Oxidative Xenobiotic Metabolism Research Articles

Synthetic Advances for Mechanistic Insights: Metal-Oxygen Intermediates with a Macrocyclic Pyridinophane System.

ConspectusMetalloenzymes, which are proteins containing earth-abundant transition-metal ions as cofactors in the active site, generate various metal-oxygen intermediates via activating a dioxygen molecule (O2) to mediate vital metabolic functions, such as the oxidative metabolism of xenobiotics and the biotransformation of naturally occurring molecules. By replicating the active sites of metalloenzymes, many bioinorganic chemists have studied the geometric and electronic properties and reactivities of model complexes to understand the nature of enzymatic intermediates and develop bioinspired metal catalysts. Among the reported model complexes, nonporphyrinic macrocyclic ligands are the predominant coordination system widely used in stabilizing and isolating diverse metal-oxygen intermediates, which allows us to extensively investigate the physicochemical characteristics of the analogs of reactive intermediates of metalloenzymes. In particular, it has been reported that the ring size of the macrocyclic ligands, defined by the number of atoms in the macrocyclic ring, drastically affects the identity of the metal-oxygen intermediate. Thus, systematic modification of the macrocyclic ligands has been a great subject being examined in various inorganic fields.In this Account, we describe synthetic advances of a macrocyclic ligand system by introducing pyridine donors into a 12-membered tetraazamacrocyclic ligand (12-TMC) that initially has 4 amine donors. Interestingly, the backbone of the pyridinophane ligand with 2 pyridine and 2 amine donors in a 12-membered ring is shown to be much more folded than in other macrocyclic ligands, thereby allowing the axial and equatorial donors to separately control the electronic structure of metal complexes. Then, we looked over independent electronic and steric effects on metal-oxygen species with thorough physicochemical analysis. The NiIII-peroxo complexes exhibit nucleophilic reactivity dependent on the steric hindrance of the second coordination sphere. Furthermore, the C-H bond strength of the second coordination sphere has also been an important factor in determining the stability of MnIV-bis(hydroxo) intermediates. Electronic tuning on CoIII-hydroperoxo intermediates results in a trend between the electron-donating abilities of para-substituents on pyridine in the pyridinophane ligand and electrophilic reactivities, from which mechanistic insights into the metal-hydroperoxo species have been gained. Importantly, the metal-oxygen intermediates supported by the pyridinophane ligand system have revealed quite challenging chemical reactions, including dioxygenase-like nitrile activation by CoIII-peroxo intermediates and the oxidation of aldehyde and aromatic compounds by manganese-oxygen intermediates. Based on the fine substitution of donors, we have addressed that those novel reactions originated from the unique framework of the pyridinophane system incorporating spin-crossover behavior and high redox potentials of the metal-oxygen intermediates. These results will be valuable for the structure-activity relationship of metal-oxygen intermediates, giving a better understanding on the enzymatic coordination system where amino acid ligands vary for specific chemical reactions.

Biochemical profiling of functionally expressed CYP6P9 variants of the malaria vector Anopheles funestus with special reference to cytochrome b5 and its role in pyrethroid and coumarin substrate metabolism

