The C-terminal domain of CblD interacts with CblC and influences intracellular cobalamin partitioning☆

PubMed Central

Gherasim, Carmen; Hannibal, Luciana; Rajagopalan, Deepa; Jacobsen, Donald W.; Banerjee, Ruma

2013-01-01

Mutations in cobalamin or B12 trafficking genes needed for cofactor assimilation and targeting lead to inborn errors of cobalamin metabolism. The gene corresponding to one of these loci, cblD, affects both the mitochondrial and cytoplasmic pathways for B12 processing. We have demonstrated that fibroblast cell lines from patients with mutations in CblD, can dealkylate exogenously supplied methylcobalamin (MeCbl), an activity catalyzed by the CblC protein, but show imbalanced intracellular partitioning of the cofactor into the MeCbl and 5′-deoxyadenosylcobalamin (AdoCbl) pools. These results confirm that CblD functions downstream of CblC in the cofactor assimilation pathway and that it plays an important role in controlling the traffic of the cofactor between the competing cytoplasmic and mitochondrial routes for MeCbl and AdoCbl synthesis, respectively. In this study, we report the interaction of CblC with four CblD protein variants with variable N-terminal start sites. We demonstrate that a complex between CblC and CblD can be isolated particularly under conditions that permit dealkylation of alkylcobalamin by CblC or in the presence of the corresponding dealkylated and oxidized product, hydroxocobalamin (HOCbl). A weak CblC·CblD complex is also seen in the presence of cyanocobalamin. Formation of the CblC·CblD complex is observed with all four CblD variants tested suggesting that the N-terminal 115 residues missing in the shortest variant are not essential for this interaction. Furthermore, limited proteolysis of the CblD variants indicates the presence of a stable C-terminal domain spanning residues ~116–296. Our results are consistent with an adapter function for CblD, which in complex with CblC·HOCbl, or possibly the less oxidized CblC·cob(II)alamin, partitions the cofactor between AdoCbl and MeCbl assimilation pathways. PMID:23415655

Deoxygenation of Unhindered Alcohols via Reductive Dealkylation of Derived Phosphate Esters

DOE PAGES

Chowdhury, Sarwat; Standaert, Robert F.

2016-09-15

Primary alcohols can be deoxygenated cleanly and in high yield by reduction of derived diphenyl phosphate esters with lithium triethylborohydride in tetrahydrofuran at room temperature. Selective deoxygenation of a primary alcohol in the presence of a secondary alcohol was demonstrated. The two-step process can be performed in one pot, making it both simple and convenient.

Comparative study of the affinity and metabolism of type I and type II binding quinoline carboxamide analogs by cytochrome P450 3A4

PubMed Central

Dahal, Upendra P.; Joswig-Jones, Carolyn; Jones, Jeffrey P.

2011-01-01

Compounds that coordinate to the heme-iron of cytochrome P450 (CYP) enzymes are assumed to increase metabolic stability. However, recently we observed that the type II binding quinoline carboxamide (QCA) compounds were metabolically less stable. To test if the higher intrinsic clearance of type II binding compounds relative to type I binding compounds is general for other metabolic transformations, we synthesized a library of QCA compounds that could undergo N-dealkylation, O-dealkylation, benzylic hydroxylation and aromatic hydroxylation. The results demonstrated that type II binding QCA analogs were metabolically less stable (2 to 12 fold) at sub-saturating concentration compared to type I binding counterparts for all the transformations. When the rates of different metabolic transformations between type I and type II binding compounds were compared, they were found to be in the order of N-demethylation>benzylic hydroxylation> O-demethylation> aromatic hydroxylation. Finally, for the QCA analogs with aza-heteroaromatic rings, we did not detect metabolism in aza-aromatic rings (pyridine, pyrazine, pyrimidine) indicating electronegativity of the nitrogen can change regioselectivity in CYP metabolism. PMID:22087535

Sunlight assisted direct amide formation via a charge-transfer complex.

PubMed

Cohen, Irit; Mishra, Abhaya K; Parvari, Galit; Edrei, Rachel; Dantus, Mauricio; Eichen, Yoav; Szpilman, Alex M

2017-09-12

We report on the use of charge-transfer complexes between amines and carbon tetrachloride, as a novel way to activate the amine for photochemical reactions. This principle is demonstrated in a mild, transition metal free, visible light assisted, dealkylative amide formation from feedstock carboxylic acids and amines. The low absorption coefficient of the complex allows deep light penetration and thus scale up to a gram scale.

Uptake, Translocation, and Biotransformation of Organophosphorus Esters in Wheat (Triticum aestivum L.).

PubMed

Wan, Weining; Huang, Honglin; Lv, Jitao; Han, Ruixia; Zhang, Shuzhen

2017-12-05

The uptake, translocation and biotransformation of organophosphate esters (OPEs) by wheat (Triticum aestivum L.) were investigated by a hydroponic experiment. The results demonstrated that OPEs with higher hydrophobicity were more easily taken up by roots, and OPEs with lower hydrophobicity were more liable to be translocated acropetally. A total of 43 metabolites including dealkylated, oxidatively dechlorinated, hydroxylated, methoxylated, and glutathione-, and glucuronide- conjugated products were detected derived from eight OPEs, with diesters formed by direct dealkylation from the parent triesters as the major products, followed with hydroxylated triesters. Molecular interactions of OPEs with plant biomacromolecules were further characterized by homology modeling combined with molecular docking. OPEs with higher hydrophobicity were more liable to bind with TaLTP1.1, the most important wheat nonspecific lipid transfer protein, consistent with the experimental observation that OPEs with higher hydrophobicity were more easily taken up by wheat roots. Characterization of molecular interactions between OPEs and wheat enzymes suggested that OPEs were selectively bound to TaGST4-4 and CYP71C6v1 with different binding affinities, which determined their abilities to be metabolized and form metabolite products in wheat. This study provides both experimental and theoretical evidence for the uptake, accumulation and biotransformation of OPEs in plants.

Optical isomer analysis of 3,4-methylene-dioxyamphetamine analogues and their stereoselective disposition in rats.

PubMed

Matsushima, K; Nagai, T; Kamiyama, S

1998-01-01

Identification of the optical activity and simultaneous analysis of racemates (+/-) of three hallucinogens, 3,4-methylenedioxy-amphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA), and the urinary excretion of their optical isomers in rats was performed by high-performance liquid chromatographic analysis. Analysis of optical enantiomers of three N-alkyl MDA derivatives was performed within 50 min using two different detectors, polarimetry (OR) and ultraviolet spectroscopy (UV). The OR detector proved suitable for identification of the optically active forms, whereas the UV detector was suitable for simultaneous analysis of the enantiomers in urine. After the administration of each of the three N-alkylated derivatives, rat urine specimens were collected over four intervals, 0-4, 4-12, 12-20, and 20-24 h. After the administration of 30 mg/kg of racemic MDA and MDMA, somewhat less of the S(+)-forms of unchanged MDA and MDMA than of the R(-)-forms in each urine specimen were detected, which gave R/S ratios greater than 1.00 (p < 0.01). Conversely, after the administration of 30 mg/kg of racemic MDEA, more of the S(+)-form than the R(-)-form was found in the urine, thus giving R/S ratios less than 1.00 (p < 0.01). The percentage of the dose excreted up to 24 h was approximately 29.4% of the administered dose for MDA [S(+) 13.40% and R(-) 15.98%], 5.8% for MDMA [S(+) 1.96% and R(-) 3.79%], and 7.3% for MDEA [S(+) 3.89% and R(-) 3.43%]. Urinary excretion of optical isomers of N-dealkylated MDA from MDMA and MDEA origin were the opposite of those of the unchanged forms, and their R/S ratios up to 24 h were 0.48 to 0.72 (p < 0.01) and 1.31 to 1.50 (p < 0.01), respectively. The urinary excretion rates up to 24 h were approximately 4.3% for N-dealkylated MDA from MDMA origin [S(+) 2.72% and R(-) 1.63%] and 0.8% for N-dealkylated MDA from MDEA origin [S(+) 0.36% and R(-) 0.47%]. The total percent of unchanged forms and N-dealkylated MDA was approximately 10.1% for MDMA [S(+) 4.68% and R(-) 5.42%] and 8.2% for MDEA [S(+) 4.25% and R(-) 3.91%]. The total R/S ratio of MDMA was found to be 1.95 (p < 0.01), whereas that of MDEA was 0.88 (p < 0.01). The total R/S ratio of MDA was 1.20 (p < 0.01 ), which was comparable with that of MDMA. These three R/S ratios did not conform to the theoretical values for three N-alkyl derivatives used and neither did the R/S ratios of urine specimens collected at the four interval. These results suggested the stereoselective disposition of three N-alkyl MDA analogues in rat. The method would be suitable for the forensic chemistry and toxicology analysis of specimens obtained from hallucinogen abusers.

Pb, Sr and Ba calix[6]arene hexacarboxylic acid octahedral complexation: a dramatic effect of dealkylation

PubMed Central

Adhikari, Birendra Babu; To, Cuong-Alexander; Iwasawa, Tetsuo; Schramm, Michael P.

2015-01-01

Calix[6]arene hexacarboxylic acid binds instantly and with low symmetry to Pb, Sr and Ba. Later a highly symmetric up-down alternating conformation emerges. The solution structures are identical to their p-tert-butylcalix[6]arene hexacarboxylic acid counterparts. With either receptor an octahedral cage is formed around the metal. The transformation from low to high symmetry however proceeds at significantly faster rates for the de-t-butylated host. PMID:26752941

Sensitive fluorescence HPLC assay for AQ-13, a candidate aminoquinoline antimalarial, that also detects chloroquine and N-dealkylated metabolites.

PubMed

Deng, Haiyan; Liu, Huayin; Krogstad, Frances M; Krogstad, Donald J

2006-04-03

A sensitive, specific and reproducible fluorescence high performance liquid chromatography (HPLC) assay has been developed for the separate or simultaneous measurement of AQ-13 (a candidate 4-aminoquinoline antimalarial), chloroquine (CQ), and their metabolites in whole blood. After liquid-solid extraction using commercially available extraction cartridges, these two aminoquinolines (AQs) and their metabolites were separated on C18 (Xterra RP18) columns using a mobile phase containing 60% borate buffer (20 mM, pH 9.0) and 40% acetonitrile with isocratic elution at a flow-rate of 1.0 ml/min. The assay uses a biologically inactive 8-chloro-4-aminoquinoline (AQ-18) as its internal standard (IS). There is a linear relationship between the concentrations of these AQs and the peak area ratio (ratio between the peak area of the AQ or metabolite and the peak area of the IS) on the chromatogram. Linear calibration curves with correlation coefficients > or = 0.997 (r2 > or = 0.995, p < 0.001) were obtained for AQ-13, CQ and their N-dealkylated metabolites. Reproducibility of the assay was excellent with coefficients of variation (CVs) < or = 3.8% for AQ-13 and its metabolites, and < or =2.5% for CQ and its metabolites. The sensitivity of the assay is 5 nM using 1.0 ml of blood and a 20 microl injection volume, and can be increased by using 5.0 ml of blood with an injection volume of 40 microl.

Engineering Herbicide Metabolism in Tobacco and Arabidopsis with CYP76B1, a Cytochrome P450 Enzyme from Jerusalem Artichoke1

PubMed Central

Didierjean, Luc; Gondet, Laurence; Perkins, Roberta; Lau, Sze-Mei Cindy; Schaller, Hubert; O'Keefe, Daniel P.; Werck-Reichhart, Danièle

2002-01-01

The Jerusalem artichoke (Helianthus tuberosus) xenobiotic inducible cytochrome P450, CYP76B1, catalyzes rapid oxidative dealkylation of various phenylurea herbicides to yield nonphytotoxic metabolites. We have found that increased herbicide metabolism and tolerance can be achieved by ectopic constitutive expression of CYP76B1 in tobacco (Nicotiana tabacum) and Arabidopsis. Transformation with CYP76B1 conferred on tobacco and Arabidopsis a 20-fold increase in tolerance to linuron, a compound detoxified by a single dealkylation, and a 10-fold increase in tolerance to isoproturon or chlortoluron, which need successive catalytic steps for detoxification. Two constructs for expression of translational fusions of CYP76B1 with P450 reductase were prepared to test if they would yield even greater herbicide tolerance. Plants expressing these constructs had lower herbicide tolerance than CYP76B1 alone, which is apparently a consequence of reduced stability of the fusion proteins. In all cases, increased herbicide tolerance results from more extensive metabolism, as demonstrated with exogenously fed phenylurea. Beside increased herbicide tolerance, expression of CYP76B1 has no other visible phenotype in the transgenic plants. Our data indicate that CYP76B1 can function as a selectable marker for plant transformation, allowing efficient selection in vitro and in soil-grown plants. Plants expressing CYP76B1 may also be a potential tool for phytoremediation of contaminated sites. PMID:12226498

A systematic study of the disposition and metabolism of mercury species in mice after exposure to low levels of thimerosal (ethylmercury)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carneiro, Maria Fernanda Hornos, E-mail: mafehoca@fcfrp.usp.br; Oliveira Souza, Juliana Maria, E-mail: souza.jmo@gmail.com; Grotto, Denise, E-mail: denise.grotto@prof.uniso.br

Thimerosal (TM) is an ethylmercury (etHg)-containing preservative used in some vaccines despite very limited knowledge on the kinetics and direct interaction/effects in mammals' tissues after exposure. Thus, this study aimed to evaluate the kinetics of Hg species in mice in a time course analysis after intramuscular injection of TM, by estimating Hg half-lives in blood and tissues. Mice were exposed to one single intramuscular dose of 20 µg of Hg as TM. Blood, brain, heart, kidney and liver were collected at 0.5 hour (h), 1 h, 8 h, 16 h, 144 h, 720 h and 1980 h after TM exposuremore » (n=4). Hg species in animal tissues were identified and quantified by speciation analysis via liquid chromatography hyphenated with inductively coupled mass spectrometry (LC–ICP-MS). It was found that the transport of etHg from muscle to tissues and its conversion to inorganic Hg (inoHg) occur rapidly. Moreover, the conversion extent is modulated in part by the partitioning between EtHg in plasma and in whole blood, since etHg is rapidly converted in red cells but not in a plasma compartment. Furthermore, the dealkylation mechanism in red cells appears to be mediated by the Fenton reaction (hydroxyl radical formation). Interestingly, after 0.5 h of TM exposure, the highest levels of both etHg and inoHg were found in kidneys (accounting for more than 70% of the total Hg in the animal body), whereas the brain contributed least to the Hg body burden (accounts for <1.0% of total body Hg). Thirty days after TM exposure, most Hg had been excreted while the liver presented the majority of the remaining Hg. Estimated half-lives (in days) were 8.8 for blood, 10.7 for brain, 7.8 for heart, 7.7 for liver and 45.2 for kidney. Taken together, our findings demonstrated that TM (etHg) kinetics more closely approximates Hg{sup 2+} than methylmercury (meHg) while the kidney must be considered a potential target for etHg toxicity. - Highlights: • Ethylmercury is rapidly converted to inorganic mercury. • Hg substantially accumulates in kidney with a terminal half-life of 45.2 d. • The dealkylation of ethylmercury occurs in red blood cells but not in plasma. • Hydroxyl radical is probably the main effector of this dealkylation. • Kidney must be considered a potential target for ethylmercury toxicity.« less

The oxidation of drugs by fishes

USGS Publications Warehouse

Buhler, Donald R.; Rasmusson, Mary E.

1968-01-01

1. Fish liver microsomal systems have been found to catalyze the hydroxylation of aniline and acetanilide, the N-demethylation of aminopyrine and the O-dealkylation of phenacetin.2. These systems are similar to the corresponding mammalian enzymes and they may be considered to be mixed function oxidase since they require NADPH and oxygen. An absolute requirement for oxygen, however, was difficult to demonstrate for the hepatic phenacetin cleavage system from fish.3. Microsomal drug metabolizing systems from fish have temperature optima which are considerably lower than those of corresponding mammalian systems

Ketene reactions with tertiary amines.

PubMed

Allen, Annette D; Andraos, John; Tidwell, Thomas T; Vukovic, Sinisa

2014-01-17

Tertiary amines react rapidly and reversibly with arylketenes in acetonitrile forming observable zwitterions, and these undergo amine catalyzed dealkylation forming N,N-disubstituted amides. Reactions of N-methyldialkylamines show a strong preference for methyl group loss by displacement, as predicted by computational studies. Loss of ethyl groups in reactions with triethylamine also occur by displacement, but preferential loss of isopropyl groups in the phenylketene reaction with diisopropylethylamine evidently involves elimination. Quinuclidine rapidly forms long-lived zwitterions with arylketenes, providing a model for catalysis by cinchona and related alkaloids in stereoselective additions to ketenes.

Reaction of cresyl saligenin phosphate, the organophosphorus agent implicated in aerotoxic syndrome, with human cholinesterases: mechanistic studies employing kinetics, mass spectrometry, and X-ray structure analysis.

PubMed

Carletti, Eugénie; Schopfer, Lawrence M; Colletier, Jacques-Philippe; Froment, Marie-Thérèse; Nachon, Florian; Weik, Martin; Lockridge, Oksana; Masson, Patrick

2011-06-20

Aerotoxic syndrome is assumed to be caused by exposure to tricresyl phosphate (TCP), an antiwear additive in jet engine lubricants and hydraulic fluid. CBDP (2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one) is the toxic metabolite of triortho-cresylphosphate, a component of TCP. Human butyrylcholinesterase (BChE; EC 3.1.1.8) and human acetylcholinesterase (AChE; EC 3.1.1.7) are irreversibly inhibited by CBDP. The bimolecular rate constants of inhibition (k(i)), determined under pseudo-first-order conditions, displayed a biphasic time course of inhibition with k(i) of 1.6 × 10(8) M(-1) min(-1) and 2.7 × 10(7) M(-1) min(-1) for E and E' forms of BChE. The inhibition constants for AChE were 1 to 2 orders of magnitude slower than those for BChE. CBDP-phosphorylated cholinesterases are nonreactivatable due to ultra fast aging. Mass spectrometry analysis showed an initial BChE adduct with an added mass of 170 Da from cresylphosphate, followed by dealkylation to a structure with an added mass of 80 Da. Mass spectrometry in (18)O-water showed that (18)O was incorporated only during the final aging step to form phospho-serine as the final aged BChE adduct. The crystal structure of CBDP-inhibited BChE confirmed that the phosphate adduct is the ultimate aging product. CBDP is the first organophosphorus agent that leads to a fully dealkylated phospho-serine BChE adduct.

Toward Improved Catholyte Materials for Redox Flow Batteries: What Controls Chemical Stability of Persistent Radical Cations?

DOE PAGES

Zhang, Jingjing; Shkrob, Ilya A.; Assary, Rajeev S.; ...

2017-10-06

We report catholyte materials are used to store positive charge in energized fluids circulating through redox flow batteries (RFBs) for electric grid and vehicle applications. Energy-rich radical cations (RCs) are being considered for use as catholyte materials, but to be practically relevant, these RCs (that are typically unstable, reactive species) need to have long lifetimes in liquid electrolytes under the ambient conditions. Only few families of such energetic RCs possess stabilities that are suitable for their use in RFBs; currently, the derivatives of 1,4- dialkoxybenzene look the most promising. In this study, we examine factors that define the chemical andmore » electrochemical stabilities for RCs in this family. To this end, we engineered rigid bis-annulated molecules that by design avoid the two main degradation pathways for such RCs, viz. their deprotonation and radical addition. The decay of the resulting RCs are due to the single remaining reaction: O-dealkylation. We establish the mechanism for this reaction and examine factors controlling its rate. In particular, we demonstrate that this reaction is initiated by the nucleophile attack of the counter anion on the RC partner. The reaction proceeds through the formation of the aroxyl radicals whose secondary reactions yield the corresponding quinones. The O-dealkylation accelerates considerably when the corresponding quinone has poor solubility in the electrolyte, and the rate depends strongly on the solvent polarity. Finally, our mechanistic insights suggest new ways of improving the RC catholytes through molecular engineering and electrolyte optimization.« less

Sequential Metabolism of Secondary Alkyl Amines to Metabolic-Intermediate Complexes: Opposing Roles for the Secondary Hydroxylamine and Primary Amine Metabolites of Desipramine, (S)-Fluoxetine, and N-Desmethyldiltiazem

PubMed Central

Hanson, Kelsey L.; VandenBrink, Brooke M.; Babu, Kantipudi N.; Allen, Kyle E.; Nelson, Wendel L.

2010-01-01

Three secondary amines desipramine (DES), (S)-fluoxetine [(S)-FLX], and N-desmethyldiltiazem (MA) undergo N-hydroxylation to the corresponding secondary hydroxylamines [N-hydroxydesipramine, (S)-N-hydroxyfluoxetine, and N-hydroxy-N-desmethyldiltiazem] by cytochromes P450 2C11, 2C19, and 3A4, respectively. The expected primary amine products, N-desmethyldesipramine, (S)-norfluoxetine, and N,N-didesmethyldiltiazem, are also observed. The formation of metabolic-intermediate (MI) complexes from these substrates and metabolites was examined. In each example, the initial rates of MI complex accumulation followed the order secondary hydroxylamine > secondary amine ≫ primary amine, suggesting that the primary amine metabolites do not contribute to formation of MI complexes from these secondary amines. Furthermore, the primary amine metabolites, which accumulate in incubations of the secondary amines, inhibit MI complex formation. Mass balance studies provided estimates of the product ratios of N-dealkylation to N-hydroxylation. The ratios were 2.9 (DES-CYP2C11), 3.6 [(S)-FLX-CYP2C19], and 0.8 (MA-CYP3A4), indicating that secondary hydroxylamines are significant metabolites of the P450-mediated metabolism of secondary alkyl amines. Parallel studies with N-methyl-d3-desipramine and CYP2C11 demonstrated significant isotopically sensitive switching from N-demethylation to N-hydroxylation. These findings demonstrate that the major pathway to MI complex formation from these secondary amines arises from N-hydroxylation rather than N-dealkylation and that the primary amines are significant competitive inhibitors of MI complex formation. PMID:20200233

Sequential metabolism of secondary alkyl amines to metabolic-intermediate complexes: opposing roles for the secondary hydroxylamine and primary amine metabolites of desipramine, (s)-fluoxetine, and N-desmethyldiltiazem.

PubMed

Hanson, Kelsey L; VandenBrink, Brooke M; Babu, Kantipudi N; Allen, Kyle E; Nelson, Wendel L; Kunze, Kent L

2010-06-01

Three secondary amines desipramine (DES), (S)-fluoxetine [(S)-FLX], and N-desmethyldiltiazem (MA) undergo N-hydroxylation to the corresponding secondary hydroxylamines [N-hydroxydesipramine, (S)-N-hydroxyfluoxetine, and N-hydroxy-N-desmethyldiltiazem] by cytochromes P450 2C11, 2C19, and 3A4, respectively. The expected primary amine products, N-desmethyldesipramine, (S)-norfluoxetine, and N,N-didesmethyldiltiazem, are also observed. The formation of metabolic-intermediate (MI) complexes from these substrates and metabolites was examined. In each example, the initial rates of MI complex accumulation followed the order secondary hydroxylamine > secondary amine > primary amine, suggesting that the primary amine metabolites do not contribute to formation of MI complexes from these secondary amines. Furthermore, the primary amine metabolites, which accumulate in incubations of the secondary amines, inhibit MI complex formation. Mass balance studies provided estimates of the product ratios of N-dealkylation to N-hydroxylation. The ratios were 2.9 (DES-CYP2C11), 3.6 [(S)-FLX-CYP2C19], and 0.8 (MA-CYP3A4), indicating that secondary hydroxylamines are significant metabolites of the P450-mediated metabolism of secondary alkyl amines. Parallel studies with N-methyl-d(3)-desipramine and CYP2C11 demonstrated significant isotopically sensitive switching from N-demethylation to N-hydroxylation. These findings demonstrate that the major pathway to MI complex formation from these secondary amines arises from N-hydroxylation rather than N-dealkylation and that the primary amines are significant competitive inhibitors of MI complex formation.

Toward Improved Catholyte Materials for Redox Flow Batteries: What Controls Chemical Stability of Persistent Radical Cations?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Jingjing; Shkrob, Ilya A.; Assary, Rajeev S.

We report catholyte materials are used to store positive charge in energized fluids circulating through redox flow batteries (RFBs) for electric grid and vehicle applications. Energy-rich radical cations (RCs) are being considered for use as catholyte materials, but to be practically relevant, these RCs (that are typically unstable, reactive species) need to have long lifetimes in liquid electrolytes under the ambient conditions. Only few families of such energetic RCs possess stabilities that are suitable for their use in RFBs; currently, the derivatives of 1,4- dialkoxybenzene look the most promising. In this study, we examine factors that define the chemical andmore » electrochemical stabilities for RCs in this family. To this end, we engineered rigid bis-annulated molecules that by design avoid the two main degradation pathways for such RCs, viz. their deprotonation and radical addition. The decay of the resulting RCs are due to the single remaining reaction: O-dealkylation. We establish the mechanism for this reaction and examine factors controlling its rate. In particular, we demonstrate that this reaction is initiated by the nucleophile attack of the counter anion on the RC partner. The reaction proceeds through the formation of the aroxyl radicals whose secondary reactions yield the corresponding quinones. The O-dealkylation accelerates considerably when the corresponding quinone has poor solubility in the electrolyte, and the rate depends strongly on the solvent polarity. Finally, our mechanistic insights suggest new ways of improving the RC catholytes through molecular engineering and electrolyte optimization.« less

REACTION OF CRESYL SALIGENIN PHOSPHATE, THE ORGANOPHOSPHORUS AGENT IMPLICATED IN THE AEROTOXIC SYNDROME, WITH HUMAN CHOLINESTERASES: MECHANISTIC STUDIES EMPLOYING KINETICS, MASS SPECTROMETRY AND X-RAY STRUCTURE ANALYSIS

PubMed Central

Carletti, Eugénie; Schopfer, Lawrence M.; Colletier, Jacques-Philippe; Froment, Marie-Thérése; Nachon, Florian; Weik, Martin; Lockridge, Oksana; Masson, Patrick

2011-01-01

The aerotoxic syndrome is assumed to be caused by exposure to tricresyl phosphate (TCP), an anti-wear additive in jet engine lubricants and hydraulic fluids. CBDP (2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one) is the toxic metabolite of tri-ortho-cresylphosphate, a component of TCP. Human butyrylcholinesterase (BChE; EC 3.1.1.8) and human acetylcholinesterase (AChE; EC 3.1.1.7) are irreversibly inhibited by CBDP. The bimolecular rate constants of inhibition (ki), determined under pseudo first-order conditions, displayed a biphasic time course of inhibition with ki 1.6×108 M−1min−1 and 2.7×107 M−1min−1 for E and E′ forms of BChE. The inhibition constants for AChE were one to two orders of magnitude slower than for BChE. CBDP-phosphorylated cholinesterases are non-reactivatable due to ultra fast “aging”. Mass spectrometry analysis showed an initial BChE adduct with an added mass of 170 Da from cresylphosphate, followed by dealkylation to a structure with an added mass of 80 Da. Mass spectrometry in 18O–water showed that 18O was incorporated only during the final aging step to form phospho-serine as the final “aged” BChE adduct. The crystal structure of CBDP-inhibited BChE confirmed that the phosphate adduct is the ultimate aging product. CBDP is the first organophosphorus agent that leads to a fully dealkylated phospho-serine BChE adduct. PMID:21438623

Comparative cytotoxicity of alachlor, acetochlor, and metolachlor herbicides in isolated rat and cryopreserved human hepatocytes.

PubMed

Kale, Vijay M; Miranda, Sonia R; Wilbanks, Mitchell S; Meyer, Sharon A

2008-02-01

Noncancerous adverse effects observed at the lowest dose for chloroacetanilide herbicides alachlor [2-chloro-2',6'-diethyl-N-(methoxymethyl)-acetanilide] and acetochlor [2-chloro-2'-methyl-6'-ethyl-N-(ethoxymethyl)acetanilide], but not metolachlor [2-chloro-2'-ethyl-6'-methyl-N-(1-methyl-2-methoxymethyl)acetanilide], are hepatotoxicity in rats and dogs. Liver microsomal N-dealkylation, a step in the putative activating pathway, of acetochlor exceeds that of alachlor and is negligible for metolachlor. In the present investigation, cytotoxicity of the three chloroacetanilides was ranked using isolated rat and cryopreserved human hepatocytes to correlate this endpoint with CYP3A-dependent metabolism. Chloroacetanilide cytotoxicity in rat hepatocyte suspensions was time dependent (e.g., LC(50 - alachlor/2 h) vs. LC(50 - alachlor/4 h) = 765 vs. 325 muM). Alachlor and acetochlor were more potent than metolachlor after 2 and 4 h, times when N-dealkylated alachlor product 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA) formation was readily detectable. Alachlor and acetochlor potencies with cryopreserved human hepatocytes at 2 h were comparable to freshly isolated rat hepatocytes, and alachlor metabolism to CDEPA was likewise detectable. Unlike rat hepatocytes, metolachlor potency was equivalent to acetochlor and alachlor in human hepatocytes. Furthermore, chloroacetanilide cytotoxicity from two sources of human hepatocytes varied inversely with CYP3A4 activity. Collectively, while cytotoxicity in rat hepatocytes was consistent with chloroacetanilide activation by CYP3A, an activating role for CYP3A4 was not supported with human hepatocytes. (c) 2008 Wiley Periodicals, Inc.

Versatile Synthesis of Stable, Functional Polypeptides via Reaction with Epoxides.

PubMed

Gharakhanian, Eric G; Deming, Timothy J

2015-06-08

Methodology was developed for efficient alkylation of methionine residues using epoxides as a general strategy to introduce a wide range of functional groups onto polypeptides. Use of a spacer between epoxide and functional groups further allowed addition of sterically demanding functionalities. Contrary to other methods to alkylate methionine residues, epoxide alkylations allow the reactions to be conducted in wet protic media and give sulfonium products that are stable against dealkylation. These functionalizations are notable since they are chemoselective, utilize stable and readily available epoxides, and allow facile incorporation of an unprecedented range of functional groups onto simple polypeptides using stable linkages.

Potential risks from UV/H2O2 oxidation and UV photocatalysis: A review of toxic, assimilable, and sensory-unpleasant transformation products.

PubMed

Wang, Wen-Long; Wu, Qian-Yuan; Huang, Nan; Xu, Zi-Bin; Lee, Min-Yong; Hu, Hong-Ying

2018-05-15

UV based advanced oxidation processes (UV-AOPs) that efficiently eliminate organic pollutants during water treatment have been the subject of numerous investigations. Most organic pollutants are not completely mineralized during UV-AOPs but are partially oxidized into transformation products (TPs), thereby adding complexity to the treated water and posing risks to humans, ecological systems, and the environment. While the degradation kinetics and mechanisms of pollutants have been widely documented, there is little information about the risks associated with TPs. In this review, we have collated recent knowledge about the harmful TPs that are generated in UV/H 2 O 2 and UV photocatalysis, two UV-AOPs that have been studied extensively. Toxic and assimilable TPs were ubiquitously observed in more than 80% of UV-AOPs of organic pollutants, of which the toxicity and assimilability levels changed with variations in the reaction conditions, such as the UV fluence and oxidant dosage. Previous studies and modeling assessments showed that toxic and assimilable TPs may be generated during hydroxylation, dealkylation, decarboxylation, and deamination. Among various reactions, TPs generated from dealkylation and decarboxylation were generally less and more toxic than the parent pollutants, respectively; TPs generated from decarboxylation and deamination were generally less and more assimilable than the parent pollutants, respectively. There is also potential concern about the sensory-unpleasant TPs generated by oxidations and subsequent metabolism of microorganisms. In this overview, we stress the need to include both the concentrations of organic pollutants and the evaluations of the risks from TPs for the quality assessments of the water treated by UV-AOPs. Copyright © 2018 Elsevier Ltd. All rights reserved.

Redox inactive metal ion triggered N-dealkylation by an iron catalyst with dioxygen activation: a lesson from lipoxygenases.

PubMed

Zhang, Jisheng; Wang, Yujuan; Luo, Nengchao; Chen, Zhuqi; Wu, Kangbing; Yin, Guochuan

2015-06-07

Utilization of dioxygen as the terminal oxidant at ambient temperature is always a challenge in redox chemistry, because it is hard to oxidize a stable redox metal ion like iron(III) to its high oxidation state to initialize the catalytic cycle. Inspired by the dioxygenation and co-oxidase activity of lipoxygenases, herein, we introduce an alternative protocol to activate the sluggish iron(III) species with non-redox metal ions, which can promote its oxidizing power to facilitate substrate oxidation with dioxygen, thus initializing the catalytic cycle. In oxidations of N,N-dimethylaniline and its analogues, adding Zn(OTf)2 to the [Fe(TPA)Cl2]Cl catalyst can trigger the amine oxidation with dioxygen, whereas [Fe(TPA)Cl2]Cl alone is very sluggish. In stoichiometric oxidations, it has also been confirmed that the presence of Zn(OTf)2 can apparently improve the electron transfer capability of the [Fe(TPA)Cl2]Cl complex. Experiments using different types of substrates as trapping reagents disclosed that the iron(IV) species does not occur in the catalytic cycle, suggesting that oxidation of amines is initialized by electron transfer rather than hydrogen abstraction. Combined experiments from UV-Vis, high resolution mass spectrometry, electrochemistry, EPR and oxidation kinetics support that the improved electron transfer ability of iron(III) species originates from its interaction with added Lewis acids like Zn(2+) through a plausible chloride or OTf(-) bridge, which has promoted the redox potential of iron(III) species. The amine oxidation mechanism was also discussed based on the available data, which resembles the co-oxidase activity of lipoxygenases in oxidative dealkylation of xenobiotic metabolisms where an external electron donor is not essential for dioxygen activation.

Effects of ketoconazole and rifampicin on the pharmacokinetics of GLS4, a novel anti-hepatitis B virus compound, in dogs.

PubMed

Zhou, Xin; Gao, Zhi-wei; Meng, Jian; Chen, Xiao-yan; Zhong, Da-fang

2013-11-01

To investigate the metabolism of GLS4, a heteroaryldihydropyrimidine compound with anti-hepatitis B virus activity, in dog and human liver microsomes in vitro and evaluate the effects of ketoconazole (a potent CYP3A inhibitor) or rifampicin (a potent CYP3A inducer) on GLS4 pharmacokinetics in dogs. Dog and human liver microsomes and CYP3A4 were incubated with [(14)C]GLS4 for 15 min and then analyzed using a HPLC-dynamic online radio flow detection method. Two groups of beagle dogs were used for in vivo studies. Group A were orally administered a single dose of GLS4 (15 mg/kg) with or without ketoconazole pretreatment (100 mg/d for 8 consecutive days). Group B were orally administered a single dose of GLS4 (15 mg/kg) with or without rifampicin pretreatment (100 mg/d for 8 consecutive days). Plasma was sampled after GLS4 dosing. GLS4 concentrations were determined by HPLC-tandem mass spectrometry. The metabolic profile of [(14)C]GLS4 in human and dog liver microsomes and CYP3A4 was similar. The major metabolites were morpholine N-dealkylated GLS4 and morpholine N,N-di-dealkylated GLS4. Pretreatment with ketoconazole or rifampicin significantly affected the plasma concentrations of GLS4 in dogs: ketoconazole increased the area under the concentration-time curve from 0 to infinity and peak concentration of GLS4 by 4.4 and 3.3 folds, respectively, whereas rifampicin decreased these parameters by 88.5% and 83.2%, respectively. GLS4 is a sensitive substrate of CYP3A. CYP3A inhibitors or inducers cause considerable change of GLS4 plasma concentrations in dogs, which should be considered in clinical practice.

Metabolism of the new psychoactive substances N,N-diallyltryptamine (DALT) and 5-methoxy-DALT and their detectability in urine by GC-MS, LC-MSn, and LC-HR-MS-MS.

PubMed

Michely, Julian A; Helfer, Andreas G; Brandt, Simon D; Meyer, Markus R; Maurer, Hans H

2015-10-01

N,N-Diallyltryptamine (DALT) and 5-methoxy-DALT (5-MeO-DALT) are synthetic tryptamine derivatives commonly referred to as so-called new psychoactive substances (NPS). They have psychoactive effects that may be similar to those of other tryptamine derivatives. The objectives of this work were to study the metabolic fate and detectability, in urine, of DALT and 5-MeO-DALT. For metabolism studies, rat urine obtained after high-dose administration was prepared by precipitation and analyzed by liquid chromatography-high-resolution mass spectrometry (LC-HR-MS-MS). On the basis of the metabolites identified, several aromatic and aliphatic hydroxylations, N-dealkylation, N-oxidation, and combinations thereof are proposed as the main metabolic pathways for both compounds. O-Demethylation of 5-MeO-DALT was also observed, in addition to extensive glucuronidation or sulfation of both compounds after phase I transformation. The cytochrome P450 (CYP) isoenzymes predominantly involved in DALT metabolism were CYP2C19, CYP2D6, and CYP3A4; those mainly involved in 5-MeO-DALT metabolism were CYP1A2, CYP2C19, CYP2D6, and CYP3A4. For detectability studies, rat urine was screened by GC-MS, LC-MS(n), and LC-HR-MS-MS after administration of low doses. LC-MS(n) and LC-HR-MS-MS were deemed suitable for monitoring consumption of both compounds. The most abundant targets were a ring hydroxy metabolite of DALT, the N,O-bis-dealkyl metabolite of 5-MeO-DALT, and their glucuronides. GC-MS enabled screening of DALT by use of its main metabolites only.

Beluga whale liver microsomal cytochrome P4501A (CYP1A) enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bullock, P.L.; Addison, R.; Lockhart, L.

