Susceptibility to oxidative stress and modulated expression of antioxidant genes in the copper-exposed polychaete Perinereis nuntia

To identify and evaluate potentially useful biomarkers for oxidative stress as early warning indices in the polychaete, Perinereis nuntia, we exposed P. nuntia to copper (Cu) and measured several biomarker enzymes (glutathione S-transferase; GST, glutathione peroxidase; GPx, Metallothionein-like protein; MTLPs, and catalase; CAT) and genes (Pn-GSTs, Pn-CAT, and Pn-MT) with a cellular oxidative index, reactive oxygen species (ROS) level. Accumulated Cu concentrations in P. nuntia increased in a time-dependent manner. Intracellular ROS reached high levels 6h after exposure in P. nuntia with an increase of GST activity and glutathione (GSH) content. Particularly, GSH in polychaetes showed a positive correlation with Cu contents accumulated in P. nuntia. Messenger RNA expressions of GST sigma and GST omega showed relatively high expressions at 50 μg/L of Cu exposure, even though the moderate increase of rest of GST isoforms was also observed. Also regarding long-term exposure, we reared P. nuntia in sediments for 15 days, and found that there was an obvious increase of Pn-GSTs, Pn-CAT, and Pn-MT genes with elevated concentrations of Cu and Cd in polychaete body, compared to initial levels, suggesting that P. nuntia in sediment was affected by metals as well as by other organic pollutants to induce oxidative stress genes and enzymes. These findings suggest that oxidative stress is a potential modulator of defense system of P. nuntia. Several potential biomarker genes are available as early warning signals for environmental biomonitoring.     

水相Cd2+在南美白对虾体内的富集及氧化应激特征

为阐明水相环境中重金属镉元素(Cadmium, Cd)在甲壳类海产品中的富集与代谢过程、亚细胞微区分布特征和生物体氧化应激效应, 文章以南美白对虾(Litopenaeus vannamei)为研究对象, 应用双箱动力学模型设置了水环境下不同Cd暴露水平的富集吸收与清水净化释放试验, 实时监测南美白对虾内脏团和肌肉组织的Cd含量及谷胱甘肽巯基转移酶(Glutathione S-transferase, GST)、谷胱甘肽过氧化物酶(Glutathione peroxidase, GPx)和过氧化氢酶(Catalase, CAT)酶活力随时间变化情况, 同时结合差速离心技术分析了Cd富集后在机体内脏组织团中的亚细胞微区分布特征。实验得出南美白对虾对水环境中Cd的富集能力与水体暴露浓度呈明显的正相关关系, 内脏团组织是南美白对虾最主要的Cd元素吸收与代谢组织, 其对Cd的吸收速率、净化速率、生物富集因子及平衡状态时Cd含量均显著高于肌肉组织, 在相同暴露剂量下前者的生物富集因子平均为后者的75.5倍, 肌肉组织的Cd生物学半衰期明显长于内脏组织; Cd主要储存于类金属硫蛋白(Metallothionein-like protein, MTLP)和细胞碎片(Cell debris, CD)组分中, 少部分存在于细胞器(Organelle, ORG)、富含金属颗粒(Metal-rich granules, MRG)和热敏感蛋白(Heat sensitive protein, HSP), 且随着富集过程的持续, Cd的亚细胞微区分布发生动态变化, MTLP、ORG和HSP中Cd含量百分比逐渐升高, CD和MRG中的Cd含量百分比呈逐渐下降; 在肝脏和肌肉组织中GST、GPx和CAT酶活力在Cd富集阶段均持续显著升高, 在净化释放阶段处于下降趋势, 同一试验组下肝脏组织中抗氧化酶酶活力明显高于对应的肌肉组织。研究旨在阐述甲壳类海洋生物对环境中Cd元素的富集与分布特征, 了解Cd富集可能性机理, 为海洋甲壳类Cd污染风险评价与环境控制提供科学依据。     

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.     

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.     

Molecular epidemiological approaches to the study of the genotoxic effects of urban air pollution.

