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CAR)-mediated mechanism of DEHP-induced cerebellar toxicity in quails (Coturnix japonica) via disrupting CYP enzyme system homeostasis
Environmental Pollution xxx (2017) 1e9
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Invited paper
A novel nuclear xenobiotic receptors (AhR/PXR/CAR)-mediated mechanism of DEHP-induced cerebellar toxicity in quails (Coturnix japonica) via disrupting CYP enzyme system homeostasis* Zheng-Hai Du a, 1, Jun Xia a, b, 1, Xiao-Chen Sun a, c, Xue-Nan Li a, b, Cong Zhang a, b, Hua-Shan Zhao a, b, Shi-Yong Zhu a, c, Jin-Long Li a, b, c, * a
College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, PR China Key Laboratory of the Provincial Education, Department of Heilongjiang for Common Animal Disease Prevention and Treatment, Northeast Agricultural University, Harbin 150030, PR China c Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine, Northeast Agricultural University, Harbin 150030, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 February 2017 Received in revised form 6 April 2017 Accepted 7 April 2017 Available online xxx
Di-(2-ethylhexyl)-phthalate (DEHP) is causing serious health hazard in wildlife animal and human through environment and food chain, including the effect of brain development and impacted neurobehavioral outcomes. However, DEHP exposure caused cerebellar toxicity in bird remains unclear. To evaluate DEHP-exerted potential neurotoxicity in cerebellum, male quails were exposed with 0, 250, 500 and 750 mg/kg BW/day DEHP by gavage treatment for 45 days. Neurobehavioral abnormality and cerebellar histopathological alternation were observed in DEHP-induced quails. DEHP exposure increased the contents of total Cytochrome P450s (CYPs) and Cytochrome b5 (Cyt b5) and the activities of NADPH-cytochrome c reductase (NCR) and aniline-4-hydeoxylase (AH) in quail cerebellum. The expression of nuclear xenobiotic receptors (NXRs) and the transcriptions of CYP enzyme isoforms were also influenced in cerebellum by DEHP exposure. These results suggested that DEHP exposure caused the toxic effects of quail cerebellum. DEHP exposure disrupted the cerebellar CYP enzyme system homeostasis via affecting the transcription of CYP enzyme isoforms. The cerebellar P450arom and CYP3A4 might be biomarkers in evaluating the neurotoxicity of DEHP in bird. Finally, this study provided new evidence that DEHP-induced toxic effect of quail cerebellum was associated with activating the NXRs responses and disrupting the CYP enzyme system homeostasis. © 2017 Elsevier Ltd. All rights reserved.
Keywords: DEHP Quail Cerebellum injury NXRs response CYP enzyme system homeostasis AhR/PXR/CAR pathway
1. Introduction Di (2-ethyl hexyl) phthalate (DEHP) is a ubiquitous environmental contaminant used worldwide as a plasticizer and solvent in medical devices, household products, pharmaceutical formulations, food packaging and industrial plastic, etc (Kelley et al., 2012; Bakir et al., 2016). Due to wide usage and constant release into the environment, DEHP existed in ground water and caused serious health hazard to the wild animals and human beings (Liu et al.,
* This paper has been recommended for acceptance by Dr. Harmon Sarah Michele. * Corresponding author. College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, PR China. E-mail address: [email protected] (J.-L. Li). 1 The two authors contributed equally to this study.
2016). Previous studies have shown that exposure to DEPH can significantly reduce the brain weight of newborns of ICR mice (Tanida et al., 2009). DEHP exposure also compromises neurons in the sexual differentiation area of the lactation phase in the rat's uterus (Moore et al., 2001), leading to neurodegeneration in the brain (Dhanya et al., 2003). DEHP treatment after the birth resulted in motor hyperactivity (Masuo et al., 2004) and markedly reduced the mesaticephalic dopaminergic neurons (Masuo et al., 2004; Tanida et al., 2009). During the last decade, potential exposure of animals to DEHP has become a growing concern. However, DEHP caused neurotoxicity in bird remains unclear. Cytochrome P450 (CYP) enzyme system is very important to metabolize xenobiotic compounds (Xia et al., 2016a, 2016b). The CYP enzymes are ubiquitously distributed throughout organisms and subserved a variety of metabolic functions in animals (Burkina et al., 2017). The members of CYP enzymes system are the products
http://dx.doi.org/10.1016/j.envpol.2017.04.015 0269-7491/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Du, Z.-H., et al., A novel nuclear xenobiotic receptors (AhR/PXR/CAR)-mediated mechanism of DEHP-induced cerebellar toxicity in quails (Coturnix japonica) via disrupting CYP enzyme system homeostasis, Environmental Pollution (2017), http:// dx.doi.org/10.1016/j.envpol.2017.04.015
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Z.-H. Du et al. / Environmental Pollution xxx (2017) 1e9
of a gene superfamily comprising over 400 members. Previous studies have shown that DEHP can cause the alternation of CYP enzymes and subject to metabolic biotransformation by CYP enzymes (Kertai et al., 2000). Besides, brain CYP enzyme system is expressed in glia cells of brain's barrier regions and in neurons throughout the brain, where they metabolize steroid hormones or fatty acids. Drug metabolising CYP enzyme isoforms have been identified in human, monkey and rodent brain (Meyer and Gehlhaus, 2010). The nuclear xenobiotic receptors (NXRs) play a critical role in the cellular regulation of CYP enzyme system in organisms. CYP enzyme system, as the primary target genetic transcription of NXRs (including constitutive androstane receptor, CAR, pregnane X receptor, PXR; aryl hydrocarbon receptor, AhR; etc), interact with NXRs controlling various pathways of xenobiotics metabolism (Cheshenko et al., 2008; Dvorak and Pavek, 2010; Vrba et al., 2014; Li-Tempel et al., 2016; Burkina et al., 2017). Despite great efforts in studying the toxicity of DEHP, relatively little consideration has been given to the DEHP-induced neurotoxicity through activating the NXRs responses and disrupting the CYP enzyme system. DEHP may be considered as an endocrine disrupting chemical contributing to nervous system disorders, indicating the need for further investigation in its mechanism of action and corresponding assessment of its risks associated with animal health. Although knowledge on the mechanism of critical organs in toxicological responses to DEHP is increasing, DEHP-induced toxic effects in the brain has not been completely elucidated. Notably, knowledge of the mechanism of the toxicological responses to DEHP in cerebellum is lacking. In this study, it was indicated that the neurotoxicity effects of DEHP on the cerebellum through activating the NXRs responses and disrupting the CYP enzyme system homeostasis. 2. Material and methods 2.1. Animals and experimental protocol
