Reductive activation of hexavalent chromium by human lung epithelial cells: generation of Cr(V) and Cr(V)-thiol species

Chromium(VI) compounds (e.g. chromates) are cytotoxic, mutagenic, and potentially carcinogenic. The reduction of Cr(VI) can yield reactive intermediates such as Cr(V) and reactive oxygen species. Bronchial epithelial cells are the primary site of pulmonary exposure to inhaled Cr(VI) and are the primary cells from which Cr(VI)-associated human cancers arise. BEAS-2B cells were used here as a model of normal human bronchial epithelium for studies on the reductive activation of Cr(VI). Cells incubated with Na₂CrO₄ exhibited two Cr(V) ESR signals, g = 1.979 and 1.985, which persisted for at least 1 h. The g = 1.979 signal is similar to that generated in vitro by human microsomes and by proteoliposomes containing P450 reductase and cytochrome b₅. Unlike many cells in culture, these cells continued to express P450 reductase and cytochrome b₅. Studies with the non-selective thiol oxidant diamide indicated that the g = 1.985 signal was thiol-dependent whereas the g = 1.979 signal was not. Pretreatment with phenazine methosulfate eliminated both Cr(V) signals suggesting that Cr(V) generation is largely NAD(P)H-dependent. ESR spectra indicated that a portion of the Cr(VI) was rapidly reduced to Cr(III). Cells incubated with an insoluble chromate, ZnCrO₄, also generated both Cr(V) signals, whereas Cr(V) was not detected with insoluble PbCrO₄. In clonogenic assays, the cells were very sensitive to Na₂CrO₄ and ZnCrO₄, but considerably less sensitive to PbCrO₄.     

Evaluation of urinary biomarkers of oxidative/nitrosative stress in adolescents and adults with Down syndrome

Urinary biomarkers of oxidative stress have been little studied in adults with Down syndrome (DS), usually no more than two biomarkers have been measured in the population studied and controversial results are reported in literature. Thus, we aimed to assess a set of oxidative and nitrosative stress biomarkers in urine samples of adolescents and adults with DS, with and without hypothyroidism, which comprise: 8-hydroxy-2′-deoxyguanosine (8-OHdG), isoprostane 15-F_2t-IsoP, thiobarbituric acid-reacting substances (TBARS), advanced glycation end products (AGEs), dityrosine (diTyr), hydrogen peroxide (H₂O₂) and nitrite/nitrate (NOx). Fluorimetric and spectrophotometric assays were performed in DS (n = 78), some of them taking levothyroxine for hypothyroidism (n = 24), and in their healthy age-matched controls (n = 65). We found that levels of AGEs, diTyr, H₂O₂ and NOx are increased in DS patients in any or in all age groups, whereas Cr levels were lower in DS than in controls in all age groups. Besides, correlations with age in DS were positive for diTyr and negative for Cr, TBARS, 15-F_2t-IsoP and NOx. We also found lower levels of Cr from 15 to 19 years, higher levels of TBARS and AGEs from 20 to 40 years and higher levels of diTyr from 15 to 40 years in DS patients receiving levothyroxine than in DS without hypothyroidism diagnosed. We conclude that AGEs, diTyr, H₂O₂ and NOx could be used as oxidative stress biomarkers in DS in contrast to 8-OHdG, 15-F_2t-IsoP and TBARS, at least with the methods used. However, renal impairment could occur in DS and Cr adjustment may bias the results, particularly in hypothyroid patients.     

