ABSTRACT
Alkenylbenzenes are aromatic compounds found in several vegetable foods that can cause genotoxicity upon bioactivation by members of the cytochrome P450 (CYP) family, forming 1'-hydroxy metabolites. These intermediates act as proximate carcinogens and can be further converted into reactive 1'-sulfooxy metabolites, which are the ultimate carcinogens responsible for genotoxicity. Safrole, a member of this class, has been banned as a food or feed additive in many countries based on its genotoxicity and carcinogenicity. However, it can still enter the food and feed chain. There is limited information about the toxicity of other alkenylbenzenes that may be present in safrole-containing foods, such as myristicin, apiole, and dillapiole. In vitro studies showed safrole as mainly bioactivated by CYP2A6 to form its proximate carcinogen, while for myristicin this is mainly done by CYP1A1. However, it is not known whether CYP1A1 and CYP2A6 can activate apiole and dillapiole. The present study uses an in silico pipeline to investigate this knowledge gap and determine whether CYP1A1 and CYP2A6 may play a role in the bioactivation of these alkenylbenzenes. The study found that the bioactivation of apiole and dillapiole by CYP1A1 and CYP2A6 is limited, possibly indicating that these compounds may have limited toxicity, while describing a possible role of CYP1A1 in the bioactivation of safrole. The study expands the current understanding of safrole toxicity and bioactivation and helps understand the mechanisms of CYPs involved in the bioactivation of alkenylbenzenes. This information is essential for a more informed analysis of alkenylbenzenes toxicity and risk assessment.
Subject(s)
Cytochrome P-450 CYP1A1 , Safrole , Safrole/metabolism , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Biotransformation , Carcinogens/toxicity , Carcinogens/metabolismABSTRACT
Safrole, a 162.2 Da natural compound belonging to the alkenylbenzenes class, is classified as a possible carcinogen to humans by IARC (group IIB) and has proven to be genotoxic and carcinogenic to rodents. Despite its use as a food or feed additive, it is forbidden in many countries due to its documented toxicity; yet, it is still broadly present within food and feed and is particularly abundant in spices, herbs and essential oils. Specifically, safrole may exert its toxicity upon bioactivation to its proximate carcinogen 1'-hydroxy-safrole via specific members of the cytochrome P450 protein family with a certain inter/intra-species variability. To investigate this variability, an in-silico workflow based on molecular modelling, docking and molecular dynamics has been successfully applied. This work highlighted the mechanistic basis underpinning differences among humans, cats, chickens, goats, sheep, dogs, mice, pigs, rats and rabbits. The chosen metric to estimate the likeliness of formation of 1'-hydroxy-safrole by the species-specific cytochrome P450 under investigation allowed for the provision of a knowledge-based ground to rationally design and prioritise further experiments and deepen the current understanding of alkenylbenzenes bioactivation and CYPs mechanics. Both are crucial for a more informed framework of analysis for safrole toxicity.
Subject(s)
Allylbenzene Derivatives , Safrole , Rats , Animals , Mice , Humans , Dogs , Rabbits , Sheep , Swine , Safrole/metabolism , Chickens/metabolism , Cytochrome P-450 Enzyme System/metabolism , Carcinogens/metabolismABSTRACT
Severe acute malnutrition (SAM), due to poor energy and/or protein intake, is associated with poor growth, depressed immune function, and long-term impacts on metabolic function. As the liver is a major metabolic organ and malnutrition poses metabolic stress, we hypothesize that SAM will be associated with alterations in the hepatic metabolome reflective of oxidative stress, gluconeogenesis, and ketogenesis. Thus, the purpose of this secondary analysis was to understand how SAM alters hepatic metabolism using a piglet model. Weanling piglets were feed either a reference (REF) or protein-energy deficient diet (MAL) for 5 weeks. After dietary treatment MAL piglets were severely underweight (weight-for-age Z-score of -3.29, Welch's t test, P = .0007), moderately wasted (weight-for-length Z-score of-2.49, Welch's t test, P = .003), and tended toward higher hepatic triglyceride content (Welch's t test, P = .07). Hematologic and blood biochemical measurements were assessed at baseline and after dietary treatment. The hepatic metabolome was investigated using 1H-NMR spectroscopy. Hepatic concentrations of betaine, cysteine, and glutathione tended to be lower in MAL (Welch's t test with FDR correction, P < .1), while inosine, lactate, and methionine sulfoxide concentrations were higher in MAL (inosine: P = .0448, lactate: P = .0258, methionine sulfoxide: P = .0337). These changes suggest that SAM is associated with elevated hepatic oxidative stress, increased gluconeogenesis, and alterations in 1-carbon metabolism.
