Impact of Heterotropic Allosteric Modulation on the Time-Dependent Inhibition of Cytochrome P450 3A4.

The current study was designed to investigate the influence of allosteric effectors on the metabolism of the prototypical cytochrome P450 (CYP) 3A4 substrate midazolam (MDZ), and on the determination in vitro time-dependent inhibition (TDI) of CYP3A4 using human liver microsomes (HLM). As the concentration of midazolam increased to 250 µM in HLMs, homotropic cooperativity resulted in a decrease in the 1'-hydroxymidazolam to 4-hydroxymidazolam ratio to a maximum of 1.1. The presence of varying concentrations of testosterone, progesterone (PGS), or carbamazepine (CBZ) in HLMs with MDZ could recapitulate the effect of homotropic cooperativity such that the formation rates of the 1'hydroxymidazolam and 4-hydroxymidazolam were equal even at low concentrations of MDZ. The presence of PGS (10 or 100 µM) and CBZ (100 or 1000 µM) in in vitro TDI determination of four known CYP3A4 time-dependent inactivators (clarithromycin, troleandomycin, mibefradil, raloxifene) simultaneously decreased potency and inactivation rate constant, resulting in fold changes in inactivation efficiency on average of 1.6-fold and 13-fold for the low and high concentrations of allosteric modulator tested, respectively. The formation of a metabolic-intermediate complex (MIC) for clarithromycin and troleandomycin decreased in the presence of the allosteric modulators in a concentration-dependent manner, reaching a new steady state formation that could not be overcome with increased incubation time. Maximum reduction of the MIC formed by clarithromycin was up to ∼91%, while troleandomycin MIC decreased up to ∼31%. These findings suggest that the absence of endogenous allosteric modulators may contribute to the poor translation of HLM-based drug-drug interaction predictions. SIGNIFICANCE STATEMENT: The reported overprediction of in vitro human liver microsome time-dependent inhibition of CYP3A4 and observed drug interactions in vivo remains an issue in drug development. We provide characterization of allosteric modulators on the CYP3A4 metabolism of the prototypical substrate midazolam, demonstrating the ability of the modulators to recapitulate the homotropic cooperativity of midazolam. Furthermore, we demonstrate that allosteric heterotropic cooperativity of CYP3A4 can impact the time-dependent inhibition kinetics of known mechanisms-based inhibitors, providing a potential mechanism to explain the overprediction.

Response of hepatic biochemical parameters and neurotoxicity to carbamazepine and ibuprofen in Oreochromis mossambicus.

Globally, the prevalence and pollution of pharmaceutical drugs in aquatic environments have been steadily increasing. This study sought to evaluate the effects of 14 days of exposure to environmental-relevant doses (ibuprofen 0.5, 5, and 50 µg/L, and carbamazepine 0.005, 1, and 10 µg/L) of the nonsteroidal anti-inflammatory drugs ibuprofen and carbamazepine in the freshwater fish Oreochromis mossambicus. The results showed a significant (P < 0.05) decrease in O. mossambicus superoxide dismutase, catalase, biotransformation enzymes, glutathione-s-transferase, glutathione peroxidase, oxidative stress lipid peroxidation, protein carbonyl activity, cellular damage metallothionine, reduced glutathione, immunological activities, and respiratory burst activity. Consequently, the acquired data revealed that O. mossambicus treated with ibuprofen and carbamazepine shows more significant alterations in metabolic depression, biochemical parameters, and oxidative stress. In addition, increased neurotoxic effects were observed in ibuprofen and carbamazepine treated O. mossambicus.

Toxicometabolomics and Biotransformation Product Elucidation in Single Zebrafish Embryos Exposed to Carbamazepine from Environmentally-Relevant to Morphologically Altering Doses.

