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The increasing industrial use of vanadium (V), as well as its recent medical use in various pathologies has intensified its environmental release, making it an emerging pollutant [1]. The sea urchin embryo has long been used to study the effects induced by metals, including V. In this study we used an integrated approach that correlates the biological effects on embryo development with proteolytic activities of gelatinases that could better reflect any metal induced imbalances. V-exposure caused morphological/morphometric aberrations, mainly concerning the correct dis-tribution of embryonic cells, the development of the skeleton and the embryo volume [2]. Moreover, V induced a concentration change in all the gelatinases expressed during embryo development and a reduction in their total proteolytic activity. The presence of three MMPs-like gelatinases (MMP-2, -9 and -14) was also demonstrated and their levels depended on V-concentration. In particular, the MMP-14-like protein modified its expression level during embryo development in a time and dose dependent manner. This enzyme also showed a specific localization on filopodia, suggesting that primary mesenchyme cells (PMCs) could be responsible for it synthesis. In conclusion, these results indicate that an integrated study among morphology/morphometry, proteolytic activity and MMP-14 expression constitutes an important response profile to V-action

Glyphosate (Gly) is a broad-spectrum herbicide widely used thanks to its high efficiency and low toxicity. However, evidence exists of its toxic effects on non-target organisms. Among these, the animals inhabiting agricultural fields are particularly threatened. Recent studies demonstrated that exposure to Gly markedly affected the morphophysiology of the liver and testis of the Italian field lizard Podarcis siculus. The present study aimed to investigate the effects of the herbicide on the female reproductive system of this lizard in order to have a full picture of Gly-induced reproductive impairment. The animals were exposed to 0.05 and 0.5 μg/kg of pure Gly by gavage for 3 weeks. The results demonstrated that Gly, at both doses tested, profoundly interfered with ovarian function. It induced germ cells’ recruitment and altered follicular anatomy by anticipating apoptotic regression of the pyriform cells. It also induced thecal fibrosis and affected oocyte cytoplasm and zona pellucida organizations. At the functional levels, Gly stimulated the synthesis of estrogen receptors, suggesting a serious endocrine-disrupting effect. Overall, the follicular alterations, combined with those found at the level of the seminiferous tubules in males, suggest serious damage to the reproductive fitness of these non-target organisms, which over time could lead to a decline in survival.

Fertility rates in animals have shown a progressive decrease in recent decades, and reproductive toxicity is considered an important regulatory endpoint in health hazard assessment [...]

The increasing industrial use of vanadium (V), as well as its recent medical use in various pathologies has intensified its environmental release, making it an emerging pollutant. The sea urchin embryo has long been used to study the effects induced by metals, including V. In this study we used an integrated approach that correlates the biological effects on embryo development with proteolytic activities of gelatinases that could better reflect any metal-induced imbalances. V-exposure caused morphological/morphometric aberrations, mainly concerning the correct distribution of embryonic cells, the development of the skeleton, and the embryo volume. Moreover, V induced a concentration change in all the gelatinases expressed during embryo development and a reduction in their total proteolytic activity. The presence of three MMP-like gelatinases (MMP-2, -9, and -14) was also demonstrated and their levels depended on V-concentration. In particular, the MMP-14-like protein modified its expression level during embryo development in a time- and dose-dependent manner. This enzyme also showed a specific localization on filopodia, suggesting that primary mesenchyme cells (PMCs) could be responsible for its synthesis. In conclusion, these results indicate that an integrated study among morphology/morphometry, proteolytic activity, and MMP-14 expression constitutes an important response profile to V-action.

The expansion of agriculture produces a steady increase in habitat fragmentation and degradation due to the increased use of pesticides and herbicides. Habitat loss and alteration associated with crop production play an important role in reptile decline, among which lizards are particularly endangered. In this study, we evaluated testicular structure, steroidogenesis, and estrogen receptor expression/localization after three weeks of oral exposure to glyphosate at 0.05 and 0.5 μg/kg body weight every other day in the field lizard Podarcis siculus. Our results show that glyphosate affected testicular morphology, reduced spermatogenesis, altered gap junctions and changed the localization of estrogen receptors in germ cells, increasing their expression; the effects were mostly dose-dependent. The result also demonstrates that glyphosate, at least at these concentrations, did not influence steroidogenesis. Overall, the data indicate that this herbicide can disturb the morphophysiology of the male lizard’s reproductive system, with obviously detrimental effects on their reproductive fitness. The effects of glyphosate must be considered biologically relevant and could endanger the reproductive capacity not only of lizards but also of other vertebrates, including humans; a more controlled and less intensive use of glyphosate in areas devoted to crop production would therefore be advisable.

