Metabolite Of Carbamazepine Research Articles

The Mechanistic Differences in HLA-Associated Carbamazepine Hypersensitivity.

Drug hypersensitivity reactions that resemble acute immune reactions are linked to certain human leucocyte antigen (HLA) alleles. Severe and life-threatening Stevens Johnson Syndrome and Toxic Epidermal Necrolysis following treatment with the antiepileptic and psychotropic drug Carbamazepine are associated with HLA-B*15:02; whereas carriers of HLA-A*31:01 develop milder symptoms. It is not understood how these immunogenic differences emerge genotype-specific. For HLA-B*15:02 an altered peptide presentation has been described following exposure to the main metabolite of carbamazepine that is binding to certain amino acids in the F pocket of the HLA molecule. The difference in the molecular mechanism of these diseases has not been comprehensively analyzed, yet; and is addressed in this study. Soluble HLA-technology was utilized to examine peptide presentation of HLA-A*31:01 in presence and absence of carbamazepine and its main metabolite and to examine the mode of peptide loading. Proteome analysis of drug-treated and untreated cells was performed. Alterations in sA*31:01-presented peptides after treatment with carbamazepine revealed different half-life times of peptide-HLA- or peptide-drug-HLA complexes. Together with observed changes in the proteome elicited through carbamazepine or its metabolite these results illustrate the mechanistic differences in carbamazepine hypersensitivity for HLA-A*31:01 or B*15:02 patients and constitute the bridge between pharmacology and pharmacogenetics for personalized therapeutics.

Soil influences on uptake and transfer of pharmaceuticals from sewage sludge amended soils to spinach

Sewage sludge from wastewater treatment plants, which may contain various contaminants including pharmaceuticals, is often used as a soil amendment. These contaminants may subsequently be taken up by plants. In the present study we examined uptake of select pharmaceuticals from sewage sludge applied to soils by spinach plants. Seven soils were amended with sewage sludge from two wastewater treatment plants (A and B). Concentrations of compounds in plant tissues (roots and leaves) of spinach planted 45 days in these soils under greenhouse conditions were evaluated after harvest. The largest bioaccumulation in the roots and leaves was observed for sertraline (bioaccumulation factors (BAF) of 3.3–37.9 and 1–13.4, respectively), tramadol (1.3–10.0 and 4.8–30.0), and carbamazepine (2.2–17.2 and 6.1–48.8) and its metabolite carbamazepine 10,11-epoxide (not-quantified to 7.3 and 9.3–96.7). Elevated bioaccumulation in spinach roots was also identified for telmisartan (3.0–20.3) and miconazole (4.3–15.1), and leaves for metoprolol acid (not-quantified to 24.3). BAF values resulting from application of sludge B were similar to or moderately higher than BAFs from sludge A. The BAF values of carbamazepine and carbamazepine 10,11-epoxide in all tissues were negatively correlated with soil cation exchange capacity (CEC). This negative correlation between BAF and CEC was also observed for tramadol (A-roots and B-leaves), citalopram (B-roots), and telmisartan (B-roots) or between BAF and clay content for metoprolol acid (A-leaves and B-roots), tramadol (B-roots and A-leaves) and venlafaxine (B-roots). However, in the case of some other compounds (i.e. sertraline, amitriptyline, mirtazapine, metoprolol), uptake and the subsequent translocation and transformation from 3 soils of a higher pH and base cation saturation (Stagnic Chernozem Siltic, Haplic Chernozem and Greyic Phaeozem) significantly differed from 4 soils with a lower pH and base cation saturation (Haplic Luvisol, Haplic Cambisol, Dystric Cambisol and Arenosol Epieutric). Such observations proved strong compound dependent influences of soil conditions on various compounds bioaccumulations in plants and necessity of studying these processes always in diverse soils.

The 2-Hydroxyiminostilbene Metabolite of Carbamazepine or the Supernatant from Incubation of Hepatocytes with Carbamazepine Activates Inflammasomes: Implications for Carbamazepine-Induced Hypersensitivity Reactions.

