Metallothioneins induction and antioxidative response in aquatic worms Tubifex tubifex (Oligochaeta, Tubificidae) exposed to copper

Metallothioneins (MTs), are low molecular weight proteins, mainly implicated in metal ion detoxification. Increase in MT contents is considered as a specific biomarker of metal exposure. Recently it has been demonstrated that MTs participate in several cellular functions such as regulation of growth, and antioxidative defences. Tubifex tubifex were exposed to different copper concentrations (50, 100, and 200 microgl(-1)) for 7 and 15 days. MT levels in exposed worms increased significantly (p<0.05) after 7 and 15 days of exposure to different concentrations of copper (maximum +208% for 100 microgl(-1) after 7 days of exposure). Also important perturbation in metal-metallothionein content occurred, along with an increase in total soluble protein content in all treated worms after 7 and 15 days (max. +88.49%). Catalase activities (CAT) in Cu treated-worms were significantly increased, and demonstrated a development of antioxidative defenses. Additionally a reduction of gulathione-S-transferase (GST) was observed in all treated worms after 7 days of exposure to Cu (max. -44.42%). The high induction of MTs observed during T. tubifex exposure to Cu make them potentially useful biomarkers to monitor metal pollution.     

Metallothionein induction, antioxidative responses, glycogen and growth changes in Tubifex tubifex (Oligochaete) exposed to the fungicide, fenhexamid

Laboratory studies were conducted to determine the effects of different concentrations of fenhexamid (0.1, 1, and 10 mg L(-1)) on growth, oxidative stress, protein, glycogen, and metallothionein (MT) contents in Tubifex tubifex after an exposure of 2, 4, and 7 days. In addition, residues of the fungicide were followed in water and in the worms. In water, fenhexamid concentration decreased slowly (maximum -2 +/- 0.03% after 2 days for 1 mg L(-1)). In the worms, it increased after 4 days and decreased thereafter, confirming that the worms were exposed to the fungicide and not to a degradation product. LC50 values were between 95.22 +/- 5.36 and 32.11 +/- 1.8 mg L(-1) depending on exposure time. Exposure to fenhexamid had a negative effect on T. tubifex growth (maximum effect -12.2 +/- 0.8% after 7 days with 10 mg L(-1)) demonstrating the toxic effect of the pesticide. This growth rate decrease was accompanied by a reduction in protein and glycogen contents. The activity of catalase (CAT), and glutathione reductase (GR) increased in response to the fungicide demonstrating an oxidative stress in the worms. In contrast glutathion-S-transferase activity (GST) decreased. Exposure to fenhexamid also induced synthesis of MT (maximum +78 +/- 8% after 2 days for 10 mg L(-1)). The specificity of MT concentration increase in response to metals is discussed.     

Effect of prochloraz fungicide on biotransformation enzymes and oxidative stress parameters in three-spined stickleback (Gasterosteus aculeatus L.)

The aim of this study was to characterize biomarker responses in three-spined sticklebacks exposed to prochloraz (Pcz). For this purpose, adult sticklebacks were exposed for 2 weeks to prochloraz at 0, 10, 50, 100 and 500 microg/L prior to one week of depuration in clean water. At days 7, 14 and 21, several hepatic biomarkers were measured including 7-ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), glutathione peroxidase (GPx), catalase (CAT), total glutathione (GSH) content and thiobarbituric acid reactive substances (TBARS). Pcz induced a transient increase of antioxidant enzymes and a depletion of glutathione content during the first 7 days of exposure. This study showed that EROD activity and antioxidants were disrupted in a transient manner. GST was rapidly induced in a dose-dependent manner and this induction was persistent and observed also after depuration. GST appeared as a valuable biomarker to assess the exposure to Pcz.     

Lead induced changes in antioxidant metabolism of horsegram (Macrotyloma uniflorum (Lam.) Verdc.) and bengalgram (Cicer arietinum L.)