Vanadium toxicology is a topic of considerable importance as this metal is widely used in industrial and biomedical fields. However, it represents a potential emerging environmental pollutant because wastewater treatment plants do not adequately remove metal compounds that are subsequently released into the environment. Vanadium applications are limited due to its toxicity, so it is urgent to define this aspect. This metal is associated with sea urchin embryo toxicity as it perturbs embryogenesis and skeletogenesis, triggering several stress responses. Here we investigated its bioaccumulation and the correlation with cellular and molecular developmental pathways. We used cytotoxic concentrations of 1 mM and 500 μM to perform quantitative analyses, showing that vanadium accumulation interferes with calcium uptake during sea urchin development and provokes a disruption in the biomineralization process. At the end of the whole treatment, the accumulation of vanadium was about 14 and 8 μg for embryos treated respectively with 1 mM and 500 μM, showing a dose-dependent response. Then, we monitored the cell signaling perturbation, analyzing key molecular markers of cell survival/cell death mechanisms and the DNA fragmentation associated with apoptosis. This paper clarifies vanadium’s trend to accumulate directly into embryonic cells, interfering with calcium uptake. In addition, our results indicate that vanadium can modulate the ERK pathway and activate a cell-selective apoptosis. These results endorse the sea urchin embryo as an adequate experimental model to study metal-related cellular/molecular responses.

Vanadium (V) is a metal widely distributed in soil, water and air. It has recently received growing interest because its compounds are often used in different applications, from industry to medicine.1 Here, using atomic absorption spectrometry, we demonstrate the predisposition of V to accumulate directly into embryonic cells, interfering with Ca uptake. At the morphological level, we observed dose- and time-dependent effects on phenotypes and on skeletal malformations. At the molecular level, V-exposed embryos showed the activation of the cellular stress response, inducing Hsp 60 and Hsp 70 synthesis and the activation of autophagy and apoptosis. The Hsps-mediated stress response to V appeared to counteract the damage induced by low (50 nM and 100 nM) and intermediate (500 nM and 1 µM) concentrations, while high cytotoxic doses (500 µM and 1 mM) induced more marked cell death mechanisms starting at 24 h of development, when the control embryos reached the gastrula stage.2 Only few cells showed nuclei with apoptotic DNA fragmentation, particularly in the ectodermal layer. Mesodermal and endodermal cells did not appear to be involved in this process of selective apoptosis.3 Microscopic fluorescence inspections indicated that primary mesenchyme cells (PMCs) were not involved in apoptotic processes, therefore their inability to carry on the skeletogenesis could be due to the Ca depletion. These results allow us to elect the sea urchin embryo as a suitable experimental model for studying the metal-correlated cellular/molecular responses.4,5

Environmental chemicals can be introduced by consuming contaminated foods. The environmental chemical dichlorodiphenyldichloroethylene (DDE), a persistent metabolite of dichlorodiphenyltrichloroethane (DDT), can affect spermatogenesis. Our study aims to evaluate, by using spectrophotometric analyses, western blot, and immunohistochemistry, the adaptive responses in testis of adult rats treated with a non-toxic dose of DDE, alone or in association with a high-fat diet (HFD). Four experimental groups were performed: N (normal diet), D (HFD), D + DDE (HFD + DDE), N + DDE (normal diet + DDE). D group showed a reduction in antioxidant capacity, and increases in lipid peroxidation, apoptosis, and proliferation associated with morphological impairment. A reduction in androgen receptor (AR) and serum testosterone levels were also found. DDE-treated groups exhibited higher lipid peroxidation levels compared to N and D, associated with pronounced defect in antioxidant capacity, apoptosis, cellular proliferation, as well as with tissue damage. Moreover, decreases in AR and serum testosterone levels were found in DDE-treated groups vs. N and D. In conclusion, HFD and DDE produced cellular stress leading to antioxidant impairment, apoptosis, and decreases in AR and serum testosterone levels associated with tissue damage. Cellular proliferation could be used as an adaptation to counterbalance the occurred damage, maintaining a pool of tubules that follow physiological maturation.

The Heat Shock Proteins (HSPs) are a superfamily of molecular chaperones that maintain cellular homeostasis under stress. HSP70 represents the major stress-inducible family member, often activated in response to changes in thermal ranges of organisms, and therefore playing an important role enhancing thermal tolerance limits in ectothermic animals. The present study aimed to investigate the presence and the localization of HSP70 through the development of Podarcis siculus, an oviparous lizard inhabiting temperate Mediterranean regions, showing a limited potential to tolerate thermal changes during embryogenesis. Immunohistochemical analysis demonstrated that HSP70 protein is constitutively present in early embryonic stages, abundantly distributed in eye, in encephalic domains (predominantly in ventricular areas and in grey matter), in grey matter of spinal cord, in lung, gut mucosa, hepatic cords and kidney tubules. Interestingly, a severe drop in incubation temperature (5 °C for 3 days) does not induce enhancements in HSP70 levels nor changes in tissues localization. These results suggest that the HSP70 found in P. siculus embryos represents a non-inducible, constitutive molecular chaperone that should be better called Heat Shock Cognate 70 (HSC70); the presence of stress-induced members of the HSP family in P. siculus has yet to be proven.

