Cytochrome P-450 Species Research Articles

Accelerating Quantum Computations of Chemistry Through Regularized Compressed Double Factorization

We propose the regularized compressed double factorization (RC-DF) method to classically compute compressed representations of molecular Hamiltonians that enable efficient simulation with noisy intermediate scale (NISQ) and error corrected quantum algorithms. We find that already for small systems with 12 to 20 qubits, the resulting NISQ measurement scheme reduces the number of measurement bases by roughly a factor of three and the shot count to reach chemical accuracy by a factor of three to six compared to truncated double factorization (DF) and we see order of magnitude improvements over Pauli grouping schemes. We demonstrate the scalability of our approach by performing RC-DF on the CpdI species of cytochrome P450 with 58 orbitals and find that using the resulting compressed Hamiltonian cuts the run time of qubitization and truncated DF based error corrected algorithms almost in half and even outperforms the lambda parameters achievable with tensor hypercontraction (THC) while at the same time reducing the CCSD(T) energy error heuristic by an order of magnitude.

Uptake of Nanoplastic particles by zebrafish embryos triggers the macrophage response at early developmental stage

Plastic pollution continues to erupt as a global ecological concern. As plastic debris is degraded into nanoscale and microscale particles via biodegradation, UV-irradiation, and mechanical processes, nanoplastic pollution arises as a threat to virtually every biological and ecological system on the planet. In this study, zebrafish (Danio rerio) embryos were exposed to fluorescently labeled plastic particles at nanoscales (30 nm and 100 nm). The uptake of both the nanoplastic particles (NPs) was found to exponentially increase with incubation time. Penetration of NPs through the natural barrier of the zebrafish embryos, the chorion, was observed prior to the hatching of the embryo. As a result, the NPs were found to accumulate on the body surface as well as inside the body of the zebrafish. The invasion of NPs into zebrafish embryos induced the upregulation of several stress and immune response genes including interleukins (il6 and il1b), cytochrome P450 (cyp1a and cyp51), and reactive oxygen species (ROS) removal protein-encoding genes (sod and cat). This suggested the initiation of ROS generation and removal as well as the activation of the immune response of zebrafish embryos. Colocalization of macrophages and NPs in zebrafish embryos indicated the involvement of macrophage response to the NP invasion at the early developmental stage of zebrafish.

Effects of alcohol-induced increase in CYP2E1 content in human liver microsomes on the activity and cooperativity of CYP3A4

We investigate the effect of the alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. Membrane incorporation of the purified CYP2E1 into HLM considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. It also eliminates the activating effect of α-naphthoflavone (ANF) seen in some HLM samples. To probe the physiological relevance of these effects, we compared three pooled preparations of HLM from normal donors (HLM-N) with a pooled preparation from ten heavy alcohol consumers (HLM-A). The composition of the P450 pool in all samples was characterized by the mass-spectrometric determination of 11 cytochrome P450 species. The fractional content of CYP2E1 in HLM-A was from 2.0 to 3.4 times higher than in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all samples. Despite that, HLM-A exhibited a much higher metabolism rate and a lower homotropic cooperativity with BQ, similar to CYP2E1-enriched HLM-N. To substantiate the involvement of interactions between CYP2E1 and CYP3A4 in these effects, we probed hetero-association of these proteins in CYP3A4-containing Supersomes™ with a technique employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments evinced the interactions between the two enzymes and revealed an inhibitory effect of ANF on their association. Our results demonstrate that the functional properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and suggest a possible impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4.

