Glutathione peroxidases in Lolium multiflorum and Festuca arundinacea: Activity, susceptibility to herbicides and characteristics

To evidence a possible mechanism of defense toward oxidative stress induced by herbicides in plants, an investigation was carried on the activity of glutathione peroxidase (GPX) in Italian ryegrass (Lolium multiflorum) and in Festuca (Festuca arundinacea) in response to atrazine (6-chloro-N-ethyl-N'-isopropyl-1,3,5-triazine-2,4-diamine) and fluorodifen (4-nitrophenyl α,α,α-trifluoro-2-nitro-p-tolyl ether). In general, the herbicide treatments significantly induced GPX activity in the shoots of Italian ryegrass, whereas inhibited it in Festuca. These opposite behaviors are examined, taking into account the accumulation and persistence of the two herbicides in the plants, and they are discussed in terms of GPX counteraction to oxidative stress in the first case, and of a lower detoxification rate unable to prevent a deleterious effect on the GPX activity in the second case. Further information on the properties of Italian ryegrass and Festuca GPX were achieved by purification and isolation of the enzymes, performed by protein liquid chromatography and by electrophoretic analyses. GPX of both the plants were found to be heterodimer with multiple function in showing also glutathione S-transferase (GST) activity.     

Festuca arundinacea,glutathione S-transferase and herbicide safeners: A preliminary case study to reduce herbicidal pollution

The expression of glutathione S-transferase (GST) activity in Festuca arundinacea was investigated in response to the following herbicide safeners: benoxacor, cloquintocet-mexyl, fenchlorazol-ethyl, fenclorim, fluxofenim and oxabetrinil. All the above compounds enhanced the GST activity tested towards the “model” substrate 1-chloro-2,4-dinitrobenzene (CDNB). Assays of GST activity towards the herbicides terbuthylazine (N ²-tert-butyl-6-chloro-N ⁴-ethyl-1,3,5-triazine-2,4-diamine) and butachlor (N-butoxymethyl-2-chloro-2′,6′-diethylacetanilide) as substrates also showed the ability of the safeners to enhance the enzyme activity towards both these herbicides, with the exception of cloquintocet-mexyl for the enzyme activity towards butachlor. As a consequence of the above effects at a macro-scale level, decreased herbicide accumulation and persistence were ascertained in response to the addition of the safener benoxacor to both terbuthylazine and butachlor treatments. These results are discussed in terms of capacity of benoxacor to induce herbicide detoxification in Festuca arundinacea with a view to utilizing them in reducing herbicide pollution.     

Anguilla anguilla L. oxidative stress biomarkers: an in situ study of freshwater wetland ecosystem (Pateira de Fermentelos, Portugal)

Pateira de Fermentelos (PF) is a natural freshwater wetland in the central region of Portugal. In the last decade, the introduction of agricultural chemicals, heavy metals, domestic wastes, as well as eutrophication and incorrect utility of resources resulted in an increased water pollution. The present research work was carried out to check the various oxidative stress biomarker responses in European eel (Anguilla anguilla L.) gill, kidney and liver due to this complex water pollution. Eels were caged and plunged at five different PF sites (A-E) for 48h. A reference site (R) was also selected at the river spring where no industrial contamination should be detected. Lipid peroxidation (LPO), catalase (CAT), glutathione peroxidase (GPX), glutathione S-transferase (GST) and reduced glutathione (GSH) were the oxidative stress biomarkers studied. In gill, site A exposure induced a significant GST activity increase and site B exposure induced CAT activity increase when compared to R. Site C exposure showed a significant CAT and GPX activity increase. Data concerning site D exposure were not determined due to cage disappearance. Site E exposure displayed a significant CAT and GST activity increase. In kidney, site A exposure induced a significant CAT and GPX decrease as well as a GST increase. Site B exposure showed a significant decrease in GPX activity and GSH content. However, site C exposure demonstrated a significant increase in CAT and a decrease in GPX. Site E exposure showed a significant decrease in GPX and increase in GST. In liver, site A exposure showed a significant GST activity decrease as well as GSH content increase. Site B exposure showed a significant CAT, GST and LPO decrease. Site C exposure showed only GST activity decrease, while site E exposure induced a significant increase in GPX. These investigation findings provide a rational use of oxidative stress biomarkers in freshwater ecosystem pollution biomonitoring using caged fish, and the first attempt reported in Portugal as a study of this particular watercourse under the previous conditions. The presence of pollutants in the PF water was denunciated even without a clear relation to the main pollution source distance. The organ specificity was evident for each parameter but without a clear pattern.     

