7-ethoxyresorufin O-deethylation Research Articles

Inhibition of CYP1 by berberine, palmatine, and jatrorrhizine: Selectivity, kinetic characterization, and molecular modeling

Cytochrome P450 (P450, CYP) 1 family plays a primary role in the detoxification and bioactivation of polycyclic aromatic hydrocarbons. Human CYP1A1, CYP1A2, and CYP1B1 exhibit differential substrate specificity and tissue distribution. Berberine, palmatine, and jatrorrhizine are protoberberine alkaloids present in several medicinal herbs, such as Coptis chinensis (Huang-Lian) and goldenseal. These protoberberines inhibited CYP1A1.1- and CYP1B1.1-catalyzed 7-ethoxyresorufin O-deethylation (EROD) activities, whereas CYP1A2.1 activity was barely affected. Kinetic analysis revealed that berberine noncompetitively inhibited EROD activities of CYP1A1.1 and CYP1B1.1, whereas palmatine and jatrorrhizine caused either competitive or mixed type of inhibition. Among protoberberines, berberine caused the most potent and selective inhibitory effect on CYP1B1.1 with the least Ki value of 44±16nM. Berberine also potently inhibited CYP1B1.1 activities toward 7-ethoxycoumarin and 7-methoxyresorufin, whereas the inhibition of benzo(a)pyrene hydroxylation activity was less pronounced. Berberine inhibited the polymorphic variants, CYP1B1.3 (V432L) and CYP1B1.4 (N453S), with IC50 values comparable to that for CYP1B1.1 inhibition. Berberine-mediated inhibition was abolished by a mutation of Asn228 to Thr in CYP1B1.1, whereas the inhibition was enhanced by a reversal mutation of Thr223 to Asn in CYP1A2.1. This result in conjugation with the molecular modeling revealed the crucial role of hydrogen-bonding interaction of Asn228 on CYP1B1.1 with the methoxy moiety of berberine. These findings demonstrate that berberine causes a selective CYP1B1-inhibition, in which Asn228 appears to be crucial. The inhibitory effects of berberine on CYP1B1 activities toward structurally diverse substrates can be different.

Metabolic detoxification pathways for 5-methoxy-sterigmatocystin in primary tracheal epithelial cells

1. The health effects of inhaled mycotoxins remain poorly documented despite their presence in bioaerosols. 5-methoxy-sterigmatocystin is produced in association with sterigmatocystin by some Aspergillus spp., sometimes in larger amounts than sterigmatocystin. Whereas sterigmatocystin can be metabolized through cytochromes P450 (CYP), UDP-glucuronosyltransferases and sulfotransferases in airway epithelial cells, little is known about 5-methoxy-sterigmatocystin.2. The 5-methoxy-sterigmatocystin metabolites were analyzed using human recombinant CYP and porcine tracheal epithelial cell (PTEC) primary cultures at an air–liquid interface. The induction of xenobiotic-metabolizing enzymes was examined by real-time quantitative PCR for mRNA expression and 7-ethoxyresorufin O-deethylation activity.3. CYP1A1 metabolized 5-methoxy-sterigmatocystin into hydroxy-nor-methoxy-sterigmatocystin, nor-methoxy-sterigmatocystin and dihydroxy-methoxy-sterigmatocystin. CYP1A2 led to monohydroxy-methoxy-sterigmatocystin. In PTEC, 5-methoxy-sterigmatocystin metabolism resulted into a glucuroconjugate of 5-methoxy-sterigmatocystin, a sulfoconjugate and a glucuroconjugate of monohydroxy-methoxy-sterigmatocystin. The exposure of PTEC for 24 h to 1 µM 5-methoxy-sterigmatocystin induced a significant increase in the mRNA levels of CYP1A1, without significant induction of the 7-ethoxyresorufin O-deethylation activity.4. These data suggest that 5-methoxy-sterigmatocystin is mainly detoxified in airway cells through conjugation, as sterigmatocystin. However, while CYP produced a reactive metabolite of sterigmatocystin, no such metabolite was detected with 5-methoxy-sterigmatocystin. Nevertheless, 5-methoxy-sterigmatocystin increases the CYP1A1 mRNA levels. The long-term consequences remain unknown.

