Biotransformation Products Research Articles

Human phase I in vitro liver metabolism of two bisphenolic diglycidyl ethers BADGE and BFDGE

Root canal sealers are commonly used to endodontically treat teeth with periapical infections. Some root canal sealers based on epoxy resin contain bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE). The presence of these chemicals is of concern due to the close contact to the blood stream at the apex and the long setting times of up to 24 h. These chemicals, or any of their degradation products or metabolites, can then exert their toxic effects before being excreted. This study aimed to identify the phase I in vitro biotransformation products of BADGE and BFDGE using human liver microsomes. During incubation with microsomal fractions, the epoxides were rapidly hydrolysed in a NADPH independent manner resulting in the formation of BADGE.2H2O and BFDGE.2H2O. Further, oxidative reactions, such as hydroxylation and carboxylation, generated other BADGE metabolites, such as BADGE.2H2O−OH and BADGE.H2O.COOH, respectively. For BFDGE, further oxidation of BFDGE.2H2O led to the newly reported carboxylic acid, BFDGE.H2O.COOH. In total, three specific metabolites have been identified which can serve in future human biomonitoring studies of BADGE and BFDGE.

Identification of Transformation Products for Benzotriazole, Triclosan, and Trimethoprim by Aerobic and Anoxic-Activated Sludge

This study identified biotransformation products formed from three anthropogenic trace organic compounds (TOrCs) (benzotriazole, triclosan, and trimethoprim) in aerobic and anoxic sludge from a biological nutrient removal (BNR) wastewater treatment system. Liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF) was used to identify intermediate transformation products. A user-defined compound library was used to target unknowns in samples with a mix of TOrCs without requiring chemical standards and developed using EAWAG-BDD predictive software, which included 37 potential transformation products. Biotransformation batch experiments were conducted using activated sludge from anoxic and aerobic redox regimes of a BNR treatment plant located in Southern Nevada, United States. Four intermediates were observed for benzotriazole in both aerobic and anoxic activated sludge, which consisted of two isomers of hydroxy benzotriazole and two isomers of methoxy benzotriazole. Four intermediates were observed for trimethoprim under aerobic conditions, which formed 2,4-diaminopyrimidin-5-yl)(3,4,5-trimethoxyphenyl)methanol (TMP 306) and 2,6-diamino-5-hydroxy-5-(3,4,5-trimethoxybenzyl)-5,6-dihydropyrimidin-4(1H)-one (TMP 324) and two demethylation isomers of desmethyl trimethoprim. Triclosan had one confirmed transformation product, triclosan-o-sulfate, formed in both conditions. The identification of these transformation products will allow for more thorough risk assessments to be performed for the target TOrCs.

Longitudinal assessment of mycotoxin co-exposures in exclusively breastfed infants

Early-life development of infants may be critically affected by man-made or natural contaminants including mycotoxins. However, data on the occurrence of food contaminants in breast milk is scarce and prohibits a comprehensive exposure and risk assessment for mothers and their infants.Here, we present a longitudinal exposure assessment over the first 211 days of a single newborn girl (studyA) by measuring multiple mycotoxins in milk. Eighty-seven consecutive breast milk samples were obtained from the newborn’s mother living in Austria and following a regular mixed diet. Mycotoxins were analyzed by utilizing a highly sensitive LC-MS/MS approach covering 29mycotoxins and key metabolites. In addition to this longitudinal study, three mothers provided breast milk samples each on five consecutive days, for a preliminary comparison of inter-day and inter-individual variation in exposures (studyB). StudyA revealed that mycotoxin occurrence in breast milk was limited to the emerging mycotoxins alternariol monomethyl ether (AME), beauvericin (BEA), enniatins (A, A1, B, B1) and to ochratoxin A (OTA), which is regulated in commercial infant food. These mycotoxins were, if present, mostly detected at very low concentrations (<10 ng/L), except AME which exceeded this concentration on two distinct days by a factor of 3x and 5x. Overall, longitudinal results indicated chronic low-dose exposure to the detected mycotoxins. Other regulated mycotoxins including the carcinogenic aflatoxins or the estrogenic zearalenone and their biotransformation products were absent in all tested samples. StudyB confirmed the results of studyA, with minimal inter-day and inter-individual variation. In addition, a preliminary correlation of OTA levels occurring in breast milk and matched urine samples was found (r = 0.64, p = 0.034) in study B. Based on the data set obtained in studyA, exposure of the infant was estimated. Exposure estimates of individual mycotoxins were on average below 1 ng/kg body weight per day.Our preliminary findings suggest that recommended maximum daily intake levels might not be exceeded in the Austrian population. However, exposure is likely to be higher in populations with lower food safety standards. In the light of co-occurrence of several emerging mycotoxins in breast milk, future studies should address low-dose mixture effects. This also includes other environmental contaminants which may be present in this bio-fluid and should involve an exposome-scale risk assessment. All these efforts must be intended to minimize exposure of mothers and infants in a window of high susceptibility.