Cytochrome P450 monooxygenases (P450s) are well studied enzymes catalyzing the oxidative metabolism of xenobiotics in insects including mosquitoes. Their duplication and upregulation in agricultural and public health pests such as anopheline mosquitoes often leads to an enhanced metabolism of insecticides which confers resistance. In the laboratory strain Anopheles funestus FUMOZ-R the duplicated P450s CYP6P9a and CYP6P9b are highly upregulated and proven to confer pyrethroid resistance. Microsomal P450 activity is regulated by NADPH cytochrome P450 oxidoreductase (CPR) required for electron transfer, whereas the modulatory role of cytochrome b5 (CYB5) on insect P450 activity is less clear. In previous studies CYP6P9a and CYP6P9b were recombinantly expressed in tandem with An. gambiae CPR using E. coli-expression systems and CYB5 added to the reaction mix to enhance activity. However, the precise role of CYB5 on substrate turn-over when combined with CYP6P9a and CYP6P9b remains poorly investigated, thus one objective of our study was to address this knowledge gap. In contrast to the CYP6P9 variants, the expression levels of both CYB5 and CPR were not upregulated in the pyrethroid resistant FUMOZ-R strain when compared to the susceptible FANG strain, suggesting no immediate regulatory role of these genes in pyrethroid resistance in FUMOZ-R. Here, for the first time we recombinantly expressed CYP6P9a and CYP6P9b from An. funestus in a baculovirus expression system using High-5 insect cells. Co-expression of each enzyme with CPR from either An. gambiae or An. funestus did not reveal noteworthy differences in catalytic capacity. Whereas the co-expression of An. funestus CYB5 – tested at different multiplicity of infection (MOI) ratios – resulted in a significantly higher metabolization of coumarin substrates as measured by fluorescence assays. This was confirmed by Michaelis-Menten kinetics using the most active substrate, 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC). We observed a similar increase in coumarin substrate turnover by adding human CYB5 to the reaction mix. Finally, we compared by UPLC-MS/MS analysis the depletion rate of deltamethrin and the formation of 4’OH-deltamethrin by recombinantly expressed CYP6P9a and CYP6P9b with and without CYB5 and detected no difference in the extent of deltamethrin metabolism. Our results suggest that co-expression (or addition) of CYB5 with CYP6P9 variants, recombinantly expressed in insect cells, can significantly enhance their metabolic capacity to oxidize coumarins, but not deltamethrin.

Effects of Diminished NADPH:cytochrome P450 Reductase in Human Hepatocytes on Lipid and Bile Acid Homeostasis.

NADPH:cytochrome P450 oxidoreductase (POR) is the obligate electron donor for microsomal cytochrome P450 (CYP) enzymes involved in the biosynthesis of endogenous substances like bile acids and other steroids as well as in the oxidative metabolism of xenobiotics. P450 oxidoreductase also supports other redox enzymes in fatty acid and cholesterol pathways. Recently, we have established CRISPR/Cas9-mediated POR knockdown in a human hepatic cell model, HepaRG, and demonstrated the differential effects of limited POR expression on CYP activity. The aim of the present work was to systematically investigate the impact of POR knockdown with a focus on the expression of ADME (absorption, distribution, metabolism, and excretion) genes and related regulators. Functional consequences have been assessed using quantitative mass spectrometry for targeted metabolomics covering bile acids, and cholesterol and its precursors, and for untargeted proteomics. In addition to the previously described alteration of RNA expression of CYP genes, we showed significant downregulation of transcriptional regulators of drug metabolism and transport, including NR1I3 (CAR), NR1I2 (PXR), NR1H4 (FXR), and NR1H3 (LXRα) in cells with POR gene disruption. Furthermore, POR knockdown resulted in deregulated bile acid and cholesterol biosynthesis demonstrated by low levels of cholic acid derivates and increased concentrations of chenodeoxycholic acid derivates, respectively. Systemic effects of POR knockdown on global protein expression were indicated by downregulation of several metabolic pathways including lipid metabolism and biological oxidation reactions. The deduced protein network map corroborates CYP enzymes as direct interaction partners, whereas changes in lipid metabolism and homeostasis are the result of indirect effects. In summary, our results emphasize a widespread role of POR in various metabolic pathways and provide the first human data on the effects of diminished POR expression on drug and endogenous metabolism in a genomeedited HepaRG cell model.

New Perspectives of CYP1B1 Inhibitors in the Light of Molecular Studies

Human cytochrome P450 1B1 (CYP1B1) is an extrahepatic heme-containing monooxygenase. CYP1B1 contributes to the oxidative metabolism of xenobiotics, drugs, and endogenous substrates like melatonin, fatty acids, steroid hormones, and retinoids, which are involved in diverse critical cellular functions. CYP1B1 plays an important role in the pathogenesis of cardiovascular diseases, hormone-related cancers and is responsible for anti-cancer drug resistance. Inhibition of CYP1B1 activity is considered as an approach in cancer chemoprevention and cancer chemotherapy. CYP1B1 can activate anti-cancer prodrugs in tumor cells which display overexpression of CYP1B1 in comparison to normal cells. CYP1B1 involvement in carcinogenesis and cancer progression encourages investigation of CYP1B1 interactions with its ligands: substrates and inhibitors. Computational methods, with a simulation of molecular dynamics (MD), allow the observation of molecular interactions at the binding site of CYP1B1, which are essential in relation to the enzyme’s functions.

