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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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily includes structurally related membrane proteins with diverse functions of widespread origin. A total of 136 proteins belonging to the MAPEG superfamily were found in database and genome screenings. The members were found in prokaryotes and eukaryotes, but not in any archaeal organism. Multiple sequence alignments and calculations of evolutionary trees revealed a clear subdivision of the eukaryotic MAPEG members, corresponding to the six families of microsomal glutathione transferases (MGST) 1, 2 and 3, leukotriene C4 synthase (LTC4), 5-lipoxygenase activating protein (FLAP), and prostaglandin E synthase. Prokaryotes contain at least two distinct potential ancestral subfamilies, of which one is unique, whereas the other most closely resembles enzymes that belong to the MGST2/FLAP/LTC4 synthase families. The insect members are most similar to MGST1/prostaglandin E synthase. With the new data available, we observe that fish enzymes are present in all six families, showing an early origin for MAPEG family differentiation. Thus, the evolutionary origins and relationships of the MAPEG superfamily can be defined, including distinct sequence patterns characteristic for each of the subfamilies. We have further investigated and functionally characterized representative gene products from Escherichia coli, Synechocystis sp., Arabidopsis thaliana and Drosophila melanogaster, and the fish liver enzyme, purified from pike (Esox lucius). Protein overexpression and enzyme activity analysis demonstrated that all proteins catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene with reduced glutathione. The E. coli protein displayed glutathione transferase activity of 0.11 micromol.min(-1).mg(-1) in the membrane fraction from bacteria overexpressing the protein. Partial purification of the Synechocystis sp. protein yielded an enzyme of the expected molecular mass and an N-terminal amino acid sequence that was at least 50% pure, with a specific activity towards 1-chloro-2,4-dinitrobenzene of 11 micromol.min(-1).mg(-1). Yeast microsomes expressing the Arabidopsis enzyme showed an activity of 0.02 micromol.min(-1).mg(-1), whereas the Drosophila enzyme expressed in E. coli was highly active at 3.6 micromol.min(-1).mg(-1). The purified pike enzyme is the most active MGST described so far with a specific activity of 285 micromol.min(-1).mg(-1). Drosophila and pike enzymes also displayed glutathione peroxidase activity towards cumene hydroperoxide (0.4 and 2.2 micromol.min(-1).mg(-1), respectively). Glutathione transferase activity can thus be regarded as a common denominator for a majority of MAPEG members throughout the kingdoms of life whereas glutathione peroxidase activity occurs in representatives from the MGST1, 2 and 3 and PGES subfamilies.

In this study, we explored the multiple heavy metal-resistant yeast isolated from heavy metal-polluted environment. The isolated yeast showed maximum growth at 30 °C, pH 7.0, and the strain was identified as Candida tropicalis through 18S ribosomal RNA (rRNA) gene sequence analysis. Yeast cells grew well in medium containing different concentrations of heavy metal ions [CdCl₂, Pb(NO₃)₂, NaAsO₂, CuSO₄ and K₂Cr₂O₇]. Minimum inhibitory concentration (MIC) against different metal ions was ranged from 5 to 19 mM, and the metal resistance value against each metal observed by yeast cells was 5 mM (Cr), 10 mM (Cd), 15 mM (As), 14 mM (Cu) and 19 mM (Pb) and increased in the following order: Pb > Cu > As ≥ Cd > Cr. The total cellular glutathione, GSH/GSSG redox couple and metallothioneins like protein (MT) were assayed by growing cultures for 24 h and exposed to 100 mg/L of each heavy metal ion. Remarkable increase in γ-glutamylcysteinylglycine (GSH) level was determined in arsenic and cadmium treatment followed by chromium, lead and copper. Stressed cells had much more oxidized GSH than unstressed cells. GSH/GSSG ratio was significantly increased in cadmium and copper treatment in contrast to chromium, arsenic and lead. Statistical analysis revealed significantly higher cysteine level in all metal-treated samples as compared to control. Antioxidant glutathione transferase activity was not detected in metal-treated and untreated yeast samples. One-dimensional electrophoresis of proteins revealed marked differences in banding pattern of heavy metal-exposed yeast samples. A prominent 20 kDa band was observed in all treated samples suggesting that some differential proteins could be over-expressed during heavy metal treatment and might be involved in cell resistance mechanisms.

