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1.
Anal Chem ; 93(3): 1749-1756, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33351590

RESUMO

Significant efforts have been made to develop robust and reliable methods for simultaneous biothiols determination in different matrices, but there still exist the problems such as easy oxidation, tedious derivatization, and difficulty in discrimination, which brings unsatisfactory results in their accuracy and fast quantification in biological samples. To overcome these problems, a simultaneous biothiols detection method combining a "selenium signature" chemical probe and paper spray mass spectrometry (PS-MS) was proposed. In the strategy, the modified-paper substrate is used to enhance the analytical performance. Chemical probe Ebselen-NH2 that has a specific response to biothiols was designed and covalently fixed on the surface of an oxidized paper substrate. By the identification of derivatized product with distinctive selenium isotope distribution and employment of the optimized PS-MS method, qualitative and quantitative analysis of five biothiols including glutathione (GSH), cysteine (Cys), cysteinylglycine (CysGly), N-acetylcysteine (Nac), and homocysteine (Hcy) were realized. Biothiols in plasma and cell lysates were measured with satisfactory results. The established method not only provides a novel protocol for simultaneous determination of biothiols, but also is helpful for understanding the biological and clinical roles played by these bioactive small molecules.


Assuntos
Acetilcisteína/análise , Cisteína/análise , Dipeptídeos/análise , Corantes Fluorescentes/química , Glutationa/análise , Papel , Selênio/química , Técnicas Biossensoriais , Cisteína/análogos & derivados , Humanos , Espectrometria de Massas
2.
Int J Mol Sci ; 21(24)2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33333739

RESUMO

Butylated hydroxytoluene (BHT) and its analogs, butylated hydroxyanisole (BHA) and tert-butyl-hydroquinone (TBHQ), are widely used synthetic preservatives to inhibit lipid oxidation in the food, cosmetic and pharmaceutical industries. Despite their widespread use, little is known about their human exposure and related biotransformation products. The metabolism of these compounds was investigated using in vitro incubations with human and rat liver fractions. Liquid chromatography coupled to high-resolution tandem mass spectrometry was employed to detect and characterize stable and reactive species formed via oxidative metabolism, as well as phase II conjugates. Several oxidative metabolites have been detected, as well as glutathione, glucuronide, and sulfate conjugates, many of which were not previously reported. A combination of accurate mass measurements, MS/MS fragmentation behavior, and isotope-labeling studies were used to elucidate metabolite structures.


Assuntos
Antioxidantes/análise , Hidroxianisol Butilado/metabolismo , Hidroxitolueno Butilado/metabolismo , Animais , Antioxidantes/química , Hidroxianisol Butilado/análogos & derivados , Hidroxianisol Butilado/química , Hidroxitolueno Butilado/análogos & derivados , Hidroxitolueno Butilado/química , Cromatografia Líquida , Glucuronídeos/análise , Glutationa/análise , Humanos , Técnicas In Vitro , Microssomos/metabolismo , Fenóis/análise , Fenóis/metabolismo , Ratos , Sulfatos/análise , Espectrometria de Massas em Tandem
3.
Plant Physiol Biochem ; 154: 538-546, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32912487

RESUMO

The sulfite reductase gene in Medicago sativa L. (MsSiR) encodes sulfite reductase (SiR) and catalyses the conversion of sulfite to sulfate in the sulfite assimilation pathway. In this study, we investigated the role of MsSiR in alfalfa by generating transgenic alfalfa that ectopically expressed MsSiR under the control of the CaMV35S promoter. The differences in alkali tolerance between the MsSiR-overexpressing and wild-type (WT) plants were analyzed, and the MsSiR-overexpressing plants exhibited an improved phenotype under alkali stress. Compared to WT plants, these plants demonstrated improved antioxidant activity as well as decreased H2O2 and O2- contents and increased glutathione reduced (GSH), Cysteine (Cys) and glutathione oxidized (GSSG) contents. MsSiR-overexpressing plants also exhibited high levels of adenosyl phosphosulfate reductases (APR), sulfite oxidase (SO) and MsSiR expression under alkali stress. It was speculated that MsSiR is involved in sulfur metabolism pathways, including the stabilization of sulfate and sulfite levels and the synthesis of GSH. These two processes achieve alkali tolerance by positively regulating the detoxification and antioxidant activities of alfalfa.


