Prenatal exposure to zearalenone disrupts reproductive potential and development via hormone-related genes in male rats

Food and Chemical Toxicology 116 (2018) 11–19

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Prenatal exposure to zearalenone disrupts reproductive potential and development via hormone-related genes in male rats

T

Xin Gaoa, Zhuohui Xiaob, Chong Lia, Jiacai Zhanga, Luoyi Zhua, Lvhui Suna,c, Niya Zhanga,c, Mahmoud Mohamed Khalila,d, Shahid Ali Rajputa, Desheng Qia,c,∗ a

College of Animal Nutrition and Feed Science, Huazhong Agricultural University, Wuhan, Hubei, 430070, China CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, China c The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, Hubei, 430070, China d Animal Production Department, Faculty of Agriculture, Benha University, Egypt b

A R T I C LE I N FO

A B S T R A C T

Keywords: Zearalenone Prenatal exposure Reproductive and developmental toxicity Hormone-related genes Male rats

The present study investigated the reproductive and developmental toxicity of male offspring induced by prenatal ZEN exposure and explored the possible mechanism. 64 pregnant rats were allocated into four groups and fed with ZEN contaminated (0, 5, 10 and 20 mg/kg) diet during the whole gestation period. The results showed that, F1 male foetal viability was not affected while newborn bodyweight (BW) was significantly decreased after prenatal exposure to ZEN. Decreased BW was found on postnatal day (PND) 21 but not on PND 63 in ZEN exposed male rats. Moreover, adult testis weight increased with seminiferous tubules atrophy as well as decreased spermatocytes and mature sperms (35% and 31%) in ZEN-treated rats. Meanwhile, circulating levels of luteinizing hormone and testosterone decreased while estradiol increased in ZEN-treated rats. These impairments concurred with down-regulations of 3β-HSD and StAR in both mRNA and protein levels in weaned and adult testis. Furthermore, gene and protein expressions of GnRHr and Esr1 were inhibited in the ZEN-treated foetal brain. These results suggested that prenatal ZEN exposure disrupted the system regulating the reproductive hormones and testis development through hormone related genes, which may result in a reproductive dysfunction in adult male offspring.

1. Introduction Over the past few decades, the increasing incidence of reproductive disorders observed in vertebrates has raised concern about the role of substances known as environmental endocrine-disrupting chemicals (EDCs). The EDCs are referred to naturally occurring substances (i.e., phytoestrogens and mycoestrogens) or synthetic chemicals that can interfere with the body's endocrine system and exert various reproductive effects (Diamanti-Kandarakis et al., 2009; Salian et al., 2011). Exposure to EDCs and in particular to xenoestrogens, leads to an increasing incidence of reproductive system disorders in animals and humans (Delbès et al., 2006). Zearalenone (ZEN), an environmental xenoestrogen, is a non-steroid estrogenic mycotoxin produced by species of Fusarium fungi (Bennett and Klich, 2003; Richard, 2007), and is one of the most prevalent mycotoxins that contaminate staple food, especially cereals in human and animal diet (Zinedine et al., 2007).

High concentrations of ZEN (up to 600 mg/kg) in feed have been reported (Herrman and Walker, 1999; Kowalska et al., 2016; Zinedine et al., 2007). For humans, food ZEN contamination levels are usually in the range of μg/kg and low mg/kg, however, there are still serious ZEN contaminations in parts of Africa, Asia and North America, with the highest reported reaching 15 mg/kg (Sangare-Tigori et al., 2006; Zhao et al., 2013; Zinedine et al., 2007). Following oral administration, ZEN is rapidly absorbed from the gastrointestinal tract to the bloodstream and distributed to organs after passing through the liver. ZEN is mostly metabolized to α- and β-zearalenol (ZOL) in the liver by 3α and 3βhydroxysteroid dehydrogenases (HSDs), respectively (Deng et al., 2012; Fink-Gremmels and Malekinejad, 2007). ZEN and its metabolites can competitively bind to estrogen receptor due to the structural similarity with 17β-estradiol (E2), and activate the transcription of oestrogen-responsive genes in many organs, especially in the gonads (Bovee et al., 2004; Etienne and Dourmad, 1994; Metzler et al., 2010; Mirocha et al.,

