Analysis of phytochelatin complexes in the lead tolerant vetiver grass [Vetiveria zizanioides (L.)] using liquid chromatography and mass spectrometry

Ethylenediamene tetraacetic acid (EDTA) has been used to mobilize soil lead (Pb) and enhance plant uptake for phytoremediation. Chelant bound Pb is considered less toxic compared to free Pb ions and hence might induce less stress on plants. Characterization of possible Pb complexes with phytochelatins (PC_n, metal-binding peptides) and EDTA in plant tissues will enhance our understanding of Pb tolerance mechanisms. In a previous study, we showed that vetiver grass (Vetiveria zizanioides L.) can accumulate up to 19,800 and 3350 mg Pb kg⁻¹ dry weight in root and shoot tissues, respectively; in a hydroponics set-up. Following the basic incubation study, a greenhouse experiment was conducted to elucidate the efficiency of vetiver grass (with or without EDTA) in remediating Pb-contaminated soils from actual residential sites where Pb-based paints were used. The levels of total thiols, PC_n, and catalase (an antioxidant enzyme) were measured in vetiver root and shoot following chelant-assisted phytostabilization. In the presence of 15 mM kg ⁻¹ EDTA, vetiver accumulated 4460 and 480 mg Pb kg⁻¹ dry root and shoot tissue, respectively; that are 15- and 24-fold higher compared to those in untreated controls. Despite higher Pb concentrations in the plant tissues, the amount of total thiols and catalase activity in EDTA treated vetiver tissues was comparable to chelant unamended controls, indicating lowered Pb toxicity by chelation with EDTA. The identification of glutathione (referred as PC₁) (m/z 308.2), along with chelated complexes like Pb-EDTA (m/z 498.8) and PC₁-Pb-EDTA (m/z 805.3) in vetiver root tissue using electrospray tandem mass spectrometry (ES-MS) highlights the possible role of such species towards Pb tolerance in vetiver grass.     

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.     

Response of two terrestrial green microalgae (Chlorophyta, Trebouxiophyceae) isolated from Cu-rich and unpolluted soils to copper stress

Some algae inhabit Cu-polluted soils. Intracellular Cu-accumulation and production of non-protein thiols in response to copper stress were compared in Stichococcus minor and Geminella terricola isolated from Cu-polluted and unpolluted soils, respectively. Cu-exposed (0.5 μM) S. minor accumulated lower amounts of copper (0.38 mM) than G. terricola (4.20 mM) and maintained 8.5-fold higher level of glutathione (GSH) than G. terricola. The ratio GSH/0.5 GSSG in the Cu-treated S. minor (7.21) was 7-times higher than in G. terricola. Reduced and oxidized forms of phytochelatins were found in both algae. Under copper stress (5 μM) the ratio -SH_total/Cu_{intracellular} in S. minor ranged from 2.3 to 6.2, while it was lower than 1.0 in G. terricola. Low intracellular Cu-accumulation and maintenance of high GSH level concomitant with PCs production seem to be responsible for a higher Cu-resistance of S. minor than G. terricola.     

MSMA Resistance Studies

Abstract

Monosodium methanearsonate (MSMA)-resistant and -susceptible common cocklebur (Xanthium strumarium L.) and cotton (Gossypium hirsutum L.) were treated with MSMA. Plant parameters analyzed were: glutathione synthetase activity, selected amino acid (arginine, glutamic acid, alanine, citrulline, glutamine, and glutathione) content and arsenic content (MSMA, total arsenic, and arsonate). No reduction of arsenic from the parent pentavalent form present in MSMA to the trivalent form was detected. Arginine, glutamic acid, and glutamine content increased in tissue three days after MSMA treatment. Glutathione content decreased during the first three days after treatment; however, five days after treatment the resistant biotype of cocklebur and cotton had elevated glutathione levels (8–20 times greater, respectively). Glutathione Synthetase activity was higher in cotton than in either of the cocklebur biotypes; MSMA did not affect its activity in cotton or either cocklebur biotype. Resistant biotypes have a slightly higher activity than the susceptible biotype. Tolerance of cotton to MSMA may be related to glutathione synthetase activity and possibly to the presence of phytochelatins. Also, increased glutathione levels in the resistant biotype may implicate phytochelatin involvement in the resistance mechanism.     

