Methoxyresorufin O-demethylase Research Articles

Effects of acaricides on the activities of monooxygenases in bovine liver microsomes.

Organophosphates (OPs), pyrethrins and fipronil, are acaricides commonly used in cattle, mainly as pour on formulations. Scant information is available on their potential interactions with hepatic xenobiotic metabolizing enzymes. This work aimed to evaluate in vitro the potential inhibitory effects of widely employed acaricides on catalytic activities mediated by hepatic cytochrome P450 (CYP) and flavin-monooxygenase (FMO) enzymes in cattle. Bovine (n = 4) liver microsomes were incubated in the absence (control assays) and in presence of different OPs (fenthion, chlorpyrifos, ethion, diazinon and dichlorvos), fipronil and cypermethrin at 0.1-100 μm. Five oxidative enzyme activities were assayed by spectrofluorimetric or HPLC methods: 7-ethoxyresorufin O-deethylase (for CYP1A1), methoxyresorufin O-demethylase (for CYP1A2), benzyloxyresorufin O-debenzylase (for CYP2B), testosterone 6-beta hydroxylase (for CYP3A) and benzydamine N-oxidase (for FMO). All acaricides, particularly phosphorothionate-containing OPs, inhibited to some extent more than one enzyme activity. The most frequent inhibitor was fenthion, which inhibited (p < .05) all enzyme activities tested (from 22% at 1 μm to 72% at 100 μm). However, low inhibitory potencies (IC50s higher than 7 μm) of all acaricides studied were observed against the catalytic activities assayed. Therefore, the risk of in vivo metabolic interactions due to inhibition of monooxygenases would be low under common husbandry conditions.

Olive leaf (Olea europaea L. folium) extract influences liver microsomal detoxifying enzymes in rats orally exposed to 2-amino-l-methyI-6-phenyI-imidazo pyridine (PhIP).

Olive tree (Olea europaea, Oleaceae) leaf extract (OLE) exerts many biological activities. One of the most common polycyclic aromatic hydrocarbons (PAHs) that pollute the environment is 2-amino-l-methyI-6-phenyI-imidazo pyridine (PhIP). It is a food-derived carcinogen that is present in fish and meat that has been cooked at high temperatures. Due to the generation of reactive electrophilic species, phase I enzymes have the potential to cause oxidative damage. In order to safely remove these reactive species from the body, phase II detoxification (conjugation) enzymes are necessary. It is not known whether OLE could influence their activities and hence reduce the carcinogenic effects of PhIP. This study evaluated whether OLE could modulate phase I detoxifying enzymes as well as phase II enzymes that metabolize PhIP in rat liver microsomes. Four groups of rats were used: group I: no treatment; group II: OLE (10mg/kg bw orally); group III: PhIP (0.1mg/kg bw orally); and group IV: PhIP followed by OLE. After 4weeks, the activities of phase I enzymes such as CYP1A1 (ethoxyresorufin O-deethylase), CYP2E1 (p-nitrophenol hydroxylase), CYP1A2 (methoxyresorufin O-demethylase), UDP-glucuronyl transferase, sulphotransferase, and glutathione-S transferase were evaluated in rat liver microsomes. Analysis of OLE by gas chromatography-mass spectrometry (GC/MS) showed various active ingredients in OLE, including 3,5-Heptadienal (C10H14O), 3,4-dimethoxy benzoic acid (C8H10O3), 4-hydroxy-3-methoxy (C8H8O4), 1,3,5-Benzenetriol (C6H6O3), hexadecanoic acid (C16H32O2), and hexadecanoic acid ethyl ester (C18H36O2). Our results showed that rats given PhIP were found to have a statistically significant (p < 0.001) reduction in the activities of CYP1A1, CYP1A2, and CYP2E1 in comparison with the control group. However, treatment with OLE enhanced their activities but not to a normal level compared with untreated groups. Administration of PhIP decreased the activities of phase II enzymes (glutathione S-transferase, UDP-glucuronyltransferase, or sulphotransferase) (p < 0.01) in comparison with the control group. Histological examination of rat livers was consistent with the biochemical changes. The administration of OLE improved the phase II enzyme activities in animals injected with PhIP. We conclude that OLE influences phase I and phase II detoxification enzymes exposed to PhIP, which may represent a new approach to attenuating carcinogenesis induced by it.

