Effects of three fungicides alone and in combination on glutathione S-transferase activity (GST) and cytochrome P-450 (CYP 1A1) in the liver and gill of brown trout (Salmo trutta)

In order to evaluate the gill glutathione S-transferase (GST) activity as a biomarker of effect of fungicide exposure in juvenile brown trout (Salmo trutta), the fungicides propiconazole [(R,S)-1-[2-(2,4-diclophenyl)-4-propyl-1,3-dioolan-2-ylmetyl]-1H-1,2,4-triazole] and fenpropimorph [(+/-)-cis-4-[3-(4-tert-butylphenyl)-2-metyl propyl]-2,6 dimetylmorfolinc] were administrated in the water separately and together in a static system (80 microg/l for each pesticide) for 5 days. The combined fungicides gave a significant decrease in gill GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB), whilst hepatic GST-activity was not significantly changed. Furthermore, continuous exposure to 540 ug/l thiabendazole[2-(thiazol-4'-yl)benzimidazole] in a flow-through system for 4 days significantly increased the gill glutathione S-transferase (GST) activity towards CDNB, whilst hepatic GST and cytochrome P450 (CYP 1A) activities were not increased by the treatment.     

Effects of β-naphthoflavone on hepatic biotransformation and glutathione biosynthesis in largemouth bass (Micropterus salmoides)

We are investigating the effects of in vivo exposure of prototypical enzyme inducing agents on hepatic biotransformation enzyme expression in largemouth bass (Micropterus salmoides), a predatory game fish found throughout the United States and Canada. The current study targeted those genes involved in biotransformation and oxidative stress that may be regulated by Ah-receptor-dependent pathways. Exposure of bass to β-naphthoflavone (β-NF, 66 mg/kg, i.p.) elicited a 7–9-fold increase in hepatic microsomal cytochrome P4501A-dependent ethoxyresorufin O-deethylase (EROD) activities, but did not affect cytosolic GST catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB) or 5-androstene-3,17-dione (ADI). Glutathione S-transferase A (GST-A) mRNA expression exhibited a transient, but non-significant increase following exposure to β-NF, and generally tracked the minimal changes observed in GST–CDNB activities. Expression of the mRNA encoding glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in glutathione (GSH) biosynthesis, was increased 1.7-fold by β-NF. Changes in GCLC mRNA expression were paralleled by increases in intracellular GSH. In summary, largemouth bass hepatic CYP1A-dependent and GSH biosynthetic pathways, and to a lesser extent GST, are responsive to exposure to β-NF.     

Glutathione S-transferase from an antarctic fish, Dissostichus mawsoni

Glutathione S-transferase activity was measured in the hepatic cytosol front Dissostichus mawsoni and Pagothenia borchgrevinki. Activity measures with 1-chloro-2,4-dinitrobenzene as substrate were 11·2 and 16·7 μmol/min/g tissue respectively. Little or no activity was detected when p-nitrobenzyl chloride or 3,4-dichloro-1-nitrobenzene were used as substrate. The hepatic glutathione S-transferases from D. mawsoni were partially purified using gel filtration and chromatofocusing. Three peaks of activity were resolved. The major isozyme (158-fold purification) eluting at pH7·1 appeared to be catalytically a homodimer. The isozyme was highly inhibited by triphenyltin chloride (IC₅₀ = 0·1 μ) while inhibition constants for Cibicron Blue 3GA, bromosulphophalein and hematin were 1·1, 20 and 34 μ respectively.     