kg is known to be able to induce adverse effects in rats without causing systemic toxicity (Foster et al., 2001; Heudorf et al., 2007). At the end of the experiment, quails were fasted for 12 h and euthanized with carbon dioxide, and their cerebellums were carefully dissected out, storing at 80 C for assays. 2.2. Histopathological analysis Cerebellum tissue was fixed with neutral formalin solution and then routinely processed for histological analysis and stained with hematoxylin and eosin (H&E). All slides were examined under a light microscope at 10 and 40 objective magnifications. The evaluations of cerebellum injury were conducted in a blinded fashion. 2.3. Determination of the protein content The protein concentration was analyzed using the protein assay kits (A045-4, Jiancheng Biochemistry Co., Nanjing, China). The protein concentration was expressed in mg/mL according the standard curve of bovine serum albumin. 2.4. Determination of the CYPs homeostasis The contents of total CYP450 (CYPs) and Cyt b5 (Cytochrome b5) were determined by monitoring difference spectra of reduced vs. oxidized and reduced vs. reduced CO-bound pigment, respectively (Xia et al., 2016a, 2016b). The activities of ERND (erythromycin Ndemethylase) and APND (aminopyrin N-demethylase) were determined via establishing HCHO (formaldehyde) standard curve. The activity of NCR (NADPH-cytochrome c reductase) was expressed in nmol/mg of protein (Xia et al., 2016a, 2016b). The activity of AH (aniline-4-hydeoxylase) was quantified by the NADPH-dependent formation of 4-aminophenol according to the manufacturer's protocol (Xia et al., 2016a, 2016b). 2.5. RNA extraction and qRT-PCR analysis
All procedures, treatments, and animal care were strictly in compliance with the guidelines of the Institutional Animal Care and Use Committee of Northeast Agricultural University (NEAU). Male quail (Coturnix japonica, 8 days old, 26.7 ± 2.5 g) chicks were obtained from Wanjia farm (Harbin, China). DEHP (C24H38O4, CAS: 117-81-7, >99.0%) was obtained from Shanghai Aladdin Biochemical Technology co., LTD, (Shanghai, China). Standard chow and water were offered ad libitum. Birds inhabited an environmentcontrolled room, consistent with previous research in our Lab (Qin et al., 2015; Lin et al., 2016b; Zhang et al., 2017a). After 7 days acclimation, birds were randomly divided into five groups (Table 1). The dosage regimen was selected to establish a dose range that would not be lethal, but may cause identification of other possible target organ effects (NTP-CERHR, 2006). After oral administration of DEHP, the lethal dose 50 (LD50) in rodent is 30 g/kg (mice), 30.6 g/kg (rats), 34 g/kg (rabbits), respectively. The dose of 750 mg/
The cerebellums were carefully dissected out, then stored in RNA locker (Tiandi, Inc. Beijing, China) and frozen at 80 C for assays. Total RNA was extracted from the cerebellum tissues using a phenol and guanidine isothiocyanate-based TRIzol kits (Tiandi, Inc. Beijing, China) according to the Operation Manual (Jiang et al., 2017; Li et al., 2017). The specific oligonucleotide primers used for the determination of mRNA levels in Table S1 were designed with the Oligo 7.22 software. The specificity of each primer was checked by dissociation curve analysis and PCR amplification. The qRT-PCR was processed using the LightCycler® 480 qPCR System. The bactin was used as housekeeping controls to normalize the amounts of cDNA among the samples. The mRNA levels of relative expression were determined according to 2DDCt method. The results were normalized to the mean of b-actin 1 and b-actin 2 (Lin et al., 2016b; Du et al., 2017; Zhang et al., 2017a). 2.6. Statistical analysis
Table 1 Animal groups. Groups Group Group Group Group Group
1 2 3 4 5
(Con) (Vcon) (D250) (D500) (D750)
Number
Drug
Concentration
60 60 60 60 60
Water Corn oil DEHP DEHP DEHP
0 mg/kg BW/day 0 mg/kg BW/day 250 mg/kg BW/day 500 mg/kg BW/day 750 mg/kg BW/day
Note: Corn oil as vehicle dissolves DEHP. Group 1 (Con): treatment control; Group 2 (Vcon): vehicle control; Group 3e5: DEHP-exposed groups. BW: Body Weight.
All experiments data presented the means ± standard deviation (S.D.) and were statistical analysis using GraphPad Prism 5.1 Software (USA). Statistical analyses were employed using one-way ANOVA and Tukey's post hoc pairwise comparison. Asterisks (*) indicated statistical differences significantly to Con, *P < 0.05, **P < 0.01 and ***P < 0.001. The expression profile of each gene derived from cerebellum tissue in quails was showed using the heatmap. It is plotted using heatmap R package (version 3.2.1). The data is presented as mean.
Please cite this article in press as: Du, Z.-H., et al., A novel nuclear xenobiotic receptors (AhR/PXR/CAR)-mediated mechanism of DEHP-induced cerebellar toxicity in quails (Coturnix japonica) via disrupting CYP enzyme system homeostasis, Environmental Pollution (2017), http:// dx.doi.org/10.1016/j.envpol.2017.04.015
Z.-H. Du et al. / Environmental Pollution xxx (2017) 1e9
The source code of heatmap package were slightly modified to improve the layout and to add some features, the R function plots was used (Lin et al., 2016a, 2016c). PCA is used as a simplifying information tool from intercorrelation variables with the SPSS 17.0 (SPSS Inc., USA). This analysis method is the key element in this study to define the most important parameters that can be used with the same software as personal changes. The relationship between the parameters observed and quantified was confirmed by the Spearman test. 3. Result and discussion DEHP is a global environmental pollutant and unusually persistent in the environment. The number of DEHP produced worldwide is over 2 million tons each year (US EPA, 2012). Its metabolites and residues have been measured in groundwater, surface water and soils. Pollution occurs through environment and food chain (Heudorf et al., 2007). In recent years, accumulating evidence in wildlife, laboratory animal and cellular systems exists on the adverse health hazard of environmental DEHP. Recent experimental evidence suggests that DEHP exposure could impact the development of brain in animals (Li et al., 2014). The main concerns associated to DEHP pollutant are linked to neurotoxicity (Tanaka, 2005; Wu et al., 2014; Yan et al., 2016), endocrine (Ye et al., 2017; Zhang et al., 2017b) and metabolic disruption (Zhang et al., 2016). There is, however, limited information concerning the neurotoxicity of DEHP in animals following DEHP exposure, especially birds. DEHP has recently attracted attention of the scientific community due to its neural toxic and developmental effects on human beings (Li et al., 2009; Cho et al., 2010). DEHP is a lipophilic compound with the greatest absorption after oral exposure. DEHP is known to cross the blood-brain barrier and accumulate in the brain (Wu et al., 2014). In animals, the growing evidence has suggested that phthalates (including DEHP) exposure was associated with neurobehavioral outcomes, including the effects of self-righting
