Rosaria Sciarrillo

Morphological and Functional Changes in the Thyroid Gland of Methyl Thiophanate-Injected Lizards, Podarcis sicula

The thyroid has been shown to be a target organ for environmental chemicals, specifically endocrine-disrupting contaminants. Reptiles are particularly suitable as contaminant biomonitors due to their persistence in a variety of habitats, wide geographic distribution, longevity, and, in many cases, site fidelity. Methyl thiophanate is a systemic broad-spectrum fungicide used to prevent and control plant diseases caused by various fungi. The aim of this study was to develop an integrated biological model for monitoring the ecotoxic effects of thiophanate-methyl fungicide on the thyroid of the lizard Podarcis sicula. The results of this study indicate that both structural and functional differences in the thyroid gland of the lizard exist in the animals exposed to methyl thiophanate. Structurally, animals exposed to methyl thiophanate showed decreased epithelial cell height; the nuclei of the thyroid cells were small and elongated with dense chromatin and a greatly reduced cytoplasm. The colloid was retracted with few reabsorption vacuoles. Functionally, the same animals exhibited decreased T4 and T3 plasma levels compared to control animals. Methyl thiophanate administration produced statistically significant inhibition on serum thyroid-stimulating hormone levels and this is the mechanism for altering thyroid function. This study highlights how thyroid gland disruption, both structural and functional, in lizard and other nontarget organisms might also have an environmental aetiology.

Endocrine-disrupting effects of nonylphenol in the newt, Triturus carnifex (Amphibia, Urodela)

The aim of our study was to verify whether environmental concentrations of nonylphenol influenced the adrenal gland of Triturus carnifex. Newts were exposed to 19 μg/L nominal concentration of nonylphenol throughout the periods of December-January and March-April, corresponding to different stages of the chromaffin cell functional cycle. The morphological features of the steroidogenic and chromaffin tissues, and the serum levels of ACTH, aldosterone, corticosterone, norepinephrine and epinephrine were evaluated. Nonylphenol did not influence ACTH serum levels. During the two periods examined, the steroidogenic tissue had the same reaction: the quantity of cytoplasmic lipids, and the corticosteroid serum levels, decreased, suggesting the inhibition of synthesis and release of corticosteroids. During the two periods examined, the chromaffin tissue reacted differently to nonylphenol. During December-January, the numeric ratio of norepinephrine granules to epinephrine granules, and the epinephrine serum levels, increased, suggesting the stimulation of epinephrine release. During March-April, the numeric ratio of norepinephrine granules to epinephrine granules did not change, and the norepinephrine serum levels decreased, suggesting the inhibition of norepinephrine release. Our results show that nonylphenol influences the activity of the newt adrenal gland; considering the physiological role of this gland, our results suggest that nonylphenol may contribute to amphibian decline.

Assessment of three approaches of bioremediation (Natural Attenuation, Landfarming and Bioagumentation - Assistited Landfarming) for a petroleum hydrocarbons contaminated soil.

Contamination with total petroleum hydrocarbons (TPH) subsequent to refining activities, is currently one of the major environmental problems. Among the biological remediation approaches, landfarming and in situ bioremediation strategies are of great interest. Purpose of this study was to verify the feasibility of a remediation process wholly based on biological degradation applied to contaminated soils from a decommissioned refinery. This study evaluated through a pot experiment three bioremediation strategies: a) Natural Attenuation (NA), b) Landfarming (L), c) Bioaugmentation-assisted Landfarming (LB) for the treatment of a contaminated soil with petroleum hydrocarbons (TPHs). After a 90-days trial, Bioagumentation - assistited Landfarming approach produced the best results and the greatest evident effect was shown with the most polluted samples reaching a reduction of about 86% of total petroleum hydrocarbons (TPH), followed by Landfarming (70%), and Natural Attenuation (57%). The results of this study demonstrated that the combined use of bioremediation strategies was the most advantageous option for the treatment of contaminated soil with petroleum hydrocarbons, as compared to natural attenuation, bioaugmentation or landfarming applied alone. Besides, our results indicate that incubation with an autochthonous bacterial consortium may be a promising method for bioremediation of TPH-contaminated soils.

