Estrogenic and antiandrogenic endocrine- disrupting compounds (EDCs) cause a wide spectrum of developmental and fertil- ity detrimental effects [for review, ...
Research Chronic Dietary Exposure to a Low-Dose Mixture of Genistein and Vinclozolin Modifies the Reproductive Axis, Testis Transcriptome, and Fertility Florence Eustache,1,* Françoise Mondon,2,3,4 Marie Chantal Canivenc-Lavier,5 Corinne Lesaffre,2,3,4 Yvonne Fulla,6 Raymond Berges,5 Jean Pierre Cravedi,7 Daniel Vaiman,2,3,4,8 and Jacques Auger 1 1Service
d’Histologie-Embryologie, Biologie de la Reproduction/CECOS (Centre d’Etude et de Conservation du Sperme Humain), Hôpital Cochin, Paris, France; 2U567, INSERM (Institut National de la Santé et de la Recherche Médicale), Institut Cochin, Département de Génétique et Développement, Equipe 21 Génomique et Epigénétique de la Pathologie Placentaire, Paris, France; 3UMR 8104, Centre National de la Recherche Scientifique (CNRS), Institut Cochin, Paris, France; 4Université Paris-Descartes, Paris, France; 5Institut National de la Recherché Agronomique (INRA) UMR 1129 FLAVIC and Université de Bourgogne, Dijon, France; 6Service de Biophysique et Médecine Nucléaire, Hôpital Cochin, Paris, France; 7INRA/Ecole Nationale Vétérinaire, UMR 1089 Xénobiotiques, Toulouse, France; 8Département de Génétique Animale, INRA, Jouy-en-Josas, France
Background: The reproductive consequences and mechanisms of action of chronic exposure to low-dose endocrine disruptors are poorly understood. O bjective : We assessed the effects of a continuous, low-dose exposure to a phytoestrogen (genistein) and/or an antiandrogenic food contaminant (vinclozolin) on the male reproductive tract and fertility. Methods: Male rats were exposed by gavage to genistein and vinclozolin from conception to adulthood, alone or in combination, at low doses (1 mg/kg/day) or higher doses (10 and 30 mg/kg/day). We studied a number of standard reproductive toxicology end points and also assessed testicular mRNA expression profiles using long-oligonucleotide microarrays. Results: The low-dose mixture and high-dose vinclozolin produced the most significant alterations in adults: decreased sperm counts, reduced sperm motion parameters, decreased litter sizes, and increased postimplantation loss. Testicular mRNA expression profiles for these exposure conditions were strongly correlated. Functional clustering indicated that many of the genes induced belong to the “neuroactive ligand-receptor interactions” family encompassing several hormonally related actors (e.g., follicle-stimulating hormone and its receptor). All exposure conditions decreased the levels of mRNAs involved in ribosome function, indicating probable decreased protein production. Conclusions: Our study shows that chronic exposure to a mixture of a dose of a phytoestrogen equivalent to that in the human diet and a low dose—albeit not environmental—of a common anti androgenic food contaminant may seriously affect the male reproductive tract and fertility. Key words: antiandrogen, endocrine disruption, male reproduction, mRNA, phytoestrogen, spermatozoa, toxicology. Environ Health Perspect 117:1272–1279 (2009). doi:10.1289/ehp.0800158 available via http://dx.doi.org/ [Online 1 April 2009]
Estrogenic and antiandrogenic endocrinedisrupting 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
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determine the effects on male reproduction of two EDCs that may be associated in the human diet: the phytoestrogen genistein and the antia ndrogenic 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 xeno biotics, particularly environmental estrogenic chemicals (You et al. 2002) and other endocrine-active dietary contaminants. Vinclozolin, a dicarboximide fungicide extensively used on volume
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 Address correspondence to J. Auger, Service d’Histologie-Embryologie, Biologie de la Reproduction/ CECOS, Hôpital Cochin, 123 Bd. de Port Royal, 75014 Paris, France. Telephone: 33-1-58-41-15-71. Fax: 33-1-58-41-15-65. E-mail: jacques.auger@cch. aphp.fr *Current address: Service d’Histologie-EmbryologieCytogénétique, Biologie de la Reproduction/CECOS, Hôpital Jean Verdier, Bondy, France. Supplemental Material is available online (doi: 10.1289/ehp.0800158.S1 via http://dx.doi.org/). We thank the animal facility and technical teams from INRA (Institut National de la Recherché Agronomique) Dijon, L. Samson from Siemens Healthcare Diagnostics, and R. Dolan from HamiltonThorne for their valuable support. This study was supported by the French Program on Endocrine Disruption (contract MEDD CV 05147), Organon France, and the French Andrology Society. The authors declare they have no competing financial interests. Received 7 September 2008; accepted 1 April 2009.
117 | number 8 | August 2009 • Environmental Health Perspectives
Male reproduction and exposure to genistein and vinclozolin
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 developm ent 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.