Ergot alkaloid mycotoxins: physiological effects, metabolism and distribution of the residual toxin in mice

The complex ergot alkaloids, ergovaline and ergotamine, cause dysregulation of physiological functions, characterised by vasoconstriction as well as thermoregulatory and cardiovascular effects in grazing livestock. To assess the effect of the mycotoxins, blood pressure and heart rate of male mice were measured, and metabolite profiling undertaken to determine relative abundances of both ergotamine and its metabolic products in body and brain tissue. Ergotamine showed similar cardiovascular effects to ergovaline, causing elevations in blood pressure and reduced heart rate. Bradycardia was preserved at low-levels of ergovaline despite no changes in blood pressure. Ergotamine was identified in kidney, liver and brainstem but not in other regions of the brain, which indicates region-specific effects of the toxin. The structural configuration of two biotransformation products of ergotamine were determined and identified in the liver and kidney, but not the brain. Thus, the dysregulation in respiratory, thermoregulatory, cardiac and vasomotor function, evoked by ergot alkaloids in animals observed in various studies, could be partially explained by dysfunction in the autonomic nervous system, located in the brainstem.

Biotransformation study of antidepressant sertraline and its removal during biological wastewater treatment

Sertraline is one of the most commonly prescribed antidepressants in the last few years. Therefore, it is not surprising that it is regularly detected in wastewaters, surface waters, sediments, biosolids and biota. Effluents from wastewater treatment plants are the main contributors to its presence in the environment. The presented study aims to elucidate the processes involved in its removal, concentrating mainly on sorption and biodegradation during wastewater treatment.We performed our laboratory scale experiments in two sets of experiments: 1) batch biodegradation and sorption experiments and 2) flow-through laboratory scale pilot wastewater treatment bioreactors. The batch experiments revealed that sorption to activated sludge was the leading removal process, eliminating up to 90% of sertraline present in the batches. Biodegradation was however the secondary removal process, influenced by the presence of alternative easily biodegradable carbon sources. We postulated chemical structures of ten detected biotransformation products. Among these, we propose the previously recognized metabolite norsertraline, sertraline ketone and hydroxy-sertraline. All the remaining biotransformation products are herein reported for the first time.The removal efficiency of approximately 94% was determined after the treatment in the flow-through bioreactors. To support our findings, we sampled influents and effluents from two wastewater treatment plants and untreated wastewater from a psychiatric hospital. Removal efficiencies of 81% and 77% were determined, and along with the parent compound sertraline, the presence of eight transformation products was confirmed in the actual wastewaters.