The glutathione transferases decrease the pKa of glutathione, allowing its deprotonation and the formation of the more reactive thiolate anion. The thiolate is maintained in the active site through a weak conventional hydrogen bond first sphere interaction donated by a Tyr hydroxyl in the Alpha, Mu, Pi, and Sigma glutathione transferase classes that can be modified by other second sphere or indirect thiolate contacts. However, the Theta and Delta class isoforms use a Ser hydroxyl for stabilizing the GSH thiolate, and as such, have a different chemical system compared with that of the Tyr possessed by other classes. We have used high level ab initio methods to investigate this interaction by using a simple methanol methanethiol system as a model. The hydrogen bond strength of this initial first sphere interaction was calculated to be less than that of the Tyr interaction. A putative second sphere interaction exists in the Theta and Delta class structures between Cys or Ser-14 and Ser-11 in the mammalian Theta subclass 1 and 2, respectively. The effect of this interaction on the first sphere interaction has also been investigated and found to significantly increase the energy of the bond.

Glutathione peroxidase is involved in scavenging free radicals in many biological systems. Its presence in plants has been implicated by identification of genes with nucleotide sequences similar to those of mammals, as well as by indirect, coupled enzyme assays. We directly quantified glutathione peroxidase activity in crude plant extracts using an organic hydroperoxide substrate and measuring GSSG, the direct reaction product, by adapting a sensitive fluorometric method. The constitutive levels of glutathione peroxidase in a paraquat-resistant biotype of Conyza bonariensis (resistant to many oxidative stresses) were almost double those of the sensitive biotype, providing correlative evidence that glutathione peroxidase is part of the system preventing oxidative damage. Glutathione peroxidase activity was separated from both glutathione transferase and glutathione reductase activities by specific antibody immunoprecipitation and by substrate-affinity chromatography. Conyza glutathione peroxidase cross-reacted with citrus polyclonal anti-phospholipid hydroperoxide glutathione peroxidase. The data suggest that this glutathione peroxidase is a component of the systems dealing with oxidative damage protection in plants and is biochemically different from glutathione peroxidases that also have glutathione transferase activity.

The effect of the mono-oxygenase inhibitors tridiphane, piperonyl butoxide and prochloraz on propanil uptake, metabolism and phytotoxicity was measured in a resistant (R) biotype of Echinochloa colona. The uptake of propanil was not significantly affected by any of the three mono-oxygenase inhibitors. The first metabolite of propanil metabolism, 3,4-dichloroaniline, was found to accumulate to higher levels in E. colona treated with each of the mono-oxygenase inhibitors mixed with formulated propanil, compared to propanil applied alone. Accumulation of further metabolites of propanil (glucosyl-3,4-dichloroaniline and bound products) was reduced in the presence of mono-oxygenase inhibitors, compared with propanil application alone. Leaf damage caused by a single drop of propanil compared to propanil + mono-oxygenase inhibitor was used to assess the degree of propanil tolerance in E. colona biotypes. Leaf damage was significantly greater in propanil + mono-oxygenase inhibitor treatments. No leaf damage was observed in mono-oxygenase inhibitor treatments alone at the concentrations used. Peroxidase activity was measured in crude extracts of the R-biotype of E. colona using 3,4-dichloroaniline as substrate, in the presence and absence of mono-oxygenase inhibitors and the specific peroxidase inhibitor salicylhydroxamic acid. Peroxidase activity was inhibited by all three mono-oxygenase inhibitors at 10 μM and by salicylhydroxamic acid at 1 μm. Glucosyl-3,4-dichloroaniline was found not to be a substrate for peroxidase activity. These results suggest that the incorporation of 3,4-dichloroaniline into bound residues involves peroxidase activity which can be inhibited by mono-oxygenase inhibitors. When peroxidase activity is inhibited, the precursor metabolite 3,4-dichloroaniline accumulates, and propanil resistance in E. colona is reduced, possibly as a consequence of phytotoxicity of this metabolite and/or product inhibition of the first step in propanil metabolism, responsible for the formation of 3,4-dichloroaniline. Glasshouse trials have demonstrated that the application of mono-oxygenase inhibitors, (particularly tridiphane which is also known to inhibit glutathione transferase activity) with propanil offers a promising approach to the control of propanil resistant biotypes of Jungle-Rice.

