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1.
Phys Chem Chem Phys ; 22(16): 8699-8712, 2020 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32270839

RESUMO

The selectivity of halogenation versus hydroxylation in α-KG de-pendent halogenases is vital to their function and has been widely studied, particularly using the halogenase SyrB2 as a model. WelO5, a new member of α-KG dependent halogenases, catalyzes the chlorination of 12-epi-fischerindole U in the welwitindolinone biosynthetic pathway. Herein, we give a detailed insight into the selectivity of WelO5 through combined quantum mechanical/molecular mechanical (QM/MM) calculations for the whole catalytic cycle. O2 activation leads to a Fe(iv)[double bond, length as m-dash]O moiety which adopts an equatorial conformation (in the plane consisting of His164, chloride and Fe atom), in contrast to axial conformation (perpendicular to the plane). Key to the conformational selectivity is a serine residue (Ser189) in the equatorial plane, that brings the precursor of the Fe(iv)[double bond, length as m-dash]O intermediate (a Fe(ii)-peracid complex) to the equatorial conformation through hydrogen bonding. Hydrogen abstraction of the substrate by the equatorial Fe(iv)[double bond, length as m-dash]O leads to a five-coordinated HO-Fe(iii)-Cl complex, where the hydroxyl ligand is still equatorial and thus relatively far from the substrate radical in the axial direction compared to the chloride ligand. This smoothly explains the extremely high selectivity of chlorination in WelO5 and provides a microscopic explanation for the experimental finding that S189A WelO5 ceases to display any chlorination selectivity versus hydroxylation. Notably, although Ser189 is vital for the selectivity of the enzyme, it is not part of the substrate binding pocket. Therefore, WelO5 serves as an excellent example how chemoselectivity can be achieved in directed evolution without the tedious redesign of the substrate binding pocket.


Assuntos
Enzimas/metabolismo , Ferroproteínas não Heme/metabolismo , Halogenação , Hidroxilação , Ferroproteínas não Heme/química , Especificidade por Substrato
2.
Xenobiotica ; 50(9): 1076-1089, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32174209

RESUMO

Coumarins have aroused high interests due to their diverse bioactivities. Understanding of its metabolism contributes to determine the druggability of coumarin in vivo.A sensitive and efficient strategy based on ultra-performance liquid chromatography-mass spectrometer (UPLC-MS) analysis combined with various data-processing techniques including metabolomics and multiple mass defect filter (MMDF) was established for the comprehensive screening and elucidation of potential coumarin metabolites.Total 20 metabolites of scoparone were identified in this study, including 14 undescribed metabolites. The metabolism of two other similar coumarins scopoletin and esculetin also could be determined using this strategy.By the established strategy, this study gives the insights about the major metabolic pathways of scoparone in vivo and in vitro metabolism, including demethylation, hydroxylation, hydration, cysteine conjugation, glucuronide conjugation and sulfate conjugation. Additionally, the metabolic pathways of scopoletin and esculetin were determined as hydroxylation, glucuronidation and sulfation. These results contribute to the understanding of metabolic characterization of coumarins, and demonstrate that the combination of UPLC-MS-based metabolomics and MMDF is a powerful approach to determine the metabolic pathways of coumarin compounds.


Assuntos
Cumarínicos/metabolismo , Metabolômica , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Hidroxilação , Redes e Vias Metabólicas , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
3.
Chimia (Aarau) ; 74(3): 108-114, 2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32197667

RESUMO

Enzymatic oxygenations initiate biodegradation processes of many organic soil and water contaminants. Even though many biochemical aspects of oxygenation reactions are well-known, quantifying rates of oxidative contaminant removal as well as the extent of oxygenation remains a major challenge. Because enzymes use different strategies to activate O2, reactions leading to substrate oxygenation are not necessarily limiting the rate of contaminant removal. Moreover, oxygenases react along unproductive pathways without substrate metabolism leading to O2 uncoupling. Here, we identify the critical features of the catalytic cycles of selected oxygenases that determine rates and extents of biodegradation. We focus most specifically on Rieske dioxygenases, a subfamily of mononuclear non-heme ferrous iron oxygenases, because of their ability to hydroxylate unactivated aromatic structures and thus initiate the transformation of the most persistent organic contaminants. We illustrate that the rate-determining steps in their catalytic cycles range from O2 activation to substrate hydroxylation, depending on the extent of O-O cleavage that is required for generating the reactive Fe-oxygen species. The extent of O2 uncoupling, on the other hand, is highly substrate-specific and potentially modulated by adaptive responses to oxidative stress. Understanding the kinetic mechanisms of oxygenases will be key to assess organic contaminant biotransformation quantitatively.


