Ecotoxocological effects of short-term exposure to a human pharmaceutical Verapamil in juvenile rainbow trout (Oncorhynchus mykiss)

Verapamil (VRP) is a calcium channel blocker that is a highly prescribed compound and commonly present in aquatic environment, but the ecotoxicological effects of this pharmaceutical in fish have not been fully documented. In this study, the toxic effects of VRP were studied in juvenile rainbow trout, Oncorhynchus mykiss, by acute static bioassay. In the acute test, the median lethal concentration (LC50, 2.72 mg/L) was evaluated and the behavioral changes were obviously intensified with increasing VRP concentrations. Compared to the control, oxidative stress was observed in fish tissues with different levels after short-term exposure to sublethal concentrations (0.27 and 1.35 mg/L) of VRP. Activities of SOD and GPx in fish brain were induced at 0.27 mg/L VRP, but all the antioxidant enzymes (SOD, GPx and GR) in fish brain were decreased at 1.35 mg/L VRP. When compared to the control, all the antioxidant enzymes in gill were decreased in both treated groups, but there was no significant change in muscle. Additional, muscle DNA/RNA ratio in fish exposed at 1.35 mg/L VRP was significantly lower than that in the control. Furthermore, through chemometrics of all parameters measured in fish exposed to sublethal VRP concentrations using principal component analysis, two groups with 89.8% of total accumulated variance were distinguished. In short, the physiological and biochemical responses in of fish indicated that VRP-induced environmental stress; but according to VRP residual status in the natural environment, more long-term experiments at lower concentrations will be necessary in the future.     

Biochemical responses in gills of rainbow trout exposed to propiconazole

The aim of this study is to investigate the toxic effect of PCZ, a triazole fungicide commonly present in surface and ground water, on the ROS defense system and Na₊-K₊-ATPase in gills of rainbow trout exposed to sublethal concentrations (0.2, 50 and 500 μg L⁻¹) for 7, 20 and 30 days. After prolonged exposure of PCZ at higher test concentrations (50 and 500 μg L⁻¹), oxidative stress was apparent as reflected by the significant higher ROS levels in fish gill, as well as the significant inhibition of SOD and CAT activities. In addition, Na₊-K₊-ATPase activities were significantly lower than those of the control with increasing PCZ concentration and prolonged exposure period. The results of this study indicate that chronic exposure to PCZ has altered multiple physiological indices in fish gill; however, before these parameters are used as unique biomarkers for monitoring residual pharmaceuticals in aquatic environments, more detailed laboratory experiments need to be performed.     

Multi-organ toxicological impact of fungicide propiconazole on biochemical and histological profile of freshwater fish Channa punctata Bloch

Fungicides are a pesticide that particularly kills or destroy fungi responsible for several diseases associated to humans and other living organisms. Assessment of toxic effects and mechanisms of fungicide action is important because humans and domesticated animals get exposed to these pesticides through a wide variety of applications. Several fungicides are being used at the large scale for the crop protection from the fungal invasion. Propiconazole (PCZ), a trazole-containing fungicide, is widely used in China and various Asian countries for food crop protection which made it easily to exposed to the aquatic system. Long term usage of PCZ may contaminate the water bodies, but its toxicity to aquatic organisms is not well studied. In this study, freshwater fish, Channa punctata Bloch was exposed to different sub-lethal concentrations of the fungicide, PCZ (0.5 and 5 ppm) for a period of 96 h. Various biochemical assays and histological alterations were measured to determine the organ toxicity caused by PCZ exposure particularly in liver, kidney and gills of the fishes. Compared to the control group, fish exposed to PCZ (96 h) showed marked dose dependent toxicity. The levels of lipid peroxidation (LPO) and protein carbonyls (PC), oxidative stress biomarker of liver, kidney and gills in the experimental group were significantly higher (P < 0.05 and P < 0.001) compared to the control group. Levels of reduced glutathione (GSH) and non-protein thiols (NP-SH) decreased significantly (P <0.05–0.001) in all analyzed intoxicated organs of the PCZ exposed fishes. Activity of glutathione-S-transferase (GST), glutathione peroxidase (GPx) and catalase (CAT) in fungicide treated groups was significantly lowered (P < 0.05–0.001). In addition, histopathological examination in the organs showed significant changes like atrophy of primary and secondary gill lamellae, infiltrations, inflammation, hepatocyte degeneration, vacuolization and necrotic kidney. Thus, PCZ exposure altered the oxidative stress homeostasis and brought about histopathological changes which may serve as potential biomarkers of the PCZ toxicity in the laboratory set-up for potential risk assessment.     

