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Limited oxidative stress in common carp (Cyprinus carpio, L., 1758) exposed to a sublethal tertiary (Cu, Cd and Zn) metal mixture
Abstract
Analyzing effects of metal mixtures is important to obtain a realistic understanding of the impact of mixed stress in natural ecosystems. The impact of a one-week exposure to a sublethal metal mixture containing copper (4.8 μg/L), cadmium (2.9 μg/L) and zinc (206.8 μg/L) was evaluated in the common carp (Cyprinus carpio). To explore whether this exposure induced oxidative stress or whether defense mechanisms were sufficiently fitting to prevent oxidative stress, indicators of apoptosis (expression of caspase 9 [CASP] gene) and of oxidative stress (malondialdehyde [MDA] level and xanthine oxidase [XO] activity) were measured in liver and gills, as well as activities and gene expression of enzymes involved in antioxidant defense (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GPx], glutathione reductase [GR] and glutathione-S-transferase [GST]). The total antioxidative capacity (T-AOC) was also quantified. No proof of oxidative stress was found in either tissue but there was indication of apoptosis in the liver. CAT, GPx, GR and GST total activities were reduced after 7 days, suggesting a potential decrease of glutathione levels and risk of increased free radicals if the exposure would have lasted longer. There were no major changes in the total activities of antioxidant enzymes in the gills, but the relative expression of the genes coding for CAT and GR were triggered, suggesting a response at the transcription level. These results indicate that C. carpio is well equipped to handle these levels of metal pollution, at least during short term exposure.
... Induction of hepatic SOD behavior was usually observed when exposed to organic pollutants; however, excess development of superoxide radicals or their conversion to H 2 O 2 induced cysteine oxidation in the enzyme and deactivated SOD (Özkan et al. 2012). The decrease in total CAT and GPX functions can become an issue in the longer term (Pillet et al. 2019). If H 2 O 2 is not converted enough into H 2 O, oxidative stress will occur (Pillet et al. 2019). ...
... The decrease in total CAT and GPX functions can become an issue in the longer term (Pillet et al. 2019). If H 2 O 2 is not converted enough into H 2 O, oxidative stress will occur (Pillet et al. 2019). Herein, the toxicity of Aceta in the present study may be induced by the unbalance between antioxidants and free radicals, which might have resulted in the reduction in SOD activity (Özkan et al. 2012). ...
... The micronucleus test and comet assay application are the most validated biomarkers for genotoxicity (Bolognesi and Cirillo 2014). Osmoregulatory dysfunctions and tissue damage might be caused by oxidative stress via the oxidation of lipids and proteins in addition to alterations of gene expression (Livingstone 2001;Pillet et al. 2019). The result of gene expression can provide insight into the biological status of fish and provide details about whether the fish is exposed to pollutants (El Megid et al. 2020). ...
On juveniles of Oreochromis niloticus, the protective potential of ascorbic acid (Asc) against oxidative stress and genotoxicity induced by acetamiprid (Aceta) sub-lethal concentrations was investigated in this study. Fishes were divided into six groups and exposed to either Asc (50 ppm), 10 and 20 ppm Aceta, 10 ppm (Aceta)+Asc, 20 ppm (Aceta)+Asc, or the unexposed control group. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities and their transcripts were assessed. DNA damage in erythrocytes, hepatocytes, and gill cells, in addition to the mitotic index (MI), and the existence of erythrocytic nuclear abnormalities (ENAs) were performed. The results showed that concentrations of Aceta (10 and 20 ppm) induced oxidative stress by altering the antioxidant enzyme activities and transcripts. There were genotoxic effects of Aceta exposure showed by the significant (P < 0.05) increase in DNA-damaged cells and ENA, meanwhile a decrease in MI. Co-exposure with Asc showed significant alleviations of oxidative status and genotoxicity. Thus, results suggest that Asc-combined exposure could be the effective treatment against Aceta-induced oxidative stress accompanied with genotoxicity in O. niloticus.
... In case of serious oxidative stress, apoptotic events might occur (Pellegrini and Baldari 2009). Apoptosis is induced by intracellular signaling molecules, such as caspase 9 (CASP), which mediates apoptosis through the mitochondrial pathway (Pillet et al., 2019;Wang et al., 2019). ...
... Nonetheless, fish have a suite of defensive mechanisms to cope with increasing ROS and oxidative stress such as the enzyme superoxide dismutase (SOD), which catalyses the conversion of the superoxide radical ( • O 2 − ) into hydrogen peroxide (H 2 O 2 ). The H 2 O 2 is further converted into water (H 2 O) and oxygen (O 2 ) by catalase (CAT) and glutathione peroxidase (GPx) (Livingstone 2001;Pillet et al., 2019). Furthermore, the presence of peroxiredoxin (Prdx), a family of peroxidases that reduce H 2 O 2 , organic peroxides and peroxynitrite by using cysteine residues helps in protecting the cells and tissues from the effects of oxidant molecules (Tolomeo et al., 2016;Tolomeo et al., 2019). ...
... The general experimental run protocol as described by Shrivastava et al. (2017), consisted of a denaturation program (3 min at 95 • C), an amplification and quantification program repeated 40 times (15 s at 95 • C, 20 s at 60 • C) followed by a melting curve program (60 • C-95 • C). Oligonucleotides primers were taken from literature: elongation factor 1α (eEF) (Sinha et al., 2012), β-actin (Wu et al., 2014); catalase (CAT) (Wu et al., 2014), superoxide dismutase Cu-Zn (SOD) (Wu et al., 2014), glutathione reductase (GR) (Wu et al., 2014), metallothionein (MT) (Reynders et al., 2006b) and caspase 9 (Casp9) (Pillet et al., 2019). Primers for the glutathione-S-transferase (GST) and glutathione peroxidase (GPx) were designed using NCBI resources Primer blast and synthesized as highly purified salt-free "OliGold" primers by Eurogentec (Eurogentec, Seraing, Belgium). ...
The aquatic environment receives a wide variety of contaminants that interact with each other, influencing their mutual toxicity. Therefore, studies of mixtures are needed to fully understand their deleterious effects on aquatic organisms. In the present experiment, we aimed to assess the effects of Cd and Zn mixtures in common carp during a one-week exposure. The used nominal waterborne metal levels were 0.02, 0.05 and 0.10 µM for Cd and 3, 7.5 and 15 µM for Zn. Our results showed on the one hand a fast Cd increase and on the other hand a delayed Zn accumulation. In the mixture scenario an inhibition of Cd accumulation due to Zn was marked in the liver but temporary in the gills. For Zn, the delayed accumulation gives an indication of the efficient homeostasis of this essential metal. Between the different mixtures, a stimulation of Zn accumulation by Cd rather than an inhibition was seen in the highest metal mixtures. However, when compared to an earlier single Zn exposure, a reduced Zn accumulation was observed. Metallothionein gene expression was quickly activated in the analysed tissues suggesting that the organism promptly responded to the stressful situation. Finally, the metal mixture did not alter tissue electrolyte levels.
... Additionally, this species is considered to be a good bioindicator for ecotoxicological studies (Altun et al., 2017;Rajeshkumar et al., 2017) and is recommended in Organization for Economic Co-operation and Development (OECD) guidelines, as one of six fish species for regulatory testing (OECD, 2019). As a model species, common carp is used to study the impact of metals both in the lab and in the field, on for example bioaccumulation (Bervoets et al., 2009;Castaldo et al., 2020;Delahaut et al., 2019;Delahaut et al., 2020), energy status (De Boeck et al., 1995a;Kunwar et al., 2009), swimming capacity (Delahaut et al., 2019) or oxidative stress (Cortes-Diaz et al., 2017;Dugmonits et al., 2013;García-Medina et al., 2017;Pillet et al., 2019). Metal is found in every aquatic ecosystem and anthropogenic activity discharges metals directly into water from mines, industry, intensive agriculture, household waste or traffic (Burger, 2008;Coufalík et al., 2019;Stohs and Bagchi, 1995). ...
... A recent study investigating the impact of a similar environmentally relevant Cu/Cd/Zn mixtures on common carp, showed a number of effects on ion-regulation (reduced Na + transport) and detoxification functions (strong induction of metallothionein expression) (Castaldo et al., 2020), as well as oxidative stress capacities (Pillet et al., 2019) reflecting different toxicity mechanisms of the Cu/Cd/Zn mixture. The present study aimed to investigate whether these mechanisms translate into changes in whole-animal physiological performance linked to bioaccumulation of metal in tissues, and whether these are affected by temperature. ...
... However, at both temperatures, complete acclimatisation of the carp is expected by the end of the experiment. To facilitate direct comparisons with previous observations (Castaldo et al., 2020;Pillet et al., 2019), the concentrations of metal used in the present study targeted the concentrations previously used (10% of the LC 50 for single metal, as defined by Delahaut et al. (2020)), and also reflected ecologically-relevant concentrations. ...
In a natural ecosystem, fish are subjected to a multitude of variable environmental factors. It is important to analyze the impact of combined factors to obtain a realistic understanding of the mixed stress occurring in nature. In this study, the physiological performance of juvenile common carp (Cyprinus carpio) exposed for one week to an environmentally relevant metal mixture (4.8 μg/L of copper; 2.9 μg/L of cadmium and 206.8 μg/L of zinc) and to two temperatures (10 °C and 20 °C), were evaluated. After 1, 3 and 7 days, standard (SMR) and maximum metabolic rate (MMR) were measured and aerobic scope (AS) was calculated. In addition, hematocrit, muscle lactate, histology of the gills and metal accumulation in gills were measured. While SMR, MMR and AS were elevated at the higher temperature, the metal mixture did not have a strong effect on these parameters. At 20 °C, SMR transiently increased, but no significant changes were observed for MMR and AS. During metal exposure, hematocrit levels were elevated in the 20 °C group. The bioaccumulation of Cd in the gills reflected the increased metabolic rate at the higher temperature, with more accumulation at 20 °C than at 10 °C. Anaerobic metabolism was not increased, which corresponds with the lack of significant histopathological damage in the gill tissue. These results show that common carp handled these metal exposures well, although increased temperature led to higher Cd accumulation and necessitated increased hematocrit levels to maintain aerobic performance.
... When metal intake is not balanced with excretion, the accumulation of metal ions might result in toxic effects. These metal ions have the capacity to increase reactive oxygen species (ROS) production and induce oxidative stress (Pillet et al., 2019;Wang, Fang, Leonard, & Rao, 2004;Zheng, Yuan, Wu, & Li, 2016). To cope with oxidative stress, fish have several defensive mechanisms. ...
... The levels of GSH are regulated by the presence of GR and GST (Dautremepuits, Marcogliese, Gendron, & Fournier, 2009;Pillet et al., 2019). ...
... Although metal ions can increase the ROS production (Rajeshkumar, Liu, Ma, Duan, & Li, 2017), defensive mechanism such as SOD, CAT, GST and GR are involved in ROS removal (Hansen, Rømma, Garmo, Olsvik, & Andersen, 2006;Pillet et al., 2019). In the present study we investigated changes in the gene expression of SOD, CAT, GST and GR in the gills as well as in the liver. ...
To improve our understanding of underlying toxic mechanisms, it is important to evaluate differences in effects that a variety of metals exert at concentrations representing the same toxic level to the organism. Therefore, the main goal of the present study was to compare the effects of waterborne copper (Cu(II)), zinc (Zn(II)) and cadmium (Cd (II)) on a freshwater fish, the common carp (Cyprinus carpio), at concentrations being 0%, 25%, 50% and 100% of the 96 h LC50 (the concentration which is lethal to 50% of the population in 96 h). All the exposures were performed for a period of 1 week at 20°C. Our results show a rapid increase in the amount of copper and cadmium accumulated in the gills, while zinc only started to increase by the end of the experiment. All three metal ions increased metallothionein gene expression in both gills and liver. However, clear adverse effects were mainly observed for the Cu exposed group. Cu caused a decrease in Na level in gill tissue; it altered the expression of genes involved in ionoregulation such as Na⁺/K⁺‐ATPase and H⁺‐ATPase as well as the expression of oxidative stress‐related genes, such as catalase, glutathione reductase and glutathione S‐transferase. Zinc and cadmium exposure did not alter the ion levels in the gills. In addition, no obvious effect of oxidative stress was observed, except for a transient increase in glutathione reductase at the highest cadmium concentration.
