1. The Impact of Prenatal Atrazine Exposure on the Reproductive Development of Male Sprague-Dawley Rats James Williams Department of Biological Sciences, York College of Pennsylvania PROJECT SUMMARY Atrazine is the most heavily used herbicide in the world. Risk assessments by the EPA have found that atrazine is non-carcinogenic in humans. However, there is growing concern that it may cause hormonal imbalances in lab animals. Endocrine-disruptors mimic natural hormones in humans and in wildlife and interfere with their actions thus affecting reproductive development. Amphibian studies have shown induced feminization in males after exposure to atrazine at low, ecologically relevant doses. Mammalian lab studies have primarily been administered on adult rats under acute atrazine dosage levels. This study will focus on the prenatal exposure of rat offspring to levels of atrazine commonly found in our drinking water. Pregnant females (GD 14-19) will be exposed to various exposure levels ranging from 0 ppb to 200 ppb which are considered safe for chronic human consumption. Male rat offspring will be raised to PND 170 at which point they will be sacrificed. Blood serum testosterone levels will be observed as well as histological identification of reproductive abnormalities among the different exposure groups. Results will be compared to the control to determine if prenatal exposure to atrazine promotes demasculinization in male rat offspring. INTRODUCTION  Atrazine(2-chloro-4-ethylamine-isopropylamino-S-triazine) has been the most widely used agricultural herbicide in the world since 1958  Atrazine is classified as an environmental contaminant and is one of the most significant water pollutants in rain, surface, marine, ground water, and drinking water  Atrazine is only slightly toxic in amphibians and mammals at high dosages; however, it may be an endocrine-disrupting contaminant in males at low ecologically relevant dosages  Endocrine-disruptors, such as atrazine, are thought to hinder male reproductive development by means of aromatase induction, which converts male androgens to female estrogens Figure 1. Figure 1. Potential pathway that atrazine may affect male reproductive development  Lab studies show atrazine exposure at levels lower than the EPA’s drinking water standards have caused hermaphroditism and demasculinization in male frogs  In utero, prenatal exposure of rat offspring to low atrazine dosages commonly found in our drinking water has not been addressed, nor have the reproductive implications HYPOTHESIS H: Atrazine-treated male rat offspring will display decreased blood serum testosterone levels and/or testicular dysgenesis at low relevant dosages commonly found in drinking water Ho: There will be no differences displayed between the dosage levels and Control REVIEW OF LITERAURE  Atrazine is the most heavily used herbicide and is the most commonly detected pesticide in drinking water in the U.S.(EPA 2003)  Currently on the restricted usage list, in addition, it is under review for re-registration pending further endocrine and toxicity research  In vitro studies show low dose atrazine induces human aromatase, the enzyme that converts androgens to estrogens (Sanderson 2000)  Recent lab studies suggest atrazine is a possible endocrine disruptor (EDC) in males, particularly during fetal stages of development (Solomon 2000)  Amphibian population studies have shown that low dose atrazine exposure in male frogs causes hermaphroditism and demasculinization (Hayes 2002) Figure 2. Figure 2. An atrazine- treated hermaphroditic frog(X. laevis). Picture shows dissected kidney-adrenal-gonadl complex. Specimen treated with 1ppb (Hayes 2002)  In utero, exposure to low levels of exogenous hormones or toxicants can result in permanent physiological changes that are not seen in adults (Bigsby 1999)  Male reproductive problems in wildlife mimic human health effects (Moline 2000)  Application of atrazine in male rats showed histological changes in testis, specifically degenerative changes in Leydig and Sertoli cells (Kniewald 2000) OBJECTIVES The objectives of this proposed study will be to determine the level of atrazine exposure in drinking water during fetal development within which male offspring exhibit the following side effects: 1)Decreased blood serum testosterone levels 2)Subsequent testicular abnormalities. RESEARCH DESIGN AND METHODS EXPECTED RESULTS Maternal Gestational Phase Twelve pregnant SD females from Charles River Labs arrive on GD 2 Will be housed in pairs of two in 30cm x 60cm x 8cm cages with corncob bedding Diet will consist of Purina Lab Diet 5008 rodent pellets ad lib Will be housed at 18-20 o C at 30-70% humidity 14-hr light: 10-hr dark lighting schedule Atrazine Administration Brand name atrazine product, Aatrex 80W®, will be purchased from Syngenta Corporation Five concentration-specific, weighed amounts of atrazine will be added to individual one liter containers of distilled water. One container will act as the control Beginning on GD 14 through GD 19, 10mL of atrazine-treated water will be administered to pregnant females by gavage daily (Wolf 2000) Postnatal Care Litters from paired mothers will be culled to male offspring on PND 3 Male pups weaned form mother on PND 21 Littermates from each group will be housed 2-3 per cage PND 170-186 mature male pups will be sacrificed using decapitation (Wolf 2004) Blood serum will be collected for radioimmunoassay of testosterone levels Castration will be performed to conduct histological examination of testis (Kniewald 2000) Reproductive abnormalities will be identified including the presence/absence of estrogen-induced oocytes Bigsby, R. 1999. Evaluating the effects of endocrine disruptors on endocrine function during development. Environmental Health Perspectives 107: 613-618. Hayes, T.B. 2002. Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. Journal of Ecology 99:5476-5480. Kniewald, J. 2000. Disorders of male reproductive tract under the influence of atrazine. Journal of Applied Toxicology 20:61-68. Moline, J.M. 2000. Exposure to hazardous substances and male reproductive health: A research framework. Environmental Health Perspectives 108:803-813. Sanderson, J.T. 2001. Effects of chloro-S-triazine herbicides and metabloites on aromatase activity in various human cell lines and on vitellogenin production In male carp hepatocytes. Environmental Health Perspectives 109:1027-1031. Sharp, P.E. 1998. The Laboratory Rat. CRC Press, New York, NY. Solomon G.M. 2000. Environment and health: Endocrine disruption and potential Health implications. Canadian Medical Journal 163:1471-1483. U.S. Environmental Protection Agency. Pesticides: Topical and Chemical Factsheet- Atrazine Re-registration Eligibility Decision. Washington, D.C. Office of Prevention, Pesticides, and Toxic Substances, 2003. Wolf, C. J. 2000. Characterization of the period of sensitivity to fetal male sexual development to vinclozolin. Toxicological Sciences 55: 152-161. Wolf, C.J. 2004. Interactive effects of vinclozolin and testosterone propionate on pregnancy and sexual differentiation of the male and female SD rat. Toxicological Sciences 76: 135-143. LITERATURE CITED Acknowledgements: Dr. Bradley Rehnberg, YCP Research Mentor Dr. Deborah Ricker Figure 4. a) b) Group 5 2 Rats 200 ppb ATR Control 2 Rats 0 ppb ATR Group 1 2 Rats 0.1 ppb ATR Group 2 2 Rats 3 ppb ATR Group 3 2 Rats 25 ppb ATR Group 4 2 Rats 100 ppb ATR Pool of Female Rats GAVAGE SACRIFICE BLOOD SERUM TESTOSTERONE LEVELS HISTOLOGICAL EXAMINATION OF TESTIS Figure 3. Expected results of prenatal atrazine exposure on serum testosterone levels in sexually mature male Sprague-Dawley rats. Figure 4. Expected histological changes of testicular tissue in sexually mature male Sprague-Dawley rats after prenatal atrazine exposure GD 14-19. A) Control; B) 10 mL per day for 6 days within range of 3 ppb(3µg/L) to 200 ppb(200µg/L). Figure 3.