Electron Spin Resonance (ESR) for the study of Reactive Oxygen Species (ROS) on the isolated frog skin (Pelophylax bergeri): A non-invasive method for environmental monitoring.

BACKGROUND: Reactive oxygen species (ROS) in biological tissues of elected biosentinels represent an optimal biomarker for eco-monitoring of polluted areas. Electron spin resonance (ESR) is the most definitive method for detecting, quantifying and possibly identifying radicals in complex systems. OBJECTIVE: A non-invasive method for monitoring polluted areas by the quantitative determination of ROS in frog skin biopsy is presented. METHODS: We assessed by ESR spectroscopy the ROS level in adult male of Pelophylax bergeri, specie not a risk of extinction, collected from the polluted Sarno River (SA, Italy) basin. The spin-trap ESR method was validated by immunohistochemical analysis of the well-assessed pollution biomarkers cytochrome P450 aromatase 1A (CYP1A) and glutathione S-transferase (GST), and by determining the poly(ADPribose) polymerase (PARP) and GST enzymatic activity. RESULTS: ROS concentration in skin samples from frogs collected in the polluted area is significantly higher than that determined for the unpolluted reference area. Immunohistochemical analysis of CYP1A and GST supported the reliability of our approach, even in the absence of evident morphological and ultrastructural differences. PARP activity assay, connected to possible oxidative DNA damage, and the detoxification index by GST enzymatic assay give statistically significant evidence that higher levels of ROS are associated to alterations of the different biomarkers. CONCLUSIONS: ROS concentration, measured by ESR on isolated frog skin, through the presented non-lethal method, is a reliable biomarker for toxicity screening and represents a useful basic datum for future modelling studies on environmental monitoring and biodiversity loss prevention.

Reactive oxygen species and glutathione antioxidants in the testis of the soil biosentinel Podarcis sicula (Rafinesque 1810).

Important toxicological achievements have been made during the last decades using reptiles. We focus our investigation on gonadal reproductive health of the soil biosentinel Podarcis sicula which is very sensitive to endocrine-disrupting chemicals. The aim of this study is to quantitatively detect, by sensitive microassays, reactive oxygen species and the glutathione antioxidants in the testis and investigate if they are differentially expressed before and after remediation of a site of the "Land of Fires" (Campania, Italy) subject to illicit dumping of unknown material. The oxidative stress level was evaluated by electron spin resonance spectroscopy applying a spin-trapping procedure able to detect products of lipid peroxidation, DNA damage and repair by relative mobility shift, and poly(ADP-ribose) polymerase enzymatic activity, respectively, the expression of glutathione peroxidase 4 transcript by real-time quantitative PCR analysis, the antioxidant glutathione S-transferase, a well-assessed pollution index, by enzymatic assay and the total soluble antioxidant capacity. Experimental evidences from the different techniques qualitatively agree, thus confirming the robustness of the combined experimental approach. Collected data, compared to those from a reference unpolluted site constitute evidence that the reproductive health of this lizard is impacted by pollution exposure. Remediation caused significant reduction of reactive oxygen species and downregulation of glutathione peroxidase 4 mRNAs in correspondence of reduced levels of glutathione S-transferase, increase of antioxidant capacity, and repair of DNA integrity. Taken together, our results indicate directions to define new screening approaches in remediation assessment.

The amphibian (Rana esculenta) brain progesterone receptor: relationship to plasma steroids and vitellogenic cycle during the gonadal recovery phase.

We report the presence of a progesterone receptor (PR) in the brain of the female amphibian Rana esculenta and changes in the levels of this PR during the gonadal recovery phase in relation to different circulating steroid levels and steroid treatment. The highest level of the PR in the nuclear brain extract corresponded to a low level of plasma progesterone and occurred when vitellogenin synthesis was at a minimum. The lowest level was found during follicular growth, concomitant with increased plasma 17beta-estradiol and progesterone levels. The PR levels were significantly higher in ovariectomized female R. esculenta than in intact and sham-operated female frogs. Treatment with 17beta-estradiol and progesterone downregulated the frog brain PR, indicating that a possible progesterone-receptor interaction is involved in the modulation of vitellogenin.

Phylogenetic conservation of cytostatic factor related genes in the ascidian Ciona intestinalis.

