Tebuconazole induced oxidative stress related hepatotoxicity in adult and larval zebrafish (Danio rerio)

Journal Pre-proof Tebuconazole induced oxidative stress related hepatotoxicity in adult and larval zebrafish (Danio rerio) Shuying Li, Yao Jiang, Qianqian Sun, Scott Coffin, Lili Chen, Kun Qiao, Wenjun Gui, Guonian Zhu PII:

S0045-6535(19)32368-9

DOI:

https://doi.org/10.1016/j.chemosphere.2019.125129

Reference:

CHEM 125129

To appear in:

ECSN

Received Date: 6 August 2019 Revised Date:

14 October 2019

Accepted Date: 14 October 2019

Please cite this article as: Li, S., Jiang, Y., Sun, Q., Coffin, S., Chen, L., Qiao, K., Gui, W., Zhu, G., Tebuconazole induced oxidative stress related hepatotoxicity in adult and larval zebrafish (Danio rerio), Chemosphere (2019), doi: https://doi.org/10.1016/j.chemosphere.2019.125129. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Graphic Abstract

1

Tebuconazole induced oxidative stress related hepatotoxicity in

2

adult and larval zebrafish (Danio rerio)

3 4

Shuying Li a, Yao Jiang a, Qianqian Sun a, Scott Coffin b, Lili Chen a, Kun Qiao a, Wenjun Gui a,*,

5

Guonian Zhu

a

6 7

a

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Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, P. R. China

b

Department of Environmental Sciences, University of California, Riverside, CA, 92521, United States

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Corresponding Author

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Name: Wenjun Gui

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Address: Institute of Pesticide and Environmental Toxicology, Zhejiang University,

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Hangzhou, 310058, P. R. China

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Phone/fax: +86 571 88982220

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E-mail: [email protected]

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Abstract:

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Tebuconazole is widely used as fungicide and has frequently been detected at elevated

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concentrations in environmental media. To characterize the potential toxicity of

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tebuconazole on vertebrate and humans. Using zebrafish as a vertebrate model, the

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toxic effects in liver that produced by low-toxic concentrations of tebuconazole were

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assessed in adult zebrafish. We further focused on tebuconazole-induced toxicity and

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its possible mechanism in larval zebrafish using a hepatotoxicity assay. The induction

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of oxidative stress in adult fish was evaluated by superoxide dismutase (T-SOD),

26

catalase (CAT), peroxidase (POD), glutathione S-transferase (GST) activity, and the

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increased aspartate aminotransferase (AST)/ alanine aminotransferase (ALT) ratio.

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Significantly increased enzyme activity was observed in the liver of male and female

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fish at both exposure and depuration stage. Exposure to maximum non-lethal (MNLC)

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concentration of tebuconazole from 72 to 120 hours post-fertilization (hpf) affected

31

the liver size and yolk retention in larval zebrafish. Decreased fluorescence intensity

32

was observed in larval Tg(Apo14:GFP) zebrafish, indicating liver degeneration after

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tebuconazole treated. Histopathological examination confirmed the alterations in liver

34

histoarchitecture in exposed zebrafish. Significant 1.28-fold and 1.65-fold increases in

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reactive oxygen species levels were observed in juveniles exposed to MNLC and

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lethal concentration 10 (LC10) group, respectively. The acridine orange staining assay

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showed that apoptotic cells occurred in the liver regions. These results indicated that

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tebuconazole exposure resulted in impacts on the ecological risk in fish and vertebrate.

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Overall, the present study suggested further research in needed to better understand

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the tebuconazole-induced toxicity mechanism that associated with oxidative stress.

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Keywords: Tebuconazole; Zebrafish; Hepatotoxicity; Apoptosis; Reactive oxygen

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species

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1.Introduction

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In recent years, the impact of environmental factors on the health of aquatic

47

environments, such as heavy metals, nutrition, chemical exposures, and physical

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variables have increased in concern (Volz et al., 2016; Liang et al., 2017; Moreira et

49

al., 2018). Notably, exposure to chemicals has been identified as a significant stressor

50

for aquatic ecosystems (Moreira et al., 2018). The liver is a sensitive target for

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toxicants due in part to the active proliferative response of hepatocytes. During the

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liver’s detoxification and excretion processes, which include synthesizing,

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concentrating, and secreting bile acids, and excreting toxicants, chemicals can induce

54

injury to hepatocytes and lead to cholestasis. In turn, cholestasis results in intrahepatic

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accumulation of toxic bile acids and excretion products, which promotes further

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hepatic injury (Jaeschke et al., 2002). Chemical-induced hepatic injury has been

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recognized as a major toxicological problem in both aquatic ecosystems and humans.

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Zebrafish are increasingly being applied as an in vivo model system for the evaluation

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of chemicals on hepatic safety (McGrath and Li, 2008; Deng et al., 2009), and studies

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have confirmed that mammalian and zebrafish toxicity profiles are strikingly similar

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(McGrath and Li, 2008; Deng et al., 2009; He et al., 2013). A useful trait of zebrafish

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as a model species is its physical transparency at early stages, which allows for in vivo

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visual observations of internal organs, including the liver. Apolipoproteins (Apo),

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which transport lipids in vertebrates, have been suggested to play significant roles

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during early development (Wang et al., 2011). The transgenic GFP zebrafish line

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Tg(Apo14:GFP) which marks liver function, could be used to trace the impacts of

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chemical exposure on the developmental processes of the liver (Wang et al., 2011).

