Effects of Excess Dietary Fluoride on Serum Biochemical Indices,Egg Quality,and Concentrations of Fluoride in Soft Organs,Eggs, and Serum of Laying Hens

According to undiscovered toxicity and safety of magnesium oxide nanoparticles (MgO NPs) in isolated pancreatic islet cells, this study was designed to examine the effects of its various concentrations on a time-course basis on the oxidative stress, viability, and function of isolated islets of rat’s pancreas. Pancreatic islets were isolated and exposed to different MgO NP (<100 nm) concentrations within three different time points. After that, oxidative stress biomarkers were investigated and the best exposure time was selected. Then, safety of MgO NPs was investigated by flow cytometry and fluorescent staining, and levels of insulin secretion and caspase activity were measured. The results illustrated a considerable decrease in oxidative stress markers such as reactive oxygen species (ROS) and lipid peroxidation (LPO) levels of pancreatic islets which were treated by MgO NPs for 24 h. Also, in that time of exposure, cell apoptosis investigation by flow cytometry and insulin test showed that MgO NPs, in a concentration of 100 μg/ml, decreased the rate of apoptotic cells via inhibiting caspase-9 activity and made a significant increase in the level of insulin secretion. Data of function and apoptosis biomarkers correlated with each other. It is concluded that the use of MgO NPs in concentration of as low as 100 μg/ml can induce antiapoptotic, antioxidative, and antidiabetic effects in rat pancreatic islets, which support its possible benefit in islet transplantation procedures.     

L-Ascorbate Protects Against Methamphetamine-Induced Neurotoxicity of Cortical Cells via Inhibiting Oxidative Stress,Autophagy, and Apoptosis

Methamphetamine (METH)-induced cell death contributes to the pathogenesis of neurotoxicity; however, the relative roles of oxidative stress, apoptosis, and autophagy remain unclear. L-Ascorbate, also called vitamin (Vit.) C, confers partial protection against METH neurotoxicity via induction of heme oxygenase-1. We further investigated the role of Vit. C in METH-induced oxidative stress, apoptosis, and autophagy in cortical cells. Exposure to lower concentrations (0.1, 0.5, 1 mM) of METH had insignificant effects on ROS production, whereas cells exposed to 5 mM METH exhibited ROS production in a time-dependent manner. We confirmed METH-induced apoptosis (by nuclear morphology revealed by Hoechst 33258 staining and Western blot showing the protein levels of pro-caspase 3 and cleaved caspase 3) and autophagy (by Western blot showing the protein levels of Belin-1 and conversion of microtubule-associated light chain (LC)3-I to LC3-II and autophagosome staining by monodansylcadaverine). The apoptosis as revealed by cleaved caspase-3 expression marked an increase at 18 h after METH exposure while both autophagic markers, Beclin 1 and LC3-II, marked an increase in cells exposed to METH for 6 and 24 h, respectively. Treating cells with Vit. C 30 min before METH exposure time-dependently attenuated the production of ROS. Vitamin C also attenuated METH-induced Beclin 1 and LC3-II expression and METH toxicity. Treatment of cells with Vit. C before METH exposure attenuated the expression of cleaved caspase-3 and reduced the number of METH-induced apoptotic cells. We suggest that the protective effect of Vit. C against METH toxicity might be through attenuation of ROS production, autophagy, and apoptosis.     

Effects of Extremely Low-Frequency Magnetic Field on Caspase Activities and Oxidative Stress Values in Rat Brain

