Multiple pollution biomarker application on tissues of Eobania vermiculata during two periods characterized by augmented and reduced snail activity.

In the present study a package of biomarkers was applied on land snails E. vermiculata collected from polluted areas, as well as from an unpolluted reference one. Snail collection was performed during two different sampling periods characterized by reduced and augmented organism activity, October and May, respectively. The biomarkers applied were lysosomal membrane stability on digestive cells (LMS), neutral red lysosomal retention assay on haemocytes (NRR), morphometric changes of the lysosomal system (VDL, NDL), morphometric alterations of the neutral lipids (VDLP, NDLP), acetylcholinesterase activity on digestive gland and hemolymph (AChE), metallothionein content on digestive gland (MTs) and cyclic AMP content on digestive gland (cAMP). The results revealed significant differences in biomarker values between the two sampling periods. Significant differences were also detected among the sampling groups. The fluctuation of the parameters applied indicated that spring is a more suitable period for sampling conduction compared to autumn and that biomonitoring studies should be performed with special attention during the last mentioned period.

Effects of organic pollutants on Eobania vermiculata measured with five biomarkers.

In the present study, the effect of organic pollution on land snails Eobania vermiculata was investigated. Five pollution biomarkers (neutral red retention assay, morphometry of lysosomes and neutral lipids, acetylcholinesterase activity and metallothioneins content), were applied on tissues of the land snails. The results showed intense differentiations between the snails treated with organic pollutants and the control ones, as indicated by the results obtained. Statistically significant correlations among the results obtained emphasize the usefulness of these biomarkers.

In vivo and in vitro effects of metals in reactive oxygen species production, protein carbonylation, and DNA damage in land snails Eobania vermiculata.

Heavy metals are known to induce oxidative damage by way of enhancement of intracellular reactive oxygen species (ROS) production, which often preludes the onset of alterations, such as protein carbonylation and DNA damage. In this study, our aim was to examine the early responses of land snails Eobania vermiculata to environmental contaminants by investigating the use of a modified enzyme-linked immunosorbent assay (ELISA) assay for the measurement of protein carbonylation as a new biomarker of terrestrial pollution as well as by measuring ROS production and DNA damage. Land snails were treated with heavy metals-cadmium, lead, or copper-in vivo (15 or 40 ppm) for 25 days or in vitro (0.5, 5, 50 or 500 µM) for 30 min in the laboratory, and the previously mentioned biomarkers were determined in digestive gland and haemolymph of the treated animals. A statistically significant increase in ROS production, protein carbonylation, and DNA damage was shown in the snails treated with pollutants compared with the untreated snails. The results indicate the effectiveness of measuring ROS production and DNA damage, as well as using the present ELISA method, as sensitive tools of terrestrial pollution biomonitoring studies. Statistically significant correlations among the previously mentioned techniques further enhance their role as promising biomarkers in terrestrial pollution studies.

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.

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 cAMP and establishment of neutral lipids alterations as pollution biomarkers using the mussel Mytilus galloprovincialis. Correlation with a battery of biomarkers.

The results of the present study suggest that the signaling molecule cAMP could constitute a reliable biomarker of pollution monitoring. Increased levels of cAMP were observed in tissues of mussels Mytilus galloprovincialis exposed to PAHs, 2,4-dichlorophenoxyacetic acid and lindane for 15 days. cAMP levels were higher in mantle/gonad complex, in relation to other tissues studied (digestive gland and gills). In support of our suggestion, cAMP was significantly correlated with established biomarkers, such as lysosomal membrane stability, AChE activity and metallothionein content. In addition, our results support the establishment of morphometrical alterations of neutral lipids as biomarker of organic pollution, since high accumulation of neutral lipids in the digestive gland of mussels exposed to organic pollutants in relation to non-exposed mussels was noted. In addition, correlation analysis between cellular and biochemical biomarkers showed that the volume density of neutral lipids negatively correlated with both lysosomal membrane stability and AChE activity. Regarding micronucleus test, our results do not support its use as a biomarker of organic pollution, since no statistical differences were found between control and exposed mussels. On the other hand, significant correlations were observed among the battery of biomarkers, such as lysosomal membrane stability, AChE activity and metallothionein content after the mussels' exposure to the specific organic compounds, thus supporting the important role of these biomarkers as reliable indicators of organic pollution. In conclusion, our results support cAMP and morphometrical alterations of neutral lipids as biomarkers of environmental pollution.