7‐Dimethylamino‐4‐trifluormethylcoumarin (Coumarin‐152) as a fluorogenic substrate of CYP3A4 and a tool for studying functional interrelationships among human cytochromes P450

In this study we probe the use of 7‐Dimethylamino‐4‐trifluromethylcoumarin (Coumarin 152, C152) as a fluorogenic substrate for in vitro studies of drug metabolism in human liver microsomes (HLM). Examining spectral properties of the formed fluorescent products and using LC‐MS/MS for monitoring C152 metabolism we demonstrate sequential formation of two N‐demethylated products – 7‐Methylamino‐4‐trifluoromethylcoumarin (desmethyl‐Coumarin 152, DC152) and amino‐4‐trifluoromethylcoumarin (Coumarin 151, C151). The predominant product formed by HLM is DC152, which accounts for >70% of C152 metabolized. Using insect cell microsomes containing recombinant P450 enzymes (Supersomes™) we found that C152 is metabolized by CYP2B6, CYP3A4, CYP2C19, CYP1A2 and CYP2C9 listed in the order of decreasing rates of turnover. The KM values exhibited by CYP2B6, CYP3A4, CYP1A2 and CYP2C19 were of the same order of magnitude (1.8 – 4.8 μM). Importantly, the major metabolizers of C152 – CYP2B6, CYP3A4, CYP2C19 and CYP1A2 – demonstrated different ability to catalyze the second demethylation step, with CYP1A2 being the most potent catalyst in this reaction. While over half of the substrate metabolized by CYP1A2 underwent the second demethylation, the fraction of C151 formed by other enzymes did not exceed 30%, similar to what is observed in HLM. Although the rate of turnover of C152 with CYP2B6 is higher than that observed with CYP3A4, the latter appears to be the major metabolizer of C152 due to its high abundance in HLM. This is confirmed by ketoconazole (KCZ) inhibition experiments. We demonstrate that in both CYP3A4 Supersomes™ and HLM over 80% of C152 turnover is suppressed by KCZ, whereas the activities of CYP1A2, CYP2B4 and CYP2C19 with C152 remain largely unaffected. Our results demonstrate that C152 can be used for probing functional interrelationships between multiple cytochrome P450 species in HLM as an example of a substrate that is almost exclusively metabolized by CYP3A4 though having similar affinities to most major human drug‐metabolizing cytochromes P450. In addition, we establish an easy and reliable fluorimetric assay for monitoring and analyzing the metabolism of C152 that allows discriminating between the two sequential steps of its N‐demethylation.Support or Funding InformationThis research was supported by the National Institute On Alcohol Abuse And Alcoholism of NlH under Award Number R21AA024548

Two‐State Reactivity: Personal Recounting of its Conception and Future Prospects

AbstractThis essay tells the story of the conception of the Two‐State Reactivity (TSR) notion. Since scientific career is part of life's flow, the story blends sub‐stories of scientific colleagues and events. This is also a story of a beguiling paradigm, which has started from a puzzling reactivity of the diatomic oxidant FeO+, has continued to larger oxidants, like the active species of Cytochrome P450 and of nonheme enzymes, and its extension to reductive processes. Finally, the essay discusses prospects of experimental probing of the reactive spin‐state, and of the transition state constitution for these reactions by means of tunneling‐augmented kinetic isotope effects.

Synergy between Experiments and Computations: A Green Channel for Revealing Metabolic Mechanism of Xenobiotics in Chemical Toxicology.

Xenobiotic metabolism can lead to metabolites with altered physicochemical and biological properties, which may differ markedly from those of their parent compounds. Thus, xenobiotic metabolism has great implication for chemical safety evaluation, which has become one of the central research areas in chemical toxicology. A plethora of analytical and in vitro methods are now available for investigating the metabolic fate of xenobiotics, especially by cytochrome P450 (CYP), at a high level of detail. However, the interpretations of metabolic reactions often face some mechanistic challenges, for example, the mechanism of the initial and rate-determining step is not easily distinguished due to the transient nature of active species of CYP, and some reactive intermediates are difficult to identify. Alternatively, computational chemistry methodologies such as quantum chemical calculations have the capacity to calculate the electronic structures for enzymatic models with hundreds of atoms, thus to be able to characterize intermediates and transition states during whole metabolic reaction course from both structural and energetics aspects, which can confront some major limitations of experimental methods. In this perspective, I first introduce state of the art experimental and computational approaches for investigating xenobiotic metabolism catalyzed by CYP, respectively. Then the strategies to harvest the synergy between experiments and computations are highlighted, which can be conducted through comparison of their analytical, kinetic, or isotope effect data at a qualitative, semiquantitative, or quantitative level to determine the metabolic mechanism. Two examples are chosen to demonstrate the synergy advantage to elucidate the metabolic mechanism of triphenyl phosphate and atrazine catalyzed by CYP, respectively, which show that the interplay between experiments and computations allows greater insight to be gained than with the isolated methods.