Accumulation and oxidative stress biomarkers in Japanese flounder larvae and juveniles under chronic cadmium exposure

This study investigated how Cd exposure affected oxidative biomarkers in Japanese flounder, Paralichthys olivaceus, at early life stages (ELS). Fish were exposed to waterborne Cd (0–48 µg L^− 1) from embryonic to juvenile stages for 80 days. Growth, Cd accumulation, activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), glutathione S-transferase (GST, EC 2.5.1.18), and levels of glutathione (GSH) and lipid peroxidation (LPO) were investigated at three developmental stages. Flounder growth decreased and Cd accumulation increased with increasing Cd concentration. In metamorphosing larvae, CAT and SOD activities were inhibited and GSH level was elevated, while LPO was enhanced by increasing Cd concentrations. CAT and GST activities of settling larvae were inhibited but GSH level was elevated at high Cd concentrations. In juveniles, SOD activity and LPO level were increased but GST activity was inhibited as Cd concentration increased. Antioxidants in flounder at ELS were able to develop ductile responses to defend against oxidative stress, but LPO fatally occurred due to Cd exposure. These biochemical parameters could be used as effective oxidative biomarkers for evaluating Cd contamination and toxicity in marine environments: CAT, SOD, GSH, and LPO for metamorphosing stage; CAT, GSH, and GST for settling stage; and SOD, GST, and LPO for juvenile stage.     

Effects of azinphos methyl and carbaryl on Rhinella arenarum larvae esterases and antioxidant enzymes

Organophosphate (OP) and carbamate pesticides are anticholinesterasic agents also able to alter antioxidant defenses in different organisms. Amphibian larvae are naturally exposed to these pesticides in their aquatic environments located within agricultural areas. We studied the effect of the carbamate carbaryl (CB) and the OP azinphos methyl (AM), compounds extensively used in Northern Patagonian agricultural areas, on reduced glutathione (GSH) levels and the activities of esterases and antioxidant enzymes of the toad Rhinella arenarum larvae. Larvae were exposed 48 h to AM 3 and 6 mg/L or CB 10 and 20 mg/L. Cholinesterase and carboxylesterases were strongly inhibited by CB and AM. In insecticide-exposed larvae, carboxylesterases may serve as alternative targets protecting cholinesterase from inhibition. GSH-S-transferase (GST) activity was significantly increased by CB and AM. Superoxide dismutase activity increased in tadpoles exposed to 6 mg/L AM. Conversely, catalase (CAT) was significantly inhibited by both pesticides. GSH levels, GSH reductase and GSH peroxidase activities were not significantly affected by pesticide exposure. GST increase constitutes an important adaptive response to CB and AM exposure, as this enzyme has been related to pesticide tolerance in amphibian larvae. Besides, the ability to sustain GSH levels in spite of CAT inhibition indicates quite a good antioxidant response. In R. arenarum larvae, CAT and GST activities together with esterases could be used as biomarkers of CB and AM exposure.     

In-situ Cr(VI) reduction with electrogenerated hydrogen peroxide driven by iron-reducing bacteria

Cr(VI) was reduced in-situ at a carbon felt cathode in an air-cathode dual-chamber microbial fuel cell (MFC). The reduction of Cr(VI) was proven to be strongly associated with the electrogenerated H₂O₂ at the cathode driven by iron-reducing bacteria. At pH 2.0, only 42.5% of Cr(VI) was reduced after 12 h in the nitrogen-bubbling-cathode MFC, while complete reduction of Cr(VI) was achieved in 4 h in the air-bubbling-cathode MFC in which the reduction of oxygen to H₂O₂ was confirmed. Conditions that affected the efficiency of the reduction of Cr(VI) were evaluated experimentally, including the cathodic electrolyte pH, the type of iron-reducing species, and the addition of redox mediators. The results showed that the efficient reduction of Cr(VI) could be achieved with an air-bubbling-cathode MFC.     

Investigation of Cr(VI) adsorption onto chemically treated Helianthus annuus: Optimization using Response Surface Methodology

In the present study, chemically treated Helianthus annuus flowers (SHC) were used to optimize the removal efficiency for Cr(VI) by applying Response Surface Methodological approach. The surface structure of SHC was analyzed by Scanning Electron Microscopy (SEM) coupled with Energy Dispersive X-ray Analysis (EDX). Batch mode experiments were also carried out to assess the adsorption equilibrium in aqueous solution. The adsorption capacity (q_e) was found to be 7.2 mg/g. The effect of three parameters, that is pH of the solution (2.0-7.0), initial concentration (10-70 mg/L) and adsorbent dose (0.05-0.5 g/100 mL) was studied for the removal of Cr(VI) by SHC. Box-Behnken model was used as an experimental design. The optimum pH, adsorbent dose and initial Cr(VI) concentration were found to be 2.0, 5.0 g/L and 40 mg/L, respectively. Under these conditions, removal efficiency of Cr(VI) was found to be 90.8%.     