Effects of tebuconazole on cytochrome P450 enzymes, oxidative stress, and endocrine disruption in male rats.

The major objective of the present study was to determine the ability of a triazole fungicide tebuconazole to induce cytochrome P450-dependent monooxygenases, oxidative stress, and endocrine-disrupting activity using male rats treated with tebuconazole at 10, 25, and 50 mg/kg p.o. once daily for 28 days. In liver, tebuconazole dose-dependently increased microsomal contents of cytochrome P450 and cytochrome b5 and the activities of NADPH-cytochrome P450 reductase, 7-ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase, pentoxyresorufin O-dealkylase, 7-ethoxycoumarin O-deethylase, aniline hydroxylase, and erythromycin N-demethylase. In kidney, tebuconazole increased 7-ethoxycoumarin O-deethylase activity without affecting other monooxygenase activities. In marked contrast to liver and kidney, tebuconazole decreased testicular 7-ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase, 7-ethoxycoumarin O-deethylase, aniline hydroxylase, and erythromycin N-demethylase activities. The results of immunoblot analysis of liver microsomes of controls and tebuconazole-treated rats revealed that tebuconazole induced CYP1A1/2, CYP2B1/2, CYP2E1, and CYP3A proteins in liver. Additions of tebuconazole to liver microsomes inhibited microsomal 7-ethoxycoumarin O-deethylase activity in vitro (IC50 = 1.50-1.69 µM). Treatment of rats with tebuconazole decreased glutathione content and increased glutathione S-transferase, superoxide dismutase, catalase, and glutathione peroxidase activities in liver; increased superoxide dismutase activities in kidney and testis; but decreased glutathione S-transferase activity in testis. Treatments with tebuconazole decreased serum testosterone concentration and cauda epididymal sperm count. The present study demonstrates that tebuconazole induces a multiplicity of CYPs and oxidative stress in liver; inhibits testicular P450 and glutathione S-transferase activities; and produces anti-androgenic effects in male rats.

Pyrethroid insecticide lambda-cyhalothrin induces hepatic cytochrome P450 enzymes, oxidative stress and apoptosis in rats

This study aimed to examine in rats the effects of the Type II pyrethroid lambda-cyhalothrin on hepatic microsomal cytochrome P450 (CYP) isoform activities, oxidative stress markers, gene expression of proinflammatory, oxidative stress and apoptosis mediators, and CYP isoform gene expression and metabolism phase I enzyme PCR array analysis. Lambda-cyhalothrin, at oral doses of 1, 2, 4 and 8mg/kg bw for 6days, increased, in a dose-dependent manner, hepatic activities of ethoxyresorufin O-deethylase (CYP1A1), methoxyresorufin O-demethylase (CYP1A2), pentoxyresorufin O-depentylase (CYP2B1/2), testosterone 7α- (CYP2A1), 16β- (CYP2B1), and 6β-hydroxylase (CYP3A1/2), and lauric acid 11- and 12-hydroxylase (CYP4A1/2). Similarly, lambda-cyhalothrin (4 and 8mg/kg bw, for 6days), in a dose-dependent manner, increased significantly hepatic CYP1A1, 1A2, 2A1, 2B1, 2B2, 2E1, 3A1, 3A2 and 4A1 mRNA levels and IL-1β, NFκB, Nrf2, p53, caspase-3 and Bax gene expressions. PCR array analysis showed from 84 genes examined (P<0.05; fold change>1.5), changes in mRNA levels in 18 genes: 13 up-regulated and 5 down-regulated. A greater fold change reversion than 3-fold was observed on the up-regulated ALDH1A1, CYP2B2, CYP2C80 and CYP2D4 genes. Ingenuity Pathway Analysis (IPA) groups the expressed genes into biological mechanisms that are mainly related to drug metabolism. In the top canonical pathways, Oxidative ethanol degradation III together with Fatty Acid α-oxidation may be significant pathways for lambda-cyhalothrin. Our results may provide further understanding of molecular aspects involved in lambda-cyhalothrin-induced liver injury.

