Targeted Quantification of Glutathione/Arginine Redox Metabolism Based on a Novel Paired Mass Spectrometry Probe Approach for the Functional Assessment of Redox Status.

Glutathione (GSH) redox control and arginine metabolism are critical in regulating the physiological response to injury and oxidative stress. Quantification assessment of the GSH/arginine redox metabolism supports monitoring metabolic pathway shifts during pathological processes and their linkages to redox regulation. However, assessing the redox status of organisms with complex matrices is challenging, and single redox molecule analysis may not be accurate for interrogating the redox status in cells and in vivo. Herein, guided by a paired derivatization strategy, we present a new ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS)-based approach for the functional assessment of biological redox status. Two structurally analogous probes, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and newly synthesized 2-methyl-6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (MeAQC), were set for paired derivatization. The developed approach was successfully applied to LPS-stimulated RAW 264.7 cells and HDM-induced asthma mice to obtain quantitative information on GSH/arginine redox metabolism. The results suggest that the redox status was remarkably altered upon LPS and HDM stimulation. We expect that this approach will be of good use in a clinical biomarker assay and potential drug screening associated with redox metabolism, oxidative damage, and redox signaling.

Determination, dissipation dynamics, terminal residues and dietary risk assessment of thiophanate-methyl and its metabolite carbendazim in cowpeas collected from different locations in China under field conditions.

BACKGROUND: Thiophanate-methyl and its metabolite carbendazim are broad-spectrum fungicides used on many crops. The residues of these chemicals could result in potential environmental and human health problems. Therefore, investigations of the dissipation and residue behaviors of thiophanate-methyl and its metabolite carbendazim on cowpeas and associated dietary risk assessments are essential for the safety of agricultural products. RESULTS: A simple analytical approach using liquid chromatography with tandem mass spectrometry was developed and validated for the determination of thiophanate-methyl and carbendazim concentrations in cowpeas. Good linearity (R2 > 0.998) was obtained, and the recoveries and relative standard deviations were 80.0-104.7% and 1.4-5.2%, respectively. The dissipation rates of thiophanate-methyl, carbendazim and total carbendazim were high (half-lives of 1.61-2.46 days) and varied in the field cowpea samples because of the different weather conditions and planting patterns. Based on the definition of thiophanate-methyl, the terminal residues of total carbendazim in cowpea samples were below the maximum residue limits set by Japan for other legumes. The acute and chronic risk quotients of three analytes were 0.0-27.6% in cowpea samples gathered from all terminal residue treatments, which were below 100%. CONCLUSION: An optimized approach for detecting thiophanate-methyl and carbendazim in cowpeas was applied for the investigation of field-trial samples. The potential acute and chronic dietary risks of thiophanate-methyl, carbendazim and total carbendazim to the health of Chinese consumers were low. These results could guide the safe and proper use of thiophanate-methyl in cowpeas and offer data for the dietary risk assessment of thiophanate-methyl in cowpeas. © 2021 Society of Chemical Industry.

Assessment of the dissipation, pre-harvest interval and dietary risk of carbosulfan, dimethoate, and their relevant metabolites in greenhouse cucumber (Cucumis sativus L.).

BACKGROUND: The dissipation behavior, pre-harvest interval and dietary risk of carbosulfan, dimethoate, and their relevant metabolites were investigated in greenhouse cucumber in Tianjin, northern China, to ensure raw consumption safety. RESULTS: Carbosulfan was metabolized to carbofuran, dibutylamine, 3-hydroxycarbofuran and 3-ketocarbofuran, and dimethoate was degraded to omethoate in cucumber fruits and leaves. The dissipation of carbosulfan, carbofuran, 3-hydroxycarbofuran and dimethoate fitted first-order kinetics well, with R2 ranging from 0.912 to 0.992, and their half-lives were 2.6, 2.7, 2.4 and 5.2 days in cucumber fruits and 2.8, 3.0, 4.6 and 2.5 days in leaves, respectively. The estimated daily intakes of the active ingredients and their relevant metabolites were 0.1-4% of the corresponding acceptable daily intakes. Acute oral exposure to carbofuran (a metabolite of carbosulfan) represented 367% of the acute reference dose (ARfD) for 1-6-year-old Chinese children and 227% for the general Chinese population. CONCLUSION: A minimum pre-harvest interval of 12 days for carbosulfan is proposed to ensure safe consumption of cucumber. The slow dissipation rate of omethoate in cucumber reveals that a longer pre-harvest interval (≥ 27 days) is necessary to prevent dietary risk when dimethoate is applied to cucumber. © 2018 Society of Chemical Industry.