Herbicide safeners: Tools for improving the efficacy and selectivity of herbicides

Abstract

Safeners (also known as antidotes) are chemical or biological agents that reduce the toxicity of herbicides to crop plants by a physiological or molecular mechanism. Commercialized safeners are mainly chemical compounds that enhance the tolerance of selected grass crops such as maize (Zea mays L.), grain sorghum [Sorghum bicolor (L.) Moench], rice (Oryza sativa L.), and wheat (Triticum aestivum L.) to chloroacetanilide, thiocarbamate, sulfonylurea, imidazolinone, and aryloxyphenoxypropionate herbicides. In practice, safeners are applied either to the crop prior to planting (seed safeners) or to the soil together with the herbicide, formulated as a prepackaged mixture. Safeners act as "bioregulators”; controlling the amount of a given herbicide that reaches its target site in an active form. A safener‐induced enhancement of the metabolic detoxification of herbicides in protected plants is the most apparent mechanism for the action of all commercialized safeners. Herbicide‐detoxifying enzymes such as glutathione transferases (GST), cytochrome P‐450 monooxygenases (CytP450), esterases, and UDP‐glucosyltransferases are induced by herbicide safeners. At the molecular level, safeners appear to act by activating or amplifying genes coding for these enzymes (e.g., GST).     

The effect of excess Zn on mineral nutrition and antioxidative response in rapeseed seedlings

Zinc (Zn) is a necessary element for plants, but excess Zn can be detrimental. To investigate Zn toxicity, rapeseed (Brassica napus) seedlings were treated with 0.07–1.12 mM Zn for 7 d. Inhibition of plant growth along with root damage, chlorosis and decreased chlorophyll (a and b) content in newly expanded leaves (the second and third leaves formed following cotyledons) were found under Zn stress. The Zn content increased in plants under external Zn stress, while concentrations of phosphorus, copper, iron, manganese and magnesium reduced significantly, especially in roots. Meanwhile, increased lipid peroxidation was detected biochemically and histochemically. Compared with controls, NADH oxidase and peroxidase (POD) activity increased in leaves and roots of plants under high Zn, but superoxide dismutase (SOD), catalase and ascorbate peroxidase activities decreased. The changes in glutathione S-transferase activity and in ascorbic acid, dehydroascorbate, non-protein thiols and glutathione contents were also measured under Zn stress. Isoforms of SOD and POD were separated using non-denaturing polyacrylamide gel electrophoresis and their activities were analyzed. Our results suggested that excess Zn exerts its toxicity partially through disturbing nutrient balance and inducing oxidative stress in plants. These data will be helpful for better understanding of toxicity of Zn and the adaptive mechanism in Zn non-hyperaccumulator plants.     

Copper and zinc induction of lipid peroxidation and effects on antioxidant enzyme activities in the microalga Pavlova viridis (Prymnesiophyceae)

The metal-induced lipid peroxidation and response of antioxidative enzymes have been investigated in the marine microalga Pavlova viridis to understand the mechanisms of metal resistance in algal cells. We have analyzed superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) activities and glutathione (GSH) contents in microalgal cells grown at different concentrations of copper and zinc. In response to each metal, lipid peroxidation was enhanced with the increase of concentrations, as an indication of the oxidative damage caused by metal concentration assayed in the microalgae cells. Exposure of P. viridis to the two metals caused changes in enzyme activities in a different manner, depending on the metal assayed: after copper treatments, total SOD activity was enhanced, while it was reduced after zinc exposure. Copper and zinc stimulated the activities of CAT and GSH whereas GPX showed a remarkable increase in activity in response to copper treatments and decrease after zinc treatments. These results suggest that an activation of some antioxidant enzymes was enhanced to counteract the oxidative stress induced by the two metals.     

