Gemfibrozil palliates adriamycin-induced testicular injury in male rats via modulating oxidative, endocrine and inflammatory changes in rats.

Adriamycin (ADR), an antineoplastic drug, is widely used to treat different types of cancers. Yet, the usage is limited because of its severe side effects on testis. On the other hand, gemfibrozil (GEM), as an anti-hyperlipidemic drug, has other pharmacological effects independent of lipid- lowering activity including anti-inflammatory and antioxidant properties. The present experiment was designed to investigate the effect of GEM on ADR-induced testicular injury in male rats. A total of 28 male Wistar rats were divided into 4 equal groups: Control; ADR; ADR + GEM; GEM. Serum level of testosterone, luteinizing hormone and follicle stimulating hormone were assessed. Also, testicular tissue oxidant/antioxidant markers (malondialdehyde, total antioxidant capacity, nitric oxide, superoxide dismutase, catalase, glutathione peroxidase and glutathione) and proinflammatory cytokines (tumor necrosis factor-α and interleukin-1ß) were measured. Histopathological studies were conducted on testes. GEM improved hormonal profile and antioxidant defenses in comparison with ADR-treated animals. GEM, significantly reduced the production of proinflammatory cytokines compared with ADR-treated animals. Hormonal and biochemical results were further supported by testicular histopathological findings. Thus, GEM might represent a promising therapeutic modality for the attenuation of testicular injury induced by ADR in clinic.

Biodegradation of emerging organic pollutant gemfibrozil: Mechanism, kinetics and pathway modelling.

The increasing population has raised the demand for pharmaceutical and personal care products to maintain a good health. Gemfibrozil (GEM), is extensively used as a lipid regulator and is frequently detected in wastewater treatment systems and poses deleterious health and ecological effects. Hence, the current study employing Bacillus sp. N2 reports the degradation of gemfibrozil via co-metabolism in 15 days. The study reported 86 % degradation with GEM (20 mgL-1) using sucrose (150 mgL-1) as a co-substrate; as compared to 42 % without a co-substrate. Further, time-profiling studies of metabolites revealed significant demethylation and decarboxylation reactions during degradation that leads to formation of six (M1, M2, M3, M4, M5, M6) metabolites as by-products. Based on the LC-MS analysis a potential degradation pathway for GEM by Bacillus sp. N2 was proposed. The degradation of GEM has not been reported so far and the study envisages eco-friendly approach to tackle pharmaceutical- active- compounds.

Gemfibrozil, a lipid-lowering drug, improves hepatorenal damages in a mouse model of aging.

Gemfibrozil (GFZ) is a medication of the fibrate category with agonistic effects on peroxisome proliferator-activated receptor-α (PPAR-α) and is effective for hypertriglyceridemia and mixed dyslipidemia. This agent also has anti-inflammatory and antioxidant properties. The current study investigated the effects of GFZ on hepatorenal damages in a D-galactose (D-gal)-induced aging model. We used 28 male mice, which were equally and randomly divided into four groups as follows: normal, D-gal (150 mg/kg/day; intraperitoneal [i.p.], for 6 weeks), GFZ (100 mg/kg/day GFZ, orally [p.o.] for 6 weeks), and the combined D-gal + GFZ. Liver and kidney function indices were measured as serum creatinine, blood urine nitrogen, alanine aminotransferase, and aspartate aminotransferase. Oxidative stress in hepatic and renal tissue was evaluated through malondialdehyde, superoxide dismutase, and glutathione peroxidase levels. Finally, the liver and kidney tissues were assessed for histopathological lesions. The results showed that D-gal-induced aging leads to abnormalities in liver and kidney function indices. D-gal also induced significant oxidative stress and histopathological lesions in these organs. GFZ improved function indices and oxidative stress compared to the D-gal-treated animals. Histological evaluations of the liver and kidney also confirmed these results. These data provide evidence for the potential therapeutic of GFZ in clinical practice for mitigating the hepatorenal damages of aging.

