Dependent Detoxification Research Articles

Oxygen tolerance and detoxification mechanisms of highly enriched planktonic anaerobic ammonium-oxidizing (anammox) bacteria

Oxygen is a key regulatory factor of anaerobic ammonium oxidation (anammox). Although the inhibitory effect of oxygen is evident, a wide range of oxygen sensitivities of anammox bacteria have been reported so far, which makes it difficult to model the marine nitrogen loss and design anammox-based technologies. Here, oxygen tolerance and detoxification mechanisms of four genera of anammox bacteria; one marine species (“Ca. Scalindua sp.”) and four freshwater anammox species (“Ca. Brocadia sinica”, “Ca. Brocadia sapporoensis”, “Ca. Jettenia caeni”, and “Ca. Kuenenia stuttgartiensis”) were determined and then related to the activities of anti-oxidative enzymes. Highly enriched planktonic anammox cells were exposed to various levels of oxygen, and oxygen inhibition kinetics (50% inhibitory concentration (IC50) and upper O2 limits (DOmax) of anammox activity) were quantitatively determined. A marine anammox species, “Ca. Scalindua sp.”, exhibited much higher oxygen tolerance capability (IC50 = 18.0 µM and DOmax = 51.6 µM) than freshwater species (IC50 = 2.7–4.2 µM and DOmax = 10.9–26.6 µM). The upper DO limit of “Ca. Scalindua sp.” was much higher than the values reported so far (~20 µM). Furthermore, the oxygen inhibition was reversible even after exposed to ambient air for 12–24 h. The comparative genome analysis confirmed that all anammox species commonly possess the genes considered to function for reduction of O2, superoxide anion (O2•-), and H2O2. However, the superoxide reductase (Sor)-peroxidase dependent detoxification system alone may not be sufficient for cell survival under microaerobic conditions. Despite the fact that anaerobes normally possess no or little superoxide dismutase (Sod) or catalase (Cat), only Scalindua exhibited high Sod activity of 22.6 ± 1.9 U/mg-protein with moderate Cat activity of 1.6 ± 0.7 U/mg-protein, which was consistent with the genome sequence analysis. This Sod-Cat dependent detoxification system could be responsible for the higher O2 tolerance of Scalindua than other freshwater anammox species lacking the Sod activity.

Boron toxicity induces sulfate transporters at transcriptional level in Arabidopsis thaliana

Plants activate glutathione (GSH)-dependent detoxification pathways at biochemical and molecular levels under boron (B) toxicity. Sulfate uptake and transport are necessary for GSH biosynthesis in plants. Therefore, the transcriptional regulation of some sulfate transporters was determined in this study to clarify the importance of these transporters in leaf and root tissues of Arabidopsis thaliana under toxic B conditions. The expression level of SULTR1;3 was dramatically increased in leaf and root tissues under moderate and severe toxic B conditions, suggesting source-to-sink sulfate translocation under B toxicity. Stable expression levels of SULTR2;1, SULTR2;2, and low SULTR3;5 expression might restrict the sulfate movement into the xylem in leaves. SULTR3;1, SULTR3;2, SULTR3;3, SULTR3;4, SULTR4;1 and SULTR4;2 were induced in root tissues under toxic B conditions, indicating an induction of root-to-shoot sulfate translocation. These results showed that B toxicity might disrupt the homogeneous distribution of sulfate and sulfur-containing compounds in both tissues of A. thaliana. Moreover, we performed in silico analysis of microarray experiments to determine the common differentially expressed genes (DEGs) under B toxicity and sulfur deficiency. Gene ontology, hierarchical clustering, and coexpression network analyses of these DEGs demonstrated the requirement of sulfate transporters under B toxicity. A set of genes involved in sulfur metabolism coexpress with sulfate transporters under B toxicity. To the best of our knowledge, this is the first report focusing on the molecular regulation of sulfate transporters in Arabidopsis thaliana under B toxicity.

