Digestive cell turnover in digestive gland epithelium of slugs experimentally exposed to a mixture of cadmium and kerosene

Slugs, Arion ater (L), have been proposed as sentinel organisms to assess soil health. In slugs under the influence of pollutants, digestive cell loss and the concomitant increase of excretory cells of the digestive gland have been described. The aim of the present work was to determine up to what extent digestive cell loss affects biomarkers and whether the affectation is reversible after exposure to a mixture of metal and organic pollutants. Slugs were dosed with a mixture of cadmium and kerosene in the food for 27 days. Apart from chemical analyses, the volume density of black silver deposits (Vv(BSD)) after autometallography, and acyl-CoA oxidase (AOX) activity were used as biomarkers of exposure to metals and organic compounds, respectively. As effect biomarkers, changes in the volume density of the cell types that constitute the digestive gland epithelium were calculated. Proliferating cells were identified by means of bromodeoxyuridine (BrdU) immunohistochemistry. Results revealed that the mixture of pollutants provoked an increase in Vv(BSD) and AOX activity and a decrease in the number of digestive cells. These changes had no effect in the digestive gland accumulation capacity or in the effect and exposure biomarkers employed. BrdU-labelling showed that exposure to pollutants provoked an enhanced digestive cell proliferation.     

Exposure of caged mussels to metals in a primary-treated municipal wastewater plume

The biological availability of metals in municipal wastewater effluents is strongly influenced by the physical and chemical conditions of both the effluent and the receiving water. Aquatic organisms are exposed to both dissolved and particulate (food ingestion) forms of these metals. In the present study, the distribution of metals in specific tissues was used to distinguish between exposure routes (i.e. dissolved vs. particulate phase) and to examine metal bioavailability in mussels exposed to municipal effluents. Caged Elliptio complanata mussels were deployed at sites located between 1.5 km upstream and 12 km downstream of a major effluent outfall in the St. Lawrence River. Metals in surface water samples were fractionated by filtration techniques to determine their dissolved, truly-dissolved (<10 kDa), total-particulate and acid-reactive-particulate forms. At the end of the exposure period (90 days), pooled mussel soft tissues (digestive gland, gills, gonad, foot and mantle) were analyzed for several metals. The results showed that gills and digestive gland were generally the most important target tissues for metal bioaccumulation, while gill/digestive gland metal ratios suggest that both exposure routes should be considered for mussels exposed to municipal effluents. We also found that Ag and Cd in the dispersion plume nearest the outfall, in contrast to other metals such as Cu and Zn, are more closely associated with colloids and were generally less bioavailable than at the reference site in the St. Lawrence River.     

Haematology, genotoxicity, enzymatic activity and histopathology as biomarkers of metal pollution in the shrew Crocidura russula

Haematological (WBC, RBC, Hgb and Hct) and genotoxicity (MNT) parameters, hepatic enzymatic activities (GST, GPx and GR), and a histopathological evaluation of liver, kidneys and gonads were assessed as general biomarkers of metal pollution in the shrew Crocidura russula inhabiting a pyrite mining area. Specimens exposed to metals presented a few significant alterations when compared with reference animals: GST activity decreased; micronuclei increased; and evident liver alterations related to metal exposure were observed. On the basis of all the parameters studied, age was an important factor that partly explained the observed variation, whereas sex was the least important factor. Significant correlations were also found between heavy metal concentrations and biomarkers evaluated, demonstrating the great influence of these metals in the metabolic alterations. To the best of our knowledge, these data constitute the first measurements of a battery of biomarkers in shrews from a mine site and are among the few available for insectivorous mammals.     

Effects of permethrin exposure on antioxidant enzymes and protein status in Mediterranean clams Ruditapes decussatus

The effects of permethrin (PER) on a panel of antoxidant enzymes; superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) and indices of protein oxidation status (carbonylation and free thiols) were determined in digestive gland and gills of the clam Ruditapes decussatus. Animals were exposed to 100 ppb PER for 2 days. These enzyme activities increased significantly in digestive gland (p?<?0.05) after PER treatment and oxidative modification of proteins was detected in both gill and digestive gland extracts using redox proteomics. PER exposure significantly reduced the amount of protein free thiol groups in digestive gland rather than in gill, when compared to controls. Conversely, digestive gland showed significantly higher levels of carbonylated proteins than gill after PER exposure. Some proteins were successfully identified by mass spectrometry of tryptic peptides. Our data suggest that digestive gland of R. decussatus can be used as a model tissue for investigating environmental risk of PER contamination.     