Multimarker Responses of Zebrafish to the Effect of Ibuprofen and Gemfibrozil in Environmentally Relevant Concentrations.

Pharmaceutical pollution of water bodies is among the top-notch environmental health risks all over the world. The aim of the present study was to investigate the effects of two common pharmaceuticals namely ibuprofen and gemfibrozil on zebrafish at environmentally relevant concentrations. In zebrafish liver, gemfibrozil caused a decrease in glutathione and glutathione transferase and an increase in catalase but had no effect on lipid peroxidation and protein carbonylation. Ibuprofen altered the antioxidant defense system, promoted protein carbonylation in zebrafish liver, and increased vitellogenin-like protein in the blood. Ibuprofen and particularly gemfibrozil induced lysosomes biogenesis. Lactate dehydrogenase in the blood was also found to be higher in the studied groups. Studied pharmaceuticals did not affect complex II of the electron respiratory chain. Ibuprofen affects zebrafish health status more profoundly than gemfibrozil. Our results showed that pharmaceuticals even in low, environmentally realistic concentrations, induced profound changes in the stress-responsive systems of zebrafish.

Inhibition of CYP2C8 by Acyl Glucuronides of Gemfibrozil and Clopidogrel: Pharmacological Significance, Progress and Challenges.

The lipid-regulating drug gemfibrozil is a useful medication for reducing high cholesterol and triglycerides in the blood. In addition to oxidation, it undergoes extensive glucuronidation to produce gemfibrozil acyl glucuronide, which is a known mechanism-based inactivator of cytochrome P450 (CYP) 2C8. Such selective and time-dependent inhibition results in clinically important drug-drug interactions (DDI) with the drugs metabolized by CYP2C8. Similarly, the acyl glucuronide of clopidogrel, a widely used antiplatelet agent, is a potent time-dependent inhibitor of CYP2C8 that demonstrated significant DDI with the substrates of CYP2C8. Current progress in atomic-level understanding mostly involves studying how different drugs bind and undergo oxidation in the active site of CYPs. It is not clear how an acyl glucuronide metabolite of the drug gemfibrozil or clopidogrel interacts in the active site of CYP2C8 and selectively inhibit the enzyme. This mini-review summarizes the current knowledge on some of the important clinical DDI caused by gemfibrozil and clopidogrel due to the inhibition of CYP2C8 by acyl glucuronide metabolites of these drugs. Importantly, it examines recent developments and potential applications of structural biology tools to elucidate the binding and orientation of gemfibrozil acyl glucuronide and clopidogrel acyl glucuronide in the active site near heme that contributes to the inhibition and inactivation of CYP2C8.

Metabolic Activation of Gemfibrozil Mediated by Cytochrome P450 Enzymes and Sulfotransferases.

Gemfibrozil (GEM), a lipid regulator, is a fibric acid derivative widely used in the treatment of hyperlipidemia. It has been reported that GEM can induce acute liver injury in the course of therapy in clinical practice, so it is necessary to elucidate the mechanisms of toxic action. The present study focused on metabolic activation of GEM, possibly participating in GEM-mediated liver injury. A benzylic alcohol metabolite (M1), along with a phenol metabolite (M2), was detected in microsomal incubations, rat primary hepatocyte culturing, and rats given GEM. A GSH conjugate (M3) was detected in cultured rat hepatocytes after exposure to GEM. Formation of M1 was found to be NADPH dependent, and generation of M3 required M1 and 3'-phosphoadenosine-5'-phosphosulfate. It is most likely that GEM was biotransformed to M1, which was further metabolized to a sulfate. The resulting sulfate was reactive to bio-thiols. Cytochrome P450 and sulfotransferases participated in the phase I and phase II reactions, respectively. M1 and M3 were chemically synthesized, and their structures were characterized by mass spectrometry and NMR. The present study has particular value for elucidating the mechanism of liver injury caused by GEM.