Subject(s)
Liver/metabolism , Metabolome , Oxidative Stress , Severe Acute Malnutrition/metabolism , Animals , Betaine/metabolism , Cysteine/metabolism , Diet , Gluconeogenesis , Glutathione/metabolism , Inosine/metabolism , Lactic Acid/metabolism , Male , Metabolomics/methods , Methionine/analogs & derivatives , Methionine/metabolism , Safrole/analogs & derivatives , Safrole/metabolism , Severe Acute Malnutrition/complications , Swine , Thinness , TriglyceridesABSTRACT
Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms. Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys-122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys-66, or by the nonconserved residue Cys-127. We noted that the overall structural changes during the disulfide cascade expose the Cys-122-Cys-66 disulfide to recycling through thioredoxin. In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the nonconserved Cys-127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure-function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys-122-Cys-66 disulfide for thioredoxin reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation.
Subject(s)
Biocatalysis , Corynebacterium diphtheriae/enzymology , Disulfides/metabolism , Methionine Sulfoxide Reductases/metabolism , Safrole/analogs & derivatives , Catalytic Domain , Conserved Sequence , Cysteine/metabolism , Glycopeptides/metabolism , Inositol/metabolism , Magnetic Resonance Spectroscopy , Methionine Sulfoxide Reductases/chemistry , Models, Molecular , Oxidation-Reduction , Safrole/metabolism , Substrate Specificity , Sulfenic Acids/metabolism , Thioredoxin-Disulfide Reductase/metabolism , Thioredoxins/metabolism , Zinc/metabolismABSTRACT
Heliotropin, a compound with important roles in the spice and fragrance industries and broad application prospects, is mainly produced through chemical methods. Here, we established a novel process for the synthesis of heliotropin by Escherichia coli whole cells through biotransformation of isosafrole. Directed evolution and high-throughput screening based on 2,4-dinitrophenylhydrazine were used to improve the activity of trans-anethole oxygenase toward isosafrole, and a mutant (TAO3G2) was obtained that had a high ability to oxidize isosafrole. Formate dehydrogenase (FDH) and TAO3G2 were coexpressed in E. coli, significantly increasing the catalytic efficiency by regenerating more NADH to promote isosafrole oxidation. Furthermore, after optimizing the molar ratio of isosafrole to the auxiliary substrate, the final concentration of heliotropin was increased from 9.15 to 19.45 g/L, and the maximum yield and space-time yield reached 96.02% and 3.89 g/L/h, respectively. These results suggest that the biosynthesis of heliotropin should have excellent industrial application value.
Subject(s)
Benzaldehydes/metabolism , Benzodioxoles/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Formate Dehydrogenases/genetics , Fungal Proteins/genetics , Oxygenases/genetics , Candida/enzymology , Formate Dehydrogenases/metabolism , Fungal Proteins/metabolism , Metabolic Engineering , NAD/metabolism , Oxygenases/metabolism , Safrole/metabolismABSTRACT
Sulfoxides occur in biology as products of the S-oxygenation of small molecules as well as in peptides and proteins and their formation is often associated with oxidative stress and can affect biological function. In bacteria, sulfoxide damage can be reversed by different types of enzymes. Thioredoxin-dependent peptide methionine sulfoxide reductases (MSR proteins) repair oxidized methionine residues and are found in all Domains of life. In bacteria MSR proteins are often found in the cytoplasm but in some bacteria, including pathogenic Neisseria, Streptococci, and Haemophilus they are extracytoplasmic. Mutants lacking MSR proteins are often sensitive to oxidative stress and in pathogens exhibit decreased virulence as indicated by reduced survival in host cell or animal model systems. Molybdenum enzymes are also known to reduce S-oxides and traditionally their physiological role was considered to be in anaerobic respiration using dimethylsulfoxide (DMSO) as an electron acceptor. However, it now appears that some enzymes (MtsZ) of the DMSO reductase family of Mo enzymes use methionine sulfoxide as preferred physiological substrate and thus may be involved in scavenging/recycling of this amino acid. Similarly, an enzyme (MsrP/YedY) of the sulfite oxidase family of Mo enzymes has been shown to be involved in repair of methionine sulfoxides in periplasmic proteins. Again, some mutants deficient in Mo-dependent sulfoxide reductases exhibit reduced virulence, and there is evidence that these Mo enzymes and some MSR systems are induced by hypochlorite produced by the innate immune system. This review describes recent advances in the understanding of the molecular microbiology of MSR systems and the broadening of the role of Mo-dependent sulfoxide reductase to encompass functions beyond anaerobic respiration.