Shift from noradrenaline to adrenaline production in the adrenal gland of the lizard, Podarcis sicula, after stimulation with vasoactive intestinal peptide (VIP)

The aim of this study was to investigate the distribution and function of VIP in the adrenal gland of the lizard, Podarcis sicula. We have shown by immunohistochemistry that VIP fibers were localized exclusively around clusters of chromaffin cells in the dorsal ribbon of the lizard adrenal gland. Moreover, a strong positivity for this peptide was observed within ganglial cells and within most chromaffin cells of the gland. To investigate the effects of VIP on the adrenal gland, we have treated lizards with several doses of this peptide and we have shown that injections of exogenous VIP increased plasma levels of catecholamines and corticosteroids, but not of ACTH. This probably suggests a direct effect of VIP on the control of adrenal hormone secretion without the involvement of the hypothalamo-hypophyseal axis. Our results also establish that the increased levels of the hormones were modulated in a time- and dose-dependent manner. Therefore, our morphological studies showed a clear increased function of steroidogenic cells. In the medullary region, VIP administration induced not only a functional enhancement of adrenaline release from adrenergic cells, but also a shift of noradrenaline cells to adrenaline ones.

Distribution of apelin, the endogenous ligand of the APJ receptor,in the lizard Podarcis sicula

Apelin is a novel bioactive peptide that has been isolated from bovine stomach extracts and identified as the endogenous ligand for the APJ receptor. Although the main physiological functions of apelin have not yet been clarified, it is known that apelin is involved in the regulation of blood pressure, central control of body fluid homeostasis and the modulation of immune response. In order to investigate the distribution of apelin in reptiles, we have performed an immunohistochemical analysis on tissues of the lizard Podarcis sicula. The peptide was found to be widely distributed, although its cellular localization differed in the various organs examined. A strong immunopositivity was found in the heart, stomach and intestine. In the spleen, an intense apelin immunopositivity was restricted to a discrete number of cells scattered throughout the red pulp and co-localized with immunoglobulin kappa and lambda chains, suggesting an analogous function of this peptide in immune responses also in reptiles. Intriguingly, apelin immunoreactivity was discretely localized in endothelial cells in the lung and thyroid gland. In the light of these data, we conclude that apelin may have multiple functions in reptiles.

Annual variations of adrenal gland hormones in the lizard Podarcis sicula

The adrenal gland regulates metabolism and maintains normal electrolyte balance. Adrenal hormones are equivalent in all vertebrates; the chromaffin tissue produces adrenaline and noradrenaline and the steroidogenic tissue produces most of the steroid hormones present in mammals. Podarcis sicula belongs to the Squamata family of lizards and it is the most abundant lizard species in southern Italy. This species shows a reproductive annual cycle and the presence of seasonal variations in the activity of the hypothalamus-hypophyseal-thyroid axis. To investigate the existence of an annual cycle of lizard adrenal gland, we have measured plasma concentrations of corticosterone, ACTH, noradrenaline and adrenaline. We have shown that corticosterone rapidly increased from January to March to reach a peak value that persisted until July, then, it slowly decreased until December. ACTH levels increased from January to May and slowly decreased from July to December. Noradrenaline levels were higher in March and then decreased until December. On the contrary, adrenaline levels increased from March to July and slowly decreased until December. Our results demonstrate the existence of an annual cycle of the lizard adrenal gland activity. This is very interesting because its activity is important to rightly regulate the reproductive status of the Podarcis sicula lizard.

Nonylphenol effects on the HPA axis of the bioindicator vertebrate, Podarcis sicula lizard

Nonylphenol (NP) is an endocrine disruptor widely distributed in the environment. It accumulates in the lipids of living organisms and enters the human food chain. The main source of human exposure is expected to be food, drinking water and foodstuff contaminated through leaching from packaging or pesticide formulation applications. NP acts as an estrogenic compound and it is able to mimic the action of estradiol 17β (E2) by binding to the estrogen receptor (ER). The aim of the present study was to investigate the NP effects on the hypothalamic-pituitary-adrenal gland (HPA) axis of the bioindicator Podarcis sicula lizard. A time-dependent stimulation of the HPA axis and variations of both catecholamine plasma levels were showed. Moreover, NP effects on adrenal gland morphology were evaluated by light and transmission electron microscopy. Clear morphological signs of adrenal gland stimulation such as an increase of steroidogenic cord diameter and vascularization, a strong escalation of adrenaline cell number and a decrease of noradrenaline cells were observed. The notably elevated levels of adrenal hormones suggested a permanent turning on of hypothalamic corticotropin releasing factor (CRF) secretion together with a lack of the negative feedback of HPA axis, perturbing systemic responses of the organism. Our data may help to predict the biological alterations induced by NP and to extend its impact upon adrenal function.

