Atrazine promotes biochemical changes and DNA damage in a Neotropical fish species

The effects of Atrazine, an herbicide used worldwide and considered as a potential contaminant in aquatic environments, were assessed on the Neotropical fish Prochilodus lineatus acutely (24 and 48 h) exposed to 2 or 10 μg L⁻¹ of atrazine by using a set of biochemical and genetic biomarkers. The following parameters were measured in the liver: activity of the biotransformation enzymes ethoxyresorufin-O-deethylase (EROD) and glutathione S transferase (GST), antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), content of reduced glutathione (GSH), generation of reactive oxygen species (ROS) and occurrence of lipid peroxidation (LPO); in brain and muscle the activity of acetylcholinesterase (AChE) and DNA damage (comet assay) on erythrocytes, gills and liver cells. A general decreasing trend on the biotransformation and antioxidant enzymes was observed in the liver of P. lineatus exposed to atrazine; except for GR, all the other antioxidant enzymes (SOD, CAT and GPx) and biotransformation enzymes (EROD and GST) showed inhibited activity. Changes in muscle or brain AChE were not detected. DNA damage was observed in the different cell types of fish exposed to the herbicide, and it was probably not from oxidative origin, since no increase in ROS generation and LPO was detected in the liver. These results show that atrazine behaves as enzyme inhibitor, impairing hepatic metabolism, and produces genotoxic damage to different cell types of P. lineatus.     

Combined effects of deltamethrin, temperature and salinity on oxidative stress biomarkers and acetylcholinesterase activity in the black tiger shrimp (Penaeus monodon)

This study aimed to investigate the interactions of two abiotic factors (temperature and salinity) and deltamethrin (pyrethroid pesticide) exposure on some oxidative stress biomarkers as well as on acetylcholinesterase activity (AChE) in hepatopancreas, gills and muscle of black tiger shrimp (Penaeus monodon). A combination of three temperatures (24, 29 and 34 °C), two salinities (15 and 25 ppt), and the absence or presence of 0.1 μg L⁻¹ deltamethrin was applied on shrimp during 4 d under laboratory conditions. Lipid peroxidation level (LPO) and glutathione S-transferase activity (GST) were not affected by combined effect of temperature, salinity and deltamethrin in any of the studied tissues. Deltamethrin impaired other tested oxidative stress biomarkers, i.e. total glutathione (tGSH), catalase (CAT), glutathione peroxidase (GPx). tGSH level significantly increased in hepatopancreas due to deltamethrin exposure mainly at 34 °C, while pesticide effects on tGSH and CAT activity in gills were influenced by both temperature and salinity. In addition, GPx activity in hepatopancreas decreased after deltamethrin treatment mainly at 24 °C. Finally, AChE in muscle was strongly inhibited by deltamethrin at all tested temperatures and salinities. These novel findings demonstrate that interactions between abiotic factors and a commonly used pesticide exposure should be taken into account when analyzing some widespread biomarkers in black tiger shrimp.     

Haematology, genotoxicity, enzymatic activity and histopathology as biomarkers of metal pollution in the shrew Crocidura russula

Haematological (WBC, RBC, Hgb and Hct) and genotoxicity (MNT) parameters, hepatic enzymatic activities (GST, GPx and GR), and a histopathological evaluation of liver, kidneys and gonads were assessed as general biomarkers of metal pollution in the shrew Crocidura russula inhabiting a pyrite mining area. Specimens exposed to metals presented a few significant alterations when compared with reference animals: GST activity decreased; micronuclei increased; and evident liver alterations related to metal exposure were observed. On the basis of all the parameters studied, age was an important factor that partly explained the observed variation, whereas sex was the least important factor. Significant correlations were also found between heavy metal concentrations and biomarkers evaluated, demonstrating the great influence of these metals in the metabolic alterations. To the best of our knowledge, these data constitute the first measurements of a battery of biomarkers in shrews from a mine site and are among the few available for insectivorous mammals.     