Abstract 2566: The potential role of licorice and its bioactive compounds in promoting a tumor preventive environment in the postmenopausal breast

Abstract Introduction: our purpose was to define if licorice and its main bioactive compounds can suppress aromatase expression/activity and Nrf2 dependent detoxification pathways in the postmenopausal breast. This chemopreventive potential has not previously been reported in preclinical models of high-risk postmenopausal breast. Methods: Inhibition of aromatase activity by three licorice extracts (Glycyrrhiza species), and their bioactive compounds, (liquiritigenin; LigF, isoliquiritigenin; LigC, 8-prenylapigenin; 8-PA, and licochalcone A; LicA) were evaluated fluorometrically, using aromatase supersomes. Computational docking was performed to assess the binding of the bioactive compounds to the binding pocket of aromatase crystal structure compared to the known aromatase inhibitors, letrozole (non-steroidal) and exemestane (steroidal). Using qPCR, the effects of treatments on the expression of aromatase (CYP19A1) mRNA and Nrf2 dependent detoxification enzyme NADPH:quinone oxidoreductase 1 (NQO1) in breast microstructures obtained from high risk postmenopausal women were evaluated. Results: Among the three medicinally used licorice species, Glycyrrhiza inflata (GI) showed the highest aromatase inhibitory potency (IC50 ≈ 1 µg/mL). Licorice phytoestrogens, LigF (400 nM or 0.1 µg/mL), and 8-PA (IC50 ≈ 590 nM or 0.2 µg/mL) exhibited the highest potency compared to the other tested licorice compounds. Computational docking suggested that these phytoestrogens bind to the aromatase binding pocket like the aromatase inhibitor, letrozole. This effect was not observed with non-estrogenic bioactive compounds of licorice, LigC and LicA (specific to GI). In breast microstructures obtained from high risk postmenopausal women, expression of aromatase mRNA was suppressed by GI (30%, P < 0.05), LigF (20%, P < 0.05), and LicA (45%, P < 0.05), while the expression of NQO1 mRNA was enhanced by LicA (200%, P < 0.0001). Conclusions: Licorice species, and their bioactive compounds inhibited aromatase activity in vitro. In the breast microstructures, GI, its compounds LigF and LicA suppressed aromatase expression, and LicA enhanced NQO1 induction, significantly. Further studies will further elucidate the potential of these natural products for promoting a breast tumor preventive environment in postmenopausal women. Citation Format: Atieh Hajirahimkhan, Caitlin Howell, Shao-Nong Chen, Susan E. Clare, Guido F. Pauli, Judy L. Bolton, Birgit M. Dietz, Seema A. Khan. The potential role of licorice and its bioactive compounds in promoting a tumor preventive environment in the postmenopausal breast [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2566.

Melatonin alleviates imidacloprid phytotoxicity to cucumber (Cucumis sativus L.) through modulating redox homeostasis in plants and promoting its metabolism by enhancing glutathione dependent detoxification

Melatonin (Mel), a powerful antioxidant that has the ability to regulate physiological and biochemical processes in plants under abiotic stresses. However, its roles in pesticide detoxification is poorly understood. Herein, selecting leaf spraying insecticide imidacloprid (IMD) as the model, we demonstrated the detoxification mechanism underlying root pretreatment of Mel on IMD in cucumber. IMD treatment affected the primary light conversion efficiency of photosystem II (Fv/Fm), reduced the quantum yield, and increased hydrogen peroxide and superoxide anions contents as well as the levels of membrane lipid peroxidation, indicating that excessive IMD treatment induces oxidative stress. Nonetheless, by increasing the appropriate levels of exogenous Mel, the photosynthesis of cucumber under IMD treatment reached the control levels, effectively removing reactive oxygen species. Furthermore, the content and ratio of ascorbate (AsA) and glutathione (GSH) were decreased under IMD treatment; Mel treatment enhanced the AsA/DHA and GSH/GSSG ratios, as well as the activities of MDHAR, DHAR and GR, suggesting that Mel could alleviate oxidative stress of cucumber treated with IMD by regulating the ascorbic acid-glutathione cycle. Importantly, IMD degradation rate and glutathione S-transferase (GST) activity increased after Mel treatment. The levels of transcripts encoding antioxidant enzymes GPX and GST (GST1,2 and 3) were also increased, indicating that Mel accelerated IMD degradation. These results suggest that Mel plays an important role in the detoxification of IMD by promoting GST activity and transcription and the AsA-GSH cycle, thus providing an approach for plants to reduce IMD residue through the plant's own detoxification mechanism.

