1. INTRODUCTION RESULTSEXPERIMENTAL DESIGN DISCUSSION ACKNOWLEDGMENTS Preliminary data suggest that acute exposure to DEHP (0.5mM and 1.0mM, 48 hours) does not affect prx3 mRNA levels, but increases Prx3 protein levels in Drosophila melanogaster adult males. Interestingly, MEHP (200  M, 24h) has no effect on prx3 mRNA levels but increases mitochondrial Prx3 protein levels in a spermatocyte-derived immortalized cell line (Onorato et al., J Androl 2008). Future experiments will include: Repeating the experiments presented here for statistical significance Determining the effects of DEHP (0.5 and 1.0mM) on adult males exposed to the toxicant from early development (i.e. embryo stage) Phthalates are infused with polymers to increase flexibility of plastics (Graham PR, EHP, 1973). Exposure to phthalates is ubiquitous, e.g., food containers, toys, baby bottles, IV tubing, blood storage bags, PVC flooring, and household dust (Bornehag CG et al., EHP, 2004; Rock G et al., EHP, 1986). Di-(2-ethylhexyl) phthalate (DEHP), one of the most commonly used phthalates, has adverse effects on the male reproductive system; germ cells undergo oxidative stress, mitochondrial dysfunction, and apoptosis (Kasahara, E et al., Biochem. J., 2002 ). DEHP may shorten the lifespan of the fruit fly, Drosophila melanogaster, as well as increase lipid peroxidation, an indicator of oxidative stress (Li SG et al., Zhonghua Yu Fang Yi Xue Za Zhi, 2005). Mono-2-ethylhexyl phthalate (MEHP), which can induce oxidative stress in the mammalian testis, increases Prx3 protein levels in a spermatocyte-derived immortalized cell line (Onorato et al., J Androl 2008). Prx 3 is a mitochondrial thioredoxin-dependent peroxidase found in both mammals and flies. Drosophila Prx3 is protective against hydrogen peroxide- and cadmium-induced apoptosis (Radyuk et al., Biochem J 2003). This study determined whether acute exposure to DEHP affects peroxiredoxin 3 expression in Drosophila melanogaster adult males. The effect acute exposure to Di-(2-ethylhexyl) phthalate (DEHP) on peroxiredoxin 3 expression in Drosophila melanogaster adult males. Cherub A. Ruiz 1, Penel L. Joseph 2, and Thomas M. Onorato 1 1 LaGuardia Community College, Long Island City, NY 11101 2 1Kingsborough Community College, Brooklyn, NY 11235. RNA Dr. Thomas M. Onorato served as faculty research mentor. Collegiate Science and Technology Entry Program (CTEP) at LaGuardia and Kingsborough Community Colleges (CAR and PLJ, respectively). American Society for Cell Biology (ASCB) Minorities Affairs Committee (MAC) Linkage Fellow Grant to TMO. ABSTRACT #2305 Phthalates are infused with polymers to increase flexibility of plastics. Exposure to phthalates is ubiquitous, e.g., food containers, toys, baby bottles, IV tubing, blood storage bags, PVC flooring, and household dust. Di-(2- ethylhexyl) phthalate (DEHP), one of the most commonly used phthalates, has adverse effects on the male reproductive system. Moreover, DEHP may shorten the lifespan of the fruit fly, Drosophila melanogaster, as well as increase lipid peroxidation, an indicator of oxidative stress. Peroxiredoxin 3 (Prx3) is a mitochondrial thioredoxin-dependent peroxidase that neutralizes excess peroxides, and helps protect against oxidative-stress- induced apoptosis. Mitochondrial Prx3 levels are significantly increased in an immortalized mouse spermatocyte- derived germ cell line, GC-2spd(ts), after 24 hour (h) exposure to 200  M mono-(2-ethylhexyl) phthalate, the biologically active metabolite of DEHP. Therefore, this study determined whether DEHP affects Prx3 expression in Drosophila adult males. Adult male flies (day 0-5) were dry starved for 3h, and exposed to filter paper soaked in 5% sucrose containing 0 mM (control), 0.5 mM, and 1 mM DEHP. After 48h, total RNA and protein were extracted from whole flies and used for reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analyses, respectively. Exposure to DEHP (0.5mM and 1mM; 48h) did not alter Prx3 mRNA expression. However, 48h treatment with 0.5 mM and 1mM DEHP resulted in a 1.5-fold increase in Prx3 protein levels. In summary, these preliminary findings show that 48h exposure to 0.5 mM and 1 mM DEHP has no affect on Prx3 mRNA levels, but causes an increase in Prx3 protein levels. The effects of acute DEHP Exposure (48h) on prx3 protein levels Left panel, Western Blot analysis, PRX3, peroxiredoxin 3; TUB,  -tubulin. Right panel, normalization of prx3 signal to  -tubulin, densitometry performed using ImageJ, free software from NIH. The effects of acute DEHP Exposure (48h) on prx3 mRNA expression Left panel, 2% agarose gel of RT-PCR analyses; Magenta arrows, molecular weight marker sizes, base pairs; prx3, peroxiredoxin 3; rp49, ribosomal protein L32. Lanes: (1) molecular weight marker, (2) control, (3) 0.5mM DEHP, (4) 1mM DEHP, (5) - reverse transcriptase control. Right panel, normalization of prx3 signal to rp49, densitometry performed using ImageJ, free software from NIH, error bars indicate standard error of mean (SEM). n=2