Effects of malathion and chlorpyrifos on acetylcholinesterase and antioxidant defense system in Oxya chinensis (Thunberg) (Orthoptera: Acrididae)

The study was undertaken to evaluate the effects of malathion and chlorpyrifos on acetylcholinesterase (AChE), esterase (EST) activity and antioxidant system after topical application with different concentration to Oxya chinensis. The results showed that malathion and chlorpyrifos inhibited EST, AChE activity and increased malondialdehyde (MDA) contents. A change in superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx), and glutathione reductase (GR) activity combined with reduced glutathione (GSH) and total glutathione (tGSH) contents was found in O. chinensis after malathion and chlorpyrifos treatments. Malathion and chlorpyrifos increased SOD and CAT activity compared with the control. With the concentrations increasing, SOD and CAT activity showed the similar tendency, namely, SOD and CAT activity increased at the lower concentrations and decreased at the higher concentrations. The results showed that malathion and chlorpyrifos decreased significantly GR activity. GST and GPx activity at the studied concentrations of chlorpyrifos was lower than that of the control. However, no significance was observed. GPx and GST activity in malathion treated grasshoppers showed a biphasic response with an initial increase followed by a decline in its activity. Malathion and chlorpyrifos decreased GSH contents and the ratio of GSH/GSSG. The present findings indicated that the toxicity of malathion and chlorpyrifos might be associated with oxidative stress.     

Effects of different inorganic arsenic species in Cyprinus carpio (Cyprinidae) tissues after short-time exposure: Bioaccumulation, biotransformation and biological responses

Differences in the toxicological and metabolic pathway of inorganic arsenic compounds are largely unknown for aquatic species. In the present study the effects of short-time and acute exposure to As^III and As^V were investigated in gills and liver of the common carp, Cyprinus carpio (Cyprinidae), measuring accumulation and chemical speciation of arsenic, and the activity of glutathione-S-transferase omega (GST Ω), the rate limiting enzyme in biotransformation of inorganic arsenic. Oxidative biomarkers included antioxidant defenses (total glutathione-S-transferases, glutathione reductase, glutathione, and glucose-6-phosphate dehydrogenase), total scavenging capacity toward peroxyl radicals, reactive oxygen species (ROS) measurement and lipid peroxidation products. A marked accumulation of arsenic was observed only in gills of carps exposed to 1000 ppb As^V. Also in gills, antioxidant responses were mostly modulated through a significant induction of glucose-6-phosphate dehydrogenase activity which probably contributed to reduce ROS formation; however this increase was not sufficient to prevent lipid peroxidation. No changes in metal content were measured in liver of exposed carps, characterized by lower activity of GST Ω compared to gills. On the other hand, glutathione metabolism was more sensitive in liver tissue, where a significant inhibition of glutathione reductase was concomitant with increased levels of glutathione and higher total antioxidant capacity toward peroxyl radicals, thus preventing lipid peroxidation and ROS production. The overall results of this study indicated that exposure of C. carpio to As^III and As^V can induce different responses in gills and liver of this aquatic organism.     

Antioxidant responses in clam Venerupis philippinarum exposed to environmental pollutant hexabromocyclododecane

The objective of this study was to assess the potential toxic effects of hexabromocyclododecane (HBCD) on tissues of clam Venerupis philippinarum using parameters of antioxidant defenses and oxidative stress. Antioxidant biomarkers including ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), superoxide dismutase (SOD), and glutathione (GSH), as well as DNA damage and lipid peroxidation (LPO) in gills and digestive glands of V. philippinarum, were analyzed after a 1-, 3-, 6-, 10-, and 15-day exposure to seawater containing HBCD at environmentally related concentrations, respectively. The results showed that the activity of most antioxidant enzymes increased, and different trends were detected with exposure time extending. The oxidative stress could be obviously caused in the gills and digestive glands under the experimental conditions. This could provide useful information for toxic risk assessment of environmental pollutant HBCD.     

Biochemical biomarkers in Nile tilapia (Oreochromis niloticus) after short-term exposure to diesel oil, pure biodiesel and biodiesel blends

