Increased Glutathione S-transferase Activity Research Articles

Understanding the main route of drug entry in adult Fasciola hepatica: Further insights into closantel pharmacological activity

Closantel (CLS) is highly effective against adult liver flukes after its oral or subcutaneous (sc) administration in ruminants. Trans-tegumental diffusion and oral ingestion are the two potential routes available for the entry of drugs into Fasciola hepatica. The work reported here contributes to improve the understanding of CLS pharmacology. The main goals of were: I) to determine the pattern of in vivo CLS accumulation into adult F. hepatica and relevant tissues in CLS-treated sheep; II) to investigate the influence of the physicochemical composition of the incubation medium on the CLS diffusion process into adult F. hepatica; III) to assess the ovicidal activity of CLS against F. hepatica eggs; and IV) to investigate the in vivo effect of CLS treatment on glutathione S-transferases activity in adult liver flukes exposed to CLS. Fourteen healthy sheep were each orally infected with 75 F. hepatica metacercariae. Sixteen (16) weeks after infection, animals were treated with CLS by oral (n = 6, 10 mg/kg) or sub-cutaneous (sc) (n = 6, 5 mg/kg) route. At 12, 24 and 36 h post-treatment, animals were sacrificed (n = 2) and samples of blood, bile and adult F. hepatica were collected. In addition, flukes recovered from non-treated sheep (n = 2) were ex vivo incubated (60 min) in the presence of CLS in either RPMI or bile as incubation medium. CLS concentration was measured by HPLC. The ovicidal activity of CLS was investigated using eggs obtained from the bile of untreated sheep. Finally, glutathione S-transferase activity in F. hepatica recovered from untreated and CLS-treated sheep was assessed. In the in vivo studies, the highest CLS concentrations were measured in plasma and adult liver flukes. A positive correlation was observed between CLS concentration in plasma and in F. hepatica. Results obtained in the current work indicate that the in vivo accumulation of CLS into adult liver flukes occurs mainly by the oral route. After ex vivo incubation, the uptake of CLS by the parasite was markedly diminished in the presence of bile compared with that observed in the presence of RPMI as incubation medium. CLS lacks ovicidal activity at therapeutically relevant concentrations. Lastly, CLS significantly increased glutathione S-transferase activity in flukes recovered at 12 h (oral treatment) and 24 h (sc treatment), compared to the control liver flukes.

The role of 17β-estradiol metabolites in chromium-induced oxidative stress

The increasing incidence of estrogen-dependent breast cancer and the presence in the environment of a large number of factors that interact with estrogen receptors have sparked interest in chemical influences on estrogen-dependent processes. In a previous work, the authors examined the interaction of estradiol with chromium. In the present article the importance of estradiol biotransformation in these interactions is investigated. There is no information in the available literature about the role of metabolites in exposure to chromium. It seems important because estradiol metabolites have various carcinogenic abilities and their formation during biotransformation could be increased or decreased by environmental enzyme inducers or inhibitors. The metabolites could play a detoxifying role or create a toxic synergism in free radical processes induced by chromium VI (CrVI). The aim of this study was to evaluate the influence of 2 17β-estradiol metabolites - 4-hydroxyestradiol (4-OHE2) and 16α-hydroxyestrone (16α-OHE1) - in conditions of oxidative stress caused by CrVI. Human blood, erythrocytes or mitochondria isolated from human placentas after natural deliveries were used in the experiments. The influence of CrVI, 4-OHE2 and 16-OHE1 on thiobarbituric acid reactive substances (TBARS), the hydroxyl radical (•OH), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST), and the interactions of the metabolites exposed to chromium expressed by these factors were examined. 4-OHE2 reduced the level of TBARS induced by CrVI in mitochondria (p < 0.05) and in erythrocytes (p < 0.05), and increased SOD activity (p < 0.05). 16α-OHE1 increased the activity of GST in erythrocytes exposed to CrVI (p < 0.05). The metabolites do not have toxic interactions with CrVI. On the contrary, they exhibited a protective effect. The mechanism of protection varied: 4-OHE2 decreased TBARS and increased SOD activity, while 16α-OHE1 increased GST activity.

Chronic toxicity of arsenic during Rhinella arenarum embryonic and larval development: Potential biomarkers of oxidative stress and antioxidant response.