Propiconazole induced toxicological alterations in brain of freshwater fish Channa punctata Bloch

Fungicides are a class of pesticides which are used indiscriminately in large amounts and pose a serious threat to the environment. Propiconazole (PCZ) is a systemic foliar fungicide with a broad range of activity. The potential of this fungicide to induce toxicity has not been fully explored. The present study was designed to investigate the dose dependent neurotoxic effect of propiconazole (PCZ), with Channa punctata Bloch as a model organism. Effect of PCZ on the brain specific enzyme activity such as acetylcholinesterase (AChE), monoamine oxidase (MAO) and Na⁺-K⁺-ATPase was determined in the fish brain tissue exposed to sub-lethal concentrations (0.5 and 5 ppm) for 96 h. Also, levels of oxidative stress reflected by various enzymatic and non-enzymatic antioxidants were measured. Neurotransmitter (epinephrine) level was also assessed. PCZ exposure induced oxidative stress as reflected by the significant increase in fish brain lipid peroxidation and protein carbonyl content with decrease in reduced glutathione levels, as well as the significant inhibition of glutathione dependent metabolizing enzymes and CAT activities. In addition, AChE, MAO and Na⁺-K⁺-ATPase activities were significantly lowered along with reduction in epinephrine levels in PCZ exposed fishes than those of the control in a dose dependent manner. Also, histopathological alterations were observed in fish brain of the treated fishes. The results point towards the potential neurotoxicity in the fish caused by PCZ exposure but the application of these findings will need more detailed study before they can be established as special biomarkers for toxicity monitoring the aquatic environment.     

Molecular cloning and characterization of cat, gpx1 and Cu/Zn-sod genes in pengze crucian carp (Carassius auratus var. Pengze) and antioxidant enzyme modulation induced by hexavalent chromium in juveniles

Hexavalent chromium (Cr^6 +) 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 Cr^6 +. To explore the influence of ROS induced directly by Cr^6 + 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 Cr^6 + at concentrations of 0.1, 1.0, 10 and 100 mg/L for 4 days. 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 Cr^6 + and selecting the organic-specific sensitive biomarkers to monitor the safety of the aquatic ecosystem.     

Modulation of antioxidant defence system in brain of rainbow trout (Oncorhynchus mykiss) after chronic carbamazepine treatment

We investigated the effect of long-term exposure to CBZ on the antioxidant system in brain tissue of rainbow trout. Fish were exposed to sublethal concentrations of CBZ (1.0 μg/L, 0.2 mg/L or 2.0 mg/L) for 7, 21, and 42 days. Oxidative stress indices (LPO and CP) and activities of antioxidant enzymes (SOD, CAT, GPx and GR) in fish brain were measured. In addition, non-enzymatic antioxidant (GSH) was determined after 42 days exposure. Carbamazepine exposure at 0.2 mg/L led to significant increases (p < 0.05) of LPO and CP after 42 days and, at 2.0 mg/L, after 21 days. Activities of the antioxidant enzymes SOD, CAT, and GPx in CBZ-treated groups slightly increased during the first period (7 days). However, activities of all measured antioxidant enzymes were significantly inhibited (p < 0.05) at 0.2 mg/L exposure after 42 days and after 21 days at 2.0 mg/L. After 42 days, the content of GSH in fish brain was significantly lower (p < 0.05) in groups exposed to CBZ at 0.2 mg/L and 2.0 mg/L than in other groups. Prolonged exposure to CBZ resulted in excess reactive oxygen species formation, finally resulting in oxidative damage to lipids and proteins and inhibited antioxidant capacities in fish brain. In short, a low level of oxidative stress could induce the adaptive responses of antioxidant enzymes, but long-term exposure to CBZ could lead to serious oxidative damage in fish brain.     