Chlorella sp. modulates the glutathione mediated detoxification and S-adenosylmethionine dependent methyltransferase to counter arsenic toxicity in Oryza sativa L.

The present study investigates the role of Chlorella sp. in the mitigation of arsenic (iAs) induced toxicity in Oryza sativa L. The study shows, co-culture of rice seedlings with Chlorella sp. reduced the iAs accumulation, simultaneously improving the growth of seedlings under iAs treatments. While treatment with As(III) and As(V) (60 µM) alone, inflicted toxicity in rice seedlings, manifested as significant enhancement in stress markers levels (TBRAS and H2O2), this coincided with the shifting of cellular reduced state to oxidized state (reduced GSH/GSSG ratio). Contrarily, co-culturing rice seedlings with Chlorella sp. under iAs toxicity, reduced these stress markers and recovered the GSH/GSSG ratio. The GSH dependent antioxidant enzymes i.e. GR and GPX activities also exhibited significant enhancement upon co-culturing rice seedlings with Chlorella sp. against iAs stress. Simultaneously, the expression of four thiol dependent GRX genes, i.e. GRX13950, GRX35340, GRX12190 and GRX07950 were enhanced against As(III) and As(V) (60 µM), which reduced upon co-culturing with Chlorella sp. A similar trend was also observed with the expression of GST genes, where the co-culture with Chlorella sp. significantly reduced the genes expression of two isoforms (GST 38600 and GST 38610). On the contrary, the expression of S-adenosylmethionine dependent methyltransferases (SAMT) gene in rice seedlings was enhanced upon co-culturing with the Chlorella sp. against iAs stress. Overall, the results demonstrate that the rice seedlings when co-culture with Chlorella sp. ameliorates iAs toxicity through GSH dependent detoxification pathway, evident from the enhanced expression of GRX, GST, SAMT genes and activity of GSH dependent antioxidant enzymes (GR and GPX) in the rice seedlings.

Amino acid dependent formaldehyde metabolism in mammals

Aldehyde dehydrogenase class 3, encoded by ADH5 in humans, catalyzes the glutathione dependent detoxification of formaldehyde. Here we show that ADH5 deficient cells turn over formaldehyde using alternative pathways starting from the reaction of formaldehyde with free amino acids. When mammalian cells are exposed to formaldehyde, the levels of the reaction products of formaldehyde with the amino acids cysteine and histidine - timonacic and spinacine - are increased. These reactions take place spontaneously and the formation of timonacic is reversible. The levels of timonacic are higher in the plasma of Adh5−/− mice relative to controls and they are further increased upon administration of methanol. We conclude that mammals possess pathways of cysteine and histidine dependent formaldehyde metabolism and that timonacic is a formaldehyde reservoir.

Overexpression of GmNAC085 enhances drought tolerance in Arabidopsis by regulating glutathione biosynthesis, redox balance and glutathione-dependent detoxification of reactive oxygen species and methylglyoxal