The objective of this study was to identify and evaluate conserved biomarkers that could be used in most species of teleost fish at most life-stages. We investigated the effects of sublethal methylmercury (MeHg) exposure on developing rainbow trout and zebrafish. Juvenile rainbow trout and young adult zebrafish were fed food with MeHg added at 0, 0.5, 5 and 50 ppm. Atomic absorption spectrometry was applied to measure whole body total Hg levels, and pathologic analysis was performed to identify MeHg-induced toxicity. Fish at six weeks were sampled from each group for microarray analysis using RNA from whole fish. MeHg-exposed trout and zebrafish did not show overt signs of toxicity or pathology, nor were significant differences seen in mortality, length, mass, or condition factor. The accumulation of MeHg in trout and zebrafish exhibited dose- and time-dependent patterns during six weeks, and zebrafish exhibited greater assimilation of total Hg than rainbow trout. The dysregulated genes in MeHg-treated fish have multiple functional annotations, such as iron ion homeostasis, glutathione transferase activity, regulation of muscle contraction, troponin I binding and calcium-dependent protein binding. Genes were selected as biomarker candidates based on their microarray data and their expression was evaluated by QPCR. Unfortunately, these genes are not good consistent biomarkers for both rainbow trout and zebrafish from QPCR evaluation using individual fish. Our conclusion is that biomarker analysis for aquatic toxicant assessment using fish needs to be based on tissue-, sex- and species-specific consideration.

Blastoschizomyces capitatus is an uncommon, opportunistic pathogenic fungus, which causes invasive and disseminated infections. This microorganism is normally present in both environmental and normal human flora. Within a host, B. capitatus is able to grow in both unicellular yeast and multicellular filamentous growth forms. In this study, we obtained in vitro morphological conversion of B. capitatus from yeast-to-mycelial phase to investigate the presence and expression of glutathione transferase (GST) enzymes in both cell forms. A protein with GST activity using the model substrate 1-chloro-2,4-dinitrobenzene was detected in both morphologies and identified by tandem mass spectrometry as a eukaryotic elongation factor 1Bγ (eEF1Bγ) protein, a member of the GST superfamily. No significant difference in GST-specific activity and kinetic constants were observed between mycelial and yeast forms, indicating that eEF1Bγ protein did not show differential expression between the two phases.

Glutathione-mediated free-radical-scavenging and plasma membrane ATPase activities increase as sinks for metabolic energy with advancing tuber age. Plasma membrane ATPase activity from 19-month-old tubers was 77% higher than that from 7-month-old tubers throughout sprouting. The higher activity was not attended by an increase in the amount of ATPase per unit plasma membrane protein. Concentrations of oxidized (GSSG) and reduced glutathione more than doubled as tuber age advanced from 6 to 30 months, but the proportion of GSSG to total glutathione remained constant with age. The activity of glutathione transferase, an enzyme that catabolizes lipid-hydroperoxides, increased by 44 and 205% on a fresh weight and protein basis, respectively, as tubers aged from 6 to 30 months. Glutathione reductase activity also increased with advancing age, by 90% on a fresh weight basis and 305% on a protein basis. Older tubers had more glutathione reductase per unit of soluble and mitochondrial protein. The age-induced increase in cytosolic glutathione transferase activity was likely due to increased availability of lipid-hydroperoxides and/or a positive effector. Synthesis of glutathione requires ATP, and the increased reduction of GSSG resulting from catalysis of lipid-hydroperoxides is NADPH-dependent. Thus, increased plasma membrane ATPase and glutathione-mediated free-radical-scavenging activities likely constitute substantial sinks for ATP in older tubers prior to and during sprouting. Increased oxidative stress and loss in membrane integrity and central features of aging that undoubtedly contribute to the enhanced respiration of sprouting older tubers.