The Argentinean autochthonous toad Rhinella arenarum was selected to study the chronic toxicity of arsenic (As) and the biochemical responses elicited by exposure to As in water during embryonic and larval development. Significant decreases in the total reactive antioxidant potential and in catalase activity were observed in individuals exposed chronically to sublethal concentrations of As, which is indicative of an oxidative stress situation. However, an antioxidant response was elicited during chronic exposure to As, as evidenced by the increase in endogenous reduced glutathione content and glutathione-related enzymatic activities such as glutathione S-transferase (GST) and glutathione reductase. This protective response might prevent a deeper decline in the antioxidant system and further oxidative damage. Alternatively, it might be linked to As conjugation with reduced glutathione for its excretion. Considering the sustained increase in GST activity and the decrease in the total antioxidant reactive potential observed, the authors propose them as good candidates to be used as biomarkers during As exposure. Interestingly, glutathione reductase activity was inhibited at a very low concentration of As considered safe for aquatic life. Environ Toxicol Chem 2017;36:1614-1621. © 2016 SETAC.

Toxicity of Cu and Cr Nanoparticles to Daphnia magna

Little is known about the potential toxicity of Cu nanoparticles (nCu), Cr nanoparticles (nCr), and their mixtures to aquatic organisms. To fill this gap, a comprehensive toxicity assessment was conducted using Daphnia magna as a test organism, including a 48-h acute toxicity test, a 21-day chronic test, and a feeding experiment. Four biomarkers were estimated after exposure to nCu, nCr, and their mixtures for 7 days, including acetylcholinesterase (AChE), catalase (CAT), superoxide dismutase (SOD), and glutathione S-transferase (GST). The results at all endpoints showed that nCu was more toxic than nCr. The 48-h median lethal concentration values of nCu and nCr were 0.63 and 1.57 mg/L, respectively. Significant inhibition of reproduction and growth of D. magna was found, and the intrinsic rate of natural increase was a sensitive parameter for nCu and nCr during the 21-day exposure. A concentration-dependent decrease in filtration and ingestion was observed which was consistent with inhibition of reproduction and growth of D. magna. The biochemical responses revealed an increase in GST activity and decrease in AChE activity, while SOD and CAT activities were increased at low concentrations and decreased at high concentrations for all exposures. Collectively, our results confirmed that nanoscale Cu and Cr can exert negative effects at different levels on D. magna.

4-Vinylcyclohexene diepoxide disrupts sperm characteristics, endocrine balance and redox status in testes and epididymis of rats

ABSTRACTObjectives: Exposure to 4-vinylcyclohexene diepoxide (VCD) was reported to induce testicular germ cell toxicity in rodents. However, there is paucity of information on the precise biochemical and molecular mechanisms of VCD-induced male reproductive toxicity.Methodology: This study investigated the influence of VCD on testicular and epidydimal functions following oral exposure of Wistar rats to VCD at 0, 100, 250 and 500 mg/kg for 28 consecutive days.Results: Administration of VCD significantly decreased the body weight gain and organo-somatic indices of the testes and epididymis. When compared with the control, VCD significantly decreased superoxide dismutase and catalase activities in the testes whereas it significantly decreased superoxide dismutase activity but increased catalase activity in the epididymis. Moreover, while glutathione peroxidase activity and glutathione level remain unaffected, exposure of rats to VCD significantly increased glutathione S-transferase activity as well as hydrogen peroxide and malondialdehyde levels in testes and epididymis of the treated rats. The spermiogram of VCD-treated rats showed significant decrease in epididymal sperm count, sperm progressive motility, testicular sperm number and daily sperm production when compared with the control. Administration of VCD significantly decreased circulatory concentrations of follicle-stimulating hormone, luteinizing hormone and testosterone along with testicular and epididymal degeneration in the treated rats. Immunohistochemical analysis showed significantly increased cyclooxygenase-2, inducible nitric oxide synthase, caspase-9 and caspase-3 protein expressions in the testes of VCD-treated rats.Conclusion: Exposure to VCD induces testicular and epidydimal dysfunctions via endocrine suppression, disruption of antioxidant enzymes activities, increase in biomarkers of oxidative stress, inflammation and apoptosis in rats.

