Effect of Cimetidine on Metformin Pharmacokinetics and Endogenous Metabolite Levels in Rats.

Tubular secretion is a primary mechanism along with glomerular filtration for renal elimination of drugs and toxicants into urine. Organic cation transporters (OCTs) and multidrug and toxic extrusion (MATE) transporters facilitate the active secretion of cationic substrates, including drugs such as metformin and endogenous cations. We hypothesized that administration of cimetidine, an Oct/Mate inhibitor, will result in increased plasma levels and decreased renal clearance of metformin and endogenous Oct/Mate substrates in rats. A paired rat pharmacokinetic study was carried out in which metformin (5 mg/kg, intravenous) was administered as an exogenous substrate of Oct/Mate transporters to six Sprague-Dawley rats with and without cimetidine (100 mg/kg, intraperitoneal). When co-administered with cimetidine, metformin area under the curve increased significantly by 3.2-fold, and its renal clearance reduced significantly by 73%. Untargeted metabolomics was performed to investigate the effect of cimetidine on endogenous metabolome in the blood and urine samples. Over 8,000 features (metabolites) were detected in the blood, which were shortlisted using optimized criteria, i.e., a significant increase (P value < 0.05) in metabolite peak intensity in the cimetidine-treated group, reproducible retention time, and quality of chromatogram peak. The metabolite hits were classified into three groups that can potentially distinguish inhibition of i) extra-renal uptake transport or catabolism, ii) renal Octs, and iii) renal efflux transporters or metabolite formation. The metabolomics approach identified novel putative endogenous substrates of cationic transporters that could be tested as potential biomarkers to predict Oct/Mate transporter mediated drug-drug interactions in the preclinical stages. SIGNIFICANCE STATEMENT: Endogenous substrates of renal transporters in animal models could be used as potential biomarkers to predict renal drug-drug interactions in early drug development. Here we demonstrated that cimetidine, an inhibitor of organic cation transporters (Oct/Mate), could alter the pharmacokinetics of metformin and endogenous cationic substrates in rats. Several putative endogenous metabolites of Oct/Mate transporters were identified using metabolomics approach, which could be tested as potential transporter biomarkers to predict renal drug-drug interaction of Oct/Mate substrates.

Differential tissue abundance of membrane-bound drug metabolizing enzymes and transporter proteins by global proteomics.

Protein abundance data of drug-metabolizing enzymes and transporters (DMETs) are critical for scaling in vitro and animal data to humans for accurate prediction and interpretation of drug clearance and toxicity. Targeted DMET proteomics which relies on synthetic stable isotope-labeled surrogate peptides as calibrators, is routinely used for the quantification of selected proteins; however, the technique is limited to the quantification of a small number of proteins. Although the global proteomics-based total protein approach (TPA) is emerging as a better alternative for large-scale protein quantification, the conventional TPA doesn't consider differential sequence coverage by identifying unique peptides across proteins. Here, we optimized the TPA approach by correcting protein abundance data by the sequence coverage (SC-TPA), which was applied to quantify 54 DMETs for characterization of i) differential tissue DMET abundance in the human liver, kidney, and intestine, and ii) interindividual variability of DMET proteins in individual intestinal samples (n=13). UGT2B7, MGST1, MGST2, MGST3, CES2, and MRP2 were expressed in all three tissues, whereas, as expected CYP3A4, CYP3A5, CYP2C9, CYP4F2, UGT1A1, UGT2B17, CES1, FMO5, MRP3, and P-gp were present in the liver and intestine. The top three DMET proteins in individual tissues were: CES1>CYP2E1>UGT2B7 (liver), CES2>UGT2B17>CYP3A4 (intestine), and MGST1>UGT1A6>MGST2 (kidney). CYP3A4, CYP3A5, UGT2B17, CES2, and MGST2 showed high interindividual variability in the intestine. These data are relevant for enhancing in vitro to in vivo extrapolation (IVIVE) of drug absorption and disposition and can be used to enhance the accuracy of physiologically based pharmacokinetic (PBPK) prediction of systemic and tissue concentration of drugs. Significance Statement We quantified the abundance and compositions of drug-metabolizing enzymes and transporters (DMETs) in pooled human liver, intestine, and kidney microsomes using an optimized sequence coverage-informed total protein approach. The quantification of DMETs revealed quantitative differences in their levels in the liver, intestine, and kidney. Further, the analysis of individual intestine samples confirmed high variability in the levels of CYP3A4, CYP3A5, UGT2B17, CES2, and MGST2. These data are applicable for the prediction of first-pass metabolism and tissue-specific drug clearance.

Comparison of Relative Activity versus Relative Expression Factors (RAF versus REF) in Predicting Glucuronidation Mediated Drug Clearance Using Recombinant UGTs.