Marine invertebrate glutathione-S-transferases: purification, characterization and induction

High glutathione-S-transferase (GST) activity was found in hepatopancreas and gill cytosol of the blue crab (Callinectes sapidus) and the digestive gland cytosol of two marine gastropods (Nassarius obsoletus and Cerithium floridanum).Purification of GST from crab hepatopancreas by Sephadex G-200, DEAE-Sephacel and chromofocusing resulted in the isolation of two isoenzymes with isoelectric points of 5·9 and 5·7 (GST 5·9 and GST 5·7). Antibodies were prepared to these two isoenzymes and the two forms cross-reacted immunologically. The two transferases had similar molecular weights, amino acid compositions, substrate specifities and kinetic parameters.Crab gill cytosol showed one isoenzyme which reacted with antibodies to GST 5·9 and GST 5·7. The major isoenzyme of N. obsoletus was a basic form while C. floridanum showed a homodimer acidic form. The gastropod GST forms did not react with antibodies to crab GST. The presence of the phenolic antioxidant, butylated hydroxytoluene, in the diet of blue crab or shrimp (Penaeus aztecus) resulted in high hepatic GST activity.     

Regulation of hepatic glutathione S-transferase expression in flounder

The major glutathione S-transferase isoform of flounder liver, an antigenically related structural homologue of plaice GST-A, also displays mRNA homology. A 901bp cRNA probe for plaice GST-A cross-hybridised to a 1100bp flounder mRNA on northern blot analysis. The plaice antibody and cRNA probes were used to study effects of inducer treatment on GST-A expression in flounder liver. Six days after PAH treatment (3-methylcholanthrene) total hepatic GST activity was halved, levels of GST-A were 80% and GST-A mRNA levels were 25% of controls. A commercial PCB mixture (Aroclor 1254^TM) had little effect on total GST or GST-A levels despite halving GST-A mRNA levels. An epoxide, trans-stilbene oxide induced total GST activity 1·4 fold and GST-A protein levels 1·8-fold and its mRNA levels 3-fold. This reduced expression of the major flounder hepatic GST by agents which induce cytochrome P4501A1 may modulate cytoxicity of environmental pollutants in this species.     

A piscine glutathione S-transferase which efficiently conjugates the end-products of lipid peroxidation

A cDNA clone for glutathione S-transferaseA (GSTA) from plaice (Pleuronectes platessa) was expressed in Eschericia coli (E. coli) and purified to homogeneity by S-hexylglutathione affinity chromatography. When compared to literature values for a variety of purified mammalian GSTs, the heterologously expressed purified plaice enzyme had moderate activity towards the model substrate 1,2-chloro-2,4-dinitrobenzene (CDNB) and exhibited a Km of 2.5 ± 2 mM and Vmax of 30.9 ± 2.3 μmol min⁻¹ mg⁻¹. It had little or no activity towards several other model GST substrates including 1,2-dinitrochloro-4-benzene (DCNB), ethacrynic acid (EA), and p-nitrobenzylchloride (NBC). However plaice GSTA was a relatively efficient catalyst for the conjugation of a series of alk-2-enals and alk-2,4-dienals and also 4-hydroxynonenal. The highest activity observed with this series of substrates was with trans-non-2-enal with a Km of 17.9 ± 2.2 μM and a Vmax of 3.01 ± 0.57 μmol min⁻¹ mg⁻¹. These unsaturated alkenals have been identified in cells and cell extracts as highly toxic products arising from peroxidation of unsaturated fatty acids particularly during periods of oxidative stress. Fish are relatively rich in polyunsaturated fatty acids and thus GSTA mediated conjugation may be an important mechanism for detoxifying peroxidised lipid breakdown products.     

GSTA is a major glutathione S-transferase gene responsible for 4-hydroxynonenal conjugation in largemouth bass liver

We have previously shown that largemouth bass (Micropterus salmoides) has a remarkable ability to conjugate 4-hydroxy-2-nonenal (4HNE), a mutagenic and cytotoxic α,β-unsaturated aldehyde produced during the peroxidation of lipids. In addition, we have isolated a glutathione S-transferase cDNA (bass GSTA) that encodes a recombinant protein which is highly active in 4HNE conjugation and structurally similar to plaice (Pleuronectes platessa) GSTA. In the present study, HPLC-GST subunit analysis revealed the presence of at least two major GST isoforms in bass liver, with one peak constituting 80% of the total bass liver GST protein. Liquid chromatography mass spectrometry (LC–MS) and electrospray ionization analysis of the major bass GST subunit yielded a molecular weight of 26,396 kDa. Endo-proteinase Lys-C digestion and Edman degradation protein sequencing of this GST peak demonstrated that this protein was encoded by bass GSTA. Analysis of genomic DNA fragments isolated by nested PCR indicated the presence of a GST gene cluster in bass liver that contained GSTA, and was similar to a GST gene cluster characterized by Leaver et al., in plaice. Collectively, our data indicates the presence of a major GST in bass liver involved in the protection against oxidative stress. This GST is part of a gene cluster that may be conserved in certain freshwater and marine fish.     