Physiologically Based Pharmacokinetic Modeling of Rosuvastatin to Predict Transporter-Mediated Drug-Drug Interactions.

PURPOSE: To build a physiologically based pharmacokinetic (PBPK) model of the clinical OATP1B1/OATP1B3/BCRP victim drug rosuvastatin for the investigation and prediction of its transporter-mediated drug-drug interactions (DDIs). METHODS: The Rosuvastatin model was developed using the open-source PBPK software PK-Sim®, following a middle-out approach. 42 clinical studies (dosing range 0.002-80.0 mg), providing rosuvastatin plasma, urine and feces data, positron emission tomography (PET) measurements of tissue concentrations and 7 different rosuvastatin DDI studies with rifampicin, gemfibrozil and probenecid as the perpetrator drugs, were included to build and qualify the model. RESULTS: The carefully developed and thoroughly evaluated model adequately describes the analyzed clinical data, including blood, liver, feces and urine measurements. The processes implemented to describe the rosuvastatin pharmacokinetics and DDIs are active uptake by OATP2B1, OATP1B1/OATP1B3 and OAT3, active efflux by BCRP and Pgp, metabolism by CYP2C9 and passive glomerular filtration. The available clinical rifampicin, gemfibrozil and probenecid DDI studies were modeled using in vitro inhibition constants without adjustments. The good prediction of DDIs was demonstrated by simulated rosuvastatin plasma profiles, DDI AUClast ratios (AUClast during DDI/AUClast without co-administration) and DDI Cmax ratios (Cmax during DDI/Cmax without co-administration), with all simulated DDI ratios within 1.6-fold of the observed values. CONCLUSIONS: A whole-body PBPK model of rosuvastatin was built and qualified for the prediction of rosuvastatin pharmacokinetics and transporter-mediated DDIs. The model is freely available in the Open Systems Pharmacology model repository, to support future investigations of rosuvastatin pharmacokinetics, rosuvastatin therapy and DDI studies during model-informed drug discovery and development (MID3).

Crystalline Sponges as a Sensitive and Fast Method for Metabolite Identification: Application to Gemfibrozil and its Phase I and II Metabolites.

Understanding the metabolism of new drug candidates is important during drug discovery and development, as circulating metabolites may contribute to efficacy or cause safety issues. In the early phase of drug discovery, human in vitro systems are used to investigate human relevant metabolism. Though conventional techniques are limited in their ability to provide complete molecular structures of metabolites (liquid chromatography mass spectrometry) or require a larger amount of material not available from in vitro incubation (nuclear magnetic resonance), we here report for the first time the use of the crystalline sponge method to identify phase I and phase II metabolites generated from in vitro liver microsomes or S9 fractions. Gemfibrozil was used as a test compound. Metabolites generated from incubation with microsomes or S9 fractions, were fractionated using online fraction collection. After chromatographic purification and fractionation of the generated metabolites, single crystal X-ray diffraction of crystalline sponges was used to identify the structure of gemfibrozil metabolites. This technique allowed for complete structure elucidation of 5'-CH2OH gemfibrozil (M1), 4'-OH gemfibrozil (M2), 5'-COOH gemfibrozil (M3), and the acyl glucuronide of gemfibrozil, 1-O-ß-glucuronide (M4), the first acyl glucuronide available in the Cambridge Crystallographic Data Centre. Our study shows that when optimal soaking is possible, crystalline sponges technology is a sensitive (nanogram amount) and fast (few days) method that can be applied early in drug discovery to identify the structure of pure metabolites from in vitro incubations. SIGNIFICANCE STATEMENT: Complete structure elucidation of human metabolites plays a critical role in early drug discovery. Low amounts of material (nanogram) are only available at this stage and insufficient for nuclear magnetic resonance analysis. The crystalline sponge method has the potential to close this gap, as demonstrated in this study.

Evaluation of bezafibrate, gemfibrozil, indomethacin, sulfamethoxazole, and diclofenac removal by ligninolytic enzymes.