1995-12-31

Beluga whale (Delphinapterus leucas) liver from the Canadian arctic was analyzed for the presence of CYP1A enzymes, as part of current studies on biomarkers for environmental contamination. CYP1A1-associated 7-ethoxyresorufin O-dealkylase activity (EROD) varied 13 fold among sixteen male whale liver microsomal samples and 31 fold among five females. Similarly, the rate of 7-methoxyresorufin O-dealkylation (MROD) varied 7 fold and 3 fold in microsomal samples from males and females, respectively. Furthermore, 7-pentoxyresorufin O-dealkylase activity (PROD) varied 10 fold in both sexes. None of these enzyme activities were sexually differentiated, and EROD and MROD were inhibited by {alpha}-naphthoflavone. There was very goodmore » correlation between EROD and MROD (r{sup 2} = .894), EROD and PROD (r{sup 2} = .909), but MROD and PROD were not as well correlated (r{sup 2} = 785). On Western immunoblots, a single band was recognized in Beluga whale liver microsomes by a polygonal antibody raised against an oligopeptide related to trout CYP1A1. This antibody also recognized purified rat CYP1A1 (56 kDa) and stained only one band (56 kDa) in liver microsomes isolated from male rats treated with {beta}-naphthoflavone. The interindividual variation in EROD paralleled differences in the amount of whale liver microsomal protein that cross-reacted with the anti-peptide antibody. The results suggest that Beluga whale liver contains at least one CYP1A enzyme which catalyzes the 0-dealkylation of 7-ethoxy, 7-methoxy and 7-pentoxyresorufin and has a molecular weight less than that of rat CYP1A1, but similar to rat CYP1A2 (52 kDa).« less

Effects of ketoconazole and rifampicin on the pharmacokinetics of GLS4, a novel anti-hepatitis B virus compound, in dogs

PubMed Central

Zhou, Xin; Gao, Zhi-wei; Meng, Jian; Chen, Xiao-yan; Zhong, Da-fang

2013-01-01

Aim: To investigate the metabolism of GLS4, a heteroaryldihydropyrimidine compound with anti-hepatitis B virus activity, in dog and human liver microsomes in vitro and evaluate the effects of ketoconazole (a potent CYP3A inhibitor) or rifampicin (a potent CYP3A inducer) on GLS4 pharmacokinetics in dogs. Methods: Dog and human liver microsomes and CYP3A4 were incubated with [14C]GLS4 for 15 min and then analyzed using a HPLC-dynamic online radio flow detection method. Two groups of beagle dogs were used for in vivo studies. Group A were orally administered a single dose of GLS4 (15 mg/kg) with or without ketoconazole pretreatment (100 mg/d for 8 consecutive days). Group B were orally administered a single dose of GLS4 (15 mg/kg) with or without rifampicin pretreatment (100 mg/d for 8 consecutive days). Plasma was sampled after GLS4 dosing. GLS4 concentrations were determined by HPLC-tandem mass spectrometry. Results: The metabolic profile of [14C]GLS4 in human and dog liver microsomes and CYP3A4 was similar. The major metabolites were morpholine N-dealkylated GLS4 and morpholine N,N-di-dealkylated GLS4. Pretreatment with ketoconazole or rifampicin significantly affected the plasma concentrations of GLS4 in dogs: ketoconazole increased the area under the concentration-time curve from 0 to infinity and peak concentration of GLS4 by 4.4 and 3.3 folds, respectively, whereas rifampicin decreased these parameters by 88.5% and 83.2%, respectively. Conclusion: GLS4 is a sensitive substrate of CYP3A. CYP3A inhibitors or inducers cause considerable change of GLS4 plasma concentrations in dogs, which should be considered in clinical practice. PMID:24056704

Monitoring Toxic Ionic Liquids in Zebrafish ( Danio rerio) with Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI-MSI)

NASA Astrophysics Data System (ADS)

Perez, Consuelo J.; Tata, Alessandra; de Campos, Michel L.; Peng, Chun; Ifa, Demian R.

2017-06-01

Ambient mass spectrometry imaging has become an increasingly powerful technique for the direct analysis of biological tissues in the open environment with minimal sample preparation and fast analysis times. In this study, we introduce desorption electrospray ionization mass spectrometry imaging (DESI-MSI) as a novel, rapid, and sensitive approach to localize the accumulation of a mildly toxic ionic liquid (IL), AMMOENG 130 in zebrafish ( Danio rerio). The work demonstrates that DESI-MSI has the potential to rapidly monitor the accumulation of IL pollutants in aquatic organisms. AMMOENG 130 is a quaternary ammonium-based IL reported to be broadly used as a surfactant in commercialized detergents. It is known to exhibit acute toxicity to zebrafish causing extensive damage to gill secondary lamellae and increasing membrane permeability. Zebrafish were exposed to the IL in a static 96-h exposure study in concentrations near the LC50 of 1.25, 2.5, and 5.0 mg/L. DESI-MS analysis of zebrafish gills demonstrated the appearance of a dealkylated AMMOENG 130 metabolite in the lowest concentration of exposure identified by a high resolution hybrid LTQ-Orbitrap mass spectrometer as the trimethylstearylammonium ion, [C21H46N]+. With DESI-MSI, the accumulation of AMMOENG 130 and its dealkylated metabolite in zebrafish tissue was found in the nervous and respiratory systems. AMMOENG 130 and the metabolite were capable of penetrating the blood brain barrier of the fish with significant accumulation in the brain. Hence, we report for the first time the simultaneous characterization, distribution, and metabolism of a toxic IL in whole body zebrafish analyzed by DESI-MSI. This ambient mass spectrometry imaging technique shows great promise for the direct analysis of biological tissues to qualitatively monitor foreign, toxic, and persistent compounds in aquatic organisms from the environment. [Figure not available: see fulltext.

Organotins' fate in lagoon sewage system: dealkylation and sludge sorption/desorption.

PubMed

Ophithakorn, Thiwari; Sabah, Aboubakr; Delalonde, Michele; Bancon-Montigny, Chrystelle; Suksaroj, Thunwadee Tachapattaworakul; Wisniewski, Christelle

2016-11-01

Organotin compounds (OTs) have been widely used for their biocidal properties and as stabilizers in various industrial applications. Due to their high toxicity, organotins are subject to many studies regarding their behavior in wastewater treatment plant and aquatic environment. However, few studies are available regarding their behavior in lagoon sewage system, although such treatment is commonly used for sewage treatment in low-population areas. The present study aimed at studying the fate of organotins (monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT)) in lagoon sewage system. Short-term experiments, carried out at lab scale, consisted in sampling sludge from aerobic stabilization ponds, and then quantifying sorption and desorption of the different organotin species, as well as their respective transformation, under defined operating conditions (e.g., tributyltin spike and dilution) simulating possible change in the surrounding environment of sludge in the lagoon. Results established that a very important percentage of the OTs was localized in the solid phase of the sludge (more than 98 %), whatever the operating conditions may be; however, transformation and locations of the three OT species differed according to the different conditions of sludge dilution, TBT spiking, and test duration. After dilution of lagoon sludge, TBT desorption from sludge was observed; it was supposed that dealkylation of TBT after desorption occurred rapidly and increased dissolved MBT and DBT in liquid phase; MBT sorbed subsequently on solid phase. The nature of the diluent (i.e., tap water or saline solution) appeared to slightly influence the sludge behavior. After TBT spiking, TBT was supposed to be rapidly sorbed but also transformed in DBT and MBT that would as well sorbed on the sludge, which explained the decrease of these species in the liquid phase. Tests aimed at studying long-term effect of TBT spiking demonstrated that the sorbed species could be remobilized and transformed after a dilution.

Investigating the stability of gadolinium based contrast agents towards UV radiation.

PubMed

Birka, Marvin; Roscher, Jörg; Holtkamp, Michael; Sperling, Michael; Karst, Uwe

2016-03-15

Since the 1980s, the broad application of gadolinium(Gd)-based contrast agents for magnetic resonance imaging (MRI) has led to significantly increased concentrations of Gd in the aqueous environment. Little is known about the stability of these highly polar xenobiotics under environmental conditions, in wastewater and in drinking water treatment. Therefore, the stability of frequently applied Gd-based MRI contrast agents towards UV radiation was investigated. The hyphenation of hydrophilic interaction liquid chromatography (HILIC) with inductively coupled plasma mass spectrometry (ICP-MS) and of HILIC with electrospray ionization mass spectrometry (ESI-MS) provided quantitative elemental information as well as structural information. The contrast agents Gd-DTPA, Gd-DOTA and Gd-BT-DO3A showed a high stability in irradiation experiments applying a wavelength range from 220 nm to 500 nm. Nevertheless, the degradation of Gd-BOPTA as well as the formation of Gd-containing transformation products was observed by means of HILIC-ICP-MS. Matrix-dependent irradiation experiments showed a degradation of Gd-BOPTA down to 3% of the initial amount in purified water after 300 min, whereas the degradation was slowed down in drinking water and surface water. Furthermore, it was observed that the sum of species continuously decreased with proceeding irradiation in all matrices. After irradiation in purified water for 300 min only 16% of the sum of species was left. This indicates a release of Gd(III) ions from the complex in course of irradiation. HILIC-ESI-MS measurements revealed that the transformation products mostly resulted from O-dealkylation and N-dealkylation reactions. In good correlation with retention times, the majority of transformation products were found to be more polar than Gd-BOPTA itself. Based on accurate masses, sum formulas were obtained and structures could be proposed. Copyright © 2016 Elsevier Ltd. All rights reserved.

CYP-dependent metabolism of PF9601N, a new monoamine oxidase-B inhibitor, by C57BL/6 mouse and human liver microsomes.

PubMed

Dragoni, Stefania; Materozzi, Giada; Pessina, Federica; Frosini, Maria; Marco, José Luis; Unzeta, Mercedes; Sgaragli, Giampietro; Valoti, Massimo

2007-01-01

The selective monoamine oxidase-B (MAO-B) inhibitor, l-deprenyl, is still used for treating Parkinson's patients, however, a disadvantage of its use lies in the formation of l-amphetamine and l-methamphetamine. Subsequently, this has promoted the design of a novel, more potent, MAO-B inhibitor PF9601N, which also has neuroprotective and antioxidant properties. The aim of this work was to investigate the effect of treatment with PF9601N on its own phase I hepatic metabolism. Kinetic parameters of PF9601N CYP-dependent N-dealkylation reaction was also studied and compared with those of l-deprenyl. C57BL/6 mice were treated with PF9601N for 4 days. After CYP content and related monooxygenase activities were assayed in liver microsomes of control and treated animals. CYP activities, cytochrome b5 content, NADPH-cytochrome P450 reductase and various monooxygenase activities were unaffected by in vivo PF9601N treatment. With microsomes from both control and treated mice, the PF9601N-dealkylation product, FA72, was the only detected metabolite with its formation rate following an hyperbolic, Michaelis-Menten curve. Among various inhibitors, only ketoconazole inhibited the FA72 formation rate, indicating a major involvement for CYP3A. Apparent Km and Vmax values generated by human liver microsomes were similar to those found with mouse microsomes. Ketoconazole inhibition indicates that CYP3A is one of the major enzymes involved in PF9601N metabolism also by human liver microsomes. In mouse liver microsomes, the intrinsic clearance of PF9601N was significantly lower than that of l-deprenyl suggestive of an improved bioavailability for the former. The observed favourable metabolic profile may suggest suitability of PF9601N for clinical use.

Dissociation of sarin on a cement analogue surface: Effects of humidity and confined geometry

DOE PAGES

O’Brien, Christopher J.; Greathouse, Jeffery A.; Tenney, Craig M.

2016-11-22

Here, first-principles molecular dynamics simulations were used to investigate the dissociation of sarin (GB) on the calcium silicate hydrate (CSH) mineral tobermorite (TBM), a surrogate for cement. CSH minerals (including TBM) and amorphous materials of similar composition are the major components of Portland cement, the binding agent of concrete. Metadynamics simulations were used to investigate the effect of the TBM surface and confinement in a microscale pore on the mechanism and free energy of dissociation of GB. Our results indicate that both the adsorption site and the humidity of the local environment significantly affect the sarin dissociation energy. In particular,more » sarin dissociation in a low-water environment occurs via a dealkylation mechanism, which is consistent with previous experimental studies.« less

Phase I metabolism of the carbazole derived synthetic cannabinoids EG-018, EG-2201 and MDMB-CHMCZCA and detection in human urine samples.

PubMed

Mogler, Lukas; Franz, Florian; Wilde, Maurice; Huppertz, Laura M; Halter, Sebastian; Angerer, Verena; Moosmann, Bjoern; Auwärter, Volker

2018-05-04

Synthetic cannabinoids (SCs) are a structurally diverse class of new psychoactive substances. Most SCs used for recreational purposes are based on indole or indazole core structures. EG-018 (naphthalen-1-yl(9-pentyl-9H-carbazol-3-yl)methanone), EG-2201 ((9-(5-fluoropentyl)-9H-carbazol-3-yl)(naphthalen-1-yl)methanone) and MDMB-CHMCZCA (methyl 2-(9-(cyclohexylmethyl)-9H-carbazole-3-carboxamido)-3,3-dimethylbutanoate) are three representatives of a structural subclass of SCs, characterized by a carbazole core system. In vitro and in vivo phase I metabolism studies were conducted to identify the most suitable metabolites for the detection of these substances in urine screening. Detection and characterization of metabolites were performed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (LC-ESI-QToF-MS). Eleven in vivo metabolites were detected in urine samples positive for metabolites of EG-018 (n=8). A hydroxypentyl metabolite, most probably the 4-hydroxypentyl isomer, and an N-dealkylated metabolite mono-hydroxylated at the carbazole core system were most abundant. In vitro studies of EG-018 and EG-2201 indicated that oxidative defluorination of the 5-fluoropentyl side chain of EG-2201 as well as dealkylation led to common metabolites with EG-018. This has to be taken into account for interpretation of analytical findings. A differentiation between EG-018 and EG-2201 (n=1) uptake is possible by the detection of compound-specific in vivo phase I metabolites evaluated in this study. Out of 30 metabolites detected in urine samples of MDMB-CHMCZCA users (n=20), one metabolite mono-hydroxylated at the cyclohexyl methyl tail is considered the most suitable compound-specific consumption marker while a biotransformation product of mono-hydroxylation in combination with hydrolysis of the terminal methyl ester function provides best sensitivity due to its high abundance. This article is protected by copyright. All rights reserved.

Methylsulfonyl benzothiazoles (MSBT) derivatives: Search for new potential antimicrobial and anticancer agents.

PubMed

Lad, Nitin P; Manohar, Yogesh; Mascarenhas, Malcolm; Pandit, Yashwant B; Kulkarni, Mahesh R; Sharma, Rajiv; Salkar, Kavita; Suthar, Ashish; Pandit, Shivaji S

2017-03-01

A series of novel 4 and 5-substituted methylsulfonyl benzothiazole (MSBT) compounds having amide, alkoxy, sulfonamide, nitro and amine functionality were synthesized from sequential reactions on 5-ethoxy-2-(methylsulfonyl)benzo[d]thiazole such as nitration, reduction, sulfonation, dealkylation, etc. All synthesized compounds were screened against antimicrobial and selected screened for anticancer activity. Antimicrobial activities studies reveled that among all compounds screened, out of MSBT-07, MSBT-11, MSBT-12, MSBT-14, MSBT-19, and MSBT-27 were found to have promising antimicrobial activity at MIC range of 4-50μg/ml against selected bacterial as well as fungal species. Compounds having good antimicrobial activity were screened for cervical cancer (HeLA cell lines). Of these MSBT-07 and MSBT-12 significantly reduced the cell growth. Consequently their calculated GI 50 values were found to be 0.1 or <0.1μM. Copyright © 2016 Elsevier Ltd. All rights reserved.

Profiling and Metabolism of Sterols in the Weaver Ant Genus Oecophylla.

PubMed

Vidkjær, Nanna H; Jensen, Karl-Martin V; Gislum, René; Fomsgaard, Inge S

2016-01-01

Sterols are essential to insects because they are vital for many biochemical processes, nevertheless insects cannot synthesize sterols but have to acquire them through their diet. Studies of sterols in ants are sparse and here the sterols of the weaver ant genus Oecophylla are identified for the first time. The sterol profile and the dietary sterols provided to a laboratory Oecophylla longinoda colony were analyzed. Most sterols originated from the diet, except one, which was probably formed via dealkylation in the ants and two sterols of fungal origin, which likely originate from hitherto unidentified endosymbionts responsible for supplying these two compounds. The sterol profile of a wild Oecophylla smaragdina colony was also investigated. Remarkable qualitative similarities were established between the two species despite the differences in diet, species, and origin. This may reflect a common sterol need/aversion in the weaver ants. Additionally, each individual caste of both species displayed unique sterol profiles.

Neutral Fragment Filtering for Rapid Identification of New Diester-Diterpenoid Alkaloids in Roots of Aconitum carmichaeli by Ultra-High-Pressure Liquid Chromatography Coupled with Linear Ion Trap-Orbitrap Mass Spectrometry

PubMed Central

Qiu, Xiao Hui; Yang, Yi Ming; Zhu, Da Yuan; Xu, Wen

2012-01-01

A rapid and effective method was developed for separation and identification of diester-diterpenoid alkaloids (DDA) in the roots of Aconitum carmichaeli by ultra-high-pressure liquid chromatography coupled with high resolution LTQ-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MSn). According to accurate mass measurement and the characteristic neutral loss filtering strategy, a total of 42 diester-diterpenoid alkaloids (DDA) were rapidly detected and characterized or tentatively identified. Meanwhile, the proposed fragmentation pathways and the major diagnostic fragment ions of aconitine, mesaconitine and hypaconitine were investigated to trace DDA derivatives in crude plant extracts. 23 potential new compounds were successfully screened and characterized in Aconitum carmichaeli, including 16 short chain fatty acyls DDA, 4 N-dealkyl DDA and several isomers of aconitine, mesaconitine and hypaconitine. PMID:23285005

Prediction of reacting atoms for the major biotransformation reactions of organic xenobiotics.

PubMed

Rudik, Anastasia V; Dmitriev, Alexander V; Lagunin, Alexey A; Filimonov, Dmitry A; Poroikov, Vladimir V

2016-01-01

The knowledge of drug metabolite structures is essential at the early stage of drug discovery to understand the potential liabilities and risks connected with biotransformation. The determination of the site of a molecule at which a particular metabolic reaction occurs could be used as a starting point for metabolite identification. The prediction of the site of metabolism does not always correspond to the particular atom that is modified by the enzyme but rather is often associated with a group of atoms. To overcome this problem, we propose to operate with the term "reacting atom", corresponding to a single atom in the substrate that is modified during the biotransformation reaction. The prediction of the reacting atom(s) in a molecule for the major classes of biotransformation reactions is necessary to generate drug metabolites. Substrates of the major human cytochromes P450 and UDP-glucuronosyltransferases from the Biovia Metabolite database were divided into nine groups according to their reaction classes, which are aliphatic and aromatic hydroxylation, N- and O-glucuronidation, N-, S- and C-oxidation, and N- and O-dealkylation. Each training set consists of positive and negative examples of structures with one labelled atom. In the positive examples, the labelled atom is the reacting atom of a particular reaction that changed adjacency. Negative examples represent non-reacting atoms of a particular reaction. We used Labelled Multilevel Neighbourhoods of Atoms descriptors for the designation of reacting atoms. A Bayesian-like algorithm was applied to estimate the structure-activity relationships. The average invariant accuracy of prediction obtained in leave-one-out and 20-fold cross-validation procedures for five human isoforms of cytochrome P450 and all isoforms of UDP-glucuronosyltransferase varies from 0.86 to 0.99 (0.96 on average). We report that reacting atoms may be predicted with reasonable accuracy for the major classes of metabolic reactions-aliphatic and aromatic hydroxylation, N- and O-glucuronidation, N-, S- and C-oxidation, and N- and O-dealkylation. The proposed method is implemented as a freely available web service at http://www.way2drug.com/RA and may be used for the prediction of the most probable biotransformation reaction(s) and the appropriate reacting atoms in drug-like compounds.Graphical abstract.

Cytochrome P450 3A expression and activity in the rabbit lacrimal gland: glucocorticoid modulation and the impact on androgen metabolism.

PubMed

Attar, Mayssa; Ling, Kah-Hiing John; Tang-Liu, Diane D-S; Neamati, Nouri; Lee, Vincent H L

2005-12-01

Cytochrome P450 3A (CYP3A) is an enzyme of paramount importance to drug metabolism. The expression and activity of CYP3A, an enzyme responsible for active androgen clearance, was investigated in the rabbit lacrimal gland. Analysis of CYP3A expression and activity was performed on lacrimal gland tissues obtained from naïve untreated and treated New Zealand White rabbits. For 5 days, treated rabbits received daily administration of vehicle or 0.1% or 1.0% dexamethasone, in the lower cul-de-sac of each eye. Changes in mRNA expression were monitored by real-time RT-PCR. Protein expression was confirmed by Western blot. Functional activity was measured by monitoring the metabolism of CYP3A probe substrates-namely, 7-benzyloxyquinoline (BQ) and [3H]testosterone. Cytochrome P450 heme protein was detected at a concentration of 44.6 picomoles/mg protein, along with its redox partner NADPH reductase and specifically CYP3A6 in the naïve rabbit lacrimal gland. Genes encoding CYP3A6, in addition to the pregnane-X-receptor (PXR) and P-glycoprotein (P-gp) were expressed in the untreated tissue. BQ dealkylation was measured in the naïve rabbit lacrimal gland at a rate of 14 +/- 7 picomoles/mg protein per minute. Changes in CYP3A6, P-gp, and androgen receptor mRNA expression levels were detected after dexamethasone treatment. In addition, dexamethasone treatment resulted in significant increases in BQ dealkylation and CYP3A6-mediated [3H]testosterone metabolism. Concomitant increases in CYP3A6-mediated hydroxylated testosterone metabolites were observed in the treated rabbits. Furthermore, ketoconazole, all-trans retinoic acid, and cyclosporine inhibited CYP3A6 mediated [3H]testosterone 6beta hydroxylation in a concentration-dependent manner, with IC50 ranging from 3.73 to 435 microM. The results demonstrate, for the first time, the expression and activity of CYP3A6 in the rabbit lacrimal gland. In addition, this pathway was shown to be subject to modulation by a commonly prescribed glucocorticoid and can be inhibited by known CYP3A inhibitors.

HI-6 assisted Catalytic Scavenging of VX by Acetylcholinesterase Choline Binding Site Mutants

PubMed Central

Hrvat, Nikolina Maček; Žunec, Suzana; Taylor, Palmer; Radić, Zoran; Kovarik, Zrinka

2016-01-01

The high toxicity of organophosphorus compounds originates from covalent inhibition of acetylcholinesterase (AChE), an essential enzyme in cholinergic neurotransmission. Poisonings that lead to life-threatening toxic manifestations require immediate treatment that combines administration of anticholinergic drugs and an aldoxime as a reactivator of AChE. An alternative approach to reduce the in vivo toxicity of OPs focuses on the use of bioscavengers against the parent organophosphate. Our previous research showed that AChE mutagenesis can enable aldoximes to substantially accelerate the reactivation of OP-enzyme conjugates, while dramatically slowing down rates of OP-conjugate dealkylation (aging). Herein, we demonstrate an efficient HI-6-assisted VX detoxification, both ex vivo in human blood and in vivo in mice by hAChE mutants modified at the choline binding site (Y337A and Y337A/F338A). The catalytic scavenging of VX in mice improved therapeutic outcomes preventing lethality and resulted in a delayed onset of toxicity symptoms. PMID:27083141

Extracellular palladium-catalysed dealkylation of 5-fluoro-1-propargyl-uracil as a bioorthogonally activated prodrug approach

PubMed Central

Weiss, Jason T.; Dawson, John C.; Macleod, Kenneth G.; Rybski, Witold; Fraser, Craig; Torres-Sánchez, Carmen; Patton, E. Elizabeth; Bradley, Mark; Carragher, Neil O.; Unciti-Broceta, Asier

2014-01-01

A bioorthogonal organometallic reaction is a biocompatible transformation undergone by a synthetic material exclusively through the mediation of a non-biotic metal source; a selective process used to label biomolecules and activate probes in biological environs. Here we report the in vitro bioorthogonal generation of 5-fluorouracil from a biologically inert precursor by heterogeneous Pd0 catalysis. Although independently harmless, combined treatment of 5-fluoro-1-propargyl-uracil and Pd0-functionalized resins exhibits comparable antiproliferative properties to the unmodified drug in colorectal and pancreatic cancer cells. Live-cell imaging and immunoassay studies demonstrate that the cytotoxic activity of the prodrug/Pd0-resin combination is due to the in situ generation of 5-fluorouracil. Pd0-resins can be carefully implanted in the yolk sac of zebrafish embryos and display excellent biocompatibility and local catalytic activity. The in vitro efficacy shown by this masking/activation strategy underlines its potential to develop a bioorthogonally activated prodrug approach and supports further in vivo investigations. PMID:24522696

A hybridization approach to efficient TiO2 photodegradation of aqueous benzalkonium chloride.

PubMed

Suchithra, Padmajan Sasikala; Carleer, Robert; Ananthakumar, Solaippan; Yperman, Jan

2015-08-15

TiO2 get positively charged upon UV-irradiation and repel the cationic pollutants away from the surface. Hybridization of AC onto TiO2 (ACT) tends catalyst surface negatively charged besides providing highly favorable adsorptions sites for cationic pollutants. The photodegradation of benzalkonium chloride (BKC), a quaternary ammonium surfactant and a pharmaceutical, is investigated with ACT. The surface charge of the catalyst in surfactant and non-surfactant aqueous dispersion under UV-irradiation is investigated and explained. The anomalous increase in COD values at the beginning of BKC-photodegradation is explained. The intermediate products formed are identified in both solution and solid phase. Trace amount of dodecane remained adsorbed on the catalyst surface after 1h UV-irradiation, but complete mineralization of BKC is achieved with 2h UV-irradiation. We propose that BKC photodegradation starts by central fission of benzyl CN bond followed by dealkylation, and demethylation steps. Copyright © 2015 Elsevier B.V. All rights reserved.

AGT Activity Towards Intrastrand Crosslinked DNA is Modulated by the Alkylene Linker.

PubMed

O'Flaherty, Derek K; Wilds, Christopher J

2017-12-05

DNA oligomers containing dimethylene and trimethylene intrastrand crosslinks (IaCLs) between the O4 and O6 atoms of neighboring thymidine (T) and 2'-deoxyguanosine (dG) residues were prepared by solid-phase synthesis. UV thermal denaturation (T m ) experiments revealed that these IaCLs had a destabilizing effect on the DNA duplex relative to the control. Circular dichroism spectroscopy suggested these IaCLs induced minimal structural distortions. Susceptibility to dealkylation by reaction with various O 6 -alkylguanine DNA alkyltransferases (AGTs) from human and Escherichia coli was evaluated. It was revealed that only human AGT displayed activity towards the IaCL DNA, with reduced efficiency as the IaCL shortened (from four to two methylene linkages). Changing the site of attachment of the ethylene linkage at the 5'-end of the IaCL to the N3 atom of T had minimal influence on duplex stability and structure, and was refractory to AGT activity. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inactivation of peptidylglycine α-hydroxylating monooxygenase by cinnamic acid analogs

PubMed Central

McIntyre, Neil R.; Lowe, Edward W.; Battistini, Matthew R.; Leahy, James W.; Merkler, David J.

2016-01-01

Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the final reaction in the maturation of α-amidated peptide hormones. Peptidylglycine α-hydroxylating monooxygenase (PHM) is the PAM domain responsible for the copper-, ascorbate- and O2-dependent hydroxylation of a glycine-extended peptide. Peptidylamidoglycolate lyase is the PAM domain responsible for the Zn(II)-dependent dealkylation of the α-hydroxyglycine-containing precursor to the final α-amidated peptide. We report herein that cinnamic acid and cinnamic acid analogs are inhibitors or inactivators of PHM. The inactivation chemistry exhibited by the cinnamates exhibits all the attributes of a suicide-substrate. However, we find no evidence for the formation of an irreversible linkage between cinnamate and PHM in the inactivated enzyme. Our data support the reversible formation of a Michael adduct between an active site nucleophile and cinnamate that leads to inactive enzyme. Our data are of significance given that cinnamates are found in foods, perfumes, cosmetics and pharmaceuticals. PMID:26024288

Biotransformation and bioactivation reactions of alicyclic amines in drug molecules.

PubMed

Bolleddula, Jayaprakasam; DeMent, Kevin; Driscoll, James P; Worboys, Philip; Brassil, Patrick J; Bourdet, David L

2014-08-01

Aliphatic nitrogen heterocycles such as piperazine, piperidine, pyrrolidine, morpholine, aziridine, azetidine, and azepane are well known building blocks in drug design and important core structures in approved drug therapies. These core units have been targets for metabolic attack by P450s and other drug metabolizing enzymes such as aldehyde oxidase and monoamine oxidase (MAOs). The electron rich nitrogen and/or α-carbons are often major sites of metabolism of alicyclic amines. The most common biotransformations include N-oxidation, N-conjugation, oxidative N-dealkylation, ring oxidation, and ring opening. In some instances, the metabolic pathways generate electrophilic reactive intermediates and cause bioactivation. However, potential bioactivation related adverse events can be attenuated by structural modifications. Hence it is important to understand the biotransformation pathways to design stable drug candidates that are devoid of metabolic liabilities early in the discovery stage. The current review provides a comprehensive summary of biotransformation and bioactivation pathways of aliphatic nitrogen containing heterocycles and strategies to mitigate metabolic liabilities.

Vitamin C: update on physiology and pharmacology

PubMed Central

Mandl, J; Szarka, A; Bánhegyi, G

2009-01-01

Although ascorbic acid is an important water-soluble antioxidant and enzyme cofactor in plants and animals, humans and some other species do not synthesize ascorbate due to the lack of the enzyme catalyzing the final step of the biosynthetic pathway, and for them it has become a vitamin. This review focuses on the role of ascorbate in various hydroxylation reactions and in the redox homeostasis of subcellular compartments including mitochondria and endoplasmic reticulum. Recently discovered functions of ascorbate in nucleic acid and histone dealkylation and proteoglycan deglycanation are also summarized. These new findings might delineate a role for ascorbate in the modulation of both pro- and anti-carcinogenic mechanisms. Recent advances and perspectives in therapeutic applications are also reviewed. On the basis of new and earlier observations, the advantages of the lost ability to synthesize ascorbate are pondered. The increasing knowledge of the functions of ascorbate and of its molecular sites of action can mechanistically substantiate a place for ascorbate in the treatment of various diseases. PMID:19508394

Tailoring partially reduced graphene oxide as redox mediator for enhanced biotransformation of iopromide under methanogenic and sulfate-reducing conditions.

PubMed

Toral-Sánchez, Eduardo; Rangel-Mendez, J Rene; Ascacio Valdés, Juan A; Aguilar, Cristóbal N; Cervantes, Francisco J

2017-01-01

This work reports the first successful application of graphene oxide (GO) and partially reduced GO (rGO) as redox mediator (RM) to increase the biotransformation of the recalcitrant iodinated contrast medium, iopromide (IOP). Results showed that GO-based materials promoted up to 5.5 and 2.8-fold faster biotransformation of IOP by anaerobic sludge under methanogenic and sulfate-reducing conditions, respectively. Correlation between the extent of reduction of GO and its redox-mediating capacity was demonstrated, which was reflected in faster removal and greater extent of biotransformation of IOP. Further analysis indicated that the biotransformation pathway of IOP involved multiple reactions including deiodination, decarboxylation, demethylation, dehydration and N-dealkylation. GO-based materials could be strategically tailored and integrated in biological treatment systems to effectively enhance the redox conversion of recalcitrant pollutants commonly found in wastewater treatment systems and industrial effluents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Process for hydrocracking carbonaceous material to provide fuels or chemical feed stock

DOEpatents

Duncan, Dennis A.

1980-01-01

A process is disclosed for hydrocracking coal or other carbonaceous material to produce various aromatic hydrocarbons including benzene, toluene, xylene, ethylbenzene, phenol and cresols in variable relative concentrations while maintaining a near constant maximum temperature. Variations in relative aromatic concentrations are achieved by changing the kinetic severity of the hydrocracking reaction by altering the temperature profile up to and quenching from the final hydrocracking temperature. The relative concentration of benzene to the alkyl and hydroxyl aromatics is increased by imposing increased kinetic severity above that corresponding to constant heating rate followed by immediate quenching at about the same rate to below the temperature at which dehydroxylation and dealkylation reactions appreciably occur. Similarly phenols, cresols and xylenes are produced in enhanced concentrations by adjusting the temperature profile to provide a reduced kinetic severity relative to that employed when high benzene concentrations are desired. These variations in concentrations can be used to produce desired materials for chemical feed stocks or for fuels.

Hit-to-lead evaluation of a novel class of sphingosine 1-phosphate lyase inhibitors.

PubMed

Dinges, Jurgen; Harris, Christopher M; Wallace, Grier A; Argiriadi, Maria A; Queeney, Kara L; Perron, Denise C; Dominguez, Eric; Kebede, Tegest; Desino, Kelly E; Patel, Hetal; Vasudevan, Anil

2016-05-01

Inhibition of sphingosine-1-phosphate lyase has recently been proposed as a potential treatment option for inflammatory disorders such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. In this report we describe our hit-to-lead evaluation of the isoxazolecarboxamide 6, a high-throughput screening hit (in vitro IC50=1.0 μM, cell IC50=1.8 μM), as a novel S1P lyase inhibitor. We were able to establish basic structure-activity relationships around 6 and succeeded in obtaining X-ray structural information which enabled structure-based design. With the discovery of 28, enzyme activity was quickly improved to IC50=120 nM and cell potency to IC50=230 nM. The main liability in the established isoxazolecarboxamide hit series was determined to be metabolic stability. In particular we identified that future lead-optimization efforts to overcome this problem should focus on blocking the N-dealkylation on the secondary amine. Copyright © 2016 Elsevier Ltd. All rights reserved.

HI-6 assisted catalytic scavenging of VX by acetylcholinesterase choline binding site mutants.

PubMed

Maček Hrvat, Nikolina; Žunec, Suzana; Taylor, Palmer; Radić, Zoran; Kovarik, Zrinka

2016-11-25

The high toxicity of organophosphorus compounds originates from covalent inhibition of acetylcholinesterase (AChE), an essential enzyme in cholinergic neurotransmission. Poisonings that lead to life-threatening toxic manifestations require immediate treatment that combines administration of anticholinergic drugs and an aldoxime as a reactivator of AChE. An alternative approach to reduce the in vivo toxicity of OPs focuses on the use of bioscavengers against the parent organophosphate. Our previous research showed that AChE mutagenesis can enable aldoximes to substantially accelerate the reactivation of OP-enzyme conjugates, while dramatically slowing down rates of OP-conjugate dealkylation (aging). Herein, we demonstrate an efficient HI-6-assisted VX detoxification, both ex vivo in human blood and in vivo in mice by hAChE mutants modified at the choline binding site (Y337A and Y337A/F338A). The catalytic scavenging of VX in mice improved therapeutic outcomes preventing lethality and resulted in a delayed onset of toxicity symptoms. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

In vivo chemistry of iofetamine HCl iodine-123 (IMP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baldwin, R.M.; Wu, J.L.

1988-01-01

Application of chemical methods for characterizing the in vivo behavior of iofetamine HCI /sup 123/I (IMP) has shed light on the metabolism of iofetamine in animals and humans. A successful technique consists of ethyl acetate extraction of the metabolites from tissue samples acidified with perchloric acid, separation of the mixture by high performance liquid chromatography, and quantitation of the radioactive components with a sensitive scintillation detector. Metabolism of iofetamine HCI /sup 123/I proceeds sequentially from the N-isopropyl group on the amphetamine side chain. The first step, dealkylation to the primary amine p-iodoamphetamine (PIA), occurs readily in the brain, lungs, andmore » liver; activity in the brain and lungs consists of only IMP and PIA even 24 hr after administration. The rate-limiting step appears to be deamination to give the transitory intermediate p-iodophenylacetone, which is rapidly degraded to p-iodobenzoic acid and conjugated with glycine in the liver to give the end product of metabolism, p-iodohippuric acid, which is excreted through the kidneys in the urine.« less

Magnetic bionanoparticles of Penicillium sp. yz11-22N2 doped with Fe3O4 and encapsulated within PVA-SA gel beads for atrazine removal.

PubMed

Yu, Jiaping; He, Huijun; Yang, William L; Yang, Chunping; Zeng, Guangming; Wu, Xin

2018-07-01

A novel magnetic bionanomaterial, Penicillium sp. yz11-22N2 doped with nano Fe 3 O 4 entrapped in polyvinyl alcohol-sodium alginate gel beads (PFEPS), was successfully synthesized. The factors including nutrient substance, temperature, pH, initial concentrations of atrazine and rotational speeds were presented and discussed in detail. Results showed that the highest removal efficiency of atrazine by PFEPS was 91.2% at 8.00 mg/L atrazine. The maximum removal capacity for atrazine was 7.94 mg/g. Meanwhile, it has been found that most of atrazine were removed by metabolism and degradation of Penicillium sp. yz11-22N2, which could use atrazine as the sole source of either carbon or nitrogen. Degradation kinetics of atrazine conformed to first-order kinetics model. The intermediates indicated that the possible pathway for atrazine degradation by PFEPS mainly included hydrolysis dechlorination, dealkylation, side-chain oxidation and ring-opening. Copyright © 2018 Elsevier Ltd. All rights reserved.

In vitro mutagenic, antimutagenic, and antioxidant activities evaluation and biotransformation of some bioactive 4-substituted 1-(2-methoxyphenyl)piperazine derivatives.

PubMed

Słoczyńska, Karolina; Pańczyk, Katarzyna; Waszkielewicz, Anna M; Marona, Henryk; Pękala, Elżbieta

2016-12-01

In vitro mutagenic, antimutagenic, and antioxidant potency evaluation and biotransformation of six novel 4-substituted 1-(2-methoxyphenyl)piperazine derivatives demonstrating antidepressant-like activity were investigated. Mutagenic and antimutagenic properties were assessed using the Ames test; free radical scavenging activity was evaluated with 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay and biotransformation was performed with liver microsomes. It was found that all tested compounds are not mutagenic in bacterial strains TA100 and TA1535 and exhibit antimutagenic effects in the Ames test. Noteworthy, compounds possessing propyl linker between phenoxyl and N-(2-methoxyphenyl)piperazine displayed more pronounced antimutagenic properties than derivatives with ethoxyethyl linker. Additionally, compounds 2 and 6 in vitro biotransformation showed that primarily their hydroxylated or O-dealkylated metabolites are formed. Some of the compounds exhibited intrinsic clearance values lower than those reported previously for antidepressant imipramine. To sum up, the results of the present study might represent a valuable step in designing and planning future studies with piperazine derivatives. © 2016 Wiley Periodicals, Inc.

Study of the photochemical transformation of 2-ethylhexyl 4-(dimethylamino)benzoate (OD-PABA) under conditions relevant to surface waters.

PubMed

Calza, P; Vione, D; Galli, F; Fabbri, D; Dal Bello, F; Medana, C

2016-01-01

We studied the aquatic environmental fate of 2-ethylhexyl 4-(dimethylamino)benzoate (OD-PABA), a widespread sunscreen, to assess its environmental persistence and photoinduced transformation. Direct photolysis is shown to play a key role in phototransformation, and this fast process is expected to be the main attenuation route of OD-PABA in sunlit surface waters. The generation of transformation products (TPs) was followed via HPLC/HRMS. Five (or four) TPs were detected in the samples exposed to UVB (or UVA) radiation, respectively. The main detected TPs of OD-PABA, at least as far as HPLC-HRMS peak areas are concerned, would involve a dealkylation or hydroxylation/oxidation process in both direct photolysis and indirect phototransformation. The latter was simulated by using TiO2-based heterogeneous photocatalysis, involving the formation of nine additional TPs. Most of them resulted from the further degradation of the primary TPs that can also be formed by direct photolysis. Therefore, these secondary TPs might also occur as later transformation intermediates in natural aquatic systems. Copyright © 2015 Elsevier Ltd. All rights reserved.

Demonstration of in vitro Resurrection of Aged Acetylcholinesterase after Exposure to Organophosphorus Chemical Nerve Agents.