The potential toxicity of glyphosate, a widely used broad-spectrum herbicide, is currently a great matter of debate. As vertebrate insectivores, lizards protect plants from herbivorous insects increasing plant biomass via the trophic cascade and represent an important link between invertebrates and higher predators. A negative effect of glyphosate on lizards’ survival could have major impacts at the ecological levels. In this study, we investigated the effects of the exposure to low doses of glyphosate on the liver of the wall lizard Podarcis siculus, a suitable bioindicator of soil pollution. Two different doses of pure glyphosate (0.05 and 0.5 μg/kg body weight) were orally administered every other day for 3 weeks to sexually mature males and females. The results demonstrated that both doses, despite being very low, are toxic for the liver that showed clear signs of suffering, regardless of sex. The histological analysis provided a scenario of severe hepatic condition, which degenerated until the appearance of fibrotic formations. The morphological observations were consistent with a loss of liver physiological functions. Immunocytochemical investigations allowed us to detect an involvement of antioxidant/cytoprotective proteins, such as superoxide dismutase 1 (Cu/Zn SOD, known as SOD1), glutathione peroxidase 1 (GPx1), metallothionein (MT), and tumor suppressor protein 53, (p53) suggesting that the liver was trying to react against stress signals and damage induced by glyphosate. Finally, in situ hybridization and Real-Time PCR analysis showed the upregulation of estrogen receptor α and vitellogenin gene expression, thus demonstrating the xenoestrogenic action of glyphosate. The imbalance of the hormonal homeostasis could threaten the lizards’ reproductive fitness and survival, altering the trophic cascade.

Metallothionein is well known for its detoxificant and anti-oxidant properties and has been shown to be effective to prevent hydroxyl radical-generated DNA degradation. The purpose of this investigation was to analyze the combined effect of two factors promoting cellular oxidative-stress, that is, the administration of the pesticide dichloro–diphenyl–dichloroethylene (DDE) and a high fat diet, on metallothionein expression and synthesis in rat liver and kidney. DDE is the main metabolite of dichloro–diphenyl–trichloroethane (DDT), and is commonly found in the food chain and in all tissues of living organisms, carried by the fats. Male Wistar rats were fed with a standard (N) or a high fat (HF) diet and exposed to DDE (10 mg/kg body mass, N + DDE and HF + DDE groups) or vehicle (corn oil, N, and HF groups) via gavage every day for 28 days. Tissues histology was determined by light microscopy analysis; differences in metallothionein gene expression and synthesis by real-time PCR and western blot, respectively. Finally, protein cellular localization was established by immunocytochemistry. The results showed a different involvement of metallothionein in defending tissues from HF- and DDE- induced oxidative stress, suggesting that hepatic and renal cells use different strategies against pro-oxidant species. In both cell types a marked increase in the metallothionein content was observed in the nucleus, with a concomitant drop of the cytoplasmatic protein, either under HF- and DDE-stress conditions; however, no synergistic or additive effects were observed between the action of fats and pesticide. These findings reinforce the role of metallothionein in protect- ing DNA from oxidative damage.

The terrestrial lizard Podarcis siculus is the most abundant reptile in Italy, where is considered a ubiquitous species. This lizard is widely distributed from the islands to the Apennines, from cultivated fields to anthropized areas such as gardens and city parks. For this reason, these animals are exposed to extensive physical and chemical stresses, as well as to the possibility of coming into contact with industrial pollutants and substances used in agricultural practices. Here, we review the health status of lizard specimens inhabiting natural non-anthropized areas and fields devoted to organic farming, considering the condition of (1) liver, representing the main detoxifying organ, directly influenced by feeding, and (2) gonads, essential for reproduction and, therefore, for the survival of the species. The morphological and biomolecular condition of these organs was then compared with those obtained from lizard specimens experimentally treated with nonylphenol, a co-formulant of many insecticides and plant protection products widely used in conventional farming, known to have harmful estrogenic effects. Taken together, data demonstrate that lizards inhabiting manured soil are in good health status and show a regular morphology of liver, testis, and ovary. Animals are found to be less exposed to the toxic heavy metals cadmium and lead if compared with specimens collected in areas not devoted to agriculture, but probably more exposed to vehicular traffic. However, manure, as well as nonylphenol, exerts a xeno-estrogenic effect, particularly evident in male specimens, more sensitive to estrogenic contamination.