Biomarker responses to pollution in two invertebrate species: Scrobicularia plana and Nereis diversicolor from the Cádiz bay (SW Spain)

The clam Scrobicularia plana and the polychaete worm Nereis diversicolor were collected in several sites from a littoral enclosure in SW Spain. The aim of our study was to relate various biomarker responses in these species to a pollution gradient caused by untreated domestic discharges and to verify the adequacy of the selected species as sentinels in this habitat. The biomarkers selected were the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPX) and DT-diaphorase (DT-D). In addition, the activities of cytochrome P450-dependent ethoxyresorufin O-deethylase (EROD) activity, the phase II detoxifying enzyme glutathione S-transferase (GST) and the neurotoxicity marker acetylcholinesterase (AChE) were measured. Metallothionein levels were selected as biomarkers of heavy metals exposure in both species. The results suggest a different response in the water filtering organism (clam) and the sediment eater (polychaete), probably as a consequent of different pollution exposure and that samples from the “Caño Sancti-Petri” were exposed to biologically active compounds that altered some of their biochemical responses. AChE was the most sensitive biomarker in both species and N. diversicolor proved to be a more robust sentinel in this ecosystem.     

Implications from a field study regarding the relationship between polycyclic aromatic hydrocarbons and glutathione S-transferase activity in mussels

An aluminium smelter on the west coast of Scotland discharges an aqueous effluent containing polycyclic aromatic hydrocarbons (PAHs) at the head of Loch Leven. The loch also supports two mussel (Mytilus edulis) farms. Data are presented on burdens of PAHs in the soft tissues of mussels and the effect of these contaminants on glutathione S-transferase (GST) activity in mussel hepatopancreas. GST activity is shown to be correlated with total PAH burden and also with the concentrations of certain individual PAHs. These field data show that high molecular weight PAHs are closely correlated to GST activity, whereas low molecular weight PAHs are not. This suggests that 5- and 6-ring PAHs have a more pronounced role than 2- to 4-ring compounds in inducing GST activity in mussels from Loch Leven. It is proposed that it may be more appropriate to link GST activity with 5- and 6-ring compounds only, rather than with the total PAH burden.     

Oxidative stress in Laeonereis acuta (Polychaeta, Nereididae): environmental and seasonal effects

Laeonereis acuta was seasonally collected in an industrially polluted site (P) and in an unpolluted site (UP) at the Patos Lagoon estuary (southern Brazil). Glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) activity (U/mg protein) was determined in five groups of worms from each site. Metallothionein (MT – μmol GSH/g ww) and lipid peroxides content (LPO – nmoles of cumene hydroperoxide/g ww) were also measured. Annual mean values for CAT (UP=3.7±0.3; P=5.7±0.6), GST (UP=0.034±0.003; P=0.045±0.004) and MT (UP=0.15±0.02; P=0.23±0.03) were higher (p<0.05) in worms from the P site. In autumn, CAT activity was higher (p<0.05) in worms from the P site (7.6 ± 1.3) than in those from the UP site (3.6 ± 0.4). In summer, MT concentration was higher in worms from the P site (0.37 ± 0.03) than in those from the UP site (0.19 ± 0.01). No significant difference (p>0.05) in the LPO content was observed in worms from the different sites or collected in different seasons. These results indicate that worms from the polluted site showed higher antioxidant responses than those from the unpolluted site, sufficient to prevent oxidative damage in terms of LPO.     