Prolonged exposure to mutagenic substances is strongly associated with an individual's risk of developing colorectal cancer. Clinical investigation of oltipraz as a chemopreventive agent is supported by its induction of the expression of detoxication enzymes in various tissues, and its protective activity against the formation of chemically induced colorectal tumors in animals. The goals of the present study were: to determine if oltipraz could induce detoxicating gene expression in human tissues; to identify effective non-toxic doses for more extensive clinical testing; and to establish a relationship between effects in the colon mucosa and those in a more readily available tissue, the peripheral mononuclear cell. 24 evaluable patients at high risk for colorectal cancer were treated in a dose-finding study with oltipraz 125, 250, 500, or 1,000 mg/m2 as a single oral dose. Biochemical analysis of sequential blood samples and colon mucosal biopsies revealed increases in glutathione transferase activity at the lower dose levels. These effects were not observed at the higher doses. More pronounced changes were observed in detoxicating enzyme gene expression in both tissues at all doses. Peripheral mononuclear cell and colon mRNA content for gamma-glutamylcysteine synthetase (gamma-GCS) and DT-diaphorase increased after dosing to reach a peak on day 2-4 after treatment, and declined to baseline in the subsequent 7-10 d. The extent of induction of gene expression in colon mucosa reached a peak of 5.75-fold for gamma-GCS, and a peak of 4.14-fold for DT-diaphorase at 250 mg/m2 ; higher doses were not more effective. Levels of gamma-GCS and DT-diaphorase correlated closely (P < or = 0.001) between peripheral mononuclear cells and colon mucosa both at baseline and at peak. These findings demonstrate that the administration of minimally toxic agents at low doses may modulate the expression of detoxicating genes in the tissues of individuals at high risk for cancer. Furthermore, peripheral mononuclear cells may be used as a noninvasive surrogate endpoint biomarker for the transcriptional response of normal colon mucosa to drug administration.

The objectives of this study were the changes of antioxidative key enzyme activities under stress conditions induced by a peroxidizing herbicide using photoheterotrophi-cally grown, suspension-cultured soybean celts (Glycine max L.). Within two days, 50 to 500 nM oxyfluorfen. a p-nitrodiphenyl ether herbicide, caused up to 100% inhibition of growth, while simultaneously, the chlorophyll was 25% to completely bleached. The major cellular antioxidants ascorbate and glutathione showed different responses. Under stress conditions with more than 250 nM oxyfluorfen, the cellular ascorbate- concentration was halved, whereas dehydroascorbate remained roughly constant. The glutathione content (approximately one-fifth of that of ascorbate in untreated control cells) increased nearly 3-fold in the presence of 250 nM oxyfluorfen. Under this condition, oxidized glutathione was 5 times above the control level. The specific activities of selected enzymes participating in cellular defence, namely ascor-bate peroxidase, glutathione reductase, rnonodehydroascorbate reductase. peroxidase and catalase increased by 40 to 70% with oxyfluorfen concentrations between 50 and 500 nM, while dehydroascorbate reductase showed a significant decrease. Glutathione transferase activity even increased 6-fold under oxyfluorfen stress.