Oxidoreductases provide a more generic response to metallic stressors (Cu and Cd) than hydrolases in soil fungi: new ecotoxicological insights

The present study investigates the effect of metals on the secretion of enzymes from12 fungal strains maintained in liquid cultures. Hydrolases (acid phosphatase, β-glucosidase, β-galactosidase, and N-acetyl-β-glucosaminidase) and ligninolytic oxidoreductases (laccase, Mn, and lignin peroxidases) activities, as well as biomass production, were measured in culture fluids from fungi exposed to Cu or Cd. Our results showed that all fungi secreted most of the selected hydrolases and that about 50 % of them produced a partial oxidative system in the absence of metals. Then, exposure of fungi to metals led to the decrease in biomass production. At the enzymatic level, Cu and Cd modified the secretion profiles of soil fungi. The response of hydrolases to metals was contrasted and complex and depended on metal, enzyme, and fungal strain considered. By contrast, the metals always stimulated the activity of ligninolytic oxidoreductases in fungal strains. In some of them, oxidoreductases were specifically produced following metal exposure. Fungal oxidoreductases provide a more generic response than hydrolases, constituting thus a physiological basis for their use as biomarkers of metal exposure in soils.     

New metallothionein assay in Scrobicularia plana: heating effect and correlation with other biomarkers

Metallothionein (MT) and other biomarker levels were measured in Scrobicularia plana clams to assess pollution of the Guadalquivir Estuary possibly affected by metals released from Aznalcóllar pyrite mine in 1998. After optimizing reagent concentrations for monobromobimane derivatization, MT levels were quantified by reversed-phase high-performance liquid chromatography coupled to fluorescence detection (RP-HPLC-FD) in heated or unheated digestive gland extracts and compared to those obtained by differential pulse polarography (DPP). MT content assayed by RP-HPLC-FD in unheated samples was higher than that obtained by DPP and correlated better with metals and anti-oxidant activities. MT assay by RP-HPLC-FD in unheated extracts would be preferable for assessing metal pollution, due to its greater sensitivity and specificity. In addition to MT induction, glyoxalase II inhibition was well correlated with metal contents. Our results suggest that metals at the estuary do not originate from Aznalcóllar spill, but from those carried along by the river and deposited at its concave bank.     

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.     

Biochemical effects in crabs (Carcinus maenas) and contamination levels in the Bizerta Lagoon: an integrated approach in biomonitoring of marine complex pollution

The biochemical effects in Carcinus maenas and contamination levels in seawater and sediments of Bizerta Lagoon (northeast of Tunisia) were investigated. The levels of metals and hydrocarbons were higher in seawater and sediments in Menzel Bourguiba and Cimentery in February and July than in the other sampling sites. Differences among sites for glutathione S-transferase, catalase, acetylcholinesterase activities, and the content of lipid peroxidation and metallothioneins in two important organs which accumulated contaminants (the gills and the digestive gland) of the C. maenas were found and possibly related to differences in metal and hydrocarbon levels. The seasonal variation of biomarkers was possibly associated with chemical contamination and also with the high fluctuation of physico-chemical characteristics of the sites. The integrated biomarker response values found in the five sites is in good agreement with hydrocarbon and trace metal concentrations detected in the water and sediments of the stressful places where crabs are living.     

Accumulation of lead and cadmium in the marine prosobranch Nerita saxtilis, chemical analysis, light and electron microscopy.

The potential value of the marine prosobranch Nerita saxtilis as an efficient biological monitor to heavy metal pollution in the Red Sea was investigated. Storage ability of lead and cadmium was compared in shell, headfoot and digestive gland of the marine snail N. saxtilis collected from Al-Hamrauin area at El-kuseir (lead, 300.35 +/- 28.53 microg/l, 1,716 +/- 16.14. cadmium 20.01 +/- 1.8 microg/l, 161.72 +/- 21.4 mean +/- S.D. for water and sediment, respectively) relative to that of inhabiting marine water and sediment employing atomic absorption spectrometry to determine the organ with highest capability of heavy metal accumulation. The influence of metal storage on light microscopic structure of that organ was investigated. Also, the ultrastructure localization of storage sites in the same organ was determined employing transmission electron microscopy. The digestive gland was shown to accumulate both metals at conccntrations that are several orders of magnitude higher than those in the surrounding marine water. The bioaccumulation capability of lead and cadmium was ranked in the following order; digestive gland > headfoot > shell for lead and digestive gland > shell > headfoot for cadmium. In spite of its evident highest metal storage capability, no histopathological changes were observed in the digestive gland of that marine prosobranch. Enlarged electron dense vesicles and many granules were observed in ultrathin sections in digestive cells of these snails and are suggested to be the sites of storage of detoxified metals. The results of that finding indicate the possibility of using the marine prosobranch N. saxtilis as biomonitor for heavy metal contaminants in the Red Sea.     

