Atrazine Triggers Mitochondrial Dysfunction and Oxidative Stress in

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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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Journal of Agricultural and Food Chemistry

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

13

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


44-46

. 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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46

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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,


China)

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

Atrazine Triggers Mitochondrial Dysfunction and Oxidative Stress in

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