... The antioxidant defence system plays a crucial role in preventing deleterious effects caused by reactive oxygen species (ROS). Enzymes such as SOD, and catalase (CAT) represent the first line of defence, converting superoxide (O 2 −% ) into hydrogen peroxide (H 2 O 2 ), and H 2 O 2 into water (H 2 O) and oxygen (O 2 ) (Pillet et al., 2019). Furthermore, GSH plays an important role in ROS defence and as a chelating agent for metal ions (Lange et al., 2002). ...
... With this work, together with all the previous studies investigating and showing the complexity of metal mixture scenarios (Komjarova and Blust, 2009;Niyogi et al., 2015;Brix et al., 2017;Pillet et al., 2019) we aim to provide new insights into understanding metal accumulation and toxicity in multi-metal exposure scenarios. We hypothesize that metal mixtures would remain sub-lethal, as our exposures were relatively short and maximum exposure concentrations were 25 % + 50 % of the 96h-LC 50 . ...
... Regarding the liver, the MT gene expression peak, observed at day three, followed by a decrease at day seven, could suggest that a temporary elevated protein synthesis was sufficient to cope with the metals, at least during this one week exposure. Common carp is known to quickly adapt to prolonged metal ion exposures, only increasing defensive mechanisms when needed (Martinez et al., 2004;Pillet et al., 2019). ...
In the aquatic environment, metals are present as mixtures, therefore studies on mixture toxicity are crucial to thoroughly understand their toxic effects on aquatic organisms. Common carp were used to assess the effects of short-term Cu(II) and Cd(II) mixtures, using a fixed concentration of one of the metals, representing 25 % of its individual 96h-LC50 (concentration lethal for 50 % of the population) combined with a variable concentration of the other metal corresponding to 10, 25 or 50 % of its 96h-LC50, and vice versa. Our results showed a fast Cu and Cd bioaccumulation, with the percentage of increase in the order gill > liver > carcass. An inhibitory effect of Cu on Cd uptake was observed; higher Cu concentrations at fixed Cd levels resulted in a decreased accumulation of Cd. The presence of the two metal ions resulted in losses of total Na, K and Ca. Fish tried to compensate for the Na loss through the induction of the genes coding for Na⁺/K⁺-ATPase and H⁺-ATPase. Additionally, a counterintuitive induction of the gene encoding the high affinity copper transporter (CTR1) occurred, while a downregulation was expected to prevent further metal ion uptake. An induction of defensive mechanisms, both metal ion binding protein and anti-oxidant defences, was observed. Despite the metal accumulation and electrolyte loss, the low mortality suggest that common carp is able to cope with these metal levels, at least during a one-week exposure.
... The induced hepatotoxicity and genotoxicity in Oreochromis niloticus exposed to a newly released. . . toxicants on transcription of mRNA or translation of corresponding protein mechanisms (Lu et al., 2013;Pillet et al.,et al., 2019). Antioxidant enzymes may be increased after a short time of exposure and then decreased with time due to the inhibition in their activity or production (Abdelazim et al., 2018;Meng et al., 2021). ...
... However, increased production of super oxide free radicals or their conversion to H 2 O 2 can deactivate the enzyme through cysteine oxidation as described by (Özkan et al., 2012). The decrease in CAT and GSH activities is an oxidative stress inducer in the long term if H 2 O 2 is not converted enough into H 2 O (Pillet et al., 2019). ...
The investigation of the toxic potential of a newly introduced herbicide, Florpyrauxifen-benzyl (FPX), on Nile tilapia (Oreochromis niloticus) was the aim of this study. For 96 h, the median lethal concentration (LC50) was assessed in fish juveniles using the Probit analysis following the exposure to five concentrations of FPX (2–3 ppm). For investigating some mechanisms of FPX toxicity, fish were allocated into three groups (0, 0.27 and 0.54 ppm of FPX) and the cut-off intervals of the exposure were at 7 and 15 days. Liver malondialdehyde (MDA) and reduced glutathione (GSH) levels were assessed. In addition, superoxide dismutase (SOD) and catalase (CAT) were evaluated at both of transcriptional and enzymatic activity levels. Histopathological effects on the liver and erythrocytic nuclear abnormalities (ENAs) were monitored too. The 96h-LC50 was found to be 2.61 ppm, revealing the toxic potential of the FPX on Nile tilapia. Concentrations of FPX induced oxidative stress in fish by altering activities of antioxidant enzymes and their transcripts. The genotoxic effect of FPX was evidenced by a significant (P < 0.05) increase in micronuclei (MNs) and ENA frequencies. Significant liver histopathological alterations were observed at both FPX concentrations, with the highest effects at a concentration of 0.54 ppm FPX. Results suggest that FPX may exert oxidative, genotoxic, and histopathological effects on non-targeted species such as Nile tilapia if it is used improperly. Although fish could be used as an indicator for toxic materials in the aquatic habitat, future studies on other organisms, FPX concentrations or durations are recommended.
... Several studies have conducted research linking the oxidative stress in fish liver and intestine [10,12,14,21,26,[29][30][31][32][33][34][35][36], in fish blood plasma [20,37,38], in fish muscle [21,32], in fish gill, kidney and brain [29][30][31]39] to heavy metals presence in the aquatic environment, proving that fish OS biomarkers can be used to determine heavy metal pollution since the trace element contamination directly affects fish welfare status. ...
... The MDA represents the end-product of lipid peroxidation degradation and high levels indicate oxidative cell damage after toxicant exposure. In the study by [33], Cyprinus carpio individuals were exposed, for one week, to a mixture of metal pollutants (Cu: 4.8 µg/L; Cd: 2.9 µg/L and Zn: 206.8 µg/L). The authors registered no sign of OS in the liver and the gill tissues during that period. ...
Heavy metal pollution is still present in the Danube River basin, due to intensive naval and agricultural activities conducted in the area. Therefore, continuous monitoring of this pivotal aquatic macro-system is necessary, through the development and optimization of monitoring methodologies. The main objective of the present study was to develop a prediction model for heavy metals accumulation in biological tissues, based on field gathered data which uses bioindicators (fish) and oxidative stress (OS) biomarkers. Samples of water and fish were collected from the lower sector of Danube River (DR), Danube Delta (DD) and Black Sea (BS). The following indicators were analyzed in samples: cadmium (Cd), lead (Pb), iron (Fe), zinc (Zn), copper (Cu) (in water and fish tissues), respectively, catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA) (in fish tissues). The pollution index (PI) was calculated to identify the most polluted studied ecosystem, which revealed that Danube River is seriously affected by the presence of Fe (IP = 4887) and strongly affected by the presence of Zn (IP = 4.49). The concentration of Cd in fish muscle tissue was above the maximum permitted level (0.05 µg/g) by the EU regulation. From all analyzed OS biomarkers, MDA registered the highest median values in fish muscle (145.7 nmol/mg protein in DR, 201.03 nmol/mg protein in DD, 148.58 nmol/mg protein in BS) and fish liver (200.28 nmol/mg protein in DR, 163.67 nmol/mg protein, 158.51 nmol/mg protein), compared to CAT, SOD and GPx. The prediction of Cd, Pb, Zn, Fe and Cu in fish hepatic and muscle tissue was determined based on CAT, SOD, GPx and MDA, by using non-linear tree-based RF prediction models. The analysis emphasizes that MDA in hepatic tissue is the most important independent variable for predicting heavy metals in fish muscle and tissues at BS coast, followed by GPx in both hepatic and muscle tissues. The RF analytical framework revealed that CAT in muscle tissue, respectively, MDA and GPx in hepatic tissues are most common predictors for determining the heavy metals concentration in both muscle and hepatic tissues in DD area. For DR, the MDA in muscle, followed by MDA in hepatic tissue are the main predictors in RF analysis.
... Exposure tanks consisted of 5 double-walled polypropylene (PP) buckets for control and 5 double-walled buckets for the treatment, each filled with 9 l of EPA medium-hard water (conductivity 308 ± 2.5 μS/cm) and containing 6 fish. Fish from the first series were used to assess bioaccumulation and electrolytes level, while fish from the second series were used for gene expression analysis and to assess oxidative stress (Pillet et al., 2019). In each bucket, oxygen was provided with an air stone. ...
... Further Na losses appeared to be prevented by the ability of common carp to cope with this situation through an increased expression of the H + -ATPase gene. In a parallel experiment Pillet et al. (2019) showed the ability of common carp to avoid oxidative stress when exposed at the same mixture. When exposed to a mixture of Cu, Cd and Zn at a concentration of 10 % of the LC 50 , the bioaccumulation pattern is similar to those described in other papers with sharp increases in Cu and Cd (De Smet et al., 2001;Celechovska et al., 2007;Tunçsoy and Erdem, 2014) followed by a drop in Na. ...
The aquatic environment is continuously under threat because it is the final receptor and sink of waste streams. The development of industry, mining activities and agriculture gave rise to an increase in metal pollution in the aquatic system. Thus a wide occurrence of metal mixtures exists in the aquatic environment. The assessment of mixture stress remains a challenge considering that we can not predict the toxicity of a mixture on the basis of single compounds. Therefore the analysis of the effects of environmentally relevant waterborne mixtures is needed to improve our understanding of the impact of metal pollution in aquatic ecosystems. Our aim was to assess whether 10 % of the concentration of the 96 h LC50 (the concentration that is lethal to 50 % of the population in 96 h) of individual metal exposures can be considered as a "safe" concentration when applied in a trinomial mixture. Therefore, common carp were exposed to a sublethal mixture of Cu 0.07 ± 0.001 μM (4.3 ± 0.6 μg/L), Zn 2.71 ± 0.81 μM (176.9 ± 52.8 μg/L) and Cd 0.03 ± 0.0004 μM (3.0 ± 0.4 μg/L) at 20 °C for a period of one week. Parameters assessed included survival rate, bioaccumulation and physiological biomarkers related to ionoregulation and defensive mechanisms such as MT induction. Our results showed a sharp increase in Cu and Cd concentration in gills within the first day of exposure while Zn levels remained stable. The accumulation of these metals led to a Na drop in gills, liver and muscle as well as a decreased K content in the liver. Biomarkers related to Na uptake were also affected: on the first day gene expression for H+-ATPase was transiently increased while a concomitant decreased gene expression of the Na+/H+ exchanger occurred. A fivefold induction of metallothionein gene expression was reported during the entire duration of the experiment. Despite the adverse effects on ionoregulation all fish survived, indicating that common carp are able to cope with these low metal concentrations, at least during a one week exposure.
... Due to this situation, when oxidative damage is severe, compensatory mechanisms fail, and antioxidant enzymes are suppressed, ultimately leading to the death of cells [32,33]. Pillet et al. [34] found that GSH-Px activity in carp liver was reduced considerably after 7 days of exposure to Cd-contaminated water, which is in agreement with what we observed in grass carp. A significant increase in GSH-Px activity was observed in groups supplemented with selenium under cadmium stress compared to control groups. ...
In order to investigate the effects of selenium (Se) against cadmium (Cd) toxicity, 180 healthy grass carp were separated into three groups and fed diets containing 0.147 (control group), 0.562, and 1.044 mg/kg of selenium Yeast throughout 60 days. In grass carp livers, malondialdehyde (MDA) content, antioxidant enzyme activities, and apoptosis-related gene expression were examined. As a result of acute exposure to cadmium, MDA content decreased significantly. With time, catalase (CAT), total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-Px) activities changed. The relative transcript levels of heavy metal scavenging genes abcc2 and mt2 were significantly reduced. The relative levels of expression of jnk, bax, caspase-3, caspase-8, and caspase-9 in apoptosis-associated factors were significantly elevated after cadmium exposure. Selenium-supplementation downregulated the expression of apoptosis-related factors. As compared to the control group, liver cells supplemented with selenium had a significantly lower apoptotic index. Comprehensive analysis showed that dietary selenium supplementation significantly attenuated cadmium-induced peroxidative damage and apoptosis in liver by increasing GSH-Px activity, and that cadmium toxicity could be alleviated by the addition of yeast selenium.