Differential induction of cytochrome P450 isoforms and peroxisomal proliferation by cyfluthrin in male Wistar rats

Cyfluthrin effects on in vivo drug metabolizing enzymes were evaluated using the oxidative substrate antipyrine. Antipyrine pharmacokinetics in plasma and urinary excretion of its major metabolites with and without cyfluthrin oral treatment (20mg/kg/day for 6 days) were investigated in rats. Cyfluthrin increased the apparent intrinsic clearance and decreased the antipyrine half-life at β phase. Cyfluthrin also increased the clearance of the antipyrine metabolites, norantipyrine, 4-hydroxyantipyrine and 3-hydroxymethylantipyrine and the formation rate constants for each of the three metabolites measured in urine. These results suggest that cyfluthrin affects hepatic cytochrome P450 (CYP) system. In order to confirm, a second experiment was carried out. We evaluated the effects of repeated exposure to cyfluthrin on hepatic and renal CYP2E, CYP1A and CYP4A subfamilies and peroxisomal proliferation in rats following oral administration (10 and 20mg/kg/day for 6 days). At the highest dose, cyfluthrin increased renal and hepatic O-deethylation of ethoxyresorufin and O-demethylation of methoxyresorufin, metabolism mediated by the CYP1A subfamily. Liver and kidney were susceptible to cyfluthrin-dependent induction of 12- and 11-hydroxylation of lauric acid, suggesting CYP4A subfamily induction. Also cyfluthrin increased the β-oxidation of palmitoyl-coenzyme A and carnitine acetyltransferase activity, supporting cyfluthrin as a peroxisome proliferator. In conclusion, the demonstration that cyfluthrin induced hepatic CYP1A, CYP4A subfamilies and peroxisomal proliferation raises the possibility of cyfluthrin could produce changes in oxidative stress.

Abstract 2580: Pharmacologic inhibition of cytochrome P450 1B1 attenuates the motility and proliferation of cultured human dysplastic oral leukoplakia cells.

Abstract Oral leukoplakia is a precancerous lesion that may progress to squamous cell carcinoma of the head and neck (HNSCC). The long latency period to invasive cancer provides an opportunity for intervention at the precancerous stage. The inability of surgical resection to prevent the recurrence of oral leukoplakia speaks clearly to the need to develop an efficacious regimen for the chemoprevention of this disease. Tobacco smoke constituents and ethanol, the major risk factors for oral leukoplakia and HNSCC, are metabolized by cytochrome P450 1B1 (CYP1B1) to form potentially carcinogenic species. Our previous data indicate that knockdown of CYP1B1 in human oral leukoplakia cells (MSK-Leuk1) by shRNA decreases cell motility and proliferation, relative to control cells overexpressing empty vector. Homoeriodictyol (HED), a flavonoid found in citrus fruits, is a selective inhibitor of CYP1B1 (IC50 0.24 μM). As a next step, we hypothesized that pharmacologic inhibition of CYP1B1 by HED would attenuate cell motility and proliferation in a manner similar to genetic knockdown. Because HED is commonly found in our diet, its toxicity is expected to be low, making it a promising agent for preventive or therapeutic intervention. The ability of HED to inhibit CYP1B1 activity in MSK-Leuk1 cells was established by incubating cells with either vehicle (0.01% ethanol) or HED (5-10 μM) for 72 hrs and quantifying 7-ethoxyresorufin o-deethylation (EROD), a measure of the activity of CYP1B1 and other enzymes of the CYP1 subfamily. HED decreased EROD activity in a dose-dependent manner, with 40% inhibition observed at 10 μM, as compared to vehicle-treated controls. Western blot analyses revealed that CYP1B1 levels were unaltered following HED treatment. The effect of CYP1B1 inhibition on cell motility was examined by exposing MSK-Leuk1 cells to HED (0-10 μM) for 96 hrs followed by a wound-healing assay, in which the ability of cell monolayers to close a gap was monitored for 23 hrs. HED inhibited cell motility in a concentration dependent manner, with maximum inhibition of gap closure reached at 10 μM (6.7 fold relative to vehicle). To assess the effect of CYP1B1 inhibition on cell growth, MSK-Leuk1 cells were exposed to HED (0-10 μM) for 5 days and proliferation was analyzed (Fluorescent DNA Quantitation kit). A direct association was observed between HED dose and inhibition of proliferation, with a 33% decrease observed at 10 μM. In summary, inhibition of CYP1B1 activity by HED resulted in attenuation of the motility and proliferation of cultured MSK-Leuk1 cells. These data confirm that CYP1B1 is a promising molecular target for intervention in HNSCC and provide strong support for the further development of CYP1B1 inhibitors as efficacious agents for the prevention of HNSCC in patients with oral leukoplakia. (Supported by NCI fellowship 1 F32 CA144199-01). Citation Format: Ekaterina G. Shatalova, Margie L. Clapper. Pharmacologic inhibition of cytochrome P450 1B1 attenuates the motility and proliferation of cultured human dysplastic oral leukoplakia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2580. doi:10.1158/1538-7445.AM2013-2580