Estrogen receptors alpha (ERα) and beta (ERβ) are transcription factors known to be involved in the regulation of many complex physiological processes in mammals. They are expressed primarily in the reproductive tract of all vertebrates females, thus indicating important and conserved functions in female reproductive success. ERs are also present in physiological different tissues as bone, brain, liver, skin and adipose tissues, in both females and males. In the latter, ERs have been found also in the genital tract, supporting the findings of a complex role for estrogen in spermatogenesis and, more generally, in male reproduction. This review provides an overview and update on ERα and ERβ expression and synthesis in male reproductive tract of non-mammalian vertebrates, with focus on their role in germ cells proliferation, maturation and survival. Data from studies on fish, amphibians, reptiles and birds were collated and common or species-specific distribution highlighted. The widespread distribution of estrogen receptors in testicular cells and ducts of all vertebrates so far investigated suggests that whatever are the roles that estrogens may exert on these structures, they are phylogenetically conserved and are possibly related to the physiological support given to achieve male reproductive success.

Metallothionein (MT) is the main protein involved in the homeostasis of metallic micronutrients and in cellular defence against heavy metals and reactive oxygen species. Found in almost all vertebrate tissues, MT presence and localization in the testis has been controversial. In the present study, by using in situ hybridization and immunohistochemical analysis we assessed the localization of both MT transcript and protein in Podarcis sicula testes during two different phases of the reproductive cycle: the autumnal resumption and the spring-early summer mating period. In addition, with the same methodological approach, we verified the effect of estradiol-17ß and nonylphenol, a potent xenoestrogen, on MT expression and synthesis. These results, the first collected in a non-mammalian oviparous vertebrate, demonstrated that the expression profile of MT mRNA and protein changes during the reproductive cycle. In the fall resumption, MT transcripts are absent in spermatogonia and present in all the other cells of tubules, including spermatozoa; vice versa, the MT protein is evident only in spermatozoa and somatic cells. In the mating period, both MT transcripts and proteins are present in spermatogonia, spermatocytes and spermatids, whereas in the spermatozoa only the proteins are detected, thus suggesting that the MTs translated in the earlier germinal stages are stored up to spermatozoa. Results also demonstrated that in lizard testis the expression of MT gene undergoes a cell-specific regulation after estrogenic exposure; the possible role and the mechanism by which this regulation occurs have been discussed.

Neurodegeneration is one of the main physiological consequences of aging on brain. Metallothioneins (MTs), low molecular weight, cysteine-rich proteins that bind heavy- metal ions and oxygen-free radicals, are commonly expressed in various tissues of mammals. MTs are involved in the regulation of cell proliferation and protection, and may be engaged in aging. Expression of the ubiquitous MTs (1 and 2) and the brain specific MT3 have been studied in many neurodegenerative disorders. The research results indicate that MTs may play important, although not yet fully known, roles in brain diseases; in addition, data lack the ability to identify the MT isoforms functionally involved. The aim of this study was to analyse the level of gene expression of selected MT isoforms during brain aging. By using real-time PCR analysis, we determined the MT1/2 and MT3 expression profiles in cerebral cortex and hippocampus of adolescent (2months), adult (4 and 8 months), and middle-aged (16 months) rats. We show that the relative abundance of all types of MT transcripts changes during aging in both hippocampus and cortex; the first effect is a generalized decrease in the content of MTs transcripts from 2- to 8-months-old rats. After passing middle age, at 16 months, we observe a huge increase in MT3 transcripts in both cortical and hippocampal areas, while the MT1/2 mRNA content increases slightly, returning to the levels measured in adolescent rats. These findings demonstrate an age- related expression of the MT3 gene. A possible link between the increasing amount of MT3 in brain aging and its different metal-binding behaviour is discussed.

Plants of the urban environment are exposed to a wide range of pollutants, including heavy metals. This research studies in situ the eco-physiological and antioxidant responses of holm oak (Q. ilex) leaves to Pb and Cd to assess the mechanisms of metal tolerance in this species, widely used as biomonitor. Leaves of plants grown at parks and roadsides were analyzed for photo- synthetic activity, Pb and Cd concentration in tissues and cell-free extracts, thiol groups, D1 and Rubisco protein content, ascorbic acid (AsA) amount, and cata- lase (CAT) activity. The main results evidenced that Cd concentration was higher in leaves collected at the park out from the downtown; whereas Pb was most abundant in leaves sampled at the roadside nearby the highway. Pb in cell-free extracts was higher in park than in road- side leaves. Although Cd in the leaf tissues was twofold lower than Pb, it was more abundant than Pb in cellular extracts deprived of all particulate matter. Leaves responded to different concentration of Cd and Pb mod- ulating some eco-physiological and biochemical traits, roadside leaves showed reduced leaf lamina, higher content of photosynthetic pigments, hydrogen peroxide, and AsA, as well as higher CAT activity compared to park leaves. In the roadside leaves, a stress condition for photosynthetic apparatus can be hypothesized on the basis of the decline of photochemical activity, the in- crease of NPQ, and the reduction of Rubisco and D1 protein content. The elevated presence of thiol groups in these leaves suggests a possible role of Pb and Cd in activation of antioxidant responses.