Carbohydrate cytochemistry of bonnet monkey (Macaca radiaca) nasal epithelium. Response to ambient levels of ozone.

The main purpose of this study was to determine whether exposures to ambient levels of ozone induced changes in the carbohydrate content in the anterior nasal cavity of bonnet monkey. Monkeys were exposed to 0.00. 0.15, or 0.30 ppm of ozone for 6 or 90 days, 8 hours/day. Monkeys treated for 90 days to 0.15 or 0.30 ppm of ozone displayed damaged ciliated cells and a new cell type in their respiratory nasal epithelium, the "intermediate" cells possessing morphological characteristics of both ciliated and secretory cells. Characterization of the secretory cell carbohydrates with the PA-TCH-SP, HID-TCH-SP and LID-TCH-SP techniques indicated the existence of periodate-reactive sulfated and carboxylated polysaccharides in both treated and control monkeys. Ozone-inhalation, however, induced the appearance of a large number of mucous cells with dilated cisternae of rough endoplasmic reticulum compared to controls, which reacted negatively for sugar presence. In addition, in the respiratory epithelium ozone induced the increase of cells with intracytoplasmic lumina containing material reacted positively for sulfated and carboxylated polysaccharides.

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 beta-glucuronidase immunoreactive, a fact that enhances the hypothesis that beta-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-beta-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-beta-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.

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.

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.

Cytochrome P-450 immunocytochemical characterization in liver cells of rabbit. Induction with phenobarbital.

Immunogold staining of Lowicryl embedded liver sections was used to examine the specific binding of antisera against two pulmonary isozymes (2 and 5) of constitutive form cytochrome P-450, as well as against NADPH-cytochrome P-450 reductase (R) on both untreated and phenobarbital-treated rabbits. In the cytoplasm of liver parenchymal cells, the antisera bound specifically to regions rich in agranular endoplasmic reticulum. In addition, the antibodies bound to plasma membranes in the endothelium of sinusoids and capillaries, as well as to plasma membranes and ground cytoplasm of the cells in bile ducts and interlobular veins. In the liver parenchymal cells, the labelling over the agranular endoplasmic reticulum was uniform throughout the lobule. On the contrary, the labelling over nucleus, mitochondria, granular endoplasmic reticulum and Golgi apparatus was comparable to that observed in control sections. Induction with phenobarbital apparently increased the amount of the agranular endoplasmic reticulum and consequently the total number of gold particles bound in each parenchymal cell compared to controls. In addition, phenobarbital increased the labelling density of isozyme 2 in parenchymal cells, and that of isozymes 2 and 5 in endothelial cells of sinusoids and capillaries.

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.

Lysosomal membrane stability and metallothionein content in Mytilus galloprovincialis (L.), as biomarkers. Combination with trace metal concentrations.

The simultaneous study of two biomarkers, the lysosomal membrane stability (LMS) of the digestive gland and the metallothionein (MT) content of the digestive gland, the gills and the mantle/gonad complex of the mussel Mytilus galloprovincialis was examined in an enclosed estuarine system in June and October 2001. Mussels were collected along the Gulf of Thermaikos (northern Greece) from stations displaying a pollution gradient, while Olympiada in the Gulf of Strymonikos was used as a reference station. In addition, the heavy metal (Cd, Pb, Cu and Zn) content, using atomic absorption spectrophotometry (AAS), were evaluated in the digestive gland, the gills and the mantle/gonad complex of mussels collected from the same sites and seasons. LMS values were significantly greater, and the MT content of the studied tissues were significantly less in mussels collected from the reference station compared to mussels from more polluted stations located in the Gulf of Thermaikos. Significant correlation was observed between the MT content of the gills and the mantle/gonad complex with the LMS values.

Tracheal epithelium of bonnet monkey (Macaca radiaca) and its response to ambient levels of ozone. A cytochemical study.

The effect of 6 or 90 days exposure to 0.15 or 0.30 ppm of ozone on the morphology or carbohydrate cytochemistry of bonnet monkey tracheal epithelium was studied. Ozone treatment induced damages and necrotic phenomena of ciliated cells, as well as a significant increase in the population density of small mucous granule (SMG) cells compared to controls. In addition, the appearance of 'intermediate' cells, a type of nonciliated cells which was not observed in controls, was noticed. Characterization of the secretory cell carbohydrates with the PA-TCH-SP, HID-TCH-SP and LID-TCH-SP sequences indicated the existence of periodate-reactive sulfated and carboxylated polysaccharides in both treated and control monkeys. The luminal surface of the affected tracheal cells, however, exhibited a glycocalyx more rich in mucosubstances than the control animals. The possible role of 'intermediate' and SMG cells in repairing the injured epithelial regions, as well as the significance of the ozone concentrations used in relation to the ozone range measured in photochemical smog are discussed.

