Methoxyresorufin O-demethylase Activities Research Articles

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.

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 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.

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.

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.

Characterization of drug metabolizing enzymes and assessment of aging in the gilthead seabream (Sparus aurata) liver

Organic anthropogenic compounds such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls, nitroaromatics, dioxins, various pesticides and natural compounds can enter the aquatic environment. These lipophilic compounds are readily taken up into the tissues of aquatic organisms where biotransformation via Phase I and Phase II metabolism can in part, determine the fate and toxicity of the xenobiotics. In this study on gilthead seabream (Sparus aurata), liver microsomal ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), penthyloxyresorufin O-depenthylase (PROD), benzyloxyresorufin O-debenzylase (BROD), aniline 4-hydroxylase (A4H), N-nitrosodimethylamine N-demethylase (NDMA-DE), aminopyrine N-demethylase (APND), caffeine N-demethylase (CN3D) and erythromycin N-demethylase (ERND) were determined. A4H, ERND, NDMA-ND, EROD and PROD activities increased in gilthead seabream (Sparus aurata) liver microsomes of different ages, while CN3D and APND activities decreased. MROD activities were barely detectable in mature fish whereas BROD activities were not detectable at all ages studied. Results were also recorded at the protein level by Western blotting using anti-CYP1A, CYP2E and CYP3A antibodies. In conclusion, elevated levels of gilthead seabream liver microsomal CYP450 enzyme activities might reveal possible exposure to various exogenous compounds, which might affect the desired responses to drugs, hormones and dietary supplements used during breeding.

Down-regulation of murine testicular 17β-HSD3 and hepatic CYP1A2 enzymes by a bovine testes extract.

We investigated the effects of a bovine testes extract (BTE), which was developed as an alternative product for andropausal men, on expression of testicular enzymes responsible for sex hormone synthesis genes and a carcinogen activation related gene. Expression of testicular CYP1A2, CYP11A1, CYP17, 3β-HSD, and 17β-HSD3 mRNAs as well as hepatic CYP1A2 mRNA were semi-quantitatively determined by RT-PCR. In addition, expression of hepatic CYP1A2 protein and methoxyresorufin O-demethylase activity were carried out. Bovine testes extract did not alter the testicular expression of CYP11A1, CYP17, and 3β-HSD mRNAs, while that of CYP11A1 was significantly down-regulated by testosterone. Interestingly, administration of BTE for 3 weeks significantly suppressed testicular 17β-HSD3 and hepatic CYP1A2 mRNA. Correspondingly, methoxyresorufin O-demethylase (MROD) activity and expression of hepatic CYP1A2 protein were significantly decreased. These findings strongly suggested considering risks versus benefits and raised concerns regarding the use of BTE as an alternative medication or health supplement in andropausal men due to its potential for suppressing expression of both 17β-HSD3 and CYP1A2 mRNAs, testicular enzymes responsible for sex hormone gene synthesis.

Alkoxyresorufin (methoxy-, ethoxy-, pentoxy- and benzyloxyresorufin) O-dealkylase activities by in vitro-expressed cytochrome P450 1A4 and 1A5 from common cormorant ( Phalacrocorax carbo)

Here we report the inter-paralog comparison of cytochrome P4501A (CYP1A) catalytic function in common cormorant ( Phalacrocorax carbo) using the recombinant proteins synthesized by yeast-based vector system. CYP1A4 and CYP1A5 proteins from common cormorant were heterologously expressed in yeast Saccaromyces cerevisiae. Kinetic analyses revealed that among alkoxyresorufin (methoxy-, ethoxy-, pentoxy- and benzyloxyresorufin) O-dealkylase (AROD) activities V max value for ethoxyresorufin O-deethylase (EROD) activity was the highest for both enzymes, reaching 0.91 ± 0.034 and 1.8 ± 0.043 nmol/min/nmol CYP for CYP1A4 and CYP1A5, respectively. Similar results were obtained for the catalytic efficiencies represented as the ratios of V max to K m ( V max/ K m). Meanwhile, distinct substrate preferences were also observed; CYP1A4 had V max and V max/ K m values for benzyloxyresorufin O-debenzylase (BROD) activity 12- and 46-fold greater than CYP1A5, respectively, while CYP1A5 was about 13- and 4.5-fold more efficient in methoxyresorufin O-demethylase (MROD) activity than CYP1A4. The K m values showed no significant change among MROD, EROD, pentoxyresorufin O-depenthylase (PROD) and BROD activities for both enzymes, except for significant differences between PROD and other three activities for CYP1A4. Comparing the results in the present study with previous studies addressing chicken and rat CYP1A enzymes, it is also clear that CYP1A orthologs have different catalytic preferences for AROD activities between cormorant and rat and even between cormorant and chicken. Variations in CYP1A catalytic function between cormorant CYP1A paralogs and between CYP1A orthologs from cormorant and other species indicate that enzymatic properties should be characterized on the basis not only of a limited model species such as chicken, but also of multiple species to further understand the mechanism underlying differences in substrate selectivity and the interaction with environmental contaminants in avian species.

