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Food Safety and Toxicology
Atrazine triggers mitochondrial dysfunction and oxidative stress in quail (Coturnix C. coturnix) cerebrum via activating xenobioticsensing nuclear receptors and modulating cytochrome P450 systems Jia Lin, Hua-Shan Zhao, Xue-Nan Li, Lei Qin, Cong Zhang, Jun Xia, and Jinlong Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01413 • Publication Date (Web): 05 Jun 2018 Downloaded from http://pubs.acs.org on June 5, 2018
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Synopsis for the Graphical Abstract: Atrazine can be absorbed into the quail cerebrum through a trophic chain that eventually results in neurotoxicity. Our findings provide evidence that mitochondria are the target organelle. Atrazine induced mitochondrial dysfunctions via oxidative stress and CYP450 disorder. 168x93mm (300 x 300 DPI)
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Atrazine triggers mitochondrial dysfunction and oxidative stress in quail
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(Coturnix C. coturnix) cerebrum via activating xenobiotic-sensing nuclear
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receptors and modulating cytochrome P450 systems
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Jia Lin1*, Hua-Shan Zhao1*, Lei Qin1,2, Xue-Nan Li1, Cong Zhang1, Jun Xia1, Jin-Long Li1,3,4**
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1
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China
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2
Laboratory animal centre, Qiqihar Medical University, Qiqihar 161006, P.R. China
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3
Key Laboratory of the Provincial Education Department of Heilongjiang for Common Animal
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Disease Prevention and Treatment, Northeast Agricultural University, Harbin 150030, P.R.
College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, P.R.
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China
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4
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Agricultural University, Harbin 150030, P.R. China
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*These authors contributed equally to this study.
Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine, Northeast
14 15 16 17 18
**Corresponding author: Jin-Long Li
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Full address: College of Veterinary Medicine, Northeast Agricultural University, NO.600
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Changjiang Street Xiangfang District, Harbin 150030, Heilongjiang Province, China
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Tel: +86 451 55190407; fax: +86 451 55190407.
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E-mail address:
[email protected] (J.L. Li)
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ABSTRACT
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The residues from the widely used broad-spectrum environmental herbicide, atrazine (ATR)
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results in the exposure of non-target organisms and persisted as a global major public health
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hazard. ATR is neurotoxic and may cause adverse health effects in mammals, birds and fishes.
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Nevertheless, the molecular mechanism of ATR induced neurotoxicity is remains unclear. To
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assess the molecular mechanisms of ATR-induced cerebral toxicity through potential
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oxidative damage, quail were treated with ATR by oral gavage administration at doses of 0,
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50, 250 and 500 mg/kg body weight daily for 45 days. Markedly, increases in the amount of
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swelling of neuronal cells, the percentage of mean damaged mitochondria, mitochondrial
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malformation, and mitochondrial vacuolar degeneration, as well as decreases in the
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mitochondrial cristae and mitochondrial volume density were observed by light and electron
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microscopy in the cerebrum of quail. ATR induced toxicities in the expression of
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mitochondrial function-related genes and promoted oxidative damage, as indicated by
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effects on oxidative stress indices. These results indicated that ATR exposure can cause
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neurological disorders and cerebral injury. ATR may initiate apoptosis by activating Bcl-2, Bax
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and Caspase3 protein expression, but failed to induce autophagy (LC3B has not cleaved to
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LC3BⅠ/Ⅱ). Furthermore, ATR induced CYP-related enzymes metabolism disorders by
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activating the nuclear xenobiotic receptors response (NXRs including AHR, CAR and PXR) and
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increased expression of several CYP isoforms (including CYP1B1 and CYP2C18) and thereby
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producing mitochondrial dysfunction. In this study, we observed ATR exposure resulted in
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oxidative stress and mitochondrial dysfunction by activating the NXR response and
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interfering the CYP450s homeostasis in quail cerebrum that supported the molecular
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mechanism of ATR induced cerebrum toxicity. In conclusion, these results provided new
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evidence on molecular mechanism of ATR induced neurotoxicity.
