Detoxification Step Research Articles

Organophosphorous Poisoning: Treatment and Analytical Methodologies Applied in Evaluation of Reactivation and Inhibition of Acetylcholinesterase

Organophosphorus derivatives inhibit the enzyme acetylcholinesterase promoting cholinergic hyperstimulation, irreversible damage and death. These compounds are used by civil and military organizations as pesticides or chemical weapons, respectively, and are responsible for over three million annual cases of poisoning, as well as more than 250,000 deaths per year from intentional self-poisoning, accounting 30% of suicides in the world. The organophosphorus sarin, soman, tabun and VX are been employed as weapons of war by different terrorist groups. Oxime derivatives (pralidoxime and obidoxima) have been used as antidote in detoxification step, but the results are still not satisfactory, because these drugs have low penetration into blood-brain barrier and an inefficient nucleophilic action against organophosphorus. Thus, different analytical methods can be used to monitor the efficiency of drugs with property to reactivate or inhibit the acetylcholinesterase enzyme. This paper provides an overview of the derivatives of oximes currently used as potential reactivators and inhibitors, as well as of the main methodologies used in monitoring or reactivation or inhibition of acetylcholinesterase. DOI: 10.5935/1984-6835.20160056

Techno‐economic analysis of cellulose dissolving ionic liquid pretreatment of lignocellulosic biomass for fermentable sugars production

AbstractPretreatment of biomass is essential to produce fermentable sugars, which can be further transformed into biofuels. Most of the common pretreatment methods, including sulfuric acid pretreatment, lead to low sugar yields and high microbial inhibitors formation, and require an additional detoxification step prior to fermentation. Ionic liquid (IL) pretreatment has the potential to reduce or eliminate these problems, and, thus, recently, has gained significant attention as an alternative pretreatment technology. However, before commercial deployment, IL pretreatment requires a thorough assessment of its techno‐economic feasibilities and bottlenecks. Thus, the main objective of this study was to assess the techno‐economic feasibility of a commercial‐scale IL pretreatment for a 113 million liter/year (30 million gal/year) cellulosic biorefinery and identify operational targets for process improvement. 1‐ethyl‐3‐methylimidazolium acetate IL was used for analyses, which is one of the mostly investigated ILs and is currently best at dissolving the lignocellulosic biomass. Corn stover, poplar, and switchgrass were used as the model feedstock. Input data were obtained from recent literatures on IL pretreatment of these feedstocks. Estimated sugar production costs ($/kg) from corn stover, switchgrass and poplar were 2.7, 3.2, and 3.0, respectively. IL recovery was identified to be the most sensitive parameter followed by IL cost and heat recovery. Furthermore, for IL pretreatment to be economically competitive with sulfuric acid pretreatment, >97% IL recovery, ≤$1/kg IL cost, and >90% waste heat recovery are necessary, all of which are very optimistic considerations at the present state of technology, thus requires further research and development efforts. © 2015 Society of Chemical Industry and John Wiley & Sons, Ltd

Measuring cytochrome P450 activity in aquatic invertebrates: a critical evaluation of in vitro and in vivo methods.

The first step in xenobiotic detoxification in aquatic invertebrates is mainly governed by the cytochrome P450 mixed function oxidase system. The ability to measure cytochrome P450 activity provides an important tool to understand macroinvertebrates' responses to chemical stressors. However, measurements of P450 activity in small aquatic invertebrates have had variable success and a well characterized assay is not yet available. The general lack of success has been scarcely investigated and it is therefore the focus of the present work. In particular, the suitability of the substrate selected for the assay, the sensitivity of the assay and the possible inhibition/attenuation of enzymatic activity caused by endogenous substances were investigated. 7-ethoxycoumarin-O-dealkylation activity of Daphnia magna, Chironomus riparius larvae and Hyalella azteca was assessed in vivo and in vitro and possible inhibition of enzymatic activity by macroinvertebrates homogenate was investigated. Activities of D. magna and C. riparius larvae measured in vivo were 1.37 ± 0.08 and 2.2 ± 0.2 pmol h(-1) organism(-1), respectively, while activity of H. azteca could not be detected. In vitro activity could be measured in C. riparius larvae only (500-1000 pmol h(-1) mg microsomal protein(-1)). The optimization of the in vitro assay has been especially long and resource consuming and particularly for D. magna, substances that inhibited cytochrome P450 activity seemed to be released during tissue homogenization preventing activity measurements in vitro. We therefore recommend testing the P450 inhibition potential of homogenate preparations prior to any investigation of P450 activity in vitro in macroinvertebrates.