PURPOSE: Predicting the quantitative fraction of glucuronidation (fgluc) by individual UDP-glucuronosyltransferase enzymes (UGTs) is challenging due to the lack of selective inhibitors and inconsistent activity of recombinant UGT systems (rUGTs). Our study compares the relative expression versus activity factors (REF versus RAF) to predict fgluc based on rUGT data to human liver and intestinal microsomes (HLM and HIM). METHODS: REF scalars were derived from a previous in-house proteomics study for eleven UGT enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17), whereas RAF was calculated by measuring activities in rUGTs to microsomes of selective UGT probe substrates. Protein-normalized activity factor (pnAF) values were generated after correcting activity of individual UGTs to their corresponding protein abundance. The utility of REF and RAF in predicting fgluc was assessed for three UGT substrates-diclofenac, vorinostat, and raltegravir. RESULTS: The REF values ranged from 0.02 to 1.75, RAF based on activity obtained in rUGTs to HLM/HIM were from 0.1 to 274. pnAF values were ~ 5 to 80-fold, except for UGT2B4 and UGT2B15, where pnAF was ~ 180 and > 1000, respectively. The results revealed confounding effect of differential specific activities (per pmol) of rUGTs in fgluc prediction. CONCLUSION: The data suggest that the activity of UGT enzymes was significantly lower when compared to their activity in microsomes at the same absolute protein amount (pmol). Collectively, results of this study demonstrate poor and variable specific activity of different rUGTs (per pmol protein), as determined by pnAF values, which should be considered in fgluc scaling.

The Plasma Membrane Monoamine Transporter is Highly Expressed in Neuroblastoma and Functions as an mIBG Transporter.

Neuroblastoma (NB) is a pediatric cancer with low survival rates in high-risk patients. 131I-mIBG has emerged as a promising therapy for high-risk NB and kills tumor cells by radiation. Consequently, 131I-mIBG tumor uptake and retention are major determinants for its therapeutic efficacy. mIBG enters NB cells through the norepinephrine transporter (NET), and accumulates in mitochondria through unknown mechanisms. Here we evaluated the expression of monoamine and organic cation transporters in high-risk NB tumors and explored their relationship with MYCN amplification and patient survival. We found that NB mainly expresses NET, the plasma membrane monoamine transporter (PMAT), and the vesicular membrane monoamine transporter 1/2 (VMAT1/2), and that the expression of these transporters is significantly reduced in MYCN-amplified tumor samples. PMAT expression is the highest and correlates with overall survival in high-risk NB patients without MYCN amplification. Immunostaining showed that PMAT resides intracellularly in NB cells and co-localizes with mitochondria. Using cells expressing PMAT, mIBG was identified as a PMAT substrate. In mitochondria isolated from NB cell lines, mIBG uptake was reduced by ∼50% by a PMAT inhibitor. Together, our data suggest that PMAT is a previously unrecognized transporter highly expressed in NB and could impact intracellular transport and therapeutic response to 131I-mIBG. SIGNIFICANCE STATEMENT: This study identified that plasma membrane monoamine transporter (PMAT) is a novel transporter highly expressed in neuroblastoma and its expression level is associated with overall survival rate in high-risk patients without MYCN amplification. PMAT is expressed intracellularly in neuroblastoma cells, transports meta-iodobenzylguanidine (mIBG) and thus could impact tumor retention and response to 131I-mIBG therapy. These findings have important clinical implications as PMAT could represent a novel molecular marker to help inform disease prognosis and predict response to 131I-mIBG therapy.

Characterization of Xenobiotic and Steroid Disposition Potential of Human Placental Tissue and Cell Lines (BeWo, JEG-3, JAR, and HTR-8/SVneo) by Quantitative Proteomics.