Assuntos
Álcalis/efeitos adversos , Glutationa/análise , Medicago sativa , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Proteínas de Plantas/genética , Antioxidantes/análise , Peróxido de Hidrogênio , Medicago sativa/enzimologia , Medicago sativa/genética , Plantas Geneticamente Modificadas/enzimologia , Estresse Fisiológico
4.
Parasitol Res ; 119(9): 2783-2798, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32683559

RESUMO

Due to the increasing consumption of platinum (Pt), especially in automobile exhaust catalysts, environmental concentrations of Pt are of emerging concern worldwide. Limited information exists on environmental concentrations, particularly in Pt mining regions, while South Africa is the world's main supplier of Pt. Moreover, other metals are also released as by-products of Pt mining, which might also cause environmental concern. Certain fish parasite taxa have the ability to accumulate metals orders of magnitude higher than their hosts and can be used to reliably detect metals with naturally low abundance. Studies on Pt accumulation in parasite-host systems are limited. Therefore, the aims of the present study were (1) to determine the accumulation of a variety of metals (cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), lead (Pb), platinum (Pt), and zinc (Zn)) in helminth fish parasites compared with their hosts from a reference site and an impoundment impacted by Pt mining activities; (2) to assess whether there is a difference between bioaccumulation of metals in infected and uninfected hosts, as well as between hosts with different infection intensities; and (3) to compare the biomarker responses (acetylcholine esterase activity (AChE), metallothionein content (MT), catalase activity (CAT), reduced glutathione content (GSH), malondialdehyde content (MDA), protein carbonyls induction (PC), superoxide dismutase activity (SOD), and cellular energy allocation (CEA)) between infected and uninfected hosts. The cestode Atractolytocestus huronensis accumulated significantly higher concentrations of Cr, Ni, and Pt than their host Cyprinus carpio, while the nematode Contracaecum sp. accumulated significantly higher concentrations of Pt and Zn than their host Clarias gariepinus. Infected fish showed lower metal concentrations compared to uninfected fish, while the parasites had no significant effects on their hosts' biomarker responses. The parasites demonstrated the bioavailability of metals derived from Pt mining activities and their ability to resist its toxic effects. Thus, these parasites are promising sensitive accumulation indicators for Cr, Ni, Pb, and Pt contaminations from Pt mining activities.


Assuntos
Bioacumulação/fisiologia , Cestoides/química , Metais Pesados/análise , Nematoides/química , Emissões de Veículos/análise , Poluentes Químicos da Água/análise , Acetilcolinesterase/metabolismo , Animais , Cádmio/análise , Cádmio/toxicidade , Carpas/parasitologia , Catalase/metabolismo , Peixes-Gato/parasitologia , Cobre/análise , Cobre/toxicidade , Ecossistema , Monitoramento Ambiental , Glutationa/análise , Malondialdeído/análise , Metalotioneína/análise , Metais Pesados/toxicidade , Parasitos , Platina/análise , Platina/toxicidade , África do Sul , Superóxido Dismutase/metabolismo , Emissões de Veículos/toxicidade , Poluentes Químicos da Água/toxicidade
5.
Clín. investig. arterioscler. (Ed. impr.) ; 32(3): 87-93, mayo-jun. 2020. tab
Artigo em Inglês | IBECS | ID: ibc-193352

RESUMO

OBJECTIVE: Post-prandial lipaemia (PL), oxidative stress (OS), and complement component C3 (C3) values are related to the atherosclerosis process. The post-prandial response of C3 after an oral fat load test (OFLT) using unsaturated fat is poorly addressed. The aim of this study was to analyze and compare the post-prandial response of OS markers and C3 values in men and women after an OFLT using unsaturated fat. METHODS: The study included a total of 22 healthy subjects with normal lipids and normal blood glucose (11 men and 11 pre-menopausal women). An oral unsaturated fat load test (OFLT: 50g fat per m2 body surface) was performed using a commercial liquid preparation of long chain triglycerides (Supracal®). OS markers and C3 were measured using standardized methods at fasting state and every 2h up to 8h after the OFLT. RESULTS: Men showed statistically significant higher C3, oxidized glutathione (GSSG), and oxidized-reduced glutathione (GSSG/GSH) ratio values at fasting state compared to that obtained in women. In addition, post-prandial C3 values and GSSG/GSH ratios were significantly higher in men compared to women. The GSSG value and GSSG/GSH ratio significantly decreased in men after the OFLT compared to fasting values. In contrast, the post-prandial OS markers decrease observed in women was not statistically significant. CONCLUSIONS: In fasting state, men showed higher statistically significant C3 values and OS markers than women. The post-prandial OS markers (GSSG and GSSG/GSH ratio) significantly decrease after the OFLT with unsaturated fat in men compared to women


OBJETIVO: Los valores de lipemia postprandial (PL), estrés oxidativo (OS) y componente C3 del complemento (C3) están relacionados con el proceso de aterosclerosis. La respuesta postprandial de C3 tras una sobrecarga oral de grasa (OFLT) utilizando grasa insaturada no es completamente conocida. Nuestro objetivo fue analizar y comparar la respuesta postprandial de los marcadores de OS y los valores de C3 en hombres y mujeres después de una OFLT utilizando grasa insaturada. MÉTODOS: Estudiamos 22 sujetos normolipidémicos y normoglicémicos (11 hombres y 11 mujeres premenopáusicas). Se realizó una sobrecarga oral con grasa insaturada (OFLT: 50g de grasa por m2 de superficie corporal) utilizando una preparación líquida comercial de triglicéridos de cadena larga (Supracal®). Los marcadores OS y C3 se midieron utilizando métodos estandarizados en estado de ayuno y cada 2 horas hasta 8 horas después de OFLT. RESULTADOS: Los hombres mostraron valores significativamente mayores de C3, glutatión oxidado (GSSG) y glutatión reducido (GSSG/GSH) en estado de ayuno en comparación con los obtenidos en mujeres. Además, los valores de C3 postprandiales y la relación GSSG/GSH fueron significativamente más altos en los hombres que en las mujeres. El valor GSSG y la relación GSSG/GSH disminuyeron significativamente en los hombres después de OFLT en comparación con los valores de ayuno. En contraste, la disminución de marcadores postprandiales de OS observada en mujeres no fue estadísticamente significativa. CONCLUSIONES: En ayunas, los hombres muestran valores estadísticamente mayores de C3 y marcadores OS que las mujeres. Los marcadores OS postprandial (GSSG y GSSG/GSH ratio) disminuyen significativamente tras OFLT con grasa insaturada en los hombres en comparación con las mujeres