This study was aimed to investigate the effect of extremely low-frequency magnetic field (ELF-MF) on apoptosis and oxidative stress values in the brain of rat. Rats were exposed to 100 and 500 µT ELF-MF, which are the safety standards of public and occupational exposure for 2 h/day for 10 months. Brain tissues were immunohistochemically stained for the active (cleaved) caspase-3 in order to measure the apoptotic index by a semi-quantitative scoring system. In addition, the levels of catalase (CAT), malondialdehyde (MDA), myeloperoxidase (MPO), total antioxidative capacity (TAC), total oxidant status (TOS), and oxidative stress index (OSI) were measured in rat brain. Final score of apoptosis and MPO activity were not significantly different between the groups. CAT activity decreased in both exposure groups (p?<?0.05), while TAC was found to be lower in ELF 500 group than those in ELF-100 and sham groups (p?<?0.05). MDA, TOS, and OSI values were found to be higher in ELF-500 group than those in ELF-100 and sham groups (p?<?0.05). In conclusion, apoptosis was not changed by long-term ELF-MF exposure, while both 100 and 500 µT ELF-MF exposure induced toxic effect in the rat brain by increasing oxidative stress and diminishing antioxidant defense system.     

A rapid in vivo zebrafish model to elucidate oxidative stress-mediated PCB126-induced apoptosis and developmental toxicity

Dioxin-like 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) is one of the most potent and widespread environmental pollutants. Although PCB126-induced toxicity is related to the aryl hydrocarbon receptor pathway, there is still no study that has constructed an in vivo visual model to clarify the role of the Nrf2/ARE signaling pathway in the oxidative stress mechanism of PCB126-induced toxicity. In the present study, an in vivo zebrafish model of nrf2a fused to enhanced green fluorescent protein (nrf2a-eGFP) was constructed. The zebrafish embryos microinjected with nrf2a-eGFP (72 h postfertilization) were exposed to various concentrations of PCB126 (0, 25, 50, 100, 200 μg/L) or 30 mM N-acetylcysteine (NAC)+200 μg/L PCB126. After 72 h exposure, PCB126 significantly increased the malformation rates and induced eGFP expression in a dose-dependent manner in several zebrafish tissue types. The distribution of eGFP fluorescence coincided with developmental deformity sites. NAC pretreatment effectively counteracted PCB126-induced developmental toxicity including heart rate, pericardial edema, and body length. The highest PCB126 dose, 200 μg/L, produced marked apoptosis in the eye, gill, and trunk detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. At 48 and 72 h exposure, 200 μg/L PCB126 affected glutathione metabolism as evidenced by decreased glutathione and increased glutathione disulfide concentrations, indicative of oxidative stress. These effects were also counteracted by NAC pretreatment. Furthermore, the Nrf2-regulated genes gclc, gpx, gstp1, and hmox1 were significantly induced at 24, 48, and 72 h at the highest PCB126 exposures but not in the NAC-pretreated group. In addition, a significant increase in ROS generation was detected in zebrafish larvae at 72 h PCB126 exposure, which might offer a link for future mechanistic studies. Collectively, these data suggest that PCB126-induced developmental toxicity and apoptosis in the nrf2a-eGFP-injected zebrafish model are due to oxidative stress mediated by disruption to glutathione metabolism and changes in Nrf2-regulated gene expression.     

Gene delivery to pancreatic exocrine cells in vivo and in vitro

Background

Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish) and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38) in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff), contains three copies of oestrogen response elements (3ERE) that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS) elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein).

Results

The response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1?C2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17?-oestradiol (E₂), the synthetic oestrogen 17??- ethinyloestradiol (EE₂), and the relatively weak environmental oestrogen nonylphenol (NP), and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE₂; 10?ng/L, E₂; 100?ng/L, after 72?h exposures). For the NP exposures, GFP expression was observed at 10???g NP/L after 72?h (100???g NP/L was toxic to the fish). We also demonstrate that our construct works in medaka, another model fish test species, suggesting the transient assay is applicable for testing oestrogenic chemicals in fish generally.

Conclusion

Our results indicate that the transient expression assay system can be used as a rapid integrated testing system for environmental oestrogens and to detect the oestrogenic target sites in developing fish embryos.     

