Cytochrome P450-dependent Monooxygenase Activities Research Articles

Biochemical and insecticidal effects of plant essential oils on insecticide resistant and susceptible populations of Musca domestica L. point to a potential cross-resistance risk

Essential oils (EOs) can provide important alternatives to chemical insecticides in the control of pests. In this study, 12 EOs of native plant species from Iran were evaluated for their adulticidal activity against the house fly. In addition, we examined the insecticidal activity of Zataria multiflora and Rosmarinus officinalis EOs on adult female house flies from pyrethroid and organophosphate resistant and susceptible populations, using both fumigant and topical bioassays. The involvement of detoxification enzymes in susceptibility was investigated with synergism experiments in vivo, while the inhibitory effects of R. officinalis and Zataria multiflora EOs on the activities of cytochrome P450-dependent monooxygenases (P450s), carboxylesterases (CarEs) and glutathione S-transferases (GSTs) were determined by enzymatic inhibition assays in vitro. The EOs of Z. multiflora, Mentha pulegium, R. officinalis and Thymus vulgaris were the most effective against adults in contact topical assays, while oils extracted from Eucalyptus cinerea, Z. multiflora, Citrus sinensis, R. officinalis, Pinus eldarica and Lavandula angustifolia where the most effective in fumigant assays. Rosmarinus officinalis and Z. multiflora EOs were selected for further investigation and showed higher toxicity against a susceptible population, compared to two insecticide-resistant populations. Correlation analysis suggested cross-resistance between these EOs and pyrethroids in the resistant populations. The toxicity of both EOs on the resistant populations was synergized by three detoxification enzyme inhibitors. Further, in vitro inhibition studies showed that R. officinalis and Z. multiflora EOs more effectively inhibited the activities of the detoxification enzymes from flies of the susceptible population compared to those of the pyrethroid resistant populations. Synergistic and enzymatic assays further revealed that increased activities of P450s, GSTs, and CarEs are possibly involved in the cross-resistance between EOs and pyrethroids. Investigating the molecular mechanisms of P450s, GSTs, and CarEs in the resistance to EOs should be subject to further studies.

Carbon monoxide releasing molecule 401 (CORM-401) modulates phase I metabolism of xenobiotics

Next to its well-studied toxicity, carbon monoxide (CO) is recognized as a signalling molecule in various cellular processes. Thus, CO-releasing molecules (CORMs) are of considerable interest for basic research and drug development. Aim of the present study was to investigate if CO, released from CORMs, inhibits cytochrome P450-dependent monooxygenase (CYP) activity and modulates xenobiotic metabolism. CORM-401 was used as a model CO delivering compound; inactive CORM-401 (iCORM-401), unable to release CO, served as control compound. CO release from CORM-401, but not from iCORM-401, was validated using the cell free myoglobin assay. CO-dependent inhibition of CYP activity was shown by 7-ethoxyresorufin-O-deethylation (EROD) with recombinant CYP and HepG2 cells. Upon CORM-401 exposure EROD activity of recombinant CYP decreased concentration dependently, while iCORM-401 had no effect. Treatment with CORM-401 decreased EROD activity in HepG2 cells at concentrations higher than 50 μM CORM-401, while iCORM-401 showed no effect. At the given concentrations cell viability was not affected. Amitriptyline was selected as a model xenobiotic and formation of its metabolite nortriptyline by recombinant CYP was determined by HPLC. CORM-401 treatment inhibited the formation of nortriptyline whereas iCORM-401 treatment did not. Overall, we demonstrate CO-mediated inhibitory effects on CYP activity when applying CORMs. Since CORMs are currently under drug development, the findings emphasize the importance to take into account that this class of compounds may interfere with xenobiotic metabolism.