Analysis of Substrate Recognition Site 2 (SRS2) in human cytochrome P450 1A2 using whole-plasmid random mutagenesis

The metabolic activation of many carcinogenic heterocyclic and aryl amines are attributed to hepatic cytochrome P450 enzymes including P450 1A2. Bioactivation mechanism analysis of P450 1A2 in the limited crystallographic data requires the generation of a large number of mutants. In this study, the SRS2 region in P450 1A2 was randomly mutated with random primers designed for the non-wild type using whole-plasmid random mutagenesis, followed the screening of the mutant library. Eight mutants in Asn222 and Glu225 were selected after Escherichia coli genotoxicity assay involving reversion to lac prototrophy as a response to activation of the heterocyclic amine 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ). Each mutant E. coli membrane was used to determine k cat and K m values for phenacetin and 7-ethoxyresorufin O-deethylation. Most mutants showed considerably increased activity for phenacetin. The Glu225Asn and Asn222Lys mutants increased catalytic efficiencies (k cat/K m) by 5- and 4-fold, respectively. However, the catalytic efficiencies of 7-ethoxyresorufin O-deethylation were not increased mainly because of the increase of K m values. The Glu225Asn and Asn222Lys mutants showed increased k cat values but exhibited greater increases in K m values, which resulted in lower catalytic efficiencies (k cat/K m) for 7-ethoxyresorufin O-deethylation. These results suggested that structural changes of metabolic enzymes in the process of mutagenesis result in different effects of activity by its substrates.

Null CYP1B1 Genotypes in Primary Congenital and Nondominant Juvenile Glaucoma

To assess the mutation spectrum, enzymatic activity, and phenotypic features associated with CYP1B1 genotypes in primary congenital glaucoma (PCG) and nondominant juvenile glaucoma (ndJG). CYP1B1 genotyping, segregation analysis, and functional evaluation of mutations in a cohort of patients. A total of 177 probands clinically diagnosed with PCG (161) or ndJG (16). Automatic DNA sequencing of the promoter (-1 to -867) and the 3 CYP1B1 exons. CYP1B1 enzymatic activity was evaluated using an ethoxyresorufin O-deethylation assay in transfected HEK-293T cells. Screening and functional evaluation of CYP1B1 mutations. Glaucoma diagnosis based on slit-lamp examination, measurement of intraocular pressure, gonioscopy, and fundus examination. Thirty-one different mutations were identified in 56 PCG and 7 ndJG index cases. To the best of our knowledge, 3 of the identified mutations were novel (-337G>T, F123L, and I399_P400del). Approximately 56% of all mutation carriers were compound heterozygotes, 25% were homozygotes, and both groups inherited glaucoma as an autosomal recessive trait. Nineteen percent of carriers were heterozygotes and showed non-Mendelian segregation. In vitro and inferred functional analysis showed that no less than approximately 74% of the recessive genotypes result in null enzymatic activity. We detected variable expressivity in relation to age of onset and a possible case of incomplete penetrance in 3 of 6 families (50%), with more than 1 affected child or more than 1 subject carrying 2 CYP1B1 mutant alleles. Altogether, these data support that PCG is not a simple monogenic disease. In addition, most patients with PCG carrying null or putative null genotypes showed severe bilateral phenotypes featured by early disease onset, frequently at birth. The mean number of trabeculectomies per eye was significantly higher in carriers than in noncarriers. This is the largest analysis of CYP1B1 mutations performed in European patients with PCG to date. Our data show that null CYP1B1 genotypes, and therefore complete absence of CYP1B1 activity, frequently lead to severe phenotypes. Our results support that CYP1B1 glaucoma is not a simple monogenic disease and that CYP1B1 activity levels could influence the phenotype.