Response of antioxidant enzymes in Nicotiana tabacum clones during phytoextraction of heavy metals

Background, aim, and scope  Tobacco, Nicotiana tabacum, is a widely used model plant for growth on heavy-metal-contaminated sites. Its high biomass and deep rooting system make it interesting for phytoextraction. In the present study, we investigated the antioxidative activities and glutathione-dependent enzymes of different tobacco clones optimized for better Cd and Zn accumulation in order to characterize their performance in the field. Main features  The improved heavy metal resistance also makes the investigated tobacco clones interesting for understanding the plant defense enzyme system in general. Freshly harvested plant material (N. tabacum leaves) was used to investigate the antioxidative cascade in plants grown on heavy metal contaminated sites with and without amendments of different ammonium nitrate and ammonium sulfate fertilizers. Materials and methods  Plants were grown on heavily polluted soils in north-east Switzerland. Leaves were harvested at the field site and directly deep frozen in liquid N₂. Studies were concentrated on the antioxidative enzymes of the Halliwell–Asada cycle, and spectrophotometric measurements of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), superoxide dismutase (SOD, EC 1.15.1.1), glutathione peroxidase (GPX, EC 1.11.1.9), glutathione reductase (GR, EC 1.6.4.2), glutathione S-transferase (GST, EC 2.5.1.18) were performed. Results and discussion  We tried to explain the relationship between fertilizer amendments and the activity of the enzymatic defense systems. When tobacco (N. tabacum) plants originating from different mutants were grown under field conditions with varying fertilizer application, the uptake of cadmium and zinc from soil increased with increasing biomass. Depending on Cd and Zn uptake, several antioxidant enzymes showed significantly different activities. Whereas SOD and CAT were usually elevated, several other enzymes, and isoforms of GST were strongly inhibited. Conclusions  Heavy metal uptake represents severe stress to plants, and specific antioxidative enzymes are induced at the cost of more general reactions of the Halliwell–Asada cycle. In well-supplied plants, the glutathione level remains more or less unchanged. The lack of certain glutathione S-transferases upon exposure to heavy metals might be problematic in cases when organic pollutants coincide with heavy metal pollution. When planning phytoremediation of sites, mixed pollution scenarios have to be foreseen and plants should be selected according to both, their stress resistance and hyperaccumulative capacity.     

Copper-induced response of physiological parameters and antioxidant enzymes in the aquatic macrophyte Potamogeton pusillus

Bioaccumulation and toxicity of copper was evaluated on Potamogeton pusillus L. The effect of copper (5–100 μg L^?1) applied for several days was assessed by measuring changes in the chlorophyll's, phaeophytin's, malondialdehyde, electrical conductivity, glutathione peroxidase (GPX), glutathione reductase (GR) and guaiacol peroxidase (POD) activities. Plants accumulated copper with a maximum of 162 μg g^?1 dw after 7-days exposure at 100 μg L^?1, however most of the metal was accumulated after 1-day exposure. The toxic effect caused by Cu was evident by the reduction of photosynthetic pigments, increase of malondialdehyde and electrical conductivity. P. pusillus shows Cu-induced oxidative stress by modulating antioxidant enzymes like GPX, GR and POD. Antioxidant enzymes activity increased significantly after exposure to 40 μg L^?1 during 24 h, followed by a drop at longer times. Thus, P. pusillus is proposed as a good biomonitor for the assessment of metal pollution in aquatic ecosystems.     