The relative sensitivity of sperm, eggs and embryos to copper in the blue mussel (Mytilus trossulus)

Copper, an essential element, is toxic at elevated concentrations, and as a result of anthropogenic activities is becoming increasingly prevalent in marine environments. In this study, we examined the effects of copper on early life stages of the blue mussel, Mytilus trossulus. We assessed the impacts of increasing copper concentrations on embryo development, egg viability, sperm fertilization capacity and, in particular, on sperm swimming speed using computer-assisted sperm analysis. Sensitivity to copper followed the pattern: embryos > sperm > eggs. A dramatic increase in abnormal embryo development was observed following exposure to copper concentrations exceeding 10 microg/L. Sperm swimming speeds decreased significantly when exposed to 100 microg/L of copper, but lower doses did not influence sperm swimming speed. Copper exposure (at any tested concentration) did not affect sperm flagellum length, or alter egg viability. Based on our results, we suggest that exposure of sperm to copper may interfere with mitochondrial activity, which reduces sperm swimming speed during the extended duration of sperm motility in blue mussel.     

4-Vinylcyclohexene diepoxide disrupts sperm characteristics,endocrine balance and redox status in testes and epididymis of rats

Objectives: Exposure to 4-vinylcyclohexene diepoxide (VCD) was reported to induce testicular germ cell toxicity in rodents. However, there is paucity of information on the precise biochemical and molecular mechanisms of VCD-induced male reproductive toxicity.

Methodology: This study investigated the influence of VCD on testicular and epidydimal functions following oral exposure of Wistar rats to VCD at 0, 100, 250 and 500?mg/kg for 28 consecutive days.

Results: Administration of VCD significantly decreased the body weight gain and organo-somatic indices of the testes and epididymis. When compared with the control, VCD significantly decreased superoxide dismutase and catalase activities in the testes whereas it significantly decreased superoxide dismutase activity but increased catalase activity in the epididymis. Moreover, while glutathione peroxidase activity and glutathione level remain unaffected, exposure of rats to VCD significantly increased glutathione S-transferase activity as well as hydrogen peroxide and malondialdehyde levels in testes and epididymis of the treated rats. The spermiogram of VCD-treated rats showed significant decrease in epididymal sperm count, sperm progressive motility, testicular sperm number and daily sperm production when compared with the control. Administration of VCD significantly decreased circulatory concentrations of follicle-stimulating hormone, luteinizing hormone and testosterone along with testicular and epididymal degeneration in the treated rats. Immunohistochemical analysis showed significantly increased cyclooxygenase-2, inducible nitric oxide synthase, caspase-9 and caspase-3 protein expressions in the testes of VCD-treated rats.

Conclusion: Exposure to VCD induces testicular and epidydimal dysfunctions via endocrine suppression, disruption of antioxidant enzymes activities, increase in biomarkers of oxidative stress, inflammation and apoptosis in rats.     

The effects of organic and inorganic phosphates on fertilization and early development in the sea urchin Lytechinus variegatus (Echinodermata: Echinoidea)

The embryos of the sea urchin Lytechinus variegatus are capable of surviving chronic exposure to inorganic sodium phosphate and organic triethyl phosphate concentrations as high as 6 and 1000 mg l(-1) seawater, respectively. However, chronic exposure to sub-lethal concentrations of these phosphates may cause arrested or abnormal embryonic development. We measured fertilization success and percentages of normal, arrested and abnormal embryos exposed to low, medium and high sub-lethal concentrations of inorganic and organic phosphate. Fertilization success was significantly reduced in all phosphate treatments. After attaining the 4-cell stage, embryos exposed to the highest phosphate concentrations displayed arrested development. Percentages of abnormally developing embryos showed a strong concentration dose-response with a significant increase in abnormal embryonic development with increasing phosphate concentration. Overall, these results indicate that the gametes and embryos of L. variegatus may provide a rapid and sensitive model bioassay for the evaluation of phosphate pollutants in marine systems. Our findings also indicate that shallow-water populations of L. variegatus spawning in areas subjected to inorganic and organic phosphate pollutants may suffer detrimental effects on fertilization and embryonic development.     

