Development of a fish in vitro cell culture model to investigate oxidative stress and its modulation by dietary vitamin E.

When cultured in Dulbecco's minimal essential medium the established epithelioma papulosum cyprini cell line from carp was found to be vitamin E-deficient due to the very low level of vitamin E in the medium and the foetal calf serum used as supplement. The toxicity of oxidative stressors to this cell line was evaluated by means of the neutral red cytotoxicity assay and it was found that an organic hydroperoxide, t-butylhydroperoxide was extremely cytotoxic and that the redox-cycling agents diquat and menadione were less toxic. When grown under vitamin E supplementation (25 microM), the toxicity of these chemicals was reduced by at least an order of magnitude in concentration demonstrating the protective effect of vitamin E. These data show the importance of vitamin E status for interpretation of in vitro and in vivo data and that this in vitro system is useful for mechanistic studies.     

Antioxidant response of the seagrass Posidonia oceanica when epiphytized by the invasive macroalgae Lophocladia lallemandii

The aim was to evaluate the antioxidant defences and the occurrence of oxidative damage in Posidonia oceanica under a stress situation due to the epiphytism of the invasive macroalgae Lophocladia lallemandii. P. oceanica leaves were collected in the absence (control station) and in the presence of the epiphytic algae L. lallemandii and the antioxidant enzyme activities, markers of oxidative damage, and hydrogen peroxide production were determined. Antioxidant enzyme - catalase, glutathione peroxidase and superoxide dismutase - activities were significantly higher in Posidonia epiphytized by L. lallemandii. Malondialdehyde, protein carbonyl derivates, and glutathione levels were also higher in L. lallemandii epiphytized P. oceanica leaves compared to control samples. The production of hydrogen peroxide was also significantly increased when Posidonia was epiphytized by L. lallemandii. The invasion of P. oceanica meadows by L. lallemandii appeared to induce oxidative stress in the seagrass as evidenced by increased levels of oxidative stress markers and antioxidant defences.     

Short-time induction of oxidative stress in hepatocytes of the European flounder (Platichthys flesus).

Oxidative stress induced by xenobiotic compounds has been studied using primary hepatocytes of juvenile European flounder (Platichthys flesus L.) caught in a low polluted area of the German Bight, Tiefe Rinne (Landwüst et al., 1996.). Cells were exposed to known oxidative stressors such as hydrogen peroxide and benzo[a]pyrene (B[a]p) in various concentrations (50 and 100 microM) up to 6 days. Cell mortality was determined using fluorescent ethidium homodimer-1 and calcein AM. Oxidative stress response was measured by image analysis using dihydrorhodamine 123, which is converted to fluorescent rhodamine 123 in the presence of intracellular ROS. Oxyradical formation was initiated already after 2 h of exposure to low concentrations of B[a]p and hydrogen peroxide. Probably due to a membrane stabilising effect of the serum factors the addition of fetal bovine serum to the culture medium had a protecting influence on the hepatocytes and resulted in (1) an increased cell viability and (2) reduced formation of intracellular ROS during exposure. In conclusion, the assay is a sensitive tool for testing the potential of various xenobiotics to induce oxidative stress in living hepatocytes.     

Effects of water-borne copper on metallothionein and lipid peroxidation in the marine amphipod Gammarus locusta

The aim of the current work was to determine over 10 days the effects of water-borne exposure of the marine amphipod Gammarus locusta to 4 microgCu l(-1) on the metallothionein (MT; measured by differential pulse polarography) protection system and lipid peroxidation (LP: thiobarbituric acid-reactive malondialdehyde equivalents) as a measure of oxidative damage. MT levels in exposed animals increased significantly at day 2 (36% > control; P < 0.001) and remained high at days 6 and 10 (55 and 38%, respectively, P < 0.001). The maximum level of MT at day 6 coincided with the highest Cu body-burden. LP increased within I day of exposure, indicative of Cu as an oxidative stressor. However, in contrast to MT, the highest LP level was seen at day 4 (68% > control, P < 0.001) before returning to control values by day 6, indicating a protective role of MT against the pro-oxidant effects of Cu.     