Chemical and genomic analyses of polycyclic aromatic hydrocarbon biodegradation in Sphingobium barthaii KK22 reveals divergent pathways in soil sphingomonads

Polycyclic aromatic hydrocarbons (PAHs) are hazardous pollutants that are biodegraded by soil bacteria and the soil sphingomonads are thought to be major contributors to PAH biodegradation. To predict PAH environmental fates it is necessary to understand the chemistry and genetics of PAH biodegradation by sphingomonads. When the soil sphingomonad Sphingobium barthaii KK22 was exposed to low molecular weight (LMW) PAHs and biotransformation products were investigated by comprehensive chemical analyses, at least twenty products were identified by different techniques and both intradiol- and extradiol-aromatic ring cleavage pathways were found to be active in this organism. Sphingomonads, a large and diverse group, have been studied in regard to PAH biodegradation, however intradiol-ring cleavage of LMW PAHs has never been reported. Whole genome sequencing and prediction of S. barthaii KK22 functional genes revealed sets of aromatic ring-hydroxylating oxygenases and PAH biotransformation genes. Combined with chemical analyses results, novel, near complete PAH biotransformation pathways for soil sphingomonads were constructed. In conjunction with quantitative assays, a comprehensive view of PAH biodegradation was obtained that revealed divergent downstream pathways that advanced our understanding of the PAH biotransformation capabilities of these versatile soil bacteria and shall aid in predictions of PAH environmental fate during soil bioremediation.

Effects of dihydro-epi-deoxyarteannuin B on artemisinin biosynthesis, transcriptional profile and associated gene expression in suspension-cultured cells of Artemisia annua

Biotechnological methods producing artemisinin (ART) directly are limited because the final stages of the ART biosynthesis pathway remain elusive. Dihydro-epi-deoxyarteannuin B (DHEDB) is synthesized from dihydroartemisinic acid (DHAA) and used as a substrate at the present research. DHEDB was fed to cultured cells of Artemisia annua 10 days post subculture under light and dark conditions. The structures of the biotransformed products were elucidated by HR-ESI-MS and NMR analysis. Three hydroxylated products, 3α-hydroxy-dihydro-epi-deoxyarteannuin B (1), 9β-hydroxy-dihydro-epi-deoxyarteannuin B (2) and 14-hydroxy-dihydro-epi-deoxyarteannuin B (3), were obtained. Among them, compound 3 is a new compound. The autoxidation experiment of DHEDB indicated that three compounds were generated by enzymatic catalysis. The content of ART increased by 23.9 % compared to the control group under dark culture. Effects of DHEDB on the transcriptome of A. annua were determined under the optimal conditions. The genes HMGR, FPS, ADS, CYP71AV1, CPR, DBR2 and ALDH1 were involved in the process of biotransformation. qRT-PCR results showed that the increase in ART content was correlated with the increasing expression of the related genes. However, DHEDB and its biotransformed products are unlikely to be the intermediates of ART production because they showed no similarities in structure. Otherwise, the increase of ART content may due to the elicitor like effect of DHEDB as exogenous substance in A. annua cells.

Comparison of Transcriptomics Changes Induced by TCS and MTCS Exposure in Human Hepatoma HepG2 Cells.

Triclosan (TCS) has been a widely used antibacterial agent in medical and personal care products in the last few decades. Methyl TCS (MTCS) is the major biotransformation product of TCS through replacement of the hydroxyl group with methoxy. Previous studies revealed that MTCS showed reduced toxicity but enhanced environmental persistence, when compared with TCS. Till date, the toxicological molecular mechanisms of TCS and MTCS remain to be clarified. This study aimed to investigate the transcriptomic changes in HepG2 cells induced by TCS and MTCS using microarray chips and to identify key target genes and related signal pathways. The microarray data showed that there were 1664 and 7144 differentially expressed genes (DEGs) in TCS- and MTCS-treated groups, respectively. Gene ontology (GO) enrichment and Kyoto Encyclopedia of genes and genomes (KEGG) analysis revealed that TCS and MTCS induced overlapping as well as distinct transcriptome signatures in HepG2 cells. Both TCS and MTCS could result in various biological responses in HepG2 cells mainly responding to biosynthetic and metabolic processes but probably through different regulatory pathways. Among the selected 50 GO terms, 9 GO terms belonging to the cellular component category were only enriched in the MTCS group, which are mainly participating in the regulation of cellular organelle’s function. KEGG analysis showed that 19 and 59 pathway terms were separately enriched in TCS and MTCS groups, with only seven identical pathways. The selected 10 TCS-specific signal pathways are mainly involved in cell proliferation and apoptosis, while the selected 10 MTCS-specific pathways mainly take part in the regulation of protein synthesis and modification. The overall data suggested that MTCS induced more enriched DEGs, GO terms, and pathway terms than TCS. In conclusion, compared with TCS, MTCS presents lower polarity and stronger lipophilicity, enabling MTCS to cause more extensive transcriptomic changes in HepG2 cells, activate differentiated signal pathways, and finally lead to differences in biological responses.