One-month old horsegram (Macrotyloma uniflorum (Lam.) Verdc. cv VZM1) and bengalgram (Cicer arietinum L. cv Annogiri) were exposed to different regimes of lead stress as Pb(NO3)2 at 0, 200, 500 and 800 ppm concentrations. The extent of oxidative damage as the rate of lipid peroxidation, antioxidative response and the accumulation of lead in roots and shoots of both plants were evaluated after 12 days of lead stress. Lead (Pb) treated plants showed increased levels of lipid peroxidation as evidenced from the increased malondialdehyde content coupled with the increase in the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione reductase (GR), glutathione S-transferase (GST) compared to control (untreated) plants. Lead stress caused significant changes in the activity of antioxidative enzymes. The effect of lead was found to be concentration dependent. Higher concentration of lead (800 ppm) resulted 2- to 3-fold increase in SOD, catalase and peroxidase activities, 3- to 5-fold increase in GR activity and 3- to 4-fold increase in GST activity in roots and leaves of both horsegram and bengalgram plants. Lead stress caused a significant increase in the rate of peroxidation as showed in the levels of malondialdehyde content in roots and leaves of both plant species. Horsegram registered lower Pb accumulation than bengalgram, however localization of Pb was greater in roots than leaves in both plants. In general, lipid peroxide levels and antioxidative enzyme activities were higher in horsegram than bengalgram and also more in roots than leaves which best concordance with the lead contents of both the plants and organs. These results suggest that Pb toxicity causes oxidative stress in plants and the antioxidative enzymes SOD, CAT, POD, GR, GST could play a pivotal role against oxidative injury.     

Antioxidant responses to benzo[a]pyrene and Aroclor 1254 exposure in the green-lipped mussel, Perna viridis

In this study, the green-lipped mussel, Perna viridis (L.), was exposed to two concentrations of benzo[a]pyrene (B[a]P) (0.3 microg l(-1); 3 microg l(-1)) and two concentrations of Aroclor 1254 (0.5 microg l(-1); 5 microg l(-1)). In addition, a mixture of the contaminants was used (0.3 microg l(-1) B[a]P+0.5 microg l(-1) Aroclor 1254; 3 microg l(-1) B[a]P+5 microg l(-1) Aroclor 1254). All concentrations were nominal. A suite of enzymes [glutathione S transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR)], glutathione (GSH) level and lipid peroxidation (LPO) in the mussel gill and hepatopancreas were monitored over 18 days. CAT and GSH in gill tissue were positively correlated with concentration of Aroclor 1254. Activity of hepatic GST and SOD was significantly related to body burden of Aroclor 1254. LPO, GR and GPx in gill and hepatopancreas and hepatic GST were positively correlated with B[a]P concentration. The results indicate the importance of using biomarkers specific to the type of contaminant(s) that are likely to be present. Controlled laboratory experiments, such as this study, are useful in ascertaining biomarkers suitable for use with complex contaminant mixtures in the marine environment.     

Glutathione and its dependent enzymes’ modulatory responses to toxic metals and metalloids in fish—a review

Toxic metals and metalloid are being rapidly added from multiple pathways to aquatic ecosystem and causing severe threats to inhabiting fauna including fish. Being common in all the type of aquatic ecosystems such as freshwater, marine and brackish water fish are the first to get prone to toxic metals and metalloids. In addition to a number of physiological/biochemical alterations, toxic metals and metalloids cause enhanced generation of varied reactive oxygen species (ROS) ultimately leading to a situation called oxidative stress. However, as an important component of antioxidant defence system in fish, the tripeptide glutathione (GSH) directly or indirectly regulates the scavenging of ROS and their reaction products. Additionally, several other GSH-associated enzymes such as GSH reductase (GR, EC 1.6.4.2), GSH peroxidase (EC 1.11.1.9), and GSH sulfotransferase (glutathione-S-transferase (GST), EC 2.5.1.18) cumulatively protect fish against ROS and their reaction products accrued anomalies under toxic metals and metalloids stress conditions. The current review highlights recent research findings on the modulation of GSH, its redox couple (reduced glutathione/oxidised glutathione), and other GSH-related enzymes (GR, glutathione peroxidase, GST) involved in the detoxification of harmful ROS and their reaction products in toxic metals and metalloids-exposed fish.     