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KEYWORDS: Atrazine; Quail cerebrum; Nuclear xenobiotic receptors response; Cytochromes
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P450 systems; Mitochondrial dysfunction
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INTRODUCTION
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Atrazine (ATR, C8H14ClN5), as a widely used herbicide, can continually be detected in soil and
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rivers, which leads to a potential ecological hazard 1, 2. It has been shown that ATR measured
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in waters across the US were
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drinking water and were all > 14 days in duration
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months
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carcinogenicity 6. Extensive study has revealed ATR has negative impacts on humans and
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animals, especially in the nervous system
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rural workers in ATR-exposed environments has been shown to be increased
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appears to cause neurotoxic effects by eliciting transient neurochemical alterations
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Furthermore, the reduced dopamine levels in the brain indicate that ATR affects the
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metabolism of dopamine, which results in neurodegenerative disorders
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P450 system (CYP450s) belong to a multi-gene family of heme proteins that, in addition to
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their role in the metabolism of xenobiotics (such as ATR) in hepatocytes, are also expressed
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in neurocytes
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products that cause biological macromolecules damage, then tissue injury 16-19. It is generally
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known that the metabolism of xenobiotic via alterations in the CYP450s provides useful
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biomarkers to the field of ecotoxicology assays 20. Several reports support an emergent role
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of CYP450s in the pathogenesis of neurological disorders. The brain CYP450s response could
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be one of the multiple factors that influence xenobiotic exposures. The transcriptional
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regulation of major xenobiotic-metabolizing CYP450s and phase II enzymes occurs through
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nuclear xenobiotic receptors (NXRs), which include aryl hydrocarbon receptor (AHR),
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pregnane X receptor (PXR), constitutive androstane receptor (CAR), the glucocorticoid
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receptor (GR) and so on
2, 5
exceeded the maximum contaminant level allowed for 3, 4
, with a half-life ranging from 1 to 3
. An important finding was that ATR and its metabolites were predicted to have
7-9
. The incidence of Parkinson's disease among
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10, 11
. ATR 12
.
. The cytochrome
14, 15
. Exogenous substances interact with the CYPs, generating oxidation
21
. NXRs exert positive effects on the metabolism, as well as
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reproductive and developmental homeostasis 15. A large body of evidence has demonstrated
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that the xenobiotic ATR induced significant CYP450 disorders
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demonstrated that ATR is metabolically biotransformed by CYP450s in mouse liver
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However, the effects of ATR on the xenobiotic metabolism via CYP450 alterations and NXR
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response in nervous system are unclear.
20, 22, 23
. Our earlier study 24
.
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CYP isoforms (CYPs) are mainly localized in microsomes, also can bind to the internal
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membranes of the mitochondria 25 because of a stretch of N-terminal chimeric signals 26-33. In
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this context, the total CYP450 content in brain mitochondria was much higher than that of
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the corresponding microsomes
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with or respond to various signaling pathways and contribute to mitochondrial injury. ATR
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exposure induces not only significant oxidative stress 35, 36 but also mitochondrial dysfunction
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damage to the cells and mitochondria. Conversely, increased ROS levels alter the glutathione
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metabolism as well as the mitochondrial dysfunction
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frequently proposed to be involved in neurodegenerative pathogenesis
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indicated that ATR induces mitochondrial degeneration and apoptosis-related damage of
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neurodegeneration in neurocytes 42. This might have been because the energy requirement
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is high in the brain, and mitochondrial dysfunction may be a serious threat to cell survival
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that may result in brain injury 43.
