7-ethoxyresorufin O-deethylation Research Articles

Differentiation-associated urothelial cytochrome P450 oxidoreductase predicates the xenobiotic-metabolizing activity of "luminal" muscle-invasive bladder cancers.

Extra‐hepatic metabolism of xenobiotics by epithelial tissues has evolved as a self‐defence mechanism but has potential to contribute to the local activation of carcinogens. Bladder epithelium (urothelium) is bathed in excreted urinary toxicants and pro‐carcinogens. This study reveals how differentiation affects cytochrome P450 (CYP) activity and the role of NADPH:P450 oxidoreductase (POR). CYP1A1 and CYP1B1 transcripts were inducible in normal human urothelial (NHU) cells maintained in both undifferentiated and functional barrier‐forming differentiated states in vitro. However, ethoxyresorufin O‐deethylation (EROD) activity, the generation of reactive BaP metabolites and BaP‐DNA adducts, were predominantly detected in differentiated NHU cell cultures. This gain‐of‐function was attributable to the expression of POR, an essential electron donor for all CYPs, which was significantly upregulated as part of urothelial differentiation. Immunohistology of muscle‐invasive bladder cancer (MIBC) revealed significant overall suppression of POR expression. Stratification of MIBC biopsies into “luminal” and “basal” groups, based on GATA3 and cytokeratin 5/6 labeling, showed POR over‐expression by a subgroup of the differentiated luminal tumors. In bladder cancer cell lines, CYP1‐activity was undetectable/low in basal PORlo T24 and SCaBER cells and higher in the luminal POR over‐expressing RT4 and RT112 cells than in differentiated NHU cells, indicating that CYP‐function is related to differentiation status in bladder cancers. This study establishes POR as a predictive biomarker of metabolic potential. This has implications in bladder carcinogenesis for the hepatic versus local activation of carcinogens and as a functional predictor of the potential for MIBC to respond to prodrug therapies.

Effects of Pterostilbene on Activities and Protein Expression of Cytochrome P450 1A1 (CYP1A1) and Glutathione S-Transferase (GST) in Benzo[a]pyrene-Induced HT-29 Colorectal Cancer Cell Line

Drug Metabolizing Enzyme (DME) has been a target of natural chemopreventive agents to inhibit, retard and reverse the process of carcinogenesis. Pterostilbene, an analog to resveratrol has been reported to possess various pharmacological benefits including chemoprevention. In our study, benzo[a]pyrene-induced HT-29 colorectal cell line was used as the DME model. The activity of phase I enzyme CYP1A as determined by the 7-ethoxyresorufin O-deethylation (EROD) assay was found to be inhibited significantly by pterostilbene at 50 µM, 75 µM and 100 µM (p ≤ 0.01, p ≤ 0.05, p ≤ 0.01 respectively) compared to the benzo[a]pyrene treated group. Meanwhile, pterostilbene induced glutathione-S-transferase (GST) activity significantly (p ≤ 0.01) at 50 µM as compared to the untreated. In addition, However, the protein expression of CYP1A1 and GST in pterostilbene treated group was not significantly affected compared to untreated. On the other hand, pterostilbene at 25 and 75 µM were able to increase the protein expression of transcription factor Nrf2 significantly (p ≤ 0.01). Results indicated that pterostilbene could reduce metabolic activation of procarcinogens and increase the detoxification process which can be potentially developed as chemopreventive agent. DOI : http://dx.doi.org./10.17576/JSKM-2018-05

Effects of Pterostilbene on Activities and Protein Expression of Cytochrome P450 1A1 (CYP1A1) and Glutathione S-Transferase (GST) in Benzo[a]pyrene-Induced HT-29 Colorectal Cancer Cell Line