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 landfill leachate effluent and bisphenol A on glutathione and glutathione-related enzymes in the gills and digestive glands of the freshwater snail Bellamya purificata

Environmental contaminants with estrogenic activity have recently attracted attention due to their potential detrimental effects on the reproduction of human and wildlife. The aim of this study was to evaluate the use of endogenous glutathione and glutathione-related enzymes as biomarkers of exposure to landfill leachate effluent and bisphenol A (BPA) in the freshwater snail, Bellamya purificata. Following exposure to 1%, 5% and 10% landfill leachate effluent and 1, 10, 50 and 100mugl(-1) BPA for 0, 2, 7 and 15d, activities of glutathione S-transferase (GST), selenium-dependent glutathione peroxidase (SeGPx) and glutathione reductase (GR) and levels of total glutathione were measured in the gills and digestive glands of the snails. GST and total glutathione were the most sensitive parameters in both exposure scenarios. GST activities increased by about 80%, while total glutathione decreased to 70% and 80% in the gills and digestive glands, respectively. In contrast, SeGPx and GR activities remained at the same levels in all the treatment groups compared with those of controls. The results indicated that among glutathione and glutathione-related enzymes, GST activity and total glutathione level, which showed dose-dependent dynamics, could be used as biomarkers of aquatic ecosystems contaminated with landfill leachate.     

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.     

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.     

Cadmium effects on the fitness-related traits and antioxidative defense of Lymantria dispar L. larvae

Cadmium, like many other pollutants, is nondegradable and can be accumulated by Lymantria dispar at a level that affects fitness components, physiology, and development, which could indicate presence of environment pollution by heavy metals. The cadmium effect on fitness-related traits in the third, fourth, fifth, and sixth instar of L. dispar L. was determined. Furthermore, activities of the following antioxidative defense components after the larvae had been fed on the artificial cadmium-supplemented diet (50 μg Cd/g dry food) were assessed: superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX), total glutathione amount (GSH), glutathione-S-transferase (GST), glutathione reductase (GR), and the amount of free sulfhydryl (SH) groups. Statistically significant delay of development in the fourth, fifth, and sixth instar and decrease of the larval mass in the third and fourth instar were estimated after the exposure to cadmium through food in comparison to the control. There were no changes in SOD activity of cadmium-treated larvae. Significantly lower CAT, APOX, and GR activities were recorded in the third, fifth, and in the third instar, respectively. At the same time, higher activity was recorded in the sixth instar, while GST activity was higher in the third. GSH content was significantly lower during all instars after treatment but the amount of SH groups was higher in older larvae. The strategy of antioxidative defense and the adjustment or modulation of fitness-related traits in presence of cadmium was dependent on the age of larvae in L. dispar, which might be used in early metal risk assessment in Lepidoptera and other insects.     

Effect of prochloraz fungicide on biotransformation enzymes and oxidative stress parameters in three-spined stickleback (Gasterosteus aculeatus L.)

The aim of this study was to characterize biomarker responses in three-spined sticklebacks exposed to prochloraz (Pcz). For this purpose, adult sticklebacks were exposed for 2 weeks to prochloraz at 0, 10, 50, 100 and 500 μg/L prior to one week of depuration in clean water. At days 7, 14 and 21, several hepatic biomarkers were measured including 7-ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), glutathione peroxidase (GPx), catalase (CAT), total glutathione (GSH) content and thiobarbituric acid reactive substances (TBARS). Pcz induced a transient increase of antioxidant enzymes and a depletion of glutathione content during the first 7 days of exposure. This study showed that EROD activity and antioxidants were disrupted in a transient manner. GST was rapidly induced in a dose-dependent manner and this induction was persistent and observed also after depuration. GST appeared as a valuable biomarker to assess the exposure to Pcz.     