The placenta is a fetal organ that performs critical functions to maintain pregnancy and support fetal development, including metabolism and transport of xenobiotics and steroids between the maternal-fetal unit. In vitro placenta models are used to study xenobiotic and steroid disposition, but how well these models recapitulate the human placenta is not well understood. We first characterized the abundance of proteins involved in xenobiotic and steroid disposition in human placental tissue. In pooled human placenta, the following xenobiotic and steroid disposition proteins were detected (highest to lowest), 1) enzymes: glutathione S-transferase P, carbonyl reductase 1, aldo-keto reductase 1B1, hydroxysteroid dehydrogenases (HSD3B1 and HSD11B1), aromatase, epoxide hydrolase 1 (EPHX1) and steryl-sulfatase, and 2) transporters: monocarboxylate transporters (MCT1 and 4), organic anion transporting polypeptide 2B1, organic anion transporter 4, and breast cancer resistance protein (BCRP). Then, the tissue proteomics data were compared with four placental cell lines (BeWo, JEG-3, JAR, and HTR-8/SVneo). The differential global proteomics analysis revealed that the tissue and cell lines shared 1420 cytosolic and 1186 membrane proteins. Although extravillous trophoblast and cytotrophoblast marker proteins were detected in all cell lines, only BeWo and JEG-3 cells expressed the syncytiotrophoblast marker, chorionic somatomammotropin hormone 1. BeWo and JEG-3 cells expressed most target proteins including aromatase, HSDs, EPHX1, MCT1, and BCRP. JEG-3 cells treated with commonly detected phthalates in human biofluids showed dysregulation of steroid pathways. The data presented here show that BeWo and JEG-3 cells are closer to the placental tissue for studying xenobiotic and steroid disposition. SIGNIFICANCE STATEMENT: This is the first study to compare proteomics data of human placental tissue and cell lines (BeWo, JAR, JEG-3, and HTR-8/SVneo). The placental cell line and tissue proteomes are vastly different, but BeWo and JEG-3 cells showed greater resemblance to the tissue in the expression of xenobiotic and steroid disposition proteins. These data will assist researchers to select an optimum cell model for mechanistic investigations on xenobiotic and steroid disposition in the placenta.

Morbidity and Mortality of Necrotizing Fasciitis and Their Prognostic Factors in Children.

Background: This is a prospective study of the clinico-etiologic profile and factors affecting outcomes in 40 children managed for necrotizing fasciitis (NF). Materials and Methods: Demographic details, clinical characteristics, and laboratory parameters were recorded, and the Laboratory Risk Indicator for Necrotizing Fasciitis (LRINEC) score was calculated. Primary outcome (survival vs. nonsurvival) was noted, and prognostic factors were identified. Results: Initiating factors included boils (45%), i.v. cannula extravasations (22.5%), and blunt trauma (17.5%). Lesion (s) were predominantly on the lower limbs (35%) and trunk (25%). Twenty-two patients (55%) had <5% body surface area (BSA) involved. Severely deranged clinical and laboratory parameters were common. Ultrasound localized fluid collections. Pus cultures showed methicillin-resistant Staphylococcus aureus (52.5%), methicillin-sensitive S. aureus [27.5%], and polymicrobial growth (20%). Blood culture was positive in 24 patients (60%). Most isolates were sensitive to clindamycin and amoxy-clavulanate. Prognostic factors for mortality (n = 6; 15%) included categorization as "Sick," BSA involvement >10%, thrombocytopenia, raised serum creatinine, late debridement, and polymicrobial blood culture isolates. All six nonsurvivors had a LRINEC score of ≥8 and positive blood cultures. Six patients (20.7%) developed unsightly scars and 5 (17.24%) contractures across joints. Conclusions: Pediatric NF has significant morbidity and mortality. Patients with adverse prognostic factors can benefit from early referral to a facility with a critical care unit. Adequate wound management is essential to minimize residual deformity.

Synthesis and characterization of a series of N,N'-substituted urea derivatives by using electrospray ionization tandem mass spectrometry: Differentiation of positional isomers.

RATIONALE: Characterization of N,N'-substituted ureas was found to be challenging by nuclear magnetic resonance (NMR) spectroscopy, particularly N-di- and tri-alkylated ureas because of the absence of adjacent protons. In the present study, electrospray ionization tandem mass spectrometry has been used to differentiate positional isomeric pairs and to characterize a series of N,N'-substituted ureas, as these compounds have significant importance for drug discovery. Additionally, urea is an essential functionality in several bioactive compounds as well as a variety of clinically approved therapies. METHODS: High-resolution electrospray ionization tandem mass spectrometry (ESI-HR-MS/MS) has been used to characterize a series of N,N'-substituted urea derivatives and differentiate two pairs of positional isomers. The data was acquired by Xcaliber application in positive ionization mode. RESULTS: ESI-HR-MS/MS spectra of [M + H]+ ions of the positional isomeric urea derivatives 8a and 8b show distinct fragmentation patterns. For example, the MS/MS spectrum of the [M + H]+ ion of isomer 8a displays the abundant fragment ion at m/z 285.1595, which was totally absent in isomer 8b. This would be plausibly formed by the cleavage of the C-N bond of the urea group with the elimination of the isocyanate moiety. In contrast, the MS/MS spectrum of the [M + H]+ ion of isomer 8b shows an intense ion at m/z 311.1389 which is completely absent in isomer 8a which would be formed by the cleavage of the C-N bond attached to the ring nitrogen. Similarly, another pair of positional isomers, 8c and 8d, have been clearly distinguished by their fragmentation behaviour. In addition, a series of N,N'-substituted urea derivatives were studied to investigate the impact of different substitution on the fragmentation behaviour. CONCLUSIONS: The present study demonstrates that ESI-HR-MS/MS can be used to differentiate pairs of N,N'-substituted urea positional isomers and characterize a series of derivatives. It was observed that a characteristic fragment ion was formed by the C-N bond cleavage with the elimination of an isocyanate moiety. The proposed mechanism of fragmentation was supported by the change in the fragmentation pathway upon alkylation of the NH. In order to generalize this fragmentation pattern, a series of N-alkylated ureas was synthesized and studied by MS/MS.