Although the pathophysiology of carbamazepine-induced idiosyncratic or hypersensitivity reactions is unclear, they are presumed to be immune mediated, involving a complex interaction between drug metabolism and activation of the immune system. Cell stress can be caused by reactive metabolites, and this has the potential to release damage-associated molecular patterns (DAMPs), which are responsible for activation of the immune system. Idiosyncratic drug reactions occur mainly in the liver because of its role in drug metabolism and reactive metabolite formation. DAMPs can activate inflammasomes, which may be a common mechanism by which DAMPs lead to an immune response. In the present study, we investigated whether carbamazepine induces the release of DAMPs by using human hepatocarcinoma functional liver cell-4 (FLC-4) cells for bioactivation of carbamazepine. THP-1 cells, a human macrophage cell line, were used for detecting inflammasome activation. We found that increased caspase-1 activity and production of interleukin-1β by THP-1 cells were caused by the supernatant from the incubation of carbamazepine with FLC-4 cells. In the supernatant, heat shock protein 60 was significantly increased. In addition, 2-hydroxyiminostilbene, which is a metabolite of carbamazepine, activated inflammasomes. These results suggest that the reactive iminoquinone metabolite can directly activate inflammasomes or that stressed hepatocytes cause the release of DAMPs, which are responsible for inflammasome activation. The activation of inflammasomes may be an important step in the immune system activation by carbamazepine, which can lead to hypersensitivity reactions in some patients. SIGNIFICANCE STATEMENT: A metabolite of carbamazepine, 2-hydroxyiminostilbene itself, and the damage-associated molecular patterns released from hepatocytes incubated with carbamazepine activated inflammasomes. The activation of inflammasomes may be an important step in the immune system activation by carbamazepine, which can lead to hypersensitivity reactions in some patients.

Human pharmaceuticals in three major fish species from the Uruguay River (South America) with different feeding habits

The accumulation of 17 human pharmaceuticals (HPs) was investigated in the muscle of three fish species characteristic of the “Rio de la Plata Basin” with different feeding habits and of relevance for human consumption: Megaleporinus obtusidens, Salminus brasiliensis, and Prochilodus lineatus. Fish were sampled in fall and spring from 8 localities distributed along 500 Km of the Uruguay River. Atenolol and carbamazepine were the most frequently detected HPs (>50%), but at concentrations always below 1 μg/kg wet weight (w/w). Hydrochlorothiazide, metoprolol, venlafaxine, propranolol, codeine, and the carbamazepine metabolite, 2-hydroxycarbamazepine, were accumulated at higher levels showing maximum concentrations between 1 and 10 μg/kg (w/w), but infrequently (<50%). The other HPs were always below 1 μg/kg (w/w) and at frequencies lower than 50%. Distinctive accumulation patterns were observed among species at different trophic levels. However, biomagnification trends were not identified for any compound. The highest number and concentration of HPs were found in M. obtusidens (omnivorous), followed by P. lineatus (detritivorous), and lastly S. brasiliensis (piscivorous). The most recurrent HPs (i.e. carbamazepine and atenolol) were present in all species, but others exclusively in one. Geographical variations were only found for carbamazepine and atenolol in M. obtusidens and P. lineatus, showing higher concentrations in localities closer to the Rio de la Plata estuary. Differences in the HPs concentrations among seasons were not identified. Acceptable daily intake and predicted no effect concentrations would indicate that measured muscle concentrations in fish from the Uruguay River do not pose a serious risk for human consumption nowadays. Further studies will be necessary for assessing the potential adverse effects on studied fish species.