Acute toxicity and cholinesterase inhibition of the nematicide ethoprophos in larvae of gar Atractosteus tropicus (Semionotiformes: Lepisosteidae)

Biomarkers are a widely applied approach in environmental studies. Analyses of cholinesterase (ChE), glutathione S-transferase (GST) and lipid peroxidation (LPO) are biomarkers that can provide information regarding early effects of pollutants at different biochemical levels on an organism. The aim of this study was to evaluate the biomarker approach on a Costa Rican native and relevant species. For this, larvae of gar (Atractosteus tropicus) were exposed to the organophosphorus nematicide, ethoprophos. Acute (96hr) exposure was conducted with pesticide concentrations ranging from 0.1 microg/L to 1 500 microg/L. The 96hr LC50 calculated was 859.7 microg/L. After exposure, three biomarkers (ChE, GST and LPO) were analyzed in fish that survived the acute test. The lowest observed effect concentration (LOEC) regarding ChE activity inhibition was 50 microg/L. This concentration produced a significant inhibition (p<0.05) of the enzyme by 20%. The highest concentration tested without showing any effect on ChE activity and therefore considered as no observed effect concentration (NOEC) was 10 microg/L. Ethoprophos concentration of 400 microg/L caused a ChE inhibition by 79%. In this study, no significant variations (p>0.05) in GST activity and LPO were observed in A. tropicus larvae after exposure to ethoprophos.     

Simultaneous Cr(VI) reduction and phenol degradation in pure cultures of Pseudomonas aeruginosa CCTCC AB91095

Simultaneous Cr(VI) reduction and phenol degradation were investigated in a reactor containing Pseudomonas aeruginosa CCTCC AB91095. Phenol was used as carbon source. P.aeruginosa utilized metabolites formed during phenol degradation as energy source for Cr(VI) reduction. Cr(VI) inhibited both Cr(VI) reduction and phenol degradation when Cr(VI) concentration exceeded the optimum value (20 mg/L), whereas phenol enhanced both Cr(VI) reduction and phenol degradation below the optimum initial concentration of 100 mg/L. Cr(III) was the predominant product of Cr(VI) reduction in cultures after incubation for 24 h. Both Cr(VI) reduction and phenol degradation were influenced by the amount of inocula. The concentration of Cr(VI) and phenol declined quickly from 20, 100 to 3.36, 29.51 mg/L in cultures containing of 5% (v/v) inoculum after incubation for 12 h, respectively. The whole study showed that P. aeruginosa is promising for the reduction of toxic Cr(VI) and degradation of organic pollutants simultaneously in the mineral liquid medium.     

Characterization of a new insect cell line (NTU-YB) derived from the common grass yellow butterfly, Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera) and its susceptibility to microsporidia

A new lepidopteran cell line, NTU-YB, was derived from pupal tissue of Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera). The doubling time of YB cells in TNM-FH medium supplemented with 8% FBS at 28 °C was 26.87 h. The chromosome numbers of YB cells varied widely from 21 to 196 with a mean of 86. Compared to other insect cell lines, the YB cells produced distinct esterase, malate dehydrogenase, and lactate dehydrogenase isozyme patterns. Identity of the internal transcribed spacer region-I (ITS-I) of YB cells to E. hecabe larvae was 96% and to Eurema blanda larvae (tissue isolated from head) was 81%. The YB cells were permissive to Nosema sp. isolated from E. blanda and the infected YB cells showed obvious cytopathic effects after 3 weeks post inoculation. The highest level of spore production was at 4 weeks post inoculation when cells were infected with the Nosema isolate, and spore production was 1.34 ± 0.9 × 10⁶ spore/ml. Ultrastructrual studies showed that YB cells can host in vitro propagation of the E. blanda Nosema isolate, and developing stages were observed in the host cell nuclei as observed in the natural host, E. blanda. The NTU-YB cell line is also susceptible to Nosema bombycis.     