Identification of novel glutathione conjugates of terbinafine in liver microsomes and hepatocytes across species.

1. Terbinafine (TBF), a common antifungal agent, has been associated with rare incidences of hepatotoxicity. It is hypothesized that bioactivation of TBF to reactive intermediates and subsequent binding to critical cellular proteins may contribute to this toxicity. In the present study, we have characterized the bioactivation pathways of TBF extensively in human, mouse, monkey, dog and rat liver microsomes and hepatocytes. 2. A total of twenty glutathione conjugates of TBF were identified in hepatocytes; thirteen of these conjugates were also detected in liver microsomes. To the best of our knowledge, only two of these conjugates have been reported previously. The conjugates were categorized into three groups based on their mechanism of formation: (a) alkene/alkyne oxidation followed by glutathione conjugation, with or without N-demethylation, (b) arene oxidation followed by glutathione conjugation, with or without N-demethylation, and (c) N-dealkylation followed by glutathione conjugation of the allylic aldehyde, alcohol and acid intermediates. 3. Differences were observed across species in the contributions of these pathways toward overall metabolic turnover. We conclude that, in addition to the glutathione conjugates known to form by Michael addition to the allylic aldehyde, there are other pathways involving the formation of arene oxides and alkene/alkyne epoxides that may be relevant to the discussion of TBF-mediated idiosyncratic drug reactions.

Immuno-protective role of biologically synthesized dietary selenium nanoparticles against multiple stressors in Pangasinodon hypophthalmus.

An environment friendly and sustainable approach is being emerged in the area of nanotechnology for accelerated growth and development of culturable aquatic animals hence green chemistry is gaining momentum in recent years. The present study has been carried out to delineate the effects of selenium nanoparticles (Se-NPs) on growth performance, antioxidative status and immunity of fish reared under lead (Pb) and high temperature (34 °C). Three hundred and fifteen fish were equally distributed in seven treatments in triplicates. Three isocaloric and isonitrogenous experimental diets viz. control (Se-NPs-0 mg/kg), Se-NPs at 1 mg/kg and Se-NPs at 2 mg/kg were formulated. The fish were reared under lead (Pb, 1/21st of LC50 (4 ppm)) and high temperature (34 °C) stress and fed with or without dietary Se-NPs. The effects of dietary Se-NPs were studied in terms of growth performance (Weight gain %, feed conversion ratio, protein efficiency ratio and specific growth rate), antioxidative status (catalase, superoxide dismutase, glutathione-S-transferase and glutathione peroxidase), neurotransmitter enzymes (AChE), stress biomarkers (heat shock protein 70, serum cortisol, blood glucose, vitamin C), immunological status (total protein, A/G ratio and respiratory burst activity) in Pangasinodon hypophthalmus post challenge with Aeromonas veronii biovar sobria. Results of the investigation demonstrated significant improvement of growth performance, antioxidative status, neurotransmitter enzyme activity, stress markers and more importantly enhanced immunity of the fish with dietary incorporation of Se-NPs at 1 mg/kg. In addition, post bacterial infection, the relative % survival increased and cumulative mortality % decreased in the group fed with Se-NPs at 1 mg/kg diet. Pb and high temperature treated and fed with control diet group showed devastating impact on the growth performance, antioxidative status, stress markers and immunity of the fish. Similarly, application of Se-NPs at 2 mg/kg showed poor growth performance and elevated level of oxidative stress and other stress biomarkers including other biochemical attributes. Inclusive results indicated that, Se-NPs at 1 mg/kg has capability to enhance overall performance and alleviate multiple stresses in P. hypophthalmus. Hence, Se-NPs at optimum level have ability to develop green chemistry in feed industry for better growth performance of the fish.

Dietary nano-silver: Does support or discourage thermal tolerance and biochemical status in air-breathing fish reared under multiple stressors?