Toward a systems approach to cytochrome P450 ensemble: interactions of CYP2E1 with other P450 species and their impact on CYP1A2.

In this study, we investigate the ability of ethanol-inducible CYP2E1 to interact with other cytochrome P450 species and affect the metabolism of their substrates. As a model system, we used CYP2E1-enriched human liver microsomes (HLM) obtained by the incorporation of purified CYP2E1. Using a technique based on homo-FRET in oligomers of CYP2E1 labeled with BODIPY 577/618 maleimide we demonstrated that the interactions of CYP2E1 with HLM result in the formation of its mixed oligomers with other P450 species present in the microsomal membrane. Incorporation of CYP2E1 results in a multifold increase in the rate of metabolism of CYP2E1-specific substrates p-Nitrophenol and Chlorzaxozone. The rate of their oxidation remains proportional to the amount of incorporated CYP2E1 up to the content of 0.3-0.4 nmol/mg protein (or ∼50% CYP2E1 in the P450 pool). The incorporated CYP2E1 becomes a fully functional member of the P450 ensemble and do not exhibit any detectable functional differences with the endogenous CYP2E1. Enrichment of HLM with CYP2E1 results in pronounced changes in the metabolism of 7-ethoxy-4-cyanocoumarin (CEC), the substrate of CYP2C19 and CYP1A2 suggesting an increase in the involvement of the latter in its metabolism. This effect goes together with an augmentation of the rate of dealkylation of CYP1A2-specific substrate 7-ethoxyresorufin. Furthermore, probing the interactions of CYP2E1 with model microsomes containing individual P450 enzymes we found that CYP2E1 efficiently interacts with CYP1A2, but lacks any ability to form complexes with CYP2C19. This finding goes inline with CYP2E1-induced redirection of the main route of CEC metabolism from CYP2C19 to CYP1A2.

Serum derived from ulcerative colitis mouse changes the metabolism of the fluorescent substrate by P450 depending on the degree of disease progression

Ulcerative colitis (UC) is characterized by erosions of the intestinal mucosa. The number of patients with UC has recently been increasing rapidly. Since the diagnosis of UC is complex and difficult, a simple, rapid, noninvasive technique for diagnosing UC is needed urgently. The expression of cytochrome P450 (P450 or CYP) species in mouse liver is known to be changed dependent on the disease. Various components such as P450 substrates and P450 metabolites in the blood may possibly change with the UC-specific way in mouse. In this study, in order to evaluate UC-specific components in UC mouse serum, we analyzed the influence of serum derived from UC mice on the results of fluorescent P450 inhibition assays based on 12 human P450 enzymes, such as CYP1A1, CYP2C8, CYP2E1,CYP3A4, CYP1A2, CYP2D6, CYP2A13, CYP2B6, CYP2C9, CYP2C18, CYP2C19, and CYP3A5. At first, in order to induce UC, mice received 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) dissolved in their drinking water for 7 days. Next, these 12 human P450 enzymes were expressed in E. coli cells. Then, P450 fluorescent competition reaction was performed using these P450 enzymes and serum of UC mice. We found that the metabolism of fluorescent substrates by CYP2B6, CYP2C19, CYP2E1, and CYP1A1 in the presence of serum obtained from DSS-treated mice was activated by 42%, 37%, 37%, and 23%, respectively, relative to that associated with sera from control mice. A receiver operating characteristic (ROC) curve analysis was carried out with the 31 samples of UC mice and healthy mice. Area under the ROC curve (AUC) value was calculated from ROC curve. AUC value of CYP2E1 and CYP2C19 showed 0.921 and 0.892, respectively. Therefore, it was shown that CYP2C19 and CYP2E1 could be used as biomarkers for evaluating ulcerative colitis. From these results, it is suggested that these simple fluorescent P450 inhibition assays have potential as a new diagnostic procedure for UC in mouse. This study is the first report on a simple non-invasive method for evaluating UC using P450 enzyme and serum interaction.