∗

Corresponding author. College of Animal Nutrition and Feed Science, Huazhong Agricultural University, Wuhan, Hubei, 430070, China. E-mail addresses: [email protected] (X. Gao), [email protected] (Z. Xiao), [email protected] (C. Li), [email protected] (J. Zhang), [email protected] (L. Zhu), [email protected] (L. Sun), [email protected] (N. Zhang), [email protected] (M.M. Khalil), [email protected] (S.A. Rajput), [email protected] (D. Qi). https://doi.org/10.1016/j.fct.2018.04.011 Received 25 September 2017; Received in revised form 25 March 2018; Accepted 4 April 2018 Available online 05 April 2018 0278-6915/ © 2018 Elsevier Ltd. All rights reserved.

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1981). α-ZOL has been proved to be the dominant ZEN derivative in humans (Adewale et al., 2009; Mirocha et al., 1981), rats, mice (Bravin et al., 2009) and pigs (Kuiper-Goodman et al., 1987). ZEN and its metabolites' oestrogenicity cause several functional and morphological changes in the reproductive organs and lead to numerous reproductive problems in both female and male animals (Belli et al., 2010; Benzoni et al., 2008; Yang et al., 2007b). As well, ZEN could disturb the prenatal development of mice and the estrous cycle, as well as increase the number of anovulatory rats (Arora et al., 1981; Nikaido et al., 2003). Studies have reported that female is more susceptible to ZEN than male (Collins et al., 2006; Kuiper-Goodman et al., 1987). It has been well documented that female foetus exposure to ZEN causes follicle damage and premature oocyte depletion, therefore comprise a health risk for young offspring (Gao et al., 2017; Schoevers et al., 2012). However, the toxicity of ZEN on male should not be underrated, lower testicular weight and decreased motility of spermatozoa were found in boars and mice after continuous ingestion of low ZEN concentrations (Yang et al., 2007a; Young and King, 1986). Reduced fertility in the offspring may be the most obvious consequence of prenatal exposure to toxic environmental chemicals. It has been reported that male mice exposed prenatally to diethylstilbestrol had an excess prevalence of malformations of the genitalia and infertility (McLachlan, 1977; McLachlan et al., 1975). Likewise, ZEN and its metabolites can transfer into the foetus during gestation period by the placenta, mediate abnormalities in foetal growth and development, so that lead to impaired development, reduced litter size and foetal malformation (Kiessling and Pettersson, 1978; Young et al., 1990; Zhang et al., 2013). In human males, an excess rate of minor malformations of the genitalia has been associated with prenatal exposure to xenoestrogens (Gill, 1988). Furthermore, foetus growth was restricted in rats with detectable ZEN residual placentas, whereas normal with no ZEN residues (Zhang et al., 2013). Despite the numerous studies about ZEN toxicity in male animals, most investigations were focused on the direct ZEN exposure toxicity, and little was known about the roles of prenatal ZEN exposure on the male reproduction. Therefore, the present study was conducted to evaluate whether prenatal exposure to ZEN was associated with reduced reproductive potential and disrupted development on male offspring. By observing testis structure, sexual hormones secretion and expressions of related genes and proteins, the detailed toxicity of prenatal ZEN were investigated in male rats.

(Collins et al., 2006); 10 and 20 mg/kg ZEN were 2 and 4 times of NOEL, which were also based on the levels reported in contaminated foods (Panel, 2011; Zinedine et al., 2007). The pregnant rats were fed on a regular diet containing no ZEN during the lactation phase of the study. Eight pregnant rats from each group were sacrificed on GD 20 by cervical dislocation in order to collect placentas and foetuses (n = 12), while the other dams (eight of each group) were allowed to deliver and care for their pups. At birth, each pup was sexed, weighed, and identified. The litter size was balanced to 8 with half males and females. All pups were breastfed, weaned on a postnatal day (PND) 21. Only male rats were investigated in the study. Two weaning rats of each dams were sacrificed by cervical dislocation (n = 16). The remaining F1 male rats were left to be sexually mature and slaughtered (n = 16) at PND 63 (9 weeks). Brains and testis of weaned and adult rats were sampled. Blood samples of adult F1 rats were immediately centrifuged following collection. All the samples were labeled and frozen at −80 °C until further analysis. 2.2. Testis histopathological analyses and germ cells quantitative analyses The testis was sampled and fixed in 10% neutral-buffered formalin for 48 h. Then, the organs were processed for paraffin embedding, sectioned (thickness, 5 mm) and stained with hematoxylin and eosin (H &E) (Nikaido et al., 2004). The slides were observed under 100× or 200× magnification using an optical microscope (Nikon, Tokyo, Japan). A quantitative analysis of germ cells in each seminiferous tubule was carried out with Image J 1.49 (Clermont and Morgentaler, 1955; Li et al., 2017). The number of germ cells (including sertoli cell, spermatogonia, spermatocyte, spermatid and mature sperm) were counted in five fields per testis (n = 8) based on H&E staining, and the results were expressed as mean number of cells per seminiferous tubule. 2.3. Hormone levels detection E2 (estradiol), LH (luteinizing hormone) and T (testosterone) levels of F1 adult male rats were determined using enzyme-linked immunosorbent assay kits (R&D Systems, Inc., Minneapolis, MO, USA) according to the manufacturer's recommended protocols. 2.4. Quantification of ZEN and its metabolites