Unexpected fluctuations in weather parameters due to global climate change have been observed in all ecosystems worldwide. The aquatic ecosystem shelters a great diversity of fishes in the upper region of the ecosystem which adversely get affected due to their poikilothermic nature. The present study was designed to elucidate the impact of critical temperature minima (CTMin), lethal temperature minima (LTMin), critical temperature maxima (CTMax), and lethal temperature maxima (LTMax) on Channa striatus. Biologically synthesized silver nanoparticles (Ag-NPs) were evaluated for their potential to enhance thermal tolerance and improve the activities of biochemical enzymes of C. striatus reared under lead (Pb) and high temperature (34 °C) for 50 days. Three iso-caloric and iso-nitrogenous diets which included a basal diet and two supplemented diets with Ag-NPs @ 0.5 mg/kg, and 1 mg/kg were used in the study. Results suggested that CTMin and LTMin were significantly (p < 0.01) reduced and CTMax and LTMax were enhanced in the group fed with 0.5 mg/kg Ag-NPs supplemented feed. Pre-exposure to high temperature led to enhanced CTMax and LTMax in C. striatus. The biochemical enzymes involved in protein metabolism, carbohydrate metabolism, acetylcholine esterase and antioxidant activities were found to be normal in fish fed with 0.5 mg/kg Ag-NPs supplemented diet. Bioaccumulation of silver and Pb was determined in different fish tissues and experimental water. Overall, the incorporation of Ag-NPs at 0.5 mg/kg in diet can confer protection to fish against Pb and thermal stress and enhance thermal tolerance of C. striatus.

Characterization of stress degradation products of amodiaquine dihydrochloride by liquid chromatography with high-resolution mass spectrometry and prediction of their properties by using ADMET Predictor™.

The degradation behavior of amodiaquine dihydrochloride, an antimalarial drug, was investigated in solution as well as solid states. The drug was subjected to hydrolytic, photolytic, oxidative, and thermal stress conditions, according to International Conference on Harmonization guideline Q1A(R2). It showed extensive hydrolysis in acidic, alkaline, and neutral solutions both with and without light, while it proved to be stable to thermal and oxidative conditions. In total, six degradation products were formed, which were separated on a C8 column, employing a gradient reversed-phase high-performance liquid chromatography method in which acetonitrile and 10 mM ammonium formate (pH 3.0) were used in the mobile phase. To characterize the degradation products, mass fragmentation behavior of the drug was established by direct infusion of solution to quadrupole time-of-flight and multiple-stage mass spectrometry systems. Liquid chromatography with high-resolution mass spectrometry studies were subsequently carried out on the stressed samples using the same gradient high-performance liquid chromatography method employed for the separation of the degradation products. Hydrogen/deuterium exchange studies were additionally conducted to determine the number of labile hydrogen atoms. The degradation pathway of the drug was delineated, justified by mechanistic explanation. Lastly, ADMET Predictor™ software was employed to predict relevant physicochemical and toxicity data for the degradation products.

Study of the forced degradation behavior of prasugrel hydrochloride by liquid chromatography with mass spectrometry and liquid chromatography with NMR detection and prediction of the toxicity of the characterized degradation products.

Prasugrel was subjected to forced degradation studies under conditions of hydrolysis (acid, base, and neutral), photolysis, oxidation, and thermal stress. The drug showed liability in hydrolytic as well as oxidative conditions, resulting in a total of four degradation products. In order to characterize the latter, initially mass fragmentation pathway of the drug was established with the help of mass spectrometry/time-of-flight, multiple stage mass spectrometry and hydrogen/deuterium exchange data. The degradation products were then separated on a C18 column using a stability-indicating volatile buffer method, which was later extended to liquid chromatography-mass spectrometry studies. The latter highlighted that three degradation products had the same molecular mass, while one was different. To characterize all, their mass fragmentation pathways were established in the same manner as the drug. Subsequently, liquid chromatography-nuclear magnetic resonance (NMR) spectroscopy data were collected. Proton and correlation liquid chromatography with NMR spectroscopy studies highlighted existence of diastereomeric behavior in one pair of degradation products. Lastly, toxicity prediction by computer-assisted technology (TOPKAT) and deductive estimation of risk from existing knowledge (DEREK) software were employed to assess in silico toxicity of the characterized degradation products.

Identification of the Biotransformation Pathways of a Potential Oral Male Contraceptive, 11ß-Methyl-19-Nortestosterone (11ß-MNT) and Its Prodrugs: An In Vitro Study Highlights the Contribution of Polymorphic Intestinal UGT2B17.

