Regio- and stereoselective oxidation of propranolol enantiomers by human CYP2D6, cynomolgus monkey CYP2D17 and marmoset CYP2D19

Toxic and pharmacokinetic profiles of drug candidates are evaluated in vivo often using monkeys as experimental animals, and the data obtained are extrapolated to humans. Well understanding physiological properties, including drug-metabolizing enzymes, of monkeys should increase the accuracy of the extrapolation. The present study was performed to compare regio- and stereoselectivity in the oxidation of propranolol (PL), a chiral substrate, by cytochrome P450 2D (CYP2D) enzymes among humans, cynomolgus monkeys and marmosets. Complimentary DNAs encoding human CYP2D6, cynomolgus monkey CYP2D17 and marmoset CYP2D19 were cloned, and their proteins expressed in a yeast cell expression system. The regio- and stereoselective oxidation of PL enantiomers by yeast cell microsomal fractions were compared. In terms of efficiency of expression in the system, the holo-proteins ranked CYP2D6 ≒ CYP2D17 ? CYP2D19. This may be caused by the bulky side chain of the amino acid residue at position 119 (leucine for CYP2D19 vs. valine for CYP2D6 and CYP2D17), which can disturb the incorporation of the heme moiety into the active-site cavity. PL enantiomers were oxidized by all of the enzymes mainly into 4-hydroxyproranolol (4-OH-PL), followed by 5-OH-PL and N-desisopropylpropranolol (NDP). In the kinetic analysis, apparent K_m values were commonly in the μM range and substrate enantioselectivity of R-PL < S-PL was observed in both K_m and V_max values for the formation of the three metabolites from PL enantiomers. The activity to produce NDP tended to be higher for the monkey enzymes, particularly CYP2D17, than for the human enzyme. These results indicate that in the oxidation of PL enantiomers by CYP2D enzymes, stereoselectivity is similar but regioselectivity is different between humans and monkeys.     

Species difference in enantioselectivity for the oxidation of propranolol by cytochrome P450 2D enzymes

We examined and compared enantioselectivity in the oxidation of propranolol (PL) by liver microsomes from humans and Japanese monkeys (Macaca fuscata). PL was oxidized at the naphthalene ring to 4-hydroxypropranolol, 5-hydroxypropranolol and side chain N-desisopropylpropranolol by human liver microsomes with enantioselectivity of [R(+)>S(-)] in PL oxidation rates at substrate concentrations of 10 microM and 1 mM. In contrast, reversed enantioselectivity [R(+)相似文献   

Investigation of cytochrome P450 inhibitory properties of maslinic acid,a bioactive compound from Olea europaea L., and its structure–activity relationship

Maslinic acid (MA), the main pentacyclic triterpene of Olea europaea L. fruit, possesses a variety of pharmacological actions, including hypoglycemic, antioxidant, cardioprotective and antitumoral activities. Despite its importance, little is known about its effects on the cytochrome P450 (CYP) activity in both humans and animals. Therefore, the aim of this study was to investigate the effects of MA on the CYP 1A2, 2C9/11, 2D1/6, 2E1 and 3A2/4 activities by human and rat liver microsomes and specific CYP isoforms. In humans, MA only weakly inhibited CYP3A4 activity in human liver microsomes and specific CYP3A4 isoform with IC₅₀ value at 46.1 and 62.3 µM, respectively. In rats, MA also exhibited weak inhibition on CYP2C11, CYP2E1 and CYP3A2 activities with IC₅₀ values more than 100 µM. Enzyme kinetic studies showed that the MA was not only a competitive inhibitor of CYP3A4 in humans, but also a competitive inhibitor of CYP2C11 and 3A2 in rats, with K_i of 18.4, 98.7 and 66.3 µM, respectively. Moreover, the presence of hydroxyl group at C-2 position of triterpenic acid in MA compared with oleanolic acid could magnify its competitive inhibition on human CYP3A4 activity. The relatively high K_i values of MA would have a low potential to cause the possible toxicity and drug interactions involving CYP enzymes, thus suggesting a sufficient safety for its putative use as a nutraceutical taken together with drugs.     