2. Materials and methods All procedures were in accordance with the National Research Council Guide (Clark et al., 1996), and approved by the Scientific Ethics Committee of Huazhong Agricultural University. The project identification code is HZAURA-2015-006.

The preliminary treatment was conducted according to the procedures outlined by (Koraichi et al., 2012). ZEN and metabolites concentrations were determined by high performance liquid chromatography (HPLC) according to (Zöllner et al., 2002) and (Koraichi et al., 2012) with modifications.

2.1. Animals and treatments

2.5. Total RNA extraction and real-time quantitative PCR

ZEN was purchased from Sigma-Aldrich (St. Louis, MO, USA). Sprague-Dawley (SD) rats were obtained from Wuhan Centers for Disease Prevention & Control (Wuhan, China). The weights of the female rats ranged from 200 to 210 g. After a period of acclimating, they were mated overnight with males of the same strain. Pregnant rats at gestational day (GD) 0 were individually housed under controlled and standardized conditions, with 12-h light-dark cycles (0700–1900 light). A total of 64 pregnant rats were divided into 4 groups (control, 5 ZEN, 10 ZEN, 20 ZEN), and received diets containing different concentrations of ZEN (0, 5, 10 and 20 mg/kg) through gestational days (GD 0–20). Water and feed were provided ad libitum during the experiment. Body weight (BW), average daily intake (ADI) and average daily gain (ADG) were recorded and calculated. The rationale for the dose selection: 5 mg/kg ZEN diet was about the dose of 0.5 mg/kg/day, it's the no-observed effect level (NOEL) of ZEN in rats

Total mRNA was extracted from tissues with Trizol® (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The quality and concentration of RNA were estimated by nucleic acid concentration analyzer (NanoDrop 2000; Thermo Fisher, Waltham, MA, USA). 1 μg of total RNA was reverse-transcribed in a 20 μL reaction using a PrimeScript™ RT reagent Kit (Takara, DRR037A). cDNA was stored at −20 °C until use in real-time quantitative PCR. Expression levels of 7 genes: Gonadotropin-releasing hormone receptor (GnRHr), estrogen receptor alpha (Esr1), 3β-hydroxysteroid dehydrogenases (3β-HSD), steroidogenic acute regulatory protein (StAR) and ATP Binding Cassette Transporters b1 (ABCb1), ABCc1, ABCc5 were analyzed using Real-time q-PCR (CFX384, Bio-Rad) as described by (Huang et al., 2015). The primer sequences used are presented in (Supplementary Table 1). A 2−ddCt method was used for the quantification with glyceraldehyde 3-phosphate dehydrogenase 12

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3.2. Testicular histological analyses and germ cells quantitation

(GADPH) as a reference gene, and the relative abundance was normalized to the control (as 1).

The histological analysis revealed pathological changes in the testis of adult F1 rats. For weaned testis (Fig. 2 A: a-d), all groups showed complete organizational structures without significant differences. For adult testis, Control and 5 ZEN groups showed a clearly visible structure of stromal cells and seminiferous tubules, a large number of mature sperms were also noted in seminiferous tubules (Fig. 2 A: e, f); in 10 ZEN samples, seminiferous tubules atrophy appeared (Fig. 2 A: g). For 20 ZEN group, decreased spermatocytes and mature sperms (35% and 31%) were observed compared with control (Fig. 2 B), given some of the seminiferous tubules an ‘empty’ appearance. Meanwhile, scarce array and reduced intercellular connections were also found (Fig. 2 A: h). The reduced intercellular connections and scarce array were mainly observed in surrounding spermatogonia and spermatocytes (especially primary spermatocytes).