Fossil fuels such as diesel are being gradually replaced by biodiesel, a renewable energy source, cheaper and less polluting. However, little is known about the toxic effects of this new energy source on aquatic organisms. Thus, we evaluated biochemical biomarkers related to oxidative stress in Nile tilapia (Oreochromis niloticus) after two and seven exposure days to diesel and pure biodiesel (B100) and blends B5 and B20 at concentrations of 0.01 and 0.1 mL L⁻¹. The hepatic ethoxyresorufin-O-deethylase activity was highly induced in all groups, except for those animals exposed to B100. There was an increase in lipid peroxidation in liver and gills in the group exposed to the higher concentration of B5. All treatments caused a significant increase in the levels of 1-hydroxypyrene excreted in the bile after 2 and 7 d, except for those fish exposed to B100. The hepatic glutathione-S-transferase increased after 7 d in animals exposed to the higher concentration of diesel and in the gill of fish exposed to the higher concentration of pure diesel and B5, but decreased for the two tested concentrations of B100. Superoxide dismutase, catalase and glutathione peroxidase also presented significant changes according to the treatments for all groups, including B100. Biodiesel B20 in the conditions tested had fewer adverse effects than diesel and B5 for the Nile tilapia, and can be suggested as a less harmful fuel in substitution to diesel. However, even B100 could activate biochemical responses in fish, at the experimental conditions tested, indicating that this fuel can also represent a risk to the aquatic biota.     

Toxicokinetics of tilapia following high exposure to waterborne and dietary copper and implications for coping mechanisms

One of the major challenges in assessing the potential metal stress to aquatic organisms is explicitly predicting the internal dose in target organs. We aimed to understand the main sources of copper (Cu) accumulation in target organs of tilapia (Oreochromis mossambicus) and to investigate how the fish alter the process of Cu uptake, depuration, and accumulation (toxicokinetics (TK)) under prolonged conditions. We measured the temporal Cu profiles in selected organs after single and combined exposure to waterborne and dietary Cu for 14 days. Quantitative relations between different sources and levels of Cu, duration of treatment, and organ-specific Cu concentrations were established using TK modeling approaches. We show that water was the main source of Cu in the gills (>94 %), liver (>89 %), and alimentary canal (>86 %); the major source of Cu in the muscle (>51 %) was food. Cu uptake and depuration in tilapia organs were mediated under prolonged exposure conditions. In general, the uptake rate, depuration rate, and net bioaccumulation ability in all selected organs decreased with increasing waterborne Cu levels and duration of exposure. Muscle played a key role in accounting for the rapid Cu accumulation in the first period after exposure. Conversely, the liver acted as a terminal Cu storage site when exposure was extended. The TK processes of Cu in tilapia were highly changed under higher exposure conditions. The commonly used bioaccumulation model might lead to overestimations of the internal metal concentration with the basic assumption of constant TK processes.     

EROD activity and antioxidant defenses of sea bass (Dicentrarchus labrax) after an in vivo chronic hydrocarbon pollution followed by a post-exposure period

Chronic concentrations of polycyclic aromatic hydrocarbons (PAHs) have been commonly detected in international estuaries ecosystems. Reliable indicators still need to be found in order to properly assess the impact of PAHs in fish. After an in vivo chronic exposure to hydrocarbons, the enzymatic activity of 7-ethoxyresorufin O-deethylase (EROD) and the antioxidant defense system were assessed in sea bass, Dicentrarchus labrax. A total of 45 fish were exposed to the water-soluble fraction of Arabian crude oil, similar to a complex pollution by hydrocarbons chronically observed in situ, while 45 other control fish sustained the same experimental conditions in clean seawater. Fish samples were made after a 21-day exposure period and after a 15-day recovery period in clean fresh water. Throughout the experiment, liver EROD activity was significantly higher in contaminated fish than in control fish. In addition, nonenzymatic (total glutathione) and enzymatic (GPx, SOD, and CAT) antioxidant defense parameters measured in liver were not significantly different in fish. Furthermore, in gills, glutathione content had significantly increased while SOD activity had significantly decreased in contaminated fish compared to controls. On the other hand, CAT and GPx activities were not affected. Chronic exposure to PAHs disturbing the first step (SOD) and inhibiting the second step (GPx and CAT) could induce oxidative stress in tissues by the formation of oxygen radicals. After the postexposure period, there was no significant difference between control and contaminated fish in any of the antioxidant defense parameters measured in gills, attesting to the reversibility of the effects.     