Metabolic activation of phenanthrene by human and mouse cytochromes P450 and pharmacokinetics in CYP1A2 knockout mice

In the present study V79 Chinese hamster cells were genetically engineered for stable expression of the cytochromes P450 1A1, 1A2, 1B1, and 2E1 from man and mouse to investigate species-specific differences in the regioselective metabolism and toxicity of phenanthrene (Phe), the simplest polycyclic aromatic hydrocarbon (PAH) forming a bay-region. Phe is present in various environmental samples and serves as a model substrate for PAH exposure in human biomonitoring studies. For this reason we explored metabolite profiles and metabolite-dependent cytotoxic activities in vitro. The total turnover of CYP-mediated transformation of Phe was as follows: human CYP1B1 > CYP1A1 > CYP1A2 ? CYP2E1, and for mouse CYP1A2 ? CYP2E1 > CYP1A1. Striking species differences were seen as mouse CYP1B1 did not activate Phe at all, but human CYP1B1 exhibited a significant metabolic turnover comparable to CYP1A1 and CYP1A2. In vivo studies monitoring the whole blood Phe elimination in CYP1A2 knockout and wild-type mice after oral administration confirmed involvement of CYP1A2 in the bioactivation of Phe, but other processes must contribute also. Our data suggest that in humans not only CYP1A2 expressed solely in the liver plays a crucial role in Phe metabolism, but also constitutively expressed extrahepatic CYP1B1 in tissues such as lung, kidney or intestine. This finding will substantially improve the validity of human biomonitoring studies using individual Phe metabolites for the assessment of PAH exposure.     

Potent inhibition of human cytochrome P450 3A isoforms by cannabidiol: role of phenolic hydroxyl groups in the resorcinol moiety

AimsIn this study, we examined the inhibitory effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD), and cannabinol (CBN), the three major cannabinoids, on the activity of human cytochrome P450 (CYP) 3A enzymes. Furthermore, we investigated the kinetics and structural requirement for the inhibitory effect of CBD on the CYP3A activity.Main methodsDiltiazem N-demethylase activity of recombinant CYP3A4, CYP3A5, CYP3A7, and human liver microsomes (HLMs) in the presence of cannabinoids was determined.Key findingsAmong the three major cannabinoids, CBD most potently inhibited CYP3A4 and CYP3A5 (IC₅₀ = 11.7 and 1.65 μM, respectively). The IC₅₀ values of Δ⁹-THC and CBN for CYP3A4 and CYP3A5 were higher than 35 μM. For CYP3A7, Δ⁹-THC, CBD, and CBN inhibited the activity to a similar extent (IC₅₀ = 23–31 μM). CBD competitively inhibited the activity of CYP3A4, CYP3A5, and HLMs (K_i = 1.00, 0.195, and 6.14 μM, respectively). On the other hand, CBD inhibited the CYP3A7 activity in a mixed manner (K_i = 12.3 μM). Olivetol partially inhibited all the CYP3A isoforms tested, whereas d-limonene showed lack of inhibition. The lesser inhibitory effects of monomethyl and dimethyl ethers of CBD indicated that the ability of CYP3A inhibition by the cannabinoid attenuated with the number of methylation on the phenolic hydroxyl groups in the resorcinol moiety.SignificanceThis study indicated that CBD most potently inhibited catalytic activity of human CYP3A enzymes, especially CYP3A4 and CYP3A5. These results suggest that two phenolic hydroxyl groups in the resorcinol moiety of CBD may play an important role in the CYP3A inhibition.     

Comparative xenobiotic metabolism capacities and pesticide sensitivity in adults of Solea solea and Solea senegalensis