Assuntos
Oxigênio/metabolismo , Dioxigenases , Hidroxilação , Cinética , Oxirredução , Oxigenases
4.
Phys Chem Chem Phys ; 22(11): 6335-6350, 2020 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-32134073

RESUMO

The major applications of carbon dots (CDs) (e.g. bio-imaging and targeted drug delivery) necessitate the latter to permeate across the lipid bilayer membrane. Unfortunately, the mechanism of permeation is poorly understood. Between the two possible routes for permeation of a nanoparticle like CDs-an endocytic pathway and direct passive permeation-the endocytic path is known to be more common, despite the fact that the passive permeation is preferred over the endocytosis for targeted drug delivery. Here, we have focused on the direct permeation of a hydroxyl functionalized CD across the POPC lipid bilayer membrane using all-atom MD simulations. We have estimated the free energy profile for the translocation of the CD across the lipid bilayer, with a barrier height of ∼170 kJ mol-1 situated at the lipid bilayer center (z = 0 nm). Using the free energy profile, we have calculated a negligible permeability coefficient value, which strongly suggests that it is almost impossible for a CD to penetrate directly across the lipid bilayer. The possible impact on the lipid bilayer structure by the CD is also investigated. Although the CD does not affect the bilayer structure up to a certain degree of penetration, the impact increases substantially when entered into the bilayer interior.


Assuntos
Carbono/química , Carbono/metabolismo , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Sistemas de Liberação de Medicamentos , Hidroxilação
5.
Chemosphere ; 247: 125844, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32069708

RESUMO

Biphenyl 2,3-dioxygenase (BphA), a Rieske-type and first enzyme in the aerobic degradation process, plays a key role in the metabolizing process of biphenyl/polychlorinated biphenyl aromatic pollutants in the environment. To understand the catalytic mechanism of biphenyl 2,3-dioxygenase, the conversions leading to the cis-diols are investigated by means of quantum mechanics/molecular mechanics (QM/MM) method. A hydroperoxo-iron (III) species is involved in the enzyme-catalyzed reaction. Herein, we explored the direct reaction mechanism of hydroperoxo-iron (III) species with biphenyl and 4-4'-dichlorobiphenyl. The reaction process involves an epoxide intermediate, it could develop into a carbocation intermediate, and ultimately evolve into a cis-diol product. The important roles of several residues during the dioxygenation process were highlighted. This study may provide theoretical support for further directed mutations and enzymatic engineering of BphA, as well as promote the development of degrading environmentally persistent biphenyl/polychlorinated biphenyl aromatic contaminants.


Assuntos
Compostos de Bifenilo/química , Dioxigenases/metabolismo , Poluentes Ambientais/química , Bifenilos Policlorados/química , Catálise , Dioxigenases/genética , Hidroxilaminas , Hidroxilação , Oxigenases/metabolismo , Bifenilos Policlorados/metabolismo
6.
Arch Biochem Biophys ; 682: 108283, 2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001245

RESUMO

Hydroxylation activity at the 6ß-position of steroid hormones (testosterone, progesterone, and cortisol) by human cytochromes P450 (CYP) 3A4, polymorphic CYP3A5, and fetal CYP3A7 were compared to understand the catalytic properties of the major forms of human CYP3A subfamily. Testosterone, progesterone, and cortisol 6ß-hydroxylation activities of recombinant CYP3A4, CYP3A5, and CYP3A7 were determined by liquid chromatography. Michaelis constants (Km) for CYP3A7-mediated 6ß-hydroxylation of testosterone, progesterone, and cortisol were similar to those of CYP3A4 and CYP3A5. The maximal velocity (kcat) and kcat/Km values for CYP3A4 were the highest, followed by CYP3A5 and those for CYP3A7 were the lowest among three CYP3A subfamily members. A decrease in Km values for progesterone 6ß-hydroxylation by CYP3A4, CYP3A5, and CYP3A7 in the presence of testosterone was observed, and the kcat values for CYP3A5 gradually increased with increasing testosterone. This indicated that testosterone stimulated progesterone 6ß-hydroxylation by all three CYP3A subfamily members. However, progesterone inhibited testosterone 6ß-hydroxylation mediated by CYP3A4, CYP3A5, and CYP3A7. In conclusion, the kcat values, rather than Km values, for 6ß-hydroxylation of three steroid hormones mediated by CYP3A7 were different from those for CYP3A4 and CYP3A5. In addition, the inhibitory/stimulatory pattern of steroid-steroid interactions would be different among CYP3A subfamily members.