Towards a System Approach to the Human Cytochrome P450 Ensemble: A New Strategy for Studying the Network of P450‐P450 Interactions in Human Liver Microsomes

The composition of the human cytochrome P450 ensemble is commonly recognized as a key factor determining the interindividual variability in drug metabolism. However, recent studies on a large selection of human liver microsomes (HLM) demonstrated the lack of a direct correlation between the composition of the cytochrome P450 ensemble and the profile of drug metabolism. At the same time, there is an emerging recognition of the functional importance of interactions between multiple cytochrome P450 species co‐localized in the microsomal membrane. A change in the content of any particular P450 enzyme alters the landscape of protein‐protein interactions and thus affects drug metabolism in a complex, hard‐to‐predict manner. Therefore, a systematic exploration of the network of P450‐P450 interactions and identification of their functional consequences represents an issue of emerging importance for revealing the interrelationships between the profile of P450 expression and the profile of drug metabolism. In a search for a model system suitable for these studies we introduced a method based on incorporation of purified cytochromes P450 into the microsomes. Here we demonstrate high potential of this approach for creating preparations of HLM with altered composition of the P450 ensemble. An important characteristic of this strategy is its adaptability for incorporating genetically or chemically modified proteins. This includes the incorporation of proteins bearing fluorescent probes and light‐activating crosslinkers. Therefore, this strategy may be used for monitoring protein‐protein interactions in HLM with a variety of biophysical techniques, as well as for searching the partners of P450 protein‐protein interactions with the technique of molecular fishing.Here we report the use of our new approach for studying the effect of enriching the HLM with ethanol‐inducible CYP2E1, one of the the most populated P450 species in human liver. Incorporation of CYP2E1 into HLM results in an increase in the rate of metabolism of CYP2E1‐specific substrates, which is proportional to the amount of the incorporated enzyme up to its content of 0.3 nmol per mg protein (or ~50% of CYP2E1 in the total P450 content). Our studies with a series of substrates of drug‐metabolizing P450s showed that CYP2E1 incorporation results in pronounced alterations in the metabolism of substrates of several other P450 species present in HLM. In particular, we observed over 2‐fold increase in the rate of metabolism and a substantial decrease in the apparent KM observed with 3‐cyano‐7‐etoxycoumarin (CEC), the substrate of CYP1A2 and CYP2C19. This finding may indicate that the increase in CYP2E1 concentration results in redirecting the main route of CEC metabolism from CYP2C19 to CYP1A2, which is characterized with lower KM and higher turnover numbers with this substrate. Similarly, increase in CYP2E1 content causes an important changes in the function of CYP3A enzymes, which is revealed in a compromised homotropic cooperativity, decreased affinity and increased Vmax, observed with 7‐benzyloxyquinoline, a selective substrate of CYP3A4 and CYP3A5.Support or Funding InformationThis research was supported by grant R01‐GM114369 from NIH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evolutionary origin of mitochondrial cytochrome P450.

Different molecular species of cytochrome P450 (P450) are distributed between endoplasmic reticulum (microsomes) and mitochondria in animal cells. Plants and fungi have many microsomal P450s, but no mitochondrial P450 has so far been reported. To elucidate the evolutionary origin of mitochondrial P450s in animal cells, available evidence is examined, and the virtual absence of mitochondrial P450 in plants and fungi is confirmed. It is also suggested that a microsomal P450 is the ancestor of animal mitochondrial P450s. It is likely that the endoplasmic reticulum-targeting sequence at the amino-terminus of a microsomal P450 was converted to a mitochondria-targeting sequence possibly by point mutations of a few amino acid residues or by an exon-shuffling/moving event shortly after animal lineage diverged from plants and fungi in the course of evolution of eukaryotes. It is suggested that the microsome-type P450 first imported into mitochondria utilized the existing ferredoxin in the matrix to receive electrons from NADPH, retained its oxygenase activity in the mitochondria, and gradually diversified to several P450s with different substrate specificities in the course of the evolution of animals.

