V.K. Dimitriadis

Evaluation of neutral red retention assay, micronucleus test, acetylcholinesterase activity and a signal transduction molecule (cAMP) in tissues of Mytilus galloprovincialis (L.), in pollution monitoring

The neutral red lysosomal retention assay (NRR) of the haemocytes, and the acetylcholinesterase activity (AChE) in the haemolymph, the digestive gland, the gills and the mantle/gonad complex have been evaluated on mussels Mytilus galloprovincialis collected from Thermaikos and Strymonikos gulfs (northern Greece) in June and October 2001. The validity of performing the above core biomarkers is supported, firstly by their ability to respond to different pollution levels and, secondly, by the significant linear correlation among them. The evaluation of the micronuclei frequency (MN) has been performed in gill tissue and haemocytes of the same mussels and, according to the results, it needs more research in order its use as stress indices to be validated. In addition, the first results on cAMP levels in the gills, the mantle/gonad complex and the digestive gland, whose concentrations correlated to both, NRR and AChE introduce this signal transduction molecule as a new, promising biomarker.

Cytochemical and Histochemical Aspects of the Digestive Gland Cells of the Mussel Mytilus galloprovincialis (L.) in Relation to Function

The epithelium of the digestive tubules of the mussel Mytilus galloprovincialis is comprised of two cell types, namely digestive and basophilic cells. In basophilic cells, the secretory granules are β-glucuronidase immunoreactive, a fact that enhances the hypothesis that β-glucuronidase is synthesized in basophilic cells. A novel observation at the ultrastructural level is the pinocytic activity associated with the formation of coated pits. This observation constitutes direct evidence for endocytic processes taking place in basophilic cells. The use of cryostat sections from the same digestive tubules reveals, in many instances, a very pronounced neutral lipid accumulation in the same structures giving a positive reaction for N-acetyl-β-hexosaminidase, indicating the association of those lipids with lysosomes. In some mussels, a high content of lipofuscin was observed in the lysosomes of the digestive cells. In these cases, the lysosomal structures show a limited neutral lipid content, and a weaker N-acetyl-β-hexosaminidase reaction. In the digestive cells, the carbohydrate content of the lysosomes, and very well-developed canal system in the apical part of cells are discussed in relation to their function.

Biomarkers in marine mussels, Mytilus galloprovincialis, exposed to environmentally relevant levels of the pesticides, chlorpyrifos and penoxsulam

The present study examines the influence of environmentally relevant concentrations of two pesticides, chlorpyrifos and penoxsulam on mussel physiological status. For this reason, lysosomal membrane stability (LMS), reactive oxygen species (ROS), DNA damage, protein carbonylation (PCC) and antioxidant capacity (TAC) in hemaolymph and hemocytes of the mussels was measured. Mussels were exposed to a range of concentrations of the pesticides chlorpyrifos and penoxsulam and the response of animals to the destabilization of lysosomal membrane in hemocytes (LMS) was studied. Subsequently, the half maximal effective concentration (EC50) for both pesticides was calculated. The animals were subsequently exposed for 0, 1, 3, 5, 7, 15 and 30 days to 10 times less concentration than EC50 of each pesticide (0.05 μg/l) and changes in LMS, ROS, DNA damage, protein carbonylation and antioxidant capacity of mussels was evaluated. Our results showed a significant change in the response of mussels for all parameters tested after 30 days exposure, in relation to the controls. The pesticides at the environmental concentrations used induced changes to the animal physiology through causing oxidative stress and lysosomal abnormalities and their usage in the agriculture demands great care. In addition, the results show that ROS, DNA damage, protein carbonylation and antioxidant capacity could constitute, after further investigation, reliable biomarkers for the evaluation of pollution or other environmental stressors.

Aspects of the usefulness of five marine pollution biomarkers, with emphasis on MN and lipid content

By using cellular biomarkers, such as the formation of the micronucleus, the morphometric alterations of lipids and the morphometric changes in the lysosomal system, we investigated effects of experimental exposure to phenanthrene, Cu, Cd, and Hg for 15 days in mussels. Concerning micronuclei, the evaluation of the total nuclear abnormalities, instead of the micronucleus only, as a biomarker of marine pollution, indicated more statistically significant differences between the control and the pollutant treated groups. Contrary to the existing knowledge showing that there is an increase in lipid content after pollutant exposure, our results showed that there was a decrease in the amount of lipid, as well as an increase in the number of neutral lipids. Furthermore, although prior studies found that fewer lysosomes formed after pollutant exposure, this was not confirmed by our work, as pollutant treated animals exhibited a decrease in the volume and an increase in the numerical density of lysosomes, compared to control groups.