34
. Certain xenobiotic-inducible CYPs are either associated
. Mitochondrial dysfunction increases the intracellular ROS levels, which lead to further
38-40
. Mitochondrial dysfunction is 41
. A recent study
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The widespread contamination and persistence of ATR residues in the environment
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results in the exposure of non-target organisms. The oxidative stress and NXR response that
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are induced by ATR play important roles in the toxicity in organisms. The quail were widely
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used as a model to assess the variations in the environmental levels of anthropogenic
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pollutants. However, little information is available about ATR-induced toxicity, especially its
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neurotoxicity. This study explores the underlying mechanism by which ATR causes oxidative
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damage, mitochondrial dysfunction, the NXR response and CYP450 disorders in the
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cerebrum. Finally, this study provided some new evidence on the mechanisms of
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ATR-induced neurotoxicity.
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MATERIALS AND METHODS
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Ethics statement
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All experimental procedures and humane endpoints for minimizing suffering were approved
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by the Guide for the Care and Use of Laboratory Animals prepared by Northeast Agriculture
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University, Harbin, China.
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Experimental animals and chemicals
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18 days old male European quail (Coturnix C. coturnix) chicks (89.17 ± 0.86 g) were employed
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in this study, obtained from Wan Jia farm (Harbin, China). To ensure the same conditions for
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all the experiments, these quail were maintained at constant laboratory temperature (26 ±
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2 °C) and under a 12-h inverted dark/light cycle. Free intake of feed and water were allowed
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during the entire experimental period. The ATR (purity≥90%), obtained from Zhonghe
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Chemical Limited Company, Binzhou, China, was dissolved in distilled water for gavage
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administration to quail.
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Experimental design
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The quail (20 quail/group) were categorised as Control group (distilled water), 50 mg/kg
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ATR-exposed group, 250 mg/kg ATR-exposed group, and 500 mg/kg ATR-exposed group. The
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dosage of ATR was employed in this study on the basis of recent studies
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doses of ATR were chosen to reveal the adverse effects and the target organ and organelle of
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ATR’s toxicity. The quail were treated with ATR by means of intragastric administration for 45
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day. After fasted for 12 h, the quail were euthanized by cervical dislocation to obtain the
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. These high
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whole brain and then isolate cerebrum from brain. The cerebrums (N=10/group) were frozen
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immediately at -80 °C to avoid repeated freeze-thaw cycles, and processed for further
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biochemical analysis, while the other cerebrums were isolated, fixed, and processed for
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histological examination (N=5/group) and electron microscopy analysis (N=5/group). The
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clinical behavior, body weight of quail was recorded every day.
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Histopathological and ultrastructural analysis
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Cerebrums (N=5/group) were preserved in 10% buffered formalin for 24 h. Then, the tissues
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was dehydrated through a graded series of ethanol, cleared in xylene and embedded in
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paraffin.
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Sections (5 μm thickness) were prepared and stained with hematoxylin and eosin (H&E) for
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microscopic examination (M3, Precipoint, Germany). For electron microscopy analysis,
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cerebral specimens (N=5/group) were treated by the method as described in previous study
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Japan).
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The measurement of mitochondrial injury and mitochondria volume density
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The measurement of mitochondrial injury
and observed in transmission electron microscopy (TEM, H-7650, Hitachi Limited, Tokyo,
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For more detailed analysis of organelle changes in the neurons, electron micrographs
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(20,000× magnification, N ≥ 5 pictures each group) were examined morphometrically under a
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grid. The severity of mitochondrial injury was graded 0 to 4 as previous study
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morphologic degree of injury. A mean mitochondrial grade for each cell was obtained by
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calculating the weighted average (0 to 4) of mitochondrial injury. 3 examiners have no idea
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whether the electron micrographs were from untreated or treated quail before they graded
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the mitochondria.
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The measurement of mitochondria volume density
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, up to its
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“Point counting grids” has been used to quantify mitochondria from transmission
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electron microscopy data recording to previous study 48.
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Measurement of ATR and its’ major metabolite DACT concentration
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Tissue extraction and analyte method were depended on previous method49.
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Measurement of CHE activity
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The protein concentration of the cerebrum cholinesterase (CHE) activity was measured using
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a detection kit (Nanjing Jiancheng bioengineering institute, Nanjing, China).