The laccase (Lac), manganese peroxidases (MnP), and lignin peroxidase enzymes produced by basidiomycete have been studied due to their potential in bioremediation, therefore, in this study, degradation of diclofenac (DCF), sulfamethoxazole (SMX), indomethacin (IND), gemfibrozil (GFB), and bezafibrate (BZF) by enzymes produced by Trametes maxima, Pleurotus sp., and Pycnosporus sanguineus grown in culture was evaluated. The degradation of drugs can mainly be attributed to MnP because a correlation between the activity of this enzyme and the degree of removal was found. The specific activity of Lac did not show correlation with drug removal, while lignin peroxidase was not expressed. Trametes maxima showed the highest specific activity of MnP (387.6 ± 67.4 U/mg) and efficiency removal 90.2% of DCF, 72.62% of SMX, 60.76% of IND, 43.39% of GFB, and 32.59% of BZF) followed by Pleurotus sp. with specific activity of MnP of 55.9 ± 8.5 U/mg and 89.47% of DCF, 47.61% of GFB and 73% of IND were removed, P. sanguineus had the lowest specific activity of 18 ± 1.3 U/mg and was able to remove only 42% of SMX and 10.59% of IND. In order to prove that MnP remove drugs instead of Lac, the pure Lac was tested and only degraded DCF.

Critical Differences between Enzyme Sources in Sensitivity to Detect Time-Dependent Inactivation of CYP2C8.

Clopidogrel acyl-ß-d-glucuronide is a mechanism-based inhibitor of cytochrome P450 2C8 in human liver microsomes (HLMs). However, time-dependent inactivation (TDI) of CYP2C8 could not be detected in an earlier study in human recombinant CYP2C8 (Supersomes). Here, we investigate whether different enzyme sources exhibit differences in detection of CYP2C8 TDI under identical experimental conditions. Inactivation of CYP2C8 by amiodarone (100 µM), clopidogrel acyl-ß-d-glucuronide (100 µM), gemfibrozil 1-O-ß-glucuronide (100 µM), and phenelzine (100 µM) was investigated in HLMs and three recombinant human CYP2C8 preparations (Supersomes, Bactosomes, and EasyCYP Bactosomes) using amodiaquine N-deethylation as the marker reaction. Furthermore, the inactivation kinetics of CYP2C8 by clopidogrel glucuronide (5-250 µM) was determined in Supersomes and Bactosomes. Amiodarone caused weak TDI in all enzyme preparations tested, while the extent of inactivation by clopidogrel glucuronide, gemfibrozil glucuronide, and phenelzine varied markedly between preparations, and even different Supersome lots. Both glucuronides caused strong inactivation of CYP2C8 in HLMs, Bactosomes and in one Supersome lot (>50% inhibition), but significant inactivation could not be reliably detected in other Supersome lots or EasyCYP Bactosomes. In Bactosomes, the concentration producing half of kinact (KI) and maximal inactivation rate (kinact) of clopidogrel glucuronide (14 µM and 0.054 minute-1) were similar to those determined previously in HLMs. Phenelzine caused strong inactivation of CYP2C8 in one Supersome lot (91% inhibition) but not in HLMs or other recombinant CYP2C8 preparations. In conclusion, different enzyme sources and different lots of the same recombinant enzyme preparation are not equally sensitive to detect inactivation of CYP2C8, suggesting that recombinant CYPs should be avoided when identifying mechanism-based inhibitors.

Effects and bioaccumulation of gold nanoparticles in the gilthead seabream (Sparus aurata) - Single and combined exposures with gemfibrozil.