Reprint of: CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to Deepwater Horizon Mississippi Canyon 252 oil

Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5ml/kg bw oil or 10ml/kg bw oil, 21day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to accurately evaluate avian exposure to oil.

Publisher's note

Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5ml/kg bw oil or 10ml/kg bw oil, 21day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to accurately evaluate avian exposure to oil.

Effects of pentachlorophenol on the quail (Coturnix japonica) liver detoxification pathway

Pentachlorophenol (PCP), an extensively used pesticide and biocide, is of critical environmental concern due to its toxicity and recalcitrance to degradation. In this study, the effect of PCP on induction of transcription factors, cytochrome P450 (CYP450) genes, and the antioxidative enzyme system were investigated in the quail liver. A total of 60 (4- to 6-week-old) male quails (Coturnix japonica) were administered 0, 0.05, 0.5, and 5 mg/kg/d PCP orally for 42 d. Following exposure, both absolute and relative liver weights were significantly lower than those of the control. Using gas chromatography-mass spectrometry, PCP accumulation was, from highest to lowest, kidney > liver > muscle for all exposure groups. The expressions of CYP1A5, CYP1B1, CYP2C18, nuclear translocator 1 (ARNT1), and aryl hydrocarbon receptor 1 (AHR1) were induced after PCP treatment, and increases were found in the activities of hepatic superoxide dismutase (SOD) and catalase (CAT), and the content of hepatic malondialdehyde (MDA). In addition, exposure to PCP induced an increase in liver 8-hydroxydeoxyguanosine (8-OHdG) and significantly elevated ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and 7-ethoxycoumarin-O-deethylase (ECOD) activity, but decreased that of glutathione peroxidase (GSH-Px), benzyloxyresorufin O-debenzylase (BROD), pentoxyresorufin O-depentylase (PROD), and erythromycin N-demethylase (END). No significant responses were observed for benzyloxy-trifluoromethyl-coumarin (BFC). The protein level of liver nuclear factor κB (NF-κB) was higher, whereas that of nuclear factor E2-related factor 2 (Nrf2) was lower for exposed quail. These results suggest that PCP affects quail oxidative stress by modulating CYP450 enzymes and nuclear transcription factors.

Comparison between in toto peach (Prunus persica L. Batsch) supplementation and its polyphenolic extract on rat liver xenobiotic metabolizing enzymes

Over the past years, there has been a growing interest in the natural constituents of foods as a potential means of cancer control. To date, epidemiology studies seem to indicate an inverse association between regular consumption of fruit and vegetables and cancer risk.Here, the potential chemopreventive activity of the polyphenolic extract (PPE) of peach (Prunus persica L. Batsch) and of the freeze-dried fruit in toto (LFT), focusing on the modulation of xenobiotic metabolizing enzymes (XMEs) in vivo, was investigated. Rats were daily supplemented with LFT at 250 and 500 mg/kg b.w. or with the corresponding amount of PPE (2.5 and 5 mL/kg b.w., respectively) for either 7 or 14 days. While PPE treatment resulted in a widespread phase-I inactivation, a complex modulation pattern with drastic decreases (7α-testosterone hydroxylase, pentoxyresorufin O-dealkylase (PROD)), coupled with marked up-regulations of ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) after LFT administration, was seen. A notable down-regulation (over 50%) following LFT or PPE treatment for the phase-II enzymes was also recorded. The observed remarkable changes in XMEs, if reproduced in humans, might have public health implications. These data suggest caution in promoting peach fruit (mono-diet) consumption or its polyphenolic extract in the field of chemoprevention.

Pharmacokinetic Interaction of Chrysin with Caffeine in Rats.