... Because of its high adaptive capability to both the environment and food, it is widely cultured and considered an important fish for commercial aquaculture in Asia and some European countries (Soltani et al. 2010;Manjappa et al. 2011;Rahman 2015). Perusal of literatures shows that this species is frequently used as a model in several toxicological studies to determine the levels and toxicity of various pollutants in the aquatic environment (Vinodhini and Narayanan 2008;Vinodhini and Narayanan 2009;Coban et al. 2013;Rajeshkumar et al. 2017;Pillet et al. 2019;Sahiti et al. 2020;Kakade et al. 2020;Gwimbi et al. 2020). ...
The purpose of this study is to assess the seasonal variation of heavy metal concentration in water and fish tissues of common carp (Cyprinus carpio L.) from the Umiam Lake reservoir located in the Ri bhoi district of Meghalaya, India, and to elucidate the possible human health risk of ingesting fish captured from the contaminated lake. Results show significant (p < 0.05) seasonal differences of heavy metal concentrations in the water and different tissues of fish Cyprinus carpio L.. The total concentration of heavy metals in the water exceeds the WHO and BIS standards and thus poses a significant threat to the aquatic flora and fauna of the reservoir. The heavy metal concentrations in fish tissues were tissue-dependent, where the average concentration of heavy metals in all the tissues of Cyprinus carpio L. was in the order of Cr > Pb > Cu > Cd. In addition, the health risk assessment suggests that the heavy metals in the fish muscle from the Umiam Lake reservoir might have adverse effects on human. Therefore, the overall results of the study provide an understanding on the seasonal distribution of heavy metals in water, provide insight on their bioaccumulation in the fish tissues, and highlights the potential health risk for the local population of long-term fish consumption from Umiam Lake reservoir.
... A metal mixture can cause different effects from those observed after exposure to the metals separately, causing greater toxicity (Abdalla et al., 2019;Oliveira et al., 2018). In addition, the sum of the individual effects is often not enough to infer the effect of the mixtures, and exposure tests are required to evaluate possible interactions between contaminants (Pillet et al., 2019). ...
Ni and Cd are widely used together in the manufacture of cells and batteries. The incorrect disposal of these products can result in environmental contamination, posing risks to the organisms exposed to these contaminants. However, the effects of the mixture of Ni and Cd in freshwater fishes are still unclear in the current literature, especially in relation to biomarkers of oxidative stress. Thus, the objective of the current work was to compare the sublethal effects caused by the mixture of cadmium (Cd) and nickel (Ni) with the effects of these metals individually, in the fish Astyanax altiparanae. The animals were exposed for 72 h to 20 μg L⁻¹ of Cd, 1.5 mg L⁻¹ of Ni, or a mixture of these two metals at the concentrations mentioned. After exposure, tissue samples were collected to evaluate hematological and plasma parameters, biomarkers of oxidative stress in the gills, and acetylcholinesterase (AChE) activity in brain and muscle. Exposure to the mixture caused alterations that were not observed in the groups exposed to the metals individually, such as increased activity of catalase and glutathione-S-transferase, and a reduction in AChE activity in the brain. Thus, we concluded that exposure to the mixture of Cd and Ni is more harmful to A. altiparanae than exposure to these metals separately.
... One of them is that the change of a specific isoenzyme at the mRNA level is not always reflected at the enzyme level due to the presence of multiple copies of genes in fish; therefore, the enzyme activity detected could be equal to the total enzyme activity of the different isoenzymes, but the level of mRNA transcription would be limited to a subtype of the antioxidant gene encoding a single isoenzyme, gpx1 in this case. Another reason they consider is the likelihood of enzyme modulation at post-translational levels, as indicated by Pillet et al. (2019) in Cyprinus carpio exposed to metal mixtures, showing impact on gill antioxidant enzymes CAT and GPx only after 3 to 7 days of exposure, although relative gene expression of CAT and glutathione reductase was indeed triggered, suggesting responses at the transcriptional level. ...
Penicillin, metformin, and aluminum are commonly used substances and their presence in the environment has increased due to their widespread use. In water bodies, the combined presence of xenobiotics leads to additive, synergistic, or antagonistic interactions that significantly modify the toxic response, being more evident in the early stages of development of individuals. These interactions can be evaluated through biomarkers such as the activity and expression of antioxidant enzymes and the production of congenital malformations in exposed organisms. However, little is known about the effects that mixtures of drugs such as penicillin, metformin, and metals such as aluminum can produce on aquatic species that are constantly exposed to these xenobiotics such as common carp. The objective of this study was to determine the toxicity and type of interaction produced on Cyprinus carpio embryos exposed to these pollutants isolated and in mixtures. Here we show that the mixture of these pollutants produces antagonism and synergism at high and low concentrations respectively. In addition, toxicity results show that in embryos subacutely exposed (NOAEL) to the mixture of contaminants, the activity of antioxidant enzymes and their gene expression (PCR-RT) is increased, embryonic development is modified, and teratogenesis occurs. As can be observed, the mixture of the contaminants influenced the toxic response, evidencing the importance of continuing to study interactions; since this is the way, they are usually found in contaminated bodies of water.
Highlights
1. Exposure of Cyprinus carpio embryos to the mixture of metformin, penicillin and aluminum at high concentrations (LC50) produces an antagonistic type of interaction.
2. Exposure of Cyprinus carpio embryos to the mixture of metformin, penicillin and aluminum at sublethal concentrations (NOAEL) produces a synergistic type interaction.
3. Antioxidant enzyme activity of Cyprinus carpio embryos exposed to NOAEL-equivalent concentrations of metformin, penicillin and aluminum is modified by simultaneous exposure to the toxicants.
4. Gene expression of antioxidant enzymes in Cyprinus carpio embryos exposed to NOAEL-equivalent concentrations of metformin, penicillin and aluminum is increased by simultaneous exposure to the toxicants during the first hours of development.
5. The embryonic developmental score of Cyprinus carpio is decreased by simultaneous exposure to metformin, penicillin and aluminum.
Graphical abstract
... Відомо, що широке коло сільськогосподарських отрутохімікатів викликає окиснювальний стрес через підвищення вмісту реактивних сполук кисню (РСК) в різних тканинах (Sharma, Jindal, 2020;Yang et al., 2020). Антиоксидантні системи повинні протидіяти пероксидації, а їх реакцію на інтоксикацію пестицидами можна використовувати як біомаркер їх токсичного впливу (Kostich et al., 2019;Sharma, Jindal, 2020) Зрозуміло, що загальні процеси клітинної реактивності на інтоксикацію мають універсальні механізми, тому модуляція антиоксидантних відповідей на ушкоджуючі фактори зазвичай не залежить від хімічної природи токсиканта (Pillet et al., 2019;Valavanidis, 2006). Незважаючи на однотипну антиоксидантну реакцію клітин на дію екотоксикантів, визначення рівня окиснювального стресу та показників стану антиоксидантної системи надають важливу інформацію щодо спряженості різних інтоксикаційних та реактивних процесів (Dos Santos, 2018). ...
The synthetic pyrethroid of systemic insecticidal and acaricidal action, λ-cyhalothrin, is characterized by typical axonal excitotoxicity. Currently, it is widely used in agriculture worldwide. In fish, it negatively affects the functional characteristics of the nervous system, causes motorial paralysis, and increases mortality. The concentrations of λ-cyhalothrin for the experiment were determined at a rate of 5% and 10% of LC50, which are 271 ng/L and 542 ng/L, respectively. Λ-cyhalothrin causes oxidative damage in the brain cells of the rainbow trout and, probably, irreversible disturbances in brain cells. The content of reduced glutathione in the fish brain treated by low doses of λ-cyhalothrin was significantly lower than in the control fish. It may indicate the breakdown of this part of antioxidant protection. One of the universal and multifunctional regulatory proteins, which plays a vital role in most cell types is the p53 protein that supports cell viability through a wide range of signalling pathways. Changes in the p53 protein expression are determined in fish of all experimental groups. Moreover, less significant suppression of its expression is found in fish that have been treated by a dose of 5% LC for 28 days. Unlike the 5% LC50, the dose of 10% LC50 induces a critical decrease in the p53 protein content after both 4 and 28 days of insecticide exposure. Detected changes in the reduced glutathione content and p53 protein expression in the fish brain are associated with the oxidative stress generation caused by low sublethal doses of λ-cyhalothrin. A significant decrease in the p53 protein content in the fish brain under the impact of the low doses of the synthetic pyrethroid can indicate irreversible disturbance of this protein functioning. The inhibition of protein p53 expression may be an adequate biomarker of the nervous cell adaptation to the toxic effect of synthetic pyrethroids in the fish brain.
... In current study, the expression level of gpx genes have been down-regulated in most cases under cd 2+ treatment. It is speculated that the excessive accumulation of cd 2+ in the organism inhibited the expression of gpx genes, which was consistent with the results of previous studies (Pillet et al., 2019;Chen et al., 2021a). ...
Glutathione peroxidase (Gpx) is an important member of antioxidant enzymes, which can play a vital role in metabolizing reactive oxygen species (ROS) and in maintaining cell homeostasis. In order to study the evolutionary dynamics of gpx gene family in allotetraploid fish species, we identified a total of 14 gpx genes in common carp Cyprinus carpio, while 9 gpx genes were discovered in the diploid progenitor-like species Poropuntius huangchuchieni. Comparative genomic analysis and phylogenetic analysis revealed that the common carp gpx genes had significant expansion and were divided into five distinct subclades. Exon-intron distribution patterns and conserved motif analysis revealed highly conserved evolutionary patterns. Transcript profiles suggested that different gpx genes had specific patterns of regulation during early embryonic development. In adult tissues, gpx genes had a relatively broad expression distribution, most of which were highly expressed in the gills, intestines, and gonads. RT-qPCR studies showed that most gpx genes were downregulated during the initial cd²⁺ treatment stage. Dietary supplementation of Bacillus coagulans at different concentrations (Group 2 of 1.0×10⁷ cfu/g, Group 3 of 1.0×10⁸ cfu/g, and Group 4 of 1.0×10⁹ cfu/g) induced different regulatory responses of gpx subclades. This result suggested that the appropriate concentration of B. coagulans can improve gpx gene expression when exposed to heavy metal cadmium treatment, which may play a vital role in the resistance to oxidative stress and immune responses. This study has expanded our understanding of the functional evolution of the gpx gene family in common carp.
... Відомо, що широке коло сільськогосподарських отрутохімікатів викликає окиснювальний стрес через підвищення вмісту реактивних сполук кисню (РСК) в різних тканинах (Sharma, Jindal, 2020;Yang et al., 2020). Антиоксидантні системи повинні протидіяти пероксидації, а їх реакцію на інтоксикацію пестицидами можна використовувати як біомаркер їх токсичного впливу (Kostich et al., 2019;Sharma, Jindal, 2020) Зрозуміло, що загальні процеси клітинної реактивності на інтоксикацію мають універсальні механізми, тому модуляція антиоксидантних відповідей на ушкоджуючі фактори зазвичай не залежить від хімічної природи токсиканта (Pillet et al., 2019;Valavanidis, 2006). Незважаючи на однотипну антиоксидантну реакцію клітин на дію екотоксикантів, визначення рівня окиснювального стресу та показників стану антиоксидантної системи надають важливу інформацію щодо спряженості різних інтоксикаційних та реактивних процесів (Dos Santos, 2018). ...