Sex steroid hormone secretion in the wall lizard Podarcis sicula testis: The involvement of VIP

Vasoactive intestinal peptide (VIP) is a pleiotropic neuropeptide involved in different functions, including testosterone synthesis. Recently, we reported the presence of VIP in the testis of Podarcis sicula, throughout the reproductive cycle. Now, we investigated the effects of the VIP on steroidogenesis in significant periods of the Podarcis reproductive cycle: winter stasis, reproductive period, and summer stasis. Using VIP treatments in testis culture in absence or presence of receptors antagonists, we demonstrated for the first time that in P. sicula, VIP is involved not only in testosterone synthesis, as in mammals, but in 17β-estradiol synthesis too. J. Exp. Zool. 323A: 714-721, 2015.

Distribution and molecular evolution of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in the lizard Podarcis sicula (Squamata, Lacertidae).

The presence of the pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors PAC1, VPAC1, and VPAC2 was studied in the lizard Podarcis sicula gastrointestinal and respiratory tissues. The expression and distribution of this neuropeptide was investigated using RT-PCR, immunohistochemistry, and in situ hybridization techniques. RT-PCR showed that several tissues of this reptile synthesize an mRNA encoding for PACAP. Performing in situ hybridization and immunohistochemistry, we found a wide distribution of PACAP and its mRNA in intestine, stomach, liver, and lung. PACAP receptors possess a specific distribution in both gastrointestinal and respiratory system. Further, we analyzed the conservation of PACAP amino acid sequence demonstrating that this peptide in the lizard is very similar to that of other vertebrates. Our findings suggest that also in reptiles an effective PACAP system is present and that it could be implicated in some essential physiological functions as a result of its high conservation amongst vertebrates.

Proteomic analysis of eucalyptus leaves unveils putative mechanisms involved in the plant response to a real condition of soil contamination by multiple heavy metals in the presence or absence of mycorrhizal/rhizobacterial additives.

Here we report on the growth, accumulation performances of, and leaf proteomic changes in Eucalyptus camaldulensis plants harvested for different periods of time in an industrial, heavy metals (HMs)-contaminated site in the presence or absence of soil microorganism (AMs/PGPRs) additives. Data were compared to those of control counterparts grown in a neighboring nonpolluted district. Plants harvested in the contaminated areas grew well and accumulated HMs in their leaves. The addition of AMs/PGPRs to the polluted soil determined plant growth and metal accumulation performances that surpassed those observed in the control. Comparative proteomics suggested molecular mechanisms underlying plant adaptation to the HMs challenge. Similarly to what was observed in laboratory-scale investigations on other metal hyperaccumulators but not on HMs-sensitive plants, eucalyptus grown in the contaminated areas showed an over-representation of enzymes involved in photosynthesis and the Calvin cycle. AMs/PGPRs addition to the soil increased the activation of these energetic pathways, suggesting the existence of signaling mechanisms that address the energy/reductive power requirement associated with augmented growth performances. HMs-exposed plants presented an over-representation of antioxidant enzymes, chaperones, and proteins involved in glutathione metabolism. While some antioxidant enzymes/chaperones returned to almost normal expression values in the presence of AMs/PGPRs or in plants exposed to HMs for prolonged periods, proteins guaranteeing elevated glutathione levels were constantly over-represented. These data suggest that glutathione (and related phytochelatins) could act as key molecules for ensuring the effective formation of HMs-chelating complexes that are possibly responsible for the observed plant tolerance to metal stresses. Overall, these results suggest potential genetic traits for further selection of phytoremediating plants based on dedicated cloning or breeding programs.