Phragmites sp. physiological changes in a constructed wetland treating an effluent contaminated with a diazo dye (DR81)

The role of Phragmites sp. in phytoremediation of wastewaters containing azo dyes is still, in many ways, at its initial stage of investigation. This plant response to the long-term exposure to a highly conjugated di-azo dye (Direct Red 81, DR81) was assessed using a vertical flow constructed wetland, at pilot scale. A reed bed fed with water was used as control. Changes in photosynthetic pigment content in response to the plant contact with synthetic DR81 effluent highlight Phragmites plasticity. Phragmites leaf enzymatic system responded rapidly to the stress imposed; in general, within 1 day, the up-regulation of foliar reactive oxygen species-scavenging enzymes (especially superoxide dismutase, ascorbate peroxidase (APX), glutathione peroxidase (GPX) and peroxidase) was noticed as plants entered in contact with synthetic DR81 effluent. This prompt activation decreased the endogenous levels of H₂O₂ and the malonyldialdehyde content beyond reference values. Glutathione S-transferase (GST) activity intensification was not enough to cope with stress imposed by DR81. GPX activity was pivotal for the detoxification pathways after a 24-h exposure. Carotenoid pool was depleted during this shock. After the imposed DR81 stress, plants were harvested. In the next vegetative cycle, Phragmites had already recovered from the chemical stress. Principal component analysis (PCA) highlights the role of GPX, GST, APX, and carotenoids along catalase (CAT) in the detoxification process.     

Nitrogen fertilizer improves boron phytoextraction by Brassica juncea grown in contaminated sediments and alleviates plant stress

In this study we evaluated the effect of different fertilizer treatments on Brassica plants grown on boron-contaminated sediments. Experiments were conducted in the laboratory and on the lysimeter scale. At laboratory scale (microcosm), five different fertilizers were tested for a 35-d period. On the lysimeter scale, nitrogen fertilization was tested at three different doses and plants were allowed to grow until the end of the vegetative phase (70 d). Results showed that nitrogen application had effectively increased plant biomass production, while B uptake was not affected. Total B phytoextracted increased three-fold when the highest nitrogen dose was applied. Phytotoxicity on Brassica was evaluated by biochemical parameters. In plants grown in unfertilized B-contaminated sediments, the activity of antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and pyrogallol peroxidase (PPX) increased, whereas catalase (CAT) decreased with respect to control plants. Addition of N progressively mitigated the alteration of enzymatic activity, thus suggesting that N can aid in alleviating B-induced oxidative stress. SOD activity was restored to control levels just at the lowest N treatment, whereas the CAT inhibition was partially restored only at the highest one. N application also lowered the B-induced increase in APX and PPX activities. Increased glutathione reductase activity indicated the need to restore the oxidative balance of glutathione. Data also suggest a role of glutathione and phytochelatins in B defense mechanisms. Results suggest that the nitrogen fertilizer was effective in improving B phytoextraction by increasing Brassica biomass and by alleviating B-induced oxidative stress.     

Lead detoxification by coontail (Ceratophyllum demersum L.) involves induction of phytochelatins and antioxidant system in response to its accumulation