Assuntos
Citocromo P-450 CYP3A/metabolismo , Hormônios/metabolismo , Esteroides/metabolismo , Catálise , Humanos , Hidrocortisona/metabolismo , Hidroxilação , Cinética , Microssomos Hepáticos/metabolismo , Progesterona/metabolismo , Proteínas Recombinantes/metabolismo , Testosterona/metabolismo
7.
Chemistry ; 26(19): 4256-4260, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32031278

RESUMO

We report the first chemical synthesis of eurysterol A, a cytotoxic and antifungal marine steroidal sulfate with a unique C8-C19 oxy-bridged cholestane skeleton. After C19 hydroxylation of cholesteryl acetate, used as an inexpensive commercial starting material, the challenging oxidative functionalization of ring B was achieved by two different routes to set up a 5α-hydroxy-7-en-6-one moiety. As a key step, an intramolecular oxa-Michael addition was exploited to close the oxy-bridge (8ß,19-epoxy unit). DFT calculations show this reversible transformation being exergonic by about -30 kJ mol-1 . Along the optimized (scalable) synthetic sequence, the target natural product was obtained in only 11 steps in 5 % overall yield. In addition, an access to (isomeric) 7ß,19-epoxy steroids with a previously unknown pentacyclic ring system was discovered.


Assuntos
Antifúngicos/síntese química , Esteroides/química , Esteróis/síntese química , Antifúngicos/química , Hidroxilação , Isomerismo , Estrutura Molecular , Oxirredução , Esteróis/química
8.
Chemosphere ; 247: 125680, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32069705

RESUMO

To examine the association between urinary metabolites of polycyclic aromatic hydrocarbons (OH-PAHs) and diabetes, online databases, including PubMed, Scopus, and Web of Science, were searched on July 17, 2019. Of the 668 articles identified through searching, six cross-sectional studies involving 24,406 participants were included. The pooled odds ratio (OR) and 95% confidence interval (CI) were calculated using a random-effect model. Heterogeneity was measured by reporting the I-square index. Moreover, subgroup analysis according to types of metabolites was performed. We found a significantly higher odds of diabetes in the highest versus the lowest category of urinary naphthalene (NAP), fluorine (FLU), phenanthrene (PHEN), and total OH-PAH metabolites. The pooled OR (95% CI) was estimated at 1.47 (1.17, 1.78), 1.50 (1.29, 1.71), 1.41 (1.21, 1.60), and 1.61 (1.01, 2.21), respectively. We also found a significant association per 1-fold increase in FLU (OR = 1.09, 95% CI [1.00, 1.19]) and PHEN (OR = 1.19, 95% CI [1.08, 1.30]) metabolites. In subgroup analysis stratified by types of OH-PAH metabolites, A significant stronger odds of diabetes was observed in the highest versus the lowest category of 2-PHEN (OR = 1.66, 95% CI [1.32, 2.00]), 2-NAP (OR = 1.66, 95% CI [1.16, 2.17]), 2-FLU (OR = 1.62, 95% CI [1.28, 1.97]), and 9-FLU (OR = 1.62, 95% CI [1.21, 2.04]) metabolites. Furthermore, there was a meaningfully greater likelihood of diabetes per 1-fold increase in 2-FLU (OR = 1.34, 95% CI [1.10, 1.57]), 2-PHEN (OR = 1.33, 95% CI [1.14, 1.51]), and 3-PHEN (OR = 1.19, 95% CI [1.04, 1.34]) metabolites. In conclusion, our study suggests the significant odds of association between urinary OH-PAH metabolites and diabetes.