Inhibition of eicosanoid signaling leads to increased lipid peroxidation in a host/parasitoid system

We posed the hypothesis that inhibition of eicosanoid biosynthesis leads to increased lipid peroxidation in insects. Here we report that rearing the greater wax moth, Galleria mellonella, on media supplemented with selected inhibitors of eicosanoid biosynthesis throughout the larval, pupal and adult life led to major alterations in selected oxidative and antioxidative parameters of wax moth and its ectoparasitoid, Bracon hebetor. The highest dietary dexamethasone (Dex), esculetin (Esc) and phenidone (Phe) led to increased malondialdehyde (MDA) levels and to elevated catalase (CAT) and glutathione-S-transferase (GST) activities in all developmental stages of host larvae. Dietary Phe resulted in increased MDA levels, and CAT activity in G. mellonella adults by about 4-fold and about 2-fold, respectively. The Phe effect on GST activity in all stages of the wax moth was expressed in a dose-dependent manner, increased to 140nmol/mg protein/min in larvae. MDA levels were increased by over 30-fold in adult wasps reared on Dex- and Esc-treated hosts. CAT and GST activities were increased in adult parasitoids reared on Esc-and Phe-treated hosts. GST activity of Dex-treated parasitoid larvae increased from about 4 to over 30nmol/mg protein/min. Dietary Phe led to increased GST activity, by about 25-fold, in adult wasps. These data indicate that chronic inhibition of eicosanoid biosynthesis leads to increased oxidative stress, strongly supporting our hypothesis. The significance of this work lies in understanding the roles of eicosanoids in insect biology. Aside from other well-known eicosanoids actions, we propose that eicosanoids mediate reductions in oxidative stress.

GST activity and membrane lipid saturation prevents mesotrione-induced cellular damage in Pantoea ananatis.

Callisto®, containing the active ingredient mesotrione (2-[4-methylsulfonyl-2-nitrobenzoyl]1,3-cyclohenanedione), is a selective herbicide that controls weeds in corn crops and is a potential environmental contaminant. The objective of this work was to evaluate enzymatic and structural changes in Pantoea ananatis, a strain isolated from water, in response to exposure to this herbicide. Despite degradation of mesotrione, probably due a glutathione-S-transferase (GST) pathway in Pantoea ananatis, this herbicide induced oxidative stress by increasing hydrogen peroxide production. Thiol fragments, eventually produced after mesotrione degradation, could be involved in increased GST activity. Nevertheless, there was no peroxidation damage related to this production, as malondialdehyde (MDA) synthesis, which is due to lipid peroxidation, was highest in the controls, followed by the mesotrione- and Callisto®-treated cultures at log growth phase. Therefore, P. ananatis can tolerate and grow in the presence of the herbicide, probably due an efficient control of oxidative stress by a polymorphic catalase system. MDA rates depend on lipid saturation due to a pattern change to a higher level of saturation. These changes are likely related to the formation of GST-mesotrione conjugates and mesotrione degradation-specific metabolites and to the presence of cytotoxic adjuvants. These features may shift lipid membrane saturation, possibly providing a protective effect to bacteria through an increase in membrane impermeability. This response system in P. ananatis provides a novel model for bacterial herbicide tolerance and adaptation in the environment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0240-x) contains supplementary material, which is available to authorized users.

Enzymatic Activity of Glutathione S-Transferase and Dental Fluorosis Among Children Receiving Two Different Levels of Naturally Fluoridated Water.

This study was conducted to measure the activity of the enzyme glutathione S-transferase (GST) in saliva and to compare the activity of this enzyme in children with and without dental fluorosis in communities with different concentrations of naturally fluoridated water. A total of 141 schoolchildren participated in this cross-sectional study. Children were selected from two communities: one with a low (0.4ppm) and the other with a high (1.8ppm) water fluoride concentration. Dental fluorosis was evaluated by applying the Thylstrup and Fejerskov Index (TFI) criteria. Stimulated saliva was obtained, and fluoride concentration and GST activity were measured. The GST activity was compared among children with different levels of dental fluorosis using multinomial logistic regression models and odds ratios (OR). The mean age of the children was 10.6 (±1.03) years. Approximately half of the children showed dental fluorosis (52.5%). The average GST activity was 0.5678 (±0.1959) nmol/min/μg. A higher concentration of fluoride in the saliva was detected in children with a higher GST activity (p=0.039). A multinomial logistic regression model used to evaluate the GST activity and the dental fluorosis score identified a strong association between TFI=2-3 (OR=15.44, p=0.007) and TFI≥4 (OR=55.40, p=0.026) and the GST activity level, compared with children showing TFI=0-1, adjusted for age and sex. Schoolchildren with higher levels of dental fluorosis and a higher fluoride concentration in the saliva showed greater GST activity. The increased GST activity most likely was the result of the body's need to inactivate free radicals produced by exposure to fluoride.