2.6. Western blotting analyses Protein expressions of GnRHr and Esr1 in F1 brains, and 3β-HSD and StAR in F1 testis were determined. The following reagents used for this study were purchased from the indicated sources: antibodies against β-Actin (Cell Signaling Technology #4967/1:2000, Boston, MA, USA), GnRHr (Abcam ab202848/1:1000, Cambridge, MA, USA), Esr1 (Abcam ab32063/1:1000, Cambridge, MA, USA), 3β-HSD (Santa Cruz sc-30820/1:500, Santa Cruz, CA, USA), StAR (Abcam ab96637/1:1000, Cambridge, MA, USA). Secondary horse-radish peroxidase labeled antibodies were goat anti-rabbit (Sigma-Aldrich, A9169/1:10,000), goat anti-rabbit (Santa Cruz sc-2004/1:6000, Santa Cruz, CA, USA) or donkey anti-goat (Santa Cruz, sc-2020/1:2000, Santa Cruz, CA, USA). Western blot analysis was performed as previously described (Xie et al., 2012). The bands were detected by a chemiluminescence WesternBright™ ECL Substrate kit (Advansta, Menlo Park, CA, USA), then visualised and quantified by Tanon-5200 Chemiluminescent Imaging Analysis System (Tanon Science & Technology, Shanghai, China).

3.3. Hormone concentrations of adult male rats To detect the effect of prenatal ZEN exposure on hormone levels, the concentrations of LH, E2, and T in the serum of F1 adult male rats on PND 63 were detected (Table 3). 20 ZEN-treated rats had significantly higher levels of serum E2 compared with the control group. Moreover, a statistically significant decrease of LH (20 ZEN) and T (10 and 20 ZEN) was noted.

2.7. Statistical analysis Statistical analysis was carried out using IBM SPSS Statistics 19 (IBM Corporation, Armonk, New York, NY, USA). Results were presented as means ± SD. We employed Student's t-test and one-way ANOVA followed by Duncan's post hoc test to access the differences between experimental groups and control on each variable within the same tissue. Values were considered significant at P < 0.05.

3.4. ZEN residues in tissues The quantification of ZEN and its metabolites in placenta, foetal brain, foetal liver as well as brain and testis of weaned and adult male rats were performed. In placentas, ZEN and α-ZOL residues increased significantly in a dose-dependent manner, as shown in (Table 4). Meanwhile, ZEN residues were also detected in 20 mg/kg ZEN-treated foetal brain and foetal liver. However, β-ZOL was not detectable in all samples. For weaned or adult rats, none of ZEN or its metabolites was detectable in brain, liver and testis.

3. Results 3.1. F1 male viability and growth performance

3.5. Gene expressions of hormone-related genes and ABC transporters As shown in Fig. 1, 20 ZEN-treated dams gave birth to offspring with significantly lower viability compared with the control. However, the litter size and male viability did not show statistical differences between control and ZEN-exposed animals. Meanwhile, significant (P < 0.05) lower BWs were observed in both neonatal and weaned male offspring receiving 20 mg/kg ZEN compared with control (Table 1). Although ADI, ADG and the adult BW showed no statistical difference, a dose-dependent decrease tendency was noticed. The relative testis and epididymis weights of adult rats were recorded (Table 2). The result showed that maternal ZEN administration of 20 mg/kg significantly increased (P < 0.05) adult testis weights, while did not affect epididymis weights.