Protective effects of Ecklonia cava extract on the toxicity and oxidative stress induced by hair dye in in-vitro and in-vivo models

Oxidative hair dyes containingρ-phenylenediamine(PPD)are reported to induce an allergic reaction by promoting oxidative stress when absorbed through the skin.Despite the associated risk,these hair dyes remain popular owing to their convenience and sharpness of color.This makes it important to minimize the cytotoxicity and oxidative stress induced by PPD-containing hair dyes.Ecklonia cava extract has been evaluated in different studies for its protective effects against external stress in fibroblasts and keratinocytes.Our study was aimed at using in-vitro and in-vivo models to investigate the extract’s effects on cytotoxicity of and oxidative stress induced by PPD-containing hair dyes.Analysis of CIEL*a*b*Color space was first used to determine the range of E.cava extract that would not interfere with the coloring ability of the dye upon addition.Subsequently,the set ranges of E.cava extract(5% and 7%)were added to the hair dye and their toxicity assessed by evaluating the viability of fibroblasts and keratinocytes.The effects on developmental phenotypes and induction of oxidative stress by hair dye were evaluated and compared with those of hair dyes containing different contents of E.cava extract using an in-vivo zebrafish model.Our results showed that E.cava extract in hair dye could significantly decrease the cytotoxicity and levels of oxidative stress caused by hair dyes containing PPD in both in-vitro and in-vivo models.These results suggest that the addition of 7% E.cava extract to 250μg/mL hair dye does not interfere with the coloring ability of the dye while showing significant protective eff ects against the hair dye.The study proposes that the use of E.cava extract as an adduct to hair dyes containing PPD reduces the cytotoxicity and oxidative stress induced by these hair dyes.     

Hydrogen peroxide concentrations in the Peru upwelling area

The concentration of hydrogen peroxide was measured in waters off the coast of Peru during June and July 1983. The study period coincided with the end of the 1982/83 El Niño and the onset of coastal upwelling. Depth profiles of hydrogen peroxide concentration exhibit surface maxima and decrease with depth to the base of the mixed layer. Surface peroxide concentrations ranged from 0.8 to 5 × 10^?8 M. Below the mixed layer hydrogen peroxide was below the detection limit (5 × 10^?9 M). Diel variations were observed, with surface peroxide levels increasing during the day and decreasing at night. The nearshore station exhibited lower hydrogen peroxide concentrations than offshore stations, a reversal of the trend found in other coastal regions. This is attributed to the lack of coastal vegetation and runoff, and to active coastal upwelling of deeper water with low hydrogen peroxide concentrations.     

Requirement of endogenous iron for cytotoxicity caused by hydrogen peroxide in Euglena gracilis

It is widely thought that redox-active metals in cells such as iron or copper catalyze the reduction of hydrogen peroxide to toxic hydroxyl radicals or their equivalent. However, this has not been directly demonstrated in vivo. To probe this requirement, the freshwater microorganism Euglena gracilis was used. Its intracellular iron content can be modulated by the concentration of iron in the defined growth medium without effect on the proliferation rate of the cells. E. gracilis contains two large storage pools of cytosolic iron which can be monitored to assess cellular iron status. The toxicity of H₂O₂ in E. gracilis was inversely related to the amount of iron in the extracellular medium. At high levels of external iron, the metal carried out the Fenton reaction with H₂O₂ outside the cell, producing hydroxyl radicals as detected by electron-spin resonance spin-trapping experiments. This reaction reduced the amount of H₂O₂ that could diffuse into cells to cause toxicity. When cells with different intracellular iron content were placed in iron-deficient media, the the toxicity of H₂O₂, measured by inhibition of proliferation, was directly related to the concentration of internal iron. Using cells deficient in iron, this oxidant did not inhibit proliferation at low concentration but was somewhat effective at higher concentrations. Although the iron chelating agents, 1,10-phenanthroline and desferrioxamine, also depressed cytosolic iron, they were cytotoxic to E. gracilis and so could not be used unambiguously to examine the role of intracellular iron in the toxic effects of hydrogen peroxide.     