Pravastatin attenuates carboplatin-induced cardiotoxicity via inhibition of oxidative stress associated apoptosis

The objective of this study was to evaluate the cardiac toxicity induced by carboplatin, a second generation platinum-containing anti-cancer drug, and to test whether pravastatin can reduce this cardio-toxicity. In the present study, infusion of carboplatin (100 mg/kg) to mice resulted in decreased survival rates and abnormal cardiac histology, concomitant with increased cardiac apoptosis. In addition, treatment of cultured rat cardiomyocytes with carboplatin (100 μM for 48 h) caused marked apoptosis and increased caspase-3, -9, and cytochrome C, but decreased BCL-XL protein expression, and this was inhibited by reactive oxygen species (ROS) scavenger n-acetylcysteine. Furthermore, pretreatment of cardiomyocytes with pravastatin (20 μM) before carboplatin exposure significantly attenuated apoptosis and decreased caspase-3, -9, cytochrome C activity. Lastly, mice pre-treated with pravastatin before carboplatin treatment showed improved survival rate and cardiac function, with reduced cardiomyocyte apoptosis via activating Akt and restoring normal mitochondrial HAX-1 in heart tissue. In summary, our results show that carboplatin can induce cardiotoxicity in vivo and in cultured cells via a mitochondrial pathway related to ROS production, whereas pravastatin administration can reduce such oxidative stress thus prevented cardiac apoptosis. Therefore, pravastatin can be used as a cytoprotective agent prior to carboplatin chemotherapy. Ching-Feng Cheng and Shu-Hui Juan contributed equally to the work.     

The Response of Metallothionein and Malondialdehyde after Exclusive and Combined Cd/Zn Exposure in the Crab Sinopotamon henanense

The purpose of this paper is to show the interactions of Cd and Zn in the freshwater crab Sinopotamon henanense through metallothionein (MT) and malondialdehyde (MDA) level measurements. Laboratory acclimated S.henanense were exposed to Cd (50 µg/L, 100 µg/L, 500 µg/L ), and Zn (100 µg/L, 1000 µg/L) alone and in combined treatments (100 µg/L Zn+50 µg/L Cd, 100 µg/L Zn+100 µg/L Cd, 100 µg/L Zn+500 µg/L Cd, 1000 µg/L Zn+50 µg/L Cd, 1000 µg/L Zn+100 µg/L Cd, 1000 µg/L Zn+500 µg/L Cd) for 7, 14, 21, 28, 35 days. The results demonstrated that the MDA contents increased with exposure time and dose and showed time- and dose-dependence in both gills and hepatopancreas of S.henanense after single Cd exposure, while the changes of MDA levels were not significant with single Zn exposure. The MDA levels decreased when the crabs were exposed to metal mixtures compared to Cd exposure alone, indicating that Zn mediated the cellular toxicity of Cd. MT contents increased after single Cd exposure and also showed a time- and dose-dependence, in a tissue-specific way. Zn showed a limited ability of MT induction both in gills and hepatopancreas of S.henanense. The MT contents represented not a simple addition of single metal exposures but were enhanced at a higher concentration of Zn combined with different Cd concentrations compared to single metal exposure. Whether MT can be used as a biomarker for complex field conditions need to be considered cautiously since different induction patterns of MT were found among single Zn, Cd and combined groups. It is suggested that several biomarkers together as a suite should be used in the monitoring of heavy metal pollution in the aquatic environment.     

Evaluation of Arthrospira platensis extracellular polymeric substances production in photoautotrophic, heterotrophic and mixotrophic conditions

