Effects of organic contaminants in reactive oxygen species, protein carbonylation and DNA damage on digestive gland and haemolymph of land snails

The present study focused on early responses of land snails Eobania vermiculata to organic environmental contaminants, by investigating the use of a newly-established method for the measurement of protein carbonylation as a new biomarker of terrestrial pollution, as well as by measuring the ROS production and the DNA damage. Land snails were exposed to different concentrations of chlorpyrifos, parathion-methyl or PAHs in vivo or in vitro in the laboratory. The susceptibility of exposed snails was increased in relation to oxidative stress induced by contaminants tested. A statistically significant increase in ROS production, protein carbonylation and DNA damage was revealed in the snails treated with pollutants, compared to the untreated ones. The results indicated the effectiveness of measuring ROS production and DNA damage and reinforce the application of the present ELISA method in organic terrestrial pollution biomonitoring studies.     

Introduction of the land snail Cornu aspersum as a bioindicator organism of terrestrial pollution with the use of a suite of biomarkers

The use of organisms in biomonitoring studies allows the determination of the pollution levels, the assessment of the quality of the enviroment and the study of toxic effects of pollutants on the organisms. By studying the responses (biomarkers) of organisms, it is possible to assess the exposure and effects of pollutants. Land gastropods are widely used in relevant studies as they possess several characteristics that make them ideal for use as bioindicators. In the present study we sought to evaluate several biomarkers in the laboratory using snails of the species Cornu aspersum after the exposure to heavy metals (Cd, Hg) and a mixture of polycyclic aromatic hydrocarbons (PAHs) (anthracene, phenanthrene and naphthalene). The biomarkers studied were the following: lysosomic membrane stability using the neutral red retention assay (NRR), the production of reactive oxygen species (ROS) using the compound DCFH-DA and flow cytometry, lipid peroxidation using the TBARs assay that measures the concentration of malondialdehyde (MDA), protein carbonylation (–CO) using ELISA, and single strand breaks of DNA using the comet assay. Our results show significant differences in the responses between the control group and the groups that were exposed to pollutans, enhancing the role of Cornu aspersum as a promising bioindicator organism in terrestrial pollution studies with the use of the biomarkers applied.     

Localization of Hg and Pb in the palps,the digestive gland and the gills in Mytilus galloprovincialis (L.) using autometallography and X-ray microanalysis

The intracellular localization of heavy metals using autometallography (AMG) and X-ray microanalysis was studied in the palps, the digestive gland and the gills of mussels Mytilus galloprovincialis, after an experimental exposure to 0.1 mg l(-1) of Hg and 0.1 mg l(-1) of Pb, for 30 and 60 days. In the examined tissues, autometallographical black silver deposits (BSD) were localized mainly in the residual bodies and heterolysosomes of the digestive cells, as well as in the dense bodies of the epithelial cells. Metal deposition after Hg exposure was much more abundant compared to Pb exposure. Using X-ray microanalysis, Hg was traced on the BSD in all examined tissues, while Pb was not traced in these deposits. The results are discussed in comparison to previous results on long-term exposure to the same metals; in addition, the palps are introduced as a new metal storing organ and, finally, the use of X-ray microanalysis under a scanning electron microscope in order to enhance the specificity of AMG is suggested.     

Lysosomal,tissue and cellular alterations in the gills,palps and intestine of the mussels Mytilus galloprovincialis,in relation to pollution

Lysosomal, tissue and cellular alterations of the gill, the palp and the intestine epithelium of the mussel Mytilus galloprovincialis (L.) collected from six stations along a closed estuarine system (Thermaikos Gulf, northern Greece) were monitored in May, August and December. Semi-quantitative evaluation of histopathological alterations was performed under the light microscope, while certain of the latter alterations were confirmed electron microscopically. In addition, morphometrical evaluation of the volume density of the lysosomes under the electron microscope was conducted. The observed alterations, as the detachment of gill epithelial cells from the basement membranes and the dilated extracellular spaces formed either between the lateral plasma membranes or between the infoldings of the basal plasma membrane in the palps and the intestine are possibly related to the degree of pollution at the examined stations. The particularly expanded extracellular spaces formed by basal plasma membrane infoldings in the intestine in spring may be attributable to red tide algal infection. On the other hand, differences in morphometric parameters of lysosomal structures indicated no direct relationships with pollution levels.Communicated by O. Kinne, Oldendorf/Luhe     

Comparative study of the accumulation and detoxification of Cu (essential metal) and Hg (nonessential metal) in the digestive gland and gills of mussels Mytilus galloprovincialis, using analytical and histochemical techniques

The aim of the present study is the comparative examination of accumulation and detoxification of Cu and Hg in digestive gland and gills of mussels Mytilus galloprovincialis, using atomic absorption spectrophotometry and autometallography. Mussels were exposed to 0.08 mg L⁻¹ Cu, 0.08 mg L⁻¹ Hg, as well as to a mixture of 0.08 mg L⁻¹ Hg and 0.08 mg L⁻¹ Cu for 11 d. After the experimental exposure, animals were kept under laboratory conditions for a detoxification period of 7 d. An antagonistic effect of Cu against to Hg accumulation was noted in the digestive gland of mussels after the experimental exposure, as well as after the detoxification period, supporting the protective role of Cu against to Hg toxicity in this tissue. Digestive gland was suggested as a main organ for Hg accumulation and gills as a target position for Cu accumulation. Additionally, lower time was evaluated for Hg detoxification in the digestive gland and gills of mussels, in relation to those addressed for Cu detoxification in the same tissues. The evaluation of black silver deposits (BSD) extent performed in digestive gland and gills was suggested as a less sensitive approach, in relation to atomic absorption spectrophotometry (AAS), to indentify the concentration of heavy metals in tissues of mussels. The toxic effects of Hg, Cu and a mixture of them on lysosomal system of the digestive cells are also discussed.     

Lysosomal and lipid alterations in the digestive gland of mussels, Mytilus galloprovincialis (L.) as biomarkers of environmental stress

Lysosomal and lipid alterations in the digestive gland cells of the common mussel Mytilus galloprovincialis collected from six stations along Thermaikos Gulf (Northern Greece) were monitored through a 1-year period. The ultrastructural observations of the digestive cells of sampled mussels indicated a higher incidence of "granular" or "dark" residual bodies in the relatively more polluted stations compared to less polluted ones. Alterations in the lysosomal system of digestive cells were assessed: (1) using the lysosomal membrane stability test (2) quantifying lysosomal structural alterations of the residual bodies and the heterolysosomes of the digestive cells at the ultrastructural level, and (3) determining the elemental composition of lysosomes by means of X-ray microanalysis on epoxy resin sections. Evaluation of neutral lipids was realized using image analysis on histochemical resin sections. Mussels from the most heavily polluted stations exhibited reduced lysosomal membrane stability, higher volume density and diameter of heterolysosomes and residual bodies, and, in some cases, relatively higher lysosomal heavy metal content compared to mussels from less polluted stations. The results showed that the lysosomal membrane stability and the volume density of the residual bodies are more sensitive to general stress compared to the other biomarkers used, and could be used successfully as early warning tools in biomonitoring programs.