Gaetano De Tommaso

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. HighlightsROS levels in frog skin biopsy were determined by spin‐trapping ESR spectroscopy.Skin of frogs collected in polluted areas contains higher ROS concentration.ROS levels determined in skin biopsy follow the same trend of those from liver samples.Immunohistochemical analyses and enzymatic activity tests validate the method.ROS concentration determined by ESR in biosentinel biopsy is an effective biomarker.

THE SOLUBILITY OF CALCIUM CITRATE HYDRATE IN SODIUM PERCHLORATE SOLUTIONS

The solubility, at 25°C, of calcium citrate, Ca3(C6H5O7)2. 4H2O, in NaClO4 solutions in the molality (m) range 0–3.5, has been determined by potentiometric titration with EDTA, using the Hg2+/Hg half-cell. The increase of solubility with m has been explained with the specific interaction theory by assuming, for Ca3(C6 H5 O7)2.4 H2 O(s) ⇄3Ca2+ + 26H55O7 3−, log Ks0= −17.81 ± 0.03 and the interaction coefficient between Na+ and C6H5O7 3− ions equal to 0.08 ± 0.03 −(0.06 ± 0.01)m. This coefficient allows the extrapolation to the infinite dilution reference state of stability constants determined in concentrated ionic media. Results of calculations compare well with those of the literature.

Homocysteine disulphides and vascular disease.

The total plasma concentration of homocysteine is a marker of this amino acids atherogenic potential. However, the homocysteine pool exists almost entirely as oxidized homocysteine equivalents (OHcyE), composed of homocystine and cysteine-homocysteine disulphides (20–30%), and protein-bound disulphide (70–80%). We have noticed that the total concentration of OHcyE in injured coronary artery tissue is higher than the aqueous solubility of homocystine (∼1.4–1.5 × 10-3 mol kg-1 versus ∼0.6 mol kg^{-1}). Based on the measurement of the solubility of homocystine in a plasma-mimetic condition (0.17 mol kg^{-1} NaCl at 37°C), we have estimated that OHcyE may really reach their saturation limit in the vascular tissue (0.93–1.02 × 10-3 mol kg-1), above which their deposition as solid phase may occur. This means that significant leakage of intracellular fluid can promote OHcyE crystallization in tissue fluids, which may serve to initiate inflammation. We speculate that deposition of OHcyE crystals could damage blood vessels and act as a primer of homocysteine-triggered inflammation, thus being along the causal pathway that leads to vascular dysfunction.

Silibinin phosphodiester glyco-conjugates: Synthesis, redox behaviour and biological investigations

New silibinin phosphodiester glyco-conjugates were synthesized by efficient phosphoramidite chemistry and were fully characterized by 2D-NMR. A wide-ranging study focused on the determination of their pKa and E° values as well as on their radical scavenging activities by different assays (DPPH, ABTS+ and HRSA) was conducted. The new glyco-conjugates are more water-soluble than silibinin, and their radical scavenging activities are higher than those of silibinin. The conjugation therefore improves both the water solubilities and antioxidant activities of the flavonolignan moieties. The serum stability was evaluated under physiological conditions, and the glyco-conjugates degraded with half-lives of 40-70 h, making them useful in pro-drug approaches. We started by treating androgen-dependent prostate cancer (PCa) LNCaP cells and then expanded our studies to androgen-independent PCa PC3 and DU145 cells. In most cases, the new derivatives significantly reduced both total and live cell numbers, albeit at different levels. Anti-HIV activities were evaluated and the glucosamine-phosphate silibinin derivative showed higher activity (IC50 = 73 μM) than silibinin.