The naturally occurring aliphatic isothiocyanates sulforaphane and erucin are weak agonists but potent non-competitive antagonists of the aryl hydrocarbon receptor

As the Ah receptor target gene products play a critical role in chemical carcinogenesis, antagonists are considered as potential chemopreventive agents. It is demonstrated in this paper that the isothiocyanates R,S-sulforaphane and erucin are non-competitive antagonists of the aryl hydrocarbon (Ah) receptor. Both isothiocyanates were poor agonists for the receptor and elevated CYP1A1 mRNA levels only modestly when incubated with precision-cut rat liver slices. In contrast, the classical Ah receptor agonist benzo[a]pyrene was a potent inducer of CYP1A1 mRNA levels, with this effect being effectively antagonized by the two isothiocyanates. In further studies, it was demonstrated that R,S-sulforaphane could both prevent the interaction of and displace already bound benzo[a]pyrene from the Ah receptor, but no concentration dependency was observed with respect to the isothiocyanate. Both erucin and R,S-sulforaphane antagonized the benzo[a]pyrene-mediated increase in the CYP1A-mediated O-deethylation of ethoxyresorufin in rat precision-cut liver slices. Of the two isomers of R,S-sulforaphane, the naturally occurring R-isomer was more effective than the S-isomer in antagonizing the activation of the Ah receptor by benzo[a]pyrene. Antagonism of the Ah receptor may be a major contributor to the established chemoprevention of aliphatic isothiocyanates.

Comparison of microbial hosts and expression systems for mammalian CYP1A1 catalysis

Mammalian cytochrome P450 enzymes are of special interest as biocatalysts for fine chemical and drug metabolite synthesis. In this study, the potential of different recombinant microorganisms expressing rat and human cyp1a1 genes is evaluated for such applications. The maximum specific activity for 7-ethoxyresorufin O-deethylation and gene expression levels were used as parameters to judge biocatalyst performance. Under comparable conditions, E. coli is shown to be superior over the use of S. cerevisiae and P. putida as hosts for biocatalysis. Of all tested E. coli strains, E. coli DH5α and E. coli JM101 harboring rat CYP1A1 showed the highest activities (0.43 and 0.42 U g⁻¹(CDW), respectively). Detection of active CYP1A1 in cell-free E. coli extracts was found to be difficult and only for E. coli DH5α, expression levels could be determined (41 nmol g⁻¹(CDW)). The presented results show that efficient expression of mammalian cyp1a1 genes in recombinant microorganisms is troublesome and host-dependent and that enhancing expression levels is crucial in order to obtain more efficient biocatalysts. Specific activities currently obtained are not sufficient yet for fine chemical production, but are sufficient for preparative-scale drug metabolite synthesis.

Effects of Etodolac on P450 Isoform-specific Activities in Human Hepatic Microsomes

The effects of etodolac (CAS 41340-25-4) on P450 isoform-specific activities in human hepatic microsomes were examined. Etodolac had little effect on 7-ethoxyresorufin O-deethylation (CYP1A2), coumarin hydroxylation (CYP2A6), 7-benzyloxyresorufin O-debenzylation (CYP2B6), S-mephenytoin hydroxylation (CYP2C19), bufuralol hydroxylation (CYP2D6), chlorzoxazone hydroxylation (CYP2E1) and nifedipine oxidation (CYP3A4) at concentrations ranging from 10 to 50 micromol/L. Etodolac inhibited tolbutamide hydroxylation (CYP2C9) with the Ki value of 64 micromol/L, suggesting that it is a weak inhibitor of CYP2C9. The in vivo drug interaction was predicted from the in vitro data using the [I]/([I] + Ki) value. Because the value was calculated to be almost 1, it is not likely that etodolac causes the drug interactions with the CYP2C9 substrates.

Effects of Irsogladine on P450-isoform Specific Activities in Human Hepatic Microsomes

The effects of irsogladine (CAS 84504-69-8) on P450-isoform specific activities in human hepatic microsomes were examined. Irsogladine had little effects on coumarin hydroxylation (CYP2A6), 7-benzyloxyresorufin O-debenzylation (CYP2B6), S-mephenytoin hydroxylation (CYP2C19), bufuralol hydroxylation (CYP2D6), chlorzoxazone hydroxylation (CYP2E1) and nifedipine oxidation (CYP3A4) at concentrations ranging from 10 to 50 pmol/L. However, it inhibited 7-ethoxyresorufin O-deethylation (CYP1A2) and tolbutamide hydroxylation (CYP2C9) with the Ki values of 276 and 156 micromol/L, respectively. This suggests that it is a weak inhibitor of these isoforms. Because the plasma concentrations of irsogladine in humans are much lower than these Ki values, it is unlikely that irsogladine causes drug interactions with other drugs.

