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Atrazine triggers developmental abnormality of ovary and oviduct in quails (Coturnix Coturnix coturnix) via disruption of hypothalamo-pituitary-ovarian axis
Environmental Pollution 207 (2015) 299e307
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Atrazine triggers developmental abnormality of ovary and oviduct in quails (Coturnix Coturnix coturnix) via disruption of hypothalamo-pituitary-ovarian axis Lei Qin a, Zheng-Hai Du a, Shi-Yong Zhu a, Xue-Nan Li a, Nan Li b, Jing-Ao Guo a, Jin-Long Li a, *, Ying Zhang c, ** a b c
College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, PR China National Research Insitiute for Family Planning, Beijing, 100081, PR China School of Resources & Environment, Northeast Agricultural University, Harbin, 150030, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 August 2015 Received in revised form 16 September 2015 Accepted 20 September 2015 Available online 29 September 2015
There has been a gradual increase in production and consumption of atrazine (ATR) in agriculture to meet the population rising demands. Female reproduction is necessary for growth and maintenance of population. However, ATR impact on females and particularly ovarian developmental toxicity is less clear. The aim of this study was to define the pathways by which ATR exerted toxic effects on ovarian development of ovary and hypothalamo-pituitary-ovarian (HPO) axis. Female quails were dosed by oral gavage from sexual immaturity to maturity with 0, 50, 250 and 500 mg ATR/kg/d for 45 days. ATR had no effect on mortality but depressed feed intake and growth and influenced the biochemical parameters. Notably, the arrested development of ovaries and oviducts were observed in ATR-exposed quails. The circulating concentrations of E2, P, LH and PRL were unregulated and FSH and T was downregulated in ATR-treated quails. The mRNA expression of GnRH in hypothalamo and LH in pituitary and FSH in ovary was downregulated significantly by ATR exposure and FSH and PRL in pituitary were upregulated. ATR exposure upregulated the level of P450scc, P450arom, 3b-HSD and 17b-HSD in ovary and downregulated ERb expression in female quails. However, ATR did not change ERa expression in ovary. This study provides new insights regarding female productive toxicology of ATR exposure. Ovary and oviduct in sexually maturing females were target organs of ATR-induced developmental toxicity. We propose that ATR-induced developmental abnormality of ovary and oviduct is associated with disruption of gonadal hormone balance and HPO axis in female quails. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Atrazine Ovarian and oviduct developmental abnormality Endocrine disrupter Quail Hypothalamo-pituitary-ovarian axis
1. Introduction The triazine herbicide ATR is widely used in agricultural to control the unwanted growth of grasses and broadleaf weeds. Being one of the most commonly used pesticides in the world (Hayes et al., 2010), ATR is widespread in the environment and a frequently detected contaminant in waterways. Like BPA and other chemicals, there are scientific indications that ATR has endocrinedisrupting potential (Hayes et al., 2010; Jin et al., 2014; Thompson et al., 2015), causing mammary gland tumors in rodents (Cooper
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (J.-L. Li), [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.envpol.2015.09.044 0269-7491/© 2015 Elsevier Ltd. All rights reserved.
et al., 2007), and altering male reproduction (Stanko et al., 2010). Despite ATR has been banned in European Union and been restricted in other countries, it is still being used in large quantities worldwide up to now. It is one of the most widely used agricultural pesticides in United States (Barr et al., 2007), and its application in Asian countries has been growing. There has been a gradual increase in production and consumption of ATR in agriculture to meet the population rising demands. Female reproduction is necessary for growth and maintenance of population. Therefore, humans and wildlife are at risk for exposure to ATR. ATR has been considered as an endocrine disruptor due to alterations caused on hormoneregulated systems in various taxa (De La Casa-Resino et al., 2012; Hayes et al., 2011; McMullin et al., 2004; Salaberria et al., 2009). However, little is known about its effects on the development of reproductive organ in females.
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In recent decades, there has been an increasing concern on clarifying the toxicological mechanisms of environmental chemicals to cause alterations in the reproductive system of humans and animals. ATR like other herbicides induces endocrine disruption and consequently interferes with various hormones physiological functions. Numerous reports have suggested that ATR might have adverse effects on the reproductive function (Cooper et al., 2007; Feyzi-dehkhargani et al., 2012; Friedmann, 2002; Kniewald et al., 2000; Stoker et al., 1999; Swan, 2006; Solomon et al., 2008; Trentacoste et al., 2001). In the male offspring a slower maturation of gonadotrophic system due to chronic expoxure to ATR was observed. In female, it has been shown that ATR could inhibit the development of ovary and oviduct in rats and cause infertility (Goldman et al., 2013; Zhao et al., 2014). It has also been reported that ATR could induce endocrine disruption and consequently interfere with physiological functions of various hormones (Jin et al., 2014; Weber et al., 2013). Consistent with these observations, Friedmann (2002) demonstrated that ATR had significantly reduced the serum and testicular testosterone (T) levels, both in acute toxicity test and chronic toxicity test, in juvenile SpragueeDawley male rats by gavage. A recent study demonstrated that the serum levels of T, follicle stimulating hormone (FSH), luteinizing hormone (LH), and inhibin-B (INHeB) had decreased by 85% after 48 d exposure to high dose (300 mg/kg BW/day) of ATR in adult male Wister rats (Feyzi-dehkhargani et al., 2012). However, there is still little knowledge on the reproductive and developmental toxicity in female animals induced by ATR. It is well known that ATR may impact endocrine activity and notably alter androgen/estrogen balance. ATR is a serious health concern, and numerous studies have been devoted to studying the effects of ATR on steroidogenic enzymes influenced steroid secretion and thus lead to reproductive toxicity (Basini et al., 2012; Payne and Youngblood, 1995; Pogrmic et al., 2009; Pogrmic-Majkic et al., 2010; Quignot et al., 2012b). In females, sex steroids are synthesized primarily in the ovaries and derived from cholesterol through a series of biochemical reactions (Foradori et al., 2013; Goldman et al., 2013; Henare et al., 2012; McMullin et al., 2004). However, the effects of ATR on the development of ovary and oviduct and the tuned balance between estrogens and androgens are not yet well clear. Assessing ATR reproductive toxicity in female animals is a challenge, given the complexity of the endocrine system and despite the increasing development of data on its workings. To explore the effects and mechanism of hormonal balance disruption and the developmental abnormality of ovary and oviduct caused by ATR, female quails (Coturnix Coturnix coturnix) were employed as the experimental model orally given ATR daily from sexual immaturity to maturity. The aim of this study was to define the pathways by which ATR exerted the effects on the hypothalamo-pituitaryovarian (HPO) axis and the developmental of ovary and oviduct, clarify the mechanism of ATR-induced toxicity in female animals. 2. Materials and methods 2.1. Animals and treatments Female European quail (Coturnix C. coturnix) chicks aged 18 days and weighted 86.7 ± 6.4 g were purchased from Wan Jia farm in Harbin, China. Chemical ATR (C8H14ClN5, CAS: 1912-24-9, 90% purity) was from Zhonghe Chemical Limited Company (Binzhou, China). Birds were housed in cages in an environmentally controlled room (temperature 24e28 C and fluorescent lights provided a photoperiod of 12 h light and 12 h dark). Feed and water were offered ad libitum during the experiment. The birds were administered ATR once a day orally by gavage for 45 days. The