PubMed

Zhuang, Qinggeng; Franjesevic, Andrew J; Corrigan, Thomas S; Coldren, William H; Dicken, Rachel; Sillart, Sydney; DeYong, Ashley; Yoshino, Nathan; Smith, Justin; Fabry, Stephanie; Fitzpatrick, Keegan; Blanton, Travis G; Joseph, Jojo; Yoder, Ryan J; McElroy, Craig A; Dogan Ekici, Ozlem; Callam, Christopher S; Hadad, Christopher M

2018-06-05

After inhibition of acetylcholinesterase (AChE) by organophosphorus (OP) nerve agents, a dealkylation reaction, referred to as aging, of the phosphylated serine can occur. When aged, known reactivators of OP-inhibited AChE are no longer effective. Realkylation of aged AChE may provide a route to reverse aging. We designed and synthesized a library of quinone methide precursors (QMPs) as proposed realkylators of aged AChE. Our lead compound (C8) from an in vitro screening, successfully resurrected 32.7% and 20.4% of the activity of methylphosphonate-aged and isopropyl phosphate-aged electric eel AChE, respectively, after 4 days. C8 displays properties of both resurrection (recovery from the aged to the native state) and reactivation (recovery from the inhibited to the native state). Resurrection of methylphosphonate-aged AChE by C8 was significantly pH-dependent, recovering 21% of activity at 4 mM and pH 9 after only 1 day. C8 is also effective against isopropyl phosphate-aged human AChE.

Metabolite profiling of RCS-4, a novel synthetic cannabinoid designer drug, using human hepatocyte metabolism and TOF-MS

PubMed Central

Gandhi, Adarsh S; Zhu, Mingshe; Pang, Shaokun; Wohlfarth, Ariane; Scheidweiler, Karl B; Huestis, Marilyn A

2014-01-01

Background Since 2009, scheduling legislation of synthetic cannabinoids prompted new compound emergence to circumvent legal restrictions. 2-(4-methoxyphenyl)-1-(1-pentyl-indol-3-yl)methanone (RCS-4) is a potent cannabinoid receptor agonist sold in herbal smoking blends. Absence of parent synthetic cannabinoids in urine suggests the importance of metabolite identification for detecting RCS-4 consumption in clinical and forensic investigations. Materials & methods & Results With 1 h human hepatocyte incubation and TOF high-resolution MS, we identified 18 RCS-4 metabolites, many not yet reported. Most metabolites were hydroxylated with or without demethylation, carboxylation and dealkylation followed by glucuronidation. One additional sulfated metabolite was also observed. O-demethylation was the most common biotransformation and generated the major metabolite. Conclusion For the first time, we present a metabolic scheme of RCS-4 obtained from human hepatocytes, including Phase I and II metabolites. Metabolite structural information and associated high-resolution mass spectra can be employed for developing clinical and forensic laboratory RCS-4 urine screening methods. PMID:25046048

In silico prediction of pharmaceutical degradation pathways: a benchmarking study.

PubMed

Kleinman, Mark H; Baertschi, Steven W; Alsante, Karen M; Reid, Darren L; Mowery, Mark D; Shimanovich, Roman; Foti, Chris; Smith, William K; Reynolds, Dan W; Nefliu, Marcela; Ott, Martin A

2014-11-03

Zeneth is a new software application capable of predicting degradation products derived from small molecule active pharmaceutical ingredients. This study was aimed at understanding the current status of Zeneth's predictive capabilities and assessing gaps in predictivity. Using data from 27 small molecule drug substances from five pharmaceutical companies, the evolution of Zeneth predictions through knowledge base development since 2009 was evaluated. The experimentally observed degradation products from forced degradation, accelerated, and long-term stability studies were compared to Zeneth predictions. Steady progress in predictive performance was observed as the knowledge bases grew and were refined. Over the course of the development covered within this evaluation, the ability of Zeneth to predict experimentally observed degradants increased from 31% to 54%. In particular, gaps in predictivity were noted in the areas of epimerizations, N-dealkylation of N-alkylheteroaromatic compounds, photochemical decarboxylations, and electrocyclic reactions. The results of this study show that knowledge base development efforts have increased the ability of Zeneth to predict relevant degradation products and aid pharmaceutical research. This study has also provided valuable information to help guide further improvements to Zeneth and its knowledge base.

Metabolism of the tryptamine-derived new psychoactive substances 5-MeO-2-Me-DALT, 5-MeO-2-Me-ALCHT, and 5-MeO-2-Me-DIPT and their detectability in urine studied by GC-MS, LC-MSn , and LC-HR-MS/MS.

PubMed

Caspar, Achim T; Gaab, Jonas B; Michely, Julian A; Brandt, Simon D; Meyer, Markus R; Maurer, Hans H

2018-01-01

Many N,N-dialkylated tryptamines show psychoactive properties and were encountered as new psychoactive substances. The aims of the presented work were to study the phase I and II metabolism and the detectability in standard urine screening approaches (SUSA) of 5-methoxy-2-methyl-N,N-diallyltryptamine (5-MeO-2-Me-DALT), 5-methoxy-2-methyl-N-allyl-N-cyclohexyltryptamine (5-MeO-2-Me-ALCHT), and 5-methoxy-2-methyl-N,N-diisopropyltryptamine (5-MeO-2-Me-DIPT) using gas chromatography-mass spectrometry (GC-MS), liquid chromatography coupled with multistage accurate mass spectrometry (LC-MS n ), and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS). For metabolism studies, urine was collected over a 24 h period after administration of the compounds to male Wistar rats at 20 mg/kg body weight (BW). Phase I and II metabolites were identified after urine precipitation with acetonitrile by LC-HR-MS/MS. 5-MeO-2-Me-DALT (24 phase I and 12 phase II metabolites), 5-MeO-2-Me-ALCHT (24 phase I and 14 phase II metabolites), and 5-MeO-2-Me-DIPT (20 phase I and 11 phase II metabolites) were mainly metabolized by O-demethylation, hydroxylation, N-dealkylation, and combinations of them as well as by glucuronidation and sulfation of phase I metabolites. Incubations with mixtures of pooled human liver microsomes and cytosols (pHLM and pHLC) confirmed that the main metabolic reactions in humans and rats might be identical. Furthermore, initial CYP activity screenings revealed that CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were involved in hydroxylation, CYP2C19 and CYP2D6 in O-demethylation, and CYP2C19, CYP2D6, and CYP3A4 in N-dealkylation. For SUSAs, GC-MS, LC-MS n , and LC-HR-MS/MS were applied to rat urine samples after 1 or 0.1 mg/kg BW doses, respectively. In contrast to the GC-MS SUSA, both LC-MS SUSAs were able to detect an intake of 5-MeO-2-Me-ALCHT and 5-MeO-2-Me-DIPT via their metabolites following 1 mg/kg BW administrations and 5-MeO-2-Me-DALT following 0.1 mg/kg BW dosage. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Removal of alachlor, diuron and isoproturon in water in a falling film dielectric barrier discharge (DBD) reactor combined with adsorption on activated carbon textile: Reaction mechanisms and oxidation by-products.

PubMed

Vanraes, Patrick; Wardenier, Niels; Surmont, Pieter; Lynen, Frederic; Nikiforov, Anton; Van Hulle, Stijn W H; Leys, Christophe; Bogaerts, Annemie

2018-07-15

A falling film dielectric barrier discharge (DBD) plasma reactor combined with adsorption on activated carbon textile material was optimized to minimize the formation of hazardous oxidation by-products from the treatment of persistent pesticides (alachlor, diuron and isoproturon) in water. The formation of by-products and the reaction mechanism was investigated by HPLC-TOF-MS. The maximum concentration of each by-product was at least two orders of magnitude below the initial pesticide concentration, during the first 10 min of treatment. After 30 min of treatment, the individual by-product concentrations had decreased to values of at least three orders of magnitude below the initial pesticide concentration. The proposed oxidation pathways revealed five main oxidation steps: dechlorination, dealkylation, hydroxylation, addition of a double-bonded oxygen and nitrification. The latter is one of the main oxidation mechanisms of diuron and isoproturon for air plasma treatment. To our knowledge, this is the first time that the formation of nitrificated intermediates is reported for the plasma treatment of non-phenolic compounds. Copyright © 2018 Elsevier B.V. All rights reserved.

Biological and nonbiological modifications of carbamates

PubMed Central

Knaak, James B.

1971-01-01

Methylcarbamate insecticides undergo hydrolysis, oxidation, dealkylation, and conjugation in animals, plants, and insects to form similar or identical products. Carbaryl is hydroxylated in biological systems to form hydroxy, dihydro-dihydroxy, and N-hydroxymethyl carbaryl and is hydrolysed to form 1-naphthol. The products are conjugated, stored, or excreted. Carbofuran is hydroxylated at the 3 position and propoxur at the 5 position to form hydroxylated derivatives. N-hydroxymethyl derivatives of these two carbamates may also be formed. Hydrolysis appears to be the major metabolic pathway of carbofuran in the animal. Aldicarb is oxidized to its sulfoxide and then hydrolysed to the oxime sulfoxide in animals and plants. Plants hydrolyse the oxime sulfoxide to form the corresponding aldehyde, which is an intermediate in the formation of 2-methyl-2-(methyl-sulfinyl)propanol. Methomyl, which is structurally similar to aldicarb, is metabolized in plants to acetonitrile, carbon dioxide, and methylamine. Bux and Meobal undergo hydrolysis and hydroxylation to form N-hydroxy methylcarbamates, as well as hydroxybutylphenyl and hydroxymethylphenyl methylcarbamates. Zectran, which contains a dimethylamino group, is converted to the methylamino, amino, and methylformamido derivatives by insects and plants. In soil and water, methylcarbamate insecticides are hydrolysed to their respective phenols or oximes. PMID:4999481

Structural Basis of Multifunctionality in a Vitamin B[subscript 12]-processing Enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koutmos, Markos; Gherasim, Carmen; Smith, Janet L.

An early step in the intracellular processing of vitamin B{sub 12} involves CblC, which exhibits dual reactivity, catalyzing the reductive decyanation of cyanocobalamin (vitamin B{sub 12}), and the dealkylation of alkylcobalamins (e.g. methylcobalamin; MeCbl). Insights into how the CblC scaffold supports this chemical dichotomy have been unavailable despite it being the most common locus of patient mutations associated with inherited cobalamin disorders that manifest in both severe homocystinuria and methylmalonic aciduria. Herein, we report structures of human CblC, with and without bound MeCbl, which provide novel biochemical insights into its mechanism of action. Our results reveal that CblC is themore » most divergent member of the NADPH-dependent flavin reductase family and can use FMN or FAD as a prosthetic group to catalyze reductive decyanation. Furthermore, CblC is the first example of an enzyme with glutathione transferase activity that has a sequence and structure unrelated to the GST superfamily. CblC thus represents an example of evolutionary adaptation of a common structural platform to perform diverse chemistries. The CblC structure allows us to rationalize the biochemical basis of a number of pathological mutations associated with severe clinical phenotypes.« less

Cytochrome P450 peroxidase/peroxygenase mediated xenobiotic metabolic activation and cytotoxicity in isolated hepatocytes.

PubMed

Anari, M R; Khan, S; Liu, Z C; O'Brien, P J

1995-12-01

Cytochrome P450 (P450) can utilize organic hydroperoxides and peracids to support hydroxylation and dealkylation of various P450 substrates. However, the biological significance of this P450 peroxygenase/peroxidase activity in the bioactivation of xenobiotics in intact cells has not been demonstrated. We have shown that tert-butyl hydroperoxide (tBHP) markedly enhances 3-20-fold the cytotoxicity of various aromatic hydrocarbons and their phenolic metabolites. The tBHP-enhanced hepatocyte cytotoxicity of 4-nitroanisole (4-NA) and 4-hydroxyanisole (4-HA) was also accompanied by an increase in the hepatocyte O-demethylation of 4-NA and 4-HA up to 7.5- and 21-fold, respectively. Hepatocyte GSH conjugation by 4-HA was also markedly increased by tBHP. An LC/MS analysis of the GSH conjugates identified hydroquinone-GSH and 4-methoxy-catechol:GSH conjugates as the predominant adducts. Pretreatment of hepatocytes with P450 inhibitors, e.g., phenylimidazole, prevented tBHP-enhanced 4-HA metabolism, GSH depletion, and cytotoxicity. In conclusion, hydroperoxides can therefore be used by intact cells to support the bioactivation of xenobiotics through the P450 peroxidase/peroxygenase system.

Inhibitors of steroidal cytochrome p450 enzymes as targets for drug development.

PubMed

Baston, Eckhard; Leroux, Frédéric R

2007-01-01

Cytochrome P450's are enzymes which catalyze a large number of biological reactions, for example hydroxylation, N-, O-, S- dealkylation, epoxidation or desamination. Their substrates include fatty acids, steroids or prostaglandins. In addition, a high number of various xenobiotics are metabolized by these enzymes. The enzyme 17alpha-hydroxylase-C17,20-lyase (P450(17), CYP 17, androgen synthase), a cytochrome P450 monooxygenase, is the key enzyme for androgen biosynthesis. It catalyzes the last step of the androgen biosynthesis in the testes and adrenal glands and produces androstenedione and dehydroepiandrosterone from progesterone and pregnenolone. The microsomal enzyme aromatase (CYP19) transforms these androgens to estrone and estradiol. Estrogens stimulate tumor growth in hormone dependent breast cancer. In addition, about 80 percent of prostate cancers are androgen dependent. Selective inhibitors of these enzymes are thus important alternatives to treatment options like antiandrogens or antiestrogens. The present article deals with recent patents (focus on publications from 2000 - 2006) concerning P450 inhibitor design where steroidal substrates are involved. In this context a special focus is provided for CYP17 and CYP19. Mechanisms of action will also be discussed. Inhibitors of CYP11B2 (aldosterone synthase) will also be dealt with.

Biodegradation of diuron by an endophytic fungus Neurospora intermedia DP8-1 isolated from sugarcane and its potential for remediating diuron-contaminated soils

PubMed Central

Feng, Guojun; Du, Liangwei; Zeng, Dongqiang

2017-01-01

A diuron-degrading endophyte DP8-1 was isolated from sugarcane root grown in diuron-treated soil in the present study. The endophyte was identified as Neurospora intermedia based on the morphological characteristics and sequence analysis. The fermentation parameters including temperature, pH, inoculation size, carbon source, and initial diuron concentration were also investigated for the optimization of degradation efficiency. The results indicated that strain DP8-1 was capable of degrading up to 99% diuron within 3 days under the optimal degrading condition. The study of degradation spectrum indicated that strain DP8-1 could also degrade and utilize fenuron, monuron, metobromuron, isoproturon, chlorbromuron, linuron, and chlortoluron as substrate for strain growth. On basis of liquid chromatography-mass spectrometry analysis for the products of the degradation of diuron, strain DP8-1 metabolized diuron to produce N-(3,4-dichlorophenyl)-urea and N-(3,4-dichlorophenyl)-N-methylurea through sequential N-dealkylations. In a soil bioaugmentation experiment, the inoculation of strain DP8-1 into diuron-treated soil effectively enhanced the disappearance rate of diuron. PMID:28809955

Investigations on the human hepatic cytochrome P450 isozymes involved in the metabolism of 3,4-methylenedioxy-amphetamine (MDA) and benzodioxolyl-butanamine (BDB) enantiomers.

PubMed

Meyer, Markus R; Peters, Frank T; Maurer, Hans H

2009-10-08

3,4-Methylenedioxy-amphetamine (MDA) and benzodioxolyl-butanamine (BDB) are chiral designer drugs distributed on the illicit drug market and they are also N-dealkyl metabolites of 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy, Adam), 3,4-methylenedioxyethylamphetamine (MDEA, Eve), and N-methyl-benzodioxolyl-butanamine (MBDB, Eden), respectively. MDA and BDB are mainly metabolized via demethylenation to the corresponding catecholamines. The aim of the present work was to elucidate the contribution of the relevant human P450s in the demethylenation of the MDA and BDB enantiomers. They were incubated using heterologously expressed human P450s and the corresponding metabolites dihydroxyamphetamine and 1,2-dihydroxy-4-[2-amino-butyl]benzene were determined. Highest contributions to the demethylenation as calculated from the enzyme kinetic data were obtained for CYP2D6 (MDA and BDB) and additionally CYP3A4 in the case of BDB at substrate concentrations corresponding to plasma concentrations of recreational users. A preferred transformation of the S-enantiomer could be observed for the CYP2D6- and CYP3A4-catalyzed reactions.

EROD activity and biliary fluorescence in Schroederichthys chilensis (Guichenot 1848): biomarkers of PAH exposure in coastal environments of the South Pacific Ocean.

PubMed

Fuentes-Rios, Daniel; Orrego, Rodrigo; Rudolph, Anny; Mendoza, Gonzalo; Gavilán, Juan F; Barra, Ricardo

2005-10-01

Schroederichthys chilensis is a common shark that lives in Chilean coastal environments. In this work, the relationship between liver 7-ethoxyresorufin-O-deethylase dealkylation (EROD) activity and Fluorescent Aromatic Compounds (FAC) in bile of S. chilensis sampled in three bays with different degrees of pollution were performed including a reference area. Sixty individuals were collected, 20 for each site; (10 males and 10 females per site) livers and bile samples were obtained and immediately frozen. EROD activity and FAC were measured according to three standard methods. EROD activity and FAC were higher in polluted areas than in the reference area. Synchronous Fluorescence Spectra of the bile from the fish collected at the most polluted area showed a peak at 347nm representing a metabolite corresponding to 1-hydroxypyrene. The low EROD activity in the reference area is likely related to the low level of PAH in sediments. We propose that this species is a good indicator of exposure to FACs, since it presents a series of characteristics that make it suitable for monitoring PAH exposure in coastal zones.

Efficient biotransformation of herbicide diuron by bacterial strain Micrococcus sp. PS-1.

PubMed

Sharma, Priyanka; Chopra, Adity; Cameotra, Swaranjit Singh; Suri, C Raman

2010-11-01

A Gram-positive, Micrococcus sp. strain PS-1 capable of utilizing phenylurea herbicide diuron as a sole carbon source at a high concentration (up to 250 ppm) was isolated from diuron storage site by selective enrichment study. The taxonomic characterization with 16S rRNA gene sequencing (1,477 bp) identified PS-1 as a member of Micrococcus sp. It was studied for the degradation of diuron and a range of its analogues (monuron, linuron, monolinuron, chlortoluron and fenuron). The shake flasks experiments demonstrated fast degradation of diuron (up to 96% at 250 ppm within 30 h incubation) with the addition of small quantity (0.01%) of non-ionic detergent. The relative degradation profile by the isolate was in the order of fenuron > monuron > diuron > linuron > monolinuron > chlortoluron. Further, the biochemical characterization of catabolic pathway by spectroscopic and chromatographic techniques demonstrated that the degradation proceeded via formation of dealkylated metabolites to form 3,4-dichloroaniline (3,4-DCA). It was the major metabolite formed, associated with profound increase in degradation kinetics in presence of appropriate additive.

Molecular evidence of heavy-oil weathering following the M/V Cosco Busan spill: insights from Fourier transform ion cyclotron resonance mass spectrometry.

PubMed

Lemkau, Karin L; McKenna, Amy M; Podgorski, David C; Rodgers, Ryan P; Reddy, Christopher M

2014-04-01

Recent studies have highlighted a critical need to investigate oil weathering beyond the analytical window afforded by conventional gas chromatography (GC). In particular, techniques capable of detecting polar and higher molecular weight (HMW; > 400 Da) components abundant in crude and heavy fuel oils (HFOs) as well as transformation products. Here, we used atmospheric pressure photoionization Fourier transform ion cyclotron resonance mass spectrometry (APPI FT-ICR MS) to identify molecular transformations in oil-residue samples from the 2007 M/V Cosco Busan HFO spill (San Francisco, CA). Over 617 days, the abundance and diversity of oxygen-containing compounds increased relative to the parent HFO, likely from bio- and photodegradation. HMW, highly aromatic, alkylated compounds decreased in relative abundance concurrent with increased relative abundance of less alkylated stable aromatic structures. Combining these results with GC-based data yielded a more comprehensive understanding of oil spill weathering. For example, dealkylation trends and the overall loss of HMW species observed by FT-ICR MS has not previously been documented and is counterintuitive given losses of lower molecular weight species observed by GC. These results suggest a region of relative stability at the interface of these techniques, which provides new indicators for studying long-term weathering and identifying sources.

Thermoactivated persulfate oxidation of pesticide chlorpyrifos in aquatic system: kinetic and mechanistic investigations.

PubMed

Zhou, Lei; Zhang, Ya; Ying, Rongrong; Wang, Guoqing; Long, Tao; Li, Jianhua; Lin, Yusuo

2017-04-01

The widespread occurrence of organophosphorus pesticides (OPPs) in the environment poses risks to both ecologic system as well as human health. This study investigated the oxidation kinetics of chlorpyrifos (CP), one of the typical OPPs, by thermoactivated persulfate (PS) oxidation process, and evaluated the influence of key kinetic factors, such as PS concentrations, pH, temperature, bicarbonate, and chloride ions. The reaction pathways and mechanisms were also proposed based on products identification by LC-MS techniques. Our results revealed that increasing initial PS concentration and temperature favored the decomposition of CP, whereas the oxidation efficiency was not affected by pH change ranging from 3 to 11. Bicarbonate was found to play a detrimental role on CP removal rates, while chloride showed no effect. The oxidation pathways including initial oxidation of P=S bond to P=O, dechlorination, dealkylation, and the dechlorination-hydroxylation were proposed, and the detailed underlying mechanisms were also discussed. Molecular orbital (MO) calculations indicated that P=S bond was the most favored oxidation site of the molecule. The toxicity of reaction solution was believed to increase due to the formation of products with P=O structures. This work demonstrates that OPPs can readily react with SO 4 ·- and provides important information for further research on the oxidation of these contaminants.

Ozone Promotes Chloropicrin Formation by Oxidizing Amines to Nitro Compounds.

PubMed

McCurry, Daniel L; Quay, Amanda N; Mitch, William A

2016-02-02

Chloropicrin formation has been associated with ozonation followed by chlorination, but the reaction pathway and precursors have been poorly characterized. Experiments with methylamine demonstrated that ozonation converts methylamine to nitromethane at ∼100% yield. Subsequent chlorination converts nitromethane to chloropicrin at ∼50% yield under the conditions evaluated. Similarly high yields from other primary amines were limited to those with functional groups on the β-carbon (e.g., the carboxylic acid in glycine) that facilitate carbon-carbon bond cleavage to release nitromethyl anion. Secondary amines featuring these reactive primary amines as functional groups (e.g., secondary N-methylamines) formed chloropicrin at high yields, likely by facile dealkylation to release the primary nitro compound. Chloropicrin yields from tertiary amines were low. Natural water experiments, including derivatization to transform primary and secondary amines to less reactive carbamate functional groups, indicated that primary and secondary amines were the dominant chloropicrin precursors during ozonation/chlorination. Ozonation followed by chlorination of the primary amine side chain of lysine demonstrated low yields (∼0.2%) of chloropicrin, but high yields (∼17%) of dichloronitrolysine, a halonitroalkane structural analogue to chloropicrin. However, chloropicrin yields increased and dichloronitrolysine yields decreased in the absence of hydroxyl radical scavengers, suggesting that future research should characterize the potential occurrence of such halonitroalkane analogues relative to natural radical scavenger (e.g., carbonate) concentrations.

Evaluation of the oxidation of enrofloxacin by permanganate and the antimicrobial activity of the products.

PubMed

Xu, Yongpeng; Liu, Shiyao; Guo, Fang; Zhang, Bo

2016-02-01

Permanganate [Mn(VII)] oxidation of the fluoroquinolone (FQ) antibiotic enrofloxacin (ENR) was investigated with respect to kinetics and mechanisms, and the products were evaluated for residual antibacterial activity. The degradation of ENR by Mn(VII) obeyed second-order kinetics. A modern liquid chromatography coupled to a hybrid quadrupole time-of-flight mass spectrometer (LC-Q-TOF) was used to determine the accurate mass of the measured degradation products. The structures of nine oxidation products were identified at a neutral pH, one of which was an N-oxide product formed from the oxidation of tertiary amines. One proposed plausible reaction pathway was that the oxidation occurred on the piperazine ring; the C-H adjacent to the amine group was attacked by Mn(VII). The identified products from ENR arose through four pathways involving two mechanisms of N-dealkylation, C-hydroxylation and the reactions of amine oxides. The quinolone core remained intact for all of the products. The residual antibacterial activity of the oxidative reaction byproducts against the nonresistant Escherichia coli (G(-)) reference strain DH5ɑ was evaluated by quantifying the bacterial colonies. The oxidation products exhibited reduced antibacterial activity compared with their parent compound. Copyright © 2015 Elsevier Ltd. All rights reserved.

GABAA receptor activity modulating piperine analogs: In vitro metabolic stability, metabolite identification, CYP450 reaction phenotyping, and protein binding.

PubMed

Zabela, Volha; Hettich, Timm; Schlotterbeck, Götz; Wimmer, Laurin; Mihovilovic, Marko D; Guillet, Fabrice; Bouaita, Belkacem; Shevchenko, Bénédicte; Hamburger, Matthias; Oufir, Mouhssin

2018-01-01

In a screening of natural products for allosteric modulators of GABA A receptors (γ-aminobutyric acid type A receptor), piperine was identified as a compound targeting a benzodiazepine-independent binding site. Given that piperine is also an activator of TRPV1 (transient receptor potential vanilloid type 1) receptors involved in pain signaling and thermoregulation, a series of piperine analogs were prepared in several cycles of structural optimization, with the aim of separating GABA A and TRPV1 activating properties. We here investigated the metabolism of piperine and selected analogs in view of further cycles of lead optimization. Metabolic stability of the compounds was evaluated by incubation with pooled human liver microsomes, and metabolites were analyzed by UHPLC-Q-TOF-MS. CYP450 isoenzymes involved in metabolism of compounds were identified by reaction phenotyping with Silensomes™. Unbound fraction in whole blood was determined by rapid equilibrium dialysis. Piperine was the metabolically most stable compound. Aliphatic hydroxylation, and N- and O-dealkylation were the major routes of oxidative metabolism. Piperine was exclusively metabolized by CYP1A2, whereas CYP2C9 contributed significantly in the oxidative metabolism of all analogs. Extensive binding to blood constituents was observed for all compounds. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural modifications of N-(1,2,3,4-tetrahydronaphthalen-1-yl)-4-aryl-1-piperazinehexanamides: influence on lipophilicity and 5-HT7 receptor activity. Part III.

PubMed

Leopoldo, Marcello; Lacivita, Enza; De Giorgio, Paola; Fracasso, Claudia; Guzzetti, Sara; Caccia, Silvio; Contino, Marialessandra; Colabufo, Nicola A; Berardi, Francesco; Perrone, Roberto

2008-09-25

Starting from the previously reported 5-HT 7 receptor agents 4-7 with N-(1,2,3,4-tetrahydronaphthalen-1-yl)-4-aryl-1-piperazinehexanamide structure, the 1-(2-methylthiophenyl)-, 1-(2-diphenyl)-, 1-(2-isopropylphenyl)-, and 1-(2-methoxyphenyl)piperazine derivatives 8-31 were designed with the primary aim to obtain new compounds endowed with suitable physicochemical properties for rapid and extensive penetration into the brain. The affinities for 5-HT 7, 5-HT 1A, and D 2 receptors of compounds 8-31 were assessed, and several compounds displayed 5-HT 7 receptor affinities in the nanomolar range. Among these, N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (25) showed high 5-HT 7 receptor affinity (Ki = 0.58 nM), high selectivity over 5-HT 1A and D 2 receptors (324- and 245-fold, respectively), and agonist properties (maximal effect = 82%, EC 50 = 0.60 microM). After intraperitoneal injection in mice, 25 rapidly reached the systemic circulation and entered the brain. Its brain concentration-time profile paralleled that in plasma, indicating that 25 rapidly and freely distributes across the blood-brain barrier. Compound 25 underwent N-dealkylation to the corresponding 1-arylpiperazine metabolite.

Reaction of β-blockers and β-agonist pharmaceuticals with aqueous chlorine. Investigation of kinetics and by-products by liquid chromatography quadrupole time-of-flight mass spectrometry.

PubMed

Quintana, José Benito; Rodil, Rosario; Cela, Rafael

2012-06-01

The degradation of two β-blockers (atenolol and propranolol) and one β-receptor agonist (salbutamol) during water chlorination was investigated by liquid chromatography-mass spectrometry (LC-MS). An accurate-mass quadrupole time-of-flight system (QTOF) was used to follow the time course of the pharmaceuticals and also used in the identification of the by-products. The degradation kinetics of these drugs was investigated at different concentrations of chlorine, bromide and sample pH by means of a Box-Behnken experimental design. Depending on these factors, dissipation half-lives varied in the ranges 68-145 h for atenolol, 1.3-33 min for salbutamol and 42-8362 min for propranolol. Normally, an increase in chlorine dosage and pH resulted in faster degradation of these pharmaceuticals. Moreover, the presence of bromide in water samples also resulted in a faster transformation of atenolol at low chlorine doses. The use of an accurate-mass high-resolution LC-QTOF-MS system permitted the identification of a total of 14 by-products. The transformation pathway of β-blockers/agonists consisted mainly of halogenations, hydroxylations and dealkylations. Also, many of these by-products are stable, depending on the chlorination operational parameters employed.

Efficient mineralization of antibiotic ciprofloxacin in acid aqueous medium by a novel photoelectro-Fenton process using a microwave discharge electrodeless lamp irradiation.

PubMed

Wang, Aimin; Zhang, Yanyu; Zhong, Huihui; Chen, Yu; Tian, Xiujun; Li, Desheng; Li, Jiuyi

2018-01-15

In this study, a novel photoelectro-Fenton (PEF) process using microwave discharge electrodeless lamp (MDEL) as a UV irradiation source was developed for the removal of antibiotic ciprofloxacin (CIP) in water. Comparative degradation of 200mgL -1 CIP was studied by direct MDEL photolysis, anodic oxidation (AO), AO in presence of electrogenerated H 2 O 2 (AO-H 2 O 2 ), AO-H 2 O 2 under MDEL irradiation (MDEL-AO-H 2 O 2 ), electro-Fenton (EF) and MDEL-PEF processes. Higher oxidation power was found in the sequence: MDEL photolysis < AO < AO-H 2 O 2 < MDEL-AO-H 2 O 2 < EF < MDEL-PEF. Effects of current density, pH, initial Fe 2+ concentration and initial CIP concentration on TOC removal in MDEL-PEF process were examined, and the optimal conditions were ascertained. The releases of three inorganic ions (F - , NH 4 + and NO 3 - ) and two carboxylic acids (oxalic and formic acids) were qualified. Seven aromatic intermediates mainly generated from hydroxylation, dealkylation and defluorination of CIP were detected by UPLC-QTOF-MS/MS technology. Therefore, plausible degradation sequences for CIP degradation in MDEL-PEF process including all detected products were proposed. Copyright © 2017 Elsevier B.V. All rights reserved.

Efficient removal of atrazine from aqueous solutions using magnetic Saccharomyces cerevisiae bionanomaterial.

PubMed

Wu, Xin; He, Huijun; Yang, William L; Yu, Jiaping; Yang, Chunping

2018-06-17

A novel bionanomaterial comprising Saccharomyces cerevisiae (S. cerevisiae) and Fe 3 O 4 nanoparticles encapsulated in a sodium alginate-polyvinyl alcohol (SA-PVA) matrix was synthesized for the efficient removal of atrazine from aqueous solutions. The effects of the operating parameters, nitrogen source, and glucose and Fe 3+ contents on atrazine removal were investigated, and the intermediates were detected by gas chromatography-mass spectrometry (GC-MS). In addition, the synthesized Fe 3 O 4 particles were characterized by XRD, EDX, HR-TEM, FTIR, and hysteresis loops, and the bionanomaterial was characterized by SEM. The results showed that the maximum removal efficiency of 100% was achieved at 28 °C, a pH of 7.0, and 150 rpm with an initial atrazine concentration of 2.0 mg L -1 and that the removal efficiency was still higher than 95.53% even when the initial atrazine concentration was 50 mg L -1 . Biodegradation was demonstrated to be the dominant removal mechanism for atrazine because atrazine was consumed as the sole carbon source for S. cerevisiae. The results of GC-MS showed that dechlorination, dealkylation, deamination, isomerization, and mineralization occurred in the process of atrazine degradation, and thus, a new degradation pathway was proposed. These results indicated that this bionanomaterial has great potential for the bioremediation of atrazine-contaminated water.

Identification of BVT.2938 metabolites by LC/MS and LC/MS/MS after in vitro incubations with liver microsomes and hepatocytes.

PubMed

Edlund, Per Olof; Baranczewski, Pawel

2004-03-10

The metabolism of the 5HT2c agonist BVT.2938, 1-(3-[2-[(2-ethoxy-3-pyridinyl)oxy]ethoxy]-2-pyrazinyl)-2(R)-methylpiperazine, was studied in vitro by incubation with rat, monkey and human liver microsomes as well as cryopreserved hepatocytes, followed by liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS analysis on a quadrupole-time of flight mass spectrometer for structural elucidation. Deuterium exchange on column was used to differentiate between hydroxylation and N-oxidation. Liver microsomes were incubated in two different buffer systems with optimum conditions for cytochrome P450 activity or UDP-glucuronosyltransferase activity. The major phase I metabolites of BVT.2938 originated from O-deethylation of the pyridine ring, O-dealkylation of the ethylene bridge, pyrazine ring hydroxylation, hydroxylation of pyridine ring and piperazine ring N-hydroxylation. When a hydrogen carbonate buffer system was supplemented with UDPGA, the piperazine carbamoyl-glucuronide from the parent compound was identified together with several glucuronides of the phase I metabolites. The metabolite pattern in hepatocytes was similar to microsomes except that the sulphate at the N-position of the piperazine ring of BVT.2938 was identified, while the carbamoyl-glucuronide was missing. Excellent correlation was obtained between radioactivity detection and the chemiluminescent nitrogen detector when the nitrogen content of the analytes was taken into account.

Copper-Hydroperoxo Mediated N-Debenzylation Chemistry Mimicking Aspects of Copper Monoxygenases

PubMed Central

Maiti, Debabrata; Narducci Sarjeant, Amy A.; Karlin, Kenneth D.

2008-01-01

Substantial oxidative N-debenzylation reaction along with PhCH=O formation occurs from a hydroperoxo copper(II) complex which has a dibenzylamino substrate (-N(CH2Ph)2 appended as a substituent on one pyridyl group of its tripodal tetradentate TMPA {≡ TPA ≡ tris(2-pyridylmethyl)amine)} ligand framework. During the course of the (LN(CH2Ph)2)CuII(−OOH) reactivity, formation of a substrate and −OOH (an oxygen atom) derived alkoxo CuII(−OR) complex occurs. The observation that the same CuII(−OR) species occurs from CuI/PhIO chemistry suggests the possibility that a copper-oxo (cupryl) reactive intermediate forms during alkoxo species formation, and new ESI-MS data obtained provides some further support for this high-valent intermediate. Net H-atom abstraction chemistry is proposed, based on kinetic isotope effect studies provided here and that previously published for a closely related CuII(−OOH) species incorporating dimethylamine (-N(CH3)2) as the internal substrate (J. Am. Chem. Soc. 2007, 129, 6720-6721); the CuI/PhIO reactivity, with similar isotope effect results, provides further support. The reactivity of these chemical systems closely resembles proposed oxidative N-dealkylation mechanisms effected by the copper-monooxygenases dopamine β-monooxygenase (DβM) or peptidylglycine-α-hydroxylating monooxygenase (PHM). PMID:18783212

Species differences in metabolism of EPZ015666, an oxetane-containing protein arginine methyltransferase-5 (PRMT5) inhibitor.

PubMed

Rioux, Nathalie; Duncan, Kenneth W; Lantz, Ronald J; Miao, Xiusheng; Chan-Penebre, Elayne; Moyer, Mikel P; Munchhof, Michael J; Copeland, Robert A; Chesworth, Richard; Waters, Nigel J

2016-01-01

1. Metabolite profiling and identification studies were conducted to understand the cross-species differences in the metabolic clearance of EPZ015666, a first-in-class protein arginine methyltransferase-5 (PRMT5) inhibitor, with anti-proliferative effects in preclinical models of Mantle Cell Lymphoma. EPZ015666 exhibited low clearance in human, mouse and rat liver microsomes, in part by introduction of a 3-substituted oxetane ring on the molecule. In contrast, a higher clearance was observed in dog liver microsomes (DLM) that translated to a higher in vivo clearance in dog compared with rodent. 2. Structure elucidation via high resolution, accurate mass LC-MS(n) revealed that the prominent metabolites of EPZ015666 were present in hepatocytes from all species, with the highest turnover rate in dogs. M1 and M2 resulted from oxidative oxetane ring scission, whereas M3 resulted from loss of the oxetane ring via an N-dealkylation reaction. 3. The formation of M1 and M2 in DLM was significantly abrogated in the presence of the specific CYP2D inhibitor, quinidine, and to a lesser extent by the CYP3A inhibitor, ketoconazole, corroborating data from human recombinant isozymes. 4. Our data indicate a marked species difference in the metabolism of the PRMT5 inhibitor EPZ015666, with oxetane ring scission the predominant metabolic pathway in dog mediated largely by CYP2D.

Integrated use of biomarkers and bioaccumulation data in Zebra mussel (Dreissena polymorpha) for site-specific quality assessment.

PubMed

Binelli, A; Ricciardi, F; Riva, C; Provini, A

2006-01-01

One of the useful biological tools for environmental management is the measurement of biomarkers whose changes are related to the exposure to chemicals or environmental stress. Since these responses might vary with different contaminants or depending on the pollutant concentration reached in the organism, the support of bioaccumulation data is needed to prevent false conclusions. In this study, several persistent organic pollutants -- 23 polychlorinated biphenyl (PCB) congeners, 11 polycyclic aromatic hydrocarbons (PAHs), six dichlorodiphenyltricholroethane (DDT) relatives, hexachlorobenzene (HCB), chlorpyrifos and its oxidized metabolite -- and some herbicides (lindane and the isomers alpha, beta, delta; terbutilazine; alachlor; metolachlor) were measured in the soft tissues of the freshwater mollusc Zebra mussel (Dreissena polymorpha) from 25 sampling sites in the Italian portions of the sub-alpine great lakes along with the measure of ethoxyresorufin dealkylation (EROD) and acetylcholinesterase (AChE) activity. The linkage between bioaccumulation and biomarker data allowed us to create site-specific environmental quality indexes towards man-made chemicals. This classification highlighted three different degrees of xenobiotic contamination of the Italian sub-alpine great lakes: a high water quality in Lake Lugano with negligible pollutant levels and no effects on enzyme activities, an homogeneous poor quality for Lakes Garda, Iseo and Como, and the presence of some xenobiotic point-sources in Lake Maggiore, whose ecological status could be jeopardized, also due to the heavy DDT contamination revealed since 1996.

Dioxygenases Catalyze O-Demethylation and O,O-Demethylenation with Widespread Roles in Benzylisoquinoline Alkaloid Metabolism in Opium Poppy*

PubMed Central

Farrow, Scott C.; Facchini, Peter J.