The kinetic study of Arthrospira platensis extracellular polymeric substances (EPS) production under different trophic modes??photoautotrophy (100???mol photons m^?2?s^?1), heterotrophy (1.5?g/L glucose), and mixotrophy (100???mol photons m^?2?s^?1 and 1.5?g/L glucose)??was investigated. Under photoautotrophic and heterotrophic conditions, the maximum EPS production 219.61?±?4.73 and 30.30?±?1.97?mg/L, respectively, occurred during the stationary phase. Under a mixotrophic condition, the maximum EPS production (290.50?±?2.21?mg/L) was observed during the early stationary phase. The highest specific EPS productivity (433.62?mg/g per day) was obtained under a photoautotrophic culture. The lowest specific EPS productivity (38.33?mg/g per day) was observed for the heterotrophic culture. The effects of glucose concentration, light intensity, and their interaction in mixotrophic culture on A. platensis EPS production were evaluated by means of 32 factorial design and response surface methodology. This design was carried out with a glucose concentration of 0.5, 1.5, and 2.5?g/L and at light levels of 50, 100, and 150???mol photons m^?2?s^?1. Statistical analysis of the model demonstrated that EPS concentration and EPS yield were mainly influenced by glucose concentration and that conditions optimizing EPS concentration were dissimilar from those optimizing EPS yield. The highest maximum predicted EPS concentration (369.3?mg/L) was found at 150???mol photons m^?2?s^?1 light intensity and 2.4?g/L glucose concentration, while the highest maximum predicted EPS yield (364.3?mg/g) was recorded at 115???mol photons m^?2?s^?1 light intensity and 1.8?g/L glucose concentration.     

Somatic embryogenesis, plant regeneration, and cryopreservation for Torreya taxifolia, a highly endangered coniferous species

Torreya taxifolia Arn., an ancient evergreen tree, is on the brink of extinction from attack by a fungal disease, recently reported to be caused by a novel isolate of Fusarium. We report the development of a somatic embryogenesis tissue culture system that can be used for cryogenic storage of T. taxifolia cultures and subsequent plant regeneration. Initiation of embryogenic tissue from immature zygotic embryos occurred on a conifer tissue culture medium containing 0.25?% activated charcoal, 43.8?mM maltose, 0.5?mM 2,4-dichlorophenoxacetic acid, 0.2?mM 6-benzylaminopurine, 0.2?mM kinetin, 0.1???M brassinolide, 3.8???M abscisic acid, 20.5???M biotin, 1.13???M folic acid, 1.28?mM 2(n-morpholino)ethanesulfonic acid and 0.69?mM pyruvic acid. Embryo induction ranged from 60 to 100?% across six seed sources. Somatic embryo development occurred on a medium containing 43.8?mM maltose, 1?% activated charcoal, 37.8???M abscisic acid, 20.5???M biotin, 0.1???M brassinolide, 0.205?mM folic acid, 1.28?mM 2(n-morpholino)ethanesulfonic acid and 0.69?mM pyruvic acid. Germination of somatic embryos ranged from 64 to 82?%. Embryogenic tissue cultures from 30 genotypes representing seed from six mother trees were cryopreserved, and culture recovery was demonstrated after freezing. In contrast to many other coniferous tree seeds, the measured water potential (?MPa) of T. taxifolia megagametophyte tissue rose greatly during seed after-ripening. Duplication of this rise in vitro allowed development of somatic embryos to the cotyledonary stage.     

Toxicity of Buprofezin on the Survival of Embryo and Larvae of African Catfish,Clarias gariepinus (Bloch)

Buprofezin is an insect growth regulator and widely used insecticide in Malaysia. The present study evaluated the toxic effects of buprofezin on the embryo and larvae of African catfish (Clarias gariepinus) as a model organism. The embryos and larvae were exposed to 7 different concentrations (0, 0.05, 0.5, 5, 25, 50 and 100 mg/L) of buprofezin. Each concentration was assessed in five replicates. Eggs were artificially fertilized and 200 eggs and larvae were subjected to a static bath treatment for all the concentrations. The mortality of embryos was significantly increased with increasing buprofezin concentrations from 5 to 100 mg/L (p< 0.05). However, the mortality was not significantly different (p<0.05) among the following concentrations: 0 (control), 0.05, 0.5 and 5 mg/L. Data obtained from the buprofezin acute toxicity tests were evaluated using probit analysis. The 24 h LC₅₀ value (with 95% confidence limits) of buprofezin for embryos was estimated to be 6.725 (3.167-15.017) mg/L. The hatching of fish embryos was recorded as 68.8, 68.9, 66.9, 66.4, 26.9, 25.1 and 0.12% in response to 7 different concentrations of buprofezin, respectively. The mortality rate of larvae significantly (p<0.05) increased with increasing buprofezin concentrations exposed to 24-48 h. The 24 and 48 h LC₅₀ values (with 95% confidence limits) of buprofezin for the larvae was estimated to be 5.702 (3.198-8.898) and 4.642 (3.264-6.287) mg/L respectively. There were no significant differences (p>0.05) in the LC₅₀ values obtained at 24 and 48 h exposure times. Malformations were observed when the embryos and larvae exposed to more than 5 mg/L. The results emerged from the study suggest that even the low concentration (5 mg/L) of buprofezin in the aquatic environment may have adverse effect on the early embryonic and larval development of African catfish.     