gavage volume was 0.5 mL and the amount of gavage was adjusted everyday according to the varied weight of each quail. After oneweek acclimation, the quails were randomly divided into four groups i.e., Group 1 (Control) treated with 0 mg/kg BW/day ATR, Group 2 (50 mg/kg ATR) treated with 50 mg/kg BW/day ATR, Group 3 (250 mg/kg ATR) treated with 250 mg/kg BW/day ATR, Group 4 (500 mg/kg ATR) treated with 500 mg/kg BW/day ATR. The ATR dose employed in the present investigation was chosen on basis of recent studies by Hussain et al. (2011) and Wilhelms et al. (2005). The current ecological risk assessment for ATR in avian species established by the USEPA reports a dietary LOAEL (lowest observable adverse effect level) of 675 mg/kg in the northern bobwhite quail, ATR exhibits modest reproductive toxicity in female birds (USEPA, 2002). In this study, all the experiments conducted in animals were in accordance with the guidance of ethical committee for research on laboratory animals. The birds were monitored daily for clinical signs and total body weights gains. At the end of the experiment, birds were fasted before the day of sacrifice, and their hypothalamo, pituitary, ovary and oviduct were carefully dissected out. The blood was collected from the heart of each bird and centrifuged at 3000 rpm for 10 min to obtain the serum. The serum were divided into two portions, one for biochemical analyses and a second to be stored at 80 C for assays. 2.2. Determination of biochemical parameters Blood samples were used to investigate changes in the serum enzymes and concentration of ions considered to be biochemical indicators of hepatobiliary, renal and myocardial enzyme. Serum Ca, Mg, P and glucose (Glu) concentrations were measured. Both the activities of creatine kinase (CK), lactate dehydrogenase (LDH), choline esterase (CHE), contents of creatinine (Cre), blood urea nitrogen (BUN), total protein (TP), albumin (ALB), total bilirubin (T.BILT) and direct bilirubin (D.BILT) were measured. The activities or contents of biochemical parameters were detected using the detection kits (Jiangsu SINNOWA Medical Technology Company, China) by a biochemical auto-analyzer. 2.3. Histopathological studies Oviducts and ovaries were washed in cold saline and soak dried on filter paper. A portion of organ was fixed in 10% buffered formalin and embedded in paraffin. Sections of 5 mm thickness were cut and stained with hematoxylin and eosin for microscopic examination. 2.4. Hormone analysis To identify ATR-induced changes in circulating concentrations of reproductive hormones, serum concentrations of E2, P, FSH, LH, PRL and T were determined. All six reproductive hormone were determined using 125I Radioimmunoassay (RIA) Kits (HAT CO. LTD. China) according to the manufacturer's protocol. Radioactivity was determined using an automatic gamma counter. All samples were run in duplicate in a single assay to avoid interassay variation. 2.5. RNA purification and quantitative real-time PCR Total mRNA was extracted from hypothalamo, pituitary and ovary using RNAout reagent (Beijing Tiandz, Inc. China), according to the manufacturer's instructions. First cDNA strand was synthesized using Oligo (dT) primers and Transcript Reverse Transcriptase (Beijing TransGen Biotech Co. Ltd., China). The primers for real-time amplification of relative cDNAs were designed using Oligo 7.22
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Table 1 Average of body weight gain (BWG) in different tests and control group. Groups
B.W.G 1d
Control 50 mg/kg 250 mg/kg 500 mg/kg
81.28 79.58 82.28 79.86
9d ± ± ± ±
3.38 8.72 2.65 5.06
117.78 130.46 123.40 114.86
18d ± ± ± ±
3.46 8.72* 7.30 8.42
156.96 162.10 137.98 121.66
27d ± ± ± ±
11.66 17.50 11.52 8.68***
183.60 189.40 159.30 139.36
36d ± ± ± ±
5.85 4.63 3.82*** 12.81***
202.40 209.04 177.56 147.24
45d ± ± ± ±
6.09 10.05 17.12** 10.08***
209.86 215.96 180.58 151.80
± ± ± ±
10.92 12.91 16.07** 3.02***
Overall changes in body weight gain and average growth rate in quails. All data are presented as mean ± SD. Compared with controls: *P < 0.05. **P < 0.01. ***P < 0.001.
(P < 0.01) (Table 2). The fasted blood Glu increased in the 250 mg/kg and 500 mg/kg groups (P < 0.05). The serum CK and LDH levels which were detected for myocardial enzyme were also significant higher (P < 0.01) (Table 2). Serum Cre and BUN levels changed differently. The content of Cre in experimental groups was higher than control group, but the differences did not reach statistical significance except the 500 mg/kg group (P < 0.05). The BUN level for all subjects decreased significantly (P < 0.001) when compared to control group (Table 2). There were no significant differences in serum D.BILT levels in experimental groups but a significant increase in T.BILT (P < 0.01) in 250 mg/kg and 500 mg/kg groups. Furthermore, the activity of CHE was increased significant (P < 0.01) in experimental groups. Similarly, the concentrations of TP and ALB were influenced by ATR (Table 2), greater ATR caused greater TP and ALB.
Software (Molecular Biology Insights, Cascade, CO) based on the deposited sequences in GenBank and primers used are given in Table S1. Quantitative real-time PCR (qRT-PCR) was conducted using a fast real-time PCR system (LightCycler® 480 Real-Time PCR System (Roche, CH)). Triplicate samples were assessed for each gene of interest, and b-actin was used as a control gene. Relative expression levels were determined by the 2 △△Ct method. 2.6. Statistical analysis The data were analyzed statistically using GraphPad Prism 5.1 (GraphPad Software Inc., USA). One-way analysis of variance (ANOVA) and least significant difference (LSD) post hoc test were used to analyze the data. Differences between the means of data were analyzed by Dunnett's multiple comparisons test and the paired T test which was utilized to determine the effects of ATR. The results were expressed as mean ± S.D. of different groups. The significant differences of all data were showed by ANOVA of each experiment. P < 0.05 was considered significant.
3.2. Histologic and morphometric analysis Next, variation of ovaries and oviducts in different groups has been observed in the quails (Fig. 1). The weight of oviduct and length of the mucosal folds observed in the transverse section of the shell gland decreased in the test groups when compared to control group. Photographs of quail ovary and oviduct in different groups presented in Fig. 1A exhibit a decrease in the number and size of ovarian follicles with the increasing dose of ATR, especially marked suppression of oviduct development in 250 mg/kg and 500 mg/kg groups. The quail ovary was characterized by a medulla and a cortex (Fig. 1B). The cortex was covered by a simple layer of cubic epithelium and contained clusters of germ cells and follicles in the different stages of development. The medulla was constituted by connective tissue with blood and lymphatic vessels. Follicles at all stages were observed in the collected ovaries in both those of the control group and those of the 50 mg/kg group (Fig. 1B). A great amount of preantral follicles presented disorganized granulosa cells and/or a degenerating oocyte in 250 mg/kg and 500 mg/kg
3. Results 3.1. Body weight and biochemical parameters analyses We first examined the effects of ATR exposure on the clinical observations of European quails. There were significant effects of ATR on body weight. ATR at 250 mg/kg and 500 mg/kg decreased total body weights gains (BWG) significantly (P < 0.001) after 27d and 18d of gavage, respectively, versus control (Table 1). The birds in all groups get the maximum growth of BWG at the 9d and it's interesting that the BWG of 50 mg/kg group was higher than control group (P < 0.05). The results were similar to what has been previously reported (Juliani et al., 2008). Regarding the biochemical analyses, serum levels of Ca, Mg, and P were higher in the test groups than control group and the concentration of Ca was significant elevated in the 500 mg/kg group
Table 2 Effects of ATR exposure on biochemical parameters in quails. Item
Unit
Control
Ca Mg P Glu CK LDH Cre BUN CHE TP ALB T.BILT D.BILT
mmol/L mmol/L mmol/L mmol/L IU/L IU/L mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L
2.67 0.43 1.26 19.97 981.67 249.50 21.30 1.98 9092.00 33.05 13.65 1.20 1.95
50 mg/kg ± ± ± ± ± ± ± ± ± ± ± ± ±
0.12 0.01 0.05 0.74 109.3 12.50 2.71 0.14 918.00 1.91 1.48 0.10 0.55
2.89 0.59 1.47 19.12 1517.5 711.50 18.87 1.15 9997.00 42.80 17.00 1.33 2.40
± ± ± ± ± ± ± ± ± ± ± ± ±
250 mg/kg 0.13 0.07 0.47 0.55 37.48** 11.5*** 2.03 0.14*** 831.66 3.54* 0.28* 0.21 0.30
All data are presented as mean ± S.D. Compared with controls: *P < 0.05; **P < 0.01; ***P < 0.001.
