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Введение. Вальпроат натрия влияет на метаболизм гамма-аминомасляной кислоты, предотвращая развитие эпилептических приступов. Многие метаболиты вальпроата натрия потенцируют его клинический эффект, однако, именно они могут приводить к развитию хронической интоксикации с поражением печени. Цель - оценка протекторного эффекта адеметионина (гептрала), цитофлавина и дигидрокверцетина у крыс, подвергнутых токсическому влиянию вальпроата натрия (депакина). Методика. Животным контрольной группы вводили вальпроат натрия в токсической дозе 600 мг/кг интрагастрально 1 раз в день в течение 28 сут. Крысам опытных групп вводили вальпроат натрия в той же дозе с одновременным введением адеметионина, цитофлавина или дигидрокверцетина. Животные выводились из эксперимента на 7-, 14-, 21- и 28-е сут. В гомогенате печени определяли стандартными спектрофотометрическими методами концентрацию диеновых конъюгатов, малонового диальдегида, восстановленного глутатиона и активность ферментов метаболизма глутатиона: глутатионредуктазы, глутатионпероксидазы и глутатионтрансферазы. Результаты. При токсическом воздействии вальпроата натрия в ткани печени развивались процессы гиперпероксидации липидов, которые подтверждались значительным повышением уровня диеновых конъюгатов и малонового диальдегида. Концентрация глутатиона снижалась начиная с 14-х сут эксперимента. Активность глутатионредуктазы повышалась к 14-м сут, а затем к 28-м сут практически достигала значений интактных животных. Активность глутатионтрансферазы начинала снижаться с 14-х сут и сохранялась низкой до конца исследования. Активность глутатионпероксидазы повышалась с 14-х сут эксперимента, достигая максимума к 28-м сут. Протекторный эффект адеметионина при токсическом воздействии вальпроата натрия выражался в снижении содержания диеновых конъюгатов и малонового диальдегида. Одновременно происходила активация системы глутатиона: повышалась активность глутатионтрансферазы и глутатионредуктазы. Антиокислительное действие цитофлавина проявлялось в основном на 1-й нед эксперимента: концентрация диеновых конъюгатов и малонового диальдегида снижалась, активность глутатионредуктазы и глутатионтрансферазы повышалась. Дигидрокверцетин также оказывал антиокислительное действие: концентрация диеновых конъюгатов и малонового диальдегида снижалась. Активность глутатионредуктазы и глутатионтрансферазы увеличивалась по сравнению с контрольными животными. Заключение. Вальпроат натрия в токсической дозе приводит к статистически значимому повышению уровня продуктов перекисного окисления липидов и снижению антиокислительной защиты системы глутатиона в печени крыс. Адеметионин, цитофлавин и дигидрокверцетин способствуют восстановлению баланса системы перекисного окисления липидов и антиоксидантной защиты в печени крыс при токсическом влиянии вальпроата натрия. Наиболее перспективным препаратом защиты является адеметионин, так как он наиболее существенно снижает концентрацию диеновых конъюгатов, малонового диальдегид и повышает активность системы глутатиона на протяжении 28 сут эксперимента. Background. Sodium valproate affects metabolism of gamma-aminobutyric acid to prevent the development of epileptic seizures. Many valproic acid metabolites potentiate its clinical effect; however, specifically these metabolites may result in chronic intoxication with liver damage. Aim: To evaluate the protective effect of ademetionine (Heptral), cytoflavin, and dihydroquircetin in rats exposed to the toxic action of sodium valproate (Depakene). Methods. Animals of the control group were administered valproate sodium at a toxic dose of 600 mg/kg, intragastrically. Rats of the experimental group were administered valproate sodium at a dose of 600 mg/kg with simultaneous administration of ademetionine, cytoflavin and dihydroquercetin. The duration of drug administration in all groups was 7 days, 14 days, 21 days, or 28 days. The contents of diene conjugates, malonic dialdehyde, reduced glutathione, and the activity of glutathione reductase, glutathione peroxidase, and glutathione transferase were measured in liver homogenates with standard spectrophotometric methods. Results. Under the toxic effects of valproate sodium in liver tissue, a significant increase in the contents of diene conjugates and malonic dialdehyde was found. The glutathione concentration decreased starting from day 14 of the experiment. The glutathione reductase activity increased by day 14 and practically reached the values of intact animals by day 28. The glutathione transferase activity began decreasing on day 14 and remained low until the end of study. The glutathione peroxidase activity increased starting from day 14 and reached maximum by day 28. The protective effect of ademetionine was manifested by a decrease in the contents of diene conjugates and malonic dialdehyde. There was a simultaneous activation of the glutathione system evident as increased activities of glutathione transferase and glutathione reductase. An antioxidant effect of cytoflavin was manifested mainly at the 1st wk of the experiment. The concentration of diene conjugates and malonic dialdehyde decreased, the activity of glutathione reductase increased, and the activity of glutathione transferase increased. Dihydroquercetin also had an antioxidant effect since the concentration of diene conjugates and malonic dialdehyde decreased. The activity of glutathione reductase and glutathione transferase increased. Conclusion. Sodium valproate significantly increased concentrations of lipid peroxidation products and impaired the glutathione antioxidant defense in the rat liver. Ademetionine, cytoflavin and dihydroquercetin help to restore the balance of lipid peroxidation and antioxidant protection in the rat liver during the toxic effects of valproate sodium. The most effective drug was ademetionine, since within 28 days, it significantly decreased the concentration of diene conjugates and malonic dialdehyde, and it increased the activity of the glutathione system.

Chronic catarrhal gingivitis (CCG) is a common stomatological disease among children suffering from diabetes mellitus. The prooxidant-antioxidant system state plays an important role in occurrence and course of chronic catarrhal gingivitis. Antioxidant protective system (AOPS) is presented by enzymatic and non-enzymatic components. Though, distribution between them is rather conditional, since the action of enzymes-antioxidants is closely connected and balanced inside and with non-enzymatic components.Glutathione system including glutathione proper and three enzymes (glutathione peroxidase – GP, glutathione reductase – GR, glutathione transferase – GT) binds free radicals, reduce oxides, lipid peroxide oxidation products, the products of membrane phospholipids, proteins, nucleic acids, and removes them from the body in the form of non-toxic conjugates. A number of methods to treat chronic catarrhal gingivitis in children including the treatment of the background of diabetes mellitus was proposed the scientists. Meanwhile, the search of new methods which able to produce a positive effect on the periodontal tissue state and regulate the body antioxidant system remains an urgent issue today. The aim of the study is to investigate the effect of our suggested therapeutic-preventive complex on the state of glutathione system and glutathione-dependent enzymes of the oral fluid in children with chronic catarrhal gingivitis accompanied by diabetes mellitus. Fifty children suffering from CCG aged from 12 to 16 years were examined and treated for stomatological issues. They were admitted to a hospital at the Children Endocrinological Department of the Municipal Institution “Regional Pediatric Clinical Hospital” in Chernivtsi due to diabetes. Eighteen children of the same age suffering from CCG without somatic pathology available were examined as well. Children with diabetes mellitus were divided into two equal groups. Thus, the first group (1st group is control one) included 18 somatically healthy children with CCG. The second group (the 2nd – the group of comparison) included 25 children with accomponied diabetes mellitus. Children from the 1st and the 2nd groups were treated according to the protocols of the Ministry of Health of Ukraine concerning giving medical aid on specialty «Pediatric Therapeutic Dentistry». The third group (the 3d – the main one) included 25 children with CCG and diabetes mellitus, who received oral complex medicine "Quertulin" in the dose of 1 tablet 3 times a day, drops «Imupret» in the dose of 25 drops 3 times a day and poly-vitamin drug "Pikovit" in the dose of 1 tablet once a day after meals till complete resorption in the oral cavity during 20 days. Topical irrigation of the oral cavity with the solution containing tooth wash "Exodent" (1 teaspoon per ¼ glass of water after every meals and cleaning teeth during 1-2 minutes). Such kind of therapy makes the foundation to prevent the formation of free radicals and decreases the concentration of LPO products. In all the groups the treatment was proceeded by professional oral hygiene, sanitization (if necessary), teaching children the method of cleaning the teeth, the floss use, choosing the means of individual oral hygiene. Effect of CCG treatment was assessed by means of comparison of the initial parameters (І examination) with the parameters immediately after its completion (ІІ examination), in 1 (ІІІ examination), 3 (IV examination) and 6 (V examination) months. The content of SH-groups was determined by means of Ellman’s reagent; activity of glutathione-S-transferase – by the amount of accumulated conjugate by means of W.H. Habig et al. method; activity of glutathione reductase – by a reduced amount of NADFH2 in the reaction medium by R.E. Pinto, V. Bartley method; activity of glutathione peroxidase – by V.M. Mogen method; activity of glucose-6-phosphate dehydrogenase – by Kornberg and Khoreker method modified by Zakharyin; level of ceruloplasmin – by the modified Revin’s method. During the first examination the parameter of reduced glutathione in the examined groups of children with diabetes mellitus did not differ reliably, but it was considerably lower than that of healthy children (р

The ecological potential of microalgae for purification of aquatic and soil ecosystems and natural restoration of their homeostatic functional state is considered to be high due to the rapid growth and development of algae, their labile and dynamic metabolism and simple growth conditions. The aim of present work was to study the effectiveness of Chlorella as a potential bioremediator to reduce the toxic effects of pesticides, roundup and chlorpyrifos after their individual and complex influence on zebrafish Danio rerio. The effect of environmental concentrations of roundup (15 μg⋅L-1) and chlorpyrifos (0,1 μg⋅L-1) provoked partial depletion of the cell thiols pool when compared to the control, which appeared as a decrease in glutathione transferase activity (under combined exposure) and total glutathione concentration. A decrease in the level of total antioxidant capacity, which was consistent with an increase in the level of reactive oxygen species in the liver tissue was also shown. Meanwhile, the studied organophosphate pesticides didn’t cause severe signs of neurotoxicity, but activated acetylcholinesterase in line with no visual manifestations of locomotion reactions. Chlorpyrifos determined an increase in the concentration of methylglyoxal and the most noticeable sign of endocrine disruption from all studied groups in terms of vitellogenin concentration. Principal component analysis allowed to identify a separate localization of each of the studied groups and the interim position of animals after combined exposure when compared to the individual action. The introduction of Chlorella vulgaris in the exposure media in the amount of about 100 thousand cells / dm3 did not show a significant corrective effect on the toxicity of pesticides for non-target species Danio rerio, which doesn’t exclude the positive impact of algae on the functioning of the ecosystem in general and requires a more detailed analysis.