Metabolite of Carbamazepine Activates NLRP3 Inflammasome in Keratinocytes and Regulates CD8+ T-Cell Skin Trafficking in SJS/TEN

Background: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are acute cutaneous adverse drug reactions involving keratinocyte death and CD8+ T lymphocyte-mediated immune response. Drugs, especially anti-epileptic drugs such as carbamazepine (CBZ) are the most important etiologic agents in patients with SJS/TEN. Carbamazepine-10,11-epoxide (CBZE), a pharmacologically active metabolite of CBZ, oxidated by cytochrome P450 enzyme 3A4 (CYP3A4) in the liver, and is also distributed in the epidermis of skin. Why do patients of SJS/TEN develop a violent CD8+ T cells immune response to the epidermis of skin is unknown. Methods: Sera and blister fluid of six patients with SJS and four patients with TEN were obtained from Xijing Hospital. The levels of interleukin-1β (IL-1β), IL-18, Granzyme B, and granulysin were assessed by ELISA and quantitative real-time PCR. The mechanism results were confirmed with transient transfection of small interfering RNAs into cultured keratinocytes, western blot, migration assay, flow cytometry, and immunohistochemistry. Findings: Increased expression of NLRP3 inflammasome in lesions of patients with SJS/TEN. Increased levels of interleukin-1β (IL-1β) and IL-18 significantly correlated with body surface area (BSA), SCORe of Toxic Epidermal Necrosis (SCORTEN), and levels of Granzyme B in sera, blister fluid and lesions of patients with SJS/TEN. CBZE induces NLRP3 inflammasome activation in SJS/TEN keratinocytes. Activation of NLRP3 inflammasome in SJS/TEN keratinocytes upregulates the expression of CXCL9/CXCL10, activates CD8+ T lymphocytes, and increases the expression of cytotoxic markers via signaling of IL-1β/IL-1R or IL-18/IL-18R. This promotes chemotactic ability in CD8+ T lymphocytes of patients with SJS/TEN. Interpretation: To our knowledge, this study shows for the first time that CBZE induces activation of NLRP3 inflammasome and increases activation, migration and cytotoxic functions of CD8+ T lymphocytes in skin, thereby contributing to the dysregulated response in patients with SJS/TEN. Funding: This work was supported by the National Natural Science Foundation of China (81371738, 31400782, 81573038, 81472885). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: The investigational protocol was approved by the Ethics Committee of the Fourth Military Medical University, according to the guidelines of Declaration of Helsinki.

Effects of Binary Mixtures and Transpiration on Accumulation of Pharmaceuticals by Spinach.

Many pharmaceuticals are present in reclaimed wastewater and effluent-dominated water bodies used to irrigate edible crops. Previous research has shown that plants irrigated with reclaimed wastewater can accumulate pharmaceuticals. However, plant-driven processes that contribute to differences in accumulation among compounds are not well understood. Here, we tested the effects of exposure to mixtures on spinach accumulation and metabolism of four psychoactive pharmaceuticals found in reclaimed wastewater: carbamazepine, fluoxetine, amitriptyline, and lamotrigine. Coexposure of plants to carbamazepine and fluoxetine or amitriptyline decreased accumulation of the toxic carbamazepine metabolite 10,11-epoxycarbamazepine. Furthermore, we tested a simple transpiration-based accumulation model and found that transpiration is a strong predictor for accumulation of the studied compounds. Amitriptyline accumulated to a larger extent than predicted from transpiration alone, and we suggest the possibility that a transporter protein may be involved in its uptake. Our findings highlight the need to consider plant physiology and mixture effects in studying accumulation of polar and ionizable organic contaminants and their metabolites.