Gold nanoparticles (AuNPs) are found in a wide range of applications and therefore expected to present increasing levels in the environment. There is however limited knowledge concerning the potential toxicity of AuNPs as well as their combined effects with other pollutants. Hence, the present study aimed to investigate the effects of AuNPs alone and combined with the pharmaceutical gemfibrozil (GEM) on different biological responses (behaviour, neurotransmission, biotransformation and oxidative stress) in one of the most consumed fish in southern Europe, the seabream Sparus aurata. Fish were exposed for 96 h to waterborne 40 nm AuNPs with two coatings - citrate and polyvinylpyrrolidone (PVP), alone or combined with GEM. Antioxidant defences were induced in liver and gills upon both AuNPs exposure. Decreased swimming performance (1600 µg.L-1) and oxidative damage in gills (4 and 80 µg.L-1) were observed following exposure to polyvinylpyrrolidone coated gold nanoparticles (PVP-AuNPs). Generally, accumulation of gold in fish tissues and deleterious effects in S. aurata were higher for PVP-AuNPs than for cAuNPs exposures. Although AuNPs and GEM combined effects in gills were generally low, in liver, they were higher than the predicted. The accumulation and effects of AuNPs showed to be dependent on the size, coating, surface charge and aggregation/agglomeration state of nanoparticles. Additionally, it was tissue' specific and dependent on the presence of other contaminants. Although, gold intake by humans is expected to not exceed the estimated tolerable daily intake, it is highly recommended to keep it on track due to the increasing use of AuNPs.

Stable Isotope Labeling-Assisted Metabolite Probing for Emerging Contaminants in Plants.

Biotransformation is a notable modulator of the fate, bioaccumulation, and toxicity of contaminants in the environment. However, it is often formidable to identify unknown biotransformation products in the absence of reference standards, and this analytical challenge is particularly true for contaminants of emerging concern (CECs) that are mostly polar molecules without characteristic structures (e.g., Cl and Br) and in complex matrices such as plants. In this study, using the fibrate drug gemfibrozil as a model CEC and Arabidopsis thaliana as a model plant, we developed and demonstrated a novel analytical framework coupling deuterium stable isotope labeling with high-resolution mass spectrometry (SILAMS) in identifying plant biotransformation products. When exposed in A. thaliana cells, gemfibrozil was quickly taken up into the cells and extensively metabolized. The use of nonlabeled and deuterated gemfibrozil at a 3:1 ratio created unique diagnostic patterns in mass spectra, enabling the identification of 11 novel phase II amino acid/peptide conjugates. Similarity in mass fragmentation patterns and chromatographic behaviors was then employed to establish the probable structures. Two major metabolites were further confirmed as glutamate and glutamine conjugates using authentic standards. Most of the identified conjugates were also detected in the whole A. thaliana plant. Therefore, SILAMS offers unique advantages by excluding false matrix positives and helping discern unknown metabolites, including polar conjugates with endogenous biomolecules, with a high degree of confidence. This novel framework may be readily applied to other CECs for high-throughput metabolite screening in plants to improve our understanding of their food safety and human health risks and potential deleterious effects on other species living on plants.

Enhanced pharmaceutical removal from water in a three step bio-ozone-bio process.

Individual treatment processes like biological treatment or ozonation have their limitations for the removal of pharmaceuticals from secondary clarified effluents with high organic matter concentrations (i.e. 17 mg TOC/L). These limitations can be overcome by combining these two processes for a cost-effective pharmaceutical removal. A three-step biological-ozone-biological (BO3B) treatment process was therefore designed for the enhanced pharmaceutical removal from wastewater effluent. The first biological step removed 38% of ozone scavenging TOC, thus proportionally reducing the absolute ozone input for the subsequent ozonation. Complementariness between biological and ozone treatment, i.e. targeting different pharmaceuticals, resulted in cost-effective pharmaceutical removal by the overall BO3B process. At a low ozone dose of 0.2 g O3/g TOC and an HRT of 1.46 h in the biological reactors, the removal of 8 out of 9 pharmaceuticals exceeded 85%, except for metoprolol (60%). Testing various ozone doses and HRTs revealed that pharmaceuticals were ineffectively removed at 0.1 g O3/g TOC and an HRT of 0.3 h. At HRTs of 0.47 and 1.46 h easily and moderately biodegradable pharmaceuticals such as caffeine, gemfibrozil, ibuprofen, naproxen and sulfamethoxazole were over 95% removed by biological treatment. The biorecalcitrant carbamazepine was completely ozonated at a dose of 0.4 g O3/g TOC. Ozonation products are likely biodegraded in the last biological reactor as a 17% TOC removal was found. No appreciable acute toxicity towards D. magna, P. subcapitata and V. fischeri was found after exposure to the influents and effluents of the individual BO3B reactors. The BO3B process is estimated to increase the yearly wastewater treatment tariff per population equivalent in the Netherlands by less than 10%. Overall, the BO3B process is a cost-effective treatment process for the removal of pharmaceuticals from secondary clarified effluents.