The research was performed to delineate how β-sitosterol laurate (β-SLE) consumption influenced serum and hepatic lipids. The results showed that 220 mg/5 mL oil/kg body weight of β-SLE robustly reduced serum total triglyceride and cholesterol levels and the epididymal adipocyte size, and efficiently protected hepatic polyunsaturated fatty acids against lipid peroxidation through superoxide dismutase and glutathione transferase activity enhancement and malondialdehyde level reduction. Based on the changes of fecal cholesterol contents, fecal and hepatic bile acid (BAs) levels, and related protein expression, it was concluded that the mechanisms for lowering serum cholesterol by β-SLE involved (i) the enhanced excretion of fecal cholesterol via down-regulation of intestinal Niemann-Pick C1-like 1 protein; (ii) the increased conversion from cholesterol to primary BAs via up-regulation of cholesterol-7α-hydroxylase and sterol 27-hydroxylase, which was induced by the reduced BAs reabsorption through up-regulating ileal apical sodium-dependent bile acid transporter and ileal bile acid-binding protein.

Although the mortality rate of papillary thyroid carcinoma (PTC) is relatively low, the recurrence rates of PTC remain high. The high recurrence rates are related to the difficulties in treatment. Gene expression profiles has provided novel insights into potential therapeutic targets and molecular biomarkers of PTC. The aim of the present study was to identify mRNA signatures which may categorize PTCs into high-and low-risk subgroups and aid with the predictions for prognoses. The mRNA expression profiles of PTC and normal thyroid tissue samples were obtained from The Cancer Genome Atlas (TCGA) database. Differentially expressed mRNAs were identified using the ‘EdgeR’ software package. Gene signatures associated with the overall survival of PTC were selected, and enrichment analysis was performed to explore the biological pathways and functions of the prognostic mRNAs using the Database for Visualization, Annotation and Integration Discovery. A signature model was established to investigate a specific and robust risk stratification for PTC. A total of 1,085 differentially expressed mRNAs were identified between the PTC and normal thyroid tissue samples. Among them, 361 mRNAs were associated with overall survival (P

Introduction: Periodontal disease (PD) is an inflammatory condition, which leads to tooth loss and promotes a systemic inflammatory state that can aggravate the nerve degeneration. As laser therapy may stimulate regeneration, this study aimed to evaluate the effect of the low-level laser (LLL) on peripheral nerve regeneration under the systemic inflammatory condition of PD. Methods: Thirry-two male rats were used, distributed in 4 groups: nerve injury (NIG); periodontal disease with nerve injury (PDNI); nerve injury and treatment (TNIG); periodontal disease with nerve injury and treatment (PDNIT). On the 7th day of the experiment, the animals had ligatures placed around the lower first molars. On the 22nd day, they underwent peripheral nerve damage, and on the 25th day, the LLL treatment was initiated, performed for two weeks. The sciatic functional index (SFI) was evaluated with subsequent euthanasia of all the animals on the 37th day of the experiment. The sciatic nerve was collected for morphological and oxidative stress analysis and the hemi jaws for radiographic analysis. Results: Regarding the SFI, there was no difference among the groups in the first evaluation (EV) pre-injury; as for theEV2, after injury, all the groups presented a decrease in these values, which remained in post-treatment. For the morphology of the PDNI, nerve tissue presented larger diameter fibers, whereas, for NIT and PDNIT, fibers had smaller diameters with endoneurial organization. When it comes to the antioxidant system, there was an increase in protein concentration, higher superoxide activity, and decreased glutathione transferase activity in the treated groups. Catalase and cholinesterase did not differ between the groups, and lipoperoxidation (LPO) increased in the PD groups. For the mandible radiographic analysis, it was possible to verify the induction of PD. Conclusion: As for the used parameters, the low-level laser was not effective in increasing the nociceptive threshold, but it contributed to the regeneration of nerve fibers, although the inflammation was still present in the site. However, the treatment was effective in protecting cells against oxidative damage due to increased SOD and increased protein, although the decrease in GST demonstrates the inhibition of this stage of the antioxidant system.

Aedes aegypti is the principal mosquito vector for many arthropod-borne viruses (arboviruses) including dengue, chikungunya, and Zika. In the United States, excessive permethrin use has led to a high frequency of resistance in mosquitoes. Insecticide resistance is a significant obstacle in the struggle against vector-borne diseases. To help overcome metabolic resistance, synergists that inhibit specific metabolic enzymes can be added to formulated pyrethroid products. Using modified CDC bottle bioassays, we assessed the effect of three inhibitors (piperonyl butoxide (PBO), which inhibits oxidase activity, S.S.S-tributyl phosphorotrithioate (DEF), which inhibits esterase activity, and diethyl maleate (DM), which inhibits glutathione transferase activity) + permethrin. We performed these against 20 Florida Ae. aegypti populations, all of which were resistant to permethrin. Our data indicated that 11 out of 20 populations (55%) exhibited metabolic resistance. Results revealed 73% of these populations had significant increases in mortality attributed to DEF + permethrin, 64% to PBO + permethrin, and 55% to DM + permethrin compared to permethrin alone. Currently, PBO is the only metabolic enzyme inhibitor added to formulated pyrethroid products used for adult mosquito control. Our results suggest that the DEF and DM inhibitors could also be useful additives in permethrin products, especially against metabolically resistant Ae. aegypti mosquitoes. Moreover, metabolic assays should be conducted to better inform mosquito control programs for designing and implementing integrated vector management strategies.

Clubroot disease is one of the major diseases affecting Brassica crops, especially Chinese cabbage (Brassica rapa L. ssp. pekinensis), which is known to be highly susceptible to the disease. In this study, the obligate biotrophic protist Plasmodiophora brassicae Woronin was used to infect the roots of Chinese cabbage seedlings. The disease symptoms were noticeable at 28 and 35 days after inoculation (DAI) in the susceptible (CM) line. Using isobaric tags for relative and absolute quantitation (iTRAQ) analysis, a total of 5,003 proteins of differential abundance were identified in the resistant/susceptible lines, which could be quantitated by dipeptide or polypeptide segments. Gene ontology (GO) analysis indicated that the differentially expressed proteins (DEPs) between the susceptible (CM) and resistant (CCR) lines were associated with the glutathione transferase activity pathway, which could catalyze the combination of glutathione and other electrophilic compounds to protect plants from disease. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the DEPs may be significantly enriched cytokinin signaling or arginine biosynthesis pathways, both of which are responses to stimuli and are plant defense reactions. The cytokinins may facilitate cell division in the shoot, resulting in the hypertrophy and formation of galls and the presentation of typical clubroot symptoms. In this study, the proteomic results provide a new perspective for creating germplasm resistance to P. brassicae, as well as a genetic basis for breeding to improve Chinese cabbage.

Background Herbicides inhibiting the synthesis of very long-chain fatty acids (HRAC group K3 , WSSA group 15), such as flufenacet, play an important role in weed management strategies, particularly when herbicide resistance to inhibitors with other modes of action, such as acetolactate synthase or acetyl coenzyme A carboxylase (ACCase), has already evolved. So far, only a few cases of resistance towards inhibitors of the synthesis of very long-chain fatty acids have been described. In this study, we characterized the level of flufenacet resistance in several Lolium spp. field populations and investigated the resistance mechanism. Results The screening for flufenacet resistance revealed the ability of Lolium spp. populations from several continents to survive flufenacet treatments at and above the field rate. This study demonstrates the way in which flufenacet is detoxified in resistant weed populations. Glutathione was found to be conjugated to flufenacet in Lolium spp. seedlings, and there was evidence that glutathione transferase activity was enhanced in protein extracts from flufenacet-resistant seedlings. A significant correlation was found between the resistance factor obtained by biotests and the degradation half-time of flufenacet in ryegrass plants obtained by high-performance liquid chromatography (HPLC). Conclusion At present, flufenacet resistance is not widespread; however, in certain Lolium spp. populations resistance levels could reach agronomic relevance due to detoxification by glutathione transferases. In Europe especially, only a few herbicide modes of action are registered for the control of Lolium spp. and therefore it is becoming increasingly important to apply best management practices to prevent the spread of flufenacet resistance. © 2019 Society of Chemical Industry.

There is limited information on the impact of dietary supplementation with separate rumen-protected (RP) amino acids (AA), or with their combination, on ewes’ oxidative status. Sixty ewes were divided into five groups, C: basal diet (control), M: basal diet + 6 g/ewe RP methionine, L: basal diet + 5 g/ewe RP lysine, LML: basal diet + 6 g methionine and 5 g lysine/ewe, and HML: basal diet + 12 g methionine + 5 g lysine/ewe. Milk’s fat content increased in RP-AA fed ewes, while that of protein in M and L only. In blood plasma, the malondialdehyde (MDA) content was reduced in the M, LML, and HML compared to C-fed ewes. An increase in glutathione transferase activity in the blood plasma of the M and LML compared to the C and HML-fed ewes were found. In milk, lower values of the ferric reducing ability of plasma (FRAP) in the LML and HML-fed ewes and of 2,2′-Azino-bis 3-ethylbenzthiazoline-6-sulfonic acid (ABTS) in LML only, were found. Lysine increased milk’s FRAP values and MDA content. Both L and HML diets increased milk’s protein carbonyls content. Methionine improves the organism’s oxidative status, without adversely affecting milk’s oxidative stability. Lysine dietary inclusion affects negatively the oxidative stability of milk.