Biological profile of medical response in alcoholic patients of different ethnic groups in Siberia

IntroductionThe individual sensitivity of a person to the effects of alcohol is defined as the possibility of adaptive reactions, which are controlled by various factors associated in their manifestation with characteristics in various ethnic populations.ObjectivesTo determine biological profile of medical response in alcoholic patients of different ethnic groups.Methods168 alcoholic men, aged 17 to 62 years were examined. For the therapeutic correction of withdrawal and post-withdrawal symptoms of patients from two different ethnic groups (Tatars and Russians in Siberia), the original anticonvulsant galodif (M-chloro-benzhydrylurea) was used. Pharmacokinetic parameters were calculated a model-independent method of statistical moments: half-life (T1/2,h), total clearance (Clt,ml/min), average time of the residual drug in the body (MRI,h), average elimination time (MET,h), the area under the pharmacokinetic curve (AUC, μg/ml).ResultsGalodif causes a reduction in almost half T1/2, a significant decrease in the average time of the residual drug in the body MRI, and the average elimination time MET. Overall clearance increased. Under the influence of the course with Galodif, antipyrine elimination intensified, which indicates the induction of the cytochrome P-450 system of microsomal liver monooxygenases in Russian alcoholic patients. Galodif causes a reduction of almost five-fold T1/2, a significant decrease MRI and MET. Activation of oxidative metabolism of xenobiotics in Tatar alcoholic patients is more significant.ConclusionsThe increased sensitivity of P-450 cytochrome system to anticonvulsants action with inductive detoxification properties reflects differences in adaptive mechanisms in human from various ethnic groups, what is significant in the therapy.DisclosureNo significant relationships.

New approaches to old problems: Removal of phospholipase A2 results in highly active microsomal membranes from the honey bee, Apis mellifera

Over the last 50 years numerous studies were published by insect toxicologists using native microsomal membrane preparations in order to investigate in vitro cytochrome P450-(P450) mediated oxidative metabolism of xenobiotics, including insecticides. Whereas the preparation of active microsomal membranes from many pest insect species is straightforward, their isolation from honey bees, Apis mellifera (Hymenoptera: Apidae) remained difficult, if not impossible, due to the presence of a yet unidentified endogenous inhibitory factor released during abdominal gut membrane isolation. Thus hampering in vitro toxicological studies on microsomal oxidative phase 1 metabolism of xenobiotics, including compounds of ecotoxicological concern. The use of microsomal membranes rather than individually expressed P450s offers advantages and allows to develop a better understanding of phase 1 driven metabolic fate of foreign compounds. Here we biochemically investigated the problems associated with the isolation of active honey bee microsomes and developed a method resulting in highly active native microsomal preparations from adult female worker abdomens. This was achieved by removal of the abdominal venom gland sting complex prior to microsomal membrane preparation. Molecular sieve chromatography of the venom sac content leads to the identification of phospholipase A2 as the enzyme responsible for the immediate inhibition of cytochrome P450 activity in microsomal preparations. The substrate specificity of functional honey bee microsomes was investigated with different fluorogenic substrates, and revealed a strong preference for coumarin over resorufin derivatives. Furthermore we were able to demonstrate the metabolism of insecticides by honey bee microsomes using an approach coupled to LC-MS/MS analysis of hydroxylated metabolites. Our work provides access to a new and simple in vitro tool to study honey bee phase 1 metabolism of xenobiotics utilising the entire range of microsomal cytochrome P450s.