In the male foetal brain, significant down regulation was found for GnRHr, Esr1 and ABCc5 in 10 and/or 20 ZEN treatments. No significant effect was observed for 3β-HSD, ABCb1 and ABCc1 mRNAs (Fig. 3A). In the brain of weaned males, a higher up modulation (more than 2-fold) of 3β-HSD was noted in 20 ZEN group, meanwhile, down regulations were observed for ABCb1 and ABCc5 mRNAs in both 10 and 20 ZEN treatments (Fig. 3B). For weaned testis, significant up-regulation was noted in Esr1 mRNA in 5 and 20 ZEN groups, however, significant inhibition in the expressions of 3β-HSD, StAR and ABCc5 mRNAs was found in 10 and/or 20 ZEN-treated animals (Fig. 3C). In the brain of adult males, no significant effect was observed for all genes detected (Fig. 3D). For adult testis, while no meaningful effect was observed for Esr1 and ABC transporters mRNAs, a significant inhibition (0.6-fold) of 3β-HSD and StAR mRNA was noted in 20 ZEN group (Fig. 3E). 3.6. Western blotting analysis of hormones-related proteins in F1 brains and testis To investigate whether the brain hormone receptors were affected by prenatal ZEN exposure, we examined the expression of GnRHr and Esr1 in brains of the F1 foetus, weaned and adult male rats (Fig. 4). Prenatal ZEN treatment at 20 mg/kg significantly inhibited the GnRHr protein expression but not affect Esr1 protein expression in foetal brain. Meanwhile, no significant changes were found for GnRHr and Esr1 protein expressions in the brain of weaned and adult F1 male rats. In weaned testis (Fig. 5 A, B), 3β-HSD protein expressions were 1.5-fold inhibited (P < 0.05) in 20 ZEN group, meanwhile, StAR were almost 2-

Fig. 1. Prenatal exposure of ZEN results in reduced foetuses viability. Data are means ± SD of n = 8 rats per treatment. *P < 0.05 compared with control. Ctrl, 0 ZEN diet; 5 ZEN, 5 mg/kg ZEN diet; 10 ZEN, 10 mg/kg ZEN diet; 20 ZEN, 20 mg/kg ZEN diet. 13

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Table 1 Body weights and feed consumptions of F1 male rats. Parameters

Treatments

Neonatal BW Weaned BW(d 21) Adult BW(d 63) ADI, g/d ADG, g/d

Control

5 ZEN

10 ZEN

20 ZEN

5.68 ± 0.53 40.26 ± 9.71 251.11 ± 24.33 20.65 ± 1.32 5.15 ± 0.36

5.47 ± 0.34 34.98 ± 1.73 250.98 ± 24.40 21.06 ± 1.51 5.10 ± 0.80

5.36 ± 0.60 36.73 ± 6.37 242.44 ± 16.35 20.15 ± 1.00 5.08 ± 0.66

4.64 ± 0.54∗ 26.70 ± 7.13∗ 223.03 ± 22.44 19.88 ± 1.45 4.70 ± 0.48

Values are means ± SD, n = 16. Statistically significant *P < 0.05 compared with control. BW, body weight; ADI, average daily intake; ADG, average daily gain; ZEN, Zearalenone. Table 2 Relative organ weights (%) of adult F1 male rats on PND 63. Parameters

Treatments Control

Testis, g Epididymis, g

Table 3 Concentrations of LH, E2, and T in the serum of F1 rats on PND 63.

1.22 ± 0.11 0.34 ± 0.03

Parameters 5 ZEN 1.21 ± 0.10 0.35 ± 0.04

10 ZEN 1.34 ± 0.05 0.35 ± 0.05

20 ZEN ∗

E2, ng/L LH, IU/L T, ng/ml

1.45 ± 0.25 0.37 ± 0.02

Values are means ± SD, n = 16. Statistically significant *P < 0.05 compared with control. PND, postnatal day; ZEN, Zearalenone.

Treatments Control

5 ZEN

10 ZEN

20 ZEN

27.64 ± 1.61 3.65 ± 0.31 5.72 ± 0.25

27.87 ± 1.16 3.21 ± 0.27 5.40 ± 0.06

29.49 ± 0.54 3.23 ± 0.52 5.14 ± 0.18∗

31.83 ± 1.99∗ 2.42 ± 0.34∗ 5.10 ± 0.10∗

Values are means ± SD, n = 10. Statistically significant *P < 0.05 compared with control. E2, estradiol; LH, luteinizing hormone; T, testosterone; ZEN, zearalenone.