Comparision of the waterborne and dietary routes of exposure on the effects of Benzo(a)pyrene on biotransformation pathways in Nile tilapia (Oreochromis niloticus)

BaP is one of the most studied PAH, due to its ubiquitous presence in aquatic environments and toxicity to aquatic organisms. The main goal of this study was to assess BaP effects in Nile Tilapia after waterborne and dietary exposures, through the evaluation of EROD and GST activities in liver, gills and intestine, and BaP metabolites in bile; and also to evaluate the usefulness of these commonly used biomarkers after two different routes of exposure. Waterborne exposure to BaP led to a significant induction of EROD in all tissues analyzed (644%, 1640% and 2880% in relation to solvent in liver, gill and intestine respectively) while in dietary exposures EROD was induced only in intestine (3143%) after exposure to high BaP concentrations. GST activities with CDNB were slightly induced in liver (40%) and in gill (66%) after water exposure to BaP, and in intestine after dietary exposure to low BaP concentrations (182%). BaP metabolites in bile increased after both exposure routes, and were highly correlated with EROD activity after water exposure. In summary, this work has shown that the effects of BaP on biotransformation pathways depend on the route of exposure. Moreover, barrier tissues like gills and intestine also have an important role in the first-pass metabolism of BaP, reducing the amount of parent compound that reaches the liver to be metabolized. For that reason, EROD activity as a biomarker of exposure should also be applied in extrahepatic organs, like gills and intestine, in monitoring studies. Biliary BaP type metabolites are good reflectors of contamination levels under both exposure routes, while GST activity with CDNB as substrate, as a phase II enzyme, does not seem a reliable biomarker of exposure to BaP regardless the route of exposure.     

Anguilla anguilla L. oxidative stress biomarkers: an in situ study of freshwater wetland ecosystem (Pateira de Fermentelos, Portugal)

Pateira de Fermentelos (PF) is a natural freshwater wetland in the central region of Portugal. In the last decade, the introduction of agricultural chemicals, heavy metals, domestic wastes, as well as eutrophication and incorrect utility of resources resulted in an increased water pollution. The present research work was carried out to check the various oxidative stress biomarker responses in European eel (Anguilla anguilla L.) gill, kidney and liver due to this complex water pollution. Eels were caged and plunged at five different PF sites (A-E) for 48h. A reference site (R) was also selected at the river spring where no industrial contamination should be detected. Lipid peroxidation (LPO), catalase (CAT), glutathione peroxidase (GPX), glutathione S-transferase (GST) and reduced glutathione (GSH) were the oxidative stress biomarkers studied. In gill, site A exposure induced a significant GST activity increase and site B exposure induced CAT activity increase when compared to R. Site C exposure showed a significant CAT and GPX activity increase. Data concerning site D exposure were not determined due to cage disappearance. Site E exposure displayed a significant CAT and GST activity increase. In kidney, site A exposure induced a significant CAT and GPX decrease as well as a GST increase. Site B exposure showed a significant decrease in GPX activity and GSH content. However, site C exposure demonstrated a significant increase in CAT and a decrease in GPX. Site E exposure showed a significant decrease in GPX and increase in GST. In liver, site A exposure showed a significant GST activity decrease as well as GSH content increase. Site B exposure showed a significant CAT, GST and LPO decrease. Site C exposure showed only GST activity decrease, while site E exposure induced a significant increase in GPX. These investigation findings provide a rational use of oxidative stress biomarkers in freshwater ecosystem pollution biomonitoring using caged fish, and the first attempt reported in Portugal as a study of this particular watercourse under the previous conditions. The presence of pollutants in the PF water was denunciated even without a clear relation to the main pollution source distance. The organ specificity was evident for each parameter but without a clear pattern.     

Histopathological and biochemical alternations of the heart induced by acute cadmium exposure in the freshwater crab Sinopotamon yangtsekiense

Cadmium (Cd) is a highly toxic element in water. Its toxicity has been attributed to oxidative stress mediated by free radicals. Here we investigated the effects of Cd on the histopathology, antioxidant enzymes and lipid peroxidation of crustacean heart. The freshwater crabs Sinopotamon yangtsekiense were exposed to different concentrations of Cd for 1, 3, 5 and 7 d. After exposure, histological abnormalities were discovered, including myocardial edema, vacuolar and vitreous degeneration, and infiltration of inflammatory cells. Additionally, alterations in nuclei, mitochondria, rough endoplasmic reticulum as well as myofibrils were observed. Meanwhile, superoxide dismutase (SOD) activity was significantly increased after Cd exposure. Catalase (CAT) activity was only increased in the group exposed to 14.50 mg L⁻¹ Cd on day 5 and decreased with increasing Cd concentration and exposure time. Glutathione peroxidase (GPx) activity was increased in groups treated with 29.00, 58.00 and 116.00 mg L⁻¹ on days 1 and 3, and decreased thereafter. Besides, malondialdehyde (MDA) levels were significantly increased after 3 d of Cd exposure at all the indicated concentrations. These results showed that acute Cd exposure led to harmful effects on the histology of crab heart, which are most likely linked to Cd-induced oxidative stress.     