The effect of pesticides and metals on acetylcholinesterase (AChE) in various tissues of blue mussel (Mytilus trossulus L.) in short-term in vivo exposures at different temperatures

The purpose of the study was to assess the impact of short-term exposure to selected toxicants as well as metal accumulation upon acetylcholinesterase (AChE) in the blue mussel, Mytilus trossulus L., in laboratory in vivo experiments. Mussels were exposed for up to 48 hours to a mixture of copper (Cu^{2 +}, 400 μ g L^?1) and cadmium (Cd^{2 +}, 200 μ g L^?1), to dichlorvos (DDVP, 100 μ g L^?1), and to carbaryl (100 μ g L^?1) at two temperatures: 5°C and 20°C. Samples were collected after 0, 6, 12, 24, and 48 hours of exposure, and AChE activity and metal concentration (where applicable) were analysed in gills, digestive gland, mantle+muscles, and the whole soft tissue. Very strong AChE inhibition was observed in response to the dichlorvos treatment, mainly in gills. Carbaryl and the metals caused a short-term inhibition effect. Considerable differences in AChE activity between the two temperatures were noticed. In particular, the metals were accumulated much faster at 20°C than at 5°C, especially in gills. No correlation between AChE activity and metal concentration was found. Gills turned out to be the optimal tissue for AChE activity analysis in short-term studies.     

In vitro effects of pesticides and metals on the activity of acetylcholinesterase (AChE) from different tissues of the blue mussel,Mytilus trossulus L.

The objective of this study was to conduct a comprehensive comparison of the effect on acetylcholinesterase (AChE) from various tissues of Mytilus trossulus caused by in vitro exposure to several pesticides and metals, because results available in the literature are inconsistent, difficult to compare, and sometimes contradict each other. For this purpose, fraction S10 extracted from gills, digestive gland, mantle and muscles, and the whole soft tissue of the mussel was exposed to several pesticides (dichlorvos, chlorpyrifos, fenitrothion, carbofuran and carbaryl) and metals (Cu, Zn, Cd, Hg, Pb) at a wide range of concentrations. AChE was inhibited in 50% or more in all the tissues exposed to dichlorvos, Cu, Hg and the mixture of Cu+Cd, and in some tissues exposed to carbaryl and carbofuran. The IC₅₀ was calculated where possible. No inhibition was found in the case of chlorpyrifos, Cd, Pb, and Zn.     

Metals bioaccumulation and histopathological biomarkers in Carcinus maenas crab from Bizerta lagoon,Tunisia

Metals concentrations and histolopathological lesions of gills and digestive gland were investigated in Carcinus maenas crabs sampled from Bizerta Lagoon and Kuriat Island (Tunisia) as control site. The concentrations of trace metals varied between tissues, sites and sampling time. The highest levels of the analysed metals in gills and digestive gland were noted in Menzel Bourguiba and Cimentery sites at both sampling times (February and July). The higher metals loads were associated with severe and various tissues alterations in contaminated crabs. We particularly noted in the gills a haemocytic infiltration, distension and enlargement of the lamellae, lifting of lamellar epithelium, necrotic lesions and fusion of lamellae in the most polluted sites (Menzel Bourguiba and Cimentery). Moreover, others pathological alterations were observed in digestive gland of crabs collected from polluted sites and with a severity site dependent. We observed necrotic tubules containing tissue debris in the lumen with more intensity in crabs collected from Cimentery site in both sampling times. The thickened basal laminae and the walling off of the tubules by haemocytes around the thickened basal laminae were more abundant at Menzel Bourguiba than at others sites. The coagulation in the thickened basal laminae was observed only at Cimentery in February. Tissues histopathological lesions were sensitive to discriminate crabs of different sites and demonstrated its usefulness in this biomonitoring study. We recommend the association of histopatholocial lesions to biochemical biomarkers in future biomonitoring studies.     