The measurement of enzymatic activities involved in xenobiotic biotransformation was carried out in adults of Solea solea and Solea senegalensis. The hepatic enzymes analysed were cytochrome P450 (CYP) related activities using eight fluorometric substrates and carboxylesterases (CbE). The conjugating activities of glutathione S-transferase (GST) and UPD-glucuronosyltransferase (UDPGT) were also assessed. Specific mammalian inhibitors were used as diagnostic tools for related activities of CYP1A (α-naphthoflavone; αNF), CYP2B6 and CYP2C19 (ticlopidine) and CYP3A4 (ketoconazole). The in vitro sensitivity to organophosphorous pesticides (OP) was tested in the S10 homogenate of brain (acetylcholinesterase-AChE) and liver (CbE). Furthermore, the pesticide chlorpyrifos oxon (CLPO) was used to explore the OP sensitivity of CbE of both species in two subcellular fractions (microsomes and cytosol), using two substrates. Overall, only two parameters confirmed species differences: EROD and cytosolic CbE being significantly elevated (p < 0.05) in the common sole, S. solea. A high inhibition of CYP1A related activities using several fluorometric substrates (ER, MR and CEC) after in vitro incubation with αNF confirmed all measure CYP1A1-related activities whereas ketoconazole was more specific for BFCOD (CYP3A4). Pesticide sensitivity was similar for brain AChE but hepatic CbE had a protective role that was species and pesticide dependent.     

Efficacy of the antagonist Aureobasidium pullulans PL5 against postharvest pathogens of peach,apple and plum and its modes of action

The efficacy of Aureobasidium pullulans PL5 against different postharvest pathogens of fruits (Monilinia laxa on plums and peaches, Botrytis cinerea and Penicillium expansum on apples) were evaluated under storage conditions when applied at 10⁸ cells ml⁻¹ and their interactions were studied in vitro and in vivo to discover the possible modes of action. Under 1.2 °C and 95% relative humidity (RH) for 28 days, the efficacy of PL5 against M. laxa on plums was 45%, reducing disease incidence from 78% to 43%. Under 1 °C and 95% RH for 21 days, the efficacy against M. laxa on peaches was 63%, reducing disease incidence from 79% to 29%. Under 4 °C and 95% RH for 45 days, the efficacy against B. cinerea and P. expansum on apples was 56% and 46%, respectively. In Lilly–Barnett minimal salt medium with the fungal cell walls of pathogens as sole carbon source, PL5 produced β-1,3-glucanase, exo-chitinase and endo-chitinase. Nutrient concentrations had significant effect on pathogen growth reduction by PL5. No attachment was observed in antagonist–pathogen interactions in vitro or in vivo. PL5 completely inhibited pathogen spore germination in PDB at 10⁸ cells ml⁻¹, whereas at 10⁶ cells ml⁻¹ the efficacy was significantly decreased. However, inactivated cells and culture filtrate of PL5 had no effect on pathogen spore germination and germ tube elongation. Our results showed that A. pullulans PL5 could be introduced in commercial formulations to control postharvest pathogens on fruits and its activity was based on secretion of lytic enzymes and competition for nutrients.     

Serum androgen level is determined by autosomal dominant inheritance and regulates sex-related CYP genes in pigs

We have previously demonstrated differences between Meishan and Landrace pigs in their serum androgen levels (Meishan > Landrace) and the expression of genes encoding hepatic cytochrome P450 (CYP) 1A subfamily enzymes (Meishan < Landrace). In the present study, to clarify whether such differences are genetically controlled, we crossbred these pigs (female Meishan × male Landrace, ML; female Landrace × male Meishan, LM) and examined the expression levels of serum androgen and hepatic CYP family genes (CYP1A1, CYP1A2, CYP2A19, and CYP2E1) among ML, LM, and their parents. In sexually mature (5-month-old) male ML or LM pigs, not only the serum androgen level, but also the hepatic expression levels of all the CYPs examined were similar to those in male Meishan pigs. In addition, there were few breed differences among the females of Meishan, Landrace, ML and LM pigs in the expression of all the CYP genes examined. Furthermore, the expression levels of these CYPs in the females of Meishan and Landrace pigs could be decreased to the corresponding levels in male Meishan pigs by administration of testosterone propionate. The present findings demonstrate that serum androgen level is determined by autosomal dominant inheritance and that the level of serum androgen is one of the host factors regulating the constitutive expression of CYP1A1, CYP1A2, CYP2A19, and CYP2E1 in the pig liver.     