Biochemical biomarkers in chronically metal-stressed daphnids

Biochemical biomarkers are a popular measure of toxic effects on organisms due to their assumed fast response, and are usually assessed after acute exposure of the organism to the stressor. However, increasing interest in the use of biochemical biomarkers in environmental pollution monitoring calls for more laboratory long-term studies of contaminants' effects on biochemical endpoints. In this study, four biochemical biomarkers (protein content, activity of cholinesterase (ChE), catalase (CAT) and glutathione S-transferase (GST), were correlated with standardised reproductive and survival endpoints of water fleas (Daphnia magna) after chronic exposure to Cr (VI) and Cd. No effect on the reproduction and survival was noticed up to the highest tested concentration of Cr (VI) (52.5 microg/L), while the protein content, and the ChE and CAT activity decreased, and GST activity increased. Cd affected reproduction of daphnids above 0.656 microg/L, but the protein content and ChE activity were changed at 0.328 microg/L and 0.082 microg/L of Cd, respectively. Biochemical biomarkers in some cases proved to be equally or more sensitive than reproduction and mortality. We recommend more frequent use of a battery of biochemical biomarkers in combination with other higher-level biomarkers also in chronic studies and not only in the acute ones.     

Effects of Cr(VI) on the performance and kinetics of the activated sludge process

The substrates removal performance, removal kinetics and the electron transport system (ETS) of sludge were investigated by sequencing batch reactors (SBR) and batch assays, respectively. Compared to the control system, significant decreases were observed in substrate removal efficiency with the Cr(VI)-feeding concentration up to 5 mg L⁻¹ in SBR system. And the recovery for NH₄⁺-N removal were more difficult than that of COD after the termination of Cr(VI)-feeding. Significant inhibitory effects of Cr(VI) on the ETS activity and substrate removal kinetics were observed in the batch assays. The inhibitory effects of Cr(VI) would be overestimated on COD removal and underestimated on NH₄⁺-N removal by the short-term batch assay as compared to the long-term operations. Additionally, significant correlations between the ETS activity and the inhibitory rates of Cr(VI) on substrate removal indicated the ETS activity can provide effective predictions on the potential performance of substrate removal in activated sludge.     

Response of blood vessels in vitro to hyperbaric oxygen (HBO): Modulation of VEGF and NOx release by external lactate or arginine

Hyperbaric oxygen therapy (HBO) is suggested to promote angiogenesis during wound healing, but the mechanisms involved are not understood. This study used a novel isolated blood vessel preparation to explore the effects of air, normobaric oxygen or hyperbaric oxygen (2.2 ATA for 90 min) on the angiogenesis factor, vascular endothelial growth factor (VEGF), nitrite and nitrate (NO_x), lactate dehydrogenase (LDH) and lactate release from the tissue in normal Krebs Ringer, and the Ringer supplemented with either l-arginine, or 15 mM lactate to mimic a wound environment, or both (l-arginine + lactate). The in vitro blood vessel preparation remained viable during all experiments. There were no effects of HBO treatment on any of the parameters measured in normal Krebs Ringer, but some treatment-dependent effects were observed in supplemented Krebs Ringer. In the lactate supplemented Krebs Ringer, medium LDH levels increased in response to either normobaric oxygen (NBO) or HBO, compared to air alone. There were also small, but statistically significant increases in total glutathione due to HBO treatment, compared to NBO or air in the lactate supplemented medium, and in the combined supplement. There were no effects of HBO on NO_x, changes in external medium lactate levels, or tissue VEGF in any of the Krebs Ringers tested. However, post treatment increases in VEGF were observed in the lactate supplemented medium, and for lactate release into the medium for the combined supplement. We conclude that HBO does not cause NO or VEGF production from the blood vessel in normal Krebs Ringer, but the data from supplemented medium show that the response of the tissue is subtly affected by the chemical environment around the blood vessel, and the tissue is more responsive to HBO when wound conditions are mimicked.     