In vitro evaluation of hepatotoxic drugs in human hepatocytes from multiple donors: Identification of P450 activity as a potential risk factor for drug-induced liver injuries

A possible risk factor for drug-induced hepatotoxicity is drug metabolizing enzyme activity, which is known to vary among individuals due to genetic (genetic polymorphism) and environmental factors (environmental pollutants, foods, and medications that are inhibitors or inducers of drug metabolizing enzymes). We hypothesize that hepatic cytochrome P450-dependent monooxygenase (CYP) activity is one of the key risk factors for drug induced liver injuries (DILI) in the human population, especially for drugs that are metabolically activated to cytotoxic/reactive metabolites. Human hepatocytes from 19 donors were evaluated for the activities of 8 major P450 isoforms: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Extensive individual variations were observed, consistent with what is known to be in the human population. As CYP3A4 is known to be one of the most important P450 isoforms for drug metabolism, studies were performed to evaluate the relationship between the in vitro cytotoxicity of hepatotoxic drugs and CYP3A4 activity. In a proof of concept study, hepatocytes from six donors (lots) representing the observed range of CYP3A4 activities were chosen for the evaluation of in vitro hepatotoxicity of four drugs known to be associated with acute liver failure: acetaminophen, cyclophosphamide, ketoconazole, and tamoxifen. The hepatocytes were cultured in collagen-coated plates and treated with the hepatotoxicants for approximately 24 h, followed by viability determination based on cellular adenosine triphosphate (ATP) contents. HH1023, the lot of hepatocytes with the highest CYP3A4 activity, was found to be the most sensitive to the cytotoxicity of all 4 hepatotoxic drugs, thereby suggesting that high CYP3A4 activity may be a risk factor. To further validate the relationship, a second study was performed with hepatocytes from 16 donors. In this study, the hepatocytes were quantified for CYP3A4 activity at the time of treatment. Results of the second study show confirm the correlation between with high CYP3A4 activity and sensitivity to hepatotoxic drugs. Our results with primary cultured hepatocytes from multiple donors support the hypothesis that elevated P450 activity may be a risk factor for drug-induced liver injuries.

Enzymes mediating resistance to lambda-cyhalothrin in Eriopis connexa (Coleoptera: Coccinellidae)

Resistance to widely used insecticide, lambda-cyhalothrin, was recently reported in the predatory lady beetle Eriopis connexa (Germar) (Coleoptera: Coccinellidae). However, to understand whether metabolic mechanisms underlie such resistance, synergism bioassays and in vitro studies were carried out by using inhibitors and model substrates for enzymatic assays, respectively. The LD50s estimated for susceptible and resistant populations ηg of lambda-cyhalothrin/insect, and thus, a 22-fold difference in resistance ratio. Synergism ratios for the susceptible population with piperonyl butoxide (PBO), diethyl maleate (DEM), triphenyl phosphate (TPP), and S,S,S-tributylphosphorotrithioate (DEF) were respectively 33.8-, 0.24-, 0.35-, and 4.25-fold, while for the resistant population, they were 1463.0-, 0.79-, 0.85-, and 282.6-fold, respectively. The synergized resistance ratios were 0.50-, 2.00-, 6.75-, and 8.77-fold with PBO, DEF, DEM, and TPP, respectively, while resistance was virtually suppressed with DEF. The esterase exhibited 4.16-, 4.03-, and 5.38-fold greater activity towards formation of α-naphthol, β-naphthol, and 4-nitrophenol in the resistant population of E. connexa than in the susceptible population. The activity of esterase depended on concentrations of DEF applied, either using α-naphthol or β-naphthol, which completely inhibited the activity at 636ηM. The PBO inhibited the β-naphthol formation in approximately 50%, suggesting it as inhibitor of esterases. The activities of glutathione-S-transferase were similar and corresponded to 0.36–0.47ηmol−1min−1μg of protein, for S and R populations, respectively. Similarly, the activities of cytochrome P450-dependent microsomal monooxygenases were 0.04 and 0.05ηmol−1min−1μg of protein. The native gel indicated that the formation of β-naphthol was completely inhibited by methyl-paraoxon, but only partially inhibited by eserine, TPP, and PBO. Although other studies with DEF and PBO have demonstrated strong inhibition of type B carboxylesterase associated with insecticide resistance, the results reported here do not rule out metabolism by cytochrome P450-dependent microsomal monooxygenases as a factor conferring E. connexa resistance to lambda-cyhalothrin and confirmed that PBO may also act by inhibiting esterases of insects.