Subject(s)
Bacteria/metabolism , Safrole/analogs & derivatives , Bacteria/enzymology , Bacteria/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Methionine Sulfoxide Reductases/genetics , Methionine Sulfoxide Reductases/metabolism , Oxidative Stress , Oxidoreductases/genetics , Oxidoreductases/metabolism , Safrole/metabolismABSTRACT
1. Safrole is a natural compound categorized as a group 2B carcinogen extracted from sassafras oil or certain other essential oils. The hepatotoxicity of safrole has always been highly concerned. So, the purpose of this study was to evaluate the role of cytochrome P450 (CYP450)-mediated reactive metabolites (RMs) formation and its induced cytotoxicity in HepaRG cells. 2. Safrole belongs to the methylenedioxyphenyl structure which could be activated to RMs. Two metabolites (M1, M2) and three new glutathione conjugates (M3-M5) of safrole ortho-oquinone RMs were found in HepaRG cells. Using human recombinant CYP450 enzymes and chemical inhibitor method, the metabolism of safrole RMs was predominantly carried out through the CYP1A2 with minor contributions by CYP2E1. 3. Induction of CYP1A2 by omeprazole (OME) enhanced safrole-induced cytotoxicity, compared with treatment with safrole alone, whereas inhibition of CYP1A2 by alpha-naphthoflavone (α-NAP) decreased the cytotoxicity. The cytotoxicity of cell induced by safrole was related to the amount of RMs formation. Besides, pretreatment with L-buthionine sulfoximine (BSO) to deplete intracellular GSH markedly enhanced safrole-induced cytotoxicity. OME induced the safrole-induced GSH exhaustion, and GSH depletion by safrole was not via oxidation of GSH and occurred prior to the increase in ROS. Furthermore, mitochondrial membrane potential (ΔΨm) could be aggravated by the inducer of CYP1A2 together with safrole. Collectively, these data suggest that the ortho-quinone RM may mediate safrole hepatotoxicity, and CYP1A2 was the core enzyme in ortho-quinone RMs-mediated safrole hepatotoxicity.
Subject(s)
Cytochrome P-450 CYP1A2/metabolism , Safrole/toxicity , Buthionine Sulfoximine/pharmacology , Cell Line , Cytochrome P-450 CYP1A2/genetics , Cytochrome P-450 Enzyme Inducers/pharmacology , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Glutathione/metabolism , Hepatocytes/drug effects , Humans , Inactivation, Metabolic , Membrane Potential, Mitochondrial/drug effects , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Reactive Oxygen Species/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Safrole/metabolism , Safrole/pharmacokineticsABSTRACT
An ascorbate peroxidase from a new source Musa paradisiaca leaf juice has been purified to homogeneity using a simple procedure involving concentration by ultra filtration and anion exchange chromatography on diethyl amino ethyl [DEAE] cellulose column. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis [SDS-PAGE] analysis of the purified enzyme has shown a single protein band of molecular mass 208.9â¯kDa which has been confirmed by native-PAGE and intact mass analysis by mass spectrometry. The Km and kcat values of the enzyme using ascorbate and H2O2 as the variable substrates were 0.13â¯mâ¯molâ¯L-1, 40.42â¯s-1 and 0.23â¯mâ¯molâ¯L-1, 27.24â¯s-1, respectively. The pH and temperature optima of the enzyme were 7.0 and 298â¯K, respectively. The enzyme transformed approximately 97% methyl phenyl sulfide to its sulfoxide. The product was racemic mixture.