In all vertebrates, mature oocytes arrest at the metaphase of the II meiotic division, while some invertebrates arrest at metaphase-I, others at prophase-I. Fertilization induces completion of meiosis and entry into the first mitotic division. Several experimental models have been considered from both vertebrates and invertebrates in order to shed light on the peculiar aspects of meiotic division, such as the regulation of the cytostatic factor (CSF) and the maturation promoting factor (MPF) in metaphase I or II. Recently, we proposed the oocytes of ascidian Ciona intestinalis as a new model to study the meiotic division. Here, taking advantage of the recent publication of the C. intestinalis genome, we presented a phylogenetic analysis of key molecular components of the CSF-related machinery. We showed that the Mos/MAP kinase pathway is perfectly conserved in ascidians. We demonstrated the presence of a CSF-like activity in metaphase-I arrested C. intestinalis oocytes able to block cell division in two-cell embryos. We further investigated the regulation of CSF by demonstrating that both CSF and MPF inactivation, at the exit of metaphase-I, are independent from protein synthesis, indicating the absence of short-lived factors that regulate metaphase stability, as in other invertebrate species. The results obtained suggest that meiotic regulation in C. intestinalis resembles that of vertebrates, such as Xenopus accordingly to the position of this organism in the evolutionary tree.

Cytotoxic and apoptogenic activity of a methanolic extract from the marine invertebrate Ciona intestinalis on malignant cell lines.

Marine invertebrates provide a series of natural products with different biological activities. Several of these compounds and their derivatives showed a potent anticancer effect. Tunicates represent an important source of bioactive agents, leading to the isolation of ecteinascidin-743 (ET-743), a compound isolated from the Caribbean sea squirt Ecteinascidia turbinata with a potent cytotoxic activity against a variety of tumours in vitro and in vivo. Current phase II clinical trials against soft tissue sarcomas in Europe and the United States indicate that ET-743 represents a highly promising anticancer agent. Another example is aplidine from the Mediterranean tunicate Aplidium albicans, with a broad spectrum activity against various types of cancers, such as colorectal, lymphoma, thyroid and renal cancers. In the present work, we reported, for the first time, that a partially purified methanolic extract prepared from the ascidian Ciona intestinalis inhibited cell proliferation in human cell lines of different origin, including Caco2, HPB-ALL, U-937 and HL-60 and induced early apoptotic events, such as caspase-3 activation and internucleosomal DNA degradation. We suggest the presence in the Ciona intestinalis extract of bioactive compounds possessing anticancer activity.

Gamma-aminobutyric acid and related molecules in the sea fan Eunicella cavolini (Cnidaria: Octocorallia): a biochemical and immunohistochemical approach.

The aim of this study has been the biochemical demonstration of the presence of gamma-aminobutyric acid (GABA) in the Mediterranean sea fan Eunicella cavolini by means of high-performance liquid chromatography, and the description of the distribution pattern of GABA and its related molecules, glutamic acid decarboxylase (GAD), vesicular GABA transporter (VGAT) and one of the GABA receptors (GABA(B) R) by immunohistochemical methods. The interrelationships of GABA, GAD and GABA receptor immunoreactivity have been established by using double-immunohistochemical methods and confocal microscopy. The immunodetection of monoclonal and/or polyclonal antibodies has revealed GABA immunoreactivity throughout the polyp tissue, both in neuronal and non-neuronal elements. GAD immunoreactivity has been mostly localized in the neuronal compartment, contacting epithelial and muscular elements. GABA(B) R immunoreactivity appears particularly intense in the nematocytes and in the oocyte envelope; its presence in GAD-immunoreactive neurons in the tentacles suggests an autocrine type of regulation. Western blot analysis has confirmed that a GABA(B) R, with a molecular weight of 142 kDa, similar to that of rat brain, is present in E. cavolini polyp tissue. The identification of the sites of the synthesis, vesicular transport, storage and reception of GABA strongly suggests the presence of an almost complete set of GABA-related molecules for the functioning of the GABAergic system in this simple nervous system. The distribution of these different immunoreactivities has allowed us to hypothesize GABA involvement in nematocyst discharge, in body wall and enteric muscular contraction, in neuronal integration and in male gametocyte differentiation.