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ability, reference memory and spatial learning, and decrease of grooming behavior (Tanaka, 2005; Li et al., 2009; Cho et al., 2010). In this study, quails exposed to DEHP showed contracture down of the head, or to the side of the reverse, standing instability, nystagmus, difficulty swallowing, feather growth insufficiency, the realization of mania and hyperreflexia. The structure of Purkinje cell layer and granular layer of quail cerebellum in Con and Vcon groups are clear (Fig. 1A and B). The normal picture of histology in two control groups was observed, including: purkinje cell layer cell rules, large cell body, homogeneous cytoplasm, slightly basophilic cytoplasm, nucleus large round, chromatin was visible, and the distribution of uniform, clear nucleolus. After exposed to DEHP, Purkinje cell shrinks, the shape is not the whole, shrinkage, and some into a small sharp-like, increased eosinophilic cytoplasm in quail cerebellum (Fig. 1CeE). Nuclear condensation deformation, narrow or irregular, volume reduction, coloring deepening, nucleolus is unclear, or even completely disappears, especially in the 750 mg/kg group (Fig. 1E). These results suggested that DEHP exposure induced the severe cerebellar injury and showed the adverse neurological symptoms. NXRs were originally defined to prevent the accumulation of toxic chemicals in the body (Wada et al., 2009). It has been known that NXRs induce the transcription of CYP enzyme isoforms through competitive interaction, which effected by exogenous substances (Ramadoss et al., 2005). PXR, CAR and AhR are ligandactivated transcription factors, and then responsible to regulate the activation of related target genes transcription (Kawamoto et al., 2000; Pascussi et al., 2000; Antos et al., 2015). They share many target genes, such as the isoenzymes (including part of CYP1 isoforms, CYP2 isoforms and CYP3 isoforms) playing a significant role in the metabolism of approximately >90% the currently all used (Williams et al., 2004; Vrba et al., 2014; Prakash et al., 2015). Of note, CYP2/3 are the most widespread enzymes in vertebrates, which regulated by the CAR and PXR, that are responsible for catalyzing up the metabolism of various exogenous toxic substances (Coon, 2005). Various studies have proved the positive
Fig. 1. Effects of DEHP exposure on histopathological change of quail cerebellum. (A) Con (H&E, 400 ); (B) Vcon (H&E, 400 ); (C) D250 (H&E, 400 ); (D) D500 (H&E, 400 ); (E) D750 (H&E, 400 ). (A and B) The structure of macularlayer (I) purkinje cell, (II) and granular layer (III) of cerebellum in Con and Vcon groups was clear. Purkinje cell layer cell rules, large cell body, homogeneous cytoplasm, slightly basophilic cytoplasm, nucleus large round, chromatin was visible, and the distribution of uniform, clear nucleolus. (C) Only the individual cells of apoptosis characteristic changes. (D and E) Purkinje cell exhibited different levels of swelling, partial necrosis. (E) Nuclear condensation deformation, narrow or irregular, volume reduction, coloring deepening, nucleolus is unclear, or even completely disappears. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Du, Z.-H., et al., A novel nuclear xenobiotic receptors (AhR/PXR/CAR)-mediated mechanism of DEHP-induced cerebellar toxicity in quails (Coturnix japonica) via disrupting CYP enzyme system homeostasis, Environmental Pollution (2017), http:// dx.doi.org/10.1016/j.envpol.2017.04.015
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Fig. 2. Effects of DEHP exposure on the NXRs (AHR/PXR/CAR pathway) response. (A) AHR; (B) PXR; (C) CAR; (D) CYPs. Values were expressed as mean ± S.D. Symbol for the significance of differences between the DEHP-treated groups and Con group: *P < 0.05, **P < 0.01, ***P < 0.001. The mRNA levels of CYPs isoforms were shown using the indicated pseudo color scale from -1x (red) to þ1 (green) relative to values for quail cerebellum in the Con group. The color scale represented the relative mRNA levels, with green indicating up-regulated genes, red indicating down-regulated genes, and black indicating unchanged genes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
effects of DEHP activating NXRs (Wang et al., 2016; Huang et al., 2016; Zhang et al., 2017b). In this study, the expressions of AhR, CAR and PXR presented significant increase in DEHP-exposed quails (Fig. 2AeC). These results suggested that DEHP activated the NXRs (AhR/PXR/CAR pathway) responses. The expressions of cerebellar CYP1 isoforms (CYP1B1), CYP2 isoforms (CYP2D6), CYP3 isoforms (CYP3A4 and CYP3A7) in quail cerebellum were upregulated by DEHP exposure significantly (Fig. 2D and Fig. 3AeF). These results indicated that DEHP metabolism in the nervous system was affected by regulating the transcription of cerebellar CYP enzyme system in quails. Aromatase, as a CYP enzyme is consists of two components: P450arom (cytochrome P450, the product of CYP19 gene) and NCR (Cheshenko et al., 2008). It is an enzyme that catalyzes the transformation of androgens such as testosterone and androstenedione into the estrogens such as estradiol and estrone, respectively, and plays an important role in reproduction and neuroprotection (Roselli, 2007). It could regulate the neuroendocrine events and behaviors. Numerous researches demonstrate that aromatase modulates the neurogenesis, synaptic plasticity and activity, the response of neural tissue to injury (Von Schassen et al., 2006). In addition, AhR-activated endocrine is likely to affect the CYP19 genes expression at the transcription (Cheshenko et al., 2008). In this study, the expressions of AhR and P450arom presented significant increase in DEHP-exposed quails (Figs. 2D and 3B). More
importantly, PCA analysis showed that P450arom and CYP3A4 play an important role in DEHP-induced cerebellum toxicity. The CYP enzymes are present in most domains of life and catalyze the metabolism of a wide variety of xenobiotic compounds. Numerous reports have investigated that the effects of xenobiotics exposure depend heavily on CYP enzyme system hemostasis (Burkina et al., 2017). The present work observed that DEHP exposure significantly increase the hepatic CYP2B1 and CYP3A4 expression and decrease the adrenal CYP11A1 expression in rodent (Liu et al., 2015; Ge et al., 2015; Wang et al., 2014). ERND is known to be at least partially related to CYP3A and APND activity is CYP2B/3A isoform-dependent (Yawetz et al., 1998; Vaccaro et al., 2003). Moreover, the activity of AH was known to be highly associated with CYP2 isoform (Wolf et al., 2004). In this work, DEHP treatment enhanced the activities of NCR and AH and increased the concentrations of total Cyt b5 and total CYP450 in quail cerebellum (Fig. 4AeD). These data suggested that DEHP caused the alternations of the levels of CYPs and Cyt b5 and the activities of CYP enzymes (NCR and AH) in cerebellum with the dose-related manner significantly. These results indicated that DEHP exposure triggered the disruption of the CYP enzyme system homeostasis in the cerebellum via activating the NXRs responses and inducing CYP enzyme isoforms expression in the nervous system of quails. Our study also provided a lot of novel evidences that DEHP changed the activities of NCR and AH via activating the AhR/PXR/CAR pathway
Please cite this article in press as: Du, Z.-H., et al., A novel nuclear xenobiotic receptors (AhR/PXR/CAR)-mediated mechanism of DEHP-induced cerebellar toxicity in quails (Coturnix japonica) via disrupting CYP enzyme system homeostasis, Environmental Pollution (2017), http:// dx.doi.org/10.1016/j.envpol.2017.04.015
5 Fig. 3. Effects of DEHP exposure on the expressions of CYPs isoforms. (A) CYP1B1; (B) P450arom; (C) CYP2D6; (D) CYP3A7; (E) CYP4V2; (F) CYP3A4. Values were expressed as mean ± S.D. Symbol for the significance of differences between the DEHP-treated groups and Con group: *P < 0.05, **P < 0.01, ***P < 0.001.