Biotransformation of organic micropollutants by anaerobic sludge enzymes

Biotransformation of organic micropollutants (OMPs) in wastewater treatment plants ultimately depends on the enzymatic activities developed in each biological process. However, few research efforts have been made to clarify and identify the role of enzymes on the removal of OMPs, which is an essential knowledge to determine the biotransformation potential of treatment technologies. Therefore, the purpose of the present study was to investigate the enzymatic transformation of 35 OMPs under anaerobic conditions, which have been even less studied than aerobic systems. Initially, 13 OMPs were identified to be significantly biotransformed (>20%) by anaerobic sludge obtained from a full-scale anaerobic digester, predestining them as potential targets of anaerobic enzymes. Native enzymes were extracted from this anaerobic sludge to perform transformation assays with the OMPs. In addition, the effect of detergents to recover membrane enzymes, as well as the effects of cofactors and inhibitors to promote and suppress specific enzymatic activities were evaluated. In total, it was possible to recover enzymatic activities towards 10 out of these 13 target OMPs (acetyl-sulfamethoxazole and its transformation product sulfamethoxazole, acetaminophen, atenolol, clarithromycin, citalopram, climbazole, erythromycin, and terbutryn, venlafaxine) as well as towards 8 non-target OMPs (diclofenac, iopamidol, acyclovir, acesulfame, and 4 different hydroxylated metabolites of carbamazepine). Some enzymatic activities likely involved in the anaerobic biotransformation of these OMPs were identified. Thereby, this study is a starting point to unravel the still enigmatic biotransformation of OMPs in wastewater treatment systems.

Simultaneous determination of carbamazepine-N-glucuronide and carbamazepine phase I metabolites in the wastewater by liquid chromatography-tandem mass spectrometry

Carbamazepine (CBZ) is one of the most frequently detected pharmaceuticals in the aquatic environment. In the human body, CBZ is metabolized and transformed into a variety of metabolites. Among metabolites, CBZ N-glucuronide (CBZ-Glu, Phase II metabolite) was discharged into sewage system together with Phase I metabolites such as 2-hydroxycarbamazepine (2-OH-CBZ), 3-hydroxycarbamazepine (3-OH-CBZ), 10-hydroxycarbamazepine (10-OH-CBZ), 10,11-Dihydroxycarbamazepine (DiOH-CBZ), and carbamazepine 10,11-epoxide (EP-CBZ). However, the occurrence of CBZ-Glu in wastewater was not reported previously due to the lack of CBZ-Glu standard. In this study, a quantitative analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the determination of CBZ-Glu as well as other CBZ metabolites in wastewater. In the developed method, sufficiently high extraction efficiencies of CBZ and its metabolites were achieved by solid phase extraction (SPE) process (recoveries ranged from 79.3% to 95.8%, and from 84.4% to 96.4% in wastewater influent and effluent, respectively). The concentration of CBZ-Glu in the wastewater influent and effluent were 59.4, and 48.1 ng/L, respectively. CBZ in the wastewater influent and effluent were 53.8, and 61.9 ng/L, respectively. Although CBZ and phase I metabolites were persistent during the A2O process, deconjugation of CBZ-Glu was suspected to contribute to the increase of CBZ.

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 abstractStudies of metabolism of carbamazepine and ibuprofen in fish

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.

Modeling of sorption and degradation of selected pharmaceuticals: Case study of Belgrade groundwater source

The application of a mathematical model that analyzes the transport of selected pharmaceuticals from the Sava River to a corresponding radial collector well at Belgrade?s groundwater source is assessed. The occurrence of the selected pharmaceuticals in surface water and the corresponding well was monitored from 2009 to 2015. The pharmaceuticals selected for the present study are carbamazepine, trimethoprim, and metamizole metabolites 4-AAA and 4-FAA. Transport is analyzed based on experimental data (sorption isotherms) and a field tracer experiment that includes injection of the selected pharmaceuticals. The analysis shows that sorption of carbamazepine is relatively low and that this pharmaceutical does not degrade under the studied conditions, so it is not possible to accurately determine the degradation half-life. Trimethoprim is detected in the Sava River with an average concentration 8.5 ng/L, but there is no positive detection in well Rb-16. The average concentration of 4-AAA in the surface water is 34 ng/L and of 4-FAA 13 ng/L. The average concentrations of 4-FAAand 4-AAA in the groundwater are in the range from 1 and 1.85 ng/L. The objective of the research is to use an existing hydrogeologic model and apply a transport model to determine the minimum degradation half-life of the investigated pharmaceuticals.

