Protamine-like proteins' analysis as an emerging biotechnique for cadmium impact assessment on male mollusk Mytilus galloprovincialis (Lamarck 1819).

Here we report the industrial pollution effects due to cadmium on the reproductive health of Mytilus galloprovincialis. Mussels were removed from the biofouling of a Conatex panel after one year exposition at a polluted site near a disposal metallurgical factory. A high cadmium bioaccumulation was observed in the testis of mussels housed at the polluted site, with respect to a control site, as determined by inductively coupled plasma-mass spectrometry, along with a 10 fold increase in metallothionein 20 kDa gene (mt20) expression levels determined by qPCR. Furthermore, mussels transferred into laboratory tanks from the reference site, and exposed to 1.5, 5 and 10 µM CdCl2, revealed a 1.7, 3.2 and 4.5 fold expression increase in the testis mt20, respectively, and a positive correlation with cadmium bioaccumulation was found. To evaluate a potential detrimental risk of such alterations on spermatozoa, we carried out electrophoretic analyses on their protamine-like proteins. As determined by AU-PAGE, after 1.5 µM CdCl2 exposure, protamine-like proteins also display major alterations with respect to those obtained after 5 and 10 µM CdCl2 exposure. All protamine-like proteins isolated from the polluted biofouling were in an aggregated form and displayed the same reduced DNA binding affinity of the protamine-like proteins obtained after 1.5 µM CdCl2 as demonstrated EMSA with sperm genomic DNA. Our results contribute to the studies concerning cadmium induced testis alterations and highlight protamine-like proteins' analysis as an emerging biotechnique for cadmium impact assessment on Mytilus galloprovincialis, for the sensitivity of the in vivo and in vitro changes of protamine-like proteins' state and their DNA binding affinity.

Frog (Pelophylax bergeri, Günther 1986) endocrine disruption assessment: characterization and role of skin poly(ADP-ribose) polymerases.

Model of the our research was the adult male amphibian anura, Pelophylax bergeri, poikilotherm species not considered threatened by the IUCN, sampled in representative sites at different degree. In the first phase, a biochemical characterization of the ADP-ribosylation on the skin of barcoded amphibian anura collected from Matese Lake (clean reference site in CE, Italy) was carried out. Two PARP isoforms were evidence: the first of 66 kDa is localized into nucleus and activated by DNA damage; the second of 150 kDa is in cytoplasm, as demonstrated by biochemical and immunohistochemical analysis. Subsequently, the PARP activity, the quantitative expression of androgen receptor gene, and the levels of arsenic and chromium in skin and testis of frog and soil, water, and sediment collected from sites at different degrees of pollution were measured. A significant variation of PARP activity and androgen receptor expression levels was detected in both tissues of barcoded frogs from Sarno and Scafati, along Sarno River (SA, Italy), suggesting that a PARP activation is correlated to pollution and to steroid-regulated physiology disruption.