4-Hydroxynonenal (HNE) is a major aldehydic product of lipid peroxidation known to exert several biological effects. Normal and malignant cells of the same origin express different sensitivity to HNE. We used human osteosarcoma cells (HOS) in different stages of differentiation in vitro, showing differences in mitosis, DNA synthesis, and alkaline phosphatase (ALP) staining. Differentiated HOS cells showed decreased proliferation (3H-thymidine incorporation), decreased viability (thiazolyl blue tetrazolium bromide-MTT), and increased apoptosis and necrosis (nuclear morphology by staining with 4′,6-diamidino-2-phenylindole-DAPI). Differentiated HOS also had less expressed c-MYC, but the same amount of c-FOS (immunocytochemistry). When exposed to HNE, differentiated HOS produced more reactive oxygen species (ROS) in comparison with undifferentiated HOS. To clarify this, we measured HNE metabolism by an HPLC method, total glutathione (GSH), oxidized GSH (ox GSH), glutathione transferase activity (GST), proteasomal activity by enzymatic methods, HNE-protein adducts by genuine ELISA and fatty acid composition by GC-MS in these cell cultures. Differentiated HOS cells had less GSH, lower HNE metabolism, increased formation of HNE-protein adducts, and lower proteasomal activity, in comparison to undifferentiated counterpart cells, while GST and oxGSH were the same. Fatty acids analyzed by GC-MS showed that there is an increase in C20:3 in differentiated HOS while the amount of C20:4 remained the same. The results showed that the cellular machinery responsible for protection against toxicity of HNE was less efficient in differentiated HOS cells. Moreover, differentiated HOS cells contained more C20:3 fatty acid, which might make them more sensitive to free radical-initiated oxidative chain reactions and more vulnerable to the effects of reactive aldehydes such as HNE. We propose that HNE might act as natural promotor of decay of malignant (osteosarcoma) cells in case of their differentiation associated with alteration of the lipid metabolism.

BACKGROUND: Gongronema latifolium leaf is used traditionally to treat diabetes and other diseases. The present study aimed to provide the modulatory effect of G. latifolium on hyperglycemia, inhibitory effect of redox imbalance and inflammation in alloxan-induced nephropathy in Wistar rats. METHODS: Alloxan monohydrate was used to induce diabetes by an intraperitoneal injection of (150 mg/kg). Three diabetic groups were administered aqueous leaf extract of G. latifolium at 6.36, 12.72 and 25.44 mg/kg bodyweight (BW) respectively; a group was administered with metformin (5 mg/kg BW), while the other two were served as positive and negative control. Thereafter, fasting blood glucose, antioxidant enzymes, malondialdehyde (MDA) level, interleukin 2 and 6 were determined. RESULTS: G. latifolium leaf significantly (p

Jasmonic acid (JA) is an important plant hormone associated with plant–pathogen defense. To study the role of JA in plant–fungal interactions, we applied a JA biosynthesis inhibitor, sodium diethyldithiocarbamate (DIECA), on wheat leaves. Our results showed that application of 10 mM DIECA 0–2 days before inoculation effectively induced resistance to powdery mildew (Bgt) in wheat. Transcriptome analysis identified 364 up‐regulated and 68 down‐regulated differentially expressed genes (DEGs) in DIECA‐treated leaves compared with water‐treated leaves. Gene ontology (GO) enrichment analysis of the DEGs revealed important GO terms and pathways, in particular, response to growth hormones, activity of glutathione metabolism (e.g., glutathione transferase activity), oxalate oxidase, and chitinase activity. Gene annotaion revealed that some pathogenesis‐related (PR) genes, such as PR1.1, PR1, PR10, PR4a, Chitinase 8, beta‐1,3‐glucanase, RPM1, RGA2, and HSP70, were induced by DIECA treatment. DIECA reduced JA and auxin (IAA) levels, while increased brassinosteroid, glutathione, and ROS lesions in wheat leaves, which corroborated with the transcriptional changes. Our results suggest that DIECA can be applied to increase plant immunity and reduce the severity of Bgt disease in wheat fields.

In olfaction, to preserve the sensitivity of the response, the bioavailability of odor molecules is under the control of odorant-metabolizing enzymes (OMEs) expressed in the olfactory neuroepithelium. Although this enzymatic regulation has been shown to be involved in olfactory receptor activation and perceptual responses, it remains widely underestimated in vertebrates. In particular, the possible activity of OMEs in the nasal mucus, i.e. the aqueous layer that lined the nasal epithelium and forms the interface for airborne odorants to reach the olfactory sensory neurons, is poorly known. Here, we used the well-described model of the mammary pheromone (MP) and behavioral response in rabbit neonates to challenge the function of nasal mucus metabolism in an unprecedented way. First, we showed, in the olfactory epithelium, a rapid glutathione transferase activity toward the MP by ex vivo real-time mass spectrometry (PTR-MS) which supported an activity in the closest vicinity of both the odorants and olfactory receptors. Indeed and second, both the presence and activity of glutathione transferases were evidenced in the nasal mucus of neonates using proteomic and HPLC analysis respectively. Finally, we strikingly demonstrated that the deregulation of the MP metabolism by in vivo mucus washing modulates the newborn rabbit behavioral responsiveness to the MP. This is a step forward in the demonstration of the critical function of OMEs especially in the mucus, which is at the nasal front line of interaction with odorants and potentially subjected to physiopathological changes.

Juvenile tegu lizards (Salvator merianae) experience gradual and mild temperature changes from autumn to winter in their habitat. This tropical/subtropical reptile enter a state of dormancy, with an 80% reduction in metabolic rate, that remains almost constant during winter. The redox metabolism in non-mammalian vertebrates that hibernate under such distinguished conditions is poorly understood. We analyzed the redox metabolism in the intestine of juvenile tegus during different stages of their first annual cycle. The effect of food deprivation (in spring) was also studied to compare with fasting during hibernation. Both winter dormancy and food deprivation caused decreases in reduced glutathione levels and glutathione transferase activity. While glutathione peroxidase and glutathione transferase activities decreased during winter dormancy, as well as glutathione (GSH) levels, other antioxidant enzymes (catalase, superoxide dismutase and glutathione reductase) remained unchanged. Notably, levels of disulfide glutathione (GSSG) were 2.1-fold higher in late autumn, when animals were in the process of depressing metabolism towards hibernation. This increased “oxidative tonus” could be due to a disruption in NADPH-dependent antioxidant systems. In dormancy, GSSG and lipid hydroperoxides were diminished by 60–70%. The results suggest that the entrance into hibernation is the main challenge for the redox homeostasis in the intestine of juvenile tegus.

Posttraumatic stress disorder (PTSD) is a prevalent psychiatric disorder and sometimes deadly consequence of exposure to severe psychological trauma. However, there has been little known about the definitive molecular changes involved in determining vulnerability to PTSD. In the current study, we used proteomics to quantify protein changes in the hippocampus of foot shocks rats. A total of 6151 proteins were quantified and 97 proteins were significantly differentially expressed. The protein-protein interaction (PPI) analysis showed that oxidation-reduction process and glutathione homeostasis may be the potential key progress of being vulnerable to PTSD. The Gene Ontology analysis revealed enriched GO terms in the protein groups of Susceptible group vs Control group rats for glutathione binding,oligopeptide binding,modified amino acid binding,and glutathione transferase activity for their molecular functions (MF) and in the process of cellular response to toxic substance,xenobiotic metabolic process, urea metabolic process, and response to drug for the biological process (BP).SIGNIFICANCE:In recent years, there has been a growing interest in mental illness associated with trauma exposure. We found that stress susceptibility was associated with increased expression of arginase 1 indicated as a potential treatment target. Our results also proposed that carbonic anhydrases 3 could be a biomarker for the development of PTSD. This research helps to explain the potential molecular mechanism in PTSD and supply a new method for ameliorating PTSD.

The pro-and antioxidant systems of the human placenta have been studied in its central and peripheral areas in the state of dysfunction. It has been shown that the intensity of free-radical oxidation (FRO) is 24% higher (p < 0.05) in mitochondria isolated from peripheral placental areas in the case of preterm termination of pregnancy than in placental mitochondria of women with normal pregnancy ending in delivery on due dates. The values of total antioxidant activity in mitochondria isolated from the central and peripheral areas of placentae of women with preterm labor exceeded 1.5-and 1.8-fold the respective values for the placental mitochondria of women with the normal duration of pregnancy. The rate of glutathione peroxidase activity in placental mitochondria of women with preterm labor was lower than in patients with normal duration of pregnancy terminated on due dates. Higher values of intensity of both the FRO processes and the active components of thiobarbituric acid (TBA) were recorded (higher by 42% and 62%, respectively) in the postmitochondrial fraction in the peripheral area of placentae of women with spontaneous termination of pregnancy, compared with the placentae of women with uncomplicated duration of pregnancy with labor on due date. No differences have been observed in the intensity of oxidative modification of placental proteins in both the periphery and the center in the placentae of women from the studied groups. The rate of glutathione peroxidase activity in the placenta of women with spontaneous termination of pregnancy was more than twice as high as the activity of this enzyme during the first trimester of normal pregnancy and remained high during the second and third trimesters. The activity of the enzyme did not depend on its localization (center or periphery) in placentae of women participating in the study. The values of glutathione transferase activity in the placentae increased in the course of normal pregnancy but remained at the level of the first trimester in the central and peripheral areas in the case of a miscarriage at different gestational terms. Our findings allow us to suggest that oxidative stress developing in placenta from its center to periphery plays a key role in the pathogenesis of placental dysfunction, mainly, due to the glutathione-dependent component of the placental antioxidant defense.