Glutathione (GSH)-dependent detoxification mechanisms are crucial for protecting plants from the adverse effects of abiotic stresses, including drought. Plant tolerance to drought is regulated by a set of transcription factors that are considered as an integral part of signaling networks in plants. Recently, there have been excellent appreciations on the drought tolerance roles of NAC (NAM, ATAF1/2, and CUC2) transcription factors in plants. In the current study, we sought the mechanisms on how the soybean GmNAC085 functions in the regulation of GSH biosynthesis and GSH-dependent detoxifications of reactive oxygen species (ROS) and methylglyoxal (MG) in order to promote drought tolerance in Arabidopsis thaliana. Our results showed that overexpression of GmNAC085 led to a marked increase in the level of reduced GSH, while decreasing oxidized GSH (GSSG) with a concomitant enhancement of the level of GSH/GSSG ratio in the leaves of Arabidopsis 35S:GmNAC085 transgenic plants. This GSH-based redox balance in transgenic lines was likely due to the increased expression of GSH-biosynthesis genes GSH1 and GSH2, as well as enhanced recycling of GSSG to GSH by glutathione reductase. The significant inductions of the activities of GSH-dependent enzymes glutathione peroxidase and glutathione-S-transferase were also evident in the 35S:GmNAC085 plants, revealing higher protection of transgenic lines against oxidative stress. We also observed that 35S:GmNAC085 plants efficiently removed toxic aldehyde MG by enhancing the activities of glyoxalase (Gly) I and Gly II, which is coincided with the increased transcript levels of Gly I and Gly II genes in the leaves. These results demonstrate a previously unknown function of GmNAC085-guided GSH-dependent detoxifications of ROS and MG, thereby providing a new insight into the mechanisms associated with GmNAC085-mediated drought tolerance. Additionally, expression of GmNAC085 under drought-inducible Arabidopsis RD29A promoter also enhanced drought tolerance without growth retardation, suggesting GmNAC085 as a potential genetic tool for developing plants with improved drought tolerance capacity.

Niacinamide and 12-hydroxystearic acid prevented benzo(a)pyrene and squalene peroxides induced hyperpigmentation in skin equivalent.

Skin surface is constantly exposed to environmental and secreted stressors such as UV, air pollution and peroxidized sebum. The current study aims to use reconstructed human skin equivalents to demonstrate topical stressor-induced hyperpigmentation and evaluate bioactives' potential protective effect. Given that polycyclic aromatic hydrocarbons are representative airborne particle-bound organic compounds with known relevance to pigmentation pathways, benzo(a)pyrene was selected as surrogate environmental toxin. On the other hand, squalene monohydroperoxides are well-characterized sebum peroxidation product under UV and pollutant exposure, thus are used as another representative skin stressor. With 3-day continuous exposure, 30pmol/cm2 of benzo(a)pyrene and 3.4nmol/cm2 of squalene monohydroperoxides induced significant viability loss, inflammatory response, and approximately 10 shades of pigmentation increase in pigmented living skin equivalents. At the same time, pretreatment and co-treatment with 12-hydroxystearic acid (12-HSA, 20μmol/L) or niacinamide (5mmol/L) ameliorated such stressor-induced consequences. Niacinamide was particularly effective against benzo(a)pyrene damage, probably as a substrate for important NAD+ dependent detoxification pathways, while 12-HSA was potent against squalene monohydroperoxides through barrier enhancing, anti-inflammatory, and anti-oxidative mechanisms. In summary, topical stressor-induced hyperpigmentation was achieved in vitro, with known bioactives showing protective benefits.

Experimental and computational modeling of interaction of kolaviron-kolaflavanone with aldehyde dehydrogenase

Aldehyde dehydrogenases (ALDHs) are a diverse family of enzymes that catalyze the NAD(P)+-dependent detoxification of toxic aldehyde compounds. ALDHs are also involved in non-enzymatic ligand binding to endobiotics and xenobiotics. Here, the enzyme crucial non-canonical and non-catalytic interaction with kolaflavanone, a component of kolaviron, and a major bioflavonoid isolated from Garcinia kola (Bitter kola) was characterized by various spectroscopic and in silico approaches under simulated physiological condition. Kolaflavanone quenched the intrinsic fluorescence of ALDH in a concentration dependent manner with an effective quenching constant (Ksv) of 1.14 × 103 L.mol−1 at 25 °C. The enzyme has one binding site for kolaflavanone with a binding constant (Ka) of 2.57 × 104 L.mol−1 and effective Forster resonance energy transfer (FRET) of 4.87 nm. The bonding process was enthalpically driven. The reaction was not spontaneous and was predominantly characterized by Van der Waals forces and hydrogen bond. The flavonoid bonding slightly perturbed the secondary and tertiary structures of ALDH that was ‘tryptophan-gated’. The interaction was regulated by both diffusion and ionic strength. Molecular docking showed the binding of kolaflavanone was at the active site of ALDH and the participation of some amino acid residues in the complex formation with −9.6 kcal mol−1 binding energy. The profiles of atomic fluctuations indicated the rigidity of the ligand-binding site during the simulation. With these, ALDH as a subtle nano-particle determinant of kolaviron bioavailability and efficacy is hereby proposed.