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Please cite this article in press as: Du, Z.-H., et al., A novel nuclear xenobiotic receptors (AhR/PXR/CAR)-mediated mechanism of DEHP-induced cerebellar toxicity in quails (Coturnix japonica) via disrupting CYP enzyme system homeostasis, Environmental Pollution (2017), http:// dx.doi.org/10.1016/j.envpol.2017.04.015
Z.-H. Du et al. / Environmental Pollution xxx (2017) 1e9 Fig. 4. Effects of DEHP on the CYPs homeostasis. (A) Total CYP450 content; (B) The Cyt b5 content; (C) The AH activity; (D) The NCR activity; (E) The APND activity; (F) The ERND activity. Values were expressed as mean ± S.D. Symbol for the significance of differences between the DEHP-treated groups and Con group: *P < 0.05, **P < 0.01, ***P < 0.001.
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Please cite this article in press as: Du, Z.-H., et al., A novel nuclear xenobiotic receptors (AhR/PXR/CAR)-mediated mechanism of DEHP-induced cerebellar toxicity in quails (Coturnix japonica) via disrupting CYP enzyme system homeostasis, Environmental Pollution (2017), http:// dx.doi.org/10.1016/j.envpol.2017.04.015
Z.-H. Du et al. / Environmental Pollution xxx (2017) 1e9
responses and increasing the transcription of CYP3A isoforms in quail cerebellum. The present study investigated the serve toxic effects of DEHP on the cerebellum and demonstrated that DEHP could cause the neurotoxicity in quail. P450arom and CYP3A4 are sensitivities and responses to DEHP in cerebellum (Fig. 5A). These results provided some new insight that P450arom and CYP3A4 are able to be biomarkers in evaluating the impact of DEHP exposure in bird. DEHP exposure triggered the AhR/PXR/CAR pathway and altered the transcription of cerebellar CYP enzymes isoforms in quails.
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Additionally, all parameters were divided into the first, second and third principle components (46.9, 27.3 and 14.1%) based on ordination plots corresponding (Table S2). Component plot indicated that DEHP-induced cerebellar toxicity presented dose-dependent effect (Fig. 5B). 4. Conclusion This study provided several novel evidences that DEHP exposure induced the toxic effects of quail cerebellum. DEHP exposure
Fig. 5. PCA of the mechanism of NXRs response to DEHP exposure. (A) PCA score plot results comparing biochemical parameters. All parameters were divided into the first, second and third principle components (46.90%, 27.37% and 14.18%) based on ordination plots corresponding. The red represented the key factors for individual variations. (B) PCA score plot results comparing biochemical parameters. There was a dose-dependent relationship between the groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. The pathway of DEHP modulated NXRs responses in quail cerebellum. DEHP regulated CYPs by activation of NXRs pathway responses and inducing CYPs isoforms transcription. Thereby NXRs responses promoted the processing of the detoxification and elimination of DEHP. Cerebellar P450arom and CYP3A4 might be biomarkers in evaluating the neurotoxicity of DEHP in bird.
Please cite this article in press as: Du, Z.-H., et al., A novel nuclear xenobiotic receptors (AhR/PXR/CAR)-mediated mechanism of DEHP-induced cerebellar toxicity in quails (Coturnix japonica) via disrupting CYP enzyme system homeostasis, Environmental Pollution (2017), http:// dx.doi.org/10.1016/j.envpol.2017.04.015
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disrupted the cerebellar CYP enzyme system homeostasis via affect the transcription of CYP enzyme isoforms. The cerebellar AhR/PXR/ CAR pathway responses were activated by expose to DEHP in quail. The results of this study show for the first time that the DEHPinduced toxic effects of quail cerebellum were associated with activating NXRs responses and disrupting the CYP enzyme system homeostasis (Fig. 6). More importantly, cerebellar P450arom and CYP3A4 might be biomarkers in evaluating the neurotoxicity of DEHP in bird. However, the further mechanism of the neurological symptoms and the role of cerebellar P450arom and CYP3A4 will be required in the DEHP-exposed birds in future research. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by China New Century Excellent Talents in University (No. NECT-1207-02), National Natural Science Foundation of China (No. 31572586) and Academic Backbone Project of 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.2017.04.015. References Antos, P.A., Blachuta, M., Hrabia, A., Grzegorzewska, A.K., Sechman, A., 2015. Expression of aryl hydrocarbon receptor 1 (AHR1), AHR1 nuclear translocator 1 (ARNT1) and CYP1 family monooxygenase mRNAs and their activity in chicken ovarian follicles following in vitro exposure to 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD). Toxicol. Lett. 237, 100e111. Bakir, A., O'Connor, I.A., Rowland, S.J., Hendriks, A.J., Thompson, R.C., 2016. Relative importance of microplastics as a pathway for the transfer of hydrophobic organic chemicals to marine life. Environ. Pollut. 219, 56e65. Burkina, V., Rasmussen, M.K., Pilipenko, N., Zamaratskaia, G., 2017. Comparison of xenobiotic-metabolising human, porcine, rodent, and piscine cytochrome P450. Toxicology 375, 10e27. Cheshenko, K., Pakdel, F., Segner, H., Kah, O., Eggen, R.I.L., 2008. Interference of endocrine disrupting chemicals with aromatase CYP19 expression or activity, and consequences for reproduction of teleost fish. Gen. Comp. Endocrinol. 155, 31e62. Cho, S.C., Bhang, S.Y., Hong, Y.C., Shin, M.S., Kim, B.N., Kim, J.W., Yoo, H.J., Cho, I.H., Kim, H.W., 2010. Relationship between environmental phthalate exposure and the intelligence of school-age children. Environ. Health Perspect. 118, 1027e1032. Coon, M.J., 2005. Cytochrome P450: nature's most versatile biological catalyst. Annu. Rev. Pharmacol. Toxicol. 45, 1e25. Dhanya, C.R., Indu, A.R., Deepadevi, K.V., Kur, P.A., 2003. Inhibition of membrane Na(þ)-Kþ Atpase of the brain, liver and RBC in rats administered di(2-ethyl hexyl) phthalate (DEHP) a plasticizer used in polyvinyl chloride (PVC) blood storage bags. Indian J. Exp. Biol. 41, 814e820. Du, Z.H., Qin, L., Lin, J., Sun, Y.C., Xia, J., Zhang, C., Li, X.N., Li, J.L., 2017. Activating nuclear xenobiotic receptors and triggering ER stress and hepatic cytochromes P450 systems in quails (Coturnix C. coturnix) during atrazine exposure. Environ. Toxicol. http://dx.doi.org/10.1002/tox.22404. Dvorak, Z., Pavek, P., 2010. Regulation of drug-metabolizing cytochrome P450 enzymes by glucocorticoids. Drug Metab. Rev. 42, 621e635. Foster, P.M., Mylchreest, E., Gaido, K.W., Sar, M., 2001. Effects of phthalate esters on the developing reproductive tract of males rats. Hum. Reprod. Update 7, 231e235. Ge, J., Han, B., Hu, H., Liu, J., Liu, Y., 2015. Epigallocatechin-3-O-Gallate protects against hepatic damage and testicular toxicity in male mice exposed to di-(2ethylhexyl) phthalate. J. Med. Food 18, 753e761. Heudorf, U., Mersch-Sundermann, V., Angerer, J., 2007. Phthalates: toxicology and exposure. Int. J. Hyg. Environ. Health 210, 623e634. Huang, Q., Zhang, H., Chen, Y.J., Chi, Y.L., Dong, S., 2016. The inflammation response to DEHP through PPARg in endometrial cells. Int. J. Environ. Res. Public Health 13, E318. Jiang, X.Q., Cao, C.Y., Li, Z.Y., Li, W., Zhang, C., Lin, J., Li, X.N., Li, J.L., 2017. Delineating