Fig. 2. Prenatal ZEN exposure induced testicular morphological changes and reduced germ cells in adult rats. The testis sections were stained with hematoxylin and eosin; photomicrographs are shown at 100 × or 200 × magnification. Empty seminiferous tubules (arrowhead) and reduced intercellular connections (black arrows) were observed. Weaned testis: (A: a-d); adult testis: (A: e-h). The number of cells per seminiferous tubule were counted in five fields per testis (n = 8) based on H & E staining and quantification in (B). Data represent the means ± SD. *P < 0.05 compared with control. ZEN, zearalenone; Ctrl, 0 ZEN diet; 5 ZEN, 5 mg/kg ZEN diet; 10 ZEN, 10 mg/kg ZEN diet; 20 ZEN, 20 mg/kg ZEN diet. 14

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Table 4 Residues of ZEN and its metabolites in different tissues of rats (n = 8). Treatments

ZEN and its metabolites concentration (ng/g) Placenta

Control 5 ZEN 10 ZEN 20 ZEN

Foetal brain

ZEN

α-ZOL

0.00 ± 0.00 1.67 ± 0.51∗ 7.41 ± 2.83∗∗ 14.07 ± 4.25∗∗∗

0.00 0.00 2.45 4.06

± ± ± ±

0.00 0.00 0.88∗ 1.05∗∗

Foetal liver

Weaned brain

ZEN

α-ZOL

ZEN

α-ZOL

ZEN

α-ZOL

ND ND ND 1.29 ± 0.57

ND ND ND ND

ND ND ND 0.79 ± 1.34

ND ND ND ND

ND ND ND ND

ND ND ND ND

Data are means ± SD. Statistically significant *P < 0.05; **P < 0.01; ***P < 0.005, compared with Control. ND, not detectable; ZEN, Zearalenone; α-ZOL, αzearalenol.

A previous study in male rats reported that direct ZEN exposure could result in testicular lesions and increase testis weights (Yang et al., 2007a), which was also found in the present work. Our work showed a significant increase in testis weight in 20 mg/kg ZEN-treated F1 adult male rats, accompanied by histological changes, including decreased number of spermatocytes and sperms and testicular interstitial expansion. Meanwhile, our results indicated spermatogonia and spermatocytes (especially spermatocytes) were more likely affected by ZEN exposure, which was also in consistent with previous research in murine (Jee et al., 2010; Kim et al., 2003). Interestingly, these pathological changes were not observed in F1 weaned testis. Furthermore, the ZEN residues were detectable in maternal placentas and foetal brain and liver. These results indicate that ZEN can be transferred into the foetal brain through the blood–brain barrier and may cause negative effects on early brain development. However, no ZEN was detectable in brain or testis of weaned and adult F1 rats. The possible explanation is that the effects of prenatal ZEN exposure manifest during puberty, in which the activation of the sex physiology and sexual hormone secretion typically occurs (Zoeller et al., 2012). The deleterious effects of ZEN on regulation of hormone synthesis, secretion, and hormone receptors could contribute to the reproductive anomalies in adulthood more than

fold inhibited (P < 0.05) in both 10 and 20 ZEN groups. Similarly, in adult testis (Fig. 5 C, D), 3β-HSD and StAR protein expressions were both inhibited significantly (P < 0.05) by 20 mg/kg ZEN (3.1 and 2.2fold inhibition, respectively). 4. Discussion In the present study, we investigated the effects of prenatal ZEN exposure on the development and reproductive potential in male offspring in rats for the first time. Previous reports indicated that endocrine disruptors administration to pregnant animals can exert detrimental effects during pregnancy (Manikkam et al., 2012). In the present study, prenatal ZEN exposure during the whole gestation caused a significant decrease in newborn viability and newborn male BW in rats fed the 20 mg/kg ZEN diet. Moreover, gestational ZEN exposure also decreased the weaned BW (PND21) of male offspring. Although no significant decrease was found in F1 male BW (PND63), the ADI and ADG decreased in a ZEN dose-dependent manner. These results indicated that prenatal ZEN exposure could influence the foetal development and result in foetal mortality, and may affect the following development of offspring in rats (Zhang et al., 2013; Zhao et al., 2013).