Sublethal responses in caged organisms exposed to sediments affected by oil spills

This study was performed to determine sublethal responses of two invertebrate species by using field deployments in areas affected by oil spills, which are acute in the Galician Coast (NNW, Spain) and chronic in the Bay of Algeciras (SSW, Spain). The organisms employed were the crab Carcinus maenas and the clam Ruditapes philippinarum, and during 28 days the animals were exposed to contaminated sediments in cages under field conditions. Different biomarkers of exposure were determined after a 28-day period exposure: ethoxyresorufin O-deethylase (EROD), phase I detoxification enzyme, glutathione-S-transferase (GST) phase II detoxification enzyme but also implicated in oxidative stress events, glutathione peroxidase (GPX) and glutathione reductase (GR), both antioxidant enzymes. In addition, histopathological effects in target tissues of the deployed organisms were evaluated. Biomarker measurements were linked with the concentration of chemicals in the sediments in order to elucidate the type, source and bioavailability of contaminants that produce adverse effects in the bioindicator species. Results obtained in this study have shown how the application of the selected battery of biomarkers under field bioassays allows for the identification of alternative sources of stress that are not observable in laboratory experiments.     

Biotransformation and antioxidant enzymes of Lumbriculus variegates as biomarkers of contaminated sediment exposure

In this study the black worm Lumbriculus variegatus was tested for suitability as biomonitor for moderately contaminated sediments. The response capacity of the biotransformation system phase II enzyme glutathione-S-transferase (GST) and the oxidative defense enzyme catalase (CAT) to contaminated sediment and atrazine was investigated to establish them as sensitive biomarkers. To get an integrated view on the enzyme activity kinetics, increasing concentrations of the herbicide atrazine were applied to stimulate GST response, and relationship between enzyme activity and herbicide concentration was observed at various exposure durations. Furthermore, animals were exposed for up to 1 week to sediments of four typical urban river sections with high anthropogenic impact. L. variegatus was capable to accomplish the environmental stress and the selected enzymes showed elevation. Significant changes of GST (membrane-bound and soluble) were detected after at least 4 days of exposure to atrazine and contaminated sediments. Although CAT increase could be observed already after 1 day of exposure to sediments, an exposure time of one week is considerable for accurate interpretation of the enzymatic response. The clear enzymatic response of especially the membrane-bound GST indicated charges with organic lipophilic substances at all sampling sites.     

Effects of chronic exposure of 2,4-dichlorophenol on the antioxidant system in liver of freshwater fish Carassius auratus

There were few reports on the antioxidant response of aquatic organisms exposed to 2,4-dichlorophenol (2,4-DCP). This research explored the hepatic antioxidant responses of fish to long-term exposure of 2,4-DCP for the first time. Freshwater fish Carassius auratus were chosen as experimental animals. The fish were exposed to six different concentrations of 2,4-DCP (0.005-1.0 mg/l) for 40 days and then liver tissues were separated for determination. As shown from the results, 40 days afterwards, the activities of catalase (CAT) and selenium-dependent glutathione peroxidase (Se-GPx) and the content of oxidized glutathione (GSSG) were induced significantly on the whole compared to control group; superoxide dismutase (SOD) responded to 2,4-DCP exposure at only 0.005 mg/l; the content of reduced glutathione (GSH) was suppressed continuously except Group 7; the activity of glutathione reductase was inhibited initially and then restored to control level from Group 4 on; glutathione S-transferase had only slight responses in Groups 3 and 4. Total glutathione (tGSH) and GSH/GSSG ratio were also calculated to analyze the occurrence of oxidative stress. Besides, good dose-effect relations, which cover most of the exposure concentration range, were found between 2,4-DCP level and CAT activity, GSSG content, Se-GPx activity, respectively. In conclusion, SOD and Se-GPx may be potential early biomarkers of 2,4-DCP contamination in aquatic ecosystems, and further studies will be necessary.     