3.07 0.76 2.02 18.24 1442.5 668.67 22.40 1.13 13,845.67 44.40 18.05 9.77 3.00
± ± ± ± ± ± ± ± ± ± ± ± ±
500 mg/kg 0.03 0.2* 0.49 0.34* 99.7** 15.01*** 3.40 0.04*** 1570.92* 2.69** 0.35** 0.46*** 0.20*
3.78 0.67 2.08 20.05 1414.33 532.00 28.83 1.08 15,655.33 44.85 14.20 3.77 2.00
± ± ± ± ± ± ± ± ± ± ± ± ±
0.52** 0.10 0.16 0.73* 192.81** 7.00** 1.33* 0.06*** 2061.28** 5.44** 0.28 0.64** 0.20
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Fig. 1. Effects of ATR exposure on the development of ovaries and oviducts in quails. (A) Progressive change has been observed in the length and diameter of the various parts of the oviduct with increasing dose and suppression in the ATR-treated quails. (B) The groups of 250 mg/kg ATR and 500 mg/kg ATR showing regression of follicles with connective tissues in the stroma. Degenerative changes can be seen in the follicle of ATR treated sexually mature quail without yolk granules in the lipid vacuoles of the ooplasm. The follicle of 50 mg/ kg ATR quail shows no specific change. (C) Photomicrograph showing the transverse section of the shell gland in high magnification. Tested birds-mucosal fold displays degenerated picture with low cuboidal luminal epithelium, very few and dispersed glands showing signs of regression with undefined structure. Center of the mucosal fold contains fibrous and cellular structure with debrises.
groups, which was not observed in the control group (Fig. 1B). Follicles of 250 mg/kg and 500 mg/kg were lined by degenerating membrana granulosa and compact theca having pyknotic nuclei. Ooplasm containing nucleus is exhibiting degenerating pattern. Sexually mature-the secretory glands are distributed
throughout the mucosal folds lined by columnar pseudo-stratified epithelium having both ciliated and non-ciliated cells. A systemic reduction in height and size was noted in the tubular gland cells and a cellular swelling were observed in the 50 mg/kg group and developed into atrophic pathology in epithelial cells (Fig. 1C). It was
Fig. 2. Effects of ATR exposure on the serum levels of gonadal hormone. Gonadal hormone concentration in samples from treatment quails was quantified by means of RIA kits. (A) FSH; (B) LH; (C) PRL; (D) P; (E) E2; (F) T. Data are presented as the mean ± SD. Compared with controls: *P < 0.05, **P < 0.01, ***P < 0.001.
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observed that oviduct loses some cytoplasmic mass and the cells involute in the test groups. The most characteristic feature observed in the shell gland of experimental quail was the involution of tubular glands and shedding of cells with increased amount of connective tissue and degenerating pigmented cells, particularly in 250 mg/kg and 500 mg/kg groups, beneath the epithelium, the leaf-like folds are characterized by the presence of small, darkly stained inclusions containing cellular debris, connective tissue and scattered involuted tubular gland cells (Fig. 1C). 3.3. Changes in gonadal hormones We first assessed the concentration of gonadal hormones. As shown in Fig. 2, the hormone levels in serum were changed significantly. The FSH level in 250 mg/kg and 500 mg/kg ATRtreated quails was decreased significantly compared with control group (P < 0.001) (Fig. 2A). The levels of LH, PRL and E2 were all significantly increased in the 250 mg/kg groups (P < 0.001), but as strange as it may seem, PRL level of the 500 mg/kg group had no similar change trend with the other two test-groups (Fig. 2BeD). The P level of 500 mg/kg groups was significantly increased compared with control group (P < 0.001) (Fig. 2E). The T level showed statistically significant differences in 50 mg/kg and 500 mg/kg groups (P < 0.05) (Fig. 2F). 3.4. Effect of ATR on gonadotropin releasing hormone (GnRH) in hypothalamo We next investigated whether ATR exposure caused HPO axis disruption. The effect of ATR on the mRNA level of GnRH in the hypothalamo is presented in (Fig. 3A). Compared with the control group, a significantly decrease (P < 0.01) in the hypothalamo of 50 mg/kg and 250 mg/kg groups were observed exposed to ATR. However, exposure to ATR of highest dose resulted in no statistically significant differences.
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3.5. Effects of ATR on gonadotropin in pituitary For each treatment group, a significantly decrease in the mRNA level of LH pituitary was observed when compared to control group (P < 0.01) (Fig. 3B). However, FSH level showed an upregulation trend but was not significantly different from control group expect the 500 mg/kg group (Fig. 3C). The FSH expression of the highest dose of treatment was up-regulated significantly (P < 0.001). No differences of PRL expression levels were found in high dose treatment groups and control group (Fig. 3D). However, a significant (P < 0.05) induction was observed in 50 mg/kg group.
3.6. Effects of ATR on gonadal hormone in ovaries We next investigated gonadal hormone gene expression levels in quail ovary. The effect of ATR on FSH expression in the ovary is presented in Fig. 4A. Compared with control group, a significantly decrease (P < 0.001) in FSH mRNA level in the ovary was observed in quail exposed to ATR. LH mRNA expressions of 250 mg/kg and 500 mg/kg groups were strongly upregulated by ATR (P < 0.01) but for 50 mg/kg group no statistically significant differences (P > 0.05) was observed when compared to control group (Fig. 4B). To further explore the effects of reproductive toxicity on ovary in ATR treated quails, we estimated the expression level of estrogen target genes. P450 cholesterol side chain cleavage (P450scc) mRNA expression was up-regulated by ATR but no significant in 50 mg/kg and 500 mg/kg groups, there was a significant increase in 250 mg/ kg group (P < 0.01) (Fig. 4C). In ATR-treated birds, aromatase cytochrome P450 (P450arom) mRNA also increased in ovaries and a significant difference was observed in the highest dose (P < 0.001) (Fig. 4D). The expressions of 3b-hydroxysteroid dehydrogenase (3bHSD) and 17b-hydroxysteroid dehydrogenase (17b-HSD) mRNA were significantly increased in a dose-dependent response in all ATR treated groups (P < 0.01 or P < 0.001) (Fig. 4E and F). The expression of ERa mRNA was approximately the same for all groups
Fig. 3. Effects of ATR exposure expression of GnRH in hypothalamo and gonadotropins in pituitary. (A) GnRH in hypothalamo; (B) FSH in pituitary; (C) LH in pituitary and (D) PRL in pituitary. Data are presented as the mean ± SD. Compared with controls: *P < 0.05, **P < 0.01, ***P < 0.001.
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Fig. 4. Expressions of gonadal hormone-related genes in ovaries of quails. (A) FSH; (B) LH; (C) P450scc; (D) P450arom; (E) 3b-HSD; (F) 17b-HSD; (G) ERa; (H) ERb; (I) StAR. Data are presented as the mean ± SD. Compared with controls: *P < 0.05, **P < 0.01, ***P < 0.001.
(Fig. 4G). Notably, the ERb expression levels in ATR-treated groups were significantly decreased in ovaries (P < 0.001) (Fig. 4H). Steroidogenic acute regulatory protein (StAR) is a rate-limiting protein for steroid biosynthesis, the levels of StAR transcripts were changed by ATR (Fig 4I) and further research was needed to explore the doseeresponse relationship between the gene level of StAR and ATR. 4. Discussion ATR have been found to interfere with reproductive function in female animals (Foradori et al., 2009, 2011, 2013, 2014; Goldman et al., 2013; Laws et al., 2000; Wilhelms et al., 2006). Evidence for the interference of ATR as mimic hormones has been reported both in vivo and in vitro (Abarikwu et al., 2011; Pogrmic et al., 2009; Pogrmic-Majkic et al., 2010; Suzawa and Ingraham, 2008; VictorCosta et al., 2010). The focus of this study was to clarify the toxicological pathways by ATR, which was an ovarian toxicants, affected the developmental abnormality of ovary and oviduct in quails in vivo. In the present study, these histologic and morphometric changes reflect the arrested development of ovaries and oviducts induced by ATR exposure. The developmental abnormality of ovary and oviduct is associated with disruption of gonadal hormone balance and HPO axis in ATR-treated quails. The present study provides new evidence that ovary and oviduct are the main targets of ATR reproductive toxicity. The widely used herbicide ATR is a potent endocrine disruptor, which alters the central nervous system regulation of the reproductive system in females. Numerous studies in experimental and wild animals all suggest that ATR can alter normal endocrine, neuroendocrine. For instance, exposure to ATR damages normal gonadal development in amphibians (Tavera-Mendoza et al., 2002).