Effects of major tanshinones isolated from Danshen (Salvia miltiorrhiza) on rat CYP1A2 expression and metabolism of model CYP1A2 probe substrates

This study explored the effects of Danshen on metabolism/pharmacokinetics of model CYP1A2 substrates and hepatic CYP1A2 expression in rats. The effects of Danshen and tanshinones on CYP1A2 activity was determined by metabolism of model substrates in vitro (phenacetin) and in vivo (caffeine). HPLC was used to determine model substrates/metabolites. The effect of Danshen on CYP1A2 expression was determined by Western blot. Tanshinones (1.25–50 μM) competitively inhibited phenacetin O-deethylation in vitro. Inhibition kinetics studies showed the K_i values were in the order: dihydrotanshinone (3.64 μM), cryptotanshinone (4.07 μM), tanshinone I (22.6 μM) and tanshinone IIA (23.8 μM), furafylline (35.8 μM), a CYP1A2 inhibitor. The Ki of Danshen extract (mainly tanshinones) was 72 μg/ml. Acute Danshen extract treatment (50–200 mg/kg, i.p.) decreased metabolism of caffeine to paraxanthine, with overall decrease in caffeine clearance (14–22%); increase in AUC (11–25%) and plasma T_1/2 (12–16%). Danshen treatment with (100 mg/kg/day, i.p. or 200 mg/kg/day, p.o.) for three or fourteen days showed similar pharmacokinetic changes of the CYP1A2 probe substrate without affecting CYP1A2 expression. This study demonstrated that major tanshinones competitively inhibited the metabolism of model CYP1A2 probe substrates but had no effect on rat CYP1A2 expression.     

Growth and antrum formation of bovine primary follicles in long-term culture in vitro

Successful antral formation in vitro from bovine preantral follicles (145–170 μm) has been described previously, but antrum formation from the primary follicle (50–70 μm) has not yet been achieved in vitro. The aim of the study was to establish an optimal culture system supporting the growth and maturation of bovine primary follicles (50–70 μm) in vitro. Bovine primary follicles were cultured in a three-dimensional culture system for 13 or 21 days in alpha-minimum essential medium. Various treatments including follicle stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (E₂), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were tested. The follicular diameter and antrum formation rate were recorded, and follicular maturation markers (P450 aromatase, CYP19A1; anti-Mullerian hormone, AMH; growth differentiation factor-9, GDF9; bone morphogenetic protein-15, BMP15; and type III transforming growth factor β receptor, TGFβR3) were analyzed by real-time RT-PCR. After 21 days of culture under each treatment condition, the follicular diameter was significantly enlarged in the presence of FSH + LH + E₂ + bFGF or FSH + LH + E₂ + bFGF + EGF (p < 0.05). An addition of 50 ng/ml bFGF or bFGF + 25 ng/ml EGF initiated antrum formation by day 19 and day 17 of culture, and the antral cavity formation rate was 16.7% and 33.3% by 21 days of culture, respectively. The expression of follicular maturation markers (CYP19A1, AMH, GDF9, BMP15 and TGFβR3) was significantly altered. We conclude that addition of 50 ng/ml bFGF + 25 ng/ml EGF to media containing FSH + LH + E₂ turned out to be the most effective optimized culture conditions to support the growth and maturation of bovine primary follicles in vitro.     

Synthesis and in vitro DMPK profiling of a 1,2-dioxolane-based library with activity against Plasmodium falciparum

A 43-member 1,2-dioxolane library was synthesized by coupling a 1,2-dioxolane-3-acetic acid derivative to a range of amines. Ten compounds had EC₅₀s ? 30 nM against Plasmodium falciparum 3D7 and Dd2 strains, and another 15 compounds had EC₅₀s ? 50 nM against both 3D7 and Dd2. The library was then subjected to a range of in vitro DMPK assays, which revealed that side chains with a heteroatom were required for favorable solubility, Log D and membrane permeability. CYP450 inhibition was isoform dependent, with 2C19 and 3A4 particularly susceptible, and the majority of compounds tested against rat and human microsomes were metabolized rapidly.     

