Disruption of Reproductive Development in Male Rat Offspring following Gestational and Lactational Exposure to Di-(2-ethylhexyl) Phthalate and Genistein

Studies of developmental eff ects of mixtures of endocrine disrupters on the male reproductive system are of great concern. In this study, the reproductive eff ects of the co-administration of di-2-(ethylhexyl) phthalate (DEHP) and genistein (GEN) during pregnancy and lactation were studied in male rat off spring. Pregnant Sprague-Dawley rats were gavaged from gestation day 3 to postnatal day 21 with vehicle control, DEHP 250 mg⁄kg body weight (bw)⁄day, GEN 50 mg⁄kg bw⁄day, GEN 400 mg⁄kg bw⁄day, and two combinations of the two compounds (DEHP 250 mg⁄kg bw⁄day + GEN 50 mg⁄kg bw⁄day, DEHP 250 mg⁄kg bw⁄day + GEN 400 mg⁄kg bw⁄day). The outcomes studied were general morphometry (weight, AGD), testicular histology, testosterone levels, and expression at the mRNA level of genes involved in steroidogenesis. Organ coeffi cient, AGD / body weight 1 / 3 , serum testosterone concentration and genes involved in steroidogenic pathway expression when exposed to DEHP (250mg/kg bw⁄day), GEN(50mg/kg bw⁄day) or GEN(400mg/kg bw⁄day) alone were not signifi cantly diff erent from the control group. When exposed to (DEHP 250mg/kg bw⁄day +GEN 50mg/kg bw⁄day) together during pregnancy and lactation, serum testosterone concentration, epididymis coeffi cient and Cypal17a1,Scarb1 m RNA expression signifi cantly decreased compared to the control and GEN(50mg/kg bw⁄day). When exposed to (DEHP 250mg/kg bw⁄day +GEN 400mg/kg bw⁄day) together during pregnancy and lactation, AGD / body weight 1 / 3 , serum testosterone concentration, testis and epididymis coeffi cient and Star, Cypal17a1 mRNA expression appeared signifi cantly decreased compared to the control and DEHP/GEN single exposure, together with developmental impairment of seminiferous tubules and seminiferous epithelium. Overall, co-administration of DEHP and GEN during gestation and lactation seem to acts in a cumulative manner to induce the most signifi cant alterations in the neonate, especially with GEN at high dose, although the eff ect of the DEHP–GEN mixture on adult off spring should be observed further.