The objective of this study was to determine the association of differentially expressed genes (DEG) in the jejunum of steers with average DMI and high or low ADG. Feed intake and growth were measured in a cohort of 144 commercial Angus steers consuming a finishing diet containing (on a DM basis) 67.8% dry-rolled corn, 20% wet distillers grains with solubles, 8% alfalfa hay, and 4.2% vitamin/mineral supplement. From the cohort, a subset of steers with DMI within ±0.32 SD of the mean for DMI and the greatest (high) and least (low) ADG were chosen for slaughter and jejunum mucosa collection ( = 8 for each group). Dry matter intake (10.1 ± 0.05 kg/d) was not different ( = 0.41) but ADG was greater in the high-gain group (2.17 and 1.72 ± 0.02 kg/d for the high- and low-ADG groups, respectively; < 0.01). A total of 13,747 genes were found to be expressed in the jejunum, of which 64 genes were differentially expressed between the 2 groups (corrected < 0.05). Ten of the DEG were upregulated in the low-ADG group and 54 were upregulated in the high-ADG group. Gene ontology analysis determined that 24 biological process terms were overrepresented ( < 0.05), including digestion, drug and xenobiotic metabolism, and carbohydrate metabolism. Additionally, 89 molecular function terms were enriched ( < 0.05), including metallopeptidase activity, transporter activity, steroid hydrolase activity, glutathione transferase activity, and chemokine receptor binding. Metabolic pathways (28 pathways) impacted by the DEG ( < 0.05) included drug and xenobiotic metabolism by cytochrome P450, carbohydrate digestion and absorption, vitamin digestion and absorption, galactose metabolism, and linoleic acid metabolism. Results from this experiment indicate that cattle with average DMI and greater ADG likely have a greater capacity to handle foreign substances (xenobiotics). It is also possible that cattle with a greater ADG have a greater potential to digest and absorb nutrients in the small intestine.

Introduction: Adequate levels of antioxidants are essential in subjects with sickle cell anaemia (SCA) to counter the effects reactive oxygen species generated during normal red cell metabolism. Objective: This study evaluated serum glutathione transferase activity and vitamin E levels in comparison with disease severity in steady state adult SCA subjects. Subjects And Methods: Five milliliters of venous blood was collected from each of 30 homozygous haemoglobin SS, 30 heterozygous haemoglobin AS and 30 haemoglobin AA subjects for glutathione transferase activity, vitamin E levels, haemoglobin phenotype and full blood count determination. The objective score of disease severity was calculated using white cell count, haemoglobin concentration and number of life time disease related complications and correlated with serum glutathione transferase activity and vitamin E levels, using the Spearman's correlation; P < 0.05 was taken as significant. Results: The median serum Glutathione transferase activity and vitamin E levels in all study participants were 22 U/L (Q1 –Q3; 15:00 U/L – 28:50 U/L) and 32:00 ug/ml (Q1 – Q3; 17.00 ug/ml – 54.00 ug/ml, respectively). Glutathione transferase enzyme activity was significantly lower in HbSS compared with HbAA (P = 0.001) and HbSS compared with HbAS study subjects (P = 0.01). Enzyme activity and vitamin E levels did not show significant correlations with disease severity in subjects with HbSS (r = 0.07; P = 0.94 and r = -0.04; P = 0.12, respectively. Conclusion: Serum glutathione transferase activity and vitamin E levels may not be predictors of disease severity in SCA patients.

Glutathione conjugation is a crucial step in xenobiotic detoxification. In the current study, we have functionally characterized an epsilon-class glutathione S-transferase (GST) from a brown planthopper Nilaparvata lugens (nlGSTE). The amino acid sequence of nlGSTE revealed approximately 36–44% identity with epsilon-class GSTs of other species. The recombinant nlGSTE was prepared in soluble form by bacterial expression and was purified to homogeneity. Mutation experiments revealed that the putative substrate-binding sites, including Phe107, Arg112, Phe118, and Phe119, were important for glutathione transferase activity. Furthermore, inhibition study displayed that nlGSTE activity was affected by insecticides, proposing that, in brown planthopper, nlGSTE could recognize insecticides as substrates.

In olfaction, to preserve the sensitivity of the response, the bioavailability of odor molecules is under the control of odorant-metabolizing enzymes (OMEs) expressed in the olfactory neuroepithelium. Although this enzymatic regulation has been shown to be involved in olfactory receptor activation and perceptual responses, it remains widely underestimated in vertebrates. In particular, the possible activity of OMEs in the nasal mucus, i.e. the aqueous layer that lined the nasal epithelium and forms the interface for airborne odorants to reach the olfactory sensory neurons, is poorly known. Here, we used the well-described model of the mammary pheromone (MP) and behavioral response in rabbit neonates to challenge the function of nasal mucus metabolism in an unprecedented way. First, we showed, in the olfactory epithelium, a rapid glutathione transferase activity toward the MP by ex vivo real-time mass spectrometry (PTR-MS) which supported an activity in the closest vicinity of both the odorants and olfactory receptors. Indeed and second, both the presence and activity of glutathione transferases were evidenced in the nasal mucus of neonates using proteomic and HPLC analysis respectively. Finally, we strikingly demonstrated that the deregulation of the MP metabolism by in vivo mucus washing modulates the newborn rabbit behavioral responsiveness to the MP. This is a step forward in the demonstration of the critical function of OMEs especially in the mucus, which is at the nasal front line of interaction with odorants and potentially subjected to physiopathological changes.

Thyroid dysfunctions, such as hypothyroidism and hyperthyroidism, are the second most prevalent endocrinopathies and are associated to reproductive disorders in men. Several genes are differentially modulated by thyroid hormones in testes and imbalances in thyroid hormone levels are also associated to alterations on sperm functionality. Imbalances on antioxidant defense mechanism and stress oxidative have been pointed out as the main factors for the impairments on male reproductive function. To clarify this issue, we investigated the expression and activity of antioxidant enzymes in testis, followed by their proteomic profile in attempt to characterize the mechanisms involved in the alterations induced by hypo- or hyperthyroidism in adult male rats. Hypothyroidism reduced the Gsr transcript expression and the activity of CAT and GSR enzymes, while the hyperthyroidism reduced the Gpx4 var2 transcript expression. Among 1082 identified proteins, 123 and 37 proteins were downregulated by hypothyroidism compared to euthyroid and hyperthyroid condition, respectively, being 36 proteins commonly reduced in both comparisons and one exclusively in hypo-hyperthyroidism comparison. A network containing 29 nodes and 68 edges was obtained in protein-protein interaction analysis and the functional enrichment analysis of differentially expressed proteins revealed significant alterations for several functions in hypo-euthyroid and hypo-hyperthyroid comparisons, such as ATP metabolic process, coenzyme binding, sperm part, peroxiredoxin activity, mitochondrial protein complex, intramolecular oxidoreductase activity, binding of sperm to zona pellucida, glutathione transferase activity, response to testosterone. Thus, there is a correlation between thyroid disorders and impaired antioxidant defense mechanism, resulting in reproductive dysfunctions, as infertility, mainly observed in hypothyroidism.

Background: Primary hyperparathyroidism (PHPT) is one of the most common endocrine disorders and defined as excessive secretion of parathormone. PHPT is a risk factor of several cardiovascular diseases, which could be caused by alterations in oxidant-antioxidant balance. Materials and methods: Blood serum collected from 52 consecutive patients with PHPT treated surgically constituted our study material, whereas 36 healthy volunteers were our control group. Oxidative stress was evaluated in both patients and control subjects by assessment of malondialdehyde (MDA) and lipid hydroperoxides (LHP). Antioxidants were evaluated by the measurement of superoxide dismutase (SOD), ceruloplasmin (CER), catalase (CAT), sulfhydryl (SH) groups, glutathione (GSH), glutathione peroxidase (GSH-Px), glutathione transferase activity (GST) and glutathione reductase (GR). Moreover total antioxidant capacity (TAC) and total oxidative status (TOS) were measured and oxidative stress index (OSI) was calculated. Results: OSI was increased in patients with PHPT when compared to normal controls, whereas TAC was lower in PHPT. The levels of CER, MnSOD, GR, SH groups and MDA were significantly decreased in PHPT. The levels of serum LHP, catalase and SOD were significantly higher in patients with PHPT than in healthy patients. The erythrocyte CAT activity and GST were significantly increased in patients after parathyroidectomy. The erythrocyte GR and GPx were up-regulated postoperatively, whereas SOD activity decreased. Conclusions: In PHPT there are several alterations in the balance between the production of reactive oxygen species and antioxidant defense system.

Although the participation of glutathione transferases (GSTs) in light-dependent pathways and the circadian changes in the whole detoxification system have been studied, there are fewer results regarding the exact daily fluctuation of GSTs. In the present study, it was demonstrated that light up-regulated, while dark period decreased the plant GST activity and the expression of the selected tau group GST genes in tomato. These findings provide additional information on our current knowledge on the circadian rhythm of GSTs in plants and could help in further defining detoxification processes.

Calmodulin1-1 (Cam1-1) gene in rice (Oryza saliva L.) is highly induced by salt stress and transgenic rice lines overexpressing OsCam1-1 are more salt-tolerant. To identify potential proteins and cellular processes that may be regulated by CaM in rice, a comparative proteomic analysis between wild type and transgenic rice was conducted, which revealed 72 differentially expressed proteins. A Gene Ontology analysis showed that several of these differentially expressed proteins were involved in the response to stimuli and signal transduction. Compared with wild type, the transgenic rice had a higher level of a putative GTPase-activating protein (GAP), a regulatory protein that binds to activated GTP-binding proteins and stimulates their GTPase activity. Real-time RT-PCR analyses verified that GAP transcript levels were higher in all transgenic rice lines than in wild type. A co-expression analysis indicated that GAP was positively and negatively co-expressed with genes encoding peroxidases and glutathione transferases. Under salt stress (150 mM NaCl), guaiacol peroxidase activity was higher in transgenic rice than in wild type. In contrast, compared with wild type, transgenic rice exhibited lower and delayed induction of glutathione transferase activity under salt stress. Under normal conditions, the H2O2 levels were lower in transgenic rice than in wild type. After 24 h of salt stress, H2O2 levels had slightly increased in both wild type and transgenic rice, leading to slightly lower H2O2 levels in transgenic rice than in wild type. Together, these results indicate that OsCam1-1 overexpression may affect redox signaling and homeostasis, resulting in an altered antioxidant system in rice plants under salt stress.