LIPID PEROXIDATION AND ANTIOXIDANT ENZYME ACTIVITY IN DIFFERENT ORGANS OF MICE EXPOSED TO LOW LEVEL OF MERCURY

The effects of mercuric chloride (Hg) on lipid peroxidation (LPO), glutathione reductase (GR), glutathione peroxidase (GPx), superoxide dismutase (SOD) and glutathione (GSH) levels in different organs of mice (CD-1) were evaluated. Mice were exposed (2 days/week) to 0.0 (control), 0.8 (low) and 8.0 (mid) and 80.0 (high) gHg/kg/day for 2 weeks. The high dose group was excluded from the study due to high mortality. LPO levels in kidney, testis and epididymus at low and mid doses; GR and GPx levels in testis at mid dose; SOD levels in brain and testis at both doses, liver and epididymus at mid dose; GSH levels in testis at both doses were significantly increased compared to their controls. However, the GR levels in kidney at both doses and in epididymus at mid dose; GPx levels in kidney and epididymus and SOD levels in kidney at both the doses; GSH levels in epididymus at mid dose were significantly decreased compared to their control. Body weight gain and food efficiency were significantly reduced (<0.05) in mid dose. These results indicated that Hg treatment enhanced LPO in all tissues, but showed significant enhancement only in kidney, testis and epididymus suggesting that these organs were more susceptible to Hg toxicity. The increase in antioxidant enzyme levels in testis could be a mechanism protecting the cells against reactive oxygen species.     

Activity of glutathione S-transferase in Spodoptera exigua larvae exposed to cadmium and zinc in two subsequent generations

The aim of our study was to establish changes in activity of important in detoxification enzyme-glutathione S-transferase (GST): in alimentary tract, fat body and Malpighian tubules of Spodoptera exigua larvae being under cadmium and zinc exposure through the first as well as the second generation. There was registered enhancement of the enzyme activity in the fat body and the Malpighian tubules caused by zinc as well as its decrease in the Malpighian tubules under cadmium action. Amounts of metals in the alimentary tract were either several times higher than in the diet ingested by larvae or than in the fat body. Metal concentration in the fat body correlated with the level of the enzyme activity (positive correlation for zinc and negative for cadmium). The effect of metal action differentiated dependently on time exposition.     

Toxicity of abamectin and doramectin to soil invertebrates

This study aimed at determining the toxicity of avermectins to soil invertebrates in soil and in faeces from recently treated sheep. Abamectin was more toxic than doramectin. In soil, earthworms (Eisenia andrei) were most affected with LC50s of 18 and 228 mg/kg dry soil, respectively, while LC50s were 67-111 and >300 mg/kg for springtails (Folsomia candida), isopods (Porcellio scaber) and enchytraeids (Enchytraeus crypticus). EC50s for the effect on reproduction of springtails and enchytraeids were 13 and 38 mg/kg, respectively for abamectin, and 42 and 170 mg/kg for doramectin. For earthworms, NOEC was 10 and 8.4 mg/kg for abamectin and doramectin effects on body weight. When exposed in faeces, springtails and enchytraeids gave LC50s and EC50s of 1.0-1.4 and 0.94-1.1 mg/kg dry faeces for abamectin and 2.2->2.4 mg/kg for doramectin. Earthworm reproduction was not affected. This study indicates a potential risk of avermectins for soil invertebrates colonizing faeces from recently treated sheep.     

The importance of glutathione and phytochelatins on the selenite and arsenate detoxification in Arabidopsis thaliana

We investigated the role of glutathione(GSH) and phytochelatins(PCs) on the detoxification of selenite using Arabidopsis thaliana. The wild-type(WT) of Arabidopsis thaliana and its mutants(glutathione deficient Cad 2–1 and phytochelatins deficient Cad 1–3) were separately exposed to varying concentrations of selenite and arsenate and jointly to both toxicants to determine their sensitivities. The results of the study revealed that, the mutants were about 20-fold more sensitive to arsenate than the WT, an indication that the GSH and PCs affect arsenate detoxification. On the contrary, the WT and both mutants showed a similar level of sensitivity to selenite, an indication that the GSH and PCs do not significantly affect selenite detoxification. However, the WT is about 8 times more sensitive to selenite than to arsenate, and the mutants were more resistant to selenite than arsenate by a factor of 2. This could not be explained by the accumulation of both elements in roots and shoots in exposure experiments. The co-exposure of the WT indicates a synergistic effect with regards to toxicity since selenite did not induce PCs but arsenic and selenium compete in their PC binding as revealed by speciation analysis of the root extracts using HPLC–ICP–MS/ESI–MS. In the absence of PCs an antagonistic effect has been detected which might suggest indirectly that the formation of Se glutathione complex prevent the formation of detrimental selenopeptides. This study, therefore, revealed that PC and GSH have only a subordinate role in the detoxification of selenite.     