Effects of beta-naphthoflavone on hepatic biotransformation and glutathione biosynthesis in largemouth bass (Micropterus salmoides)

We are investigating the effects of in vivo exposure of prototypical enzyme inducing agents on hepatic biotransformation enzyme expression in largemouth bass (Micropterus salmoides), a predatory game fish found throughout the United States and Canada. The current study targeted those genes involved in biotransformation and oxidative stress that may be regulated by Ah-receptor-dependent pathways. Exposure of bass to beta-naphthoflavone (beta-NF, 66 mg/kg, i.p.) elicited a 7-9-fold increase in hepatic microsomal cytochrome P4501A-dependent ethoxyresorufin O-deethylase (EROD) activities, but did not affect cytosolic GST catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB) or 5-androstene-3,17-dione (ADI). Glutathione S-transferase A (GST-A) mRNA expression exhibited a transient, but non-significant increase following exposure to beta-NF, and generally tracked the minimal changes observed in GST-CDNB activities. Expression of the mRNA encoding glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in glutathione (GSH) biosynthesis, was increased 1.7-fold by beta-NF. Changes in GCLC mRNA expression were paralleled by increases in intracellular GSH. In summary, largemouth bass hepatic CYP1A-dependent and GSH biosynthetic pathways, and to a lesser extent GST, are responsive to exposure to beta-NF.     

Expression and activity of glutathione S-transferases and catalase in the shrimp Litopenaeus vannamei inoculated with a toxic Microcystis aeruginosa strain

Microcystin (MC) produced during cyanobacteria blooms is notably toxic to human and wildlife. Conjugation with reduced glutathione (GSH) by glutathione S-transferase (GST) and the antioxidant enzymes defenses (e.g. catalase, CAT) are important biochemical defense mechanisms against MCs toxicity. We investigated the enzymatic activity of CAT and GST and the gene expression levels of CAT and eight GST isoforms in the hepatopancreas of the globally farmed shrimp Litopenaeus vannamei 48-h after injection with a sub-lethal dose of 100 μg kg⁻¹ of a toxic Microcystis aeruginosa extract. MCs caused up-regulation for GST Ω, μ and a MAPEG isoform, by 12-, 2.8- and 1.8-fold, respectively, and increases in the total GST enzyme activity and CAT enzyme activity. The study points to the importance of further characterization for the L. vannamei GST isoforms and GST/CAT post-translational regulation processes to better understand the key mechanisms involved in the shrimp’s defense against MC exposure.     

Differential expression of alpha-like glutathione S-transferase (GST) isoforms in catfish intestine.

Previous studies suggested that dietary composition affected glutathione S-transferase (GST) activity in catfish intestine, and this activity varied along the intestine. In this study, catfish were fed a semi-purified diet or a commercial chow for at least 2 weeks. GST activity, percent protein cross-reacting with anti-catfish GST pi antibody, and immuno-cross-reactivity with antibodies specific for human alpha, mu, pi and theta class GSTs were determined in cytosol prepared from sections of proximal, medial, and distal intestine. The bulk of GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid, and the percent protein cross-reacting with anti-catfish GST pi were in the more proximal segments and dropped off distally in the two diet groups. The percent of cross-reacting GST protein in the proximal section of fish fed on commercial chow was significantly higher (4.3 +/- 1.7%) than in fish fed purified diet (2.3 +/- 0.2%). Further Western blot analysis revealed a differential expression of GST isoforms only in the distal segment of fish fed commercial chow that recognized human anti-alpha GST antibody. Distal intestinal segments of catfish exposed to 3,3',4,4'-tetrachlorobiphenyl (TCB) and beta-naphthoflavone (BNF) also revealed expression of distinct alpha-like GST isoforms. Results strongly suggest the distal segment as a site for potential biomarkers for polycyclic aromatic hydrocarbon (PAH)- and co-planar polychlorinated biphenyl (PCB)-type contaminants.     