Drug Metabolizing Enzyme (DME) has been a target of natural chemopreventive agents to inhibit, retard and reverse the process of carcinogenesis. Pterostilbene, an analog to resveratrol has been reported to possess various pharmacological benefits including chemoprevention. In our study, benzo[a]pyrene-induced HT-29 colorectal cell line was used as the DME model. The activity of phase I enzyme CYP1A as determined by the 7-ethoxyresorufin O-deethylation (EROD) assay was found to be inhibited significantly by pterostilbene at 50 µM, 75 µM and 100 µM (p ≤ 0.01, p ≤ 0.05, p ≤ 0.01 respectively) compared to the benzo[a]pyrene treated group. Meanwhile, pterostilbene induced glutathione-S-transferase (GST) activity significantly (p ≤ 0.01) at 50 µM as compared to the untreated. In addition, However, the protein expression of CYP1A1 and GST in pterostilbene treated group was not significantly affected compared to untreated. On the other hand, pterostilbene at 25 and 75 µM were able to increase the protein expression of transcription factor Nrf2 significantly (p ≤ 0.01). Results indicated that pterostilbene could reduce metabolic activation of procarcinogens and increase the detoxification process which can be potentially developed as chemopreventive agent. DOI : http://dx.doi.org./10.17576/JSKM-2018-05

Variation in the Response of Clozapine Biotransformation Pathways in Human Hepatic Microsomes to CYP1A2- and CYP3A4-selective Inhibitors.

The atypical antipsychotic agent clozapine (CLZ) is effective in many patients who are resistant to conventional antipsychotic drugs. Cytochromes P450 (CYPs) 1A2 and 3A4 oxidize CLZ to norCLZ and CLZ N-oxide in human liver. Concurrent treatment with inducers and inhibitors of CYP1A2 modulates CLZ elimination that disrupts therapy. Drug-drug interactions involving CYP3A4 are also significant but less predictable. To further characterize the factors underlying these interactions, we used samples from a cohort of human livers to assess variation in CLZ oxidation pathways in relation to intrinsic CYP3A4 and CYP1A2 activities and the effects of the corresponding selective inhibitors ketoconazole (0.2 and 2 μM) and fluvoxamine (1 and 10 μM). The CYP3A4-selective inhibitor ketoconazole (2 μM) impaired CLZ N-oxide formation in all 14 of the livers used in inhibition studies (≥50% inhibition) while the CYP1A2-selective inhibitor fluvoxamine (10 μM) decreased norCLZ formation in nine. Ketoconazole effectively inhibited CLZ metabolism in five of seven livers that catalysed CYP3A4-dependent testosterone 6β-hydroxylation at or above the median rate and in four other livers with lower intrinsic CYP3A4 activity. Similarly, fluvoxamine (10 μM) readily inhibited CLZ oxidation in seven livers with high CYP1A2-mediated 7-ethoxyresorufin O-deethylation activity (at or above the median) and three livers with lower intrinsic CYP1A2 activity. In three livers, CLZ biotransformation was impaired by both ketoconazole and fluvoxamine, consistent with a major role for both CYPs. These findings suggest that the intrinsic activities of CYPs 1A2 and 3A4 are unrelated to the response to CYP-selective inhibitors and that assessment of the activities in vivo may not assist the prediction of drug-drug interactions.

Thyrotoxic rubber antioxidants, 2-mercaptobenzimidazole and its methyl derivatives, cause both inhibition and induction of drug-metabolizing activity in rat liver microsomes after repeated oral administration

We examined the effects of thyrotoxic rubber antioxidants, 2-mercaptobenzimidazole (MBI, 0.3 mmol/kg/day) and its methyl derivatives, methyl-MBIs [4-methyl-MBI (4-MeMBI, 0.6 mmol/kg/day), 5-methyl-MBI (5-MeMBI, 0.6 mmol/kg/day), and 4(or 5)-methyl-MBI (4(5)-MeMBI, 0.6 or 1.2 mmol/kg/day)], on the drug-metabolizing activity in male rat liver microsomes by 8-day repeated oral administration. The weight of liver and thyroid were increased by all the test chemicals; MBI was most potent, and there was no additive or synergistic effect between 4-MeMBI and 5-MeMBI. MBI decreased the cytochrome P450 (CYP) content, NADPH-cytochrome P450 reductase (POR) activity, 7-ethoxycoumarin O-deethylation (ECOD) activity, and flavin-containing monooxygenase (FMO) activity, but increased the 7-pentoxyresorufin O-depentylation (PROD) activity, suggesting inhibition of the drug-metabolizing activity on the whole but induce some activities such as the CYP2B activity. On the contrary, all the methyl-MBIs increased the CYP content, CYB5 content, ECOD activity, 7-ethoxyresorufin O-deethylation (EROD) activity, and PROD activity, indicating that they are mostly inducible of the CYP activity. However, the methyl-MBIs decreased the FMO activity, and 5-MeMBI and 4(5)-MeMBI appeared inhibitory for CYPs 2C11 and 2C13. Between 4-MeMBI and 5-MeMBI, there was no additive or synergistic effect on the drug-metabolizing activity, but was counteraction. It was concluded that MBI and methyl-MBIs had both inhibitory and inducible effects on the drug-metabolizing activity in rat liver microsomes at thyrotoxic doses. The effects of 4(5)-MeMBI indicated that the increased liver weight alone can be a hepatotoxic sign but not an adaptive no-adverse response in toxicity studies. The present results were related to the toxicokinetic profiles of MBI and 4(5)-MeMBI in the repeated toxicity studies.