Effects of the genotoxic compounds, benzo[a]pyrene and cyclophosphamide on phase 1 and 2 activities in EpiDerm™ models

The micronucleus assay in the 3D human reconstructed EpiDerm™ skin model (RSMN) is a promising new assay for evaluating genotoxicity of dermally applied chemicals.To complement the testing of metabolically activated chemicals, such as cyclophosphamide (CPA) and benzo[a]pyrene (B[a]P), we measured phase 1 (ethoxyresorufin O-deethylation (EROD) and testosterone metabolism) and 2 activities (UGTs and GSTs) in non-treated and genotoxin treated EpiDerm™ models in a study design which mimics the RSMN assay. The assay involved a three-dose dosing regimen over 72 h to take into account effects e.g. enzyme induction, which requires longer than the standard 2 dose 48-h assay.These studies demonstrated the presence of basal phase 1 and 2 activities of EpiDerm™ models.With the exception of GST, all of the activities measured did not reproducibly change over time.It was possible to measure enzyme induction using this assay design. EROD activity was significantly induced by B[a]P but not by CPA. CPA and B[a]P had little or no reproducible effects on GST and UGT activities.In conclusion, a number of metabolic enzyme activities were present in the EpiDerm™ skin model and at least the CYP1 family was inducible.

Phenethyl isothiocyanate, a naturally occurring phytochemical, is an antagonist of the aryl hydrocarbon receptor

The aryl hydrocarbon (Ah) receptor is a ligand-activated transcription factor that is activated by many carcinogens, and its target gene products play a major role in tumour development, so that antagonists of the Ah receptor represent potential chemopreventive agents. Experimental evidence is presented herein that phenethyl isothiocyanate (PEITC), a phytochemical present in cruciferous vegetables, is such an antagonist. PEITC was a very weak ligand to the Ah receptor, as assessed using the chemical-activated luciferase expression (CALUX) assay, and a poor inducer of CYP1A1 mRNA levels when incubated in precision-cut rat liver slices for 24 h. It antagonised effectively, however, the interaction of benzo[a]pyrene to the receptor, being capable of preventing its binding as well as displacing it from the receptor. Moreover, PEITC suppressed in concentration-dependent manner the benzo[a]pyrene-mediated rise in rat hepatic CYP1A1 mRNA levels in rat slices. Finally, PEITC antagonised the benzo[a]pyrene-mediated increase in the O-deethylation of ethoxyresorufin in both rat and human precision-cut liver slices. It is concluded that PEITC is an effective antagonist of the Ah receptor in rat and human liver, and this potential may contribute to its established chemopreventive activity.

Application of an integrated biomarker response index (IBR) to assess temporal variation of environmental quality in two Portuguese aquatic systems

Estuaries are extremely productive systems faced with great anthropogenic pressure, namely from contaminants of both metallic and organic nature which may affect the ecological quality of these systems. Moreover, it is known that contaminants fluctuate seasonally and this could influence the health status of organisms. In order to identify the most affected seasons and areas in two Portuguese aquatic systems with strong anthropogenic influences, a method that combines several biomarker responses into a general “stress index” denominated “integrated biomarker response” (IBR) was applied using the stress responses in the liver of common goby Pomatoschistus microps collected from two sites within both estuaries (Ria de Aveiro and Tagus). Parameters included in the IBR were the biomarkers superoxide dismutase (SOD), catalase (CAT), ethoxyresorufin-O-deethylation (EROD), glutathione s-transferase (GST), metallothioneins (MT), lipid peroxidation (LPO) and condition indices (RNA:DNA ratio and Fulton's condition factor K. Pollutant concentrations in the sediment (metals and PAHs) were also analyzed. Biomarkers in P. microps in Ria de Aveiro lagoon (sites A and B) showed distinct seasonal variation. In site A, IBR highlighted the spring as the season with highest biomarker responses, and RNA:DNA as the most contributing biomarker, while in site B the most impacted season was the summer, associated to the responses of MT and antioxidant enzymes (CAT and SOD), probably as a result of levels of Hg in the sediments. In both sites within the Tagus estuary (C and D) the IBR showed that biomarker responses were higher in autumn, with CAT, EROD, GST and LPO as the most altered biomarkers. In this season the levels of PAHs, Pb and Cr were the highest. Overall, the IBR index provides an integrated approach for assessing the health status of organisms and to identify temporal patterns of contamination.