Direct epidemiological observations suggest that exposure to high levels of urban air pollution may result in increased risk of lung cancer, sufficient to account for a few (approximately 1-3) percent of total lung cancer incidence. Extrapolation from occupational exposure and risk data suggests that among potential carcinogens present in polluted urban air, polycyclic aromatic hydrocarbons (PAHs) may make a major contribution to air pollution-associated lung cancer risks. The use of biomarkers of genotoxocity in large-scale population studies may help to reduce the uncertainty involved in the assessment of such risks, especially those associated with relatively low pollution levels such as nowadays found in many Western cities. Increases in biomarkers of exposure to urban air PAHs as well as biomarkers of early effects have been detected in situations of relatively high levels of air pollution (e. g., ambient PAH concentrations of the order of a few tens of micrograms per cubic meter). Evidence has also been found about the modulation genetic damage accumulation in different individuals by polymorphisms in genes involved in the activation or detoxification of PAHs, especially of polymorphisms GSTM1 and CYP1A1 genes. However, the inconsistencies in the currently reported effects of genetic polymorphisms suggest that additional factors may also be important in the modulation of individual susceptibility to the accumulation of PAH-derived genetic damage. Biomarkers studies in populations exposed to relatively low ambient PAH concentrations (below 20 microg/m(3)) have not demonstrated clear dose-related effects (e.g., on DNA adduct levels), possibly because of the existence of multiple sources and routes of human exposure to PAHs in addition to inhalation of urban air (including, for example, home heating, environmental tobacco smoke and diet), and the consequent difficulty of adequately and specifically assessing atmospheric air-related exposure. This makes it imperative that molecular epidemiology studies be designed in such a way as to allow adequate assessment of exposure to urban air PAHs at the individual level and over short-, medium- and long-term time periods which correspond to the expression times of different biomarkers.     

Alterations in the expression of translation factors as molecular markers in cadmium-exposed workers

Eukaryotic translation initiation factor 3 (eIF3) and elongation factor 1δ (eEF-1δ) are novel cadmium (Cd) responsive proto-oncogenes. This research investigated the expression of these genes in Cd-exposed workers (n = 58), and to evaluate their usefulness as biomarkers of Cd exposure. According to urinary Cd concentration, the subjects were divided into four groups (urinary Cd concentration ≥0.1 μg/g.Cr, ≥1.0 μg/g.Cr, ≥5.0 μg/g.Cr and ≥50.0 μg/g.Cr). Subjects exhibited increased severe health problems with higher urinary Cd concentrations. The eIF3 and eEF-1δ expression in the blood were investigated with real-time PCR. PCR data showed a strong positive correlation between blood eEF-1δ and urinary Cd concentrations (r = 0.788, p < 0.01), and a weak positive correlation between blood eIF3 expression and urinary Cd concentrations (r = 0.569, p < 0.05). These findings, for the first time, demonstrate that the blood eEF-1δ overexpression can be used as a molecular biomarker of Cd-exposed population.     

Enzymatic activities as potential stress biomarkers of two substituted benzene compounds in Propsilocerus akamusi (Diptera: Chironomidae)

Chironomids are a globally distributed family of insects that can serve as biological indicators of environmental pollution. Substituted benzenes are a group of serious environmental pollutants and severely threaten biological and human health. In order to investigate potential stress biomarkers of substituted benzenes, the effects of 4-chlorophenol and p-phenylene diamine on superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), carboxylesterase (CarE), acid phosphatase (ACP) and alkaline phosphatase (ALP) activities of 4th-instar larval Propsilocerus akamusi were investigated. During 48 h exposure to 4-chlorophenol and p-phenylene diamine, the larval CAT, SOD, GST and CarE activities responded to 4-chlorophenol and p-phenylene diamine in a concentration-and time-dependent way. The ACP and ALP activities of larval P. akamusi responded slowly to 4-chlorophenol and p-phenylene diamine, and only the ALP activities were inhibited significantly under the exposure to p-phenylene diamine for 6 and 24 h. These results suggest that CAT, SOD, GST and CarE activities of chironomids can be used as potential stress biomarkers to monitor 4-chlorophenol and p-phenylene diamine pollution.     