Effects of lemongrass oil and citral on hepatic drug-metabolizing enzymes, oxidative stress, and acetaminophen toxicity in rats

The essential oil from a lemongrass variety of Cymbopogon flexuosus [lemongrass oil (LO)] is used in various food and aroma industry products and exhibits biological activities, such as anticancer and antimicrobial activities. To investigate the effects of 200 LO (200 mg/kg) and 400 LO (400 mg/kg) and its major component, citral (240 mg/kg), on drug-metabolizing enzymes, oxidative stress, and acetaminophen toxicity in the liver, male Sprague-Dawley rats were fed a pelleted diet and administered LO or citral by gavage for 2 weeks. After 2 weeks of feeding, the effects of LO and citral on the metabolism and toxicity of acetaminophen were determined. The results showed that rats treated with 400 LO or citral had significantly reduced hepatic testosterone 6β-hydroxylation and ethoxyresorufin O-deethylation activities. In addition, NAD(P)H:quinone oxidoreductase 1 activity was significantly increased by citral, and Uridine 5′-diphospho (UDP) glucurosyltransferase activity was significantly increased by 400 LO in the rat liver. Treatment with 400 LO or citral reduced lipid peroxidation and reactive oxygen species levels in the liver. After acetaminophen treatment, however, LO and citral treatment resulted in little or no change in plasma alanine aminotransferase activity and acetaminophen-protein adducts content in the liver. Our results indicate that LO and citral may change the activities of drug-metabolizing enzymes and reduce oxidative stress in the liver. However, LO and citral may not affect the detoxification of acetaminophen.

CYP1A1 I462V polymorphism is associated with reduced genotoxicity in yeast despite positive association with increased cancer risk

CYP1A1 functions in detoxifying xenobiotics but occasionally converts compounds into potent genotoxins. CYP1A1 activates polyaromatic hydrocarbons, such as benzo[a]pyrene 7,8 dihydrodiol (BaP-DHD), rendering them genotoxic. Particular alleles of CYP1A1, such as CYP1A1 I462V have been correlated with a higher incidence of breast and lung cancer, but it is unknown whether these variants express enzymes in vivo that are more potent in generating genotoxins. We individually expressed CYP1A1 (CYP1A1.1), CYP1A1 T461N (CYP1A1.4) and I462V (CYP1A1.2) alleles in wild-type and DNA repair deficient mutant strains of Saccharomyces cerevisiae (budding yeast) and asked which yeast strains exhibited the highest levels of carcinogen-associated genotoxicity after exposure to BaP-DHD, aflatoxin B1 (AFB1), and heterocyclic aromatic amines (HAAs). We measured carcinogen-associated recombination, Rad51 foci, and carcinogen-associated toxicity in a DNA repair mutant deficient in both nucleotide excision repair and recombinational repair. CYP1A1 activity was confirmed by measuring ethoxyresorufin-O-deethylation (EROD) activities. Our data indicate that CYP1A1 I462V allele confers the least carcinogen-associated genotoxicity, compared to CYP1A1; however, results vary depending on the chemical carcinogen and the genotoxic endpoint. We speculate that the cancer-associated risk of CYP1A1 I462V may be caused by exposure to other xenobiotics.