Pharmacokinetic interaction of chrysin, a flavone present in honey, propolis and herbs, with caffeine was investigated in male Sprague-Dawley rats. Because chrysin inhibited CYP1A-selective ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase activities in enriched rat liver microsomes, the pharmacokinetics of caffeine, a CYP 1A substrate, was studied following an intragastric administration with 100 mg/kg chrysin. In addition to the oral bioavailability of chrysin, its phase 2 metabolites, chrysin sulfate and chrysin glucuronide, were determined in rat plasma. As results, the pharmacokinetic parameters for caffeine and its three metabolites (i.e., paraxanthine, theobromine and theophylline) were not changed following chrysin treatment in vivo, despite of its inhibitory effect on CYP 1A in vitro. The bioavailability of chrysin was found to be almost zero, because chrysin was rapidly metabolized to its sulfate and glucuronide conjugates in rats. Taken together, it was concluded that the little interaction of chrysin with caffeine might be resulted from the rapid metabolism of chrysin to its phase 2 metabolites which would not have inhibitory effects on CYP enzymes responsible for caffeine metabolism.

Effects of pentachlorophenol on the detoxification system in white-rumped munia (Lonchura striata)

Pentachlorophenol (PCP), a priority pollutant due to its persistence and high toxicity, has been used worldwide as a pesticide and biocide. To understand the adverse effects of PCP, adult male white-rumped munias (Lonchura striata) were orally administrated commercial PCP mixed with corn oil at dosages of 0, 0.05, 0.5, and 5mg/(kg·day) for 42day. Gas chromatography–mass spectrometry (GC–MS) analysis found that PCP was preferentially accumulated in the kidney rather than in the liver and muscle in all exposure groups. To examine the function of CYP1A in pollutant metabolism, we isolated two full-length cDNA fragments (designated as CYP1A4 and CYP1A5) from L. striata liver using reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. PCP induced the expression of CYP1A5, although no obvious change was observed in CYP1A4 expression. Furthermore, PCP significantly elevated the activities of ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase and decreased the activity of benzyloxy-trifluoromethyl-coumarin, with no significant responses observed in benzyloxyresorufin O-debenzylase. PCP induced significant changes in antioxidant enzyme (superoxide dismutase and catalase) activities and malondialdehyde content, but decreased glutathione peroxidase (GSH-Px) and glutathione S-transferase activities and GSH content in the liver of L. striata. The present study demonstrated that PCP had hepatic toxic effects by affecting CYP1As and anti-oxidative status.

Effects of baicalin on oral pharmacokinetics of caffeine in rats.

Scutellaria baicalensis is one of the most widely used herbal medicines in East Asia. Because baicalein and baicalin are major components of this herb, it is important to understand the effects of these compounds on drug metabolizing enzymes, such as cytochrome P450 (CYP), for evaluating herb-drug interaction. The effects of baicalin and baicalein on activities of ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), benzyloxyresorufin O-debenzylase (BROD), p-nitrophenol hydroxylase and erythromycin N-demethylase were assessed in rat liver microsomes in the present study. In addition, the pharmacokinetics of caffeine and its three metabolites (i.e., paraxanthine, theobromine and theophylline) in baicalin-treated rats were compared with untreated control. As results, EROD, MROD and BROD activities were inhibited by both baicalin and baicalein. However, there were no significant differences in the pharmacokinetic parameters of oral caffeine and its three metabolites between control and baicalin-treated rats. When the plasma concentration of baicalin was determined, the maximum concentration of baicalin was below the estimated IC50 values observed in vitro. In conclusion, baicalin had no effects on the pharmacokinetics of caffeine and its metabolites in vivo, following single oral administration in rats.

Effects of Oral Administration of Non-genotoxic Hepato-hypertrophic Compounds on Metabolic Potency of Rat Liver