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Conventional tests for evaluating chemical-induced hepatotoxicity in animals include

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enzyme assays, hepatic excretory tests, alterations in liver assessment, histological

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analyses, as well as apoptosis (Wiegand et al., 2000; Jaeschke et al., 2002; McGrath

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and Li, 2008; He et al., 2013) . Recently, studies have shown that zebrafish exhibit

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xenobiotic defense mechanisms in highly similar ways to mammals, such as oxidative

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stress, and enzyme induction (Carney et al., 2004).

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Tebuconazole, [(RS)-1-p-chlorophenyl-4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)

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pentan-3-ol], is a sterol demethylation inhibitor fungicide that is used to control

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numerous pathogens in crops such as fruits, vegetables and cereals, with an

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environmental half-life from 300 to 600 days in soil (Strickland et al., 2004; Cui et al.,

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2018; Li et al., 2019a). Due to its relative aqueous solubility, tebuconazole can be

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transported into aquatic ecosystems through runoff, and coupled with widespread use,

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results in environmental aqueous concentrations ranging from 0.6 to 200 µg/L

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(Richardson and Kimura, 2007; Rabiet et al., 2010; Zhang et al., 2015). Previous

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studies have demonstrated that exposure to tebuconazole induced various toxic effects

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in aquatic organisms, such as thyroid toxicity (Yu et al., 2013; Li et al., 2019b),

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developmental toxicity (Bernabò et al., 2016; Li et al., 2019a), genotoxicity (Castro et

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al., 2018), neurotoxicity (Altenhofen et al., 2017) and reproductive toxicity (Sancho et

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al., 2016; Li et al., 2019a). Hepatotoxicity induced by tebuconazole has been observed

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in vitro and in vivo (Schmidt et al., 2016; Knebel et al., 2019). In vivo effects of

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tebuconazole exposure include increases in liver weight and centrilobular hypertrophy

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in rats and mice (Schmidt et al., 2016). Additionally, tebuconazole activates

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transcription of aryl hydrocarbon receptor dependent genes in human cell lines and in

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rat liver in vivo (Knebel et al., 2019). As tebuconazole persists within environmental

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media and exhibits developmental toxicity effects (Bernabò et al., 2016; Li et al.,

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2019b), exposure to tebuconazole may pose a health risk to humans and ecosystems,

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particularly during sensitive windows of development.

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Using zebrafish as a model, we previously demonstrated in bioaccumulation tests

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that the highest enrichment of tebuconazole was observed in livers of adult zebrafish

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(Li et al., 2019a), with results indicating that tebuconazole may induce hepatotoxic

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effects in zebrafish. Despite several studies on tebuconazole’s biological effects,

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including several toxicological studies in fish, and tebuconazole’s behavior in the

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environment, few studies have investigated the potential impacts and mechanisms of

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tebuconazole on hepatotoxicity within adult and larval zebrafish. Therefore, the

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objectives of this study were to determine whether (1) tebuconazole induces

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hepatotoxicity in adult zebrafish; (2) uptake of tebuconazole from 72 hpf to 120 hpf

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leads to impacts on the hepatic development during larval zebrafish stage; and (3)

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tebuconazole-induced impact on liver are related to reactive oxygen species (ROS)

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and the apoptosis pathway.

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2 Materials and Methods

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2.1 Chemicals and Materials

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Tebuconazole (CAS 107534-96-3) standard (purity > 98 %) was purchased from

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Shanghai Yuanji Chemical Co., Ltd. (Shanghai, China). Tebuconazole stock solution

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(1000 mg/L) was dissolved in dimethyl sulfoxide (DMSO) and stored at -20

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more than three months. Tricaine (CAS 886-86-2; “MS-222”) was obtained from

113

Sigma (St. Louis, MO, USA). Methanol and acetonitrile were obtained from Merck

114

(Darmstadt, Germany). Acridine orange was obtained from Sigma. TRIzolTM,

115

RNase-free water, PrimeScriptTM RT reagent with gDNA Eraser Kit and TB Green

116

Premix Ex TapTM II were purchased from Takara (Dalian, China).

117

2.2 Zebrafish Maintenance

118

The cultivation of zebrafish (AB strain zebrafish purchased from the Institute of

119

Hydrobiology, Chinese Academy of Sciences; Tg(Apo14:GFP) strain zebrafish

120

obtained from Core Facilities, Zhejiang University School of Medicine) and artificial

121

insemination of eggs were performed according to previous methods, with minor

122

modifications (Wang et al., 2015a; Ma et al., 2016). Fish were maintained as

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described in Supporting Information (SI-1). All procedures were conducted in

124

accordance with The Guide for the Care and Use of Laboratory Animals (Council,

125

2010).

126

2.3 Adult zebrafish exposure

127

Adult zebrafish (AB strain; 4 months old) were exposed to 0.18, 0.92 and 1.84 mg/L

for no

128

tebuconazole solution (representing 1/100 LC50 (lethal concentration 50), 1/20 LC50

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and 1/10 LC50 of adult lethality, respectively) for 28 d with exposure solution renewal

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every 3 d. Following the 28 d exposure, ten fish (five male and five female) were

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randomly sampled from each tank and were anesthetized using 160 mg/L MS-222,

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followed by measurements of wet body weight. Fish were then dissected, with

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individual livers divided into three parts. One part of each divided sample from fish of

134

the same gender was pooled into a single composite sample for biochemical

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determinations and mRNA transcript analyses, respectively. The remaining one-third

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of each divided liver sample was then used for histopathological examination. The

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remaining fish were transferred to tebuconazole-free water and cultured for 30 d with

138

daily renewal of water. Following the 30 d depuration, ten fish (five males and five

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females) were sampled using the same procedure described above, with the same

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biochemical, mRNA and histopathological analyses. Each treatment was conducted in

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triplicate. Zebrafish in tebuconazole-free exposure solution (containing 0.01% DMSO,

142

v/v) served as vehicle control. Exposures were conducted in a barrel-type glass tank

143

(40 cm inside diameter × 40 cm) filled with 15L solution (12 male and 12 female fish

144

were put in each tank). The quantification of measured tebuconazole in exposure

145

solution is described in Supporting Information (SI-2).

