In this work, we describe results of the reproductive health monitoring studies in Mytilus galloprovincialis following spermatozoa hsp70 expression and protamine-like protein properties. Mussels control (ctr) were released within cages for 30 days in three different marine sites near Naples (Campania, Italy): Bagnoli south (BAs) and Bagnoli north (BAn), both close to a disposal metallurgical factory and in Capo Miseno (CM). Studies of hsp70 gene expression carried out, by RT-qPCR, in mussel spermatozoa have shown varied expression levels, particularly 5, 13, and 15-fold more than ctr in CM, BAs, and BAn, respectively, indicating highest involvement of stress proteins in spermatozoa of mussels in Bagnoli. In order to evaluate the possible risk on Mytilus galloprovincialis sustainability loss, electrophoretic analyses were performed on protamine-like proteins (PL) of collected spermatozoa. The results showed that CM PL were apparently unaltered with respect to ctr PL, while BAs and BAn PL appeared in part in the form of peptides and in part as bands with low mobility. Further, CM and BAs PL showed, by electrophoretic mobility shift assay, a decrease in DNA binding ability and a change in their DNA binding mode. The results of this investigation show the usefulness of the study of alterations of spermatozoa hsp70 expression and protamine-like protein properties for eco-toxicological evaluation using Mytilus galloprovincialis as a bioindicator.
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.