The distribution dynamics and desorption behaviour of mobile pharmaceuticals and caffeine to combined sewer sediments

Pharmaceuticals are discharged to the environment from wastewater resource recovery facilities, sewer overflows, and illicit sewer connections. To understand the fate of pharmaceuticals, there is a need to better understand their sorption dynamics to suspended sediments (SS) and settled sediments (StS) in sewer systems. In this study, such sorption dynamics to both SS and StS were assessed using a batch equilibrium method under both static and dynamic conditions. Experiments were performed with natively occurring and artificially modified concentrations of sewer pharmaceuticals (acetaminophen, theophylline, carbamazepine, and a metabolite of carbamazepine) and caffeine. Differences in apparent distribution coefficients, Kd,app, between SS and StS were related to differences in their organic carbon (OC) content, and the practice of artificially modifying the concentration. Kd,app values of modified contaminant concentrations and high OC sediments were substantially higher. Pseudo-second order desorption rates for these mobile compounds were also quantified. Successive flushing events to simulate the addition of stormwater to sewer networks revealed that aqueous concentrations would not necessarily decrease, because the added water will rapidly return to equilibrium concentrations with the sediments. Sorption and desorption kinetics must be considered in addition to dilution, to avoid underestimating the influence of dilution on concentrations of pharmaceuticals discharged to the environment.

Pharmaceuticals, Their Metabolites, and Other Polar Pollutants in Field-Grown Vegetables Irrigated with Treated Municipal Wastewater.

The reuse of treated municipal wastewater for crop irrigation is a necessity in arid and semiarid regions but a potential entrance for emerging contaminants into the food chain. However, little attention has yet been paid to the detection of micropollutants and possible metabolites in vegetables grown under realistic field conditions. In this study, the uptake of 28 micropollutants and carbamazepine metabolites in 10 different field-grown vegetable species (among them carrot, lettuce, potato, and zucchini) from Jordan was studied. A total of 12 micropollutants and six carbamazepine metabolites, four of which have never been analyzed before in plant-uptake studies, could be detected in all of the samples in concentrations ranging from 1.7 to 216 ng per g of dry weight. In edible tissues, the total concentration of micropollutants decreased in the order of leafy (247-533) > root (73-126) > fruit-bearing (5-76 ng per g of dry weight) vegetables. A preliminary health-risk assessment for nine compounds according to the TTC concept shows no risk for seven of the micropollutats; for ciprofloxacin and 10,11-epoxycarbamazepine, however, more-specific toxicity data would be required for a refined risk assessment.

Development, validation and clinical application of an online-SPE-LC-HRMS/MS for simultaneous quantification of phenobarbital, phenytoin, carbamazepine, and its active metabolite carbamazepine 10,11-epoxide

A simple and efficient bioanalytical method for simultaneous determination of phenobarbital (PB), phenytoin (PHT), carbamazepine (CBZ), and its active metabolite carbamazepine 10,11-epoxide (CBZE) in human plasma using online solid phase extraction (SPE)-liquid chromatography (LC) coupled with high resolution mass spectrum (HRMS) under targeted MS/MS (t-MS2) analysis mode has been developed. The procedure integrated an automated sample clean-up of human plasma by Oasis®HLB SPE cartridge, a separation by ZORBAX SB-C18 analysis column, and a quantification by Q-Exactive Hybrid Quadrupole-Orbitrap. The total running time was 13min. The lower limit of quantification (LLOQ) of PB, PHT, CBZ, and CBZE were 0.008, 0.008, 0.0016 and 0.0016μgmL−1 respectively and the linearities were in the range of 0.008–2.500, 0.008–2.500, 0.0016–0.500 and 0.0016–0.500μgmL−1 respectively. The mean recovery was between 91.82% and 108.27% and the matrix effect was between 93.29% and 102.09%. The relative standard deviations of interday and intraday were less than 6.41%. The method has been successfully applied in therapeutic drug monitoring (TDM) of four Chinese epilepsy patients. This fully automated, simple, sensitive and reliable online-SPE-LC-HRMS/MS method serves well for TDM of PB, PHT, CBZ and CBZE at clinics for either single or combination treatment.