Differential Action between Schisandrin A and Schisandrin B in Eliciting an Anti-Inflammatory Action: The Depletion of Reduced Glutathione and the Induction of an Antioxidant Response.

Schisandrin A (Sch A) and schisandrin B (Sch B) are active components of Schisandrae Fructus. We compared the biochemical mechanism underlying the anti-inflammatory action of Sch A and Sch B, using cultured lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and concanavalin (ConA)-stimulated mouse splenocytes. Pre-incubation with Sch A or Sch B produced an anti-inflammatory action in LPS-stimulated RAW264.7 cells, as evidenced by the inhibition of the pro-inflammatory c-Jun N-terminal kinases/p38 kinase/nuclear factor-κB signaling pathway as well as the suppression of various pro-inflammatory cytokines and effectors, with the extent of inhibition by Sch A being more pronounced. The greater activity of Sch A in anti-inflammatory response was associated with a greater decrease in cellular reduced glutathione (GSH) level and a greater increase in glutathione S-transferase activity than corresponding changes produced by Sch B. However, upon incubation, only Sch B resulted in the activation of the nuclear factor (erythroid-derived 2)-like factor 2 and the induction of a significant increase in the expression of thioredoxin (TRX) in RAW264.7 cells. The Sch B-induced increase in TRX expression was associated with the suppression of pro-inflammatory cytokines and effectors in LPS-stimulated macrophages. Studies in a mouse model of inflammation (carrageenan-induced paw edema) indicated that while long-term treatment with either Sch A or Sch B suppressed the extent of paw edema, only acute treatment with Sch A produced a significant degree of inhibition on the inflammatory response. Although only Sch A decreased the cellular GSH level and suppressed the release of pro-inflammatory cytokines and cell proliferation in ConA-simulated splenocytes in vitro, both Sch A and Sch B treatments, while not altering cellular GSH levels, suppressed ConA-stimulated splenocyte proliferation ex vivo. These results suggest that Sch A and Sch B may act differentially on activating GST/ depleting cellular GSH and inducing an antioxidant response involved in their anti-inflammatory actions.

Use of an integrated biomarker-based strategy to evaluate physiological stress responses induced by environmental concentrations of caffeine in the Mediterranean mussel Mytilus galloprovincialis

The occurrence of caffeine (CF), a biologically active drug, has widely been documented in coastal waters, and whether its environmental concentrations do represent a threat for marine organisms is unclear. The present study aimed at assessing sub-lethal effects induced by a 7-day exposure to environmentally relevant concentrations of CF (5, 50 and 500ng/L) in the Mediterranean mussel, Mytilus galloprovincialis. CF in water and mussel tissues, and a battery of biomarkers, including lysosomal parameters of general stress, oxidative stress responses and endpoints of neurological and genetic damages, were evaluated and tested for significance vs controls (p<0.05). CF exposure triggered a significant decrease of lysosomal membrane stability in both haemocytes and digestive gland (at 50 and 500ng/L CF) and a significant increase of lysosomal content of neutral lipids (at 500ng/L CF), indicating the onset of a stress syndrome. No effects were noted on lipid peroxidation parameters, such as malondialdehyde and lipofuscin content. The activity of the antioxidant enzymes glutathione S-transferase (GST) and catalase was unmodified in gills, while a significant increase of GST activity was observed in digestive gland (at 5 and 500ng/L CF), suggesting the occurrence of GST-mediated phase II detoxifying processes. CF did not induce geno/neurotoxicity, as shown by the lack of effects on primary DNA damages and acetylcholinesterase activity. In line with its high hydrophilicity, CF did not bioaccumulate in mussel tissues. Data were integrated using the Mussel Expert System, which assigned a low stress level to mussels exposed to 500ng/L CF, whereas no alterations of animal health status were highlighted at lower dosages. This study revealed a low profile of toxicity for environmental concentrations of CF, and confirmed the suitability of an integrated biomarker-based approach to provide a comprehensive picture of the degree of stress induced by emerging contaminants in marine invertebrates.

A de novo transcriptome of the Malpighian tubules in non-blood-fed and blood-fed Asian tiger mosquitoes Aedes albopictus: insights into diuresis, detoxification, and blood meal processing.