Electrochemical generation of selegiline metabolites coupled to mass spectrometry

The metabolic pathways of selegiline (a drug used for the treatment of early-stage Parkinson's disease) were analyzed by electrochemical oxidation with application of the flow electrochemical cell consisting of three electrodes (ROXY™, Antec, the Netherlands). Two types of working electrodes were applied: glassy carbon (GC) and boron-doped diamond (BDD). The potential applied at working electrode and composition of the solvent were optimized for the best conditions for oxidation and identification processes. All products were directly analyzed on-line by mass spectrometry. For further characterization of electrochemical oxidation products, the novel approach involving reversed phase chromatography linked to mass spectrometry with dielectric barrier discharge ionization (DBDI-MS) was used. In this manuscript, we report a novel technique for simulation of drug metabolism by electrochemical system (EC) connected to liquid chromatography (LC) and dielectric barrier discharge ionization (DBDI) mass spectrometry (MS) for direct on-line detection of electrochemical oxidation products. Here, we linked LC/DBDI-MS system with an electrochemical flow cell in order to study metabolic pathways via identification of drug metabolites generated electrochemically. The DBDI source has never been used before for identification of psychoactive metabolites generated in an electrochemical flow cell. Our knowledge on the biological background of xenobiotics metabolism and its influence on human body is constantly increasing, but still many mechanisms are not explained. Nowadays, metabolism of pharmaceuticals is mainly studied using liver cells prepared from animals or humans. Cytochrome P450, present in microsomes, is primarily responsible for oxidative metabolism of xenobiotics. It was also shown, that breakdown of popular medicines may be successfully simulated by electrochemistry under appropriate conditions. The presented experiments allow for comparison of these two entirely distinct techniques using selegiline as the model xenobiotic with well-described metabolic pathway in human body. The obtained results for selegiline oxidation show that it is possible to generate the most important selegiline metabolites present in human body – some of them with psychoactive properties, such as methamphetamine and amphetamine. These metabolites, serving as an evidence of the xenobiotic intake, can also be produced, among a larger group of metabolites, by incubation of selegiline with rat and human liver microsomes. The EC/LC/DBDI-MS system provides novel, promising platform for drugs screening of the phase I metabolism. The metabolites can be detected directly by MS or collected and separated by liquid chromatography.

Development of multifunctional metabolic synergists to suppress the evolution of resistance against pyrethroids in insects that blood feed on humans.

Pyrethroids are the insecticides of choice when exposure to humans is likely, such as occurs in vector and public-health-related control programs. Unfortunately, the pyrethroids share a common resistance mechanism with dichlorodiphenyltrichloroethane (DDT), knockdown resistance (kdr), and prior extensive use of DDT has predisposed the pyrethroids to cross-resistance via kdr. Given the widespread occurrence of kdr, the use of synergists with pyrethroids is considered to be prudent to guard against the selection of multiply resistant insects. 3-Phenoxybenzyl hexanoate (PBH) was synthesized as a multifunctional pyrethroid synergist that, besides being a surrogate substrate for sequestration/hydrolytic carboxylesterases, now also functions as a substrate for oxidative xenobiotic metabolism. The addition of PBH to permethrin-treated females of the ISOP450 strain of Culex pipiens quinquefasciatus resulted in a threefold increase in synergism, as judged by the synergistic ratio. Similarly, PBH synergized the action of deltamethrin sixfold on females of the common bed bug, Cimex lectularius, and was 2.8-fold more synergistic than piperonyl butoxide (PBO). PBH synergized the action of both type I and type II pyrethroids in a mosquito vector (Cx. p. quinquefasciatus) and in a public-health pest, C. lectularius, respectively, indicating a broad spectrum of action on blood-feeding insects. PBH appears to have residual properties similar to permethrin and is itself non-toxic, unlike PBO, and therefore should be compatible with existing pyrethroid formulations used for insecticide-treated nets and home/residential sprays.

Mononuclear Metal–O2 Complexes Bearing Macrocyclic N-Tetramethylated Cyclam Ligands