Computational approach to characterization of human liver drug-metabolizing enzymes

Cytochromes P450 are the key enzymes for activating and inactivating many drugs; individual expression levels of CYPs may play a crucial role in drug safety and drug efficacy. Statistical comparison of biochemical profiles of 23 human liver microsomes have been used to characterize human liver samples. The profile included 12 parameters, namely activity of NADPH-cytochrome P450 reductase, cytochrome P450 content and cytochrome P450-dependent monooxygenase activities with marker substrates. Unsupervised statistical methods including cluster analysis and principal component analysis revealed with very high confidence the presence of two groups. Difference between the groups was explained by peculiarities of reductase activity and cytochrome P450 enzyme activities with 7-ethoxyresorufin, 7-methoxyresorufin, 7-methoxycoumarin, 7-benzyloxyresorufin and 7-benzyloxyquinoline. Results of biochemical assays coupled with multidimensional data analysis can be further used for targeted proteomic profiling of microsome oxidation mechanisms.

Precision-cut liver slices from rats of different ages: basal cytochrome P450-dependent monooxygenase activities and inducibility

The biotransformation capacity - of the cytochrome P450 (CYP) system for example - is lower but inducibility is more pronounced in neonates than in adults. On the other hand, both enzyme activities and inducibility decline with senescence. Precision-cut rat liver slices are widely used as an in vitro tool for the examination of drug toxicity, xenobiotic metabolism or enzyme induction. The aim of the present study was to assess whether age-related changes in CYP activities and induction observed in vivo are also mirrored in vitro in liver slices. For this purpose, different CYP model reactions were measured in precision-cut liver slices from one-day-old, 40-day-old and one-year-old rats after in vitro exposure to various inducers. Similar to the in vivo situation, basal CYP activities were distinctly lower and inducibility was much more pronounced in liver slices from neonatal than in those from adult animals. Also, enzyme activities were mostly somewhat lower in liver slices from aged rats compared to those from 40-day-old rats. However, CYP inducibility was less pronounced than with younger animals too. Thus, precision-cut rat liver slices are a suitable in vitro tool for investigating age-related changes in CYP activities and induction as well as developmental differences in drug metabolism and toxicity.

Effects of Cu 2+ and Pb 2+ on different fish species: Liver cytochrome P450-dependent monooxygenase activities and FTIR spectra

The effects of Cu 2+-sulfate and Pb 2+-acetate on carp ( Cyprinus carpio L.), silver carp ( Hypopthalmichtys molitrix V.) and wels ( Silurus glanis L.) were studied. The liver microsomal Cyt P450 content, the EROD, ECOD and APND monooxygenase activities were measured. In vivo treatment with 1 mg L − 1 Cu 2+ significantly elevated the activities of these enzymes and Cyt P450 content in silver carp livers. The high-dose Cu 2+ treatment (10 mg L − 1 ) on silver carp caused two-fold higher induction in the P450 dependent monooxygenase isoensymes than in wels. Although the 2 mg kg − 1 treatment with Pb 2+ in carp elevated significantly the P450 content, the EROD isoenzyme activities were significantly decreased after 1 day, showing the destructive effect of metal ion on the enzyme system. In vitro, Cu 2+ and Pb 2+ decreased the Cyt P450 content in the carp liver microsomes and the absorption peak shifted to higher wavelength. Fourier Transform Infrared (FTIR) spectroscopy was used to detect the damaging effects of the heavy metals. According to the inhibitory potency to Cu 2+, the most sensitive isoenzyme was the EROD in wels, the least was the silver carp's isoenzyme. The investigated fish P450 isoenzymes showed, that the Cu 2+ was a stronger inhibitor than Pb 2+.

In vivo effects of Urtica urens (dwarf nettle) on the expression of CYP1A in control and 3-methylcholanthrene-exposed rats