Oxidative stress on land snail Helix aspersa as a sentinel organism for ecotoxicological effects of urban pollution with heavy metals

The oxidative stress in the digestive gland of the land snail Helix aspersa was considered as a bioindicator for atmospheric pollution with heavy metals from several industries and vehicular traffic in Kafr El-Zayat city. Regional means of heavy metals concentration of all sites were 0.71, 7.09, 0.71, 2.68, 41.44 and 18.01 mg kg⁻¹ wet mass for Cd, Mn, Ni, Pb, Zn and Cu, respectively. In addition, the highest values of Cd concentrations were found 1.22 and 1.73 mg kg⁻¹ wet mass in S1 (Potato International Center) and S4 (The Nile bank), respectively. Lactate dehydrogenase (D-LDH(and recorded lipid peroxidation (LPO) levels were significantly high in S1 and S2 (Traffic station). On the other hand, the highest activity of catalase (CAT) was found in S2 (194.04% of control), while the activity of glutathione peroxidase (GP_x) reached the highest significant value in S1.     

Sub-cellular partitioning of Cd, Cu and Zn in tissues of indigenous unionid bivalves living along a metal exposure gradient and links to metal-induced effects

We studied organ and sub-cellular distributions of several trace metals in a freshwater bivalve that has been proposed for use as a metal biomonitor. Specimens of Pyganodon grandis were collected from nine lakes located along a Cd, Cu and Zn concentration gradient (Rouyn-Noranda area, Quebec). Gills and digestive gland were isolated, homogenized and six sub-cellular fractions were separated by differential centrifugation and analysed for their Cd, Cu and Zn content. Metallothionein was quantified independently. Gill tissues contained abundant calcium concretions that accounted for over 60% of the total gill burden of each metal. Cadmium and Zn concentrations in this granule fraction reflected ambient metal concentrations. Metal concentrations in the digestive gland also responded to the metal contamination gradient, but to a lesser extent than the gills, reflecting the lower abundance of granules in the digestive gland. Metals (Cd, Cu) in this organ were present largely in the "heat-stable proteins" fraction, and metal concentrations in this fraction were strongly correlated with those of both metallothionein and, to a lesser extent, the "lysosomes+microsomes" and "mitochondria" fractions. In both organs, Cd concentrations in the "heat-denaturable protein" fraction remained low and constant, suggesting reasonably effective metal detoxification. Some evidence for oxidative stress was noted in the gills but not in the digestive gland. Overall, we conclude that in nature metals in P. grandis are bound differently in the gills and in the digestive gland and that metal detoxification in the former organ may be less effective than in the latter.     

Transcriptional response of stress genes to metal exposure in zebra mussel larvae and adults

Development of stress markers for the invader freshwater zebra mussel (Dreissena polymorpha) is of great interest for both conservation and biomonitoring purposes. Gene expression profiles of several putative or already established gene expression stress markers (Metallothionein, Superoxide dismutase, Catalase, Glutathione S transferase, Glutathione peroxidase, Cytochrome c oxidase, the multixenobiotic resistance P-gp1, and heat shock proteins HSP70 and HSP90) were analyzed by quantitative Real-Time PCR in adults and pediveliger larvae after exposure to metals (Hg, Cu, Cd). A defined pattern of coordinated responses to metal exposure and, presumably, to oxidative stress was observed in gills and digestive gland from adults. A similar, albeit partial response was observed in larvae, indicating an early development of stress-related gene responses in zebra mussel. The tools developed in this study may be useful both for future control strategies and for the use of zebra mussel as sentinel species in water courses with stable populations.     

Experimental exposure to cadmium affects metallothionein-like protein levels but not survival and growth in wolf spiders from polluted and reference populations

Both local adaptation and acclimation in tolerance mechanisms may allow populations to persist under metal pollution. However, both mechanisms are presumed to incur (energetic) costs and to trade-off with other life-history traits. To test this hypothesis, we exposed Pardosa saltans (Lycosidae) spiderlings originating from metal-polluted and unpolluted sites to a controlled cadmium (Cd) treatment, and compared contents of metal-binding metallothionein-like proteins (MTLPs), internal metal concentrations, and individual survival and growth rates with a reference treatment. While increased MTLP concentrations in offspring originating from both polluted and unpolluted populations upon exposure indicates a plastic tolerance mechanism, survival and growth rates remain largely unaffected, independent of the population of origin. However, MTLP and Cd concentrations were not significantly correlated. We suggest that MTLP production may be an important mechanism enabling P. saltans populations to persist in ecosystems polluted with heavy metals above a certain level.     

