ToxSci Advance Access originally published online on July 14, 2004
Toxicological Sciences 2004 81(2):430-442; doi:10.1093/toxsci/kfh218
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Toxicological Sciences vol. 81 no. 2 © Society of Toxicology 2004; all rights reserved.
Haloacid Induced Alterations in Fertility and the Sperm Biomarker SP22 in the Rat Are Additive: Validation of an ELISA
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* North Carolina State University, Department of Environmental and Molecular Toxicology, Raleigh, North Carolina 27606; and United States Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Reproductive Toxicology Division, MD #72, Research Triangle Park, North Carolina 27711
Received May 10, 2004; accepted July 1, 2004
Dibromoacetic acid (DBA) and bromochloroacetic acid (BCA) are prevalent disinfection by-products of drinking water that produce defects in spermatogenesis and fertility in adult rats. Previously we demonstrated that BCA compromises the fertility of cauda epididymal rat sperm and SP22, a sperm membrane protein that is highly correlated with the fertility of these sperm. Herein, we administered DBA and BCA, individually and in combination, to determine whether fertility and levels of SP22 on sperm were diminished in an additive fashion. Moreover, we wished to validate an immunoassay for quantitation of SP22. In a dose finding study, animals were exposed by oral gavage daily for 14 days to: BCA alone at 1.6, 4, and 8 mg/kg; DBA at equimolar levels of 2, 5, and 10 mg/kg; and two binary mixtures of 1.6 mg/kg BCA + 2 mg/kg DBA and 4 mg/kg BCA + 5 mg/kg DBA. The ED50s for the decrease in SP22 quantified by two-dimensional SDS–PAGE were 7.2 and 4.6 mg/kg for DBA and BCA. The ED50s for the decrease in SP22 quantified by ELISA were 8.1 and 5.9 mg/kg for DBA and BCA. The definitive study consisted of 2 and 4 mg/kg DBA, 1.6 and 3.2 mg/kg BCA, and a 2 mg/kg DBA + 1.6 mg/kg BCA mixture. The ED50s for decreases in fertility assessed by intrauterine insemination were 3.5 mg/kg and 2.7 mg/kg for DBA and BCA. Immunolocalization of SP22 in spermatocytes and spermatids, as well as on the cytoplasmic droplet and the equatorial segment of luminal sperm, was decreased by the DBA + BCA mixture. The decrease in SP22 in testicular parenchyma was comparable to that observed for sperm extracts. Based on 2D SDS–PAGE, ELISA, or fertility the haloacid-induced decreases in SP22 or fertility were additive or synergistic. The correlation between SP22 levels by ELISA and fertility was r2 = 0.72 compared to 0.82 for SP22 levels by 2D SDS–PAGE and fertility, validating SP22 quantitation by ELISA.
Key Words: disinfection by-products; additivity; fertility; sperm biomarker.
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