Residues and dissipation kinetics of carbendazim and diethofencarb in tomato (Lycopersicon esculentum Mill.) and intake risk assessment.

Dissipation behaviors and residues of carbendazim and diethofencarb in combination in tomato were investigated. The half-lives were 2.1-3.4 days for carbendazim, and 1.8-3.2 days for diethofencarb at a dose of 1.5 times of the recommended dosage. The residues of carbendazim and diethofencarb were below the maximum residue limits (MRLs) in China one day after application of the combination. The ultimate residues were significantly lower than the maximum permissible intake (MPI) in China at the recommended high dose for both child and adult. The values of the maximum dietary exposure for carbendazim and diethofencarb were 0.26 and 0.27 mg per person per day, respectively. The theoretical maximum daily intake (TMDI) values for carbendazim and diethofencarb were 1.5 and 0.5 mg/day, respectively. The dietary exposure was lower than the MPI, which indicates the harvested tomato samples under the experimental conditions (open field) are safe for human consumption at the recommended high dosage of the wettable powder.

The influence of effective microorganisms (EM) and yeast on the degradation of strobilurins and carboxamides in leafy vegetables monitored by LC-MS/MS and health risk assessment.

The aim of this study was to determine the behaviour of strobilurin and carbocyamides commonly used in chemical protection of lettuce depending on carefully selected effective microorganisms (EM) and yeast (Y). Additionally, the assessment of the chronic health risk during a 2-week experiment was performed. The statistical method for correlation of physico-chemical parameters and time of degradation for pesticides was applied. In this study, the concentration of azoxystrobin, boscalid, pyraclostrobin and iprodione using liquid chromatography-mass spectrometry (LC-MS/MS) in the matrix of lettuce plants was performed, and there was no case of concentration above maximum residues levels. Before harvest, four fungicides and their mixture with EM (1 % and 10 %) and/or yeast 5 % were applied. In our work, the mixtures of 1%EM + Y and 10%EM + Y both stimulated and inhibited the degradation of the tested active substances. Adding 10%EM to the test substances strongly inhibited the degradation of iprodione, and its concentration decreased by 30 %, and in the case of other test substances, the degradation was approximately 60 %. Moreover, the addition of yeast stimulated the distribution of pyraclostrobin and boscalid in lettuce leaves. The risk assessment for the pesticides ranged from 0.4 to 64.8 % on day 1, but after 14 days, it ranged from 0.0 to 20.9 % for children and adults, respectively. It indicated no risk of adverse effects following exposure to individual pesticides and their mixtures with EM and yeast.

[(3) H]-L685,458 binding sites are abundant in multiple peripheral organs in rats: implications for safety assessment of putative γ-secretase targeting drugs.

γ-Secretase is a multimeric enzyme complex that carries out proteolytic processing to a variety of cellular proteins. It is currently explored as a therapeutic target for Alzheimer's disease (AD) and cancer. Mechanism-based toxicity needs to be thoroughly evaluated for γ-secretase inhibitory and/or modulatory drugs. This study comparatively assessed putative γ-secretase catalytic sites in rat peripheral tissues relative to brain and explored an effort of its pharmacological inhibition on hair regeneration. Using [(3) H]-labelled L685,458, a potent γ-secretase inhibitor, as probe, we found more abundant presence of γ-secretase binding sites in the liver, gastrointestinal tract, hair follicle, pituitary gland, ovary and testis, as compared to the brain. Local application of L658,458 delayed vibrissal regrowth following whisker removal. These results suggest that γ-secretase may execute important biological functions in many peripheral systems, as in the brain. The development of γ-secretase inhibitors/modulators for AD and cancer therapy should include close monitoring of toxicological panels for hepatic, gastrointestinal, endocrinal and reproductive functions.

A comprehensive assessment of repaglinide metabolic pathways: impact of choice of in vitro system and relative enzyme contribution to in vitro clearance.