Enhancement of phase II enzyme activity by purpurin resulting in the suppression of MeIQx–DNA-adduct formation in mice

We previously demonstrated using a bacterial system that the antigenotoxic activity of the anthraquinone compounds purpurin and alizarin was due to the suppression of microsomal enzyme activity involved in the activation of mutagens. In the present study we determined the effect of purpurin and alizarin on (i) MeIQx–DNA-adduct formation in mouse tissues and (ii) the activity of phases I and II enzymes in liver fractions, the liver being the target tissue of MeIQx. The amount of MeIQx–DNA adduct formed was determined using 32P-postlabeling methods. Methoxyresorufin- O-demethylase (MROD) and ethoxyresorufin- O-deethylase (EROD) enzyme activities, which reflect CYP 1A activity, were measured as markers for phase I enzymes, and UDP–glucuronyltransferase (UGT) and glutathione S-transferase (GST) activities were determined as markers for phase II enzymes. Mice fed with a diet containing 0.5% purpurin for 3 days prior to MeIQx administration had 70% fewer MeIQx–DNA adducts in the lung and kidney, and fewer DNA adducts (insignificant, statistically) in the liver compared with mice fed a diet lacking purpurin. MROD and EROD activities in the liver of these mice increased six- and eight-fold, respectively, and were higher than those determined for the control mice within 1 day following commencement of purpurin treatment. These elevated activities were maintained during treatment and declined immediately following removal of purpurin from the diet. GST and UGT activities gradually increased 2.5- and 3-fold, respectively, following purpurin treatment, and were maintained at significantly high levels even after purpurin administration ceased. Alizarin did not significantly affect DNA-adduct formation and enzyme activity, except in the case of UGT. Taken together, our results show that purpurin reduced MeIQx–DNA-adduct formation by maintaining elevated phase II enzyme activities, thereby facilitating accelerated excretion of MeIQx.

Apiaceous vegetable constituents inhibit human cytochrome P-450 1A2 (hCYP1A2) activity and hCYP1A2-mediated mutagenicity of aflatoxin B 1

In humans, apiaceous vegetables (carrots, parsnips, celery, parsley, etc.) inhibit cytochrome P-450 1A2, a biotransformation enzyme known to activate several procarcinogens, including aflatoxin B 1 (AFB). We evaluated eight phytochemicals from apiaceous vegetables for effects on human cytochrome P-450 1A2 (hCYP1A2) activity using a methoxyresorufin O-demethylase (MROD) assay and a trp-recombination assay. Saccharomyces cerevisiae was used for heterologous CYP1A2 expression and this yeast strain is also diploid and auxotrophic for tryptophan due to mutations in the trp5 alleles. When these two alleles undergo AFB-induced mitotic recombination, gene conversion occurs, allowing yeast to grow in the absence of tryptophan. The apiaceous constituents psoralen, 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), and apigenin were potent inhibitors of hCYP1A2-mediated MROD activity in yeast microsomes, whereas quercetin was a modest hCYP1A2 inhibitor. Naringenin, caffeic acid, and chlorogenic acid did not inhibit hCYP1A2-mediated MROD activity. The 2-h pretreatment of intact yeast cells with psoralen, 5-MOP, and 8-MOP significantly improved cell survival after subsequent 4-h AFB treatment and reduced hCYP1A2-mediated mutagenicity of AFB. Apigenin also significantly decreased mutagenicity. These results suggest that in vivo CYP1A2 inhibition by apiaceous vegetables may be due to the phytochemicals present and imply that apiaceous vegetable intake may be chemopreventive by inhibiting CYP1A2-mediated carcinogen activation.