Effects of cadmium exposure on digestive enzymes, antioxidant enzymes, and lipid peroxidation in the freshwater crab Sinopotamon henanense

In this study, the effects of cadmium (Cd) stress on the activities of disaccharidases (sucrase, lactase, and maltase), amylase, trypsin, pepsase, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) content in the alimentary system of freshwater crabs Sinopotamon henanense were studied. Results showed that the enzyme activities in the stomach, intestine, and hepatopancreas changed with Cd concentration. In terms of digestive enzymes, Cd exposure had an inhibitory effect on the activities of the disaccharidases, amylase, and pepsase (only in the stomach). Significant induction of trypsin activity by Cd at a lower concentration was observed, but as Cd concentration increased, trypsin activity decreased. Maltase activity showed a slight recovery after inhibition by Cd. The activities of SOD and CAT increased initially and decreased subsequently. Cd significantly inhibited the activity of GPx. MDA content increased with increasing concentration of Cd. These results showed that acute Cd exposure led to harmful effects on the alimentary system of crabs, which are likely linked to Cd induced oxidative stress.     

Effect of heavy metals on tissue-specific antioxidant response in Indian major carps

Concentrations of heavy metals (Cu, Ni, Zn, Cd and Pb) were measured in sediments, water and liver and kidney tissues of three Indian major carps (Labeo rohita, Catla catla and Cirrhinus cirrhosus), belonging to two different weight groups (250 and 500 g), collected from ponds at two different sites (Nalban bheri and Diamond Harbour). The tissues were analysed for the levels of different antioxidant defence systems such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GRd), glutathione S-transferase (GST), glutathione (GSH) and malondialdehyde (MDA). Concentrations of all the metals were significantly higher (P < 0.05) in sediment, water and the tissues from Nalban bheri compared to those in Diamond Harbour. Metal concentrations were the lowest in C. cirrhosus, which increased with an increase in fish weight, and the liver accumulated higher amount of metals than the kidney. Activities of all enzymatic and non-enzymatic antioxidant parameters except GPx and GRd were significantly higher (P < 0.05) in the tissues from Nalban bheri than those in Diamond Harbour. Significant multicollinearity was found in the values of SOD, CAT, GST, GRd, GPx and MDA with Pb, Cu and Ni in all three fish species at Nalban and with Cd in L. rohita and C. catla. Principal component analysis results revealed that stress response in a polluted site was directly regulated by an amalgamation of GSH profile and the levels of MDA in a synchronized manner. The study indicated a tissue-specific and species-specific difference for heavy metal-induced oxidative stress response in fish and a correlation between different heavy metals and individual oxidative stress markers.     

Assessment of typical pollutants in waterborne by combining active biomonitoring and integrated biomarkers response

Organic pollutants, heavy metals and pharmaceuticals are continuously dispersed into the environment and have become a relevant environmental emerging concern. In this study, a situ assay to assess ecotoxicity of mixed pollutants was carried out in three typical sites with different priority contaminations in Guangzhou, China. Chemical analysis of organic pollutants, metals and quinolones in three exposure sites were determined by GC-ECD/MS, ICP-AES and HPLC, as well as, a combination of biomarkers including: ethoxyresorufin O-deethylase (EROD); aminopyrine N-demethylase (APND); erythromycin N-demethylase (ERND); glutathione S-transferase (GST); malondialdehyde (MDA); CYP1A; and P-glycoprotein (P-gp) mRNA expressions were evaluated in Mugilogobius abei. Results of chemical analysis in sediment samples revealed that the dominant chemicals were organic pollutants and heavy metals in Huadi River while quinolones in the pond. Bioassays indicated that differences among sites were in relation to some specific biomarkers. EROD and GST activities significantly increased after 72 h in situ exposure, but no difference was observed among the exposure sites. APND, ERND and MDA exhibited dissimilar change patterns for different priority pollutants. CYP1A and P-gp mRNA expressions were significantly induced at all exposure sites, whilst P-gp activity was typical for S2 with the highest levels of quinolones. The molecular biomarkers seemed to be more susceptible than enzyme activities. These assays confirmed the usefulness of applying a large array of various combined biomarkers at different levels, in assessing the toxic effects of mixed pollutants in a natural aquatic environment.     