Metalloenzymes activate dioxygen to carry out a variety of biological reactions, including the biotransformation of naturally occurring molecules, oxidative metabolism of xenobiotics, and oxidative phosphorylation. The dioxygen activation at the catalytic sites of the enzymes occurs through several steps, such as the binding of O(2) at a reduced metal center, the generation of metal-superoxo and -peroxo species, and the O-O bond cleavage of metal-hydroperoxo complexes to form high-valent metal-oxo oxidants. Because these mononuclear metal-dioxygen (M-O(2)) adducts are implicated as key intermediates in dioxygen activation reactions catalyzed by metalloenzymes, studies of the structural and spectroscopic properties and reactivities of synthetic biomimetic analogues of these species have aided our understanding of their biological chemistry. One particularly versatile class of biomimetic coordination complexes for studying dioxygen activation by metal complexes is M-O(2) complexes bearing the macrocyclic N-tetramethylated cyclam (TMC) ligand. This Account describes the synthesis, structural and spectroscopic characterization, and reactivity studies of M-O(2) complexes bearing tetraazamacrocyclic n-TMC ligands, where M ═ Cr, Mn, Fe, Co, and Ni and n = 12, 13, and 14, based on recent results from our laboratory. We have used various spectroscopic techniques, including resonance Raman and X-ray absorption spectroscopy, and density functional theory (DFT) calculations to characterize several novel metal-O(2) complexes. Notably, X-ray crystal structures had shown that these complexes are end-on metal-superoxo and side-on metal-peroxo species. The metal ions and the ring size of the macrocyclic TMC ligands control the geometric and electronic structures of the metal-O(2) complexes, resulting in the end-on metal-superoxo versus side-on metal-peroxo structures. Reactivity studies performed with the isolated metal-superoxo complexes reveal that they can conduct electrophilic reactions such as oxygen atom transfer and C-H bond activation of organic substrates. The metal-peroxo complexes are active oxidants in nucleophilic reactions, such as aldehyde deformylation. We also demonstrate a complete intermolecular O(2)-transfer from metal(III)-peroxo complexes to a Mn(II) complex. The results presented in this Account show the significance of metal ions and supporting ligands in tuning the geometric and electronic structures and reactivities of the metal-O(2) intermediates that are relevant in biology and in biomimetic reactions.

Proposed new nomenclature for Bos taurus cytochromes P450 involved in xenobiotic drug metabolism

The cytochrome P450 (CYP) superfamily of drug metabolizing enzymes (DMEs) plays a central role in the oxidative metabolism of xenobiotics to which living organisms are exposed. In Bos taurus (cattle), a definitive nomenclature for CYP proteins is still lacking, and to unambiguously settle cattle nomenclature a phylogenetic analysis of proteins belonging to CYP 1-4 families was performed. Sequences collected from GenBank and Dr Nelson's P450 homepage databases were analyzed according to the maximum likelihood method. Phylogenetic outputs showed that CYPs sharing the same name and collected from different species did not form, in several instances, monophyletic groups. Some cattle CYPs did not group with their supposed human orthologous counterparts, thus requiring a new nomenclature. Name changes mostly mirrored the orthologous counterparts established for other species, and new names were created when no clear orthologous sequences were identified. The new nomenclature will allow a more appropriate investigation of biochemical and molecular mechanisms involved in the expression and regulation of these DMEs.

Role of microsomal epoxide hydrolase in methamphetamine‐induced drug dependence in mice

Microsomal epoxide hydrolase (mEH) and cytochrome P-450 (CYP) ensure the rapid detoxification of epoxides generated during the oxidative metabolism of xenobiotics. Although CYP has been demonstrated to modulate methamphetamine (METH)-induced behavioral effects, little is known about the role of the mEH gene on these effects. We examined the role of mEH gene expression in METH-induced conditioned place preference and behavioral sensitization by using mEH(-/-) and wild-type (WT) mice. Extracellular dopamine (DA) levels and DA uptake into synaptosomes were assessed by using an in vivo microdialysis and [(3)H]DA uptake assay. We applied double-label immunocytochemistry to characterize mEH-positive cellular types. METH-induced behavioral responses paralleled striatal c-Fos-like immunoreactivity. METH treatment resulted in increased extracellular DA levels in the nucleus accumbens but decreased synaptosomal DA uptake in the striatum. These behavioral and neurochemical changes were more pronounced in the mEH(-/-) mice than in WT mice. In WT mice, mEH-like immunoreactivity was expressed in astrocytes labeled by GFAP or S100B after METH treatment. The results suggest that epoxide intermediates mediate METH drug dependence and that astrocytic reactions of mEH protein are important in the endogenous modulation in response to METH drug dependence.