Up-regulation of CYP1A1 and phase II enzymes by 5-ring isomeric polycyclic aromatic hydrocarbons in precision-cut rat hepatic slices: Importance of molecular shape

The objectives of the present study were two-fold: (a) to evaluate the role of molecular shape on the interaction of polycyclic aromatic hydrocarbons (PAHs) with the Ah receptor and CYP1A1 upregulation, and (b) to evaluate the potential of PAHs to induce epoxide hydrolase and glutathione S-transferase, two major enzymes involved in their metabolism. In order to achieve these objectives, precision-cut rat liver slices were incubated with a range of concentrations of seven 5-ring isomeric PAHs, namely benzo[c]chrysene, benzo[b]chrysene, benzo[g]chrysene, dibenzo[a,j]anthracene, dibenzo[a,c]anthracene, picene and pentacene, for 24h. All compounds, with the exception of pentacene, elevated the O-deethylation of ethoxyresorufin, an activity associated with CYP1A1; induction of this enzyme by the various PAHs correlated with their avidity for the Ah receptor. None of the PAHs studied increased epoxide hydrolase activity, monitored using benzo[a]pyrene 4,5-oxide. Of the seven PAHs, only benzo[g]chrysene elevated glutathione S-transferase activity, measured using 1-chloro-2,4-dinitrobenzene or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole as substrates. No relationship could be established between length or length/width and interaction with the Ah receptor and CYP1A1 up-regulation indicating that other structural or electronic factors are likely to be more important. Finally, 5-ring PAHs are poor inducers of the epoxide hydrolase and glutathione S-transferase enzyme systems.

Effectiveness of the liver micronucleus assay using juvenile mice.

This study investigated the effectiveness of the liver micronucleus (MN) assay using juvenile mice. Therefore, we analyzed various hepatic cytochrome P450 (CYP)- mediated activities of ethoxyresorufin O-deethylation, pentoxyresorufin O-dealkylation, tolbutamide hydroxylation, bufuralol 1’-hydroxylation, aniline hydroxylation and midazolam 4-hydroxylation by CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A, respectively, in non-treated male ICR mice aged between 3 and 8 weeks. The enzyme efficiency levels in 3- and 4-week-old mice were approximately similar to or higher than those in 8-week-old mice, except for CYP1A and CYP2E in 3- and 4-week-old mice, respectively. Since these results suggest that juvenile mice have sufficient activities for most CYP enzymes, we also conducted a liver MN assay using diethylnitrosamine (DEN), a rodent hepatocarcinogen, on male ICR mice aged between 3 and 6 weeks. A peripheral blood (PB) MN assay was performed simultaneously in 4-week-old mice. Assays incorporating DEN produced positive results in 3- and 4-week-old mice and showed a dose-dependent increase in the micronucleated hepatocyte frequencies at 4 weeks. Both the liver MN assay in 5- and 6-week-old mice and the PB MN assay had negative results when using DEN. These results suggest that 3- and 4-week-old mice have micronuclei-inducing potential in the liver to detect genotoxic compounds using the liver MN assay.

Influence of Sulforaphane Metabolites on Activities of Human Drug-Metabolizing Cytochrome P450 and Determination of Sulforaphane in Human Liver Cells.

The influence of metabolites of sulforaphane, natural compounds present in broccoli (Brassica oleracea var. botrytis italica) and in other cruciferous vegetables, on drug-metabolizing cytochrome P450 (CYP) enzymes in human liver microsomes and possible entry of sulforaphane into human hepatic cells were investigated. Metabolites studied are compounds derived from sulforaphane by the mercapturic acid pathway (conjugation with glutathione and by following reactions), namely sulforaphane glutathione and sulforaphane cysteine conjugates and sulforaphane-N-acetylcysteine. Their possible effect on four drug-metabolizing CYP enzymes, CYP3A4 (midazolam 1'-hydroxylation), CYP2D6 (bufuralol 1'-hydroxylation), CYP1A2 (7-ethoxyresorufin O-deethylation), and CYP2B6 (7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation), was tested. Inhibition of four prototypical CYP activities by sulforaphane metabolites was studied in pooled human liver microsomes. Sulforaphane metabolites did not considerably affect biological function of drug-metabolizing CYPs in human liver microsomes except for CYP2D6, which was found to be inhibited down to 73-78% of the original activity. Analysis of the entry of sulforaphane into human hepatocytes was done by cell disruption by sonication, methylene chloride extraction, and modified high-performance liquid chromatography method. The results have shown penetration of sulforaphane into the human hepatic cells.