11ß-Methyl-19-nortestosterone dodecylcarbonate (11ß-MNTDC) is a prodrug of 11ß-MNT and is being considered as a promising male oral contraceptive candidate in clinical development. However, the oral administration of 11ß-MNTDC exhibits an ~200-fold lower serum concentration of 11ß-MNT compared to 11ß-MNTDC, resulting in the poor bioavailability of 11ß-MNT. To elucidate the role of the first-pass metabolism of 11ß-MNT in its poor bioavailability, we determined the biotransformation products of 11ß-MNT and its prodrugs in human in vitro models. 11ß-MNT and its two prodrugs 11ß-MNTDC and 11ß-MNT undecanoate (11ß-MNTU) were incubated in cryopreserved human hepatocytes (HHs) and subjected to liquid chromatography-high resolution tandem mass spectrometry analysis, which identified ten 11ß-MNT biotransformation products with dehydrogenated and glucuronidation (11ß-MNTG) metabolites being the major metabolites. However, 11ß-MNTG formation is highly variable and prevalent in human intestinal S9 fractions. A reaction phenotyping study of 11ß-MNT using thirteen recombinant UDP-glucuronosyltransferase (UGT) enzymes confirmed the major role of UGT2B17 in 11ß-MNTG formation. This was further supported by a strong correlation (R2 > 0.78) between 11ß-MNTG and UGT2B17 abundance in human intestinal microsomes, human liver microsomes, and HH systems. These results suggest that 11ß-MNT and its prodrugs are rapidly metabolized to 11ß-MNTG by the highly polymorphic intestinal UGT2B17, which may explain the poor and variable bioavailability of the drug.

Effects of alcohol consumption and tobacco smoking on the composition of the ensemble of drug metabolizing enzymes and transporters in human liver.

We examined the effect of alcohol consumption and smoking on the abundance of drug-metabolizing enzymes and transporters (DMET) in human liver microsomes (HLM) isolated from liver tissues of 94 donors. Global proteomics analysis was performed and DMET protein levels were analyzed in relation to alcohol consumption levels, smoking history, and sex using non-parametric tests (p-value ≤ 0.05; cutoff of 1.25-fold change, FC). The examination of the alcohol-induced changes was further enforced by correlational analysis, where we used arbitrary alcohol consumption grade (ACG) scaling from 0 to 4 to establish a set of protein markers. We elaborated a provisional index of alcohol exposure (PIAE) based on a combination of relative abundances of four proteins (ER chaperone HSPA5, protein disulfide isomerases PDIA3 and P4HB, and cocaine esterase CES2) best correlating with ACG. The PIAE index was then used to find its correlations with the abundances of DMET proteins. Our results demonstrate considerable alcohol-induced changes in composition of the pool of cytochrome P450 enzymes in HLM. We observed significantly increased abundances of CYP2E1, CYP2B6, CYP2J2, and NADPH-cytochrome P450 reductase. In contrast, CYP1A2, CYP2C8, CYP2C9, CYP4A11, and cytochrome b5 protein levels were downregulated. Significant alteration in abundances of UDP-glucuronosyltransferase (UGT) were also detected, comprising of elevated UGT1A6, UGT1A9, and UGT2A1, and reduced UGT1A3, UGT1A4, UGT2B7, UGT2B10, and UGT2B15 levels. Important alcohol-induced changes were also observed in the expression of non-CYP and non-UGT DMET. Additionally, tobacco smoke was associated with elevated CYP1A2, UGT1A6, UGT2A1, and UGT2B4 and decreased FMO3, FMO4, and FMO5 levels.

Health risk assessment and metal contamination in fish, water and soil sediments in the East Kolkata Wetlands, India, Ramsar site.

East Kolkata Wetlands (EKW) is an important site for fish culture in sewage-fed areas, which are major receivers of pollutants and wastages from Kolkata. EKW is internationally important as the Ramsar site was declared on Aug 2002 with an area of 125 km2. EKW is a natural water body where wastewater-fed natural aquaculture has been practiced for more than 70 years. It is ecologically vulnerable due to the discharge of toxic waste through sewage canals from cities. Assessing the EKW to understand the inflow and load of the toxic metal (s) in fish, water, and sediments samples is essential. The field (samples collection from 13 sites) and lab (determination of toxic level of metals) based research were carried out to assess metal toxicity and health risk assessment in EKW. The levels of eighteen metals (18), namely Chromium, Vanadium, Cobalt, Manganese, Copper, Nickel, Zinc, Silver, Molybdenum, Arsenic, Selenium, Tin, Gallium, Germanium, Strontium, Cadmium, Mercury, and Lead, were determined using Inductively coupled plasma mass spectrometry (ICP-MS) in five fish tissues viz. muscle, liver, kidney, gill and brain, along with the water samples and soil sediments in 13 sampling sites. The bioaccumulation and concentration of metals in fish tissues, soil sediments, and water samples were well within the safe level concerning the recommendation of different national and international agencies except for a few metals in a few sampling sites like Cd, As, and Pb. The geoaccumulation index (Igeo) was also determined in the soil sediments, indicating moderate arsenic, selenium, and mercury contamination in a few sites. The contamination index in water was also determined in 13 sampling sites. The estimated daily intake (EDI), reference dose (RfD), target hazard quotient (THQ), slope factor and cancer risk of Cr, Mn, Co, Ni, Cu, Zn, As, Se, Cd, Pb and Hg from fish muscle were determined. Based on the results of the present investigation, it is concluded that fish consumption in the East Kolkata Wetland (EKW) is safe. The effects of bioaccumulation of metals in muscle tissue were well within the safe level for consumption as recommended by WHO/FAO.