Assuntos
Humanos , Masculino , Feminino , Adulto , Estresse Oxidativo/efeitos dos fármacos , Complemento C3/efeitos dos fármacos , Aterosclerose/diagnóstico , Gorduras Insaturadas/administração & dosagem , Biomarcadores , Glutationa/sangue , Gorduras Insaturadas/farmacologia , Glutationa/análise , Glutationa Peroxidase/análise , Pré-Menopausa/sangue , Índice de Massa Corporal , Antropometria , Lipoproteínas/análise , Lipídeos/análise , Gorduras na Dieta/administração & dosagem
6.
J Chromatogr A ; 1622: 461160, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32450990

RESUMO

The glutathione (GSH) trapping assay is commonly utilized for the screening and characterization of reactive metabolites produced by drug metabolism. This study describes a fluorous derivatization method for a more sensitive and selective analysis of reactive metabolites trapped by GSH using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, the GSH-trapped reactive metabolites, which were obtained after incubation of the test compounds with human liver microsome (HLM) in the presence of GSH and NADPH, were derivatized using the perfluoroalkylamine reagent through oxazolone chemistry. Since this reaction enabled the selective modification of the α-carboxyl group in GSH, the structural compositions of the metabolites were not affected by the derivatization. Furthermore, the selective analysis of the resulting derivatives could be performed using perfluoroalkyl-modified stationary phase LC separation via the interaction between the perfluoroalkyl-containing compounds, such as fluorous affinity, followed by detection with the precursor ion and/or enhanced product ion scan modes in MS/MS. Finally, we demonstrated the applicability of this method by analyzing perfluoroalkyl derivatives of some drug metabolites trapped by GSH in HLM incubation.


Assuntos
Cromatografia Líquida/métodos , Flúor/química , Glutationa/análise , Espectrometria de Massas em Tandem/métodos , Glutationa/química , Humanos , Microssomos Hepáticos/metabolismo , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo
7.
Anal Chim Acta ; 1113: 43-51, 2020 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-32340668

RESUMO

This study fabricated a dual-emission probe consisting of monolayer MoS2 quantum dots (M - MoS2 QDs) and L-cystine-linked boron-dipyrromethene (L-Cys-BODIPY) molecules for ratiometric sensing of biothiols, thiol product-related enzyme reactions, and ratiometric imaging of glutathione (GSH)-related reactions in HeLa cells. The formation of L-Cys-BODIPY-adsorbed M - MoS2 QDs (named as BODIPY-M-MoS2 QDs) was demonstrated by comparing them with M - MoS2 QDs using transmission electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. The BODIPY-M-MoS2 QDs exhibited dual-emission bands, excellent biocompatibility, and good resistance to photobleaching. It was found that the adsorbed L-Cys-BODIPY molecules rarely quenched the fluorescence of M - MoS2 QDs, and meanwhile, they were self-quenched by π-π stacking between each BODIPY backbones. The presence of biothiols induced the reduction of weakly fluorescent L-Cys-BODIPY to strongly fluorescent of L-cysteine-conjugated BODIPY. Since having a much higher molar absorption coefficient than L-Cys-BODIPY, the liberated L-cysteine-conjugated BODIPY behaved as an effective inner filter to absorb the excitation light and subsequently quenched the fluorescence of M - MoS2 QDs. The appearance of L-cysteine-conjugated BODIPY could barely affected to the fluorescence lifetime of M - MoS2 QDs, confirming the inner filter effect of L-cysteine-conjugated BODIPY onto the fluorescence of M - MoS2 QDs. The present probe not only provided a linear ratiometric response to 1-10 mM GSH, 1-10 µM cysteine, and 1-10 µM of homocysteine but also remarkably showed the ratiometric detection of thiol products from the reactions of 1-900 units L-1 S-adenosylhomocysteine (SAH) hydrolase and SAH as well as 1-850 units L-1 GSH reductase and disulfide GSH. Additionally, the present probe was well-suited for ratiometric imaging of intracellular GSH levels in non-treated and drug-treated HeLa cells.


Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Glutationa/análise , Homocisteína/análise , Pontos Quânticos/química , Compostos de Boro/química , Cistina/química , Dissulfetos/química , Células HeLa , Humanos , Limite de Detecção , Molibdênio/química , Espectrometria de Fluorescência/métodos
8.
Org Biomol Chem ; 18(13): 2468-2474, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32167516

RESUMO

A new N2O-type BODIPY probe (LF-Bop) has been proposed for the selective and sensitive detection of biologically relevant small molecular thiols. This detection is based on the Michael addition reaction between the thiol and nitrostyrene groups in the probe, which decreases the quenching effect from the nitro group, thus resulting in the recovery of the deep-red fluorescence from the BODIPY structure. The results show that LF-Bop is able to detect all tested free thiols through a fluorescence turn-on assay. The lowest limit of detection (LOD) for glutathione was found to be down to nanomolar levels (220 nM). Based on this probe, we have developed a new fluorescence assay for the screening of acetylcholinesterase inhibitors. In total, 11 natural and synthetic alkaloids have been evaluated. Both experimental measurements and theoretical molecular docking results reveal that both natural berberine and its synthetic derivative dihydroberberine are potential inhibitors of acetylcholinesterase.


Assuntos
Compostos de Boro/química , Inibidores da Colinesterase/química , Corantes Fluorescentes/química , Glutationa/análise , Estirenos/química , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Animais , Berberina/análogos & derivados , Berberina/química , Berberina/metabolismo , Compostos de Boro/síntese química , Inibidores da Colinesterase/metabolismo , Avaliação Pré-Clínica de Medicamentos , Elasmobrânquios , Peixe Elétrico , Corantes Fluorescentes/síntese química , Glutationa/química , Limite de Detecção , Simulação de Acoplamento Molecular , Ligação Proteica , Estirenos/síntese química , Tacrina/química , Tacrina/metabolismo
9.
J Anim Sci ; 98(4)2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32207523

RESUMO

Broilers stocked in high densities may be prone to oxidative and inflammatory insults, resulting in impaired health status, growth performance, and meat quality. This study was to determine if 30% extra supplemental dl-methionine alleviated or prevented those adverse effects of a higher stocking density in broiler chickens. A total of 560 male Cornish Cross cockerels (day old) were divided into four groups: two stocking densities (9 and 12 birds/m2) and two supplementations of methionine (grower: 2.90 or 3.77 g/kg and finisher: 2.60 or 3.38 g/kg). Growth performance was recorded weekly. Blood and tissues were sampled at the end of each period. High stocking density decreased (P < 0.05) body weight and growth performance of growers and (or) finishers. Those differences were partially attenuated by the extra methionine supplementation. The high methionine elevated (P < 0.05) glutathione (GSH) concentration in the thigh at both ages (> 24%). The high stocking density elevated (>28%, P < 0.05) glutathione concentration in the plasma, breast, and thigh of growers, but decreased (P < 0.05) it in the liver of growers and thigh of finishers. Interaction effects (P < 0.05) between dietary methionine and stocking density were found on activities of the antioxidant enzyme glutathione S-transferase in the liver of growers and breast, thigh, and adipose tissue of finishers. The interaction effect was also found on activities of glutathione peroxidase and superoxide dismutase in the thigh of growers. The extra methionine decreased (P < 0.05) hepatic gene expression of heat shock protein 90 (18%) and thigh and breast malondialdehyde concentrations of the finishers (35%). In conclusion, the 30% extra dl-methionine supplementation was able to partially mitigate adverse effects caused by the higher stocking density and to improve the redox status of the broilers.


Assuntos
Antioxidantes/metabolismo , Galinhas/fisiologia , Suplementos Nutricionais/análise , Ácidos Graxos/análise , Metionina/farmacologia , Ração Animal/análise , Animais , Peso Corporal/efeitos dos fármacos , Galinhas/crescimento & desenvolvimento , Dieta/veterinária , Glutationa/análise , Glutationa Peroxidase/metabolismo , Fígado/metabolismo , Masculino , Malondialdeído/análise , Oxirredução , Superóxido Dismutase/metabolismo
10.
Mikrochim Acta ; 187(4): 229, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170465

RESUMO

Black phosphorus quantum dots (BP QDs) with small size are synthesized using an easy to operate thermal method. It was found that BP QDs possess oxidase-mimicking activity. They can catalyze the oxidation of the substrate 3,3',5,5'-tetramethylbenzidine to produce a blue-colored product even in the absence of hydrogen peroxide. Active oxygen species are proved to be involved in the reaction through the experiments of radical scavenging and electron spin resonance. Biothiols including reduced glutathione and cysteine inactivate the oxidase-mimicking activity of BP QDs, concomitant to the fading of the blue solution. This provides the  base for a colorimetric method for the determination of glutathione and cysteine. The decreased absorbance at 652 nm displays linear response to the concentrations of glutathione ranging from 0.1 to 5.0 µmol L-1, and cysteine from 0.1 to 10.0 µmol L-1. The detection limits are 0.02 µmol L-1 and 0.03 µmol L-1 for glutathione and cysteine, respectively. Successive determinations of 1.0 µmol L-1 glutathione and 5.0 µmol L-1 cysteine solution give relative standard deviations of 0.8% and 1.7% (n = 11), respectively. As a preliminary application, the practicability of the method was evaluated by the determination of glutathione in pharmaceutical preparations. This work not only discovers a useful oxidase mimics but also sets up a reliable platform based on BP QDs in colorimetric detection. Graphical abstract Schematic representation of colorimetric determination for biothiols through inactivating oxidase mimetic-like catalytic activity of black phosphorus quantum dots (BP QDs) on the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) with dissolved oxygen to produce its blue oxidized product (oxTMB).