P.3.c.005 - Potent inhibition of CYP1A2 and CYP2D6 by the novel antipsychotic drug asenapine in human liver

In this study, we performed a screening of the specificities of rat cytochrome P450 (CYP) isoforms for metabolic reactions known as the specific probes of human CYP isoforms, using 13 rat CYP isoforms expressed in baculovirus-infected insect cells or B-lymphoblastoid cells. Among the metabolic reactions studied, diclofenac 4-hydroxylation (DFH), dextromethorphan O-demethylation (DMOD) and midazolam 4-hydroxylation were specifically catalyzed by CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. These results suggest that diclofenac 4-hydroxylation, dextromethorphan O-demethylation and midazolam 4-hydroxylation are useful as catalytic markers of CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. On the other hand, phenacetin O-deethylation and 7-ethoxyresorufin O-deethylation were catalyzed both by CYP1A2 and by CYP2C6. Benzyloxyresorufin O-dealkylation and pentoxyresorufin O-dealkylation were also catalyzed by CYP1A2 in addition to CYP2B1. Bufuralol 1′-hydroxylation was extensively catalyzed by CYP2D2 but also by CYP2C6 and CYP2C11. p-Nitrophenol 2-hydroxylation and chlorzoxazone 6-hydroxylation were extensively catalyzed by CYP2E1 but also by CYP1A2 and CYP3A1. Therefore, it is necessary to conduct further study to clarify whether these activities in rat liver microsomes are useful as probes of rat CYP isoforms. In contrast, coumarin 7-hydroxylation and S- and R-mephenytoin 4′-hydroxylation did not show selectivity toward any isoforms of rat CYP studied. Therefore, activities of coumarin 7-hydroxylation and S- and R-mephenytoin 4′-hydroxylation are not able to be used as catalytic probes of CYP isoforms in rat liver microsomes. These results may provide useful information regarding catalytic probes of rat CYPs for studies using rat liver microsomal samples.

Impaired dacarbazine activation and 7-ethoxyresorufin deethylation in vitro by polymorphic variants of CYP1A1 and CYP1A2: implications for cancer therapy.

To extend our understanding of how interindividual variability mediates the efficacy of cancer treatment. The kinetics of dacarbazine (DTIC) N-demethylation by the most frequent polymorphic variants of CYP1A1 (T461N, I462V) and CYP1A2 (F186L, D348N, I386F, R431W, R456H) were characterized, along with kinetic parameters for the O-deethylation of the prototypic CYP1A substrate 7-ethoxyresorufin, using recombinant protein expression and high-performance liquid chromatographic techniques. A reduction of ∼30% in the catalytic efficiencies (measured as in-vitro intrinsic clearance, CLint) was observed for DTIC N-demethylation by the two CYP1A1 variants relative to wild type. Although a modest increase in the CLint value for DTIC N-demethylation was observed for the CYP1A2 D348N variant relative to the wild type, the CLint for the F186L variant was reduced and the I386F, R431W, and R456H variants all showed loss of catalytic function. Comparison of the kinetic data for DTIC N-demethylation and 7-ethoxyresorufin O-deethylation indicated that alterations in the kinetic parameters (Km, Vmax, CLint) observed with each of the CYP1A1 and CYP1A2 polymorphic variants were substrate dependent. These data indicate that cancer patients treated with DTIC who possess any of the CYP1A1-T461N and I462V variants or the CYP1A2-F186L, D348N, I386F, R431W, and R456H variants are likely to have decreased prodrug activation, and hence may respond less favorably to DTIC treatment compared with individuals with wild-type CYP1A alleles.

Effects of inducers of cytochrome P450s on enrofloxacin N-deethylation in crucian carp Carassius auratus gibelio.