Subject(s)
Ascorbate Peroxidases/metabolism , Musa/enzymology , Safrole/analogs & derivatives , Sulfides/metabolism , Amino Acid Sequence , Ascorbate Peroxidases/chemistry , Biotransformation , Hydrogen-Ion Concentration , Plant Leaves/enzymology , Safrole/metabolism , TemperatureABSTRACT
Sulfoxides and sulfones are commonly found in nature as a result of thioether oxidation, whereas only a very few enzymes have been found to metabolize these compounds. Utilizing the strong reduction potential of the [4Fe-4S] cluster of radical S-adenosyl-l-methionine (SAM) enzymes, we herein report the first enzyme-catalyzed reductive cleavage of sulfoxide and sulfone. We show two radical SAM enzymes, tryptophan lyase NosL and the class C radical SAM methyltransferase NosN, are able to act on a sulfoxide SAHO and a sulfone SAHO2, both of which are structurally similar to SAM. NosL cleaves all of the three bonds (i.e., S-C(5'), S-C(γ), and S-O) connecting the sulfur center of SAHO, with a preference for S-C(5') bond cleavage. Similar S-C cleavage activity was also found for SHAO2, but no S-O cleavage was observed. In contrast to NosL, NosN almost exclusively cleaves the S-C(5') bonds of SAHO and SAHO2 with much higher efficiencies. Our study provides valuable insights into the [4Fe-4S] cluster-mediated reduction reactions and highlights the remarkable catalytic promiscuity of radical SAM enzymes.
Subject(s)
Carbon-Carbon Lyases/metabolism , Methyltransferases/metabolism , S-Adenosylmethionine/chemistry , Safrole/analogs & derivatives , Sulfones/chemistry , Tryptophan/metabolism , S-Adenosylmethionine/metabolism , Safrole/chemistry , Safrole/metabolism , Sulfones/metabolismABSTRACT
1. Safrole is the main component of the volatile oil in Xixin, which has a strong antifungal effect. However, safrole has been shown to be associated with the development of hepatocellular carcinoma. Methylenedioxyphenyl and allyl-benzene substructures of safrole may cause a mechanism-based inhibition (MBI) of CYP450 enzymes (CYPs) and produce reactive metabolites (RMs), resulting in inhibition of enzyme activity and toxic effects. 2. Based on the experiments of CYPs cocktail screening, glutathione (GSH) capture and the IC50 data, we found that safrole had an inhibitory effect on CYP1A2. The test of enzyme activity recovery when adding GSH may help to verify the MBI of safrole. 3. Two metabolites, 1,2-dihydroxy-4-allylbenzene (M1) and 1'-hydroxy safrole (M2) could be captured by GSH. The ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS) method was used to identify the RMs through a detailed characterization of the safrole cleavage processes and the GSH-M1 adduct. The RMs identified are quinone and its tautomer. Thus, preliminary conclusion can be obtained that safrole is a mechanism-based inhibitor of CYP1A2. 4. The cleavage process of the GSH-M1/M2 adduct was analyzed in further detail. We believe the safrole hepatotoxicity mechanism is related to the RMs mediated by CYP1A2. This work provides important information on predicting in vivo drug induced liver injury.
Subject(s)
Chemical and Drug Induced Liver Injury/metabolism , Microsomes, Liver/drug effects , Safrole/pharmacokinetics , Safrole/toxicity , Chromatography, High Pressure Liquid/methods , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/metabolism , Cytochrome P-450 Enzyme System/pharmacology , Glutathione/metabolism , Humans , Inactivation, Metabolic , Inhibitory Concentration 50 , Microsomes, Liver/metabolism , Molecular Structure , Safrole/metabolism , Tandem Mass SpectrometryABSTRACT
The direct in vitro fungitoxicity and metabolism of safrole and dillapiole (isolated from Piper auritum and Piper holtonii, respectively) by Botryodiplodia theobromae and Colletotrichum acutatum were investigated. Higher values of mycelial growth inhibition for both fungi were obtained for dillapiole, as compared with safrole. B. theobromae was able to metabolize both compounds to their respective vicinal diols, reaching 65 percent relative abundance during the biotransformation of dillapiole; while C. acutatum only transformed safrole to various metabolites with relative abundances under 5 percent. According to the low antifungal activity of the major metabolic products (< 5 percent for vicinal diols), a detoxification process was implied. Studies on the influence of some substituents in the aromatic ring of safrole and dillapiole on the antifungal activity against B. theobromae were also carried out. As result, the safrole nitrated derivative, 6-nitrosafrole, showed a fungitoxicity level similar to that displayed by the commercial fungicide Carbendazim® under the conditions used. In light of this, safrole and dillapiole could be suggested as feasible structural templates for developing new antifungal agents.