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2.4 Zebrafish larval exposure

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Maximum non-lethal concentration (MNLC) and lethal concentration for 10% of the

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population (LC10) was determined for tebuconazole by exposing zebrafish

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Tg(apo14:GFP) to tebuconazole (concentrations were set as 2.5, 5, 10, 20, 40, 60 and

150

80 mg/L) from 72 hpf to 120 hpf and mortality was recorded every 24 h. 10 larvae per

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exposure tank, each treatment was conducted in triplicate, and exposure solutions

152

were exchanged every day. MNLC is defined as the maximum concentration at which

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there is no statistically significant increase in mortality relative to vehicle control.

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Dead zebrafish were removed three times a day. Four concentrations (1/10 MNLC,

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1/3 MNLC, MNLC and LC10) of tebuconazole were used to assess hepatotoxicity.

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Briefly, 90 larvae (72 hpf) were distributed into 15 cm glass culture dish (90 fish/dish)

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and then treated with tebuconazole from 72 hpf to 120 hpf. Zebrafish treated with

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0.01% DMSO served as vehicle controls. Each concentration was conducted in

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triplicate. Following treatment, 20 zebrafish from each culture dish were randomly

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selected and subjected to visual observation and image acquisition under a

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fluorescence microscope (Nikon, Japan). A subset of larvae (60 larvae from each

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culture dish) were sampled at random and frozen immediately at - 80

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subsequent analysis of gene expression, aspartate aminotransferase (AST) and alanine

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aminotransferase (ALT) enzyme activity. Several phenotypic endpoints, including

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liver size, yolk sac retention and liver degeneration were used for assessing

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hepatotoxicity. Morphometric analysis was performed using Image-Pro Plus 6.0

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(Media Cybernetics inc., USA). Liver size, yolk sac retention and liver degeneration

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were calculated via Eq. 1, 2 and 3, respectively.

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Liver size (% of control) = liver area (A) / liver area (B) × 100%

(Eq. 1)

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Yolk sac retention (% of control) = yolk sac area (A) / yolk sac area (B) × 100%

(Eq. 2)

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Liver degeneration (%) =

for

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liver optical density (A) / liver optical density (B) × 100%

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where (A) and (B) refer to tebuconazole treatments and vehicle control (VC),

174

respectively.

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2.5 Biochemical determinations

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After splitting into thirds, one part of fish liver was homogenized in 0.15 M NaCl on

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ice and centrifuged at 2500 rpm (4

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for biochemical measurements. The enzyme activity of superoxide dismutase

179

(T-SOD), catalase (CAT), peroxidase (POD), glutathione S-transferase (GST),

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aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured

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using corresponding commercial kits following the manufacturer’s instructions

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(Nanjing Jiancheng Bioengineering Institute, China).

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2.6 Reactive Oxygen Species Assay

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The ROS in exposed 120 hpf larvae were measured using 2′,7′-dichlorofluorescein

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diacetate (DCFH-DA). Briefly, 30 larvae (AB strain) form different concentrations of

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tebuconazole (VC, 1/10 MNLC, 1/3 MNLC, MNLC, LC10) were washed three times

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with cold phosphate-buffered saline (PBS; pH 7.4), and incubated with trypsin and

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0.2% ethylenediaminetetraacetic acid (EDTA) at 37

189

(pH 7.4, incubated at 4

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collection. Each concentration was conducted in triplicate. Fluorescence intensity was

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measured using a microplate reader (Molecular Device, CA) with excitation and

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emission wavelengths set at 500 and 530 nm, respectively. The ROS level was

193

normalized to protein mass, and results of treatment fish are expressed as fold change

(Eq. 3)

) for 15 min, with the resulting supernatants used

for 15 min. Then, cold PBS

before use) was added and filtered for cell suspension

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relative to control.

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2.7 Acridine orange staining

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After 48 h (from 72 hpf to 120 hpf) of exposure to the concentrations of tebuconazole

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(VC, 1/10 MNLC, 1/3 MNLC, MNLC, LC10), 20 larvae (AB strain) from each beaker

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(n = 3) were washed twice in 30% Danieau’s solution (Deng et al., 2009), then

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transferred to acridine orange (AO) solution and incubated for 20 min at room

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temperature. Then, larvae were washed with 30% Danieau’s solution three times for 5

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min. Apoptotic cells were observed with a fluorescence microscope (Nikon, Japan).

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2.8 Histopathology

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To confirm that visually observed abnormalities in liver represent pathological liver

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damage, we quantitatively assessed histopathology in livers obtained from exposed

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larval zebrafish Tg(Apo14:GFP) and adult zebrafish (AB strain). Detailed information

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was provided in Supporting Information (SI-3).