Toxicometabolomics and biotransformation product (bioTP) elucidation were carried out in single zebrafish (ZF) embryos exposed to carbamazepine (CBZ). Exposures were conducted in 96-well plates containing six CBZ concentrations ranging from 0.5 µg/L to 50 mg/L (n = 12 embryos per dose). In the 50 mg/L dose group, 33% of embryos developed edema during the exposure (120 hpf), while hatching was significantly delayed in three of the lower-dose groups (0.46, 3.85, and 445 µg/L) compared to the control at 48 hpf. Toxicometabolomic analysis together with random forest modeling revealed a total of 80 significantly affected metabolites (22 identified via targeted lipidomics and 58 via nontarget analysis). The wide range of doses enabled the observation of both monotonic and nonmonotonic dose responses in the metabolome, which ultimately produced a unique and comprehensive biochemical picture that aligns with existing knowledge on the mode of action of CBZ. The combination of high dose exposures and apical endpoint assessment in single embryos also enabled hypothesis generation regarding the target organ for the morphologically altering insult. In addition, two CBZ bioTPs were identified without additional exposure experiments. Overall, this work showcases the potential of toxicometabolomics and bioTP determination in single ZF embryos for rapid and comprehensive chemical hazard assessment.

Assessment of hepatic prostaglandin E2 level in carbamazepine induced liver injury.

Objective. Carbamazepine (CBZ), a widely used antiepileptic drug, is one major cause of the idiosyncratic liver injury along with immune reactions. Conversely, prostaglandin E2 (PGE2) demonstrates a hepatoprotective effect by regulating immune reactions and promoting liver repair in various types of liver injury. However, the amount of hepatic PGE2 during CBZ-induced liver injury remains elusive. In this study, we aimed to evaluate the hepatic PGE2 levels during CBZ-induced liver injury using a mouse model. Methods. Mice were orally administered with CBZ at a dose of 400 mg/kg for 4 days, and 800 mg/kg on the 5th day. Results. Plasma alanine transaminase (ALT) level increased in some of mice 24 h after the last CBZ administration. Although median value of hepatic PGE2 amount in the CBZ-treated mice showed same extent as vehicle-treated control mice, it exhibited significant elevated level in mice with severe liver injury presented by a plasma ALT level >1000 IU/L. According to these results, mice had a plasma ALT level >1000 IU/L were defined as responders and the others as non-responders in this study. Even though, the hepatic PGE2 levels increased in responders, the hepatic expression and enzyme activity related to PGE2 production were not upregulated when compared with vehicle-treated control mice. However, the hepatic 15-hydroxyprostaglandin dehydrogenase (15-PGDH) expression and activity decreased significantly in responders when compared with control mice. Conclusions. These results indicate that elevated hepatic PGE2 levels can be attributed to the downregulation of 15-PGDH expression under CBZ-induced liver injury.

The effect of carbamazepine, which increases oestrogen destruction, on the endometriotic implants; an experimental rat model.

We planned this experimental study to investigate the effect of carbamazepine (CMZ) on the endometriotic implants. Rats were randomised into four groups after endometriosis surgery. Drinking water was given to the sham group, 0.2 mg/kg oestradiol valerate (EV) to the EV group, 100 mg/kg/day CMZ to the CMZ group, and 0.2 mg/kg EV and 100 mg/kg/day CMZ to the EV-CMZ group. The endometrium of the rats using CMZ stained more intensely with cytochrome P450-3A4 (CYP3A4) enzyme. No endometrial hyperplasia was found in these rats. Endometriotic implants weight was found to be higher in these rats. There was no difference between the groups in terms of staining of the endometriotic implants with CYP3A4 enzyme. Endometriotic implants were less stained with the CYP3A4 enzyme than the endometrium. According to our results, CMZ does not increase the destruction of oestrogen in the endometriotic implants, unlike the endometrium. It may even cause the lesion to enlarge.Impact statementWhat is already known on this subject? Endometriosis is an oestrogen-dependent, progressive disease. Carbamazepine (CMZ) is known to increase oestrogen degradation by activating the cytochrome P450-3A4 (CYP3A4) enzyme. CMZ can be used in the treatment of endometriosis because it increases oestrogen breakdown in tissues.What do the results of this study add? CMZ can protect the endometrium against hyperplasia by increasing the amount of CYP3A4 enzyme in the endometrium. This effect could not be demonstrated in the endometriotic implants. The presence of CYP3A4 enzyme less in the endometriotic implants than in the endometrium may explain this situation. In addition, the fact that CMZ does not increase the enzyme in the endometriotic implants may contribute to this situation.What are the implications of these findings for clinical practice and/or further research? CMZ may not be a suitable alternative in the treatment of endometriosis. However, it may protect against endometrial hyperplasia. Clinical studies are needed for this effect.

Uptake of atenolol, carbamazepine and triclosan by crops irrigated with reclaimed water in a Mediterranean scenario.