Coontail (Ceratophyllum demersum L.) plants when exposed to various concentrations of Pb (1-100microM) for 1-7days, exhibited both phytotoxic and tolerance responses. The specific responses were function of concentration and duration. Plants accumulated 1748mugPbg(-1) dw after 7d which reflected its metal accumulation ability, however most of the metal (1222microgg(-1) dw, 70%) was accumulated after 1d exposure only. The toxic effect and oxidative stress caused by Pb were evident by the reduction in biomass and photosynthetic pigments and increase in malondialddehyde (MDA) content and electrical conductivity with increase in metal concentration and exposure duration. Morphological symptoms of senescence phenomena such as chlorosis and fragmentation of leaves were observed after 7d. The metal tolerance and detoxification strategy adopted by the plant was investigated with reference to antioxidant system and synthesis of phytochelatins. Protein and antioxidant enzymes viz., superoxide dismutase (SOD, EC 1.15.1.1), guaiacol peroxidase (GPX, EC 1.11.1.7) ascorbate peroxidase (APX, EC 1.11.1.11), catalase (CAT, EC 1.11.1.6) and glutathione reductase (GR, EC 1.6.4.2) showed induction at lower concentration and duration followed by decline. All enzymes except GPX showed maximum activity after 1d. An increase in cysteine, non-protein thiols (NP-SH) and glutathione (GSH) content was observed at moderate exposure conditions followed by decline. Phytochelatins (PC(2) and PC(3)) were synthesized to significant levels at 10 and 50microM Pb with concomitant decrease in GSH levels. Thus production of PCs seems important for the detoxification of metal, however it may lead to depletion of GSH and consequently oxidative stress. Results suggest that plants responded positively to moderate Pb concentrations and accumulated high amount of metal. Due to metal accumulation coupled with detoxification potential, the plant appears to have potential for its use as phytoremediator species in aquatic environments having moderate pollution of Pb.     

Antioxidative responses of duckweed (<Emphasis Type="Italic">Lemna minor</Emphasis> L.) to short-term copper exposure

Goal, Scope and Background

Elevated concentrations of copper in the environment result in accumulation of the metal in plants and cause an increase in reactive oxidative species (ROS). The first response to elevated amounts of ROS is increased levels of enzymatic and non-enzymatic antioxidants that reduce oxidative stress. The aim of our study was to evaluate the early stages of antioxidative responses to the low copper concentrations usually present in moderately polluted environments. In addition, some other parameters were examined to evaluate the effect of copper on plants.

Methods

Duckweed (Lemna minor L.) was exposed to different concentrations of copper sulphate for up to 24 hours. Glutathione concentration and enzymatic activities of catalase, guaiacol peroxidase and glutathione reductase were measured spectrophotometrically. Additionally, delayed and prompt chlorophyll fluorescence was measured by luminometry and fluorometry, respectively. The accumulation of copper in plants exposed for 24 hours to various concentrations of copper sulphate was measured by flame atomic absorption spectrophotometry.

Results

The treatment of plants with copper sulphate resulted in an immediate decrease of the glutathione pool, which was replenished after 24 hours at CuSO₄ concentrations lower than 2 μM. Higher CuSO₄ concentrations caused a decrease of reduced glutathione. The responses of the antioxidant enzymes glutathione reductase, guaiacol peroxidase and catalase to CuSO₄ differed during the first six hours of exposure, but their enzyme activities all increased after 24 hours of exposure. All these enzymes displayed biphasic activity curves with maximum values between 0.5 μM and 1 μM CuSO₄. The response of guaiacol peroxidase was the most pronounced and statistically significantly specific and that of catalase the least. Delayed chlorophyll fluorescence decreased after exposure to 1 μM CuSO₄, but no significant effect on maximum quantum yield of photosystem II (Fv/Fm) was observed. L. minor accumulated relatively high concentrations of copper. The accumulation rate was higher at lower concentrations of copper in the test medium (up to 2 μM CuSO₄) than at concentrations above 2 μM CuSO₄.

Discussion

One of the most pronounced antioxidative responses to copper exposure was modified levels of oxidized and reduced forms of glutathione. The decrease of the glutathione pool is most probably coupled with induced production of phytochelatins. Antioxidative enzymes showed the biphasic enzyme activity characteristic of stress response. Guaiacol peroxidase exhibited the greatest significant increase of activity, even at higher CuSO₄ concentrations at which the activity of catalase and glutathione reductase dropped. The intensity of delayed chlorophyll fluorescence decreased, indicating reduced photosynthesis of plants under stress. All the measured parameters showed that plants respond to even low copper concentrations very soon after exposure. The accumulation rate of copper in duckweed tissues indicates that L. minor is an accumulator species.