In this study, our aim was to determine whether caspase 3 plays a role, during previtellogenesis, in the ovarian follicular epithelium of the lizard Podarcis sicula. We investigated the presence and localization of proform and active caspase 3 by enzyme assay, Western blotting and immunocytochemistry. In parallel, a fragment of caspase 3 was cloned for the first time in this species, sequenced and used for in situ hybridization to localize messengers and analysed by a phylogenetic survey to shed light on its homology with reptilian caspases. Results demonstrated that: (1) the follicle cells expressed a caspase of the 3/7 group and the mRNA for caspase 3 was transcribed in the stem phase and was completely translated during cell differentiation; (2) the proform protein was stored during the differentiated (nurse) stage and activated at the end of previtellogenesis provoking the degeneration of cells; (3) the predicted protein sequence, although partial, had a strong similarity with the known reptilian caspases 3. The epithelial cells of the ovarian follicle, therefore, do not employ caspase 3 during the nurse stage but, instead, prepare for apoptosis long before the process actually begins. The relevance of this strategy is discussed.

The effects of an exposure to cadmium chloride 0.47 μM for 150 days were studied in kidneys of juveniles Sparus aurata by a multidisciplinary approach so to correlate uptake and detoxification potential to changes in brush border and glycocalyx sugar composition. Results demonstrated that cadmium concentration in kidney significantly increased from day 30 reaching a plateau on day 120 while metallothioneins reached a peak on day 90 and by day 120 were already decreasing to control values. Cytological damage was extensive on day 90, clearly detectable at both structural and ultrastructural levels, in tubular cells and brush-border. Staining with a panel of four lectins revealed a significant increase in N-Ac-Gal and a decrease in mannose in the glycocalyx and the tubular basal membranes. From day 120, when cadmium concentration was high and metallothionein concentration decreasing, a clear recovery was observed in tubular cells morphology and sugar composition. Possible significance of these apparently contrasting data are discussed.

Chelates of Gadolinium (Gd), a lanthanide metal, are employed as contrast agents for magnetic resonance imaging and are released into the aquatic environment where they are an emerging contaminant. We studied the effects of environmentally relevant Gd concentrations on the development of two phylogenetically and geographically distant sea urchin species: the Mediterranean Paracentrotus lividus and the Australian Heliocidaris tuberculata. We found a general delay of embryo development at 24 h post-fertilization, and a strong inhibition of skeleton growth at 48 h. Total Gd and Ca content in the larvae showed a time- and concentration-dependent increase in Gd, in parallel with a reduction in Ca. To investigate the impact of Gd on the expression of genes involved in the regulation of skeletogenesis, we performed comparative RT-PCR analysis and found a misregulation of several genes involved in the skeletogenic and left-right axis specification gene regulatory networks. Species-specific differences in the biomineralization response were evident, likely due to differences in the skeletal framework of the larvae and the amount of biomineral produced. Our results highlight the hazard of Gd for marine organisms.

Metallothioneins (MTs) are an evolutionary conserved multigene family of proteins whose role was initially identified in binding essential metals. The physiological role of MT, however, has been revealed to be more complex than expected, since not only are MTs able to bind to toxic heavy metals, but many isoforms have shown specialized and alternative functions. Within this uncertainty, the information available on MTs in non-mammalian vertebrates, particularly in neglected tetrapods such as the reptiles, is even more scant. In this review, we provide a summary of the current understanding on metallothionein presence and function in the oviparous lizard Podarcis sicula, highlighting the results obtained by studying MT gene expression in most representative adult and embryonic tissues. The results demonstrate that in adults, cadmium induces MT transcription in a dose- and tissue-specific manner. Thus, the MT mRNAs appear, at least in some cases, to be an unsuitable tool for detecting environmental ion contamination. In early embryos, maternal RNAs sustain developmental needs for MT protein until organogenesis is well on its way. At this time, transcription starts, but again in a tissue- and organ-specific manner, suggesting an involvement in alternative roles. In conclusion, the spatiotemporal distribution of transcripts in adults and embryos definitively confirms that MT has deserved the title of elusive protein.

In the last years, worldwide organic farming has grown exponentially; as a consequence, the use of animal manure as a soil fertility source has become the principal agricultural choice. However, the use of manure as fertilizer can increase the amount of steroid hormone metabolites in the soil. In southern Italy, lacertidae lizards are the most abundant vertebrate group in agroecosystems and have been identified as potential model species for ecotoxicological studies. The aim of this study was to understand if the manure applied in organic farming has estrogen-like effects in the lizard Podarcis sicula. Adult male lizards were captured in two organic agricultural fields (manure-treated sites) and in an uncultivated field (control site). Lizards from the two organic farms displayed hepatic biosynthetic alterations typical of an estrogenic contamination; hepatocytes contained both vitellogenin and estrogen receptor alpha transcripts and proteins, detected by in situ hybridization and immunocytochemistry. The same cells did not show cadmium, lead and metallothionein accumulation, indicative of the lack of inorganic contamination. These findings suggest that exogenous estrogens, arising from the use of manure, could affect the welfare of wild animals and animal breeding, leading to bioaccumulation of estrogens in food chain, with possible risk for human consumers. For this reason, organic farming should implement the use of sustainable practices such as crop rotation to preserve the soil biological activity, rather than organic manure as fertilizer.

