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IDENTIFICATION OF PROTEINS INVOLVED IN TESTICULAR TOXICITY INDUCED BY HALOACID BY-PRODUCTS OF DRINKING WATER DISINFECTION
Citation:
KAYDOS, E., MICHAEL HOLMES, J. D. SUAREZ, N. L. ROBERTS, AND G. R. KLINEFELTER. IDENTIFICATION OF PROTEINS INVOLVED IN TESTICULAR TOXICITY INDUCED BY HALOACID BY-PRODUCTS OF DRINKING WATER DISINFECTION. Presented at Society of Toxicology, Salt Lake City, UT, March 09 - 13, 2003.
Description:
Dibromoacetic acid (DBA), a prevalent disinfection by-product in drinking water, perturbs spermiogenesis in adult rats suggesting that Sertoli-germ cell communication is compromised. When isolated seminiferous tubules from rats exposed to DBA in vivo were cultured, quantitative analysis of autoradiograms following two-dimensional gel electrophoresis (2D SDS-PAGE) revealed that synthesis of multiple cytosolic proteins in tubules representing stages I-V was diminished; four of these cytosolic proteins were also diminished by in vitro DBA exposure. We sought to identify these four proteins to determine whether Sertoli cells and/or germ cells are target cells in haloacid-induced toxicity. Seminiferous tubules representing stages I-V of spermatogenesis were isolated from adult rats and cultured overnight. Tubule homogenate was centrifuged and supernatant was concentrated and assayed for protein. Proteins resolved by 2D SDS-PAGE gels were stained with SyproRuby and gel punches of each of the four proteins were frozen at -70oC. After tryptic digestion and extraction, proteins were resolved and analyzed by electrospray ionization /ion trap mass spectrometry. Data were analyzed using the Sequest search algorithm. The major proteins represented were: 14-3-3 Epsilon, 14-3-3 Theta/ Zeta, cAMP-dependent Protein Kinase Regulatory Subunit, and 1-Cys Peroxiredoxin. While cAMP-dependent Protein Kinase Regulatory Subunit is involved with FSH-mediated signaling in Sertoli cells, 14-3-3 Theta and 14-3-3 Epsilon modulate protein kinase C activity and are also found in Sertoli cells. 1-Cys Peroxiredoxin and 14-3-3 Zeta have phopholipase A2 activity. These data reveal disruption in Sertoli cells and imply that germ cell compromise may be secondary to Sertoli cell insult.