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We examined, from a morphological and ultrastructural point of view, the liver of the Italian newt (Lissotriton italicus), under basal conditions and after exposure to nonylphenol ethoxylates (NPEs). Nonylphenol ethoxylates are surfactants widely used in a variety of industrial and agricultural processes that may pose a significant risk to aquatic fauna. NPEs, and their degradation intermediates, are known to affect reproductive biology acting as endocrine disruptors; besides estrogenic effects, nonylphenolic compounds may induce organ toxicity, particularly in liver and gonads. We investigated the effects of a nonylphenol ethoxylate (NPE10-ETO) on L. italicus liver using two low concentrations, consistent with the environmental concentrations. For this purpose, animals were exposed to nominal concentrations of 50 and 100 μg/L in a short-term experiment (96 h). A morpho-functional analysis was performed in order to investigate the amphibian responses to NPEs thus contributing to elucidate other potential mode of action of these compounds; indeed very little attention has been dedicated to amphibians though they are often exposed to such contaminants in aquatic ecosystems. Pathological alterations on liver histology and ultrastructure were observed at both tested concentrations; the main effects recorded were: increase of intercellular spaces, accumulation of large lipid droplets, increase in melanin content, and a degeneration phenomenon. We also detected, through confocal analysis, the induction of caspase-3, a key mediator of apoptosis, and an up-regulation of cytochrome P450-1A. By using both ultrastructural and a morpho-functional approach, we found that a short-term exposure to NPEs negatively affected the amphibian liver.

Endosulfan is a persistent organic pollutant (POP) that has lethal and sublethal effects on non-target organisms, including amphibians. In a laboratory study, we investigated direct and post-exposure effects of endosulfan on Bufo bufo tadpoles. For this purpose we exposed the tadpoles to a single short-term contamination event (96 h) at an environmentally-realistic concentration (200 μg endosulfan/L). This was followed by a recovery period of 10 days when the experimental animals were kept in pesticide-free water. The endpoints were assessed in terms of mortality, incidence of deformity, effects on behavior, and the morpho-functional features of the epidermis. We found that a short-term exposure to the tested concentration of endosulfan did not cause mortality but induced severe sublethal effects, such as hyperactivity, convulsions, and axis malformations. Following relocation to a pesticide-free environment, we noted two types of response within the experimental sample, in terms of morphological and behavioral traits. Moreover, by using both ultrastructural and a morpho-functional approach, we found that a short-term exposure to endosulfan negatively affected the amphibian epidermis. We also observed several histo-pathological alterations: increased mucous secretion, an increase in intercellular spaces and extensive cell degeneration, together with the induction of an inducible isoform of nitric oxide synthase (iNOS). Following the post-exposure period, we found large areas of epidermis in which degeneration phenomena were moderate or absent, as well as a further increase in iNOS immunoreactivity. Thus, after 10 days in a free-pesticide environment, the larval epidermis was able to partially replace elements that had been compromised due to a physiological and/or a pathological response to the pesticide. These results highlight the need for both exposure and post-exposure experiments, when attempting to assess pollutant effects.

Nonylphenols ethoxylates (NPEs) are surface active agents (surfactants) commonly used in cleaning products, in industrial processes, agricultural formulations and paints. They are found in sewage, municipal wastewaters and industrial effluents, and as contaminants in water bodies. Accumulating data suggest that exposure to NPEs can adversely affects functional properties of the neurologic, reproductive, immune, and endocrine systems. In order to examine whether NPEs exert similar damaging effects on the cardiovascular system, we used an amphibian model to examine the ultrastructural alterations of the ventricular myocardium following exposure to NPEs. Adult Lissotriton italicus in the aquatic phase were exposed to NPE10 (100 μg/L, ppb) for 96 h. Heart specimens were collected from a total of 10 individuals and processed for scanning and transmission electron microscopy. Our ultrastructural examinations demonstrated that amphibian ventricle is susceptible to the effects of NPEs. The most pronounced alterations were observed in the membrane compartments of both myocardial and endothelial cells as demonstrated by the presence of swollen mitochondria with disrupted cristae and dilated rough endoplasmic reticulum. We suggest that destabilization of the lipid milieu within membranes might represent one of the potential mechanisms by which NPEs exert their toxic effects on amphibian heart.

