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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

Dichlorodiphenyldichloroethylene (DDE) is an environmental pollutant that accumulates in adipose tissue through the food chain. Hypercaloric, high-fat diet is considered to promote the accumulation of toxic lipophilic substances in tissues, whereas the loss of body fat through caloric restriction results in a recirculation of these substances. In rats, oral administration of DDE causes the onset of tissues damage; the concomitant intake of a high-fat diet ameliorates tissues status, probably because of the entrapment of the lipophilic substance in fat depots. Recent evidence demonstrates that DDE alters the expression of metallothioneins, proteins involved in cellular defense from oxidative stress, in a diet- and tissue-specific manner. This study is aimed to verify if 2 weeks of caloric restriction after the oral DDE treatment can modify metallothionein gene expression in tissues of high-fat fed rats. Real-time PCR analysis demonstrates that metallothionein gene expression after calorie restriction is tissue-specific and strongly influenced by both previous dietary conditions and DDE exposure. To avoid misleading conclusions on the interference of toxic xenobiotics on metallothionein gene expression is particularly important to consider the tissue, the cellular conditions, and the nutritional status of the animals, especially when the protein is used as an index of cells health.

The expansion of agriculture causes a constant increase in habitat fragmentation and degradation due to the increased use of pesticides and herbicides. Habitat loss and alteration associated with agricultural expansion play an important role in the decline of reptiles and, among them, lizards are particularly endangered. This chapter describes the toxic effects of pure glyphosate on the liver of the common field lizard Podarcis siculus. Glyphosate (GLY) is the most widely used active ingredient in agrochemical formulations worldwide to maximize crop yields. The onset of liver damage was evaluated after oral administration of GLY to simulate a realistic situation of herbicide exposure in the lizard habitat. The results demonstrate a severe liver condition, with fibrotic formations that become scarring with increased exposure. Hepatocytes enhance their defense mechanism by activating their antioxidant systems and increasing the expression of p53. The results also confirm the xenoestrogenic nature of GLY. Altogether, the effects of GLY must be considered biologically relevant and could endanger the viability and survival of lizard populations, also unbalancing the trophic cascade. A more controlled and less intensive use of the herbicide GLY in areas devoted to crop production would therefore be advisable.

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.

Spermatogenesis is a genetically driven differentiation process that occurs in the testis and leads to the formation of spermatozoa. This process is extensively studied in several experimental models, particularly in vertebrates that share the morphological structure and functionality of the mammalian testis. Although reptiles are not generally considered biological models, the lizard

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.

In aquatic organisms, cadmium exposure occurs from ovum to death and the route of absorption is particularly wide, being represented by skin, gills and gastrointestinal tract, through which contaminated water and/or preys are ingested. It is known that cadmium interferes with the gut; however, less information is available on cadmium effects on an important component of the gut, namely goblet cells, specialized in mucus synthesis. In the present work, we studied the effects of two sublethal cadmium concentrations on the gut mucosa of

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

Aluminium, despite being extremely widespread in the world, is a non-essential metal to human metabolism. This metal is known to have toxic effects on a variety of organs including the brain and is considered an etiological factor in neurodegenerative diseases. However, the molecular mechanisms by which aluminium exerts neurotoxic effects are not yet completely understood. Zebrafish is an animal model also used to study neurodegenerative diseases since the overall anatomical organization of the central nervous system is relatively conserved and similar to mammals. Adult zebrafish were exposed to 11 mg/L of Al for 10, 15, and 20 days and the neurotoxic effects of aluminium were analysed by histological, biochemical, and molecular evaluations. Histological stainings allowed to evaluation of the morphology of the brain parenchyma, the alteration of myelin and the activation of neurodegenerative processes. The expression of the Glial Fibrillary Acidic Protein, a marker of glial cells, was evaluated to observe the quantitative alteration of this important protein for the nervous system. In addition, the poly(ADP-ribose) polymerase activity was measured to verify a possible oxidative DNA damage caused by exposure to this metal. Finally, the evaluation of the markers involved in Parkinsonism was assessed by Real-Time PCR to better understand the role of aluminium in the regulation of genes related to Parkinson's neurodegenerative disease. Data showed that aluminium significantly affected the histology of cerebral tissue especially in the first periods of exposure, 10 and 15 days. This trend was also followed by the expression of GFAP. At longer exposure times, there was an improvement/stabilization of the overall neurological conditions and decrease in PARP activity. In addition, aluminium is involved in the deregulation of the expression of genes closely related to Parkinsonism. Overall, the data confirm the neurotoxicity induced by aluminium and shed a light on its involvement in neurodegenerative processes.