Short-term adaptation during propagation improves the performance of xylose-fermenting Saccharomyces cerevisiae in simultaneous saccharification and co-fermentation.

BackgroundInhibitors that are generated during thermochemical pretreatment and hydrolysis impair the performance of microorganisms during fermentation of lignocellulosic hydrolysates. In omitting costly detoxification steps, the fermentation process relies extensively on the performance of the fermenting microorganism. One attractive option of improving its performance and tolerance to microbial inhibitors is short-term adaptation during propagation. This study determined the influence of short-term adaptation on the performance of recombinant Saccharomyces cerevisiae in simultaneous saccharification and co-fermentation (SSCF). The aim was to understand how short-term adaptation with lignocellulosic hydrolysate affects the cell mass yield of propagated yeast and performance in subsequent fermentation steps. The physiology of propagated yeast was examined with regard to viability, vitality, stress responses, and upregulation of relevant genes to identify any links between the beneficial traits that are promoted during adaptation and overall ethanol yields in co-fermentation.ResultsThe presence of inhibitors during propagation significantly improved fermentation but lowered cell mass yield during propagation. Xylose utilization of adapted cultures was enhanced by increasing amounts of hydrolysate in the propagation. Ethanol yields improved by over 30 % with inhibitor concentrations that corresponded to ≥2.5 % water-insoluble solids (WIS) load during the propagation compared with the unadapted culture. Adaptation improved cell viability by >10 % and increased vitality by >20 %. Genes that conferred resistance against inhibitors were upregulated with increasing amounts of inhibitors during the propagation, but the adaptive response was not associated with improved ethanol yields in SSCF. The positive effects in SSCF were observed even with adaptation at inhibitor concentrations that corresponded to 2.5 % WIS. Higher amounts of hydrolysate in the propagation feed further improved the fermentation but increased the variability in fermentation outcomes and resulted in up to 20 % loss of cell mass yield.ConclusionsShort-term adaptation during propagation improves the tolerance of inhibitor-resistant yeast strains to inhibitors in lignocellulosic hydrolysates and improves their ethanol yield in fermentation and xylose-fermenting capacity. A low amount of hydrolysate (corresponding to 2.5 % WIS) is optimal, whereas higher amounts decrease cell mass yield during propagation.

Structure of human carbamoyl phosphate synthetase: deciphering the on/off switch of human ureagenesis

Human carbamoyl phosphate synthetase (CPS1), a 1500-residue multidomain enzyme, catalyzes the first step of ammonia detoxification to urea requiring N-acetyl-L-glutamate (NAG) as essential activator to prevent ammonia/amino acids depletion. Here we present the crystal structures of CPS1 in the absence and in the presence of NAG, clarifying the on/off-switching of the urea cycle by NAG. By binding at the C-terminal domain of CPS1, NAG triggers long-range conformational changes affecting the two distant phosphorylation domains. These changes, concerted with the binding of nucleotides, result in a dramatic remodeling that stabilizes the catalytically competent conformation and the building of the ~35 Å-long tunnel that allows migration of the carbamate intermediate from its site of formation to the second phosphorylation site, where carbamoyl phosphate is produced. These structures allow rationalizing the effects of mutations found in patients with CPS1 deficiency (presenting hyperammonemia, mental retardation and even death), as exemplified here for some mutations.

Characterization of hemicellulase and cellulase from the extremely thermophilic bacterium Caldicellulosiruptor owensensis and their potential application for bioconversion of lignocellulosic biomass without pretreatment.