Consistent with these phenotypes, acute exposure to ATR lowers T et al., levels and impairs gonadal development in young fish (Spano 2004), and in female rats (Laws et al., 2000). Other studies suggest that reduced T after ATR exposure results from a marked drop in body weight and food consumption. These latter effects are observed for female rats and potentially reflect an unknown role of ATR in neuroendocrine signaling (Eldridge et al., 1999). ATR exhibits low acute toxicity to birds with a dietary LD50 > 5000 mg/kg in Japanese quail (USEPA, 2006). Wilhelms et al. (2006) indicates no overt toxicity or endocrine disruptor effect/estrogenicity of ATR in quail at dietary concentrations up to 1000 ppm (109 mg/kg/day). However, our results showed that exposure to ATR had no effect on mortality but depressed feed intake and growth. ATR influenced the biochemical parameters and resulted in the damage of the liver, heart and kidney function in female quails. Notably, overt developmental abnormality of ovary and oviduct were observed in quails exposed to more than 250 mg ATR/kg/day. Our result is not consistent with Wilhelms et al. (2006). Female reproductive toxicity of ATR depended on the dose and duration of exposure in quails. This present study results suggest that the development of ovary and oviduct in sexually maturing female quails is more sensitive to ATR exposure. However, the mechanism of ATR-induced developmental toxicity of the female reproductive organs further needs to be clear in quails. The development of female reproductive organs are regulated by the HPO system through a complex feedback loops. These feedback mechanisms are perturbed by ovarian toxicants, especially ATR. In rodents, repeated exposure to ovarian toxicants produces identifiable histopathological changes in the reproductive tract as well as abnormal hormone secretion (Sanbuissho et al., 2009). In this study, the serum E2, LH, PRL and P levels of ATRexposed female quails were upregulated significantly, and the
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FSH and T levels were obviously downregulated. A number of previous studies demonstrated that daily ATR treatments administered rats can cause a significant attenuation in the LH and PRL surge (Cooper et al., 2000; Foradori et al., 2009; McMullin et al., 2004). However, this is the first report to show that ATR will increase the amount of LH released in female animals exposed to ATR, and it is similar to the report which demonstrated that exposure to ATR will increase the amount of LH released if a female rat is exposed in the hours immediately preceding the expected rise in LH (Goldman et al., 2013). This augmentation is in agreement with the synergistic relationship between E2 and P and demonstrates that the increase in circulating P in response to an ATR-induced activation of the HPO axis has a clear impact on the central regulation of LH. Furthermore, LH can stimulate the production of T de novo from cholesterol, whereas in the present study there was no clear pattern of T secretion in females. ATR stimulates PRL secretion, and this is in contrast to previous studies showing suppression of PRL secretion following ATR treatment of female rats (Cooper et al., 2000; Stoker et al., 1999). Fa et al. (2013) found that FSH can stimulate LHR mRNA level in granulosa cells decreases and can be a novel target of ATR in immature granulosa cells. In our results, the serum FSH level and mRNA expression in the ovary was decreased significantly, it is similar to the antecedent research. Besides, maybe it's because of negative feedback regulating role of HPO axis that the level FSH in the pituitary was upregulated. Moreover, our results illustrate that ATR may directly suppress the ovary by stimulating steroidogenic factor expressions, including P450scc, P450arom, 3b-HSD, 17b-HSD and by inhibiting ERb. Steroidogenesis starts with the transfer of cholesterol into the mitochondria to the site of action of P450scc which then converts cholesterol to pregnenolone. Pregnenolone is converted to progesterone by 3b-HSD and the conversion of progesterone to androstenedione is catalyzed by P450arom (Payne and Youngblood, 1995). Estrogens act via two types of receptors (ERa and ERb), which are members of a large super family of proteins that function as ligand-activated transcription factors (Katzenellenbogen and Katzenellenbogen, 1996) and estrogen signaling is selectively stimulated or inhibited depending upon a balance between ERa and ERb activities in target organs. However, our results showed that exposure to ATR only affected the ERb expression in ovary. Endocrine-related tissues with a capacity for steroidogenesis appear to be especially sensitive to ATR, as demonstrated by the specificity of the ATR response (Victor-Costa et al., 2010). A significant contrast results arised in doses of 250 mg/kg and 500 mg/kg groups that the level of mRNA expression of StAR had a significant downregulation in the group of 250 mg/kg but a significant upregulation in the group of 500 mg/kg. Additional studies are needed to determine the exact dosage that caused the different influence of the StAR expression by ATR and further understand the impact on reproductive function. Therefore, exposure to ATR leads to accumulation of the parent compound in the ovary where it changes the balance of the steroid hormones, thus negatively affecting ovulation and female fertility. Our results were similar to the previous research (Quignot et al., 2012a, 2012b). Although the pathway of this disruption is unclear, our results represent new evidence that ATR, via the HPO axis, directly and indirectly induced developmental abnormality of ovary and oviduct. The experimental inhibition of the formation or action of estrogen in the female chicken and Japanese quail embryos can result in almost complete phenotypic sex-reversal, such as formation of testis-like ovaries, development of male secondary sex characteristics, lack of oviductal development and male-like growth of the cloacal gland in response to T (Elbrecht and Smith, 1992). Therefore,
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Fig. 5. Overview of the pathways mediating the toxic effects of ATR on ovary and the HPO axis. Red arrows represent the increases or decreases of the hormone secretion in serum. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the objective of many previous studies was to investigate if ATR produces any alteration in profiles of different biomarkers (De La Casa-Resino et al., 2013; Fa et al., 2013; Quignot et al., 2012b) and the results were inconsistent. An overview of the pathways mediating the toxic effects of ATR on ovary and the HPO axis is summarized in Fig 5. We hypothesize that ATR affect the HPO axis by both direct and indirect pathways. ATR stimulates P secretion by increased LH from pituitary and by upregulating P450scc and 3bHSD expression levels. This results in an increase in E2 secretion in serum, which is regulated by the upregulation of steroidogenic factors through transformation from P. Furthermore, ATR stimulates PRL secretion sustainably by suppressing the dopaminergic activity in the anterior pituitary (Taketa et al., 2011) and stimulate the above disorder. And at the same time, the serum FSH is downregulated through negative feedback because of the high level of E2 in serum. Similarly, besides the negative feedback suppresses the expression of GnRH in hypothalamo, ATR directly inhibit expression of GnRH mRNA (Foradori et al., 2013). 5. Conclusion The present study provides new insights regarding the female productive toxicology of ATR exposure. Ovary and oviduct in sexually maturing female quails were the target organs of ATRinduced developmental toxicity. ATR upregulates steroidogenic factor and downregulates ERb factor in ovary of quails. We propose that ATR-induced developmental abnormality of ovary and oviduct is associated with disruption of gonadal hormone balance and HPO axis in female quails. In the future, additional studies further need to confirm the direct effect of ATR on ovarian cell and to elucidate its mechanism. Acknowledgments This work was supported by Program for New Century Excellent Talents in University (No. NECT-1207-02); Program for New Century Excellent Talents In Heilongjiang Provincial University (No. 1252-NCET-009); China Postdoctoral Science Foundation (No. 2012T50301), Research Special funds for Scientific Innovation Talents of Haerbin (No. 2012RFQXN006), National Science and Technology Major Project of the Ministry of Science and Technology of China (No. 2012ZX07201003) and Academic Backbone Project of