It remains uncertain why non-genotoxic compounds that result in liver hypertrophy cause liver tumors. In an effort to resolve this issue, we examined whether liver post-mitochondrial fraction (S9) prepared from rats treated with non-genotoxic compounds affected the genotoxicity of pro-mutagens. Known hepatotoxic compounds, such as piperonyl butoxide (PBO), decabromodiphenyl ether (DBDE), beta-naphthoflavone (BNF), indole-3-carbinol (I3C) and acetaminophen (AA), were orally administered to male and female F344 rats at doses sufficient to cause liver hypertrophy. Rats received diets containing each test compound for 3 days, 4 weeks or 13 weeks, and were then kept for 4 weeks without the test chemical. S9 prepared from the livers of each group was used for the Ames test with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), benzo[a]pyrene (BaP) and N-nitrosodimethylamine (NDMA). In both sexes, liver hypertrophy was observed following administration of all test compounds, and was then reversed to the control state when administration ceased. The mutagenicity of MeIQx, BaP and NDMA increased with the use of S9 derived from rats treated with non-genotoxic compounds other than AA. DBDE administration had a marked effect on the mutagenicity of BaP (over a 30-fold increase in females) and NDMA (about a 20-fold increase in males). To estimate the involvement of metabolic enzymes in the alteration of mutagenicity, we measured the activity of ethoxyresorufin-O-deethylase (EROD) and methoxyresorufin-O-demethylase (MROD) (phase I enzymes), and UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) (phase II enzymes) in each S9 sample. The activity of phase I enzymes increased, even at the 3rd day following administration, and then decreased gradually, except in the case of AA, while the activity of phase II enzymes increased slightly. These results suggest that non-genotoxic hepato-hypertrophic compounds may be partly involved in carcinogenesis by modulating the metabolism of pre-carcinogens incorporated from the environment, in a manner that is dependent on sex and pre-incorporated chemicals.

Characterization of chicken cytochrome P450 1A4 and 1A5: Inter-paralog comparisons of substrate preference and inhibitor selectivity

The chicken (Gallus gallus) is one of the most economically important domestic animals and also an avian model species. Chickens have two CYP1A genes (CYP1A4 and CYP1A5) which are orthologous to mammalian CYP1A1 and CYP1A2. Although the importance of chicken CYP1As in metabolism of endogenous compounds and xenobiotics is well recognized, their enzymatic properties, substrate preference and inhibitor selectivity remain poorly understood. In this study, functional enzymes of chicken CYP1A4 and CYP1A5 were successfully produced in Escherichia coli (E. coli). The substrate preference and inhibitor specificity of the two chicken CYP1As were compared. Kinetic results showed that the enzymatic parameters (Km, Vmax, Vmax/Km) for ethoxyresorufin O-deethylase (EROD) and benzyloxyresorufin O-debenzylase (BROD) differed between CYP1A4 and CYP1A5, while no significant difference was observed for methoxyresorufin O-demethylase (MROD). Lower Km of CYP1A4 for BROD suggests that CYP1A4 has a greater binding affinity to benzyloxyresorufin than either ethoxyresorufin or methoxyresorufin. The highest Vmax/Km ratio was seen in BROD activity for CYP1A4 and in MROD for CYP1A5 respectively. These results indicate that substrate preference of chicken CYP1As is more notably distinguished by BROD activity and CYP1A5 prefers shorter alkoxyresorufins resembling its mammalian ortholog CYP1A2. Differential patterns of MROD inhibition were observed between CYP1As and among the five CYP inhibitors (α-naphthoflavone, furafylline, piperonyl butoxide, erythromycin and ketoconazole). α-Naphthoflavone was determined to be a potent MROD inhibitor of both CYP1A4 and CYP1A5. In contrast, no or only a trace inhibitory effect (<15%) was observed by erythromycin at a concentration of 500μM. Stronger inhibition of MROD activity was found in CYP1A5 than CYP1A4 by relatively small molecules α-naphthoflavone, piperonyl butoxide and furafylline. AROD kinetics and inhibition profiles between chicken CYP1A4 and CYP1A5 demonstrate that the two paralogous members of the CYP1A subfamily have distinct enzymatic properties, reflecting differences in the active site geometry between CYP1A4 and CYP1A5. These findings suggest that CYP1A4 and CYP1A5 play partially overlapping but distinctly different physiological and toxicological roles in the chicken.