The in vivo effects of the intraperitoneal administration of an Urtica urens L. (dwarf nettle) seed extract were examined on the hepatic, pulmonary, and renal cytochrome P450-dependent monooxygenase activities of rats co-administered with 3-methylcholanthrene (MC). Urtica extract was administered by intraperitoneal injection to male Wistar rats at 200mgkg−1day−1 for 4 days from which were also co-administered with intraperitoneal injection of 50mg of MCkg−1 of body weight twice on days 1 and 3. MC treatment increased the 7-ethoxyresorufin O-deethylase (EROD) activity of the liver, lung, and kidney 54-, 21-, and 119-fold, respectively. Urtica treatment substantially reduced the 3MC induction of hepatic, lung, and renal EROD activity by 79, 42, and 50%, respectively. Similarly, compared with the control, MROD activities in liver and kidney were increased after MC administration, and these increases were significantly inhibited by Urtica. reverse transcriptase-polymerase chain reaction (RT-PCR) analysis clearly showed that the hepatic CYP1A1 and CYP1A2 mRNA levels substantially increased after treatment with MC, which was suppressed by Urtica supplementation. Western blotting studies also supported the alterations observed in the catalytic activities and mRNA levels. In conclusion, substantial reduction in CYP1A1 and CYP1A2 expression levels and related activities with Urtica are possibly associated with a potential chemoprotective ability of the Urtica due to the anticipated decrease in the activation of environmental chemical carcinogens through modulation of the CYP1A enzymes.

Cloning and expression of an atrazine inducible cytochrome P450, CYP4G33, from Chironomus tentans (Diptera: Chironomidae)

Previous studies performed in our laboratory have measured the effect of atrazine exposure on cytochrome P450-dependent monooxygenase activity and have found increased activity in midge larvae ( Chironomus tentans) as a result of atrazine exposure (1–10 ppm). Here we report the cloning and expression of a specific C. tentans CYP4 gene that is responsive to atrazine induction with an open reading frame of 1678 bp which encodes a putative protein of 559 amino acid residues. Alignments of deduced amino acid sequences with other insect P450 genes and phylogenetic analysis indicated a high degree of similarity to other insect CYP4 genes. Northern blotting analysis employing a fragment of 1200 bp from the CYP4 gene as a probe indicated that the CYP4 gene was expressed in all developmental stages, but was expressed at highest levels in late instar larvae. Additionally, over-expression of CYP4 in C. tentans exposed to atrazine (10 mg/l) confirms the ability of atrazine to induce specific P450 genes and provides insight into potential consequences of atrazine exposure in aquatic organisms.

Monooxygenase Activity in Methidathion Resistant and Susceptible Populations of Amblyseius womersleyi (Acari: Phytoseiidae)

The purpose of this study was to evaluate the cytochrome P450-dependent monooxygenase activities in methidathion resistant and susceptible strains of Amblyseius womersleyi Schicha. Artificial laboratory selections for resistance and susceptibility to methidathion were performed in an organophosphate resistant strain of A. womersleyi (Kanaya strain). Selections for susceptibility were also performed in a susceptible strain of this predaceous mite (Ishigaki Strain). After the selection process, the LC(50) of methidathion for the selected strains of A. womersleyi were 816 mg/l (Kanaya R), 4.61 mg/l (Kanaya S) and 1.59 mg/l (Ishigaki S). The monooxygenase activities were determined biochemically by the O-deethylation of 7-ethoxycoumarin (7-EC). The monooxygenase activity in adult females of Kanaya R strain (51.1 pmol/30 min/mg protein) was 3.60- and 5.42-fold higher than the activity observed for Kanaya S and Ishigaki S strains, respectively. Significant correlation between monooxygenase activity and LC(50) (mg/l) of methidathion was observed analyzing 16 populations of A. womersleyi with different susceptibilities to methidathion. Monooxygenase activity was also evaluated in different life stages (egg, larva, protonymph, deutonymph and adult) of A. womersleyi. The lowest activity was observed for the larval stage, which presented the highest susceptibility to methidathion. Protonymph, deutonymph and adult presented the highest monooxygenase activities. These stages were the most tolerant to methidathion. Monooxygenase activities of the Kanaya R strain were higher than of the Kanaya S strain in all developmental stages. The present study can be helpful for the implementation of a program involving release of insecticide-resistant populations of A. womersleyi in the field. The monooxygenase activity determination is easier and quicker than the estimation of LC(50), requiring fewer mites.