Water scarcity is a natural condition in the Mediterranean rim countries. In this region, reuse of reclaimed water (RW) from wastewater treatment plants (WWTPs) is becoming a potential source for highly water-demanding activities such as agriculture. However, the removal capacity of contaminants in regular WWTPs has been found to be limited. Considering a Mediterranean scenario, this research investigated the plant uptake and translocation of three representative pharmaceuticals and personal care products (PPCPs) typically present in RW samples from a WWTP located in an urban area in Spain, and assessed the potential risk to humans from plant consumption. The RW samples were collected and analyzed for three representative PPCPs (atenolol -ATN-, carbamazepine -CBZ- and triclosan -TCS-). The target contaminants were also spiked at two levels in the RW samples to consider two worst-case scenarios. Three plant models (lettuce, maize and radish) were grown outdoors and irrigated with four treatments: tap water; RW samples, and the two spiked RW samples. Generally speaking, results revealed an efficient root uptake for the three PPCPs regardless of plant species and fortification level, and suggested an interaction effect of treatment and plant organ. Different bioaccumulation and translocation potentials of the three PPCPs were seen into the aerial organs of the plants. Overall, these observations support the idea that factors including the physico-chemical properties of the PPCPs and physiological plant variables, could be responsible for the differential accumulation and translocation potentials observed. These variables could be critical for crops irrigated with RW in regions with extended dry seasons, high solar incidence and low annual rainfall such as those in the Mediterranean rim where plants are subjected to high transpiration rates. However, the results obtained from this experimental approach suggested a negligible risk to humans from consumption of edible plants irrigated with RW samples with presence of PPCPs, despite the fact that the three representative PPCPs under study accumulated efficiently in the plants.

Biological uptake, depuration and biochemical effects of diclofenac and carbamazepine in Carassius carassius.

The uptake and depuration kinetics of diclofenac and carbamazepine alone at an environmentally relevant nominal concentration of 2 µg/L and in combination at a concentration ratio of 1:1 with total concentration of 4 µg/L were evaluated in Carassius carassius after 7 d uptake and depuration. Also, the biochemical effects of both drugs alone at nominal concentrations of 2 and 10 µg/L as well as in combination with total concentrations of 4 and 20 µg/L were investigated in Carassius carassius after 7 d exposure followed by 10 d recovery. In the single treatments, steady-state BCFs measured after the 7 d exposure were 73.05, 49.71, 38.01 and 24.93 L/kg for diclofenac and 9.25, 8.99, 5.29 and 4.11 L/kg for carbamazepine in the liver, brain, gill and muscle of Carassius carassius, respectively. Comparatively lower BCFs were measured in the tissues of Carassius carassius for both drugs in the combined treatments. Acetylcholinesterase activity in the brain was significantly induced by diclofenac while carbamazepine and the mixtures significantly inhibited it during all the exposure days as well as after the 10 d recovery in all treatments. This indicates that Carassius carassius could not recover from the neurotoxic effects caused by carbamazepine unlike the inductive effect caused by diclofenac which was recoverable after 10 days. A significant increase in the activities of 7-ethoxyresorufin O-deethylase and glutathione s-transferase for individual and mixed pharmaceuticals suggest that metabolism and detoxification of both drugs took place in the liver of Carassius carassius. Also, a significant increase in the activities of superoxide dismutase, catalase, glutathione reductase and malondialdehyde contents in the individual and mixture treatments mean that the antioxidant defence system of Carassius carassius was triggered to fight against oxidative stress but lipid peroxidation still occurred. However, Carassius carassius recovered from all these increases (superoxide dismutase, catalase, glutathione reductase and malondialdehyde) after the 10 d recovery, suggesting that oxidative damage is reversible. Our results indicate that both drugs at environmentally relevant concentrations might cause adverse effects in Carassius carassius and other fish species.

Methyl Paraben and Carbamazepine in Water and Striped Catfish (Pseudoplatystoma magdaleniatum) in the Cauca and Magdalena Rivers.

Between 2017 and 2019, samplings were carried out in the San Jorge, Cauca and Magdalena River basins in Colombia, to determine the presence of methyl paraben and carbamazepine in water and Pseudoplatystoma magdaleniatum. For the analysis of the samples, a validation of the analytical method was performed, following the EPA method 1694 (Pharmaceutical and personal care products in water), with slight modifications. This was done by liquid-chromatography tandem mass spectrometry, for quantification of methyl paraben and carbamazepine, including parameters of linearity, accuracy precision and veracity. Carbamazepine was found in the Magdalena River at 8.03 ± 0.01 µg/L in transition season. In fish samples, methyl paraben and carbamazepine were detected in a range between 32 and 90.80 µg/kg in transition and dry seasons.