BackgroundPretreatment is currently the common approach for improving the efficiency of enzymatic hydrolysis on lignocellulose. However, the pretreatment process is expensive and will produce inhibitors such as furan derivatives and phenol derivatives. If the lignocellulosic biomass can efficiently be saccharified by enzymolysis without pretreatment, the bioconversion process would be simplified. The genus Caldicellulosiruptor, an obligatory anaerobic and extreme thermophile can produce a diverse set of glycoside hydrolases (GHs) for deconstruction of lignocellulosic biomass. It gives potential opportunities for improving the efficiency of converting native lignocellulosic biomass to fermentable sugars.ResultsBoth of the extracellular (extra-) and intracellular (intra-) enzymes of C. owensensis cultivated on corncob xylan or xylose had cellulase (including endoglucanase, cellobiohydrolase and β-glucosidase) and hemicellulase (including xylanase, xylosidase, arabinofuranosidase and acetyl xylan esterase) activities. The enzymes of C. owensensis had high ability for degrading hemicellulose of native corn stover and corncob with the conversion rates of xylan 16.7 % and araban 60.0 %. Moreover, they had remarkable synergetic function with the commercial enzyme cocktail Cellic CTec2 (Novoyzmes). When the native corn stover and corncob were respectively, sequentially hydrolyzed by the extra-enzymes of C. owensensis and CTec2, the glucan conversion rates were 31.2 and 37.9 %,which were 1.7- and 1.9-fold of each control (hydrolyzed by CTec2 alone), whereas the glucan conversion rates of the steam-exploded corn stover and corncob hydrolyzed by CTec2 alone on the same loading rate were 38.2 and 39.6 %, respectively. These results show that hydrolysis by the extra-enzyme of C. owensensis made almost the same contribution as steam-exploded pretreatment on degradation of native lignocellulosic biomass. A new process for saccharification of lignocellulosic biomass by sequential hydrolysis is demonstrated in the present research, namely hyperthermal enzymolysis (70–80 °C) by enzymes of C. owensensis followed with mesothermal enzymolysis (50–55 °C) by commercial cellulase. This process has the advantages of no sugar loss, few inhibitors generation and consolidated with sterilization.ConclusionsThe enzymes of C. owensensis demonstrated an enhanced ability to degrade the hemicellulose of native lignocellulose. The pretreatment and detoxification steps may be removed from the bioconversion process of the lignocellulosic biomass by using the enzymes from C. owensensis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0313-0) contains supplementary material, which is available to authorized users.

Hemicellulose-derived sugars solubilisation of rape straw. Cofermentation of pentoses and hexoses by Escherichia coli

Bioconversion of hemicellulose sugars is essential for increasing fuel ethanol yields from lignocellulosic biomass. We report for the first time with rape straw, bioethanol production from hemicellulose sugars. Rape straw was pretreated at mild conditions with sulfuric acid to solubilize the hemicellulose fraction. This pretreatment allows obtaining a prehydrolysate, consisting basically in a solution of monomeric hemicellulosic sugars, with low inhibitor concentrations. The remaining water insoluble solid constitutes a cellulose-enriched, free of extractives material. The influence of temperature (120ºC and 130ºC), acid concentration (2-4% w/v) and pretreatment time (30-180 min) on hemicellulose-derived sugars solubilisation was evaluated. The highest hemicellulosic sugars recovery, 72.3%, was achieved at 130ºC with 2% sulfuric acid and 60 min. At these conditions, a concentrated sugars solution, 52.4 g/L, was obtained after three acid consecutive contacts, with 67% xylose and acetic acid concentration above 4.5 g/L. After a detoxification step by activated charcoal or ion-exchange resin, prehydrolysate was fermented by ethanologenic Escherichia coli. An alcoholic solution of 25 g/L and 86% of theoretical ethanol yield was attained after 144 h when the prehydrolysate was detoxified by ion-exchange resin. The results obtained in the present work show sulfuric acid pretreatment under mild conditions and E. coli as an interesting process to exploit hemicellulosic sugars in rape straw.

A γ-lactamase from cereal infecting Fusarium spp. catalyses the first step in the degradation of the benzoxazolinone class of phytoalexins

The benzoxazolinone class of phytoalexins are released by wheat, maize, rye and other agriculturally important species in the Poaceae family upon pathogen attack. Benzoxazolinones show antimicrobial effects on plant pathogens, but certain fungi have evolved mechanisms to actively detoxify these compounds which may contribute to the virulence of the pathogens. In many Fusarium spp. a cluster of genes is thought to be involved in the detoxification of benzoxazolinones. However, only one enzyme encoded in the cluster has been unequivocally assigned a role in this process. The first step in the detoxification of benzoxazolinones in Fusarium spp. involves the hydrolysis of a cyclic ester bond. This reaction is encoded by the FDB1 locus in F. verticillioides but the underlying gene is yet to be cloned. We previously proposed that FDB1 encodes a γ-lactamase, and here direct evidence for this is presented. Expression analyses in the important wheat pathogen F. pseudograminearum demonstrated that amongst the three predicted γ-lactamase genes only the one designated as FDB1, part of the proposed benzoxazolinone cluster in F. pseudograminearum, was strongly responsive to exogenous benzoxazolinone application. Analysis of independent F. pseudograminearum and F. graminearum FDB1 gene deletion mutants, as well as biochemical assays, demonstrated that the γ-lactamase enzyme, encoded by FDB1, catalyses the first step in detoxification of benzoxazolinones. Overall, our results support the notion that Fusarium pathogens that cause crown rot and head blight on wheat have adopted strategies to overcome host-derived chemical defences.