Studies of anthelminthic benzimidazole derivatives on cytochrome P450 1A (CYP1A) dependent detoxification mechanism in Labeo rohita

Fenbendazole (FBZ) and Mebendazole (MBZ) are considered the most safe and potent broad spectrum anthelminthic agents used against intestinal helminthic infection in humans and animals. In the present study, the induction and expression of Cytochrome p4501A (CYP1A) was observed against the benzimidazole derivatives (FBZ and MBZ) administrated at 20mg/kg body weight of fish (Labeo rohita). The enzymatic activity of CYP1A induction was assayed by Ethoxyresorufin-O-deethylase (EROD) method from hepatic microsomal fraction at 0, 24, 48, 72 and 96h post drug administration. EROD analysis showed the induction of CYP1A activity from 1.9 to 3.8 with FBZ and 3.8 to 6.0 times higher with MBZ group than control. The CYP1A gene induction was found significant in all time point (p<0.05) with highest level at 48h in FBZ fed group, while at 72h in case of MBZ fed group of fishes. The overall expression level was high in MBZ group in compared to FBZ administered group and control. Increase in the CYP1A induction and expression caused by MBZ may lead to the acceleration of drug elimination and thus to the loss of efficacy in compare to FBZ fed fishes which showed less induction of CYP1A. Thus, present study revealed that detoxification metabolism for FBZ in Labeo rohita is efficient as compared to MBZ. The information establishes the interaction of drugs with CYP1A gene and metabolization in fish.

Mechanisms and Control of NO2- Inhibition of Anaerobic Ammonium Oxidation (Anammox).

Nitrite (NO2-), one of the main substrates in the anaerobic ammonium oxidation (anammox) process, has the potential to inhibit anammox bacteria. The sensitivity of anammox cells with different energy status to NO2- was evaluated, and addition of nitrate (NO3-) inhibition on the basis of narK gene with the putative function of facilitating NO3-/NO2- antiporter. The results showed that the resistance of anammox bacteria to NO2- inhibition follows the order: active-cells > starved-cells > resting-cells > starved-/resting-cells. Anammmox resting cells have increasing tolerance to NO2- in the pH range from 7.0 to 7.5. Dissipating the proton gradient by using carbonyl cyanide m-chlorophenyl hydrazine (CCCP) caused severe inhibition at all pH values including pH = 7.5. Addition of NO3- enabled activity recovery of NO2--inhibited anammox bacteria regardless of whether the proton gradient was disrupted or not, supporting the hypothesis of NO3--dependent detoxification via a secondary transport system.

The Skp1 Homologs SKR-1/2 Are Required for the Caenorhabditis elegans SKN-1 Antioxidant/Detoxification Response Independently of p38 MAPK.