INTRODUCTION
It is well recognized that humans and wild animals are exposed to multiple endocrine-disrupting chemicals (EDCs) simultaneously (Main et al., 2010;TM et al., 2006;Toppari et al., 2010).Di-2-(ethylhexyl) phthalate (DEHP), the most widely used plasticizer in polyvinylchloride plastics, is used in cosmetics, pharmaceuticals, lubricant oils and solvents, comprising about 80% of the phthalate consumption in the world (Kamrin, 2009).Since phthalate plasticizers are not chemically bound to PVC, they can leach, migrate or evaporate into indoor air and atmosphere, foodstuff s, other materials, etc. Especially since the "Plasticizer Crisis" occurred in May 2011 in Taiwan, DEHP arouses concern about its antiandrogen eff ects (Fisher, 2004).Genistein, a soy-derived product, is widely present in the diet of the eastern world (Duncan et al., 2003).Phytoestrogen consumption during pregnancy and in infants through soy-based formulas is of great concern (Cederroth et al., 2010;Strom et al., 2001).
DEHP exerts anti-androgen effect by suppressing fetal testosterone biosynthesis via peroxisome proliferatoractivated receptor (PPARs) activation (Culty et al., 2008), resulting in a series of reproductive tract defects in male off spring (Christiansen et al., 2010;Foster, 2006;Lagos-Cabre & Moreno, 2012;Pocar et al., 2012) .The spectrum of eff ects is characterized by disrupted androgen-dependent development and increased incidence of hypospadias, cryptorchidism, impaired spermatogenesis and testicular cancer, which collectively comprise the testicular dysgenesis syndrome (TDS) (Hu et al., 2009).In both humans and rodents, fetal Leydig and Sertoli cell dysfunction seems to play a vital role in the development of TDS or TDS-like symptoms.In the rat model, DEHP exerts an anti-androgen eff ect mainly by suppressing fetal testosterone biosynthesis (Culty et al., 2008).
Genistein can mimic the actions of estrogen and behave as an antioxidant.Exposure to genistein, especially during pregnancy and lactation, can cause disruption of the reproductive system development in a dose-dependent way (Lehraiki et al., 2011;Wisniewski et al., 2003;Wisniewski et al., 2005).It is also interesting to note that genistein could enhance fertility by promoting acrosome reaction at lower doses, but potentially suppress male fertility via suppressing acrosome reaction at higher doses, having no signifi cant eff ect on sperm morphology (Kumi-Diaka et al., 1999).It has also been shown that isofl avones can reduce the negative eff ects of cypermethrin on reproductive characteristics of mature male rabbits (Yousef et al., 2003).
Although many studies have examined the effects of single EDC using in vitro or in vivo assays (Christiansen et al., 2010;Roberts et al., 2000), few studies have been conducted to examine the eff ects of mixtures of EDCs on mammalian reproductive development, especially for those EDCs that act via diff erent mechanisms.So as a result of growing concerns about mixture exposure in reality recently, research on EDC mixture exposure during pregnancy and lactation have became a focus in andrology and toxicology (Kortenkamp, 2008).Studies evaluating the eff ect of DEHP in combination with genistein have been lacking.In this study, we mainly investigated the changes produced by the co-administration of DEHP and genistein during pregnancy and lactation on male off spring testis histology, serum testosterone, anogenital distance and mRNA expression on postnatal day1 and postnatal day 22.

Animals
Prior to initiation of the study, the experimental protocol was reviewed and approved by the Committee on Animal Research and Ethics of Xi'an Jiaotong University (Xi'an, P.R.China).Specifi c pathogen-free (SPF) Sprague-Dawley rats were obtained from the Experimental Animal Center of Xi'an Jiaotong University and kept under 12-h light/ dark cycle within the temperature range of 21±2 ºC, relative humidity held constant at 50±5%.Soy-and alfalfa-free diet (SAFD) and purifi ed water were provided ad libitum.
Forty-eight eight-week-old virgin SD female rats (F0 generation) were housed two per cage on arrival.Each pair of female rats was mated overnight with one male adult rat.Vaginal smears were collected early in the morning daily and the day of sperm detection was considered as gestation day 0 (GD0).Once insemination was confi rmed, the fi rst thirty-six sperm positive female rats were weighed, housed individually and randomly allocated to fi ve treatment groups.F0 pregnant rats were treated by daily gavage from gestation day 3 (GD3) to postnatal day 21(PND21) with peanut oil (vehicle control), DEHP 250 mg/kg body weight (bw)/day (DEHP 250), GEN 50 mg/kg bw/day (GEN 50), GEN 400 mg/kg bw/day (GEN 400), a combination of DEHP 250 mg/kg bw/day and GEN 50 mg/kg bw/day (DEHP 250+GEN 50), or a combination of DEHP 250 mg/kg bw/day and GEN 400 mg /kg bw/ day (DEHP 250+GEN 400).DEHP and GEN were dissolved in peanut oil and peanut oil was administrated at a dose of 2ml/kg.There was a 1-day interruption of treatment on the day of parturition (PND0).The dose of each chemical was calculated daily according to body weight for each dam before dosing.Off spring were weaned on PND21 when F0 rats were euthanized.
The doses of DEHP and genistein were chosen on the basis of previous reports (Shirota et al., 2005;Michael et al., 2006); we expected that the selected doses of DEHP 250 mg/kg/ day would produce small but not signifi cant changes, if any, on endpoints such as testicular testosterone level decrease or anogenital distance decrease, therefore any potentially cumulative effects would be better manifested.Serum concentration of phytoestrogen under a classical Asian diet is equivalent to that of a rat at the dose of 40-50 mg/kg (H et al., 1993;KS et al., 2001) and the no observed adverse eff ect level (NOAEL) of genistein is considered to be 50 mg/kg/ day (Michael et al., 2006).No treatment-related teratogenic eff ects were noted in body weight, normalized anogenital distance, serum testosterone or sperm concentration in the male offspring after exposure to GEN 300 mg /kg bw/ day (Wisniewski et al., 2005) and GEN 500 mg /kg bw/day (McClain et al., 2007) during pregnancy.