Heavy metal deposition in the Italian “triangle of death” determined with the moss Scorpiurum circinatum

In this study, a biomonitoring project using the moss Scorpiurum circinatum was carried out to evaluate the deposition and biological effects of heavy metals in the area of Acerra (Naples, S Italy), one of the vertices of the sadly called “Italian triangle of death” owing to the dramatic increase in tumours. The results clearly indicated that the study area is heavily polluted by heavy metals, a large proportion of which is likely present in the atmosphere in particulate form. The ultrastructural organization of exposed samples was essentially preserved, but cell membrane pits, cytoplasm vesicles and concentric multilamellar/multivesicular bodies, probably induced by pollution, were found, which may be involved in the tolerance mechanisms to metal pollution in this moss species. Although severe biological effects were not found at the ultrastructural level in the exposed moss, effects on humans, especially after long-term exposure, are to be expected.     

Effects of environmentally relevant concentrations of metallic compounds on the flatfish Scophthalmus maximus: biomarkers of neurotoxicity,oxidative stress and metabolism

Flatfish species, such as the turbot (Scophthalmus maximus), are common targets for toxic effects, since they are exposed through the food chain (ingestion of contaminated preys) and are in direct contact with the waterborne contaminant and sediments. Furthermore, these fish species live in close proximity to interstitial water that frequently dissolves high amounts of contaminants, including metals. Despite this significant set of characteristics, the present knowledge concerning flatfish contamination and toxicity by metals is still scarce. To attain the objective of assessing the effects of metals on a flatfish species, S. maximus specimens were chronically exposed to lead, copper and zinc, at ecologically relevant concentrations, and biochemical (oxidative stress: catalase and glutathione S-transferases activities, and lipid peroxidation; neurotoxicity: cholinesterase activity) parameters were assessed on selected tissues (gills and liver). Copper had no significant effects on all tested parameters; lead was causative of significant increases in liver GSTs activities and also in lipoperoxidation of gill tissue; exposure to zinc caused a significant increase in catalase activity of gill tissue. None of the tested metals elicited noteworthy effects in terms of neurotoxicity. The obtained results showed that only the metal lead is of some environmental importance, since it was able to cause deleterious modifications of oxidative nature at relevant concentrations.     

Effects of atmospheric exposure to naphthalene on xenobiotic-metabolising enzymes in the snail Helix aspersa

Polycyclic aromatic hydrocarbons are xenobiotics whose elevated toxicity for living organisms requires to efficiently monitor air pollution, either by evaluating their levels in the environment, or by assessing their biological impacts on sentinel organisms. We investigated the effects of naphthalene exposure on some xenobiotic-metabolising enzyme activities in three organs of Helix aspersa. Particular activities depending on cytochrome P450 (ethoxyresorufin O-deethylase, EROD; ethoxycoumarin O-deethylase, ECOD; pentoxyresorufin O-dealkylase, PROD) and associated with glutathione (glutathione S-transferase, GST; glutathione peroxidase, GPX; glutathione reductase, GR) were assessed. In control animals, the P450-dependent specific activities were distributed according to the range kidney > digestive gland > mantle cavity forming tissues (MCFT). Neither ECOD nor PROD activities could be detected in MCFT. In the two other organs, the major phase I activities were due to ECOD, the level of PROD being very low or null. The glutathione-associated activities showed comparable levels in the three organs, except GPX activity that was higher in the digestive gland. Naphthalene (NAP) exposure did not affect any activity in MCFT, but it significantly decreased EROD and ECOD activities in the kidney as opposed to their increase in the digestive gland, whereas PROD activities were not influenced by the treatment. Glutathione-dependent activities were not significantly affected by NAP exposure, except for GPX which activity diminished in the digestive gland. This study demonstrates that complex detoxification pathways should exist in Helix aspersa as in mammals and that they could be used as potential biomarkers of NAP exposure.