Gut microbiota dysbiosis might be responsible to different toxicity caused by Di-(2-ethylhexyl) phthalate exposure in murine rodents

Journal Pre-proof Gut microbiota dysbiosis might be responsible to different toxicity caused by Di-(2ethylhexyl) phthalate exposure in murine rodents Gang Wang, Qian Chen, Peijun Tian, Linlin Wang, Xiu Li, Yuan-kun Lee, Jianxin Zhao, Hao Zhang, Wei Chen PII:

S0269-7491(19)35404-1

DOI:

https://doi.org/10.1016/j.envpol.2020.114164

Reference:

ENPO 114164

To appear in:

Environmental Pollution

Received Date: 19 September 2019 Revised Date:

23 January 2020

Accepted Date: 10 February 2020

Please cite this article as: Wang, G., Chen, Q., Tian, P., Wang, L., Li, X., Lee, Y.-k., Zhao, J., Zhang, H., Chen, W., Gut microbiota dysbiosis might be responsible to different toxicity caused by Di-(2-ethylhexyl) phthalate exposure in murine rodents, Environmental Pollution (2020), doi: https://doi.org/10.1016/ j.envpol.2020.114164. 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. © 2020 Published by Elsevier Ltd.

CRediT author statement Gang Wang: Conceptualization, Methodology, Validation, Investigation, Writing - Original Draft, Writing - Review & Editing, Visualization, Project administration, Funding acquisition Qian Chen: Software, Formal analysis, Investigation, Writing - Original Draft, Visualization Peijun Tian: Software, Validation, Visualization Linlin Wang: Validation, Formal analysis, Data Curation Xiu Li: Data Curation Yuan-kun Lee: Writing - Original Draft, Writing - Review & Editing Jianxin Zhao: Validation, Resources, Data Curation Hao Zhang: Methodology, Validation, Resources Wei Chen: Conceptualization, Resources, Writing - Review & Editing, Supervision, Project administration, Funding acquisition

1

Gut microbiota dysbiosis might be responsible to different toxicity caused by

2

Di-(2-ethylhexyl) phthalate exposure in murine rodents

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Gang Wang1, 2, Qian Chen1, 2, Peijun Tian1, 2, Linlin Wang1, 2, Xiu Li1, 2, Yuan-kun Lee3, Jianxin Zhao1, 2, 4, 5, Hao

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Zhang1, 2, 5, 6, 7, Wei Chen1, 2, 6, 8*

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1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, P. R. China

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2. School of Food Science and Technology, Jiangnan University, Wuxi214122, P. R. China

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3. Department of Microbiology & Immunology, National University of Singapore, Singapore 117597, Singapore

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4. International Joint Research Laboratory for Probiotics, Jiangnan University, Wuxi 214122, P. R. China

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5. (Yangzhou) Institute of Food Biotechnology, Jiangnan University, Yangzhou 225004, P. R. China

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6. National Engineering Research Center for Functional Food, Jiangnan University, Wuxi 214122, P. R. China

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7. Wuxi Translational Medicine Research Center and Jiangsu Translational Medicine Research Institute Wuxi

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Branch, Wuxi 214122, P. R. China

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8. Beijing Innovation Centre of Food Nutrition and Human Health, Beijing Technology and Business University

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(BTBU), Beijing 100048, P. R. China

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* Corresponding author: Wei Chen, State Key Laboratory of Food Science and Technology, Jiangnan University,

1＜

Wuxi 214122, P. R. China

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Phone: (86)510-85912155

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Fax: (86)510-85912155

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E-mail address: [email protected] (W. Chen)

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Abstract

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Di-(2-ethylhexyl) phthalate (DEHP) is widely used as a plasticizer, which can enter the body through a variety of

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ways and exerted multiple harmful effects, including liver toxicity, reproductive toxicity and even glucose

2＜

metabolism disorder. Many studies have suggested that changes of gut microbiota are closely related to the