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Northeast Agricultural University (No. 15XG16). We also acknowledge the valuable help provided by Prof. Shi-Wen Xu in Northeast Agricultural University and all of the workers involved. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2015.09.044. References Abarikwu, S.O., Farombi, E.O., Kashyap, M.P., Pant, A.B., 2011. Atrazine induces transcriptional changes in marker genes associated with steroidogenesis in primary cultures of rat Leydig cells. Toxicol. Vitr. 25, 1588e1595. Barr, D.B., Panuwet, P., Nguyen, J.V., Udunka, S., Needham, L.L., 2007. Assessing exposure to atrazine and its metabolites using biomonitoring. Environ. Health Perspect. 115, 1474e1478. Basini, G., Bianchi, F., Bussolati, S., Baioni, L., Ramoni, R., Grolli, S., Conti, V., Bianchi, F., Grasselli, F., 2012. Atrazine disrupts steroidogenesis, VEGF and NO production in swine granulosa cells. Ecotoxicol. Environ. Saf. 85, 59e63. 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Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Atrazine triggers developmental abnormality of ovary and oviduct in quails (Coturnix Coturnix coturnix) via disruption of hypothalamo-pituitary-ovarian axis Lei Qin a, Zheng-Hai Du a, Shi-Yong Zhu a, Xue-Nan Li a, Nan Li b, Jing-Ao Guo a, Jin-Long Li a, *, Ying Zhang c, ** a b c
College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, PR China National Research Insitiute for Family Planning, Beijing, 100081, PR China School of Resources & Environment, Northeast Agricultural University, Harbin, 150030, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 August 2015 Received in revised form 16 September 2015 Accepted 20 September 2015 Available online 29 September 2015
There has been a gradual increase in production and consumption of atrazine (ATR) in agriculture to meet the population rising demands. Female reproduction is necessary for growth and maintenance of population. However, ATR impact on females and particularly ovarian developmental toxicity is less clear. The aim of this study was to define the pathways by which ATR exerted toxic effects on ovarian development of ovary and hypothalamo-pituitary-ovarian (HPO) axis. Female quails were dosed by oral gavage from sexual immaturity to maturity with 0, 50, 250 and 500 mg ATR/kg/d for 45 days. ATR had no effect on mortality but depressed feed intake and growth and influenced the biochemical parameters. Notably, the arrested development of ovaries and oviducts were observed in ATR-exposed quails. The circulating concentrations of E2, P, LH and PRL were unregulated and FSH and T was downregulated in ATR-treated quails. The mRNA expression of GnRH in hypothalamo and LH in pituitary and FSH in ovary was downregulated significantly by ATR exposure and FSH and PRL in pituitary were upregulated. ATR exposure upregulated the level of P450scc, P450arom, 3b-HSD and 17b-HSD in ovary and downregulated ERb expression in female quails. However, ATR did not change ERa expression in ovary. This study provides new insights regarding female productive toxicology of ATR exposure. Ovary and oviduct in sexually maturing females were target organs of ATR-induced developmental toxicity. We propose that ATR-induced developmental abnormality of ovary and oviduct is associated with disruption of gonadal hormone balance and HPO axis in female quails. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Atrazine Ovarian and oviduct developmental abnormality Endocrine disrupter Quail Hypothalamo-pituitary-ovarian axis
1. Introduction The triazine herbicide ATR is widely used in agricultural to control the unwanted growth of grasses and broadleaf weeds. Being one of the most commonly used pesticides in the world (Hayes et al., 2010), ATR is widespread in the environment and a frequently detected contaminant in waterways. Like BPA and other chemicals, there are scientific indications that ATR has endocrinedisrupting potential (Hayes et al., 2010; Jin et al., 2014; Thompson et al., 2015), causing mammary gland tumors in rodents (Cooper
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (J.-L. Li), [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.envpol.2015.09.044 0269-7491/© 2015 Elsevier Ltd. All rights reserved.
et al., 2007), and altering male reproduction (Stanko et al., 2010). Despite ATR has been banned in European Union and been restricted in other countries, it is still being used in large quantities worldwide up to now. It is one of the most widely used agricultural pesticides in United States (Barr et al., 2007), and its application in Asian countries has been growing. There has been a gradual increase in production and consumption of ATR in agriculture to meet the population rising demands. Female reproduction is necessary for growth and maintenance of population. Therefore, humans and wildlife are at risk for exposure to ATR. ATR has been considered as an endocrine disruptor due to alterations caused on hormoneregulated systems in various taxa (De La Casa-Resino et al., 2012; Hayes et al., 2011; McMullin et al., 2004; Salaberria et al., 2009). However, little is known about its effects on the development of reproductive organ in females.
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In recent decades, there has been an increasing concern on clarifying the toxicological mechanisms of environmental chemicals to cause alterations in the reproductive system of humans and animals. ATR like other herbicides induces endocrine disruption and consequently interferes with various hormones physiological functions. Numerous reports have suggested that ATR might have adverse effects on the reproductive function (Cooper et al., 2007; Feyzi-dehkhargani et al., 2012; Friedmann, 2002; Kniewald et al., 2000; Stoker et al., 1999; Swan, 2006; Solomon et al., 2008; Trentacoste et al., 2001). In the male offspring a slower maturation of gonadotrophic system due to chronic expoxure to ATR was observed. In female, it has been shown that ATR could inhibit the development of ovary and oviduct in rats and cause infertility (Goldman et al., 2013; Zhao et al., 2014). It has also been reported that ATR could induce endocrine disruption and consequently interfere with physiological functions of various hormones (Jin et al., 2014; Weber et al., 2013). Consistent with these observations, Friedmann (2002) demonstrated that ATR had significantly reduced the serum and testicular testosterone (T) levels, both in acute toxicity test and chronic toxicity test, in juvenile SpragueeDawley male rats by gavage. A recent study demonstrated that the serum levels of T, follicle stimulating hormone (FSH), luteinizing hormone (LH), and inhibin-B (INHeB) had decreased by 85% after 48 d exposure to high dose (300 mg/kg BW/day) of ATR in adult male Wister rats (Feyzi-dehkhargani et al., 2012). However, there is still little knowledge on the reproductive and developmental toxicity in female animals induced by ATR. It is well known that ATR may impact endocrine activity and notably alter androgen/estrogen balance. ATR is a serious health concern, and numerous studies have been devoted to studying the effects of ATR on steroidogenic enzymes influenced steroid secretion and thus lead to reproductive toxicity (Basini et al., 2012; Payne and Youngblood, 1995; Pogrmic et al., 2009; Pogrmic-Majkic et al., 2010; Quignot et al., 2012b). In females, sex steroids are synthesized primarily in the ovaries and derived from cholesterol through a series of biochemical reactions (Foradori et al., 2013; Goldman et al., 2013; Henare et al., 2012; McMullin et al., 2004). However, the effects of ATR on the development of ovary and oviduct and the tuned balance between estrogens and androgens are not yet well clear. Assessing ATR reproductive toxicity in female animals is a challenge, given the complexity of the endocrine system and despite the increasing development of data on its workings. To explore the effects and mechanism of hormonal balance disruption and the developmental abnormality of ovary and oviduct caused by ATR, female quails (Coturnix Coturnix coturnix) were employed as the experimental model orally given ATR daily from sexual immaturity to maturity. The aim of this study was to define the pathways by which ATR exerted the effects on the hypothalamo-pituitaryovarian (HPO) axis and the developmental of ovary and oviduct, clarify the mechanism of ATR-induced toxicity in female animals. 2. Materials and methods 2.1. Animals and treatments Female European quail (Coturnix C. coturnix) chicks aged 18 days and weighted 86.7 ± 6.4 g were purchased from Wan Jia farm in Harbin, China. Chemical ATR (C8H14ClN5, CAS: 1912-24-9, 90% purity) was from Zhonghe Chemical Limited Company (Binzhou, China). Birds were housed in cages in an environmentally controlled room (temperature 24e28 C and fluorescent lights provided a photoperiod of 12 h light and 12 h dark). Feed and water were offered ad libitum during the experiment. The birds were administered ATR once a day orally by gavage for 45 days. The