Mercury is a widespread heavy metal that causes a stable and prolonged environmental pollution. Low concentrations of inorganic and organic mercury compounds are found in almost all water bodies. The high level of mercury bioaccumulation is a cause of tissue-specific toxicity, including neurotoxicity. Absorbed in nervous tissue mercury can cause brain disorders both in neural and glial cells. The brain of fish is considered one of the most susceptible targets for cytotoxicity of mercury in aquatic ecosystems. Taking into account that different forms of mercury have widespread distribution and exhibit a strong neurotoxic effect, the assessment of mercury cytotoxicity in the brain of fish is relevant and extremely important. Rainbow trout Oncorhynchus mykiss was exposed to mercury chloride in the dose range of 5-20 μg/L for 60 days to study the chronic exposure of low doses. In this paper, we studied the influence of inorganic mercury on oxidative stress, DNA repair proteins – ERCC1 and PARP1 in the trout’s brain. The results obtained have shown that the chronic effect of inorganic mercury causes dose-dependent oxidative stress in the fish brain. In addition, low concentrations of mercury (10 and 20 μg/L) caused a decrease in the content of ERCC1 in the brain of fish. On the contrary, the same doses have caused an increase in PARP1 expression. That is the chronic influence of low concentrations of inorganic mercury has a negative effect in the fish brain. Observed results showed that inorganic mercury has a potential for suppressing DNA repair and, therefore, increases the instability of genome. Thus, ERCC1 and PARP1 can be considered as the sensitive biomarkers of mercury cytotoxicity in the fish brain. A further study of mercury neurotoxicity is needed to find out the hazard of mercury environmental pollution as well as a validation of biomarkers of their impact.
... Antioxidant defense responses can be regulated at different levels, transcriptional, translational, and post-translational, and these alterations can occur at the same time or independently of each other, depending on the pollutant, dose and time of exposure, generating different patterns of gene transcription levels, protein abundance and enzyme activities (Defo et al., 2015;Regoli et al., 2011;Zheng et al., 2016a, b). Increased levels of gene expression without post-transcriptional changes under low contamination conditions can represents a pre-adaptation mechanism through which, if the contaminant concentration increases or extends over time, organisms would be prepared to increase the activity of antioxidant defenses when they need it (Pillet et al., 2019). This mechanism could be related to the cellular content of ROS; we can hypothesize that, under low contamination conditions, the intracellular ROS level (produced in normal metabolic pathways such as processes involved in the control of pollutant accumulation and excretion) may increases reaching levels which are not high enough to produce oxidative stress (and consequently activation of antioxidant defense system) but sufficient to induce transcriptional factors that regulate the expression of different genes. ...
The knowledge about the effects of pharmaceuticals on aquatic organisms has been increasing in the last decade. However, due to the variety of compounds presents in the aquatic medium, exposure scenarios and exposed organisms, there are still many gaps in the knowledge on how mixtures of such bioactive compounds affect exposed non target organisms. The crayfish Procambarus clarkii was used to analyze the toxicity effects of mixtures of ciprofloxacin, flumequine and ibuprofen at low and high concentrations (10 and 100 μg/L) over 21 days of exposure and to assess the recovery capacity of the organism after a depuration phase following exposure during additional 7 days in clean water. The crayfish accumulated the three compounds throughout the entire exposure in the hepatopancreas. The exposure to the mixture altered the abundance of proteins associated with different cells functions such as biotransformation and detoxification processes (i.e. catalase and glutathione transferase), carbohydrate metabolism and immune responses. Additionally changes in expression of genes encoding antioxidant enzymes and in activity of the corresponding enzymes (i.e. superoxide dismutase, glutathione peroxidase and glutathione transferase) were reported. Alterations at different levels of biological organization did not run in parallel under all circumstances and can be related to changes in the redox status of the target tissue. No differences were observed between control and exposed organisms for most of selected endpoints after a week of depuration, indicating that exposure to the drug mixture did not produce permanent damage in the hepatopancreas of P. clarkii.
... Реакцію антиоксидантних систем на вплив пестицидів запропоновано використовувати як біомаркери токсичного впливу (Sharma & Jindal, 2020). Однак модуляція активності антиоксидантних ферментів ушкоджуючими факторами має спільні риси, що не залежить від природи токсичної речовини (Pillet et al., 2019;Valavanidis, 2006). Незважаючи на те, що різні токсиканти можуть викликати схожу відповідь антиоксидантної системи, реєстрація показників окиснювального стресу, а також активності антиоксидантної системи є важливим доповненням до вивчення дії екотоксикантів на живі організми (Dos Santos, 2018). ...
Anthropogenic pollution of natural waters by pesticides became a global prob-lem with the beginning of their widespread use in the 1960s of the twentieth century. The most studied cells’ response to the pesticides is an increase in the content of reactive oxygen species (ROS) and, as a consequence, the development of oxidative stress. The proliferative activity of cells is regulated by various factors and largely depends on the expression of proteins involved in the DNA replication. Among such proteins are replicative proteins A (RPA), which binds sin-gle-stranded DNA (ssDNA) of eukaryotes. Disruption of RPA-associated cellular activity initi-ates genomic imbalance, so the expression and content of RPA1 serves as a marker not only of the cell cycle stability, but also the relevant response to DNA damage caused by ecotoxicants.The aim of our study is to determine the oxidative stress development and the RPA1 protein state as a response of rainbow trout’s brain cells to the influence of sublethal concentrations of the lambda-cyhalothrin. A model experiment on the neurotoxicity of lambda-cyhalothrin was performed on the rainbow trout (Oncorhynchus mykiss) under experimental conditions in a la-boratory. The obtained results indicate that sublethal concentrations of lambda-cyhalothrin in the water induce a significant increase in ROS generation in the rainbow trout’s brain. It was found that the content of ROS in the fish brain in all experimental groups depends on both the lambda-cyhalothrin concentration and the duration of exposure to the pesticide. Oxidative stress under the influence of lambda-cyhalothrin increases the content of stress-regulating protein RPA1 in the fish brain, inhibits transcriptional activity and limits the DNA breaks repair, which, in turn, can lead to genomic instability and activation of apoptosis. This indicates a high sensitivity of the mechanism of DNA replication to the neurotoxic effect of synthetic pyrethroids. Thus, the detected increased expression of RPA1 protein may be an adequate biomarker of cellular response to the toxic effects of synthetic pyrethroids in the brain of fish. Further studies of the effect of pyrethroids on key proteins of the aquatic organisms’ reaction to intoxication are of great importance for the determi-nation of indicative biomarkers of pesticide pollution and its potential risk.
... The degree of oxidative stress in the body of the organism could be indirectly reflected by the changes in these biomarkers, which can be used as an indicator of environmental pollution (Gerber et al. 2018). Domestic and international researches of sublethal pollutant stress on aquatic animals have focused on single heavy metals (Rihab et al. 2017), organic pollutants (Xiu et al. 2014), the combination of heavy metals (Pillet et al. 2019;Vellingera et al. 2013), the combination of organic pollutants (Zhang et al., 2012), and the combination of heavy metals and organic pollutants (Maria et al. 2013;Chen et al. 2018). However, there are only a few reports on the combination of heavy metal and organic pollutant stress. ...
Both polychlorinated biphenyls (PCBs) and cadmium (Cd) can be frequently found in marine ecosystems and have detrimental effects on marine organisms, especially on filter-feeding marine mussels. Although biological responses to single metal or PCB exposure in mussels have been well-studied, information about oxidative stress is still limited, especially in different tissues in mussels. Considering the variety of contaminants existing in the actual marine environment, the exposures of the marine mussel Mytilus coruscus to Cd2+ alone (0.194, 0.388, and 0.775 mg/L) and Aroclor 1254 alone (0.005, 0.010, and 0.050 mg/L) and the co-exposures of the marine mussel Mytilus coruscus to Cd2+ (0.194 and 0.388 mg/L) and Aroclor 1254 (0.005 and 0.010 mg/L) were tested in an 8-day exposure experiment followed by a 7-day acclimation experiment. The alterations in superoxide dismutase, catalase, glutathione peroxidase, and malondialdehyde levels in the gills of the mussels were assessed. The effects of the depressed antioxidant were induced by the exposures of Cd2+ and Aroclor 1254 and their co-exposures. All exposures resulted in an initial increase and then a reduction in antioxidant enzyme activities. The range and rate of the antioxidant enzyme activities were positively correlated with stress duration and the concentration of the stress material. The effect of combined stress was stronger than that of each individual stressor. The valuable information for future investigations of stress response mechanisms, especially in relation to tissue functions in marine organisms, has been provided by the results and experimental model. The study of combined pollution effects has more scientific significance for marine pollution monitoring.
This study investigates the potential of Escherichia coli and Aeromonas hydrophila as bioremediation agents for removing copper (Cu) and zinc (Zn) from contaminated water. Although Cu and Zn are necessary in trace levels, excessive amounts can be harmful and can linger in aquatic environments, endangering the food chain. Bioremediation using microorganisms offers an alternative method for mitigating heavy metal pollution. In this study, 126 tilapia fish (Oreochromis mossambicus) were exposed to CuSO4 and ZnCl2 for 15 days, followed by treatment with E. coli and A. hydrophila. Atomic absorption spectrometry (AAS) revealed that both bacterial treatments reduced copper and zinc accumulation in fish organs, though they did not fully heal external lesions. Histopathological analysis showed significant reductions in melanomacrophage centers (MMC), cell necrosis, cell dissociation, and vacuolization in fish liver tissue after bacterial treatment, particularly at concentrations of 2.5 mg.L⁻¹ and 5 mg.L⁻¹ for CuSO4 and 7.5 mg.L⁻¹ for ZnCl2. These findings suggest that E. coli and A. hydrophila have the potential to be developed as effective bioremediation agents for CuSO4 and ZnCl2 pollution in aquatic environments.
Graphical abstract
The primary objective of this study is to investigate the effects of Zinc toxicity when fishes are exposed in a controlled environment using two-dimensional correlation spectral analysis. This analysis is superior as subtle spectral changes induced by an external perturbation are not readily noticeable in conventional one-dimensional spectra. A higher decrease in protein and carbohydrates and a lesser decrease in lipids (20%–28%) against the control sample occurs due to Zn toxicity. The lowest value of the bioconcentration factor for Zn shows carbohydrate utilization, which was resolved well in our two-dimensional spectral analysis. Synchronous maps show the predominant changes in protein (1668 cm−1), phospholipids (1771 cm−1), and fatty acids (1707 cm−1). The asynchronous two-dimensional spectra show alteration in phosphodiester stretching of glycogen followed by Amide I. In addition, we explored the importance of principal component analysis coupled two-dimensional spectra analysis which shows weak amide III protein changes. The hetero asynchronous shows well-resolved cross peaks of +(1047, 1389), resulting in improved resolution. The study helps in understanding biomolecular changes of muscle tissues of G. affinis under Zn toxicity using two-dimensional correlation infrared spectral analysis.
Green evolutionary products such as biologically fabricated nanoparticles (NPs) pose a hazard to aquatic creatures. Herein, biogenic silver nanoparticles (AgNPs) were synthesized by the reaction between ionic silver (AgNO3) and aqueous onion peel extract (Allium cepa L). The synthesized biogenic AgNPs were characterized with UV–Visible spectrophotometer, XRD, FT-IR, and TEM with EDS analysis; then, their toxicity was assessed on common carp fish (Cyprinus carpio) using biomarkers of haematological alterations, oxidative stress, histological changes, differential gene expression patterns, and bioaccumulation. The 96 h lethal toxicity was analysed with various concentrations (2, 4, 6, 8, and 10 mg/l) of biogenic AgNPs. Based on 96 h LC50, sublethal concentrations (1/15th, 1/10th, and 1/5th) were given to C. carpio for 28 days. At the end of experiment, the bioaccumulations of Ag content were accumulated mainly in the gills, followed by the liver and muscle. At an interval of 7 days, the haematological alterations showed significance (p < 0.05) and elevation of antioxidant defence mechanism reveals the toxicity of biogenic synthesized AgNPs. Adverse effects on oxidative stress were probably related to the histopathological damage of its vital organs like gill, liver, and muscle. Finally, the fish treated with biogenic synthesized AgNPs were significantly (p < 0.05) downregulates the oxidative stress genes such as Cu–Zn SOD, CAT, GPx1a, GST-α, CYP1A, and Nrf-2 expression patterns. The present study provides evidence of biogenic synthesized AgNPs influence on the aquatic life through induction of oxidative stress.