Physical and Functional Interaction between Human Oxidized Base-specific DNA Glycosylase NEIL1 and Flap Endonuclease 1

The S phase-specific activation of NEIL1 and not of the other DNA glycosylases responsible for repairing oxidatively damaged bases in mammalian genomes and the activation of NEIL1 by proliferating cell nuclear antigen (PCNA) suggested preferential action by NEIL1 in oxidized base repair during DNA replication. Here we show that NEIL1 interacts with flap endonuclease 1 (FEN-1), an essential component of the DNA replication. FEN-1 is present in the NEIL1 immunocomplex isolated from human cell extracts, and the two proteins colocalize in the nucleus. FEN-1 stimulates the activity of NEIL1 in vitro in excising 5-hydroxyuracil from duplex, bubble, forked, and single-stranded DNA substrates by up to 5-fold. The disordered region near the C terminus of NEIL1, which is dispensable for activity, is necessary and sufficient for high affinity binding to FEN-1 (K(D) approximately = 0.2 microm). The interacting interface of FEN-1 is localized in its disordered C-terminal region uniquely present in mammalian orthologs. Fine structure mapping identified several Lys and Arg residues in this region that form salt bridges with Asp and Glu residues in NEIL1. NEIL1 was previously shown to initiate single nucleotide excision repair, which does not require FEN-1 or PCNA. The present study shows that NEIL1 could also participate in strand displacement repair synthesis (long patch repair (LP-BER)) mediated by FEN-1 and stimulated by PCNA. Interaction between NEIL1 and FEN-1 is essential for efficient NEIL1-initiated LP-BER. These studies strongly implicate NEIL1 in a distinct subpathway of LP-BER in replicating genomes.

Differential regulation of human hepatic flavin containing monooxygenase 3 ( FMO3) by CCAAT/enhancer-binding protein β (C/EBPβ) liver inhibitory and liver activating proteins

Flavin-containing monooxygenase 3 (FMO3) is important for oxidative xenobiotic metabolism, but regulation of the FMO3 gene remains poorly understood. FMO3 is not expressed in HepG2 cells, a commonly employed model for hepatic gene regulation studies. Transcription factor transient expression and treatment with histone deacetylase or DNA methylase inhibitors identified decreased hepatic nuclear factor (HNF) 4α levels and DNA hypermethylation as mechanisms suppressing HepG2 FMO3 expression. The absence of major deficiencies in transcriptional machinery suggested that within limits, the HepG2 model is suitable for the study of FMO3 regulation. DNA–protein binding studies with HepG2 cell and hepatic tissue nuclear protein extracts and reporter construct transient expression experiments were performed to characterize FMO3 sequences from position −494 to −439 (domain I), previously demonstrated to significantly impact promoter function. Although both HNF3β and CCAAT enhancer-binding protein (C/EBP) were observed to specifically interact with this element using HepG2 cell nuclear proteins, only C/EBP DNA–protein interactions were observed using adult liver nuclear proteins. No specific DNA/protein interactions were observed using fetal liver nuclear proteins. Mutation of a putative HNF3β element had no effect on FMO3 promoter activity, while mutagenesis of a distinct, but overlapping C/EBP element resulted in a 55% reduction in activity. Furthermore, promoter activity was regulated as a function of defined C/EBPβ liver activating protein:liver inhibitory protein ratios through this same element. Chromatin immunoprecipitation demonstrated C/EBPβ binding to the FMO3 domain I element in intact cells and adult liver tissue. These results are consistent with C/EBPβ being important for regulating hepatic FMO3 expression.

Neuroendocrine disruption and health effects in Elliptio complanata mussels exposed to aeration lagoons for wastewater treatment

