Chronic Dietary Exposure to a Low-Dose Mixture of Genistein and Vinclozolin Modifies the Reproductive Axis, Testis Transcriptome, and Fertility

Estrogenic and antiandrogenic endocrine-disrupting compounds (EDCs) cause a wide spectrum of developmental and fertility detrimental effects [for review, see Sharpe (2003) and Gray et al. (2001), respectively]. Most studies have used high doses of a single compound and short exposure periods, generally during the critical uterine or neonatal period. However, humans and wild animals are exposed simultaneously to various environmental and food EDCs, generally at low levels, throughout their lives. Therefore, it would be valuable to determine the effects of a chronic exposure to low doses of EDCs on the reproductive axis and to identify the mechanisms involved. In particular, it is not known whether lifetime exposures to low (environmental) doses of estrogenic “feminizing” and antiandrogenic “demasculinizing” EDCs can have adverse effects on male reproductive function of the same magnitude as those of acute exposure to high nonenvironmental doses of these compounds in isolation. In this study, we used a rat model of prolonged exposure to EDCs by gavage to determine the effects on male reproduction of two EDCs that may be associated in the human diet: the phytoestrogen genistein and the antiandrogenic fungicide vinclozolin. Genistein is an estrogenic isoflavonoid found in leguminous plants (Breinholt et al. 2000). It is particularly abundant in diets containing soya or soya-derived products, leading to a dietary exposure of up to 2 mg/kg body weight/day; for infants fed milk formulas containing soya, dietary exposure can reach 1 mg/kg body weight/day (Setchell et al. 1998). In previous studies involving transient gestational, lactational, or adult intakes (Santti et al. 1998), rodents have been exposed to various doses of genistein but the findings are equivocal: some report male reproductive anomalies (Wisniewski et al. 2003), whereas other do not (Faqi et al. 2004; Jung et al. 2004). Human exposure to genistein may affect the responsiveness and sensitivity to other xenobiotics, particularly environmental estrogenic chemicals (You et al. 2002) and other endocrine-active dietary contaminants. Vinclozolin, a dicarboximide fungicide extensively used on fruit and vegetables, is recognized as a human diet contaminant acting—essentially through its two main metabolites M1 and M2—as an androgen-receptor (AR) binding antagonist (Kelce et al. 1994, 1997; Nellemann et al. 2003). A recent French study reported that 20% of 139 meal samples from work canteens contained measurable levels of vinclozolin (Leblanc et al. 2000). In addition, assays of metabolites in urine revealed that > 80% of a population in central Italy was exposed to noticeable levels of vinclozolin and similar pesticides (Turci et al. 2006). Vinclozolin administered to experimental animals in vivo at various doses, by various routes, and for exposure periods (gestation, lactation, puberty, adulthood) produces a wide spectrum of reproductive defects: reduced anogenital distance (AGD); persistent nipples; cleft phallus; hypospadias; cryptorchidism; reduced weights of the ventral prostate, seminal vesicles, and epididymis; and reduced sperm counts (Gray et al. 1999; Monosson et al. 1999; Yu et al. 2004). It is highly plausible that vinclozolin can induce such anomalies of the reproductive tract in humans (Kavlock and Cummings 2005). However, most studies used doses 100 times the U.S. Environmental Protection Agency (EPA) no observed adverse effect level (NOAEL) of 1.2 mg/kg body weight/day based on a combination of chronic toxicity, carcinogenicity, and reproductive toxicity in rats (U.S. EPA 2003). To our knowledge, only one recent study has investigated the reproductive consequences (the frequency of hypospadias) of in utero exposure to both genistein and vinclozolin (Vilela et al. 2007). Using a lifelong exposure scheme, we found significant alterations of reproductive development and impairment of several fertility end points by these compounds, the most severe effect resulting from combined exposure to a dietary level of genistein and a level of vinclozolin lower than the U.S. EPA-proposed NOAEL. In addition, we found that mRNA expression profiles in the adult testis are notably and differentially modified according to the exposure protocol. We also describe functional clustering of the genes affected into ontologic families.