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RNA purification and qRT-PCR
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Analysis of the mRNA levels of CYPs, NXRs and mitochondria-related genes in the cerebrum
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was done by using quantitative real-time PCR (qRT-PCR). Total RNA was extracted from
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cerebral tissue homogenate and transcribed into cDNA as described by previous study50. The
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amplified products were quantified by spectrophotometry at 260 and 280 nm.
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The specific oligonucleotide primers for the CYPs, NXRs and mitochondria-related genes
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were list in Table S1, designing by Oligo 7.22. To screen qRT-PCR primers, the expected
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fragment size should be confirmed by gel electrophoresis after PCR reactions. After that,
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qRT-PCR was performed using LightCycler® 480 (Roche, CH). To calculate the mRNA relative
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expression, the 2-ΔΔCt method were used and normalized to the mean of the values for
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β-Actin 1 and β-Actin 2.
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Measurement of protein content
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The protein concentration of the cerebrum samples was measured using a quantitative
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protein detection kit (Nanjing Jiancheng bioengineering institute, Nanjing, China),
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constructed with bovine serum albumin (BSA, Beyotime, Shanghai, China) as the standard
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curve.
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Measurement of oxidative stress indices
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The oxidative stress indices including total antioxidant capacity (T-AOC), total superoxide
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dismutase (T-SOD), catalase (CAT), glutathione S-transferase (GST), hydrogen peroxide (H2O2)
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and malondialdehyde (MDA) were determined in 10% tissue homogenates using detection
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kits
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manufacturer's instructions. The levels of T-AOC, CAT, GST, H2O2, MDA were assayed by
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colorimetric method at 520 nm, 405 nm, 412 nm, 405 nm and 532 nm, respectively. The
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activity of T-SOD was assayed at 550 nm using the hydroxylamine method.
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Measurement of the CYP450 contents and CYP450 enzymatic activities
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The difference spectra were determined by recording the reduced vs. oxidized and the
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reduced vs. CO-bound reduced pigments for the measurement of The contents of total
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CYP450 and Cytb5 as described by the method of Omura and Sato51 Furthermore, the
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activities of cerebrum aminopyrin N-demethylase (APND), erythromycin N-demethylase
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(ERND), aniline-4-hydeoxylase (AH) and the NADPH-cytochrome c reductase (NCR) were also
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measured in this study according to the methods described in our previous study 24.
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The heatmap of CYP expression
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The expression profile for each gene derived from cerebral tissue in the quail is shown using
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heatmaps. They were plotted using R package (version 3.2.1), presented as the mean value
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of data.
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Western Blotting Assay
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Cerebrum tissue specimens were lysed in cold RIPA lysis buffer (Beyotime, Shanghai, China)
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plus 1: 100 volume of protease inhibitor cocktail (MedChem Express, HY-K0010, USA),
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pelleted by centrifugation at 16,000 xg for 15 minutes at 4 °C and boiled in 5× SDS-PAGE
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sample loading buffer (Beyotime, P0015, Shanghai, China). Proteins were separated on 12%
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SDS-PAGE gels, transferred to PVDF membranes (Biotopped, IPVH00010, Beijing, China) and
(Nanjing
Jiancheng
bioengineering
institute,
Nanjing,
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following
the
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immunoblotted with commercial antibodies. The antibodies were purchased by GoodHere,
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Hangzhou, China, Bioss, bs-0061, Beijing, China, ABclonal, Wuhan, China. The immune
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complex was detected by ECL kit (Beyotime, P0018, Shanghai, China) and quantified by
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Image J software.
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Statistical analysis
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GraphPad Prism 5.1 software was applied to statistically analyze the effect of treatment on
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the incidence of neurotoxicity in ATR exposed animals. The experimental data were
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presented as the means ± standard deviation (S.D.). One-way ANOVAs followed by Tukey’s
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post hoc pairwise comparisons to meet statistical demand. Statistical significance from
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control group was considered for *P < 0.05, **P < 0.01 and ***P < 0.001. SPSS 17.0 (SPSS Inc.,
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USA) was used for principal component analysis (PCA).