Exposure of the blue mussel, Mytilus edulis, to gold nanoparticles and the pro-oxidant menadione

Relatively little is known about how gold nanoparticles (GNP) might interact in vivo with marine organisms. Mytilus edulis was exposed (24 h) to ~ 15 nm GNP, menadione and both compounds simultaneously (GNP/menadione). GNP was detected by inductively coupled plasma-optical emission spectroscopy mainly in digestive gland of samples exposed to GNP though not GNP/menadione, perhaps due to impaired feeding. Thioredoxin reductase activity and malondialdehyde levels were determined in all tissues. Thioredoxin reductase inhibition was detected only in digestive gland exposed to menadione whilst malondialdehyde levels did not vary in response to treatment in all tissues. GNP caused a decrease in the reduced/oxidized glutathione ratio in digestive gland, but no difference was found in other tissues or for other treatments. One dimensional electrophoresis of proteins containing thiol groups was performed in all tissues and revealed a reduction in protein thiols for all treatments in digestive gland. Two dimensional electrophoresis of digestive gland extracts, from GNP and control groups, showed decreased levels of thiol proteins in response to GNP which we attribute to oxidation. Our results suggest that GNP causes a modest level of oxidative stress sufficient to oxidize thiols in glutathione and proteins but without causing lipid peroxidation or induction of thioredoxin reductase activity.     

In vitro effects of bisphenol A on the quality parameters,oxidative stress,DNA integrity and adenosine triphosphate content in sterlet (Acipenser ruthenus) spermatozoa

Among endocrine disruptors, the xenoestrogen bisphenol A (BPA) deserves particular attention due to widespread human exposure. Besides hormonal effects, BPA has been suspected to be responsible for adverse effect on reproductive ability of various species. In the present study the effect of BPA on the quality parameters, oxidative stress, the DNA integrity and intracellular ATP content of sterlet (Acipenser ruthenus) spermatozoa were investigated in vitro. Fish spermatozoa were exposed to concentrations of BPA possibly occurring in nature (0.5, 1.75, 2.5, 5 and 10 μg/L) for 2 h. Results revealed that BPA significantly decreased spermatozoa motility and velocity of spermatozoa at concentration of BPA 2.5–10 μg/L. Significant positive correlation (r = 0.713, P < 0.05) was found between percent motile spermatozoa and ATP content. Oxidative stress was observed at concentrations 1.75–10 μg/L, as reflected by significantly higher levels of protein and lipid oxidation and superoxide dismutase activity. Intracellular ATP content of spermatozoa decreased with increasing concentrations of BPA. A dramatic increase in DNA fragmentation expressed as percent tail DNA (2.2% ± 0.46) and Olive tail moment (0.37 ± 0.09 arbitrary units) was recorded at concentrations of 1.75 μg/L and above. The present study confirms that concentrations of BPA that can be encountered in nature are capable to induce oxidative stress, leading to impaired sperm quality, DNA fragmentation and intracellular ATP content.     

Creatine prevents the imbalance of redox homeostasis caused by homocysteine in skeletal muscle of rats

Homocystinuria is a neurometabolic disease caused by severe deficiency of cystathionine beta-synthase activity, resulting in severe hyperhomocysteinemia. Affected patients present several symptoms including a variable degree of motor dysfunction, being that the pathomechanism is not fully understood. In the present study we investigated the effect of chronic hyperhomocysteinemia on some parameters of oxidative stress, namely 2′7′dichlorofluorescein (DCFH) oxidation, levels of thiobarbituric acid-reactive substances (TBARS), antioxidant enzyme activities (SOD, CAT and GPx), reduced glutathione (GSH), total sulfhydryl and carbonyl content, as well as nitrite levels in soleus skeletal muscle of young rats subjected to model of severe hyperhomocysteinemia. We also evaluated the effect of creatine on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injection of homocysteine (0.3–0.6 μmol/g body weight), and/or creatine (50 mg/kg body weight) from their 6th to the 28th days age. Controls and treated rats were decapitated at 12 h after the last injection. Chronic homocysteine administration increased 2′7′dichlorofluorescein (DCFH) oxidation, an index of production of reactive species and TBARS levels, an index of lipoperoxidation. Antioxidant enzyme activities, such as SOD and CAT were also increased, but GPx activity was not altered. The content of GSH, sulfhydril and carbonyl were decreased, as well as levels of nitrite. Creatine concurrent administration prevented some homocysteine effects probably by its antioxidant properties. Our data suggest that the oxidative insult elicited by chronic hyperhomocystenemia may provide insights into the mechanisms by which homocysteine exerts its effects on skeletal muscle function. Creatine prevents some alterations caused by homocysteine.     