In vitro biotransformation of pharmaceuticals and pesticides by trout liver S9 in the presence and absence of carbamazepine.

The aim of the present study was to develop (i) a technique for identifying metabolites of organic contaminants by using an in vitro system of trout S9 and liquid chromatography-high-resolution mass spectrometry-based identification method and (ii) to apply this technique to identify the interactive potential of carbamazepine on the formation rate of other metabolites. The pharmaceuticals carbamazepine and propranolol and the pesticides azoxystrobin, diazinon, and fipronil were selected as test contaminants. As a result, a total of ten metabolites were identified for the five parent substances, six of which were confirmed using reference standards. Metabolic reactions included hydroxylation, epoxidation, S-oxidation, and dealkylation. The metabolic transformation rate ranged from 0.2 to 3.5 pmol/mg protein/min/µmol substrate. In the binary exposure experiment with increasing carbamazepine concentration, the formation rates of diazinon and fipronil metabolites (MDI2 and MFP2, respectively) increased, while formation of metabolites of propranolol and azoxystrobin (MPR1, MPR2, MPR3, and MAZ1) slowed down. Meanwhile, S9 pre-exposed to carbamazepine produced diazoxon, a toxic metabolite of diazinon, and pyrimidinol, a less toxic metabolite, more rapidly. These results suggest that carbamazepine, a perennial environmental pollutant, might modulate the toxicity of other substances such as diazinon but further in vivo studies are needed.

Bioconcentration of the antiepileptic drug carbamazepine and its physiological and biochemical effects on Daphnia magna.

Owing to its persistence, carbamazepine an antiepileptic drug is regularly detected in the aquatic environment. The motive for our research was to assess the bioconcentration, physiological and biochemical effects of carbamazepine in Daphnia magna. A 48 h aqueous exposure of carbamazepine yielded bioconcentration factors of 202.56 and 19.95 in Daphnia magna for the respective nominal treatments of 5 and 100 µg/L. Apparently, the inhibition of the capability of Daphnia magna to obtain food attributable to carbamazepine exposure will reduce their fitness to reproduce as well as to grow. Also, a significant alteration in the phototactic behaviour of Daphnia magna exposed to carbamazepine is maladaptive since it will increase their chance of being preyed upon in the surface water during daylight. Again, a significant decline in the acetylcholinesterase activity observed herein brings to light the neurotoxicity of carbamazepine to Daphnia magna. Moreover, significant inhibition of the superoxide dismutase, catalase and glutathione reductase activities coupled with the simultaneous induction of the malondialdehyde content imply that carbamazepine evoked a life-threatening oxidative stress that overpowered the antioxidant defence system of Daphnia magna. These observations confirm that carbamazepine can accumulate and consequently cause negative physiological and biochemical changes to wild Daphnia magna populations.

Utilization of naproxen by Amycolatopsis sp. Poz 14 and detection of the enzymes involved in the degradation metabolic pathway.

The pollution of aquatic environments by drugs is a problem for which scarce research has been conducted in regards of their removal. Amycolatopsis sp. Poz 14 presents the ability to biotransformation naphthalene at high efficiency, therefore, in this work this bacterium was proposed as an assimilator of naproxen and carbamazepine. Growth curves at different concentrations of naproxen and carbamazepine showed that Amycolatopsis sp. Poz 14 is able to utilize these drugs at a concentration of 50 mg L-1 as a source of carbon and energy. At higher concentrations, the bacterial growth was inhibited. The transformation kinetics of naproxen showed the total elimination of the compound in 18 days, but carbamazepine was only eliminated in 19.9%. The supplementation with cometabolites such as yeast extract and naphthalene (structure similar to naproxen) at 50 mg L-1, showed that the yeast extract shortened the naproxen elimination to 6 days and reached a higher global consumption rate compared to the naphthalene cometabolite. The biotransformation of carbamazepine was not improved by the addition of cometabolites. The partial sequencing of the genome of Amycolatopsis sp. Poz 14 detected genes encoding putative enzymes for the degradation of cyclic aromatic compounds and the activities of aromatic monooxygenase, catechol 1,2-dioxygenase and gentisate 1,2-dioxygenase exhibited their involving in the naproxen biodegradation. The HPLC-MS analysis detected the 5-methoxysalicylic acid at the end of the biotransformation kinetics. This work demonstrates that Amycolatopsis sp. Poz 14 utilizes naproxen and transforms it to 5-methoxysalicylic acid which is the initial compound for the catechol and gentisic acid metabolic pathway.