Photochemical activity in waters of the Great Barrier Reef

Photochemical activity in waters of the Great Barrier Reef was investigated through studies on the vertical, horizontal and temporal distribution of hydrogen peroxide and factors influencing its generation and decay processes. Surface hydrogen peroxide concentrations varied from 15 to 110 nM and generally decreased with depth, though a number of anomalies were detected. Photochemical activity decreased with increasing distance from the coast reflecting the positive influence of terrestrial inputs to the hydrogen peroxide generation and decay processes. Increases in photochemical activity were observed in the proximity of coral reefs. Hydrogen peroxide concentrations in the region were influenced by wind-induced mixing processes, atmospheric inputs, anthropogenic activity and seasonal light regimes.     

Effects of water-borne copper on metallothionein and lipid peroxidation in the marine amphipod gammarus locusta

The aim of the current work was to determine over 10 days the effects of water-borne exposure of the marine amphipod Gammarus locusta to 4 μgCu l⁻¹ on the metallothionein (MT; measured by differential pulse polarography) protection system and lipid peroxidation (LP; thiobarbituric acid-reactive malondialdehyde equivalents) as a measure of oxidative damage. MT levels in exposed animals increased significantly at day 2 (36% > control; P<0.001) and remained high at days 6 and 10 (55 and 38%, respectively, P<0.001). The maximum level of MT at day 6 coincided with the highest Cu body-burden. LP increased within 1 day of exposure, indicative of Cu as an oxidative stressor. However, in contrast to MT, the highest LP level was seen at day 4 (68% > control, P<0.001) before returning to control values by day 6, indicating a protective role of MT against the pro-oxidant effects of Cu.     

Alterations in hepatic metallothionein content in perch, Perca fluviatilis, environmentally exposed to cadmium

During recent years laboratory studies have demonstrated the presence of metallothionein in several species of teleosts.^1–3 The role of metallothionein (MT) in protection against cadmium toxicity has been investigated by exposing fish to high levels of cadmium in the water.^4–6 So far, however, few field studies have focused on the existence of MT and its role in subcellular distribution of cadmium.⁷ In the present study the synthesis of MT as a possible mechanism by which perch adapt to cadmium contamination was investigated in a wild population of perch. An increased MT content of perch liver was found to correlate to an increase in cadmium present in the liver.     

斑节对虾GPx3a基因的分子克隆及表达分析

谷胱甘肽过氧化物酶(Glutathione peroxidase,GPx)是生物体内重要的抗氧化酶,能防止过氧化氢对生物体的氧化应激。该研究利用RACE技术获得了斑节对虾(Penaeus monodon)GPx3a(PmGPx3a)的全长c DNA序列,进行了相关生物信息学分析。作者利用荧光定量PCR方法研究了PmGPx3a在斑节对虾不同组织的表达情况。探究了PmGPx3a在不同胁迫条件下(盐度、重金属和细菌)的表达情况。结果表明PmGPx3a c DNA全长1135 bp,其中开放阅读框(ORF)长651 bp,预测编码216个氨基酸。PmGPx3a推导的氨基酸序列与其他动物的GPx3a氨基酸序列具有高度一致性。实时定量PCR结果显示,在高低盐胁迫下,PmGPx3a在肝胰腺中相对表达量都为上升的(p0.05)。在铜、锌、铬胁迫中,鳃中的PmGPx3a的相对表达量总体呈现下降趋势,在肝胰腺中呈现上升趋势。在哈维弧菌(Vibrio harveyi)刺激下,PmGPx3a在血淋巴中的相对表达量总体呈现上升趋势,24 h后表达量最大,显著高于对照组2.2倍(P0.05)。以上研究结果表明,PmGPx3a基因参与了斑节对虾对环境胁迫和氧化应激的适应性反应。     

