ANF), an inhibitor of P450 1A1/1A2 catalysed reactions and antibody for hepatic P450 1A1/1A2 isoenzymes produced a concentration-dependent inhibition of EROD activity, metyrapone, an inhibitor of P450 2B1/2B2 and antibody for hepatic P450 2B1/2B2 significantly inhibited the activity of PROD and BROD in vitro. The data suggest that, as in the case of liver, dealkylation of alkoxyresorufins can be used as a biochemical tool to characterise the xenobiotic metabolising P450s and substrate selectivity of P450 isoenzymes in rat brain microsomes.

A comparison of basal and induced hepatic microsomal cytochrome P450 monooxygenase activities in the cynomolgus monkey (Macaca fascicularis) and man

1. The specific activities of hepatic microsomal cortisol 6β-hydroxylase, coumarin 7-hydroxylase, S-mephenytoin 4'-hydroxylase and phenoxazone hydroxylase and the O-dealkylations of seven homologous alkoxyresorufins were < 3-fold different between the untreated (UT) cynomolgus monkey and man. 2. Heptoxy- and octoxyresorufin O-dealkylase, S-mephenytoin N-demethylase and dextromethorphan O-demethylase specific activities were > 6-fold higher, whereas tolbutamide hydroxylase was almost 5-fold lower in the UT monkey than in man. 3. Phenobarbitone induced (2-6-fold) coumarin 7-hydroxylase, cortisol 6β-hydroxylase, S-mephenytoin N-demethylase, phenoxazone hydroxylase and benzyloxyresorufin O-dealkylase activities, but not the O-dealkylations of pentoxyresorufin or other alkoxyresorufins, in monkey. 4. Rifampicin induced (2-3-fold) cortisol 6β-hydroxylase, S-mephenytoin 4′-hydroxylase, S-mephenytoin N-demethylase and tolbutamide hydroxylase activities, the O-dealkylations of methoxy-, ethoxy- and propoxyresorufin and CYP2C- and CYP3A- immunorelated proteins in monkey. 5. Dextromethorphan O-demethylase was significantly reduced by both phenobarbitone and rifampicin treatment in monkey. 6. β-Naphthoflavone induced (8-39-fold) the O-dealkylations of several alkoxyresorufins, the greatest effect being on propoxyresorufin, but had no effect on the other activities measured in monkey. 7. Constitutive hepatic microsomal CYP2D6-immunorelated proteins were expressed at apparently much higher levels in monkey than in man.