Obesity and overweight are frequently associated with male subfertility. To address new findings on the players involved in the obesity-induced impairment of spermatogenesis, we used a high-fat diet-induced overweight-rat model. Following four weeks of high-fat diet, the organization of seminiferous epithelium was affected, and tubules lumen showed immature/degenerated cells, typical signs of hormonal imbalance and testicular damage. Real-time PCR analysis allowed us to detect increased levels of ERα and decreased levels of aromatase CYP19 transcripts in testis, suggesting an increase in circulating estrogens derived from the accumulating adipose tissue rather than the induction of testicular estrogen synthesis. Moreover, in situ hybridization analysis showed an increased susceptibility towards estrogens in testis from high-fat fed rats, being ERs expressed not only in spermatogonia, as in control testis, but also in spermatids. Western blot and immunohistochemical analyses revealed an increase in the amount of p53 and PCNA, together with a change in their immunodetection, being the proteins localised on germ cells at different stages of maturation. Differences in p53 and PCNA expression may give evidence and be part of a cellular response to stress conditions and damage caused by the excessive intake of saturated fatty acids.

The biological mechanisms linking nutrition and antioxidants content of the diet with cardiovascular protection are subject of intense investigation. It has been demonstrated that dietary supplementation with cow, donkey or human milk, characterized by distinct nutritional properties, triggers significant differences in the metabolic and inflammatory status through the modulation of hepatic and skeletal muscle mitochondrial functions. Cardiac mitochondria play a key role for energy-demanding heart functions, and their disfunctions is leading to pathologies. Indeed, an altered heart mitochondrial function and the consequent increased reactive oxygen species (ROS) production and inflammatory state, is linked to several cardiac diseases such as hypertension and heart failure. In this work it was investigated the impact of the milk consumption on heart mitochondrial functions, inflammation and oxidative stress. In addition, it was underlined the crosstalk between mitochondrial metabolic flexibility, lipid storage and redox status as control mechanisms for the maintenance of cardiovascular health.

Nonessential metal cadmium is widely used and released in the environment, causing cell toxicity and posing a severe threat to wildlife. Zebrafish (Danio rerio) is one of the most commonly used animals in the investigation of environmental cadmium toxicity in vertebrates. In this study, we identified two cadmium-responsive genes, RBBP6 and CRYL1, in the early phases of zebrafish development, at the gastrula stage. The retinoblastoma binding protein 6 is associated with increased protein degradation and cell proliferation; crystallin-lambda 1 is a lens protein with redox activity. In situ hybridization analysis performed on adult zebrafish exposed to 1.5-40 μM cadmium for 30 days confirmed the ability of cadmium to up-regulate the expression of both genes in retinal cells in a dose-dependent manner. The over-expression was transient, being switched off when cadmium was removed. The involvement of RBBP6 and CRYL1 in the onset of cadmium-induced morphological alterations in adult zebrafish retina is discussed.

Spermatogenesis is a fascinating biological process aiming to generate haploid spermatozoa from diploid spermatogonia through a specific hormonal network between gonadotropins and steroids. Increasing evidence suggests that the primary female sex hormone estrogen plays an active role in this process. This research points out on the role of estrogen during lizard spermatogenesis by using three experimental approaches: (1) exposure to an analogue of nonsteroidal estrogen as Clomiphene citrate that acts both as estrogen agonist and antagonist; (2) exposure to the gonadotropin FSH; and (3) exposures to FSH followed by Clomiphene. Histological and immunohistochemical results demonstrate that in the lizardPodarcis siculaduring the mating period, Clomiphene as well as FSH determines the breakdown of spermatogenesis and the epididymal regression, presumably through estrogens input as indirectly demonstrated by the appearance of ERαand vitellogenin in the liver. The ability of Clomiphene to restore the gonadal natural condition after FSH treatment is also demonstrated. Finally, data indicate that lizard testis and epididymis control their morphophysiology regulating the intracellular presence of ERα.

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 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.

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.

The cleidoic eggs of oviparous reptiles are protected from the external environment by membranes and a parchment shell permeable to water and dissolved molecules. As a consequence, not only physical but also chemical insults can reach the developing embryos, interfering with gene expression. This review provides information on the impact of the exposure to cadmium contamination or thermal stress on gene expression during the development of Italian wall lizards of the genus Podarcis. The results obtained by transcriptomic analysis, although not exhaustive, allowed to identify some stress-reactive genes and, consequently, the molecular pathways in which these genes are involved. Cadmium-responsive genes encode proteins involved in cellular protection, metabolism and proliferation, membrane trafficking, protein interactions, neuronal transmission and plasticity, immune response, and transcription regulatory factors. Cold stress changes the expression of genes involved in transcriptional/translational regulation and chromatin remodeling and inhibits the transcription of a histone methyltransferase with the probable consequence of modifying the epigenetic control of DNA. These findings provide transcriptome-level evidence of how terrestrial vertebrate embryos cope with stress, giving a key to use in population survival and environmental change studies. A better understanding of the genes contributing to stress tolerance in vertebrates would facilitate methodologies and applications aimed at improving resistance to unfavourable environments.