Pyrochars from bioenergy residue as novel bio-adsorbents for lignocellulosic hydrolysate detoxification

The robust supramolecular structure of biomass often requires severe pretreatments conditions to produce soluble sugars. Nonetheless, these processes generate some inhibitory compounds (i.e. furans compounds and aliphatic acids) deriving mainly from sugars degradation. To avoid the inhibition of the biological process and to obtain satisfactory sugars conversion level into biofuels, a detoxification step is required. This study investigates the use of two pyrochars derived from solid anaerobic digestates for the detoxification of lignocellulosic hydrolysates. At a pyrochar concentration of 40gL−1, more than 94% of 5-HMF and 99% of furfural were removed in the synthetic medium after 24h of contact time, whereas sugars concentration remained unchanged. Furfural was adsorbed faster than 5-HMF by both pyrochars and totally removed after 3h of contact. Finally, the two pyrochars were found efficient in the detoxification of corn stalks and Douglas fir wood chips hydrolysates without affecting the soluble sugars concentrations.

Ralstonia solanacearum uses inorganic nitrogen metabolism for virulence, ATP production, and detoxification in the oxygen-limited host xylem environment.

ABSTRACTGenomic data predict that, in addition to oxygen, the bacterial plant pathogen Ralstonia solanacearum can use nitrate (NO3−), nitrite (NO2−), nitric oxide (NO), and nitrous oxide (N2O) as terminal electron acceptors (TEAs). Genes encoding inorganic nitrogen reduction were highly expressed during tomato bacterial wilt disease, when the pathogen grows in xylem vessels. Direct measurements found that tomato xylem fluid was low in oxygen, especially in plants infected by R. solanacearum. Xylem fluid contained ~25 mM NO3−, corresponding to R. solanacearum’s optimal NO3− concentration for anaerobic growth in vitro. We tested the hypothesis that R. solanacearum uses inorganic nitrogen species to respire and grow during pathogenesis by making deletion mutants that each lacked a step in nitrate respiration (ΔnarG), denitrification (ΔaniA, ΔnorB, and ΔnosZ), or NO detoxification (ΔhmpX). The ΔnarG, ΔaniA, and ΔnorB mutants grew poorly on NO3− compared to the wild type, and they had reduced adenylate energy charge levels under anaerobiosis. While NarG-dependent NO3− respiration directly enhanced growth, AniA-dependent NO2− reduction did not. NO2− and NO inhibited growth in culture, and their removal depended on denitrification and NO detoxification. Thus, NO3− acts as a TEA, but the resulting NO2− and NO likely do not. None of the mutants grew as well as the wild type in planta, and strains lacking AniA (NO2− reductase) or HmpX (NO detoxification) had reduced virulence on tomato. Thus, R. solanacearum exploits host NO3− to respire, grow, and cause disease. Degradation of NO2− and NO is also important for successful infection and depends on denitrification and NO detoxification systems.

LC3 overexpression reduces Aβ neurotoxicity through increasing α7nAchR expression and autophagic activity in neurons and mice