There is a growing concern nowadays over the exposure of nanomaterials and their effects in aquatic life. In spite of reporting the changes in physiology, reproduction and behaviour in fish by different nanoparticles, the molecular events underlying in the aquatic bodies due to the toxicity of zinc oxide nanoparticles (ZnO NPs) are mainly unexplored. Therefore, the present study carried out an ex vivo exposure of ZnO NPs at various concentrations (0.382, 0.573 and 1.146 mg L⁻¹) in Cyprinus carpio fish to investigate the potential adverse effects. The results revealed that ZnO NPs exposure altered the haematological parameter and induces the reactive oxygen species (ROS) and leads to elevation of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidise (GPx), glutathione S-transferase (GST) and reduced glutathione (GSH) activity in C. carpio. Furthermore, histopathological analysis exhibited that the ZnO NPs caused lamellar fusion, aneurism, cytoplasmic vacuolation, nuclear alteration, necrotic muscle fiber and pyknotic nuclei in the gills, liver and muscles of C. carpio. ZnO NPs exposure significantly up-regulated the overlapping expressions of SOD1, CAT, GPx1a, GST-α, CYP1A, and Nrf-2 genes. A higher level of Zn bioaccumulation was observed in the following order: gill (35.03 ± 2.500 μg g⁻¹), liver (5.335 ± 0.73 μg g⁻¹) and muscle (2.303 ± 0.2 μg g⁻¹) at 1.146 mg L⁻¹ exposure of ZnO NPs. Hence, the current study indicated that the biogenic ZnO NPs generate toxicity in fishes by modifying the antioxidant defense mechanisms, histomorphology, and oxidative stress encoding genes.
Исследование проводили с использованием в качестве тест-объекта радужной форели, подвергнутой воздействию неорганической ртути в концентрациях 25% ЛК50 и 50% ЛК50 в течение 2 и 7 дней. Полученные результаты показали, что снижение уровня белка р53 сопровождается повышением уровня протеина RPA1 в головном мозге рыб, обитающих в среде с сублетальными концентрациями препарата. Повышенная экспрессия RPA1 может быть одним из критических факторов клеточной адаптации при стрессе в головном мозге радужной форели, вызванном неорганической ртутью. Полученные результаты позволяют предположить, что Hgиндуцированная генерация АФК связана с модуляцией экспрессии как р53, так и RPA1 при клеточном ответе на цитотоксическое действие ртути. Снижение содержания протеина р53 и повышение уровня белка RPA1 в тканях головного мозга рыб под воздействием неорганической ртути установлено впервые. Выявленная регуляция белка RPA1 может служить частью клеточного ответа на разрывы ДНК, вызванные ионами неорганической ртути.
Lead (Pb) is a known nephrotoxicant that causes damage to proximal tubular cells. Autophagy has an important protective role in various renal injuries, but the role of autophagy in Pb-elicited nephrotoxicity remains largely unknown. In this study, Pb promoted the accumulation of autophagosomes in primary rat proximal tubular (rPT) cells, and subsequent findings revealed that this autophagosome accumulation was caused by the inhibition of autophagic flux. Moreover, Pb exposure did not affect the autophagosome–lysosome fusion in rPT cells. Next, we found that Pb caused lysosomal alkalinization, may be through suppression of two V-ATPase subunits. Simultaneously, Pb inhibited lysosomal degradation capacity by affecting the maturation of cathepsin B (CTSB) and cathepsin D (CTSD). Furthermore, translocation of CTSB and CTSD from lysosome to cytoplasm was observed in this study, suggesting that lysosomal membrane permeabilization (LMP) occurred in Pb-exposed rPT cells. Meanwhile, Pb-induced caspase-3 activation and apoptosis were significantly but not completely inhibited by CTSB inhibitor (CA 074) and CTSD inhibitor (pepstatin A), respectively, demonstrating that LMP-induced lysosomal enzyme release was involved in Pb-induced apoptosis in rPT cells. In conclusion, Pb-mediated autophagy blockade in rPT cells is attributed to the impairment of lysosomal function. Both inhibition of autophagic flux and LMP-mediated apoptosis contribute to Pb-induced nephrotoxicity in rPT cells.
Metal uptake by biota due to elevated environmental concentrations elicits oxidative stress and could lead to pathological outcomes. The relationship between the histopathological profile of hepatopancreas and gills and altered biochemical features (antioxidant enzymes i.e. GSH, GPx, CAT, SOD, lipid peroxidation (MDA) and serum protein) in the blue crab, Callinectes amnicola from contaminated parts of the Lagos Lagoon was investigated. Monthly crab, sediment and surface water samples were taken from effluent receiving areas of the Lagos lagoon i.e. Makoko, Okobaba, Iddo, Ikoyi and Mid-lagoon (control site) over an 18-month period and analyzed for metal levels (Pb, Cd, Zn and Cu). Significantly higher levels of GPx and lower levels of Pb, Zn and Cu was recorded in gills and hepatopancreas of crabs from the mid-lagoon compared to crabs from other sites. Reaction patterns of gills across the different sites of the lagoon included regressive (ranging from epithelial lifting, disruption of pilaster cells, detached cuticle to focal necrosis) and circulatory disruptions (oedema); increased activity of GSH and GPx in gills were positively correlated with lesions of lower importance factor. Reaction patterns in hepatopancreas were more regressive including vacuolation/ infiltration of fatty lobules, necrosis, granuloma, disintegrated lumen, atrophied tubules and loss of lobular hepatocyte structure; increased activity of GSH, GPx and CAT were positively correlated with lesions of low importance factor in the hepatopancreas. Findings show that lesions in both gills and hepatopancreas of the blue crab could be associated with uptake of metals, depleted antioxidant activity and incidence of LPO in tissue.
This study was carried out to determine the median lethal concentrations (LC50) of Zinc nanoparticles (ZnNPs) on Oreochromis niloticus and Tilapia zillii.The biochemical and molecular potential effect of ZnNPs (500μgL-1 and 2000μgL-1) on antioxidant system in the brain tissue of O. niloticus and T. zillii were invetigated. Four hundred fish were used for acute and sub-acute study. The ZnNPs LC50 was investigated in O. niloticus and T. zillii. The effect of 500 and 2000μgL-1 ZnNPs on brain antioxidant of O. niloticus and T. zillii was investigated. The result indicated that 69h LC50 was 5.5±0.6 and 5.6±0.4 for O. nilotica and T. zillii respectively. Fish exposed to 500 μgL-1 ZnNPs showed a significant increase of reduced glutathione (GSH), total glutathione (tGSH) levels, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activity and gene expression. On the contrary, malondialdehyde (MDA) levels significantly decreased. Meanwhile, Fish exposed to 2000μgL-1 ZnNPs showed a significant decrease of GSH, tGSH levels, SOD, CAT, GR, GPx and GSTactivity and gene expression. On the contrary, MDA levels significantly increased. It concluded that, the 96 h LC50 of ZnNPs was 5.5±0.6 and 5.6±0.4 for O. nilotica and T. zillii respectively. ZnNPs in exposure concentration of 2000μg/L induced a deleterious effect on brain antioxidant system of O. nilotica and T. zillii. In contrast, ZnNPs in exposure concentration of 500μgL-1 produced an inductive effect on brain antioxidant system of O. nilotica and T. zillii.
Nile tilapia; Oreochromis niloticus was used as a bio-indicator to evaluate the impact of zinc metal. The potential impacts of zinc nanoparticles (Zn NPs) on aquatic ecosystems have attracted special attention due to their unique properties. The present investigation was designed to evaluate and compare between the possible ecotoxicological effects of zinc bulk particles (BPs) and Zn NPs on O. niloticus. LC50/96h of Zn (BPs & NPs) were (1.36 & 0.18g/l), respectively. The concentration equivalent to 1/2 LC50/96h of Zn BPs (0.68g/l) and Zn NPs (0.09g/l) was selected for 7, 14 & 28days. Exposure of the studied fish to 1/2 LC50/96h of Zn BPs & Zn NPs elicited a significant decrease in total lipids, total protein and globulin contents, coinciding with an enhancement in serum glucose, albumin, creatinine and uric acid concentrations, as well as activities of liver enzymes named AST, ALT, and ALP after the experimental periods. Moreover, Zn NPs significantly induced an increase in liver and gills glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD) activities and malondialdehyde (MDA) levels and with concomitant decreases in the liver and gill (GSH) level in the studied fish. The adverse effects of NPs were observed to be time dependent with increasing extent during the studied time intervals, while fish groups treated with BPs showed more or less time dependent effects. In conclusion, Zn NPs have stronger toxic impacts than that of the Zn BPs, and increased with prolonged exposure time.
The contamination of aquatic systems with heavy metals is affecting the fish population and hence results in a decline of productivity rate. River Kabul is a transcountry river originating at Paghman province in Afghanistan and inters in Khyber Pakhtunkhwa province of Pakistan and it is the major source of irrigation and more than 54 fish species have been reported in the river. Present study aimed at the estimation of heavy metals load in the fish living in River Kabul. Heavy metals including chromium, nickel, copper, zinc, cadmium, and lead were determined through atomic absorption spectrophotometer after tissue digestion by adopting standard procedures. Concentrations of these metals were recorded in muscles and liver of five native fish species, namely,
Wallago attu
,
Aorichthys seenghala
,
Cyprinus carpio
,
Labeo dyocheilus
, and
Ompok bimaculatus
. The concentrations of chromium, nickel, copper, zinc, and lead were higher in both of the tissues, whereas the concentration of cadmium was comparatively low. However, the concentration of metals was exceeding the RDA (Recommended Dietary Allowance of USA) limits. Hence, continuous fish consumption may create health problems for the consumers. The results of the present study are alarming and suggest implementing environmental laws and initiation of a biomonitoring program of the river.
Publisher Summary Glutathione reductase is a flavoprotein catalyzing the NADPH-dependent reduction of glutathione disulfide (GSSG) to glutathione (GSH). The reaction is essential for the maintenance of glutathione levels. Glutathione has a major role as a reductant in oxidation–reduction processes, and serves in detoxication and several other cellular functions of great importance. A purification method of this enzyme from calf liver and rat liver is described in this chapter. Similar methods are used for the purification of the enzyme from yeast, porcine, and human erythrocytes. All the steps are carried out at about 5 ° . The purification method from calf liver consists of various steps including preparation of cytosol fraction, chromatography on DEAE-sephadex, precipitation with ammonium sulfate, and chromatography on hydroxyapatite. The purification of glutathione reductase from rat liver is usually combined with the preparation of glutathione transferases, thioltransferase, and glyoxalase I.
This review summarizes the current knowledge on the contribution of metals to the development of oxidative stress in fish. Metals are important inducers of oxidative stress in aquatic organisms, promoting formation of reactive oxygen species through two mechanisms. Redox active metals generate reactive oxygen species through redox cycling, while metals without redox potential impair antioxidant defences, especially that of thiol-containing antioxidants and enzymes. Elevated levels of reactive oxygen species lead to oxidative damage including lipid peroxidation, protein and DNA oxidation, and enzyme inactivation. Antioxidant defences include the enzyme system and low molecular weight antioxidants. Metal-binding proteins, such as ferritin, ceruloplasmin and metallothioneins, have special functions in the detoxification of toxic metals and also play a role in the metabolism and homeostasis of essential metals. Recent studies of metallothioneins as biomarkers indicate that quantitative analysis of mRNA expression of metallothionein genes can be appropriate in cases with elevated levels of metals and no evidence of oxidative damage in fish tissue. Components of the antioxidant defence are used as biochemical markers of oxidative stress. These markers may be manifested differently in the field than in results found in laboratory studies. A complex approach should be taken in field studies of metal contamination of the aquatic environment.