Structural analysis of the regulatory mechanism of MarR protein Rv2887 in M. tuberculosis.

MarR family proteins are transcriptional regulators that control expression of bacterial proteins involved in metabolism, virulence, stress responses and multi-drug resistance, mainly via ligand-mediated attenuation of DNA binding. Greater understanding of their underlying regulatory mechanism may open up new avenues for the effective treatment of bacterial infections. To gain molecular insight into the mechanism of Rv2887, a MarR family protein in M. tuberculosis, we first showed that it binds salicylate (SA) and para-aminosalicylic acid (PAS), its structural analogue and an antitubercular drug, in a 1:1 stoichiometry with high affinity. Subsequent determination and analysis of Rv2887 crystal structures in apo form, and in complex with SA, PAS and DNA showed that SA and PAS bind to Rv2887 at similar sites, and that Rv2887 interacts with DNA mainly by insertion of helix α4 into the major groove. Ligand binding triggers rotation of the wHTH domain of Rv2887 toward the dimerization domain, causing changes in protein conformation such that it can no longer bind to a 27 bp recognition sequence in the upstream region of gene Rv0560c. The structures provided here lay a foundation for the design of small molecules that target Rv2887, a potential new approach for the development of anti-mycobacterials.

Transport behavior of the pharmaceutical compounds carbamazepine, sulfamethoxazole, gemfibrozil, ibuprofen, and naproxen, and the lifestyle drug caffeine, in saturated laboratory columns.

Despite the large number of pharmaceutically active compounds found in natural environments little is known about their transport behavior in groundwater, which is complicated by their wide range of physical and chemical properties. The transport behavior of five widely used and often detected pharmaceutical compounds and one lifestyle drug has therefore been investigated, using a set of three column experiments. The investigated compounds were the anticonvulsant carbamazepine, the lifestyle drug caffeine, the antibiotic sulfamethoxazole, the lipid regulator gemfibrozil, and the nonsteroidal anti-inflammatories ibuprofen and naproxen. The columns were filled with three different types of sand. The substrates consisted of artificially prepared iron-coated sand, artificially prepared organic carbon sand (with 5% leaf compost), and natural aquifer sand from Long Point, Ontario (Canada). The experiments were conducted simultaneously under the same hydraulic conditions and with the same input solution of about 1µg·L-1 of each compound. The transport behavior of the organic compounds differed significantly between both the different columns and the different compounds. A strong correlation was observed between the retardation factors for carbamazepine, gemfibrozil, and ibuprofen and the organic carbon content of the substrate. While the retardation increased with increasing organic carbon content, no direct relationship was observed between the organic carbon content and the removal of these compounds. In contrast, the retardation factors for sulfamethoxazole and naproxen showed no correlation with the organic carbon content but these compounds were significantly removed in the presence of organic matter. The influence of the Fe3+ surfaces in the iron-coated sand was less significant than expected, with all compounds except for sulfamethoxazole having retardation factors <1.8. Caffeine was so strongly removed during transport through those substrates containing organic carbon that no reliable retardation factor could be determined.

Role of gemfibrozil as an inhibitor of CYP2C8 and membrane transporters.