Assuntos
Diabetes Mellitus/urina , Hidrocarbonetos Policíclicos Aromáticos/urina , Feminino , Flúor/urina , Humanos , Hidroxilação , Masculino , Naftalenos/urina , Razão de Chances , Fenantrenos/urina , Hidrocarbonetos Policíclicos Aromáticos/metabolismo
9.
J Biosci Bioeng ; 129(5): 552-557, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31983661

RESUMO

The gut bacterium Gordonibacter urolithinfaciens DSM 27213 metabolizes ellagic acid into three polyphenol compounds, namely, urolithin M5, urolithin M6, and urolithin C, which are collectively called urolithin. The key reactions of this metabolic pathway are the dehydroxylation of the phenolic hydroxy group, i.e., conversion of urolithin M5 to urolithin M6, and successive conversion of urolithin M6 to urolithin C. By testing the effects of various electron-transferring compounds on the dehydroxylation reactions, methylviologen was found to effectively support the dehydroxylation catalyzed by the cell free extracts. The urolithin dehydroxylating enzymes were found in the soluble fraction of the cell free extracts. The urolithin dehydroxylation was found to be coupled with reduction of dicationic methylviologen to a cation radical form catalyzed by enzymes with hydrogen as an electron donor, which was also found with the soluble fraction. Further investigation of the reaction in the presence of natural cofactors with or without methylviologen and hydrogen revealed the involvement of NADPH and FAD in the electron transportation systems of the urolithin dehydroxylation.


Assuntos
Actinobacteria/enzimologia , Proteínas de Bactérias/metabolismo , Coenzimas/metabolismo , Taninos Hidrolisáveis/metabolismo , Actinobacteria/química , Actinobacteria/metabolismo , Proteínas de Bactérias/genética , Elétrons , Taninos Hidrolisáveis/química , Hidroxilação
10.
J Agric Food Chem ; 68(6): 1563-1570, 2020 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-31927998

RESUMO

Ethanamizuril(N-{4-[4-(3,5-dioxo-4,5-dihydro-3H-[1,2,4]triazin-2-yl)-2-methyl-phenoxy]-phenyl}-acetamide, EZL) is a new anticoccidiosis compound and belongs to the class of triazines. In this study, the metabolism, distribution, and excretion of EZL were evaluated in chickens after administration of EZL at a single dosage. According to the relevant drug biotransformation rules, the exact molecular mass detection, the fragmentation characteristics, and the retention times, a total of five metabolites were identified in vivo in chickens, including two phase I metabolites and three phase II conjugated metabolites. The major metabolic pathways of EZL in chickens were deacetylation, hydroxylation, and glucuronidation. Regarding 14C-tissue residues after administration, kidney was considered to be the target tissue, as 14C-tissue residues could be detected at 240 h postdose. DeacetylEZL (M3) was the main metabolite, accounting for 68.65% and 25.62% of 14C in kidney at 6 and 24 h, respectively. In heart, muscle, skin+fat, and lung tissues, EZL was the main radioactive substance accounting for 94.88%, 97.32%, 96.23%, and 91.3% of 14C, respectively. In the liver, EZL and M3 were 20.76% and 54.65% of 14C, respectively. In chicken tissues the ratio of M5 was too low to be quantitated and it was mainly detected in chicken fecal and bile samples. In chicken excreta, EZL, M3, and glucuronidation of EZL (M5) accounted for 7.02%, 12.33%, and 10.32% of the dose, respectively and were eliminated primarily. This study presents the first detection of EZL metabolites, which is helpful for further understanding of the metabolic mechanism and in vivo intermediate processes of EZL. The results of this study will be good bases for better understanding EZL's anticoccidiosis mechanism and will serve as a helpful reference for assessing the risks to animals and humans.


Assuntos
Coccidiostáticos/farmacocinética , Triazinas/farmacocinética , Animais , Biotransformação , Galinhas , Coccidiostáticos/administração & dosagem , Coccidiostáticos/metabolismo , Hidroxilação , Rim/química , Rim/metabolismo , Fígado/química , Fígado/metabolismo , Pulmão/química , Pulmão/metabolismo , Músculos/química , Músculos/metabolismo , Triazinas/administração & dosagem , Triazinas/metabolismo
11.
Nat Commun ; 11(1): 174, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924757

RESUMO

mTORC1 is an important regulator of muscle mass but how it is modulated by oxygen and nutrients is not completely understood. We show that loss of the prolyl hydroxylase domain isoform 1 oxygen sensor in mice (PHD1KO) reduces muscle mass. PHD1KO muscles show impaired mTORC1 activation in response to leucine whereas mTORC1 activation by growth factors or eccentric contractions was preserved. The ability of PHD1 to promote mTORC1 activity is independent of its hydroxylation activity but is caused by decreased protein content of the leucyl tRNA synthetase (LRS) leucine sensor. Mechanistically, PHD1 interacts with and stabilizes LRS. This interaction is promoted during oxygen and amino acid depletion and protects LRS from degradation. Finally, elderly subjects have lower PHD1 levels and LRS activity in muscle from aged versus young human subjects. In conclusion, PHD1 ensures an optimal mTORC1 response to leucine after episodes of metabolic scarcity.