The glutathione S-transferase (GST) supergene family comprises gene families that encode isoenzymes that are widely expressed in mammalian tissue cytosols and membranes. Both cytosolic (particularly the isoenzymes encoded by the alpha, mu and theta gene families) and microsomal GST catalyse the conjugation of reduced glutathione (GSH) with a wide variety of electrophiles which include known carcinogens as well as various compounds that are products of oxidative stress including oxidised DNA and lipid. Indeed, several lines of evidence suggest certain of these isoenzymes play a pivotal role in protecting cells from the consequences of such stress. An assessment of the importance of these GST in humans is presently difficult however, because the number of alpha and theta class genes is not known and, the catalytic preferences of even identified isoforms is not always clear.
Links among metal exposure, metal accumulation, and metal-induced effects were explored in indigenous yellow perch (Perca flavescens) collected from eight lakes located along a metal concentration gradient in two mining regions. Fish exposure to Cd, Cu, Ni, and Zn was estimated on the basis of calculated free metal ion concentrations in lake waters. Hepatic metal concentrations were determined and various markers of oxidative stress were measured to assess metal effects in liver cells. Accumulated metals were not consistently related to ambient free metal ion concentrations, possibly because of unaccounted for fluctuations in lake water metal concentrations. Accumulated metals were associated with limited oxidative stress in perch livers, as evidenced by reductions in glutathione concentrations and glutathione reductase activities. However, this stress appeared to be minor, since increasing hepatic Cu concentrations were associated with reduced lipid peroxidation, a response opposite to that predicted from basic principles. Our results suggest that oxidative stress will not have direct repercussions on the health of the perch at the individual level. We speculate that the observed increase in metallothionein concentrations with increasing accumulated metals might afford protection against reactive oxygen species.
Among the cellular molecules, lipids that contain unsaturated fatty acids with more than one double bond are particularly susceptible to action of free radicals. The resulting reaction, known as lipid peroxidation, disrupts biological membranes and is thereby highly deleterious to their structure and function. Lipid peroxidation is being studied extensively in relation to disease, modulation by antioxidants and other contexts. A large number of by-products are formed during this process. These can be measured by different assays. The most common method used is the estimation of aldehydic products by their ability to react with thiobarbituric acid (TBA) that yield 'thiobarbituric acid reactive substances' (TBARS), which can be easily measured by spectrophotometry. Though this assay is sensitive and widely used, it is not specific and TBA reacts with a number of components present in biological samples. Hence caution should be used while employing this method. Wherever possible this assay should be combined with other assays for lipid peroxidation. Such methods are measurement of conjugated dienes, lipid hydroperoxides, individual aldehydes, exhaled gases like pentane, isoprostanes, etc. The modern methods also involve newer techniques involving HPLC, spectrofluorimetry, mass spectrometry, chemiluminescence etc. These and other modern methods are more specific and can be applied to measure lipid peroxidation. There are certain restraints, in terms of high cost and certain artifacts, and these should be considered while selecting the method for estimation. This review analyses the merits and demerits of various assays to measure lipid peroxidation.
Several components of branchial copper uptake were identified in juvenile freshwater rainbow trout (Oncorhynchus mykiss) using 64Cu. On the basis of competitive interactions between sodium and copper uptake,inhibition of copper uptake by a proton pump inhibitor (bafilomycin A1, 2μmol l-1) and a Na+ channel blocker (phenamil, 100μmol l-1), it appears that a proportion of the branchial copper uptake occurs via an apical Na+ channel. This sodium-sensitive copper uptake demonstrates saturation kinetics, with a Km of 7.1 nmol l-1 and a Jmax of 21.2 pmol g-1 h-1, and is characterized by an IC50 of 104 μmol l-1 sodium. On the basis of residual copper uptake in the presence of high sodium concentrations (20 mmol l-1) and differential inhibition of sodium and copper uptake by phenamil (100 μmol l-1), a sodium-insensitive component of copper uptake is also present in trout gills. It demonstrates saturation kinetics with a comparably low Km (9.6 nmol l-1) but a lower maximum transport capacity (Jmax=3.5 pmol g-1 h-1)than the sodium-insensitive system. Sodium uptake exhibits saturation kinetics with a Km of 69 μmol l-1. Copper reduced branchial sodium transport affinity but increased the maximal sodium transport capacity.
Due to the excessive pursuit of crop yields and the abuse of herbicides, water pollution caused by atrazine (ATR) has become one of the most severe environmental issues threatening the health of fish and aquatic animals. However, no detailed report has been conducted on the mechanisms of ATR immunotoxicity in fish neutrophils. To investigate these mechanism, we exposed peripheral blood neutrophils to 25 μg/ml atrazine for 1, 2, and 3 h. The results showed that ATR induced the mRNA expression of CYPs enzymes (CYP1A1, CYP1B1, CYP1C and CYP3A138), which increased the ROS levels, and inhibited the SOD and CAT activities, GSH content and spurred the accumulation of MDA. Additionally, a significant decline in the OXPHOS, Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase activities of mitochondria was observed after ATR exposure. Concurrently, ATR activated Caspase3 and induced apoptosis by changing the expression of mitochondrial pathway factors (Bcl-2, BAX, Caspase9) and death receptor pathway major genes (TNF-α TNFR, Fas, FasL, and Caspase8). The results reported here indicate that the oxidative stress and mitochondrial damage caused by ATR metabolism may play a crucial role in the apoptosis of carp neutrophils, and enrich the immunotoxicological mechanisms of ATR observed in fish.
Atrazine (ATR), a selective herbicide, is consistently used worldwide and has been confirmed to be harmful to the health of aquatic organisms. The release of neutrophil extracellular traps (NETs) is one of the newly discovered antimicrobial mechanisms. Although several immune functions have been analyzed under ATR exposure, the effect of ATR on NETs remains mainly unexplored. In the present study, we treated carp neutrophils using 5 μg/ml ATR and 5 μg/ml ATR combined with 100 nM rapamycin to elucidate the underlying mechanisms and to clarify the effect of ATR on phorbol myristate acetate (PMA)-induced NETs. The results of the morphological observation and quantitative analysis of extracellular DNA and myeloperoxidase (MPO) showed that NETs formation were significantly inhibited by ATR exposure. Moreover, we found that in the NETs process, ATR downregulated the expression of the anti-apoptosis gene B-cell lymphoma-2 (Bcl-2), increased the expression of the pro-apoptosis factors Bcl-2-Associated X (BAX), cysteinyl aspartate specific proteinases (Caspase3, 9), and anti-autophagy factor mammalian target of rapamycin (mTOR), decreased the expression of autophagy-related protein light chain 3B (LC3B) and glucose transport proteins (GLUT1, 4), disturbed the activities of phosphofructokinase (PFK), pyruvate kinase (PKM), and hexokinase (HK) and limited reactive oxygen species (ROS) levels, indicating that the reduced NETs release was a consequence of increased apoptosis and diminished ROS burst, autophagy and down-regulated glycolysis under ATR treatment. Meanwhile, rapamycin restored the inhibited autophagy and glycolysis and thus resisted the ATR-suppressed NETs. The present study perfects the mechanism theory of ATR immunotoxicity to fish and has a certain value for human health risk assessment.
The present study was conducted to determine the effects of waterborne copper exposure on the lipid metabolism and intestinal microbiota of juvenile common carp (Cyprinus carpio L.). Common carp were exposed to four waterborne copper (Cu) concentrations (0 (control), 0.07 (low), 0.14 (medium), and 0.28 (high) mg Cu/L) for 8 weeks. Exposure to a high concentration of Cu had a negative effect on growth indices (weight gain rate (WGR) and specific growth rate (SGR)). The biochemical indices measured in serum (low-density lipoprotein (LDL) and triglycerides (TGs)) were significantly affected by exposure to medium concentration levels of Cu. The mRNA levels of lipogenic enzymes (acetyl-CoA carboxylase 1 (ACC-1) and fatty acid synthase (FAS)) and sterol-regulator element-binding protein-1 (SREBP-1) in liver tissue and tight binding protein genes (ZO-1 and occludin) in intestinal epithelial tissue were significantly downregulated in the 0.14 and 0.28 mg/L Cu treatment groups, accompanied by upregulated mRNA levels of lipolysis enzymes (lipoprotein lipase (LPL) and carnitine palmitoyl transferase 1 (CPT-1)) in the liver. The data also showed that the composition of intestinal microbiota was changed following Cu exposure and could alter the α-diversity and β-diversity. The abundances of few putative short-chain fatty acid (SCFA)-producing bacteria, including Allobaculum, Blautia, Coprococcus, Faecalibacterium, Roseburia, and Ruminococcus, decreased significantly. More specifically, Roseburia sequences were positively associated with lipogenic enzymes, total protein (TP), and TGs and negatively associated with lipolysis enzymes. Other sequences related to probiotics (Lactobacillus, Bacillus and Akkermansia) were also found to decrease, accompanied by an increase in sequences related to pathogens (Pseudomonas and Acinetobacter). To the best of our knowledge, the present study provides the first evidence that waterborne, chronic Cu exposure can disturb the composition of intestinal microbiota related to lipid metabolism and immunity in freshwater fish, thereby increasing the risk of pathogen invasion.
The present study was performed to determine the effect of waterborne CdCl2exposure influencing lipid deposition and metabolism, oxidative stress and mitochondrial dysfunction, and explore the underlying molecular mechanism of cadmium (Cd)-induced disorder of hepatic lipid metabolism in fish. To this end, adult zebrafish were exposed to three waterborne CdCl2concentrations (0(control), 5 and 25 μg Cd/l, respectively) for 30 days. Lipid accumulation, the activities of enzymes related to lipid metabolism and oxidative stress, as well as the expression level of genes involved in lipid metabolism and mitophagy were determined in the liver of zebrafish. Waterborne CdCl2exposure increased hepatic triglyceride (TG) and Cd accumulation, the activities of fatty acid synthase (FAS), 6-phosphogluconate dehydrogenase (6PGD), glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME), and the mRNA level of fatty acid synthase (fas), acetyl-CoA carboxylase alpha (acaca), glucose 6-phosphate dehydrogenase (g6pd) and malic enzyme (me), but reduced the mRNA level of carnitine palmitoyl transferase 1 (cpt1), hormone-sensitive lipase alpha (hsla), and adipose triacylglyceride lipase (atgl). The activities of superoxide dismutase (SOD), glutathoinine peroxidase (GPx) and cytochrome c oxidase (COX) and the ATP level were significantly reduced after CdCl2exposure. CdCl2exposure significantly increased the mRNA level of genes (microtubule-associated protein light chain 3 alpha (lc3a), PTEN-induced putative kinase 1 (pink1), NIP3-like protein X (nix) and PARKIN (parkin)) related to mitophagy. To elucidate the mechanism, reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) and the mitochondrial permeability transition (MPT) inhibitor cyclosporine A (CsA) were used to verify the role of ROS and mitochondrial dysfunction in Cd-induced disorder of lipid metabolism. NAC pretreatment reversed the Cd-induced up-regulation of TG accumulation and activities of lipogenic enzymes, and the Cd-induced down-regulation of mRNA levels of lipolytic genes. Meanwhile, NAC pretreatment also blocked the mitochondrial membrane potential (MMP) collapse and decreased the ATP level, suggesting that ROS played a crucial role in regulating the Cd-induced mitochondrial dysfunction. Taken together, our findings, for the first time, highlight the importance of the oxidative stress and mitochondrial dysfunction in Cd-induced disorder of hepatic lipid metabolism, which proposed a novel mechanism for elucidating metal element exposure inducing the disorder of lipid metabolism in vertebrates.