Regulation of vinblastine biosynthesis in cell suspension cultures of catharanthus roseus

Various elicitors of hydroxylase, peroxidase, acetyltransferase and inhibitors of oxygenase were added to a Catharanthus roseus cell culture medium to investigate the regulatory effects on tabersonine, vindoline and vinblastine biosynthesis. Hydrogen peroxide was found to be the most effective agent for enhancing the biosynthesis of tabersonine. By adding 20???g/L hydrogen peroxide, the tabersonine concentration reached 9.02?mg/g dry weight (DW) after culturing cell suspensions for 7?days. With the addition of 30???g/L acetyl CoA, the most vindoline (final cell content of 0.33?mg/g DW) was produced. By effective inhibition of lochnericine biosynthesis with the addition of 0.5???mol/L benzotriazole, the cell content of vindoline was increased to 0.42?mg/g DW. An orthogonal experiment consisting of multiple regulation factors was carried out to optimize vinblastine biosynthesis. It was shown that optimal vinblastine biosynthesis was achieved by addition of 5?mg/L acetyl CoA, 20???g/L hydrogen peroxide, 0.5???mol/L benzotriazole, 100?mg/L tryptophan, 100?mg/L loganin and 30?mg/L cerium chloride. Under these conditions, the cell content of vinblastine reached 0.81?mg/g DW. Simultaneous changes in cell content and enzyme activities of Cytochrome P-450 monooxygenases, Deacetylvindoline-O-acetyltransferase and Peroxidase enzyme indicated that these enzymes were closely linked to vinblastine biosynthesis.     

Induction of embryonic dysmorphogenesis by high glucose concentration, disturbed inositol metabolism, and inhibited protein kinase C activity

BACKGROUND: Exposure to a diabetic environment causes excess reactive oxygen species (ROS), decreased prostaglandin E(2) (PGE(2)) concentration, and increased embryonic maldevelopment. The aim of the present work was to study whether embryonic dysmorphogenesis is also dependent on alterations of inositol and associated intracellular metabolites. METHODS: Day 9 rat embryos were cultured for 24 or 48 hr and evaluated for gene expression. Day 10 and day 11 embryos from normal and diabetic rats were also examined. RT-PCR was used to study embryonic gene expression of protein kinase C (PKC) and cytosolic phospholipase A(2) (cPLA(2)). RESULTS: Embryos exposed to 30 mmol/L glucose (30G), 500 or 750 micromol/L of scyllo-inositol (500SI or 750SI) had higher malformation score than control embryos cultured in 10 mmol/L glucose (10G). Adding 1.6 mmol/L inositol to the 30G or 750SI culture medium partly corrected these embryos, and completely normalized 500SI embryonic development. Adding 0.5 mmol/L N-acetylcysteine (NAC) or 280 nmol/L PGE(2) protected, and failed to protect, the SI-exposed embryos, respectively. 10G embryos exposed to the PKC inhibitor GF-109203X displayed dose-dependent dysmorphogenesis. Addition of 1.6 mmol/L inositol or 0.5 mmol/L NAC to the PKC-inhibitor-exposed 10G embryos largely normalized the outcome, whereas PGE(2) again failed to protect embryonic development. 30G culture tended to decrease the expression of cPLA(2) after 24 hr in vitro. We also found decreased mRNA levels of cPLA(2) in offspring of diabetic rats on gestational day 10 and of PKC on day 11, as compared with normal offspring. CONCLUSIONS: High glucose concentration causes dysmorphogenesis in embryos by an interaction of oxidative stress and inositol depletion.     