Conclusions

The synchronized and prompt inducibility of antioxidants indicates their involvement in a general plant defence strategy for coping with metal-induced oxidative stress. Glutathione concentration and guaiacol peroxidase activity were found to be the most sensitive of the early indicators of exposure to copper concentrations present in polluted water bodies.

Recommendation and Perspectives

The experimental design of the present study allowed us to compare the sensitivity of various methods and parameters for detecting plant responses to heavy metal-induced oxidative stress. The level of glutathione and the enzyme activities of guaiacol peroxidase and glutathione reductase could be used as a rapidly determined early warning system in toxicity studies.

     

Poor efficacy of herbicides in biochar-amended soils as affected by their chemistry and mode of action

We evaluated wheat straw biochar produced at 450 °C for its ability to influence bioavailability and persistence of two commonly used herbicides (atrazine and trifluralin) with different modes of action (photosynthesis versus root tip mitosis inhibitors) in two contrasting soils. The biochar was added to soils at 0%, 0.5% and 1.0% (w/w) and the herbicides were applied to those soil-biochar mixes at nil, half, full, two times, and four times, the recommended dosage (H₄). Annual ryegrass (Lolium rigidum) was grown in biochar amended soils for 1 month. Biochar had a positive impact on ryegrass survival rate and above-ground biomass at most of the application rates, and particularly at H₄. Within any given biochar treatment, increasing herbicide application decreased the survival rate and fresh weight of above-ground biomass. Biomass production across the biochar treatment gradient significantly differed (p < 0.01) and was more pronounced in the case of atrazine than trifluralin. For example, the dose-response analysis showed that in the presence of 1% biochar in soil, the value of GR₅₀ (i.e. the dose required to reduce weed biomass by 50%) for atrazine increased by 3.5 times, whereas it increased only by a factor of 1.6 in the case of trifluralin. The combination of the chemical properties and the mode of action governed the extent of biochar-induced reduction in bioavailability of herbicides. The greater biomass of ryegrass in the soil containing the highest biochar (despite having the highest herbicide residues) demonstrates decreased bioavailability of the chemicals caused by the wheat straw biochar. This work clearly demonstrates decreased efficacy of herbicides in biochar amended soils. The role played by herbicide chemistry and mode of action will have major implications in choosing the appropriate application rates for biochar amended soils.     

Exposure to low doses of endosulfan and chlorpyrifos modifies endogenous antioxidants in tissues of rats

Two experiments were conducted in male SD rats (225-250 g) to determine changes in the activities of endogenous antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and concentrations of glutathione (GSH) in tissues after exposure to low doses of endosulfan and chlorpyrifos using a whole body exposure technique. In both experiments, 6 rats/group were exposed 3 hr/day, 5 days/week for 30 days to: 0 (control), 5, 10, 20, 40 and 60% of LD50 of either pesticide in 50% ethanol; actual concentrations were: endosulfan = 0, 0.5, 1.0, 2.0, 4.0, 6.0 mg/250 g body weight; chlorpyrifos = 0, 1.9, 3.8, 7.6, 15.2, and 22.8 mg/250 g body weight. Endosulfan decreased erythrocyte SOD by 21% in all groups and chlorpyrifos increased SOD by 18% in groups 40 and 60. Liver SOD was 12%-20% lower after endosulfan exposure; lung SOD was altered: endosulfan decreased activity by 21% and 51% and chlorpyrifos by 58 and 75% in the 40 and 60 groups, respectively (P < or = 0.05). Both pesticides increased plasma GPX activity at lower levels and reduced it by 26% and 19% in groups 40 and 60, respectively (P < or = 0.05). Liver GPX increased in the 60 group and lung GPX declined between 20% and 38% after endosulfan exposure. GSH in the liver and lung: endosulfan reduced GSH by about 30% at lower levels and increased by 41% or 70% at higher levels; chlorpyrifos decreased GSH by 28-40% in 20 and 60 groups, respectively (P < or = 0.05). Exposure to low, increasing levels of endosulfan and chlorpyrifos can differentially modify endogenous antioxidants SOD, GPX and GSH, which may lead to the development of oxidative stress in some tissues.     