Background Several lines of clinical and experimental evidence have demonstrated that reactive oxygen species and nitrogen species are generated in unregulated amounts during diverse cardiovascular disorders. It has been previously reported by our group and others that augmented expression of nitric oxide synthase isoforms is associated with human atherogenesis and that the activity of the enzymes in an atherosclerotic environment may promote the formation of peroxynitrite. Among the downstream mechanisms triggered by oxidants, poly(ADP-ribose) polymerase-1 activation has recently been implicated in the pathogenesis of acute and chronic myocardial dysfunction, diabetes, hypertension, aging, and various forms of shock. Methods Based on these observations, we performed immunohistochemical and immunogold labeling analyses to evaluate the expression profile and the subcellular localization of inducible nitric oxide synthase and poly(ADP-ribose) polymerase-1 in healthy and atherosclerotic human aortae. Results We have demonstrated that inducible nitric oxide synthase colocalizes with poly(ADP-ribose) polymerase-1 within vascular cells of atherosclerotic human aortae. We have reported for the first time, to our knowledge, the ultrastructural localization of poly(ADP-ribose) polymerase-1 within the nuclei of lesional smooth muscle cells. Finally, we have evidenced that poly(ADP-ribose) polymerase-1 induction within cells of the diseased aorta strongly correlates with alterations in mitochondrial morphology. Conclusions Our data imply the possibility of a significant role for cross-talk between inducible nitric oxide synthase and poly(ADP-ribose) polymerase-1 in human atherosclerotic lesions. We conclude that the prooxidant milieu of the plaque might exert damaging effects on mitochondria via a poly(ADP-ribose) polymerase-1-mediated mechanism since the absence of the enzyme results in a corresponding lack of changes in mitochondrial morphology. The present report may open avenues for further researches that could have important therapeutic consequences for the treatment of atherosclerosis and its clinical sequelae.

Endosulfan is a widely used organochlorine pesticide with well-documented neurotoxic effects in both humans and laboratory animals (mammals and fish). Neurotoxicity has been implied also in amphibians after short-term exposure to endosulfan. Little is known about effects of chronic exposure of endosulfan in amphibians. Previously, we examined the short-term toxicity of endosulfan in common toad (Bufo bufo) tadpoles and determined the LC50 value to 0.43 mg/L. In the present study, we investigated the effects of endosulfan on B. bufo tadpoles after chronic exposure to ecologically relevant concentrations. Tadpoles were exposed in a static renewal test, from shortly after hatching (Gosner stage 25) to completed metamorphosis, to 0.01, 0.05 and 0.1mg endosulfan/L (nominal). The exposure period lasted 43-52 days. Mortality, larval growth (mass), development (reached Gosner stage at various times and deformities presence), metamorphosis and behaviour (swimming activity) were monitored regularly over the entire course of larval development. Our results show that 0.05 and 0.1mg endosulfan/L caused impaired behaviour, prolonged time to metamorphosis, increased incidences of mouth and skeletal malformations as well as mortality, and reduced body weight (observed also at 0.01 mg/L) in B. bufo tadpoles. Behavioural effects occurred at exposure day 4, before any other effects occurred, indicating a neurotoxic effect. Endosulfan levels found in groundwater and surface water range from 0.1 to 100 microg/L and after extraordinary runoff events, concentrations exceed 0.5 mg/L in surface water. Our results indicate that endosulfan may negatively affect wild frog populations in agricultural areas.