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2.9 Quantitative Real-Time PCR (qRT-PCR) Assay

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Total RNA was extracted using TRIzol reagent as described by Li et al. (2016 and

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2019a). Extraction of RNA and analysis of gene expression are described in the

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Supporting Information (SI-4). qRT-PCR was performed on select genes (p53, bax,

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caspase8) related to hepatotoxicity with previously determined primer sequences

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(Table S3) (Wang et al., 2015a, 2015b; Ma et al., 2016). Gene expression data was

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calculated as fold change relative to control (2-∆∆Ct).

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2.10 Statistical Analysis.

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Statistical analysis was performed using SPSS® version 20.0 (SPSS, USA). The data

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were initially verified for normality and homogeneity of variance using the

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Kolmogorov-Smirnov test and Levene’s test. Data are expressed as mean ± standard

218

deviation (SD). Statistical differences in gene expression, enzyme activity and ROS

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concentrations were evaluated by one-way analysis of variance (ANOVA) followed

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by Tukey’s Honest Significant Difference post-hoc analysis. A p-value <0.05 was

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considered statistically significant.

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3. Results

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3.1 Toxicities of tebuconazole in zebrafish

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Percentages of fish mortality were calculated for each tebuconazole concentration for

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fish exposed from 72 to 120 hpf (Fig. S1). Based on the results, LC10 and MNLC

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were calculated as 6.9 mg/L (95% CI: 4.9 - 8.8 mg/L) and 3.3 mg/L (95% CI: 2.0 - 4.7

227

mg/L), respectively. During tebuconazole exposure, measured aqueous tebuconazole

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concentrations ranged from 0.13 to 0.20 mg/L, 0.73 to 0.96 mg/L, 1.50 to 1.86 mg/L

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for 0.18, 0.94 and 1.84 mg/L groups respectively (Fig. S2). Zebrafish mortality in

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exposure groups was lower than 11.1%, which conformed to the requirements of

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OECD (Organization for Economic Cooperation and development) Guideline 203.

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(OECD, 1992) Several enzymatic biomarkers (Table 1) were significantly altered in

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exposed fish, indicating toxicity. ALT activity was strongly increased in both female

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(70.54% relative to control; p < 0.001) and male (56.74% relative to control; p =

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0.020) zebrafish exposed to 1.84 mg/L tebuconazole following the 28 d exposure.

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ALT activity remained significantly increased at the end of the 30 d depuration time

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(p = 0.015, p = 0.018 for female and male, respectively). AST activity was altered in a

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dose-dependent manner which was statistically significant in fish sacrificed

239

immediately following tebuconazole exposure (1.84 mg/L), but was not significantly

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different relative to control following the 30 d depuration. After exposure to

241

tebuconazole, significant increases in CAT activity relative to VC were observed in

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1.84 mg/L exposure group up to 175 % in female fish and up to 207 % in male fish.

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Significant increases in GST activity (up to 243%) were found in male fish exposed to

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1.84 mg/L tebuconazole, and was recovered following depuration. Compared with VC,

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T-SOD and POD activities were significantly increased in zebrafish at all

246

tebuconazole treatments. Following depuration, slight recovery of enzyme activity

247

was observed (Table 1).

248

3.2. Hepatotoxicity analysis in larvae

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Based on visual assessment, vehicle control-exposed zebrafish displayed transparent

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liver tissue (Fig. 1A). After treatment with tebuconazole, zebrafish liver were no

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longer translucent and became dark/brown and liver blood flow was no longer

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visually observable (Fig. 1B, C, D and E). In Tg(Apo14:GFP) zebrafish exposed to

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VC and tebuconazole, fluorescence expression was observed primarily in the liver

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region (Fig. 1F). Following exposure to tebuconazole, mean fluorescence intensity

255

decreased gradually at treatments (Fig. 1G, H, I and J). Compared with VC, liver size

256

was significantly reduced in zebrafish treated with tebuconazole at MNLC (9.89%

257

reduction, p = 0.022) and LC10 (17.12% reduction, p = 0.002). Relative to VC,

258

statistically significant liver degeneration was observed in zebrafish exposed to

259

tebuconazole concentrations of 1/3 MNLC (23.39% reduction, p = 0.016), MNLC

260

(34.07% reduction, p < 0.001) and LC10 (44.20% reduction, p < 0.001). At the same

261

time, significant delays in yolk sac absorption was found in zebrafish exposed to

262

tebuconazole (Fig. 2), the yolk sac size increased relative to control by 32.26% (p <

263

0.001) and 53.53% (p < 0.001) at MNLC and LC10 tebuconazole concentrations,

264

respectively.

265

3.3. ROS and Apoptosis analysis

266

Following exposure from 72 hpf to 120 hpf, ROS concentrations were measured in

267

zebrafish larvae (Fig. 3). Exposure to tebuconazole led to an increase in

268

protein-normalized ROS levels by 1.28-fold and 1.65-fold relative to VC for the

269

MNLC and LC10 groups, respectively.

270

The larvae were also stained with AO. Apoptotic cells were not visually observed in

271

the vehicle control larvae, however a visually apparent number of apoptotic cells were

272

identified in around the liver and heart region, head and submaxillary area in

273

tebuconazole-treated larvae (Fig. S3).