Fig. 3. Effects of prenatal ZEN exposure on relative mRNA abundance of hormones-related genes or ABC transporters in foetal, weaned and adult F1 tissues. Values are means ± SD, n = 12. Statistically significant *P < 0.05; **P < 0.01; ***P < 0.005. ZEN, zearalenone; Esr1, oestrogen receptor-alpha; GnRHr, gonadotropinreleasing hormone receptor; 3β-HSD, 3β-hydroxysteroid dehydrogenase; StAR, Steroidogenic acute regulatory protein; ABCb1, ATP binding cassette transporters b1; ABCc1, ATP binding cassette transporters c1; ABCc5, ATP binding cassette transporters c5. (A) foetal brain; (B) weaned F1 brain; (C) weaned F1 testis; (D) adult F1 brain; (E) adult F1 testis. 15

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Fig. 4. Effects of prenatal ZEN exposure on protein expressions of GnRHr and Esr1 in brains of the F1 foetus, weaned and adult male rats. For a given protein, the band was a representative image of 3–6 independent analyses, and the value shows the relative density of protein bands (mean ± SDs, n = 3–6). Statistically significant *P < 0.05 compared with control. ZEN, zearalenone; GnRHr, Gonadotropin-releasing hormone receptor; Esr1, oestrogen receptor alpha; (A) GnRHr and Esr1expressions in F1 foetal brain; (B) GnRHr and Esr1expressions in weaned brain; (C) GnRHr and Esr1 expressions in adult brain.

glucuronide estrodiol is a potent inhibitor of cGMP transport (Jedlitschky et al., 2000; Sundkvist et al., 2002). While the metabolites of ZEN (α-ZOL and β-ZOL) can be further transformed into glucuronide conjugates by uridine diphosphate glucuronyl transferases (UDPGT) (Biehl et al., 1993; Mirocha et al., 1982). Due to the structural similarity of ZEN and 17β-estrodiol, the ZEN glucuronide conjugate may also inhibit the cGMP transport which could induce the downregulation of ABCc5. Inhibition of the ABCC5 may cause developmental retardation of embryos (Long et al., 2011). Disruption of the testicular ABCc5 also has the potential to impair male gonadal function and be detrimental to male reproductive performance (Bloise et al., 2015). However, no difference of ABCc5 was found in adult tissues. This may because ABCc5 is highly expressed in foetal but little in adult tissues (McAleer et al., 1999). For ABCb1 and ABCc1, no significant difference was found in all analyzed tissues except a decrease of ABCb1 in the weaned brain. The possible explanation is that the effect of ZEN on mRNA expression of ABC transporters is varying according to the type of tissues (Heneweer et al., 2007; Koraichi et al., 2012). Besides the study on major ABC transporters, we explored the effects of ZEN exposure on mRNA expression of hormone related genes. The estrogenic activity of ZEN and its metabolites stems from their 17β estradiol (E2)-like conformation therefore binding to estrogen receptors (Esrs) (Shier et al., 2001). It is reported that ZEN could inhibit testosterone biosynthesis in mouse Leydig cells via the crosstalk of estrogen receptor signaling (Liu et al., 2014). ZEN can also disturb the expression of GnRHr in gilts' hypothalamus, which plays an important part in hypothalamic pituitary gonadal (HPG) axis (Yang et al., 2007b). Our previous study showed alerted Esr1 and GnRHr genes expression after ZEN administration in both hypothalamus and ovaries in prepubertal gilts as well as female rats (Gao et al., 2017; Wang et al., 2010). In the present study, ZEN was able to down-regulate the mRNA expressions of GnRHr and Esr1 in the male foetal brain, and the protein expressions were also in good agreement with those of mRNA expression even though in lower amplitudes. This may be associated with the toxicity effect of ZEN residues in the male foetal brain, which indicates a risk of a hormone disorder. Furthermore, as an endocrine-disruptor, ZEN can interfere with steroid metabolism by altering enzyme activity of 3β-HSD and StAR, which are key enzymes involved in the biosynthesis of gonadal steroid hormones (Bravin et al., 2009). Soma et al. have reported that estrogen treatment can increase the mRNA expression of 3β-HSD and activity in rat