Se investigó la fungitoxicidad directa in vitro y el metabolismo de safrol y dilapiol (obtenidos desde Piper auritum and Piper holtonii, respectivamente) por Botryodiplodia theobromae y Colletotrichum acutatum. Los valores mayores de inhibición del crecimiento micelial de ambos hongos se obtuvieron para dilapiol, en comparación con safrol. B. theobromae metabolizó ambos compuestos a sus respectivos dioles vecinales, alcanzando abundancias relativas del 65 por ciento durante la biotransformación del dilapiol; mientras que C. acutatum solo transformó safrol en varios metabolitos con abundancias relativas menores al 5 por ciento. De acuerdo con la baja actividad antifúngica de los productos metabólicos mayoritarios (< 5 por ciento para los dioles vecinales), se sugiere un proceso de desintoxicación. Adicionalmente, se evaluó la influencia de algunos sustituyentes en el anillo aromático de safrol y dilapiol sobre la actividad antifúngica contra B. theobromae. Como resultado, el derivado nitrado del safrol, el 6nitro safrol, presentó un nivel de fungitoxicidad similar al exhibido por el fungicida comercial Carbendazim® bajo las condiciones usadas. A la luz de lo anterior, safrol y dilapiol podrían ser sugeridos como plantillas estructurales adecuadas para el desarrollo de nuevos agentes antifúngicos.
Subject(s)
Antifungal Agents/pharmacology , Dioxoles/pharmacology , Mitosporic Fungi , Safrole/pharmacology , Antifungal Agents/metabolism , Biotransformation , Colletotrichum , Dioxoles/metabolism , In Vitro Techniques , Safrole/metabolismABSTRACT
Methionine (Met) oxidation is a major modification of proteins, which converts Met to Met sulfoxide as the common product. It is challenging to determine the level of Met sulfoxide, because it can be generated during sample preparation and analysis as an artifact. To determine the level of Met sulfoxide in proteins accurately, an isotope labeling and LC-MS peptide mapping method was developed. Met residues in proteins were fully oxidized using hydrogen peroxide enriched with (18)O atoms before sample preparation. Therefore, it was impossible to generate Met sulfoxide as an artifact during sample preparation. The molecular weight difference of 2 Da between Met sulfoxide with the (16)O atom and Met sulfoxide with the (18)O atom was used to differentiate and calculate the level of Met sulfoxide in the sample originally. Using a recombinant monoclonal antibody as a model protein, much lower levels of Met sulfoxide were detected for the two susceptible Met residues with this new method compared to a typical peptide mapping procedure. The results demonstrated efficient elimination of the analytical artifact during LC-MS peptide mapping for the measurement of Met sulfoxide. This method can thus be used when accurate determination of the level of Met sulfoxide is critical.
Subject(s)
Chromatography, Liquid/methods , Isotope Labeling/methods , Mass Spectrometry/methods , Methionine/chemistry , Methionine/metabolism , Proteins/chemistry , Proteins/metabolism , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , CHO Cells , Cricetulus , Oxidation-Reduction , Safrole/analogs & derivatives , Safrole/chemistry , Safrole/metabolismABSTRACT
Ergothioneine and ovothiol A are sulfur-containing histidine derivatives produced by microorganisms including Mycobacterium tuberculosis, Trypanosoma cruzi or Erwinia amylovora and may also play important roles in human physiology. Based on our recent identification of thiohistidine biosynthetic enzymes from Mycobacterium smegmatis and Erwinia tasmaniensis we investigate several aspects of sulfur-based redox biochemistry. For example, we are characterizing the catalytic mechanism of two thiohistidine biosynthetic enzymes which afford O2-dependent sulfur insertion into the C(5)-H and C(2)-H bonds of the imidazolyl side chain of histidine.
Subject(s)
Histidine/biosynthesis , Safrole/analogs & derivatives , Sulfhydryl Compounds/chemistry , Catalysis , Histidine/chemistry , Ligases/metabolism , Methylhistidines/metabolism , Oxidation-Reduction , Safrole/metabolismABSTRACT
L-Leucine 5-hydroxylase (LdoA) previously found in Nostoc punctiforme PCC 73102 is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase. LdoA catalyzed regio- and stereoselective hydroxylation of L-leucine and L-norleucine into (2S,4S)-5-hydroxyleucine and (2S)-5-hydroxynorleucine, respectively. Moreover, LdoA catalyzed sulfoxidation of L-methionine and L-ethionine in the same manner as previously described L-isoleucine 4-hydroxylase. Therefore LdoA should be a promising biocatalyst for effective production of industrially useful amino acids.