Introduction of the land snail Eobania vermiculata as a bioindicator organism of terrestrial pollution using a battery of biomarkers

The present study aimed to enrich the group of sentinel organisms of terrestrial pollution biomonitoring, by investigating the efficacy of the land snail Eobania vermiculata. For this reason, a package of biomarkers was performed on land snails E. vermiculata collected from polluted areas in the field or treated with heavy metals in the laboratory. The biomarkers used were neutral red lysosomal retention assay of the haemocytes, acetylcholinesterase activity in the digestive gland and the haemolymph, and metallothionein content of the digestive gland. Moreover, the morphometric changes in the lysosomal system and the morphometric alterations of the neutral lipids were also investigated. In addition, the content of cadmium, lead and copper was evaluated in the digestive gland of the snails. The results revealed appreciable alterations in the biomarker values both in field- and laboratory-conditions, accompanied by significant correlations among the biomarkers. Therefore, this exploratory study suggests the utility of E. vermiculata as a sentinel organism for biomonitoring the biologic impact of terrestrial pollution, and supports the packages efficacy of the selected biomarkers.

Fine structure of the midgut of adult Drosophila auraria and its relationship to the sites of acidophilic secretion

Abstract Electron microscopic and morphometric examination of the Drosophila auraria midgut allowed its subdivision into three main regions, anterior, middle and posterior midgut. Among these regions, the anterior and the posterior midgut display similar quantities of rough endoplasmic reticulum and of Golgi complexes. The posterior midgut can be considered to be the most important site for secretory granule accumulation and secretion. The cup-shaped cells of the middle midgut may be primarily involved in generating and managing the acid pH in the middle midgut lumen. These cells display small amounts of rough endoplasmic reticulum, a small number of Golgi complexes, a large number of mitochondria located throughout the cytoplasm and few infoldings of the basal plasma membrane. The cup-shaped cells indicate negative carbonic anhydrase activity, an enzyme involved in tissues mediating active transport of acids or bases. However, these cells show a high concentration of Cl − ions in their cytoplasm and apical microvilli compared to the rest of the middle midgut and other midgut cells. It is proposed that the transport of the acidophilic fluids from the intrato the extra-cellular space takes place through vesicles, which are pinched off from the tip of the microvilli of the cup-shaped cells and are partially dissolved in the highly acidic midgut lumen.

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.

Assessment of the health status of mussels Mytilus galloprovincialis along Thermaikos Gulf (Northern Greece): An integrative biomarker approach using ecosystem health indices

Pollution biomarkers were assessed in the digestive gland of mussels Mytilus galloprovincialis collected from Greek coasts (Thermaikos and Strymonikos Gulf, Northern Greece) on July 2004. The values of biomarkers were integrated using three ecosystem health indices: health status index (HIS) calculated by the expert system, lysosomal response index (LRI) and integrated biomarker response (IBR). The HIS is based on the integration of the biomarker values, classifying the health status of mussels according to pollution degree. The LRI combines two lysosomal responses (lysosomal membrane stability and lysosomal structural changes) in a single index, which is associated with the ecosystem contamination. The IBR is a useful tool for the visualization of biological responses in marine environments. The results support the use of HIS and LRI as powerful tools for the qualitative and quantitative evaluation of environmental stress, while enhance the use of IBR as a constructive method for the qualitative discrimination of contaminated sites. HIS and LRI revealed high stress conditions in all sampling stations of Thermaikos gulf, whereas unstressed conditions were detected in Olympiada (reference station) by the use of HIS and low stress conditions were shown by the use of LRI. The IBR illustrated higher levels of stress in mussels collected from Thermaikos Gulf, compared to mussels gathered from the reference station. The advantages and disadvantages of the HIS, LRI and IBR are also discussed.

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 7d. 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 identify 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.

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.