Background. In adult female mosquitoes, the renal (Malpighian) tubules play an important role in the post-prandial diuresis, which removes excess ions and water from the hemolymph of mosquitoes following a blood meal. After the post-prandial diuresis, the roles that Malpighian tubules play in the processing of blood meals are not well described.Methods. We used a combination of next-generation sequencing (paired-end RNA sequencing) and physiological/biochemical assays in adult female Asian tiger mosquitoes (Aedes albopictus) to generate molecular and functional insights into the Malpighian tubules and how they may contribute to blood meal processing (3–24 h after blood ingestion).Results/Discussion. Using RNA sequencing, we sequenced and assembled the first de novo transcriptome of Malpighian tubules from non-blood-fed (NBF) and blood-fed (BF) mosquitoes. We identified a total of 8,232 non-redundant transcripts. The Malpighian tubules of NBF mosquitoes were characterized by the expression of transcripts associated with active transepithelial fluid secretion/diuresis (e.g., ion transporters, water channels, V-type H+-ATPase subunits), xenobiotic detoxification (e.g., cytochrome P450 monoxygenases, glutathione S-transferases, ATP-binding cassette transporters), and purine metabolism (e.g., xanthine dehydrogenase). We also detected the expression of transcripts encoding sodium calcium exchangers, G protein coupled-receptors, and septate junctional proteins not previously described in mosquito Malpighian tubules. Within 24 h after a blood meal, transcripts associated with active transepithelial fluid secretion/diuresis exhibited a general downregulation, whereas those associated with xenobiotic detoxification and purine catabolism exhibited a general upregulation, suggesting a reinvestment of the Malpighian tubules’ molecular resources from diuresis to detoxification. Physiological and biochemical assays were conducted in mosquitoes and isolated Malpighian tubules, respectively, to confirm that the transcriptomic changes were associated with functional consequences. In particular, in vivo diuresis assays demonstrated that adult female mosquitoes have a reduced diuretic capacity within 24 h after a blood meal. Moreover, biochemical assays in isolated Malpighian tubules showed an increase in glutathione S-transferase activity and the accumulation of uric acid (an end product of purine catabolism) within 24 h after a blood meal. Our data provide new insights into the molecular physiology of Malpighian tubules in culicine mosquitoes and reveal potentially important molecular targets for the development of chemical and/or gene-silencing insecticides that would disrupt renal function in mosquitoes.

Gills as a glutathione-dependent metabolic barrier in Pacific oysters Crassostrea gigas: Absorption, metabolism and excretion of a model electrophile

The mercapturic acid pathway (MAP) is a major phase II detoxification route, comprising the conjugation of electrophilic substances to glutathione (GSH) in a reaction catalyzed by glutathione S-transferase (GST) enzymes. In mammals, GSH-conjugates are exported from cells, and the GSH-constituent amino acids (Glu/Gly) are subsequently removed by ectopeptidases. The resulting Cys-conjugates are reabsorbed and, finally, a mercapturic acid is generated through N-acetylation. This pathway, though very well characterized in mammals, is poorly studied in non-mammalian biological models, such as bivalve mollusks, which are key organisms in aquatic ecosystems, aquaculture activities and environmental studies. In the present work, the compound 1-chloro-2,4-dinitrobenzene (CDNB) was used as a model electrophile to study the MAP in Pacific oysters Crassostrea gigas. Animals were exposed to 10μM CDNB and MAP metabolites were followed over 24h in the seawater and in oyster tissues (gills, digestive gland and hemolymph). A rapid decay was detected for CDNB in the seawater (half-life 1.7h), and MAP metabolites peaked in oyster tissues as soon as 15min for the GSH-conjugate, 1h for the Cys-conjugate, and 4h for the final metabolite (mercapturic acid). Biokinetic modeling of the MAP supports the fast CDNB uptake and metabolism, and indicated that while gills are a key organ for absorption, initial biotransformation, and likely metabolite excretion, hemolymph is a possible milieu for metabolite transport along different tissues. CDNB-induced GSH depletion (4h) was followed by increased GST activity (24h) in the gills, but not in the digestive gland. Furthermore, the transcript levels of glutamate-cysteine ligase, coding for the rate limiting enzyme in GSH synthesis, and two phase II biotransformation genes (GSTpi and GSTo), presented a fast (4h) and robust (∼6–70 fold) increase in the gills. Waterborne exposure to electrophilic compounds affected gills, but not digestive gland, while intramuscular exposure was able to modulate biochemical parameters in both tissues. This study is the first evidence of a fully functional and interorgan MAP pathway in bivalves. Hemolymph was shown to be responsible for the metabolic interplay among tissues, and gills, acting as a powerful GSH-dependent metabolic barrier against waterborne electrophilic substances, possibly also participating in metabolite excretion into the sea water. Altogether, experimental and modeled data fully agree with the existence of a classical mechanism for phase II xenobiotic metabolism and excretion in bivalves.