Exposure to Cr(VI) induces organ dependent MSI in two loci related with photophosphorylation and with glutamine metabolism

Chromium (Cr), as a mutagenic agent in plants, has received less attention than other metal pollutants. To understand if Cr induces microsatellite instability (MSI), Pisum sativum seedlings were exposed for 28 days to different concentrations of Cr(VI) up to 2000 mg L⁻¹, and the genetic instability of ten microsatellites (SSRs) was analyzed. In plants exposed to Cr(VI) up to 1000 mg  L⁻¹, MSI was never observed. However, roots exposed to 2000 mg L⁻¹ displayed MSI in two of the loci analyzed, corresponding to a mutation rate of 8.3%. SSR2 (inserted in the locus for plastid photosystem I 24 kDa light harvesting protein) and SSR6 (inserted in the locus for P. sativum glutamine synthetase) from Cr(VI)-treated roots presented alleles with, respectively, less 6 bp and more 3 bp than the corresponding controls. This report demonstrates that: (a) SSRs technique is sensitive to detect Cr-induced mutagenicity in plants, being Cr-induced-MSI dose and organ dependent (roots are more sensitive); (b) two Cr-sensitive loci are related with thylakoid photophosphorylation and with glutamine synthetase, respectively; (c) despite MSI is induced by Cr(VI), it only occurs in plants exposed to concentrations higher than 1000 mg L⁻¹ (values rarely found in real scenarios). Considering these data, we also discuss the known functional changes induced by Cr(VI) in photosynthesis and in glutamine synthetase activity.     

Long-term exposure to chromium(VI) oxide leads to defects in sulfate transport system in chinese hamster ovary cells

Chromium(VI) resistant Chinese hamster ovary (CHO) cell lines were established in this study by exposing parental CHO-K1 cells to sequential increases in CrO₃ concentration. The final concentration of CrO₃ used for selection was 7 μM for Cr7 and 16 μM for Cr16 cells. Cr16-1 was a subclone derived from Cr16 cells. Next, these resistant cells were cultured in media without CrO₃ for more than 6 months. The resistance of these cells to CrO₃ was determined by colony-forming ability following a 24-h treatment. The LD₅₀ of CrO₃ for chromium(VI) resistant cells was at least 25-fold higher than that of the parental cells. The cellular growth rate, chromosome number, and the hprt mutation frequency of these chromium(VI) resistant cells were quite similar to their parental cells. The glutathione level, glutathione S-transferase, catalase activity, and metallothionine mRNA level in Cr7 and Cr16-1 cells were not significantly different from their parental cells. Furthermore, Cr16-1 cells were as sensitive as CHO-K1 cells to free-radical generating agents, including hydrogen peroxide, nickel chloride, and methanesulfonate methyl ester, and emetine, i.e., a protein synthesis inhibitor. The uptake of chromium(VI) and the remaining amount of this metal in these resistant and the parental cell lines were assayed by atomic absorption spectrophotometry. Experimental results indicated that a vastly smaller amount of CrO₃ entered the resistant cell lines than their parental cells did. A comparison was made of the sulfate uptake abilities of CHO-K1 and chromium(VI) resistant cell lines. These results revealed that the uptake of sulfate anion was substantially reduced in Cr7 and Cr16-1 cells. Extracellular chloride reduced sulfate uptake in CHO-K1 but not in Cr16-1 cells. Therefore, the major causative for chromium(VI) resistance in these resistant cells could possibly be due to the defects in SO₄^2-/C1^? transport system for uptake chromium(VI).     