In this study with crucian carp (Carassius auratus gibelio), the effect on enrofloxacin (EF) and its metabolite ciprofloxacin (CF) and on the activity of cytochrome P450 1A (CYP1A) and cytochrome P450 3A (CYP3A) was estimated following the oral administration of rifampicin (RIF) (12mg/kg) and β-naphthoflavone (BNF) (12mg/kg), respectively. First, reversed-phase high-performance liquid chromatography (RP-HPLC) was used to detect the pharmacokinetics of EF with continual blood sampling. In RIF-treated, BNF-treated and control groups, the value of the CmaxCF/CmaxEF ratio was 4.41, 0.81 and 0.95, and the corresponding value of the AUC0-t-CF/AUC0-t-EF ratio was 3.69, 1.84 and 1.76, respectively. In the RIF-treated, BNF-treated and control groups, the MRT values of EF were 26.57, 27.45 and 30.88h, and the corresponding values for CF were 5.79, 35.18 and 38.11h, respectively. Based on these results for crucian carp, the accumulation and elimination of EF and CF in the RIF-treated group were more rapid than in BNF-treated and control groups. Second, liver microsomes were pretreated with the inducer of CYP1A for BNF and that of CYP3A for RIF, and then the enzymatic activities of CYP1A and CYP3A were measured, respectively. The activities of ethoxyresorufin-O-deethylation (EROD) and erythromycin-N-demethylation (ERND) increased significantly (P<0.05) for CYP1A and CYP3A, respectively. However, in further experiments on the formation of CF, the level of EF N-deethylation was significantly induced by RIF and inhibited by ketoconazole (KTZ) for CYP3A but had no influence for CYP1A, BNF and berberine chloride (BER). We concluded that CYP3A might be responsible for the N-deethylation of EF and because of this activity, could also serve as a toxicity biomarker in crucian carp.

Fipronil induces CYP isoforms in rats

The goal of the present study was to evaluate fipronil effects on the activities of drug metabolizing enzymes in rat liver microsomes. Rats were orally treated with fipronil at doses of 1, 5, 10 and 15 mg/kg bw/day for 6 days. Determinations of cytochrome P450 (CYP) enzyme activities were carried out in hepatic microsomes isolated from treated rats. The activities of some members of CYP2E, CYP1A, CYP2A, CYP2B and CYP3A subfamilies significantly increased after fipronil treatment in a dose-dependent manner as compared to control. The major effects were observed in the O-deethylation of ethoxyresorufin and O-demethylation of methoxyresorufin (reflecting CYP1A1/2 activities), in the O-depenthylation of pentoxyresorufin and 16β-hydroxylation of testosterone (reflecting CYP2B1/2 activities), and in the N-demethylation of erythromycin and 6β-hydroxylation of testosterone (reflecting CYP3A1/2 activities). Immunoblot studies revealed that fipronil increased the apoprotein levels of CYP1A1. Our results suggest that fipronil is an inducer of hepatic phase I CYP enzymes, causing an increased potential to interact with a wide range of xenobiotics or endogenous chemicals that are substrates of the CYP1A, CYP2B and CYP3A subfamilies. Further investigations are required to in vivo evaluate the potential of the metabolite fipronil sulfone as an inducer of phase I CYP enzymes.

Interaction of selected platinum(II) complexes containing roscovitine-based CDK inhibitors as ligands with human liver microsomal cytochrome P450

We studied the interaction of oxaliplatin derivatives involving cytotoxic adenine-based cyclin-dependent kinase inhibitors, with human liver microsomal cytochrome P450. The activities of 9 human liver microsomal CYP forms (CYPs 1A2, 7-ethoxyresorufin O-deethylation; 2A6, coumarin 7-hydroxylation; 2B6, 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation; 2C8, luciferin-6´ methyl ether demethylation; 2C9, diclofenac 4´-hydroxylation, 6´-deoxyluciferin hydroxylation; 2C19, (S)-mephenytoin 4´-hydroxylation; 2D6, bufuralol 1´-hydroxylation, 2E1, chlorzoxazone 6-hydroxylation; 3A4, testosterone 6β-hydroxylation, luciferin-6´ benzyl ether debenzylation) were tested using HPLC, fluorescence and luminescence product detection. At 100 µM platinum(II) oxalato complex concentration, CYP inhibition was in general 25%-50%, except for the CYP3A4 form which showed roughly twice the inhibition (72%-95%). At low complex concentration (10 µM), the difference in inhibition of CYP3A4 and other forms was even more pronounced. Dixon and Lineweaver-Burk plots indicated a partially noncompetitive mechanism of CYP3A4 inhibition. The tested complexes significantly inhibit human liver microsomal CYP3A4 activity even at clinically relevant concentrations. This could be a serious drawback for the use of these compounds in clinical practice.

