Cobalt-induced oxidative stress in brain, liver and kidney of goldfish Carassius auratus.

Cobalt is an essential element, but at high concentrations it is toxic. In addition to its well-known function as an integral part of cobalamin (vitamin B12), cobalt has recently been shown to be a mimetic of hypoxia and a stimulator of the production of reactive oxygen species. The present study investigated the responses of goldfish, Carassius auratus, to 96 h exposure to 50, 100 or 150 mg L⁻¹ Co²âº in aquarium water (administered as CoCl2). The concentrations of cobalt in aquaria did not change during fish exposure. Exposure to cobalt resulted in increased levels of lipid peroxides in brain (a 111% increase after exposure to 150 mg L⁻¹ Co²âº) and liver (30-66% increases after exposure to 50-150 mg L⁻¹ Co²âº), whereas the content of protein carbonyls rose only in kidney (by 112%) after exposure to 150 mg L⁻¹ cobalt. Low molecular mass thiols were depleted by 24-41% in brain in response to cobalt treatment. The activities of primary antioxidant enzymes, superoxide dismutase (SOD) and catalase, were substantially suppressed in brain and liver as a result of Co²âº exposure, whereas in kidney catalase activity was unchanged and SOD activity increased. The activities of glutathione-related enzymes, glutathione peroxidase and glutathione-S-transferase, did not change as a result of cobalt exposure, but glutathione reductase activity increased by ∼40% and ∼70% in brain and kidney, respectively. Taken together, these data show that exposure of fish to Co²âº ions results in the development of oxidative stress and the activation of defense systems in different goldfish tissues.

Chromium effects on free radical processes in goldfish tissues: comparison of Cr(III) and Cr(VI) exposures on oxidative stress markers, glutathione status and antioxidant enzymes.

The present study directly compared the effects of exposure to Cr6+ and Cr3+ (10 mg/L) over 24, 48 and 96 h on indices of oxidative stress and activities of antioxidant and related enzymes in goldfish brain, liver, kidney and gills. Glutathione status clearly demonstrated the development of oxidative stress, whereas changes in protein carbonyls and lipid peroxides were less pronounced. The activity of superoxide dismutase (SOD) was virtually unaffected after 24 or 96 h exposure, but 48 h exposure to Cr6+ reduced SOD activity in brain (by 30%), enhanced activity in kidney (by 28%) and had no effect on liver SOD. Chromium exposure for shorter times had no effect on catalase activity, whereas 96 h exposure depressed activity in liver, kidney and gills. Exposure to Cr6+ reduced catalase activity in liver by 53% and in kidney by 21%, while in gills it was reduced by 20 and 38% by exposure to Cr3+ and Cr6+, respectively. Exposure to chromium for 24 h did not affect glutathione-S-transferase activity, but treatment with Cr6+ for 48 h enhanced it in brain by 1.5-fold, whereas exposure to Cr3+ decreased activity by 29% in kidney. Fish treatment with chromium ions for 96 h decreased glutathione-S-transferase activity in liver by 51 and 25%, respectively. Chromium exposure had very little effect on the activities of GR or G6PDH. These data show that both chromium ions induced oxidative stress in goldfish tissues and affected the activity of antioxidant and associated enzymes.

Cytotoxicity of chromium ions may be connected with induction of oxidative stress.

Chromium ions are frequently found in aquatic ecosystems and are known to be inducers of oxidative stress in fish solid tissues. The present study was designed to determine whether fish blood samples can be used to allow nonlethal diagnostic testing for chromium intoxication. First, we confirmed that 96 h exposures to water containing 10.0 mg L(-1) chromium ions, either Cr3+ or Cr6+, induced oxidative stress in brain of goldfish (Carassius auratus). Multiple blood parameters were then evaluated. Cr6+ exposure triggered a 579% increase in the number of erythrocytes containing micronuclei, a frequently used marker of cellular toxicity. Leucocyte numbers were also perturbed by exposure to either Cr3+ or Cr6+ indicating that chromium ions could impair the immune system as well. The content of protein carbonyl groups, a marker of oxidative damage to proteins, was enhanced in fish plasma by exposure to either chromium ion and activities of catalase and lactate dehydrogenase also were affected. The data demonstrate that chromium ions induced oxidative stress in goldfish blood and were cytotoxic for erythrocytes. This indicates that analysis of plasma can be used as a good early nonlethal diagnostic marker of fish intoxication by transition metal ions.