In oviparous vertebrates, vitellogenin (VTG) is the major yolk precursor synthesized in the liver of sexually mature females during the reproductive period. In males, the VTG gene is silent, but it may be activated by estradiol-17β (E2) or estrogen-like substances. Until now, extra-hepatic expression and synthesis of VTG after estrogen exposure has been reported only for aquatic vertebrates. This study demonstrates the ability of testis and epididymis of the terrestrial oviparous lacertid Podarcis sicula to synthesize VTG following E2 exposure. The results of in situ hybridization and immunohistochemistry analysis show the presence of both VTG mRNA and protein in these districts besides the known induction in the liver. The possible contemporaneous uptake of the E2-induced hepatic VTG by means of the specific vitellogenin receptor has been also evaluated. Finally, histological analysis shows that the E2-treatment during the mating season impairs spermatogenesis.

Ocular malformations are observed in embryos and adults of aquatic species after exposure to toxicants. In this study, cadmium, a chemical stressor, was used to determine the toxic effects on the retinal development and visual ability of adult zebrafish (Danio rerio). Our results demonstrated that Cd-exposure (100 μM)from sphere (4 hpf) to gastrula stage (8hpf) led to the up-regulation of the expression of lambda1-crystallin and Retinoblastoma Binding Protein 6 transcripts in embryos. Cryl1 is a lens protein that acts as a molecular chaperone; Rbbp6 is a protein regulating cellular proliferation. In situ hybridization demonstrated that Cd induced the up-regulation of these genes also in adult retina, in the inner and outer nuclear layers. The Cd-induced over-expression was dose-dependent and reversible. Interestingly, metallothionein, the cysteine-rich, low molecular mass metal-binding protein considered a key molecule involved in cellular protection against toxic metals, did not seem to be up-regulated. Adult retinal morpho-cytological alterations were investigated by light and electron microscopy, while the functionality of Cd-exposed retina was assessed by re-illumination behavioral tests. After Cd-treatment, dose-dependent alterations were clearly observed. The nerve fiber layer was clearly thickened and vacuolated, several ganglion cells had compact pycnotic nuclei. Changes occurred in the thickness of the pigmented epithelium and at the level of the cones inner segments. TEM observations oCd-treated retina showed ganglion cells with highly vacuolated cytoplasm and cells with ultrastructural apoptotic features. Apoptotic cells were also evident in the inner nuclear and in the photoreceptor layers. Many polymorphic Müller cells were observed both in inner and outer nuclear layers. Behavioral tests demonstrated that re-illumination with white or colored light induced a significant escape response after Cd-treatment, not observed in untreated animals. All together, data demonstrated that Cd toxicity caused: 1) dysregulation of gene expression; 2) degeneration and loss of organization at both macro and microscopic levels; 3) impairment of the functional response, particularly through increased light sensitivity. The increased amount of Müller cells with irregular shape suggested that the recovery of visual acuity is due to a regenerative process involving these retinal macroglial cells, that are able to dedifferentiate and function as neural stem cells.

Alteration in cholesterol metabolism has been implicated in the pathogenesis of several neurodegenerative disorders. Apolipoprotein E (ApoE) is the major component of brain lipoproteins supporting cholesterol transport. We previously reported that the acute-phase protein Haptoglobin (Hpt) binds ApoE, and influences its function in blood cholesterol homeostasis. Major aim of this study was to investigate whether Hpt influences the mechanisms by which cholesterol is shuttled from astrocytes to neurons. In detail it was studied Hpt effect on ApoE-dependent cholesterol efflux from astrocytes and ApoE-mediated cholesterol incorporation in neurons. We report here that Hpt impairs ApoE-mediated cholesterol uptake in human neuroblastoma cell line SH-SY5Y, and limits the toxicity of a massive concentration of cholesterol for these cells, while it does not affect cholesterol efflux from the human glioblastoma-astrocytoma cell line U-87 MG. As aging is the most important non-genetic risk factor for various neurodegenerative disorders, and our results suggest that Hpt modulates ApoE functions, we evaluated the Hpt and ApoE expression profiles in cerebral cortex and hippocampus of adolescent (2 months), adult (5 and 8 months), and middle-aged (16 months) rats. Hpt mRNA level was higher in hippocampus of 8 and 16 month-old than in 2-month old rats (p < 0.05), and Hpt concentration increased with the age from adolescence to middle-age (p < 0.001). ApoE concentration, in hippocampus, was higher (p < 0.001) in 5 month-old rats compared to 2 month but did not further change with aging. No age-related changes of Hpt (protein and mRNA) were found in the cortex. Our results suggest that aging is associated with changes, particularly in the hippocampus, in the Hpt/ApoE ratio. Age-related changes in the concentration of Hpt were also found in human cerebrospinal fluids. The age-related changes might affect neuronal function and survival in brain, and have important implications in brain pathophysiology.