Subject(s)
Dioxygenases/isolation & purification , Dioxygenases/metabolism , Iron/metabolism , Ketoglutaric Acids/metabolism , Leucine/analogs & derivatives , Leucine/metabolism , Nostoc/enzymology , Ethionine/metabolism , Methionine/metabolism , Norleucine/metabolism , Safrole/analogs & derivatives , Safrole/metabolismABSTRACT
Thioether-containing pesticides are more toxic in certain anadromous and catadromous fish species that have undergone acclimation to hypersaline environments. Enhanced toxicity has been shown to be mediated through the bioactivation of these xenobiotics by one or more flavin-containing monooxygenases (FMOs), which are induced by hyperosmotic conditions. To better understand the number of FMO genes that may be regulated by hyperosmotic conditions, rainbow trout (Oncorhynchus mykiss) were maintained and acclimated to freshwater (<0.5 g/L salinity) and to 18 g/L salinity. The expression of 3 different FMO transcripts (A, B and C) and associated enzymatic activities methyl p-tolyl sulfoxidation (MTSO) and benzydamine N-oxigenation (BZNO) were measured in four tissues. In freshwater-acclimated organisms FMO catalytic activities were as follows: liver>kidney>gills=olfactory tissues; in hypersaline-acclimated animals activities were higher in liver>gills>olfactory tissues>kidney. Acclimation to 18 g/L caused a significant induction in the stereoselective formation of R-MTSO in gill. In olfactory tissues, stereoselective (100%) formation of S-MTSO was observed and was unaltered by acclimation to hypersaline water. When specific transcripts were evaluated, salinity-acclimation increased FMO A in liver (up to 2-fold) and kidney (up to 3-fold) but not in olfactory tissues and gills. FMO B mRNA was significantly down-regulated in all tissues, and FMO C was unchanged by hypersaline acclimation. FMO B and C failed to correlate with any FMO catalytic activity, but FMO A mRNA expression linearly correlated to both FMO catalytic activities (MTSO and BZNO) in liver (r(2)=0.92 and r(2)=0.88) and kidney microsomes (r(2)=0.93 and r(2)=90). FMO A only correlated with MTSO activity in gills (r(2)=0.93). These results indicate unique tissue specific expression of FMO genes in salmonids and are consistent with salinity-mediated enhancement of thioether-containing pesticide bioactivation by FMO which may occur in liver or kidney after salinity acclimation.
Subject(s)
Adaptation, Physiological , Fish Proteins/metabolism , Oncorhynchus mykiss/metabolism , Oxygenases/metabolism , Pesticides/metabolism , Amino Acid Sequence , Animals , Benzydamine/metabolism , Biocatalysis/drug effects , Fish Proteins/genetics , Gene Expression Regulation, Enzymologic/drug effects , Gills/enzymology , Gills/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Kidney/enzymology , Kidney/metabolism , Liver/enzymology , Liver/metabolism , Molecular Sequence Data , Olfactory Bulb/enzymology , Olfactory Bulb/metabolism , Oncorhynchus mykiss/genetics , Oxidoreductases/metabolism , Oxygenases/genetics , Reverse Transcriptase Polymerase Chain Reaction , Safrole/analogs & derivatives , Safrole/metabolism , Salinity , Sequence Homology, Amino Acid , Sodium Chloride/pharmacology , Substrate Specificity , Sulfides/metabolismABSTRACT
Culture conditions with Pseudomonas putida strain HKT554, expressing naphthalene dioxygenase, known as the biocatalyst showing wide substrate specificity, were optimized for high cell density cultivation (HCDC). Culture in a medium TK-B modified from that for HCDC of Escherichia coli with glucose fed-batch and dissolved oxygen stat resulted in a high cell density growth of 114 g dry cell/l at 40 h of cultivation. This system was further applied for S-(+)-methyl phenyl sulfoxide (MPSO) production from methyl phenyl sulfide. Addition of nonpolar organic solvent, such as n-hexadecane, greatly enhanced the MPSO production due to the prevention of substrate evaporation, resulting in a MPSO production up to 39 mM in 30 h with a conversion rate of 95.7 mol%.