Assuntos
Técnicas Biossensoriais , Colorimetria , Cisteína/análise , Glutationa/análise , Oxirredutases/química , Fósforo/química , Pontos Quânticos/química , Cisteína/metabolismo , Glutationa/metabolismo , Estrutura Molecular , Oxirredutases/metabolismo , Fósforo/metabolismo
11.
Planta ; 251(3): 69, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32076872

RESUMO

MAIN CONCLUSION: This report proves a cross talk between H2S and IAA in cold stress response, which has presented strong evidence that IAA acts as a downstream signal mediating the H2S-induced stress tolerance in cucumber seedlings. We evaluated changes in endogenous hydrogen sulfide (H2S) and indole-3-acetic acid (IAA) emission systems, and the interactive effect of exogenous H2S and IAA on chilling tolerance in cucumber seedlings. The results showed that chilling stress increased the activity and relative mRNA expression of L-/D-cysteine desulfhydrase (L-/D-CD), which in turn induced the accumulation of endogenous H2S. Similarly, the endogenous IAA system was triggered by chilling stress. We found that 1.0 mM sodium hydrosulfide (NaHS, an H2S donor) significantly enhanced the activity of flavin monooxygenase (FMO) and relative expression of FMO-like proteins (YUCCA2), which in turn elevated endogenous IAA levels in cucumber seedlings. However, IAA had little effects on activities of L-/D-CD and endogenous H2S levels. H2S-induced IAA production accompanied by increase in chilling tolerance, as shown by the decrease in stress-induced electrolyte leakage (EL) and reactive oxygen species (ROS) accumulation, and increase in gene expressions and enzyme activities of photosynthesis. 1-naphthylphthalamic acid (NPA, an IAA polar transport inhibitor) declined H2S-induced chilling tolerance and defense genes' expression. However, scavenging of H2S had a little effect on IAA-induced chilling tolerance. These results suggest that IAA acting as a downstream signaling molecule is involved in the H2S-induced chilling tolerance in cucumber seedlings.


Assuntos
Aclimatação/efeitos dos fármacos , Cucumis sativus/fisiologia , Sulfeto de Hidrogênio/farmacologia , Ácidos Indolacéticos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Aclimatação/genética , Aclimatação/fisiologia , Ácido Ascórbico/análise , Temperatura Baixa , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glutationa/análise , Espécies Reativas de Oxigênio/metabolismo , Plântula/genética , Plântula/fisiologia , Estresse Fisiológico/efeitos dos fármacos , Sulfetos , Transcriptoma
12.
Life Sci ; 246: 117400, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32032645

RESUMO

AIMS: Comparative sub-acute toxicity, including tolerance and dependence potential of fentanyl and its three novel and potent analogues was determined in mice. MAIN METHODS: Comparative sub-acute (21 d, intraperitoneal; 1/10 LD50) toxicity of fentanyl and its three novel analogues viz., N-(1-(2-phenoxyethyl)-4-piperidinyl) propionanilide (2), N-isopropyl-3-(4-(N-phenylpropionamido)piperidin-1-yl)propanamide (5), and N-t-butyl-3-(4-(N-phenylpropionamido)piperidin-1-yl)propanamide (6) was determined in mice. Animals were observed for additional seven days to assess the recovery. The brain, liver and kidney toxicity was assessed on the basis of various biochemical, oxidative, histological, and neuroadaptive markers. The expression levels of key neuronal markers associated with drug tolerance and dependence were investigated by western blot and immunohistochemistry. KEY FINDINGS: Fentanyl and its analogues caused abnormal levels of liver and kidney specific biomarkers in plasma. Brain malondialdehyde (MDA) levels were raised by all the treatments and kidney MDA level by analogue 6 (21 d). Reduced glutathione levels in brain, liver, and kidney were diminished by all the treatments (21 & 28 d) and a significant change in the levels of antioxidant enzymes was also produced mainly after 21 d. The deleterious effects of fentanyl and its analogues were further substantiated by corresponding histopathological changes in brain, liver and kidney (21 & 28 d). These compounds were also found to produce neuroadaptive changes as evidenced by the increased expression levels of c-Fos, glucocorticoid receptor, N-methyl-d-aspartate receptor1 and µ-opioid receptor (21 & 28 d). SIGNIFICANCE: Three novel analogues of fentanyl were envisaged to have alternative therapeutic potentials. However, their comparative sub-acute toxicity revealed undesirable side effects, thereby masking their therapeutic ability.