Assuntos
Leucina-tRNA Ligase/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Músculos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Adulto , Idoso , Envelhecimento/metabolismo , Aminoácidos/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Hidroxilação , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Leucina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Desenvolvimento Muscular , Músculos/patologia , Oxigênio/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Transdução de Sinais
12.
Environ Sci Technol ; 54(5): 2902-2912, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-31967796

RESUMO

Phenols are ubiquitous environmental pollutants, whose biotransformation involving phenol coupling catalyzed by cytochromes P450 may produce more lipophilic and toxic metabolites. Density functional theory (DFT) computations were performed to explore the debated phenol-coupling mechanisms, taking triclosan as a model substrate. We find that a diradical pathway facilitated by compound I and protonated compound II of P450 is favored vs alternative radical addition or electron-transfer mechanisms. The identified diradical coupling resembles a "two-state reactivity" from compound I characterized by significantly high rebound barriers of the phenoxy radicals, which can be formulated into three equations for calculating the ratio [coupling]/[hydroxylation]. A higher barrier for rebound than for H-abstraction in high-spin triclosan can facilitate the phenoxy radical dissociation and thus enable phenol coupling, while H-abstraction/radical rebound causing phenol hydroxylation via minor rebound barriers mostly occurs via the low-spin state. Therefore, oxidation of triclosan by P450 fits the first equation with a ratio [coupling]/[hydroxylation] of 1:4, consistent with experimental data indicating different extents of triclosan coupling (6-40%). The high rebound barrier of phenoxy radicals, as a key for the mechanistic identification of phenol coupling vs hydroxylation, originates from their weak electron donor ability due to spin aromatic delocalization. We envision that the revealed mechanism can be extended to the cross-coupling reactions between different phenolic pollutants, and the coupling reactions of several other aromatic pollutants, to infer unknown metabolites.


Assuntos
Poluentes Ambientais , Fenol , Biotransformação , Hidroxilação , Fenóis
13.
J Chromatogr A ; 1617: 460828, 2020 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-31911001

RESUMO

In this study a heart-cutting 2D-LC method was successfully developed and optimized in order to discriminate and quantitate (S)-propranolol, (R)-propranolol, and its hydroxy metabolites, namely the isomeric (S)-4'­hydroxy propranolol, (R)-4'­hydroxy propranolol, (S)-5'­hydroxy propranolol, (R)-5'­hydroxy propranolol, (S)-7'-hydroxy propranolol, and (R)-7'­hydroxy propranolol in one chromatographic run. Thereby, experiments investigating chiral discrimination in ring hydroxylation of propranolol were made feasible. Analysis of human urine samples after administration of a single oral dose of 40 mg of propranolol clearly revealed considerable chiral shifts in propranolol and its 4'-, 5'-, and 7'-hydroxy metabolites. Furthermore, the excretion rates of the individual (S)- and (R)-enantiomers were continuously monitored over 24 h post administration. Studies were performed utilizing a 2D-LC system hyphenated to a triple quadrupole mass spectrometer. The chromatographic system was endued with a reversed phase column (phenyl-hexyl) in first dimension and a teicoplanin based chiral column in second dimension. The method was basically validated and successfully evaluated as robust. Calibration was performed achieving accuracy between 80% and 120%. Maximal excretion rates of (S)-propranolol, (R)-propranolol, (S)-4'­hydroxy propranolol, (R)-4'­hydroxy propranolol, (S)-5'­hydroxy propranolol, (R)-5'­hydroxy propranolol, and (R)-7'­hydroxy propranolol were 237 ng/min, 281 ng/min, 4 ng/min, 4 ng/min, 1 ng/min, 9 ng/min, and 3 ng/min, respectively.