We analysed the morphology and ultrastructure of the gill apparatus of the ornate wrasse, Thalassoma pavo , under normal conditions and after exposure to a sublethal concentration of sodium lauryl sulphate (3.5 mg/l, which is one-third of the 96LC99 value). To identify the biochemical mechanisms affected by this pollutant, we evaluated and compared the localisation of Na + /K + ATPase in normal and experimental conditions. Immunocytochemical analysis revealed that this enzyme was active in the chloride cells (CCs), which were distributed in clusters in the interlamellar region of the filament. Ultrastructural analysis revealed conspicuous alterations on the epithelium after 96 and 192 h of exposure to sodium lauryl sulphate: structural features of the surface cells were lost, the appearance of intercellular lacunae changed, and cellular degeneration occurred. Statistical analysis comparing the number and dimensions of CCs in normal conditions and after 96 h of exposure showed that the CC area decreased after exposure to the detergent.

The morphology and development of the larval oral apparatus of Rana dalmatina , Bombina variegata , Bufo bufo , and Bufo viridis are described and compared using scanning electron microscopy. The species show different arrangements of the mouthparts. The small oral apparatus of R. dalmatina larvae has three labial tooth rows on the upper labium, while there are four tooth rows on the lower labium with a medial gap in row proximal to the mouth. The margins of the oral apparatus are defined by papillae that encircle the lower labium. B. variegata tadpoles have two upper labial tooth rows and three lower labial tooth rows that are uninterrupted, unlike the ones of R. dalmatina . The mouth is encircled by papillae that are larger than those of R. dalmatina . The oral discs of tadpoles of both B. bufo and B. viridis are similar. They are defined by two upper labial tooth rows (the second of which is interrupted by a medial gap) and by three lower tooth rows that differ in lengths in the two Bufo species. Both species develop papillae on the mouth angles and in two rows on the upper labium. Some morphological differences among the oral discs of R. dalmatina , B. variegata , B. bufo , and B. viridis tadpoles can be attributed to phylogenetic differences, but most can be related to their varying feeding habits and/or to their dietary specializations.

Endosulfan is an organochlorine pesticide used in agriculture for a wide range of crops. Endosulfan concentrations of up to 0.7 mg/L can be found in ponds and streams near sprayed agricultural fields. We investigated the short-term toxicity of endosulfan in common toad (Bufo bufo) tadpoles after 24, 48, and 96 h of exposure. Acute toxicity was evaluated at nominal concentrations ranging from 0.01 to 0.6 mg/L: concentrations that could be found after the application of pesticide. Our results show that 0.43 mg/L of endosulfan caused 50% mortality (LC(50)). The effects of a sublethal endosulfan concentration (0.2mg/L) on gill apparatus morphology were evaluated by scanning and transmission electron microscopy. Immunohistochemical methods were also applied to detect the expression pattern of the inducible isoform of nitric oxide synthase (iNOS) in the gills using the confocal laser scanner microscope. Exposure to 0.2mg/L of endosulfan caused an apparent increase in mucus production, the occurrence of secretory vesicles and lamellar bodies, a widening of intercellular spaces and additionally there was evidence of an inflammatory response in the gill apparatus. The morphological alterations occurred after 24h and were more pronounced after 48 and 96 h of exposure. Altered morphology and increased mucus secretion indicate impaired gas exchange and osmoregulation in the gills. In addition, there was an increase of iNOS expression after 24 and 48 h which may reflect hypoxia and inflammation in the gill epithelium. Our results clearly indicate that short-term exposure to a sublethal concentration of endosulfan, near the high end of the environmental range, disrupts gill morphology and function in B. bufo tadpoles.

Labial and maxillary palps in 22 species of ground beetle larvae revealed the presence of four different types of sensilla: sensilla digitiformia, sensilla campaniformia, sensilla basiconica and sensilla chaetica. Using transmission electron microscopy techniques, the role of most of them was defined: sensilla campaniformia and sensilla chaetica are mechanoreceptors; while sensilla basiconica are chemoreceptors. Sensilla digitiformia can be both mechanoreceptors and chemoreceptors, though hygroreception may also be postulated on the basis of their abundance on the mouthparts of some hygrophilous species that depend on damp or wet patchy biotopes/seasons. The number and location of the sensory structures has been found to vary among the investigated taxa. The differences are related to the larval behavioural types, and are an adaptive response to different lifestyles, feeding habits and use of space.