Rickettsiae-like microorganisms in the midgut and other visceral tissues during development of Drosophila auraria.

Rickettsiae-like organisms (RLOs) were identified for the first time in midgut, Malpighian tubules, wreath cells (ventral nephrocytes), spermatogonia and gut muscles in a species of Drosophila. Their number in the midgut cells of Drosophila auraria significantly increased at the late third larval instar and at the beginning of pupation. The RLO population in the larval midgut followed the fate of their host cells and was destroyed during metamorphosis. The RLOs pass from the larval to the adult midgut via the RLOs existing in the 'regenerative cells', which will form the adult midgut during population. The total volume of RLOs per anterior midgut cell increased analogically in relation to the absolute volume of the host cells till the 130 h larval stage. However, during the late-third instar and at the beginning of pupation the total volume of RLOs per host cell significantly increased. The physiological significance of the latter data as well as the relationship between RLOs and host cells are discussed.

Effect of He-Ne laser on the midgut cells of Drosophila auraria larvae and its correlation with acupuncture.

The He-Ne laser is generally considered as a "cold" or "soft" laser. However, the present study indicates that doses from 0.3 to 0.5 J/cm2 of He-Ne laser caused intensive damage that was mainly observed on the plasma membrane and the mitochondria of the midgut cells of Drosophila auraria larvae. In contrast, doses less than 0.3 J/cm2 of the He-Ne laser didn't cause any visible effect, as measured by the electron microscope. The possible use of the He-Ne laser and the subsequent effect in relation to its application in acupuncture and therapeutic research is discussed.

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.

Effect of starvation and hibernation on the values of five biomarkers of general and specific stress using the land snail Eobania vermiculata.

Values of five biomarkers related to cell stress or pollution were evaluated in tissues of the land snail Eobania vermiculata under starvation or hibernation conditions. The biomarkers applied were lysosomal membrane stability in digestive gland cells (LMS) or in haemocytes (neutral red retention assay (NRR)), acetylcholinesterase activity (AchE; EC 3.1.1.7), metallothionein content (MTs), and cyclic AMP content (cAMP). Three groups of snails were studied that were kept under starvation, hibernation and normal conditions, respectively. The results indicated statistically lower values of LMS and NRR in snails kept under starvation or hibernation compared to control ones. Higher values of AChE activity were measured in snails under hibernation compared to controls. MT contents were statistically higher in snails under starvation compared to controls. Measurement of cAMP contents showed no significant differences among the tested groups. The values of the first four biomarkers may be affected by factors other than pollution, such as starvation or hibernation. Therefore, these factors should be taken into consideration when biomonitoring studies are performed in time intervals of hibernation or starvation.

Aspects of the digestive gland cells of the mussel Mytilus galloprovincialis, in relation to lysosomal enzymes, lipofuscin presence and shell size: contribution in the assessment of marine pollution biomarkers.

The present study investigates the histochemical localization of N-acetyl-ß-hexozaminidase (Hex), acid phosphatase (AcP) and ß-glucuronidase (ß-Gus) in the digestive gland of mussels Mytilus galloprovincialis, as well as the clarification of suitable enzyme for biomarkers' application dealing with lysosomes. The results show more intense and homogenous localization of Hex, in relation to AcP and ß-Gus and, thus, Hex histochemistry is supported as more suitable procedure for the evaluation of "lysosomal membrane stability" and "morphometrical alterations of lysosomes". The affection of lipofuscin granules on lysosomal enzymes' activity is also discussed. Additionally, the present study examines the response of small- and large-sized mussels M. galloprovincialis by assessing the "lysosomal membrane stability", "morphometrical alterations of lysosomes", "lysosomal response index (LRI)" and "structural epithelial changes in digestive tubules". The results indicate appreciable alterations of the above parameters in large-sized mussels, supporting their greater influence by the environmental factors, in relation to small-sized ones.

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 package's efficacy of the selected biomarkers.