274

3.4. Histolopathology

275

Histopathological examinations demonstrated alterations in liver histoarchitecture

276

following exposure to tebuconazole. In VC-exposed adult fish, liver appeared normal

277

and healthy (Fig. 4). After 28 days of tebuconazole exposure, boundaries between

278

hepatocytes became more difficult to characterize. Specifically, liver nuclei trended

279

towards the periphery, and in the cytoplasm, karyolysis and vacuole formation were

280

observed. After 30 days of depuration, the aforementioned pathological changes

281

slightly reversed, however structural alterations were still observed in hepatocytes. In

282

VC-exposed larval zebrafish (Fig. 5), liver tissue appeared normal in both cell

283

structure and shape, while liver from zebrafish treated with tebuconazole appeared

284

dissociated, with irregular cell shape, and various vacuoles were observed.

285

3.5. Gene expression

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In larval zebrafish (Fig. 6), mRNA expression of p53 was significantly upregulated

287

(2.67-fold, p = 0.008) in the LC10 tebuconazole treatment group relative to VC. Bax

288

was significantly up-regulated 2.00-fold (p = 0.031), 2.43-fold (p = 0.038) and

289

2.16-fold (p = 0.018) in the 1/10 MNLC, 1/3 MNLC and LC10 tebuconazole treatment

290

groups, respectively. Expression of caspase8 was significantly up-regulated 1.88-fold

291

(p = 0.016) and 2.30-fold (p = 0.002) in 1/10 MNLC and LC10 exposure groups

292

relative to VC, respectively. In adult fish liver (Fig. 6), mRNA expression of p53 was

293

significantly upregulated in both female (1.91-fold, 0.92 mg/L, p = 0.037; 2.79-fold,

294

1.84 mg/L, p = 0.047) and male fish exposed to (2.30-fold, 0.92 mg/L, p = 0.040;

295

3.54-fold, 1.84 mg/L, p = 0.021) tebuconazole. Compared with VC, bax was

296

significantly up-regulated in both female (2.07-fold, 0.92 mg/L, p = 0.040; 3.26-fold,

297

1.84 mg/L, p = 0.033) and male (2.20-fold, 0.92 mg/L, p = 0.039; 3.32-fold, 1.84

298

mg/L, p = 0.020) zebrafish. The gene expression of caspase8 was significantly

299

up-regulated in a concentration-dependent manner (Fig. 6).

300

Discussion

301

In the present study, the impacts of tebuconazole on hepatotoxicity in adult and larval

302

zebrafish were assessed. The results show that aqueous tebuconazole exposure

303

significantly affects enzyme activity (e.g. T-SOD, CAT, POD, GST, AST and ALT) in

304

adult zebrafish. In larval zebrafish, tebuconazole induced changes in specific

305

phenotypic endpoints including liver degeneration, reduced liver size and delayed

306

yolk absorption. Liver-induced toxicity was confirmed through histopathological

307

examination, which revealed alterations in liver histoarchitecture in exposed zebrafish.

308

AO staining, ROS and qRT-PCR analyses implicated apoptosis as a possible

309

mechanistic pathway for the observed liver toxicity.

310

In many organisms, the liver transforms toxicants using various enzymes and

311

detoxification systems, and may consequently experience damage (Mukhopadhyay

312

and Chattopadhyay, 2014). Our previous study showed that tebuconazole body loads

313

were enriched in the liver of adult zebrafish relative to other compartments (Li et al.,

314

2019a). In the present study, we determined that tebuconazole modulated the activities

315

of antioxidant enzymes such as T-SOD, CAT, POD and GST, which corroborated the

316

earlier report. These antioxidant enzymes are essential for the conversion of ROS to

317

harmless metabolites and may be increased or inhibited under chemical stress (Toni et

318

al., 2011). In general, exposure to azole fungicides induces increased antioxidant

319

enzyme activity as a result of the liver’s detoxification mechanisms (Jiang et al.,

320

2017). For example, triadimefon induced significantly increased activity of SOD,

321

CAT and glutathione peroxidase (GPX) during development in rare minnow

322

(Gobiocypris rarus) and medaka (Oryzias latipes) (Jiang et al., 2017). However,

323

decreased GST activity was observed in common carp (Cyprinus carpio) exposed to

324

tebuconazole (Toni et al., 2011), perhaps due to reduced liver function. The present

325

study showed significant increases in activities of antioxidant enzymes in zebrafish

326

liver following exposure to tebuconazole, including in adult fish that were depurated,

327

indicating activation of the antioxidant defense system. Additionally, the ratio of

328

AST/ALT was significantly increased in both female and male fish exposed to

329

tebuconazole. A previous study demonstrated that increased AST and ALT are linked

330

to liver damage, with the ratio (AST/ALT) being one of the most well-established

331

biomarkers of liver damage (McGill, 2016). Taken together, the present results clearly

332

showed persistent hepatotoxicity following tebuconazole exposure in adult zebrafish.

333

In this study, the tendency in significantly changed toxicity indicator was consistent in

334

both male and female zebrafish, while different degrees of change in those indicators

335

were found, indicating further study need to explore the different toxicities induced by

336

tebuconazole in a gender-manner.

337

While the liver of normal zebrafish are transparent, exposure to hepatotoxic chemicals

338

makes the liver darker with a brown or gray coloration, and the texture of liver tissue

339

can become amorphous, indicating degeneration and/ or necrosis (He et al., 2013). In

340

zebrafish line Tg(Apo14:GFP), the Apo-14 promoter-driven GFP is sustainably

341

expressed from hepatoblasts and liver progenitor cells in liver primordium to

342

hepatocytes in the larvae (Wang et al., 2011). Therefore, Tg(Apo14:GFP) zebrafish

343

offer a reliable visual method to monitor the developmental process of the liver

344

following tebuconazole exposure. In the present study, significantly reduced liver size

345

was

346

tebuconazole-exposed zebrafish liver were amorphous and gray, indicating necrosis.