Detection of toxic effects of Cd2+ on different fish species via liver cytochrome P450-dependent monooxygenase activities and FTIR spectroscopy

The in vivo and in vitro effects of Cd2+ and the CYP1A inductor beta-naphthoflavone(beta-NF) on the hepatic cytochrome P450 (Cyt 450) monooxygenases were studied in silver carp (Hypophthalmichtys molitrix V.), wels (Silurus glanis L.), and carp (Cyprinus carpio). In vivo treatment of carp with a high dose of Cd2+ (10 mg kg(-1), for 3 days) caused a strong inhibition of 7-ethoxyresorufin-O-deethylase (EROD) and a lower inhibition of 7-ethoxycoumarin-O-deethylase (ECOD) activity. The low-dose cadmium treatment (2 mg kg(-1) Cd2+, for 6+3 days) resulted in 4-fold increase in EROD and a 3-fold increase in ECOD activity. The combined treatment with Cd2+ and beta-NF in both cases led to a loss of EROD inducibility. The silver carp and wels were treated with 10 mg L(-1) Cd2+ for 72 h in water. The Cyt P450 content in the wels liver microsomes was increased significantly after treatment for 48 h, whereas there was only a slight, not significant increase in Cyt P450 content in the silver carp microsomes. While the Cd2+ treatment resulted in inhibition of the CYP1A isoenzymes (EROD and ECOD), the APND (aminopyrene-N-demethylase, CYP2B or CYP3A isoenzyme) activity was increased 3- to 4-fold in both fish species. In vitro experiments of the effect of Cd2+ led to a concentration-dependent inhibition in all three investigated fish species. The ECOD isoenzyme of silver carp was the most sensitive to Cd2+. The lowest concentration of Cd2+ resulted in 50% inhibition. The APND isoenzyme was similarly sensitive to Cd2+ in all three investigated fish species. The most sensitive species was the wels, and the least sensitive were the carp isoenzyme. FTIR spectroscopy confirmed that cadmium caused damage to the protein structure. These results support the enzyme activity measurements measured in vivo and in vitro.

Changes in hepatic drug metabolizing enzymes and lipid peroxidation by methanol extract and major compound of Orostachys japonicus

The effects of methanol extract and gallic acid (3,4,5-trihydroxybenzoic acid) of Orostachys japonicus A. Berger on hepatic drug metabolizing enzymes and lipid peroxidation were investigated in rats treated with bromobenzene. The methanol extract of Orostachys japonicus reduced the activities of phase I enzymes, aminopyrine N-demethylase and aniline hydroxylase, that had been increased by i.p. injection of bromobenzene. Gallic acid isolated from Orostachys japonicus also reduced the aniline hydroxylase activity, while it did not affect the aminopyrine N-demethylase activity. The methanol extract and gallic acid restored the activity of epoxide hydrolase which had been decreased by bromobenzene. Hepatic glutathione content was lowered, along with increase in hepatic lipid peroxide, by bromobenzene administration. The hepatic lipid peroxidation induced by bromobenzene was prevented with the methanol extract and gallic acid of Orostachys japonicus. However, the decrease in glutathione was not altered by gallic acid. The present results suggest that the methanol extract and gallic acid of Orostachys japonicus may protect liver from bromobenzene toxicity through, at least in part, inhibiting the cytochrome P450-dependent monooxygenase activities and enhancing the activity of epoxide hydrolase. Antioxidant effect also may contribute to the protection of Orostachys japonicus against the bromobenzene-induced hepatotoxicity.

Inhibition of Cytochrome P450 Mediated Enzyme Activity by Alkylphosphocholines

The inhibitory potency of four alkylphospholipids: rac-1-O-phosphocholine-2-hydroxy-octadecane (rac-2-OH), rac-1-O-phosphocholine-2-O-acetyl-octadecane (rac-2-O-acetyl), rac-1-O-phosphocholine-2-amino-octadecane (rac-2-NH2) and rac-1-O-phosphocholine-2-N-acetyloctadecane (rac-2-N-acetyl), on the cytochrome P450-dependent monooxygenase activity has been evaluated. The IC50 values of the alkylphosphocholines with 7-ethoxycoumarin as substrate in liver microsomal fractions of PB-treated rats and with a reconstituted CYP2B1: NADPH-P450-reductase system are in the range of 3.2-5.0 microM and 2.8-3.5 microM, respectively. Lineweaver-Burk plots with the inhibitors in concentrations that were found to cause roughly a 50% inhibition and with 7-ethoxycoumarin as substrate revealed for all four alkylphospholipids a competitive inhibition type. The degree of the competitive inhibition is quantified by the Ki values. With liver microsomal fractions of PB-treated rats, the Ki values of rac-2-OH (Ki = 1.36 microM) and rac-2-O-acetyl (Ki = 1.33 microM) differs slightly from those of rac-2-NH2 (Ki = 2.2 microM) and rac-2-N-acetyl (Ki = 2.2 microM), but with the reconstituted CYP2B1: NADPH-P450-reductase system all Ki values are in the small range of 1.8 - 2.6 microM, indicating that the short substituted group at the 2-position (OH; O-acetyl; NH2; N-acetyl) of the long chain octadecanol part of the phosphodiesters exhibit no essential role on the strong inhibitory potency of these alkylphosphocholines on the 7-ethoxycoumarin-O-deethylase activity.