The freshwater fish Galaxias maculatus (inanga) is a widespread Southern hemisphere species, but despite its habitation of lowland near-coastal waters with a high potential for cadmium contamination, nothing is known regarding its sensitivity to this toxic trace metal. Acute (96h) exposures were therefore performed to determine sublethal responses of inanga to waterborne cadmium at a regulatory trigger value (nominally 0.2μgL(-1); measured 1μgL(-1)), an environmental level (measured at 2.5μgL(-1)), and an effect level (measured at 10μgL(-1)). Whole body (tissue remaining following excision of kidney and liver) cadmium burden remained constant up until an exposure concentration of 10μgL(-1), at which point cadmium concentration increased significantly. A transient effect of cadmium on metabolic rate was observed, with an impaired oxygen consumption noted at 2.5, but not 1 or 10, μg L(-1). Cadmium did not impair influx rates of either sodium or calcium, and no effects of cadmium on oxidative stress parameters (catalase activity, lipid peroxidation) were noted in the kidney. However, at cadmium concentrations of 2.5 and 10μgL(-1), lipid peroxidation in the liver increased, concomitant with a decline in hepatic catalase activity. These data indicate that there are significant differences in the mechanisms of cadmium toxicity in inanga, relative to better-studied Northern hemisphere species, especially with respect to ionoregulatory impacts. However, effects were induced at cadmium concentrations unlikely to be encountered in any but the most highly contaminated waterways, and thus our data suggest that current trigger values for cadmium concentrations in Australian and New Zealand waters are likely to be protective of inanga.
Cadmium (Cd) is an increasingly important environmental pollutant which causes irreversible toxicity to fish. To understand how Cd impacts the immune response and oxidative stress in common carp, we performed transcriptomic profiles for head kidney, the immune organ of common carp which were underwent Cd exposure. Totally there are 42,489,124 and 48,562,526 high quality clean reads obtained from the Cd exposure groups, and 44,677,578 and 44,106,696 clean reads from the control groups. Among them, 308 genes were differently expressed, including 101 upregulated and 207 down-regulated genes. The identified genes were enriched using databases of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Antioxidant systems and immune function genes and pathways were identified and validated by quantitative real-time RT-PCR. Our results showed that Cd exposure leads to oxidative stress and immunosuppression in head kidney of common carp. These results provide new insights for unveiling the biological effects of Cd in common carp.
Mercury is one of the most toxic metals in aquatic systems since it is able to induce neurobehavioral disorders as well as renal and gastrointestinal tract damage. The common carp Cyprinus carpio is an important species from both an ecological and economic viewpoint as it is consumed in many countries, the top producers being Mexico, China, India and Japan. The present study aimed to evaluate the relation between Hg-induced oxidative stress and genotoxicity in diverse tissues of C. carpio. Specimens were exposed to 0.01mgHg/L (the maximum permissible limit for aquatic life protection), and lipid peroxidation, protein carbonyl content and the activity of antioxidant enzymes were evaluated at 96h. Micronuclei frequency and DNA damage by comet assay were determined at 12, 24, 48, 72 and 96h. Hg induced oxidative stress and genotoxicity on exposed fish, since inhibition of antioxidant enzymes activity and increases in lipid peroxidation, DNA damage and micronuclei frequency occurred. Blood, gill and liver were more susceptible to oxidative stress, while blood were more sensitive to genotoxicity. In conclusion, Hg at concentrations equal to the maximum permissible limit for aquatic life protection induced oxidative stress and genotoxicity on C. carpio, and these two effects prove to be correlated.
Heavy metals are frequently encountered as mixtures of essential and non-essential elements. Therefore, evaluation of their toxic effects individually does not offer a realistic estimate of their impact on biological processes. We studied effects of exposure to mixtures of essential and toxic metals (Cr, Cd and Pb) on biochemical, immunotoxicity level and morphological characteristics of the various tissues of a biomarker freshwater fish common carp using environmentally relevant concentrations. Fish were exposed to metal mixture through tank water for 7, 15 and 30 days, under controlled laboratory conditions. Tissue accumulation of the metals was measured using Atomic Absorption Spectrophotometric techniques. Chromium, cadmium and lead accumulation in muscle, gills, liver, kidney and intestine, tissue of common carp exposed to mixture metals for 30 days increased significant compared with control group (p < 0.001). However, the activity of antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD) levels was significant altered in various tissues of exposed fish. Besides, the lipid peroxidation (LPO) was significant (p < 0.001) increased. Moreover, the tumor necrosis factor - α (TNF-α), interleukin (IL-6), and interferon-γ (IFN-γ) contents in tissues of muscle, gills, liver, kidney and intestine were increased significant compared with control fish (p < 0.001). In addition, microscopic examination of the main alterations in general morphology of fish gills included spiking and fusion of secondary lamellae, formation of club-shaped filaments epithelium in the interlamellar regions and hepatocytes showed damage of central vein and rupture of irregular hepatic plate with more number of vacuoles in the fish exposed to metal mixture for a longer duration (30 days). These results of this study clearly demonstrate that concentration individual and mixtures of metals in aquatic systems will greatly influence the cytokine alterations may result in an immune suppression or excessive activation in the treated common carp as well as may cause immune dysfunction or reduced immunity. In conclusion, toxicity of multiple metal mixtures of Cr, Cd and Pb has antioxidant and immunotoxic effects on C. carpio.
A comprehensive approach to chemical accumulation and biological effects of short-term Cu exposure in juveniles of European seabass (Dicentrarchus labrax) has been achieved. Fish were exposed to 0.01–10 mg L− 1 nominal Cu concentrations for 24–96 h. Metal concentrations in water and gills, liver, muscle and brain tissues were studied along with oxidative stress biomarkers (superoxide dismutase, catalase, glutathione peroxidase, lipid peroxidation). Induction of oxidative damage was observed in all the organs with differential antioxidant responses; gills appearing as the most sensitive from low environmentally water Cu concentrations as 0.01 mg L− 1. Histopathological alterations were also observed in liver and gills, even without a significant Cu accumulation.
The results show that the combination of oxidative stress parameters, particularly lipid peroxidation and glutathione peroxidase activities, and histopathological alterations provide a good model fish and reliable early biomarkers for monitoring Cu pollution in seawater and might call for the protection agencies to revise the Cu environmental standards.
To our knowledge, this is the first study to evaluate captopril-induced oxidative stress in fish, and specifically in the common carp Cyprinus carpio. At present, very few studies in the international literature evaluate the sublethal effects of captopril on aquatic organisms such as fish, and available ones focus on determination of median lethal concentration in crustaceans and algae. Also, studies evaluating these effects do not make reference to the mechanism of action of this pharmaceutical or its toxicokinetics. This limits our knowledge of the characterization of the sublethal effects of this medication and of its potential ecological impact. The present study aimed to evaluate the sublethal effects induced by three different concentrations of captopril, on C. carpio), by determination of activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), as well as indicators of cellular oxidation: hydroperoxide content (HPC), lipid peroxidation (LPX) and protein carbonyl content (PCC). Specimens were exposed for 12, 24, 48, 72 and 96 h to three different captopril concentrations: 1 μg L− 1, 1 mg L− 1 and 100 mg L− 1 (the first one has been detected environmentally, the other two have been associated with diverse toxic effects in aquatic species), and brain, gill, liver, kidney and blood samples were evaluated. Significant increases in HPC and LPX were observed mainly in kidney and gill, while PCC also increased in brain. Modifications were found in the activity of SOD (mostly in kidney, brain and blood), CAT (all organs) and GPx (kidney and gill). In conclusion, captopril induces oxidative stress in C. carpio.
In this study, we aimed to identify the toxic effects of chlorpyrifos exposure on the tissues of common carp. For this purpose, we evaluated histopathological changes in the brain, gills, liver, kidney, testis, and ovaries after 21 days of chlorpyrifos exposure. Activation of 8-OHdG, cleaved caspase-3, and iNOS were assesed by immunofluorescence assay in chlorpyrifos-exposed brain and liver tissue. Additionally, we measured the expression levels of caspase-3, caspase-8, iNOS, MT1, CYP1A, and CYP3A genes in chlorpyrifos-exposed brain tissue, as well as the expression levels of FSH and LH genes in chlorpyrifos-exposed ovaries, using qRT-PCR. We observed severe histopathological lesions, including inflammation, degeneration, necrosis, and hemorrhage, in the evaluated tissues of common carp after both high and low levels of exposure to chlorpyrifos. We detected strong and diffuse signs of immunofluorescence reaction for 8-OHdG, iNOS, and cleaved caspase-3 in the chlorpyrifos-exposed brain and liver tissues. Furthermore, we found that chlorpyrifos exposure significantly upregulated the expressions of caspase-3, caspase-8, iNOS, and MT1, and also moderately upregulated CYP1A and CYP3A in the brain tissue of exposed carp. We also noted downregulation of FSH and LH gene expressions in chlorpyrifos-exposed ovary tissues. Based on our results, chlorpyrifos toxication caused crucial histopathological lesions in vital organs, induced oxidative stress, inflammation, and apoptosis in liver and brain tissues, and triggered reproductive sterility in common carp. Therefore, we can propose that chlorpyrifos toxication is highly dangerous to the health of common carp. Moreover, chlorpyrifos pollution in the water could threaten the common carp population. Use of chlorpyrifos should be restricted, and aquatic systems should be monitored for chlorpyrifos pollution.
Native mussels Mytilus galloprovincialis are used as bioindicator organisms to assess the concentration levels and toxic effects of persistent chemicals, polychlorobiphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and heavy metals using biomarker responses, such as catalase (CAT), glutathione s-transferase (GST), and condition indices, for the Algerian coast. The results show that mussels of Oran Harbour are extremely polluted by PCBs and PAHs, i.e., 97.6 and 2892.1μg/kg d.w., respectively. Other sites present low levels of pollution. Furthermore, high concentrations of zinc, lead and cadmium are found in mussels from fishing, agricultural and estuarine sites, respectively, while low concentrations of copper are found in all of the sites studied. CAT activity is negatively correlated with Cd and Cu, and Zn is positively correlated with GST and CAT. Site classification tools reveal the potential toxicity of coastal areas exposed to anthropogenic pressure and a gradient of toxicity along the Algerian west coast.
The present study was performed to validate the suitability of using gene expression in zebra mussels, Dreissena polymorpha, for biomonitoring of freshwater environment. Mussels were collected in four French rivers (Meuse, Moselle, Oise and Vilaine) in spring and autumn. Relative gene expression of 9 candidate genes involved in cellular metabolic activities (Cytochrome-c-oxidase - cox, and ATP synthase - atp), detoxification process (Metallothionein - mt and Glutathion-S-Transferase - gst), oxidative stress (Catalase - cat, Superoxyde Dismutase - sod and Glutathion peroxidase - gpx) and digestive functions (Amylase - amy and Cellulase - ghf) were measured in digestive gland. Metal bioaccumulation in tissues and morphometric parameters were also analyzed to interpret molecular responses. All our results are consistent with different physiological reactions to environmental condition between zebra mussel populations. In spring, the levels of mt, sod, gpx, cat, atp, amy and ghf relative expression were significantly higher in mussels with the lowest metal bioaccumulation (the Meuse) compared to at least one of the other sites. In autumn, this higher expression levels in Meuse River were still observed for gpx, cat, atp and amy. This study has also pointed out different sources of variability in gene expression (individual size, season, trophic resources and origin of mussels) which are inevitable in natural fluctuant environment. This underlines the importance to take them into account in field study to propose a correct interpretation of biomarker responses.
Based on the same toxic level of 0.6% LC50 for 96-hour and the severe situation of water pollution, we compared effects of chronic Zn (180 μg L−1) and Cd exposures (30 μg L−1) on growth, survival, histology, ultrastructure, and oxidative stress in the liver of zebrafish for 5 weeks. Growth performance and survival rate remained relatively constant under Zn stress, but was reduced under Cd exposure. Cd exposure also induced severe pyknotic nuclei, evident ultrastructure damage, and considerable lipid inclusions in the hepatocytes. However, these phenomena were not pronounced under Zn exposure. The negative effects caused by Cd may be explained by an increase in hepatic oxidative damage, as reflected by the enhanced levels of lipid peroxidation (LPO) and protein carbonylation (PC). The reduced activity of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and catalase (CAT) may result in the enhanced hepatic oxidative damage, though the mRNA and protein levels of both genes increased and remained unchanged respectively. On the contrary, Zn up-regulated the levels of mRNA, protein and activity of Cu/Zn-SOD, which may contribute to the decreased LPO levels. Nonetheless, the sharply up-regulated mRNA levels of CAT did not induce an increase in the protein and activity levels of CAT under Zn stress. Furthermore, transcription factor NF-E2-related factor 2 (Nrf2) expression parelleled with its target genes, suggesting that Nrf2 is required for the protracted induction of antioxidant genes. In conclusion, our data demonstrated that essential and non-essential metals induced some differences in oxidative damage in fish. The differences were not caused by the transcriptional level of related genes but depended on post-transcriptional modifications.