Assuntos
Fentanila/toxicidade , Animais , Biomarcadores/análise , Western Blotting , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Dano ao DNA/efeitos dos fármacos , Fentanila/análogos & derivados , Glutationa/análise , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Malondialdeído/análise , Camundongos , Estresse Oxidativo/efeitos dos fármacos
13.
Nat Commun ; 11(1): 793, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034152

RESUMO

Fluorescence-based technologies have revolutionized in vivo monitoring of biomolecules. However, significant technical hurdles in both probe chemistry and complex cellular environments have limited the accuracy of quantifying these biomolecules. Herein, we report a generalizable engineering strategy for dual-emission anti-Kasha-active fluorophores, which combine an integrated fluorescein with chromene (IFC) building block with donor-π-acceptor structural modification. These fluorophores exhibit an invariant near-infrared Kasha emission from the S1 state, while their anti-Kasha emission from the S2 state at around 520 nm can be finely regulated via a spirolactone open/closed switch. We introduce bio-recognition moieties to IFC structures, and demonstrate ratiometric quantification of cysteine and glutathione in living cells and animals, using the ratio (S2/S1) with the S1 emission as a reliable internal reference signal. This de novo strategy of tuning anti-Kasha-active properties expands the in vivo ratiometric quantification toolbox for highly accurate analysis in both basic life science research and clinical applications.


Assuntos
Bioquímica/métodos , Corantes Fluorescentes/química , Imagem Molecular/métodos , Células A549 , Animais , Benzopiranos/química , Cisteína/análise , Feminino , Fluoresceína/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Glutationa/análise , Células Hep G2 , Humanos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Neoplasias Experimentais/diagnóstico por imagem , Piranos/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Espironolactona/química
14.
Anal Chim Acta ; 1097: 245-253, 2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-31910966

RESUMO

In this work, we based on an iminocoumarin borate complex to design and synthesize a red-emitting fluorescent probe, THQ-BF2-O-NBD, to discriminatively detect Cys/Hcy and GSH with a large Stokes shift (100 nm). This probe responded to GSH to display red fluorescence with λmax = 630 nm. In contrast, two well-separated emission bands with λmax = 565 and 630 nm were observed in the solution of THQ-BF2-O-NBD with Cys/Hcy. The probe was capable to distinguish Cys/Hcy from GSH in real biological systems.


Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Glutationa/análise , Homocisteína/análise , Animais , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Cinética , Estrutura Molecular , Espectrometria de Fluorescência , Células Tumorais Cultivadas , Peixe-Zebra
15.
Sci Rep ; 10(1): 1209, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31988350

RESUMO

In this study, the toxic effects of phenoxyethanol (Phy-Et), which is widely used in cosmetic industry, has been investigated with Allium test by means of physiological, cytogenetic, anatomical and biochemical parameters. To determine the changes in physiological reactions weight gain, relative injury rate, germination percentage and root length were investigated. Malondialdehyde, superoxide dismutase, glutathion and catalase levels were analyzed as biochemical parameters for determining the presence of oxidative stress. Mitotic index, micronucleus and chromosomal abnormality frequencies were studied as cytogenetic evaluation and the anatomical changes in root tip cells were investigated by cross sections. Changes in surface polarity and wettability were investigated by taking contact angle measurements of pressed root preparations. The mechanism of toxicity has been tried to be explained by these contact angles and this is the first study using contact angle measurements in toxicity tests. Consequently, exposure to Phy-Et resulted in a decrease in all measured physiological parameters and in mitotic index. In contrast, significant increases in the micronucleus and chromosomal abnormality frequencies were observed and the most significant toxic effect was found in 10 mM Phy-Et treated group. Phy-Et application induced oxidative damage and caused a significant increase in malondialdehyde level and a decrease in glutathione level compared to control group. Also a response occured against oxidative damage in superoxide dismutase and catalase activity and the activities increased in 2.5 mM and 5 mM Phy-Et treated groups and decreased in 10 mM Phy-Et treated groups. Furthermore, Phy-Et treatment resulted in some anatomical damages and changes such as necrosis, cell deformation and thickening of the cortex cell wall in root tip meristem cells of A. cepa. In the contact angle measurements taken against water, it was found that the wettability and hydrophilicity of the root preparations treated with Phy-Et were reduced, and this was the explanation of the growth abnormalities associated with water uptake. As a result, it was found that Phy-Et application caused toxic effects on many viability parameters and A. cepa test material was a reliable biomarker in determining these effects.