A study has been carried out to investigate the cellular distribution and levels of glutathione-S-transferase isoenzymes (GST), acidic (pi), basic (alpha) and neutral (mu), in ovarian tumour biopsies, and to measure GST activity in the same tumour specimens. Two methods of assessing isoenzyme levels (immunohistochemistry and Western blot) were compared. Well-known important clinicopathological features were correlated with response to treatment, overall survival and progression-free survival for each of 97 patients from whom biopsies had been obtained. The glutathione-S-transferase isoenzyme levels were also correlated with overall and progression-free survival, and with the important clinicopathological features. As expected, there was a significant correlation between FIGO stage, histological grade of tumour, amount of residual disease after staging laparotomy, response to chemotherapy, and both overall and progression-free survival. Glutathione-S-transferase isoenzyme levels (acidic, basic and neutral) measured by Western blot were not found to be significantly correlated with any of the clinicopathological parameters tested. Using the immunohistochemistry method of detection there was a correlation between the GST acidic isoenzyme level and the amount of residual disease remaining after initial debulking surgery (higher levels were detected in the group with no residual disease, P=0.034), and also between the GST acidic isoenzyme level and the type of chemotherapy regimen used. Higher levels of the acidic isoenzyme were present in tumour biopsies taken from the patient group who had received a combination regimen (cyclophosphamide, carboplatin, ifosfamide and doxorubicin). The neutral and basic GST isoenzyme levels were not significantly correlated with any of the clinicopathological parameters. None of the GST isoenzyme levels were significantly correlated with response to treatment, overall survival or progression-free survival (using either method of detection). Similarly, glutathione transferase activity showed no significant correlation with prognosis or survival. Images Figure 1

The microsomal mixed function oxidase system metabolizes xenobiotics (Phase I) to products that, if not activated and conjugated for excretion (Phase II), are capable of forming conjugates with cellular macromolecules, including DNA, resulting in toxic, mutagenic, or carcinogenic events. Benzo(a)pyrene (BP), a polycyclic aromatic hydrocarbon, is a model carcinogen for this system. Vitamin K1 (phylloquinone) is a regulator of BP metabolism. These studies demonstrate that K1 is capable of increasing Phase I metabolism and decreasing glutathione transferase activity (Phase II) in chick embryo liver; that deprivation of K1 reduces BP/DNA adducts in mouse liver and reduces tumor formation in mice given intraperitoneal BP; and that K1 supplementation increases BP induced tumor formation in mice. However, epidemiologic studies indicate that children of mothers who smoke during pregnancy may not be at increased risk of cancer. It is known that the placentas from these pregnancies exhibit markedly increased levels of arylhydrocarbon hydroxylase induced by the polycyclic aromatic hydrocarbons in tobacco smoke, but there is no corresponding increase in this enzyme activity in the fetus in such pregnancies. We suggest that the low vitamin K level is a secondary protective mechanism for xenobiotics, such as BP, that may escape the primary placental screen. The recently described role of vitamin K-dependent Gla protein as ligands for receptor tyrosine kinases, also establishes K as a link in cell growth and transformation. It is proposed that the small total body pool of K1 in the adult, which is sufficient only to meet continuing needs, and the even smaller pool in the fetus are protective. This protective effect of low K1 levels is particularly important in the presence of the high mitotic rates and rapid cell turnover in the avian embryo and mammalian fetus.

International audience; Although trypanothione [T(S)2] is the major thiol component in trypanosomatidae, significant amounts of glutathione are present in Trypanosoma cruzi. This could be explained by the existence of enzymes using glutathione or both glutathione and T(S)2 as cofactors. To assess these hypotheses, a cytosolic fraction of T. cruzi epimastigotes was subjected to affinity chromatography columns using as ligands either S-hexylglutathione or a non-reducible analogue of trypanothione disulphide. A similar protein of 52 kDa was eluted in both cases. Its partial amino acid sequence indicated that it was identical with the protein encoded by the TcAc2 cDNA previously described [Schoneck, Plumas-Marty, Taibi et al. (1994) Biol. Cell 80, 1-10]. This protein showed no significant glutathione transferase activity but surprisingly catalysed the thiol-disulphide exchange between dihydrotrypanothione and glutathione disulphide. The kinetic parameters were in the same range as those determined for trypanothione reductase toward its natural substrate. This trypanothione-glutathione thioltransferase provides a new target for a specific chemotherapy against Chagas' disease and may constitute a link between the glutathione-based metabolism of the host and the trypanothione-based metabolism of the parasite.