Carcinogen-Metabolizing Enzymes and Insecticides

Abstract

The present study was under taken to demonstrate the effect of some commonly used insecticides on the activity of cytochrome P450 system including cytochrome b5, aryl hydrocarbon [benzo(a)pyrene] hydroxylase (AHH), N-nirosodimethylamine N-demethylase I [NDMA-dI] and NADPH-cytochrome c reductase as phase I of drug oxidation. In addition, the activity of glutathione S-transferase (GST), glutathione reductase (GR), and the level of glutathione (GSH) were determined in the liver of male mice after oral administration of sumithion, dursban, chlordane, methoxychlor, heptachlor epoxide, and lindane as single (24 h) or as repeated doses for six consecutive days. Oral administration of sumithion, dursban, chlordane, methoxychlore, and heptachlor epoxide as repeated doses decreased: (i) the hepatic content of cytochrome P450 by 36, 37, 47, 37, and 67%, respectively, (ii) AHH activity by 28, 29, 70, 31, and 79%, respectively, (iii) NDMA-dI activity by 43, 44, 32, 27, and 31, respectively. On the other hand, sumithion, chlordane, and methoxychlore induced the activity of NADPH-cytochrome c reductase by 45, 62, and 43 respectively after repeated dose treatments. In addition, single and repeated-dose treatments of mice with lindane induced; (i) cytochrome P450 by 23 and 65%, respectively, (ii) cytochrome b5 by 49 and 131%, respectively, (iii) AHH activity by 64 and 50%, respectively. Repeated-dose treatments of mice with chlordane, methoxychlore, and heptachlor epoxide decreased the GSH level by 42, 38, and 68%, respectively and GST activity by 44, 44, and 55% respectively. Moreover, single- and repeated-dose treatments of mice with lindane decreased the GSH levels by 40 and 54%, respectively, and induced GST activity by 25 and 41%, respectively. Interestingly, single-dose treatments with chlordane, methoxychlore, and heptachlor epoxide decreased the activity of GR by 32, 38, and 31, respectively whereas repeated doses of these compounds induced such activity by 83, 50, and 64%, respectively. It is concluded that modifications in cytochrome P450 system by pesticides could potentiate the toxicity and carcinogenicity of environmental carcinogens such as polycyclic aromatic hydrocarbon and N-nirosodimethylamine (NDMA).     

Reproductive toxicity and underlying mechanisms of di(2-ethylhexyl) phthalate in nematode Caenorhabditis elegans

DEHP (di(2-ethylhexyl) phthalate) is an endocrine disruptor commonly found in plastic products that has been associated with reproduction alterations, but the effect of DEHP on toxicity is still widely unknown. Using DEHP concentrations of 10, 1, and 0.1 mg/L, we showed that DEHP reduced the reproductive capacity of Caenorhabditis elegans after 72 hr. of exposure. DEHP exposure reduced the reproductive capacity in terms of decreased brood sizes, egg hatchability (0.1, 1 and 10 mg/L), and egg-laying rate (1 and 10 mg/L), and increased numbers of fertilized eggs in the uterus (1 and 10 mg/L). DEHP also caused damage to gonad development. DEHP decreased the total number of germline cells, and decreased the relative area of the gonad arm of all exposure groups, with worms in the 1 mg/L DEHP exposure group having the minimum gonad arm area. Additionally, DEHP caused a significant concentration‐dependent increase in the expression of unc-86. Autophagy and ROS contributed to the enhancement of DEHP toxicity in reducing reproductive capacity, and glutathione peroxidase and superoxide dismutase were activated as the antioxidant defense in this study. Hence, we found that DEHP has a dual effect on nematodes. Higher concentration (10 mg/L) DEHP can inhibit the expression of autophagy genes (atg-18, atg-7, bec-1, lgg-1 and unc-51), and lower concentrations (0.1 and 1 mg/L) can promote the expression of autophagy genes. Our data highlight the potential environmental risk of DEHP in inducing reproductive toxicity toward the gonad development and reproductive capacity of environmental organisms.