27

occurrence of various diseases, but the effects of DEHP exposure on gut microbiota are still unclear. It was found in

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this study that the damage to different tissues by DEHP on two strains each from two different species of male

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rodents before puberty was dose and time of exposure dependent, and also depending on the strain and species of

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rodent. Sprague-Dawley (SD) rats showed highest sensitivity to DEHP exposure, with most severe organ damage,

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highest Th1 inflammatory response and most significant body weight gain. Correspondingly, the gut microbiota of

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SD rats showed most significant changes after DEHP exposure. Only SD rats, but not Wistar rat, BALB/c and

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C57BL/6J mice showed an increase in Firmicutes/Bacteroidetes ratio and Proteobacteria abundance in the fecal

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samples, which are known to associate with obesity and diabetes. This is consistent with the increasing body weight

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gain which was only found in SD rats. In addition, the decrease in the level of butyrate, increase in the abundance

3＜

of potential pathogens and microbial genes linked to colorectal cancer, Parkinson's disease, and type 2 diabetes in

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the SD rats were associated with issue and functional damages and Th1 inflammatory response caused by DEHP

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exposure. We postulate that the differential effects of DEHP on gut microbiota may be an important cause of the

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differences in the toxicity on different strains and species of rodents to DEHP.

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Keywords: Di-(2-ethylhexyl) phthalate; murine rodents; toxicity; gut microbiota

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Main finding

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Among 2 strains each from 2 species of male murine rodents before puberty, SD rats showed highest sensitivity to

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DEHP exposure. The susceptibility of SD rats to DEHP exposure probably be related to the changes in gut

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microbiota.

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1 Introduction

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The plasticizer, di-(2-ethylhexyl) phthalate (DEHP) is widely used in medical equipment, chemical and plastic

5＜

products (Sampson and De, 2011; Shelby, 2002). Human are exposed to DEHP because of leaching and migration

57

into atmosphere, water, foods or even body fluids (Fay and Donohue, 1999; Koch et al., 2010). As an

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environmental endocrine disruptor, DEHP has multiple toxicities to the body and manifests in different forms.

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DEHP imparts an estrogen effect in the body (Okubo et al., 2003). Testicular tissue being the main target organ for

＜0

DEHP causing destruction to the male reproductive system (Stenz et al., 2017). In addition, female ovarian tissue is

＜1

the main target organ of DEHP for reproductive toxicity in females (Yang et al., 2008). Previous studies have found

＜2

that DEHP inhibits the activity of antioxidant enzymes in the body, inducing cytotoxicity and causing liver damage

＜3

via hypertrophy and hyperplasia of liver parenchymal cells. Also, DEHP can induce liver tumors, so it is considered

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a carcinogen (Rusyn et al., 2006). Moreover, low-dose chronic exposure to DEHP can cause insulin resistance in

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female rats affecting maternal glucose metabolism, and also causing insulin resistance in offspring (Lin et al., 2011).

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Epidemiological investigations show that DEHP can disrupt the body's glucose metabolism and trigger the

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occurrence of type 2 diabetes (James-Todd et al., 2012; Stahlhut et al., 2007; Svensson et al., 2011).

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Animal studies demonstrated that early periods of development (in uterus until adolescence) were more sensitive

＜9

to DEHP exposure (Tonk et al., 2012). In addition to the decisive exposure time, species and strain also respond

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differently to DEHP. In the body, DEHP primarily metabolized to mono-(2-ethylhexyl) phthalate (MEHP) by

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unspecific lipases. Species and strain difference in the lipase activity may result in differences in DEHP metabolism

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in different animals. For example, DEHP was primarily excreted as glucuronide conjugates in mice urine but

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unconjugated metabolites in rats (Albro, 1986; Frederiksen et al., 2007; Schulte et al., 2010). In addition, in male

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Sprague-Dawley (SD) and Wistar rat, DEHP exposure led to significant differences in the incidence rates of

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epididymal and gubernacular pathological changes (Wilson et al., 2007). Thus, species and strain should play an

7＜

important role in effects of DEHP exposure (Ito et al., 2014).