Determination of the ß-glycosylate fraction of contaminants of emerging concern in lettuce (Lactuca sativa L.) grown under controlled conditions.

The uptake of a large variety of contaminants of emerging concern (CECs) by crops has already been reported, and the occurrence of phase II metabolites or conjugates has only been detected in plant cell cultures. However, the extent of their formation under cropping conditions is largely unknown. In this study, an analytical strategy to assess the conjugation of 11 CECs in lettuce (Lactuca sativa L.) irrigated with different concentrations (0, 0.05, 0.5, 5, and 50 µg L-1) of CECs was developed. The methodology involved enzymatic digestion with ß-glucosidase to obtain the total fraction (free form + conjugates) of CECs. The conjugation fraction was then obtained based on the difference. The highest extent of conjugation (i.e., 27 to 83%) was found with the most hydrophobic compounds, such as bisphenol A, carbamazepine, methyl paraben, and triclosan. So, the CEC conjugate fraction cannot be neglected in the estimate of human daily intake.

Frequency of drug combinations between enzyme-inducing first-generation antiepileptic drugs and inducible drugs in patients with epilepsy.

OBJECTIVE: The objective of the study was to systematically assess, through the analysis of administrative data, the frequency of combinations of first-generation enzyme-inducing (EI) antiepileptic drugs (AEDs) with drugs frequently prescribed in patients with epilepsy whose metabolism is induced by EIAEDs. METHODS: From the population of Tuscany (a region in Italy of about 3,750,000 habitants), patients who had been treated with at least one first-generation EIAEDs (carbamazepine, phenytoin, phenobarbital, and primidone) and had received prescriptions of an inducible non-AED (NON-AED) included in a prespecified list of 103 inducible drugs were identified. RESULTS: At the index date, 9221 patients with epilepsy were treated with at least one traditional EIAED, and there were 2538 drug combinations between EIAEDs and NON-AEDs, which may result in potentially serious clinical consequences, and 3317 combinations with NON-AEDs that have their metabolism consistently increased. CONCLUSIONS: Patients with epilepsy treated with traditional EIAEDs are at a very high risk of drug interactions.

Uptake and metabolism of carbamazepine (CBZ) by clam Ruditapes decussatus and its effects in biochemical responses.

1. Laboratory experiments were carried out to assess uptake and metabolism of the epilepsy drug, carbamazepine and its consequent biological responses in marine clam (Ruditapes decussatus) a model non-target organism in ecotoxicology. 2. Clams were exposed to two nominal concentrations (C1 = 30 µg/L and C2 = 50 µg/L) of CBZ for a maximum period of 14 days. Analysis of CBZ and their metabolites in clam and water after exposure to two nominal concentrations of the pharmaceutical drug were performed using UPLC-HRMS analysis. CBZ accumulation reached an average tissue concentration of 1241.59 ng/g dw and 1664.33 ng/g dw at low and high nominal concentration, respectively. 3. Furthermore, a metabolite (3-hydroxy-CBZ) was detected in tissues indicating carbamazepine translocation and metabolism inside clam, suspect screening of CBZ glucuronides was also performed by accurate mass extraction but it could not be detected. 4. Activities of antioxidant enzymes superoxide dismutase, catalase and gluthatione-S-transferase generally increased. Change in the contents of glutathione, malondialdehyde and protein carbonyl were also studied. 5. Results indicated that the bioaccumulation of CBZ resulted in the changes of the antioxidant defense system and the production of ROS with the oxidative stress, ultimately induced alteration in lipid peroxidation and protein carbonyl.

[Progress in synaptic mechanism in epileptogenesis].