SKN-1/Nrf are the primary antioxidant/detoxification response transcription factors in animals and they promote health and longevity in many contexts. SKN-1/Nrf are activated by a remarkably broad-range of natural and synthetic compounds and physiological conditions. Defining the signaling mechanisms that regulate SKN-1/Nrf activation provides insights into how cells coordinate responses to stress. Nrf2 in mammals is regulated in part by the redox sensor repressor protein named Keap1. In C. elegans, the p38 MAPK cascade in the intestine activates SKN-1 during oxidative stress by promoting its nuclear accumulation. Interestingly, we find variation in the kinetics of p38 MAPK activation and tissues with SKN-1 nuclear accumulation among different pro-oxidants that all trigger strong induction of SKN-1 target genes. Using genome-wide RNAi screening, we identify new genes that are required for activation of the core SKN-1 target gene gst-4 during exposure to the natural pro-oxidant juglone. Among 10 putative activators identified in this screen was skr-1/2, highly conserved homologs of yeast and mammalian Skp1, which function to assemble protein complexes. Silencing of skr-1/2 inhibits induction of SKN-1 dependent detoxification genes and reduces resistance to pro-oxidants without decreasing p38 MAPK activation. Global transcriptomics revealed strong correlation between genes that are regulated by SKR-1/2 and SKN-1 indicating a high degree of specificity. We also show that SKR-1/2 functions upstream of the WD40 repeat protein WDR-23, which binds to and inhibits SKN-1. Together, these results identify a novel p38 MAPK independent signaling mechanism that activates SKN-1 via SKR-1/2 and involves WDR-23.

Glutathione protects Candida albicans against horseradish volatile oil

Horseradish essential oil (HREO; a natural mixture of different isothiocyanates) had strong fungicide effect against Candida albicans both in volatile and liquid phase. In liquid phase this antifungal effect was more significant than those of its main components allyl, and 2-phenylethyl isothiocyanate. HREO, at sublethal concentration, induced oxidative stress which was characterized with elevated superoxide content and up-regulated specific glutathione reductase, glutathione peroxidase, catalase and superoxide dismutase activities. Induction of specific glutathione S-transferase activities as marker of glutathione (GSH) dependent detoxification was also observed. At higher concentration, HREO depleted the GSH pool, increased heavily the superoxide production and killed the cells rapidly. HREO and the GSH pool depleting agent, 1-chlore-2,4-dinitrobenzene showed strong synergism when they were applied together to kill C. albicans cells. Based on all these, we assume that GSH metabolism protects fungi against isothiocyanates.

Proline and Betaine Upregulated Glutathione Dependent Detoxification Enzymes in Tolerant Maize Seedlings under Saline Stress

Glutathione S-transferases (GSTs, EC: 2.5.1.18) and Glyoxalases [Glyoxalase-I (Gly-I, EC: 4.4.1.5) and glyoxalase-II (Gly-II, EC: 3.1.2.6)] are major glutathione dependent detoxification as well as important antioxidants enzymes in plants. On the other hand, proline and betaine are important osmoprotectants in plants under abiotic stresses including salinity. In this study, roles of GST, Gly-I, Gly-II and glutathione were investigated on cytotoxic metabolites in presence of 15 mM proline and betaine under 16 dSm-1 salinity in leaves of seedlings of a saline tolerant maize inbred CZ-10 to understand the underlying saline tolerant mechanism. The salinity stress increased the contents of H2O2, melondialdehyde (MDA), methylglyoxal (MG) along with decreased reduced glutathione (GSH) and glutathione redox state over control. The activities of GST and Gly-I increased under saline stress. However, activity of Gly-II decreased with stress duration. The application of proline and betaine in saline water reduced the contents of H2O2, MDA and MG. Conversely, proline and betaine increased the activities of GST, Gly-I and Gly-II, and GSH and glutathione-redox state over salinity stress. The western blotting of the soluble protein also suggested the accumulation of maize GST in leaf under salinity stress. The accumulation of GST along with reduced contents of H2O2 and MDA suggested its detoxification roles on organic hydroperoxides under saline stress. The higher activities of Gly-I and Gly-II concurrently with lower content of MG indicated their protective roles from cytotoxic MG. Considering all, this study concluded that both proline and betaine provided protective roles in maize seedlings under salinity stress by maintaining GSH and its related detoxification enzymes.