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Gut microbiota dysbiosis have been shown associated with obesity, diabetes and the disorders of digestive tract,

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immune and nervous system (Collins et al., 2015; Cryan and Dinan, 2015; Michail et al., 2012; Qin et al., 2012).

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Gut microbiota dysbiosis occur in humans and animals after exposure to chemical contaminants in the environment.

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For example, low-dose exposures to diethyl phthalate (DEP), methylparaben (MPB) and triclosan (TCS) can cause

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gut microbiota dysbiosis in adolescent rats. After low-dose exposure of DEP or MPB, the body weight of

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adolescent rats decreased, which is consistent with the decreased Firmicutes/Bacteroidetes ratio in weight loss

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study (Hu et al., 2016). In addition, gut microbiota changes caused by bisphenol (BPA) or ethinyl estradiol (EE)

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exposure are also gender related (Javurek et al., 2016). Whether DEHP exposure leads to dose-dependent gut

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microbiota dysbiosis and the sensitivity of gut microbiota in different animal species and strains are rarely studied.

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Beside the roles of gut microbiota on DEHP toxicity are still unclear. Therefore, the aim of this study was to

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investigate the relationship between physiological injuries in different animal species/strains and changes in their

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gut microbiota. Two strains each from two species of male murine rodents (Sprague-Dawley (SD) rats, Wistar rats,

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BALB/c mice, C57BL/6J mice), which were widely used in the researches of toxicology, immunology and even gut

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microbiota were engaged in this study.

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2. Materials and methods

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2.1 Animals and DEHP exposure

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Four-weeks-old male rodents (rat: Wistar, SD, initial body weight 100 to 104 g; mice: BALB/c, C57BL/6J, initial

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body weight 17 to 19 g) were kept in a barrier environment with controlled temperature (22±1ºC) and humidity

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(55±10%) under a 12h/12h light–dark cycle, and with free access to food and water. Before study, the experimental

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animals were acclimatized for seven days. This study was approved by the Ethics Committee of Experimental

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Animals in Jiangnan University, China (JN. No 20170627-20170803 (82)), and the procedures were conducted in

98

accordance with the European Community Guidelines for the Care and Use of Experimental Animals (Directive

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2010/63/EU).

100

Each species/strain of rodents were randomly divided into four experimental groups (n=6). DEHP was

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administered to rodents in drinking water at doses of 0 (control), 300 (low), 1000 (median), 3000 (high)

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mg/kgBW/day for 30 days. Body and water intake were weighted weekly and the doses of DEHP were adjusted to

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their body weight and drinking water volume. For DEHP emulsion preparing, a certain amount of sucrose fatty acid

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monoesters (SE, food grade, Tianjia biotechnology, Nanjing, China) powder was dissolved firstly with a small

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amount of warm water. After cooling, a certain amount of DEHP (99% pure) purchased from Sigma-Aldrich (St.

10＜

Louis, MO, USA) was added to the water containing SE. After shaking to produce an emulsion, a certain volume of

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water was added to prepare DEHP stock emulsion containing 0.5% SE. Before administering to rodents, the stock

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emulsion was diluted to a certain concentration with water containing 0.5% SE and shook to homogenize the DEHP

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water solution.

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2.2 Measurement of organ mass and tissue samples

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All animals were sacrificed under ether anesthesia. Organ samples including liver, kidney, spleen, testis and

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epididymis were harvested and wet organ weights were measured on day 30. The liver index (LI) , kidney index

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(KI), spleen index (SI), testis index (TI) and epididymis index (EI) were calculated as the ratio of the organ weight

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to the body weight(Suna et al., 2013).