Multiple biomarkers of pollution effects in caged mussels on the Greek coastline

A suite of biomarkers was measured in caged mussels at areas impacted by different anthropogenic activities along the Greek coastline to assess biological effects of environmental pollution. Mussels were caged at coastal sites in the vicinity of major cities, in areas influenced by major industries, agricultural practices and in islands away from known sources of pollution. Biomarkers indicative of neurotoxicity (acetylcholinesterase, AchE), oxidative stress (catalase, CAT), phase II biotransformation of xenobiotics (glutathione S-transferase, GST), metal exposure (metallothioneins, MTs) and protein synthesis (RNA:DNA ratio) were measured to assess effects of various types of pollutants. AchE activity proved to be the most responsive biomarker with decreased values at sites influenced by agricultural, urban and industrial activities. Decreased CAT and GST activities and increased MTs levels were recorded at a number of anthropogenic-impacted sites. RNA:DNA ratio showed a biphasic response as both high and low values were found at impacted sites. Principal component analysis clearly distinguished sites receiving pollution inputs from non-polluted sites. The combination of the selected biomarkers used in caged mussels resulted useful in the assessment of the effects of environmental pollution.     

Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies

Background  

Odontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR) has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers.     

铜对梨形环棱螺抗氧化酶活性和金属硫蛋白含量的影响

本实验采用暴露重金属的方法,研究了不同浓度硫酸铜（Cu2+ 分别为0、0.005、0.01、0.02、0.05 mg/L）在不同暴露时间（0—14d）下对梨形环棱螺（Bellamya purificata）过氧化氢酶(CAT)、超氧化物歧化酶（SOD）、谷胱甘肽硫转移酶（GST）的活性、还原性谷胱甘肽（GSH）和金属硫蛋白（MT）含量的影响,以探讨Cu2+ 对梨形环棱螺的氧化损伤及其防御作用的机理,并为水环境Cu2+ 污染的早期诊断及生态风险评价提供科学的依据。结果表明：Cu2+对梨形环棱螺肝脏和鳃中CAT、SOD、GST、GSH 和MT 均有明显影响,表现出时间剂量效应。SOD在前4天、CAT在前3天酶活性总体上表现出诱导趋势, GST在前4天酶活性处于诱导状态,随着暴露时间的延长,酶活性下降,到第5天时表现出抑制趋势;随着时间的进一步增长,至14d时, 0.005 mg/L剂量组酶活性维持在正常值附近波动, 0.01 mg/L剂量组酶活性被诱导, 0.02 mg/L剂量组酶活性在肝脏中表现为诱导而在鳃中则被抑制,0.05 mg/L剂量组酶活性被抑制。肝脏和鳃GSH含量的变化与GST相似,在短时间内表现出诱导效应,肝脏GSH在暴露的前5天、鳃GSH在暴露的前4天均处于诱导状态,随着暴露时间的延长,0.005 mg/L剂量组表现出诱导,0.05 mg/L剂量组则受到抑制。MT在整个实验期间均处于诱导状态,各剂量组在0.5d被极显著诱导,随后MT含量出现起伏波动,有上升和下降,至第14天时达到一稳定水平。其中,0.01 mg/L剂量组肝脏的MT在整个实验期间均被极显著地诱导（P <0.01）,0.01 mg/L 剂量组的鳃组织MT除第10天外也被极显著诱导（P <0.01）。在暴露14d时,除0.05 mg/L剂量组的肝脏MT外,其余处于极显著诱导状态（P <0.01）。     

Influence of some metal concentrations on the activity of antioxidant enzymes and concentrations of vitamin E and SH-groups in the digestive gland and gills of the freshwater bivalve Unio tumidus from the Serbian part of Sava River

We examined whether the freshwater bivalve Unio tumidus from the Sava River can serve as a bioindicator organism for long-term biomonitoring of river ecosystems for the presence of metal pollutants. To this end, we assessed in the digestive glands and gills of mussels, changes in activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), the phase II biotransformation enzyme glutathione-S-transferase (GST)), and changes in the concentrations of the non-enzymatic components of the antioxidant system (vitamin E and sulfhydryl groups (-SH), after exposure to metals in the environment. Mussels were collected at four sites where the concentrations of dissolved metals (Cu, Cd, Zn, Fe, Mn, Hg, Ni, As, Pb) were quantified. Cu, Ni and As exerted concentration-dependent inhibitory effects on CAT and GST activities. Increasing concentrations of Cd promoted increases in GSH-Px activity and -SH concentration. In response to increased Zn concentration GR activity increased whereas Fe promoted decreased enzymatic activity. Negative correlations between the concentrations of Cu and Cd and vitamin E, and a positive correlation between Mn and vitamin E concentrations were detected. The described correlations between components of the antioxidant system and metal levels in the environment reveal a high physiological sensitivity of freshwater mussels to pollution, supporting their use in biomonitoring of metal contamination in river ecosystems.     