Pulmonary CYP2A13 levels are associated with early occurrence of lung cancer—Its implication in mutagenesis of non-small cell lung carcinoma

CYP2A13, a human pulmonary specific cytochrome P450 enzyme, plays an important role in susceptibility to tobacco-specific nitrosamines (TSNAs)-induced lung cancer in humans. The pattern of CYP2A13 distribution in respiratory tract affects the susceptibility of the lung to carcinogens. CYP2A13 is expressed in the epithelium of trachea and bronchi; however its pattern of expression in human lung cancer remains largely unknown. This study aimed to determine the CYP2A13 expression in specimens from human non-small cell lung carcinomas (NSCLCs), i.e., adenocarcinoma and squamous carcinoma, by immunohistochemical (IHC) analysis and to identify the potential linkage between tumor CYP2A13 levels and some clinicopathological characteristics of NSCLC patients in Taiwan. The tumor CYP2A13 IHC staining signal was strong in 76% of the 112 study subjects. Study subjects (especially non-smoking or lung adenocarcinoma patients) with higher tumor CYP2A13 levels were younger. Multiple logistic regression analysis revealed that in younger subjects (age ≤ 66) and heavy smokers (pack-years ≥ 40), the odds ratio (OR) for positive tumor CYP2A13 staining was significantly higher than that for negative tumor CYP2A13 staining. Moreover, the association of EGFR gene mutations and positive tumor CYP2A13 staining was also revealed. In conclusion, these findings suggest the potential involvement of pulmonary CYP2A13 in the early occurrence of NSCLC as well as in the development of EGFR gene mutations.     

Metabolism of carbosulfan II. Human interindividual variability in its in vitro hepatic biotransformation and the identification of the cytochrome P450 isoforms involved

This study aims to characterize interindividual variability and individual CYP enzymes involved in the in vitro metabolism of the carbamate insecticide carbosulfan. Microsomes from ten human livers (HLM) were used to characterize the interindividual variability in carbosulfan activation. Altogether eight phase I metabolites were analyzed by LC–MS. The primary metabolic pathways were detoxification by the initial oxidation of sulfur to carbosulfan sulfinamide (‘sulfur oxidation pathway’) and activation via cleavage of the nitrogen sulfur bond (N–S) to give carbofuran and dibutylamine (‘carbofuran pathway’). Differences between maximum and minimum carbosulfan activation values with HLM indicated nearly 5.9-, 7.0, and 6.6-fold variability in the k_m, V_max and CL_int values, respectively. CYP3A5 and CYP2B6 had the greatest efficiency to form carbosulfan sulfinamide, while CYP3A4 and CYP3A5 were the most efficient in the generation of the carbofuran metabolic pathway. Based on average abundances of CYP enzymes in human liver, CYP3A4 contributed to 98% of carbosulfan activation, while CYP3A4 and CYP2B6 contributed 57 and 37% to detoxification, respectively. Significant correlations between carbosulfan activation and CYP marker activities were seen with CYP3A4 (omeprazole sulfoxidation), CYP2C19 (omeprazole 5-hydroxylation) and CYP3A4 (midazolam 1′-hydroxylation), displaying r² = 0.96, 0.87 and 0.82, respectively. Activation and detoxification pathways were inhibited by ketoconazole, a specific CYP3A4 inhibitor, by 90–97% and 47–94%, respectively. Carbosulfan inhibited relatively potently CYP3A4 and moderately CYP1A1/2 and CYP2C19 in pooled HLM. These results suggest that the carbosulfan activation pathway is more important than the detoxification pathway, and that carbosulfan activation is predominantly catalyzed in humans by CYP3A4.     

In vitro characterization of CYP102G4 from Streptomyces cattleya: A self-sufficient P450 naturally producing indigo

Self-sufficient CYP102As possess outstanding hydroxylating activity to fatty acids such as myristic acid. Other CYP102 subfamily members share substrate specificity of CYP102As, but, occasionally, unusual characteristics of its own subfamily have been found. In this study, only one self-sufficient cytochrome P450 from Streptomyces cattleya was renamed from CYP102A_scat to CYP102G4, purified and characterized. UV–Vis spectrometry pattern, FAD/FMN analysis, and protein sequence comparison among CYP102s have shown that CYP102 from Streptomyces cattleya belongs to CYP102G subfamily. It showed hydroxylation activity toward fatty acids generating ω-1, ω-2, and ω-3-hydroxyfatty acids, which is similar to the general substrate specificity of CYP102 family. Unexpectedly, however, expression of CYP102G4 showed indigo production in LB medium batch flask culture, and high catalytic activity (k_cat/K_m) for indole was measured as 6.14 ± 0.10 min^− 1 mM^− 1. Besides indole, CYP102G4 was able to hydroxylate aromatic compounds such as flavone, benzophenone, and chloroindoles. Homology model has shown such ability to accept aromatic compounds is due to its bigger active site cavity. Unlike other CYP102s, CYP102G4 did not have biased cofactor dependency, which was possibly determined by difference in NAD(P)H binding residues (Ala984, Val990, and Tyr1064) compared to CYP102A1 (Arg966, Lys972 and Trp1046). Overall, a self-sufficient CYP within CYP102G subfamily was characterized using purified enzymes, which appears to possess unique properties such as an only prokaryotic CYP naturally producing indigo.     