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Invited paper
A novel nuclear xenobiotic receptors (AhR/PXR/CAR)-mediated mechanism of DEHP-induced cerebellar toxicity in quails (Coturnix japonica) via disrupting CYP enzyme system homeostasis* Zheng-Hai Du a, 1, Jun Xia a, b, 1, Xiao-Chen Sun a, c, Xue-Nan Li a, b, Cong Zhang a, b, Hua-Shan Zhao a, b, Shi-Yong Zhu a, c, Jin-Long Li a, b, c, * a
College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, PR China Key Laboratory of the Provincial Education, Department of Heilongjiang for Common Animal Disease Prevention and Treatment, Northeast Agricultural University, Harbin 150030, PR China c Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine, Northeast Agricultural University, Harbin 150030, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 February 2017 Received in revised form 6 April 2017 Accepted 7 April 2017 Available online xxx
Di-(2-ethylhexyl)-phthalate (DEHP) is causing serious health hazard in wildlife animal and human through environment and food chain, including the effect of brain development and impacted neurobehavioral outcomes. However, DEHP exposure caused cerebellar toxicity in bird remains unclear. To evaluate DEHP-exerted potential neurotoxicity in cerebellum, male quails were exposed with 0, 250, 500 and 750 mg/kg BW/day DEHP by gavage treatment for 45 days. Neurobehavioral abnormality and cerebellar histopathological alternation were observed in DEHP-induced quails. DEHP exposure increased the contents of total Cytochrome P450s (CYPs) and Cytochrome b5 (Cyt b5) and the activities of NADPH-cytochrome c reductase (NCR) and aniline-4-hydeoxylase (AH) in quail cerebellum. The expression of nuclear xenobiotic receptors (NXRs) and the transcriptions of CYP enzyme isoforms were also influenced in cerebellum by DEHP exposure. These results suggested that DEHP exposure caused the toxic effects of quail cerebellum. DEHP exposure disrupted the cerebellar CYP enzyme system homeostasis via affecting the transcription of CYP enzyme isoforms. The cerebellar P450arom and CYP3A4 might be biomarkers in evaluating the neurotoxicity of DEHP in bird. Finally, this study provided new evidence that DEHP-induced toxic effect of quail cerebellum was associated with activating the NXRs responses and disrupting the CYP enzyme system homeostasis. © 2017 Elsevier Ltd. All rights reserved.
Keywords: DEHP Quail Cerebellum injury NXRs response CYP enzyme system homeostasis AhR/PXR/CAR pathway
1. Introduction Di (2-ethyl hexyl) phthalate (DEHP) is a ubiquitous environmental contaminant used worldwide as a plasticizer and solvent in medical devices, household products, pharmaceutical formulations, food packaging and industrial plastic, etc (Kelley et al., 2012; Bakir et al., 2016). Due to wide usage and constant release into the environment, DEHP existed in ground water and caused serious health hazard to the wild animals and human beings (Liu et al.,
* This paper has been recommended for acceptance by Dr. Harmon Sarah Michele. * Corresponding author. College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, PR China. E-mail address: [email protected] (J.-L. Li). 1 The two authors contributed equally to this study.
2016). Previous studies have shown that exposure to DEPH can significantly reduce the brain weight of newborns of ICR mice (Tanida et al., 2009). DEHP exposure also compromises neurons in the sexual differentiation area of the lactation phase in the rat's uterus (Moore et al., 2001), leading to neurodegeneration in the brain (Dhanya et al., 2003). DEHP treatment after the birth resulted in motor hyperactivity (Masuo et al., 2004) and markedly reduced the mesaticephalic dopaminergic neurons (Masuo et al., 2004; Tanida et al., 2009). During the last decade, potential exposure of animals to DEHP has become a growing concern. However, DEHP caused neurotoxicity in bird remains unclear. Cytochrome P450 (CYP) enzyme system is very important to metabolize xenobiotic compounds (Xia et al., 2016a, 2016b). The CYP enzymes are ubiquitously distributed throughout organisms and subserved a variety of metabolic functions in animals (Burkina et al., 2017). The members of CYP enzymes system are the products
http://dx.doi.org/10.1016/j.envpol.2017.04.015 0269-7491/© 2017 Elsevier Ltd. All rights reserved.
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Z.-H. Du et al. / Environmental Pollution xxx (2017) 1e9
of a gene superfamily comprising over 400 members. Previous studies have shown that DEHP can cause the alternation of CYP enzymes and subject to metabolic biotransformation by CYP enzymes (Kertai et al., 2000). Besides, brain CYP enzyme system is expressed in glia cells of brain's barrier regions and in neurons throughout the brain, where they metabolize steroid hormones or fatty acids. Drug metabolising CYP enzyme isoforms have been identified in human, monkey and rodent brain (Meyer and Gehlhaus, 2010). The nuclear xenobiotic receptors (NXRs) play a critical role in the cellular regulation of CYP enzyme system in organisms. CYP enzyme system, as the primary target genetic transcription of NXRs (including constitutive androstane receptor, CAR, pregnane X receptor, PXR; aryl hydrocarbon receptor, AhR; etc), interact with NXRs controlling various pathways of xenobiotics metabolism (Cheshenko et al., 2008; Dvorak and Pavek, 2010; Vrba et al., 2014; Li-Tempel et al., 2016; Burkina et al., 2017). Despite great efforts in studying the toxicity of DEHP, relatively little consideration has been given to the DEHP-induced neurotoxicity through activating the NXRs responses and disrupting the CYP enzyme system. DEHP may be considered as an endocrine disrupting chemical contributing to nervous system disorders, indicating the need for further investigation in its mechanism of action and corresponding assessment of its risks associated with animal health. Although knowledge on the mechanism of critical organs in toxicological responses to DEHP is increasing, DEHP-induced toxic effects in the brain has not been completely elucidated. Notably, knowledge of the mechanism of the toxicological responses to DEHP in cerebellum is lacking. In this study, it was indicated that the neurotoxicity effects of DEHP on the cerebellum through activating the NXRs responses and disrupting the CYP enzyme system homeostasis. 2. Material and methods 2.1. Animals and experimental protocol