Dimethoate-induced oxidative stress and DNA damage in Oncorhynchus mykiss

The present study was conducted in order to investigate pro-oxidant activity of dimethoate in liver and brain tissues following sublethal pesticide exposure for 5, 15 and 30 d by using SOD, GPx, CAT enzyme activities and lipid peroxidation as biomarkers as well as DNA damaging potential via detecting% Tail DNA, Tail moment and Olive tail moment as endpoints in erythrocytes of Oncorhynchus mykiss in an in vitro experiment. Antioxidant enzyme activities were found to elicit two staged response which was an initial induction followed by a sharp inhibition in liver tissue while a sustained increase in GPx activity and slight stimulation in SOD activity were detected in brain tissue. Lipid peroxidation showed an ascending pattern throughout the exposure period in both tissues and a decreasing trend was determined in tissue protein levels which was proved to be positively correlated with duration. Similar findings were obtained from outcomes preferred to quantify DNA damage and TM was decided to reflect the extent of damage more sensitively because of determined positive correlation with concentrations applied. Considering these results, it can be concluded that oxidative stress condition evoked by dimethoate could not be responded effectively and genotoxic nature of pesticide was proven by determined clastogenic effect possibly via being an alkylation agent or stimulating the production of reactive species.     

Effects of hexachlorobenzene on antioxidant status of liver and brain of common carp (Cyprinus carpio)

Hexachlorobenzene (HCB)-induced oxidative damages have been published in rats while the effects have not yet been reported in fishes. Juvenile common carps (Cyprinus carpio) were exposed to waterborne HCB from 2 to 200 microg l-1 for 5, 10 or 20 days. Liver and brain were analyzed for various parameters of oxidative stress. There were no significant changes of glutathione (GSH) content and superoxide dismutase (SOD) activity in liver after 5 or 10 days exposure, whereas obvious drops were observed at higher concentrations after 20 days exposure. Significant decreases of GSH content and SOD activity in brain were found during all the exposure days. In brain, HCB also significantly elevated the contents of reactive oxygen species (ROS), thiobarbituric acid- reactive substances (TBARS, as an indicator of lipid peroxidation products), glutathione disulfide (GSSG), and activities of nitric oxide synthase (NOS), glutathione peroxidase (GPx), and glutathione reductase (GR), and inhibited activities of acetylcholinesterase (AchE) and glutathione S-transferase (GST). The results clearly demonstrated that environmentally possible level of HCB could result in oxidative stress in fish and brain was a sensitive target organ of HCB toxicity.     

Effects of chronic exposure to lead,copper, zinc,and cadmium on biomarkers of the European eel,Anguilla anguilla

Exposure to specific metallic compounds can cause severe deleterious modifications in organisms. Fishes are particularly prone to toxic effects from exposure to metallic compounds via their environment. Species that inhabit estuaries or freshwater environments can be chronically affected by persistent exposure to a large number of metallic compounds, particularly those released by industrial activities. In this study, we exposed yellow eels (European eel, Anguilla anguilla) for 28 days to environmentally relevant concentrations of four specific metals; lead (300, 600, and 1,200 μg/l), copper (40, 120, and 360 μg/l), zinc (30, 60, and 120 μg/l) and cadmium (50, 150, and 450 μg/l). The selected endpoints to assess the toxicological effects were neurotransmission (cholinesterasic activity in nervous tissue), antioxidant defense, and phase II metabolism (glutathione-S-transferase [GST] activity, in both gills and liver tissues), and peroxidative damage. The results showed an overall lack of effects on acetylcholinesterase for all tested metals. Lead, copper, and cadmium exposure caused a significant, dose-dependent, increase in GST activity in gill tissue. However, liver GST only significantly increased following zinc exposure. No statistically significant effects were observed for the thiobarbituric acid reactive substances assay, indicating the absence of peroxidative damage. These findings suggest that, despite the occurrence of an oxidative-based response after exposure to lead, copper, and cadmium, this had no consequence in terms of peroxidative membrane damage; furthermore, cholinergic neurotoxicity caused by lead, copper, and cadmium did not occur. The implications of these results are further discussed.