347

We therefore speculated that the observed reduction in liver size could be due to liver

observed

in

zebrafish

treated

with

tebuconazole.

Additionally,

348

degeneration or necrosis, which we then confirmed through histopathology.

349

In zebrafish, the yolk sac is maternally derived and represents the sole source of

350

energy for the larva during early development (Jones et al., 2008). In zebrafish, the

351

yolk sac is metabolized mainly in the liver, and is a quantifiably finite source that is

352

consumed primarily during the first week of larval development (Jones et al., 2008).

353

Therefore, yolk sac size is regarded as a reliable indicator of liver function in larvae

354

zebrafish (Hill et al., 2012). Furthermore, it has been demonstrated that impairment of

355

liver function can cause yolk sac absorption to be delayed (He et al., 2013). In our

356

study, tebuconazole increased the yolk sac size in larval zebrafish- a response which is

357

consistent with the effects observed on liver size and degeneration.

358

It has been demonstrated that contaminant-stimulated ROS production is closely

359

associated with oxidative damage and multiple toxicities in aquatic organisms

360

(Livingstone, 2001). In the present study, it was found that tebuconazole exposure

361

induced ROS production with pronounced effects at higher concentrations of

362

tebuconazole at MNLC and LC10. Consistent with the results of the present study, a

363

previous study reported a significant induction of ROS after tebuconazole exposure in

364

fish (common carp) (Toni et al., 2011). It has also been previously demonstrated that

365

exposure to tebuconazole causes lipid peroxidation and apoptosis in embryo larval

366

zebrafish (Kumar et al., 2019). Meanwhile, an in vitro study on

367

fungicide, difenoconazole, showed that the generation of ROS may result from the

368

induction of apoptosis through an ROS-mediated mitochondrion signaling pathway

369

(Zhuang et al., 2015). ROS-induced oxidative stress has been indicated to contribute

another azole

370

to abnormal development during embryogenesis (Yamashita, 2003). Therefore, the

371

observed liver damage in larvae in the present study may be partly explained by the

372

generation of ROS.

373

As discussed above, the generation of ROS has been shown to be an important signal

374

of apoptosis (Livingstone, 2001). AO is an intercalating nucleic acid-specific

375

fluorochrome, which emits a green fluorescence when it is bound to DNA (Shi et al.,

376

2008). To measure cell apoptosis in larval zebrafish, AO staining was used, which is a

377

simple technique capable of showing apoptosis patterns (Qi et al., 2016). The AO

378

staining assay showed that apoptotic cells occurred in the liver regions, suggesting

379

that the developing liver may be an important potential target for tebuconazole

380

toxicity in zebrafish. This result was possibly due to the increased ROS levels in

381

larval zebrafish and may partially explain the observed liver damage in zebrafish.

382

To further investigate the relationship between ROS and apoptosis, we measured

383

transcripts of p53, bax and caspase8, which are genetic biomarkers downstream of a

384

apoptotic signaling pathway that occurs whenever oxidative stress persists (Kumar et

385

al., 2019). p53 is important in programmed cell death during zebrafish embryo

386

development, and the pathway of p53-mediated cell apoptosis has been extensively

387

reported (Deng et al., 2009; Holley and St Clair, 2009; Kumar et al., 2019). Normally,

388

p53 is expressed at low concentrations and is activated only when the cell is under

389

stress. Stresses that activate p53 include heat shock, hypoxia as well as reactive

390

oxygen species-induced damage (Holley and St Clair, 2009). In the present study,

391

up regulation of p53 was observed in adult zebrafish exposed to tebuconazole at 0.92

392

and 1.84 mg/L and in larval zebrafish exposed to tebuconazole at LC10. Changes in

393

ROS production were consistent with p53 mRNA induction, suggesting that p53 gene

394

involvement in cell apoptosis due to tebuconazole exposure is likely associated with

395

an ROS-mediated pathway. However, further study is needed to reveal the

396

relationship between ROS and p53 pathway.

397

In addition, p53 regulates the transcription of a myriad of proteins involved in

398

apoptosis, and can also induce apoptosis in a transcription-independent manner by

399

directly targeting the mitochondria of the cell (Holley and St Clair, 2009). For

400

example, p53 can upregulate Bax as well as interact with Bax to induce apoptosis

401

(Chipuk et al., 2004; Holley and St Clair, 2009). Correspondingly, results from the

402

present study showed that tebuconazole significantly induced the mRNA expression

403

of the pro-apoptotic bax accompanied with an up-regulation of p53 transcripts,

404

suggesting that tebuconazole could induce apoptosis. Further supporting this linkage,

405

tebuconazole was found to significantly activate another apoptosis related gene

406

caspase-8. The Caspase family has been identified as a key executor of apoptosis, and

407

Caspase-8 is one of the most important caspases activated downstream in apoptosis

408

pathways (Zhuang et al., 2015). Caspase activity is also regarded as a useful marker

409

for stress-induced apoptosis in the early-life stages of fish (Deng et al., 2009). A

410

previous study reported that caspase-8/9 activity were related to ROS-mediated

411

mitochondria apoptotic signals (Zhuang et al., 2015). In the present study, caspase-8

412

gene expression was up-regulated, supporting this apoptosis model in zebrafish larvae.

413

However, the mechanisms for caspase activation and whether caspase activity is

414

mediated through mitochondrial apoptotic signaling requires further investigation.