F1 treatment
Body weights of pups were determined on PND 1 and PND 22. Anogenital distances were measured using a Vernier caliper by a single investigator in a blind manner on PND 1 and PND22.The AGD of each animal was divided by the cube root of body weight to avoid errors caused by diff erences in body size.On PND 21, pups were moved from their mothers and housed by litter and sex.One litter of the (DEHP 250+GEN 400) group died because there was no milk secreted by the dam, so that only fi ve dams remained in this group.On PND22, two to three F1 male off spring per litter were anaesthetized using 10% chloral hydrate.Blood samples were taken from the inferior vena cava and centrifuged at 3500 rpm for 10min.Serum was exacted and stored at −80 ºC.Serum testosterone was tested with a testosterone radioimmunoassay kit (Tianjin Nine Tripods Medical &Bioengineering Co., Ltd, P.R.China) according to manufacturer ' s instructions.The right testis of each pup was removed and stored in liquid nitrogen and then transferred to a −80 ºC freezer for later analysis of gene expression.The left testis, liver, left kidney and spleen of these pups were removed and weighted and the testis was immediately placed in Bouin's fi xative solution for 12 h and routinely processed for histology.

Testicular Histology
After fi xation in Bouin's fi xative solution, testes collected on PND22 were transferred to 75% ethanol, embedded in paraffi n and cut at 5μm.Sections were stained with haematoxylin and eosin and evaluated by light microscopy.The diameter of at least twenty randomly selected round cords per testis was measured via Image-Pro-Plus 5.0 version (Media Cybernetics, USA) and the ratio of seminiferous cords/seminiferous tubules was calculated at the same time.Those evaluations were performed by an experienced investigator blind to the treatment group.

Testicular RNA isolation and RT-PCR
Right testes of male off spring were taken from the −80 ºC freezer and immediately ground on ice homogeneously in a plate containing 2ml DEPC-treated PBS.For total RNA isolation, the suspension later was transferred to RNAasefree Eppendorf tubes.Total RNA was isolated according to instructions of the Fast 200 RNA isolation kit (Fastgen biotechnology Co, Shanghai, P.R .China).Total RNA was quantifi ed (A260) and assessed for purity (A260/A280) by spectrophotometry.Then reverse transcription was carried out using a RevertAid First Strand cDNA Synthesis Kit (Fermentas, Lithuania) according to manufacturer's instructions.
Reverse transcription polymerase chain reaction (RT-PCR) was performed on a PTC-200 DNA Engine from MJ Research (now Bio-Rad).The genes, primer sequences and optimal amplifi cation conditions are listed in Table 1.Amplifi cation of cDNA was performed using the PCR mix (Applied Biosystems) according to the manufacturer's instructions.The relative level of mRNA was standardized to the housekeeping genes β-actin in order to control for diff erences in RNA loading, quality and cDNA synthesis.RT-PCR was performed in a reaction volume of 25 μl containing 12.5μl 2×PCR Mix,1μl forward primer, 1 μl reverse primer,1μl cDNA template and 9.5μl ddH 2 O.

Statistical analysis
Data were expressed (mean ± SD) and analyzed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA).Normality and homogeneity of variances were evaluated prior to statistical analysis.Data were analyzed by one-way analysis of variance (ANOVA) followed by the Games-Howell post-hoc test when the results of the ANOVA were signifi cant.In all analyses, litters were used as the statistical units.Diff erences were considered to be statistically signifi cant at a probability level of 5% (P< 0.05).