Effect of different wavelengths of light on the antioxidant and immunity status of juvenile rock bream, <Emphasis Type="Italic">Oplegnathus fasciatus</Emphasis>, exposed to thermal stress

We investigated the effect of light wavelengths on antioxidant and immunity parameters in juvenile rock bream, Oplegnathus fasciatus, exposed to thermal stress (25 and 30°C). We exposed the fish to light emitting diodes (LEDs) emitting green (520 nm) and red light (630 nm) of 0.25 and 0.5 W/m² intensity, and measured the activity, and mRNA and protein expression levels of the antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase. We also determined the levels of plasma hydrogen peroxide (H₂O₂), melatonin, and lysozyme. Furthermore, the mRNA and protein levels of caspase-3 were measured and terminal transferase dUTP nick end labeling (TUNEL) assays were performed. We observed that mRNA expression and activities of antioxidant enzymes and plasma H₂O₂ levels were significantly higher after exposure to high temperatures. However, increases in these parameters were significantly lower after exposure to green LED light. The plasma melatonin and lysozyme levels were significantly lower in the different groups after exposure to high temperatures; however, in groups exposed to green LED light, their levels were significantly higher than those in the control group. The expression pattern of caspase-3 mRNA was similar to that of H₂O₂. The TUNEL assay showed that apoptosis was markedly higher at higher water temperatures than that at 20°C. These results indicate that high water temperatures induce oxidative stress and decrease the immunity in juvenile rock bream but green LED light inhibits the rise in oxidative stress and combats the decrease in immunity and should, thus, be useful in the culture of rock bream.     

Cloning of a chub metallothionein cDNA and development of competitive RT-PCR of chub metallothionein mRNA as a potential biomarker of heavy metal exposure

Metallothionein has been assayed in a range of aquatic animal tissues as an indicator of metal exposure. We sequenced chub (Leuciscus cephalus) metallothionein cDNA which showed over 90% homology to common carp, goldfish and stone loach and 77% homology to rainbow trout sequences for metallothionein. We then used the extended primer method to develop an accurate quantitative competitive RT-PCR assay for metallothionein mRNA. RT-PCR was used to measure metallothionein mRNA in feral chub from a range of field sites, with different levels of heavy metal pollution, in the West Midlands, UK. Measurements were complemented by analysis of liver and gill metallothionein protein by capillary electrophoresis. There was no significant difference in the metallothionein protein levels between fish of different rivers and there was no evidence of elevation of mRNA at the sites of highest metal exposure. The level of metal exposure (e.g. zinc, nickel and cadmium each ranging between 15 and 28 microg/l ) at the pH (7.5-8.5) of these rivers appears insufficient to elevate hepatic or gill metallothionein in chub. A lack of elevation of hepatic metallothionein mRNA in chub exposed to zinc, copper and manganese for 24 h and 10 days in the laboratory also suggests a non-responsiveness of this species.     

Metallothionein and heavy metal levels in rainbow trout (Salmo gairdneri) during exposure to cadmium in the water

Rainbow trout were exposed to 200 ppb cadmium in the water during four months at 5°C. The liver, kidney and gills were analyzed for cadmium, copper, zinc and metallothionein. Cadmium accumulated in all three organs and reached the highest concentration in the kidney. The zinc and copper concentrations were not altered during the experiment. The metallothionein concentration was significantly higher in liver of exposed fish than in control fish after three months. The kidneys reached significantly elevated levels of metallothionein in the exposed group after four months. These results demonstrate that metallothionein is induced by cadmium after exposure to the metal via water.     