415

Results from the present study suggest that tebuconazole is capable of inducing

416

hepatotoxicity in adult and larval zebrafish, predominately through the generation of

417

ROS beginning with the activation of p53, followed by induction of genes that encode

418

pro-apoptotic proteins (bax, caspase-8), finally resulting in liver damage. As a model

419

vertebrate animal, zebrafish have a high degree of genetic conservation and their

420

molecular basis of tissue development is either identical or similar to other

421

vertebrates- including humans (He et al., 2013). Therefore, while the tebuconazole

422

concentrations that induced hepatotoxicity in the present study are likely to be higher

423

than concentrations found in the environment, further consideration should be given

424

to the potential adverse effects induced by tebuconazole exposure at different life

425

stages.

426 427

Conflicts of interest

428

The authors declare no competing financial interest.

429 430

Acknowledge

431

The present study was financially supported by the National Natural Science

432

Foundation of China (No. 31572026), the Zhejiang Provincial Natural Science

433

Foundation of China (No. LZ18C030001), the National Natural Science Foundation

434

of China (No. 31801756) and the China Postdoctoral Science Foundation

435

(2017M621947). We would also like to thank Core Facilities, Zhejiang University

436

School of Medicine for the support of Tg(Apo14:GFP) zebrafish.

437 438

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Zhang, Q., Hua, X., Yang, Y., Yin, W., Tian, M., Shi, H., Wang, M., 2015.

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Rehman, K., Naranmandura, H., 2015. The involvement of ER-stress and ROS

575

generation in difenoconazole-induced hepatocellular toxicity. Toxicol. RES-UK. 4,

576

1195-1203.

577

578

Figure captions

579

Figure 1. Visual phenotype of hepatotoxicity in zebrafish at 120 hpf after exposure to

580

tebuconazole from 72 to 120 hpf. Larval zebrafish exposed to vehicle control

581

exhibited translucent, healthy liver (A and F). Larval zebrafish treated with

582

tebuconazole exhibited lower mean fluorescence in liver tissue (G, H, I and J),

583

suggesting hepatotoxicity- which was visually apparent (B, C, D and E). Changes in

584

liver size (K) and liver degeneration (L) in response to tebuconazole exposure were

585

quantitatively assessed, and are expressed as a percentage relative to control (mean ±

586

SD; n=3). Asterisks indicate significant differences between exposure groups and the

587

corresponding control group (*p < 0.05, ** p<0.01, ***p<0.001).

588

Figure 2. Changes in zebrafish yolk sac size relative to vehicle control after exposure

589

to tebuconazole from 72 to 120 hpf. Data are expressed as percentages relative to

590

control (mean ± SD; n=3). Asterisks indicate significant differences between exposure

591

groups and the corresponding control group (*p < 0.05, ** p<0.01, ***p<0.001).

592

Figure 3. ROS in zebrafish larvae (72 hpf to 120 hpf) following 48 hr exposure to VC

593

or tebuconazole (1/10 MNLC, 1/3 MNLC, MNLC, LC10). Data are expressed as fold

594

change to control (mean ± SD; n=3). Asterisks indicate significant differences

595

between exposure groups and the corresponding control group (*p < 0.05).

596

Figure 4. Representative histological pictures of zebrafish (Danio rerio) liver. A

597

(vehicle control of female) and C (vehicle control of male) have normal nucleus (NN).

598

B (female fish after 1.84 mg/L tebuconazole exposure) and D (male fish after 1.84

599

mg/L tebuconazole exposure) show histopathological changes such as vacuole

600

formation (V), karyolysis (K), and nucleus tends to periphery (NP).

601

Figure 5. Representative histological pictures of larval zebrafish at 120 hpf. A and B

602

were the liver histopathology from vehicle control. C and D were the liver

603

histopathology from zebrafish treated with tebuconazole.

604

Figure 6. Bi-directional heatmap showing relative mRNA expression profiles of select

605

genes in liver of adult zebrafish and larval zebrafish after exposure to tebuconazole.

606

Data are Normalized to housekeeping gene and displayed as mean fold change in

607

gene expression relative to control, with red hue indicating upregulation greater than

608

1.5-fold change. Asterisks indicate significant differences between exposure groups

609

and the corresponding control group (*p < 0.05, ** p<0.01, ***p<0.001).

610

Tables

611

Table 1. Liver enzyme activities of adult zebrafish Tebuconazole concentration Toxicity index

ALT b (U/gprot) AST b (U/gprot) AST/ALT b CAT b (U/mgprot) GST b (U/mgprot) T-SOD b(U/mgprot) POD b (U/mgprot) ALT c (U/gprot) AST c (U/gprot) AST/ALT c CAT c (U/mgprot) GST c (U/mgprot) T-SOD c (U/mgprot) POD c (U/mgprot) 612 613 614 615 616 617 618