Integrated assessment of biochemical markers in premetamorphic tadpoles of three amphibian species exposed to glyphosate- and methidathion-based pesticides in single and combination forms.

In this study, we evaluated the toxic effects of a glyphosate-based herbicide (GBH) and a methidathion-based insecticide (MBI), individually and in combination, on premetamorphic tadpoles of three anuran species: Pelophylax ridibundus, Xenopus laevis, and Bufotes viridis. Based on the determined 96-h LC50 values of each species, the effects of a series of sublethal concentrations of single pesticides and their mixtures after 96-h exposure and also the time-related effects of a high sublethal concentration of each pesticide were evaluated, with determination of changes in selected biomarkers: glutathione S-transferase (GST), glutathione reductase (GR), acetylcholinesterase (AChE), carboxylesterase (CaE), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). Also, the integrated biomarker response (IBR) was used to assess biomarker responses and quantitatively evaluate toxicological effects. Isozyme differences in CaE inhibition were assessed using native page electrophoresis; results showed that GBH to cause structural changes in the enzyme but not CaE inhibition in P.ridibundus. In general, single MBI and pesticide mixture exposures increased GST activity, while single GBH exposures decreased GST activity in exposed tadpoles. The AChE and CaE activities were inhibited after exposure to all single MBI and pesticide mixtures. Also, higher IBR values and GST, GR, AST, and LDH activities were determined for pesticide mixtures compared with single-pesticide exposure. This situation may be indicative of a synergistic interaction between pesticides and a sign of a more stressful condition.

Biochemical responses in freshwater fish after exposure to water-soluble fraction of gasoline.

The water-soluble fraction of gasoline (WSFG) is a complex mixture of mono-polycyclic aromatic hydrocarbons. The study aimed to evaluate the effects of WSFG diluted 1.5% on freshwater fish. Astyanax altiparanae were exposed to the WSFG for 96 h, under a semi-static system, with renewal of 25% of the gasoline test solution every 24 h. In addition, a decay of the contamination (DC) was carried out. During DC, the fish was exposed to the WSFG for 8 d, followed by another 7 d with renewal of 25% of volume aquaria with clean water every 24 h. For depuration, fish were transferred to aquaria with clean water, and in addition, 25% of the water was replaced every 24 h. The liver and kidney biotransformation, antioxidant defenses and lipid peroxidation (LPO) levels were evaluated. In the liver, the WSFG 1.5% caused reduction of glutathione S-transferase (GST) after 96 h and DC. In the kidney, only in depuration an increased GST activity was observed, and after DC a higher LPO levels. An increase of the superoxide dismutase (SOD) activity occurred at 96 h in both tissues; however, in the liver was also observed during the depuration. In WSFG 96 h, the glutathione peroxidase (GPx) activity in the kidney increased. As biomarkers of neurotoxicity, the brain and muscle acetylcholinesterase activities were measured, but the WSFG 1.5% did not change them. Therefore, this study brought forth more data about WSFG effects on freshwater fish after lower concentrations exposure and a DC, simulating an environmental contamination.

Activity and Transcriptional Responses of Hepatopancreatic Biotransformation and Antioxidant Enzymes in the Oriental River Prawn Macrobrachium nipponense Exposed to Microcystin-LR.

Microcystins (MCs) are a major group of cyanotoxins with side effects in many organisms; thus, compounds in this group are recognized as potent stressors and health hazards in aquatic ecosystems. In order to assess the toxicity of MCs and detoxification mechanism of freshwater shrimp Macrobrachium nipponense, the full-length cDNAs of the glutathione S-transferase (gst) and catalase (cat) genes were isolated from the hepatopancreas. The transcription level and activity changes in the biotransformation enzyme (glutathione S-transferase (GST)) and antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)) in the hepatopancreas of M. nipponense exposed to MC-LR (0.2, 1, 5, and 25 μg/L) for 12, 24, 72 and 96 h were analyzed. The results showed that the isolated full-length cDNAs of cat and gst genes from M. nipponense displayed a high similarity to other crustaceans, and their mRNAs were mainly expressed in the hepatopancreas. MC-LR caused significant increase of GST activity following 48–96 h (p < 0.05) and an increase in SOD activity especially in 24- and 48-h exposures. CAT activity was activated when exposed to MC-LR in 12-, 24- and 48-h exposures and then it was inhibited at 96-h exposure. There was no significant effect on GPx activity after the 12- and 24-h exposures, whereas it was significantly stimulated after the 72- and 96-h exposures (p < 0.05). The transcription was altered similarly to enzyme activity, but the transcriptional response was generally more immediate and had greater amplitude than enzymatic response, particularly for GST. All of the results suggested that MC-LR can induce antioxidative modulation variations in M. nipponense hepatopancreas in order to eliminate oxidative damage.