INTRODUCTION: Cytochrome P450 (CYP) 2C8 is a drug metabolizing enzyme of major importance. The lipid-lowering drug gemfibrozil has been identified as a strong inhibitor of CYP2C8 in vivo. This effect is due to mechanism-based inhibition of CYP2C8 by gemfibrozil 1-O-ß-glucuronide. In vivo, gemfibrozil is a fairly selective CYP2C8 inhibitor, which lacks significant inhibitory effect on other CYP enzymes. Gemfibrozil can, however, have a smaller but clinically meaningful inhibitory effect on membrane transporters, such as organic anion transporting polypeptide 1B1 and organic anion transporter 3. Areas covered: This review describes the inhibitory effects of gemfibrozil on CYP enzymes and membrane transporters. The clinical drug interactions caused by gemfibrozil and the different mechanisms contributing to the interactions are reviewed in detail. Expert opinion: Gemfibrozil is a useful probe inhibitor of CYP2C8 in vivo, but its effect on membrane transporters has to be taken into account in study design and interpretation. Moreover, gemfibrozil could be used to boost the pharmacokinetics of CYP2C8 substrate drugs. Identification of gemfibrozil 1-O-ß-glucuronide as a potent mechanism-based inhibitor of CYP2C8 has led to recognition of glucuronide metabolites as perpetrators of drug-drug interactions. Recently, also acyl glucuronide metabolites of clopidogrel and deleobuvir have been shown to strongly inhibit CYP2C8.

Simultaneous analysis of gemfibrozil, morphine, and its two active metabolites in different mouse brain structures using solid-phase extraction with ultra-high performance liquid chromatography and tandem mass spectrometry with a deuterated internal standard.

A rapid and sensitive bioassay was established and validated to simultaneously determine gemfibrozil, morphine, morphine-3ß-glucuronide, and morphine-6ß-glucuronide in mouse cerebrum, epencephalon, and hippocampus based on ultra-high performance liquid chromatography and tandem mass spectrometry. The deuterated internal standard, M6G-d3, was mixed with the prepared samples at 10 ng/mL as the final concentration. The samples were transferred into the C18 solid-phase extraction columns with gradient elution for solid-phase extraction. The mobile phase consisted of methanol and 0.05% formic acid (pH 3.2). Multiple reaction monitoring has been applied to analyze gemfibrozil (m/z 249.0 → 121.0) in anion mode, and M6G-d3 (m/z 465.1 → 289.1), morphine (m/z 286.0 → 200.9), and M3G and M6G (m/z 462.1 → 286.1) in the positive ion mode. The method has a linear calibration range from 0.05 to 10 ng for gemfibrozil, morphine, and M3G and M6G with correlation coefficients >0.993. The lower limit of quantitation for all four analytes was 0.05 ng/mL, relative standard deviation of intra- and interday precision was less than 10.5%, and the relative error of accuracy was from -8.2 to 8.3% at low, medium, and high concentrations for all the analytes. In conclusion, gemfibrozil can influence the morphine antinociception after coronary heart disease induced chronic angina by the change in one of morphine metabolites', M3G, distribution in mouse brain.

Evaluation of the simultaneous removal of recalcitrant drugs (bezafibrate, gemfibrozil, indomethacin and sulfamethoxazole) and biodegradable organic matter from synthetic wastewater by electro-oxidation coupled with a biological system.