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas , Propranolol/química , Propranolol/urina , Humanos , Hidroxilação , Propranolol/metabolismo , Estereoisomerismo , Teicoplanina
14.
Nat Prod Res ; 34(2): 204-209, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30580617

RESUMO

A new oleanane triterpenoid, 2α,3ß,6ß,23,29-pentahydroxyolean-12-en-28- oic acid (1), was isolated from the roots of Rhodomyrtus tomentosa, together with four known oleanane triterpenoids (2-5) and two known ursane triterpenoids (6-7). The structure of compound 1 was determined by extensive NMR and HR-ESI-MS data analysis. Compounds 4-5 showed cytotoxicity against PC12 cell lines at a concentration of 50 µM, and compound 1 exhibited moderate neuroprotective activity against corticosterone induced PC12 cell death at the same concentration.


Assuntos
Myrtaceae/química , Ácido Oleanólico/análogos & derivados , Raízes de Plantas/química , Triterpenos/isolamento & purificação , Animais , Citotoxinas/química , Citotoxinas/isolamento & purificação , Humanos , Hidroxilação , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/isolamento & purificação , Ácido Oleanólico/química , Ácido Oleanólico/isolamento & purificação , Células PC12 , Ratos , Espectrometria de Massas por Ionização por Electrospray , Triterpenos/química , Triterpenos/farmacologia
15.
Artigo em Inglês | MEDLINE | ID: mdl-31707907

RESUMO

Among three monofluoroanilines, 2-fluoroaniline (2-FA) and 3-fluoroaniline (3-FA) exhibit relatively poor biodegradability. This work examined their degradation characteristics in a mixed culture system and also analyzed the microorganism community. After acclimation for 58 d and 43 d, the high removal efficiency of 100% of 2-FA and 95.3% of 3-FA was obtained by adding 25 mg L-1 of 2-FA or 3-FA to the two reactors, respectively. In addition, the high defluorination rates of 2-FA and 3-FA were observed to be 87.0% and 89.3%, respectively. The degradation kinetics showed that the maximum specific degradation rates of 2-FA and 3-FA were (21.23 ± 0.91) mg FA (g•VSS·h)-1, and (11.75 ± 0.99) mg FA (g•VSS·h)-1, respectively. PCR-DGGE analysis revealed that the unique bacteria degrading 2-FA were mainly composed of six genera (Novosphingobium, Bradyrhizobium, Aquaspirillum, Aminobacter, Ochrobactrum, and Labrys), and five genera that degraded 3-FA (Ochrobactrum, Aquaspirillum, Lachnobacterium, Bradyrhizobium, and Variovorax). Analysis of the key catabolic enzyme activities indicated that the simultaneous hydroxylation and dehalogenation were involved in monooxygenase elimination of 2-FA and conversion of 3-FA to 4-fluorocatechol by dioxygenase, indicating that enriched mixed cultures were effective to metabolize 2-FA or 3-FA by unconventional pathways to prevent the accumulation of toxic metabolites.


Assuntos
Compostos de Anilina/metabolismo , Fluorbenzenos/metabolismo , Microbiota , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/metabolismo , Biodegradação Ambiental , Reatores Biológicos/microbiologia , Halogenação , Hidroxilação , Cinética , Microbiota/genética
16.
Xenobiotica ; 50(6): 640-653, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31596164

RESUMO

Temsirolimus, a derivative of sirolimus, exhibits potent antitumor properties. It was the goal of this study to identify yet unknown temsirolimus metabolites generated after incubation with human liver microsomes. Previously, 23-hydroxy-, 24-hydroxy, 12-hydroxy, hydroxy-piperidine and 27-O-desmethyl temsirolimus had been described.Metabolite structures were identified using high-resolution mass spectrometry, MS/iontrap (MSn) and comparison of fragmentation patterns of the metabolites with those of temsirolimus and other known sirolimus derivatives. Moreover, enzyme kinetic parameters of temsirolimus metabolite formation as well as the contribution of individual recombinant cytochrome P450 (CYP) enzymes to temsirolimus metabolism were investigated.Human liver microsomes mainly hydroxylated and/or demethylated temsirolimus. The structures of the following metabolites were identified: O-demethylated metabolites: 39-O-desmethyl, 16-O-desmethyl and 27-O-desmethyl temsirolimus; hydroxylated metabolites: hydroxy piperidine temsirolimus, 11-hydroxy, 12-hydroxy, 14-hydroxy, 23-hydroxy, 24-hydroxy, 25-hydroxy, 45/46-hydroxy and 49-hydroxy temsirolimus; demethylated-hydroxylated metabolites: 16-O-desmethyl, 24-hydroxy; 16-O-desmethyl, 23-hydroxy and 16-O-desmethyl 46-hydroxy temsirolimus; didemethylated metabolite: 27,39-O-didesmethyl temsirolimus; and dihydroxylated metabolite: 12,24-dihydroxy temsirolimus. It was confirmed that CYP3A4 represents the predominant enzyme responsible for temsirolimus metabolism. Moreover, CYP3A5 as well as CYP2C8 also showed significant activities especially resulting in the formation of 27-O-desmethyl, 25-hydroxy and hydroxy-piperidine temsirolimus.It is concluded that temsirolimus is metabolized to more than 20 metabolites, not counting metabolism via the sirolimus pathway. Eighteen of these metabolites could be structurally identified using ion trap MSn and high-resolution mass spectrometry. Moreover, the present study showed that, in addition to CYP3A4, metabolism via CYP3A5 and CYP2C8 also represent significant metabolic pathways.