Investigation of insecticide-resistance status of Cydia pomonella in Chinese populations.

The codling moth Cydia pomonella (L.) is an economically important fruit pest and it has been directly targeted by insecticides worldwide. Serious resistance to insecticides has been reported in many countries. As one of the most serious invasive pest, the codling moth has populated several areas in China. However, resistance to insecticides has not been reported in China. We investigated the insecticide-resistance status of four field populations from Northwestern China by applying bioassays, enzyme activities, and mutation detections. Diagnostic concentrations of lambda-cyhalothrin, chlorpyrifos-ethyl, carbaryl, and imidacloprid were determined and used in bioassays. Field populations were less susceptible to chlorpyrifos-ethyl and carbaryl than laboratory strain. Insensitive populations displayed an elevated glutathione S-transferases (GSTs) activity. Reduced carboxylesterase (CarE) activity was observed in some insecticide insensitive populations and reduced acetylcholinesterase activity was observed only in the Wuw population. The cytochrome P450 polysubstrate monooxygenases activities in four field populations were not found to be different from susceptible strains. Neither the known-resistance mutation F399V in the acetylcholinesterase (AChE) gene, ace1, nor mutations in CarE gene CpCE-1 were found in adult individuals from our field populations. Native-PAGE revealed that various CarE isozymes and AChE insensitivity were occurring among Chinese populations. Our results indicate that codling moth populations from Northwestern China were insensitivity to chlorpyrifos-ethyl and carbaryl. Increased GST activity was responsible for insecticides insensitivity. Decreased CarE activity, as well as the presence of CarE and AChE polymorphisms might also be involved in insecticides insensitivity. New management strategies for managing this pest are discussed.

Effect of fluoxetine treatment on mitochondrial bioenergetics in central and peripheral rat tissues.

Recent investigations have focused on the mitochondrion as a direct drug target in the treatment of metabolic diseases (obesity, metabolic syndrome). Relatively few studies, however, have explicitly investigated whether drug therapies aimed at changing behavior by altering central nervous system (CNS) function affect mitochondrial bioenergetics, and none has explored their effect during early neonatal development. The present study was designed to evaluate the effects of chronic treatment of newborn male rats with the selective serotonin reuptake inhibitor fluoxetine on the mitochondrial bioenergetics of the hypothalamus and skeletal muscle during the critical nursing period of development. Male Wistar rat pups received either fluoxetine (Fx group) or vehicle solution (Ct group) from the day of birth until 21 days of age. At 60 days of age, mitochondrial bioenergetics were evaluated. The Fx group showed increased oxygen consumption in several different respiratory states and reduced production of reactive oxygen species, but there was no change in mitochondrial permeability transition pore opening or oxidative stress in either the hypothalamus or skeletal muscle. We observed an increase in glutathione S-transferase activity only in the hypothalamus of the Fx group. Taken together, our results suggest that chronic exposure to fluoxetine during the nursing phase of early rat development results in a positive modulation of mitochondrial respiration in the hypothalamus and skeletal muscle that persists into adulthood. Such long-lasting alterations in mitochondrial activity in the CNS, especially in areas regulating appetite, may contribute to permanent changes in energy balance in treated animals.

The monoterpenoid (S)-carvone massively up-regulates several classes of glutathione S-transferase genes in tobacco leaf discs

The monoterpenoid (S)-carvone was shown earlier to induce glutathione S-transferase (GST) activity and to attenuate necrotic lesion formation in Tobacco mosaic virus (TMV) inoculated, hypersensitively reacting tobacco Xanthi-nc plants. To explore structure-activity relationships, three monoterpenes and five monoterpenoids were tested for their ability to induce increased GST activity in tobacco leaf discs. (S)-Carvone proved to be the most potent inducer. (S)-Carvone treatments markedly up-regulated the expression of several tobacco GST genes belonging to the phi, tau and theta classes, particularly in the case of tau class GSTs. Furthermore, TMV inoculation slightly induced the transcript abundance of the GSTT2 gene. The expression of three catalase (CAT) genes was also examined in (S)-carvone-treated tobacco leaf discs. The expression of CAT1 was not influenced by (S)-carvone treatments, whereas that of CAT2 was significantly repressed. (S)-Carvone very weakly induced the transcription of the CAT3 gene....