The expressions of protooncogenes and CYP1A in lungs of rats exposed to sulfur dioxide and benzo( a)pyrene

Sulfur dioxide (SO 2) is a ubiquitous air pollutant, present in low concentrations in the urban air, and in higher concentrations in the working environment. Benzo( a)pyrene (B( a)P), a polycyclic aromatic hydrocarbon, is a ubiquitous environmental contaminant with diverse toxicological effects. To investigate the interactions between SO 2 and B( a)P, male Wistar rats were exposed to intratracheally instilled with benzo( a)pyrene (B( a)P; 3 mg) or SO 2 (20 ppm) inhalation alone or together. The mRNA of CYP1A1 and 1A2, c-fos, and c-jun and protein levels of c-fos and c-jun were analyzed in lungs using a real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) assay and Western blot analysis, respectively. And 7-ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities were detected. In lungs of rats exposed to SO 2 alone, the gene transcription of CYP1A1 and 1A2, the EROD and MROD activities were decreased. Meanwhile, the mRNA and protein levels of c-jun and c-fos were increased significantly. Exposure to B( a)P alone induced CYP1A1, CYP1A2 mRNA levels, the protein levels of c-jun, and the EROD and MROD activities in lungs. However, exposure to B( a)P plus inhaled SO 2 neither increased nor decreased CYP1A1/2 mRNA expressions, EROD, and MROD activities in lungs, versus exposure to B( a)P alone. Nevertheless, exposure to B( a)P plus inhaled SO 2 increased the mRNA and protein levels of c-jun and c-fos in lungs compared with lungs exposed to SO 2 alone. Accordingly, the SO 2-induced decreases of CYP1A1/2 might not influence the metabolic activation of B( a)P. However, when B( a)P and SO 2 were given in the combinations, one might postulate that a synergistic effect on the expressions of c-fos and c-jun between SO 2 and B( a)P, which might be one of the possible mechanisms of combination effects between B( a)P and the air pollutants.

Effect of sulfur dioxide inhalation on CYP1A1 and CYP1A2 in rat liver and lung

Sulfur dioxide (SO 2) is a ubiquitous air pollutant, presents in low concentrations in the urban air, and in higher concentrations in the working environment. In the present study, male Wistar rats were housed in exposure chambers and treated with 14.00 ± 1.53, 28.00 ± 2.12 and 56.00 ± 4.28 mg/m 3 SO 2 for 6 h/day for 7 days, while control rats were exposed to filtered air in the same condition. Highly specific substrates were used as probes of cytochrome P4501A1 (CYP1A1) and cytochrome P4501A2 (CYP1A2). The mRNA levels of CYP1A1 and 1A2 were analyzed in livers and lungs by using a real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) assay. Our results showed that the activities and mRNA levels of P450 were decreased in livers and lungs of rats exposed to SO 2. In the liver, a decrease down to 0.68- and 0.64-fold in the CYP1A1 activity, probed by the O-deethylation of ethoxyresorufin (EROD), was observed at higher dose of SO 2 (28 and 56 mg/m 3); Similarly, CYP1A1 mRNA levels were reduced in livers of rats exposed to SO 2 at 28 and 56 mg/m 3. For livers, CYP1A2-mediated methoxyresorufin O-demethylase activity (MROD) was unaltered by SO 2 at low concentrations, except for a significant decrease in the rats exposed to SO 2 at 56 mg/m 3 (0.79-fold); however, SO 2 at higher concentrations significantly decreased levels of CYP1A2 (28 and 56 mg/m 3) ( p < 0.05). Significant inhibition of both EROD and MROD was observed in lungs of rats exposed to SO 2 at 28 and 56 mg/m 3. CYP1A1 activity was repressed 0.62- and 0.53-fold, while CYP1A2 activity was reduced to 0.74- and 0.55-fold in lungs, respectively. SO 2 at higher concentrations (28 and 56 mg/m 3) decreased significantly pulmonary CYP1A1 and 1A2 mRNA levels relative to control animals. Furthermore, the decreases of activities and mRNA levels of these P450 enzymes caused by SO 2 at different concentrations in lungs and livers of rats followed linear dose–response curves. These results lead to the conclusion that SO 2 exposure can reduce CYP1A1 and 1A2 in lungs and livers of rats and ROS and/or cytokines might act as mediators of this effect according to previous studies performed in mice. Reduction of hepatic and pulmonary CYPlAl and lA2 expression during SO 2 exposure may be part of an adaptive response by the liver and lung to minimize cell damage.