Vitellogenesis and other physiological responses induced by 17-β-estradiol in males of freshwater fish Rhamdia quelen

This study investigated the effects of different doses of 17-β-estradiol (E₂) in Rhamdia quelen. Groups of males exposed to different doses of E₂ (0.1 mg kg^− 1, 1 mg kg^− 1 and 10 mg kg^− 1) were compared with non-exposed male and female fish groups. Among the considered biomarkers, no significant differences were observed for micronuclei test, reduced glutathione concentration and lipid peroxidation. All E₂-treated individuals had decreased glutathione S-transferase activity. Increased catalase and superoxide dismutase activities, increased vitellogenin expression and decreased metallothionein concentration were observed in males treated with the highest dose. Liver of all test groups showed necrotic areas, but cytoplasm vacuolization was again found only in the individuals exposed to highest dose. E₂ causes deleterious hepatic effects to R. quelen, and vitellogenin expression, catalase and superoxide dismutase activity and metallothionein concentration represent appropriate biomarkers for studying E₂ effects. Additionally, the response of some biomarkers was similar in males exposed to E₂ and unexposed females, and therefore exposure to endocrine disruptors may cause consequences for fish populations.     

Adsorption of Cr(VI) and As(V) ions by modified magnetic chitosan chelating resin

Cross-linked magnetic chitosan anthranilic acid glutaraldehyde Schiff's base (CAGS) was prepared for adsorption of both As(V) and Cr(VI) ions and their determination by ICP-OES. Prepared cross-linked magnetic CAGS was investigated by means of SEM, FTIR, wide angle X-ray diffraction (WAXRD) and TGA analysis. The adsorption properties of cross-linked magnetic CAGS resin toward both As(V) and Cr(VI) were evaluated. Various factors affecting the uptake behavior such as pH, temperature, contact time, initial concentration of metal ions, effect of other ions and desorption were studied. The equilibrium was achieved after about 110 min and 120 min for As(V) and Cr(VI), respectively at pH = 2. The adsorption kinetics followed the mechanism of the pseudo-second order equation for all systems studied, evidencing chemical sorption as the rate-limiting step of adsorption mechanism and not involving a mass transfer in solution. The equilibrium data were analyzed using the Langmuir, Freundlich, and Tempkin isotherm models. The best interpretation for the equilibrium data was given by Langmuir isotherm, and the maximum adsorption capacities were 58.48 and 62.42 mg/g for both Cr(VI) and As(V), respectively. Cross-linked magnetic CAGS displayed higher adsorption capacity for Cr(VI). The adsorption capacity of the metal ions increased with increasing temperature under optimum conditions in case of Cr(VI), but decreased in case of As(V). The metal ion-loaded cross-linked magnetic CAGS were regenerated with an efficiency of greater than 88% using 0.2 M sodium hydroxide (NaOH).     

Evaluation and elimination of inhibitory effects of salts and heavy metal ions on biodegradation of Congo red by Pseudomonas sp. mutant

In this study, it was attempted to evaluate the influences and also recommended some elimination methods for inhibitory effects offered by salts and heavy metal ions. Congo red dye solution treated with mutant Pseudomonas sp. was taken as a model system for study. The salts used in this study are NaCl, CaCl₂ and MgSO₄·7H₂O. Though the growth was inhibited at concentrations above 4 g/l, toleration was achieved by acclimatization process. In case of heavy metal ions, Cr (VI) showed low inhibition up to 500 mg/l of concentration, compared to Zn (II) and Cu (II). It was due to the presence of chromium reductase enzyme which was confirmed by SDS-PAGE. Zn (II) and Cu (II) ion inhibitions were eliminated by chelation with EDTA. The critical ion concentrations obtained as per Han-Levenspiel model for Cr (VI), Zn (II) and Cu (II) were 0.8958, 0.3028 and 0.204 g/l respectively.     

Remediation of chromite ore processing residue by pyrolysis process with sewage sludge

The present work developed a novel technique to treat chromite ore processing residue (COPR). The process involved mixing the COPR with sewage sludge followed by pyrolysis. The gaseous organic fraction generated during pyrolysis of sludge was beneficial to Cr(VI) reduction. Process variables, such as the amount of sludge added to COPR (sludge-to-COPR (S/C) ratio), heating temperature, reaction time and particle size, were systematically varied, and their influences on the Cr(VI) reduction in COPR were investigated. Cr(VI) content had decreased greatly, from 3384 mg kg⁻¹ for untreated COPR to less than 30 mg kg⁻¹ for COPR treated at 600 °C.     