Effect of oil pollution on gluthione and relative enzyme in oyster(Saccostrea cuculiata)

The oysters ( Saccostrea cuculiata ) were collected from four stations around Xiamen Island (Lundu Port, Xinglin Bay, Tong" an Bay, Huangcuo). The relation between the level of petroleum hydrocarbon in whole tissue and the contents of glutathione (GSH), the activity of selenium-dependent glutathione peroxidase (Se-GPx) and glutathione S-transferase (GST) in digestive gland and gill were investigated. The results showed: (1) The contents of petroleum hydrocarbon in oyster collected from four stations (Lundu Port, Xinglin Bay, Tong'an Bay, Huangcuo) were 380.68, 112.34, 27.31, 20.37μg/g wet weight, respectively; (2) the activity of Se-GPx and GST in digestive gland was lower than that in gill, and the content of GSH seemed reversibly; (3) among the four stations, both Se-GPx and GST activity of digestive gland and gill in Saccostrea cuculiata sampled from the four stations showed a good correlation with whole tissue petroleum hydrocarbon, could be as biomarkers of sea oil pollution.     

Effects of environmental pollution in caged mussels (Mytilus galloprovincialis)

Biological effects of environmental pollution, mainly related to presence of PAHs, were assessed in mussels Mytilus galloprovincialis caged in Priolo, an anthropogenically-impacted area, and Vendicari, a reference site, both located along the eastern coastline of Sicily (Italy). PAHs concentration and histopathological changes were measured in digestive gland tissues. Expression of cytochrome P4504Y1 (CYP4Y1) and glutathione S-transferase (GST), indicative of xenobiotic detoxification, and activity of catalase (CAT) as oxidative stress index, were evaluated.The results show a direct correlation between the high concentrations of PAHs in digestive glands of mussels from Priolo and the significantly altered activity of phase I (P < 0.001) and phase II (P < 0.0001) biotransformation enzymes, along with increased levels of CAT activity (P < 0.05). These findings show the enhancement of the detoxification and antioxidant defense systems. The mussel caging approach and selected biomarkers demonstrated to be reliable for the assessment of environmental pollution effects on aquatic organisms.     

Biomarkers in flounder (Platichthys flesus): an evaluation of their use in pollution monitoring

Flounder (Platichthys flesus) is among the most common fish-species in Norwegian and European estuaries. It lives in or on sediments from which it also finds most of its food. The aim of the present work was to evaluate biomarkers in flounder for possible future use in monitoring programmes. There were clear biomarker responses in flounder following injection of model contaminants benzo[a]pyrene (B[a]P), PCB #156 and Cd, singly or in sequence. Cytochrome P4501A responded following injection of the organic contaminants and metallothionein (MT) following Cd injection. All groups receiving B[a]P, either singly or in combination with other contaminants, accumulated high levels of B[a]P-metabolites in bile. There was little change in glutathione-S-transferase activity (measured using CDNB as substrate) following the treatments. Starvation appeared to affect the response of hepatic MT to Cd, but none of the other biomarkers. PAH in sediments elicited strong biomarker responses in caged flounder, whereas sediment-associated metals appeared to be largely unavailable to flounder in this study.     

Altered glutathione S-transferase catalytic activities in female brown bullheads from a contaminated central Florida lake.

Brown bullheads (Ameriurus nebulosus) are a demersal freshwater species that can be found in a number of polluted ecosystems. The purpose of the present study was to determine the overall capacity for in vitro glutathione S-transferase (GST) detoxification by brown bullheads, and to see if bullhead GST catalysis was altered in bullheads from a polluted site. Brown bullhead liver cytosolic GSTs catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) over a large range of substrate concentrations, with apparent Km and Vmax for CDNB at fixed nucleophile (glutathione, GSH) concentrations of 1.8-1.9 mM and 12.1-14.6 mumol CDNB conjugated/min/mg, respectively. Bullhead GSTs were also highly active toward other substrates such as ethacrynic acid (ECA), delta 5-androstene-3,17-dione (ADI), and nitrobutyl chloride (NBC). Initial rate GST catalytic activities toward CDNB, NBC, ECA, and ADI were significantly lower in female bullheads from a contaminated lake (Lake Apopka Marsh) as compared to female bullheads inhabiting a nearby control site (Lake Woodruff). No site differences were observed with respect to male bullhead GST activities. These studies suggest that brown bullheads efficiently carry out GST conjugation of diverse electrophilic substrates. However, bullhead GST catalysis may be compromised in bullheads inhabiting polluted ecosystems.     