Assuntos
Microssomos Hepáticos/metabolismo , Sirolimo/análogos & derivados , Citocromo P-450 CYP2C8/metabolismo , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Hidroxilação , Espectrometria de Massas , Redes e Vias Metabólicas , Sirolimo/metabolismo
17.
J Biosci Bioeng ; 129(1): 41-46, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31375401

RESUMO

Hydroxy fatty acids (HFAs) are highly valued industrial materials. Pseudomonas sp. NRRL B-2994 was used for stereospecific microbial biotransformation to hydroxylate unsaturated fatty acids (UFAs). As Pseudomonas sp. was continuously subcultured, the hydroxylation capability (both conversion rate and productivity) decreased. A morphology change was observed from large to small colonies. To produce stereospecific 10-hydroxy-12(Z)-octadecenoic acid from plant oils by using Pseudomonas sp. NRRL B-2994, the effect of phenotypic variations related to microbial hydroxylation of UFAs was confirmed. The conversion rate and the total productivity of creating HFAs from UFAs by microbial hydroxylation were highly dependent upon colony phenotype variations of Pseudomonas sp. NRRL B-2994. The morphological change was responsible for a lower rate of hydroxylation. The small colony variants showed increased hydrophobicity of the cell surface resulting in cell aggregation in liquid culture and lower hydroxylation due to limited exposure of substrates, UFAs. Small colony variants could be reverted to typical large colony variants. An economically feasible process was established for microbial hydroxylation using large colony variants with 50% HFA conversion rate and 10-15 g/L of productivity.


Assuntos
Ácidos Graxos/metabolismo , Pseudomonas/metabolismo , Variação Biológica da População , Biotransformação , Ácidos Graxos/química , Ácidos Graxos Insaturados/química , Ácidos Graxos Insaturados/metabolismo , Hidroxilação , Óleos Vegetais/química , Óleos Vegetais/metabolismo , Pseudomonas/química , Pseudomonas/crescimento & desenvolvimento
18.
Oncogene ; 39(2): 414-427, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31477841

RESUMO

Drug resistance is a major problem limiting the efficacy of chemotherapy in cancer treatment, and the hypoxia-induced stabilization of HIF-1α plays a role in this process. HIF-1α overexpression has been observed in a variety of human cancers, including colorectal cancer (CRC). Therefore, targeting HIF-1α is a promising strategy for overcoming chemoresistance to enhance the efficacy of chemotherapies in CRC. Here, we show that DNMT inhibitors can induce HIF-1α degradation to overcome oxaliplatin resistance and enhance anti-CRC therapy. We found that a low-toxicity DNMT inhibitor, zebularine, could downregulate HIF-1α expression and overcome hypoxia-induced oxaliplatin resistance in HCT116 cells and showed efficacy in HCT116 xenograft models and AOM/DSS-induced CRC mouse models. Zebularine could induce the degradation of HIF-1α protein through hydroxylation. LC-MS analysis showed a decrease in succinate in various CRC cells under hypoxia and in colon tissues of AOM/DSS-induced CRC mice. The decrease was reversed by zebularine. Tumor angiogenesis was also reduced by zebularine. Furthermore, zebularine potentiated the anticancer effect of oxaliplatin in AOM/DSS-induced CRC models. This finding provides a new strategy in which an increase in HIF-1α hydroxylation could overcome oxaliplatin resistance to enhance anti-CRC therapy.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Terapia de Alvo Molecular , Oxaliplatina/farmacologia , Animais , Antineoplásicos/uso terapêutico , Neoplasias Colorretais/irrigação sanguínea , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Citidina/análogos & derivados , Citidina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Hidroxilação/efeitos dos fármacos , Camundongos , Neovascularização Patológica/tratamento farmacológico , Oxaliplatina/uso terapêutico , Estabilidade Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Int J Phytoremediation ; 22(2): 224-225, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31385520