Subject(s)
Dioxygenases/metabolism , Multienzyme Complexes/metabolism , Pseudomonas putida/growth & development , Pseudomonas putida/metabolism , Safrole/analogs & derivatives , Culture Media/chemistry , Dioxygenases/genetics , Multienzyme Complexes/genetics , Safrole/metabolism , Solvents/metabolism , Sulfides/metabolismABSTRACT
CYP 2A6 is a human enzyme that metabolizes many xenobiotics including coumarin, indole, nicotine and carcinogenic nitrosamines. The gene for CYP2A6 is polymorphic. There are few data available to clarify the relationship between P450 genetic variants and the metabolism of materials in food. The CYP 2A6 wild-type protein and 13 mutants (CYP2A6.1, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.7, CYP2A6.8, CYP2A6.11, CYP2A6.15, CYP2A6.16, CYP2A6.17, CYP2A6.18, CYP2A6.21, CYP2A6.23 and CYP2A6.25) were co-expressed with NADPH-cytochrome P450 reductase in E. coli. The hydroxylase activities toward 7-ethoxycoumarin, coumarin, safrole, flavanone and hydroxyflavanone were examined. Ten types of CYP2A6 variants except for CYP2A6.2, CYP2A6.5 and CYP2A6.6 showed Soret peaks (450 nm) typical of P450 in the reduced CO-difference spectra and had 7-ethoxycoumarin O-deethylase activities. CYP2A6.15 and CYP2A6.18 showed higher activities for safrole 1'-hydroxylation than CYP2A6.1. CYP2A6.25 and CYP2A6.7 had lower safrole 1'-hydroxylase activities. CYP2A6.7 had lower flavanone 6- and 2'-hydroxylase activities, whereas CYP2A6.25 had higher 6-hydroxylase activity and lower 2'-hydroxylase activity. Hydroxyflavanone was metabolized by CYP2A6.25, but was not metabolized by wild-type CYP2A6.1. These results indicate that CYP2A6.25 possessed new substrate specificity toward flavonoids.
Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , Coumarins/metabolism , Flavanones/metabolism , Safrole/metabolism , Aryl Hydrocarbon Hydroxylases/genetics , Cytochrome P-450 CYP2A6 , Escherichia coli/genetics , Genetic Variation , Humans , Hydroxylation , NADPH-Ferrihemoprotein Reductase/genetics , NADPH-Ferrihemoprotein Reductase/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
BACKGROUND: N-Desalkylquetiapine may be a pharmacologically active quetiapine metabolite. However, information on plasma concentrations of N-desalkylquetiapine and other quetiapine metabolites attained during quetiapine therapy is scant. The aim of this study was to investigate plasma concentrations of quetiapine, N-desalkylquetiapine, O-desalkylquetiapine, 7-hydroxyquetiapine, and quetiapine sulfoxide attained during therapy and analyze the data with respect to prescribed dose and other variables. METHOD: Quetiapine and its metabolites were measured in plasma samples submitted for quetiapine therapeutic drug monitoring (2009-2011). Concentration, metabolic ratio, and concentration corrected for dose (C/D) were investigated against quetiapine dose, age, sex, and formulation. Sample results were excluded if nonadherence with therapy was queried. RESULTS: There were 99 samples from 59 patients. N-Desalkylquetiapine plasma concentrations showed the strongest correlation with dose of all analytes, but O-desalkylquetiapine and quetiapine sulfoxide were strongly correlated to plasma quetiapine concentrations. There was no significant difference in C/D for any analyte between males and females and no correlation to age. Quetiapine and quetiapine sulfoxide C/D were significantly different (P < 0.01) between patients prescribed immediate- and extended-release formulations. Quetiapine, 7-hydroxyquetiapine and quetiapine sulfoxide C/D showed significant variation (P < 0.02) between those samples taken 10-14 hours postdose as compared with that of 16-24 hours postdose, but there was no significant effect as regards N-desalkylquetiapine. CONCLUSIONS: Plasma quetiapine, O-desalkylquetiapine, 7-hydroxyquetiapine, and quetiapine sulfoxide concentrations were significantly affected by formulation and/or time since last dose. Plasma N-desalkylquetiapine concentrations were not affected by either factor therefore may be a better marker for quetiapine exposure than plasma quetiapine concentrations.