Maternal data and Off spring data on PND1
As expected, no signs of general toxicity were observed in any experimental group.Briefly, there was no significant effect on maternal weight gain, litter size, sex ratio, number of viable pups or AGD/(bodyweight) 1/3 at any dose tested.But body weight of male offspring significantly decreased in group DEHP250 +GEN50 (compared to the control) and group DEHP250 +GEN400 (compared to the other five groups) (Table 2).

Serum testosterone concentration
Exposure to either DEHP 250 mg/kg bw/day, GEN 400 mg/kg bw/day or GEN 400 mg/kg bw/day resulted in slight but nonsignifi cant reductions in serum testosterone levels compared to the control (Fig. 1).In contrast, co-administration of the two chemicals resulted in a signifi cant reduction of testosterone when exposed to DEHP250mg/kg bw/d+GEN50mg/kg

TABLE 1
Genes, primer sequences and amplifi cation size for RT-PCR  bw/d (compared to the control, GEN50 and GEN400) and DEHP250mg/kg bw/d+GEN400 mg/kg bw/d (compared to the control, DEHP250, GEN50 and GEN400) .

Off spring data on PND22
Body weight, organ weight and organ coeffi cient and AGD/ body weight 1/3 of male rat off spring on postnatal day 22 are shown in Table 3.The organ coefficient was calculated as (weight of organ)/(body weight) .When exposed to DEHP 250 mg/kg bw/day + GEN 50 mg/kg bw/day, a signifi cant decrease was observed in both epididymis weight and epididymis coeffi cient compared to the control, GEN 50 and GEN 400; no signifi cant diff erences were observed in AGD/ body weight 1/3 in this group.Exposure to DEHP 250 mg/kg bw/day + GEN 400 mg/kg bw/day caused signifi cant decrease in body weight and testicular weight and testicular coeffi cient (compared to the control and GEN 50), epididymis weight and epididymis coeffi cient (compared to the control, DEHP250 and GEN 50) and kidney coeffi cient (compared to the control).
Signifi cant diff erences in AGD/body weight 1/3 were observed in DEHP250+GEN400 compared to the control, DEHP250 and GEN 50.Malformations of the external genitalia or cryptorchidism were not observed at any dose tested (Table 3).

Testicular histology
Testicular sections of male offspring rats, seminiferous cord diameter of the testis and ratio of seminiferous cord/ seminiferous tubules on postnatal day 22 are shown in Figure 2 and Figure 3 separately.Diameter and germ cell development of groups DEHP250 and DEHP250 +GEN50 were not signifi cantly diff erent from the control, but cell layers of the two groups were thinner than control layers.The diameter of the seminiferous cords of group DEHP250 +GEN400 was signifi cantly reduced with relatively few cell layers compared to the control and GEN 400, indicating delayed development of the testis.The ratios of