Inhibitory Potentials of Five Phytoestrogens from Pueraria candollei var. mirifica on CYP1A1 and CYP1A2 Proteins in Mouse Liver Microsomes and in silico

Pueraria candollei var. mirifica (PM) is a Thai traditional medicinal plant for rejuvenation and estrogen replacement therapy in menopausal women. CYP1A1 and CYP1A2 proteins are the members of hepatic cytochrome P450 (CYP) enzymes to activate a procarcinogen, in which ethoxyresorufin O -deethylase (EROD) and methoxyresorufin O -demethylase (MROD) activities are the specific markers for CYP1A1 and CYP1A2, respectively. In the present study, the effects of five phytoestrogens isolated form the bark of PM tuberous roots namely miroestrol, deoxymiroestrol, khawkhurin, isomiroestrol, and methoxyisomiroestrol, on EROD and MROD activities were examined in mouse hepatic microsomes, compared to a typical CYP1A1/2 inducer and substrate beta-napthoflavone (BNF). The bindings of these five compounds to either CYP1A1 or CYP1A2 enzymes were analyzed using molecular docking with homology modeling technique. Rank of the median inhibitory concentration (IC 50 ) of these compounds on EROD activity corresponded to that of MROD, namely BNF > miroestrol > kwakhurin > deoxymiroestrol > methoxyisomiroestrol> isomiroestrol, respectively. Interestingly, the binding pose energy of these compounds to CYP1A1 and CYP1A2 proteins were consistent to those of inhibitory effects on EROD and MROD activities. The observations suggested for the first time that the active phytoestrogens from PM possessed inhibitory potentials on CYP1A1 and CYP1A2 via EROD and MROD activities, respectively. Furthermore, the binding energy of the compounds to CYP1A1 and CYP1A2 proteins might be a useful tool to predict the effects of a compound on these two CYP enzymes.

Impact of six fruits—banana, guava, mangosteen, pineapple, ripe mango and ripe papaya—on murine hepatic cytochrome P450 activities

The effects of six Thai fruits, namely banana, guava, mangosteen, pineapple, ripe mango and ripe papaya, on cytochrome P450 (P450) activities were investigated. The median inhibitory concentrations (IC(50) ) of each of the fruit juices on CYP1A1, CYP1A2, CYP2E1 and CYP3A11 activities were determined. Pineapple juice showed the strongest inhibitory effect against all the evaluated P450 isozyme activities in mouse hepatic microsomes, followed by mangosteen, guava, ripe mango, ripe papaya and banana. The study was further performed in male ICR mice given pineapple juice intragastrically at doses of 10, 20 and 40 mg kg(-1) per day for 7 or 28 days. In a concentration-dependent fashion, the pineapple juice raised ethoxyresorufin O-deethylase, aniline hydroxylase and erythromycin N-demethylase activities, which are marker enzymatic reactions responsible for CYP1A1, CYP2E1 and CYP3A11, respectively. The effect of pineapple juice on the expression of CYP1A1, CYP2E1 and CYP3A11 mRNAs corresponded to their enzymatic activities. However, the pineapple juice significantly decreased methoxyresorufin O-demethylase activity. These observations supported that the six Thai fruits were a feasible cause of food-drug interaction or adverse drug effects owing to their potential to modify several essential P450 activities. Individuals consuming large quantities of pineapple for long periods of time should be cautioned of these potential adverse effects.

Diverse action of acrylamide on cytochrome P450 and glutathione S-transferase isozyme activities, mRNA levels and protein levels in human hepatocarcinoma cells

Humans are exposed to acrylamide in their diet and cigarette smoke. Acrylamide is metabolized into glycidamide by CYP2E1. However, very few studies regarding the effects of acrylamide on cytochrome P450 and Glutathione S-Transferase (GST) isozymes have been pursued. The aim of this study is to elucidate the effects of acrylamide on cytochrome P450 and GST isozymes in HepG2 cell line. Treatment with 1.25 and 2.5mM acrylamide caused 9.5- and 3.7-fold increases and 4.0- and 3.3-fold increases in CYP1A-associated ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities, respectively. These increases were consistent with increases in mRNA and protein levels of these isozymes. Similarly, CYP2E1-associated aniline 4-hydroxylase (ANH) activity, protein levels, and mRNA levels increased 2.1- and 2.6-fold, 2.4- and 3.2-fold, and 1.4- and 1.9-fold following 1.25 and 2.5mM acrylamide treatments, respectively. In addition, GST-mu activity was increased 2.4- and 5.1-fold by acrylamide. Moreover, GST-mu mRNA and protein levels increased twofold as a result of acrylamide treatment. In contrast, GST-pi protein and mRNA levels decreased significantly. In conclusion, human cell exposure to acrylamide causes an increase in the levels of carcinogenicity and toxicity and a disturbance in drug metabolism, possibly due to complex effects on P450 and GST isozymes.