EFFECTS OF THE NONSTEROIDAL ECDYSONE AGONIST, TEBUFENOZIDE, ON LIVER CYP ACTIVITIES

The non-steroidal ecdysone agonist tebufenozide was orally administered to Wistar rats for 5 days to examine their effects on the liver cytochrome P450 (CYP) activities. Also Michaelis-Menten kinetics of the metabolic reactions catalyzed by liver CYPs were studied after adding tebufenozide to the assay system to investigate its possible inhibitory effects on liver CYPs activities. These reactions included ethoxyresorufin O-deethylation, tolbutamide hydroxylation, bufuralol 1-hydroxylation, and midazolam 1,4-hydroxylation, for CYP1A, 2C, 2D, and 3A activities, respectively. Results revealed no any significant alteration in the activities of the tested microsomal enzymes after oral administration of tebufenozide. none of the examined CYPs was inhibited by tebufenozide as the Ki values were very high (more than 9000 M). We can conclude that tebufenozide is considered safe and can be a good alternate for the traditional insecticides.

Effects of Teratogenic Drugs on CYP1A1 Activity in Differentiating Rat Embryo Cells

CYP1A1, a P450 isoenzyme, is involved in the phase I xenobiotic metabolism including teratogen drugs. In the present study, the ability of teratogens to elevate the embryonic expression of CYP1A1 was examined. Micromass cell cultures prepared from day 13 rat embryo limb buds (LB). LB cells were cultivated and exposed for 5 days to retinoic acid (RA), hydrocortisone (HC), caffeine (CA) and quinine (QN). CYP1A1 protein expression and activity were measured using immunofluorescence staining and ethoxyresorufin O-deethylation (EROD) assay, respectively. The EROD activity increased significantly following LB cells exposure to RA and HC (p<0.05) but the expression of CYP1A1 protein was reduced by these drugs, whereas the expression of CYP1A1 protein and EROD activity decreased significantly following the addition of CA and QN (p<0.05, p<0.01). Our findings show that studied teratogens have potency to increase CYP1A1 activity.

The multikinase inhibitor axitinib is a potent inhibitor of human CYP1A2

The tyrosine kinase inhibitors (TKIs) and multikinase inhibitors (MKIs) are oncology drugs of increasing importance that have improved the treatment of multiple tumors types. In some patients these agents produce adverse effects, including pharmacokinetic drug–drug interactions, due to cytochrome P450 (CYP) inhibition. Information on the propensity of the drugs to elicit such effects often only becomes evident as the drugs enter clinical use. The present study assessed 18 kinase inhibitors (1 and 50μM) for the inhibition of major drug metabolizing CYPs 1A2, 2C9, 2D6 and 3A4 in human liver microsomes. Most TKIs and MKIs inhibited CYP reactions at the higher concentration but axitinib also potently inhibited CYP1A2-dependent 7-ethoxyresorufin O-deethylation activity at the lower concentration. Kinetic analyses of CYP1A2 inhibition by axitinib were undertaken in microsomes and found a Ki of 0.11±0.01μM, which was 7.5-fold lower than the Km for 7-ethoxyresorufin oxidation (0.83±0.06μM); the inhibition mechanism was linear-mixed. From computational modeling two potential binding modes for axitinib were identified in the active site of CYP1A2: one in which the oxidizable axitinib thioether sulfur atom is within ∼4.45Å of the CYP1A2 heme, and is likely to favor biotransformation of the drug, and a second in which the pyridine moiety is in proximity to the heme, which may contribute to inhibition. The applicability of these findings to potential pharmacokinetic interactions in patients during axitinib treatment should now be assessed.

