Biomonitoring is an important subject within environmental sciences. Biomonitoring tests are required to be quick, relatively inexpensive, accurate, and reproducible. No genetic test currently fulfils all of these requirements. The chromosome aberration and sister chromatid exchange tests are very time consuming, the DNA adduct technique is rather expensive, and the micronucleus test has not inconclusively proven its use as a reliable monitoring tool. This work is focused on the validation of the comet assay as a candidate for monitoring marine ecosystems. For the comet assay, this work deals with the effectiveness of tissue dissociation, storage of cells in lysing buffer and in liquid nitrogen, different electrophoretic conditions, neutralisation and fixation of slides, interindividual variation between samples, and responsiveness of four tissue types to ethyl methanesulphonate (EMS). The main conclusions are: (i) dissociation of solid tissues in a phosphate buffer supplemented with 200 mM N-t-butyl-alpha-phenylnitrone provides cells with an acceptable background DNA damage; (ii) freezing of cells or tissues in liquid nitrogen generally leads to an increase in DNA breakage, especially for liver, gill and kidney tissue; (iii) storage of slides in the lysing solution for up to one week gives minor changes in comet tails; (iv) differences in protocols for neutralisation and fixation may influence the results; (v) high intra- and interindividual variations in comets (length and DNA content) may obscure the interpretation of comet results; (vi) blood, gill, liver and kidney all showed a statistically significant increase of DNA damage after exposure to 50 mg EMS/l; (vii) electrophoresis at low voltage for longer periods is to be preferred to high voltage and short electrophoresis times. The simplicity and sensitivity of the comet assay make it an adequate test system for biomonitoring of chronic low level exposure. However, protocols and experimental conditions have to be chosen carefully.
Polychlorinated biphenyls (PCBs) are stable pollutants, which can be found in almost every compartment of terrestrial and aquatic ecosystems. They are very lipophilic and therefore have the potency of accumulating in the fat stores of animals. The mechanisms by which PCBs exert their adverse effects are still unclear. It is known that PCBs induce some important biotransformation enzymes, but their mutagenic properties are still controversial. The DNA breakage and clastogenic potency of a planar PCB77 (3,3',4,4'-tetrachlorobiphenyl) was determined in vivo in fish, using the single cell gel electrophoresis or comet assay and the micronucleus test, on erythrocytes of the brown trout exposed for 3, 9 and 14 days to initial PCB concentrations of 780 and 918 pg/ml, dissolved in the water. Blood was taken by a caudal puncture and the erythrocytes were either deposited in an agarose gel (0.6%) for the comet assay or smeared directly on slides for the micronucleus test. Five fish were studied per treatment and 50 and 2000 erythrocytes per concentration and per animal were analysed for the comet assay and the micronucleus test respectively. Ethyl methanesulphonate (EMS) at a concentration of 25 mg/l water was used as a positive control. Although EMS induced a statistically significant increase of single strand breaks in the comet assay, in neither of the two tests used, were mutagenic effects due to PCB exposure observed.
Hard metals (WC-Co) are made of a mixture of cobalt metal (Co, 5-10%) and tungsten carbide particles (WC, >80%). Excessive inhalation of WC-Co is associated with the occurrence of different lung diseases including an excess of lung cancers. The elective toxicity of hard metal is based on a physico-chemical interaction between cobalt metal and tungsten carbide particles to produce activated oxygen species. The aim of the present study was to assess the genotoxic activity of hard metal particles as compared with Co and WC alone. In human peripheral lymphocytes incubated with Co or WC-Co, a dose- and time-dependent increased production of DNA single strand breaks (ssb) was evidenced by alkaline single cell gel electrophoresis (SCGE) and modified alkaline elution (AE) assays. Addition of 1 M formate, a hydroxyl radical scavenger, had a protective effect against the production of ssb by both WC-Co or Co alone. On the basis of an equivalent cobalt-content, WC-Co produced significantly more ssb than Co. WC alone did not produce DNA ssb detectable by the AE assay, but results obtained with the SCGE assay may suggest that it either allows some uncoiling of the chromatin loops or induces the formation of slowly migrating fragments. Overall, this in vitro study is the first demonstration of the clastogenic property of cobalt metal-containing dusts. The results are consistent with the implication of an increased production of hydroxyl radicals when Co is mixed with WC particles. The SCGE results also suggest that WC may modify the structure of the chromatin, leading to an increased DNA sensitivity to clastogenic effects. Both mechanisms are not mutually exclusive and may concurrently contribute to the greater clastogenic activity of WC-Co dust. This property of WC-Co particles may account for the excess of lung cancers observed in hard metal workers.
Polychlorinated biphenyls (PCBs) are classified by IARC as non-mutagenic in vivo. However, despite almost 20 years of research, their mutagenicity in vitro is still debatable. In this work the in vitro cytochalasin B micronucleus test and the alkaline comet assay applied to human lymphocytes were used to study the genotoxicity of a PCB. PCB77, at concentrations ranging from 0.01 to 100 microg/ml, was used in whole blood or isolated lymphocyte cultures, with final dimethylsulfoxide percentages of 0.5-2%. In the micro-nucleus test lymphocytes were exposed for 48 h, and in the alkaline comet assay for 30 min, 1 h and 3 h. No increases of single strand breaks or micronucleus frequencies was found, in contrast to previously reported data. Our data indicate that PCB77 has no clastogenic properties in human lymphocytes.
Coastal mapping plays an important role in informing decision makers on issues such as national sovereignty, resource management, maritime safety and hazard assessment. A key aspect of this trend has been the development of coastal web atlases (CWAs), based on web enabled geographic information systems (GIS). CWAs contain spatial data and thematic information on environmental, social and economic aspects of the coast and provide an integrated visualisation, download and sometimes analysis environment.An important element of CWA development is to ensure that the needs of the target audience are being addressed. Sometimes there can be a break in developer-user communication after the initial atlas design phase is completed. Identifying how to maintain effective engagement by CWA providers with end users is a key task for development teams. A key initial task is to profile the target end users. Workshops and surveys can be effective at this stage. Launch events and media exposure is used effectively to build user communities, however usage drops when such publicity events are discontinued. During the atlas operations phase baseline web statistics are often gathered, but these give a somewhat limited idea of the end user community and their use of the atlases.Additional means of remaining in touch with users and incorporating their requirements in CWAs need to be used. Web 2.0 applications such as "like/dislike" buttons and content comment boxes are used with success in social networking and some news media sites and their utility should be considered for CWAs. Focus groups have also been used with success to complete semi-structured surveys when trying to gather answers to specific questions. It is important to demonstrate to users that the survey has a goal such as atlas improvements in order to motivate them to provide information.Enhancing users' sense of ownership achieved by web applications that allow more direct user input has been accomplished by some CWAs. The strengths and weaknesses of such an approach should be further explored by individual CWAs, as this could enhance the understanding of user needs and improve the relationship of the atlas developer to the user community. Atlas developers within the International Coastal Atlas Network (ICAN) who have worked on the Marine Irish Digital Atlas, the Belgian Coastal Atlas, Chesapeake Bay Atlases, the Washington Coastal Atlas and the Wisconsin Coastal Atlas have significant experience in gathering and analysing user feedback and are investigating means of enhancing their interaction with the end-user community.
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