Toxicity and mutagenicity of waste waters from Baikalsk Pulp and Paper Mill: Evaluation of pollutant contamination in Lake Baikal

Lake Baikal has no agricultural and only little municipal pollution. Instead a potential source of pollution is the Baikalsk Pulp and Paper Mill (BPPM). All waste waters of the mill are mechanically, biologically and chemically purified and there are sedimentation and aeration ponds at the final stage. In this study the efficiency of the waste water purification was detected by testing toxicity and mutagenicity. Waters and sediments were collected along the cellulose bleaching process and waste water treatment. Free and bound polychlorinated phenols, guaiacols, catechols, anisoles and veratroles were analysed. Cytotoxicity of diethylether-extracted waste waters was screened by measuring total protein content in a fish hepatoma cell line (PLHC-1). The mutagenicity of the water, sediment and tissue extracts of selected aquatic species was determined by a modified Ames test. Diethylether fractions of the effluents from BPPM were cytotoxicfor the PLHC-1 cells when concentrated. Biological purification of the waste waters did not affect the cytotoxicity (ED₅₀ ˜2.2 and ˜2.0 μml⁻¹ before and after). The cytotoxicity was decreased after chemical purification (ED₅₀ ˜3.7μml⁻¹) and even more after the aeration pond (ED₅₀ ˜4.2 μml⁻¹). Mutagens were found in nearly all water samples released after the pulp chlorination. However, the mutagenic activity was effectively decreased during both biological and chemical treatment of waste waters. Only the tissue extracts of seals and a few roach possessed mutagenic activity. In conclusion, even the modern waste water purification systems do not totally abolish potential toxicity and/or mutagenicity of the effluents.     

Effect of intertidal compared to subtidal exposure on the uptake,loss and oxidative toxicity of water-born benzo[a]pyrene in the mantle and whole tissues of the mussel,Mytilus edulis L

Blue mussels (Mytilus edulis, L.) were exposed to a single dose of 1 ppb benzo[a]pyrene (BaP) under subtidal (SC) or tidal conditions (TC; 6 h immersion, 6 h emersion) in order to follow its bioaccumulation in whole mussel and mantle tissue, and to compare BaP-mediated toxicity on lipids (malonaldehyde formation, MDA) in the mantle. Rapid uptake of BaP (70-80% of BaP initially introduced in tanks) was observed in both conditions after 12 h, but subsequent depuration in clean water was slower in TC mussels. BaP levels decreased in whole tissue in both conditions between 12 and 24 h, but increased in mantle. The mantle BaP levels were similar during the first 4 days in SC and TC, but whereas they decreased in SC after 7 days. BaP was retained at high levels in mantle in TC until the end of the study (14 days). In both conditions, significant increases (P < 0.05) in lipid peroxidation were observed after 4 days, but MDA levels were approximately 3 times higher in the mantle of TC than SC mussels, although BaP tissue concentrations were similar. These observations suggested that increased BaP-mediated toxicity in mantle lipid was due to the interactive effect of the tidal cycle of immersion/emersion on BaP-mediated oxidative damage.     

The correlation between bioaccumulation and pattern of stress-related genes expression of black sea bream (<Emphasis Type="Italic">Acanthopagrus schlegeli</Emphasis>) by cadmium exposure

In order to correlate the expression of detoxifying enzyme genes and Cd accumulation in black sea bream, we analyzed four tissues (brain, gills, liver, and muscle) from black sea breams that were exposed to four different concentrations of Cd (0, 2, 13, and 25 mg/L) for various durations (0, 24, 48, 72, and 96 h). The highest level of Cd was accumulated in the liver, followed by the gills, brain, and muscle. The accumulation of Cd was significantly correlated with the duration of exposure and the concentration in brain, gill, and liver tissue, but not in muscle tissue, and the rate of accumulation increased with Cd concentration. The expression of metallothionein II (MT II) mRNA exhibited a similar pattern as Cd accumulation, especially in that the expression of MT II mRNA decreased in muscle tissue with increases in exposure duration. In contrast, the expression of cytochrome P450 1A (CYP1A) mRNA was highest in the liver, followed by brain, muscle, and gill tissues, and in gills and muscle tissue of Cd-exposed fish, the expression of CYP1A mRNA fell below that of the control fish. Overall, the liver of black sea bream was the most sensitive to Cd exposure, and the expression of MT II mRNA was 200-fold greater than the control fish. These findings indicate that the detoxification mechanisms of black sea bream are influenced by both MT II and CYP1A and that the genes participate in the detoxification of different tissues.     