Anguilla anguilla L. oxidative stress biomarkers responses to copper exposure with or without beta-naphthoflavone pre-exposure

The redox cycling of heavy metals as well as their interactions with organic pollutants is a major contributor to the oxidative stress resulting from aquatic pollution. Therefore, in order to evaluate beta-naphthoflavone (BNF), Cu and BNF/Cu-induced oxidative stress with single and subsequent exposures, research was carried out in European eel (Anguilla anguilla L.). Eel gill and kidney oxidative stress biomarker responses such as lipid peroxidation (LPO), glutathione peroxidase (GPX), catalase (CAT), glutathione S-transferase (GST) and total reduced glutathione (GSH) to a single 24 h exposure to two copper concentrations (Cu-1 microM, 2.5 microM) and BNF (2.7 microM) with or without 24 h BNF (2.7 microM) pre-exposure were investigated. Cu exposure alone showed a significant gill GST increase at the lowest concentration and GSH content decrease for the highest concentration. Double BNF exposure in gill demonstrated a significant increase in LPO, CAT, GPX and GST, as well as a decrease in GSH content. However, in sequential BNF/Cu exposures, only the highest Cu concentration exhibited a significant increase in LPO and GSH as well as a decrease in GPX (vs. BNF + CW). In kidney, Cu exposure alone showed a significant CAT and GSH contents decrease for both concentrations, and at highest concentration in GPX; as well as GST increase at the lowest concentration. Double BNF exposure showed a significant increase in LPO and GST. Nevertheless, in sequential BNF/Cu exposures, both concentrations exhibited a significant increase in LPO and decrease in GSH contents. Moreover, LPO was also increased significantly in comparison to BNF+CW and the equivalent Cu exposures without BNF pre-exposure. Concerning GPX, a significant increase was observed at highest Cu concentration. In GST, a significant decrease at the lowest Cu concentration and increase at the highest Cu concentration was observed. Summarizing, a simple copper or BNF exposures have no ability to induce LPO in both gill and kidney. However, double BNF exposure induced LPO in both organs and sequential BNF/Cu exposures potentiated the risk of peroxidative damage occurrence in both organs. BNF/Cu interference on antioxidant responses differs between the studied organs. In gill, antagonistic effects were denoted with probable reflex in terms of peroxidative damage increase. In kidney, BNF pre-exposure prevented CAT and GPX inhibition by copper; though, no advantage of this effect was perceptible as defence against LPO generation. Considering BNF as a surrogate for a PAH and the detected interactions with copper, as well as the likelihood that these effects would be observed in polluted ecosystems, current results demonstrate their relevance to actual ecological exposures contributing to a better knowledge on oxidative stress mechanisms in fish.     

Assays of PCB congeners and organochlorine insecticides with the transgenic Arabidopsis and tobacco plants carrying recombinant guinea pig AhR and GUS reporter genes

Certain congeners of polychlorinated biphenyls (PCBs) and organochlorine insecticides are ligands of aryl hydrocarbon receptors (AhRs) in animals. A recombinant guinea pig (g) AhR, XgDV, was constructed by fusing the ligand-binding domain of gAhR, the DNA-binding domain of LexA, and the transactivating domain of VP16. Then, the expression unit of β-glucuronidase (GUS) reporter gene regulated by XgDV was introduced into Arabidopsis and tobacco plants. When the transgenic Arabidopsis XgDV plants were cultured on Murashige–Skoog (MS) medium containing PCB congeners, the GUS activity in the plants increased toxic equivalent (TEQ)-dependently. The GUS activity in the transgenic Arabidopsis XgDV plants cultured on MS medium containing the organochlorine insecticide dieldrin was also induced. On the other hand, in the case of DDT, the GUS activity induced by 3-methylcholanthere in the plants decreased. The transgenic Arabidopsis XgDV plants detected 1000 ng g^?1 PCB126 in 1 g of soils. Thus the XgDV plants seemed to be useful for convenient assays of PCB congeners and organochlorine insecticides, without any extraction and purification steps.     