E2 potentializes benzo(a)pyrene-induced hepatic cytochrome P450 enzyme activities in Nile tilapia at high concentrations.

In the aquatic environment, biotransformation enzymes are established biomarkers for assessing PAH exposure in fish, but little is known about the effect of 17β-estradiol (E2) on these enzymes during exposure to benzo(a)pyrene (BaP). In this study, Nile tilapia (Oreochromis niloticus) were exposed for 3, 5, and 10 days to BaP (300 μg L(-1)) and E2 (5 μg L(-1)). These substances were applied isolated or mixed. In the mixture experiment, fish were analyzed pre- and postexposure in order to better understand whether preexposure to the hormone masks the responses activated by PAH or vice versa. Phase I enzymes ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-depenthylase (PROD), and benzyloxyresorufin-O-debenzylase (BROD) activities as well as the phase II enzyme glutathione S-transferase (GST) were analyzed. Isolated E2 treatment decreased EROD activity after 3 days, but this enzyme activity returned to control values after 5 and 10 days of exposure. Isolated BaP treatment significantly induced EROD activity after 3 and 5 days, and the activity returned to control levels after ten exposure days. Combined treatment (E2 + Bap) significantly increased EROD activity, both in the pre- and postexposure. This increase was even higher than in the isolated BaP treatment, suggesting a synergism between these two compounds. When E2 and BaP were used singly, they did not change BROD and PROD activities. However, combined treatment (E2 + Bap) significantly increased PROD activity. Isolated BaP treatment increased GST activity after 10 days. However, this response was not observed in the mixture treatment, suggesting that E2 suppressed the GST induction modulated by BaP. The results put together indicated that E2 altered the biotransformation pathway regarding enzymes activated by BaP in Nile tilapia.

Interplay between GST and nitric oxide in the early response of soybean (Glycine max L.) plants to salinity stress

Glutathione-s-transferases (GSTs) and nitric oxide (NO) have both been implicated in the response of plants to salinity stress. However, their interplay and underlying mechanisms are relatively unknown. The present study attempts to provide new insight into the time course effects of NO application on GST biosynthesis regulation in Glycine max L. leaves under salt stress. A 150μM concentration of sodium nitroprusside (SNP), a widely used NO donor, was sprayed on soybean seedlings for two days at 24h intervals, followed by application of 200mM NaCl. The relative water content (RWC), total chlorophyll content (CHL), stomatal conductance (gs), ABA content, malondialdehyde (MDA), hydrogen peroxide content (H2O2), along with GST enzyme and isoenzyme activities and GST1 and GST4 transcript levels were determined at 0h, 6h and 12h after stress imposition. The results indicated that salt treatment alone did not alter MDA, H2O2 or ABA content and stomatal conductance in soybean leaves, most likely due to short-term (6h and 12h) application, although lower RWC and CHL were recorded. SNP treatment alone increased ABA content and reduced stomatal conductance, but did not change RWC, CHL, MDA (except at 12h) and H2O2. However, exogenous SNP application protected soybean leaves from salt stress by increasing RWC, CHL and ABA content, as well as by lowering stomatal conductance in order to maintain water balance. A significant increase in GST activity was recorded under salt stress alone at 6h. Conversely, SNP application lowered GST activity in soybean leaves at 0h and 12h, while it increased at 6h, supported by GST isoenzyme activities. Thus, it could be suggested that exogenous NO application induced GST activity in an ABA-dependent manner, while GST activity could also be induced by salt stress independent of ABA. In addition, SNP pre-treatment in salt-stressed seedlings lowered GST activity at 6h and 12h, in line with the GST isoenzyme expression profile. Finally, GST1 and GST4 transcript levels were significantly induced in both salt-stressed and SNP pre-treated and subsequently stressed samples at 6h and 12h, while a more variable regulation pattern was observed in plants treated only with SNP. Overall, our findings suggest that both NO and salt stress act as potent regulators of GST gene and enzyme expression through both ABA-dependent and independent pathways.