Autophagy is an intracellular degradation pathway with dynamic interactions for eliminating damaged organelles and protein aggregates by lysosomal digestion. The EGFP-conjugated microtubule-associated protein 1 light chain 3 (EGFP-LC3) serves to monitor autophagic process. Extracellular β-amyloid peptide accumulation is reported as a major cause in Alzheimer's disease (AD) pathogenesis; large numbers of autophagic vacuoles accumulate in patients' brains. We previously demonstrated that extracellular Aβ (eAβ) induces strong autophagic response and α7nAChR acts as a carrier to bind with eAβ; which further inhibits Aβ-induced neurotoxicity via autophagic degradation. In the present study, we overexpressed LC3 in both neuroblastoma cells (SH-SY5Y/pEGFP-LC3) and mice (TgEGFP-LC3) to assess the effect of LC3 overexpression on Aβ neurotoxicity. SH-SY5Y/pEGFP-LC3 cells and primary cortical neuron cultures derived from E17 (embryonic day 17) TgEGFP-LC3 mice showed not only better resistance against Aβ neurotoxicity but also higher α7nAChR expression and autophagic activity than control. Administration of α-bungarotoxin (α-BTX) to block α7nAChR antagonized the neuroprotective action of SH-SY5Y/pECGF-LC3 cells, suggesting that eAβ binding with α7nAChR is an important step in Aβ detoxification. LC3 overexpression thus exerts neuroprotection through increasing α7nAChR expression for eAβ binding and further enhancing autophagic activity for Aβ clearance in vitro and in vivo.

Determining trace amounts and the origin of formaldehyde impurity in Neisseria meningitidis A/C/Y/W-135-DT conjugate vaccine formulated in isotonic aqueous 1× PBS by improved C18-UPLC method

The ability to accurately measure and report trace amounts of residual formaldehyde impurity in a vaccine product is not only critical in the product release but also a regulatory requirement. In many bacterial or viral vaccine manufacturing procedures, formaldehyde is used either at a live culture inactivation step or at a protein de-toxification step or at both. Reported here is a validated and improved C18-UPLC method (developed based on previously published C-8 HPLC method) to determine the traces of formaldehyde process impurity in a liquid form Neisseria meningitidis A/C/Y/W-135-DT conjugate vaccine formulated in isotonic aqueous 1× PBS. UPLC C-18 column and the conditions described distinctly resolved the 2,4-DNPH–HCHO adduct from the un-reacted 2,4-DNPH as detected by TUV detector at 360nm. This method was shown to be compatible with PBS formulation and extremely sensitive (with a quantitation limit of 0.05ppm) and aided to determine formaldehyde contamination sources by evaluating the in-process materials as a track-down analysis. Final nanogram levels of formaldehyde in the formulated single dose vialed vaccine mainly originated from the diphtheria toxoid carrier protein used in the production of the conjugate vaccine, whereas relative contribution from polysaccharide API was minimal.

Concentration and Detoxification of Kraft Prehydrolysate by Combining Nanofiltration with Flocculation

The prehydrolysate stream from a Kraft dissolving pulp mill can be valorized by fermentation of the hemicellulosic sugars into biofuels or bioproducts, such as ethanol or butanol, instead of the typical practice of combustion to produce energy. An obstacle facing the use of Kraft hemicelluloses prehydrolysate for biofuels production is the low sugar concentration and the presence of fermentation inhibitors that include organic acids, furans and phenolic compounds. A precondition to ensure the survival of the fermentation microorganisms and to have high fermentation yields is to remove the inhibitors. Concentration of the prehydrolysate is also necessary to reduce the size of the processing equipment and decrease the energy cost. The purpose of this study was to develop a strategy for the concentration and detoxification of hemicelluloses prehydrolysate prior to its conversion into biofuels. Experiments were conducted to screen and select suitable organic membranes among 7 samples of reverse osmosis, nanofiltration, and ultrafiltration membranes. Three membranes (Dow NF270, Trisep TS40, and Trisep XN45) showed the highest sugar retentions relative to inhibitors removal. They were however not efficient for the removal of the phenolic compounds. It was also found that flocculation with ferric sulfate as coagulant could be utilized as a secondary detoxification step that can be combined with nanofiltration. The optimization of the flocculation step with a jar test showed that the highest phenolics removal (∼80%) can be obtained when the ratio of ferric ions to phenols is 1 g/g, and the pH is between 6.5 and 7.5. A new process concept for the detoxification and concentration has been developed based on these experimental results.

The importance of incorporating a waste detoxification step in analytical methodologies

Waste detoxification of copper determination in pesticide formulations improves the green character of the method. In this work: copper was determined in pesticide formulations. Electrogravimetry and atomic spectrometry methods were evaluated from their environmental point of view. The addition of waste decontamination steps improves the greenness of the method.