Subject(s)
Dibenzothiazepines/administration & dosage , Dibenzothiazepines/blood , Adolescent , Adult , Age Factors , Aged , Aged, 80 and over , Chemistry, Pharmaceutical/methods , Dibenzothiazepines/chemistry , Drug Monitoring/methods , Female , Humans , Male , Middle Aged , Quetiapine Fumarate , Safrole/analogs & derivatives , Safrole/metabolism , Sex Factors , Young AdultABSTRACT
Fexinidazole is a 5-nitroimidazole drug currently in clinical development for the treatment of human sleeping sickness (human African trypanosomiasis [HAT]), caused by infection with species of the protozoan parasite Trypanosoma brucei. The compound and its two principal metabolites, sulfoxide and sulfone, have been assessed for their ability to kill a range of T. brucei parasite strains in vitro and to cure both acute and chronic HAT disease models in the mouse. The parent molecule and both metabolites have shown trypanocidal activity in vitro in the 0.7-to-3.3 µM (0.2-to-0.9 µg/ml) range against all parasite strains tested. In vivo, fexinidazole is orally effective in curing both acute and chronic diseases in the mouse at doses of 100 mg/kg of body weight/day for 4 days and 200 mg/kg/day for 5 days, respectively. Pharmacokinetic data indicate that it is likely that the sulfoxide and sulfone metabolites provide most, if not all, of the in vivo killing activity. Fexinidazole and its metabolites require up to 48 h exposure in order to induce maximal trypanocidal efficacy in vitro. The parent drug and its metabolites show no in vitro cross-reactivity in terms of trypanocidal activity with either themselves or other known trypanocidal drugs in use in humans. The in vitro and in vivo antitrypanosomal activities of fexinidazole and its two principal metabolites provide evidence that the compound has the potential to be an effective oral treatment for both the T. b. gambiense and T. b. rhodesiense forms of human sleeping sickness and both stages of the disease.
Subject(s)
Nitroimidazoles/administration & dosage , Trypanocidal Agents/administration & dosage , Trypanosoma brucei brucei/drug effects , Trypanosoma brucei rhodesiense/drug effects , Trypanosomiasis, African/drug therapy , Animals , Female , Humans , Mice , Nitroimidazoles/pharmacokinetics , Nitroimidazoles/pharmacology , Nitroimidazoles/therapeutic use , Parasitic Sensitivity Tests/methods , Safrole/analogs & derivatives , Safrole/metabolism , Sulfones/metabolism , Treatment Outcome , Trypanocidal Agents/pharmacokinetics , Trypanocidal Agents/pharmacology , Trypanocidal Agents/therapeutic use , Trypanosoma brucei brucei/growth & development , Trypanosoma brucei rhodesiense/growth & development , Trypanosomiasis, African/parasitologyABSTRACT
A physiologically based biokinetic (PBBK) model for alkenylbenzene safrole in rats was developed using in vitro metabolic parameters determined using relevant tissue fractions. The performance of the model was evaluated by comparison of the predicted levels of 1,2-dihydroxy-4-allylbenzene and 1'-hydroxysafrole glucuronide to levels of these metabolites reported in the literature to be excreted in the urine of rats exposed to safrole and by comparison of the predicted amount of total urinary safrole metabolites to the reported levels of safrole metabolites in the urine of safrole exposed rats. These comparisons revealed that the predictions adequately match observed experimental values. Next, the model was used to predict the relative extent of bioactivation and detoxification of safrole at different oral doses. At low as well as high doses, P450 mediated oxidation of safrole mainly occurs in the liver in which 1,2-dihydroxy-4-allylbenzene was predicted to be the major P450 metabolite of safrole. A dose dependent shift in P450 mediated oxidation leading to a relative increase in bioactivation at high doses was not observed. Comparison of the results obtained for safrole with the results previously obtained with PBBK models for the related alkenylbenzenes estragole and methyleugenol revealed that the overall differences in bioactivation of the three alkenylbenzenes to their ultimate carcinogenic 1'-sulfooxy metabolites are limited. This is in line with the generally less than 4-fold difference in their level of DNA binding in in vitro and in vivo studies and their almost similar BMDL(10) values (lower confidence limit of the benchmark dose that gives 10% increase in tumor incidence over background level) obtained in in vivo carcinogenicity studies. It is concluded that in spite of differences in the rates of specific metabolic conversions, overall the levels of bioactivation of the three alkenylbenzenes are comparable which is in line with their comparable carcinogenic potential.