Spectral Modification and Catalytic Inhibition of Human Cytochromes P450 1A1, 1A2, 1B1, 2A6, and 2A13 by Four Chemopreventive Organoselenium Compounds

Several organoselenium compounds including benzyl selenocyanate (BSC), 1,2-phenylenebis(methylene)selenocyanate (o-XSC), 1,3-phenylenebis(methylene)selenocyanate (m-XSC), and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) have been shown to prevent cancers caused by polycyclic aromatic hydrocarbons (PAHs) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in experimental animals; these chemical carcinogens are activated by human P450 1 and 2A family enzymes, respectively, to carcinogenic metabolites. In this study, we examined whether these selenium compounds interact with and inhibit human P450 1 and 2A enzymes in vitro. Four organoselenium compounds induced reverse Type I binding spectra with P450 1A1, 1A2, and 1B1 and Type I binding spectra with P450 2A6 and 2A13. The spectral dissociation constants (K(s)) for the interaction of P450 1B1 with these chemicals were 3.6-5.7 μM; the values were lower than those with seen with P450 1A1 (19-30 μM) or 1A2 (6.3-13 μM). The K(s) values for Type I binding of P450 2A13 with m-XSC and BSC were both 0.20 μM; the values were very low compared to those for the interaction of P450 2A6 with m-XSC (5.7 μM) and BSC (2.0 μM). Four selenium compounds directly inhibited 7-ethoxyresorufin O-deethylation activities catalyzed by P450 1A1, 1A2, and 1B1 with IC(50) values <1.0 μM, except for the inhibition of P450 1A2 by BSC (1.3 μM). Coumarin 7-hydroxylation activities of P450 2A13 were more inhibited by four selenium compounds than those of P450 2A6, with IC(50) values of 0.22-1.4 μM for P450 2A13 and 2.4-6.2 μM for P450 2A6. Molecular docking studies of the interaction of four organoselenium compounds with human P450 enzymes suggest that these chemicals can be docked into the active sites of these human P450 enzymes and that the sites of the selenocyanate functional groups of these chemicals differ between the P450 1 and 2A family enzymes.

Suppressive Effect of the Ethanolic Extract of Adlay Bran on Cytochrome P-450 Enzymes in Rat Liver and Lungs

Adlay ( Coix lachryma-jobi L. var. ma-yuen Stapf) is a grass crop and is reported to protect against various diseases such as cancer. To investigate the effect of the ethanolic extract of adlay bran (ABE) on drug-metabolizing enzymes and glutathione-related antioxidant enzymes in rats, three groups of eight male Sprague-Dawley rats each were fed a control diet or a diet containing 5 or 10% ABE for 4 weeks. Significant decreases in microsomal cytochrome P-450 (CYP) 1A1-catalyzed ethoxyresorufin O-deethylation, CYP2C-catalyzed diclofenac 4-hydroxylation, CYP2D-catalyzed dextromethorphan O-demethylation, and CYP3A-catalyzed testosterone 6β-hydroxylation in the liver and CYP1A1-catalyzed ethoxyresorufin O-deethylation in the lungs of rats fed ABE were observed. Immunoblot analyses also showed decreases of CYP1A1, 1A2, 2C6, 2C11, 2D1, 2E1, 3A1, and 3A2 in the liver and CYP1A1 in the lungs. Furthermore, rats fed the 10% ABE diet had a higher glutathione content and glutathione peroxidase, glutathione reductase, and glutathione S-transferase activities in the lungs, but such an increase was not noted in the liver. Inhibition of various CYP-catalyzed enzyme reactions by ABE in rat and human liver microsomes had also been shown. The results of this study indicate that ABE feeding may suppress CYP enzyme activities and CYP protein expression in the liver and lungs of rats. Moreover, the increase of the antioxidant potential by ABE is tissue-specific.