Measure of stress response induced by temperature and salinity changes on hatched larvae of three marine gastropod species

To better understand the cascade of molecular reactions leading to delayed development and mortality of early life stages of marine intertidal gastropods, in response to temperature and salinity changes associated with climate change, three biomarkers: total antioxidant capacity, lipid peroxidation and lysosomal stability were investigated on hatched larvae. Encapsulated embryos of three marine gastropod species (Bembicium nanum, Siphonaria denticulata and Dolabrifera brazieri), which have already proven responsive to thermal and osmotic variations, were exposed to six combinations of temperature (22 °C and 30 °C) and salinity (25‰, 35‰ and 45‰) until the larvae hatched. Time to hatching was affected by salinity and temperature in all three species. High salinity (45‰) generally retarded the hatching process although the response was species-specific for temperature. Total antioxidant capacity and lipid peroxidation were also highly species-specific with the general trend showing that these biomarkers were adversely affected by high temperature (30 °C) at salinities of 25‰ and 45‰. Bembicium nanum lysosomal destabilisation increased significantly with an increase in temperature and salinity (30 °C and 45‰) and this was associated with delayed development and increased mortality. Investigations on the additional biomarker, lysosomal stability, gave a clearer picture of the numerous and complex molecular and cellular mechanisms leading to mortality and underdevelopment in response to environmental stress for this species. As few differences were observed in the enzymatic biomarkers total antioxidant capacity and lipid peroxidation between hatched larvae and the previously investigated encapsulated embryo response to thermal and osmotic stress, it is suggested that further studies could be undertaken using embryos encapsulated in egg masses, as it is less time consuming than working on hatched larvae.     

Uptake and Translocation of Tri- and Hexa-Valent Chromium and Their Effects on Black Gram (Vigna mungo L. Hepper cv. Co4) Roots

An equal concentration (100 μM) of Cr(III)- and Cr(VI)-induced changes in activities of antioxidative enzymes and metabolites of ascorbate-glutathione cycle was studied in 7-d-old black gram (Vigna mungo L Hepper cv. Co4) seedlings for 5-d after infliction of Cr stress. Seeds were germinated and grown in the presence or absence of Cr under controlled environmental conditions. Uptake and translocation of Cr rate was relatively higher during first 12 h of treatment with both speciation of Cr, Cr(III)- and Cr(VI)-treated black gram roots retained 15 times more Cr than the shoots. Significantly increased lipid peroxidation was observed in the form of accumulation of malondialdehyde (MDA) and production of hydrogen peroxide (H₂O₂) molecule and superoxide (O₂ ) radical after 6 h of infliction with Cr(VI) and after 12 h in Cr(III)-treated black gram roots. Superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities were significantly increased under Cr(VI)-treatment after 12 and 6 h, respectively. However, catalase (CAT) and monodehydroascorbate reductase (MDHAR) activities were not significantly increased under Cr(Ill)-treatment. There was a steep increase of 2.71 μmol g^-1 FW in ascorbic acid (AA) content was observed between 6 and 24 h of Cr(VI)-treatment. Oxidized glutathione (GSSG) content was steadily increased through the course of Cr(III)- and Cr(VI)-treatments, where as reduced glutathione (GSH) level was decreased after 24 h of treatment. GSH/GSSG ratio was rapidly decreased in treatment with Cr(III) than the Cr(VI). There was significant increase of 99 nmol g^-1 FW in non-protein thiol (NPT) content was recorded between 6 and 24 h of Cr(VI)-treatment. The present results showed differential response to AA and H₂O₂ signaling by Cr(III) and Cr(VI), AA in combination with APX was more effective in mitigating oxidative stress as against the role of GSH as an antioxidant.