Some investigations were made on the Satha canal water and health of dwelling fish Channa punctatus at Satha village, district Aligarh (U.P). Metal bioaccumulation and induction of biomarkers such as lipid peroxidation (LPO), superoxide dismutase (SOD), catalase (CAT), glutathione S transferase (GST), reduced glutathione (GSH), DNA damage and histopathology are potential indicators of stress in C. punctatus exposed to effluents. In canal water Cr, Mn, Fe and Ni concentrations were exceeding the permissible limits set by both Bureau of Indian standards (BIS) and WHO. Fe (74%) was highly bioavailable and accumulated in all organs (gill, liver, kidney, muscle and integument). The highest metal pollution index (MPI) value of 53 was observed in gills and the lowest 6 in liver tissue. SOD and LPO were significantly higher in all tissues, whereas CAT, GST and GSH levels declined significantly compared to fish from the reference site. Concomitant damage to DNA was observed with significantly higher mean tail length in the exposed fish gill cells (19 μm) and in liver (12.7 μm) compared to reference fish (5 and 4 μm respectively). Histopathology in gill and liver also show significant damage. Therefore, it can be concluded that the sugar mill effluent has the potential to cause oxidative stress, DNA damage and histopathology in C. punctatus. This canal is a prime source of water and fish food to the local residents of the area. Therefore, the consumers may suffer adverse health effects like that in indicator organism.
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
Real-time reverse transcription followed by polymerase chain reaction (RT–PCR) is the most suitable method for the detection
and quantification of mRNA. It offers high sensitivity, good reproducibility and a wide quantification range. Today, relative
expression is increasingly used, where the expression of a target gene is standardised by a non-regulated reference gene.
Several mathematical algorithms have been developed to compute an expression ratio, based on real-time PCR efficiency and
the crossing point deviation of an unknown sample versus a control. But all published equations and available models for the
calculation of relative expression ratio allow only for the determination of a single transcription difference between one
control and one sample. Therefore a new software tool was established, named REST© (relative expression software tool), which
compares two groups, with up to 16 data points in a sample and 16 in a control group, for reference and up to four target
genes. The mathematical model used is based on the PCR efficiencies and the mean crossing point deviation between the sample
and control group. Subsequently, the expression ratio results of the four investigated transcripts are tested for significance
by a randomisation test. Herein, development and application of REST© is explained and the usefulness of relative expression
in real-time PCR using REST© is discussed. The latest software version of REST© and examples for the correct use can be downloaded
at http://www.wzw.tum.de/gene-quantification/.
Cadmium and copper accumulations in gills of zebrafish were measured during a 48 h exposure to 0.025 mu M Cd-106 and 0.05 or 0.5 mu M Cu-65 as a single metal or their mixtures. The gill transcript levels of genes involved in the transport of Cu (CTR1 and ATP7A), Na (NHE-2), Ca (ECaC), divalent metals (DMT1), and Zn (ZIP8) were also compared between treatments at 24 and 48 h. Cd uptake was significantly suppressed in the presence of Cu, indicating interaction between Cu and Cd at uptake sites, but Cu uptake was unaffected by Cd. The decrease in Cd accumulation rates in the presence of Cu was associated with an increase in transcript abundance of ECaC at 24 h and DMT1 at 48 h and a decrease in Zip8 transcript levels, all known as routes for Cd uptake. Fish exposed to 0.5 mu M Cu-65 show an increase in gill ATP7a transcript abundance, suggesting that Cu is removed from the gill and is transferred to other organs for detoxification. A reduction in gill CTR1 transcript abundance was observed during the Cu-Cd exposure; this may be a regulatory mechanism to reduce Cu loading if Cu is entering the gills by other uptake routes, such as ECaC and DMT1.
Reduced growth occurred in yearlings and adults of the minnow Phoxinus phoxinus L. following exposure to zinc nitrate in fresh water over a 150-day period. Yearlings were more sensitive than adults and showed reduced growth at 0.13 ppm zinc (corresponding to 1/25 of the estimated 96-hr LC50). Suppressed growth was associated with reduced Tubifex consumption. /// У пескарей Phoxinus phoxinus L. наблюдалось замедление роста у годовалых и взрослых особей после воздействия нитрата цинка в пресной воде, в течение более 150 дней. Годовалые особи более чувствительны, чем взрослые. У них происходит замедление роста при 0,13 частей/млн цинка (соответствует 1/25 ранее установленной 96-часовой LC50). Подавление роста связано со снижением потребления Tubifex.
The present work evaluates the effects of various levels of dietary choline on antioxidant defenses and gene expressions of Nrf2 signaling molecule in spleen and head kidney of juvenile Jian carp (Cyprinus carpio var. Jian). Fish were fed with six different experimental diets containing graded levels of choline at 165 (choline-deficient control), 310, 607, 896, 1167 and 1820 mg kg(-1) diet for 65 days. At the end of the feeding trail, fish were challenged with Aeromonas hydrophila and mortalities were recorded over 17 days. Dietary choline significantly decreased malondialdehyde and protein carbonyl contents in spleen and head kidney. However, anti-superoxide anion and anti-hydroxyl radical activities in spleen and head kidney also decreased. Interestingly, activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) in spleen, GPx activity in head kidney, and glutathione contents in spleen and head kidney were decreased with increase of dietary choline levels up to a certain point, whereas, activities of SOD, GST and GR in head kidney showed no significantly differences among groups. Similarly, expression levels of CuZnSOD, MnSOD, CAT, GPx1a, GPx1b and GR gene in spleen and head kidney were significantly lower in group with choline level of 607 mg kg(-1) diet than those in the choline-deficient group. The relative gene expressions of Nrf2 in head kidney and Keap1a in spleen and head kidney were decreased with increasing of dietary choline up to a certain point. However, the relative gene expression of Nrf2 in spleen were not significantly affected by dietary choline. In conclusion, dietary choline decreased the oxidant damage and regulated the antioxidant system in immune organs of juvenile Jian carp.
Serotonin (5-hydroxytryptamine, 5-HT) and dopamine (DA) are two major monoamine neurotransmitters with a multitude of functions in the vertebrate brain. In fish, the 5-HT system has been shown to be sensitive to various forms of stress, but very few studies have examined the effects of toxic metals on these monoamine systems. Juvenile common carp were exposed to copper levels of 0.22, 0.34 and 0.84 μM during 1 week. In telencephalon, dose-dependent falls in 5-HT and DA levels were observed, with approximately 50% losses of these neurotransmitters at the highest copper concentration. Although less dramatic, falls were also seen in 5-HT and DA levels in hypothalamus and brain stem. No changes in 5-hydroxyindoleacetic acid (5-HIAA, the main 5-HT metabolite), AMP, ADP, ATP, adenylate energy charge or lactate levels were observed in brain. However, lactate levels in blood plasma increased with copper concentration. A significant copper accumulation only occurred in the liver, while no changes in brain or muscle were seen. It is concluded that copper exposure of common carp causes decreased brain 5-HT and DA levels, two neurotransmitters involved in, for example, feeding behaviour and locomotor control in fish. In fact, in telencephalon a fall in 5-HT levels was seen already at a copper concentration below that where food intake and movement were impaired.
Hexavalent chromium (Cr6+) is a common pollutant transient metal with high toxicity in the environment. The toxicological effects partly result from oxidative damage due to the production of excessive reactive oxygen species (ROS) in the reductive process of Cr6+. To explore the influence of ROS induced directly by Cr6+ on the oxidative stress generation and antioxidant system, the full length cDNAs of antioxidant-related genes cat, gpx1 and Cu/Zn-sod were successfully acquired from pengze crucian carp first and analyzed. Furthermore, the mRNA expression of the antioxidant genes encompassing catalase (cat), copper/zinc superoxide dismutase (Cu/Zn-sod) and glutathione peroxidase (gpx1), antioxidant enzyme activities of CAT, SOD, and GPx and total protein content were further studied in the gill, intestine and liver of pengze crucian carp (Carassius auratus var. Pengze) juveniles upon acute exposure to Cr6+ at concentrations of 0.1, 1.0, 10 and 100mg/L for 4days. Differential significant changes of the antioxidant enzymes and gene expression were observed in different tissues. The findings contribute to better understanding the antioxidant mechanisms induced by Cr6+ and selecting the organic-specific sensitive biomarkers to monitor the safety of the aquatic ecosystem.
The effects of cadmium were assessed in the freshwater alga Chlamydomonas reinhardtii. Algae were exposed to concentrations of 0, 8.1 or 114.8μM of cadmium and growth rates, gene transcription and metabolite profiles were examined after 48 and 72h of exposure. In algae exposed to 8.1μM Cd, several genes were differentially transcribed after 48h but no adverse growth related effects were detected. A transient effect on both gene transcription patterns and metabolite profiles could be discerned after 48h of exposure but the majority of these changes disappeared after 72h. In contrast, all effects were more pronounced at the 114.8μM cadmium exposure. Here growth was clearly reduced and transcription of a large number of genes involved in oxidative stress defense mechanisms was differentially increased. Metabolites involved in the glutathione synthesis pathway (an important antioxidant defense) were also affected but the effects of cadmium were found to be more pronounced at the transcript level than in the metabolome, suggesting that the former exhibits greater sensitivity toward cadmium exposure.
Waterborne ammonia has become a persistent pollutant of aquatic habitats. During certain periods (e.g. winter), food deprivation may occur simultaneously in natural water. Additionally, under such stressful circumstances, fish may be enforced to swim at a high speed in order to catch prey, avoid predators and so on. Consequently, fish need to cope with all these stressors by altering physiological processes which in turn are controlled by their genes. In this present study, toxicogenomic analyses using real time PCR was used to characterize expression patterns of potential biomarker genes controlling growth, ion regulation and stress responses in common carp subjected to elevated ammonia (1 mg/L; Flemish water quality guideline for surface water) following periods of feeding (2% body weight) and fasting (unfed for 7 days prior to sampling). Both feeding groups of fish were exposed to high environment ammonia (HEA) for 0 h (control), 3h, 12h, 1 day, 4 days, 10 days, 21 days and 28 days, and were sampled after performing swimming at different speeds (routine versus exhaustive). Results show that the activity and expression of Na(+)/K(+)-ATPase, an important branchial ion regulatory enzyme, was increased after 4-10 days of exposure. Effect of HEA was also evident on expression patterns of other ion-regulatory hormone and receptor genes; prolactin and cortisol receptor mRNA level(s) were down-regulated and up-regulated respectively after 4, 10 and 21 days. Starvation and exhaustive swimming, the additional challenges in present study significantly further enhanced the HEA effect on the expression of these two genes. mRNA transcript of growth regulating hormone and receptor genes such as Insulin-like growth factor I, growth hormone receptor, and the thyroid hormone receptor were reduced in response to HEA and the effect of ammonia was exacerbated in starved fish, with levels that were remarkably reduced compared to fed exposed fish. However, the expression of the growth hormone gene itself was up-regulated under the same conditions. Expression of somatolactin remained unaltered. Stress representative genes, cytochrome oxidase subunit 1 showed an up-regulation in response to HEA and starvation while the mRNA level of heat shock protein 70 was increased in response to all the three stressors. The expression kinetics of the studied genes could permit to develop a "molecular biomarker system" to identify the underlying physiological processes and impact of these stressors before effects at population level occur.