Regional Distribution of Drug-metabolizing Enzyme Activities in the Liver and Small Intestine of Cynomolgus Monkeys

The cynomolgus monkey is an animal species widely used to study drug metabolism because of its evolutionary closeness to humans. However, drug-metabolizing enzyme activities have not been compared in various parts of the liver and small intestine in cynomolgus monkeys. In this study, therefore, drug-metabolizing enzyme activities were analyzed in the liver (the five lobes) and small intestine (six sections from the duodenum to the distal ileum). 7-Ethoxyresorufin O-deethylation, coumarin 7-hydroxylation, paclitaxel 6α-hydroxylation, diclofenac 4'-hydroxylation, tolbutamide methylhydroxylation, S-mephenytoin 4'-hydroxylation, bufuralol 1'-hydroxylation, chlorzoxazone 6-hydroxylation, midazolam 1'-hydroxylation, and testosterone 6β-, 16α-, 16β-, and 2α-hydroxylation were used as the probe reactions for this investigation. In liver, all probe reactions were detected and enzyme activity levels were similar in all lobes, whereas, in the small intestine, all enzyme activities were detected (except for coumarin 7-hydroxylase and testosterone 16α-hydroxylase activity), but from jejunum to ileum there was a decrease in the level of enzyme activity. This includes midazolam 1'-hydroxylation and testosterone 6β-hydroxylation, which are catalyzed by cynomolgus monkey cytochrome P450 (CYP) 3A4/5, orthologs of human CYP3A4/5, which are important drug-metabolizing enzymes. The data presented in this study are expected to facilitate the use of cynomolgus monkeys in drug metabolism studies.

Comparison of the Ability of Myricetin and Quercetin to Modulate the Oxidative DNA Damage Induced by Heterocyclic Amines

The aim of the present study was to compare the ability of the myricetin and quercetin to modulate the oxidative DNA damage induced by 2-amino-3, 8- dimethylimidazo [4,5-f] quinoxaline (8-MeIQx), 2-amino- 3, 4, 8- trimethylimidazo [4, 5-f]-quinoxaline (4,8-diMeIQx) and 2-amino-1-methyl-6-phenyl-imidazo [4,5-b] pyridine (PhIP), in human hepatoma cells. DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single-cell gel electrophoresis or comet assay. None of the myricetin and quercetin concentrations tested protected against 8-MeIQx, 4, 8-diMeIQx and PhIP-induced DNA strand breaks. The oxidized pyrimidines induced by 4, 8-diMeIQx and PhIP were reduced by myricetin but not by quercetin. Quercetin reduced the oxidized purines induced by 8-MeIQx and PhIP, while myricetin also reduced the induced by 4, 8-diMeIQx. One feasible mechanism by which myricetin and quercetin exert their protective effect towards HCAs-induced oxidative DNA could be related in part to the reduction of human CYP1A1. Another mechanism claimed to be responsible for the protective effect of myricetin and quercetin is the induction of phase II metabolizing enzymes such as UDP-glucuronyltrasferase (UGT). The ethoxyresorufin O-deethylation (CYP1A1) activity was moderately inhibited by myricetin, while little effect was observed by quercetin. On the contrary, quercetin showed the greatest increase on UDP-glucuronyltransferase activity. However, these are not the only mechanisms by which myricetin and quercetin exert their protective effect, other mechanisms such as stimulation of the repair of carcinogen-induced DNA damage and or the free radical scavenging efficiency have been also implicated. In conclusion, our results clearly indicate that myricetin was more efficient than quercetin to prevent DNA damage (oxidized purines and pyrimidines) induced by the three HCAs evaluated. This protective effect depends on the chemical structure of flavonoid and the mutagen studied.

Effects of (+)catechin and (−)epicatechin on heterocyclic amines-induced oxidative DNA damage

The aim of the present study was to evaluate the protective effect of (+)-catechin and (-)-epicatechin against 2-amino-3,8- dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]-quinoxaline (4,8-diMeIQx) and 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP)-induced DNA damage in human hepatoma cells (HepG2). DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single-cell gel electrophoresis or comet assay. Increasing concentrations of 8-MeIQx, 4,8-diMeIQx and PhIP induced a significant increase in DNA strand breaks and oxidized purines and pyrimidines in a dose-dependent manner. Among those, PhIP (300 µm) exerted the highest genotoxicity. (+)-Catechin exerted protection against oxidized purines induced by 8-MeIQx, 4,8-diMeIQx and PhIP. Oxidized pyrimidines and DNA strand breaks induced by PhIP were also prevented by (+)-catechin. Otherwise, (-)-epicatechin protected against the oxidized pyrimidines induced by PhIP and the oxidized purines induced by 8-MeIQx and 4,8-diMeIQx. One feasible mechanism by which (+)-catechin and (-)-epicatechin exert their protective effect towards heterocyclic amines-induced oxidative DNA damage may be by modulation of phase I and II enzyme activities. The ethoxyresorufin O-deethylation (CYP1A1) activity was moderately inhibited by (+)-catechin, while little effect was observed by (-)-epicatechin. However, (+)-catechin showed the greatest increase in UDP-glucuronyltransferase activity. In conclusion, our results clearly indicate that (+)-catechin was more efficient than (-)-epicatechin in preventing DNA damage (strand breaks and oxidized purines/pyrimidines) induced by PhIP than that induced by 8-MeIQx and 4,8-diMeIQx.