2013-01-01

In opium poppy, the antepenultimate and final steps in morphine biosynthesis are catalyzed by the 2-oxoglutarate/Fe(II)-dependent dioxygenases, thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM). Further investigation into the biochemical functions of CODM and T6ODM revealed extensive and unexpected roles for such enzymes in the metabolism of protopine, benzo[c]phenanthridine, and rhoeadine alkaloids. When assayed with a wide range of benzylisoquinoline alkaloids, CODM, T6ODM, and the functionally unassigned paralog DIOX2, renamed protopine O-dealkylase, showed novel and efficient dealkylation activities, including regio- and substrate-specific O-demethylation and O,O-demethylenation. Enzymes catalyzing O,O-demethylenation, which cleave a methylenedioxy bridge leaving two hydroxyl groups, have previously not been reported in plants. Similar cleavage of methylenedioxy bridges on substituted amphetamines is catalyzed by heme-dependent cytochromes P450 in mammals. Preferred substrates for O,O-demethylenation by CODM and protopine O-dealkylase were protopine alkaloids that serve as intermediates in the biosynthesis of benzo[c]phenanthridine and rhoeadine derivatives. Virus-induced gene silencing used to suppress the abundance of CODM and/or T6ODM transcripts indicated a direct physiological role for these enzymes in the metabolism of protopine alkaloids, and they revealed their indirect involvement in the formation of the antimicrobial benzo[c]phenanthridine sanguinarine and certain rhoeadine alkaloids in opium poppy. PMID:23928311

The possible role of hydroxylation in the detoxification of atrazine in mature vetiver (Chrysopogon zizanioides Nash) grown in hydroponics.

PubMed

Marcacci, Sylvie; Raventon, Muriel; Ravanel, Patrick; Schwitzguébel, Jean-Paul

2005-01-01

The resistance mechanism of vetiver (Chrysopogon zizanioides) to atrazine was investigated to evaluate its potential for phytoremediation of environment contaminated with the herbicide. Plants known to metabolise atrazine rely on hydroxylation mediated by benzoxazinones, conjugation catalyzed by glutathione-S-transferases and dealkylation probably mediated by cytochromes P450. All three possibilities were explored in mature vetiver grown in hydroponics during this research project. Here we report on the chemical role of benzoxazinones in the transformation of atrazine. Fresh vetiver roots and leaves were cut to extract and study their content in benzoxazinones known to hydroxylate atrazine, such as 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA), 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) and their mono- and di-glucosylated forms. Identification of benzoxazinones was performed by thin layer chromatography (TLC) and comparison of retention factors (Rf) and UV spectra with standards: although some products exhibited the same Rf as standards, UV spectra were different. Furthermore, in vitro hydroxylation of atrazine could not be detected in the presence of vetiver extracts. Finally, vetiver organs exposed to [14C]-atrazine did not produce any significant amount of hydroxylated products, such as hydroxyatrazine (HATR), hydroxy-deethylatrazine (HDEA), and hydroxy-deisopropylatrazine (HDIA). Altogether, these metabolic features suggest that hydroxylation was not a major metabolic pathway of atrazine in vetiver.

A Mechanism-Based Model for the Prediction of the Metabolic Sites of Steroids Mediated by Cytochrome P450 3A4.

PubMed

Dai, Zi-Ru; Ai, Chun-Zhi; Ge, Guang-Bo; He, Yu-Qi; Wu, Jing-Jing; Wang, Jia-Yue; Man, Hui-Zi; Jia, Yan; Yang, Ling

2015-06-30

Early prediction of xenobiotic metabolism is essential for drug discovery and development. As the most important human drug-metabolizing enzyme, cytochrome P450 3A4 has a large active cavity and metabolizes a broad spectrum of substrates. The poor substrate specificity of CYP3A4 makes it a huge challenge to predict the metabolic site(s) on its substrates. This study aimed to develop a mechanism-based prediction model based on two key parameters, including the binding conformation and the reaction activity of ligands, which could reveal the process of real metabolic reaction(s) and the site(s) of modification. The newly established model was applied to predict the metabolic site(s) of steroids; a class of CYP3A4-preferred substrates. 38 steroids and 12 non-steroids were randomly divided into training and test sets. Two major metabolic reactions, including aliphatic hydroxylation and N-dealkylation, were involved in this study. At least one of the top three predicted metabolic sites was validated by the experimental data. The overall accuracy for the training and test were 82.14% and 86.36%, respectively. In summary, a mechanism-based prediction model was established for the first time, which could be used to predict the metabolic site(s) of CYP3A4 on steroids with high predictive accuracy.

Investigation of chemical modifications of micro- and macromolecules in bio-oil during hydrodeoxygenation with Pd/C catalyst in supercritical ethanol.

PubMed

Oh, Shinyoung; Hwang, Hyewon; Choi, Hang Seok; Choi, Joon Weon

2014-12-01

Miscanthus bio-oil was subjected to hydrodeoxygenation (HDO) with Pd/C at different temperatures (250, 300 and 350°C) and times (30, 45 and 60 min) to investigate the chemical modification of micro- and macromolecules in bio-oil. Four main products - char, gas and two immiscible oils (light and heavy oil) - were obtained from the HDO reaction. Yields of heavy oil as a targeting product of HDO varied from 60% to 13%, whereas those of gas and char were ranged from 7% to 36% and 6% to 17%, respectively. Water content was estimated to<1% and heating value was 26-31 MJ kg(-1). Reduction of unstable oxygen-containing compounds such as acids (2-hydroxy-butanoic acid), aldehydes (furfural), alcohols (butanedial) and sugars (levoglucosan) were characteristic in heavey oil. Apart from hydrogenation and deoxygenation, micromolecules in bio-oil were plausibly modified to stable ketones, esters and saturated components via demethoxylation, dealkylation, decarbonylation, dehydroxylation and ring opening. Macromolecular lignin fragments (referred to as pyrolytic lignins in bio-oil and phenol polymers in heavy oil) were extracted and subjected to several analyses. Approximately 60% of the pyrolytic lignins were decomposed into low molecular weight compounds during HDO reaction. Moreover, essential functional groups, OCH3 and phen-OH groups attached to pyrolytic lignin, were severely modified during HDO reaction. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification of metabolites of vindoline in rats using ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

PubMed

Zhang, Yuqian; Sun, Yupeng; Mu, Xiyan; Yuan, Lin; Wang, Qiao; Zhang, Lantong

2017-08-15

Vindoline (VDL) is an indole alkaloid, possessing hypoglycemic and vasodilator effects, and it is also the prodrug of many vinca alkaloids. In this paper, we analyzed in vivo (including plasma, urine, bile and faeces) and in vitro metabolic profile of VDL in rat with ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). The chromatographic separation was performed on a C 18 column with a mobile phase consisted of 3mM ammonium acetate buffer and acetonitrile at a flow rate of 300μL/min. The mass spectral analysis was conducted in a positive electrospray ionization mode, and on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) were used in the biological samples analysis to trace all the potential metabolites of VDL. Twenty-five metabolites of VDL were detected by comparing with the blank sample, of which there were 2 sulfate conjugates. These data suggested that the biotransformation of VDL was deacetylation, oxidation, deoxidization, methylation, dealkylation and sulfate conjugation. This study provides useful information for further study of the pharmacology and mechanism of VDL, meanwhile, the research method can be widely applied to speculate structural features of the metabolites of other vinca alkaloids. Copyright © 2017 Elsevier B.V. All rights reserved.

Degradation and mineralization of atrazine by a soil bacterial isolate.

PubMed Central

Radosevich, M; Traina, S J; Hao, Y L; Tuovinen, O H

1995-01-01

An atrazine-degrading bacterial culture was isolated from an agricultural soil previously impacted by herbicide spills. The organism was capable of using atrazine under aerobic conditions as the sole source of C and N. Cyanuric acid could replace atrazine as the sole source of N, indicating that the organism was capable of ring cleavage. Ring cleavage was confirmed in 14CO2 evolution experiments with [U-14C-ring]atrazine. Between 40 and 50% of ring-14C was mineralized to 14CO2. [14C]biuret and [14C]urea were detected in spent culture media. Cellular assimilation of 14C was negligible, in keeping with the fully oxidized valence of the ring carbon. Chloride release was stoichiometric. The formation of ammonium during atrazine degradation was below the stoichiometric amount, suggesting a deficit due to cellular assimilation and metabolite-N accumulation. With excess glucose and with atrazine as the sole N source, free ammonium was not detected, suggesting assimilation into biomass. The organism degraded atrazine anaerobically in media which contained (i) atrazine only, (ii) atrazine and glucose, and (iii) atrazine, glucose, and nitrate. To date, this is the first report of a pure bacterial isolate with the ability to cleave the s-triazine ring structure of atrazine. It was also concluded that this bacterium was capable of dealkylation, dechlorination, and deamination in addition to ring cleavage. PMID:7887609

Tributyltin (TBT) and dibutyltin (DBT) differently inhibit the mitochondrial Mg-ATPase activity in mussel digestive gland.

PubMed

Nesci, Salvatore; Ventrella, Vittoria; Trombetti, Fabiana; Pirini, Maurizio; Borgatti, Anna Rosa; Pagliarani, Alessandra

2011-02-01

Tri-n-butyltin (TBT) has long been considered as the most toxic among organotins, especially to membrane systems. The partially dealkylated derivative di-n-butyltin (DBT) has up to now received poor attention and, whenever considered, shown to be less toxic than TBT except on the immune system. The present kinetic approach evidences that both TBT and DBT in vitro inhibit the Mg-ATPase in mussel digestive gland mitochondria by a different mechanism. DBT even displays a higher efficiency than TBT (IC(50)=0.32 μM for TBT vs. 0.19 μM for DBT) in inhibiting the enzyme hydrolytic activity. Differently from TBT which at high concentrations (>1 μM) apparently decreases the oligomycin-sensitivity of the Mg-ATPase, DBT at any concentration tested does not affect the oligomycin sensitivity. TBT probably binds to F(0), either in the form of free enzyme or of enzyme-substrate complex (Ki=K'i), acting as non-competitive inhibitor with respect to the ATP substrate. Conversely DBT, which acts as uncompetitive inhibitor of ATP and as competitive inhibitor of Mg(2+) cofactor, may bind strongly to F(1) subunit, thus preventing ATP hydrolysis. The Mg-ATPase inhibition by both organotins warns against a potential threat to crucial cell energy metabolism processes even after years from contamination and partial TBT debutylation. Copyright © 2010 Elsevier Ltd. All rights reserved.

Toxicology of tetramethyltin and other organometals used in photovoltaic cell manufacture

NASA Astrophysics Data System (ADS)

Hamilton, L. D.; Medeiros, W. H.; Moskowitz, P. D.; Rybicka, K.

1988-07-01

In photovoltaic cell fabrication, organometals (alkyl metals) may be used in such processes as metalorganic chemical vapor deposition, transparent contact oxide deposition, doping, and ion implantation. Although these compounds offer potential performance advantages over earth metals and possibly greater safety in handling than metal hydrides, they are not without risk to health and property. Most organometals can ignite spontaneously in air. Some also react violently with water. Oxidation by-products from these reactions are hazardous to health. Of the organometals used in photovoltaic cell fabrication, only the toxicology of organotins (triethyl-, trimethyl- and tetramethyltin) was studied extensively. In mammalian systems, tetramethyltin is rapidly dealkylated to trimethyltin. Although tin was classified by some investigators as an essential trace element, the effects of organotin compounds on humans are poorly known. Animal studies show that the most prominent effects of trimethyltin are on the central nervous system. Several observations of poisoning were reported; effects ranged from reversible neurologic disorders to death. Limited available data suggest that humans respond to single acute doses and more alarmingly to repeated sub-toxic doses, suggesting a cumulative effect. Toxicologic properties of diethyltelluride also were evaluated in animal experiments. The compound had toxic effects on the blood, liver, kidney, heart, and skin. Based on these studies and others of related compounds (e.g., methylmercury, tributyltin) extreme caution should be exercised in using organometal compounds in photovoltaic cell manufacturing.

Dioxygenases catalyze O-demethylation and O,O-demethylenation with widespread roles in benzylisoquinoline alkaloid metabolism in opium poppy.

PubMed

Farrow, Scott C; Facchini, Peter J

2013-10-04

In opium poppy, the antepenultimate and final steps in morphine biosynthesis are catalyzed by the 2-oxoglutarate/Fe(II)-dependent dioxygenases, thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM). Further investigation into the biochemical functions of CODM and T6ODM revealed extensive and unexpected roles for such enzymes in the metabolism of protopine, benzo[c]phenanthridine, and rhoeadine alkaloids. When assayed with a wide range of benzylisoquinoline alkaloids, CODM, T6ODM, and the functionally unassigned paralog DIOX2, renamed protopine O-dealkylase, showed novel and efficient dealkylation activities, including regio- and substrate-specific O-demethylation and O,O-demethylenation. Enzymes catalyzing O,O-demethylenation, which cleave a methylenedioxy bridge leaving two hydroxyl groups, have previously not been reported in plants. Similar cleavage of methylenedioxy bridges on substituted amphetamines is catalyzed by heme-dependent cytochromes P450 in mammals. Preferred substrates for O,O-demethylenation by CODM and protopine O-dealkylase were protopine alkaloids that serve as intermediates in the biosynthesis of benzo[c]phenanthridine and rhoeadine derivatives. Virus-induced gene silencing used to suppress the abundance of CODM and/or T6ODM transcripts indicated a direct physiological role for these enzymes in the metabolism of protopine alkaloids, and they revealed their indirect involvement in the formation of the antimicrobial benzo[c]phenanthridine sanguinarine and certain rhoeadine alkaloids in opium poppy.

Selection of electrogenic bacteria for microbial fuel cell in removing Victoria blue R from wastewater.

PubMed

Chen, Chih-Yu; Tsai, Teh-Hua; Wu, Pei-Ssu; Tsao, Shuo-En; Huang, Yu-Shan; Chung, Ying-Chien

2018-01-28

This study was conducted to select electrogenic bacteria from wastewater sludge. Phylogenetic analysis revealed that Proteobacteria was the dominant phylum in the microbial fuel cell (MFC) during the decomposition process of organic pollutants. Five culturable bacteria strains - namely, Bacillus subtilis, Flavobacterium sp., Aeromonas hydrophila, Citrobacter freundii, and Stenotrophomonas sp. - have a double potential in dye removal and electricity generation. We inoculated the mixed electrogenic bacteria at a specific ratio and treated them with a triphenylmethane dye, Victoria blue R (VBR), to evaluate their electricity generation ability for the artificial and real wastewater. The results of the VBR shock-loading experiment indicated that the inoculated MFC could adapt to shock loading in 1-2 days and exhibited high removal efficiency (95-100%) for 100-800 mg L -1 VBR with a power density of 8.62 ± 0.10 to 34.81 ± 0.25 mW m -2 . The selected electrogenic bacteria in the MFC could use VBR as only electron donor for power generation. The matrix effects of the real wastewater on VBR removal and electricity generation of MFC were insignificant. VBR degradation by the electrogenic bacteria involves a stepwise demethylation process to yield partially dealkylated VBR species. In addition, these results demonstrate the feasibility of inoculating culturable bacteria strains to develop an efficient MFC for purifying wastewater.

RS-predictor: a new tool for predicting sites of cytochrome P450-mediated metabolism applied to CYP 3A4.

PubMed

Zaretzki, Jed; Bergeron, Charles; Rydberg, Patrik; Huang, Tao-wei; Bennett, Kristin P; Breneman, Curt M

2011-07-25

This article describes RegioSelectivity-Predictor (RS-Predictor), a new in silico method for generating predictive models of P450-mediated metabolism for drug-like compounds. Within this method, potential sites of metabolism (SOMs) are represented as "metabolophores": A concept that describes the hierarchical combination of topological and quantum chemical descriptors needed to represent the reactivity of potential metabolic reaction sites. RS-Predictor modeling involves the use of metabolophore descriptors together with multiple-instance ranking (MIRank) to generate an optimized descriptor weight vector that encodes regioselectivity trends across all cases in a training set. The resulting pathway-independent (O-dealkylation vs N-oxidation vs Csp(3) hydroxylation, etc.), isozyme-specific regioselectivity model may be used to predict potential metabolic liabilities. In the present work, cross-validated RS-Predictor models were generated for a set of 394 substrates of CYP 3A4 as a proof-of-principle for the method. Rank aggregation was then employed to merge independently generated predictions for each substrate into a single consensus prediction. The resulting consensus RS-Predictor models were shown to reliably identify at least one observed site of metabolism in the top two rank-positions on 78% of the substrates. Comparisons between RS-Predictor and previously described regioselectivity prediction methods reveal new insights into how in silico metabolite prediction methods should be compared.

Metabolic profiles of neratinib in rat by using ultra-high-performance liquid chromatography coupled with diode array detector and Q-Exactive Orbitrap tandem mass spectrometry.

PubMed

Liu, Wen; Li, Sha; Wu, Yangke; Yan, Xiao; Zhu, Y-M; Huang, J-H; Chen, Zhuo

2018-05-03

Neratinib is a tyrosine kinase inhibitor that has been approved by the US Food and Drug Administration for the treatment of breast cancer. However, its metabolism remains unknown. This study was carried out to investigate the in vitro and in vivo metabolism of neratinib using an UHPLC-DAD-Q Exactive Orbitrap-MS instrument with dd-MS 2 on-line data acquisition mode. The post-acquisition data was processed using MetWorks software. Under the current conditions, a total of 12 metabolites were detected and structurally identified based on their accurate masses, fragment ions and chromatographic retention times. Among these metabolites, M3, M10 and M12 were unambiguously identified using chemically synthesized reference standards. M6 and M7 (GSH conjugates) were the major metabolites. The metabolic pathways of neratinib were proposed accordingly. Our findings suggested that neratinib was mainly metabolized via O-dealkylation (M3), oxygenation (M8), N-demethylation (M10), N-oxygenation (M12), GSH conjugation (M1, M2, M4, M5, M6 and M7) and N-acetylcysteine conjugation (M9 and M11). The α,β-unsaturated ketone was the major metabolic site and GSH conjugation was the predominant metabolic pathway. In conclusion, this study provided valuable metabolic data and would benefit the assessment of the contributions to the overall activity or toxicity from the key metabolites. Copyright © 2018 John Wiley & Sons, Ltd.

Sex difference in the principal cytochrome P-450 for tributyltin metabolism in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohhira, Shuji; Enomoto, Mitsunori; Matsui, Hisao

Tributyltin is metabolized by cytochrome P-450 (CYP) system enzymes, and its metabolic fate may contribute to the toxicity of the chemical. In the present study, it is examined whether sex differences in the metabolism of tributyltin exist in rats. In addition, the in vivo and in vitro metabolism of tributyltin was investigated using rat hepatic CYP systems to confirm the principal CYP involved. A significant sex difference in metabolism occurred both in vivo and in vitro, suggesting that one of the CYPs responsible for tributyltin metabolism in rats is male specific or predominant at least. Eight cDNA-expressed rat CYPs, includingmore » typical phenobarbital (PB)-inducible forms and members of the CYP2C subfamily, were tested to determine their capability for tributyltin metabolism. Among the enzymes studied, a statistically significant dealkylation of tributyltin was mediated by CYP2C6 and 2C11. Furthermore, the sex difference in metabolism disappeared in vitro after anti-rat CYP2C11 antibody pretreatment because CYP2C11 is a major male-specific form in rats. These results indicate that CYP2C6 is the principal CYP for tributyltin metabolism in female rats, whereas CYP2C11 as well as 2C6 is involved in tributyltin metabolism in male rats, and it is suggested that CYP2C11 is responsible for the significant sex difference in the metabolism of tributyltin observed in rats.« less

Chronic methylmercurialism in a horse

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seawright, A.A.; Roberts, M.C.; Costigan, P.

1978-02-01

Chronic methylmercurialism was produced in a horse given 10 g methylmercury chloride over 10 weeks. Neurological signs, particularly proprioceptive disturbances, were apparent by the final week of dosing and became more severe thereafter. An exudative dermatitis, a reluctance to move, weight loss, reduced appetite and dullness were among the earlier clinical signs, and renal changes characterized by a steadily increasing BUN and glucosuria were detected later. Pathological lesions were confined to the kidneys and the nervous system. There was mild neuronal degeneration in the cerebral cortex and in the cerebellar cortex, axonal demyelination in the dorsal columns of the spinalmore » cord and extensive degeneration of ganglion cells in the dorsal root ganglia. The blood organic mercury level, which had plateaued in the second month, increased rapidly in the last weeks of dosing with a sharp rise terminally. This pattern was repeated for the much lower inorganic mercury levels except for a terminal decrease. The proportion of inorganic mercury was five times greater in the dorsal root ganglia than elsewhere in the CNS, although total mercury levels were similar. Highest tissue mercury levels were found in the liver and kidneys, over 50% being in the form of inorganic mercury. As dealkylation of the methylmercury appeared to be more efficient in the dorsal root ganglia and the kidneys, inorganic mercury derived therefrom may have been responsible for some of the clinical and pathological features of this intoxication in the horse. 21 references, 6 figures, 2 tables.« less

Coordination chemistry controls the thiol oxidase activity of the B12-trafficking protein CblC

PubMed Central

Li, Zhu; Shanmuganathan, Aranganathan; Ruetz, Markus; Yamada, Kazuhiro; Lesniak, Nicholas A.; Kräutler, Bernhard; Brunold, Thomas C.; Koutmos, Markos; Banerjee, Ruma

2017-01-01

The cobalamin or B12 cofactor supports sulfur and one-carbon metabolism and the catabolism of odd-chain fatty acids, branched-chain amino acids, and cholesterol. CblC is a B12-processing enzyme involved in an early cytoplasmic step in the cofactor-trafficking pathway. It catalyzes the glutathione (GSH)-dependent dealkylation of alkylcobalamins and the reductive decyanation of cyanocobalamin. CblC from Caenorhabditis elegans (ceCblC) also exhibits a robust thiol oxidase activity, converting reduced GSH to oxidized GSSG with concomitant scrubbing of ambient dissolved O2. The mechanism of thiol oxidation catalyzed by ceCblC is not known. In this study, we demonstrate that novel coordination chemistry accessible to ceCblC-bound cobalamin supports its thiol oxidase activity via a glutathionyl-cobalamin intermediate. Deglutathionylation of glutathionyl-cobalamin by a second molecule of GSH yields GSSG. The crystal structure of ceCblC provides insights into how architectural differences at the α- and β-faces of cobalamin promote the thiol oxidase activity of ceCblC but mute it in wild-type human CblC. The R161G and R161Q mutations in human CblC unmask its latent thiol oxidase activity and are correlated with increased cellular oxidative stress disease. In summary, we have uncovered key architectural features in the cobalamin-binding pocket that support unusual cob(II)alamin coordination chemistry and enable the thiol oxidase activity of ceCblC. PMID:28442570

Plant Expression of a Bacterial Cytochrome P450 That Catalyzes Activation of a Sulfonylurea Pro-Herbicide.

PubMed Central

O'Keefe, D. P.; Tepperman, J. M.; Dean, C.; Leto, K. J.; Erbes, D. L.; Odell, J. T.

1994-01-01

The Streptomyces griseolus gene encoding herbicide-metabolizing cytochrome P450SU1 (CYP105A1) was expressed in transgenic tobacco (Nicotiana tabacum). Because this P450 can be reduced by plant chloroplast ferredoxin in vitro, chloroplast-targeted and nontargeted expression were compared. Whereas P450SU1 antigen was found in the transgenic plants regardless of the targeting, only those with chloroplast-directed enzyme performed P450SU1-mediated N-dealkylation of the sulfonylurea 2-methylethyl-2,3-dihydro-N-[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]-1, 2-benzoisothiazole- 7-sulfonamide-1,1-dioxide (R7402). Chloroplast targeting appears to be essential for the bacterial P450 to function in the plant. Because the R7402 metabolite has greater phytotoxicity than R7402 itself, plants bearing active P450SU1 are susceptible to injury from R7402 treatment that is harmless to plants without P450SU1. Thus, P450SU1 expression and R7402 treatment can be used as a negative selection system in plants. Furthermore, expression of P450SU1 from a tissue-specific promoter can sequester production of the phytotoxic R7402 metabolite to a single plant tissue. In tobacco expressing P450SU1 from a tapetum-specific promoter, treatment of immature flower buds with R7402 caused dramatically lowered pollen viability. Such treatment could be the basis for a chemical hybridizing agent. PMID:12232216

Capture of the volatile carbonyl metabolite of flecainide on 2,4-dinitrophenylhydrazine cartridge for quantitation by stable-isotope dilution mass spectrometry coupled with chromatography

PubMed Central

Prokai, Laszlo; Szarka, Szabolcs; Wang, Xiaoli; Prokai-Tatrai, Katalin

2012-01-01

Carbonyl compounds are common byproducts of many metabolic processes. These volatile chemical entities are usually derivatized before mass spectrometric analysis to enhance the sensitivity of their detections. The classically used reagent for this purpose is 2,4-dinitrophenylhydrazine (DNPH) that forms the corresponding hydrazones. When DNPH is immobilized on specific cartridges it permits solvent-free collection and simultaneous derivatization of aldehydes and ketones from gaseous samples. The utility of this approach was tested by assembling a simple apparatus for the in vitro generation of trifluoroacetaldehyde (TFAA) and its subsequent capture on the attached DNPH cartridge. TFAA was generated via cytochrome P450-catalyzed dealkylation of flecainide, an antiarrhythmic agent, in pooled human liver microsomes. Stable-isotope dilution mass spectrometry coupled with GC and LC using negative chemical ionization (NCI) and electrospray ionization (ESI) was evaluated for quantitative analyses. To eliminate isotope effects observed with the use of deuterium-labeled DNPH, we selected its 15N4-labeled analog to synthesize the appropriate TFAA adduct, as internal standard. Quantitation by GC–NCI-MS using selected-ion monitoring outperformed LC–ESI-MS methods considering limits of detection and linearity of the assays. The microsomal metabolism of 1.5 μmol of flecainide for 1.5 h resulted in 2.6 ± 0.5 μg TFAA-DNPH, corresponding to 9.3 ± 1.7 nmol TFAA, captured by the cartridge. PMID:22342210

Capture of the volatile carbonyl metabolite of flecainide on 2,4-dinitrophenylhydrazine cartridge for quantitation by stable-isotope dilution mass spectrometry coupled with chromatography.

PubMed

Prokai, Laszlo; Szarka, Szabolcs; Wang, Xiaoli; Prokai-Tatrai, Katalin

2012-04-06

Carbonyl compounds are common byproducts of many metabolic processes. These volatile chemicals are usually derivatized before mass spectrometric analysis to enhance the sensitivity of their detections. The classically used reagent for this purpose is 2,4-dinitrophenylhydrazine (DNPH) that forms the corresponding hydrazones. When DNPH is immobilized on specific cartridges it permits solvent-free collection and simultaneous derivatization of aldehydes and ketones from gaseous samples. The utility of this approach was tested by assembling a simple apparatus for the in vitro generation of trifluoroacetaldehyde (TFAA) and its subsequent capture on the attached DNPH cartridge. TFAA was generated via cytochrome P450-catalyzed dealkylation of flecainide, an antiarrhythmic agent, in pooled human liver microsomes. Stable-isotope dilution mass spectrometry coupled with GC and LC using negative chemical ionization (NCI) and electrospray ionization (ESI) was evaluated for quantitative analyses. To eliminate isotope effects observed with the use of deuterium-labeled DNPH, we selected its (15)N(4)-labeled analog to synthesize the appropriate TFAA adduct, as internal standard. Quantitation by GC-NCI-MS using selected-ion monitoring outperformed LC-ESI-MS methods considering limits of detection and linearity of the assays. The microsomal metabolism of 1.5 μmol of flecainide for 1.5h resulted in 2.6 ± 0.5 μg TFAA-DNPH, corresponding to 9.3 ± 1.7 nmol TFAA, captured by the cartridge. Copyright © 2012 Elsevier B.V. All rights reserved.

Phosphonic acid: preparation and applications

PubMed Central

Sevrain, Charlotte M; Berchel, Mathieu; Couthon, Hélène

2017-01-01

The phosphonic acid functional group, which is characterized by a phosphorus atom bonded to three oxygen atoms (two hydroxy groups and one P=O double bond) and one carbon atom, is employed for many applications due to its structural analogy with the phosphate moiety or to its coordination or supramolecular properties. Phosphonic acids were used for their bioactive properties (drug, pro-drug), for bone targeting, for the design of supramolecular or hybrid materials, for the functionalization of surfaces, for analytical purposes, for medical imaging or as phosphoantigen. These applications are covering a large panel of research fields including chemistry, biology and physics thus making the synthesis of phosphonic acids a determinant question for numerous research projects. This review gives, first, an overview of the different fields of application of phosphonic acids that are illustrated with studies mainly selected over the last 20 years. Further, this review reports the different methods that can be used for the synthesis of phosphonic acids from dialkyl or diaryl phosphonate, from dichlorophosphine or dichlorophosphine oxide, from phosphonodiamide, or by oxidation of phosphinic acid. Direct methods that make use of phosphorous acid (H3PO3) and that produce a phosphonic acid functional group simultaneously to the formation of the P–C bond, are also surveyed. Among all these methods, the dealkylation of dialkyl phosphonates under either acidic conditions (HCl) or using the McKenna procedure (a two-step reaction that makes use of bromotrimethylsilane followed by methanolysis) constitute the best methods to prepare phosphonic acids. PMID:29114326

Toxicokinetics of new psychoactive substances: plasma protein binding, metabolic stability, and human phase I metabolism of the synthetic cannabinoid WIN 55,212-2 studied using in vitro tools and LC-HR-MS/MS.

PubMed

Mardal, Marie; Gracia-Lor, Emma; Leibnitz, Svenja; Castiglioni, Sara; Meyer, Markus R

2016-10-01

The new psychoactive substance WIN 55,212-2 ((R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone) is a potent synthetic cannabinoid receptor agonist. The metabolism of WIN 55,212-2 in man has never been reported. Therefore, the aim of this study was to identify the human in vitro metabolites of WIN 55,212-2 using pooled human liver microsomes and liquid chromatography-high resolution-tandem mass spectrometry (LC-HR-MS/MS) to provide targets for toxicological, doping, and environmental screening procedures. Moreover, a metabolic stability study in pooled human liver microsomes (pHLM) was carried out. In total, 19 metabolites were identified and the following partly overlapping metabolic steps were deduced: degradation of the morpholine ring via hydroxylation, N- and O-dealkylation, and oxidative deamination, hydroxylations on either the naphthalene or morpholine ring or the alkyl spacer with subsequent oxidation, epoxide formation with subsequent hydrolysis, or combinations. In conclusion, WIN 55,212-2 was extensively metabolized in human liver microsomes incubations and the calculated hepatic clearance was comparably high, indicating a fast and nearly complete metabolism in vivo. This is in line with previous findings on other synthetic cannabinoids. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Phosphonic acid: preparation and applications.

PubMed

Sevrain, Charlotte M; Berchel, Mathieu; Couthon, Hélène; Jaffrès, Paul-Alain

2017-01-01

The phosphonic acid functional group, which is characterized by a phosphorus atom bonded to three oxygen atoms (two hydroxy groups and one P=O double bond) and one carbon atom, is employed for many applications due to its structural analogy with the phosphate moiety or to its coordination or supramolecular properties. Phosphonic acids were used for their bioactive properties (drug, pro-drug), for bone targeting, for the design of supramolecular or hybrid materials, for the functionalization of surfaces, for analytical purposes, for medical imaging or as phosphoantigen. These applications are covering a large panel of research fields including chemistry, biology and physics thus making the synthesis of phosphonic acids a determinant question for numerous research projects. This review gives, first, an overview of the different fields of application of phosphonic acids that are illustrated with studies mainly selected over the last 20 years. Further, this review reports the different methods that can be used for the synthesis of phosphonic acids from dialkyl or diaryl phosphonate, from dichlorophosphine or dichlorophosphine oxide, from phosphonodiamide, or by oxidation of phosphinic acid. Direct methods that make use of phosphorous acid (H 3 PO 3 ) and that produce a phosphonic acid functional group simultaneously to the formation of the P-C bond, are also surveyed. Among all these methods, the dealkylation of dialkyl phosphonates under either acidic conditions (HCl) or using the McKenna procedure (a two-step reaction that makes use of bromotrimethylsilane followed by methanolysis) constitute the best methods to prepare phosphonic acids.

Cunninghamella Biotransformation--Similarities to Human Drug Metabolism and Its Relevance for the Drug Discovery Process.

PubMed

Piska, Kamil; Żelaszczyk, Dorota; Jamrozik, Marek; Kubowicz-Kwaśny, Paulina; Pękala, Elżbieta

2016-01-01

Studies of drug metabolism are one of the most significant issues in the process of drug development, its introduction to the market and also in treatment. Even the most promising molecule may show undesirable metabolic properties that would disqualify it as a potential drug. Therefore, such studies are conducted in the early phases of drug discovery and development process. Cunninghamella is a filamentous fungus known for its catalytic properties, which mimics mammalian drug metabolism. It has been proven that C. elegans carries at least one gene coding for a CYP enzyme closely related to the CYP51 family. The transformation profile of xenobiotics in Cunninghamella spp. spans a number of reactions catalyzed by different mammalian CYP isoforms. This paper presents detailed data on similar biotransformation drug products in humans and Cunninghamella spp. and covers the most important aspects of preparative biosynthesis of metabolites, since this model allows to obtain metabolites in sufficient quantities to conduct the further detailed investigations, as quantification, structure analysis and pharmacological activity and toxicity testing. The metabolic activity of three mostly used Cunninghamella species in obtaining hydroxylated, dealkylated and oxidated metabolites of different drugs confirmed its convergence with human biotransformation. Though it cannot replace the standard methods, it can provide support in the field of biotransformation and identifying metabolic soft spots of new chemicals and in predicting possible metabolic pathways. Another aspect is the biosynthesis of metabolites. In this respect, techniques using Cunninghamella spp. seem to be competitive to the chemical methods currently used.

In vitro metabolism of a novel synthetic cannabinoid, EAM-2201, in human liver microsomes and human recombinant cytochrome P450s.

PubMed

Kim, Ju Hyun; Kim, Hee Seung; Kong, Tae Yeon; Lee, Joo Young; Kim, Jin Young; In, Moon Kyo; Lee, Hye Suk

2016-02-05

In vitro metabolism of a new synthetic cannabinoid, EAM-2201, has been investigated with human liver microsomes and major cDNA-expressed cytochrome P450 (CYP) isozymes using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Incubation of EAM-2201 with human liver microsomes in the presence of NADPH resulted in the formation of 37 metabolites, including nine hydroxy-EAM-2201 (M1-M9), five dihydroxy-EAM-2201 (M10-M14), dihydrodiol-EAM-2201 (M15), oxidative defluorinated EAM-2201 (M16), two hydroxy-M16 (M17 and M18), three dihydroxy-M16 (M19-M21), N-dealkyl-EAM-2201 (M22), two hydroxy-M22 (M23 and M24), dihydroxy-M22 (M25), EAM-2201 N-pentanoic acid (M26), hydroxy-M26 (M27), dehydro-EAM-2201 (M28), hydroxy-M28 (M29), seven dihydroxy-M28 (M30-M36), and oxidative defluorinated hydroxy-M28 (M37). Multiple CYPs, including CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2J2, 3A4, and 3A5, were involved in the metabolism of EAM-2201. In conclusion, EAM-2201 is extensively metabolized by CYPs and its metabolites can be used as an indicator of EAM-2201 abuse. Copyright © 2015 Elsevier B.V. All rights reserved.

Metabolites of 5F-AKB-48, a synthetic cannabinoid receptor agonist, identified in human urine and liver microsomal preparations using liquid chromatography high-resolution mass spectrometry.

PubMed

Holm, Niels Bjerre; Pedersen, Anders Just; Dalsgaard, Petur Weihe; Linnet, Kristian

2015-03-01

New types of synthetic cannabinoid designer drugs are constantly introduced to the illicit drug market to circumvent legislation. Recently, N-​(1-Adamant​yl)-​1-​(5-​fluoropentyl)-​1H-​indazole-​3-​carboxamide (5F-AKB-48), also known as 5F-APINACA, was identified as an adulterant in herbal products. This compound deviates from earlier JHW-type synthetic cannabinoids by having an indazole ring connected to an adamantyl group via a carboxamide linkage. Synthetic cannabinoids are completely metabolized, and identification of the metabolites is thus crucial when using urine as the sample matrix. Using an authentic urine sample and high-resolution accurate-mass Fourier transform Orbitrap mass spectrometry, we identified 16 phase-I metabolites of 5F-AKB-48. The modifications included mono-, di-, and trihydroxylation on the adamantyl ring alone or in combination with hydroxylation on the N-fluoropentylindazole moiety, dealkylation of the N-fluoropentyl side chain, and oxidative loss of fluorine as well as combinations thereof. The results were compared to human liver microsomal (HLM) incubations, which predominantly showed time-dependent formation of mono-, di-, and trihydroxylated metabolites having the hydroxyl groups on the adamantyl ring. The results presented here may be used to select metabolites specific of 5F-AKB-48 for use in clinical and forensic screening. Copyright © 2014 John Wiley & Sons, Ltd.

Thermal hydrocracking of coal derived liquid from mild gasification (production of phenols, BTX and naphthalenes)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sato, Yoshiki; Kodera, Yoichi; Kamo, Tohru

1997-12-31

Coal derived liquid from mild gasification contains more than 40% of alkylphenols with alkylnaphthalenes and a small amount of benzofuran, naphthol and condensed aromatic derivatives. In this study, thermal hydrocracking of the coal derived liquid, and related model compounds have been investigated using a small atmospheric flow apparatus at temperatures of 700--770 C with residence time of 3--10 sec, and hydrogen-to-reactant molar ratio of about 3--10. In the experiments using m-cresol and 2,5-dimethylphenol, dealkylation and dehydroxylation proceeded concurrently with high selectivity in the presence of excess hydrogen. The rates of hydrocracking of m-cresol and 2,5-dimethylphenol were in accordance with first-ordermore » rate law with respect to reactant. This indicates that the same kinetic equation for the demethylation of alkylbenzenes can be applied. Rates of demethylation and dehydroxylation for cresols and dimethylphenols have been measured at 700 C by using toluene as an internal reference, and discussed in terms of chemical structure. Thermal hydrocracking of the coal derived liquid produced 20--40 wt% gases and 60--80 wt% liquids. Gaseous products mainly consisted of carbon monoxide and methane with small amounts of C{sub 2} and C{sub 3} hydrocarbon gases. Liquid products, obtained at 770 C, contained 24 wt% of BTX, 40 wt% of phenol and cresols, and 12 wt% of naphthalene and methylnaphthalenes. Yield of useful chemicals, having simple aromatic structure in the liquid product increased with temperature and residence time.« less

Biodegradation of the X-ray contrast agent iopromide and the fluoroquinolone antibiotic ofloxacin by the white rot fungus Trametes versicolor in hospital wastewaters and identification of degradation products.