115

2.3 Biochemical measurements in serum

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Serum testosterone concentration was measured with Testosterone Elisa Kit (Jiancheng Institute of

117

Biotechnology, Nanjing, China) according to the manufacturer's protocol. Alanine amino transferase (ALT),

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aspartate aminotransferase (AST), alkaline phosphatase (ALP) were measured with an automatic biochemistry

119

meter (SELECRTA-E, Vital Scientific). The levels of serum cytokines were detected with A Luminex MAGPIX

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system (Luminex, USA) according to the manufacturer's instructions.

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2.4 Cecal and feacal short-chain fatty acids (SCFAs) measurement

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Fecal and cecal samples were snap-frozen once collected from each mouse and stored at -80ºC for no longer than

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48 hours before DNA extraction or metabolites measurement. All animals were sacrificed under ether anesthesia.

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As previously described (Samuel and Gordon, 2006; Zhu et al., 2018), the contents of acetic acid, propionic acid,

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butyric acid, isobutyric acid, and n-valeric acid in cecal contents or feces were determined by gas

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chromatograph-mass spectrometer (GCMS-QP2010 Ultra system, Shimadzu Corporation, Japan).

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2.5 Gut microbiota analysis

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A Fast DNA Stool Kit (MP Biomedicals, Santa Ana, CA, USA) was used for DNA extraction from the cecal

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contents or feces samples. The V3-V4 region of the bacteria’s 16S rDNA was amplified with barcode-indexed

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primers (341F and 806R) by PCR, products were then purified by gel extraction (TIANgel Mini Purification Kit,

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TIANGEN, Beijing, China) and pooled in equimolar concentrations. Paired-end sequencing was performed on the

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Illumina MiSeq PE300 platform (Illumina, San Diego, CA, USA).

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2.6 Statistical analysis

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Statistical analysis was performed by SPSS Statistics Version 21 (IBM) and GraphPad Prism. Values are

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presented as mean ± standard deviations (SD). One-way ANOVA with Tukey's multiple comparisons test was

13＜

aimed to determine the correlations between the variables. Microbial data were analyzed by QIIME and R (version

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3.5.0) software. Criterion for significance was set to P 0.05 in all comparisons.

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3. Results

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3.1 SD rats showed the most significant changes in body weight and organ index after DEHP exposure

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As is shown in Fig. 1A, statistically significant changes in body weight gain (BWG) were only observed in SD

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rats. Meanwhile, in SD rats, liver index (Fig. 1B) significantly increased in medium and high dose groups

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compared with control group. Also, testis (Fig.1C), epididymis (Fig.1D) and spleen index (Fig.1E) in SD rats

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decreased significantly in varying degree after treatment of DEHP in different doses. In the mice, only C57LB/6J

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mice in high dose group had higher liver index (Fig. 1B). Except for this, no other significant organ differences

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were observed between DEHP-treated groups and control group in mice (C57LB/6J and BALB/c). Moreover, SD

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rats also showed the most obvious changes on total cholesterol (TC) and superoxide dismutase (SOD) upon

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exposure to DEHP (Fig. S1, S2). Furthermore, only SD rats showed the increasing level of leptin, decreasing level

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of insulin and activation of PPAR-α and PPAR-β (Fig. S3).

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3.2 DEHP induced different changes of liver enzymes level in blood of rodents

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In SD rats, AST, ALT and ALP levels increased markedly in highest DEHP dose group (Fig. 2A). However,

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DEHP treatment only induced significant elevation of ALT in both low and medium dose groups in Wistar rats.

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Whilst in Wistar rats, both ALT and ALP showed decreasing tendency in highest dose group, which indicated that

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different rats responded differently to the exposure of different doses of DEHP. In contrast, the liver enzymes in

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both kind of mice were not sensitive to DEHP exposure except elevation of ALP in medium and high dose groups

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of BALB/c mice. The histopahtological changes in liver also indicated the different tissue damage caused by DEHP

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in different rodents (Fig. S4A).