Assessment of the mechanisms of detoxification of chemical compounds and antioxidant enzymes in the digestive gland of mussels, Mytilus galloprovincialis, from Mediterranean coastal sites

In this study the effects of the main marine pollutants (metals, PAHs, PCBs and DDTs) were assessed in native mussels from the Mediterranean coast of Spain. For this purpose several biomarkers such as benzo[a]pyrene hydroxylase (BPH), DT-diaphorase (DTD), glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPs), glutathione reductase (GR), metallothionein (MT) and lipid peroxidation (LPO) were measured in the digestive gland. Results showed increased LPO levels in mussels which accumulated high loads of organic compounds and arsenic in their tissues. BPH levels correlated to the concentrations of organic compounds in mussel tissues, though the range of BPH response was low in relation to the high gradient of accumulation of organic pollutants. Increased BPH levels, concomitant to low DTD and GST activities, were detected in mussels which presented high levels of organic pollutants in their tissues. This suggests that signs of LPO present in these organisms are related to the imbalance between phase I and phase II biotransformation processes. Furthermore, the increased levels of MT and CAT detected in mussels which showed high levels of Cd in their tissues appear to reflect a coordinated response which protects against the toxicity of this metal. The application of these biomarkers in environmental assessment is discussed.     

Oxidative stress and detoxification biomarker responses in aquatic freshwater vertebrates exposed to microcystins and cyanobacterial biomass

Cyanobacterial blooms represent a serious threat to the aquatic environment. Among other effects, biochemical markers have been studied in aquatic vertebrates after exposures to toxic cyanobacteria. Some parameters such as protein phosphatases may serve as selective markers of exposure to microcystins, but under natural conditions, fish are exposed to complex mixtures, which affect the overall biomarker response. This review aims to provide a critical summary of biomarker responses in aquatic vertebrates (mostly fish) to toxic cyanobacteria with a special focus on detoxification and oxidative stress. Detoxification biomarkers such as glutathione (GSH) and glutathione-S-transferase (GST) showed very high variability with poor general trends. Often, stimulations and/or inhibitions and/or no effects at GSH or GST have been reported, even within a single study, depending on many variables, including time, dose, tissue, species, etc. Most of the oxidative stress biomarkers (e.g., superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase) provided more consistent responses, but only lipid peroxidation (LPO) seemed to fulfill the criteria needed for biomarkers, i.e., a sufficiently long half-life and systematic response. Indeed, reviewed papers demonstrated that toxic cyanobacteria systematically elevate levels of LPO, which indicates the important role of oxidative damage in cyanobacterial toxicity. In summary, the measurement of biochemical changes under laboratory conditions may provide information on the mode of toxic action. However, comparison of different studies is very difficult, and the practical use of detoxification or oxidative stress biomarkers as diagnostic tools or early warnings of cyanobacterial toxicity is questionable.     

Field validation of a battery of biomarkers to assess sediment quality in Spanish ports

Two marine invertebrates, the crab Carcinus maenas and the clam Ruditapes philippinarum, were used as bioindicator species to assess contamination when exposed in situ to sediment from different sites from four Spanish ports Cadiz (SW Spain), Huelva (SW Spain), Bilbao (NE Spain) and Pasajes (NE Spain). In an attempt to determine sediments toxicity, a combination of exposure biomarkers was analyzed in both species: metallothionein-like-proteins (MTLPs), ethoxyresorufin O-deethylase (EROD), glutathione S-transferase activity (GST), glutathione peroxidase (GPX) and glutathione reductase (GR). In parallel, physical and chemical characterization of the different sediments was performed and biological responses related to the contaminants. Significant induction of MTLPs was observed when organisms were exposed to metal contaminated sediments (port of Huelva), and EROD and GPX activities after exposure to sediments containing organic compounds (port of Bilbao and Pasajes). No significant interspecies differences were observed in biomarker responses except for the GST and GR.