Gerardino D'Errico1, Giuseppe Vitiello2, Gaetano De Tommaso3, Fagr Kh Abdel-Gawad4, Maria Violetta Brundo5, Margherita Ferrante6, Anna De Maio7, Samantha Trocchia7, Anna Rita Bianchi7, Gaetano Ciarcia8, Giulia Guerriero8. 1. Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario di Monte S. Angelo, via Cintia 4, I-80126 Naples, Italy; CSGI, Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Florence, Italy. Electronic address: gerardino.derrico@unina.it. 2. CSGI, Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Florence, Italy; Department of Chemical, Materials and Production Engineering, University of Naples Federico II, Piazzale Tecchio 80, I-80125 Naples, Italy. 3. Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario di Monte S. Angelo, via Cintia 4, I-80126 Naples, Italy. 4. Water Pollution Research Department, Centre of Excellence for Advanced Sciences (CEAS), National Research Centre, El Buhout St., Dokki, ET-12622 Giza, Egypt. 5. Department of Biological, Geological and Environmental Science, University of Catania, Corso Italia 57, I-95129 Catania, Italy. 6. Department of Medical, Surgical and Advanced Technologies "G.F. Ingrassia", University of Catania, Via Santa Sofia 78, I-95123 Catania, Italy. 7. Department of Biology, University of Naples Federico II, Complesso Universitario di Monte S. Angelo, via Cintia 4, I-80126 Naples, Italy. 8. Department of Biology, University of Naples Federico II, Complesso Universitario di Monte S. Angelo, via Cintia 4, I-80126 Naples, Italy; Interdepartmental Research Center for Environment (I.R.C.Env.), University of Naples Federico II, Via Mezzocannone 16, I-80134 Naples, Italy.
Abstract
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.
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.