Construction of the Heterologous Laccase Producer Aspergillus nidulans lac№4 (argB–) and Its Application for the Progesterone Transformation

A novel Aspergillus nidulans lac№4 (argB–) strain, a producer of recombinant laccase A from basidiomycete Trametes hirsuta 072, was obtained. This strain was used for the biocatalytic transformation of progesterone (PG). The major biotransformation products are 11α-hydroxy-PG, 11α-acetoxy-PG and 6β, 11α-dihydroxy-PG. Macronet MN-200 sorbent was used to study the adsorption kinetics of PG, and the main biotransformation products from the transformation medium. The solid-phase extraction of steroids without preliminary removal of the mycelium was shown to be efficient.

Emerging siRNA Design Principles and Consequences for Biotransformation and Disposition in Drug Development.

After two decades teetering at the intersection of laboratory tool and therapeutic reality, with two siRNA drugs now clinically approved, this modality has finally come into fruition. Consistent with other emerging modalities, initial proof-of-concept efforts concentrated on coupling pharmacologic efficacy with desirable safety profiles. Consequently, thorough investigations of siRNA absorption, distribution, metabolism, and excretion (ADME) properties are lacking. Advancing ADME knowledge will aid establishment of in vitro-in vivo correlations and pharmacokinetic-pharmacodynamic relationships to optimize candidate selection through discovery and translation. Here, we outline the emerging siRNA design principles and discuss the consequences for siRNA disposition and biotransformation. We propose a conceptual framework for siRNA ADME evaluation, contextualizing the site of biotransformation product formation with PK-PD modulation, and end with a discussion around safety and regulatory considerations and future directions for this modality.

Salicylate sensitizes oral squamous cell carcinoma to chemotherapy through targeting mTOR pathway.

Oral squamous cell carcinoma (OSCC) is an extremely aggressive neoplasm, which is usually diagnosed in the advanced stage of the disease. Extensive studies have shown a link between chronic inflammation and various types of cancer, including OSCC. Salicylate is a biotransformation product of aspirin, with similar anti-inflammatory ability to aspirin but lacks aspirin's inhibitory effect on the isolated cyclooxygenase activity. Our study indicates that salicylate sensitizes OSCC to anti-cancer drugs, but the mechanisms of its action are unclear. Here, OSCC cells were used to evaluate the cytotoxicity of salicylate alone or in combination with cisplatin (CDDP). RPPA proteomic array and Western blotting were employed to determine the signaling pathways affected by salicylate. Salicylate decreased cell survival rate and induced cell apoptosis in OSCC cells but not human normal oral mucosal epithelial cells (hTERT-OME). The use of sodium salicylate (SS) dramatically sensitized OSCC cells to CDDP. RPPA array showed that SS reduced many oncogenes such as PI3K/mTOR signaling and cancer stem cell (CSC)-related genes versus control. Western and transcriptional analyses substantiated that salicylate down-regulated these CSC-associated genes and the mTOR pathway dose dependently. Salicylate preferentially repressed the ability of sorted ALDH1+ cells to form tumor spheres. Finally, salicylate suppressed tumor growth in vivo, and the combination of salicylate and CDDP further synergistically reduced the growth of tumors. Salicylate hinders OSCC cell growth and sensitizes OSCC cells to CDDP through targeting CSCs and the mTOR signaling pathway. We propose that salicylate is beneficial for OSCC patients, and salicylate may be combined with chemotherapies to effectively treat OSCC patients.