kg is known to be able to induce adverse effects in rats without causing systemic toxicity (Foster et al., 2001; Heudorf et al., 2007). At the end of the experiment, quails were fasted for 12 h and euthanized with carbon dioxide, and their cerebellums were carefully dissected out, storing at 80 C for assays. 2.2. Histopathological analysis Cerebellum tissue was fixed with neutral formalin solution and then routinely processed for histological analysis and stained with hematoxylin and eosin (H&E). All slides were examined under a light microscope at 10 and 40 objective magnifications. The evaluations of cerebellum injury were conducted in a blinded fashion. 2.3. Determination of the protein content The protein concentration was analyzed using the protein assay kits (A045-4, Jiancheng Biochemistry Co., Nanjing, China). The protein concentration was expressed in mg/mL according the standard curve of bovine serum albumin. 2.4. Determination of the CYPs homeostasis The contents of total CYP450 (CYPs) and Cyt b5 (Cytochrome b5) were determined by monitoring difference spectra of reduced vs. oxidized and reduced vs. reduced CO-bound pigment, respectively (Xia et al., 2016a, 2016b). The activities of ERND (erythromycin Ndemethylase) and APND (aminopyrin N-demethylase) were determined via establishing HCHO (formaldehyde) standard curve. The activity of NCR (NADPH-cytochrome c reductase) was expressed in nmol/mg of protein (Xia et al., 2016a, 2016b). The activity of AH (aniline-4-hydeoxylase) was quantified by the NADPH-dependent formation of 4-aminophenol according to the manufacturer's protocol (Xia et al., 2016a, 2016b). 2.5. RNA extraction and qRT-PCR analysis
All procedures, treatments, and animal care were strictly in compliance with the guidelines of the Institutional Animal Care and Use Committee of Northeast Agricultural University (NEAU). Male quail (Coturnix japonica, 8 days old, 26.7 ± 2.5 g) chicks were obtained from Wanjia farm (Harbin, China). DEHP (C24H38O4, CAS: 117-81-7, >99.0%) was obtained from Shanghai Aladdin Biochemical Technology co., LTD, (Shanghai, China). Standard chow and water were offered ad libitum. Birds inhabited an environmentcontrolled room, consistent with previous research in our Lab (Qin et al., 2015; Lin et al., 2016b; Zhang et al., 2017a). After 7 days acclimation, birds were randomly divided into five groups (Table 1). The dosage regimen was selected to establish a dose range that would not be lethal, but may cause identification of other possible target organ effects (NTP-CERHR, 2006). After oral administration of DEHP, the lethal dose 50 (LD50) in rodent is 30 g/kg (mice), 30.6 g/kg (rats), 34 g/kg (rabbits), respectively. The dose of 750 mg/
The cerebellums were carefully dissected out, then stored in RNA locker (Tiandi, Inc. Beijing, China) and frozen at 80 C for assays. Total RNA was extracted from the cerebellum tissues using a phenol and guanidine isothiocyanate-based TRIzol kits (Tiandi, Inc. Beijing, China) according to the Operation Manual (Jiang et al., 2017; Li et al., 2017). The specific oligonucleotide primers used for the determination of mRNA levels in Table S1 were designed with the Oligo 7.22 software. The specificity of each primer was checked by dissociation curve analysis and PCR amplification. The qRT-PCR was processed using the LightCycler® 480 qPCR System. The bactin was used as housekeeping controls to normalize the amounts of cDNA among the samples. The mRNA levels of relative expression were determined according to 2DDCt method. The results were normalized to the mean of b-actin 1 and b-actin 2 (Lin et al., 2016b; Du et al., 2017; Zhang et al., 2017a). 2.6. Statistical analysis
Table 1 Animal groups. Groups Group Group Group Group Group
1 2 3 4 5
(Con) (Vcon) (D250) (D500) (D750)
Number
Drug
Concentration
60 60 60 60 60
Water Corn oil DEHP DEHP DEHP
0 mg/kg BW/day 0 mg/kg BW/day 250 mg/kg BW/day 500 mg/kg BW/day 750 mg/kg BW/day
Note: Corn oil as vehicle dissolves DEHP. Group 1 (Con): treatment control; Group 2 (Vcon): vehicle control; Group 3e5: DEHP-exposed groups. BW: Body Weight.
All experiments data presented the means ± standard deviation (S.D.) and were statistical analysis using GraphPad Prism 5.1 Software (USA). Statistical analyses were employed using one-way ANOVA and Tukey's post hoc pairwise comparison. Asterisks (*) indicated statistical differences significantly to Con, *P < 0.05, **P < 0.01 and ***P < 0.001. The expression profile of each gene derived from cerebellum tissue in quails was showed using the heatmap. It is plotted using heatmap R package (version 3.2.1). The data is presented as mean.
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The source code of heatmap package were slightly modified to improve the layout and to add some features, the R function plots was used (Lin et al., 2016a, 2016c). PCA is used as a simplifying information tool from intercorrelation variables with the SPSS 17.0 (SPSS Inc., USA). This analysis method is the key element in this study to define the most important parameters that can be used with the same software as personal changes. The relationship between the parameters observed and quantified was confirmed by the Spearman test. 3. Result and discussion DEHP is a global environmental pollutant and unusually persistent in the environment. The number of DEHP produced worldwide is over 2 million tons each year (US EPA, 2012). Its metabolites and residues have been measured in groundwater, surface water and soils. Pollution occurs through environment and food chain (Heudorf et al., 2007). In recent years, accumulating evidence in wildlife, laboratory animal and cellular systems exists on the adverse health hazard of environmental DEHP. Recent experimental evidence suggests that DEHP exposure could impact the development of brain in animals (Li et al., 2014). The main concerns associated to DEHP pollutant are linked to neurotoxicity (Tanaka, 2005; Wu et al., 2014; Yan et al., 2016), endocrine (Ye et al., 2017; Zhang et al., 2017b) and metabolic disruption (Zhang et al., 2016). There is, however, limited information concerning the neurotoxicity of DEHP in animals following DEHP exposure, especially birds. DEHP has recently attracted attention of the scientific community due to its neural toxic and developmental effects on human beings (Li et al., 2009; Cho et al., 2010). DEHP is a lipophilic compound with the greatest absorption after oral exposure. DEHP is known to cross the blood-brain barrier and accumulate in the brain (Wu et al., 2014). In animals, the growing evidence has suggested that phthalates (including DEHP) exposure was associated with neurobehavioral outcomes, including the effects of self-righting
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ability, reference memory and spatial learning, and decrease of grooming behavior (Tanaka, 2005; Li et al., 2009; Cho et al., 2010). In this study, quails exposed to DEHP showed contracture down of the head, or to the side of the reverse, standing instability, nystagmus, difficulty swallowing, feather growth insufficiency, the realization of mania and hyperreflexia. The structure of Purkinje cell layer and granular layer of quail cerebellum in Con and Vcon groups are clear (Fig. 1A and B). The normal picture of histology in two control groups was observed, including: purkinje cell layer cell rules, large cell body, homogeneous cytoplasm, slightly basophilic cytoplasm, nucleus large round, chromatin was visible, and the distribution of uniform, clear nucleolus. After exposed to DEHP, Purkinje cell shrinks, the shape is not the whole, shrinkage, and some into a small sharp-like, increased eosinophilic cytoplasm in quail cerebellum (Fig. 1CeE). Nuclear condensation deformation, narrow or irregular, volume reduction, coloring deepening, nucleolus is unclear, or even completely disappears, especially in the 750 