The effects on drug metabolizing enzymes of cyclopropenoid fatty acids present in baobab seed oil were evaluated in rats fed either a diet with baobab seed oil (1.27% cyclopropenoid fatty acids in the diet) or a diet with heated baobab seed oil (0.046% cyclopropenoid fatty acids in the diet). Comparison was made with rats fed a mixture of oils that contained no cyclopropenoid fatty acid. Rats fed baobab oil showed retarded growth. In comparison with the other groups, the relative liver weights were markedly increased whereas cytochrome P-450 content and NADPH cytochrome c reductase and NADH cytochrome c reductase activities were decreased. In rats fed the heated baobab oil the relative liver weight was decreased and the cytochrome P-450 level and reductase activities were increased relative to levels in rats fed the unheated oil. Ethoxycoumarin deethylase, ethoxyresorufin deethylase and pentoxyresorufin depentylase activities, expressed on the basis of cytochrome P-450, were greater in the group fed unheated baobab seed oil. Cytosolic glutathione transferase activity was markedly decreased in rats fed fresh baobab seed oil and heating the oil, which reduced the content of cyclopropenoid fatty acids, led to a considerable increase of this activity. UDP-glucuronyl transferase activities were not modified by the type of oil included in the diet. It is possible that the mechanisms of action of cyclopropenoid fatty acids are related to alterations of membrane lipid composition or microsomal proteins.

Glutathione transferases (GSTs; EC. 2.1.5.18) activity was measured in maternal liver and conceptal tissues during gestation. In maternal liver, maximum activity was found at gestational day (GD) 9 after which it slowly decreased up to the end of gestation. The placental GSTs activity at GD18 was three times lower than that found at GD14. Conversely, fetal liver GSTs at GD14 was about 75% that at GD18. It was also observed that GSTs activity at GD9 and GD10 was higher in visceral yolk sac than in embryo proper. Substrate specificity measurements, SDS PAGE analysis and HPLC runs, carried out on GSH-affinity purified fractions, revealed that with the progress of gestation in maternal liver an increase in pi class GSTs subunit occurs, with a concomitant decrease in alpha class GSTs. With respect to the time of gestation, a significant change in alpha, mu and pi class GSTs expression also occurred in fetal liver and in chorioallantoic placenta. It was concluded that during gestation the GSTs system is subjected to a time-dependent and tissue-specific modulation which may play a protective role against developmental toxicants.

Potential mechanisms of resistance to azinphosmethyl, an organophosphate insecticide, were examined in five field populations of the tufted apple bud moth Platynota idaeusalis (Walker). Adult male moths from resistant and susceptible populations were collected on sex pheromone traps and analyzed for glutathione transferase, general carboxylesterase, and cytochrome P 450 monooxygenase activity. Kinetics and inhibition profiles for acetylcholinesterase were also compared among populations. Significant intra- and interpopulation variation was demonstrated in these detoxification enzyme systems, but no resistance correlation was found to carboxylesterases, cytochrome P 450 monooxygenases, or glutathione transferase using the substrate 1-chloro-2,4-dinitrobenzene or for acetylcholinesterase activity. However, elevated glutathione transferase activity toward the model substrate 1,2-dichloro-4-nitrobenzene was consistently associated with azinphosmethyl resistance.

The dihydrochalcone glycoside phloridzin is a dominant allelochemical in foliage of apple. This allelochemical varies within and among apple cultivars and can influence distribution and abundance of tufted apple bud moth, Platynota idaeusalis (Walker), in orchards. Laboratory bioassays were used to investigate the influence of phloridzin on tolerance of both susceptible and resistant colonies of P. idaeusalis to the organophosphate insecticide azinphosmethyI. Addition of phloridzin to diet did not influence tolerance of first ins tars of the susceptible strain to azinphosmethyI. Third instars of the susceptible strain exhibited reduced tolerance to azinphosmethyl in the presence of phloridzin, and this response was associated with inhibition of the activities of glutathione transferase, esterase, and aniline hydroxylation enzymes. Neonate larvae of the resistant strain fed on diet with phloridzin were more tolerant to azinphosmethyl at high insecticide concentrations only. Third instars of the resistant strain were equally tolerant to azinphosmethyl in the presence and absence of phloridzin. Although phloridzin inhibited glutathione transferase activity in resistant larvae, as in the susceptible strain, it increased activity of esterases. Our data provide further evidence that both host-plant allelochemistry and insect selection history influence results of pesticide bioassays.