CYP1D1, pseudogenized in human, is expressed and encodes a functional drug-metabolizing enzyme in cynomolgus monkey

Cytochrome P450 (P450 or CYP) 1 family consists of the CYP1A, CYP1B, CYP1C, and CYP1D subfamilies. In the human genome, CYP1A1, CYP1A2, and CYP1B1 are expressed and encode functional enzymes, whereas CYP1D1P (formerly known as CYP1A8P) is present as a pseudogene due to five nonsense mutations in the putative coding region. In this study, we identified CYP1D1 cDNA, highly identical (nearly 95%) to human CYP1D1P sequence, in cynomolgus monkey, a species frequently used in drug metabolism studies due to its evolutionary closeness to human. The amino acid sequence deduced from cynomolgus monkey CYP1D1 cDNA shared the high sequence identity (91%) with human CYP1D1P (postulated from the gene sequence), and the highest sequence identity (44–45%) with CYP1A1 and CYP1A2 among cynomolgus monkey P450s. CYP1D1 mRNA was most abundantly expressed in liver, followed by kidney, and jejunum. The hepatic expression level of CYP1D1 mRNA was comparable to that of CYP1A1 mRNA and much higher than that of CYP1A2 mRNA. CYP1D1 was barely detectable in immunoblots of cynomolgus monkey liver. Cynomolgus monkey CYP1D1 mRNA was induced in primary hepatocytes with omeprazole. Cynomolgus monkey CYP1D1 protein heterologously expressed in Escherichia coli catalyzed ethoxyresorufin O-deethylation and caffeine 8-hydroxylation, which CYP1As also catalyze. Finally, no nonsense mutations, corresponding to those found in human CYP1D1P, were found in the 20 cynomolgus monkeys and 10 rhesus monkeys used in this study. These results suggest that CYP1D1 plays a role as a functional, drug-metabolizing enzyme in cynomolgus monkey liver.

Structure−Function Relationships of Inhibition of Human Cytochromes P450 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 Flavonoid Derivatives

Structure-function relationships for the inhibition of human cytochrome P450s (P450s) 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives were studied. Thirty-two of the 33 flavonoids tested produced reverse type I binding spectra with P450 1B1, and the potencies of binding were correlated with the abilities to inhibit 7-ethoxyresorufin O-deethylation activity. The presence of a hydroxyl group in flavones, for example, 3-, 5-, and 7-monohydroxy- and 5,7-dihydroxyflavone, decreased the 50% inhibition concentration (IC50) of P450 1B1 from 0.6 μM to 0.09, 0.21, 0.25, and 0.27 μM, respectively, and 3,5,7-trihydroxyflavone (galangin) was the most potent, with an IC50 of 0.003 μM. The introduction of a 4'-methoxy- or 3',4'-dimethoxy group into 5,7-dihydroxyflavone yielded other active inhibitors of P450 1B1 with IC50 values of 0.014 and 0.019 μM, respectively. The above hydroxyl and/or methoxy groups in flavone molecules also increased the inhibition activity with P450 1A1 but not always toward P450 1A2, where 3-, 5-, or 7-hydroxyflavone and 4'-methoxy-5,7-dihydroxyflavone were less inhibitory than flavone itself. P450 2C9 was more inhibited by 7-hydroxy-, 5,7-dihydroxy-, and 3,5,7-trihydroxyflavones than by flavone but was weakly inhibited by 3- and 5-hydroxyflavone. Flavone and several other flavonoids produced type I binding spectra with P450 3A4, but such binding was not always related to the inhibitiory activities toward P450 3A4. These results indicate that there are different mechanisms of inhibition for P450s 1A1, 1A2, 1B1, 2C9, and 3A4 by various flavonoid derivatives and that the number and position of hydroxyl and/or methoxy groups highly influence the inhibitory actions of flavonoids toward these enzymes. Molecular docking studies suggest that there are different mechanisms involved in the interaction of various flavonoids with the active site of P450s, thus causing differences in inhibition of these P450 catalytic activities by flavonoids.