Atrazine induction of a family 4 cytochrome P450 gene in Chironomus tentans (Diptera: Chironomidae)

Cytochrome P450-dependent microsomal monooxygenase (P450) activity was measured in control and atrazine-exposed third instar midge larvae, Chironomus tentans. Significantly elevated O-demethylase activity was observed in gut homogenates taken from midges exposed to atrazine concentrations from 1 to 10 ppm for 90 h. No significant induction was observed at atrazine concentrations below 1 ppm. A region of a cytochrome P450 family 4 gene was amplified and sequenced from C. tentans larvae. Alignments of inferred amino acid sequences with other insect CYP4 gene homologues indicate a high degree of similarity. Northern blot analysis employing the CYP4 gene fragment as a probe showed an over-expression in C. tentans exposed to atrazine. The results support the previously identified inducibility of cytochrome P450-dependent activity and provide insight into the potential consequences of atrazine exposure to aquatic organisms.

In vivo and in vitro metabolism of fipronil by larvae of the European corn borer Ostrinia nubilalis.

In vivo and in vitro metabolism of [14C]fipronil was examined in a susceptible European corn borer (Ostrinia nubilalis, Hübner) laboratory strain. [14C]Fipronil penetrated the larval integument slowly, with 71.5% of the applied radioactivity recovered from surface rinses 24 h after topical application. Despite this slow penetration, radioactivity was detected in both the excrement and internal organo-soluble fractions. Radioactivity in the internal aqueous fraction and tissue pellet accounted for less than 0.8% of total radioactivity. The in vivo studies suggest that fipronil oxidation to its sulfone metabolite is the major route of metabolic conversion. In vitro studies were performed using subcellular microsomal fractions isolated from European corn borer larval midguts. Cytochrome P450-dependent monooxygenase activity (methoxyresorufin O-demethylase) was consistently observed in midgut preparations, and formation and detection of the sulfone metabolite in the same midgut preparations was also NADPH-dependent and inhibited by piperonyl butoxide. In vitro metabolism results indicate microsomal monooxygenases are responsible for the conversion of fipronil to its sulfone form in the European corn borer.

EFFECT OF ALACHLOR ON HEPATIC CYTOCHROME P450 ENZYMES IN RATS

Alachlor ((2-chloro-N-methoxymethyl)-N-(2,6-diethylphenyl)acetamide) is a widely used preemergence herbicide which has been classified by the USEPA as a probable human carcinogen. The herbicide has been suggested to be metabolized by hepatic cytochrome P450 system. We examined the effects of alachlor on cytochrome P450 enzymes in rat liver microsomes. Rats were treated intraperitoneally with alachlor daily for 5 days, at doses of 25, 50 and 100 mg/kg. Among the cytochrome P450-dependent monooxygenase activities, 7-pentoxyresorufin O-depentylase, which is associated with CYP2B1, was dose-dependently increased by alachlor. The induction relative to control activity was 1.7–4.2-fold. The activities of CYP1A-dependent monooxygenases such as 7-ethoxyresorufin O-deethylase and acetanilide 4-hydroxylase were also significantly increased by alachlor at doses of 50 and 100 mg/kg (1.7–2.1-fold). Furthermore, immunoblotting showed that alachlor significantly increased CYP2B1/2 and CYP1A1/2 protein levels by 4.2–6.3- and 1.8-fold, respectively. Although 7-ethoxycoumarin O-deethylase, bufuralol 1′-hydroxylase and 4-nitrophenol 2-hydroxylase activities were significantly increased by alachlor at higher doses (≥ 50 mg/kg), the induction ratios were less than 1.6-fold. The activities of other cytochrome P450-dependent monooxygenases, namely testosterone 7α-hydroxylase, testosterone 2α-hydroxylase, testosterone 6β-hydroxylase and lauric acid ω-hydroxylase, were not affected by alachlor at any dose. In addition, there was no significant change in the protein levels of CYP2C11/6, CYP2D1, CYP2E1, CYP3A2/1 and CYP4A1/2/3. These results suggest that alachlor selectively induces cytochrome P450 isoforms of the CYP1A and CYP2B subfamilies in rat liver microsomes, and that the expression of these isoforms is closely related to the toxicity of alachlor.

