Volodymyr I. Lushchak

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.

Catalase inhibition by amino triazole induces oxidative stress in goldfish brain

The effects of in vivo inhibition of catalase by 3-amino 1,2,4-triazole (AMT) on the levels of damage products resulting from reactive oxygen species attack on proteins and lipids as well as on the activities of five antioxidant and associated enzymes were studied in the brain of goldfish, Carassius auratus. Intraperitoneal injection of AMT at a concentration of 0.1 mg/g wet weight caused a gradual decrease in brain catalase activity over 72 h, whereas higher AMT concentrations (0.5 or 1.0 mg/g) reduced catalase activity by about two-thirds within 5-10 h. AMT effects on antioxidant enzyme activities and oxidative stress markers were studied in detail using fish treated with 0.5 mg/g AMT for 24 or 168 h. The levels of thiobarbituric acid-reactive substances (a lipid damage product) increased 6.5-fold by 24 h after AMT injection but fell again after 168 h. The content of carbonylproteins (CP) also rose within 24 h (by approximately 2-fold) and remained 1.5-fold higher compared with respective sham-injected fish after 168 h. CP levels correlated inversely with catalase activity (R(2) = 0.83) suggesting that catalase may protect proteins in vivo against oxidative modification. The activities of both glutathione peroxidase and glutathione-S-transferase increased by approximately 50% and 80%, respectively, in brain of AMT-treated fish and this might represent a compensatory response to lowered catalase activity. Possible functions of catalase in the maintenance of prooxidant/antioxidant balance in goldfish brain are discussed.

Hydrogen peroxide increases the activities of soxRS regulon enzymes and the levels of oxidized proteins and lipids in Escherichia coli

The effects of hydrogen peroxide treatments on Escherichia coli KS400 and AB1157 cells were assessed by monitoring the accumulation of oxidative damage products, carbonyl proteins and thiobarbituric acid‐reactive substances (TBARS), as well as the activities of selected antioxidant enzymes. H2O2 treatment stimulated increases in both TBARS and carbonyl protein levels in dose‐ and time‐dependent manners in KS400 cells. The accumulation of TBARS was much more variable with H2O2 treatment; TBARS content was significantly increased in response to 5 μM H2O2, whereas a significant increase in carbonyl protein content occurred at 100 μM H2O2. Similarly, treatment with 20 μM hydrogen peroxide for different lengths of time resulted in peak TBARS accumulation by 20 min, whereas carbonyl protein levels were significantly elevated only after 60 min. In AB1157 cells, treatment with 20 μM hydrogen peroxide for 20 min led to strong increases in both carbonyl protein and TBARS levels. This treatment also triggered increased activities of enzymes of the oxyR regulon (catalase, peroxidase, and glutathione reductase) in both strains. In the AB1157 strain, H2O2 exposure also increased the activities of two enzymes of the soxRS regulon (superoxide dismutase and glucose‐6‐phosphate dehydrogenase) by 50–60%. The data show differential variability of lipids versus proteins to oxidative damage induced by H2O2, as well as strain‐specific differences in the accumulation of damage products and the responses by antioxidant enzymes to H2O2 stress.

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.

Catalases protect cellular proteins from oxidative modification in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae cells had higher antioxidant enzyme activities under growth in ethanol than that in glucose as a carbon and energy source. The correlations between catalase activity and protein carbonyl level (r 2 = 0.857), between catalase and glucose‐6‐phosphate dehydrogenase activities (r 2 = 0.924) and between protein carbonyl levels and glucose‐6‐phosphate dehydrogenase activity (r 2 = 0.988) under growth in ethanol were found. Growing in ethanol the strain deficient in cytosolic and peroxisomal catalases had 7.1‐fold higher level of carbonyl proteins than that of wild‐type strain. Our data suggest that in vivo catalases may protect glucose‐6‐phosphate dehydrogenase against oxidative inactivation.

Tocopherol biosynthesis: chemistry, regulation and effects of environmental factors

Tocopherols are lipophilic antioxidants and together with tocotrienols belong to the vitamin-E family. The four forms of tocopherols (α-, β-, γ- and δ-tocopherols) consist of a polar chromanol ring and lipophilic prenyl chain with differences in the position and number of methyl groups. The biosynthesis of tocopherols takes place mainly in plastids of higher plants from precursors derived from two metabolic pathways: homogentisic acid, an intermediate of degradation of aromatic amino acids, and phytyldiphosphate, which arises from methylerythritol phosphate pathway. The regulation of tocopherol biosynthesis in photosynthetic organisms occurs, at least partially, at the level of key enzymes as such including p-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27), homogentisate phytyltransferase (HPT, EC 2.5.1.-), tocopherol cyclase (TC, EC 5.4.99.-), and two methyltransferases. Tocopherol biosynthesis changes during plant development and in response toward different stresses induced by high-intensity light, drought, high salinity, heavy metals, and chilling. It is supposed that scavenging of lipid peroxy radicals and quenching of singlet oxygen are the main functions of tocopherols in photosynthetic organisms. The antioxidant action of tocopherols is related to the formation of tocopherol quinone and its following recycling or degradation. However, until now, the mechanisms of tocopherol degradation in plants have not been established in detail. This review focuses on mechanisms of tocopherols biosynthesis and its regulation in photosynthetic organisms. In addition, available information on tocopherol degradation is summarized.

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.

Inactivation of genes, encoding tocopherol biosynthetic pathway enzymes, results in oxidative stress in outdoor grown Arabidopsis thaliana

Tocopherols (alpha-, beta-, gamma- and delta-tocopherols) represent a group of lipophilic antioxidants which are synthesized only by photosynthetic organisms. It is widely believed that protection of pigments and proteins of photosynthetic system and polyunsaturated fatty acids from oxidative damage caused by reactive oxygen species (ROS) is the main function of tocopherols. The wild type Columbia and two mutants of Arabidopsis thaliana with T-DNA insertions in tocopherol biosynthesis genes - tocopherol cyclase (vte1) and gamma-tocopherol methyltransferase (vte4) - were analyzed after long-term outdoor growth. The concentration of total tocopherol was up to 12-fold higher in outdoor growing wild type and vte4 plant lines than in plants grown under laboratory conditions. The vte4 mutant plants had a lower concentration of chlorophylls and carotenoids, whereas the mutant plants had a higher level of total glutathione than of wild type. The activities of antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate oxidase (AO, EC 1.10.3.3) were lower in both mutants, whereas activities of catalase (EC 1.11.1.6) and ascorbate peroxidase (APx, EC 1.11.1.11) were lower only in vte1 mutant plants in comparison to wild type plants. However, the activity of guaiacol peroxidase (GuPx, EC 1.11.1.7) was higher in vte1 and vte4 mutants than that in wild type. Additionally, both mutant plant lines had higher concentration of protein carbonyl groups and oxidized glutathione compared to the wild type, indicating the development of oxidative stress. These results demonstrate in plants that tocopherols play a crucial role for growth of plants under outdoor conditions by preventing oxidation of cellular components.

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.