Human Exposure to Wastewater-Derived Pharmaceuticals in Fresh Produce: A Randomized Controlled Trial Focusing on Carbamazepine.

Fresh water scarcity has led to increased use of reclaimed wastewater as an alternative and reliable source for crop irrigation. Beyond microbiological safety, concerns have been raised regarding contamination of reclaimed wastewater by xenobiotics including pharmaceuticals. This study focuses on carbamazepine, an anticonvulsant drug which is ubiquitously detected in reclaimed wastewater, highly persistent in soil, and taken up by crops. In a randomized controlled trial we demonstrate that healthy individuals consuming reclaimed wastewater-irrigated produce excreted carbamazepine and its metabolites in their urine, while subjects consuming fresh water-irrigated produce excreted undetectable or significantly lower levels of carbamazepine. We also report that the carbamazepine metabolite pattern at this low exposure level differed from that observed at therapeutic doses. This "proof of concept" study demonstrates that human exposure to xenobiotics occurs through ingestion of reclaimed wastewater-irrigated produce, providing real world data which could guide risk assessments and policy designed to ensure the safe use of wastewater for crop irrigation.

Metabolism of pharmaceutical and personal care products by carrot cell cultures

With the increasing use of treated wastewater and biosolids in agriculture, residues of pharmaceutical and personal care products (PPCPs) in these reused resources may contaminate food produce via plant uptake, constituting a route for human exposure. Although various PPCPs have been reported to be taken up by plants in laboratories or under field conditions, at present little information is available on their metabolism in plants. In this study, we applied carrot cell cultures to investigate the plant metabolism of PPCPs. Five phase I metabolites of carbamazepine were identified and the potential metabolism pathways of carbamazepine were proposed. We also used the carrot cell cultures as a rapid screening tool to initially assess the metabolism potentials of 18 PPCPs. Eleven PPCPs, including acetaminophen, caffeine, meprobamate, primidone, atenolol, trimethoprim, DEET, carbamazepine, dilantin, diazepam, and triclocarban, were found to be recalcitrant to metabolism. The other 7 PPCPs, including triclosan, naproxen, diclofenac, ibuprofen, gemfibrozil, sulfamethoxazole, and atorvastatin, displayed rapid metabolism, with 0.4–47.3% remaining in the culture at the end of the experiment. Further investigation using glycosidase hydrolysis showed that 1.3–20.6% of initially spiked naproxen, diclofenac, ibuprofen, and gemfibrozil were transformed into glycoside conjugates. Results from this study showed that plant cell cultures may be a useful tool for initially exploring the potential metabolites of PPCPs in plants as well as for rapidly screening the metabolism potentials of a variety of PPCPs or other emerging contaminants, and therefore may be used for prioritizing compounds for further comprehensive evaluations.

Biocatalytic degradation of carbamazepine with immobilized laccase-mediator membrane hybrid reactor

Carbamazepine (CBZ) is one of the most recalcitrant pharmaceutically active compounds routinely detected in wastewater effluent-impacted environment. Biocatalytic degradation with enzymes such as laccase provides a promising approach for the elimination of CBZ. However, the relatively low redox potential of laccase makes its efficient CBZ degradation difficult. Therefore, an environmentally benign and effective mediator is required. In this study, three natural phenolic compounds, namely p-coumaric acid (PCA), syringaldehyde (SYR), and acetosyringone (ACE), were investigated as redox mediators for the enzymatic removal of CBZ by both free and immobilized laccase. Among the tested mediators, PCA resulted in the optimal CBZ removal performance with 60% removal rate (20μM initial CBZ) after 96h incubation with immobilized laccase in a conventional suspension system. The degradation of CBZ was then carried out in a membrane hybrid reactor with the effluent recirculating through suspended biocatalytic TiO2 nanoparticles. The effect of operation parameters including PCA concentrations, initial enzyme activity and operational flux on CBZ removal were investigated. Further CBZ metabolites study identified 10,11-dihydro-10,11-dihydroxy-CBZ (CBZD), 10,11-dihydro-10,11-epoxy-CBZ (CBZE) and acridone as the major metabolites of CBZ oxidation by laccase. The toxicity tests determined by algal viability using the fluorometric indicator alamarBlue indicated that the CBZ treatment via the hybrid reactor could effectively remove the toxicity of parent CBZ compound.

