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(22-48) Exposure to pollutant mixtures of chub (Leuciscus cephalus): effects on mRNA stability and translational activation of connexin-43, connexin-32 and Hsp-70 (CITYFISH). Cillo, F*,1, Brevini, T1, Francisci, C1, Ceradini, S2, Chipman, J3, Taylor, E3, 1 Department of Anatomy of Domestic Animals, Embryology Unit, Milan, Italy2 CESI Environment Unit, Milan, Italy3 School of Biosciences, Birmingham, United Kingdom ABSTRACT- The CITYFISH project focuses on establishing correlation between the chemical and ecological quality of urban rivers and the population biology, biochemical and physiological status of resident fish species. Two sites on the River Lambro (Lombardy, Italy) were selected because of their similarity in hydrological characteristics and difference in chemical and ecological quality. Levels of bioavailable polyaromatic hydrocarbons and heavy metals, measured by passive samples, were higher at Brugherio (urban site, sumPAHs 13.2 ng/L; Ni 2.72 ug/L) than at Merone (upstream site, sumPAHs 2.11 ng/L; Ni 0.67 ug/L). Brugherio had a lower extended biotic index and reduced diversity of fish species relative to Merone. Wild chub (mass approx. 150g) were collected at Merone and exposed in cages to the prevailing water conditions at each site for 3 weeks, in May 2001. Reproductive tracts were removed from chub for RNA extraction, Poly(A) test RT-PCR (mRNA stability studies) and protein evaluation (translational activation/disactivations studies). RNA was denatured in the presence of phosphorylated oligo(dT)-anchor. It was ligated to the extreme 3 prime end of the polyadenylated tail and was then reverse transcribed using the ligated poly(dT)-anchor stretch as the template primer. cDNAs obtained were amplified by PCR; 5 prime primers were specifically designed for Cx-43 and Hsp70 and the products were detected by autoradiography. I addition, expression of constitutive protein, extracted from reproductive chub tissues, was evaluated after SDS-PAGE using antibodies for Cx-32, Cx-43 and Hsp-70. The results obtained demonstrate the pollutant ability to affect mRNA stability in reproductive tissues, and indicate an interesting correlation between water pollution and availability of Cx-43, Cx-32 and Hsp-70. Specifically, a decrease in mRNA stability was detected for Cx-43 and Hsp-70 in chub caged at Brugherio. Consistent with this observation, a related decreased in concentration of the encoded proteins was observed at the same site. By contrast Cx-32 appeared to be present at higher amount at the polluted site (Brugherio) which may represent an adaptive response. Key words: RNA stability, connexins, Hsp-70, chub reproductive tract |