Assuntos
Etilenoglicóis/farmacologia , Cebolas/efeitos dos fármacos , Extratos Vegetais/farmacologia , Raízes de Plantas/efeitos dos fármacos , Catalase/análise , Aberrações Cromossômicas/efeitos dos fármacos , Etilenoglicóis/administração & dosagem , Etilenoglicóis/toxicidade , Germinação/efeitos dos fármacos , Glutationa/análise , Malondialdeído/análise , Índice Mitótico , Cebolas/crescimento & desenvolvimento , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/administração & dosagem , Extratos Vegetais/toxicidade , Raízes de Plantas/crescimento & desenvolvimento , Medição de Risco , Superóxido Dismutase/análise , Chá/química , Molhabilidade/efeitos dos fármacos
16.
Talanta ; 209: 120566, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31892018

RESUMO

In this work, a simple device for extraction glutathione (GSH) in biological fluids using a hybrid monolithic material within a polypropylene syringe is developed. For this purpose, glycidyl methacrylate-based monolith was firstly prepared within this housing material, and the polymer was modified with different ligands (ammonia, cysteamine and cystamine). The resulting materials (containing amine or thiol groups, respectively) were then functionalized with gold nanoparticles (AuNPs). The hybrid material that gave the largest AuNPs coverage was selected as solid-phase (SPE) sorbent and several variables affecting the extraction recovery of this compound were investigated. Under optimal conditions, GSH was quantitatively retained at pH 6.0, and then it was desorbed with aqueous dithiothreitol solution and determined, after derivatization with o-phthaldialdehyde, via reversed-phase LC with fluorometric detection. The limit of detection was ca. 1.5 ng mL-1, and the reproducibility between extraction units was below 8% (expressed as relative standard deviation), which demonstrates the robustness of the method. The developed material was also applied for the extraction of GSH in saliva and urine samples yielding recoveries ranging from 86 to 105%.


Assuntos
Glutationa/isolamento & purificação , Ouro/química , Nanopartículas Metálicas/química , Extração em Fase Sólida/métodos , Cromatografia Líquida de Alta Pressão/métodos , Compostos de Epóxi/química , Glutationa/análise , Glutationa/urina , Humanos , Limite de Detecção , Metacrilatos/química , Reprodutibilidade dos Testes , Saliva/química , Seringas
17.
Biochim Biophys Acta Mol Basis Dis ; 1866(5): 165665, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31918005

RESUMO

The present study designed to investigate the protective effect of curcumin nanoparticles (CUR-NPs) on the cardiotoxicity induced by doxorubicin. Rats were divided into four groups; control, rats treated daily with CUR-NPs (50 mg/kg) for 14 days, rats treated with an acute dose of doxorubicin (20 mg/kg) and rats treated daily with CUR-NPs for 14 days injected with doxorubicin on the 10th day. After electrocardiogram (ECG) recording from rats at different groups, rat decapitation was carried out and the heart of each rat was excised out to measure the oxidative stress parameters; lipid peroxidation (MDA), nitric oxide (NO) and reduced glutathione (GSH) and the activities of Na,K,ATPase and acetylcholinesterase (AchE). In addition, the levels of dopamine (DA), norepinephrine (NE) and serotonin (5-HT) were determined in the cardiac tissues. Lactate dehydrogenase (LDH) activity was measured in the serum. The ECG recordings indicated that daily pretreatment with CUR- NPs has prevented the tachycardia (i.e. increase in heart rate) and ameliorated the changes in ST wave and QRS complex induced by doxorubicin. In addition, CUR-NPs prevented doxorubicin induced significant increase in MDA, NO, DA, AchE and LDH and doxorubicin induced significant decrease in GSH, NE, 5-HT and Na,K,ATPase. According to the present findings, it could be concluded that CUR-NPs have a protective effect against cardiotoxicity induced by doxorubicin. This may shed more light on the importance of CUR-NPs pretreatment before the application of doxorubicin therapy.


Assuntos
Cardiotônicos/administração & dosagem , Cardiotoxicidade/prevenção & controle , Curcumina/administração & dosagem , Doxorrubicina/toxicidade , Coração/efeitos dos fármacos , Acetilcolinesterase/metabolismo , Administração Oral , Animais , Cardiotoxicidade/diagnóstico , Cardiotoxicidade/etiologia , Cardiotoxicidade/patologia , Modelos Animais de Doenças , Dopamina/análise , Dopamina/metabolismo , Eletrocardiografia , Proteínas Ligadas por GPI/metabolismo , Glutationa/análise , Glutationa/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Miocárdio/química , Miocárdio/metabolismo , Miocárdio/patologia , Nanopartículas/administração & dosagem , Norepinefrina/análise , Norepinefrina/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Serotonina/análise , Serotonina/metabolismo
18.
Spectrochim Acta A Mol Biomol Spectrosc ; 229: 118003, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31923786

RESUMO

Biological thiols including homocysteine (Hcy), cysteine (Cys), hydrogen sulfide (H2S) and glutathione (GSH) play crucial roles in various pathological and physiological processes. The development of optical probes for biothiols has been an active research area in recent years. Herein, a new turn-on fluorescence probe (HD-NBD) was designed and synthesized by fusing tetrahydro[5]helicene and 7-nitro-2,1,3-benzoxadiazole (NBD) for simultaneous discrimination of Hcy/Cys, H2S and GSH in aqueous solution. This probe is able to show unique absorbance enhancement at 548 nm for H2S and additional fluorescence enhancement at 536 nm only for Cys/Hcy, which can be used to discriminate H2S, Cys/Hcy and GSH simultaneously. In addition, HD-NBD also shows low background without any self-fluorescence, as well as high selectivity toward common biothiols. The low detection limits of this probe are about 0.15 µM for Hcy with a wide linear range (1-80 µM), 0.36 µM for Cys (linear range: 1-45 µM), 0.79 µM for H2S (linear range: 1-80 µM) and 4.44 µM for GSH (linear range: 1-60 µM). Moreover, HD-NBD can identify Hcy/Cys, H2S from GSH and other amino acids with high sensitivity and selectivity, therefore it could be used for detecting endogenous and exogenous Hcy/Cys under biological condition.