Materials and Methods Chemicals. Genistein with a purity of 99% was synthesized at the Laboratoire de Chimie Organique et Organométallique (Université Bordeaux 1, Talence, France). We extracted vinclozolin from the commercial formulation Ronilan (BASF France, Levallois-Perret, France) according to Bursztyka et al. (2008). The extract was dried under vacuum and then recrystallized from methanol. Vinclozolin has a melting point of 108–109°C and its purity, as verified by HPLC-diode-array detection (from 192 to 400 nm) and gas chromatography/ mass spectrometry analyses, was > 96% (data not shown). In addition, we tested the absence of the degradation products M1 and M2 by liquid chromatography/mass spectrometry as previously described (Bursztyka et al. 2008). Doses used. The exposure scheme consisted of a “high” and a “low” dose for each compound, and the corresponding combinations. The high doses we used were higher than the reported NOAEL of vinclozolin and the plausible levels of genistein in the human diet; we chose these doses to be sufficiently low to maintain normal growth, as well as food and water intake. The genistein high dose, 10 mg/kg body weight (G10), was greater than the genistein levels found in human diets in Southeast Asia (Tanaka et al. 2008) and was several times lower than the doses used in some reproductive studies; the low dose, 1 mg/kg body weight (G1), was similar to that in soya-based diets (Tanaka et al. 2008). The vinclozolin high dose, 30 mg/kg body weight (V30), was substantially greater than real-life exposure levels but was 3–10 times lower than the doses used in several male reproductive studies (Gray et al. 2001). The acceptable daily intake (ADI) of vinclozolin is 600 µg/day/person, corresponding to
an exposure of 0.01 mg/kg body weight/day (Food and Agriculture Organization/World Health Organization 1998). In France, the estimated daily intake is 3.3 µg/kg/person (Leblanc et al. 2000), which is 8. Pools of RNAs from six testes were prepared with strict equilibration of their quantity to ensure equal representation of each individual RNA. After quality control, we sent the samples to the microarray platform of NimbleGen (Reykjavik, Iceland). NimbleGen rat microarrays cover 23,456 transcripts represented by eight 60-mer oligonucleotides spotted in duplicate on the glass slide. The oligonucleotides are isothermic, which allows hybridization at a high temperature acceptable for the complete set of transcripts (70°C). The correlation between the homologous oligo nucleotides in each hybridization was > 0.99.
Table 1. Developmental anomalies, incidence of reproductive malformations, and reproductive organ weights in adult rats. Observation Development Anogenital distance [mean ± SD (mm)]a Immature penile developmenta Hypospadias (%) Cryptorchidism (%) Epididymal anomalies (%)b Other (%) Overall incidence of anomalies (%) Organ weight (mean ± SE) Body (g) Testisf Epididymisf Seminal vesiclesf Ventral prostatef Liverf
Control
G1
G10
Treatment V1
V30
G1 + V1
G10 + V30
19.4 ± 0.8 1/5 0 (0) 1 (4) 0 (0) 0 (0) 1 (4)
20.0 ± 1.1 1/5 0 (0) 1 (4) 1 (4) 0 (0) 2 (8)
19.7 ± 0.9 5/5 0 (0) 3 (13) 0 (0) 0 (0) 3 (13)
17.8 ± 0.7* 5/5 0 (0) 0 (0) 1 (4) 1 (4)c 2 (8)
17.5 ± 0.7* 2/5 7 (24) 3 (10) 1 (3) 2 (7)d 13 (44)
17.8 ± 0.7* 4/5 0 (0) 1 (5) 4 (19) 0 (0) 5 (24)
18.0 ± 1.0 2/5 2 (10) 0 (0) 0 (0) 1 (5)e 3 (15)
309 ± 7 9.7 ± 0.3 1.56 ± 0.07 3.54 ± 0.26 2.28 ± 0.14 30.9 ± 0.7
350 ± 7# 9.8 ± 0.2 1.30 ± 0.03# 3.26 ± 0.18 2.19 ± 0.09 31.7 ± 0.8
333 ± 6** 9.9 ± 0.3 1.36 ± 0.07# 3.24 ± 0.20 2.14 ± 0.13 33.1 ± 0.5**
327 ± 8 9.9 ± 0.3 1.32 ± 0.05# 3.20 ± 0.15 2.08 ± 0.11 30.8 ± 0.6
320 ± 5 10.0 ± 0.3 1.21 ± 0.09# 2.78 ± 0.11# 1.90 ± 0.07** 31.5 ± 0.7
322 ± 9 9.7 ± 0.4 1.16 ± 0.06# 2.93 ± 0.12** 2.22 ± 0.14 28.9 ± 0.7*
335 ± 6# 10.6 ± 0.2# 1.30 ± 0.04# 2.93 ± 0.17** 1.92 ± 0.11** 30.9 ± 0.5
and observations on PND25 for five randomly selected animals from each group. bMacroscopically irregular, cystic, and/or enlarged epididymis. cMicropenis. dTwo pups from the same litter had multiple anomalies: sexual ambiguity, abnormal seminal vesicles, and absence of vas deferens. eSexual ambiguity. fRelative weights (g/kg). *Tendency, but not significant (p