In the present study, aqueous extracts from eight Chlorella strains were evaluated towards selected cosmeceutical properties such as enzymatic and non-enzymatic antioxidant capacity, glutathione content, inhibition potency towards skin regulatory enzymes, and cytotoxic effect on human cell lines. The results showed that the Chlorella strains display wide catalytic diversity with different strains exhibiting quite varied antioxidant enzymatic activities. The enzymes glutathione reductase, peroxidase, and superoxide dismutase were found in all strains, whereas only Chlorella minutissima displays glutathione transferase activity. Total antioxidant capacity (TAC) measurements, using the FRAP, ABTS, and DPPH assays, indicated significant differences among strains suggesting a large chemical diversity of antioxidants in Chlorella species. Chlorella minutissima extract showed high inhibitory potency with IC50 23.3 ± 2.5 and 29.2±2.7 μg towards tyrosinase and elastase, respectively, suggesting a potential activity in controlling skin aging, inflammatory process, and pigmentation. Furthermore, the effect of C. minutissima extract on a human hepatoma cell line (Huh-7) was evaluated using cell proliferation assay and qRT-PCR to analyze the expression of marker genes (SOD1, GPx1, GPx2) involved in oxidative stress response. The results established the absence of cytotoxic effects of the aqueous extracts. SOD1, GPx1, and GPx4 expression levels showed a specific profile and appear to be slightly upregulated, downregulated, and remained unaffected, respectively. Taken together, these results demonstrate that Chlorella species can provide promising extracts rich in antioxidants, anti-aging, and skin-whitening ingredients with the potential for use as raw material in cosmetics industry.

Glutathione transferase (GST) activity revealed in vacuoles of red beetroot (Beta vulgaris L.) cells was investigated in comparison with the GST activity of plastids and extracts of tissues. The level of GST activity determined by spectrophotometric method proved fairly high in water extracts and membrane fractions of isolated vacuoles and plastids, as well as in water extracts of tissues. In the objects studied, pH dependence of the GST activity slightly differed. Optimal pH for the vacuolar GST activity was in the range 7.0–7.5, for the GST of plastids and tissue extracts it was 7.5. The GSTs differed in specificity to the substrates fluorodifen and ethacrynic acid. The activity of the vacuolar and tissue extract GSTs with fluorodifen was significantly higher than that of the GST from plastids. Ethacrynic acid, often used as a competitive inhibitor of GST, almost completely inhibited the GST activity assayed with 1-chloro-2,4-dinitrobenzene as a main substrate. However, ethacrynic acid was a substrate only for the GSTs of vacuoles and tissue extract, but not for the GST of plastids. Using zymography allowing estimation of the GST activity in a gel after electrophoresis of proteins, several zones of enzymatic activity were revealed in all objects that may correspond to different isozymes. It was found that the composition of the vacuolar GST isoforms and their substrate specificity may differ from the GSTs of other cellular structures. It is assumed that vacuole, having quite high activity of GST, should make a significant contribution to intracellular detoxification processes.

A metal-resistant Rhodotorula mucilaginosa strain was isolated from an industrial wastewater. Effects on reduced/oxidized glutathione (GSSG/GSH), antioxidant enzymes and proteome were assessed on metal challenge (100 mg/L). Increased GSH (mM/g) was found with CdCl 2 (18.43 ± 3.34), NaAsO 2 (14.76 ± 2.14), CuSO 4 (14.73 ± 2.49), and Pb(NO 3 ) 2 (15.74 ± 5.3) versus control (7.67 ± 0.95). GSH:GSSG ratio decreased with CdCl 2 , NaAsO 2 , and Pb(NO 3 ) 2 but not with CuSO 4 and cysteine-containing protein levels increased with CdCl 2 and NaAsO 2 . NaAsO 2 exposure enhanced glutathione transferase activity but this decreased with CdCl 2 . Both metals significantly increased glutathione reductase and catalase activities. Metabolism-dependent uptake of Cd and As (12-day exposure) of approximately 65 mg/g was observed in live cells with greater cell surface interaction for As compared to Cd. A particular role for arsenic oxidase in As resistance was identified. One dimensional electrophoresis revealed higher oxidation of protein thiols in response to NaAsO 2 than to CdCl 2 . Two dimensional electrophoresis showed altered abundance of some proteins on metal treatment. Selected spots were excised for mass spectrometry and seven proteins identified. Under oxidative stress conditions, xylose reductase, putative chitin deacetylase, 20S proteasome subunit, eukaryotic translation elongation factor 2, valine-tRNA ligase and a metabolic enzyme F0F1 ATP synthase alpha subunit were all expressed as well as a unique hypothetical protein. These may comprise a protein expression signature for metal-induced oxidation in this yeast. Significance Fungi are of widespread importance in agriculture, biodegradation and often show extensive tolerance to heavy metals. This makes them of interest from the perspective of bioremediation. In this study an environmental isolate of R. mucilaginosa showing extensive tolerance of a panel of heavy metals, in particular cadmium and arsenic, was studied. Several biochemical parameters such as activity of antioxidant enzymes, status of reduced and oxidized glutathione and thiols associated with proteins were all found to be affected by metal exposure. A detailed analysis with arsenic and cadmium pointed to a particular role for arsenic oxidase in arsenic bioaccumulation and tolerance. This is the first time this has been reported in R. mucilaginosa , and suggests that this isolate may have potential in biosorption of these metals in the environment. Proteomic analysis revealed that seven proteins with a variety of roles - ATP synthesis, protein degradation/synthesis, and metabolism of xylose and chitin - were differentially affected by metal exposure in a manner consistent with oxidative stress. These may therefore represent a protein expression signature for exposure to cadmium and arsenic.

Alzheimer disease (AD) is one of the most common neurodegenerative disorders widely occurring among the elderly. The pathogenic mechanisms involved in the development of this disease are still unknown. In AD, in addition to brain, a number of peripheral tissues and cells are affected, including erythrocytes. In this study, we analyzed glycolytic energy metabolism, antioxidant status, glutathione, adenylate and proteolytic systems in erythrocytes from patients with AD and compared with those from age-matched controls and young adult controls. Glycolytic enzymes hexokinase, phosphofructokinase, bisphosphoglycerate mutase and bisphosphoglycerate phosphatase displayed lower activities in agematched controls, and higher activities in AD patients, as compared to those in young adult control subjects. In both aging and AD, oxidative stress is increased in erythrocytes whereas elevated concentrations of hydrogen peroxide and organic hydroperoxides as well as decreased glutathione/glutathione disulfide ratio and glutathione transferase activity can be detected. These oxidative disturbances are also accompanied by reductions in ATP levels, adenine nucleotide pool size and adenylate energy charge. Caspase-3 and calpain activities in age-matched controls and AD patients were about three times those of young adult controls. 2,3-diphosphoglycerate levels were significantly decreased in AD patients. Taken together these data suggest that AD patients are associated with chronic disturbance of 2,3-diphosphoglycerate metabolism in erythrocytes. These defects may play a central role in pathophysiological processes predisposing elderly subjects to dementia.

The paper deals with the influence of genetic polymorphisms on the features of folate metabolism and antioxidant protection in newborns with critical conditions in neonatal period. The objective of the study was to examine changes in folate metabolism, antioxidant defense indicators and their relationship with genetic polymorphism to determine the contribution of genetically determined folate metabolism disorders in the development of critical conditions in newborns. To perform the work 45 infants were examined (gestational age 38-40 weeks) with critical conditions of the early neonatal period (the main group) and 30 infants (gestational age 38-40 weeks) with physiological early neonatal period (the comparison group). Examined children were in obstetric and neonatal intensive care units of the Regional Maternity Hospital in Poltava. There were significant differences in the indices of homocysteine, folate and serum glutathione transferase activity in newborns of the examined groups during the first three days of life, with varying strength correlation found. Dynamics of the indices of folate metabolism on the third day of life as compared to the first day was significantly different in the newborns from the main groups and correlated with the indices of antioxidant protection. The peculiarities of the dynamics of folate metabolism indices, characterizing antioxidant and folate dependent metabolic processes in the perinatal period, were determined. The analysis of the intergenic interaction impact on the development of folate metabolism disorders and critical conditions revealed association with polymorphisms of GSTT1, GSTM1, MTHFR, ACE, TNF-a, AT2R1 genes. Synergistic interaction between polymorphic variants of genes GSTT1 and GSTM1, MTHFR and ACE was found. Polymorphic variants of these genes associated with changes in the dynamics of folate metabolism indicators. In the course of the study the best predictive model of intergenic interaction in the development of critical conditions and genetically determined folate metabolism disorders in newborns was found (predictive value - 91.47%). Analysis of unfavorable polymorphic variants in GSTT1, GSTM1, MTHFR, ACE, TNF-a, AT2R1 genes is a perspective one to predict unfavourable early neonatal period in newborns in clinical practice.

There is substantial agreement that the unbalance between oxidant and antioxidant species may affect the onset and/or the course of a number of common diseases including Parkinson's and Alzheimer's diseases. Many studies suggest a crucial role for oxidative stress in the first phase of aging, or in the pathogenesis of various diseases including neurological ones. Particularly, the role exerted by glutathione and glutathione-related enzymes (Glutathione Transferases) in the nervous system appears more relevant, this latter tissue being much more vulnerable to toxins and oxidative stress than other tissues such as liver, kidney or muscle. The present review addresses the question by focusing on the results obtained by specimens from patients or by in vitro studies using cells or animal models related to Parkinson's and Alzheimer's diseases. In general, there is an association between glutathione depletion and Parkinson's or Alzheimer's disease. In addition, a significant decrease of glutathione transferase activity in selected areas of brain and in ventricular cerebrospinal fluid was found. For some glutathione transferase genes there is also a correlation between polymorphisms and onset/outcome of neurodegenerative diseases. Thus, there is a general agreement about the protective effect exerted by glutathione and glutathione transferases but no clear answer about the mechanisms underlying this crucial role in the insurgence of neurodegenerative diseases.