Testicular gene expression
Exposure to DEHP250 mg/kg bw/day or GEN400 mg/kg bw/day did not signifi cantly aff ect genes involved in the steroidogenic pathway Star, Cyp17a1, Scarb1 or Cyp11a1 mRNA levels on PND22.Expression of the Star of group DEHP 250 +GEN400 was less than in the control, group GEN50 and group GEN400; expression of Cypal17a1 of the two mixture exposed groups was lower than that of the control group (both groups) and group GEN50 (group DEHP 250 +GEN50) (P <0.05); Scarb1 of group DEHP 250 +GEN50 expressed less than the control and group GEN50 (P <0.05); there was no signifi cant diff erence in Cyp11a1 between groups (P >0.05), which was not altered after exposure in any group (Figure 4).(B), GEN 50 mg/kg bw/day(C), GEN400 mg/kg bw/day(D),DEHP 250 mg/kg bw/day+GEN50 mg/kg bw/day(E) and DEHP 250 mg/ kg bw/day+GEN400 mg/kg bw/day (F).There were 14, 14, 13, 15, 13 and10 male offspring rats in each group, respectively.Note the signifi cant decrease of diameter of the seminiferous cords as well as reduced cell layers in group DEHP250 +GEN400 .400×magnification.Scale bars indicate 100µm.
Figure 3. Seminiferous cord diameter of the testes (A) and ratio of seminiferous cords/seminiferous tubules (B) of male offspring on PND 22 after gestational and lactational exposure to vehicle, DEHP 250 mg/kg bw/day, GEN 50 mg/kg bw/day, GEN 400 mg/kg bw/day, DEHP 250 mg/kg bw/day+GEN50 mg/kg bw/day and DEHP 250 mg/kg bw/day+GEN400 mg/kg bw/day.A) Note the considerable decrease in group DEHP 250 +GEN400.B) Note the signifi cantly lower ratio of seminiferous cords/seminiferous tubules in group DEHP 250 +GEN50 and group DEHP 250 +GEN400 compared with group GEN50.There were 14, 14, 13, 15, 13 and10 male offspring rats in each group, respectively.Results were expressed as litter mean± standard deviation.Statistical differences between control and treated groups were determined using an ANOVA followed by Games-Howell post-hoc test when results of the ANOVA were signifi cant.*:Signifi cantly different from control group at P<0.05, : Signifi cantly different from GEN50 at P<0.05, : Signifi cantly different from GEN400 at P<0.05.