Production and properties of alpha-amylase from Bacillus sp. BKL20.

As a result of screening Bacillus sp. strains isolated from different natural substrates, strain BKL20 was identified as a producer of a thermostable alkaline alpha-amylase. Maximum production of this alpha-amylase was achieved by optimizing culture conditions. Production of alpha-amylase seemed to be independent of the presence of starch in the culture medium and was stimulated by the presence of peptone (0.3%, m/v) and yeast extract (0.2%, m/v). The enzyme was thermostable and retained amylolytic activity after 30 min of incubation at 60 and 70 degrees C. High activity was maintained over a broad pH range, from 6.0 to 11.0, and the enzyme remained active after alkaline incubation for 24 h. Bacillus sp. BKL20 alpha-amylase was not stimulated by Ca2+ or other bivalent metal cations and was not inhibited by EGTA or EDTA at 1-10 mmol/L, suggesting that this alpha-amylase is a Ca2+-independent enzyme. It also showed good resistance to both oxidizing (H2O2) and denaturating (urea) agents.

Low toxic herbicide Roundup induces mild oxidative stress in goldfish tissues.

The formulation of Roundup consists of the herbicide glyphosate as the active ingredient with polyethoxylene amine added as a surfactant. The acute toxicity of Roundup (particularly of glyphosate) to animals is considered to be low according to the World Health Organization, but the extensive use of Roundup may still cause environmental problems with negative impact on wildlife, particularly in an aquatic environment where chemicals may persist for a long time. Therefore, we studied the effects of Roundup on markers of oxidative stress and antioxidant defense in goldfish, Carassius auratus. The fish were given 96 h exposure to Roundup at concentrations of 2.5-20 mg L(-1). Exposure to Roundup did not affect levels of lipid peroxides (LOOH) in goldfish brain or liver, and in kidney only the 10 mg L(-1) treatment elevated LOOH by 3.2-fold. Herbicide exposure also had no effect on the concentrations of protein thiols or low molecular mass thiols in kidney, but selective suppression of low molecular mass thiols by 26-29% occurred at some treatment levels in brain and liver. Roundup exposure generally suppressed the activities of superoxide dismutase (SOD), glutathione S-transferase (GST), glutathione reductase and glucose-6-phosphate dehydrogenase in fish tissues. For example, SOD activities were reduced by 51-68% in brain, 58-67% in liver and 33-53% in kidney of Roundup treated fish. GST activity decreased by 29-34% in liver. However, catalase activity increased in both liver and kidney of herbicide-exposed fish. To our knowledge this is the first study to demonstrate a systematic response by the antioxidant systems of fish to Roundup exposure.

Chromium(III) induces oxidative stress in goldfish liver and kidney.

In the environment chromium is found mainly in two valence states-hexavalent chromium (Cr6+) and trivalent chromium (Cr3+). The present study evaluates the effects of Cr3+ exposure on goldfish by analyzing parameters of oxidative stress and antioxidant defense in liver and kidney of fish given 96 h exposures to Cr3+ concentrations of 1, 2.5, 5 or 10 mg/l in aquarium water. Cr3+ exposure did not alter two parameters of oxidative stress-protein carbonyl content and lipid peroxide concentrations in either organ. However, Cr3+ exposure did decrease total glutathione concentration in liver by 34-69% and in kidney to 36-49% of the respective control values. Oxidized GSSG content fell by similar percentages so that the ratio [GSSG]/[total glutathione] remained constant at all Cr3+ exposure levels except in liver under the highest, 10 mg/l, exposure level. In liver, exposure to 1-5 mg/l Cr3+ led to a decrease in the activity of superoxide dismutase (SOD) by 29-36%, and at 10 mg/l Cr3+ the reduction was 54%, whereas in kidney approximately 30% reductions in SOD activity were seen at concentrations 1 and 10 mg/l Cr3+. Catalase activity was not significantly affected by 1-5 mg/l Cr3+, but was reduced by 57 and 42% in liver and kidney, respectively. Chromium exposure also reduced the activity of glutathione-S-transferase in both organs by 17-50% but did not affect glutathione reductase or glucose-6-phosphate dehydrogenase activities. A comparison of Cr3+ effects on goldfish liver and kidney metabolism indicates that the trivalent ion induces stronger oxidative stress than Cr6+ at the same concentrations.