Rapid biotransformation of the insensitive munitions compound, 3-nitro-1,2,4-triazol-5-one (NTO), by wastewater sludge.

As the use of the new insensitive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO) increases, wastewaters, runoff and groundwater containing NTO will be generated. Little is known about the fate of NTO during biological wastewater treatment. The objective of this study was to explore the ability of wastewater sludges to promote the biotransformation of NTO. Three different sludges, i.e., anaerobic granular sludge, anaerobic digested sludge, and return activated sludge, were used to study the biotransformation of NTO under anaerobic conditions. Three different electron donor amendments were compared- hydrogen, ethanol, and acetate. Mixed microbial communities in each of the three sludge sources were effective in the reductive biotransformation of NTO. 3-amino-1,2,4-triazol-5-one (ATO) was observed as the major product of NTO biotransformation. The highest maximum specific rate of NTO reduction, about 120mg NTO/g volatile suspended solids/d, was observed in anaerobic granular sludge with hydrogen or ethanol supplied as electron donors. NTO biotransformation to ATO by anaerobic digested sludge was also studied under denitrifying conditions. In this case, reduction of NTO started only after complete denitrification of added nitrate. An important implication of this paper is that sludge from wastewater treatment plants can rapidly and readily reduce NTO to ATO.

A case report of cholinesterase inhibitor poisoning: cholinesterase activities and analytical methods for diagnosis and clinical decision making

Suicidal ingestion of organophosphorus (OP) or carbamate (CM) compounds challenges health care systems worldwide, particularly in Southeast Asia. The diagnosis and treatment of OP or CM poisoning is traditionally based on the clinical appearance of the typical cholinergic toxidrome, e.g. miosis, salivation and bradycardia. Yet, clinical signs might be inconclusive or even misleading. A current case report highlights the importance of enzymatic assays to provide rapid information and support clinicians in diagnosis and rational clinical decision making. Furthermore, the differentiation between OP and CM poisoning seems important, as an oxime therapy will most probably not provide benefit in CM poisoning, but—as every pharmaceutical product—it might result in adverse effects. The early identification of the causing agent and the amount taken up in the body are helpful in planning of the therapeutic regimen including experimental strategies, e.g. the use of human blood products to facilitate scavenging of the toxic agent. Furthermore, the analysis of biotransformation products and antidote levels provides additional insights into the pathophysiology of OP or CM poisoning. In conclusion, cholinesterase activities and modern analytical methods help to provide a more effective treatment and a thorough understanding of individual cases of OP or CM poisoning.

Phytotoxicity and metabolic responses induced by tetrachlorobiphenyl and its hydroxylated and methoxylated derivatives in rice (Oryza sative L.)

Polychlorinated biphenyls (PCBs) and their biotransformation products, hydroxylated (OH-PCBs) and methoxylated derivatives (MeO-PCBs), have been detected in the environment and biota, especially crops. However, to date, little information is available on the phytotoxicity and metabolic responses induced by these chemicals in crops. In this study, we exposed rice (Oryza sative L.) seedlings to 2,3,4,5-tetrachlorobiphenyl (CB-61) and its hydroxylated (4′-OH-CB-61) and methoxylated derivatives (4′-MeO-CB-61) at 0, 10, 50, 100 and 500 μg/L, respectively. After exposure for 14 days, significantly growth inhibition and oxidative damage were observed, among which the toxicities of 4′-OH-CB-61 and 4′-MeO-CB-61 were greater than that of the parent PCBs. Metabolomics analysis indicated that exposure to the three chemicals induced different metabolic responses. 4′-MeO-CB-61 mainly affected the saccharide catabolism, including pyruvate metabolism, the TCA cycle, the transfer of acetyl groups into mitochondria and the Warburg effect, resulting in a greater energy consumption. Moreover, both CB-61 and 4′-OH-CB-61 promoted several amino acid metabolism and fatty acid biosynthesis, thereby alleviating the potential ROS damage. This study for the first time evaluates and reveals the phytotoxicity of OH-PCBs and MeO-PCBs at the metabolic level, which attempts to provide important information for accurately evaluating the environmental risks of PCBs from the perspective of metabolism.