DISCUSSION
The endocrine disruptor hypothesis states that early developmental exposure to environmental disrupting chemicals can disrupt male reproductive development and impair fertility (Casals-Casas & Desvergne, 2011;Skakkebaek et al., 2001;Yiee and Baskin, 2010).In the present study we evaluated the eff ects of gestational and lactational exposure to DEHP and/ or genistein on the sexual development of offspring rats.Exposure to DEHP250 mg/kg bw/day, GEN50 mg/ kg bw/day or GEN400 mg/kg bw/day alone did not have a signifi cant detrimental eff ect on male off spring development, including body weight, serum testosterone, corrected AGD, organ coeffi cient and histology of testis, although exposure to single chemicals may cause a slight but not significant eff ect.We also did not fi nd any signifi cant alteration of the transcripts of key proteins of the steroidogenic pathway such as Star, Cyp17a1, Scarb1 and Cyp11a1 and we may suppose that these alterations were too small to be detected.Vo et al. (2009) demonstrated that in utero administration of DEHP at 100 mg/ kg/day and 500 mg/kg/day to SD rats from GD11 to GD21 was able to reduce the sperm concentration, viability and motility of the male off spring, but no signifi cant reduction of testis weight, epididymis weight, prostate weight or testosterone level of the male off spring were observed at PND63, or for gene expression such as Star, Cyp11a1, Hsd3b1, etc. on GD21.Mariko Shirota et al. (2005) found that after exposure to DEHP at doses of 125, 250 and 500 mg/kg/day during GD7-GD18 there was no abnormality in the testes at 5 and 10 weeks after birth in any of the treated groups, which is in agreement with our study.Although variability might exist in the response of rats to DEHP across diff erent laboratories, individual doses of DEHP that are similar to the dose selected in our study have been demonstrated to produce responses that are at borderline significance for several endpoints such as testosterone production, anogenital distance and nipple retention.But those studies mostly focused on exposure during either gestation or lactation and did not cover both the gestation and lactation periods, which are two critical periods for masculinization.Recently Pocar et al (2012) reported more severe reproductive toxicity of male offspring mice on PND 42 after maternal exposure to DEHP at relatively low doses from GD 0.5 to the end of lactation.The diff erences with our study may largely due to diff erent end points of observation and diff erent species; DEHP administration cannot produce immediate reproductive toxicity because of its delayed effect on HPG and Leydig cell maturation, so it is necessary to observe the long-term reproductive outcome.However, DEHP administration via chow intake cannot assure accurate DEHP dosage because body weight and chow intake do not have linear correlation.E) the expression of Cyp11a1 showed no signifi cant difference between the groups (P >0.05).There were 14, 14, 13, 15, 13 and10 male offspring rats in each group, respectively.Results were expressed as litter mean± standard deviation.Statistical differences between control and treated groups were determined using an ANOVA followed by Games-Howell post-hoc test when results of the ANOVA were signifi cant.*:Significantly different from control group at P<0.05, : Signifi cantly different from GEN50 at P<0.05, : Signifi cantly different from GEN400 at P<0.05 Dietary exposure to isofl avones plays an important role in various physiological processes in the body.It is well known that genistein, an isofl avone found in soybeans and soy products, has both estrogenic (Cederroth et al., 2010) and antioxidant eff ects (Qian et al., 2012).In our study, the eff ect of genistein on the male off spring also appears to be negligible.This is in accordance with previous reports of no major alterations in the reproductive system of SD rats exposed during gestation and/or lactation to high or low phytoestrogen diets.Wisniewski et al. (2005) showed that after receiving a phytoestrogen-free diet supplemented with 0, 5 or 300 mg/kg of genistein throughout gestation and lactation, timing of preputial separation, adult reproductive behavior, sperm concentrations and testosterone production were not infl uenced by genistein treatment at either dose.But previous studies did not examine the toxicity of genistein exposure in utero and lactation at 400mg/kg bw/day.In an in vitro study (Lehraiki et al., 2011) in organ cultures of fetal testes from wild type and ER alpha or ER beta knockout mice showed that genistein inhibits testosterone secretion by fetal Leydig cells during early fetal development, while in this in vivo study we did not observe any modifi cation of serum testosterone production in the neonate.
Co-administration of DEHP and GEN (at 50 mg/kg bw/ day or 400mg/kg bw/day) from gestation day 3 to postnatal day 22 signifi cantly reduced the body weight of male off spring, serum testosterone levels, epididymis coefficient, testis coeffi cient (Group DEHP+GEN400) and AGD/(body weight) 1/3 (Group DEHP+GEN400) in male off spring on PND22 at doses that individually had no signifi cant eff ect on this variable.The DEHP and genistein (at low and high doses) mixture produced more signifi cant alterations than DEHP or genistein alone, especially when DEHP was co-administrated with genistein at 400mg/kg bw/day.This evokes a cumulative eff ect after exposure to the mixture.Genistein has been reported to exhibit in vitro anti-androgenic activity in addition to its well-established estrogenic activity especially at high dose (Rosenberg et al., 2000).This type of mechanism may act in vivo and contribute to some of the anti-androgenic demasculinizing effects observed here.Eustache et al. (2009) observed a deleterious synergistic eff ect of low doses of genistein and vinclozolin on fertility using a similar experimental procedure.The steroidogenic acute regulatory protein Star, a protein involved in the transfer of cholesterol from the cytosol to the outer mitochondrial membrane, plays a key role in the acute regulation of steroid hormone synthesis by enhancing the conversion of cholesterol into pregnenolone.The combination of DEHP and high-dose GEN, down-regulated the expression of Star, as well as 17-αhydroxylase (Cypal17a1).Scavenger receptor class B (Scarb1) Cypal17a1 was also down-regulated with DEHP co-administrated with GEN at low dose.The modifi cation of genes involved in the steroidogenic pathway was probably responsible for the following events, which regulated testosterone production, epididymis and testis development.AGD or corrected AGD, which is a non-invasive index of masculinization, hypospadias and cryptorchidism, may be associated with reduced anogenital distance as a result of endocrine disruption (MH et al., 2008) .AGD/(body weight) 1/3 in Group DEHP+GEN400 were signifi cantly reduced compared to the control and DEHP exposure alone, which means deterioration of the masculinization process and may further impair the fertility of male off spring.
Several studies have been recently published on possible cumulative eff ects of endocrine disruptors in rats.However, their focus has been mostly on exposure to endocrine disruptors of the same category or which act as anti-androgens, such as phthalates (Hass et al., 2007;Rider et al., 2008;Rider et al., 2009;Rider et al., 2010).Research on diff erent species of endocrine disruptors has been seldom published.Our research demonstrated fi rstly that a natural phytoestrogen, genistein, can exacerbate the deleterious eff ect of the most commonly used plasticizer DEHP when exposed during gestation and lactation, especially when genistein was co-administrated at a high dose.It is also necessary to observe the reproductive eff ect on the adult male off spring, especially when exposed to the mixture.Previously Yousef et al (2003) demonstrated the benefi cial infl uence of isofl avones in reducing the negative eff ects of cypermethrin on thr reproductive characteristics of mature male New Zealand white rabbits when given every other day for 12 weeks.While previous studies mainly focused on genistein exposure at relatively lower dose, especially when co-administrated (Eustache et al., 2009;Lehraiki et al., 2011;You et al., 2002), in our study for the fi rst time genistein was co-administrated at high dose with DEHP.This emphasizes the importance of further observation and continuous study of reproductive toxicology evaluation of co-administration.