The defense potential of glutathione–ascorbate dependent detoxification pathway to sulfur dioxide exposure in Tagetes erecta

Sulfur dioxide (SO2) exposure is associated with increased risk of various damages to plants. However, little is known about the defense response in ornamental plants. In this study, an artificial fumigation protocol was carried out to study the defense potential of the glutathione (GSH)–ascorbate (AsA) dependent detoxification pathway to SO2 exposure in Tagetes erecta. The results show that when the plants were exposed to different doses of SO2 (0, 15, 30, 50 or 80mgm−3) for different times (6, 12, 18, 24 or 33h), SO2 induced oxidative stress was confirmed by the increased hydrogen peroxide (H2O2), malondialdehyde (MDA) and relative conductivity of membrane (RC) in a dose-dependent manner for different exposure times. However, the increased levels for H2O2, MDA and RC were not significant vis-a-vis the control when SO2 doses and exposure times were lower than 15mgm−3/33h, 30mgm−3/24h or 50mgm−3/12h (p>0.05). The results could be explained by the increases in the content of reduced form of glutathione (GSH), total glutathione (TGSH), ascorbate (AsA), ratio of GSH/GSSG (oxidized form of glutathione), activities of ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione S-transferases (GST). On the other hand, exposure to higher doses of SO2 and longer exposure times, the values of the GSH–AsA dependent antioxidative indices decreased significantly (p<0.01), manifested by increased levels of H2O2. Furthermore, the levels of H2O2, MDA and RC varied little when SO2 doses and exposure times reached a ‘critical’ value (50mgm−3/24h). The defense ability of T. erecta to SO2 reached nearly extremity. To summarize, the response of T. erecta to elevated SO2 was related to higher H2O2 levels. GSH–AsA dependent detoxification pathway played an important role in against SO2-induced toxicity, although the defense response could not sufficiently alleviate oxidative damage when SO2 doses and exposure times reached critical value.

In silico molecular modeling and docking studies on the leishmanial tryparedoxin peroxidase

Leishmaniasis is one of the most common form of neglected parasitic disease that affects about 350 million people worldwide. Leishmanias have a trypanothione mediated hydroperoxide metabolism to eliminate endogenous or exogenous oxidative agents. Both of 2-Cys peroxiredoxin (Prx) and glutathione peroxidase type tryparedoxin peroxidase (Px) are the terminal enzymes in the trypanothione dependent detoxification system. Therefore absence of trypanothione redox system in mammals and the sensitivity of trypanosomatids against oxidative stress, enzymes of this pathway are drug targets candidates. In this study, 3D structure of tryparedoxin peroxidase (2-Cys peroxiredoxin type) from Leishmania donovani (LdTXNPx) was described by homology modeling method based on the template of tryparedoxin peroxidase from Crithidia fasciculata and selected compounds were docked to the active site pocket. The quality of the 3D structure of the model was confirmed by various web based validation programs. When compared secondary and tertiary structure of the model, it showed a typical thioredoxin fold containing a central beta-sheet and three alpha-helices. Docking study showed that the selected compound 2 (CID 16073813) interacted with the active site amino acids and binding energy was -118.675 kcal/mol.