CYP17 and CYP19 genetic variants are not associated with age at natural menopause in Polish women

The aim of the study was to investigate associations between two common polymorphisms of CYP17 and CYP19, encoding key enzymes of estrogen biosynthesis, and age at menopause in Polish women. One hundred fifty women after menopause (49.5 ± 3.8 years), with no previous history of hormone replacement therapy took part in the study. The genetic control group consisted of 150 newborns from the same population. We investigated an association between the age at menopause and the single nucleotide polymorphism T → C in the 5′ untranslated region (promoter) of the CYP17 gene (c.-34T>C; rs743572 – MspA1) or the number of tetranucleotide repeats [TTTA]_n (rs60271534) including deletion/insertion (D/I) of a 3 bp sequence in intron 4 of the CYP19 gene. CYP17 polymorphism was analyzed by PCR-RFLP and CYP19 by PCR and capillary electrophoresis. In the case of CYP17 polymorphism, 28.7% and 36.7% wild homozygous (TT), 50.7% and 46.0% heterozygous (TC), as well as 20.6% and 17.3% mutated homozygous (CC) types were identified in the subjects and controls, respectively. The frequency of mutated alleles (C) was 46.0% vs. 40.3% (p = 0.19). In the case of CYP19 polymorphism, 34.0% and 32.0% of homozygotes (1_1), 50.7% and 51.3% of heterozygotes (1_2), 15.3% and 16.7% of homozygotes (2_2) were identified in the subjects and controls, respectively. No association between the studied CYP17 or CYP19 polymorphisms and age at menopause was found in Polish women.     

Lead optimization of 5-amino-6-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)-4-hydroxyhexanamides to reduce a cardiac safety issue: Discovery of DS-8108b,an orally active renin inhibitor

With the aim to address an undesired cardiac issue observed with our related compound in the recently disclosed novel series of renin inhibitors, further chemical modifications of this series were performed. Extensive structure–activity relationships studies as well as in vivo cardiac studies using the electrophysiology rat model led to the discovery of clinical candidate trans-adamantan-1-ol analogue 56 (DS-8108b) as a potent renin inhibitor with reduced potential cardiac risk. Oral administration of single doses of 3 and 10 mg/kg of 56 in cynomolgus monkeys pre-treated with furosemide led to significant reduction of mean arterial blood pressure for more than 12 h.     

Challenges and pitfalls of P450-dependent (+)-valencene bioconversion by Saccharomyces cerevisiae

Natural nootkatone is a high value ingredient for the flavor and fragrance industry because of its grapefruit flavor/odor, low sensorial threshold and low availability. Valencene conversion into nootkatol and nootkatone is known to be catalyzed by cytochrome P450 enzymes from both prokaryotic and eukaryotic organisms, but so far development of a viable bioconversion process using either native microorganisms or recombinant enzymes was not successful. Using an in silico gene-mining approach, we selected 4 potential candidate P450 enzymes from higher plants and identified two of them that selectively converted (+)-valencene into β-nootkatol with high efficiency when tested using recombinant yeast microsomes in vitro. Recombinant yeast expressing CYP71D51v2 from tobacco and a P450 reductase from arabidopsis was used for optimization of a bioconversion process. Bioconversion assays led to production of β-nootkatol and nootkatone, but with low yields that decreased upon increase of the substrate concentration. The reasons for this low bioconversion efficiency were further investigated and several factors potentially hampering industry-compatible valencene bioconversion were identified. One is the toxicity of the products for yeast at concentrations exceeding 100 mg L⁻¹. The second is the accumulation of β-nootkatol in yeast endomembranes. The third is the inhibition of the CYP71D51v2 hydroxylation reaction by the products. Furthermore, we observed that the formation of nootkatone from β-nootkatol is not P450-dependent but catalyzed by a yeast component. Based on these data, we propose new strategies for implementation of a viable P450-based bioconversion process.     