The purpose of this study was to examine neuroendocrine-disrupting effects of two domestic wastewater aeration lagoons on freshwater mussels. Mussels were caged and placed in two final aeration lagoons for treating domestic wastewaters for 60 days, at a site 1km downstream of the dispersion plume on the eastern shores of the Richelieu River; the western shore served as the reference site. The mussels were analysed for gonad activity, oxidative metabolism of xenobiotics, stress biomarkers and neuroendocrine status (monoamine and arachidonic acid metabolism). The domestic wastewaters produced many different effects at all levels examined. The gonado-somatic index and vitellogenin-like proteins were significantly induced in both aeration lagoons and gonad pyrimidine synthesis (aspartate transcarbamylase activity) was significantly reduced, indicating that vitellogenin-like proteins were produced while DNA synthesis in gametes remained constant. Biomarkers of oxidative metabolism revealed that global heme oxidase (HO), glutathione S-transferase and xanthine (caffeine) oxydoreductase (XOR) activities were significantly induced in at least one of the aeration lagoons, but not downstream of the dispersion plume. The activities of 7-ethoxyresorufin (cytochrome P4501A1), dibenzoylfluorescein (cytochrome P450 3A4 and 3A5) and benzoyloxyresorurufin (cytochrome P450 3A4 and 2B6) dealkylases were readily induced by substances sharing structural similarities with coplanar polyaromatic hydrocarbons and hydroxylated or aminated aromatic or cyclic hydrocarbon compounds such as pharmaceuticals or steroids in the domestic wastewaters. Biomarkers of toxic stress revealed that exposure to aeration lagoons led to increased production of metallothioneins, lipid peroxidation and DNA strand breaks, with decreased heme oxygenase activity. LPO was significantly correlated with XOR, HO and cytochrome P4501A1 activities. Neuroendocrine effects included significant increases in dopamine and serotonin levels and in monoamine oxidase (MAO). Dopamine transport in synaptosome was significantly increased while serotonin transport activity was significantly decreased, suggesting the mussels were in a state of serotonergicity. Moreover, arachidonic acid cyclooxygenase (COX) activity was also readily increased in one aeration lagoon. Aeration lagoons for the treatment of domestic wastewaters are toxic, estrogenic and disrupt the metabolism of monoamines and COX in freshwater mussels.

Involvement of enzymes other than CYPs in the oxidative metabolism of xenobiotics

Although the majority of oxidative metabolic reactions are mediated by the CYP superfamily of enzymes, non-CYP-mediated oxidative reactions can play an important role in the metabolism of xenobiotics. The (major) oxidative enzymes, other than CYPs, involved in the metabolism of drugs and other xenobiotics are: the flavin-containing monooxygenases, the molybdenum hydroxylases (aldehyde oxidase and xanthine oxidase), the prostaglandin H synthase, the lipoxygenases, the amine oxidases (monoamine, polyamine, diamine and semicarbazide-sensitive amine oxidases) and the alcohol and aldehyde dehydrogenases. In a similar manner to CYPs, these oxidative enzymes can also produce therapeutically active metabolites and reactive/toxic metabolites, modulate the efficacy of therapeutically active drugs or contribute to detoxification. Many of them have been shown to be important in endobiotic metabolism, and, consequently, interactions between drugs and endogenous compounds might occur when they are involved in drug metabolism. In general, most non-CYP oxidative enzymes appear to be noninducible or much less inducible than the CYP system, although some of them may be as inducible as some CYPs. Some of these oxidative enzymes exhibit polymorphic expression, as do some CYPs. It is possible that the contribution of non-CYP oxidative enzymes to the overall metabolism of xenobiotics is underestimated, as most investigations of drug metabolism in discovery and lead optimisation are performed using in vitro test systems optimised for CYP activity.

Oxidative metabolic profiling of xenobiotics by human P450s expressed in tobacco cell suspension cultures

Elucidation of metabolic pathways of xenobiotics (pesticides, pharmaceuticals and industrial pollutants) in human, animals and plants and chemical identification of corresponding metabolites are required for comprehensive (eco-) toxicological evaluation of the compounds prior to their usage. The most important metabolic products are oxidized metabolites, and most of these are formed by catalytic activity of P450s (cytochrome P450 mono-oxygenases). In human, 11 P450 isoenzymes exhibiting broad and overlapping substrate specificities are responsible for approx. 90% of drug metabolism. As support for inevitable metabolism studies with intact organisms under relevant conditions, tobacco cell cultures were transformed separately with cDNA sequences of human P450 isoenzymes CYP1A1, CYP1A2 and CYP3A4. The resulting P450-transgenic cell suspensions were used for metabolism studies with pesticides, industrial pollutants, a secondary plant metabolite and human sex hormones. A summary of basic results is provided; these are discussed regarding application of the method for screening of the oxidative metabolism of xenobiotics and the large-scale production of metabolites.