Ferritin is a major intracellular iron storage protein in higher vertebrates and plays an important role in iron metabolism. This study reports the identification from the Antarctic icefish Chionodraco rastrospinosus of a complete mRNA sequence and four partial mRNA sequences, all encoding the ferritin M subunit and sharing a clear homology with the ferritin M-chain of other fish species. The open reading frame of the complete ferritin M transcript is of 528 base pairs and encodes a protein of 176 amino acids that retains the residues involved in the ferroxidase diiron center and in the ferrihydrite nucleation center. Despite the absence of hemoglobin and of any appreciable amount of iron in the icefish blood, RT-PCR analysis shows that H and M ferritin subunits are expressed both in blood and in other tissues, such as spleen, head kidney, liver and kidney. Phylogenetic analysis shows that the H and M subunits form two well separated clusters. Basal to these two clusters emerges a heterogeneous cluster, formed by two Danio rerio M, a Salmo salar M and an Orechromis niloticus H isoforms; these forms maybe represent the heritage of ancestral forms from which arose the two major H and M subunits of the fishes.

Lizard embryos are nutritionally independent from their environment. During the early phases of oogenesis, the egg prepares for development by storing reserve organelles, proteins, and RNAs sufficient to allow the zygote to transform into a juvenile. This preparation also includes the storage of metallothionein (MT) transcripts. This study investigated the localization of these transcripts by in situ hybridization throughout Podarcis sicula developmental stages. Our data show that MT expression undergoes shifts in both regional and cellular localization. MT transcripts were detected early in the central nervous system, later in tissues implicated in metabolic processes. Results are discussed highlighting differences in lizard embryonic spatial and temporal MT expression compared with piscine, amphibian, and mammalian embryos. We hypothesize that, under natural conditions, the nutritionally closed system represented by the lizard egg protects the developing embryo from an unwanted excess of metals. This mechanism would make MT expression and accumulation in detoxifying organs in developing animals unnecessary until hatching and food intake begins. Conversely, the presence of MT transcripts during brain development may ensure the correct final architecture of this organ.

Screening for differentially expressed genes is a straightforward approach to study the molecular basis of contaminant toxicity. In this paper, the mRNA differential display technique was applied to analyze transcriptional regulation in response to cadmium exposure in the lizard embryos. Lizard eggs may be particularly susceptible to soil contamination and in ovo exposure may interfere or disrupt normal physiological function in the developing embryo, including regulation of gene expression. Fertilized eggs of the lizard Podarcis sicula were incubated in cadmium-contaminated soil at 25 degrees C for 20 days. Gene expression profiling showed 5 down- and 9 up-regulated genes. Four cDNAs had no homology to known gene sequences, thus suggesting that may either encode not yet identified proteins, or correspond to untranslated regions of mRNA molecules. Four fragments exhibited significant sequence similarity with genes encoding novel proteins or ESTs derived from other vertebrates. The remaining genes are mainly involved in molecular pathways associated with processes such as membrane trafficking, signal transduction, cytoskeletal organization, cell proliferation and differentiation. Cadmium also affected the expression of factors actively involved in the regulation of the transcription machinery. Down-regulated genes are mainly associated with cellular metabolism and cell-cycle regulation and apoptosis. All of these differentially expressed genes may represent candidates that function in cadmium responses. The present study leads to an increased understanding of genes and/or the biochemical pathways involved in perturbation of embryo development following cadmium exposure.

This study examined the cytological and molecular effects of cadmium, a toxic heavy metal, in the liver of the Italian wall lizard Podarcis sicula. Cadmium was administered in single dose, by diet, to induce a concentration comparable with that measured in animals living in contaminated sites. For comparison, cadmium was also administered in multiple doses by food (chronic) or in a single dose intraperitoneally (i.p.); the effects were followed at regular time intervals up to 30 days post treatments. Atomic absorption spectrometry analysis demonstrated cadmium ion uptake and accumulation in the parenchyma with an estimated half-life of approximately 8 days. Cytological analyses revealed that the metal induced oedema, activated metallothionein expression in Kupffer cells and extracellular matrix production in fat storing cells. It also caused swelling and alteration in lipid and sugar metabolism in hepatocytes. In conclusion, in the wall lizard cadmium is toxic to the liver even at very low concentrations, the response is not strictly dose and time dependent and almost no recovery occurs in short (30 days) time periods.