Assuntos
Neoplasias da Mama/metabolismo , Cisteína/análise , Corantes Fluorescentes/química , Glutationa/análise , Homocisteína/análise , Sulfeto de Hidrogênio/análise , Neoplasias da Mama/patologia , Dinitrobenzenos/química , Feminino , Humanos , Oxidiazóis/química , Compostos Policíclicos/química , Células Tumorais Cultivadas
19.
Talanta ; 208: 119934, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31816805

RESUMO

Biothiols, such as glutathione (GSH) and cysteine (Cys), play important roles in many physiological processes, and variations in their levels are related to different diseases. Many fluorescence probes have been developed to understand the function of biothiols, but only few of such probes can detect both Cys and GSH. Herein, a new strategy for specific colorimetric and fluorescent detection of Cys and GSH by different cascade reactions was developed. By utilizing this strategy, we designed and synthesized two fluorescent probes, namely, CR1 and CR2, for detection of Cys and GSH under physiological conditions. CR1 contains a stronger electron-withdrawing substituent group and provides high selectivity and sensitivity for Cys and GSH. This probe is based on the mechanism of Cys-induced native-chemical-ligation-cyclization and GSH-induced transthioesterification-cyclization cascade reactions, with detection limits of 0.029 and 0.371 µM, respectively. CR1 can be successfully applied for imaging Cys and GSH in living cells with low cell toxicity.


Assuntos
Técnicas Biossensoriais/métodos , Cisteína/análise , Corantes Fluorescentes/química , Glutationa/análise , Imagem Molecular/métodos , Células HeLa , Humanos , Limite de Detecção , Espectrometria de Fluorescência
20.
J Nutr ; 150(4): 722-729, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31773161

RESUMO

BACKGROUND: Cys is limiting for reduced glutathione (GSH) synthesis and can be synthesized from Met. We hypothesized that the dietary Met hydroxyl analogue dl-2-hydroxy-4-methylthiobutyric acid (dl-HMTBA) affects Cys and GSH metabolism and oxidative stress defense differently than Met. OBJECTIVE: The objective was to elucidate whether dl-HMTBA supplementation of a Met-deficient diet affects Cys flux, GSH fractional synthetic rate (FSR), and the basal oxidative stress level relative to Met supplementation in pigs. METHODS: Twenty-nine male German Landrace piglets aged 28 d were allocated to 3 dietary groups: a basal diet limiting in Met (69% of Met plus Cys requirement) supplemented with either 0.15% l-Met (LMET; n = 9), 0.15% dl-Met (DLMET; n = 11), or 0.17% dl-HMTBA (DLHMTBA; n = 9) on an equimolar basis. At age 54 d the pigs received a continuous infusion of [1-13C]-Cys to calculate Cys flux and Cys oxidation. After 3 d, GSH FSR was determined by [2,2-2H2]-glycine infusion, and RBC GSH and oxidized GSH concentrations were measured. At age 62 d the animals were killed to determine hepatic mRNA abundances of enzymes involved in GSH metabolism, GSH concentrations, and plasma oxidative stress defense markers. RESULTS: The Cys oxidation was 21-39% and Cys flux 5-15% higher in the fed relative to the feed-deprived state (P < 0.001). On average, GSH FSR was 49% lower (P < 0.01), and RBC GSH and total GSH concentrations were 12% and 9% lower, respectively, in DLHMTBA and DLMET relative to LMET pigs (P < 0.05). In the feed-deprived state, Gly flux, the GSH:oxidized glutathione (GSSG) ratio, RBC GSSG concentrations, plasma oxidative stress markers, and the hepatic GSH content did not differ between groups. CONCLUSIONS: Although GSH FSR was higher in LMET compared with DLMET or DLHMTBA feed-deprived pigs, these differences were not reflected by lower oxidative stress markers and antioxidant defense enzymes in LMET pigs.


Assuntos
Aminoácidos Sulfúricos/administração & dosagem , Dieta/veterinária , Glutationa/biossíntese , Metionina/análogos & derivados , Sus scrofa/metabolismo , Aminoácidos/sangue , Animais , Antioxidantes/análise , Biomarcadores/sangue , Cisteína/sangue , Suplementos Nutricionais , Eritrócitos/química , Glutationa/análise , Glutationa/sangue , Glicina/sangue , Fígado/química , Masculino , Metionina/administração & dosagem , Estresse Oxidativo/fisiologia , Desmame
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