Favism is an acute hemolytic syndrome caused by the ingestion of fava bean (FB) in glucose 6-phosphate dehydrogenase (G6PD) deficient individuals. However, little is known about the global transcripts alteration in liver tissue after FB ingestion in G6PD-normal and -deficient states. In this study, deep sequencing was used to analyze liver genes expression alterations underlying the effects of FB in C3H (Wild Type, WT) and G6PD-deficient (G6PDx) mice and to evaluate and visualize the collective annotation of a list of genes to Gene Ontology (GO) terms associated with favism. Our results showed that FB resulted in a decrease of glutathione (GSH)-to-oxidized glutathione (GSSG) ratio and an increase of malondialdehyde (MDA) both in the G6PDx and WT-control check (CK) mice plasma. Significantly, liver transcript differences were observed between the control and FB-treated groups of both WT and G6PDx mice. A total of 320 differentially expressed transcripts were identified by comparison of G6PDx-CK with WT-CK and were associated with immune response and oxidation-reduction function. A total of 149 differentially expressed genes were identified by comparison of WT-FB with WT-CK. These genes were associated with immune response, steroid metabolic process, creatine kinase activity, and fatty acid metabolic process. A total of 438 differential genes were identified by comparing G6PDx-FB with G6PD-CK, associated with the negative regulation of fatty acid metabolic process, endoplasmic reticulum, iron binding, and glutathione transferase activity. These findings indicate that G6PD mutations may affect the functional categories such as immune response and oxidation-reduction.

Tuberculosis is one of the top causes of death among curable infectious diseases; it is an airborne infectious disease that killed 1.1 million people worldwide in 2010. Anti-tuberculosis drug-induced liver injury is the primary cause of drug-induced liver injury (DILI). Rifampicin is one of the most common anti-tuberculosis therapies and has well-known hepatotoxicity. To understand the mechanism of rifampicin-induced liver injury, we performed a global proteomic analysis of liver proteins by LC-MS/MS in a mouse model after the oral administration of 177 and 442.5 mg/kg rifampicin (LD10 and LD25) for 14 days. Based on the biochemical parameters in the plasma after rifampicin treatment, the hepatotoxic effect of rifampicin in the mouse liver was defined as a mixed liver injury. In the present study, we identified 1101 proteins and quantified 1038 proteins. A total of 29 and 40 proteins were up-regulated and 27 and 118 proteins were down-regulated in response to 177 and 442.5 mg/kg rifampicin, respectively. Furthermore, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses to characterize the mechanism of rifampicin-induced hepatotoxicity. In the molecular function category, glutathione transferase activity was up-regulated and proteins related to arachidonic acid metabolism were down-regulated. In the KEGG pathway enrichment-based clustering analysis, the peroxisome proliferator-activated receptor-γ (PPARγ) signaling pathway, cytochrome P450, glutathione metabolism, chemical carcinogenesis, and related proteins increased dose-dependently in rifampicin-treated livers. Taken together, this study showed in-depth molecular mechanism of rifampicin-induced liver injury by comparative toxicoproteomics approach.

We studied the effects of consumption of low-alcohol drinks (no more than 2 doses) during pregnancy on the state of LPO-antioxidant defense system and glutathione level in the blood serum and erythrocytes of pregnant women and newborns. It was found that even single alcohol intake was followed by the development of oxidative stress in women and newborns. In pregnant women, the decrease in reduced glutathione content and glutathione reductase activity associated with increased level of oxidized glutathione and glutathione transferase activity were typical changes. In newborns, the decrease in reduced glutathione concentration was accompanied by a decrease in glutathione reductase, glutathione transferase, and glutathione peroxidase activities. It should be emphasized that even small alcohol doses produce delayed negative effects on metabolism of the mother and newborns.

In a subacute toxicological and sanitary experiment, the hydroxylated fullerene C60, fullerenol C60(OH)24 is intragastrally administered to male Wistar rats at doses of 0.1, 1, and 10 mg/kg daily for 28 days. The integrated, biochemical, hematological, physiological, microbiological, and immunological indices that characterize the state of animals are determined. It is shown that a series of statistically reliable changes in indices occurs in rats during the daily administration of fullerenol at doses of 0.1 mg/kg and more for a month: a decrease in activity of cytochrome P450 CYP1A1 isoform at a dose of 0.1 mg/kg, a decrease in activity of cytochrome P450 CYP1A2 isoform at a dose of 0.1mg/kg and an increase in this parameter at 10 mg/kg, increased glutathione transferase activity at doses of 1 and 10 mg/kg, a decrease in the content of diene conjugates of polyunsaturated fatty acids (PUFAs) in plasma, and decreased activity of glutathione reductase in red blood cells at doses of 0.1 and 1 mg/kg. The level of uric acid, percentage content of monocytes, and relative content of immature granulocytes increases in a dose-dependent manner when fullerenol is administered at doses of 1 and 10 mg/kg, whereas the mean corpuscular hemoglobin concentration decreases within all ranges of the doses studied. Thus, based on these studies, we can conclude that the changes taking place in rats when fullerenol is administered at a dose of 0.1 mg/kg are small in absolute value and fall in the limits of the physiological norm variability. Consequently, it can be assumed that a fullerenol dose of less than 0.1 mg/kg may be considered maximally ineffective in a monthly experiment.

Erythrocyte glutathione transferase (e-GST) displays increased activity in patients with renal damage and positive correlation with homocysteine (Hcy) in patients under maintenance hemodialysis. Here, we determined e-GST, Hcy, and erythrocyte catalase (e-CAT) in 328 patients affected by type 2 diabetes mellitus (T2DM), 61 diabetic non-nephropathic patients and 267 affected by diabetes and by chronic kidney disease (CKD) under conservative therapy subdivided into four stages according to K-DOQI lines. e-GST activity was significantly higher in all T2DM patients compared to the control group (7.90 ± 0.26 vs. 5.6 ± 0.4 U/g(Hb)), and we observed an enhanced activity in all subgroups of CKD diabetic patients. No significant correlation or increase has been found for e-CAT in all patients tested. Mean Hcy in diabetic patients is higher than that in healthy subjects (33.42 ± 1.23 vs. 13.6 ± 0.8 μM), and Hcy increases in relation to the CKD stage. As expected, a significant correlation was found between e-GST and Hcy levels. These findings suggest that e-GST hyperactivity is not caused directly by diabetes but by its consequent renal damage. e-GST, as well as Hcy, may represent an early biomarker of renal failure.

Aim. To determine the activity of glutathione transferase (GT) and the content of reduced glutathione (GSH) in the cytosol of the gastric mucosa cells in experimental gastrocarcinogenesis. Methods. The activity of GT was determined spectrophotometrically, the content of GSH was measured spectrofluorimetrically. Gastrocarcinogenesis was initiated by 10-week replacement of drinking water by 0.01 % solution of carcinogen N-methyl-N'-nitro-N-nitrosoguanidine, at the same time the rats were given a diet containing 5 % NaCl. Results. It was established that at the end of the 4th and 6th weeks of consumption of carcino- gen and NaCl, the activity of GT increased by 26 and 94 %, whereas the content of GSH increased by 135 and 85 %, respectively. After 12 weeks there was a decrease in the activity of GT by 50 % and the maximum decrease in the GSH concentration by 69 %. At the end of the 18th and 24th weeks it was recorded the increase in the activity of GT by 44 and 47 % and the decrease in the GSH content by 55 and 52 %. Conclusions. The changes in the activity of GT and GSH-content are evidence of the violation of glutathione homeostasis, which may cause the delay as well as initiation of development of the pathology. The reduction of GSH is established at the early stages of tumors formation. Keywords: glutathione transferase, reduced glutathione, gastric cancer. Мета. Визначити глутатіонтрансферазну (ГТ) активність і вміст відновленого глутатіону (GSH) у цитозолі клітин слизової оболонки шлунка за умов експериментального гастроканцерогенезу. Методи. ГТ-активність визначали спектрофотометричним методом, вміст GSH – методом спектрофлуориметрії. Гастроканцерогенез ініціювали 10-тижневою заміною питної води на 0,01 %-й розчин канцеро- гену N-метил-N'-нітро-N-нітрозогуанідину з одночасним переведенням щурів на корм, який містить 5 % NaCl. Результати. По закінченні 4- і 6-тижневого споживання щурами канцерогену і NaCl ГТ-активність зростала відповідно на 26 і 94 %, тоді як вміст GSH – на 135 і 85 %. Через 12 тижнів спостерігали зниження активності ГТ на 50 % і максимальне зменшення концентрації GSH на 69 %. Після 18 і 24 тижнів зафіксовано зростання ГТ-активності на 44 і 47 % та зниження вмісту GSH на 55 і 52 %. Висновки. Зміни ГТ-активності і концентрації GSH свідчать про порушення глутатіонового гомеостазу, що може призводити як до затримки, так і ініціації розвитку патології. Встановлено зниження вмісту GSH на ранніх стадіях формування пухлин. Ключові слова: глутатіонтрансфераза, відновлений глутатіон, рак шлунка. Цель. Определить глутатионтрансферазную (ГТ) активность и содержание восстановленного глутатиона (GSH) в цитозоле клеток слизистой оболочки желудка в условиях экспериментального гастроканцерогенеза. Методы. ГТ-активность определяли спектрофотометрическим методом, содержание GSH – методом спектрофлуориметрии. Гастроканцерогенез инициировали 10-недельной заменой питьевой воды на 0,01 %-й раствор канцерогена N-метил-N'-нитро-N-нитрозогуанидина с одновременным переводом крыс на корм, содержащий 5 % NaCl. Результаты. По окончании 4 и 6 недель потребления крысами канцерогена и NaCl ГТ-активность увеличилась соответственно на 26 и 94 %, тогда как содержание GSH – на 135 и 85 %. Через 12 недель наблюдали снижение ГТ-активности на 50 % и максимальное уменьшение концентрации GSH на 69 %. Спустя 18 и 24 недели зафиксировано возрастание активности ГТ на 44 и 47 %, а также снижение содержания GSH на 55 и 52 %. Выводы. Изменения ГТ- активности и концентрации GSH свидетельствуют о нарушении гомеостаза глутатиона, что может вызывать как задержку, так и инициацию развития патологии. Установлено снижение содержания GSH на ранних стадиях формирования опухолей. Ключевые слова: глутатионтрансфераза, восстановленный глутатион, рак желудка.