Pharmaceutical degradation in conventional wastewater treatment plants (WWTP) represents a challenge since municipal wastewater and hospital effluents contain pharmaceuticals in low concentrations (recalcitrant and persistent in WWTP) and biodegradable organic matter (BOM) is the main pollutant. This work shows the feasibility of coupling electro-oxidation with a biological system for the simultaneous removal of recalcitrant drugs (bezafibrate, gemfibrozil, indomethacin and sulfamethoxazole (BGIS)) and BOM from wastewater. High removal efficiencies were attained without affecting the performance of activated sludge. BGIS degradation was performed by advanced electrochemical oxidation and the activated sludge process for BOM degradation in a continuous reactor. The selected electrochemical parameters from microelectrolysis tests (1.2 L s(-1) and 1.56 mA cm(-2)) were maintained to operate a filter press laboratory reactor FM01-LC using boron-doped diamond as the anode. The low current density was chosen in order to remove drugs without decreasing BOM and chlorine concentration control, so as to avoid bulking formation in the biological process. The wastewater previously treated by FM01-LC was fed directly (without chemical modification) to the activated sludge reactor to remove 100% of BGIS and 83% of BOM; conversely, the BGIS contained in wastewater without electrochemical pre-treatment were persistent in the biological process and promoted bulking formation.

Oxidative stress, energy storage, and swimming performance of Limnodynastes peronii tadpoles exposed to a sub-lethal pharmaceutical mixture throughout development.

Pharmaceutical contaminants represent emerging threats to aquatic animals and ecosystem health, and research exploring toxicological outcomes associated with these compounds in non-target wildlife has been flagged for prioritization. Amphibians represent particularly vulnerable organisms and many populations around the world are currently at risk of extinction. However, to date, relatively few studies have explored the consequences of exposures to common non-steroidal pharmaceuticals during sensitive amphibian life-stages. To address existing knowledge gaps, tadpoles of the Australian striped-marsh frog (Limnodynastes peronii) were exposed to control water and a mixture of the common pharmaceutical contaminants diclofenac, naproxen, atenolol and gemfibrozil at 0.1, 1, 10, 100 and 1000 µg/L throughout the developmental period. Effects on detoxification pathways, energy storage, growth and development, and swimming performance were assessed following exposure. Developmental rates and liver-somatic index (LSI) were significantly reduced in the highest exposure concentration, and condition factor (K) was increased at concentrations as low as 10 µg/L. Morphological endpoints were associated with significantly altered levels of hepatic triglycerides, which in turn were correlated with increased peroxidase activity in animals exposed to the highest concentration (1000 µg/L). The mixture had no significant effect on swimming performance, but a trend of decreased swimming velocity (average and maximum) was observed with increasing concentration, and this was correlated with effects on LSI. Results demonstrate that mixtures of common non-steroidal pharmaceuticals can elicit a range of physiological, metabolic and morphological responses in larval amphibians, and more research is therefore warranted to explore possible relationships between endpoints at different levels of organization.

Genomic, Proteomic, and Metabolite Characterization of Gemfibrozil-Degrading Organism Bacillus sp. GeD10.

Gemfibrozil is a widely used hypolipidemic and triglyceride lowering drug. Excess of the drug is excreted and discharged into the environment primarily via wastewater treatment plant effluents. Bacillus sp. GeD10, a gemfibrozil-degrader, was previously isolated from activated sludge. It is the first identified bacterium capable of degrading gemfibrozil. Gemfibrozil degradation by Bacillus sp. GeD10 was here studied through genome sequencing, quantitative proteomics and metabolite analysis. From the bacterial proteome of Bacillus sp. GeD10 1974 proteins were quantified, of which 284 proteins were found to be overabundant by more than 2-fold (FDR corrected p-value ≤0.032, fold change (log2) ≥ 1) in response to gemfibrozil exposure. Metabolomic analysis identified two hydroxylated intermediates as well as a glucuronidated hydroxyl-metabolite of gemfibrozil. Overall, gemfibrozil exposure in Bacillus sp. GeD10 increased the abundance of several enzymes potentially involved in gemfibrozil degradation as well as resulted in the production of several gemfibrozil metabolites. The potential catabolic pathway/modification included ring-hydroxylation preparing the substrate for subsequent ring cleavage by a meta-cleaving enzyme. The identified genes may allow for monitoring of potential gemfibrozil-degrading organisms in situ and increase the understanding of microbial processing of trace level contaminants. This study represents the first omics study on a gemfibrozil-degrading bacterium.