Effects of dietary inulin, statin, and their co-treatment on hyperlipidemia, hepatic steatosis and changes in drug-metabolizing enzymes in rats fed a high-fat and high-sucrose diet

BackgroundRats fed a high-fat and high-sucrose (HF) diet develop hepatic steatosis and hyperlipidemia. There are several reports that a change in nutritional status affects hepatic levels of drug-metabolizing enzymes. Synthetic inulin is a dietary component that completely evades glucide digestion. Supplementing a HF diet with inulin ameliorates hypertriglycemia and hepatic steatosis, but not hypercholesterolemia. This study aimed at distinguishing the effects of synthetic inulin and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (statin), which inhibit cholesterol biosynthesis.MethodsWe examined effects of co-treatment with synthetic inulin (5%) and fluvastatin (0, 4, and 8 mg/kg, per os) on body weight, epidydimal white adipose tissue weight, serum and hepatic lipid profiles, and hepatic cytochrome P450 (CYP) mRNA and protein profiles in rats fed a standard diet or a HF diet for 3 weeks.ResultsTreatment with the synthetic inulin (5%) or fluvastatin at 4 mg/kg (lethal dose in rats fed the HF diet, 8 mg/kg) ameliorated the elevation in hepatic triacylglycerol and total cholesterol levels in rats fed the HF diet. Whereas co-treatment with the inulin (5%) and fluvastatin (4 mg/kg) had a tendency to more strongly suppress the elevation in serum levels of very low density lipoprotein triacylglycerol than either treatment alone, no additive or synergistic effect was found in decrease in hepatic lipid levels. Hepatic levels of CYP1A1/2 and CYP2E1 mRNA and protein and methoxyresorufin O-demethylase and ethoxyresorufin O-deethylase activities were reduced in rats fed the HF diet. The synthetic inulin alleviated the reduction in hepatic levels of CYP1A1/2 and CYP2E1 mRNA and protein more strongly than fluvastatin, and no synergistic effects were observed on co-treatment. Furthermore, hepatic levels of aryl hydrocarbon receptor mRNA were decreased in rats fed the HF diet and recovered to near normal values with the intake of dietary inulin, which correlated with change in CYP1A1/2.ConclusionsDietary inulin alone was effective to prevent the development of hepatic steatosis, ameliorate nutritional effects, and alleviate the hepatic change in the expression of CYP1A1/2 and CYP2E1, while co-treatment with statin did not have additive or synergistic effects and statin may cause adverse effects in rats fed the HF diet.

Effects of Furanocoumarins from Apiaceous Vegetables on the Catalytic Activity of Recombinant Human Cytochrome P-450 1A2

Inhibition of cytochrome P-450 1A2 (CYP1A2)-mediated activation of procarcinogens may be an important chemopreventive mechanism. Consumption of apiaceous vegetables (rich in furanocoumarins) inhibits CYP1A2 in humans. Because many furanocoumarins are potent inhibitors of several CYPs, we characterized the effects of three furanocoumarins from apiaceous vegetables on human CYP1A2 (hCYP1A2). We assessed hCYP1A2 methoxyresorufin O-demethylase (MROD) activity using microsomes from Saccharomyces cerevisiae expressing hCYP1A2. Isopimpinellin exhibited mechanism-based inactivation (MBI) of hCYP1A2 (K(i) = 1.2 μM, k (inact) = 0.34 min⁻¹, and partition ratio = 8). Imperatorin and trioxsalen were characterized as mixed inhibitors with K(i) values of 0.007 and 0.10 μM, respectively. These results indicate that even if present at low levels in apiaceous vegetables, imperatorin, trioxsalen and isopimpinellin may contribute significantly to CYP1A2 inhibition and potentially decreased procarcinogen activation. Moreover, the in vivo effect of isopimpinellin on CYP1A2 may be longer lasting compared to reversible inhibitors.