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

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

CYP1AI, glutathione S-transferase gene polymorphisms and risk of Polycythemia vera

Background: Associations between polymorphisms for gene encoding enzymes involved in biotransformation of xenobiotics and susceptibility to several cancers have been shown in several studies. The aim of the present study was to evaluate the association of polymorphisms of cytochrome P450 (CYP1A1) and GST deletions with the incidence of Polycythemia vera (PV) among the Jordanian population. Methods: The study included 61 PV patients and 70 cancer-free healthy controls. CYP1A1 (m1, m2, m3, m4) and GST (T1, M1) genotypes were determined by polymerase chain reaction and restriction fragment length polymorphism. The risk of cancer associated with gene polymorphisms was estimated by calculations of odds ratio (ORs) and confidence intervals (95% CIs) using Mantel–Haenszel statistics. Results: A statistically significant difference between the PV group and the control group was observed in the case of GSTM1 null genotype with 3.38 fold increase in risk of developing PV (95% CI = 1.63–7.01, p = 0.001) while GSTT1 null genotype showed no significance (OR = 1.11; 95% CI = 0.50–2.44, p = 0.38). No significant association was found between the CYP1A1 mutant genotypes (m1, m2, m4) and PV. The m3 genotype was absent in both patients and controls. Interestingly, a substantial significant increase of PV risk for the combination of GSTM1 null genotype and CYP1A1 m1 (T6235C) genotype was observed (OR = 4.38; 95% CI = 1.15–16.73, p = .02). Furthermore, the present case–control study showed that the studied Jordanian population generally resembles Caucasian populations with respect to the frequencies of CYP1A1 polymorphisms. Conclusion: Our data suggests that GSTM1 null genotype alone and in combination with CYP1A1 m1 genotype may be predisposing risk factors for PV in the Jordanian population.     

Molecular cloning and functional analysis of cytochrome P450 1A2 from Japanese monkey liver: comparison with marmoset cytochrome P450 1A2

A cDNA encoding a novel cytochrome P450 1A2 (CYP1A2) was cloned from the liver of an adult female Japanese monkey. The CYP1A2 protein was expressed in yeast cells and its enzymatic properties were compared with those of marmoset CYP1A2 using ethoxyresorufin (ER) and phenacetin (PN) as substrates. The nucleotide sequence of Japanese monkey CYP1A2 revealed 94.7, 99.5 and 93.5% identities to those of human, cynomolgus monkey and marmoset monkey CYP1A2, respectively. Multiple amino acid sequence alignment of Japanese monkey CYP1A2 with CYP1A2 of humans, cynomolgus monkeys and marmosets showed that Japanese monkey CYP1A2 had 92.4, 99.0 and 91.9% identities to the human, cynomolgus monkey and marmoset enzymes, respectively. Kinetic studies demonstrated that the enzymatic properties as ER and PN O-deethylases were considerably different between the Japanese monkey and the marmoset CYP1A2. Furthermore, both of these reactions in liver microsomal fractions from the Japanese monkey and marmoset showed biphasic kinetics. On the basis of the kinetic parameters, it is suggested that Japanese monkey CYP1A2 is a high-K(m) enzyme in both ER and PN O-deethylations, whereas marmoset CYP1A2 is a high-K(m) and low-K(m) enzyme in ER and PN O-deethylations, respectively. alpha-Naphthoflavone, an inhibitor of human CYP1A1 and CYP1A2, did not completely inhibit the liver microsomal oxidations of ER and PN even at the highest concentration (50muM), supporting the notion that CYP1A2 enzymes are not the sole ER or PN O-deethylase in Japanese monkey and marmoset liver microsomes. Inhibitory effects of furafylline, an inhibitor of human CYP1A2, on ER O-deethylation by recombinant CYP1A2 enzymes were much lower than those of alpha-naphthoflavone, but marmoset CYP1A2 was more sensitive to furafylline than Japanese monkey CYP1A2. These results indicate that the properties of Japanese monkey CYP1A2 are considerably different from those of marmoset CYP1A2.