In Vitro Study of Bisphenol A (BPA) in the early human placenta

In Vitro Study of Bisphenol A (BPA) in the early human placenta

The role of placental carbonyl-reducing enzymes in biotransformation of bupropion and 4-methylnitrozamino-1-(3-pyridyl)-1-butanone (NNK)

Background: Bupropion (BUP) has a potential to be an effective pharmacotherapy for smoking cessation during pregnancy. Smoking during pregnancy stimulates placental carbonyl reductases that catalyze the biotransformation of BUP. 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen of cigarette smoke. Carbonyl reduction of NNK into 4-methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) constitutes a major step in NNK detoxification. Thus, placentas of pregnant smokers on BUP therapy can become a site of drug-drug interaction. Therefore, we investigated the effect of continuous exposure to BUP and cigarette smoke on the activity of placental carbonyl reductases in the formation of NNAL from NNK. Methods: The reductive metabolism of NNK was determined using microsomal and cytosolic subcellular fractions of placentas obtained from non-smoking women treated with BUP for depression, and women not exposed to BUP: non-smokers (control) and smokers. The effect of BUP and its metabolites on the reductive metabolism of NNK was investigated using subcellular fractions of control placentas. Results: The formation of NNAL from NNK by placental cytosolic fractions of heavy smokers (≥20 cigarettes per day) was lower than that of control (12.1±3.5 nmol.mgP-1 vs 16.5±6.0 nmol.mgP-1, P<0.05). While exposure to BUP did not affect the activity of placental carbonyl reductases, the formation of NNAL in the placental cytosolic fraction was decreased only in the presence of high concentrations of BUP metabolites. Conclusion: Smoking during pregnancy decrease the detoxifying capacity of soluble carbonyl reductases towards NNK. Given the experimental conditions, exposure to BUP and its metabolites should not impede the reductive metabolism of NNK by placenta in vivo.

Crystal structures of γ-glutamyltranspeptidase in complex with inhibitors

γ-Glutamyltranspeptidase (GGT; EC 2.3.2.2) is involved in the degradation of γ-glutamyl compounds such as glutathione (GSH; γ-glutamyl-cysteinyl-glycine) . A major physiological role of this enzyme is to cleave the extracellular GSH as a source of cysteine for intracellular glutathione biosynthesis. Another crucial role of GGT is to cleave glutathione-S-conjugates as a key step in detoxification of xenobiotics and drug metabolism. In mammals, GGT has been implicated in physiological disorders such as Parkinson's disease, other neurodegenerative diseases including Alzheimer's disease and cardiovascular disease. The indispensable roles played by GGT in GSH-mediated detoxification and cellular response to oxidative stress suggest that GGT is an attractive pharmaceutical target. We here report the binding mode of acivicin, a well-known glutamine antagonist, to B. subtilis GGT at 1.8 Å resolution showing that acivicin is bound to the Oγ atom of Thr403, the catalytic nucleophile of the enzyme, through its C3 atom [1]. The observed electron density around the C3 atom was best fitted to the planar and sp2 hybridized carbon, consistent with a simple nucleophilic substitution of Cl at the imino carbon by Oγ atom of Thr403. Furthermore, comparison of three bacterial enzymes, the GGTs from E. coli, H. pylori and B. subtilis in complex with acivicin, showed significant diversity in the orientation of the dihydroisoxazole ring among three GGTs. The differences are discussed in terms of the recognition of the α-amino and α-carboxy groups in preference to the dihydroisoxazole ring as observed in time-lapse soaking crystal structures of B. subtilis GGT with acivicin.

Dietary exposure of Daphnia to microcystins: No in vivo relevance of biotransformation

