Oleh V. 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.

Factors that regulate insulin producing cells and their output in Drosophila

Insulin-like peptides (ILPs) and growth factors (IGFs) not only regulate development, growth, reproduction, metabolism, stress resistance, and lifespan, but also certain behaviors and cognitive functions. ILPs, IGFs, their tyrosine kinase receptors and downstream signaling components have been largely conserved over animal evolution. Eight ILPs have been identified in Drosophila (DILP1-8) and they display cell and stage-specific expression patterns. Only one insulin receptor, dInR, is known in Drosophila and most other invertebrates. Nevertheless, the different DILPs are independently regulated transcriptionally and appear to have distinct functions, although some functional redundancy has been revealed. This review summarizes what is known about regulation of production and release of DILPs in Drosophila with focus on insulin signaling in the daily life of the fly. Under what conditions are DILP-producing cells (IPCs) activated and which factors have been identified in control of IPC activity in larvae and adult flies? The brain IPCs that produce DILP2, 3 and 5 are indirectly targeted by DILP6 and a leptin-like factor from the fat body, as well as directly by a few neurotransmitters and neuropeptides. Serotonin, octopamine, GABA, short neuropeptide F (sNPF), corazonin and tachykinin-related peptide have been identified in Drosophila as regulators of IPCs. The GABAergic cells that inhibit IPCs and DILP release are in turn targeted by a leptin-like peptide (unpaired 2) from the fat body, and the IPC-stimulating corazonin/sNPF neurons may be targeted by gut-derived peptides. We also discuss physiological conditions under which IPC activity may be regulated, including nutritional states, stress and diapause induction.

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.

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.

Identified peptidergic neurons in the Drosophila brain regulate insulin-producing cells, stress responses and metabolism by coexpressed short neuropeptide F and corazonin

Insulin/IGF-like signaling regulates the development, growth, fecundity, metabolic homeostasis, stress resistance and lifespan in worms, flies and mammals. Eight insulin-like peptides (DILP1-8) are found in Drosophila. Three of these (DILP2, 3 and 5) are produced by a set of median neurosecretory cells (insulin-producing cells, IPCs) in the brain. Activity in the IPCs of adult flies is regulated by glucose and several neurotransmitters and neuropeptides. One of these, short neuropeptide F (sNPF), regulates food intake, growth and Dilp transcript levels in IPCs via the sNPF receptor (sNPFR1) expressed on IPCs. Here we identify a set of brain neurons that utilizes sNPF to activate the IPCs. These sNPF-expressing neurons (dorsal lateral peptidergic neurons, DLPs) also produce the neuropeptide corazonin (CRZ) and have axon terminations impinging on IPCs. Knockdown of either sNPF or CRZ in DLPs extends survival in flies exposed to starvation and alters carbohydrate and lipid metabolism. Expression of sNPF in DLPs in the sNPF mutant background is sufficient to rescue wild-type metabolism and response to starvation. Since CRZ receptor RNAi in IPCs affects starvation resistance and metabolism, similar to peptide knockdown in DLPs, it is likely that also CRZ targets the IPCs. Knockdown of sNPF, but not CRZ in DLPs decreases transcription of Dilp2 and 5 in the brain, suggesting different mechanisms of action on IPCs of the two co-released peptides. Our findings indicate that sNPF and CRZ co-released from a small set of neurons regulate IPCs, stress resistance and metabolism in adult Drosophila.

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.

Drosophila melanogaster larvae fed by glucose and fructose demonstrate difference in oxidative stress markers and antioxidant enzymes of adult flies