Prenatal administration of the cytochrome P4501A inducer, Β-naphthoflavone (BNF), attenuates hyperoxic lung injury in newborn mice: Implications for bronchopulmonary dysplasia (BPD) in premature infants

Supplemental oxygen contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. In this investigation, we tested the hypothesis that prenatal treatment of pregnant mice (C57BL/6J) with the cytochrome P450 (CYP)1A1 inducer, ß-napthoflavone (BNF), will lead to attenuation of lung injury in newborns (delivered from these dams) exposed to hyperoxia by mechanisms entailing transplacental induction of hepatic and pulmonary CYP1A enzymes. Pregnant mice were administered the vehicle corn oil (CO) or BNF (40 mg/kg), i.p., once daily for 3 days on gestational days (17–19), and newborns delivered from the mothers were either maintained in room air or exposed to hyperoxia (> 95% O 2) for 1–5 days. After 3–5 days of hyperoxia, the lungs of CO-treated mice showed neutrophil infiltration, pulmonary edema, and perivascular inflammation. On the other hand, BNF-pretreated neonatal mice showed decreased susceptibility to hyperoxic lung injury. These mice displayed marked induction of ethoxyresorufin O-deethylase (EROD) (CYP1A1) and methoxyresorufin O-demethylase (MROD) (CYP1A2) activities, and levels of the corresponding apoproteins and mRNA levels until PND 3 in liver, while CYP1A1 expression alone was augmented in the lung. Prenatal BNF did not significantly alter gene expression of pulmonary NAD(P)H quinone reductase (NQO1). Hyperoxia for 24–72 h resulted in increased pulmonary levels of the F 2-isoprostane 8-iso-PGF 2α, whose levels were decreased in mice prenatally exposed to BNF. In conclusion, our results suggest that prenatal BNF protects newborns against hyperoxic lung injury, presumably by detoxification of lipid hydroperoxides by CYP1A enzymes, a phenomenon that has implications for prevention of BPD in infants.

Hepatic Ethoxy‐, Methoxy‐ and Pentoxyresorufin O‐Dealkylase Activities in Landrace and Duroc Pigs Stimulated with hCG

The effect of human chorionic gonadotropin (hCG) stimulation on the activities of ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD) and pentoxyresorufin O-depentylase (PROD) was studied in intact male pigs of purebred Landrace and Duroc breeds. Pigs were divided into four groups: two control groups of each breed, without hCG stimulation (n = 20 for each breed), and two experimental groups (n = 18 for each breed), with hCG stimulation (Pregnyl(®); N.V. Organon, Oss, The Netherlands, 30 IU/kg live weight). Pigs were slaughtered 3 days after hCG stimulation and enzyme activities were measured in hepatic microsomes using two approaches. First, only one substrate concentration was used for the analysis of each enzyme activity. We found that EROD activity was suppressed by hCG-stimulation in Landrace (p = 0.004), but not Duroc pigs (p > 0.05). Generally, EROD activity was higher in Duroc pigs compared with Landrace (p = 0.017). Methoxyresorufin O-demethylase and PROD activities did not differ between groups (p > 0.05). To further characterize EROD, MROD and PROD, enzyme kinetic studies were performed. V(max) values for EROD and MROD in both breeds were lower after hCG stimulation (p < 0.001 for Landrace and p < 0.05 for Duroc). Additionally, V(max) values for EROD significantly differed between Landrace and Duroc pigs being higher in Duroc pigs (p < 0.05). We concluded that both hCG stimulation and breed differences may be important in the regulation of EROD and MROD activities. This study provides the first data on the effect of hCG stimulation and thus high testicular steroids, on EROD, MROD and PROD activities. Further studies are needed to investigate individual CYP450 enzymes and their regulation in porcine tissues.