Effects of exposure to sublethal propiconazole on intestine-related biochemical responses in rainbow trout,Oncorhynchus mykiss

The effect of long-term (30 days) exposure to PCZ (0.2, 50, and 500 μg l^?1) on intestine-related biochemical markers in rainbow trout was investigated. Multiple biomarkers were measured, including digestive enzymes (proteolytic enzymes and amylase), antioxidant responses (TBARS, CP, SOD, CAT, GR and GPx) and energy metabolic parameters (RNA/DNA ratio, Na⁺-K⁺-ATPase). Exposure to 500 μg l^?1 PCZ led to significantly inhibited (p < 0.01) proteolytic enzyme and amylase activity. Activities of the antioxidant enzymes SOD, CAT, and GPx gradually increased at lower PCZ concentrations (0.2 and 50 μg l^?1). At the highest concentration (500 μg l^?1), oxidative stress was apparent as significant higher (p < 0.05) lipid peroxidation and protein carbonyls, associated with an inhibition of antioxidant enzymes activity. Moreover, energy metabolic parameters (RNA/DNA ratio, Na⁺-K⁺-ATPase) were significantly inhibited (p < 0.01) in the intestines of fish exposed to 500 μg l^?1 PCZ, compared with controls. We suggest that long-term exposure to PCZ could result in several responses in intestine-related biochemical markers, which potentially could be used as indicators for monitoring residual PCZ present in the aquatic environment.     

Short term exposure of pendimethalin induces biochemical and histological perturbations in liver,kidney and gill of freshwater fish

Our study was designed to evaluate effects of an herbicide, pendimethalin on biochemical biomarkers and histopathological indices of the freshwater fish Channa punctata Bloch. Fish were acutely exposed (96 h) to sub-lethal concentrations (0.5 and 0.8 ppb of pendimethalin). Various oxidative stress indicators such as thiobarbituric acid reactive substances levels and protein carbonyl content, as well as antioxidant defenses parameters, such as glutathione-S-transferase (GST), catalase (CAT), reduced glutathione (GSH) and non-protein thiols (NP-SH) levels were studied, using the liver, kidney and gill tissues. Pendimethalin exposure increased lipid peroxidation and protein oxidation processes. There was significant inhibition in levels of GSH and NP-SH. The activity of antioxidant enzymes GST and CAT depleted in all the tissues in a dose dependent manner. The histopathological change in the gill showed necrosis and atrophy of primary and secondary gill lamellae. The tissue damages like degeneration of cytoplasm in hepatocytes, atrophy, formation of vacuoles, are some histopathological changes observed in the liver. The changes in histoarchitechture observed in the kidney included necrosis, cellular hypertrophy and granular cytoplasm. The present study demonstrates the disturbances in antioxidant armamentarium and importance of study in the potential risk assessment of herbicides on fish species.     

Changes in phytohormones and oxidative stress markers in buried seeds of Vellozia alata

Little information is still available on the mechanisms underlying seed persistence in the soil in several species, and most particularly in vegetation of the rupestrian fields of the Espinhaço Range in Brazil, where ca. 90% of their species are endemic and are of interest for conservation biology. Here we aimed at examining the putative physiological and biochemical changes seeds of one of this species (Vellozia alata L.B.Sm., Velloziaceae) may experience after burial under natural conditions. Endogenous concentrations of phytohormones and oxidative stress markers were measured in seeds buried in the soil for 12 months. Buried seeds experienced a significant loss of germination capacity, which decreased from 58 to 29% during the first 6 months. This was associated with a decline in gibberellins (by 65%), abscisic acid (by 98%), cytokinins (up to 75%) and jasmonic acid (by 97%) during the first 3 months, while salicylic acid increased at 6 months of burial. Malondialdehyde and tocopherol levels also decreased dramatically to non-detectable values during this period, while all tocotrienol homologues decreased by 30%. By contrast, germination capacity remained constant around 30% between 6 and 12 months of burial. During this period, concentrations of all phytohormones examined remained unaltered, except for salicylic acid, whose levels returned to initial values. Tocotrienols decreased significantly, particularly between 9 and 12 months of burial, while the extent of lipid peroxidation remained constant. It is concluded that in V. alata, (i) seed burial causes dramatic changes in phytohormones and in the extent of lipid peroxidation, while the potential for germination decreases; (ii) after 6 months of burial, seed germination capacity remains constant, at least until one year, which is associated with absence of oxidative damage.     