To support the key role of glutathione (GSH) in the mechanisms of tolerance and accumulation of arsenic in plants, this work examines the impact of several effectors of GSH synthesis or action in the response of maize ( Zea mays L.) to arsenic. Maize was exposed in hydroponics to iso-toxic rates of 150 μM arsenate or 75 μM arsenite for 9 days and GSH effectors, flurazole (an herbicide safener), l -buthionine-sulfoximine (BSO, a known inhibitor of GSH biosynthesis), and dimercaptosuccinate (DMS) and dimercaptopropanesulfonate (DMPS) (two thiols able to displace GSH from arsenite-GSH complexes) were assayed. The main responses of plants to arsenic exposure consisted of a biomass reduction (fresh weight basis) of about 50%, an increase of non-protein thiol (NPTs) levels (especially in the GSH precursor γ-glutamylcysteine and the phytochelatins PC 2 and PC 3 ) in roots, with little effect in shoots, and an accumulation of between 600 and 1000 ppm of As (dry weight basis) in roots with very little translocation to shoots. Growth inhibition caused by arsenic was partially or completely reversed in plants co-treated with flurazole and arsenate or arsenite, respectively, highly exacerbated in plants co-treated with BSO, and not modified in plants co-treated with DMS or DMPS. These responses correlated well with an increase of both NPTs levels in roots and glutathione transferase activity in roots and shoots due to flurazole treatment, the decrease of NPTs levels in roots caused by BSO and the lack of effect on NPT levels caused by both DMS and DMPS. Regarding to arsenic accumulation in roots, it was not modified by flurazole, highly reduced by BSO, and increased between 2.5- and 4.0-fold by DMS and DMPS. Therefore, tolerance and accumulation of arsenic by maize could be manipulated pharmacologically by chemical effectors of GSH.

An early step in the intracellular processing of vitamin B(12) involves CblC, which exhibits dual reactivity, catalyzing the reductive decyanation of cyanocobalamin (vitamin B(12)), and the dealkylation of alkylcobalamins (e.g. methylcobalamin; MeCbl). Insights into how the CblC scaffold supports this chemical dichotomy have been unavailable despite it being the most common locus of patient mutations associated with inherited cobalamin disorders that manifest in both severe homocystinuria and methylmalonic aciduria. Herein, we report structures of human CblC, with and without bound MeCbl, which provide novel biochemical insights into its mechanism of action. Our results reveal that CblC is the most divergent member of the NADPH-dependent flavin reductase family and can use FMN or FAD as a prosthetic group to catalyze reductive decyanation. Furthermore, CblC is the first example of an enzyme with glutathione transferase activity that has a sequence and structure unrelated to the GST superfamily. CblC thus represents an example of evolutionary adaptation of a common structural platform to perform diverse chemistries. The CblC structure allows us to rationalize the biochemical basis of a number of pathological mutations associated with severe clinical phenotypes.

Four isatin-isoniazid derivatives with proven efficacy against resistant strains of Mycobacterium tuberculosis, that had greater lipophilicity than isoniazid were evaluated for effects on hepatic drug metabolizing and chemoprotective enzymes and compared to isoniazid. Following intragastric administration to mice (75 or 150 mg/kg×3 days), none of the four compounds exhibited hepatotoxicity, and only isoniazid was found to elevate CYP2E1 activity. All compounds slightly elevated Cyp1a1/2 mRNA levels, but these did not result in increased methoxyresorufin demethylase activity. With the exception of isoniazid, approximately twofold elevations in Ugt1a mRNAs (seen with isatin-isoniazid [Ugt1a1, Ugt1a6, Ugt1a9], 1-propynylisatin-isoniazid [Ugt1a1, Ugt1a9], and 1-propylisatin-isoniazid [Ugt1a1]) also did not result in changes in 4-nitrophenol glucuronidation activity. 1-Benzylisatin-isoniazid was the only compound that (1) elevated Ugt2b5 mRNA, and (2) like isoniazid, had no effect on glutathione transferase activities and mRNAs. 1-Propynylisatin-isoniazid elevated glutathione transferase activity toward 1-chloro-2,4-dinitrobenzene, 1-propylisatin-isoniazid decreased the 2.4-kb Gstp transcript, and isatin-isoniazid increased Gsta, Gstm, and the 1.2-kb Gstp transcripts. None of the four compounds affected glutathione peroxidase activity, although 1-propynylisatin-isoniazid and 1-benzylisatin-isoniazid elevated mRNA transcripts. None of the four compounds affected microsomal epoxide hydrolase or thioredoxin reductase activities but, in parallel, isoniazid, 1-propynylisatin-isoniazid, and 1-propylisatin-isoniazid elevated mRNAs of each. 1-Propynylisatin-isoniazid, 1-propylisatin-isoniazid, and 1-benzylisatin-isoniazid, and isoniazid, elevated quinone oxidoreductase (Nqo1) mRNA, but only 1-propylisatin-isoniazid elevated the activity. Altogether, and despite some changes in mRNA expression, the novel isatin-isoniazid derivatives had only minor effects on mouse hepatic enzyme activities and are worthy of further investigation as possible therapeutic agents likely free of major induction-related drug–drug interactions. Drug Dev Res 2010. © 2010 Wiley-Liss, Inc.

Background Of the five human Alpha-class glutathione transferases, expression of hGSTA5 has not been experimentally documented, even though in silico the hGSTA5 sequence can be assembled into a mRNA and translated. The present work was undertaken to determine whether hGSTA5 is functional. Methods Human K562 cells were transfected with the hGSTA5 gene driven by the CMV promoter, and hGSTA5 cDNA was recovered from mature mRNA by reverse transcription. The cDNA was used in bacterial and eukaryotic protein expression systems. The resulting protein, after purification by glutathione affinity chromatography where appropriate, was tested for glutathione transferase activity. Results Human K562 cells transfected with the hGSTA5 gene under control of a CMV promoter produced a fully spliced mRNA which, after reverse transcription and expression in E. coli, yielded a protein that catalyzed the conjugation of the lipid peroxidation product 4-hydroxynonenal to glutathione. Similarly, transfection of human HEK-293 cells with the hGSTA5 gene driven by the CMV promoter led to an elevated 4-hydroxynonenal-conjugating activity in the cell lysate. In addition, translation of hGSTA5 cDNA in a cell-free eukaryotic system gave rise to a protein with 4-hydroxynonenal-conjugating activity. Conclusions hGSTA5 can be processed to a mature mRNA which is translation-competent, producing a catalytically active enzyme. General significance Because a functional gene would not be maintained in the absence of selective pressure, we conclude that the native hGSTA5 promoter is active but has a spatially or temporally restricted expression pattern, and/or is expressed only under specific (patho)physiological conditions.

Pathways for tailoring and processing vitamins into active cofactor forms exist in mammals that are unable to synthesize these cofactors de novo. A prerequisite for intracellular tailoring of alkylcobalamins entering from the circulation is removal of the alkyl group to generate an intermediate that can subsequently be converted into the active cofactor forms. MMACHC, a cytosolic cobalamin trafficking chaperone, has been shown recently to catalyze a reductive decyanation reaction when it encounters cyanocobalamin. In this study, we demonstrate that this versatile protein catalyzes an entirely different chemical reaction with alkylcobalamins using the thiolate of glutathione for nucleophilic displacement to generate cob(I)alamin and the corresponding glutathione thioether. Biologically relevant thiols, e.g. cysteine and homocysteine, cannot substitute for glutathione. The catalytic turnover numbers for the dealkylation of methylcobalamin and 5'-deoxyadenosylcobalamin by MMACHC are 11.7 +/- 0.2 and 0.174 +/- 0.006 h(-1) at 20 degrees C, respectively. This glutathione transferase activity of MMACHC is reminiscent of the methyltransferase chemistry catalyzed by the vitamin B(12)-dependent methionine synthase and is impaired in the cblC group of inborn errors of cobalamin disorders.

Total glutathione S-transferase (GST, EC 2.5.1.18) and glutathione peroxidase (GPOX) activity were measured spectrophotometrically in Triticum aestivum cv. MV Emese and cv. Plainsman (drought tolerant) and cv. GK Elet and Cappelle Desprez (drought-sensitive) flag leaves under control and drought stress conditions during the grain-filling period, in order to reveal possible roles of different GST classes in the senescence of flag leaves. Six wheat GSTs, members of 3 GST classes, were selected and their regulation by drought and senescence was investigated. High GPOX activity (EC 1.11.1.9) was observed in well-watered controls of the drought-tolerant Plainsman cultivar. At the same time, TaGSTU1B and TaGSTF6 sequences, investigated by real-time PCR, showed high-expression levels that increased with time, indicating that the gene products of these genes may play important roles in monocarpic senescence of wheat. Expression of these genes was also induced by drought stress in all of the four investigated cultivars, but extremely high transcript amounts were detected in cv. Plainsman. Our data indicate genotypic variations of wheat GSTs. Expression levels and early induction of two senescence-associated GSTs under drought during grain filling in flag leaves correlated with high yield stability.

The antimutagenic and antioxidant enzyme activities of captopril (CAP), cysteine (CYS), and glutathione (GSH) were evaluated for finding concentration-dependent inhibitory effects against: (1) the mutagenicity of 2-amino-3-methylimidazo [4,5- f ] quinoline (IQ), an indirect mutagen; and (2) N -methyl- N ′-nitrosoguanidine (MNNG), a direct mutagen toward Salmonella typhimurium TA98 and TA100. Of the three thiols, CYS and GSH exhibited better inhibitory effects against IQ-induced mutation toward TA98 and TA100, respectively. GSH also showed a protective effect against MNNG-induced mutation toward TA98 and TA100, meanwhile, CAP showed the least inhibitory effect. CYS, GSH, and CAP also dose-dependently increased the activities of glutathione transferase, glutathione peroxidase, and glutathione reductase in hepatic BNL cells. CAP showed the superior inducing effects on glutathione transferase activity. These data suggested that the bioactive properties of biological thiols might contribute to their effects of antimutagenic activities as well as regulation on activities of antioxidant enzymes.