Oxygen requirements of zebrafish (Danio rerio) embryos in embryo toxicity tests with environmental samples

The zebrafish embryo test is a widely used bioassay for the testing of chemicals, effluents and other types of environmental samples. Oxygen depletion in the testing of sediments and effluents is especially important and may be a confounding factor in the interpretation of apparent toxicity. In order to identify oxygen levels critical to early developmental stages of zebrafish, oxygen consumption of zebrafish embryos between 0 and 96h post-fertilization, minimum oxygen levels required by the embryos for survival as well as the effects of oxygen depletion following exposure to model sediments were determined. No significant effects on zebrafish embryo development were observed for oxygen concentrations between 7.15 and 3.33mg/L, whereas at concentrations between 3.0and 2.0mg/L minor developmental retardations were observed, yet without any pathological consequences. Oxygen concentrations lower than 0.88mg/L were 100% lethal. In the sediment contact tests with zebrafish embryos, native sediments rich in organic materials rapidly developed strongly hypoxic conditions, particularly at the sediment-water interface (0 to 500μm distance to the sediment).     

Determination of a Threshold Dose to Reduce or Eliminate CdTe-Induced Toxicity in L929 Cells by Controlling the Exposure Dose

With the widespread use of quantum dots (QDs), the likelihood of exposure to quantum dots has increased substantially. The application of quantum dots in numerous biomedical areas requires detailed studies on their toxicity. In this study, we aimed to determine the threshold dose which reduced or eliminated CdTe-induced toxicity in L929 cells by controlling the exposure dose. We established a cellular model of acute exposure to CdTe QDs. Cells were exposed to different concentrations of CdTe QDs (2.2 nm and 3.5 nm) followed by illustrative cytotoxicity analysis. The results showed that low concentrations of CdTe QDs (under 10 µg/mL) promoted cell viability, caused no obvious effect on the rate of cell apoptosis, intracellular calcium levels and changes in mitochondrial membrane potential, while high concentrations significantly inhibited cell viability. In addition, reactive oxygen species in the 10 µg/mL-treated group was significantly reduced compared with the control group. In summary, the cytotoxicity of CdTe QDs on L929 cell is dose-dependent, time-dependent and size-dependent. Low concentrations of CdTe QDs (below 10 µg/mL) may be nontoxic and safe in L929 cells, whereas high concentrations (above 10 µg/mL) may be toxic resulting in inhibition of proliferation and induction of apoptosis in L929 cells.     

Single-strand breaks, cell cycle arrest and apoptosis in HL-60 and LLCPK1 cells exposed to 1,2-dibromo-3-chloropropane

We investigated 1,2-dibromo-3-chloropropane (DBCP)-induced DNA damage, cell cycle alterations and cell death in two cell lines, the human leukemia HL-60 and the pig kidney LLCPK₁, both of which are derived from potential target sites for DBCP-induced toxicity. DBCP (30–300 µmol/L) caused a concentration-dependent increase in the levels of DNA single-strand breaks in both cell lines as well as in cultured human renal proximal tubular cells. After extended DBCP exposure in LLCPK₁ cells (100 µmol/L, 30 h), the level of DNA breaks returned almost to control values. Incubation for 48 h showed a clear reduction of growth with DBCP concentrations as low as 10 µmol/L. Flow cytometric analysis showed that DBCP (1–10 µmol/L) exposure for 24 h caused an accumulation of LLCPK₁ cells in the G₂/M-phase. In HL-60 cells the accumulation in G₂/M-phase was less marked, and at higher concentrations the cells accumulated in S-phase. Flow cytometric studies of HL-60 and LLCPK₁ cells exposed to 100–500 µmol/L DBCP showed increased number of apoptotic cells/bodies with a lower DNA content than that of the G₁ cells. Microscopic studies revealed that there were increased numbers of cells with nuclear condensation and fragmentation, indicating that apoptosis was the dominant mode of death in these cell lines, following exposure to DBCP. The characteristic ladder pattern of apoptotic cells was observed when DNA from DBCP-treated HL-60 cells and LLCPK₁ cells was electrophoresed in agarose. The finding that DBCP can cause an accumulation of cells in G₂/M-phase and induce apoptosis in vitro may be of importance for the development of DBCP-induced toxicity in vivo.     