PmMGST3, a novel microsomal glutathione S-transferase gene in the dinoflagellate Prorocentrum minimum, is a potential biomarker of oxidative stress

In this study, we evaluated a novel microsomal glutathione S-transferase3 (MGST3) gene from the dinoflagellate Prorocentrum minimum, and examined its expression pattern in response to copper-and nickel-induced stresses. The full length of PmMGST3 was 732 bp, ranging from the dinoflagellate splice leader (DinoSL) sequence to the poly (A) tail, covering a 441-bp ORF, 97-bp 5′UTR, and 194-bp 3′UTR. The PmMGST3 was up-regulated by metals, including copper and nickel. The highest up-regulation levels of the PmMGST3 were found under 0.1 mg/L copper and 0.5 mg/L nickel treatment, respectively. In addition, the PmMGST3 was gradually up-regulated by 0.1 mg/L copper with increasing exposure time. Furthermore, ROS production and reduced GSH was measured in the copper treated cells. A significant increased ROS production and reduced GSH were found in the copper treated cells. These results suggest that PmMGST3 may be related to defense mechanisms associated with oxidative stress in dinoflagellates.     

Evaluation of urinary biomarkers of oxidative/nitrosative stress in adolescents and adults with Down syndrome

Urinary biomarkers of oxidative stress have been little studied in adults with Down syndrome (DS), usually no more than two biomarkers have been measured in the population studied and controversial results are reported in literature. Thus, we aimed to assess a set of oxidative and nitrosative stress biomarkers in urine samples of adolescents and adults with DS, with and without hypothyroidism, which comprise: 8-hydroxy-2′-deoxyguanosine (8-OHdG), isoprostane 15-F_2t-IsoP, thiobarbituric acid-reacting substances (TBARS), advanced glycation end products (AGEs), dityrosine (diTyr), hydrogen peroxide (H₂O₂) and nitrite/nitrate (NOx). Fluorimetric and spectrophotometric assays were performed in DS (n = 78), some of them taking levothyroxine for hypothyroidism (n = 24), and in their healthy age-matched controls (n = 65). We found that levels of AGEs, diTyr, H₂O₂ and NOx are increased in DS patients in any or in all age groups, whereas Cr levels were lower in DS than in controls in all age groups. Besides, correlations with age in DS were positive for diTyr and negative for Cr, TBARS, 15-F_2t-IsoP and NOx. We also found lower levels of Cr from 15 to 19 years, higher levels of TBARS and AGEs from 20 to 40 years and higher levels of diTyr from 15 to 40 years in DS patients receiving levothyroxine than in DS without hypothyroidism diagnosed. We conclude that AGEs, diTyr, H₂O₂ and NOx could be used as oxidative stress biomarkers in DS in contrast to 8-OHdG, 15-F_2t-IsoP and TBARS, at least with the methods used. However, renal impairment could occur in DS and Cr adjustment may bias the results, particularly in hypothyroid patients.     

Hemoglobin attenuates the effects of inspired oxygen on plasma isofurans in humans during upper-limb surgery

Reperfusion injury is characterized by significant oxidative stress. F₂-isoprostanes (F₂-IsoP's) and isofurans (IsoF's), the latter preferentially produced during increased oxygen tension, are recognized markers of in vivo oxidative stress. We aimed to determine whether increasing oxygen tension during reperfusion modified levels of plasma total IsoF's and F₂-IsoP's. Forty-five patients undergoing upper-limb surgery were randomized to receive inspired oxygen concentrations of 30, 50, or 80% during the last 15 min of surgery. Venous blood samples were taken before the change in inspired oxygen, after 10 min (before reperfusion), and after 15 min (5 min after reperfusion). IsoF's and F₂-IsoP's were measured by gas chromatography-mass spectrometry. Venous oxygen tension and hemoglobin concentrations were also measured. Plasma IsoF and F₂-IsoP levels in the 50 and 80% O₂ groups were not significantly different from those of the 30% O₂ group. In secondary analyses, using data combining all groups, levels of IsoF's, but not F₂-IsoP's, associated with higher venous oxygen tension (P = 0.038). Hemoglobin negatively modified the influence of oxygen tension on levels of IsoF's (P = 0.014). This study has shown, for the first time, that plasma IsoF levels associate with higher oxygen tension in a human model of reperfusion, and this effect is significantly attenuated by hemoglobin.