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RESULTS
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Effects of ATR on clinical behavior and body weight
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The quail in 500mg/kg atrazine group were easier to be scared and sensitive to external
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stimulus than the control group. ATR decreased the body weight of quail significantly (Fig 1A,
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B).
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ATR and DACT concentration in cerebrum tissues.
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The content of ATR and DACT were markedly upregulated in the cerebrum of quail exposed
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to ATR, as compared to that of controls (Fig. 1C). Moreover, the DACT levels were higher than
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ATR levels (Fig. 1C).
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Effects of ATR on CHE activity
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CHE activity in quail’s cerebrum and serum were showed in Fig. 1D. ATR significantly
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decreased cerebral CHE activity in 250/500mg/kg groups and serum CHE activity in
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500mg/kg groups, as compared to that of controls (P < 0.01, P < 0.001; P < 0.001).
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Effects of ATR on histologic and morphometric features in the cerebrum
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Changes in the cerebral cortex neuronal cells in the various groups were observed (Fig. 1E).
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The neuronal cells in the control group revealed normal structure and staining. In contrast,
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swollen neuronal cells were observed in all ATR-treated groups. Additionally, some of the
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neurons showed lighter staining for H&E in the 50 and 250 mg/kg ATR-treated groups, which
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revealed a decreased affinity. More of the neuronal cells showed lighter and uneven staining
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in the 500 mg/kg ATR-treated group. A normal density of the mitochondrial cristae, cell
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membrane integrity, no swelling, and normal structures of the neuronal cells were observed
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in the control group (Fig. 1F). In the ATR-exposed groups, the cristae of the mitochondria in
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the cytoplasm decreased and appeared to be fractured, and mitochondrial vacuolar
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degeneration and malformation were observed by TEM in the ATR-treated group. All these
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characteristics occurred in cells that were undergoing an injury process.
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Effects of ATR on mitochondrial dysfunction
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Fig. 1I shows that there were no massive injury with disruption of cristae and rupture of
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inner and outer mitochondrial membranes (Grade 4) in all experimental groups. In the
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control group, percentage of mean mitochondrial grade 0-3 is 80.37%, 14.70%, 3.91%, 1.02%.
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In the 50 mg/kg ATR-treated group, percentage of mean mitochondrial grade 0-3 is 63.52%,
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25.99%, 7.51%, 2.98%. In the 250 mg/kg ATR-treated group, percentage of mean
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mitochondrial grade 0-3 is 61.79%, 25.61%, 7.63%, 4.97%. In the 500 mg/kg ATR-treated
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group, percentage of mean mitochondrial grade 0-3 is 46.92%, 17.80%, 19.26%, 16.02%.
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Percentage of mitochondria volume density was significantly decreased in 250, 500 mg/kg
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ATR-treated groups (P < 0.01, P < 0.001) compared to the control group (Fig. 1J).
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To investigate the effects of ATR on mitochondrial dysfunction, the mRNA levels of
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mitochondrial function-related genes were measured using qRT-PCR (Fig. 2). There were no
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overt alterations between the control and 50 kg/mg ATR-exposed group for the Raf-1
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proto-oncogene (RAF1) and peroxiredoxin3 (PRDX3) levels. 250 and 500 kg/mg ATR lowered
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the mRNA levels of RAF1 and PRDX3 (RAF1, 0.036 and 0.086-fold, P < 0.001, P < 0.001;
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PRDX3, 0.551 and 0.475-fold, P < 0.05, P < 0.05, respectively) (Fig. 2A-B). Interestingly, ATR
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significantly reduced the cytochrome c (CYCS), cytochrome c oxidase assembly factor 6
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(COA6) and Sirtuin1 (SIRT1) mRNA expression (CYCS, 0.220, 0.309, 0.454-fold, P < 0.01, P