Simultaneous Quantitative Analysis of Multiple Biotransformation Products of Xian-Ling-Gu-Bao, a Traditional Chinese Medicine Prescription, with Rat Intestinal Microflora by Ultra-Performance Liquid Chromatography Tandem Triple Quadrupole Mass Spectrometry.

Xian-Ling-Gu-Bao (XLGB), a famous traditional Chinese medicine prescription consisted of six herbal medicines, was used for prevention and treatment of osteoporosis in China. As an oral formulation, the multiple components contained in XLGB were inevitably biotransformed by the intestinal microflora before absorption via the gastrointestinal tract. However, the dynamic profiles of biotransformation products of XLGB remain unknown. In this paper, a rapid and sensitive ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry method was developed for the simultaneous quantitative analysis of multiple biotransformation products of XLGB with rat intestinal microflora. For 10 selected quantitative compounds, all calibration curves revealed good linearity (r2>0.99) within the sampling ranges considered. The whole intra- and inter-day precisions (as relative standard deviation) of all analytes were <13.5%, and the accuracies (as relative error) were in the range from -11.3 to 11.2%. The lower limits of quantification were 20, 10, 5, 20, 2, 2, 2, 5, 2 and 2ng/mL for sweroside, timosaponin BII, epimedin C, asperosaponin VI, psoralen, isobavachin, icariside II, timosaponin AIII, isobavachalcone and icaritin, respectively. The matrix effects, extraction recoveries and stabilities were all satisfactory. Meanwhile, dynamic profiles of 21 additional biotransformation products were also monitored by their area-time curves. The analytical method was successfully applied to describe dynamic profiles of 31 biotransformation products of XLGB and the recipes with removal of a definite composed herbal medicine (Anemarrhenae Rhizoma or Rehmanniae Radix).

Regioselective Biotransformation of Phloretin Using Streptomyces avermitilis MA4680

Streptomyces avermitilis MA4680 was used for the biotransformation of phloretin, one of the dihydrochalcone found in apple bark. After LC analysis, two main biotransformed products were identified and they were further analyzed using GC/MS. After BSTFA derivatization of biotransformed product, they were interpreted as regioselectively hydroxylated products of phloretin in Bring. Maximum conversion of phloretin was 6.7% with 1 h of reaction, and the phloretin and reaction products were completely metabolized after 3 h of reaction due to cellular metabolism. Addition of 0.5 mM quinidine completely blocked hydroxylation of phloretin, which means the hydroxylation proceed by P450 monoxygenase dependent metabolism. Addition of Brij 35 detergent resulted in 150% increase of hydroxylated product due to facilitating transport of phloretin and its reaction product across cellular membrane.

Enzymatic Biotransformation of Pomegranate Ellagitannins: Initial Approach to Reaction Conditions.

Background: Ellagitannase (Ellagitannin acyl hydrolase) is an inducible enzyme with great potential use in food industry since allows the ellagic acid release from ellagitannins.Objective: In this work, ellagitannase was produced by the fungus Aspergillus niger GH1 in solid state fermentation using polyurethane foam as solid support and pomegranate husk ellagitannins as sole carbon source and ellagitannase inducer and an initial approach to the enzymatic reaction conditions was reached.Materials and Methods: Ellagitannase was produced by Aspergillus niger GH1 in solid state fermentation and the ideal reaction conditions for ellagitannase activity based on ellagic acid quantification as ellagitannins biotransformation product by high performance liquid chromatographic are reported.Results:The enzyme ideal reaction conditions were substrate concentration of 1 mg.mL-1, 60 °C and pH 5.0, during 10 min of reaction. The kinetic enzyme constants (Vmax = 30.34 mM.mL-1.min-1 and Km = 1.48 x 103 mM) using punicalagin assubstrate were determined. Conclusion: The assay was completed in a short time and may find application in future studies of ellagic acid production.