Cyanamide-mediated Inhibition of N-acetyltransferase 1

Cyanamide has been used for decades for medical intentions in the treatment of alcoholism and for agricultural purposes as a plant growth regulator and bud-breaking agent. Its therapeutic effect is mediated by reversible inhibition of aldehyde dehydrogenase and it was reported to be metabolized in vivo mainly via coenzyme A dependent N-acetylation by N-acetyltransferases. Although described to be a substrate for N-acetyltransferases (NATs), cyanamide has a different molecular structure to arylamines and hydrazines, the preferred substrates for N-acetyltransferases. Therefore, a more detailed investigation of its interrelations with N-acetyltransferases was performed. We analyzed the impact of cyanamide on NAT1 activities of human monocytes (monocytic THP-1 cells) using the classical substrate p-aminobenzoic acid. We found that a 24h treatment with physiologically relevant concentrations of cyanamide decreased the NAT1 activity significantly. Based on this observation we performed additional experiments using recombinant human NAT1 and NAT2 to achieve further insights. In detail a significant dose- and time-dependent inhibition of NAT1 activity was observed for 100 and 1000μM cyanamide using recombinant human NAT1*4. However, cyanamide did not inhibit recombinant NAT2*4. Experiments testing cyanamide as substrate did not provide evidence that cyanamide is metabolized via coenzyme A dependent N-acetylation in vitro by human NAT1 or NAT2, THP-1 or human liver cytosol. Therefore we can conclude that the observed enzyme inhibition (around 50% and 25% after treatment with 0.5 and 0.25mM CA, respectively) is not based on substrate-dependent down-regulation of NAT1. Further mechanistic and kinetic studies indicated that cyanamide reacts with the active site cysteine residue of NAT1, leading to its rapid inhibition (significant inhibition after 30min and 2h for 1000 and 100μM CA, respectively). Addition of the reduction agent dithiothreitol (DTT) did not modify the effect, indicating that oxidative processes that can be reversed by 5mM DTT are not likely involved in the inhibition. Taken together our results show that cyanamide is able to inhibit NAT1 most likely via interaction with the active site cysteine residue. Thereby cyanamide might modulate NAT1 dependent detoxification and activation of arylamines.

Macroinvertebrate community response to repeated short-term pulses of the insecticide imidacloprid

Small streams in agricultural landscape can experience short and repeated pulses of fluctuating pesticide concentrations. A single pesticide pulse may not have adverse effects on macrozoobenthos species but repeated pulses may have, especially if the organisms have not yet fully recovered when the second pesticide pulse occurs. Against this background, a comprehensive indoor stream mesocosm study was carried out in order to evaluate the cumulative effects of repeated insecticide pulses on a macrozoobenthos community. Weekly 12h pulses of 12μg/L of the insecticide imidacloprid were set 3 times in 4 stream mesocosms in 2 series, one in spring and one in summer. Another 4 mesocosms served as controls. Prior to each pulse series, the mesocosms were stocked with macroinvertebrates from an uncontaminated reference stream using straw bags as attraction devices. The straw bag method proved suitable for establishing a functional macroinvertebrate community in the stream mesocosms. The caddisfly species Neureclipsis sp. reacted immediately and most sensitively after a single imidacloprid pulse whilst insect larvae such as ephemerids and dipteran larvae were negatively affected only after repeated imidacloprid pulses. Effects on insect larvae were more pronounced in the summer series most likely due to increased temperature. Abundance was a less sensitive endpoint than sublethal endpoints such as emergence. The results of the study underline that pulse effects are driven by a number of variables like pulse height, pulse duration, number of pulses, time in between pulses and by the species and live stage specific ability of temperature dependent detoxification which all should be taken into account in the risk assessment of pesticides.

Phytoremediation of organic xenobiotics – Glutathione dependent detoxification in Phragmites plants from European treatment sites

Studies on the uptake of several organic xenobiotics and on their subsequent conjugation to biomolecules have been performed to elucidate the use of reed plants in phytoremediation of polluted water. Phragmites australis plants were able to accumulate organic xenobiotics in their rhizomes. The uptake was correlated to the logKOW and pKa of the xenobiotics and highest with compounds exhibiting logKOWs between 1 and 3. Detoxification of xenobiotics was demonstrated when the activity of glutathione S-transferase was determined in plants from various treatment sites. Enzyme activities were strongly dependent on the provenience of the plant and the history of the stand. Detoxification enzymes were also inducible. Naphthylic acetic acid (NAA), 2,4-dichlorophenol and BION were tested as potential inducers. BION was able to induce the GST activity 5-fold, albeit only for a short period of hours. The mechanism of induction and the flexibility of the detoxification system of certain ecotypes of reed toward stress or the pollution level will require further investigation.

A physiologically based biokinetic model for estragole in rats providing more detailed insight in dose dependent bioactivation and detoxification

A physiologically based biokinetic model for estragole in rats providing more detailed insight in dose dependent bioactivation and detoxification