Effects of Cypermethrin Selection on Expression of Insecticide Resistance Mechanisms in the German Cockroach (Blattaria: Blattellidae)

A cross-resistant strain of German cockroach, Blattella germanica (L.), (Marietta) expressing multiple resistance mechanisms was subjected to selection pressure with cypermethrin. Resistance to cypermethrin increased incrementally from 3.6-fold in the parental strain to 35-fold after 4 rounds of selection. No significant changes were observed in cytochrome P450 content, aldrin epoxidase activity, α-naphthyl acetate hydrolysis (cytosolic fraction), or glutathione S-transferase (CDNB conjugation) activities. Although no significant differences were observed in cypermethrin metabolism, a trend toward greater detoxification among each successive generation was observed. Furthermore, methoxyresorufin O-demethylase activity (often associated with pyrethroid resistance) increased incrementally with each successive selection iteration. The average knockdown time increased incrementally from 37 min (parental strain) to 177 min, and there was a corresponding increase in the kdr allele frequency from 19% to 99% after 4 rounds of selection. The data indicated that kdr-type nerve insensitivity and enhanced metabolism by cytochromes P450 and hydrolases were the principle mechanisms of resistance after selection with cypermethrin.

Potent antimutagenic activity of white tea in comparison with green tea in the Salmonella assay

There is growing interest in the potential health benefits of tea, including the antimutagenic properties. Four varieties of white tea, which represent the least processed form of tea, were shown to have marked antimutagenic activity in the Salmonella assay, particularly in the presence of S9. The most active of these teas, Exotica China white tea, was significantly more effective than Premium green tea (Dragonwell special grade) against 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) and four other heterocyclic amine mutagens, namely 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethyl-3 H-imidazo[4,5- f]quinoxaline (4,8-DiMeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), and 3-amino-1-methyl-5 H-pyrido[4,3- b]indole (Trp-P-2). Mechanism studies were performed using rat liver S9 in assays for methoxyresorufin O-demethylase (MROD), a marker for the enzyme cytochrome P4501A2 that activates heterocyclic amines, as well as Salmonella assays with the direct-acting mutagen 2-hydroxyamino-3-methylimidazo[4,5- f]quinoline ( N-hydroxy-IQ). White tea at low concentrations in the assay inhibited MROD activity, and attenuated the mutagenic activity of N-hydroxy-IQ in the absence of S9. Nine of the major constituents found in green tea also were detected in white tea, including high levels of epigallocatechin-3-gallate (EGCG) and several other polyphenols. When these major constituents were mixed to produce ‘artificial’ teas, according to their relative levels in white and green teas, the complete tea exhibited higher antimutagenic potency compared with the corresponding artificial tea. The results suggest that the greater inhibitory potency of white versus green tea in the Salmonella assay might be related to the relative levels of the nine major constituents, perhaps acting synergistically with other (minor) constituents, to inhibit mutagen activation as well as ‘scavenging’ the reactive intermediate(s).

Inducibility of Hepatic CYP1A Enzymes by 3-Methylcholanthrene and Isosafrole Differs in Male Rats Fed Diets Containing Casein, Soy Protein Isolate or Whey from Conception to Adulthood