Cadmium is a heavy metal toxic for living organisms even at low concentrations. It does not have any biological role, and since it is a permanent metal ion, it is accumulated by many organisms. In the present paper we have studied the apoptotic effects of continuous exposure to subacute/sublethal cadmium concentrations on a model system: Paracentrotus lividus embryos. We demonstrated, by atomic absorption spectrometry, that the intracellular amount of metal increased during exposure time. We found, using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, that long treatments with cadmium triggered a severe DNA fragmentation. We demonstrated, by immunocytochemistry on whole-mount embryos, that treatment with cadmium causes activation of caspase-3 and cleavage of death substrates alpha-fodrin and lamin A. Incubating the embryos since fertilization with Z-DEVD FMK, a caspase-3 inhibitor, we found, by immunocytochemistry, that cleavage by caspase-3 and cleavage of death substrates were inactivated.

Organisms from yeast to mammals contain cysteine-rich, heavy metal binding proteins termed metallothioneins. The putative roles of these proteins are trace metal homeostasis and detoxification of poisonous heavy metals. The highly conserved chemical composition and the structural constraints led to the conclusion that metallothioneins of different origin must display remarkably similar features. The present review aims at surveying the studies carried out on the metallothioneins of Antarctic Notothenioidei, a dominating fish group endowed of a number of striking adaptive characters, including reduced (or absent) hematocrit and presence of antifreeze glycoproteins. Given the unique peculiarities of the Antarctic environment, a comparative study of the features of notothenioid metallothioneins could provide new insights into the role of these proteins in physiology and toxicology. The results summarized here show that the metallothioneins of this fish group display a number of features at the level of evolution, expression pattern, structure and function remarkably different from those of mammal metallothioneins.

Cold-adapted organisms have developed a number of adjustments at the molecular level to maintain metabolic functions at low temperatures. Among other features, they can produce enzymes characterized by a high turnover number or a high catalytic efficiency. The present work is aimed at investigating the process of food digestion at low temperature through the study of pepsins in Antarctic notothenioids. For such a purpose, we have cloned and sequenced three forms of pepsin A and a single form of gastricsin from the gastric mucosa of Trematomus bernacchii (rock cod). Phylogenetic analysis has suggested that the three pepsin A isotypes arose from two gene duplication events leading to the most ancestral pepsin A3 and to the most recent forms represented by pepsin A1 and pepsin A2. Molecular modeling has unraveled significant structural differences in these enzymes with respect to their mesophilic counterparts. Hydropathy and flexibility determined on the substrate-binding subsites of Antarctic and mesophilic pepsins have shown for pepsin A2 reduced hydropathy and increased flexibility at the level of the substrate cleft, features typical of cold-adapted enzymes. Northern blot analysis of RNA from rock cod gastric mucosa hybridized with molecular probes designed on specific regions of different pepsin forms has shown that rock cod pepsin genes are expressed at comparable levels. The present results suggest that the Antarctic rock cod adopted two different strategies to accomplish efficient protein digestion at low temperature. One mechanism is the gene duplication that increases enzyme production to compensate for the reduced kinetic efficiency, the other is the expression of a new enzyme provided with features typical of cold-adapted enzymes.

In vertebrates, a large proportion of genes is organized in gene families. Paralogous gene groups generated by gene duplication are related by homology, high degree of sequence identity and similar structural architecture of their products. Aspartic proteinases form a widely distributed protein superfamily including cathepsins, pepsins, renin and napsin. In the present study, the nucleotide sequences coding for various pepsins in 30 vertebrate species have been used to derive a gene phylogeny. Gene duplication and losses have been inferred from a reconciled tree, reconstructed by combining information from gene tree and species tree. Our findings based on the results of the relative rate ratio test and maximum likelihood analysis suggest that each round of gene duplication is characterized by adaptive evolution, although instances of evolution under positive selection have been found also long after divergence of gene families. The results of functional divergence analysis provided statistical evidence for shifted evolutionary rate after gene duplication.

In the present report, we investigated zinc, copper and metallothionein (MT) contents in zebrafish oocytes and embryos. Our results demonstrate that the metal content increases during oocytes maturation. Zinc increases from 30 ng/oocyte (stage-1 oocytes) to 100 ng/oocyte (stage-3 oocytes); copper varied from 1 ng/oocyte (stage-1 oocytes) to 3.5 ng/oocyte (stage-3 oocytes). During embryogenesis, zinc and copper contents dramatically increase after fertilisation around the 512-cells stage, then slowly decrease until the mid-gastrula stage. During oocyte growth, the changes in the MT level are proportional to metal content, whereas during embryogenesis the pattern of MT accumulation does not parallel that of the two metals. Indeed, the maternal pool of MT decreases steadily during the early stages of the development until the gastrula stage. We have examined the effect of cadmium on the expression of MT during zebrafish development. After cadmium exposure, MT content increases in embryos at the blastula stage, whereas no induction occurs in embryos at the gastrula stage. However, pre-treatment of embryos at the gastrula stage with 5-aza-2'-deoxycytidine induces MT synthesis following exposure to cadmium. These observations show that changes in metal levels are not correlated to MT content in the embryo, whereas DNA methylation is one of the factors regulating MT expression.