Research | Article Perinatal Exposure to Low Levels of the Environmental Antiandrogen Vinclozolin Alters Sex-Differentiated Social Play and Sexual Behaviors in the Rat Nathan K.W. Colbert, Nicole C. Pelletier, Joyce M. Cote, John B. Concannon, Nicole A. Jurdak, Sara B. Minott, and Vincent P. Markowski Maine Center for Toxicology and Environmental Health, University of Southern Maine, Portland, Maine, USA In this study we examined the effects of exposure to the antiandrogenic fungicide vinclozolin (Vz) on the development of two sex-differentiated behaviors that are organized by the perinatal actions of androgens. Pregnant Long-Evans rats were administered a daily oral dose of 0, 1.5, 3, 6, or 12 mg/kg Vz from the 14th day of gestation through postnatal day (PND)3. The social play behavior of juvenile offspring was examined on PND22 and again on PND34 during play sessions with a same-sex littermate. After they reached adulthood, the male offspring were examined with the ex copula penile reflex procedure to assess erectile function. Vz did not produce any gross maternal or neonatal toxicity, nor did it reduce the anogenital distance in male pups. We observed no effects of Vz on play behavior on PND22. However, the 12-mg/kg Vz dose significantly increased play behavior in the male offspring on PND34 compared with controls. The most dra- matic increases were seen with the nape contact and pounce behavior components of play. The Vz effect was more pronounced in male than in female offspring. As adults, male offspring showed a significant reduction of erections at all dose levels during the ex copula penile reflex tests. The 12-mg/kg dose was also associated with an increase in seminal emissions. These effects demon- strate that perinatal Vz disrupts the development of androgen-mediated behavioral functions at exposure levels that do not produce obvious structural changes or weight reductions in androgen- sensitive reproductive organs. Key words: antiandrogen, penile reflexes, prenatal exposure, rat, social play, vinclozolin. Environ Health Perspect 113:700–707 (2005). doi:10.1289/ehp.7509 available via http://dx.doi.org/ [Online 16 March 2005] Fungicides are applied to many foods to con- trol plant diseases such as Sclerotinis sclerotio- rum (white mold) and Botrytis cinerea (gray mold) (Papadopoulou-Mourkidou 1991). After application, fungicides have been shown to volatilize and circulate through air and water and on untreated foods, increasing their distribution (Baumeister et al. 2002). Consumers cannot readily reduce their expo- sure because fungicides are not removed from fresh produce by rinsing with tap water (Krol et al. 2000), and commercial process- ing increases their concentrations (Will and Kruger 1999). Fungicides are also widely used on golf courses, industrial landscapes, lawn turf, and ornamental plants, where they can enter water supplies in contaminated runoff (Haith and Rossi 2003). The dicarboximide fungicide vinclozolin (Vz) is used in a number of commercial formu- lations to treat fruits and vegetables such as let- tuce, snap beans, canola, and grapes [U.S. Environmental Protection Agency (EPA) 2003]. Vz belongs to a group of environmental endocrine disruptors known as the anti- androgens. These compounds share a common, clearly defined hormone-receptor–mediated mechanism of action. Vz is biotransformed into at least two active metabolites that bind com- petitively to the human, monkey, and rat androgen receptor (Kelce and Monosson 1995; Kelce et al. 1994). Exposure to antiandrogens 700 during development could have serious effects on sexual development. They are already recognized as one of the factors responsible for the recent increase of hypospadias, a male reproductive disorder where the urethral opening is on the ventral surface of the penis (Baskin et al. 2001; Davis et al. 1998; Egeland et al. 1994; Jensen et al. 1995; Sharpe and Skakkebaek 1993). To date, most investigations have focused on the impact of the environmental anti- androgens on the development of androgen- sensitive male reproductive organs. For example, adult male rats have reduced ano- genital distances (AGDs), reduced seminal vesicle and ventral prostate weights, and lower epididymal sperm counts after perinatal exposure to Vz (Gray et al. 1994; Hellwig et al. 2000). Vz is not the only fungicide that acts as an environmental antiandrogen. Procymidone and iprodione are fungicides that are structurally similar to Vz, and they produce a nearly identical proﬁle of effects on the reproductive system (Gray et al. 1999b; Ostby et al. 1999). All three fungicides have the same ﬁnal metabolite. However, the doses of Vz that have been shown to affect the weights of reproductive organs in animal studies are quite high, often 5–10 times the U.S.EPA’s lowest observed adverse effect level (LOAEL). High doses are associated with measures of gross toxicity such as lowered body weights and increased mortality rate due to granulomas and bladder stones (Gray et al. 1994; Hellwig et al. 2000). Thus, although effects such as reductions in rat reproductive organ weights demonstrate that environmental antiandrogens can produce long-term effects after perinatal exposure, they do not necessarily represent the most sensitive end points. Investigations of the functional effects of low- level environmental antiandrogen exposure are needed to complement the high-dose studies and place organ deﬁcits into the larger context of male reproductive health. One of the questions examined in the pre- sent study was whether much lower levels of Vz during the perinatal period affect repro- duction via a disruption of male copulatory behavior. An androgen-sensitive neuro- muscular system that is critical for normal male copulatory behavior is the levator ani and bulbocavernosus (BC) skeletal muscles and their motor neuron control centers in the lumbar spinal cord [the spinal nucleus of the BC (SNB)]. In rats, contraction of the levator ani and BC muscles, as well as vascular mechanisms, produces penile erections (Hart and Melese-D’Hospital 1983; Leipheimer and Sachs 1993; Sachs 1982). The sex-specific development of the BC/SNB system is orga- nized during the perinatal period by the non- aromatizable androgen dihydrotestosterone (DHT) (Hart 1979; Thomas et al. 1982). In developing males, the presence of DHT reduces motor neuron death in the SNB and promotes retention of the BC (Mills and Sengelaub 1993). In the adult male, there are two to three times more SNB motor neurons than in females (Sengelaub et al. 1989). However, environmental antiandrogen expo- sure can disrupt the development of the Address correspondence to V.P. Markowski, Department of Psychology, University of Southern Maine, 96 Falmouth St., 178 Science Building, Portland, ME 04104-9300 USA. Telephone: (207) 228-8174. Fax: (207) 228-8057. E-mail: markowski@ usm.maine.edu We thank W.D. Thompson for his assistance with the statistical analyses and S.E. Frankel for his efforts proofreading the manuscript. This study was supported by a research grant from the Bioscience Research Institute of Southern Maine to V.P.M. The authors declare they have no competing ﬁnancial interests. Received 18 August 2004; accepted 15 March 2005. VOLUME 113 | NUMBER 6 | June 2005 • Environmental Health Perspectives