The growing number of studies on metallothioneins (MTs), cysteine-rich metal-binding proteins, have been disclosing new functions of these proteins. Thanks to their inducibility, they were considered to play a pivotal role in regulating trace metals homeostasis and in detoxification from heavy metals; nowadays, it is known that they are involved in various physiological and pathological processes, such as regulation of apoptosis, elimination of free radicals, and protection of nucleic acids against toxic insults. MT induction has been demonstrated following stress factors other than heavy metals, such as endocrine-disrupting chemicals, insecticides, and herbicides. However, retrieved data are often controversial: in some cases, xenobiotics elicit MT expression and synthesis; under different conditions, they lead to a decrease in cellular MT content. This review describes the MT response to dichlorodiphenyltrichloroethane (DDT) contamination in mammalian tissues. In particular, attention focuses on changes in MT expression, synthesis, and localization in rat liver, kidneys, and testes following oral administration of dichlorodiphenyldichloroethylene (DDE), the main metabolite of DDT, under normal dietary conditions or in combination with a high fat diet potentially able to increase the cellular uptake of this lipophilic pesticide. The potential connection between MT expression and synthesis, lipophilic substances and trace metals availability is also discussed.

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.

Exploiting the annotation of the western clawed frog Silurana tropicalis genome, we identified a new metallothionein (MT) gene, exhibiting all the features to be considered an active gene, but with an atypical coding region, showing only 17 cysteine residues instead of the canonical 20 cysteines of vertebrate metallothioneins and two anomalous cysteine triplets. However, the presence of a gene in the genome does not ensure its effective expression. By using conventional and Real-Time PCR analyses, we demonstrated that this atypical MT is constitutively expressed throughout the life cycle of the African clawed frog Xenopus laevis; moreover, this gene is highly expressed in the adult liver, the major site of MT expression and synthesis in vertebrates. To our knowledge, the X. laevis MT described in this paper is the first sequence of a vertebrate MT showing only 17 cysteine residues, arranged in two Cys–Cys–Cys motifs. Phylogenetic analyses also demonstrated that the atypical X. laevis MT merges in the anuran clade, but is the most derived sequence among tetrapods MTs. Finally, Tajima's Relative Rate Test suggested a different evolutionary rate between the canonical X. laevis MT and this novel isoform.

The "sex-hormone" estrogen-17β promotes several cognitive functions and is a master regulator of brain bioenergetics via the estrogen receptors α and β (ERα and ERβ). In this work, by using Real-Time PCR analysis, we evaluated the effect of aging and high fat diet (HFD) on ERα and ERβ expression in rat hippocampus and cortex. In young rats, ERβ is abundant in cortex and ERα in hippocampus. During the aging, in the cortex, we observe a general decrease in ERα and ERβ expression; in hippocampus ERα increases and ERβ decreases. ER expression patterns in rat brain are also affected by the administration of an HFD. In cortex, after 4weeks of HFD, ERβ transcripts are down-regulated, whereas ERα levels remain unchanged; after 12weeks, both ERα and ERβ expression is up-regulated. In the hippocampus, the level of ERβ transcripts does not change following HDF, whereas ERα expression is affected by HDF, in a time-dependent manner: it increases after the 4-week treatment and decreases after 12weeks. Possible involvements of these receptors in the control of cortex and hippocampus functions during aging and in the modulation of energetic metabolism and feeding behaviour are discussed.

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.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide involved in multiple functions, including vertebrate reproduction. Recently, we reported the presence of PACAP in the testis of Italian wall lizard Podarcis sicula during reproductive period (May–June). Herein we investigated the PACAP mRNA expression and the localization of PACAP/PACAP receptor system, in the other periods of the Podarcis reproductive cycle, namely in summer stasis, early autumnal resumption, mid-autumnal resumption, winter stasis, and spring resumption. Using biomolecular and immunohistochemical investigations, we demonstrated that PACAP mRNA was widely expressed in all germ and somatic cells; in summer stasis (July–August) and early autumnal resumption (September) in particular, the mRNA was always found in Sertoli cells while was transiently expressed in germ and in Leydig cells. Differently from the mRNA, the protein was always present in germ and somatic cells independently from the reproductive cycle phase. As PACAP, the PAC1 receptor was always present in the testis, except for the summer stasis (July–August) and the early autumnal resumption (September), when PACAP was lacking in germ and somatic cells (Leydig and Sertoli cells). The present results strongly suggest that PACAP/PAC1 receptor system is widely represented during the reproductive cycle of male lizard. The possible involvement of PACAP/PACAP receptor system in the control of spermatogenesis is discussed.