Recurrent seizures lead to abnormal expression of synaptic proteins, reorganization of synapse and formation of abnormal neuron network. Recently, it is identified that the synaptic proteins are involved in epileptogenesis. The abnormal expression of several synaptic regulatory proteins and postsynaptic membrane receptor proteins may result in epilepsy. The ion channels usually are the action target of most antiepileptic drugs. However, carbamazepine and zonisamide may interact with syntaxin and/or soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex to exert its function of anti-epilepsy. The synaptic vesicle protein 2A is the action target for new anti-epileptic drugs, including levetiracetam, brivaracetam and seletracetam.

Mass Spectrometric Characterization of Circulating Covalent Protein Adducts Derived from Epoxide Metabolites of Carbamazepine in Patients.

Carbamazepine (CBZ) is an effective antiepileptic drug that has been associated with hypersensitivity reactions. The pathogenesis of those reactions is incompletely understood but is postulated to involve a complex interplay between the drug's metabolism, genetic variation in human leukocyte antigens, and adverse activation of the immune system. Multiple T-cell activation mechanisms have been hypothesized including activation by drug-peptide conjugates derived from proteins haptenated by reactive metabolites. However, definitive evidence of the drug-protein adducts in patients has been lacking. In this study, mass spectrometry was used to characterize protein modifications by microsomally-generated metabolites of CBZ and in patients taking CBZ therapy. CBZ 10,11-epoxide (CBZE), a major electrophilic plasma metabolite of CBZ, formed adducts with glutathione-S-transferase pi (GSTP; Cys47) and human serum albumin (HSA; His146 and His338, but not Cys34) in vitro via notably divergent side-chain selectivity. Both proteins were adducted at the same residues by undefined monoxygenated metabolites ([O]CBZ) when they were incubated with human liver microsomes, NADPH and CBZ. There was also evidence for formation of a CBZ adduct at His146 and His338 of HSA derived via dehydration from an intermediate arene oxide adduct. Glutathione trapping of reactive metabolites confirmed microsomal production of CBZE and indicated simultaneous production of arene oxides. In 15 patients prescribed CBZ therapy, [O]CBZ-modified HSA (His146) was detected in all subjects. The relative amount of adduct was moderately positively correlated with plasma concentrations of CBZ (r2 = 0.44, p = 0.002) and CBZE (r2 = 0.35, p = 0.006). Our results have provided the first chemical evidence for microsomal production of [O]CBZ species that are able to escape the microsomal domain to react covalently with soluble proteins. This study has also demonstrated the presence of circulating [O]CBZ-modified HSA in patients without hypersensitivity reactions who were receiving standard CBZ therapy. The implications of those circulating adducts for susceptibility to CBZ hypersensitivity merit further immunological investigation in hypersensitive patients.

Extensive Transformation of the Pharmaceutical Carbamazepine Following Uptake into Intact Tomato Plants.

Carbamazepine (CBZ) is an antiepileptic drug which is persistent in wastewater treatment plants and the environment. It has been frequently detected in plant material after irrigation with treated wastewater. To date, little information is, however, available on the transformation of CBZ in plants. In the present study, the uptake, translocation, and transformation of CBZ was studied in hydroponically grown tomato plants. After 35 days of exposure >80% of the total spiked amount of CBZ was taken by the tomato plants and mainly stored in the leaves. A total of 11 transformation products (TP) (mainly phase-I) were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and their total amount corresponded to 33% of the CBZ taken up. The ratio of CBZ metabolites to CBZ was highest in fruits (up to 2.5) and leaves (0.5), suggesting an intensive transformation of CBZ in these compartments. Further 10 TPs (phase-I and II) were identified by LC-high resolution mass spectrometry screening, likely comprising another 12% of CBZ. On the basis of these experiments and on an experiment with CBZ-10,11-epoxide a transformation pathway of CBZ in intact tomato plants is proposed that involves epoxidation, hydrolysis, hydroxylation, ring contraction, or loss of the carbamoyl group, followed by conjugation to glucose or cysteine, but also reduction of CBZ. This transformation pathway and analytical data of CBZ transformation products allow for their determination also in field grown vegetable and for the generation of more accurate exposure data of consumers of vegetable irrigated with treated municipal wastewater.

In Silico Identification of Bioremediation Potential: Carbamazepine and Other Recalcitrant Personal Care Products.