Reponses of the Hepatic Microsomal Cytochrome P450 Monooxygenase System in Rock Bream Oplegnathus fasciatus Exposed to Tributyltin (TBT)

Abstract The study was conducted to investigate the responses of the hepatic microsomal cytochrome P450 monooxygenase system in the rock bream Oplegnathus fasciatus after chronic exposure to 0, 1, 2, 4, and 8 μg/L tributyltin (TBT) concentrations for 4 weeks. Hepatic cytochrome 450 content and ethoxyresorufin O -deethylation (EROD) activity were found to significantly increase in fish treated with the higher concentration of TBT (≥4 μg/L); however, no significant changes were observed in penthoxyresorufin O -deethylation (PROD) activity in all treated groups compared to the control group. These findings suggest that exposure to a low TBT concentration (≥4 μg/L) has the potential to induce cytochrome 450 content and EROD enzyme activity in hepatic tissue in the rock bream. Key words: Oplegnathus fasciatus , Tributyltin, Cytochrome 450, EROD, PROD Introduction Many aquatic ecosystems are faced with spatially or tem-porally alarmingly high levels of xenobiotic chemicals (Brack et al., 2002; Diez et al., 2002). Tributyltin (TBT) is a typi-cal synthetic pollutant, which was selected for investigation in this study. TBT is an organotin compound used primarily as a biocide in antifouling paints for ships, boats, and fishing nets. The use of TBT as an antifouling agent in paint has been banned for small boats and fishing nets in most countries as it has been shown to be toxic to aquatic life and is an endo-crine disrupting chemical that generates severe reproductive effects in aquatic animals (Horiguchi et al., 1994). Reproduc-tive impairment in gastropods has been reported at low TBT concentrations (Gibbs et al., 1988). TBT has been shown to induce imposex and intersex in brosobranchs (Oehlmann et al., 1998), and impaired reproductive function in fish has also been described (Mc Allister and Kime, 2003).Some studies have reported toxic effects of TBT such as morphological and functional alterations of teleost gills in aquatic media (Byrne et al., 1989; Schwaiger et al., 1992; Tsu -da et al., 1992; Wang and Huang, 1998). TBT has also been shown to markedly inhibit fish hepatic CYP and in particu-lar the isozyme cytochrome P4501A1 (CYP1A), both in vivo and vitro (Fent and Bucheli, 1994; Morcillo and Porte, 1997). The CYP1A isozyme plays a key role in the biotransformation of xenobiotic compounds, leading to either their detoxifica-tion or bioactivation, while other CYP forms are important

Effect of baicalin and glycyrrhizin on enrofloxacin in vivo in crucian carp (Carassius auratus gibelio)

Cytochrome P450s(CYPs) widely distribute in the liver of aquatic organisms and play a key role in drug metabolism.CYPs catalyze the N-deethylation of Enrofloxacin(ENR) to Ciprofloxacin(CIP).However,there is limited information regarding the action of CYPs in ?sh.We evaluated the metabolism of ENR following oral administrated of baicalin(100 mg/kg) and glycyrrhizin(100 mg/kg) in crucian carp,Carassium auratus gibelio.We measured the enzymatic activity of cytochrome P450 1A(CYP1A) and 3A(CYP3A) in liver microsomes.In addition,we documented the pharmacokinetics of ENR by continuous blood sampling.The absorption of ENR decreased significantly over time and both Cmax and AUC also deceased.AUC and t1/2z decreased signficantly whereas CL inceased after oral administrated of baicalin and glycyrrhizin,suggesting that ENR elimination was accelerated.The Cmax CIP/Cmax ENR was 1.48% and 2.22% for the BL and GZ groups,respectively(control: 0.95%).The AUC0-t-CIP/AUC0-t-ENR was 2.16% and 1.76% for the BL and GZ groups,respectively(control: 1.7%).Thus,BL and GZ contributed to induce N-deethylation of ENR.Pretreatment with BL and GZ was associated with a significant(P0.05) incease in ethoxyresorufin-O-deethylation(EROD) and erythromycin-N-demethylation(ERND) activity,the specific probes for CYP1A and CYP3A,respectively.In summary,BL and GZ accelerated the elimination of ENR.Furthermore,production of the ENR metabolite was related to the induction of CYP1A and CYP3A enzymatic activity.