Hepatic cytochrome P450 (CYP)1A1 and 1A2 enzymes were studied in male Sprague-Dawley rats derived from 5-7 litters fed diets in which the protein source was casein, soy protein isolate or whey. At age 65 d, rats were gavaged with corn oil (vehicle), 40 mg/kg 3-methylcholanthrene (3-MC) or 75 mg/kg isosafrole (ISO). Hepatic expression of CYP1A1 and CYP1A2 mRNA, apoprotein and associated monooxygenase activities were measured 17 h later. No significant dietary effects were observed on basal expression of either enzyme. However, interactions between diet and the two inducers (3-MC and ISO) were observed in soy-fed rats for ethoxy- and methoxyresorufin O-dealkylase activity, CYP1A1 and CYP1A2 apoprotein and mRNA (P < 0.05). The level of induction of CYP1A1 mRNA and apoprotein was lower in rats fed soy diets than in rats fed casein diets (P < 0.05), and the level of induced CYP1A2 mRNA was lower in rats fed soy or whey (P < 0.05) after treatment with the aryl hydrocarbon (Ah) receptor-dependent inducer 3-MC. This was accompanied by a 50% reduction in constitutive levels of the Ah receptor in liver cytosol of soy-fed, relative to casein-fed rats, and a slightly smaller reduction in whey-fed rats. Expression of the Ah receptor correlated with 3-MC-inducibility of CYP1A1 mRNA in rats fed the three diets. In contrast, in rats induced with ISO, which does not bind to the Ah receptor and induces CYP1As via different mechanisms than 3-MC, ethoxyresorufin O-deethylase activity and levels of CYP1A1 apoprotein and mRNA were elevated to a greater degree in soy-fed than in casein- or whey-fed rats (P < 0.05). Moreover, after ISO treatment, induction of methoxyresorufin O-demethylase activity, CYP1A2 apoprotein and mRNA levels was observed only in rats fed soy (P < 0.05). These data suggest potential effects of dietary protein source on metabolism of a wide variety of CYP1A substrates, including environmental and dietary carcinogens, many of which induce their own metabolism.

Acetaminophen produces cataract in DBA2 mice by Ah receptor-independent induction of CYP1A2.

The metabolic transformation of acetaminophen to N-acetyl-p-benzoquinone imine by cytochrome P450 enzymes (e.g., cytochrome P450 1A2) is a prerequisite for acetaminophen-induced cataract formation in mice. Aromatic hydrocarbons, such as beta-naphthoflavone, induce cytochrome P450 1A2 in C57BL6 mice via the mediation of the aromatic hydrocarbon receptor and render the animals susceptible to cataract formation by acetaminophen administration but not in DBA2 mice which do not respond to cytochrome P450 1A2 induction by these compounds. Polycyclic hydrocarbons, such as acenaphthylene, were recently found to induce cytochrome P450 1A2 gene expression in young DBA2 mice by aromatic hydrocarbon receptor-independent pathways. In this work, we investigated whether enhanced metabolism of acetaminophen to N-acetyl-p-benzoquinone by cytochrome P450 1A2 induction by acenaphthylene could produce cataract in young DBA2 mice. Fifteen-day-old DBA2 mice were pretreated with two intraperitoneal injections of acenaphthylene and, 24 hr later, with one injection of acetaminophen. In most mice, cataract developed 18-24 hr after acenaphthylene injection. Acenaphthylene treatment of young DBA2 mice resulted in a 2-fold increase in cytochrome P450 1A2-dependent methoxyresorufin O-demethylase activity in the liver. These results support the hypothesis that the aromatic hydrocarbon receptor-independent induction of cytochrome P450 1A2 enzyme leads to accumulation of sufficient N-acetyl-p-benzoquinone in the liver and cataract development in the eye.

Determination of cytochrome P450 1A activities in mammalian liver microsomes by high-performance liquid chromatography with fluorescence detection

A sensitive method for the determination of cytochrome P450 (P450 or CYP) 1A activities such as ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) in liver microsomes from human, monkey, rat and mouse by high-performance liquid chromatography with fluorescence detection is reported. The newly developed method was found to be more sensitive than previous methods using a spectrofluorimeter and fluorescence plate reader. The detection limit for resorufin (signal-to-noise ratio of 3) was 0.80 pmol/assay. Intra-day and inter-day precisions (expressed as relative standard deviation) were less than 6% for both enzyme activities. With this improved sensitivity, the kinetics of EROD and MROD activities in mammalian liver microsomes could be determined more precisely. EROD activities in human and monkey liver microsomes, and MROD activities in liver microsomes from all animal species exhibited a monophasic kinetic pattern, whereas the pattern of EROD activities in rat and mouse liver microsomes was biphasic. In addition, the method could determine the non-inducible and 3-methylcholanthrene-inducible activities of EROD and MROD in rat and mouse liver microsomes under the same assay conditions. Therefore, this method is applicable to in vivo and in vitro studies on the interaction of xenobiotic chemicals with cytochrome CYP1A isoforms in mammals.