Regulation of nodularin-induced apoptosis by epigallocatechin-3-gallate on fish lymphocytes in vitro

Nodularin is one of the most conspicuous and widespread pollutants that elicit water ecological hazards to fish, causing serious damage on the immune system and physiological functions. Nodularin can cause oxidative stress-induced apoptosis on fish lymphocytes. The regulatory effects of epigallocatechin-3-gallate (EGCG) at 10, 100, and 1000 μg/L levels on the antioxidant defense system and apoptosis of Carassius auratus lymphocytes exposed to a high dose of nodularin (100 μg/L) were quantified in vitro. EGCG reduced nodularin-induced oxidative damage on fish immune cells. This compound significantly increased the activities of superoxide dismutase and catalase and the level of glutathione but decreased the levels of intracellular reactive oxygen species and malondialdehyde. Flow cytometry results showed that the percentages of apoptotic cells after treatment with 10, 100, and 1000 μg/L EGCG for 12 h reached 27.9%, 19.1%, and 13.7%, respectively. By contrast, the nodularin alone-induced group showed a high percentage of apoptosis (44.2%). Western blot analysis showed the increased expression of bcl-2 and the decreased expression of bax and caspase-3 in EGCG-treated fish lymphocytes. EGCG also inhibited the potential collapse of the mitochondrial membrane. Overall, EGCG can inhibit nodularin-induced apoptosis and protect the normal immunity of fish by regulating bax/bcl-2 and blocking the downstream of mitochondrial apoptosis pathway with increased intracellular antioxidant enzyme activity.     

Hydroalcoholic Seed Extract of Coriandrum sativum (Coriander) Alleviates Lead-Induced Oxidative Stress in Different Regions of Rat Brain

Lead exposure is known to cause apoptotic neurodegeneration and neurobehavioral abnormalities in developing and adult brain by impairing cognition and memory. Coriandrum sativum is an herb belonging to Umbelliferae and is reported to have a protective effect against lead toxicity. In the present investigation, an attempt has been made to evaluate the protective activity of the hydroalcoholic extract of C. sativum seed against lead-induced oxidative stress. Male Wistar strain rats (100–120 g) were divided into four groups: control group: 1,000 mg/L of sodium acetate; exposed group: 1,000 mg/L lead acetate for 4 weeks; C. sativum treated 1 (CST1) group: 250 mg/kg body weight/day for seven consecutive days after 4 weeks of lead exposure; C. sativum treated 2 (CST2) group: 500 mg/kg body weight/day for seven consecutive days after 4 weeks of lead exposure. After the exposure and treatment periods, rats were sacrificed by cervical dislocation, and the whole brain was immediately isolated and separated into four regions: cerebellum, hippocampus, frontal cortex, and brain stem along with the control group. After sacrifice, blood was immediately collected into heparinized vials and stored at 4 °C. In all the tissues, reactive oxygen species (ROS), lipid peroxidation products (LPP), and total protein carbonyl content (TPCC) were estimated following standard protocols. An indicator enzyme for lead toxicity namely delta-amino levulinic acid dehydratase (δ-ALAD) activity was determined in the blood. A significant (p?<?0.05) increase in ROS, LPP, and TPCC levels was observed in exposed rat brain regions, while δ-ALAD showed a decrease indicating lead-induced oxidative stress. Treatment with the hydroalcoholic seed extract of C. sativum resulted in a tissue-specific amelioration of oxidative stress produced by lead.