Figure 1 .
Figure1.Serum testosterone levels collected on PND 22 from control DEHP and/or GEN-exposed rats.Values are litter mean ± standard deviation.Note the considerable decrease in group DEHP 250 +GEN50, DEHP 250 +GEN400.There were 14, 14, 13, 15, 13 and 10 male offspring rats in each group, respectively.Statistical differences between control and treated groups were determined using an ANOVA followed by Games-Howell post-hoc test when results of the ANOVA were signifi cant.*:Signifi cantly different from control group at P<0.05, #:Signifi cantly different from DEHP250 at P<0.05, : Signifi cantly different from GEN50 at P<0.05, : Signifi cantly different from GEN400 at P<0.05

Figure 4 .
Figure 4. Testicular gene expression of control, DEHP and/or GEN-exposed male offspring on PND 22 after gestational and lactational exposure.(A) gene expression determined by RT-PCR.(B) the expression of Star in group DEHP 250 +GEN400 was signifi cantly lower than the control , group GEN50 and group GEN400(P <0.05).(C) the expression of Cyp17a1 in group DEHP 250 +GEN50, DEHP 250 +GEN400 showed signifi cant decrease compared to the control (group DEHP 250 +GEN50, DEHP 250 +GEN400)and group GEN50 (group DEHP 250 +GEN50) (P <0.05).(D) the expression of Scarb1 in group DEHP 250 +GEN50 was decreased signifi cantly compared to the control and group GEN50 (P <0.05).(E) the expression of Cyp11a1 showed no signifi cant difference between the groups (P >0.05).There were 14, 14, 13, 15, 13 and10 male offspring rats in each group, respectively.Results were expressed as litter mean± standard deviation.Statistical differences between control and treated groups were determined using an ANOVA followed by Games-Howell post-hoc test when results of the ANOVA were signifi cant.*:Significantly different from control group at P<0.05, : Signifi cantly different from GEN50 at P<0.05, : Signifi cantly different from GEN400 at P<0.05

TABLE 2
Maternal data and offspring data following oral exposure to DEHP and/or GEN on PND1 Values are litter mean ± standard deviation.Games-Howell post-hoc test: *: Signifi cantly different from control group at P<0.05, **: Signifi cantly different from control group at P<0.01, #: Signifi cantly different from DEHP250 at P<0.05; ÷: Signifi cantly different from GEN50 at P<0.05; ◊: Signifi cantly different from GEN400 at P<0.05; ♦: Signifi cantly different from DEHP250+GEN50 at P<0.05.

TABLE 3
Organ weight, organ coeffi cient and AGD/body weight 1/3 on PND22 in DEHP and/or GEN-exposed male offspring rats Values are litter mean ± standard deviation.Organ weights are presented as absolute and relative (%) weights (organ coeffi cient).Games-Howell post-hoc test: *: S ignifi cantly different from control group at P<0.05, **: Signifi cantly different from control group at P<0.01, #: Signifi cantly different from DEHP250 at P<0.05; ÷: Signifi cantly different from GEN50 at P<0.05.