P.8.b.009 Inhaled loxapine for the acute treatment of agitation: clinical experience from eastern and northern Finland

Ellipticine is an antineoplastic agent considered a pro-drug, the pharmacological and genotoxic effects of which are dependent on cytochrome P450 (CYP)- and/or peroxidase-mediated activation to covalent DNA adducts. We investigated whether ellipticine–DNA adducts are formed in human hepatic microsomes and human hepatocytes. We then identified which human CYPs oxidize ellipticine to metabolites forming DNA adducts and the effect of cytochrome b5 on this oxidation. 13-Hydroxyellipticine, the metabolite forming the major ellipticine–DNA adduct, was generated mainly by CYP3A4 and 1A1, followed by CYP2D6 > 2C19 > 1B1 > 1A2 > 2E1 and >2C9. Cytochrome b5 increased formation of this metabolite by human CYPs, predominantly by CYP1A1, 3A4, 1A2 and 2C19. Formation of 12-hydroxyellipticine is generated mainly by CYP2C19, followed by CYP2C9 > 3A4 > 2D6 > 2E1 and >2A6. Other CYPs were less active (CYP2C8 and 2B6) or did not oxidize ellipticine to this metabolite (CYP1A1, 1A2 and 1B1). CYP2D6 was the most efficient enzyme generating ellipticine N2-oxide. CYP3A4 and 1A1 in the presence of cytochrome b5 are mainly responsible for bioactivation of ellipticine to DNA adduct 1 (formed by ellipticine-13-ylium from 13-hydroxyellipticine), while 12-hydroxyellipticine generated during the CYP2C19-mediated ellipticine oxidation is the predominant metabolite forming ellipticine-12-ylium that generates ellipticine–DNA adduct 2. These ellipticine–DNA adducts were also generated by human hepatic microsomes and in primary human hepatocytes exposed to ellipticine. Ellipticine is toxic to these hepatocytes, decreasing their viability; the IC50 value of ellipticine in these cells was 0.7 μM. In liver CYP3A4 is the predominant ellipticine activating CYP species, which is expected to result in efficient metabolism after oral ingestion of ellipticine in humans.

Expression of CYP1A1 and GSTP1 in Human Brain Tumor Tissues in Pakistan

Most of the exogenous and endogenous chemical compounds are metabolized by enzymes of xenobiotic processing pathways, including the phase I cytochrome p450 species. Carcinogens and their metabolites are generally detoxified by phase II enzymes like glutathione-S-transferases (GST). The balance of enzymes determines whether metabolic activation of pro-carcinogens or inactivation of carcinogens occurs. Under certain conditions, deregulated expression of xenobiotic enzymes may also convert endogenous substrates to metabolites that can facilitate DNA adduct formation and ultimately lead to cancer development. In this study, we aimed to test the association between deregulation of metabolizing genes and brain tumorigenesis. The expression profile of metabolizing genes CYP1A1 and GSTP1 was therefore studied in a cohort of 36 brain tumor patients and controls using Western blotting. In a second part of the study we analyzed protein expression of GSTs in the same study cohort by ELISA. CYP1A1 expression was found to be significantly high (p<0.001) in brain tumor as compared to the normal tissues, with ~4 fold (OR=4, 95%CI=0.43-37) increase in some cases. In contrast, the expression of GSTP1 was found to be significantly low in brain tumor tissues as compared to the controls (p<0.02). This down regulation was significantly higher (OR=0.05, 95%CI=0.006-0.51; p<0.007) in certain grades of lesions. Furthermore, GSTs levels were significantly down-regulated (p<0.014) in brain tumor patients compared to controls. Statistically significant decrease in GST levels was observed in the more advanced lesions (III-IV, p<0.005) as compared to the early tissue grades (I-II). Thus, altered expression of these xenobiotic metabolizing genes may be involved in brain tumor development in Pakistani population. Investigation of expression of these genes may provide information not only for the prediction of individual cancer risk but also for the prevention of cancer.

