Screening of Biomarkers for the Histological Examination of Cellular Proliferation and Death in the Livers of the Western Mosquitofish Gambusia Affinis

Abstract

The western mosquitofish, G. affinis, is an excellent aquatic organism for the biomonitoring of aquatic ecosystems for environmental carcinogens. However, proteinspecific biomarkers are not readily available for this species. Therefore, protein antibody probes for cellular proliferation and cell death were examined to determine their specificity in G. affinis. proliferating cell nuclear antigen (PCNA) antibodies for cellular proliferation and p53, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP) antibodies for apoptosis were investigated using Western blot analysis for use in immunohistochemical staining, along with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) immunoperoxidase staining for cellular death. PCNA antibody (PC10) was specific to G. affinis putative PCNA protein and can be readily used in immunohistochemical staining of liver tissues for cellular proliferation and that TUNEL immunoperoxidase staining specifically labeled apoptotic and/or necrotic cells in G. affinis liver sections. The remaining antibodies tested (p53 and cleaved caspase-3 and PARPI did react with components of G. afinis protein extracts; however, they require further studies to demonstrate their specificity for the proteins of interest. The use of PCNA and TUNEL biomarkers for cellular proliferation and cell death, respectively, were demonstrated in subsequent environmental carcinogenesis studies using G. afinis. (The J Histotechnol 26:157, 2003)Submitted: July 10, 2003; Accepted July 29, 2003