Emerging contaminants are principally personal care products not readily removed by conventional wastewater treatment and, with an increasing reliance on water recycling, become disseminated in drinking water supplies. Carbamazepine, a widely used neuroactive pharmaceutical, increasingly escapes wastewater treatment and is found in potable water. In this study, a mechanism is proposed by which carbamazepine resists biodegradation, and a previously unknown microbial biodegradation was predicted computationally. The prediction identified biphenyl dioxygenase from Paraburkholderia xenovorans LB400 as the best candidate enzyme for metabolizing carbamazepine. The rate of degradation described here is 40 times greater than the best reported rates. The metabolites cis-10,11-dihydroxy-10,11-dihydrocarbamazepine and cis-2,3-dihydroxy-2,3-dihydrocarbamazepine were demonstrated with the native organism and a recombinant host. The metabolites are considered nonharmful and mitigate the generation of carcinogenic acridine products known to form when advanced oxidation methods are used in water treatment. Other recalcitrant personal care products were subjected to prediction by the Pathway Prediction System and tested experimentally with P. xenovorans LB400. It was shown to biodegrade structurally diverse compounds. Predictions indicated hydrolase or oxygenase enzymes catalyzed the initial reactions. This study highlights the potential for using the growing body of enzyme-structural and genomic information with computational methods to rapidly identify enzymes and microorganisms that biodegrade emerging contaminants.

Metabolite profiling of carbamazepine and ibuprofen in Solea senegalensis bile using high-resolution mass spectrometry.

The widespread occurrence of pharmaceuticals in the aquatic environment has raised concerns about potential adverse effects on exposed wildlife. Very little is currently known on exposure levels and clearance mechanisms of drugs in marine fish. Within this context, our research was focused on the identification of main metabolic reactions, generated metabolites, and caused effects after exposure of fish to carbamazepine (CBZ) and ibuprofen (IBU). To this end, juveniles of Solea senegalensis acclimated to two temperature regimes of 15 and 20 °C for 60 days received a single intraperitoneal dose of these drugs. A control group was administered the vehicle (sunflower oil). Bile samples were analyzed by ultra-high-performance liquid chromatography-high-resolution mass spectrometry on a Q Exactive (Orbitrap) system, allowing to propose plausible identities for 11 metabolites of CBZ and 13 metabolites of IBU in fish bile. In case of CBZ metabolites originated from aromatic and benzylic hydroxylation, epoxidation, and ensuing O-glucuronidation, O-methylation of a catechol-like metabolite was also postulated. Ibuprofen, in turn, formed multiple hydroxyl metabolites, O-glucuronides, and (hydroxyl)-acyl glucuronides, in addition to several taurine conjugates. Enzymatic responses after drug exposures revealed a water temperature-dependent induction of microsomal carboxylesterases. The metabolite profiling in fish bile provides an important tool for pharmaceutical exposure assessment. Graphical abstract Studies of metabolism of carbamazepine and ibuprofen in fish.

Influence of diosmin on the metabolism and disposition of carbamazepine in healthy subjects.

1. Carbamazepine (CBZ) is an antiepileptic drug with narrow therapeutic window and administration in humans receiving long-term therapy with diosmin (DSN) may occur, which leads to CYP3A4-mediated drug interactions. The purpose of the present study was to assess the influence of DSN on the metabolism and pharmacokinetics of CBZ in healthy volunteers. 2. An open-label, sequential, two-period study was conducted in 12 healthy male volunteers. A single dose of DSN 500 mg was administered once daily for 10 days during treatment phase. A single dose of CBZ 200 mg was administered during control and after treatment phases under fasting conditions. The blood samples were collected after CBZ dosing at predetermined time intervals and analyzed by LC-MS/MS. 3. Treatment with DSN significantly enhanced the maximum plasma concentration (Cmax), area under the curve (AUC), half-life (t1/2) and significantly decreased the apparent oral clearance (CL/F) and elimination rate constant (Kel) of CBZ. On the other hand, treatment with DSN significantly decreased the Cmax and AUC of carbamazepine 10, 11-epoxide (CBZE). Furthermore, treatment with DSN significantly decreased the metabolite to parent ratios of Cmax and AUC, indicating the reduced metabolism of CBZ to CBZE. 4. The results suggest that the altered CYP3A4 enzyme activity and pharmacokinetics of CBZ might be attributed to DSN-mediated inhibition of CYP3A4 enzyme, which indicates pharmacokinetic interaction present between DSN and CBZ. Therefore, we conclude that DSN has an inhibiting effect on the metabolism and disposition of CBZ.