PubMed

Gros, Meritxell; Cruz-Morato, Carles; Marco-Urrea, Ernest; Longrée, Philipp; Singer, Heinz; Sarrà, Montserrat; Hollender, Juliane; Vicent, Teresa; Rodriguez-Mozaz, Sara; Barceló, Damià

2014-09-01

This paper describes the degradation of the X-ray contrast agent iopromide (IOP) and the antibiotic ofloxacin (OFLOX) by the white-rot-fungus Trametes versicolor. Batch studies in synthetic medium revealed that between 60 and 80% of IOP and OFLOX were removed when spiked at approximately 12 mg L(-1) and 10 mg L(-1), respectively. A significant number of transformation products (TPs) were identified for both pharmaceuticals, confirming their degradation. IOP TPs were attributed to two principal reactions: (i) sequential deiodination of the aromatic ring and (ii) N-dealkylation of the amide at the hydroxylated side chain of the molecule. On the other hand, OFLOX transformation products were attributed mainly to the oxidation, hydroxylation and cleavage of the piperazine ring. Experiments in 10 L-bioreactor with fungal biomass fluidized by air pulses operated in batch achieved high percentage of degradation of IOP and OFLOX when load with sterile (87% IOP, 98.5% OFLOX) and unsterile (65.4% IOP, 99% OFLOX) hospital wastewater (HWW) at their real concentration (μg L(-1) level). Some of the most relevant IOP and OFLOX TPs identified in synthetic medium were also detected in bioreactor samples. Acute toxicity tests indicated a reduction of the toxicity in the final culture broth from both experiments in synthetic medium and in batch bioreactor. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification of AB-FUBINACA metabolites in authentic urine samples suitable as urinary markers of drug intake using liquid chromatography quadrupole tandem time of flight mass spectrometry.

PubMed

Vikingsson, Svante; Gréen, Henrik; Brinkhagen, Linda; Mukhtar, Shahzabe; Josefsson, Martin

2016-09-01

Synthetic cannabinoids are a group of psychoactive drugs presently widespread among drug users in Europe. Analytical methods to measure these compounds in urine are in demand as urine is a preferred matrix for drug testing. For most synthetic cannabinoids, the parent compounds are rarely detected in urine. Therefore urinary metabolites are needed as markers of drug intake. AB-FUBINACA was one of the top three synthetic cannabinoids most frequently found in seizures and toxicological drug screening in Sweden (2013-2014). Drug abuse is also reported from several other countries such as the USA and Japan. In this study, 28 authentic case samples were used to identify urinary markers of AB-FUBINACA intake using liquid chromatography quadrupole tandem time of flight mass spectrometry and human liver microsomes. Three metabolites suitable as markers of drug intake were identified and at least two of them were detected in all but one case. In total, 15 urinary metabolites of AB-FUBINACA were reported, including hydrolxylations on the indazole ring and the amino-oxobutane moiety, dealkylations and hydrolysis of the primary amide. No modifications on the fluorobenzyl side-chain were observed. The parent compound was detected in 54% of the case samples. Also, after three hours of incubation with human liver microsomes, 77% of the signal from the parent compound remained. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Metabolite profiling of bendamustine in urine of cancer patients after administration of [14C]bendamustine.

PubMed

Dubbelman, Anne-Charlotte; Jansen, Robert S; Rosing, Hilde; Darwish, Mona; Hellriegel, Edward; Robertson, Philmore; Schellens, Jan H M; Beijnen, Jos H

2012-07-01

Bendamustine is an alkylating agent consisting of a mechlorethamine derivative, a benzimidazole group, and a butyric acid substituent. A human mass balance study showed that bendamustine is extensively metabolized and subsequently excreted in urine. However, limited information is available on the metabolite profile of bendamustine in human urine. The objective of this study was to elucidate the metabolic pathways of bendamustine in humans by identification of its metabolites excreted in urine. Human urine samples were collected up to 168 h after an intravenous infusion of 120 mg/m(2) (80-95 μCi) [(14)C]bendamustine. Metabolites of [(14)C]bendamustine were identified using liquid chromatography (high-resolution)-tandem mass spectrometry with off-line radioactivity detection. Bendamustine and a total of 25 bendamustine-related compounds were detected. Observed metabolic conversions at the benzimidazole and butyric acid moiety were N-demethylation and γ-hydroxylation. In addition, various other combinations of these conversions with modifications at the mechlorethamine moiety were observed, including hydrolysis (the primary metabolic pathway), cysteine conjugation, and subsequent biotransformation to mercapturic acid and thiol derivatives, N-dealkylation, oxidation, and conjugation with phosphate, creatinine, and uric acid. Bendamustine-derived products containing phosphate, creatinine, and uric acid conjugates were also detected in control urine incubated with bendamustine. Metabolites that were excreted up to 168 h after the infusion included products of dihydrolysis and cysteine conjugation of bendamustine and γ-hydroxybendamustine. The range of metabolic reactions is generally consistent with those reported for rat urine and bile, suggesting that the overall processes involved in metabolic elimination are qualitatively the same in rats and humans.

Metabolism of bepridil in laboratory animals and humans.

PubMed

Wu, W N; Hills, J F; Chang, S Y; Ng, K T

1988-01-01

The metabolism of bepridil was studied in the Swiss mouse, Sprague-Dawley rat, New Zealand rabbit, rhesus monkey, and healthy human. After oral administration of bepridil-14C-hydrochloride, recoveries of total radioactivity in urine and feces (7 days) were greater than or equal to 80% of the administered dose in all five species. Bepridil and 25 metabolites have been isolated by HPLC and TLC from representative plasma, urine, and fecal extract pools from all species and identified on the basis of TLC, HPLC, and mass spectrometry. The identified metabolites explained 60-99% of the total radioactivity in each sample for rabbit plasma, in which only 17% of the total radioactivity was characterized. Metabolic pathways involving oxidative reactions at seven sites on the bepridil molecule are proposed for each species. Metabolite formation in the five species is described by four interrelated pathways. The metabolic pathway involving aromatic hydroxylation followed by N-dealkylation, N-debenzylation, and N-acetylation was important in all species. Major metabolites produced by this pathway included 4-hydroxy(at N-phenyl)-bepridil (Ia), N-benzyl-4-amino-phenol (IV), and N-acetyl-4-aminophenol (Vy). Metabolite Ia was isolated in significant amounts (greater than or equal to 5% of sample) in all fecal and urine samples except rat urine. Metabolite IV was a major circulating metabolite in all species and a major urinary metabolite in humans. Metabolite Vy was present in significant quantities in urine in all species except rabbit. Other important pathways involved primary reactions such as iso-butyl hydroxylation, pyrrolidine ring oxidation, and N-debenzylation.(ABSTRACT TRUNCATED AT 250 WORDS)

CYP3A4 activity in four different animal species liver microsomes using 7-benzyloxyquinoline and HPLC/spectrofluorometric determination.

PubMed

Baririan, Narine; Desager, Jean-Pierre; Petit, Martine; Horsmans, Yves

2006-01-23

Some microplate-based direct assays with different fluorometric substrates have been developed, among which 7-benzyloxyquinoline (BOQ) has demonstrated the highest degree of selectivity for CYP3A subfamily. In our study, we firstly developed and validated an efficient, fast and cheap HPLC/spectrofluorometric analytical method to quantify 7-hydroxyquinoline (BOQ metabolite). Secondly, BOQ oxidation rate (1.95 +/- 0.24 microM/mg protein/min) was compared to that of midazolam (MDZ) (1.4 +/- 0.21 microM/mg protein/min), an other specific CYP3A probe. However, the difference did not reach statistically significance (test of Sign; p = 0.125, two tailed). Thirdly, the potential use of BOQ in other species than the rat (mouse, dog and monkey) was studied. The highest BOQ activity was observed in rat microsomes (3.75 micromol/mg protein/min) with lower P450 content (0.3 nmol/mg protein) compared to other species. Finally, the effect of CYP3A enzymes-selective inhibitor ketoconazole on the dealkylation of BOQ in control and dexamethasone (DM)-treated rat microsomes was studied. Ketoconazole inhibition potency was greater in control (IC(50) approximately 21.6 microM) compared to DM induced (IC(50) approximately 32.3 microM) microsomes. At concentrations greater than that considered to be enzyme-selective (e.g., 10-30 microM), ketoconazole inhibitory activity did not rise significantly, and at the maximal concentration tested (1,000 microM) a nearly similar inhibition (76%) was observed than that at 50 microM concentration (68.2%).

Buprenorphine is protective against the depressive effects of norbuprenorphine on ventilation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Megarbane, Bruno; Marie, Nicolas; Pirnay, Stephane

2006-05-01

High dose buprenorphine is used as substitution treatment in heroin addiction. However, deaths have been reported in addicts using buprenorphine. The role of norbuprenorphine, an N-dealkyl metabolite of buprenorphine, was hypothesized to explain these fatal cases. We determined the median intravenous lethal dose (LD{sub 5}) of norbuprenorphine in male Sprague-Dawley rats. The effects of a single intravenous dose of 3 or 9 mg/kg norbuprenorphine alone on arterial blood gases were studied. Finally, the effect of pre- and post-administrations of buprenorphine on norbuprenorphine-induced changes on arterial blood gases were analyzed. Norbuprenorphine's LD{sub 5} was 10 mg kg{sup -1}. Norbuprenorphine 3 mgmore » kg{sup -1} produces the rapid onset of sustained respiratory depression, as demonstrated at 20 min by a maximal significant increase in PaCO{sub 2} (8.4 {+-} 0.9 versus 5.7 {+-} 0.1 kPa), decrease in arterial pH (7.25 {+-} 0.06 versus 7.44 {+-} 0.01), and hypoxia (8.3 {+-} 0.6 versus 11.1 {+-} 0.2 kPa). Buprenorphine not only protected against the effects of 3 mg kg{sup -1} norbuprenorphine in a dose-dependent manner but also reversed the effects when given afterward. Binding experiments suggest a role for mu- and to a lesser extent for delta-opioid receptors in buprenorphine protective effect against norbuprenorphine-induced respiratory depression. In conclusion, our data clearly show that norbuprenorphine alone causes important deleterious effects on ventilation in rats. However, buprenorphine protective effect calls into question the role for norbuprenorphine in respiratory toxicity associated with buprenorphine use.« less

Determination of alpidem, an imidazopyridine anxiolytic, and its metabolites by column-switching high-performance liquid chromatography with fluorescence detection.

PubMed

Flaminio, L; Ripamonti, M; Ascalone, V

1994-05-13

Alpidem, 6-chloro-2-(4-chlorophenyl)-N,N-dipropylimidazo[1,2-a]pyridine- 3-acetamide, is an anxiolytic imidazopyridine that undergoes a first-pass elimination after oral administration to humans; it is actively metabolized and three circulating metabolites have been identified in plasma due to N-dealkylation, oxidation or a combination of both processes. For the determination of the unchanged drug and its metabolites in human plasma, a column-switching HPLC method was developed. The method, based on solid-phase extraction (performed on-line), involves the automatic injection of plasma samples (200 microliters) on to a precolumn filled with C18 material, clean-up of the sample with water in order to remove protein and salts and transfer of the analytes to the analytical column (after valve switching) by means of the mobile phase. All the processes were performed in the presence of an internal standard, a compound chemically related to alpidem. During the analytical chromatography, the precolumn was flushed with different solvents and after regeneration with water, it was ready for further injections. The analytical column was a C8 type and the mobile phase was acetonitrile-methanol-phosphate buffer solution (45:15:45, v/v/v) at a flow-rate of 1.5 ml min-1. The column was connected to a fluorimetric detector operating at excitation and emission wavelengths of 255 and 423 nm, respectively. The limits of quantitation of alpidem and three metabolites were 2.5 and 1.5 ng ml-1, respectively, in human plasma.

Bioactivation of tamoxifen to metabolite E quinone methide: reaction with glutathione and DNA.

PubMed

Fan, P W; Bolton, J L

2001-06-01

Despite the beneficial effects of tamoxifen in the treatment and prevention of breast cancer, long-term usage of this popular antiestrogen has been linked to an increased risk of developing endometrial cancer in women. One of the suggested pathways leading to the potential toxicity of tamoxifen involves its oxidative metabolism to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. Alternatively, tamoxifen could undergo O-dealkylation to give cis/trans-1,2-diphenyl-1-(4-hydroxyphenyl)-but-1-ene, which is commonly known as metabolite E. Because of its structural similarity to 4-hydroxytamoxifen, metabolite E could also be biotransformed to a quinone methide, which has the potential to alkylate DNA and may contribute to the genotoxic effects of tamoxifen. To further probe the chemical reactivity/toxicity of such an electrophilic species, we have prepared metabolite E quinone methide chemically and enzymatically and examined its reactivity with glutathione (GSH) and DNA. Like 4-hydroxytamoxifen quinone methide, metabolite E quinone methide is quite stable; its half-life under physiological conditions is around 4 h, and its half-life in the presence of GSH is approximately 4 min. However, unlike the unstable GSH adducts of 4-hydroxytamoxifen quinone methide, metabolite E GSH adducts are stable enough to be isolated and characterized by NMR and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Reaction of metabolite E quinone methide with DNA generated exclusively deoxyguanosine adducts, which were characterized by LC/MS/MS. These data suggest that metabolite E has the potential to cause cytotoxicity/genotoxicity through the formation of a quinone methide.

Metabolism of the new designer drug alpha-pyrrolidinopropiophenone (PPP) and the toxicological detection of PPP and 4'-methyl-alpha-pyrrolidinopropiophenone (MPPP) studied in rat urine using gas chromatography-mass spectrometry.

PubMed

Springer, Dietmar; Fritschi, Giselher; Maurer, Hans H

2003-11-05

R,S-alpha-pyrrolidinopropiophenone (PPP) is a new designer drug with assumed amphetamine-like effects which has appeared on the illicit drug market. The aim of this study was to identify the PPP metabolites using solid-phase extraction, ethylation or acetylation as well as to develop a toxicological detection procedure in urine using solid-phase extraction, trimethylsilylation and gas chromatography-mass spectrometry (GC-MS). Analysis of urine samples of rats treated with PPP revealed that PPP was extensively metabolized by hydroxylation of the pyrrolidine ring with subsequent dehydrogenation to the corresponding lactam, hydroxylation of the aromatic ring in position 4' or double dealkylation of the pyrrolidine ring to the corresponding primary amine (cathinone) partly followed by reduction of the keto group to the corresponding secondary alcohol (norephedrines). As cathinone and the norephedrine diastereomers are also formed after intake of other drugs of abuse or medicaments, special attention must be paid to the detection of the unequivocal metabolite 2"-oxo-PPP as an unambiguous proof for the intake of PPP. The hydroxy groups were found to be partly conjugated. Based on these data, PPP could be detected in urine via its metabolites by full-scan GC-MS using mass chromatography for screening and library search for identification by comparison of the spectra with reference spectra. The same toxicological detection procedure can be applied to other designer drugs of the pyrrolidinophenone type, like MOPPP, MDPPP, MPHP, and MPPP. The detection of the latter will also be presented here.

Synthesis of a selective ratiometric fluorescent probe based on Naphthalimide and its application in human cytochrome P450 1A.

PubMed

Zhang, Xiuxuan; Zhou, Yan; Gu, Xiaofei; Cheng, Yu; Hong, Manxin; Yan, Liqiang; Ma, Fulong; Qi, Zhengjian

2018-08-15

Cytochrome P450s have brought considerable attention to researchers for their significant correlations with metabolic behaviors of procarcinogenic chemicals. To better understand the roles of CYP1A in biological and physiological systems, we developed a novel ratiometric fluorescence probe N-((2-hydroxyl ethoxy) ethyl)- 4-methoxy-1, 8-naphthalimide (NEMN) allowing for selectively and sensitively monitoring the target enzymes under physiological conditions and living cells. The probe was designed based on substrate predilection of CYP1A and its outstanding O-dealkylation capacity, and 1, 8-naphthalimide was chosen as fluorophore on account of its desirable photophysical properties. Absorption and emission spectra of the probe solution and reacted metabolism showed obvious red-shift with remarkable colour changes, which indicated that NEMN could be a promising ratiometric detector of CYP1A. Additionally, the selectivity assays displayed that NEMN only sensitive to CYP1A1 and CYP1A2 enzymes with scarce interference of other CYPs. Furthermore, the excellent linear relationships between the ratio of fluorescent intensities and incubation time and enzymes concentration signified time- and concentration- dependence of the probe, which were of desire benefit to quantify and monitor the CYP1A-involved biological behaviors in physiological conditions. The assay in real living samples (Human liver microsomes) further proved the analytical utility of the probe. Finally, the cytotoxicity assay and confocal fluorescence imaging demonstrated that this probe was of great promise for detecting the activity of endogenous CYP1A in human living cells. Copyright © 2018 Elsevier B.V. All rights reserved.

An open-label, single-dose, phase 1 study of the absorption, metabolism and excretion of quizartinib, a highly selective and potent FLT3 tyrosine kinase inhibitor, in healthy male subjects, for the treatment of acute myeloid leukemia.

PubMed

Sanga, Madhu; James, Joyce; Marini, Joseph; Gammon, Guy; Hale, Christine; Li, Jianke

2017-10-01

1. Quizartinib absorption, metabolism and excretion were characterized in six healthy men receiving a single oral dose of 60 mg (≈100 μCi) of [ 14 C]-quizartinib. Blood, plasma, urine and faeces were collected ≤336 h postdose. 2. Four hours postdose, maximum mean ± SD blood radioactivity concentrations were 296 ± 67.4 ng equivalents/g. A mean ± SD of 1.64 ± 0.482% and 76.3 ± 6.23% of the dose was recovered in urine and faeces, respectively, within 336 h postdose. 3. Radio-detector high-performance liquid chromatography (radio-HPLC) and liquid chromatography-mass spectrometry (LC-MS) showed two main radioactive peaks in plasma, unchanged quizartinib and mono-oxidative metabolite, AC886. Five additional metabolites in plasma were identified by LC-MS, but low levels prevented radio-HPLC detection. Although unchanged quizartinib was the main radioactive component in faeces (mean, 4.0% of administered dose), 15 metabolites representing a mean of 1.0-3.5% of administered dose were found. Quizartinib was predominantly metabolized by phase I biotransformations (oxidation, reduction, dealkylation, deamination, hydrolysis and combinations thereof). 4. This study indicated that quizartinib was rapidly and orally bioavailable, extensively metabolized, with AC886 as the major circulating metabolite, and predominantly eliminated in faeces. Quizartinib was well tolerated in the subjects.

Chlorofluorocarbon dating of herbicide-containing well waters in Fresno and Tulare counties, California

USGS Publications Warehouse

Spurlock, F.; Burow, K.; Dubrovsky, N.

2000-01-01

Simazine, diuron, and bromacil are the most frequently detected currently registered pesticides in California groundwater. These herbicides have been used for several decades in Fresno and Tulare counties, California; however, previous data are inadequate to determine whether the detections are a result of recent or historical applications (i.e., within the last decade, or 20-30 yr ago). Chlorofluorocarbon (CFC) groundwater age-dating was used in conjunction with one-dimensional transport modeling to address this question. The estimated times between herbicide application and subsequent detection in groundwater samples from 18 domestic wells ranged from 3 to 33 yr; the aggregate data indicate that more than half of the detections are associated with applications in the last decade. The data also suggest that changes in groundwater quality arising from modified management practices will probably not be discernible for at least a decade. A secondary objective of this study was to evaluate the contribution of simazine degradates deethylsimazine (DES; 2-amino-4-chloro-6-ethylamino-s-triazine) and diaminochlorotriazine (DACT; 2,4-diamino-6-chloro-s-triazine) to total triazine concentrations (defined here as simazine + DES + DACT) in 30 domestic wells. The N-dealkylated s- chlorotriazine degradates DES and DACT substantially contribute to total triazine concentrations in Fresno and Tulare County groundwater, composing 24 to 100% of the total triazines, with a median of 82%. If s-chlorotriazines display a common mode of toxicological action, the prevalence of triazine degradates in water samples found in this and other studies indicate that drinking water standards based on total s-chlorotriazine concentrations may be most appropriate.

Identification of AKB-48 and 5F-AKB-48 Metabolites in Authentic Human Urine Samples Using Human Liver Microsomes and Time of Flight Mass Spectrometry.

PubMed

Vikingsson, Svante; Josefsson, Martin; Gréen, Henrik

2015-01-01

The occurrence of structurally related synthetic cannabinoids makes the identification of unique markers of drug intake particularly challenging. The aim of this study was to identify unique and abundant metabolites of AKB-48 and 5F-AKB-48 for toxicological screening in urine. Investigations of authentic urine samples from forensic cases in combination with human liver microsome (HLM) experiments were used for identification of metabolites. HLM incubations of AKB-48 and 5F-AKB-48 along with 35 urine samples from authentic cases were analyzed with liquid chromatography quadrupole tandem time of flight mass spectrometry. Using HLMs 41 metabolites of AKB-48 and 37 metabolites of 5F-AKB-48 were identified, principally represented by hydroxylation but also ketone formation and dealkylation. Monohydroxylated metabolites were replaced by di- and trihydroxylated metabolites within 30 min. The metabolites from the HLM incubations accounted for on average 84% (range, 67-100) and 91% (range, 71-100) of the combined area in the case samples for AKB-48 and 5F-AKB-48, respectively. While defluorinated metabolites accounted for on average 74% of the combined area after a 5F-AKB-48 intake only a few identified metabolites were shared between AKB-48 and 5F-AKB-48, illustrating the need for a systematic approach to identify unique metabolites. HLMs in combination with case samples seem suitable for this purpose. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

In vitro characterization of sarizotan metabolism: hepatic clearance, identification and characterization of metabolites, drug-metabolizing enzyme identification, and evaluation of cytochrome p450 inhibition.

PubMed

Gallemann, Dieter; Wimmer, Elmar; Höfer, Constance C; Freisleben, Achim; Fluck, Markus; Ladstetter, Bernhard; Dolgos, Hugues

2010-06-01

In vitro biotransformation studies of sarizotan using human liver microsomes (HLM) showed aromatic and aliphatic monohydroxylation and dealkylation. Recombinant cytochromes P450 (P450) together with P450-selective inhibitors in HLM/hepatocyte cultures were used to evaluate the relative contribution of different P450s and revealed major involvement of CYP3A4, CYP2C9, CYP2C8, and CYP1A2 in sarizotan metabolism. The apparent K(m, u) and V(max) of sarizotan clearance, as investigated in HLM, were 9 microM and 3280 pmol/mg/min, predicting in vivo hepatic clearance of 0.94 l/h, which indicates that sarizotan is a low-clearance compound in humans and suggests nonsaturable metabolism at the targeted plasma concentration (< or =1 microM). This finding is confirmed by the reported human clearance (CL/F of 3.6-4.4 l/h) and by the dose-linear area under the curve increase observed with doses up to 25 mg. The inhibitory effect of sarizotan toward six major P450s was evaluated using P450-specific marker reactions in pooled HLM. K(i, u) values of sarizotan against CYP2C8, CYP2C19, and CYP3A4 were >10 microM, whereas those against CYP2D6 and CYP1A2 were 0.43 and 8.7 microM, respectively. Based on the estimates of sarizotan concentrations at the enzyme active sites, no clinically significant drug-drug interactions (DDIs) due to P450 inhibition are expected. This result has been confirmed in human DDI studies in which no inhibition of five major P450s was observed in terms of marker metabolite formation.

Crystal structures of human group-VIIA phospholipase A2 inhibited by organophosphorus nerve agents exhibit non-aged complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Samanta, Uttamkumar; Kirby, Stephen D.; Srinivasan, Prabhavathi

The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of P{submore » R} and P{sub S} stereoisomers at the P-chiral center. The tabun complex displayed only the P{sub R} stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix-assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long-term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents.« less

In vitro and in vivo metabolic investigation of the Palbociclib by UHPLC-Q-TOF/MS/MS and in silico toxicity studies of its metabolites.

PubMed

Chavan, Balasaheb B; Tiwari, Shristy; G, Shankar; Nimbalkar, Rakesh D; Garg, Prabha; R, Srinivas; Talluri, M V N Kumar

2018-05-14

Palbociclib (PAB) is a CDK4/6 inhibitor and U. S Food and Drug Administration (FDA) granted regular approval for the treatment of hormone receptor (HR) positive, metastatic breast cancer in combination with an aromatase inhibitor in postmenopausal women. Metabolite identification is a crucial aspect during drug discovery and development as the drug metabolites may be pharmacologically active or possess toxicological activity. As there are no reports on the metabolism studies of the PAB, the present study focused on investigation of the in vitro and in vivo metabolic fate of the drug. The in vitro metabolism studies were carried out by using microsomes (HLM and RLM) and S9 fractions (Human and rat). The in vivo metabolism of the drug was studied by administration of the PAB orally to the Sprague-Dawley rats followed by analysis of urine, faeces and plasma samples. The sample preparation includes simple protein precipitation (PP) followed by solid phase extraction (SPE). The extracted samples were analyzed by ultrahigh-performance liquid chromatography-quadruple time-of-flight tandem mass spectrometry (UHPLC/Q-TOF/MS/MS). A total of 14 metabolites were detected in in vivo matrices. The PAB was metabolized via hydroxylation, oxidation, sulphation, N-dealkylation, acetylation and carbonylation pathways. A few of the metabolites were also detected in in vitro samples. Metabolite identification and characterization were performed by using UHPLC/Q-TOF/MS/MS in combination with HRMS data. To identify the toxicity potential of these metabolites, in silico toxicity assessment was carried out using TOPKAT and DEREK softwares. Copyright © 2018. Published by Elsevier B.V.

Catalytic soman scavenging by Y337A/F338A acetylcholinesterase mutant assisted with novel site-directed aldoximes

PubMed Central

Kovarik, Zrinka; Hrvat, Nikolina Maček; Katalinić, Maja; Sit, Rakesh K.; Paradyse, Alexander; Žunec, Suzana; Musilek, Kamil; Fokin, Valery V.; Taylor, Palmer; Radić, Zoran

2016-01-01

Exposure to the nerve agent soman is difficult to treat due to the rapid dealkylation of soman-acetylcholinesterase (AChE) conjugate known as aging. Oxime antidotes commonly used to reactivate organophosphate inhibited AChE are ineffective against soman, while the efficacy of the recommended nerve agent bioscavenger butyrylcholinesterase is limited by strictly stoichiometric scavenging. To overcome this limitation, we tested ex vivo, in human blood, and in vivo, in soman exposed mice, the capacity of aging-resistant human AChE mutant Y337A/F338A in combination with oxime HI-6 to act as a catalytic bioscavenger of soman. HI-6 was previously shown in vitro to efficiently reactivate this mutant upon soman, as well as VX, cyclosarin, sarin and paraoxon inhibition. We here demonstrate that ex vivo, in whole human blood, 1 μM soman was detoxified within 30 minutes when supplemented with 0.5 μM Y337A/F338A AChE and 100 μM HI-6. This combination was further tested in vivo. Catalytic scavenging of soman in mice improved the therapeutic outcome and resulted in the delayed onset of toxicity symptoms. Furthermore, in a preliminary in vitro screen we identified an even more efficacious oxime than HI-6, in a series of forty-two pyridinium aldoximes, and five imidazole 2-aldoxime N-propyl pyridinium derivatives. One of the later imidazole aldoximes, RS-170B, was a 2–3 –fold more effective reactivator of Y337A/F338A AChE than HI-6 due to the smaller imidazole ring, as indicated by computational molecular models, that affords a more productive angle of nucleophilic attack. PMID:25835984

High-resolution chromatography/time-of-flight MSE with in silico data mining is an information-rich approach to reactive metabolite screening.

PubMed

Barbara, Joanna E; Castro-Perez, Jose M

2011-10-30

Electrophilic reactive metabolite screening by liquid chromatography/mass spectrometry (LC/MS) is commonly performed during drug discovery and early-stage drug development. Accurate mass spectrometry has excellent utility in this application, but sophisticated data processing strategies are essential to extract useful information. Herein, a unified approach to glutathione (GSH) trapped reactive metabolite screening with high-resolution LC/TOF MS(E) analysis and drug-conjugate-specific in silico data processing was applied to rapid analysis of test compounds without the need for stable- or radio-isotope-labeled trapping agents. Accurate mass defect filtering (MDF) with a C-heteroatom dealkylation algorithm dynamic with mass range was compared to linear MDF and shown to minimize false positive results. MS(E) data-filtering, time-alignment and data mining post-acquisition enabled detection of 53 GSH conjugates overall formed from 5 drugs. Automated comparison of sample and control data in conjunction with the mass defect filter enabled detection of several conjugates that were not evident with mass defect filtering alone. High- and low-energy MS(E) data were time-aligned to generate in silico product ion spectra which were successfully applied to structural elucidation of detected GSH conjugates. Pseudo neutral loss and precursor ion chromatograms derived post-acquisition demonstrated 50.9% potential coverage, at best, of the detected conjugates by any individual precursor or neutral loss scan type. In contrast with commonly applied neutral loss and precursor-based techniques, the unified method has the advantage of applicability across different classes of GSH conjugates. The unified method was also successfully applied to cyanide trapping analysis and has potential for application to alternate trapping agents. Copyright © 2011 John Wiley & Sons, Ltd.

Assessing the phytoremediation potential of crop and grass plants for atrazine-spiked soils.

PubMed

Sánchez, Virtudes; López-Bellido, Francisco Javier; Cañizares, Pablo; Rodríguez, Luis

2017-10-01

Pollution of soil and groundwater by atrazine has become an increasing environmental concern in the last decade. A phytoremediation test using plastic pots was conducted in order to assess the ability of several crops and grasses to remove atrazine from a soil of low permeability spiked with this herbicide. Four plant species were assessed for their ability to degrade or accumulate atrazine from soils: two grasses, i.e., ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea), and two crops, i.e., barley (Hordeum vulgare) and maize (Zea mays). Three different doses of atrazine were used for the contamination of the pots: 2, 5 and 10 mg kg -1 . 16 days after spiking, the initial amount of atrazine was reduced by 88.6-99.6% in planted pots, while a decrease of only 63.1-78.2% was found for the unplanted pots, thus showing the contribution of plants to soil decontamination. All the plant species were capable of accumulating atrazine and its N-dealkylated metabolites, i.e., deethylatrazine and deisopropylatrazine, in their tissues. Some toxic responses, such as biomass decreases and/or chlorosis, were observed in plants to a greater or lesser extent for initial soil doses of atrazine above 2 mg kg -1 . Maize was the plant species with the highest ability to accumulate atrazine derivatives, reaching up to 38.4% of the initial atrazine added to the soil. Rhizosphere degradation/mineralization by microorganisms or plant enzymes, together with degradation inside the plants, have been proposed as the mechanisms that contributed to a higher extent than plant accumulation to explain the removal of atrazine from soils. Copyright © 2017 Elsevier Ltd. All rights reserved.

Screening for the synthetic cannabinoid JWH-018 and its major metabolites in human doping controls.

PubMed

Möller, Ines; Wintermeyer, Annette; Bender, Katja; Jübner, Martin; Thomas, Andreas; Krug, Oliver; Schänzer, Wilhelm; Thevis, Mario

2011-09-01

Referred to as 'spice', several new drugs, advertised as herbal blends, have appeared on the market in the last few years, in which the synthetic cannabinoids JWH-018 and a C(8) homologue of CP 47,497 were identified as major active ingredients. Due to their reported cannabis-like effects, many European countries have banned these substances. The World Anti-Doping Agency has also explicitly prohibited synthetic cannabinoids in elite sport in-competition. Since urine specimens have been the preferred doping control samples, the elucidation of the metabolic pathways of these substances is of particular importance to implement them in sports drug testing programmes. In a recent report, an in vitro phase-I metabolism study of JWH-018 was presented yielding mainly hydroxylated and N-dealkylated metabolites. Due to these findings, a urine sample of a healthy man declaring to have smoked a 'spice' product was screened for potential phase-I and -II metabolites by high-resolution/high-accuracy mass spectrometry in the present report. The majority of the phase-I metabolites observed in earlier in vitro studies of JWH-018 were detected in this urine specimen and furthermore most of their respective monoglucuronides. As no intact JWH-018 was detectable, the monohydroxylated metabolite being the most abundant one was chosen as a target analyte for sports drug testing purposes; a detection method was subsequently developed and validated in accordance to conventional screening protocols based on enzymatic hydrolysis, liquid-liquid extraction, and liquid chromatography/electrospray tandem mass spectrometry analysis. The method was applied to approximately 7500 urine doping control samples yielding two JWH-018 findings and demonstrated its capability for a sensitive and selective identification of JWH-018 and its metabolites in human urine. Copyright © 2010 John Wiley & Sons, Ltd.

Environmental fate and effect assessment of thioridazine and its transformation products formed by photodegradation.

PubMed

Wilde, Marcelo L; Menz, Jakob; Trautwein, Christoph; Leder, Christoph; Kümmerer, Klaus

2016-06-01

An experimental and in silico quantitative structure-activity relationship (QSAR) approach was applied to assess the environmental fate and effects of the antipsychotic drug Thioridazine (THI). The sunlight-driven attenuation of THI was simulated using a Xenon arc lamp. The photodegradation reached the complete primary elimination, whereas 97% of primary elimination and 11% of mineralization was achieved after 256 min of irradiation for the initial concentrations of 500 μg L(-1) and 50 mg L(-1), respectively. A non-target approach for the identification and monitoring of transformation products (TPs) was adopted. The structure of the TPs was further elucidated using liquid chromatography-high resolution mass spectrometry (LC-HRMS). The proposed photodegradation pathway included sulfoxidation, hydroxylation, dehydroxylation, and S- and N-dealkylation, taking into account direct and indirect photolysis through a self-sensitizing process in the higher concentration studied. The biodegradability of THI and photolytic samples of THI was tested according to OECD 301D and 301F, showing that THI and the mixture of TPs were not readily biodegradable. Furthermore, THI was shown to be highly toxic to environmental bacteria using a modified luminescent bacteria test with Vibrio fischeri. This bacteriotoxic activity of THI was significantly reduced by phototransformation and individual concentration-response analysis confirmed a lowered bacterial toxicity for the sulfoxidation products Thioridazine-2-sulfoxide and Thioridazine-5-sulfoxide. Additionally, the applied QSAR models predicted statistical and rule-based positive alerts of mutagenic activities for carbazole derivative TPs (TP 355 and TP 339) formed through sulfoxide elimination, which would require further confirmatory in vitro validation tests. Copyright © 2016 Elsevier Ltd. All rights reserved.

Oxidation of fluoroquinolone antibiotics and structurally related amines by chlorine dioxide: Reaction kinetics, product and pathway evaluation.

PubMed

Wang, Pei; He, Yi-Liang; Huang, Ching-Hua

2010-12-01

Fluoroquinolones (FQs) are a group of widely prescribed antibiotics and have been frequently detected in the aquatic environment. The reaction kinetics and transformation of seven FQs (ciprofloxacin (CIP), enrofloxacin (ENR), norfloxacin (NOR), ofloxacin (OFL), lomefloxacin (LOM), pipemidic acid (PIP) and flumequine (FLU)) and three structurally related amines (1-phenylpiperazine (PP), N-phenylmorpholine (PM) and 4-phenylpiperidine (PD)) toward chlorine dioxide (ClO(2)) were investigated to elucidate the behavior of FQs during ClO(2) disinfection processes. The reaction kinetics are highly pH-dependent, can be well described by a second-order kinetic model incorporating speciation of FQs, and follow the trend of OFL > ENR > CIP ∼ NOR ∼ LOM > > PIP in reactivity. Comparison among FQs and related amines and product characterization indicate that FQs' piperazine ring is the primary reactive center toward ClO(2). ClO(2) likely attacks FQ's piperazinyl N4 atom followed by concerted fragmentation involving piperazinyl N1 atom, leading to dealkylation, hydroxylation and intramolecular ring closure at the piperazine moiety. While FQs with tertiary N4 react faster with ClO(2) than FQs with secondary N4, the overall reactivity of the piperazine moiety also depends strongly on the quinolone ring through electronic effects. The reaction rate constants obtained in clean water matrix can be used to model the decay of CIP by ClO(2) in surface water samples, but overestimate the decay in wastewater samples. Overall, transformation of FQs, particularly for those with tertiary N4 amines, could be expected under typical ClO(2) disinfection conditions. However, the transformation may not eliminate antibacterial activity because of little destruction at the quinolone ring. Copyright © 2010 Elsevier Ltd. All rights reserved.

Effect of blueberry juice on clearance of buspirone and flurbiprofen in human volunteers

PubMed Central

Hanley, Michael J; Masse, Gina; Harmatz, Jerold S; Cancalon, Paul F; Dolnikowski, Gregory G; Court, Michael H; Greenblatt, David J

2013-01-01

Aim The present study evaluated the possibility of drug interactions involving blueberry juice (BBJ) and substrate drugs whose clearance is dependent on cytochromes P4503A (CYP3A) and P4502C9 (CYP2C9). Methods A 50:50 mixture of lowbush and highbush BBJ was evaluated in vitro as an inhibitor of CYP3A activity (hydroxylation of triazolam and dealkylation of buspirone) and of CYP2C9 activity (flurbiprofen hydroxylation) using human liver microsomes. In clinical studies, clearance of oral buspirone and oral flurbiprofen was studied in healthy volunteers with and without co-treatment with BBJ. Results BBJ inhibited CYP3A and CYP2C9 activity in vitro, with 50% inhibitory concentrations (IC50) of less than 2%, but without evidence of mechanism-based (irreversible) inhibition. Grapefruit juice (GFJ) also inhibited CYP3A activity, but inhibitory potency was increased by pre-incubation, consistent with mechanism-based inhibition. In clinical studies, GFJ significantly increased area under the plasma concentration−time curve (AUC) for the CYP3A substrate buspirone. The geometric mean ratio (GMR = AUC with GFJ divided by AUC with water) was 2.12. In contrast, the effect of BBJ (GMR = 1.39) was not significant. In the study of flurbiprofen (CYP2C9 substrate), the positive control inhibitor fluconazole significantly increased flurbiprofen AUC (GMR = 1.71), but BBJ had no significant effect (GMR = 1.03). Conclusion The increased buspirone AUC associated with BBJ is quantitatively small and could have occurred by chance. BBJ has no effect on flurbiprofen AUC. The studies provide no evidence for concern about clinically important pharmacokinetic drug interactions of BBJ with substrate drugs metabolized by CYP3A or CYP2C9. PMID:22943633

Transformation products formation of ciprofloxacin in UVA/LED and UVA/LED/TiO2 systems: Impact of natural organic matter characteristics.

PubMed

Li, Si; Hu, Jiangyong

2018-04-01

The role of natural organic matter (NOM) in contaminants removal by photolysis and photocatalysis has aroused increasing interest. However, evaluation of the influence of NOM characteristics on the transformation products (TPs) formation and transformation pathways of contaminants has rarely been performed. This study investigated the decomposition kinetics, mineralization, TPs formation and transformation pathways of antibiotic ciprofloxacin (CIP) during photolysis and photocatalysis in the presence of three commercial NOM isolates (Sigma-Aldrich humic acid (SAHA), Suwannee River humic acid (SRHA) and Suwannee River NOM (SRNOM)) by using UVA light emitting diode (UVA/LED) as an alternative light source. NOM isolates insignificantly affected CIP photolysis but strongly inhibited CIP photocatalysis due to competitive radical quenching. The inhibitory effect followed the order of SAHA (49.6%) > SRHA (29.9%) > SRNOM (21.2%), consistent with their •OH quenching abilities, SUVA 254 values and orders of aromaticity. Mineralization rates as revealed by F - release were negatively affected by NOM during CIP photocatalysis. TPs arising from hydroxylation and defluorination were generally suppressed by NOM isolates in UVA/LED and UVA/LED/TiO 2 systems. In contrast, dealkylation and oxidation of piperazine ring were promoted by NOM. The enhancement in the apparent formation kinetics (k app ) of TP245, TP291, TP334a, TP334b and TP362 followed the order of SRNOM > SRHA > SAHA. k app values were positively correlated with O/C ratio, carboxyl content, E2/E3 and fluorescence index (FI) of NOM and negatively related with SUVA 254 values. The observed correlations indicate that NOM properties are important in determining the fate and transformation of organic contaminants during photolysis and photocatalysis. Copyright © 2018 Elsevier Ltd. All rights reserved.