P.6.064 Influence of alcohol dependence on oxidative metabolism of xenobiotics in humans

P.6.064 Influence of alcohol dependence on oxidative metabolism of xenobiotics in humans

P450 structures and oxidative metabolism of xenobiotics.

This review focuses on the structural models for cytochrome P450, which are improving our knowledge and understanding of the P450 catalytic cycle, and the way in which substrates bind to the enzyme leading to catalytic conversion and subsequent formation of mono-oxygenated metabolites. Various stages in the P450 reaction cycle have now been investigated using X-ray crystallography and electronic structure calculations, whereas homology modeling of mammalian P450s is currently revealing important aspects of pharmaceutical and other xenobiotic metabolism mediated by P450 involvement. These features are explored in the current review on P450-based catalysis, which emphasizes the importance of structural modeling to our understanding of this enzyme's function. In addition, the results of various quantitative structure-activity relationships (QSAR) analyses on series of chemicals, which are metabolized via P450 enzymes, are presented such that the importance of electronic and other structural factors in explaining variations in rates of metabolism can be appreciated. As an important example of biocatalysis, the P450 system has a major future as an enzyme for use in many biotechnological applications, including biodegradation and bioremediation.

Up-regulation of cytochrome P450 1A2, 2C9, and 2E1 in chronic pancreatitis.

The oxidative metabolism of xenobiotics is effected mainly by cytochrome P450 enzymes (CYP), which are expressed as a family of genetically related enzymes primarily in hepatocytes. The pancreas is among the extrahepatic tissues expressing CYP, and it has been suggested that intermediates generated by them might be of pathogenetic significance for diseases of the pancreas such as chronic pancreatitis. We studied 10 surgical resection specimens by immunohistochemistry with polyclonal antibodies against recombinant human CYP 1A1, 1A2, 2C9, 2E1, and 3A and used tissues from 11 normal pancreata as controls. In addition, we assayed microsomal preparations for their capacity to metabolize verapamil. In normal pancreata weak to moderate expression of all enzymes was demonstrated immunohistochemically in up to 50% of duct epithelia, acinar cells, and islet cells. In contrast, in chronic pancreatitis an up-regulation was observed, with immunohistochemical positivity in some cases in up to 100% of duct epithelia and acinar cells. The oxidative capacity of microsomal preparations from chronic pancreatitis was higher than that of preparations obtained from control tissues; compared to liver microsomes, however, it was low. The up-regulation of CYP may have pathogenetic significance for chronic pancreatitis. Yet considering the pancreas' capacity for conjugation reactions, conceivably low levels of reactive intermediates could effectively undergo inactivation.

Monoamine oxidases and related amine oxidases as phase I enzymes in the metabolism of xenobiotics.

To date most of the interest in oxidative metabolism of xenobiotics has been devoted to the role of the microsomal cytochrome P-450 system and to establish the basis for classifying and naming P450 enzymes. The contribution of amine oxidases to the metabolism of xenobiotics has been largely neglected, with the exception of the contribution of monoamine oxidases (MAOs) to the metabolism of exogenous tyramine and the studies of the "cheese effect" produced as the result of ingestion of large amounts of tyramine-containing foods under particular conditions. A review of the involvement of the mitochondrial MAOs in drug metabolism was published in 1988. Since that time, considerable additional evidence has appeared in the literature to support the contribution of MAOs to drug metabolism. In addition, the involvement of other amine oxidases in the metabolism of foreign compounds has been established. A second review on the contribution of amine oxidases to the metabolism of xenobiotics was therefore published in 1994. On an arbitrary basis, the heterogeneous class of amine oxidases can be divided into two types according to their prosthetic group: the flavineadenine dinucleotide (FAD)-dependent amine oxidases (Monoamine Oxidase and Polyamine Oxidase) and the amine oxidases not containing FAD (Semicarbazide-sensitive amine oxidases). In this overview, the contributions of these two types in xenobiotic metabolism are considered separately.