褐菖鲉肝CYP 1A作为生物标志物监测厦门海域石油污染状况

以褐菖鲉为实验鱼类,以鱼肝微粒体CYP1A 生物标志物(EROD活性和CYP1A蛋白表达量)为指标,在厦门海域开展了两次野外监测实验,研究EROD活性和CYP1A蛋白表达量的变化,以及它们与海水和沉积物中石油类和重金属含量之间的相关性。结果表明,在现场属于一类海水的石油类浓度(0.0121-0.0242 mg/L)条件下,石油类就能够显著诱导褐菖鲉肝EROD活性和CYP1A蛋白表达量,鱼肝EROD活性和CYP1A蛋白表达量与海水中石油类含量均呈现极显著正相关,CYP1A蛋白表达量比EROD活性较为敏感和稳定。此外,在监测实验中,尚未发现这两种生物标志物受所监测海区的海水和沉积物重金属含量的影响。因此,利用褐菖鲉肝微粒体EROD活性和CYP1A蛋白表达量作为生物标志物监测海洋石油类及其PAHs污染是可行的,在海洋环境石油类污染监测及其生化效应评价中具有重要的应用价值。而且,把这两种生物标志物结合起来加以研究并推广应用将更有意义。     

Alterations in the expression of translation factors as molecular markers in cadmium-exposed workers

Eukaryotic translation initiation factor 3 (eIF3) and elongation factor 1δ (eEF-1δ) are novel cadmium (Cd) responsive proto-oncogenes. This research investigated the expression of these genes in Cd-exposed workers (n?=?58), and to evaluate their usefulness as biomarkers of Cd exposure. According to urinary Cd concentration, the subjects were divided into four groups (urinary Cd concentration ≥0.1 μg/g.Cr, ≥1.0 μg/g.Cr, ≥5.0 μg/g.Cr and ≥50.0 μg/g.Cr). Subjects exhibited increased severe health problems with higher urinary Cd concentrations. The eIF3 and eEF-1δ expression in the blood were investigated with real-time PCR. PCR data showed a strong positive correlation between blood eEF-1δ and urinary Cd concentrations (r?=?0.788, p?<?0.01), and a weak positive correlation between blood eIF3 expression and urinary Cd concentrations (r?=?0.569, p?<?0.05). These findings, for the first time, demonstrate that the blood eEF-1δ overexpression can be used as a molecular biomarker of Cd-exposed population.     

Differential response of wheat roots to Cu, Ni and Cd treatment: oxidative stress and defense reactions

Wheat seedlings cv. Zyta were treated with Cu, Ni and Cd at the concentrations causing approximately 50 % root growth inhibition, i.e. 12.5, 50 and 60 μM, respectively. Tissue metal accumulation, membrane permeability, lipid peroxidation, protein oxidation, concentration of thiol compounds as well as protease, glutathione S-transferase (GST) and peroxidase (POD) activities were studied in roots after 7 days of metal exposure. The metals showed different concentrations in root tissues with Cu and Cd being accumulated to the smallest and to the greatest extent, respectively. Membrane permeability was significantly enhanced by Cu and Ni but not by Cd treatment. All metals induced similar increase in protein oxidation, while significant enhancement of lipid peroxidation was observed only in the case of Cu treatment. The detected thiol compounds: cysteine (Cys), homocysteine (Hcy), γ-glutamylcysteine (γ-GluCys) and glutathione (GSH) were differently influenced by the metal treatment. Ni appeared to be the most effective inductor of GSH accumulation while both Cu and Ni similarly increased Cys content in the roots. Accumulation of γ-GluCys was found in response to Cu and Cd applications. Concentration of Hcy was enhanced by Cd treatment but exposure to Ni decreased its content below the level of detection. The activity of GST was considerably elevated by Cd and Ni treatments, while POD activity was increased only in response to Cu application. Our study showed that wheat roots differently responded to treatment with metals used at the concentrations having similar impact on growth.