Detecting genotoxicity using the Comet assay following chronic exposure of Manila clam Tapes semidecussatus to polluted estuarine sediments

Sediments frequently cause damage to biota due to the accumulation of toxic compounds and the bioavailability of sediment-bound contaminants. Damage can be assessed using biomarkers, such as the degree of genotoxic impact following in vivo exposure to pollutants. Genotoxic damage, expressed as single-strand DNA breaks, was measured in cells isolated from haemolymph, gill and digestive gland from the clam Tapes semidecussatus, using the single cell gel electrophoresis (Comet assay). Clams were exposed for three weeks to sediment samples collected from a polluted site and a ‘clean’ reference site.

The level of DNA damage was assessed using an image analysis package and expressed as Tail Moment. Throughout the study, significant differences in DNA damage were recorded for each tissue type between clams exposed to the two sediment samples. We conclude that the Comet assay is a useful tool for the detection of DNA damage in clams chronically exposed to polluted sediments.     

Use of Plant Bioassays for the Detection of Genotoxins in the Aquatic Environment

The present article describes the use of plant bioassays for the detection of genotoxins in the aquatic environment and gives an overview of test methods, their detection spectrum for environmental mutagens and carcinogens and their limitations and pitfalls. The most widely used test systems are micronucleus assays with meiotic cells of Tradescantia and with meristematic root tip cells of Allium and Vicia. In the last years, protocols for single cell gel electrophoresis assays have been developed, which can be conducted with a variety of species. Also various gene mutation test procedures have been developed with plants but they have hardly ever been used in environmental studies. Plants detect a broad variety of environmentally relevant genotoxins, in particular directly acting compounds. Many pesticides and industrial chemicals caused positive results; plant assays are also a unique tool for the detection of DNA‐reactive carcinogenic heavy metals in the environment. In many studies with complex mixtures, positive results were obtained which indicates that plants are sufficiently sensitive to detect effects without concentration of water samples. One of the shortcomings of the use of plants as indicators is their lack of sensitivity towards certain classes of promutagens such as nitrosamines, heterocyclic amines, and polycyclic aromatic hydrocarbons (PAHs). However, the former compounds are hardly encountered in the environment and PAHs can easily be detected chemically and in other mutagenicity tests. Taken together, the currently available data show that plant bioassays are a useful component of test batteries for environmental monitoring.     

Prüfstrategie zur Bewertung potentiell erbgutverändernder Wasserinhaltsstoffe unter Einsatz gentoxikologischer Methoden

Assessment and Characterization of Genotoxic Water Contaminants with Modern Genotoxicological Methods The necessity of genotoxicological studies in water requires modern and practicable concepts for a prospective environmental protection. Up to now, genotoxicological studies prefer only the observation and detection of basic genotoxic effects in enriched surface water samples and the fruitless search for the involved water contaminants. In this publication, a new test strategy for the detection and the assessment of genotoxicity in water will be introduced. This strategy allows the detection and the verification of positive results and is therefore able to assess the evidence of these results for the water quality more accurate. The aim of this strategy is the detection of basic genotoxic effects (primary DNA damages), the clarification of the biological mechanisms, and the search for the reasons with modern analytical methods. The involved water contaminants and their mode of action will then be better understood. A test strategy in three steps will be proposed. In the first step genotoxicological indicator tests such as comet assay, alkaline filter elution, or the SOS-umu-test are used to get information about the basic status of the tested water samples. In the second step, the positive results of the primary DNA damages will be verified with mutation tests such as the micronucleus assay. The involved biological mechanisms will be biochemically characterized, and the biological active substances will be preclassified. Finally, in the third step the observed mechanisms will be confirmed with modern chemical methods such as HPLC MS-MS. These modern techniques are also able to detect and quantify the genotoxic compounds preclassified in step two.     

长江口表层水体的生态遗传毒性初步研究

采用Ames试验方法,结合浮游动物多样性数据和一些水质理化参数的分析,对长江口表层水体的生态遗传毒性进行初步研究。结果表明,长江口南支的水样有明显的遗传毒性,而河口外海滨区的部分样品在加入S9活化后也表现出一定的遗传毒性,口外海区样品没有遗传毒性。浮游动物的调查结果显示,长江口南支水样中浮游动物的密度及多样性都较低,口外海区较高。统计分析也表明,遗传毒性数据和生物多样性数据存在着一定的线形关系。这说明长江口表层水体污染物的毒性效应在分子水平和生物群落水平上有相关性。而二者与水质参数的相关分析表明,遗传毒性与多数所测水质理化因子之间没有相关性,但浮游动物多样性与部分理化参数有一定的相关性。同时,本文也从实践上证明,在河口、近岸海域的环境监测中开展遗传毒性检测具有可行性和必要性。     

Comet assay for the detection of genotoxicity in blood cells of flounder (Paralichthys olivaceus) exposed to sediments and polycyclic aromatic hydrocarbons