Ellipticine oxidation and DNA adduct formation in human hepatocytes is catalyzed by human cytochromes P450 and enhanced by cytochrome b5

Ellipticine is an antineoplastic agent considered a pro-drug, the pharmacological and genotoxic effects of which are dependent on cytochrome P450 (CYP)- and/or peroxidase-mediated activation to covalent DNA adducts. We investigated whether ellipticine–DNA adducts are formed in human hepatic microsomes and human hepatocytes. We then identified which human CYPs oxidize ellipticine to metabolites forming DNA adducts and the effect of cytochrome b5 on this oxidation. 13-Hydroxyellipticine, the metabolite forming the major ellipticine–DNA adduct, was generated mainly by CYP3A4 and 1A1, followed by CYP2D6>2C19>1B1>1A2>2E1 and >2C9. Cytochrome b5 increased formation of this metabolite by human CYPs, predominantly by CYP1A1, 3A4, 1A2 and 2C19. Formation of 12-hydroxyellipticine is generated mainly by CYP2C19, followed by CYP2C9>3A4>2D6>2E1 and >2A6. Other CYPs were less active (CYP2C8 and 2B6) or did not oxidize ellipticine to this metabolite (CYP1A1, 1A2 and 1B1). CYP2D6 was the most efficient enzyme generating ellipticine N2-oxide. CYP3A4 and 1A1 in the presence of cytochrome b5 are mainly responsible for bioactivation of ellipticine to DNA adduct 1 (formed by ellipticine-13-ylium from 13-hydroxyellipticine), while 12-hydroxyellipticine generated during the CYP2C19-mediated ellipticine oxidation is the predominant metabolite forming ellipticine-12-ylium that generates ellipticine–DNA adduct 2. These ellipticine–DNA adducts were also generated by human hepatic microsomes and in primary human hepatocytes exposed to ellipticine. Ellipticine is toxic to these hepatocytes, decreasing their viability; the IC50 value of ellipticine in these cells was 0.7μM. In liver CYP3A4 is the predominant ellipticine activating CYP species, which is expected to result in efficient metabolism after oral ingestion of ellipticine in humans.

Photoregulation of Cytochrome P450 Activity by Using Caged Compound

Cytochrome P450 (P450) species play an important role in the metabolism of xenobiotics, and assaying the activities of P450 is important for evaluating the toxicity of chemicals in drugs and food. However, the lag time caused by the introduction and mixing of sample solutions can become sources of error as the throughput is heightened by increasing the sample number and decreasing the sample volume. To amend this technological obstacle, we developed a methodology to photoregulate the activity of P450 by using photoprotected (caged) compounds. We synthesized caged molecules of nicotinamide adenine dinucleotide phosphate (NADP(+)) and glucose 6-phosphate (G6P), which are involved in the generation of NADPH (cofactor of P450). The use of caged-G6P completely blocked the P450 catalysis before the UV illumination, whereas caged-NADP(+) resulted in a little background reaction. Upon UV illumination, more than 90% of the enzymatic activity could be restored. The use of caged-G6P enabled assays in isolated microchambers (width, 50 μm; height, 50 μm) by encapsulating necessary ingredients in advance and initiating the reaction by UV illumination. The initiation of enzymatic reaction could be observed in a single microchamber. Minimizing uncertainties caused by the introduction and mixing of solutions led to significantly reduced errors of obtained kinetic constants.

Simultaneous Ion-Pair LC Analysis of Ephedrine and Pseudoephedrine in Rat Liver Microsomal Incubations: Application to In-Vitro Metabolism