gavage volume was 0.5 mL and the amount of gavage was adjusted everyday according to the varied weight of each quail. After oneweek acclimation, the quails were randomly divided into four groups i.e., Group 1 (Control) treated with 0 mg/kg BW/day ATR, Group 2 (50 mg/kg ATR) treated with 50 mg/kg BW/day ATR, Group 3 (250 mg/kg ATR) treated with 250 mg/kg BW/day ATR, Group 4 (500 mg/kg ATR) treated with 500 mg/kg BW/day ATR. The ATR dose employed in the present investigation was chosen on basis of recent studies by Hussain et al. (2011) and Wilhelms et al. (2005). The current ecological risk assessment for ATR in avian species established by the USEPA reports a dietary LOAEL (lowest observable adverse effect level) of 675 mg/kg in the northern bobwhite quail, ATR exhibits modest reproductive toxicity in female birds (USEPA, 2002). In this study, all the experiments conducted in animals were in accordance with the guidance of ethical committee for research on laboratory animals. The birds were monitored daily for clinical signs and total body weights gains. At the end of the experiment, birds were fasted before the day of sacrifice, and their hypothalamo, pituitary, ovary and oviduct were carefully dissected out. The blood was collected from the heart of each bird and centrifuged at 3000 rpm for 10 min to obtain the serum. The serum were divided into two portions, one for biochemical analyses and a second to be stored at 80 C for assays. 2.2. Determination of biochemical parameters Blood samples were used to investigate changes in the serum enzymes and concentration of ions considered to be biochemical indicators of hepatobiliary, renal and myocardial enzyme. Serum Ca, Mg, P and glucose (Glu) concentrations were measured. Both the activities of creatine kinase (CK), lactate dehydrogenase (LDH), choline esterase (CHE), contents of creatinine (Cre), blood urea nitrogen (BUN), total protein (TP), albumin (ALB), total bilirubin (T.BILT) and direct bilirubin (D.BILT) were measured. The activities or contents of biochemical parameters were detected using the detection kits (Jiangsu SINNOWA Medical Technology Company, China) by a biochemical auto-analyzer. 2.3. Histopathological studies Oviducts and ovaries were washed in cold saline and soak dried on filter paper. A portion of organ was fixed in 10% buffered formalin and embedded in paraffin. Sections of 5 mm thickness were cut and stained with hematoxylin and eosin for microscopic examination. 2.4. Hormone analysis To identify ATR-induced changes in circulating concentrations of reproductive hormones, serum concentrations of E2, P, FSH, LH, PRL and T were determined. All six reproductive hormone were determined using 125I Radioimmunoassay (RIA) Kits (HAT CO. LTD. China) according to the manufacturer's protocol. Radioactivity was determined using an automatic gamma counter. All samples were run in duplicate in a single assay to avoid interassay variation. 2.5. RNA purification and quantitative real-time PCR Total mRNA was extracted from hypothalamo, pituitary and ovary using RNAout reagent (Beijing Tiandz, Inc. China), according to the manufacturer's instructions. First cDNA strand was synthesized using Oligo (dT) primers and Transcript Reverse Transcriptase (Beijing TransGen Biotech Co. Ltd., China). The primers for real-time amplification of relative cDNAs were designed using Oligo 7.22
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Table 1 Average of body weight gain (BWG) in different tests and control group. Groups
B.W.G 1d
Control 50 mg/kg 250 mg/kg 500 mg/kg
81.28 79.58 82.28 79.86
9d ± ± ± ±
3.38 8.72 2.65 5.06
117.78 130.46 123.40 114.86
18d ± ± ± ±
3.46 8.72* 7.30 8.42
156.96 162.10 137.98 121.66
27d ± ± ± ±
11.66 17.50 11.52 8.68***
183.60 189.40 159.30 139.36
36d ± ± ± ±
5.85 4.63 3.82*** 12.81***
202.40 209.04 177.56 147.24
45d ± ± ± ±
6.09 10.05 17.12** 10.08***
209.86 215.96 180.58 151.80
± ± ± ±
10.92 12.91 16.07** 3.02***
Overall changes in body weight gain and average growth rate in quails. All data are presented as mean ± SD. Compared with controls: *P < 0.05. **P < 0.01. ***P < 0.001.
(P < 0.01) (Table 2). The fasted blood Glu increased in the 250 mg/kg and 500 mg/kg groups (P < 0.05). The serum CK and LDH levels which were detected for myocardial enzyme were also significant higher (P < 0.01) (Table 2). Serum Cre and BUN levels changed differently. The content of Cre in experimental groups was higher than control group, but the differences did not reach statistical significance except the 500 mg/kg group (P < 0.05). The BUN level for all subjects decreased significantly (P < 0.001) when compared to control group (Table 2). There were no significant differences in serum D.BILT levels in experimental groups but a significant increase in T.BILT (P < 0.01) in 250 mg/kg and 500 mg/kg groups. Furthermore, the activity of CHE was increased significant (P < 0.01) in experimental groups. Similarly, the concentrations of TP and ALB were influenced by ATR (Table 2), greater ATR caused greater TP and ALB.
Software (Molecular Biology Insights, Cascade, CO) based on the deposited sequences in GenBank and primers used are given in Table S1. Quantitative real-time PCR (qRT-PCR) was conducted using a fast real-time PCR system (LightCycler® 480 Real-Time PCR System (Roche, CH)). Triplicate samples were assessed for each gene of interest, and b-actin was used as a control gene. Relative expression levels were determined by the 2 △△Ct method. 2.6. Statistical analysis The data were analyzed statistically using GraphPad Prism 5.1 (GraphPad Software Inc., USA). One-way analysis of variance (ANOVA) and least significant difference (LSD) post hoc test were used to analyze the data. Differences between the means of data were analyzed by Dunnett's multiple comparisons test and the paired T test which was utilized to determine the effects of ATR. The results were expressed as mean ± S.D. of different groups. The significant differences of all data were showed by ANOVA of each experiment. P < 0.05 was considered significant.
3.2. Histologic and morphometric analysis Next, variation of ovaries and oviducts in different groups has been observed in the quails (Fig. 1). The weight of oviduct and length of the mucosal folds observed in the transverse section of the shell gland decreased in the test groups when compared to control group. Photographs of quail ovary and oviduct in different groups presented in Fig. 1A exhibit a decrease in the number and size of ovarian follicles with the increasing dose of ATR, especially marked suppression of oviduct development in 250 mg/kg and 500 mg/kg groups. The quail ovary was characterized by a medulla and a cortex (Fig. 1B). The cortex was covered by a simple layer of cubic epithelium and contained clusters of germ cells and follicles in the different stages of development. The medulla was constituted by connective tissue with blood and lymphatic vessels. Follicles at all stages were observed in the collected ovaries in both those of the control group and those of the 50 mg/kg group (Fig. 1B). A great amount of preantral follicles presented disorganized granulosa cells and/or a degenerating oocyte in 250 mg/kg and 500 mg/kg
3. Results 3.1. Body weight and biochemical parameters analyses We first examined the effects of ATR exposure on the clinical observations of European quails. There were significant effects of ATR on body weight. ATR at 250 mg/kg and 500 mg/kg decreased total body weights gains (BWG) significantly (P < 0.001) after 27d and 18d of gavage, respectively, versus control (Table 1). The birds in all groups get the maximum growth of BWG at the 9d and it's interesting that the BWG of 50 mg/kg group was higher than control group (P < 0.05). The results were similar to what has been previously reported (Juliani et al., 2008). Regarding the biochemical analyses, serum levels of Ca, Mg, and P were higher in the test groups than control group and the concentration of Ca was significant elevated in the 500 mg/kg group
Table 2 Effects of ATR exposure on biochemical parameters in quails. Item
Unit
Control
Ca Mg P Glu CK LDH Cre BUN CHE TP ALB T.BILT D.BILT
mmol/L mmol/L mmol/L mmol/L IU/L IU/L mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L
2.67 0.43 1.26 19.97 981.67 249.50 21.30 1.98 9092.00 33.05 13.65 1.20 1.95
50 mg/kg ± ± ± ± ± ± ± ± ± ± ± ± ±
0.12 0.01 0.05 0.74 109.3 12.50 2.71 0.14 918.00 1.91 1.48 0.10 0.55
2.89 0.59 1.47 19.12 1517.5 711.50 18.87 1.15 9997.00 42.80 17.00 1.33 2.40
± ± ± ± ± ± ± ± ± ± ± ± ±
250 mg/kg 0.13 0.07 0.47 0.55 37.48** 11.5*** 2.03 0.14*** 831.66 3.54* 0.28* 0.21 0.30
All data are presented as mean ± S.D. Compared with controls: *P < 0.05; **P < 0.01; ***P < 0.001.