SCN1A and SCN1B gene polymorphisms and their association with plasma concentrations of carbamazepine and carbamazepine 10, 11 epoxide in Iranian epileptic patients.

From a genetic point of view, epilepsy is a polygenic multifactorial syndrome. The SCN1A and B genes belong to a family of genes that provide instructions for making sodium channels. Understanding the relevance of SCN1A and SCN1B gene polymorphisms to plasma concentration of carbamazepine (CBZ) and 'its active metabolite carbamazepine 10, 11 epoxide (CBZE), may shed more light on inter-individual variations in response to CBZ. In this cross-sectional study, genotype distribution and allele frequency of six non-synonymous exonic single nucleotide polymorphisms (SNPs) of the SCN1A and B genes were selected and determined using PCR-RFLP in 70 epileptic patients treated with CBZ for at least 6 months. The patients had no hepatic or renal diseases and received no medications known to have a major interaction with CBZ. Serum concentrations of CBZ and CBZE were measured using High-Performance Liquid Chromatography (HPLC). The AA, AG and GG alleles of SCN1A were found in 23, 37 and 10 patients, respectively. There were no statistically significant differences in the mean (± standard deviation) of plasma concentrations of CBZ (P=0.8) and CBZE (P=0.1) among these 3 groups. Likewise, there was no statistically significant relationship between SCN1A polymorphisms and CBZ concentration/dose ratio (P=0.7). A significant association was found between CBZ plasma level and CBZ concentration/dose with CBZ daily dose. All patients had the same genotype of SCN1B gene(CC). and no statistical analysis was performed. No significant association between SCN1A gene polymorphisms and plasma levels of CBZ and CBZE were found[u1].

Transformation Pathways of the Recalcitrant Pharmaceutical Compound Carbamazepine by the White-Rot Fungus Pleurotus ostreatus: Effects of Growth Conditions.

The widely used anticonvulsant pharmaceutical carbamazepine is recalcitrant in many environmental niches and thus poses a challenge in wastewater treatment. We followed the decomposition of carbamazepine by the white-rot fungus Pleurotus ostreatus in liquid culture compared to solid-state fermentation on lignocellulosic substrate where different enzymatic systems are active. Carbamazepine metabolites were identified using liquid chromatography-high-resolution mass spectrometry (LC-Q-TOF-MS). In liquid culture, carbamazepine was only transformed to 10,11-epoxy carbamazepine and 10,11-dihydroxy carbamazepine as a dead-end product. During solid-state fermentation, carbamazepine metabolism resulted in the generation of an additional 22 transformation products, some of which are toxic. Under solid-state-fermentation conditions, 10,11-epoxy carbamazepine was further metabolized via acridine and 10,11-dihydroxy carbamazepine pathways. The latter was further metabolized via five subpathways. When (14)C-carbonyl-labeled carbamazepine was used as the substrate, (14)C-CO2 release amounted to 17.4% of the initial radioactivity after 63 days of incubation. The proposed pathways were validated using metabolites (10,11-epoxy carbamazepine, 10,11-dihydroxy carbamazepine, and acridine) as primary substrates and following their fate at different time points. This work highlights the effect of growth conditions on the transformation pathways of xenobiotics. A better understanding of the fate of pollutants during bioremediation treatments is important for establishment of such technologies.