Activities of antioxidant and associated enzymes, and oxidative stress markers were assessed in newly enclosed adult fruit flies Drosophila melanogaster developed on diets with 4 and 10% glucose or fructose. In fly males, 10% fructose promoted higher content of protein carbonyls and catalase activity, but lower superoxide dismutase (SOD) activity than 4%, while in females-lower levels of high molecular mass thiols (H-SH). Females at all diets had virtually the same level of lipid peroxides, low-molecular-mass thiols, catalase, and superoxide dismutase activities. Fed with 4% fructose and glucose males demonstrated 24 and 26% lower H-SH level than females, respectively. On diets with 4% glucose, 10% glucose and fructose females had 32, 26 and 27% lower catalase activity than respective males, and 1.3-1.5-fold lower glucose-6-phosphate dehydrogenase activity on glucose-containing diets. Strong positive correlations between H-SH level and G6PD activity, as well as between catalase and G6PDH activity were found. These results suggest that type and concentration of dietary carbohydrate affect antioxidant defense in fruit flies. It also substantially depends on fly sex, comprising presumably levels of protein carbonyls and lipid peroxides, as well as catalase and SOD activities in males and G6PDH activity in females.

Drosophila Insulin-Producing Cells Are Differentially Modulated by Serotonin and Octopamine Receptors and Affect Social Behavior

A set of 14 insulin-producing cells (IPCs) in the Drosophila brain produces three insulin-like peptides (DILP2, 3 and 5). Activity in IPCs and release of DILPs is nutrient dependent and controlled by multiple factors such as fat body-derived proteins, neurotransmitters, and neuropeptides. Two monoamine receptors, the octopamine receptor OAMB and the serotonin receptor 5-HT1A, are expressed by the IPCs. These receptors may act antagonistically on adenylate cyclase. Here we investigate the action of the two receptors on activity in and output from the IPCs. Knockdown of OAMB by targeted RNAi led to elevated Dilp3 transcript levels in the brain, whereas 5-HT1A knockdown resulted in increases of Dilp2 and 5. OAMB-RNAi in IPCs leads to extended survival of starved flies and increased food intake, whereas 5-HT1A-RNAi produces the opposite phenotypes. However, knockdown of either OAMB or 5-HT1A in IPCs both lead to increased resistance to oxidative stress. In assays of carbohydrate levels we found that 5-HT1A knockdown in IPCs resulted in elevated hemolymph glucose, body glycogen and body trehalose levels, while no effects were seen after OAMB knockdown. We also found that manipulations of the two receptors in IPCs affected male aggressive behavior in different ways and 5-HT1A-RNAi reduced courtship latency. Our observations suggest that activation of 5-HT1A and OAMB signaling in IPCs generates differential effects on Dilp transcription, fly physiology, metabolism and social interactions. However the findings do not support an antagonistic action of the two monoamines and their receptors in this particular system.

Aconitase post-translational modification as a key in linkage between Krebs cycle, iron homeostasis, redox signaling, and metabolism of reactive oxygen species.

Abstract Aconitase, an enzyme possessing an iron–sulfur cluster that is sensitive to oxidation, is involved in the regulation of cellular metabolism. There are two isoenzymes of aconitase (Aco) – mitochondrial (mAco) and cytosolic (cAco) ones. The primary role of mAdco is believed to be to control cellular ATP production via regulation of intermediate flux in the Krebs cycle. The cytosolic Aco in its reduced form operates as an enzyme, whereas in the oxidized form it is involved in the control of iron homeostasis as iron regulatory protein 1 (IRP1). Reactive oxygen species (ROS) play a central role in regulation of Aco functions. Catalytic Aco activity is regulated by reversible oxidation of [4Fe-4S]2+ cluster and cysteine residues, so redox-dependent posttranslational modifications (PTMs) have gained increasing consideration as regards possible regulatory effects. These include modifications of cysteine residues by oxidation, nitrosylation and thiolation, as well as Tyr nitration and oxidation of Lys residues to carbonyls. Redox-independent PTMs such as phosphorylation and transamination also have been described. In the presence of a sustained ROS flux, redox-dependent PTMs may lead to enzyme damage and cell stress by impaired energy and iron metabolism. Aconitase has been identified as a protein that undergoes oxidative modification and inactivation in aging and certain oxidative stress-related disorders. Here we describe possible mechanisms of involvement of the two aconitase isoforms, cAco and mAco, in the control of cell metabolism and iron homeostasis, balancing the regulatory, and damaging effects of ROS.