Effects of environmental pollution in caged mussels (Mytilus galloprovincialis)

Biological effects of environmental pollution, mainly related to presence of PAHs, were assessed in mussels Mytilus galloprovincialis caged in Priolo, an anthropogenically-impacted area, and Vendicari, a reference site, both located along the eastern coastline of Sicily (Italy). PAHs concentration and histopathological changes were measured in digestive gland tissues. Expression of cytochrome P4504Y1 (CYP4Y1) and glutathione S-transferase (GST), indicative of xenobiotic detoxification, and activity of catalase (CAT) as oxidative stress index, were evaluated.The results show a direct correlation between the high concentrations of PAHs in digestive glands of mussels from Priolo and the significantly altered activity of phase I (P < 0.001) and phase II (P < 0.0001) biotransformation enzymes, along with increased levels of CAT activity (P < 0.05). These findings show the enhancement of the detoxification and antioxidant defense systems. The mussel caging approach and selected biomarkers demonstrated to be reliable for the assessment of environmental pollution effects on aquatic organisms.     

Expression of heat shock protein 70 in a permanent cell line (EPC) exposed to sediment extracts from the North Sea and the Baltic Sea

Heat shock proteins 70 (hsp70) are known to be induced by a great variety of chemical stressors. The effects of different environmental contaminants, which were identified in sediments of the North Sea and the Baltic Sea as main contaminants or as contaminants of special toxicological interest, on the extent of expression of hsp70 in a permanent cell line Epithelioma papulosum cyprini (EPC) were investigated. Concentrations of the single contaminants leading to elevated hsp70 levels were compared to the respective concentrations of these contaminants encountered in the sediment extracts thus allowing an assessment of their contribution to the toxicity of these sediments. Analysis of hsp70 was performed using western blotting techniques with subsequent comparative quantification by densitometry. Eleven of thirteen contaminants have shown a dose-dependent increase in hsp70 levels. An expression of hsp70 was observed in most cases between 0.1 and 10 microg/mL test solution. Some of the investigated stressors led to extremely high hsp70 contents even at low concentrations. However, in most cases contaminant concentrations in sediments were too low to cause an effect. For this reason, effects of the tested sediments are attributed to other contaminants or rather to mixtures of stressors.     

不同粒径TiO_2颗粒对海洋微藻的毒性效应

本实验以新月菱形藻为受试生物,研究了低浓度不同粒径TiO2颗粒(21nm、60nm和400nm)对海洋微藻生长、抗氧化酶活性(超氧化物歧化酶SOD、过氧化氢酶CAT和过氧化物酶POD)、脂质过氧化产物(MDA)含量的影响,并测定了相应的活性氧自由基(ROS)的含量,初步探讨了TiO2颗粒对海洋微藻的作用机制。结果表明,1mg/L TiO2颗粒对新月菱形藻生长的抑制作用随着粒径的减小而逐渐增强,第48h、72h、96h呈现出显著的纳米效应。TiO2颗粒可以诱导藻细胞内ROS的含量增加,对藻细胞产生氧化胁迫,新月菱形藻的抗氧化酶活性发生应激响应,以清除过量的ROS,但剩余的ROS对藻细胞产生氧化损伤,导致MDA含量升高,并且纳米级TiO2颗粒对新月菱形藻的氧化损伤大于微米级颗粒。在不同粒径TiO2颗粒的胁迫下,藻细胞SOD和CAT活性的响应也存在差异。本研究将为开展人工纳米材料对海洋生态系统影响的潜在风险评估提供科学依据。