mg/kg group (Fig. 1E). These results suggested that DEHP exposure induced the severe cerebellar injury and showed the adverse neurological symptoms. NXRs were originally defined to prevent the accumulation of toxic chemicals in the body (Wada et al., 2009). It has been known that NXRs induce the transcription of CYP enzyme isoforms through competitive interaction, which effected by exogenous substances (Ramadoss et al., 2005). PXR, CAR and AhR are ligandactivated transcription factors, and then responsible to regulate the activation of related target genes transcription (Kawamoto et al., 2000; Pascussi et al., 2000; Antos et al., 2015). They share many target genes, such as the isoenzymes (including part of CYP1 isoforms, CYP2 isoforms and CYP3 isoforms) playing a significant role in the metabolism of approximately >90% the currently all used (Williams et al., 2004; Vrba et al., 2014; Prakash et al., 2015). Of note, CYP2/3 are the most widespread enzymes in vertebrates, which regulated by the CAR and PXR, that are responsible for catalyzing up the metabolism of various exogenous toxic substances (Coon, 2005). Various studies have proved the positive
Fig. 1. Effects of DEHP exposure on histopathological change of quail cerebellum. (A) Con (H&E, 400 ); (B) Vcon (H&E, 400 ); (C) D250 (H&E, 400 ); (D) D500 (H&E, 400 ); (E) D750 (H&E, 400 ). (A and B) The structure of macularlayer (I) purkinje cell, (II) and granular layer (III) of cerebellum in Con and Vcon groups was clear. Purkinje cell layer cell rules, large cell body, homogeneous cytoplasm, slightly basophilic cytoplasm, nucleus large round, chromatin was visible, and the distribution of uniform, clear nucleolus. (C) Only the individual cells of apoptosis characteristic changes. (D and E) Purkinje cell exhibited different levels of swelling, partial necrosis. (E) Nuclear condensation deformation, narrow or irregular, volume reduction, coloring deepening, nucleolus is unclear, or even completely disappears. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. Effects of DEHP exposure on the NXRs (AHR/PXR/CAR pathway) response. (A) AHR; (B) PXR; (C) CAR; (D) CYPs. Values were expressed as mean ± S.D. Symbol for the significance of differences between the DEHP-treated groups and Con group: *P < 0.05, **P < 0.01, ***P < 0.001. The mRNA levels of CYPs isoforms were shown using the indicated pseudo color scale from -1x (red) to þ1 (green) relative to values for quail cerebellum in the Con group. The color scale represented the relative mRNA levels, with green indicating up-regulated genes, red indicating down-regulated genes, and black indicating unchanged genes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
effects of DEHP activating NXRs (Wang et al., 2016; Huang et al., 2016; Zhang et al., 2017b). In this study, the expressions of AhR, CAR and PXR presented significant increase in DEHP-exposed quails (Fig. 2AeC). These results suggested that DEHP activated the NXRs (AhR/PXR/CAR pathway) responses. The expressions of cerebellar CYP1 isoforms (CYP1B1), CYP2 isoforms (CYP2D6), CYP3 isoforms (CYP3A4 and CYP3A7) in quail cerebellum were upregulated by DEHP exposure significantly (Fig. 2D and Fig. 3AeF). These results indicated that DEHP metabolism in the nervous system was affected by regulating the transcription of cerebellar CYP enzyme system in quails. Aromatase, as a CYP enzyme is consists of two components: P450arom (cytochrome P450, the product of CYP19 gene) and NCR (Cheshenko et al., 2008). It is an enzyme that catalyzes the transformation of androgens such as testosterone and androstenedione into the estrogens such as estradiol and estrone, respectively, and plays an important role in reproduction and neuroprotection (Roselli, 2007). It could regulate the neuroendocrine events and behaviors. Numerous researches demonstrate that aromatase modulates the neurogenesis, synaptic plasticity and activity, the response of neural tissue to injury (Von Schassen et al., 2006). In addition, AhR-activated endocrine is likely to affect the CYP19 genes expression at the transcription (Cheshenko et al., 2008). In this study, the expressions of AhR and P450arom presented significant increase in DEHP-exposed quails (Figs. 2D and 3B). More
importantly, PCA analysis showed that P450arom and CYP3A4 play an important role in DEHP-induced cerebellum toxicity. The CYP enzymes are present in most domains of life and catalyze the metabolism of a wide variety of xenobiotic compounds. Numerous reports have investigated that the effects of xenobiotics exposure depend heavily on CYP enzyme system hemostasis (Burkina et al., 2017). The present work observed that DEHP exposure significantly increase the hepatic CYP2B1 and CYP3A4 expression and decrease the adrenal CYP11A1 expression in rodent (Liu et al., 2015; Ge et al., 2015; Wang et al., 2014). ERND is known to be at least partially related to CYP3A and APND activity is CYP2B/3A isoform-dependent (Yawetz et al., 1998; Vaccaro et al., 2003). Moreover, the activity of AH was known to be highly associated with CYP2 isoform (Wolf et al., 2004). In this work, DEHP treatment enhanced the activities of NCR and AH and increased the concentrations of total Cyt b5 and total CYP450 in quail cerebellum (Fig. 4AeD). These data suggested that DEHP caused the alternations of the levels of CYPs and Cyt b5 and the activities of CYP enzymes (NCR and AH) in cerebellum with the dose-related manner significantly. These results indicated that DEHP exposure triggered the disruption of the CYP enzyme system homeostasis in the cerebellum via activating the NXRs responses and inducing CYP enzyme isoforms expression in the nervous system of quails. Our study also provided a lot of novel evidences that DEHP changed the activities of NCR and AH via activating the AhR/PXR/CAR pathway
Please cite this article in press as: Du, Z.-H., et al., A novel nuclear xenobiotic receptors (AhR/PXR/CAR)-mediated mechanism of DEHP-induced cerebellar toxicity in quails (Coturnix japonica) via disrupting CYP enzyme system homeostasis, Environmental Pollution (2017), http:// dx.doi.org/10.1016/j.envpol.2017.04.015
5 Fig. 3. Effects of DEHP exposure on the expressions of CYPs isoforms. (A) CYP1B1; (B) P450arom; (C) CYP2D6; (D) CYP3A7; (E) CYP4V2; (F) CYP3A4. Values were expressed as mean ± S.D. Symbol for the significance of differences between the DEHP-treated groups and Con group: *P < 0.05, **P < 0.01, ***P < 0.001.
Z.-H. Du et al. / Environmental Pollution xxx (2017) 1e9
Please cite this article in press as: Du, Z.-H., et al., A novel nuclear xenobiotic receptors (AhR/PXR/CAR)-mediated mechanism of DEHP-induced cerebellar toxicity in quails (Coturnix japonica) via disrupting CYP enzyme system homeostasis, Environmental Pollution (2017), http:// dx.doi.org/10.1016/j.envpol.2017.04.015
Z.-H. Du et al. / Environmental Pollution xxx (2017) 1e9 Fig. 4. Effects of DEHP on the CYPs homeostasis. (A) Total CYP450 content; (B) The Cyt b5 content; (C) The AH activity; (D) The NCR activity; (E) The APND activity; (F) The ERND activity. Values were expressed as mean ± S.D. Symbol for the significance of differences between the DEHP-treated groups and Con group: *P < 0.05, **P < 0.01, ***P < 0.001.