Many carbonyl metabolizing enzymes are equally involved in xenobiotic and endogenous metabolism, but few have been investigated in terms of substrate competition and interference between different cellular pathways. Mammalian alcohol dehydrogenase 3 (ADH3) represents the key enzyme in the formaldehyde detoxification pathway by oxidation of S-hydroxymethylglutathione [HMGSH; the glutathione (GSH) adduct of formaldehyde]. In addition, several studies have established ADH3 as S-nitrosoglutathione (GSNO) reductase in endogenous NO homeostasis during the last decade. GSNO depletion associates with various diseases including asthma, and evidence for a causal relationship between ADH3 and asthma pathology has been put forward. In a recent study, we showed that ADH3-mediated alcohol oxidation, including HMGSH oxidation, is accelerated in presence of GSNO which is concurrently reduced under immediate cofactor recycling [C.A. Staab, J. Ålander, M. Brandt, J. Lengqvist, R. Morgenstern, R.C. Grafström, J.-O. Höög, Reduction of S-nitrosoglutathione by alcohol dehydrogenase 3 is facilitated by substrate alcohols via direct cofactor recycling and leads to GSH-controlled formation of glutathione transferase inhibitors, Biochem. J. 413 (2008) 493–504]. Thus, considering the usually low cytosolic free NADH/NAD+ ratio, formaldehyde may trigger and promote GSNO reduction by enzyme-bound cofactor recycling. These findings provided evidence for formaldehyde-induced, ADH3-mediated GSNO depletion with potential direct implications for asthma. Furthermore, analysis of product formation as a function of GSH concentrations suggested that, under conditions of oxidative stress, GSNO reduction can lead to the formation of glutathione sulfinamide and its hydrolysis product glutathione sulfinic acid, both potent inhibitors of glutathione transferase activity.

Organisms present a series of cellular mechanisms to avoid the effects of toxic compounds. Such mechanisms include the increase in activity of detoxification enzymes [e.g., 7-ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST)I, which could explain the low retention of ingested toxins generally observed in copepods. In addition, decreasing gross growth efficiency (GGE) of copepods with increasing concentration of toxic diets could be caused either by a high expenditure coping with toxins (e.g., increase in the activity of detoxification enzymes) or by a deterioration of reproductive tissues. To assess the effect of toxic phytoplankton on the activity of detoxification enzymes and on oocyte maturation of Acartia tonsa and Temora longicornis, feeding and egg production experiments were carried out with a variety of toxic diets and an adequate non-toxic food control (Rhodomonas spp.) all provided as single species diets. Toxic diets included the nodularin-producing cyanobacterium Nodularia spumigena, the dinoflagellates Alexandrium minutum, and A. tamarense, which contained Paralytic Shellfish Poisoning (PSP) toxins, the dinoflagellate Prorocentrum lima with Diarrhetic Shellfish Poisoning (DSP) toxins and the haptophyte Prymnesium parvum, which produces ichtyotoxins with haemolytic activity. Feeding on toxic diets was lower than on Rhodomonas spp., except for A. minutum and A. tamarense. In addition, toxic diets negatively affected reproduction in both copepod species with the production of oocytes and oocyte development impaired with A. minutum and N. spumigena. While the negative effect of N. spumigena seemed to be connected to gonad atresia likely caused by severe food limitation (starvation), the negative effect of A. minutum could have been either caused by a direct effect of saxitoxins or nutritional inadequacy on oocyte production. We could not detect EROD activity in the copepods, while the activity of GST was generally higher with the non-toxic food control and positively related to the feeding and egestion rates, suggesting relation to feeding conditions rather than to exposure to toxic diets. No relationship was found between GGE and CST activity. Our results refute the hypothesis that toxic diets, provided at ecologically relevant levels, would induce cellular mechanisms in copepods regarding GST activity. GST activity thus seems to play no role in detoxification of copepods confronted with toxic phytoplankton. Toxin detoxification and its cost for copepods still remain an open question. (C) 2008 Elsevier B.V. All rights reserved. (Less)

The yeast prion Ure2p is composed of an N-terminal prion domain, and a C-terminal globular domain, which shows similarity to glutathione transferases (GSTs) in both sequence and structure. Ure2p protects Saccharomyces cerevisiae cells from heavy metal ion and oxidant toxicity. Ure2p shows glutathione-dependent peroxidase (GPx) activity, which is often an adjunct activity of GSTs, but wild-type Ure2p shows no detectable GST activity toward the standard substrate 1-chloro-2,4-dinitrobenzene (CDNB). The structural basis for the substrate specificity of Ure2p enzymatic activity is an interesting problem that is fundamental to understanding the in vivo roles of Ure2p and its relationship to the GST structural family. The critical catalytic residue in the other known GSTs is Ser, Tyr or Cys. Here, we demonstrate that residue N124 is important for the GPx activity of Ure2p, and a wild-type level of activity is maintained in N124S, but not in N124Y/C. Interestingly, we found that the single-site mutations A122C and N124A/V (but not N124S/Y/C) "restore" the GST activity of Ure2p toward CDNB, while causing a substantial reduction in GPx activity. The steady-state kinetics for the GST activity of A122C appears to follow a ping-pong mechanism. In contrast, the GST activity of 124-site mutants shows a sequential mechanism, as is observed for the native GPx activity of Ure2p, and typical GST enzymes. These findings shed light on the evolutionary relationship of Ure2p with other GST family members, and contribute to our understanding of catalytic promiscuity and divergent evolution.

Azathioprine is a prodrug that is widely used clinically as an immunosuppressive agent. The pharmacological action of azathioprine is associated with the release of 6-mercaptopurine by a reaction involving glutathione. This biotransformation of azathioprine is catalyzed by glutathione transferases (GSTs). The nonenzymatic reaction with glutathione is minimal in comparison with the GST-catalyzed process, but azathioprine is still a slow substrate in comparison with the most effective GST substrates. Novel GSTs with higher catalytic efficiency toward azathioprine could be useful in novel therapeutic applications; therefore, directed evolution of GSTs for enhanced activities is desirable. However, screening for variants having higher catalytic activity with azathioprine is a time-consuming process due to the low activity with this substrate. A new chromogenic and faster substrate, 1-methyl-4-nitro-5-(4-nitrophenylthio)-1H-imidazole (NPTI), has been synthesized and characterized by assays with several GSTs. The novel substrate mimicked azathioprine in the reaction with glutathione catalyzed by alpha class GSTs and, therefore, is a valuable surrogate in the screening of large mutant libraries. NPTI may also find use in the elucidation of the exact mechanism of immunosuppression effected by azathioprine where there is evidence that the imidazole moiety of azathioprine, rather than 6-mercaptopurine, is involved.

International audience; Nivalenol (NIV) is a toxic Fusarium secondary trichothecene metabolite occurring naturally in cereal grains. In order to evaluate the no observed adverse effect level (NOAEL), we tested the effects of a large array of oral doses of this toxin for responses on plasma biochemistry, the immune system and hepatic drug metabolism in mice. C57Bl6 mice received oral doses of toxin (0.014, 0.071, 0.355, 1.774 or 8.87 mg/kg bw) 3 days a week for 4 weeks. Only the highest dose of NIV induced an increase in plasma phosphate, decreases in plasma urea and immunoglobulin M and additional changes like increases in plasma alkaline phosphatase and immunoglobulin G. Interleukin 4 production was increased in cultured murine splenocytes. Regarding liver drug metabolising enzymes, the only glutathione transferase activity accepting 1-chloro-2,4-dinitro-benzene as substrate was transiently increased in mice receiving low doses (0.071 and 0.355 mg/kg bw) of NIV. Regarding the cytochrome P450 monooxygenases, no significant change was observed in ethoxyresorufin $O$-deethylase activity whereas both methoxyresorufin and pentoxyresorufin $O$-dealkylase activities were decreased by 38-45% for the highest dose (8.87 mg/kg bw) of NIV. However, when analysed by Western blot analysis, the protein expression of mouse P450 1a, 2b, 2c, 3a and 4a subfamilies was unchanged in animals receiving NIV. In conclusion, the NOAEL of this toxin in our study was 1.774 mg/kg bw, corresponding to an exposure to 5 ppm contaminated food. Indeed hepatotoxicity appears in the only mice treated with a five fold higher oral dose of 8.87 mg/kg bw of NIV. Such exposure levels appear to be by far higher than the maximal natural occurrence measured in European cereals, known to range from 0.34 to 1.86 ppm.

This study examined the effects of dietary vitamin C and ammonia concentration on the cellular defense response of Macrobrachium nipponese. The response of the cellular defense was determined by monitoring changes in reactive oxygen intermediates in hemocytes, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, glutathione reductase and glutathione transferase activity in muscle. The results showed that prawns fed the basal diet had a significant increase in reactive oxygen intermediates in response to exposure to ammonia-N. When dietary vitamin C was added to the feed, there was a significant decrease in reactive oxygen intermediates in response to exposure to ammonia-N. Total superoxide dismutase and glutathione peroxidase activity of prawns fed the basal diet decreased as ammonia-N increased. This decrease in activity was significantly less pronounced in prawns that were fed the dietary vitamin C supplement. In prawns that were fed the dietary vitamin C supplement there was no significant variation in catalase reductase or gluxathione reductase activity in response to variations in ammonia-N concentration. The glutathione transference activity increased at a low concentration of ammonia-N in prawns that were fed the dietary vitamin C supplement, but there was no significant variation in the prawns fed the basal diet. Overall the results indicate that dietary vitamin C supplements enhance the capacity of M. nipponese to respond to elevations in ammonia-N. This response involved several biochemical reactions known to be involved in cellular defense.