To investigate the genotoxic effect of marine sediments on aquatic organism, sediment samples were collected from 13 sites along the coast of Gwangyang Bay (Korea). Concentrations of polycyclic aromatic hydrocarbons (PAHs) in sediments were determined and the relationship between exposure of flounder blood cells to sediment extracts and DNA single-strand breakage in the blood cells was examined using the comet assay. Levels of DNA damage were proportionally increased by exposure concentration and the highest sediment-associated DNA damage was observed at the station showing the highest PAHs contamination. DNA damage in blood cells exposed to five types of PAHs (benzo[a]pyrene, fluoranthene, anthracene, pyrene and phenanthrene) and in flounder (Paralichthys olivaceus) exposed to benzo[a]pyrene (BaP) for 0, 2 and 4 days were assessed by measuring comet tail length. The tail lengths of five PAHs-exposed groups at 50 and 100 ppb were significantly different from the non-exposed group, and the genotoxic effect of BaP correlated with both concentration and duration of exposure. Throughout the study, significant differences in DNA breakage were recorded between cells exposed to sediment extracts or PAHs and non-exposed control. This study demonstrated the comet assay as a successful tool in monitoring contamination of marine sediments and assessing genotoxicity of PAHs in marine organisms, either in vitro or in vivo.     

Temporal estimates of genetic diversity in some Mytilus galloprovincialis populations impacted by the Prestige oil-spill

The sinking of the tanker Prestige in November 2002 off the coast of Galicia resulted in the release of about 60,000 tons of heavy oil. The oil-spill provoked a serious environmental impact in Spanish and French coasts, which biological consequences are still being assessed. In this study we address the temporal dynamics of genetic diversity in some mussel populations impacted by the oil-spill. Changes in genetic diversity can be measured in natural populations provided that serial samples are available from before (year 2000) and after (years 2003, 2005) the oil-spill. Analyses of seven microsatellites indicate a weak but significant increase of genetic variation after the spill. This phenomenon is interpreted herein in terms of a balance between a enhanced genome mutability on microsatellite variation and a low genetic drift due to toxicants and asphyxia although other stochastic phenomena cannot be ruled out. Per locus annotation showed that in spite of the allelic changes observed in the period 2000-2005, the final size of most allelic series remained quite alike to those of year 2000. Present genetic data suggest that the genotoxic impact of the Prestige spill did not compromise the genetic diversity of studied mussel populations, at least regarding the genetic markers analysed.     

Toxicity of tributyltin in the marine mollusc Mytilus edulis

Our previous studies have demonstrated that tributyltin (TBT) is genotoxic to the early life stages of marine mussels and worms. Here, the toxicity of TBT to adult organisms was determined using a suite of biomarkers designed to detect cytotoxic, immunotoxic and genotoxic effects. Exposure of adult mussels, Mytilus edulis, to environmentally realistic concentrations of TBTO for 7 days resulted in a statistically significant decrease in cell viability at concentrations of 0.5 μg/l and above. TBT had no effect on phagocytic activity or antioxidant capacity (FRAP assay). There was a statistically significant increase in DNA damage detected using the comet and micronucleus assays between the controls and 0.5, 1 and 5 μg/l of TBTO (P > 0.0005). Furthermore there was a strong correlation between DNA strand breaks (comet assay) and formation of micronuclei (P = 0.0005; R² = 61.5%). Possible mechanisms by which TBT could damage DNA either directly or indirectly are discussed including the possibility that TBT is genotoxic due to its ability to disrupt calcium homeostasis.     

化学诱变剂在实验海洋食物链中的流动以及遗传毒性的检测

于１９８９年３月－１９８９年９月，利用极谱测定法研究了无机诱变剂Ｃｄ在３级实验食物（链从褐指藻经中国对虾至欧氏六线鱼）中的传递，并对积累Ｃｄ的中国对虾和欧氏六线鱼肌肉和内脏灰样进行了紫露草微核测定。结果表明，Ｃｄ浓度在传递中依次减少；经过６ｄ喂食，Ｃｄ从褐指藻到中国对虾的传递比例为６６．６％；经过７ｄ喂食，Ｃｄ从中国对虾到欧氏六线鱼的传递比例为２９．９％。ｔ测验发现，处理组对虾内脏灰样与肌肉灰样之     

B[a]P–DNA binding in early life-stages of Atlantic tomcod: population differences and chromium modulation

Atlantic tomcod (Microgadus tomcod) from the Hudson River (HR) are resistant at the molecular and organismic levels to the effects of exposure to dioxin-like aromatic hydrocarbon (AH) compounds, but much less so to benzo[a]pyrene (B[a]P). The aims of this study were to determine in early life-stages of tomcod exposed to B[a]P: (1) if DNA binding levels differed between fish from the HR and Miramichi River (MR), and (2) if co-exposure to chromium could modulate this genotoxic effect. After exposure to [³H]B[a]P alone, DNA-bound radioactivity was 5–10-fold higher in embryos and larvae of MR than HR descent. Co-exposure to chromium modulated DNA binding levels in offspring of both populations. In MR embryos, co-exposure to chromium inhibited B[a]P uptake. These results demonstrated resistance to the genotoxic effects of B[a]P in early life stages of HR tomcod at an ecologically important endpoint and suggest the ability of chromium to modulate AH-induced genotoxicity.