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Please cite this article in press as: Du, Z.-H., et al., A novel nuclear xenobiotic receptors (AhR/PXR/CAR)-mediated mechanism of DEHP-induced cerebellar toxicity in quails (Coturnix japonica) via disrupting CYP enzyme system homeostasis, Environmental Pollution (2017), http:// dx.doi.org/10.1016/j.envpol.2017.04.015
Z.-H. Du et al. / Environmental Pollution xxx (2017) 1e9
responses and increasing the transcription of CYP3A isoforms in quail cerebellum. The present study investigated the serve toxic effects of DEHP on the cerebellum and demonstrated that DEHP could cause the neurotoxicity in quail. P450arom and CYP3A4 are sensitivities and responses to DEHP in cerebellum (Fig. 5A). These results provided some new insight that P450arom and CYP3A4 are able to be biomarkers in evaluating the impact of DEHP exposure in bird. DEHP exposure triggered the AhR/PXR/CAR pathway and altered the transcription of cerebellar CYP enzymes isoforms in quails.
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Additionally, all parameters were divided into the first, second and third principle components (46.9, 27.3 and 14.1%) based on ordination plots corresponding (Table S2). Component plot indicated that DEHP-induced cerebellar toxicity presented dose-dependent effect (Fig. 5B). 4. Conclusion This study provided several novel evidences that DEHP exposure induced the toxic effects of quail cerebellum. DEHP exposure
Fig. 5. PCA of the mechanism of NXRs response to DEHP exposure. (A) PCA score plot results comparing biochemical parameters. All parameters were divided into the first, second and third principle components (46.90%, 27.37% and 14.18%) based on ordination plots corresponding. The red represented the key factors for individual variations. (B) PCA score plot results comparing biochemical parameters. There was a dose-dependent relationship between the groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. The pathway of DEHP modulated NXRs responses in quail cerebellum. DEHP regulated CYPs by activation of NXRs pathway responses and inducing CYPs isoforms transcription. Thereby NXRs responses promoted the processing of the detoxification and elimination of DEHP. Cerebellar P450arom and CYP3A4 might be biomarkers in evaluating the neurotoxicity of DEHP in bird.
Please cite this article in press as: Du, Z.-H., et al., A novel nuclear xenobiotic receptors (AhR/PXR/CAR)-mediated mechanism of DEHP-induced cerebellar toxicity in quails (Coturnix japonica) via disrupting CYP enzyme system homeostasis, Environmental Pollution (2017), http:// dx.doi.org/10.1016/j.envpol.2017.04.015
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Z.-H. Du et al. / Environmental Pollution xxx (2017) 1e9
disrupted the cerebellar CYP enzyme system homeostasis via affect the transcription of CYP enzyme isoforms. The cerebellar AhR/PXR/ CAR pathway responses were activated by expose to DEHP in quail. The results of this study show for the first time that the DEHPinduced toxic effects of quail cerebellum were associated with activating NXRs responses and disrupting the CYP enzyme system homeostasis (Fig. 6). More importantly, cerebellar P450arom and CYP3A4 might be biomarkers in evaluating the neurotoxicity of DEHP in bird. However, the further mechanism of the neurological symptoms and the role of cerebellar P450arom and CYP3A4 will be required in the DEHP-exposed birds in future research. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by China New Century Excellent Talents in University (No. NECT-1207-02), National Natural Science Foundation of China (No. 31572586) and Academic Backbone Project of 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.2017.04.015. References Antos, P.A., Blachuta, M., Hrabia, A., Grzegorzewska, A.K., Sechman, A., 2015. Expression of aryl hydrocarbon receptor 1 (AHR1), AHR1 nuclear translocator 1 (ARNT1) and CYP1 family monooxygenase mRNAs and their activity in chicken ovarian follicles following in vitro exposure to 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD). Toxicol. Lett. 237, 100e111. Bakir, A., O'Connor, I.A., Rowland, S.J., Hendriks, A.J., Thompson, R.C., 2016. Relative importance of microplastics as a pathway for the transfer of hydrophobic organic chemicals to marine life. Environ. Pollut. 219, 56e65. Burkina, V., Rasmussen, M.K., Pilipenko, N., Zamaratskaia, G., 2017. Comparison of xenobiotic-metabolising human, porcine, rodent, and piscine cytochrome P450. Toxicology 375, 10e27. Cheshenko, K., Pakdel, F., Segner, H., Kah, O., Eggen, R.I.L., 2008. Interference of endocrine disrupting chemicals with aromatase CYP19 expression or activity, and consequences for reproduction of teleost fish. Gen. Comp. Endocrinol. 155, 31e62. Cho, S.C., Bhang, S.Y., Hong, Y.C., Shin, M.S., Kim, B.N., Kim, J.W., Yoo, H.J., Cho, I.H., Kim, H.W., 2010. Relationship between environmental phthalate exposure and the intelligence of school-age children. Environ. Health Perspect. 118, 1027e1032. Coon, M.J., 2005. Cytochrome P450: nature's most versatile biological catalyst. Annu. Rev. Pharmacol. Toxicol. 45, 1e25. Dhanya, C.R., Indu, A.R., Deepadevi, K.V., Kur, P.A., 2003. Inhibition of membrane Na(þ)-Kþ Atpase of the brain, liver and RBC in rats administered di(2-ethyl hexyl) phthalate (DEHP) a plasticizer used in polyvinyl chloride (PVC) blood storage bags. Indian J. Exp. Biol. 41, 814e820. Du, Z.H., Qin, L., Lin, J., Sun, Y.C., Xia, J., Zhang, C., Li, X.N., Li, J.L., 2017. Activating nuclear xenobiotic receptors and triggering ER stress and hepatic cytochromes P450 systems in quails (Coturnix C. coturnix) during atrazine exposure. Environ. Toxicol. http://dx.doi.org/10.1002/tox.22404. Dvorak, Z., Pavek, P., 2010. Regulation of drug-metabolizing cytochrome P450 enzymes by glucocorticoids. Drug Metab. Rev. 42, 621e635. Foster, P.M., Mylchreest, E., Gaido, K.W., Sar, M., 2001. Effects of phthalate esters on the developing reproductive tract of males rats. Hum. Reprod. Update 7, 231e235. Ge, J., Han, B., Hu, H., Liu, J., Liu, Y., 2015. Epigallocatechin-3-O-Gallate protects against hepatic damage and testicular toxicity in male mice exposed to di-(2ethylhexyl) phthalate. J. Med. Food 18, 753e761. Heudorf, U., Mersch-Sundermann, V., Angerer, J., 2007. Phthalates: toxicology and exposure. Int. J. Hyg. Environ. Health 210, 623e634. Huang, Q., Zhang, H., Chen, Y.J., Chi, Y.L., Dong, S., 2016. The inflammation response to DEHP through PPARg in endometrial cells. Int. J. Environ. Res. Public Health 13, E318. Jiang, X.Q., Cao, C.Y., Li, Z.Y., Li, W., Zhang, C., Lin, J., Li, X.N., Li, J.L., 2017. Delineating
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Please cite this article in press as: Du, Z.-H., et al., A novel nuclear xenobiotic receptors (AhR/PXR/CAR)-mediated mechanism of DEHP-induced cerebellar toxicity in quails (Coturnix japonica) via disrupting CYP enzyme system homeostasis, Environmental Pollution (2017), http:// dx.doi.org/10.1016/j.envpol.2017.04.015