(−)-Ephedrine (ephedrine, EPH) and (+)-ephedrine (pseudoephedrine, PEPH) are metabolized by the liver, but the species of hepatocyte cytochrome P450 (CYP450) responsible is not yet clear. To investigate which subtype of CYP450 is involved in the metabolism of EPH and PEPH, a rapid and reliable reversed-phase ion-pair liquid chromatographic method for simultaneous analysis of EPH and PEPH in rat liver microsomes has been established and validated. Matrine was selected as a suitable internal standard (IS) for calibration. After liquid–liquid extraction of liver microsomal samples with methyl tert-butyl ether, EPH and PEPH were separated on a C18 reversed-phase column (200 mm × 4.6 mm, 5 μm) with methanol–0.5% sodium dodecyl sulfate–phosphoric acid–triethylamine 60:40:1.25:1 (v/v) as mobile phase at a flow rate of 1.0 mL min. Detection was by UV absorbance at 210.5 nm. For both EPH and PEPH, calibration curves were linear over the range 1.5–60.0 μg mL−1, the limit of quantification was 1.5 μg mL−1, and intra-day and inter-day variability was 73%. The validated method was successfully used to study the in-vitro metabolism of EPH and PEPH. In rat liver microsomes induced by dexamethasone, enzyme activity in the metabolism of EPH and PEPH was higher than that for metabolism of phenobarbital and β-naphthoflavone.

Trends in Aromatic Oxidation Reactions Catalyzed by Cytochrome P450 Enzymes: A Valence Bond Modeling.

The mixed density functional theory (DFT) and valence bond study described herein focuses on the activation of 17 benzene derivatives by the active species of Cytochrome P450, so-called Compound I (Cpd I), as well as by the methoxy radical, as a potentially simple model of Cpd I (Jones, J. P.; Mysinger, M.; Korzekwa, K. R. Drug Metab. Dispos. 2002, 30, 7-12). Valence bond modeling is employed to rationalize the P450 mechanism and its spin-state selectivity from first principles of electronic structure and to predict activation energies independently, using easily accessible properties of the reactants: the singlet-triplet excitation energies, the ionization potentials of the aromatics, and the electron affinity of Cpd I and/or of the methoxy radical. It is shown that the valence bond model rationalizes all the mechanistic aspects and predicts activation barriers (for 35 reactions) with reasonable accuracy compared to the DFT barriers with an average deviation of ±1.0 kcal·mol(-1) (for DFT barriers, see: Bathelt, C. M.; Ridder, L.; Mulholland, A. J.; Harvey, J. N. Org. Biomol. Chem. 2004, 2, 2998-3005). The valence bond modeling also reveals the mechanistic similarities between the P450 Cpd I and methoxy reactions and enables one to make predictions of barriers for reactions from other studies.

Effect of Tea Beverages on Aldehyde Oxidase Activity

Aldehyde oxidase (AO) plays an important role in metabolizing antitumor and antiviral drugs, including methotrexate, cyclophosphamide and acyclovir. Green tea and its catechins have been shown to modulate the activities of various xenobiotic-metabolizing cytochrome P450 species, both in vivo and in vitro, but their effect on AO has not been studied. Therefore, we evaluated the effect of tea beverages on AO activity in rat and human liver cytosol. We also investigated the influence of several catechins on AO activity in rat liver cytosol. AO activity was evaluated in terms of oxidation of N-1-methylnicotinamide to N-1-methyl-2-pyridone-5-carboxamide and N-1-methyl-4-pyridone-3-carboxamide. Bottled green tea beverages at 10% (vol/vol) inhibited AO activity by 90.0-93.5%, while at 1.0% (vol/vol), they reduced AO activity by 73.9-90.0%. At 0.1% (vol/vol), green tea II and III, which have high contents of catechins and their derivatives, inhibited AO activity by 24.3% and 38.8%, respectively. Bottled mineral water had no effect. AO activity was inhibited potently by epicatechin and epicatechin gallate. These results indicate that the AO-inhibitory activity of tea beverages is predominantly due to catechins and their derivatives. Thus, consumption of tea beverages may cause a decrease of AO activity, which may result in reduced clearance of drugs that are AO substrates.

Transcriptomic analysis of chilling stress in Phaseolus spp.