a

VCa

0.18 mg/L

0.92 mg/L

1.84 mg/L

♀

15.28±0.34

15.02±7.03

19.60±1.70

26.07±0.83***

♂

23.25±0.24

16.90±2.43

27.77±1.61

36.44±4.34*

♀

40.15±3.22

121.21±26.70*

111.47±21.58*

187.72±47.44*

♂

47.47±15.35

92.75±3.37*

147.70±25.75*

216.74±19.93**

♀

2.62±0.15

12.08±8.60

5.70±0.99*

7.26±2.06

♂

2.04±0.66

5.60±0.76**

5.33±0.93*

6.11±1.38

♀

45.27±1.75

54.44±4.01

84.17±4.26***

124.5±5.56**

♂

50.84±1.67

62.39±1.81**

104.04±2.46**

155.9±6.42**

♀

27.83±0.71

30.78±2.26

37.44±0.77***

81.95±1.52***

♂

33.31±2.23

52.26±0.83***

66.13±1.47***

114.3±2.14***

♀

49.29±1.45

60.46±2.21***

65.65±1.40***

117.01±1.95***

♂

59.5±3.19

92.12±1.32***

109.66±3.00***

174.31±2.56***

♀

3.42±0.10

4.15±0.09***

4.76±0.13***

7.84±0.14***

♂

4.9±0.18

6.87±0.13**

9.68±0.45***

12.00±0.19***

♀

12.81±0.91

8.91±3.18

17.85±2.58

23.25±2.37*

♂

20.16±3.24

16.93±3.04

25.22±2.53

31.44±4.02*

♀

42.14±2.95

48.23±6.71

90.51±28.28

103.25±17.42*

♂

43.83±12.95

78.88±53.92

73.20±31.76

128.11±12.92*

♀

3.32±0.45

5.99±1.95

5.04±1.40

4.43±0.53

♂

2.36±1.14

4.96±3.53

3.05±1.46

4.17±0.86

♀

40.19±1.20

83.48±2.83*

37.28±1.95

62.61±1.85

♂

63.66±1.44

117.85±2.49***

62.26±1.32**

81.63±3.83***

♀

35.09±0.87

37.57±1.27

41.92±2.69*

50.23±0.83***

♂

37.89±0.82

41.84±1.82

43.14±3.39

37.44±1.19

♀

44.64±2.65

83.26±11.31*

56.41±0.80*

72.45±7.11*

♂

51.02±2.09

74.51±9.03*

69.05±7.43

83.64±7.70*

♀

15.74±0.65

16.34±1.20

18.26±1.46

26.64±1.06**

♂

22.98±1.48

27.36±2.43

28.52±1.99*

39.16±1.76***

b

VC means vehicle control. Data were obtained after 28 day exposure to tebuconazole. c Data were obtained at the end of 30 day depuration. All data are expressed as the mean ± SD. Significant differences between exposure groups and the corresponding vehicle control group are denoted with asterisks (*p<0.05 and **p<0.01. Gender is denoted with symbols (♀ = female; ♂ = male). GST: glutathione S-transferase. CAT: catalase. T-SOD: superoxide dismutase. POD: peroxidase. AST: aspartate aminotransferase, ALT: alanine aminotransferase.

619 620 621 622 623 624 625 626 627 628

Figure 1. Visual phenotype of hepatotoxicity in zebrafish at 120 hpf after exposure to tebuconazole from 72 to 120 hpf. Larval zebrafish exposed to vehicle control exhibited translucent, healthy liver (A and F). Larval zebrafish treated with tebuconazole exhibited lower mean fluorescence in liver tissue (G, H, I and J), suggesting hepatotoxicity- which was visually apparent (B, C, D and E). Changes in liver size (K) and liver degeneration (L) in response to tebuconazole exposure were quantitatively assessed, and are expressed as a percentage relative to control (mean ± SD; n=3). Asterisks indicate significant differences between exposure groups and the corresponding control group (*p < 0.05, ** p<0.01, ***p<0.001).

629 630 631 632 633 634

Figure 2. Changes in zebrafish yolk sac size relative to vehicle control after exposure to tebuconazole from 72 to 120 hpf. Data are expressed as percentages relative to control (mean ± SD; n=3). Asterisks indicate significant differences between exposure groups and the corresponding control group (*p < 0.05, ** p<0.01, ***p<0.001).

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Figure 3. ROS in zebrafish larvae (72 hpf to 120 hpf) following 48 hr exposure to VC or tebuconazole (1/10 MNLC, 1/3 MNLC, MNLC, LC10). Data are expressed as fold change to control (mean ± SD; n=3). Asterisks indicate significant differences between exposure groups and the corresponding control group (*p < 0.05).

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Figure 4. Representative histological pictures of zebrafish (Danio rerio) liver. A (vehicle control of female) and C (vehicle control of male) have normal nucleus (NN). B (female fish after 1.84 mg/L tebuconazole exposure) and D (male fish after 1.84 mg/L tebuconazole exposure) show histopathological changes such as vacuole formation (V), karyolysis (K), and nucleus tends to periphery (NP).

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Figure 5. Representative histological pictures of larval zebrafish at 120 hpf. A and B were the liver histopathology from vehicle control. C and D were the liver histopathology from zebrafish treated with tebuconazole.

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Figure 6. Bi-directional heatmap showing relative mRNA expression profiles of select genes in liver of adult zebrafish and larval zebrafish after exposure to tebuconazole. Data are Normalized to housekeeping gene and displayed as mean fold change in gene expression relative to control, with red hue indicating upregulation greater than 1.5-fold change. Asterisks indicate significant differences between exposure groups and the corresponding control group (*p < 0.05, ** p<0.01, ***p<0.001).

Highlights Tebuconazole induced hepatotoxicity in both adult and larval zebrafish. Tg(Apo14: GFP) zebrafish was used for hepatotoxicity assay. Histopathological results showed alterations in liver histoarchitecture after exposure. ROS-mediated pathway might induce apoptosis in zebrafish after tebuconazole exposure.

Conflicts of interest The authors declare no competing financial interest.