Characterization of Gla proteoforms and non-Gla peptides of gamma carboxylated proteins: Application to quantification of prothrombin proteoforms in human plasma.

Gamma (γ) carboxylation is an essential post-translational modification in vitamin K-dependent proteins (VKDPs), involved in maintaining critical biological homeostasis. Alterations in the abundance or activity of these proteins have pharmacological and pathological consequences. Importantly, low levels of fully γ-carboxylated clotting factors increase plasma des-γ-carboxy precursors resulting in little or no biological activity. Therefore, it is important to characterize the levels of γ-carboxylation that reflect the active state of these proteins. The conventional enzyme-linked immunosorbent assay for protein induced by vitamin K absence or antagonist II (PIVKA-II) quantification uses an antibody that is not applicable to distinguish different γ-carboxylation states. Liquid chromatography-mass spectrometry (LC-MS) approaches have been utilized to distinguish different γ-carboxylated proteoforms, however, these attempts were impeded by poor sensitivity due to spontaneous neutral loss of CO2 and simultaneous cleavage of the backbone bond in the collision cell. In this study, we utilized an alkaline mobile phase in combination with polarity switching (positive and negative ionization modes) to simultaneously identify and quantify γ-carboxylated VKDPs. The method was applied to compare Gla proteomics of prothrombin (FII) in 10 µL plasma samples of healthy control and warfarin-treated adults. We also identified surrogate non-Gla peptides for seven other VKDPs to quantify total (active plus inactive) protein levels. The total protein approach (TPA) was used to quantify absolute levels of the VKDPs in human plasma.

High-Throughput Assay of Cytochrome P450-Dependent Drug Demethylation Reactions and Its Use to Re-Evaluate the Pathways of Ketamine Metabolism.

In a search for a reliable, inexpensive, and versatile technique for high-throughput kinetic assays of drug metabolism, we elected to rehire an old-school approach based on the determination of formaldehyde (FA) formed in cytochrome P450-dependent demethylation reactions. After evaluating several fluorometric techniques for FA detection, we chose the method based on the Hantzsch reaction with acetoacetanilide as the most sensitive, robust, and adaptable to high-throughput implementation. Here we provide a detailed protocol for using our new technique for automatized assays of cytochrome P450-dependent drug demethylations and discuss its applicability for high-throughput scanning of drug metabolism pathways in the human liver. To probe our method further, we applied it to re-evaluating the pathways of metabolism of ketamine, a dissociative anesthetic and potent antidepressant increasingly used in the treatment of alcohol withdrawal syndrome. Probing the kinetic parameters of ketamine demethylation by ten major cytochrome P450 (CYP) enzymes, we demonstrate that in addition to CYP2B6 and CYP3A enzymes, which were initially recognized as the primary metabolizers of ketamine, an important role is also played by CYP2C19 and CYP2D6. At the same time, the involvement of CYP2C9 suggested in the previous reports was deemed insignificant.

Nano­zinc enhances gene regulation of non­specific immunity and antioxidative status to mitigate multiple stresses in fish.

The toxicity of ammonia surged with arsenic pollution and high temperature (34 °C). As climate change enhances the pollution in water bodies, however, the aquatic animals are drastically affected and extinct from nature. The present investigation aims to mitigate arsenic and ammonia toxicity and high-temperature stress (As + NH3 + T) using zinc nanoparticles (Zn-NPs) in Pangasianodon hypophthalmus. Zn-NPs were synthesized using fisheries waste to developing Zn-NPs diets. The four isonitrogenous and isocaloric diets were formulated and prepared. The diets containing Zn-NPs at 0 (control), 2, 4 and 6 mg kg-1 diets were included. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-s-transferase (GST) were noticeably improved using Zn-NPs diets in fish reared under with or without stressors. Interestingly, lipid peroxidation was significantly reduced, whereas vitamin C and acetylcholine esterase were enhanced with supplementation of Zn-NPs diets. Immune-related attributes such as total protein, globulin, albumin, myeloperoxidase (MPO), A:G ratio, and NBT were also improved with Zn-NPs at 4 mg kg-1 diet. The immune-related genes such as immunoglobulin (Ig), tumor necrosis factor (TNFα), and interleukin (IL1b) were strengthening in the fish using Zn-NPs diets. Indeed, the gene regulations of growth hormone (GH), growth hormone regulator (GHR1), myostatin (MYST) and somatostatin (SMT) were significantly improved with Zn-NPs diets. Blood glucose, cortisol and HSP 70 gene expressions were significantly upregulated by stressors, whereas the dietary Zn-NPs downregulated the gene expression. Blood profiling (RBC, WBC and Hb) was reduced considerably with stressors (As + NH3 + T), whereas Zn-NPs enhanced the RBC, WBC, and Hb count in fish reread in control or stress conditions. DNA damage-inducible protein gene and DNA damage were significantly reduced using Zn-NPs at 4 mg kg-1 diet. Moreover, the Zn-NPs also enhanced the arsenic detoxification in different fish tissues. The present investigation revealed that Zn-NPs diets mitigate ammonia and arsenic toxicity, and high-temperature stress in P. hypophthalmus.