RESUMO

In response to Dr. Yang et al.'s comments on our article "Transcriptomic response of Arabidopsis thaliana exposed to hydroxylated polychlorinated biphenyls (OH-PCBs)", additional details were provided regarding the analysis of the gene expression level (One-Way Between-Subject ANOVA) and correction for false discovery rate (FDR) (Benjamini-Hochberg). The gene expression analysis was performed again using the new release of the Transcriptome Analysis Console™ (version 4.0.1, Life Technologies - not available at the time our initial study was conducted), which integrates the Limma differential expression portion of the Bioconductor package. Overall similar results were obtained regarding the number of genes differentially expressed and the enrichment of genes in different Gene Ontology (GO) categories. The transcriptomic profiles induced in response to the three OH-derivatives were shown, again, to be similar to those induced by inhibitors of the brassinosteroid synthesis (i.e., brassinazole, propiconazole, and uniconazole), potentially resulting in iron deficiency in exposed plants. The new (and improved) method used for the selection of differentially expressed genes did not change the conclusion of our initial study, which suggested that the higher phytotoxicity of OH-derivatives, as compared to the parent compound 2,5-dichlorobiphenyl (2,5-DCB), may be explained by the inhibition of the brassinosteroid synthesis pathway.


Assuntos
Arabidopsis , Poluentes Ambientais , Bifenilos Policlorados , Biodegradação Ambiental , Hidroxilação , Transcriptoma
20.
J Anal Toxicol ; 44(2): 140-148, 2020 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-31788682

RESUMO

New psychoactive substances are emerging on the illegal drug market. Synthetic opioids including fentanyl analogues are of special concern due to their high potency. This indicates the possibility of low drug concentrations in vivo and calls for sensitive analytical methods and identification of the most appropriate analytical targets. In this study the in vitro metabolism of ortho-, meta- and para-fluorofentanyl, three fluorinated derivatives of fentanyl, has been investigated using human hepatocytes and compared to the results from an authentic human urine sample. Based on knowledge on the metabolism of similar fentanyl analogues N-dealkylation and hydroxylation was hypothesized to be the most central pathways. The three fluorofentanyl isomers were incubated with pooled human hepatocytes at 1, 3 and 5 h. Liquid chromatography quadrupole time of flight mass spectrometry operating in data-dependent mode was used to analyse the hepatocyte samples, as well as the hydrolysed and non-hydrolysed authentic urine sample. Data were analysed by a targeted approach with a database of potential metabolites. The major metabolite formed in vitro was the N-dealkylation product norfluorofentanyl. In addition various hydroxylated metabolites, a N-oxide, dihydrodiol metabolites and a hydroxymethoxy metabolite were found. In total, 14 different metabolites were identified for each fluorofentanyl isomer. In the authentic urine sample, three metabolites were detected in addition to the ortho-fluorofentanyl parent compound, with hydroxymethoxy metabolite having the highest abundance followed by norfluorofentanyl and a metabolite hydroxylated on the ethylphenyl ring. This in vitro study showed that the metabolic pattern for ortho-, meta-, and para-fluorofentanyl was close to those previously reported for other fentanyl analogues. We suggest that the hydroxymethoxy metabolite and the metabolite hydroxylated on the ethylphenyl ring should be the metabolites primarily investigated in further studies to determine the most appropriate marker for intake of fluorofentanyl derivatives in urine drug screening for human subjects.


Assuntos
Fentanila/metabolismo , Hepatócitos/metabolismo , Drogas Ilícitas/metabolismo , Detecção do Abuso de Substâncias , Analgésicos Opioides , Cromatografia Líquida , Drogas Desenhadas , Fentanila/análogos & derivados , Fentanila/química , Humanos , Hidrólise , Hidroxilação , Drogas Ilícitas/química , Espectrometria de Massas , Microssomos Hepáticos
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