The wild bean species Phaseolus angustissimus has shown a greater ability to survive chilling temperatures than the cultivated bean Phaseolus vulgaris. Arrays of cDNA clones from cold acclimated crowns of Medicago sativa were used to measure low temperature-induced changes in the expression profiles of both bean species and compare them to each other. Macroarray hybridization results indicate that P. vulgaris failed to sustain homeostatic expression levels of more genes than did P. angustissimus after 3 days of chilling temperature exposure. Expression patterns of selected genes were further characterized with RT-PCR using plants exposed to various durations of chilling. The most notable difference between the two species in these gene expression experiments was that P. angustissimus was able to recover the expression of a cytochrome P450 species under chilling temperature exposure whereas P. vulgaris failed to sustain levels of cytochrome P450 gene expression levels. Results of these experiments suggest that a key to chilling tolerance in Phaseolus is maintenance of homeostatic gene expression levels under chilling conditions.

Prednisone reduces ketoconazole-induced skeletal defects in rat fetuses

Ketoconazole (KT) is a broad-spectrum antifungal agent whose pharmacological activity is based on the capability to interfere with steroid biosynthesis through an interaction with fungal cytochrome P-450 enzymes and thereby avoiding the formation of fungal walls. As the inhibition of fungal cytochrome P-450 by KT is not specific, the mammalian cytochrome P-450 species, which play an important role in the biosynthesis of steroidogenesis, are also affected. The reproductive and developmental toxicity of KT have been assessed. This antimycotic agent has been reported as embryotoxic and teratogenic when administered in high doses (80 mg/kg) to pregnant rats. The mechanisms by which KT exert teratogenic effects remains to be elucidated. When considering the potential inhibitory effect of KT on mammalian steroid biosynthesis as a possible responsible for the skeletal anomalies induced by this drug, this study aimed at determining whether steroid maternal supplementation may prevent the skeletal anomalies induced by KT. To test this hypothesis, maternal supplementation with prednisone (PRED) (0.1, 0.2 or 0.4 mg/kg) and 80 mg/kg of KT were administered to pregnant Wistar rats (n = 10) during organogenesis period. On gestational day 21, the dams were euthanized and examined for standard parameters of reproductive outcome. In summary, the results showed that PRED supplementation therapy may cause reductions in the incidence of KT-induced cranial and appendicular skeletal anomalies as well as cleft palate in the rat, being these results more consistent with 0.4 mg/kg of this drug. These results suggest an important role for glucocorticoids in KT-induced teratogenesis.

Bioconversion of small molecules by cytochrome P450 species expressed in Escherichia coli

P450 (cytochrome P450) enzymes catalyse the mono-oxygenation of a wide range of compounds such as steroids, fatty acids, vitamins and drugs. In the present paper we demonstrate a system for bioconverting diverse compounds [flavanone, DHEA (dehydroepiandrosterone) and 7-ethoxycoumarin] using P450 species expressed in Escherichia coli. First, we expressed four P450 species: rabbit CYP2B (P450 family 2, subfamily B), fruitfly (Drosophila) CYP317A, rat CYP3A23 and mouse CYP2J5. Next, we added substrates directly to the incubation medium. The resulting metabolites were extracted and analysed by HPLC and spectrofluorimetry. The first substrate, 7-ethoxycoumarin, was de-ethylated by CYP2B; CYP2J5 and CYP3A23 showed weak activity, and CYP317A had no activity for 7-ethoxycoumarin. We next used flavanone, a flavonoid, as a substrate for these four P450 species and other P450 species expressed previously. As a result, CYP2B, CYP2C43 and CYP2C29 catalysed flavanone 2-hydroxylation. CYP2A5 catalysed 2- and 4-hydroxylations. Finally, to produce diverse modified compounds, variants of CYP2A5 with point mutations were incubated with a steroid (DHEA) and an antioxidant (flavanone) in vivo. HPLC analysis indicated that two P450 species produced a 7-beta-hydroxy-DHEA and two P450 species produced a 2-alpha-hydroxy-DHEA. Four P450 species catalysed flavanone 2- and 4-hydroxylations. These results indicate that bioconversion by P450 is a useful technique to modify small molecules (steroids, coumarin and flavanone) and produce new, diverse hydroxylated compounds, which could be used for high-throughput screening for drug discovery.