childhood. Likewise, the effects of EDCs were also often delayed and thus may not be manifested until adulthood, even though critical exposure occurred during early embryonic, or foetal life (Colborn et al., 1993; Otrocka-Domagala et al., 2003). Similar delayed effects were reported in adult women with prenatal diethylstilbestrol (DES) exposure (Gore, 2008). Thus, the adverse effects of EDCs exposures during development may require an extended period to be manifested, some of which do not become manifested until adulthood (Gore and Patisaul, 2010). ZEN and its metabolites can act as potential endocrine disruptors at the level of nuclear receptor signaling by altering the sexual hormone production, including testosterone production (Frizzell et al., 2011). In the adult, testosterone supports spermatogenesis, sperm maturation, and sexual function, the disruption of testosterone biosynthesis can adversely affect male fertility (Ewing and Keeney, 1993). In the present study, ZEN significantly decreased LH and testosterone in adult F1 male rats, which were vital in the development of testis and spermatogenesis (Singh and Handelsman, 1996; Singh et al., 1995). Also, the inhibition of LH in our work is in agreement with a previous study in which ZEN suppressed GnRH-stimulated LH secretion in bovine anterior pituitary cells (Nakamura and Kadokawa, 2015). Previous studies point that estradiol plays a suppressive role in Leydig cells development by inhibiting androgens production in mice (Delbès et al., 2005; Jin et al., 2005). The results presented in this study also showed significantly increased serum E2 in ZEN treated rats, indicating that prenatal exposure of ZEN may inhibit the testosterone production by affecting E2 secretion via Esr1, therefore, developing negative effects on testis development and reproductive ability. The ABC transporters are known to be helpful in protecting tissues from xenobiotic accumulation and thus resulting toxicity (Weaver et al., 2005; Wijnholds et al., 2000), thus, the effects of ZEN exposure on mRNA expression of major ABC transporters were explored in the present study. Attention was paid to ABCc5 because it is reported to be essential in the development, maintenance of normal physiological functions, and transport of toxicants in brain and testis (Long et al., 2011; Middendorff et al., 2000). In our work, a significant decrease in the expression of ABCc5 genes was observed in male foetal brain and weaned brain and testis. ABCc5 has been considered to play important roles in intracellular signaling by exporting cyclic guanosine monophosphatec (cGMP) (Jedlitschky et al., 2000). It's reported that 17β16

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Fig. 5. Effects of prenatal ZEN exposure on protein expressions of 3β-HSD and StAR in testis of F1 weaned and adult male rats. For a given protein, the band was a representative image of 3–6 independent analyses, and the value shows the relative density of protein bands (mean ± SDs, n = 3–6). Statistically significant *P < 0.05 compared with control. ZEN, zearalenone; 3β-HSD, 3β-hydroxysteroid dehydrogenase; StAR, steroidogenic acute regulatory protein; (A) 3β-HSD expressions in F1 weaned testis; (B) StAR expressions in weaned testis; (C) 3β-HSD expressions in adult testis; (D) StAR expressions in adult testis.

5. Conclusion

hypothalamus (Soma et al., 2005). Likewise, other studies also have reported the increase of 3β-HSD expression in brain tissues in response to exogenous toxins, which is considered an adaptive response by the brain to lessen the extent of damage (Sadasivam et al., 2014; Saldanha et al., 2009). Thus, the increase of 3β-HSD in rat brain may be a neuroprotective action to ZEN treatment. It's reported that LH stimulates the synthesis of steroidogenic enzymes, thereby regulating testosterone production, while StAR is required in the regulatory mechanism (Tsuchiya et al., 2003). In agreement with the results in mouse Leydig cells in vitro (Yang et al., 2007a), the present study showed a significant decrease in 3β-HSD and StAR expressions in both mRNA and protein levels in weaned and adult testis. The down-regulation of two enzymes strongly indicate poor testosterone production in male rats, eventually leading to a great decline in reproductive function (Liu et al., 2012).

In summary, the present study showed that prenatal exposure to ZEN could delay the male foetal development and induced long-term toxicity on male offspring, including disrupted circulating concentrations of sexual hormones (E2, LH and T) and impaired testis development. These reproduction dysfunctions observed in ZEN-treated male offspring are closely related to altered GnRHr and Esr1 expressions in the foetal brain and down-regulation of 3β-HSD and StAR expressions in developing testis. Humans are easily exposed and more susceptible to ZEN contaminated food, gestational ZEN exposure may have significant implications for human health and fertility, especially during development, and in sensitive populations. Noteworthy, the transgenerational effect of ZEN might be enhanced by other oestrogenic food 17

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contaminants such as phthalates, bisphenol A and pesticides through dietary intake. The present study may raise the awareness of the potential adverse health consequences in children's later life for those pregnant women exposed to ZEN by dietary intake during their gestation period. With the evidence from this work, further study is ongoing to characterize the mechanisms by which ZEN may contribute to the hormones disorder and related genes down-regulation.

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