3.07 0.76 2.02 18.24 1442.5 668.67 22.40 1.13 13,845.67 44.40 18.05 9.77 3.00
± ± ± ± ± ± ± ± ± ± ± ± ±
500 mg/kg 0.03 0.2* 0.49 0.34* 99.7** 15.01*** 3.40 0.04*** 1570.92* 2.69** 0.35** 0.46*** 0.20*
3.78 0.67 2.08 20.05 1414.33 532.00 28.83 1.08 15,655.33 44.85 14.20 3.77 2.00
± ± ± ± ± ± ± ± ± ± ± ± ±
0.52** 0.10 0.16 0.73* 192.81** 7.00** 1.33* 0.06*** 2061.28** 5.44** 0.28 0.64** 0.20
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Fig. 1. Effects of ATR exposure on the development of ovaries and oviducts in quails. (A) Progressive change has been observed in the length and diameter of the various parts of the oviduct with increasing dose and suppression in the ATR-treated quails. (B) The groups of 250 mg/kg ATR and 500 mg/kg ATR showing regression of follicles with connective tissues in the stroma. Degenerative changes can be seen in the follicle of ATR treated sexually mature quail without yolk granules in the lipid vacuoles of the ooplasm. The follicle of 50 mg/ kg ATR quail shows no specific change. (C) Photomicrograph showing the transverse section of the shell gland in high magnification. Tested birds-mucosal fold displays degenerated picture with low cuboidal luminal epithelium, very few and dispersed glands showing signs of regression with undefined structure. Center of the mucosal fold contains fibrous and cellular structure with debrises.
groups, which was not observed in the control group (Fig. 1B). Follicles of 250 mg/kg and 500 mg/kg were lined by degenerating membrana granulosa and compact theca having pyknotic nuclei. Ooplasm containing nucleus is exhibiting degenerating pattern. Sexually mature-the secretory glands are distributed
throughout the mucosal folds lined by columnar pseudo-stratified epithelium having both ciliated and non-ciliated cells. A systemic reduction in height and size was noted in the tubular gland cells and a cellular swelling were observed in the 50 mg/kg group and developed into atrophic pathology in epithelial cells (Fig. 1C). It was
Fig. 2. Effects of ATR exposure on the serum levels of gonadal hormone. Gonadal hormone concentration in samples from treatment quails was quantified by means of RIA kits. (A) FSH; (B) LH; (C) PRL; (D) P; (E) E2; (F) T. Data are presented as the mean ± SD. Compared with controls: *P < 0.05, **P < 0.01, ***P < 0.001.
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observed that oviduct loses some cytoplasmic mass and the cells involute in the test groups. The most characteristic feature observed in the shell gland of experimental quail was the involution of tubular glands and shedding of cells with increased amount of connective tissue and degenerating pigmented cells, particularly in 250 mg/kg and 500 mg/kg groups, beneath the epithelium, the leaf-like folds are characterized by the presence of small, darkly stained inclusions containing cellular debris, connective tissue and scattered involuted tubular gland cells (Fig. 1C). 3.3. Changes in gonadal hormones We first assessed the concentration of gonadal hormones. As shown in Fig. 2, the hormone levels in serum were changed significantly. The FSH level in 250 mg/kg and 500 mg/kg ATRtreated quails was decreased significantly compared with control group (P < 0.001) (Fig. 2A). The levels of LH, PRL and E2 were all significantly increased in the 250 mg/kg groups (P < 0.001), but as strange as it may seem, PRL level of the 500 mg/kg group had no similar change trend with the other two test-groups (Fig. 2BeD). The P level of 500 mg/kg groups was significantly increased compared with control group (P < 0.001) (Fig. 2E). The T level showed statistically significant differences in 50 mg/kg and 500 mg/kg groups (P < 0.05) (Fig. 2F). 3.4. Effect of ATR on gonadotropin releasing hormone (GnRH) in hypothalamo We next investigated whether ATR exposure caused HPO axis disruption. The effect of ATR on the mRNA level of GnRH in the hypothalamo is presented in (Fig. 3A). Compared with the control group, a significantly decrease (P < 0.01) in the hypothalamo of 50 mg/kg and 250 mg/kg groups were observed exposed to ATR. However, exposure to ATR of highest dose resulted in no statistically significant differences.
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3.5. Effects of ATR on gonadotropin in pituitary For each treatment group, a significantly decrease in the mRNA level of LH pituitary was observed when compared to control group (P < 0.01) (Fig. 3B). However, FSH level showed an upregulation trend but was not significantly different from control group expect the 500 mg/kg group (Fig. 3C). The FSH expression of the highest dose of treatment was up-regulated significantly (P < 0.001). No differences of PRL expression levels were found in high dose treatment groups and control group (Fig. 3D). However, a significant (P < 0.05) induction was observed in 50 mg/kg group.
3.6. Effects of ATR on gonadal hormone in ovaries We next investigated gonadal hormone gene expression levels in quail ovary. The effect of ATR on FSH expression in the ovary is presented in Fig. 4A. Compared with control group, a significantly decrease (P < 0.001) in FSH mRNA level in the ovary was observed in quail exposed to ATR. LH mRNA expressions of 250 mg/kg and 500 mg/kg groups were strongly upregulated by ATR (P < 0.01) but for 50 mg/kg group no statistically significant differences (P > 0.05) was observed when compared to control group (Fig. 4B). To further explore the effects of reproductive toxicity on ovary in ATR treated quails, we estimated the expression level of estrogen target genes. P450 cholesterol side chain cleavage (P450scc) mRNA expression was up-regulated by ATR but no significant in 50 mg/kg and 500 mg/kg groups, there was a significant increase in 250 mg/ kg group (P < 0.01) (Fig. 4C). In ATR-treated birds, aromatase cytochrome P450 (P450arom) mRNA also increased in ovaries and a significant difference was observed in the highest dose (P < 0.001) (Fig. 4D). The expressions of 3b-hydroxysteroid dehydrogenase (3bHSD) and 17b-hydroxysteroid dehydrogenase (17b-HSD) mRNA were significantly increased in a dose-dependent response in all ATR treated groups (P < 0.01 or P < 0.001) (Fig. 4E and F). The expression of ERa mRNA was approximately the same for all groups
Fig. 3. Effects of ATR exposure expression of GnRH in hypothalamo and gonadotropins in pituitary. (A) GnRH in hypothalamo; (B) FSH in pituitary; (C) LH in pituitary and (D) PRL in pituitary. Data are presented as the mean ± SD. Compared with controls: *P < 0.05, **P < 0.01, ***P < 0.001.
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Fig. 4. Expressions of gonadal hormone-related genes in ovaries of quails. (A) FSH; (B) LH; (C) P450scc; (D) P450arom; (E) 3b-HSD; (F) 17b-HSD; (G) ERa; (H) ERb; (I) StAR. Data are presented as the mean ± SD. Compared with controls: *P < 0.05, **P < 0.01, ***P < 0.001.
(Fig. 4G). Notably, the ERb expression levels in ATR-treated groups were significantly decreased in ovaries (P < 0.001) (Fig. 4H). Steroidogenic acute regulatory protein (StAR) is a rate-limiting protein for steroid biosynthesis, the levels of StAR transcripts were changed by ATR (Fig 4I) and further research was needed to explore the doseeresponse relationship between the gene level of StAR and ATR. 4. Discussion ATR have been found to interfere with reproductive function in female animals (Foradori et al., 2009, 2011, 2013, 2014; Goldman et al., 2013; Laws et al., 2000; Wilhelms et al., 2006). Evidence for the interference of ATR as mimic hormones has been reported both in vivo and in vitro (Abarikwu et al., 2011; Pogrmic et al., 2009; Pogrmic-Majkic et al., 2010; Suzawa and Ingraham, 2008; VictorCosta et al., 2010). The focus of this study was to clarify the toxicological pathways by ATR, which was an ovarian toxicants, affected the developmental abnormality of ovary and oviduct in quails in vivo. In the present study, these histologic and morphometric changes reflect the arrested development of ovaries and oviducts induced by ATR exposure. The developmental abnormality of ovary and oviduct is associated with disruption of gonadal hormone balance and HPO axis in ATR-treated quails. The present study provides new evidence that ovary and oviduct are the main targets of ATR reproductive toxicity. The widely used herbicide ATR is a potent endocrine disruptor, which alters the central nervous system regulation of the reproductive system in females. Numerous studies in experimental and wild animals all suggest that ATR can alter normal endocrine, neuroendocrine. For instance, exposure to ATR damages normal gonadal development in amphibians (Tavera-Mendoza et al., 2002).