IMPROVED CATALYSTS FOR HEAVY OIL UPGRADING BASED ON ZEOLITE Y NANOPARTICLES ENCAPSULATED IN STABLE NANOPOROUS HOST

DOE Office of Scientific and Technical Information (OSTI.GOV)

Conrad Ingram; Mark Mitchell

2005-11-15

Composite materials of SBA-15/zeolite Y were synthesized from zeolite Y precursor and a synthesis mixture of mesoporous silicate SBA-15 via a hydrothermal process in the presence of a slightly acidic media of pH 4-6 with 2M H{sub 2}SO{sub 4}. The SBA-15/ZY composites showed Type IV adsorption isotherms, narrow BJH average pore size distribution of 4.9 nm, surface areas up to 800 m{sup 2}2/g and pore volumes 1.03 cm{sup 3}, all comparable to pure SBA-15 synthesized under similar conditions. Chemical analysis revealed Si/Al ratio down to 8.5 in the most aluminated sample, and {sup 27}AlSS MAS NMR confirmed aluminum was inmore » tetrahedral coordination. This method of introduction of Al in pure T{sub d} coordination is effective in comparison to other direct and post synthesis alumination methods. Bronsted acid sites were evident from a pyridinium peak at 1544 cm-1 in the FTIR spectrum after pyridine adsorption, and from NH{sub 3} -TPD experiments. SBA-15/ZY composites showed significant catalytic activities for the dealkylation of isopropylbenzene to benzene and propene, similar to those of commercial zeolite Y. It was observed that higher conversion for catalysts synthesized with high amount of ZY precursor mixture added to the SBA-15. Over all the composites has shown good catalytic activity. Further studies will be focused on gaining a better understand the nature of the precursor, and to characterize and to locate the acid sites in the composite material. The composite will also be evaluated for heavy oil conversion to naphtha and middle distillates.« less

Efficacy, Pharmacokinetics, and Metabolism of Tetrahydroquinoline Inhibitors of Plasmodium falciparum Protein Farnesyltransferase▿ †

PubMed Central

Van Voorhis, Wesley C.; Rivas, Kasey L.; Bendale, Pravin; Nallan, Laxman; Hornéy, Carolyn; Barrett, Lynn K.; Bauer, Kevin D.; Smart, Brian P.; Ankala, Sudha; Hucke, Oliver; Verlinde, Christophe L. M. J.; Chakrabarti, Debopam; Strickland, Corey; Yokoyama, Kohei; Buckner, Frederick S.; Hamilton, Andrew D.; Williams, David K.; Lombardo, Louis J.; Floyd, David; Gelb, Michael H.

2007-01-01

New antimalarials are urgently needed. We have shown that tetrahydroquinoline (THQ) protein farnesyltransferase (PFT) inhibitors (PFTIs) are effective against the Plasmodium falciparum PFT and are effective at killing P. falciparum in vitro. Previously described THQ PFTIs had limitations of poor oral bioavailability and rapid clearance from the circulation of rodents. In this paper, we validate both the Caco-2 cell permeability model for predicting THQ intestinal absorption and the in vitro liver microsome model for predicting THQ clearance in vivo. Incremental improvements in efficacy, oral absorption, and clearance rate were monitored by in vitro tests; and these tests were followed up with in vivo absorption, distribution, metabolism, and excretion studies. One compound, PB-93, achieved cure when it was given orally to P. berghei-infected rats every 8 h for a total of 72 h. However, PB-93 was rapidly cleared, and dosing every 12 h failed to cure the rats. Thus, the in vivo results corroborate the in vitro pharmacodynamics and demonstrate that 72 h of continuous high-level exposure to PFTIs is necessary to kill plasmodia. The metabolism of PB-93 was demonstrated by a novel technique that relied on double labeling with a radiolabel and heavy isotopes combined with radiometric liquid chromatography and mass spectrometry. The major liver microsome metabolite of PB-93 has the PFT Zn-binding N-methyl-imidazole removed; this metabolite is inactive in blocking PFT function. By solving the X-ray crystal structure of PB-93 bound to rat PFT, a model of PB-93 bound to malarial PFT was constructed. This model suggests areas of the THQ PFTIs that can be modified to retain efficacy and protect the Zn-binding N-methyl-imidazole from dealkylation. PMID:17606674

Binding and hydrolysis of soman by human serum albumin.

PubMed

Li, Bin; Nachon, Florian; Froment, Marie-Thérèse; Verdier, Laurent; Debouzy, Jean-Claude; Brasme, Bernardo; Gillon, Emilie; Schopfer, Lawrence M; Lockridge, Oksana; Masson, Patrick

2008-02-01

Human plasma and fatty acid free human albumin were incubated with soman at pH 8.0 and 25 degrees C. Four methods were used to monitor the reaction of albumin with soman: progressive inhibition of the aryl acylamidase activity of albumin, the release of fluoride ion from soman, 31P NMR, and mass spectrometry. Inhibition (phosphonylation) was slow with a bimolecular rate constant of 15 +/- 3 M(-1) min (-1). MALDI-TOF and tandem mass spectrometry of the soman-albumin adduct showed that albumin was phosphonylated on tyrosine 411. No secondary dealkylation of the adduct (aging) occurred. Covalent docking simulations and 31P NMR experiments showed that albumin has no enantiomeric preference for the four stereoisomers of soman. Spontaneous reactivation at pH 8.0 and 25 degrees C, measured as regaining of aryl acylamidase activity and decrease of covalent adduct (pinacolyl methylphosphonylated albumin) by NMR, occurred at a rate of 0.0044 h (-1), indicating that the adduct is quite stable ( t1/2 = 6.5 days). At pH 7.4 and 22 degrees C, the covalent soman-albumin adduct, measured by MALDI-TOF mass spectrometry, was more stable ( t1/2 = 20 days). Though the concentration of albumin in plasma is very high (about 0.6 mM), its reactivity with soman (phosphonylation and phosphotriesterase activity) is too slow to play a major role in detoxification of the highly toxic organophosphorus compound soman. Increasing the bimolecular rate constant of albumin for organophosphates is a protein engineering challenge that could lead to a new class of bioscavengers to be used against poisoning by nerve agents. Soman-albumin adducts detected by mass spectrometry could be useful for the diagnosis of soman exposure.

Aging pathways for organophosphate-inhibited human butyrylcholinesterase, including novel pathways for isomalathion, resolved by mass spectrometry.

PubMed

Li, He; Schopfer, Lawrence M; Nachon, Florian; Froment, Marie-Thérèse; Masson, Patrick; Lockridge, Oksana

2007-11-01

Some organophosphorus compounds are toxic because they inhibit acetylcholinesterase (AChE) by phosphylation of the active site serine, forming a stable conjugate: Ser-O-P(O)-(Y)-(XR) (where X can be O, N, or S and Y can be methyl, OR, or SR). The inhibited enzyme can undergo an aging process, during which the X-R moiety is dealkylated by breaking either the P-X or the X-R bond depending on the specific compound, leading to a nonreactivatable enzyme. Aging mechanisms have been studied primarily using AChE. However, some recent studies have indicated that organophosphate-inhibited butyrylcholinesterase (BChE) may age through an alternative pathway. Our work utilized matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry to study the aging mechanism of human BChE inhibited by dichlorvos, echothiophate, diisopropylfluorophosphate (DFP), isomalathion, soman, sarin, cyclohexyl sarin, VX, and VR. Inhibited BChE was aged in the presence of H2O18 to allow incorporation of (18)O, if cleavage was at the P-X bond. Tryptic-peptide organophosphate conjugates were identified through peptide mass mapping. Our results showed no aging of VX- and VR-treated BChE at 25 degrees C, pH 7.0. However, BChE inhibited by dichlorvos, echothiophate, DFP, soman, sarin, and cyclohexyl sarin aged exclusively through O-C bond cleavage, i.e., the classical X-R scission pathway. In contrast, isomalathion aged through both X-R and P-X pathways; the main aged product resulted from P-S bond cleavage and a minor product resulted from O-C and/or S-C bond cleavage.

Crystal structures of human group-VIIA phospholipase A2 inhibited by organophosphorus nerve agents exhibit non-aged complexes.

PubMed

Samanta, Uttamkumar; Kirby, Stephen D; Srinivasan, Prabhavathi; Cerasoli, Douglas M; Bahnson, Brian J

2009-08-15

The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of P(R) and P(S) stereoisomers at the P-chiral center. The tabun complex displayed only the P(R) stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix-assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long-term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents.

Detection of metabolites of the new synthetic cannabinoid CUMYL-4CN-BINACA in authentic urine samples and human liver microsomes using high-resolution mass spectrometry.

PubMed

Öztürk, Yeter Erol; Yeter, Oya; Öztürk, Serkan; Karakus, Goksun; Ates, Ismail; Buyuk, Yalçın; Yurdun, Turkan

2018-03-01

CUMYL-4CN-BINACA(1-(4-cyanobutyl)-N-(2-phenylpropan-2-yl)-1H-indazole-3-carboxamide) is a recently introduced indazole-3-carboxamide-type synthetic cannabinoid (SC) that was detected in herbal incense seized by of the Council of Forensic Medicine, Istanbul Narcotics Department, in May 2016 in Turkey. Recently introduced SCs are not detected in routine toxicological analysis; therefore, analytical methods to measure these compounds are in demand. The present study aims to identify urinary marker metabolites of CUMYL-4CN-BINACA by investigating its metabolism in human liver microsomes and to confirm the results in authentic urine samples (n = 80). In this study, 5 μM CUMYL-4CN-BINACA was incubated with human liver microsomes (HLMs) for up to 3 hours, and metabolites were identified using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Less than 21% of the CUMYL-4CN-BINACA parent compound remained after 3 hours of incubation. We identified 18 metabolites that were formed via monohydroxylation, dealkylation, oxidative decyanation to aldehyde, alcohol, and carboxylic acid formation, glucuronidation or reaction combinations. CUMYL-4CN-BINACA N-butanoic acid (M16) was found to be major metabolite in HLMs. In urine samples CUMYL-4CN-BINACA was not detected; CUMYL-4CN-BINACA N-butanoic acid (M16) was major metabolite after β-glucuronidase hydrolysis. Based on these findings, we recommend using M16 (CUMYL-4CN-BINACA N-butanoic acid), M8 and M11 (hydroxylcumyl CUMYL-4CN-BINACA) as urinary marker metabolites to confirm CUMYL-4CN-BINACA intake. Copyright © 2017 John Wiley & Sons, Ltd.

Summer cover crops reduce atrazine leaching to shallow groundwater in southern Florida.

PubMed

Potter, Thomas L; Bosch, David D; Joo, Hyun; Schaffer, Bruce; Muñoz-Carpena, Rafael

2007-01-01

At Florida's southeastern tip, sweet corn (Zea Mays) is grown commercially during winter months. Most fields are treated with atrazine (6-chloro-N-ethyl-N'-[1-methylethyl]-1,3,5-triazine-2,4-diamine). Hydrogeologic conditions indicate a potential for shallow groundwater contamination. This was investigated by measuring the parent compound and three degradates--DEA (6-chloro-N-[1-methylethyl]-1,3,5-triazine-2,4-diamine), DIA (6-chloro-N-ethyl)-1,3,5-triazine-2,4-diamine, and HA (6-hydroxy-N-[1-methylethyl]-1,3,5-triazine-2,4-diamine)--in water samples collected beneath sweet corn plots treated annually with the herbicide. During the study, a potential mitigation measure (i.e., the use of a cover crop, Sunn Hemp [Crotalaria juncea L.], during summer fallow periods followed by chopping and turning the crop into soil before planting the next crop) was evaluated. Over 3.5 yr and production of four corn crops, groundwater monitoring indicated leaching of atrazine, DIA, and DEA, with DEA accounting for more than half of all residues in most samples. Predominance of DEA, which increased after the second atrazine application, was interpreted as an indication of rapid and extensive atrazine degradation in soil and indicated that an adapted community of atrazine degrading organisms had developed. A companion laboratory study found a sixfold increase in atrazine degradation rate in soil after three applications. Groundwater data also revealed that atrazine and degradates concentrations were significantly lower in samples collected beneath cover crop plots when compared with concentrations below fallow plots. Together, these findings demonstrated a relatively small although potentially significant risk for leaching of atrazine and its dealkylated degradates to groundwater and that the use of a cover crop like Sunn Hemp during summer months may be an effective mitigation measure.

In situ experiments for element species-specific environmental reactivity of tin and mercury compounds using isotopic tracers and multiple linear regression.

PubMed

Rodriguez-Gonzalez, Pablo; Bouchet, Sylvain; Monperrus, Mathilde; Tessier, Emmanuel; Amouroux, David

2013-03-01

The fate of mercury (Hg) and tin (Sn) compounds in ecosystems is strongly determined by their alkylation/dealkylation pathways. However, the experimental determination of those transformations is still not straightforward and methodologies need to be refined. The purpose of this work is the development of a comprehensive and adaptable tool for an accurate experimental assessment of specific formation/degradation yields and half-lives of elemental species in different aquatic environments. The methodology combines field incubations of coastal waters and surface sediments with the addition of species-specific isotopically enriched tracers and a mathematical approach based on the deconvolution of isotopic patterns. The method has been applied to the study of the environmental reactivity of Hg and Sn compounds in coastal water and surface sediment samples collected in two different coastal ecosystems of the South French Atlantic Coast (Arcachon Bay and Adour Estuary). Both the level of isotopically enriched species and the spiking solution composition were found to alter dibutyltin and monomethylmercury degradation yields, while no significant changes were measurable for tributyltin and Hg(II). For butyltin species, the presence of light was found to be the main source of degradation and removal of these contaminants from surface coastal environments. In contrast, photomediated processes do not significantly influence either the methylation of mercury or the demethylation of methylmercury. The proposed method constitutes an advancement from the previous element-specific isotopic tracers' approaches, which allows for instance to discriminate the extent of net and oxidative Hg demethylation and to identify which debutylation step is controlling the environmental persistence of butyltin compounds.

Single-particle spectroscopy on large SAPO-34 crystals at work: methanol-to-olefin versus ethanol-to-olefin processes.

PubMed

Qian, Qingyun; Ruiz-Martínez, Javier; Mokhtar, Mohamed; Asiri, Abdullah M; Al-Thabaiti, Shaeel A; Basahel, Suliman N; van der Bij, Hendrik E; Kornatowski, Jan; Weckhuysen, Bert M

2013-08-19

The formation of hydrocarbon pool (HCP) species during methanol-to-olefin (MTO) and ethanol-to-olefin (ETO) processes have been studied on individual micron-sized SAPO-34 crystals with a combination of in situ UV/Vis, confocal fluorescence, and synchrotron-based IR microspectroscopic techniques. With in situ UV/Vis microspectroscopy, the intensity changes of the λ=400 nm absorption band, ascribed to polyalkylated benzene (PAB) carbocations, have been monitored and fitted with a first-order kinetics at low reaction temperatures. The calculated activation energy (Ea ) for MTO, approximately 98 kJ mol(-1) , shows a strong correlation with the theoretical values for the methylation of aromatics. This provides evidence that methylation reactions are the rate-determining steps for the formation of PAB. In contrast for ETO, the Ea value is approximately 60 kJ mol(-1) , which is comparable to the Ea values for the condensation of light olefins into aromatics. Confocal fluorescence microscopy demonstrates that during MTO the formation of the initial HCP species are concentrated in the outer rim of the SAPO-34 crystal when the reaction temperature is at 600 K or lower, whereas larger HCP species are gradually formed inwards the crystal at higher temperatures. In the case of ETO, the observed egg-white distribution of HCP at 509 K suggests that the ETO process is kinetically controlled, whereas the square-shaped HCP distribution at 650 K is indicative of a diffusion-controlled process. Finally, synchrotron-based IR microspectroscopy revealed a higher degree of alkylation for aromatics for MTO as compared to ETO, whereas high reaction temperatures favor dealkylation processes for both the MTO and ETO processes. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Degradation mechanism of alachlor during direct ozonation and O(3)/H(2)O(2) advanced oxidation process.

PubMed

Qiang, Zhimin; Liu, Chao; Dong, Bingzhi; Zhang, Yalei

2010-01-01

The degradation of alachlor by direct ozonation and advanced oxidation process O(3)/H(2)O(2) was investigated in this study with focus on identification of degradation byproducts. The second-order reaction rate constant between ozone and alachlor was determined to be 2.5+/-0.1M(-1)s(-1) at pH 7.0 and 20 degrees C. Twelve and eight high-molecular-weight byproducts (with the benzene ring intact) from alachlor degradation were identified during direct ozonation and O(3)/H(2)O(2), respectively. The common degradation byproducts included N-(2,6-diethylphenyl)-methyleneamine, 8-ethyl-3,4-dihydro-quinoline, 8-ethyl-quinoline, 1-chloroacetyl-2-hydro-3-ketone-7-acetyl-indole, 2-chloro-2',6'-diacetyl-N-(methoxymethyl)acetanilide, 2-chloro-2'-acetyl-6'-ethyl-N-(methoxymethyl)-acetanilide, and two hydroxylated alachlor isomers. In direct ozonation, four more byproducts were also identified including 1-chloroacetyl-2,3-dihydro-7-ethyl-indole, 2-chloro-2',6'-ethyl-acetanilide, 2-chloro-2',6'-acetyl-acetanilide and 2-chloro-2'-ethyl-6'-acetyl-N-(methoxymethyl)-acetanilide. Degradation of alachlor by O(3) and O(3)/H(2)O(2) also led to the formation of low-molecular-weight byproducts including formic, acetic, propionic, monochloroacetic and oxalic acids as well as chloride ion (only detected in O(3)/H(2)O(2)). Nitrite and nitrate formation was negligible. Alachlor degradation occurred via oxidation of the arylethyl group, N-dealkylation, cyclization and cleavage of benzene ring. After O(3) or O(3)/H(2)O(2) treatment, the toxicity of alachlor solution examined by the Daphnia magna bioassay was slightly reduced. 2009 Elsevier Ltd. All rights reserved.

Differences in hepatic microsomal cytochrome P-450 isoenzyme induction by pyrazole, chronic ethanol, 3-methylcholanthrene, and phenobarbital in high alcohol sensitivity (HAS) and low alcohol sensitivity (LAS) rats.

PubMed

Lucas, D; Ménez, J F; Berthou, F; Cauvin, J M; Deitrich, R A

1992-10-01

High and low alcohol sensitivity (HAS and LAS) rats have been selected for their differences in ethanol-induced sleep time. Liver monooxygenase activities were studied in HAS and LAS rats before and after treatments with known inducers such as chronic ethanol, pyrazole, 3-methylcholanthrene (3-MC) and phenobarbital (PB) to determine whether the selection procedure also selected for differences in the cytochrome P-450 (P-450) inducibility. This previously has been shown with long sleep (LS) and short sleep (SS) mice, which were selected using a similar criterion. 3-MC and PB, in conjunction with chronic ethanol treatment, were used in order to evaluate the interactions of ethanol with these inducers. Prior to treatment, total P-450 content was slightly lower in LAS than in HAS rats. However, both lines displayed the same microsomal monooxygenase activities related to different P-450 isozymes. This was demonstrated by ethoxyresorufin deethylation (EROD) for cytochrome P-450 1A1 (CYP1A1), acetanilide hydroxylation (ACET) for CYP1A2, pentoxyresorufin dealkylation (PROD) for CYP2B, 1-butanol oxidation (BUTAN) and N-nitrosodimethylamine demethylation (NDMA) for CYP2E1. After the different treatments, HAS rats did not differ from LAS rats in their CYP2E1 inducibility. However, pyrazole, PB and 3-MC treatment led to differences in CYP1A and CYP2B monooxygenase activities between the two lines. The enhancement of PROD by pyrazole treatment was less prominent in LAS (1.7-fold of the control value) than in HAS rats (3.8-fold).(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of anxiolytics and hypnotics: benzodiazepines, buspirone, zoplicone, and zolpidem.

PubMed

Chouinard, G; Lefko-Singh, K; Teboul, E

1999-08-01

1. The benzodiazepines are among the most frequently prescribed of all drugs and have been used for their anxiolytic, anticonvulsant, and sedative/hypnotic properties. Since absorption rates, volumes of distribution, and elimination rates differ greatly among the benzodiazepine derivatives, each benzodiazepine has a unique plasma concentration curve. Although the time to peak plasma levels provides a rough guide, it is not equivalent to the time to clinical onset of effect. The importance of alpha and beta half-lives in the actions of benzodiazepines is discussed. 2. The role of cytochrome P450 isozymes in the metabolism of benzodiazepines and in potential pharmacokinetic interactions between the benzodiazepines and other coadministered drugs is discussed. 3. Buspirone, an anxiolytic with minimal sedative effects, undergoes extensive metabolism, with hydroxylation and dealkylation being the major pathways. Pharmacokinetic interactions of buspirone with other coadministered drugs seem to be minimal. 4. Zopiclone and zolpidem are used primarily as hypnotics. Both are extensively metabolized; N-demethylation, N-oxidation, and decarboxylation of zopiclone occur, and zolpidem undergoes oxidation of methyl groups and hydroxylation of a position on the imidazolepyridine ring system. Zopiclone has a chiral centre, and demonstrates stereoselective pharmacokinetics. Metabolic drug-drug interactions have been reported with zopiclone and erythromycin, trimipramine, and carbamazepine. Reports to date indicate minimal interactions of zolpidem with coadministered drugs; however, it has been reported to affect the Cmax and clearance of chlorpromazepine and to decrease metabolism of the antiviral agent ritonavin. Since CYP3A4 has been reported to play an important role in metabolism of zolpidem, possible interactions with drugs which are substrates and/or inhibitors of that CYP isozyme should be considered.

Identification of the cytochrome P450 enzymes involved in the metabolism of cisapride: in vitro studies of potential co-medication interactions

PubMed Central

Bohets, H; Lavrijsen, K; Hendrickx, J; van Houdt, J; van Genechten, V; Verboven, P; Meuldermans, W; Heykants, J

2000-01-01

Cisapride is a prokinetic drug that is widely used to facilitate gastrointestinal tract motility.Structurally, cisapride is a substituted piperidinyl benzamide that interacts with 5-hydroxytryptamine-4 receptors and which is largely without central depressant or antidopaminergic side-effects.The aims of this study were to investigate the metabolism of cisapride in human liver microsomes and to determine which cytochrome P-450 (CYP) isoenzyme(s) are involved in cisapride biotransformation. Additionally, the effects of various drugs on the metabolism of cisapride were investigated.The major in vitro metabolite of cisapride was formed by oxidative N-dealkylation at the piperidine nitrogen, leading to the production of norcisapride.By using competitive inhibition data, correlation studies and heterologous expression systems, it was demonstrated that CYP3A4 was the major CYP involved. CYP2A6 also contributed to the metabolism of cisapride, albeit to a much lesser extent.The mean apparent Km against cisapride was 8.6±3.5 μM (n=3). The peak plasma levels of cisapride under normal clinical practice are approximately 0.17 μM; therefore it is unlikely that cisapride would inhibit the metabolism of co-administered drugs.In this in vitro study the inhibitory effects of 44 drugs were tested for any effect on cisapride biotransformation. In conclusion, 34 of the drugs are unlikely to have a clinically relevant interaction; however, the antidepressant nefazodone, the macrolide antibiotic troleandomycin, the HIV-1 protease inhibitors ritonavir and indinavir and the calcium channel blocker mibefradil inhibited the metabolism of cisapride and these interactions are likely to be of clinical relevance. Furthermore, the antimycotics ketoconazole, miconazole, hydroxy-itraconazole, itraconazole and fluconazole, when administered orally or intravenously, would inhibit cisapride metabolism. PMID:10780971

Development, validation and application of the liquid chromatography tandem mass spectrometry method for simultaneous quantification of azilsartan medoxomil (TAK-491), azilsartan (TAK-536), and its 2 metabolites in human plasma.

PubMed

Kuze, Yoji; Kogame, Akifumi; Jinno, Fumihiro; Kondo, Takahiro; Asahi, Satoru

2015-09-15

Azilsartan medoxomil potassium salt (TAK-491) is an orally administered angiotensin II type 1 receptor blocker for the treatment of hypertension and is an ester-based prodrug that is rapidly hydrolyzed to the pharmacologically active moiety, azilsartan (TAK-536), during absorption. TAK-536 is biotransformed to the 2 metabolites M-I by decarboxylation and M-II by dealkylation. In this study, we developed and validated a LC/MS/MS method which can simultaneously determine 4 analytes, TAK-491, TAK-536, M-I and M-II. The bioanalytical method can be outlined as follows: two structural analogues are used as the internal standards. The analytes and the IS are extracted from human plasma using solid phase extraction. After evaporating, the residue is reconstituted and injected into a LC/MS/MS system with an ESI probe and analyzed in the positive ion mode. Separation is performed through a conventional reversed-phase column with a mobile phase of water/acetonitrile/acetic acid (40:60:0.05, v/v/v) mixture at a flow rate of 0.2mL/min. The total run time is 8.5min. The calibration range is 1-2500ng/mL in human plasma for all the analytes. Instability issues of the prodrug, TAK-491, were overcome and all the validation results met the acceptance criteria in accordance with the regulatory guideline/guidance. As a result of the clinical study, the human PK profiles of TAK-536, M-I and M-II were successfully obtained and also it was confirmed that TAK-491 was below the LLOQ (1ng/mL) in the human plasma samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Hydrous pyrolysis of polycyclic aromatic hydrocarbons and implications for the origin of PAH in hydrothermal petroleum

NASA Technical Reports Server (NTRS)

McCollom, T. M.; Simoneit, B. R.; Shock, E. L.

1999-01-01

Polycyclic aromatic hydrocarbons (PAH) are found at high concentrations in thermally altered organic matter and hydrothermally generated petroleum from sediment-covered seafloor hydro-thermal systems. To better understand the factors controlling the occurrence of PAH in thermally altered environments, the reactivities of two PAH, phenanthrene and anthracene, were investigated in hydrothermal experiments. The compounds were heated with water at 330 degrees C in sealed reaction vessels for durations ranging from 1 to 17 days. Iron oxide and sulfide minerals, formic acid, or sodium for-mate were included in some experiments to vary conditions within the reaction vessel. Phenanthrene was unreactive both in water alone and in the presence of minerals for up to 17 days, while anthracene was partially hydrogenated (5-10%) to di- and tetrahydroanthracene. In the presence of 6-21 vol % formic acid, both phenanthrene and anthracene reacted extensively to form hydrogenated and minor methylated derivatives, with the degree of hydrogenation and methylation increasing with the amount of formic acid. Phenanthrene was slightly hydrogenated in sodium formate solutions. The hydrogenation reactions could be readily reversed; heating a mixture of polysaturated phenanthrenes resulted in extensive dehydrogenation (aromatization) after 3 days at 330 degrees C. While the experiments demonstrate that reaction pathways for the hydrogenation of PAH under hydrothermal conditions exist, the reactions apparently require higher concentrations of H2 than are typical of geologic settings. The experiments provide additional evidence that PAH may be generated in hydrothermal systems from progressive aromatization and dealkylation of biologically derived polycyclic precursors such as steroids and terpenoids. Furthermore, the results indicate that PAH initially present in sediments or formed within hydrothermal systems are resistant to further thermal degradation during hydrothermal alteration.

Cytotoxicity of chloroacetanilide herbicide alachlor in HepG2 cells independent of CYP3A4 and CYP3A7.

PubMed

Miranda, Sonia R; Meyer, Sharon A

2007-05-01

Alachlor is cytotoxic to human hepatoblastoma HepG2s, a cell line that expresses constitutive CYP3A7 and dexamethasone (DEX)-inducible CYP3A4 and CYP3A7. CYP3A4 catalyzes alachlor N-dealkylation to 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA), precursor of 2,6-diethylbenzoquinoneimine, putative reactive metabolite for rat nasal carcinogenicity. We hypothesized that HepG2 alachlor cytotoxicity would be mediated by CYP3A4/7 and increased with DEX. Here, we report time-dependent alachlor cytotoxicity (EC(50) approximately 500 microM and 264+/-17 microM at 6 and 24h, respectively) as assessed by lactate dehydrogenase leakage. DEX pretreatment (25 microM, 48 h) significantly increased CYP3A7-catalyzed luciferin 6' benzylether O-debenzylation, but had no effect on alachlor toxicity. Further, CYP3A4/7 inhibitor triacetyloleandomycin did not prevent, but rather potentiated, alachlor cytotoxicity. In agreement, CDEPA was less toxic than parent alachlor. HepG2 CYP3A4 activity was unaffected by 48 h DEX pretreatment; therefore, studies were done in DPX-2 cells, a HepG2 derivative engineered to overexpress pregnane-X receptor (PXR) that exhibits rifampicin (RIF)-inducible endogenous CYP3A4. Alachlor cytotoxicity in DPX-2 cells occurred over a concentration range equivalent to that in HepG2. CYP3A4 activity of DPX-2 cells treated with RIF (10 microM, 48 h) was twice that of untreated cells, but RIF did not increase alachlor toxicity. These results demonstrate that neither CYP3A4 nor CYP3A7 initiate a pathway leading to a toxic alachlor metabolite.

Defective Cytochrome P450-Catalysed Drug Metabolism in Niemann-Pick Type C Disease

PubMed Central

Wassif, Christopher A.; Gray, James; Burkert, Kathryn R.; Smith, David A.; Morris, Lauren; Cologna, Stephanie M.; Peer, Cody J.; Sissung, Tristan M.; Uscatu, Constantin-Daniel; Figg, William D.; Pavan, William J.; Vite, Charles H.; Porter, Forbes D.; Platt, Frances M.

2016-01-01

Niemann-Pick type C (NPC) disease is a neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 gene. NPC is characterised by storage of multiple lipids in the late endosomal/lysosomal compartment, resulting in cellular and organ system dysfunction. The underlying molecular mechanisms that lead to the range of clinical presentations in NPC are not fully understood. While evaluating potential small molecule therapies in Npc1-/- mice, we observed a consistent pattern of toxicity associated with drugs metabolised by the cytochrome P450 system, suggesting a potential drug metabolism defect in NPC1 disease. Investigation of the P450 system in the context of NPC1 dysfunction revealed significant changes in the gene expression of many P450 associated genes across the full lifespan of Npc1-/- mice, decreased activity of cytochrome P450 reductase, and a global decrease of multiple cytochrome P450 catalysed dealkylation reactions. In vivo drug metabolism studies using a prototypic P450 metabolised drug, midazolam, confirmed dysfunction in drug clearance in the Npc1-/- mouse. Expression of the Phase II enzyme uridinediphosphate-glucuronosyltransferase (UGT) was also significantly reduced in Npc1-/- mice. Interestingly, reduced activity within the P450 system was also observed in heterozygous Npc1+/- mice. The reduced activity of P450 enzymes may be the result of bile acid deficiency/imbalance in Npc1-/- mice, as bile acid treatment significantly rescued P450 enzyme activity in Npc1-/- mice and has the potential to be an adjunctive therapy for NPC disease patients. The dysfunction in the cytochrome P450 system were recapitulated in the NPC1 feline model. Additionally, we present the first evidence that there are alterations in the P450 system in NPC1 patients. PMID:27019000

Metabolism of halofantrine to its equipotent metabolite, desbutylhalofantrine, is decreased when orally administered with ketoconazole.

PubMed

Khoo, S M; Porter, J H; Edwards, G A; Charman, W N

1998-12-01

Halofantrine (Hf) is a highly lipophilic antimalarial with poor and erratic absorption. Published data indicates that the oral bioavailability of Hf was increased 3-fold in humans and 12-fold in dogs when administered postprandially; however, the proportional formation of the active desbutyl metabolite (desbutylhalofantrine, Hfm) decreased 2.4-fold in humans and 6.8-fold in dogs (Milton et al., Br. J. Clin. Pharmacol. 1989, 28, 71-77; Humberstone et al., J. Pharm. Sci. 1996, 85, 525-529). The current study was undertaken to confirm the putative involvement of CYP3A4 in the N-dealkylation of Hf to Hfm by administering Hf with and without ketoconazole (KC), a specific CYP3A4 inhibitor, and measuring the resulting plasma concentration profiles of Hf and Hfm. The plasma Hfm/Hf AUC(0-72 h) ratio after fasted oral administration of Hf without KC was 0.56, whereas the ratio after fasted oral administration with KC was less than 0.05. It is likely that both hepatic and prehepatic (enterocyte-based) CYP3A4 contributed to metabolism of Hf to Hfm after oral administration. Interestingly, the low plasma Hfm/Hf AUC ratios observed after fasted administration of Hf with KC were similar to the low values previously observed when Hf was administered postprandially (despite increased Hf absorption). The mechanism(s) by which postprandial administration of Hf led to a decrease in its metabolism are unknown, but based on the current data, could include inhibition of CYP3A4-mediated metabolism by components of the ingested meal. Other possibilities include a lipid-induced postprandial recruitment of intestinal lymphatic transport or avoidance of metabolism during transport through the enterocyte into the portal blood. Further studies are required to determine the relative contributions by which these different processes may decrease the presystemic metabolism of Hf.

The Mechanism of Autoinduction of Methadone N-demethylation in Human Hepatocytes

PubMed Central

Campbell, Scott D.; Crafford, Amanda; Williamson, Brian L.; Kharasch, Evan D.

2013-01-01

Background There is considerable inter-and intraindividual variability in methadone metabolism and clearance. Methadone dosing is particularly challenging during initiation of therapy, due to time-dependent increases in hepatic clearance (autoinduction). Although methadone N-demethylation is catalyzed in vitro by cytochrome P4502B6 (CYP2B6) and CYP3A4, and clearance in vivo depends on CYP2B6, mechanism(s) of autoinduction are incompletely understood. In this investigation we determined mechanism(s) of methadone autoinduction using human hepatocytes. Methods Fresh human hepatocytes were exposed to 0.1-10 μM methadone for 72 hr. Cells were washed and methadone N-demethylation assessed. CYP2B6, CYP3A4, and CYP3A5 mRNA, protein expression (by gel-free high performance liquid chromatography-mass spectrometry) and catalytic activity (bupropion hydroxylation and alfentanil dealkylation for CYP2B6 and CYP3A4/5, respectively) were measured. Mechanisms of CYP induction were characterized using pregnane X receptor and constitutive androstane receptor reporter gene assays. Results Methadone (10 μM) increased methadone N-demethylation 2-fold, CYP2B6 and CYP3A4 mRNA 3-fold, and protein expression 2-fold. CYP3A5 mRNA was unchanged. CYP2B6 and CYP3A4/5 activities increased 2-fold. Induction by methadone enantiomers (R- vs S-methadone) did not differ. Induction was relatively weak compared with maximum induction by phenobarbital and rifampin. Lower methadone concentrations had smaller effects. Methadone was an agonist for the pregnane X receptor but not the constitutive androstane receptor. Conclusions Methadone caused concentration-dependent autoinduction of methadone N-demethylation in human hepatocytes, related to induction of CYP2B6 and CYP3A4 mRNA expression, protein expression, and catalytic activity. Induction was related to pregnane X receptor but not constitutive androstane receptor activation. These in vitro findings provide mechanistic insights into clinical autoinduction of methadone metabolism and clearance. PMID:23733841

Characterization of the biotransformation pathways of clomiphene, tamoxifen and toremifene as assessed by LC-MS/(MS) following in vitro and excretion studies.

PubMed

Mazzarino, Monica; Biava, Mariangela; de la Torre, Xavier; Fiacco, Ilaria; Botrè, Francesco

2013-06-01

The use of selective oestrogen receptor modulators has been prohibited since 2005 by the World Anti-Doping Agency regulations. As they are extensively cleared by hepatic and intestinal metabolism via oxidative and conjugating enzymes, a complete investigation of their biotransformation pathways and kinetics of excretion is essential for the anti-doping laboratories to select the right marker(s) of misuse. This work was designed to characterize the chemical reactions and the metabolizing enzymes involved in the metabolic routes of clomiphene, tamoxifen and toremifene. To determine the biotransformation pathways of the substrates under investigation, urine samples were collected from six subjects (three females and three males) after oral administration of 50 mg of clomiphene citrate or 40 mg of tamoxifen or 60 mg of toremifene, whereas the metabolizing enzymes were characterized in vitro, using expressed cytochrome P450s and uridine diphosphoglucuronosyltransferases. The separation, identification and determination of the compounds formed in the in vivo and in vitro experiments were carried out by liquid chromatography coupled with mass spectrometry techniques using different acquisition modes. Clomiphene, tamoxifen and toremifene were biotransformed to 22, 23 and 18 metabolites respectively, these phase I reactions being catalyzed mainly by CYP3A4 and CYP2D6 isoforms and, to a lesser degree, by CYP3A5, CYP2B6, CYP2C9, CYP2C19 isoforms. The phase I metabolic reactions include hydroxylation in different positions, N-oxidation, dehalogenation, carboxylation, hydrogenation, methoxylation, N-dealkylation and combinations of them. In turn, most of the phase I metabolites underwent conjugation reaction to form the corresponding glucuro-conjugated mainly by UGT1A1, UGT1A3, UGT1A4, UGT2B7, UGT2B15 and UGT2B17 isoenzymes.

Characterization of the hepatic cytochrome P450 enzymes involved in the metabolism of 25I-NBOMe and 25I-NBOH.

PubMed

Nielsen, Line Marie; Holm, Niels Bjerre; Leth-Petersen, Sebastian; Kristensen, Jesper Langgaard; Olsen, Lars; Linnet, Kristian

2017-05-01

The dimethoxyphenyl-N-((2-methoxyphenyl)methyl)ethanamine (NBOMe) compounds are potent serotonin 5-HT2A receptor agonists and have recently been subject to recreational use due to their hallucinogenic effects. Use of NBOMe compounds has been known since 2011, and several non-fatal and fatal intoxication cases have been reported in the scientific literature. The aim of this study was to determine the importance of the different cytochrome P450 enzymes (CYP) involved in the metabolism of 2-(4-iodo-2,5-dimethoxyphenyl)-N-(2methoxybenzyl)ethanamine (25I-NBOMe) and 2-[[2-(4-iodo-2,5dimethoxyphenyl)ethylamino]methyl]phenol (25I-NBOH) and to characterize the metabolites. The following approaches were used to identify the main enzymes involved in primary metabolism: incubation with a panel of CYP and monoamine oxidase (MAO) enzymes and incubation in pooled human liver microsomes (HLM) with and without specific CYP chemical inhibitors. The study was further substantiated by an evaluation of 25I-NBOMe and 25I-NBOH metabolism in single donor HLM. The metabolism pathways of 25I-NBOMe and 25I-NBOH were NADPHdependent with intrinsic clearance values of (CLint) of 70.1 and 118.7 mL/min/kg, respectively. The biotransformations included hydroxylation, O-demethylation, N-dealkylation, dehydrogenation, and combinations thereof. The most abundant metabolites were all identified by retention time and spectrum matching with synthesized reference standards. The major CYP enzymes involved in the metabolism of 25I-NBOMe and 25INBOH were identified as CYP3A4 and CYP2D6, respectively. The compound 25I-NBOH was also liable to direct glucuronidation, which may diminish the impact of CYP2D6 genetic polymorphism. Users of 25I-NBOMe may be subject to drug-drug interactions (DDI) if 25I-NBOMe is taken with a strong CYP3A4 inhibitor. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.