Mitigating multiple stresses in Pangasianodon hypophthalmus with a novel dietary mixture of selenium nanoparticles and Omega-3-fatty acid.

Effects of a novel dietary mixture of selenium nanoparticles (Se-NPs) and omega-3-fatty acids i.e., Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on mitigating arsenic pollution, high-temperature stress and bacterial infection were investigated in Pangasianodon hypophthalmus. To aim this, four isocaloric and iso-nitrogenous diets were prepared: control feed (no supplementation), Se-NPs at 0.2 mg kg-1 diet with EPA + DHA at 0.2, 0.4 and 0.6% as supplemented diets. Fish were reared under normal condition or concurrent exposure to arsenic (2.65 mg L-1), and temperature (34 °C) (As + T) stress for 105 days. The experiment was conducted with eight treatments in triplicates. Response to various stresses i.e., primary (cortisol), secondary (oxidative stress, immunity, and stress biomarkers) and tertiary stress response (growth performance, bioaccumulation and mortality due to bacterial infection) were determined. Supplementation of dietary Se-NPs at 0.2 mg kg-1 diet and EPA + DHA at 0.2 and 0.4% reduced the primary stress level. Exposure to arsenic and temperature (As + T) and fed with control diet and EPA + DHA at 0.6% aggravated the cortisol level. Anti-oxidative enzymes (Catalase, superoxide dismutase, glutathione peroxidase and glutathione-s-transferase) and immunity (Nitroblue tetrazolium, total protein, albumin, globulin, A:G ratio, total immunoglobulin and myeloperoxidase) of the fish were augmented by supplementation of Se-NPs and EPA + DHA at 0.2 and 0.4%. Neurotransmitter enzyme, HSP 70, Vitamin C were significantly enhanced (p < 0.01) with supplementation of Se-NPs at 0.2 mg kg-1 and EPA + DHA at 0.2 and 0.4%. Whereas total lipid, cholesterol, phospholipid, triglyceride and very low-density lipoprotein (VLDL) were reduced (p < 0.01) with the supplementation of Se-NPs at 0.2 mg kg-1 diet and EPA + DHA at 0.2 and 0.4%. Tertiary stress response viz. growth performance was also significantly enhanced with supplementation of Se-NPs at 0.2 mg kg-1 and EPA + DHA at 0.2 and 0.4% reared under As + T. Whereas arsenic bioaccumulation in fish tissues was significantly reduced with dietary supplementation of Se-NPs and EPA + DHA. Cumulative mortality and relative percentage survival were reduced with Se-NPs at 0.2 mg kg-1 and EPA + DHA at 0.2 and 0.4%. The investigation revealed that a novel combination of Se-NPs at 0.2 mg kg-1 and EPA + DHA at 0.4% followed by 0.2% has the potential to alleviate temperature stress, bacterial infection and arsenic pollution. Whereas diet containing Se-NPs at 0.2 mg kg-1 diet and EPA + DHA at 0.6% was noticeably enhanced the stress in P. hypophthalmus.

Characterization of degradation products of celiprolol hydrochloride using hyphenated mass and NMR techniques.

Stress degradation studies were carried out on celiprolol hydrochloride under the ICH prescribed hydrolysis (acidic, basic and neutral), photolytic, oxidative and thermal conditions. Maximum degradation was observed upon hydrolysis, especially in the basic condition. In oxidative condition, the drug degraded only upon severe exposure to H2O2, but it remained stable when challenged with AIBN. It also degraded significantly under photolytic conditions. However, the drug was stable to thermal stress. A total of seven degradation products were formed, whose separation was successfully achieved on an Inertsil ODS-3V C-18 HPLC column employing a gradient mobile phase. A comprehensive mass fragmentation pattern of the drug was initially established through the support of high resolution mass spectrometry (HR-MS), multi-stage tandem mass spectrometry (MSn) and on-line H/D exchange MS data. The same approach was then extended to characterization of the degradation products. Additionally, two degradation products were isolated and subjected to 1D/2D NMR studies for their structural confirmation. One of the degradation products showed instability during isolation, therefore, it was subjected to LC-NMR studies for its structural confirmation.