Trivalent chromium induces oxidative stress in goldfish brain.

Although information on the effects of Cr(6+) in biological systems is abundant, Cr(3+) has received less attention. Toxic effects of chromium compounds are partially associated with activation of redox processes. Recently we found that Cr(6+) induced oxidative stress in goldfish tissues and the glutathione system was shown to play a protective role. The present study aimed to investigate free radical processes in brain of goldfish exposed to CrCl(3). Trivalent chromium at a concentration of 50 mg L(-1) was lethal and therefore we chose to examine sublethal dosages of 1.0-10.0 mg L(-1) in aquarium water. The levels of lipid peroxides and protein carbonyls (measures of oxidative damage to lipids and proteins) in brain increased after 96 h exposure of goldfish to Cr(3+). However, exposure to 1.0-10.0 mg L(-1) Cr(3+) decreased total glutathione concentration in brain by approximately 50-60%. Oxidized glutathione levels also fell by approximately 40-60% except at the 10.0 mg L(-1) dosage where they decreased by 85%. Therefore, 10.0 mg L(-1) Cr(3+) significantly reduced the ratio [GSSG]/[totalGSH] to 35% of the control value. Chromium treatment did not affect the activity of superoxide dismutase, but reduced the activities of catalase by 55-62% and glutathione-S-transferase by 14-21%. The activities of glucose-6-phosphate dehydrogenase and glutathione reductase were unchanged under any experimental conditions used. Therefore, it can be concluded that although Cr(3+) exposure induced oxidative stress in goldfish brain, it failed to enhance the efficiency of the antioxidant system in the organ.

The effect of potassium dichromate on free radical processes in goldfish: possible protective role of glutathione.

The effects of 96 h exposure to Cr(6+) (added as potassium dichromate) on the status of antioxidant defenses and markers of oxidative damage were evaluated in three tissues of goldfish, Carassius auratus. Fish exposure to high dichromate concentrations, 10 and 50mg/l, increased protein carbonyl levels in brain and liver, but not in kidney. Chromium exposure also increased concentrations of lipid peroxides in brain (at 5mg/l) and liver (10mg/l), but not in kidney. The concentrations of reduced glutathione (GSH) were higher in the liver of goldfish treated with 5-50mg/l Cr(6+) than in controls, but in kidney only the 5mg/l-treated group showed increased GSH levels. Dichromate at 1mg/l increased the concentration of oxidized glutathione (GSSG) in liver and kidney by 80% and 60%, respectively, whereas at 10 and 50mg/l the levels of GSSG decreased by 50% in kidney. These results indicate that the dichromate concentrations used induced oxidation of lipids and proteins in goldfish tissues in a concentration- and tissue-specific manner. Also, the redox status of fish tissues was affected in a concentration- and tissue-specific manner. The activities of glutathione reductase increased in all three tissues in response to dichromate treatment, increasing by approximately 2-fold in brain and liver in goldfish treated with 50mg/l Cr(6+). Dichromate treatment did not change the activities of SOD, catalase or GST in brain, but reduced the activities of SOD in liver and kidney, and catalase in liver. The results suggest that the glutathione system may be responsible for protecting against the deleterious effects of dichromate in fish and indicate the possible development of an adaptive response during the 96 h treatment with the toxicant.