Consistent with these phenotypes, acute exposure to ATR lowers T et al., levels and impairs gonadal development in young fish (Spano 2004), and in female rats (Laws et al., 2000). Other studies suggest that reduced T after ATR exposure results from a marked drop in body weight and food consumption. These latter effects are observed for female rats and potentially reflect an unknown role of ATR in neuroendocrine signaling (Eldridge et al., 1999). ATR exhibits low acute toxicity to birds with a dietary LD50 > 5000 mg/kg in Japanese quail (USEPA, 2006). Wilhelms et al. (2006) indicates no overt toxicity or endocrine disruptor effect/estrogenicity of ATR in quail at dietary concentrations up to 1000 ppm (109 mg/kg/day). However, our results showed that exposure to ATR had no effect on mortality but depressed feed intake and growth. ATR influenced the biochemical parameters and resulted in the damage of the liver, heart and kidney function in female quails. Notably, overt developmental abnormality of ovary and oviduct were observed in quails exposed to more than 250 mg ATR/kg/day. Our result is not consistent with Wilhelms et al. (2006). Female reproductive toxicity of ATR depended on the dose and duration of exposure in quails. This present study results suggest that the development of ovary and oviduct in sexually maturing female quails is more sensitive to ATR exposure. However, the mechanism of ATR-induced developmental toxicity of the female reproductive organs further needs to be clear in quails. The development of female reproductive organs are regulated by the HPO system through a complex feedback loops. These feedback mechanisms are perturbed by ovarian toxicants, especially ATR. In rodents, repeated exposure to ovarian toxicants produces identifiable histopathological changes in the reproductive tract as well as abnormal hormone secretion (Sanbuissho et al., 2009). In this study, the serum E2, LH, PRL and P levels of ATRexposed female quails were upregulated significantly, and the
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FSH and T levels were obviously downregulated. A number of previous studies demonstrated that daily ATR treatments administered rats can cause a significant attenuation in the LH and PRL surge (Cooper et al., 2000; Foradori et al., 2009; McMullin et al., 2004). However, this is the first report to show that ATR will increase the amount of LH released in female animals exposed to ATR, and it is similar to the report which demonstrated that exposure to ATR will increase the amount of LH released if a female rat is exposed in the hours immediately preceding the expected rise in LH (Goldman et al., 2013). This augmentation is in agreement with the synergistic relationship between E2 and P and demonstrates that the increase in circulating P in response to an ATR-induced activation of the HPO axis has a clear impact on the central regulation of LH. Furthermore, LH can stimulate the production of T de novo from cholesterol, whereas in the present study there was no clear pattern of T secretion in females. ATR stimulates PRL secretion, and this is in contrast to previous studies showing suppression of PRL secretion following ATR treatment of female rats (Cooper et al., 2000; Stoker et al., 1999). Fa et al. (2013) found that FSH can stimulate LHR mRNA level in granulosa cells decreases and can be a novel target of ATR in immature granulosa cells. In our results, the serum FSH level and mRNA expression in the ovary was decreased significantly, it is similar to the antecedent research. Besides, maybe it's because of negative feedback regulating role of HPO axis that the level FSH in the pituitary was upregulated. Moreover, our results illustrate that ATR may directly suppress the ovary by stimulating steroidogenic factor expressions, including P450scc, P450arom, 3b-HSD, 17b-HSD and by inhibiting ERb. Steroidogenesis starts with the transfer of cholesterol into the mitochondria to the site of action of P450scc which then converts cholesterol to pregnenolone. Pregnenolone is converted to progesterone by 3b-HSD and the conversion of progesterone to androstenedione is catalyzed by P450arom (Payne and Youngblood, 1995). Estrogens act via two types of receptors (ERa and ERb), which are members of a large super family of proteins that function as ligand-activated transcription factors (Katzenellenbogen and Katzenellenbogen, 1996) and estrogen signaling is selectively stimulated or inhibited depending upon a balance between ERa and ERb activities in target organs. However, our results showed that exposure to ATR only affected the ERb expression in ovary. Endocrine-related tissues with a capacity for steroidogenesis appear to be especially sensitive to ATR, as demonstrated by the specificity of the ATR response (Victor-Costa et al., 2010). A significant contrast results arised in doses of 250 mg/kg and 500 mg/kg groups that the level of mRNA expression of StAR had a significant downregulation in the group of 250 mg/kg but a significant upregulation in the group of 500 mg/kg. Additional studies are needed to determine the exact dosage that caused the different influence of the StAR expression by ATR and further understand the impact on reproductive function. Therefore, exposure to ATR leads to accumulation of the parent compound in the ovary where it changes the balance of the steroid hormones, thus negatively affecting ovulation and female fertility. Our results were similar to the previous research (Quignot et al., 2012a, 2012b). Although the pathway of this disruption is unclear, our results represent new evidence that ATR, via the HPO axis, directly and indirectly induced developmental abnormality of ovary and oviduct. The experimental inhibition of the formation or action of estrogen in the female chicken and Japanese quail embryos can result in almost complete phenotypic sex-reversal, such as formation of testis-like ovaries, development of male secondary sex characteristics, lack of oviductal development and male-like growth of the cloacal gland in response to T (Elbrecht and Smith, 1992). Therefore,
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Fig. 5. Overview of the pathways mediating the toxic effects of ATR on ovary and the HPO axis. Red arrows represent the increases or decreases of the hormone secretion in serum. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the objective of many previous studies was to investigate if ATR produces any alteration in profiles of different biomarkers (De La Casa-Resino et al., 2013; Fa et al., 2013; Quignot et al., 2012b) and the results were inconsistent. An overview of the pathways mediating the toxic effects of ATR on ovary and the HPO axis is summarized in Fig 5. We hypothesize that ATR affect the HPO axis by both direct and indirect pathways. ATR stimulates P secretion by increased LH from pituitary and by upregulating P450scc and 3bHSD expression levels. This results in an increase in E2 secretion in serum, which is regulated by the upregulation of steroidogenic factors through transformation from P. Furthermore, ATR stimulates PRL secretion sustainably by suppressing the dopaminergic activity in the anterior pituitary (Taketa et al., 2011) and stimulate the above disorder. And at the same time, the serum FSH is downregulated through negative feedback because of the high level of E2 in serum. Similarly, besides the negative feedback suppresses the expression of GnRH in hypothalamo, ATR directly inhibit expression of GnRH mRNA (Foradori et al., 2013). 5. Conclusion The present study provides new insights regarding the female productive toxicology of ATR exposure. Ovary and oviduct in sexually maturing female quails were the target organs of ATRinduced developmental toxicity. ATR upregulates steroidogenic factor and downregulates ERb factor in ovary of quails. We propose that ATR-induced developmental abnormality of ovary and oviduct is associated with disruption of gonadal hormone balance and HPO axis in female quails. In the future, additional studies further need to confirm the direct effect of ATR on ovarian cell and to elucidate its mechanism. Acknowledgments This work was supported by Program for New Century Excellent Talents in University (No. NECT-1207-02); Program for New Century Excellent Talents In Heilongjiang Provincial University (No. 1252-NCET-009); China Postdoctoral Science Foundation (No. 2012T50301), Research Special funds for Scientific Innovation Talents of Haerbin (No. 2012RFQXN006), National Science and Technology Major Project of the Ministry of Science and Technology of China (No. 2012ZX07201003) and Academic Backbone Project of
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Northeast Agricultural University (No. 15XG16). We also acknowledge the valuable help provided by Prof. Shi-Wen Xu in Northeast Agricultural University and all of the workers involved. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2015.09.044. References Abarikwu, S.O., Farombi, E.O., Kashyap, M.P., Pant, A.B., 2011. Atrazine induces transcriptional changes in marker genes associated with steroidogenesis in primary cultures of rat Leydig cells. Toxicol. Vitr. 25, 1588e1595. Barr, D.B., Panuwet, P., Nguyen, J.V., Udunka, S., Needham, L.L., 2007. Assessing exposure to atrazine and its metabolites using biomonitoring. Environ. Health Perspect. 115, 1474e1478. Basini, G., Bianchi, F., Bussolati, S., Baioni, L., Ramoni, R., Grolli, S., Conti, V., Bianchi, F., Grasselli, F., 2012. Atrazine disrupts steroidogenesis, VEGF and NO production in swine granulosa cells. Ecotoxicol. Environ. Saf. 85, 59e63. 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