Propranolol as an inhibitor of some cytochrome P450-dependent monooxygenase activities in native and induced rat liver microsomes.

The in vitro effect of propranolol (10(-3) M and 10(-4) M), a nonselective and extensively metabolized beta-adrenergic blocking agent, on rat liver drug metabolism in native and induced (with phenobarbital and beta-naphthoflavone [beta-NF]) microsomes was studied. The type of inhibition and the inhibitory constants of some cytochrome P450-dependent microsomal enzyme reactions (hexobarbital oxidation [HBO], ethylmorphine-N-demethylation [EMND], aniline hydroxylation [AH], ethoxycoumarin-O-deethylation [ECOD], ethoxyresorufin-O-dealkylation [EROD] and penthoxyresorufin-O-dealkylation [PROD]) were estimated. The results showed that propranolol competitively inhibited AH activity in native microsomes. The type of inhibition was changed from competitive to noncompetitive in all other enzyme activities studied. This inhibition was more pronounced after phenobarbital induction in PROD (Ki = 0.11 +/- 0.01 mM), ECOD (Ki = 0.40 +/- 0.09 mM) and EMND (Ki = 0.59 +/- 0.1 mM), and after beta-NF induction in AH (Ki = 0.28 +/- 0.05 mM) and in HBO (Ki = 0.35 +/- 0.1 mM) in native microsomes. It was assumed that the noncompetitive type of inhibition is due to the covalent binding of reactive metabolites derived from propranolol to hepatic microsomal proteins. The competitive type of inhibition of AH suggested a common P450 isoenzyme in the metabolism of propranolol and aniline. Thus, in this study, propranolol has been found to be not only a selective inhibitor of CYP2D6 isoenzyme-dependent reactions, but also a nonspecific inhibitor of other cytochrome P450 isoenzymes.

Bioavailability of PAHs in the Biobio river (Chile): MFO activity and biliary fluorescence in juvenile Oncorhynchus mykiss

Cytochrome P450-dependent monooxygenase (MFO) activity and levels of bile PAH metabolites were measured in fish ( Oncorhynchus mykiss) exposed to Biobio river (Chile) water. Experiments were performed simultaneously in both the field and laboratory to investigate whether the river water contained notable bioavailable PAH fractions. The field experiment was conducted using fish caged for 41 days at La Mochita (Biobio river mouth) whereas the laboratory experiment involved a 21-day exposure treatment with water collected in the same area. Induction of hepatic MFO, assayed by benzo(a)pyrene monooxygenase activity (BaPMO), and levels of PAH metabolites in the bile were measured in groups of eight (field experiment) and four specimens (laboratory experiment) sampled after 5, 10, 21 and 41 days of exposure. BaPMO was induced by a factor of 23 and 25 in fish caged for 21 and 41 days, respectively. Likewise, a significant BaPMO induction was found in the fish group experimentally exposed to river water for 21 days. Biliary fluorescence measurements, recorded by fixed wavelength fluorescence (FF) and synchronous fluorescence spectroscopy (SFS), revealed significantly high levels of PAH metabolites in bile samples of fish caged for 21 and 41 days. A positive relationship was found between BaPMO activity and biliary PAH metabolites. This study provides evidences that (i) Biobio river water contains a significant bioavailable PAH fraction in terms of marked effects on fish and (ii) the biological indicators MFO activity and bile PAH metabolites represent an excellent screening methodology to assess PAH exposure.