Starting from the knowledge that in the reproductive period the Vasoactive Intestinal Peptide (VIP) is widely distributed in Podarcis sicula testis, we studied VIP expression and the localization of the neuropeptide and its receptors in the testis of the Italian wall lizard P. sicula in the other phases of its reproductive cycle (summer stasis, autumnal resumption, winter stasis, spring resumption). By Real Time-PCR, we demonstrated that testicular VIP mRNA levels change during the reproductive cycle, showing a cyclic trend with two peaks, one in the mid-autumnal resumption and the other in the reproductive period. By in situ hybridization and immunohistochemistry, we demonstrated that both VIP mRNA and protein were widely distributed in the testis in almost all the phases of the cycle, except in the early autumnal resumption. As regards the receptors, the VPAC 1 R was localized mainly in Leydig cells, while the VPAC 2 R showed the same distribution of VIP. Our results demonstrate that, differently from mammals, where VIP is present only in nerve fibres innerving the testis, an endotesticular synthesis takes place in the lizard and the VIP synthesis changes throughout the reproductive cycle. Moreover, the VIP/VPAC receptor system distribution observed in germ and somatic cells in various phases of the cycle, and particularly in the autumnal resumption and the reproductive period, strongly suggests its involvement in both spermatogenesis and steroidogenesis. Finally, the wider distribution of VIP in lizards with respect to mammals leads us to hypothesize that during the evolution the synthesis sites have been transferred from the testis to other districts, such as the brain.

Lizards are soil surface animals that represent an important link between invertebrates and higher predators. Being part of wild fauna, they can be affected by contamination from anthropic activities and in particular, pesticides and chemical substances of various nature that reach the soil surface directly or through fall out. Among these substances, heavy metals such as cadmium may exert particularly marked toxic effect on both adult and embryos. In lizards, recent studies show that cadmium may cause developmental defects, including alteration of eye development, with appearance of unilateral microphthalmia and retinal folding. In the present study, the effects of cadmium incubation on retinal development were investigated demonstrating that cadmium interferes with cell cycle regulation by increasing proliferation. An increased expression of Otx2 and Pax6 genes, markers of retinal differentiation, was also found. However, the cellular localization of Pax6 and Otx2 transcripts did not change in treated embryos: in the early stages of retinogenesis, the two genes were expressed in all retinal cells; in the differentiated retina, Otx2 remained in the cellular bodies of retinal cells forming the nuclear and the ganglion layers, whereas Pax6 was expressed only in the cells of the inner nuclear and the ganglion layers. Data suggest that the increased expression of Pax6 and Otx2 could be ascribed to the hyperproliferation of retinal cells rather than to an effective gene overexpression.

Oxidative stress plays a key role in steatohepatitis induced by both xenobiotic agents and high fat diet (HFD). The present study aimed to evaluate hepatic oxidative stress and anti- oxidant systems response in rats exposed to HFD and/or non-toxic dose of dichlorodiphe- nyldichloroethylene (DDE), the first metabolite of dichlorodiphenyltrichloroethane. Groups of 8 rats were so treated for 4 weeks: 1- standard diet (N group); 2- standard diet plus DDE (10 mg/kg b.w.) (N+DDE group); 3- HFD (D group); 4- HFD plus DDE (D+DDE group). Oxi- dative stress was analyzed by determining malondialdehyde as lipid peroxidation product, while the anti-oxidant systems were evaluating by measuring the levels of the principal cyto- solic and mitochondrial antioxidant proteins and enzymes, namely superoxide dismutase 1 and 2 (SOD1, SOD2), glutathione peroxidase 1 (GPx1) and uncoupling protein 2 (UCP2) involved in the control of hepatic reactive oxygens species (ROS) accumulation. The results showed malondialdehyde accumulation in livers of all groups, confirming the pro-oxidant effects of both HFD and DDE, but with a greater effect of DDE in absence of HFD. In addi- tion, we found different levels of the analyzed anti-oxidant systems in the different groups. DDE mainly induced UCP2 and SOD2, while HFD mainly induced GPx1. Noteworthy, in the condition of simultaneous exposure to DDE and HFD, the anti-oxidant response was more similar to the one induced by HFD than to the response induced by DDE. Present findings confirmed that both HFD and xenobiotic exposure induced hepatic oxidative stress and showed that the anti-oxidant defense response was not the same in the diverse groups, sug- gesting that UCP2 induction could be an adaptive response to limit excessive ROS damage, mainly in condition of xenobiotic exposure.