PubMed

Schwarz, D; Kisselev, P; Honeck, H; Cascorbi, I; Schunck, W H; Roots, I

2001-06-01

1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (1462V) and CYP1A1.4 (T461N), were co-expressed with human NADPH-P450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus co-infection to elaborate a suitable system for studying the role of CYPA1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. tinder optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-co-infection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CY1A1 variants.

Cytosine Methylation Effects on the Repair of O6-Methylguanines within CG Dinucleotides*

PubMed Central

Guza, Rebecca; Ma, Linan; Fang, Qingming; Pegg, Anthony E.; Tretyakova, Natalia

2009-01-01

O6-Alkyldeoxyguanine adducts induced by tobacco-specific nitrosamines are repaired by O6-alkylguanine DNA alkyltransferase (AGT), which transfers the O6-alkyl group from the damaged base to a cysteine residue within the protein. In the present study, a mass spectrometry-based approach was used to analyze the effects of cytosine methylation on the kinetics of AGT repair of O6-methyldeoxyguanosine (O6-Me-dG) adducts placed within frequently mutated 5′-CG-3′ dinucleotides of the p53 tumor suppressor gene. O6-Me-dG-containing DNA duplexes were incubated with human recombinant AGT protein, followed by rapid quenching, acid hydrolysis, and isotope dilution high pressure liquid chromatography-electrospray ionization tandem mass spectrometry analysis of unrepaired O6-methylguanine. Second-order rate constants were calculated in the absence or presence of the C-5 methyl group at neighboring cytosine residues. We found that the kinetics of AGT-mediated repair of O6-Me-dG were affected by neighboring 5-methylcytosine (MeC) in a sequence-dependent manner. AGT repair of O6-Me-dG adducts placed within 5′-CG-3′ dinucleotides of p53 codons 245 and 248 was hindered when MeC was present in both DNA strands. In contrast, cytosine methylation within p53 codon 158 slightly increased the rate of O6-Me-dG repair by AGT. The effects of MeC located immediately 5′ and in the base paired position to O6-Me-dG were not additive as revealed by experiments with hypomethylated sequences. Furthermore, differences in dealkylation rates did not correlate with AGT protein affinity for cytosine-methylated and unmethylated DNA duplexes or with the rates of AGT-mediated nucleotide flipping, suggesting that MeC influences other kinetic steps involved in repair, e.g. the rate of alkyl transfer from DNA to AGT. PMID:19531487

Cytosine methylation effects on the repair of O6-methylguanines within CG dinucleotides.

PubMed

Guza, Rebecca; Ma, Linan; Fang, Qingming; Pegg, Anthony E; Tretyakova, Natalia

2009-08-21

O(6)-alkyldeoxyguanine adducts induced by tobacco-specific nitrosamines are repaired by O(6)-alkylguanine DNA alkyltransferase (AGT), which transfers the O(6)-alkyl group from the damaged base to a cysteine residue within the protein. In the present study, a mass spectrometry-based approach was used to analyze the effects of cytosine methylation on the kinetics of AGT repair of O(6)-methyldeoxyguanosine (O(6)-Me-dG) adducts placed within frequently mutated 5'-CG-3' dinucleotides of the p53 tumor suppressor gene. O(6)-Me-dG-containing DNA duplexes were incubated with human recombinant AGT protein, followed by rapid quenching, acid hydrolysis, and isotope dilution high pressure liquid chromatography-electrospray ionization tandem mass spectrometry analysis of unrepaired O(6)-methylguanine. Second-order rate constants were calculated in the absence or presence of the C-5 methyl group at neighboring cytosine residues. We found that the kinetics of AGT-mediated repair of O(6)-Me-dG were affected by neighboring 5-methylcytosine ((Me)C) in a sequence-dependent manner. AGT repair of O(6)-Me-dG adducts placed within 5'-CG-3' dinucleotides of p53 codons 245 and 248 was hindered when (Me)C was present in both DNA strands. In contrast, cytosine methylation within p53 codon 158 slightly increased the rate of O(6)-Me-dG repair by AGT. The effects of (Me)C located immediately 5' and in the base paired position to O(6)-Me-dG were not additive as revealed by experiments with hypomethylated sequences. Furthermore, differences in dealkylation rates did not correlate with AGT protein affinity for cytosine-methylated and unmethylated DNA duplexes or with the rates of AGT-mediated nucleotide flipping, suggesting that (Me)C influences other kinetic steps involved in repair, e.g. the rate of alkyl transfer from DNA to AGT.

Metabolic Profile of Synthetic Cannabinoids 5F-PB-22, PB-22, XLR-11 and UR-144 by Cunninghamella elegans.

PubMed

Watanabe, Shimpei; Kuzhiumparambil, Unnikrishnan; Nguyen, My Ann; Cameron, Jane; Fu, Shanlin

2017-07-01

The knowledge of metabolic profile of synthetic cannabinoids is important for the detection of drugs in urinalysis due to the typical absence or low abundance of parent cannabinoids in human urine. The fungus Cunninghamella elegans has been reported to be a useful tool for metabolism study and thus applicability to synthetic cannabinoid metabolism was examined. In this study, 8-quinolinyl 1-(5-fluoropentyl)-1H-indole-3-carboxylate (5F-PB-22), 8-quinolinyl 1-pentyl-1H-indole-3-carboxylate (PB-22), [1-(5-fluoropentyl)-1H-indol-3-yl](2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11) and (1-pentyl-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone (UR-144) were incubated with C. elegans and the metabolites were identified using liquid chromatography-quadrupole time-of-flight mass spectrometry. The obtained metabolites were compared with reported human metabolites to assess the suitability of the fungus to extrapolate human metabolism. 5F-PB-22 underwent dihydroxylation, dihydrodiol formation, oxidative defluorination, oxidative defluorination to carboxylic acid, ester hydrolysis and glucosidation, alone and/or in combination. The metabolites of PB-22 were generated by hydroxylation, dihydroxylation, trihydroxylation, dihydrodiol formation, ketone formation, carboxylation, ester hydrolysis and glucosidation, alone and/or in combination. XLR-11 was transformed through hydroxylation, dihydroxylation, aldehyde formation, carboxylation, oxidative defluorination, oxidative defluorination to carboxylic acid and glucosidation, alone and/or in combination. UR-144 was metabolised by hydroxylation, dihydroxylation, trihydroxylation, aldehyde formation, ketone formation, carboxylation, N-dealkylation and combinations. These findings were consistent with previously reported human metabolism except for the small extent of ester hydrolysis observed and the absence of glucuronidation. Despite the limitations, C. elegans demonstrated the capacity to produce a wide variety of metabolites including some major human metabolites of XLR-11 and UR-144 at high abundance, showing the potential for metabolism of newly emerging synthetic cannabinoids.

Effect of Ginkgo biloba extract on procarcinogen-bioactivating human CYP1 enzymes: Identification of isorhamnetin, kaempferol, and quercetin as potent inhibitors of CYP1B1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Thomas K.H.; Chen Jie; Yeung, Eugene Y.H.

2006-05-15

In the present study, we investigated the effect of Ginkgo biloba extracts and some of its individual constituents on the catalytic activity of human cytochrome P450 enzymes CYP1B1, CYP1A1, and CYP1A2. G. biloba extract of known abundance of terpene trilactones and flavonol glycosides inhibited 7-ethoxyresorufin O-dealkylation catalyzed by human recombinant CYP1B1, CYP1A1, and CYP1A2, and human liver microsomes, with apparent K {sub i} values of 2 {+-} 0.3, 5 {+-} 0.5, 16 {+-} 1.4, and 39 {+-} 1.2 {mu}g/ml (mean {+-} SE), respectively. In each case, the mode of inhibition was of the mixed type. Bilobalide, ginkgolides A, B, C,more » and J, quercetin 3-O-rutinoside, kaempferol 3-O-rutinoside, and isorhamentin 3-O-rutinoside were not responsible for the inhibition of CYP1 enzymes by G. biloba extract, as determined by experiments with these individual chemicals at the levels present in the extract. In contrast, the aglycones of quercetin, kaempferol, and isorhamentin inhibited CYP1B1, CYP1A1, and CYP1A2. Among the three flavonol aglycones, isorhamentin was the most potent in inhibiting CYP1B1 (apparent K {sub i} = 3 {+-} 0.1 nM), whereas quercetin was the least potent in inhibiting CYP1A2 (apparent K {sub i} 418 {+-} 50 nM). The mode of inhibition was competitive, noncompetitive, or mixed, depending on the enzyme and the flavonol. G. biloba extract also reduced benzo[a]pyrene hydroxylation, and the effect was greater with CYP1B1 than with CYP1A1 as the catalyst. Overall, our novel findings indicate that G. biloba extract and the flavonol aglycones isorhamnetin, kaempferol, and quercetin preferentially inhibit the in vitro catalytic activity of human CYP1B1.« less

Cometabolism of Methyl tertiary Butyl Ether and Gaseous n-Alkanes by Pseudomonas mendocina KR-1 Grown on C5 to C8 n-Alkanes

PubMed Central

Smith, Christy A.; O'Reilly, Kirk T.; Hyman, Michael R.

2003-01-01

Pseudomonas mendocina KR-1 grew well on toluene, n-alkanes (C5 to C8), and 1° alcohols (C2 to C8) but not on other aromatics, gaseous n-alkanes (C1 to C4), isoalkanes (C4 to C6), 2° alcohols (C3 to C8), methyl tertiary butyl ether (MTBE), or tertiary butyl alcohol (TBA). Cells grown under carbon-limited conditions on n-alkanes in the presence of MTBE (42 μmol) oxidized up to 94% of the added MTBE to TBA. Less than 3% of the added MTBE was oxidized to TBA when cells were grown on either 1° alcohols, toluene, or dextrose in the presence of MTBE. Concentrated n-pentane-grown cells oxidized MTBE to TBA without a lag phase and without generating tertiary butyl formate (TBF) as an intermediate. Neither TBF nor TBA was consumed by n-pentane-grown cells, while formaldehyde, the expected C1 product of MTBE dealkylation, was rapidly consumed. Similar Ks values for MTBE were observed for cells grown on C5 to C8 n-alkanes (12.95 ± 2.04 mM), suggesting that the same enzyme oxidizes MTBE in cells grown on each n-alkane. All growth-supporting n-alkanes (C5 to C8) inhibited MTBE oxidation by resting n-pentane-grown cells. Propane (Ki = 53 μM) and n-butane (Ki = 16 μM) also inhibited MTBE oxidation, and both gases were also consumed by cells during growth on n-pentane. Cultures grown on C5 to C8 n-alkanes also exhibited up to twofold-higher levels of growth in the presence of propane or n-butane, whereas no growth stimulation was observed with methane, ethane, MTBE, TBA, or formaldehyde. The results are discussed in terms of their impacts on our understanding of MTBE biodegradation and cometabolism. PMID:14660389

Effects of chloro-s-triazine herbicides and metabolites on aromatase activity in various human cell lines and on vitellogenin production in male carp hepatocytes.

PubMed Central

Sanderson, J T; Letcher, R J; Heneweer, M; Giesy, J P; van den Berg, M

2001-01-01

We investigated a potential mechanism for the estrogenic properties of three chloro-s-triazine herbicides and six metabolites in vitro in several cell systems. We determined effects on human aromatase (CYP19), the enzyme that converts androgens to estrogens, in H295R (adrenocortical carcinoma), JEG-3 (placental choriocarcinoma), and MCF-7 (breast cancer) cells; we determined effects on estrogen receptor-mediated induction of vitellogenin in primary hepatocyte cultures of adult male carp (Cyprinus carpio). In addition to atrazine, simazine, and propazine, two metabolites--atrazine-desethyl and atrazine-desisopropyl--induced aromatase activity in H295R cells concentration-dependently (0.3-30 microM) and with potencies similar to those of the parent triazines. After a 24-hr exposure to 30 microM of the triazines, an apparent maximum induction of about 2- to 2.5-fold was achieved. The induction responses were confirmed by similar increases in CYP19 mRNA levels, determined by reverse-transcriptase polymerase chain reaction. In JEG-3 cells, where basal aromatase expression is about 15-fold greater than in H295R cells, the induction responses were similar but less pronounced; aromatase expression in MCF-7 cells was neither detectable nor inducible under our culture conditions. The fully dealkylated metabolite atrazine-desethyl-desisopropyl and the three hydroxylated metabolites (2-OH-atrazine-desethyl, -desisopropyl, and -desethyl-desisopropyl) did not induce aromatase activity. None of the triazine herbicides nor their metabolites induced vitellogenin production in male carp hepatocytes; nor did they antagonize the induction of vitellogenin by 100 nM (EC(50) 17beta-estradiol. These findings together with other reports indicate that the estrogenic effects associated with the triazine herbicides in vivo are not estrogen receptor-mediated, but may be explained partly by their ability to induce aromatase in vitro. PMID:11675267

Suppressive effects of caraway (Carum carvi) extracts on 2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin-dependent gene expression of cytochrome P450 1A1 in the rat H4IIE cells.

PubMed

Naderi-Kalali, B; Allameh, A; Rasaee, M J; Bach, H-J; Behechti, A; Doods, K; Kettrup, A; Schramm, K-W

2005-04-01

Cytochrome P450 1A1 (CYP1A1) is among the cytochrome P450 classes known to convert xenobiotics and endogenous compounds to toxic and/or carcinogenic metabolites. Suppression of CYP1A1 over expression by certain compounds is implicated in prevention of cancer caused by chemical carcinogens. Chemopreventive agents containing high levels of flavonoids and steroids-like compounds are known to suppress CYP1A1. This study was carried out for assessment of the genomic and proteomic effects of caraway (Carum carvi) extracts containing high levels of both flavonoids and steroid-like substances on ethoxy resorufin dealkylation (EROD) activity and CYP1A1 at mRNA levels. Rat hepatoma cells co-treated with a CYP1A1 inducer i.e. TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin) and different preparations of caraway extracts at concentrations of 0, 0.13, 1.3, and 13 microM in culture medium. After incubation (37 degrees C and 7% CO2 for 20 h), changes in EROD specific activity recorded and compared in cells under different treatments. The results show that caraway seed extract prepared in three different organic solvents suppressed the enzyme activity in hepatoma cells in a dose-dependent manner. The extracts added above 0.13 microM could significantly inhibit EROD activity and higher levels of each extract (1.3 and 13 microM) caused approximately 10-fold suppression in the enzyme activity. Accordingly, data obtained from the RT-PCR (TaqMan) clearly showed the suppressive effects of plant extract on CYP1A1-related mRNA expression. These data clearly show that substances in caraway seeds extractable in organic solvents can potentially reverse the TCDD-dependent induction in cytochrome P450 1A1.

High-resolution mass spectrometric metabolite profiling of a novel synthetic designer drug, N-(adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135) using cryopreserved human hepatocytes and assessment of metabolic stability with human liver microsomes

PubMed Central

Gandhi, Adarsh S.; Wohlfarth, Ariane; Zhu, Mingshe; Pang, Shaokun; Castaneto, Marisol; Scheidweiler, Karl B.; Huestis, Marilyn A.

2014-01-01

N-(Adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135) is a new synthetic cannabinoid in herbal incense products discussed on internet drug user forums and identified in police seizures. To date, there are no STS-135 clinical or in vitro studies identifying STS-135 metabolic profiles. However, characterizing STS-135 metabolism is critical because synthetic cannabinoid metabolites can possess pharmacological activity and parent compounds are rarely detectable in urine. To characterize the metabolite profile, human hepatocytes were incubated with 10 μmol/L STS-135 for up to 3 h. High-resolution mass spectrometry with software-assisted data mining identified 29 STS-135 metabolites. Less than 25% of STS-135 parent compound remained after 3 h incubation. Primary metabolites were generated by mono-, di- or trihydroxylation with and without ketone formation, dealkylation and oxidative defluorination of N-fluoropentyl side chain or possible oxidation to carboxylic acid, some of them further glucuronidated. Hydroxylations occurred mainly on the aliphatic adamantane ring and less commonly on the N-pentyl side chain. At 1 h phase I metabolites predominated, while at 3 h phase II metabolites were present in higher amounts. The major metabolites were monohydroxy STS-135 (M25) and dihydroxy STS-135 (M21), both hydroxylated on the adamantane system. Moreover, metabolic stability of STS-135 (1 μmol/L) was assessed in human liver microsomes experiments. The in vitro half-life of STS-135 was 7.2±0.6 min and intrinsic clearance (CLint) was 93.6 mL·min−1·kg−1. This is the first report characterizing STS-135 hepatic metabolic pathways. These data provide potential urinary targets to document STS-135 intake in clinical and forensic settings and potential candidates for pharmacological testing. PMID:24827428

Toxicity profiles in mice treated with hepatotumorigenic and non-hepatotumorigenic triazole conazole fungicides: Propiconazole, triadimefon, and myclobutanil.

PubMed

Allen, James W; Wolf, Douglas C; George, Michael H; Hester, Susan D; Sun, Guobin; Thai, Sheau-Fung; Delker, Don A; Moore, Tanya; Jones, Carlton; Nelson, Gail; Roop, Barbara C; Leavitt, Sharon; Winkfield, Ernest; Ward, William O; Nesnow, Stephen

2006-01-01

Conazoles comprise a class of fungicides used in agriculture and as pharmaceutical products. The fungicidal properties of conazoles are due to their inhibition of ergosterol biosynthesis. Certain conazoles are tumorigenic in rodents; both propiconazole and triadimefon are hepatotoxic and hepatotumorigenic in mice, while myclobutanil is not a mouse liver tumorigen. As a component of a large-scale study aimed at determining the mode(s) of action for tumorigenic conazoles, we report the results from comparative evaluations of liver and body weights, liver histopathology, cell proliferation, cytochrome P450 (CYP) activity, and serum cholesterol, high-density lipoprotein and triglyceride levels after exposure to propiconazole, triadimefon, and myclobutanil. Male CD-1 mice were treated in the feed for 4, 30, or 90 days with triadimefon (0, 100, 500, or 1800 ppm), propiconazole (0, 100, 500, or 2500 ppm) or myclobutanil (0, 100, 500, or 2000 ppm). Alkoxyresorufin O-dealkylation (AROD) assays indicated that all 3 chemicals induced similar patterns of dose-related increases in metabolizing enzyme activity. PROD activities exceeded those of MROD, and EROD with propiconazole inducing the highest activities of PROD. Mice had similar patterns of dose-dependent increases in hepatocyte hypertrophy after exposure to the 3 conazoles. High-dose exposures to propiconazole and myclobutanil, but not triadimefon, were associated with early (4 days) increases in cell proliferation. All the chemicals at high doses reduced serum cholesterol and high-density lipoprotein (HDL) levels at 30 days of treatment, while only triadimefon had this effect at 4 days of treatment and only myclobutanil and propiconazole at 90 days of treatment. Overall, the tumorigenic and nontumorigenic conazoles induced similar effects on mouse liver CYP enzyme activities and pathology. There was no specific pattern of tissue responses that could consistently be used to differentiate the tumorigenic conazoles, propiconazole, and triadimefon, from the nontumorigenic myclobutanil. These findings serve to anchor other transcriptional profiling studies aimed at probing differences in key events and modes of action for tumorigenic and nontumorigenic conazoles.

Identification of phase-I metabolites and chronic toxicity study of the Kv1.3 blocker PAP-1 (5-(4-phenoxybutoxy)psoralen) in the rat.

PubMed

Hao, B; Chen, Z-W; Zhou, X-J; Zimin, P I; Miljanich, G P; Wulff, H; Wang, Y-X

2011-03-01

1. PAP-1 (5-(4-phenoxybutoxy)psoralen), a potent small-molecule blocker of the voltage-gated potassium Kv1.3 channel, is currently in preclinical development for psoriasis. This study was undertaken to identify the major phase I metabolites of PAP-1 in Sprague-Dawley (SD) rats. 2. Five phase I metabolites, that is 5-(oxybutyric-acid)psoralen (M1), 5-[4-(4-hydroxybutoxy)]psoralen (M2), 5-[4-(4-hydroxyphenoxy)butoxy]psoralen (M3), 5-[4-(3-hydroxyphenoxy)butoxy]psoralen (M4), and 8-hydroxyl-5-(4-phenoxybutoxy)psoralen (M5), were isolated from the bile of rats and identified by mass spectrometry and NMR spectroscopy. The last four metabolites are new compounds. 3. Incubation of PAP-1 with SD rat liver microsomes rendered the same five major metabolites in a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent manner suggesting that cytochrome P450 (CYP) enzymes are involved in PAP-1 metabolism. Inhibitors of rat CYP1A1/2 (alpha-naphthoflavone) and CYP3A (ketoconazole) but not CYP2D6 (quinidine), CYP2E (diethyldithiocarbamate), or CYP2C9 (sulphaphenazole) blocked the metabolism of PAP-1 in rat microsomes. 4. Of the five metabolites M3, M4, and M5 were found to inhibit Kv1.3 currents with nanomolar IC50s, while M1 and M2 were inactive. Our results identified the Kv1.3-inactive M1 as the major phase I metabolite, and suggest that hydroxylation and O-dealkylation are the major pathways of PAP-1 metabolism. 5. We further conducted a 6-month repeat-dose toxicity study with PAP-1 at 50 mg/kg in both male and female Lewis rats and did not observe any toxic effects.

Effects of carbon amendment on in situ atrazine degradation and total microbial biomass.

PubMed

Ngigi, Anastasiah N; Getenga, Zachary M; Dörfler, Ulrike; Boga, Hamadi I; Kuria, Benson; Ndalut, Paul; Schroll, Reiner

2013-01-01

This study elucidates the effects of carbon amendment on metabolic degradation of atrazine (6-chloro-N(2)-ethyl-N(4)-isopropyl-1,3,5-triazine-2,4-diamine) and total microbial biomass in soil. Degradation of (14)C-ring-labelled atrazine was monitored in laboratory incubations of soils supplemented with 0, 10, 100 and 1000 μg g(-1) sucrose concentrations. An experiment to determine the effect of carbon amendment on total microbial biomass and soil respiration was carried out with different concentrations of sucrose and non-labelled atrazine. The soils were incubated at a constant temperature and constant soil moisture at water potential of -15 kPa and a soil density of 1.3 g cm(-3). Mineralization of (14)C-ring-labelled atrazine was monitored continuously over a period of 59 d in the first experiment. The CO(2) production was monitored for 62 d in the second experiment and microbial biomass determined at the end of the incubation period. The addition of 1000 μg g(-1) sucrose reduced atrazine mineralization to 43.5% compared to 51.7% of the applied amount for the treatment without sucrose. The addition of 1000 μg g(-1) sucrose modified the transformation products to 1.08 μg g(-1) deisopropylatrazine (DIA), 0.32 μg g(-1) desethylatrazine (DEA) and 0.18 μg g(-1) deisopropyl-2-hydroxyatrazine (OH-DIA). Treatment without sucrose resulted in formation of 0.64 μg g(-1) hydroxyatrazine (HA), 0.28 μg g(-1) DIA and 0.20 μg g(-1) OH-DIA. Atrazine dealkylation was enhanced in treatments with 100 and 1000 μg g(-1) of sucrose added. HA metabolite was formed in the control (no sucrose) and in the presence of 10 μg g(-1) of sucrose, whereas DEA was only detected in treatment with 1000 μg g(-1) sucrose. Results indicate that total microbial biomass increased significantly (P < 0.001) with the addition of 1000 μg g(-1) sucrose.

Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Seung Heon; Kang, Sukmo; Dong, Mi Sook

2015-06-15

Cytochrome P450 (CYP) 1A1 is a heme-containing enzyme involved in detoxification of hydrophobic pollutants. Its Ala62Pro variant has been identified previously. Ala62 is located in α-helix A of CYP1A1. Residues such as Pro and Gly are α-helix breakers. In this study, the Ala62Pro variant was characterized using heterologous expression. E. coli expressing the Ala62Pro variant, and the purified variant protein, had lower CYP (i.e. holoenzyme) contents than their wild-type (WT) equivalents. The CYP variant from E. coli and mammalian cells exhibited lower 7-ethoxyresorufin O-dealkylation (EROD) and benzo[a]pyrene hydroxylation activities than the WT. Enhanced supplementation of a heme precursor during E.more » coli culture did not increase CYP content in E. coli expressing the variant, but did for the WT. As for Ala62Pro, E. coli expressing an Ala62Gly variant had a lower CYP content than the WT counterpart, but substitution of Ala62 with α-helix-compatible residues such as Ser and Val partially recovered the level of CYP produced. Microsomes from mammalian cells expressing Ala62Pro and Ala62Gly variants exhibited lower EROD activities than those expressing the WT or Ala62Val variant. A region harboring α-helix A has interactions with another region containing heme-interacting residues. Site-directed mutagenesis analyses suggest the importance of interactions between the two regions on holoenzyme expression. Together, these findings suggest that the Ala62Pro substitution leads to changes in protein characteristics and function of CYP1A1 via structural disturbance of the region where the residue is located. - Highlights: • Ala62 is located in α-helix A of the carcinogen-metabolizing enzyme CYP1A1. • Pro acts as an α-helix breaker. • A variant protein of CYP1A1, Ala62Pro, had lower heme content than the wild-type. • The variant of CYP1A1 had lower enzyme activities than the wild-type.« less

CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to Deepwater Horizon Mississippi Canyon 252 oil

USGS Publications Warehouse

Alexander, Courtney R.; Hooper, Michael J.; Cacela, Dave; Smelker, Kim D.; Calvin, Caleshia S.; Dean, Karen M.; Bursian, Steve J.; Cunningham, Fred L.; Hanson-Dorr, Katie C.; Horak, Katherine E.; Isanhart, John P.; Link, Jane E.; Shriner, Susan A.; Godard-Codding, Céline A.J.

2017-01-01

Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20 ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5 ml/kg bw oil or 10 ml/kg bw oil, 21 day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10 ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to accurately evaluate avian exposure to oil.

In vitro biotransformation of tris(2-butoxyethyl) phosphate (TBOEP) in human liver and serum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van den Eede, Nele, E-mail: nele.vandeneede@uantwerpen.be; Erratico, Claudio; Exarchou, Vassiliki

Tris(2-butoxyethyl) phosphate (TBOEP) is a plasticizer present in indoor dust, reaching levels of several micrograms per gram. Such levels could lead to significant daily exposure of adults and children. Currently, no toxicokinetic data are available to estimate TBOEP clearance in humans after uptake and therefore, one objective of this study was to investigate intrinsic clearance of TBOEP by human liver microsome (HLM) and serum enzymes. Another objective was to generate information to identify and prioritize several metabolites of TBOEP for investigation of human exposure by biomonitoring. 1D and 2D-NMR methodologies were successfully applied on a mixture of the metabolites tomore » confirm the structure of 3-HO-TBOEP (bis(2-butoxyethyl) 3-hydroxyl-2-butoxyethyl phosphate) and to tentatively assign structures to 1-HO-TBOEP and 2-HO-TBOEP. HO-TBOEP isomers and bis(2-butoxyethyl) phosphate (BBOEP), bis(2-butoxyethyl) hydroxyethyl phosphate (BBOEHEP) were further monitored by liquid chromatography–tandem mass spectrometry. Rates of formation of BBOEHEP and HO-TBOEP metabolites by liver enzymes were best described by the Michaelis–Menten model. Apparent K{sub m} values for BBOEHEP, 3-HO-TBOEP, and sum of 1- and 2-HO-TBOEP isomer formation were 152, 197 and 148 μM, respectively. Apparent V{sub max} values for the formation of BBOEHEP, 3-HO-TBOEP, and the sum of 1- and 2-HO-TBOEP isomers were 2560, 643, and 254 pmol/min/mg protein, respectively. No detectable formation of BBOEP occurred with liver or serum enzymes. Our findings indicate that intrinsic clearance of TBOEP is mainly catalyzed by oxidative enzymes in the liver and that its major in vitro metabolite is BBOEHEP. These findings can be applied in human biomonitoring studies and risk assessment. - Highlights: • First steps in the elucidation of TBOEP toxicokinetics • Quantification of TBOEP metabolites in human serum and liver microsomes • No detectable formation of BBOEP occurred with liver or serum enzymes. • Oxidative dealkylation to BBOEHEP was likely the major metabolic pathway. • 1D-NMR and 2D-NMR were used to tentatively assign structures of HO-TBOEP isomers.« less

A systematic study of the disposition and metabolism of mercury species in mice after exposure to low levels of thimerosal (ethylmercury).

PubMed

Carneiro, Maria Fernanda Hornos; Oliveira Souza, Juliana Maria; Grotto, Denise; Batista, Bruno Lemos; de Oliveira Souza, Vanessa Cristina; Barbosa, Fernando

2014-10-01

Thimerosal (TM) is an ethylmercury (etHg)-containing preservative used in some vaccines despite very limited knowledge on the kinetics and direct interaction/effects in mammals׳ tissues after exposure. Thus, this study aimed to evaluate the kinetics of Hg species in mice in a time course analysis after intramuscular injection of TM, by estimating Hg half-lives in blood and tissues. Mice were exposed to one single intramuscular dose of 20 µg of Hg as TM. Blood, brain, heart, kidney and liver were collected at 0.5 hour (h), 1 h, 8 h, 16 h, 144 h, 720 h and 1980 h after TM exposure (n=4). Hg species in animal tissues were identified and quantified by speciation analysis via liquid chromatography hyphenated with inductively coupled mass spectrometry (LC-ICP-MS). It was found that the transport of etHg from muscle to tissues and its conversion to inorganic Hg (inoHg) occur rapidly. Moreover, the conversion extent is modulated in part by the partitioning between EtHg in plasma and in whole blood, since etHg is rapidly converted in red cells but not in a plasma compartment. Furthermore, the dealkylation mechanism in red cells appears to be mediated by the Fenton reaction (hydroxyl radical formation). Interestingly, after 0.5 h of TM exposure, the highest levels of both etHg and inoHg were found in kidneys (accounting for more than 70% of the total Hg in the animal body), whereas the brain contributed least to the Hg body burden (accounts for <1.0% of total body Hg). Thirty days after TM exposure, most Hg had been excreted while the liver presented the majority of the remaining Hg. Estimated half-lives (in days) were 8.8 for blood, 10.7 for brain, 7.8 for heart, 7.7 for liver and 45.2 for kidney. Taken together, our findings demonstrated that TM (etHg) kinetics more closely approximates Hg(2+) than methylmercury (meHg) while the kidney must be considered a potential target for etHg toxicity. Copyright © 2014 Elsevier Inc. All rights reserved.

Toxico-pathological effects in rats induced by lambda-cyhalothrin.

PubMed

Dahamna, S; Harzallah, D; Guemache, A; Sekfali, N

2009-01-01

Pesticides are widely used chemicals making human exposure to pesticides a realistic possibility. Biomonitoring is a common and useful tool for assessing human exposure to pesticides. Pyrethroids are effective insecticides that are often used in household sprays, aerosol bombs, insect repellents, pet shampoos, and lice treatments. Using products containing these compounds will expose people to these chemicals. Since these compounds frequently are used on crOPs, they are often detected in fruits and vegetables. Biomonitoring of exposure is a useful tool for assessing exposure to pesticides. Biomonitoring involves the measurement of the parent pesticide, its metabolite or reaction product in biological media, typically blood or urine, to determine if an exposure has occurred and the extent of that exposure. Although not without its limitations, biomonitoring has great utility in integrating all routes of exposure allowing for one exposure measurement. Pesticides have much shorter environmental half-lives and tend not to bioaccumulate. In fact, from humans within 24 hr as the parent pesticide, a mercapturic acid detoxification product, oxidative or dealkylation metabolites, and/or glucuronide or sulphate-bound metabolites. However, because of the heavy agricultural and residential use of these chemicals, humans are continually exposed to many of these chemicals. The objective of the present study was to explore modification in toxico-pathological responses of rats treated with lambda-cyhalothrin (commercially called karate). Rats (250 g weight), were gavaged by 1/100 LD50 for 4 weeks (one dose every week). Blood was collected before dosing and after 48 hours from the treatment. Enzyme activities were assayed in the plasma samples obtained. Glutamate oxaloacetate transaminase (GOT), Glutamate pyruvate transaminase (GPT), Alkaline phosphatase (ALPH) and Glucose. The results showed a decrease in RBC; WBC and Hb. This probably explained by the effect of lambda cyhalothrin on the erythropoiesis and the destruction of cells. An increase of plasma enzyme activities in GOT and GPT were recorded, explain a high energy-generating product. The histopathological results showed alteration on the target organs such as liver and kidney, these biochemical and histological modifications are probably due to the effect of lambda cyhalothrin or their metabolites.

Enantiomeric metabolic interactions and stereoselective human methadone metabolism.

PubMed

Totah, Rheem A; Allen, Kyle E; Sheffels, Pamela; Whittington, Dale; Kharasch, Evan D

2007-04-01

Methadone is administered as a racemate, although opioid activity resides in the R-enantiomer. Methadone disposition is stereoselective, with considerable unexplained variability in clearance and plasma R/S ratios. N-Demethylation of methadone in vitro is predominantly mediated by cytochrome P450 CYP3A4 and CYP2B6 and somewhat by CYP2C19. This investigation evaluated stereoselectivity, models, and kinetic parameters for methadone N-demethylation by recombinant CYP2B6, CYP3A4, and CYP2C19, and the potential for interactions between enantiomers during racemate metabolism. CYP2B6 metabolism was stereoselective. CYP2C19 was less active, and stereoselectivity was opposite that for CYP2B6. CYP3A4 was not stereoselective. With all three isoforms, enantiomer N-dealkylation rates in the racemate were lower than those of (R)-(6-dimethyamino-4,4-diphenyl-heptan-3-one) hydrochloride (R-methadone) or (S)-(6-dimethyamino-4,4-diphenyl-heptan-3-one) hydrochloride (S-methadone) alone, suggesting an enantiomeric interaction and mutual metabolic inhibition. For CYP2B6, the interaction between enantiomers was stereoselective, with S-methadone as a more potent inhibitor of R-methadone N-demethylation than R-of S-methadone. In contrast, enantiomer interactions were not stereoselective with CYP2C19 or CYP3A4. For all three cytochromes P450, methadone N-demethylation was best described by two-site enzyme models with competitive inhibition. There were minor model differences between cytochromes P450 to account for stereoselectivity of metabolism and enantiomeric interactions. Changes in plasma R/S methadone ratios observed after rifampin or troleandomycin pretreatment in humans in vivo were successfully predicted by CYP2B6- but not CYP3A4-catalyzed methadone N-demethylation. CYP2B6 is a predominant catalyst of stereoselective methadone metabolism in vitro. In vivo, CYP2B6 may be a major determinant of methadone metabolism and disposition, and CYP2B6 activity and stereoselective metabolic interactions may confer variability in methadone disposition.

Peroxidase-mediated dealkylation of tamoxifen, detected by electrospray ionization-mass spectrometry, and activation to form DNA adducts.

PubMed

Gaikwad, Nilesh W; Bodell, William J

2012-01-15

Tamoxifen (TAM) is extensively used for the treatment and prevention of breast cancer. Associated with TAM treatment is a two- to eightfold increase in risk of endometrial cancer. To understand the mechanisms associated with this increased risk several pathways for TAM metabolism and DNA adduct formation have been studied. The purpose of this study was to investigate the role of peroxidase enzymes in the metabolism of TAM and its activation to form DNA adducts. Using advanced tandem mass spectrometry we have investigated the peroxidase-mediated metabolism of TAM. Incubation of TAM with horseradish peroxidase (HRP) and H(2)O(2) produced multiple metabolites. Electrospray ionization-MS/MS analysis of the metabolites demonstrated a peak at 301.3m/z with daughter ions at 183.0, 166.9, 128.9, and 120.9m/z, which identified the metabolite as metabolite E (ME). The levels of ME were significantly inhibited by the addition of ascorbic acid to the incubation mixture. Co-incubation of either TAM or ME and DNA with HRP and H(2)O(2) produced three DNA adducts with a RAL of 1.97±0.01×10(-7) and 8.45±2.7×10(-7). Oxidation of ME with MnO(2) produced metabolite E quinone methide (MEQM). Furthermore, incubation of either TAM or ME with HRP and H(2)O(2) resulted in formation of MEQM. Reaction of calf thymus DNA with MEQM produced three DNA adducts with a RAL of 9.8±1.0×10(-7). Rechromatography analyses indicated that DNA adducts 1, 2, and 3 formed in the HRP activation of either TAM or ME were the same as those formed by the chemical reaction of DNA with MEQM. The results of these studies demonstrate that peroxidase enzymes can both metabolize TAM to form the primary metabolite ME and activate ME to a quinone methide intermediate, which reacts with DNA to form adducts. It is possible that peroxidase enzymes or peroxidase-like activity in endometrium could contribute to the formation of DNA damage and genotoxic effects in endometrium after TAM administration. Published by Elsevier Inc.

Functional characterization of cytochromes P450 2B from the desert woodrat Neotoma lepida

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilderman, P. Ross, E-mail: pwilderman@ucsd.edu; Jang, Hyun-Hee; Malenke, Jael R.

Mammalian detoxification processes have been the focus of intense research, but little is known about how wild herbivores process plant secondary compounds, many of which have medicinal value or are drugs. cDNA sequences that code for three enzymes of the cytochrome P450 (CYP) 2B subfamily, here termed 2B35, 2B36, and 2B37 have been recently identified from a wild rodent, the desert woodrat (Malenke et al., 2012). Two variant clones of each enzyme were engineered to increase protein solubility and to facilitate purification, as reported for CYP2B enzymes from multiple species. When expressed in Escherichia coli each of the woodrat proteinsmore » gave the characteristic maximum at 450 nm in a reduced carbon monoxide difference spectrum but generally expressed at lower levels than rat CYP2B1. Two enzymes, 2B36 and 2B37, showed dealkylation activity with the model substrates 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin, whereas 2B35 was inactive. Binding of the monoterpene (+)-α-pinene produced a Type I shift in the absorbance spectrum of each enzyme. Mutation of 2B37 at residues 114, 262, or 480, key residues governing ligand interactions with other CYP2B enzymes, did not significantly change expression levels or produce the expected functional changes. In summary, two catalytic and one ligand-binding assay are sufficient to distinguish among CYP2B35, 2B36, and 2B37. Differences in functional profiles between 2B36 and 2B37 are partially explained by changes in substrate recognition site residue 114, but not 480. The results advance our understanding of the mechanisms of detoxification in wild mammalian herbivores and highlight the complexity of this system. - Highlights: • Three CYP2B enzymes from Neotoma lepida were cloned, engineered, and expressed. • A mix of catalytic and binding assays yields unique results for each enzyme. • Mutational analysis indicates CYP{sub 2}B substrate recognition remains to be clarified. • Reported N. lepida gene sequences allow for larger scale analyses of CYP{sub 2}B enzymes.« less

Photolysis of the herbicide bispyribac sodium in aqueous medium under the influence of UV and sunlight in presence or absence of sensitizers.