Removal of nonylphenol polyethoxylates by adsorption on polyurethane foam and biodegradation using immobilized Trametes versicolor

Nonylphenol polyethoxylates (NPEOs) are banned in EU due to their endocrine disrupting properties. In a proof of concept study including continuous reactor lab-scale experiments, polyurethane foam (PUF)-immobilized Trametes versicolor was used to reduce the concentration levels of these compounds in an acidic nutrient solution over an 18-day period. Biodegradation and adsorption were identified as the major removal principles. A 90% removal was achieved by solely biodegradation in an experimental setup in which steady state conditions occurred, including NPEO-saturated glass and PUF surfaces. Biotransformation products containing mono- and di-ethoxylated nonylphenol, nonylphenol (NP1EO, NP2EO, NP) and nonylphenol polyethoxy carboxylates (NPECs) were tentatively identified.The maximum static NPEO adsorption capacity of PUF (determined with Erlenmeyer flask experiment) was calculated to 106 mg g−1, and the adsorption was described by the Langmuir isotherm equation. The corresponding maximum dynamic adsorption capacity (determined by continuous reactor experiment) was 100 mg g−1. These findings show that PUF is an excellent adsorbent to NPEOs. Therefore, PUF can either be used as a stand-alone adsorbent to NPEOs or as an immobilizing agent for Trametes versicolor through which a highly efficient biodegradation of these potentially harmful compounds can be achieved. The findings can be of importance in the search for alternative methods to remove NPEOs in process effluents.

Influence of Selected Antidepressants on the Ciliated Protozoan Spirostomum ambiguum: Toxicity, Bioaccumulation, and Biotransformation Products.

The present study aimed to evaluate the effect of the most common antidepressants on aquatic protozoa. Spirostomum ambiguum was used as the model protozoan. The biological activity of four antidepressants, namely fluoxetine, sertraline, paroxetine, and mianserin, toward S. ambiguum was evaluated. Sertraline was found to be the most toxic drug with EC50 values of 0.2 to 0.7 mg/L. The toxicity of the antidepressants depended on the pH of the medium and was the highest in alkaline conditions. Sertraline was also the most bioaccumulating compound tested, followed by mianserin. Slow depuration was observed after transferring the protozoa from the drug solutions to a fresh medium, which indicated possible lysosomotropism of the tested antidepressants in the protozoa. The biotransformation products were identified using a high-resolution mass spectrometer after two days of incubation of the protozoa with the tested antidepressants. Four to six potential biotransformation products were observed in the aqueous phase, while no metabolites were detected in the protozoan cells. Because of the low abundance of metabolites in the medium, their structure was not determined.

Stable Isotope-Assisted Metabolomics for Deciphering Xenobiotic Metabolism in Mammalian Cell Culture.

Xenobiotics are ubiquitous in the environment and modified in the human body by phase I and II metabolism. Liquid chromatography coupled to high resolution mass spectrometry is a powerful tool to investigate these biotransformation products. We present a workflow based on stable isotope-assisted metabolomics and the bioinformatics tool MetExtract II for deciphering xenobiotic metabolites produced by human cells. Its potential was demonstrated by the investigation of the metabolism of deoxynivalenol (DON), an abundant food contaminant, in a liver carcinoma cell line (HepG2) and a model for colon carcinoma (HT29). Detected known metabolites included DON-3-sulfate, DON-10-sulfonate 2, and DON-10-glutathione as well as DON-cysteine. Conjugation with amino acids and an antibiotic was confirmed for the first time. The approach allows the untargeted elucidation of human xenobiotic products in tissue culture. It may be applied to other fields of research including drug metabolism, personalized medicine, exposome research, and systems biology to better understand the relevance of in vitro experiments.