Phytotoxicity and antioxidative enzymes of green microalga (Desmodesmus subspicatus) and duckweed (Lemna minor) exposed to herbicides MCPA,chloridazon and their mixtures

In this study, we evaluate the toxicity of MCPA (auxin-like growth inhibitor), chloridazon (CHD) (PSII-inhibitor) and their mixtures to floating plants and planktonic algae. Toxicity of MCPA (4-chloro-2-methylphenoxyacetic acid) and CHD (5-amino-4-chloro-2-phenyl-3(2H)-pyridazinone) was first assessed in two growth inhibition tests with Lemna minor (ISO/DIS 20079) and Desmodesmus subspicatus (ISO 8692). Next, herbicide mixtures at concentrations corresponding to the EC values were used to assess their interactive effects, and the biomarkers were: for duckweed fresh weight, frond area, chlorophyll content and number of fronds, and for algae cell count and cell volume. The 3d EC₁₀ and EC₅₀ values using cell counts of D. subspicatus were 142.7 and 529.1 mg/L for MCPA and 1.7 and 5.1 mg/L for CHD. The 7d EC₁₀ and EC₅₀ values using frond number of L. minor amounted to 0.8 and 5.4 mg/L for MCPA and 0.7 and 10.4 mg/L for CHD. Higher sensitivity of reproductive (number of cells/fronds) than growth processes (cell volume/frond area) to herbicides applied individually and in mixtures was especially pronounced in the responses of Desmodesmus. Herbicide interactions were assessed by the two-way ANOVA and Abbott's formula. Generally, an antagonistic interaction with Lemna was revealed by MCPA and chloridazon, whereas additive effect of both herbicides was observed for Desmodesmus. A significant stimulation of SOD and APX activity by binary mixtures was noted in algal cells mainly after 24 and 48 hours of exposure. The extremely high stimulation of the activity of both enzymes was induced by the combination EC₁₀CHD + EC₅₀MCPA (48 h). Presumably due to oxidative stress, the treatment with CHD at concentration EC₅₀ after 72h was lethal for algae grown in aerated cultures, in contrast to standardized test conditions. Taking into account the consequences of risk assessment for herbicide mixtures we can state that a relatively low toxicity, as well as the lack of significant synergy between MCPA and CHD to non-target plants appears to be the most important result.     

Investigation of low-level <Superscript>242</Superscript>Pu contamination on nutrition disturbance and oxidative stress in <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.

Plutonium associated with higher molecular weight molecules is presumed to be poorly mobile and hardly plant available. In our present study, we investigate the uptake and effects of Pu treatments on Solanum tuberosum plants in amended Hoagland medium at concentrations of [²⁴²Pu] = 100 and 500 nm, respectively. We found a direct proof of oxidative stress in the plants caused by these rather low concentrations. For the confirmation of oxidative stress, we explored the production of nitric oxide (NO) and hydrogen peroxide (H₂O₂) by epifluorescence microscopy. Oxidative stress markers like lipid peroxidation and superoxide radicals (O₂ ^??) are monitored through histochemical analysis. The biochemical parameters i.e. chlorophyll and carotenoids are measured as an indicator of cellular damage in the tested plants including the enzymatic parameters such as catalase and glutathione reductase. From our work, we conclude that Pu in low concentration has no significant effects on the uptake of many trace and macroelements. In contrast, the content of O₂ ^?? , malondialdehyde (MDA), and H₂O₂ increases with increasing Pu concentration in the solution, while the opposite effects was found for NO, catalase, and glutathione reductase. These findings prove that even low concentration of Pu regulates ROS production and generate oxidative stress in S. tuberosum L.