PubMed

Kanrar, Bappaditya; Bhattacharyya, Anjan

2009-11-01

The photolysis of a rice herbicide Bispyribac sodium (Sodium 2, 6-bis [(4, 6-dimethoxypyrimidin-2-yl) oxy] benzoate) has been studied in different aqueous medium (distilled water, pond water and Irrigation water) under the influence of UV (lambda max > or = 250 nm) and sunlight in presence or absence of sensitizers (TiO(2) and KNO(3)). The study was conducted under laboratory simulated condition which made it possible to evaluate the contribution of different factors viz. source of irradiation, solvent and sensitizers towards the photolysis of bispyribac sodium. The photodegradation proceeds via first order reaction Kinetics in all the cases. Five photo metabolites (M(1)-M(5)) were isolated in pure form by column chromatographic method from the irradiation system under UV influenced and TiO(2) as sensitizer. From the different spectral data (IR, NMR, UV-VIS, Mass) the structure of these five metabolites were assigned as M(1) (Phenol), M(2) [2, 6-Dihydroxy benzoic acid], M(3) [2, 6-bis [(4, 6 dimethoxypyrimidin-2yl) oxy] benzoic acid], M(4) [2-(3-Hydroxy-phenoxy)-pyrimidine-4, 6-diol] and M(5) as [2,4-Dihydroxy-3, 5-dimethoxy-6-(4-methoxy pyrimidine-2-yloxy)-benzoic acid]. Moreover, another six photometabolites (M(6)-M(11)) were identified from the different irradiation system on the basis of Micromass analysis. On the basis of MS/MS data analysis, the structure of these six photometabolites were assigned as M(6) [2-(4, 6-Dimethoxy-pyrimidin-2-yloxy)-6-hydroxy-benzoic acid], M(7) [2-Hydroxy-6-(4-hydroxy-6-methoxy-pyrimidin-2-yloxy)-benzoic acid], M(8) [4, 6-Dimethoxy-pyrimidin-2-ol], M(9) [6-Methoxy-pyrimidine-2, 4-diol], M(10) [2-Hydroxy-6-(pyrimidin-2-yloxy)-benzoic acid] and M(11) [2, 4, 6-Trimethoxy-pyrimidine]. The plausible Photodegradation pathways of bispyribac sodium in the present investigation were portrayed which proceeds via hydrolysis, hydrolytic cleavage, O-dealkylation, decarboxylation, dehydroxylation, O-alkylation and hydroxylation.

Metabolism and pharmacokinetics of novel selective vascular endothelial growth factor receptor-2 inhibitor apatinib in humans.

PubMed

Ding, Juefang; Chen, Xiaoyan; Gao, Zhiwei; Dai, Xiaojian; Li, Liang; Xie, Cen; Jiang, Haoyuan; Zhang, Lijia; Zhong, Dafang

2013-06-01

Apatinib is a new oral antiangiogenic molecule that inhibits vascular endothelial growth factor receptor-2. The present study aimed to determine the metabolism, pharmacokinetics, and excretion of apatinib in humans and to identify the enzymes responsible for its metabolism. The primary routes of apatinib biotransformation included E- and Z-cyclopentyl-3-hydroxylation, N-dealkylation, pyridyl-25-N-oxidation, 16-hydroxylation, dioxygenation, and O-glucuronidation after 3-hydroxylation. Nine major metabolites were confirmed by comparison with reference standards. The total recovery of the administered dose was 76.8% within 96 hours postdose, with 69.8 and 7.02% of the administered dose excreted in feces and urine, respectively. About 59.0% of the administered dose was excreted unchanged via feces. Unchanged apatinib was detected in negligible quantities in urine, indicating that systemically available apatinib was extensively metabolized. The major circulating metabolite was the pharmacologically inactive E-3-hydroxy-apatinib-O-glucuronide (M9-2), the steady-state exposure of which was 125% that of the apatinib. The steady-state exposures of E-3-hydroxy-apatinib (M1-1), Z-3-hydroxy-apatinib (M1-2), and apatinib-25-N-oxide (M1-6) were 56, 22, and 32% of parent drug exposure, respectively. Calculated as pharmacological activity index values, the contribution of M1-1 to the pharmacology of the drug was 5.42 to 19.3% that of the parent drug. The contribution of M1-2 and M1-6 to the pharmacology of the drug was less than 1%. Therefore, apatinib was a major contributor to the overall pharmacological activity in humans. Apatinib was metabolized primarily by CYP3A4/5 and, to a lesser extent, by CYP2D6, CYP2C9, and CYP2E1. UGT2B7 was the main enzyme responsible for M9-2 formation. Both UGT1A4 and UGT2B7 were responsible for Z-3-hydroxy-apatinib-O-glucuronide (M9-1) formation.

Metabolic characterization of (1-(5-fluoropentyl)-1H-indol-3-yl)(4-methyl-1-naphthalenyl)-methanone (MAM-2201) using human liver microsomes and cDNA-overexpressed cytochrome P450 enzymes.

PubMed

Kong, Tae Yeon; Kim, Ju-Hyun; Choi, Won Gu; Lee, Joo Young; Kim, Hee Seung; Kim, Jin Young; In, Moon Kyo; Lee, Hye Suk

2017-02-01

MAM-2201 is a synthetic cannabinoid that is increasingly found in recreational drug abusers and cases of severe intoxication. Thus, characterization of the metabolic pathways of MAM-2201 is necessary to predict individual pharmacokinetics and toxicity differences, and to avoid toxic drug-drug interactions. Collectively, 19 phase 1 metabolites of MAM-2201 were identified using liquid chromatography-Orbitrap mass spectrometry following human liver microsomal incubations in the presence of NADPH: 7 hydroxy-MAM-2201 (M1-M7), 4 dihydroxy-MAM-2201 (M8-M11), dihydrodiol-MAM-2201 (M12), N-(5-hydroxypentyl)-MAM-2201 (M13), hydroxy-M13 (M14), N-dealkyl-MAM-2201 (M15), 2 hydroxy-M15 (M16, M17), MAM-2201 N-pentanoic acid (M18), and hydroxy-M18 (M19). On the basis of intrinsic clearance values in human liver microsomes, hydroxy-MAM-2201 (M1), N-(5-hydroxypentyl)-MAM-2201 (M13), and hydroxy-M13 (M14) were the major metabolites. Based on an enzyme kinetics study using human cDNA-expressed cytochrome P450 (CYP) enzymes and an immunoinhibition study using selective CYP antibodies in human liver microsomes, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 enzymes were responsible for MAM-2201 metabolism. The CYP3A4 enzyme played a prominent role in MAM-2201 metabolism, and CYP1A2, CYP2B6, CYP2C8, and CYP2C9 enzymes played major roles in the formation of some metabolites. MAM-2201 is extensively metabolized by multiple CYP enzymes, indicating that MAM-2201 and its metabolites should be used as markers of MAM-2201 abuse and toxicity. Graphical abstract In vitro metabolic pathways of MAM-2201 were characterized in human liver microsomes and recombinant CYPs using LC-HRMS analysis. Total 19 phase I metabolites were identified with predominant contribution of CYP3A4.

Fenton process on single and mixture components of phenothiazine pharmaceuticals: Assessment of intermediaries, fate, and preliminary ecotoxicity.

PubMed

Wilde, Marcelo L; Schneider, Mandy; Kümmerer, Klaus

2017-04-01

Pharmaceuticals do not occur isolated in the environment but in multi-component mixtures and may exhibit antagonist, synergistic or additive behavior. Knowledge on this is still scarce. The situation is even more complicated if effluents or potable water is treated by oxidative processes or such transformations occur in the environment. Thus, determining the fate and effects of parent compounds, metabolites and transformation products (TPs) formed by transformation and degradation processes in the environment is needed. This study investigated the fate and preliminary ecotoxicity of the phenothiazine pharmaceuticals, Promazine (PRO), Promethazine (PRM), Chlorpromazine (CPR), and Thioridazine (THI) as single and as components of the resulting mixtures obtained from their treatment by Fenton process. The Fenton process was carried out at pH7 and by using 0.5-2mgL -1 of [Fe 2+ ] 0 and 1-12.5mgL -1 of [H 2 O 2 ] 0 at the fixed ratio [Fe 2+ ] 0 :[H 2 O 2 ] 0 of 1:10 (w:w). No complete mineralization was achieved. Constitutional isomers and some metabolite-like TPs formed were suggested based on their UHPLC-HRMS n data. A degradation pathway was proposed considering interconnected mechanisms such as sulfoxidation, hydroxylation, N-dealkylation, and dechlorination steps. Aerobic biodegradation tests (OECD 301 D and OECD 301 F) were applied to the parent compounds separately, to the mixture of parent compounds, and for the cocktail of TPs present after the treatment by Fenton process. The samples were not readily biodegradable. However, LC-MS analysis revealed that abiotic transformations, such hydrolysis, and autocatalytic transformations occurred. The initial ecotoxicity tested towards Vibrio fischeri as individual compounds featured a reduction in toxicity of PRM and CPR by the treatment process, whereas PRO showed an increase in acute luminescence inhibition and THI a stable luminescence inhibition. Concerning effects of the mixture components, reduction in toxicity by the Fenton process was predicted by concentration addition and independent action models. Copyright © 2017 Elsevier B.V. All rights reserved.

Stereoselective Inhibition of CYP2C19 and CYP3A4 by Fluoxetine and Its Metabolite: Implications for Risk Assessment of Multiple Time-Dependent Inhibitor Systems

PubMed Central

Lutz, Justin D.; VandenBrink, Brooke M.; Babu, Katipudi N.; Nelson, Wendel L.; Kunze, Kent L.

2013-01-01

Recent guidance on drug-drug interaction (DDI) testing recommends evaluation of circulating metabolites. However, there is little consensus on how to quantitatively predict and/or assess the risk of in vivo DDIs by multiple time-dependent inhibitors (TDIs) including metabolites from in vitro data. Fluoxetine was chosen as the model drug to evaluate the role of TDI metabolites in DDI prediction because it is a TDI of both CYP3A4 and CYP2C19 with a circulating N-dealkylated inhibitory metabolite, norfluoxetine. In pooled human liver microsomes, both enantiomers of fluoxetine and norfluoxetine were TDIs of CYP2C19, (S)-norfluoxetine was the most potent inhibitor with time-dependent inhibition affinity constant (KI) of 7 μM, and apparent maximum time-dependent inhibition rate (kinact,app) of 0.059 min−1. Only (S)-fluoxetine and (R)-norfluoxetine were TDIs of CYP3A4, with (R)-norfluoxetine being the most potent (KI = 8 μM, and kinact,app = 0.011 min−1). Based on in-vitro-to-in-vivo predictions, (S)-norfluoxetine plays the most important role in in vivo CYP2C19 DDIs, whereas (R)-norfluoxetine is most important in CYP3A4 DDIs. Comparison of two multiple TDI prediction models demonstrated significant differences between them in in-vitro-to-in-vitro predictions but not in in-vitro-to-in-vivo predictions. Inclusion of all four inhibitors predicted an in vivo decrease in CYP2C19 (95%) and CYP3A4 (60–62%) activity. The results of this study suggest that adequate worst-case risk assessment for in vivo DDIs by multiple TDI systems can be achieved by incorporating time-dependent inhibition by both parent and metabolite via simple addition of the in vivo time-dependent inhibition rate/cytochrome P450 degradation rate constant (λ/kdeg) values, but quantitative DDI predictions will require a more thorough understanding of TDI mechanisms. PMID:23785064

Modulation of cytochrome P450 and induction of DNA damage in Cyprinus carpio exposed in situ to surface water treated with chlorine or alternative disinfectants in different seasons.

PubMed

Canistro, Donatella; Melega, Simone; Ranieri, Dario; Sapone, Andrea; Gustavino, Bianca; Monfrinotti, Monica; Rizzoni, Marco; Paolini, Moreno

2012-01-03

Epidemiological studies have shown an association between consumption of disinfected drinking water and adverse health outcomes. The chemicals used to disinfect water react with occurring organic matter and anthropogenic contaminants in the source water, resulting in the formation of disinfection by-products (DBPs). The observations that some DBPs are carcinogenic in animal models have raised public concern over the possible adverse health effects for humans. Here, the modulation of liver cytochrome P450-linked monooxygenases (MFO) and the genotoxic effects in erythrocytes of Cyprinus carpio fish exposed in situ to surface drinking water in the presence of disinfectants, such as sodium hypochlorite (NaClO), chlorine dioxide (ClO(2)) and peracetic acid (PAA), were investigated in winter and summer. A complex induction/suppression pattern of CYP-associated MFOs in winter was observed for all disinfectants. For example, a 3.4- to 15-fold increase was recorded of the CYP2B1/2-linked dealkylation of penthoxyresorufin with NaClO (10 days) and PAA (20 days). In contrast, ClO(2) generated the most notable inactivation, the CYP2E1-supported hydroxylation of p-nitrophenol being decreased up to 71% after 10 days' treatment. In summer, the degree of modulation was modest, with the exception of CYP3A1/2 and CYP1A1 supported MFOs (62% loss after 20 days PAA). The micronucleus (MN) induction in fish circulating erythrocytes was also analysed as an endpoint of genotoxic potential in the same fish population. Significant increases of MN induction were detected at the latest sampling time on fish exposed to surface water treated with chlorinate-disinfectants, both in winter (NaClO) and summer (NaClO and ClO(2)), while no effect was observed in fish exposed to PAA-treated water. These results show that water disinfection may be responsible for harmful outcomes in terms of MFO perturbation and DNA damage; if extrapolated to humans, they ultimately offer a possible rationale for the increased urinary cancer risk recorded in regular drinking water consumers. Copyright © 2011 Elsevier B.V. All rights reserved.

Biotransformation and detectability of the new psychoactive substances N,N-diallyltryptamine (DALT) derivatives 5-fluoro-DALT, 7-methyl-DALT, and 5,6-methylenedioxy-DALT in urine using GC-MS, LC-MSn, and LC-HR-MS/MS.

PubMed

Michely, Julian A; Brandt, Simon D; Meyer, Markus R; Maurer, Hans H

2017-02-01

Derivatives of N,N-diallyltryptamine (DALT) can be classified as new psychoactive substances. Biotransformation and detectability of 5-fluoro-DALT (5-F-DALT), 7-methyl-DALT (7-Me-DALT), and 5,6-methylenedioxy-DALT (5,6-MD-DALT) are described here. Their metabolites detected in rat urine and pooled human liver microsomes were identified by liquid chromatography (LC)-high resolution (HR)-tandem mass spectrometry (MS/MS). In addition, the human cytochrome-P450 (CYP) isoenzymes involved in the main metabolic steps were identified and detectability tested in urine by the authors' urine screening approaches using GC-MS, LC-MS n , or LC-HR-MS/MS. Aromatic and aliphatic hydroxylations, N-dealkylation, N-oxidation, and combinations could be proposed for all compounds as main pathways. Carboxylation after initial hydroxylation of the methyl group could also be detected for 7-Me-DALT and O-demethylenation was observed for 5,6-MD-DALT. All phase I metabolites were extensively glucuronidated or sulfated. Initial phase I reactions were catalyzed by CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5. Rat urine samples were analyzed following two different low-dose administrations. GC-MS was not able to monitor consumption reliably, but all three compounds are predicted to be detectable in cases of overdose. The LC-MS n and LC-HR-MS/MS approaches were suitable for detecting an intake of all three compounds mainly via their metabolites. However, after the lowest dose, a reliable monitoring could only be achieved for 5-F-DALT via LC-MS n and LC-HR-MS/MS and for 7-Me-DALT via LC-HR-MS/MS. The most abundant targets in both LC-MS screenings were one of two hydroxy-aryl metabolites and both corresponding glucuronides for 5-F-DALT, one N-deallyl hydroxy-aryl, the carboxy, and one dihydroxy-aryl metabolite for 7-Me-DALT, and the demethylenyl metabolite, its oxo metabolite, and glucuronide for 5,6-MD-DALT.

Toxic effects of tributyltin and its metabolites on harbour seal (Phoca vitulina) immune cells in vitro.

PubMed

Frouin, Héloïse; Lebeuf, Michel; Saint-Louis, Richard; Hammill, Mike; Pelletier, Emilien; Fournier, Michel

2008-11-21

The widespread environmental contamination, bioaccumulation and endocrine disruptor effects of butyltins (BTs) to wildlife are well documented. Although suspected, potential effects of BTs exposure on the immune system of marine mammals have been little investigated. In this study, we assessed the effects of tributyltin (TBT) and its dealkylated metabolites dibutyltin (DBT) and monobutyltin (MBT) on the immune responses of harbour seals. Peripheral blood mononuclear cells isolated from pup and adult harbour seals were exposed in vitro to varying concentrations of BTs. DBT resulted in a significant decrease at 100 and 200 nM of phagocytotic activity and reduced significantly phagocytic efficiency at 200 nM in adult seals. There was no effect in phagocytosis with TBT and MBT. In pups, the highest concentration (200 nM) of DBT inhibited phagocytic efficiency. A reduction of tumor-killing capacity of adult natural killer (NK) cells occurred when leukocytes were incubated in vitro with 50 nM DBT and 200 nM TBT for 24h. In adult seals, T-lymphocyte proliferation was significantly suppressed when the cells were exposed to 200 nM TBT and 100 nM DBT. In pups, the proliferative response increased after an exposure to 100 nM TBT and 50 nM DBT, but decreased with 200 nM TBT and 100 nM DBT. The immune functions were more affected by BTs exposure in adults than in pups, suggesting that other unsuspected mechanisms could trigger immune parameters in pups. The toxic potential of BTs followed the order of DBT>TBT>MBT. BT concentrations of harbour seal pups from the St. Lawrence Estuary (Bic National Park) ranged between 0.1-0.4 ng Sn/g wet weight (ww) and 1.2-13.4 ng Sn/g ww in blood and blubber, respectively. For these animals, DBT concentrations were consistently below the quantification limit of 0.04 ng Sn/g ww in blood and 0.2 ng Sn/g ww in blubber. Results suggest that concentrations measured in pups are considered too low to induce toxic effects to their immune system during first days of life. However, based on our in vitro results, we hypothesize that BTs, and DBT in particular, could pose a serious threat to the immune functions in free-ranging harbour seal adults.

Chronic ecotoxic effects to Pseudomonas putida and Vibrio fischeri, and cytostatic and genotoxic effects to the hepatoma cell line (HepG2) of ofloxacin photo(cata)lytically treated solutions.

PubMed

Vasquez, M I; Garcia-Käufer, M; Hapeshi, E; Menz, J; Kostarelos, K; Fatta-Kassinos, D; Kümmerer, K

2013-04-15

Ofloxacin (OFL), a broad-spectrum and widespread-used photolabile fluoroquinolone, is frequently found in treated wastewaters, aquatic and terrestrial ecosystems leading to increasing concern during the past decades regarding its effects to the environment and human health. The elimination of OFL and other xenobiotics by the application of advanced oxidation processes using photolytic (PL) and photocatalytic (PC) treatments seems promising. However, an integrated assessment scheme is needed, in which, not only the removal of the parent compound, but also the effects of the photo-transformation products (PTPs) are investigated. For this purpose, in the present study, a chronic ecotoxic assessment using representative bacteria of marine and terrestrial ecosystems and a cytostatic and genotoxic evaluation using hepatoma cell line were performed. PL and PC treatments of OFL were applied using UV radiation. The photo-transformation of OFL during the treatments was monitored by DOC measurements and UPLC-MS/MS analysis. The chronic ecotoxicity of OFL and treated samples was evaluated using Pseudomonas putida and Vibrio fischeri; whereas the cytostasis and genotoxicity were estimated by the cytokinesis-block micronucleus assay (CBMN). The main results suggest that photo-transformation of OFL took place during these treatments since the concentration of OFL decreased when the irradiation time increased, as quantified by UPLC-MS/MS analysis, and this was not coupled with an analogous DOC removal. Furthermore, nine compounds were identified as probable PTPs formed through piperazinyl dealkylation and decarboxylation. The ecotoxicity of treated solutions to the bacteria studied decreased while the cytostasis to the hepatoma cell line remained at low levels during both treatments. However, the genotoxicity to the hepatoma cell line demonstrated a different pattern in which treated samples induced a greater number of MNi for the 4-16 min of irradiation (p<0.05) during both treatments. After 64 min of irradiation, the effects decreased to non genotoxic levels (p<0.05). These findings suggest that UV radiation for various treatment processes (catalytic or not), such as disinfection, may create genotoxic by-products. Therefore, in relevant technical applications, the residence time during treatment should receive special attention. Copyright © 2012. Published by Elsevier B.V.

MD simulations and QM/MM calculations show that single-site mutations of cytochrome P450BM3 alter the active site’s complexity and the chemoselectivity of oxidation without changing the active species† †Electronic supplementary information (ESI) available: The RMS deviation of the protein during 100 ns (and 200 ns) of simulation and the total QM/MM and ZPE energies, as well as the Cartesian coordinates of all of the computed species. See DOI: 10.1039/c7sc01932g

PubMed Central

Dubey, Kshatresh Dutta; Wang, Binju; Vajpai, Manu

2017-01-01

It is a long-standing mechanistic consensus that the mutation of the proton-shuttle mediator Threonine (T) in Cytochrome P450 enzymes severs the water channel and thereby quenches the formation of the active species: the high-valent iron(iv)-oxo porphyrin π-cation radical species, compound I (Cpd I). Using MD simulations and hybrid QM/MM calculations of P450BM3 we demonstrate that this is not the case. Thus, while the original water channel is disrupted in the T268A mutant of the enzyme, a new channel is formed that generates Cpd I. With this new understanding, we address the puzzling regiochemical and kinetic-isotope effect (KIE) results (Volz et al., J. Am. Chem. Soc., 2002, 124, 9724–9725) on the sulfoxidation and N-dealkylation of dimethyl-(4-methylsulfanyl-phenyl)-amine by wild type (WT) P450BM3 and its T268A vs. F87A mutants. We show that the observed variable ratio of S/Me oxidation for these enzymes, vis-à-vis the constant KIE, originates from Cpd I being the sole oxidant. Thus, while the conserved KIE probes the conserved nature of the transition state, the variable regiochemical S/Me ratio reflects the active-site reorganization in the mutants: the shifted location of the new water channel in T268A tightens the binding of the S-end by Cpd I and increases the S/Me ratio, whereas the absence of π-interaction with the S-end in F87A creates a looser binding that lowers the S/Me ratio. Our results match the experimental findings. As such, this study sheds light on puzzling experimental results, and may shift a central paradigm in P450 research. The broader implication on enzymatic research is that a single-site mutation is not a localised alteration but one that may lead to a profound change in the active site, sufficiently so as to change the chemoselectivity of catalyzed reactions. PMID:29568477

Probing the Compound I-like reactivity of a bare high-valent oxo iron porphyrin complex: the oxidation of tertiary amines.

PubMed

Chiavarino, Barbara; Cipollini, Romano; Crestoni, Maria Elisa; Fornarini, Simonetta; Lanucara, Francesco; Lapi, Andrea

2008-03-12

The mechanisms of oxidative N-dealkylation of amines by heme enzymes including peroxidases and cytochromes P450 and by functional models for the active Compound I species have long been studied. A debated issue has concerned in particular the character of the primary step initiating the oxidation sequence, either a hydrogen atom transfer (HAT) or an electron transfer (ET) event, facing problems such as the possible contribution of multiple oxidants and complex environmental effects. In the present study, an oxo iron(IV) porphyrin radical cation intermediate 1, [(TPFPP)*+ Fe(IV)=O]+ (TPFPP = meso-tetrakis (pentafluorophenyl)porphinato dianion), functional model of Compound I, has been produced as a bare species. The gas-phase reaction with amines (A) studied by ESI-FT-ICR mass spectrometry has revealed for the first time the elementary steps and the ionic intermediates involved in the oxidative activation. Ionic products are formed involving ET (A*+, the amine radical cation), formal hydride transfer (HT) from the amine ([A(-H)]+, an iminium ion), and oxygen atom transfer (OAT) to the amine (A(O), likely a carbinolamine product), whereas an ionic product involving a net initial HAT event is never observed. The reaction appears to be initiated by an ET event for the majority of the tested amines which included tertiary aliphatic and aromatic amines as well as a cyclic and a secondary amine. For a series of N,N-dimethylanilines the reaction efficiency for the ET activated pathways was found to correlate with the ionization energy of the amine. A stepwise pathway accounts for the C-H bond activation resulting in the formal HT product, namely a primary ET process forming A*+, which is deprotonated at the alpha-C-H bond forming an N-methyl-N-arylaminomethyl radical, A(-H)*, readily oxidized to the iminium ion, [A(-H)]+. The kinetic isotope effect (KIE) for proton transfer (PT) increases as the acidity of the amine radical cation increases and the PT reaction to the base, the ferryl group of (TPFPP)Fe(IV)=O, approaches thermoneutrality. The ET reaction displayed by 1 with gaseous N,N-dimethylaniline finds a counterpart in the ET reactivity of FeO+, reportedly a potent oxidant in the gas phase, and with the barrierless ET process for a model (P)*+ Fe(IV)=O species (where P is the porphine dianion) as found by theoretical calculations. Finally, the remarkable OAT reactivity of 1 with C6F5N(CH3)2 may hint to a mechanism along a route of diverse spin multiplicity.

Quantitative estimation of α-PVP metabolites in urine by GC-APCI-QTOFMS with nitrogen chemiluminescence detection based on parent drug calibration.

PubMed

Mesihää, Samuel; Rasanen, Ilpo; Ojanperä, Ilkka

2018-05-01

Gas chromatography (GC) hyphenated with nitrogen chemiluminescence detection (NCD) and quadrupole time-of-flight mass spectrometry (QTOFMS) was applied for the first time to the quantitative analysis of new psychoactive substances (NPS) in urine, based on the N-equimolar response of NCD. A method was developed and validated to estimate the concentrations of three metabolites of the common stimulant NPS α-pyrrolidinovalerophenone (α-PVP) in spiked urine samples, simulating an analysis having no authentic reference standards for the metabolites and using the parent drug instead for quantitative calibration. The metabolites studied were OH-α-PVP (M1), 2″-oxo-α-PVP (M3), and N,N-bis-dealkyl-PVP (2-amino-1-phenylpentan-1-one; M5). Sample preparation involved liquid-liquid extraction with a mixture of ethyl acetate and butyl chloride at a basic pH and subsequent silylation of the sec-hydroxyl and prim-amino groups of M1 and M5, respectively. Simultaneous compound identification was based on the accurate masses of the protonated molecules for each compound by QTOFMS following atmospheric pressure chemical ionization. The accuracy of quantification of the parent-calibrated NCD method was compared with that of the corresponding parent-calibrated QTOFMS method, as well as with a reference QTOFMS method calibrated with the authentic reference standards. The NCD method produced an equally good accuracy to the reference method for α-PVP, M3 and M5, while a higher negative bias (25%) was obtained for M1, best explainable by recovery and stability issues. The performance of the parent-calibrated QTOFMS method was inferior to the reference method with an especially high negative bias (60%) for M1. The NCD method enabled better quantitative precision than the QTOFMS methods To evaluate the novel approach in casework, twenty post- mortem urine samples previously found positive for α-PVP were analyzed by the parent calibrated NCD method and the reference QTOFMS method. The highest difference in the quantitative results between the two methods was only 33%, and the NCD method's precision as the coefficient of variation was better than 13%. The limit of quantification for the NCD method was approximately 0.25μg/mL in urine, which generally allowed the analysis of α-PVP and the main metabolite M1. However, the sensitivity was not sufficient for the low concentrations of M3 and M5. Consequently, while having potential for instant analysis of NPS and metabolites in moderate concentrations without reference standards, the NCD method should be further developed for improved sensitivity to be more generally applicable. Copyright © 2018 Elsevier B.V. All rights reserved.

Determination of the sulfate and glucuronide conjugates of levornidazole in human plasma and urine, and levornidazole and its five metabolites in human feces by high performance liquid chromatography-tandem mass spectrometry.

PubMed

He, Gaoli; Guo, Beining; Zhang, Jing; Li, Yi; Wu, Xiaojie; Fan, Yaxin; Chen, Yuancheng; Cao, Guoying; Yu, Jicheng

2018-04-01

Levornidazole is a novel third-generation nitroimidazoles antibiotic which metabolism and disposition in human are not well known. We have previously developed two methods to quantify levornidazole and its phase I metabolites, Ml (Hydroxylation metabolite), M2 (N-dealkylation metabolite) and M4 (Oxidative dechlorination metabolite), in human plasma and urine. In this study, we developed three novel liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods and analyzed its phase II metabolites, sulfate conjugate (M6) and glucuronide conjugate (M16), in human plasma and urine, and the parent drug and above-mentioned five metabolites in human feces samples. Analytes and internal standard (IS) in human plasma were extracted by a solid-phase extraction procedure and separated on an ACQUITY UPLC CSH C18 column in gradient elution using acetonitrile and 0.1% formic acid aqueous solution as the mobile phase. The pretreatment procedures for urine and feces homogenate samples involved a protein precipitation followed by liquid-liquid extraction, and chromatographic separations were performed on the Atlantis T3 columns of different lengths and particle sizes (2.1 × 50 mm, 3 μm and 2.1 × 150 mm, 5 μm), respectively. The mobile phases consisted of formic acid and acetonitrile-methanol solution (v/v, 50:50) in gradient elution. The MS/MS analysis was conducted on TSQ Quantum triple quadrupole mass spectrometer using electrospray ionization with selected reaction monitoring (SRM) in the positive ion mode. The calibration curves for all analytes were linear and the validation ranges were as follows: 0.005-0.500 μg/mL for M6 and 0.005-2.500 μg/mL for M16 in plasma; 0.010-10.000 μg/mL for M6 and M16 in urine; 0.005-1.000 μg/mL for levornidazole, M2, M4 and M16, and 0.010-2.000 μg/mL for M1 and M6 in human feces homogenate. Across these matrices, mean intra- and inter- batch accuracy values were in the ranges of 80.0%-120.0%, and intra- and inter-batch precision values did not exceed 20%. It was fully validated including selectivity, linearity, matrix effect, extraction recovery, stability, dilution integrity, carryover and incurred sample analysis (ISR). These newly developed methods were successfully applied in pharmacokinetics, metabolism and disposition study of levornidazole in 12 healthy Chinese subjects. Copyright © 2018 Elsevier B.V. All rights reserved.

Toxicity profiles in rats treated with tumorigenic and nontumorigenic triazole conazole fungicides: Propiconazole, triadimefon, and myclobutanil.

PubMed

Wolf, Douglas C; Allen, James W; George, Michael H; Hester, Susan D; Sun, Guobin; Moore, Tanya; Thai, Sheau-Fung; Delker, Don; Winkfield, Ernest; Leavitt, Sharon; Nelson, Gail; Roop, Barbara C; Jones, Carlton; Thibodeaux, Julie; Nesnow, Stephen

2006-01-01

Conazoles are a class of azole based fungicides used in agriculture and as pharmaceutical products. They have a common mode of antifungal action through inhibition of ergosterol biosynthesis. Some members of this class have been shown to be hepatotoxic and will induce mouse hepatocellular tumors and/or rat thyroid follicular cell tumors. The particular mode of toxic and tumorigenic action for these compounds is not known, however it has been proposed that triadimefon-induced rat thyroid tumors arise through the specific mechanism of increased TSH. The present study was designed to identify commonalities of effects across the different conazoles and to determine unique features of the tissue responses that suggest a toxicity pathway and a mode of action for the observed thyroid response for triadimefon. Male Wistar/Han rats were treated with triadimefon (100, 500, 1800 ppm), propiconazole (100, 500, 2500 ppm), or myclobutanil (100, 500, 2000 ppm) in feed for 4, 30, or 90 days. The rats were evaluated for clinical signs, body and liver weight, histopathology of thyroid and liver, hepatic metabolizing enzyme activity, and serum T3, T4, TSH, and cholesterol levels. There was a dose-dependent increase in liver weight but not body weight for all treatments. The indication of cytochrome induction, pentoxyresorufin O-dealkylation (PROD) activity, had a dose-related increase at all time points for all conazoles. Uridine diphopho-glucuronosyl transferase (UDPGT), the T4 metabolizing enzyme measured as glucuronidation of 1-naphthol, was induced to the same extent after 30 and 90 days for all three conazoles. Livers from all high dose treated rats had centrilobular hepatocyte hypertrophy after 4 days, while only triadimefon and propiconazole treated rats had hepatocyte hypertrophy after 30 days, and only triadimefon treated rats had hepatocyte hypertrophy after 90 days. Thyroid follicular cell hypertrophy, increased follicular cell proliferation, and colloid depletion were present only after 30 days in rats treated with the high dose of triadimefon. A dose-dependent decrease in T4 was present after 4 days with all 3 compounds but only the high doses of propiconazole and triadimefon produced decreased T4 after 30 days. T3 was decreased after high-dose triadimefon after 4 days and in a dose-dependent manner for all compounds after 30 days. Thyroid hormone levels did not differ from control values after 90 days and TSH was not increased in any exposure group. A unique pattern of toxic responses was not identified for each conazole and the hypothesized mode of action for triadimefon-induced thyroid gland tumors was not supported by the data.

Novel Organophosphate Ligand O-(2-Fluoroethyl)-O-(p-Nitrophenyl)Methylphosphonate: Synthesis, Hydrolytic Stability and Analysis of the Inhibition and Reactivation of Cholinesterases.

PubMed

Chao, Chih-Kai; Ahmed, S Kaleem; Gerdes, John M; Thompson, Charles M

2016-11-21

The organophosphate O-(2-fluoroethyl)-O-(p-nitrophenyl) methyphosphonate 1 is the first-in-class, fluorine-18 radiolabeled organophosphate inhibitor ([ 18 F]1) of acetylcholinesterase (AChE). In rats, [ 18 F]1 localizes in AChE rich regions of the brain and other tissues where it likely exists as the (CH 3 )( 18 FCH 2 CH 2 O)P(O)-AChE adduct (ChE-1). Characterization of this adduct would define the inhibition mechanism and subsequent postinhibitory pathways and reactivation rates. To validate this adduct, the stability (hydrolysis) of 1 and ChE-1 reactivation rates were determined. Base hydrolysis of 1 yields p-nitrophenol and (CH 3 ) (FCH 2 CH 2 O)P(O)OH with pseudo first order rate constants (k obsd ) at pH 7.4 (PBS) of 3.25 × 10 -4 min -1 (t 1/2 = 35.5 h) at 25 °C and 8.70 × 10 -4 min -1 (t 1/2 = 13.3 h) at 37 °C. Compound 1 was a potent inhibitor of human acetylcholinesterase (HuAChE; k i = 7.5 × 10 5 M -1 min -1 ), electric eel acetylcholinesterase (EEAChE) (k i = 3.0 × 10 6 M -1 min -1 ), and human serum butyrylcholinesterase (HuBChE; 1.95 × 10 5 M -1 min -1 ). Spontaneous and oxime-mediated reactivation rates for the (CH 3 ) (FCH 2 CH 2 O)P(O)-serine ChE adducts using 2-PAM (10 μM) were (a) HuAChE 8.8 × 10 -5 min -1 (t 1/2 = 131.2 h) and 2.41 × 10 -2 min -1 (t 1/2 = 0.48 h), (b) EEAChE 9.32 × 10 -3 min -1 (t 1/2 = 1.24 h) and 3.33 × 10 -2 min -1 (t 1/2 = 0.35 h), and (c) HuBChE 1.16 × 10 -4 min -1 (t 1/2 = 99.6 h) and 4.19 × 10 -2 min -1 (t 1/2 = 0.27 h). All ChE-1 adducts undergo rapid and near complete restoration of enzyme activity following addition of 2-PAM (30 min), and no aging was observed for either reactivation process. The fast reactivation rates and absence of aging of ChE-1 adducts are explained on the basis of the electron-withdrawing fluorine group that favors the nucleophilic reactivation processes but disfavors cation-based dealkylation aging mechanisms. Therefore, the likely fate of radiolabeled compound 1 in vivo is the formation of (CH 3 )(FCH 2 CH 2 O)P(O)-serine adducts and monoacid (CH 3 )(FCH 2 CH 2 O)P(O)OH from hydrolysis and reactivation.

Pyrolytic indices of diagenetic transformation of lignin as biogeochemical proxies for soil organic matter quality and C storage potential

NASA Astrophysics Data System (ADS)

Jiménez-González, Marco A.; Almendros, Gonzalo; Álvarez, Ana M.; Jiménez-Morillo, Nicasio T.; González-Vila, Francisco J.

2017-04-01

The environmental factors involved in soil organic carbon sequestration remain unclear. The functional relationships between the macromolecular structure of the soil organic matter (SOM) and its resilience has been a constant in classical biogeochemical models. Other more recent hypotheses have postulated that preservation by soil minerals may play a chief role in the accumulation of stable SOM forms. However, additional experimental data are required to demonstrate a cause-to-effect relationship between preservation and stabilization. Some authors might consider that models neglecting the role of macromolecular structure are swapping cause and effect i.e., that SOM structurally flexible, weakly condensed and having 'open' structures is the one with high potential to interact with the soil mineral matrix, leading to stable microaggregates. In this study up to 35 topsoil samples (0-5 cm) were collected from different Spanish soils with contrasted values of organic C (the dependent variable), geological substrate and vegetation type. A wide array of uni- and multivariate chemometric models were applied to independent variables consisting of total abundances of the major aromatic compounds, i.e., alkylbenzenes and methoxyphenols released from whole soil samples using pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS). These two families of compounds were selected since they are classically considered to inform on the degree of microbial reworking of lignins, which is an important precursor of the aromatic moiety of the SOM. A series of pyrolytic surrogate indices (aiming to express SOM diagenetic transformation in relation to the original biogenic molecular composition) were especially successful in forecasting SOC, viz: a) ratio between alkylbenzenes and methoxyphenols, b) ratio between short-chain (C0-C4) and long-chain (>C4) alkylbenzenes, c) ratio between methoxyphenols and short-chain alkylbenzenes, and d) ratios between methoxyphenols with different side-chain length, i.e., methyl-, ethyl-, vinyl-, propenyl- and aceto- derivatives of guaiacyl and syringyl rings. In particular partial least squares regression (PLS) models led to very significant prediction of SOC based exclusively on the major aromatic pyrolytic compounds as predictors, i.e, methoxyphenols and alkylbenzenes. The PLS coefficients showed high positive loadings for methoxyphenols (i.e., these compounds were diagnostic for soils with high C-storage potential), and negative for alkylbenzenes (which were characteristic for C-depleted soils). This may be explained as if selective preservation of lignin were the most efficient process in soils behaving as active C sinks. Conversely, the progressive smoothing in the lignin signature and the accumulation of methoxyl-lacking aromatics (i.e., short-chain alkylbenzenes) was typical in the soils with low SOC content but high levels of diagenetic alteration of lignin. The progressive alteration of C6-C3 phenylpropanoid lignin units also corresponded to the fact that heavily altered lignin, presumptively accumulated in SOM depleted soils, tended to release typical pyrolytic markers consisting of C0-C1 methoxyphenols instead of its vinyl and propenyl counterparts, in addition to major proportions of short-chain (