Evidence from heterologous expression of glutathione S-transferases A and A1 of the plaice (Pleuronectes platessa) that their endogenous role is in detoxification of lipid peroxidation products

cDNA clones for glutathione S-transferases A (GST-A) and A1 (GST-A1) from plaice (Pleuronectes platessa) were expressed as N-terminally 6XHis tagged proteins in Escherichia coli and purified to homogeneity from Ni-NTA silica. GST-A was an efficient catalyst for conjugation of unsaturated alkenals derived from peroxidation of polyunsaturated fatty acids with the highest activity observed with trans-non-2-enal (8 micromol min(-1) mg(-1)). GST-A1 was a very efficient Se-independent glutathione peroxidase with an activity towards cumene hydroperoxide of 25 micromol min(-1) mg(-1). Although the enzymes exhibited moderately high activities towards the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) they exhibited little or no activity towards other common prototypical xenobiotic substrates. Together with data for ontogeny, tissue distribution and inducibility of these enzymes, we contend that a primary function of these enzymes is protection from the harmful effects of lipid peroxidation products generated naturally or exacerbated by xenobiotic exposure.     

Effects of the inhibitor ellipticine on cytochrome P450-reductase and cytochrome P450 (1A) function in hepatic microsomes of flounder (Platichthys flesus)

The effects of the mammalian inhibitor ellipticine (5,11-dimethyl-[6H]-pyrido[4,3b] carbazole) were examined in a mechanistic study of the cytochrome P450 monooxygenase system of control and β-naphthoflavone (βNF)-induced hepatic microsomes of Platichthys flesus. Ellipticine was indicated to bind to the haem moiety of cytochrome P450s (gave type II binding spectra) and to inhibit the transfer of electrons from both the hydrophobic binding site of cytochrome P450 reductase (P450R) to P450 (inhibited P450R reductase activity) and the hydrophilic binding site of P450R to soluble electron acceptors (inhibited NAD(P)H-cytochrome c reductase activity). No effect was seen on cytochrome b₅ reductase activity. Ellipticine inhibition indicated the involvement of (i) P450R (possibly also P450s) in NADPH- but not NADH- dependent hydroxyl radical production, and (ii) electron transfer and P450/P450R interaction in NADPH-dependent cytochrome P450 1A-catalysed monooxygenation (7-ethoxyresorufin O-deethylase activity and benzo(a)pyrene (BaP) metabolism). Differential effects of ellipticine on cumene hydroperoxide (CHP)-dependent BaP metabolism (P450 peroxidase activity) with CHP concentration indicated the existence of at least two forms of P450 with different substrate affinities for CHP, and different mechanisms of formation for protein adducts and free metabolites. Overall, the studies indicate the primary site of action of ellipticine in P. flesus is binding between Fe³⁺-P450 and P450R.     

Relationship between metallothionein, copper and zinc in perch (Perca fluviatilis) environmentally exposed to heavy metals

Hepatic levels of Cu, Zn and metallothionein (MT) in perch, caught in a Cu/Zn gradient from a brassworks, reflected the water concentration of Cu (1·0–9·4 ppb) and Zn (0·56–59 ppb). Significant correlations were found between hepatic Cu and MT levels (r = 0·72), and between Zn and MT levels (r = 0·69). There was an increase of the amount of Cu and Zn in the cytosolic fraction of the liver with increased hepatic levels of the metals. When liver samples. from perch caught at the most contaminated location, were run on a gel filtration column (Sephadex G-75) 78% of the cytosolic Cu and 24% of the Zn in the cytosol eluted together with MT.