Anthropogenic nutrient input into lakes has contributed to the increased frequency of toxic cyanobacterial blooms. Daphnia populations have been shown to be locally adapted to toxic cyanobacteria and are able to suppress bloom formation; little is known about the physiology behind this phenomenon. Microcystin-LR (MCLR) is the most widespread cyanobacterial toxin, and, based on in vitro experiments, it is assumed that the enzyme glutathione-S-transferase (GST) might act as the first step of detoxification in Daphnia by conjugating MCLR with glutathione.In the present study Daphnia magna was fed a diet of 100% Microcystis aeruginosa PCC7806, a cyanobacterial strain that contains MCLR in high amounts (4.8–5.6fgcell−1), in order to test for a possible conjugation of MCLR with GST in Daphnia in vivo. We used high-resolution LCMS to analyze incubation water, cyanobacterial cells and Daphnia tissue for the presence of MCLR conjugation products as well as unconjugated MCLR.Newly formed conjugation products were detected neither in Daphnia tissue nor in the incubation water. Moreover, the presence of Daphnia led to a decrease in unconjugated MCLR in the cyanobacterial cell fraction due to grazing, in comparison to a control without daphnids, which was well reflected by a similar increase of MCLR in the respective incubation water. As a consequence, the MCLR content did not change due to Daphnia presence within the entire experimental setup. In summary, MCLR ingestion by Daphnia led neither to the formation of conjugation products, nor to a decrease of unconjugated MCLR.GST-mediated conjugation thus seems to be of minor relevance for microcystin (MC) tolerance in Daphnia in vivo. This finding is supported by the fact that GST activity in Daphnia feeding on the MC-containing wildtype or a MC-free mutant of M. aeruginosa PCC7806 revealed an identical increase of specific activity in comparison to a cyanobacteria-free diet. Therefore, the frequently observed induction of GST activity upon exposure to toxic cyanobacteria is not a specific MC effect but a general cyanobacterial effect. This suggests that GST in Daphnia is involved in an oxidative stress response rather than in the specific detoxification of MCs. Furthermore, our results indicate the presence of an efficient transport mechanism which efficiently removes unconjugated MCLR from the Daphnia tissue. Further studies are needed to elucidate the nature of this transport mechanism.

Toxicity of the mycotoxin citrinin and its metabolite dihydrocitrinone and of mixtures of citrinin and ochratoxin A in vitro

Citrinin (CIT) and ochratoxin A (OTA) are mycotoxins produced by several species of the genera Aspergillus, Penicillium and Monascus. Both can be present as contaminants in various food commodities and in animal feed. The occurrence and toxicity of OTA and human exposure have been intensively studied, but for CIT such data are scarce by comparison. Recently, dihydrocitrinone (DH-CIT) was detected as main metabolite of CIT in human urine, and co-occurrence of CIT and OTA was shown in human blood plasma (Blaszkewicz et al. in Arch Toxicol 87:1087-1094, 2013). In light of these new findings, we have now investigated the toxicity of the metabolite DH-CIT in comparison with CIT and analysed the effects of mixtures of CIT and OTA in vitro. The cytotoxic potency of DH-CIT (IC50 of 320/200μM) was distinctly lower compared with CIT (IC50 of 70/62μM) after treatment of V79 cells for 24 and 48h. Whereas CIT induced a concentration-dependent increase in micronucleus frequencies at concentrations ≥30μM, DH-CIT showed no genotoxic effect up to 300μM. Thus, conversion of CIT to DH-CIT in humans can be regarded as a detoxification step. Mixtures of CIT and OTA exerted additive effects in cytotoxicity assays. The effect of CIT and OTA mixtures on induction of micronuclei varied dependent on the used concentrations between additive for low μM concentrations and more-than-additive for high μM concentrations. Effects on cell cycle were mostly triggered by OTA when both mycotoxins were used in combination. The implications of our and related in vitro studies are discussed with respect to in vivo concentrations of CIT and OTA, which are found in animals and in humans.

CYP3A4 activity reduces the cytotoxic effects of okadaic acid in HepaRG cells

The biotoxin okadaic acid (OA), produced by dinoflagellates in marine environment, can accumulate in sponges and shellfish. Consumption of contaminated shellfish induces acute toxic effects such as diarrhea, nausea, vomiting, and abdominal pain. CYP3A4, one of the most important human xenobiotic metabolizing enzymes, is supposed to be involved in the metabolism of OA. Aim of our study was to evaluate the role of CYP3A4 in OA in vitro metabolism as well as in cell cytotoxicity in parallel. Therefore, a metabolic competent HepaRG cell line was exposed to OA with and without addition of the CYP3A4 inhibitor ketoconazole. Without the inhibitor, two mono-hydroxylated metabolites could be identified, whereas in its presence, no metabolites could be detected. Confirmation of the formed metabolites was accomplished by measuring the exact masses and investigating the fragmentation pattern. Data obtained from cytotoxicity assays showed that OA cytotoxicity is reduced when CYP3A4 is active. Thus, hydroxylation appears to be a crucial step for metabolic OA detoxification.