Complexation of Membrane-Bound Enzyme Systems

The effect of changes in the N-terminal membrane-binding domain of cytochrome P450 forms and NADPH-cytochrome P450 reductase types on the cytochrome P450-dependent monooxygenase activities, has been examined. The nifedipine oxidase activity of two human P450 forms (CYP3A4, CYP3A4NF14) which differ only in their primary structure by ten amino acid residues in the N-terminal membrane-binding domain, yields nearly the same catalytic cycle time tau =2.65 +/- 0.15 s, due to their identical cytosolic catalytic protein structure. In contrast, the complex formation process ([P450]+[reductase] <--> [complex]) described by the dissociation constant KD, at high substrate concentration ([S]>>KS) and low product concentration ([P]<<KP) is determined to be KD/[P4501]o = 0.3 and 2.0, respectively. These values reflect large differences in the affinity of both P450 forms for the same type of reductase which is only due to their modified membrane-binding domains. In the present work, it has been shown for the first time, that the membrane-binding domain of cytochrome P450 enzymes determines the complexation process of the binary P450:reductase system. Furthermore, the nifedipine oxidase activity of the human CYP3A4 form reconstituted with two different types of reductase from human and rabbit also has the same catalytic cycle time tau = 2.65 +/- 0.15 s. This result is based on the similarity of the primary structure of the cytosolic catalytic domain of both reductase types. However, the complex was formed with different dissociation constants of KD/[P450]o = 0.3 and 4.7, respectively. This different affinity of both reductase types to the same P450 form is interpreted as a consequence of the substantial alteration of the amino acids in the N-terminal primary structure of their membrane-binding domains. 7-Ethoxycoumarin O-deethylase activity of two rat P450 forms (CYP2B1 and CYP1A1) were reconstituted with the same rat reductase. The catalytic cycle time for each P450 form is tau = 1.8 and 0.6 s, respectively. Correspondingly, the complex formation process controlled by the dissociation constant KD has changed from KD/[P450]o = 2.3 to 1.7, respectively. This is because both forms differ in their cytosolic as well as in their membrane-binding domains.

Cytochrome P450-Dependent Monooxygenase Activities and Their Inducibility by Classic P450 Inducers in the Liver, Kidney, and Nasal Mucosa of Male Adult Ring-Necked Pheasants

In this study, several P450-dependent monoxygenase activities in the liver, kidney, and nasal mucosa of ring-necked pheasants were examined. In addition, the presence and inducibility of P450 isoenzymes in the hepatic and renal tissues of pheasants were examined by using typical substrates and inducers of P450s along with polyclonal antibodies raised against mammalian isoforms. Anti-rat P450 1A1 recognized in microsomes of both pheasant liver and kidney a protein that was markedly induced by β-naphthoflavone and accompanied by an increase of various monooxygenases, in particular, methoxyresorufin-O-demethylase (MROD) activity. Anti-rat P450 2E1 revealed in microsomes of the pheasant liver but not in kidney an immunoreactive protein that was slightly induced by acetone but not accompanied by an increase of para-nitrophenol hydroxylase activity. On the other hand, acetone treatment caused an induction of other hepatic monoxygenases including MROD, erythromycin N-demethylase, and 6β-testosterone hydroxylase. These two latter activities, known to be markers for 3A isoenzymes in rodents, were also enhanced in pheasant liver by phenobarbital but not by dexamethasone. The treatment with these two inducers also lacked to point out hepatic and renal proteins immunorelated to P450 3A or 2B subfamily, suggesting that these isoforms may be not expressed in pheasant. On the other hand, anti-rat P450 2C11 recognized two immunorelated proteins in the liver of both control and treated pheasants. The treatment with clofibrate, a mammalian inducer of 4A subfamily, induced both in liver and kidney of pheasant: i) a protein that cross-reacted with anti rat P450 4A1 and ii) the (ω) and (ω−1) lauric acid hydroxylase activities, known to be associated in mammals to this P450 subfamily. In the nasal mucosa of pheasant, a protein immunorelated to P450 2A and some monooxygenase activities (i.e., 7-ethoxycoumarin O-deethylase) linked, in mammals, to this isoform have been found; by contrast a protein immunoreactive with anti P450 2G1 was not found. In conclusion, the immunochemical properties and monooxygenase activities of constitutive and inducible P450s in pheasants were different not only from those of mammals but also from those of chickens. The findings of the present work also suggest that the P450 induction profiles might provide a potential biomarker of pheasant exposure to chemicals or environmental pollutants in the wild-field or in the stock-farm.