Repaglinide is presently recommended by the U.S. Food and Drug Administration as a clinical CYP2C8 probe, yet current in vitro and clinical data are inconsistent concerning the role of this enzyme in repaglinide elimination. The aim of the current study was to perform a comprehensive investigation of repaglinide metabolic pathways and assess their contribution to the overall clearance. Formation of four repaglinide metabolites was characterized using in vitro systems with differential complexity. Full kinetic profiles for the formation of M1, M2, M4, and repaglinide glucuronide were obtained in pooled cryopreserved human hepatocytes, human liver microsomes, human S9 fractions, and recombinant cytochrome P450 enzymes. Distinct differences in clearance ratios were observed between CYP3A4 and CYP2C8 for M1 and M4 formation, resulting in a 60-fold M1/M4 ratio in recombinant (r) CYP3A4, in contrast to 0.05 in rCYP2C8. Unbound K(m) values were within 2-fold for each metabolite across all in vitro systems investigated. A major system difference was seen in clearances for the formation of M2, which is suggested to be a main metabolite of repaglinide in vivo. An approximately 7-fold higher unbound intrinsic clearance was observed in hepatocytes and S9 fractions in comparison to microsomes; the involvement of aldehyde dehydrogenase in M2 formation was shown for the first time. This systematic analysis revealed a comparable in vitro contribution from CYP2C8 and CYP3A4 to the metabolism of repaglinide (<50%), whereas the contribution of glucuronidation ranged from 2 to 20%, depending on the in vitro system used. The repaglinide M4 metabolic pathway is proposed as a specific CYP2C8 probe for the assessment of drug-drug interactions.

Assessment of acetylcholinesterase activity in Clarias gariepinus as a biomarker of organophosphate and carbamate exposure.

The objective of this study was to investigate the response of acetylcholinesterase (AChE) activities in Clarias gariepinus in response to Organophosphates (Ops) and carbamate exposure. The AChE activities were determined in plasma, and eye and brain homogenates of unexposed and exposed fish using Ellman's method and 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) chromophore. The baseline AChE activities in plasma, eyes and brain tissues in unexposed fish were comparable between males and females (P > 0.05). Concentrations of pesticides that inhibited 50% (IC(50)) of AChE activities in brain homogenates following in vitro exposures were 0.003, 0.03, 0.15, 190, 0.2, 0.003 and 0.002 microM for carbaryl, chlorfenvinphos, diazinon, dimethoate, fenitrothion, pirimiphosmethyl and profenofos, respectively. The in vivo dose-effect relationships were assessed using chlorfenvinphos and carbaryl at different concentrations that ranged from 0.0003 to 0.06 microM and 0.0005 to 0.05 microM, respectively. Acetylcholinesterase activities were comparable in plasma, and eye and brain homogenates from control and carbaryl-exposed fish. Following exposure of fish to chlorfenvinphos at concentrations above 0.03 microM, a significant inhibition of AChE activities in plasma (84%) and eye homogenate (50%) was observed. The AChE activities in brain homogenate were comparable between chlorfenvinphos-exposed fish and controls. Because carbaryl cause reversible inhibition of AChE activities was found to be more potent than chlorfenvinphos that cause irreversible inhibition following in vitro exposure. Contrary, carbaryl was less potent than chlorfenvinphos after in vivo exposure possibly due to more rapid biotransformation of carbaryl than chlorfenvinphos. Findings from this study have demonstrated that inhibition of AChE activity in C. gariepinus is a useful biomarker in assessing aquatic environment contaminated by anticholinesterases.

Application of liquid chromatography/thermospray mass spectrometry to studies on the formation of glutathione and cysteine conjugates from monomethylcarbamate metabolites of bambuterol.

Liquid chromatography/thermospray mass spectrometry (LC/TSP-MS) has been used to identify and study the rates of formation of S-(N-methylcarbamoyl)cysteine and S-(N-methylcarbamoyl)glutathione as products of the in vitro reaction of cysteine and glutathione, respectively, with two monomethylcarbamate metabolites of the bronchodilator pro-drug bambuterol. The conjugates of interest afforded MH+ species and yielded abundant structurally informative fragment ions which were employed in the development of quantitative, selected-ion monitoring assays. It is concluded that LC/TSP-MS represents a rapid and convenient approach to the direct aqueous-phase analysis of the class of S-(N-alkylcarbamoyl) conjugates of cysteine and glutathione.