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3.3 High dose exposure of DEHP caused significant decrease of serum testosterone level in SD rats and BALB/c

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mice

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As is shown in Fig. 2B, testosterone level in SD rats treated with 3000mg/kg/day was 60% less than that in

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control group, and there was a tendency of dose dependent decrease of testosterone level following DEHP exposure

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in SD rats. Similarly, testosterone concentration in BALB/c mice also decreased significantly on DEHP exposure.

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In contrast, no significant variations of serum testosterone level was found in both Wistar rats and C57LB/6J mice.

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The histopahtological changes in testicular tissue also confirmed that SD rats and BALB/c mice had higher

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sensitivity to DEHP reproductive toxicity (Fig. S4B).

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3.4 DEHP exposure led to significant Th1 response in SD rats

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As is shown in Fig. 3, IL-1α increased significantly only in C57LB/6J mice exposed to all doses of DEHP.

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Besides, with the increasing dose of DEHP, MCP-1 level increased in C57LB/6J and BALB/c mice. All these

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suggested the inflammation responses in these rodents upon exposure to DEHP. Moreover, in the serum of SD rats,

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the levels of IL-2 (high dose exposure group), IFN-γ (high dose exposure group), TNF-α (low and middle dose

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exposure groups) showed significant elevation, as well as the level of IL-12 (p70, all dose exposure groups), which

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indicated highten Th1 response in SD rat. However, according to the levels of other cytokines, it was indicated that

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no significant Th2 response occured in rodents and only mice showed Th17 response (Fig. S5).

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3.5 DEHP exposure led to vary changes on gut SCFAs level and microflora diversity in rodent

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Histopathological sections showed that DEHP exposure did not result in visible tissue damage in the intestinal

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tract of rodents (data not shown). However, it was found that DEHP exposure could have a significant differential

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effect on the SCFAs levels in the intestinal tract of rodents. The results of the cecal contents did not show a

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significant difference (Fig. S6), however, feces showed significant changes in the levels of acetate and butyrate (Fig.

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4A), and these changes are diametrically opposed in mice and rats. The highest concentration of DEHP exposure

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resulted in a significant increase in acetate concentration in BALB/c mice and C57LB/6J mice, whilst resulted in a

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significant decrease in acetate concentration in SD rats. Similarly, exposure to the highest concentration of DEHP

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resulted in an increase in butyrate in C57LB/6J mice, but all concentrations of DEHP resulted in a significant

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decrease in the butyrate level in the feces of SD rats. It is believed that decreased levels of SCFAs are often

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associated with damage to the gut microbiota (Morrison and Preston, 2016; Unger et al., 2016; Yang et al., 2015),

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and these results correlated with the highest sensitivity of SD rats to DEHP exposure.

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Correspondingly, DEHP exposure caused significant specific changes in the gut microbiota of the various

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rodents. From the intestinal microbiota diversity of the four rodents, there was no significant change in the α

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diversity of gut microbiota in mice, but a significant change in the rats (Fig. 4B). Interestingly, changes in SD rats

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were distinct from those in Wistar rats. There was a significant increase in the estimated richness (chao-1) and

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Observed operational taxonomic units (OTUs) in the cecal contents of SD rats after the median- and high-dose

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DEHP exposure (P < 0.001), whilst in Wistar rats, although significant increase (P < 0.05, median-dose exposure)

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in the Observed OTUs was found, the opposite trend could be seen in the chao-1 and Observed OTUs (both cecal

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content and feces). Furthermore, a significant decrease in the Shannon index was found in the Wistar rats’ fecal

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microbiota after the high-dose DEHP exposure (P < 0.05), which was not found in SD rats. In addition, higher dose

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DEHP exposures resulted in more significant changes in β diversity while in the feces of both strains of rats, the

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lowest dose exposure showed the most significant changes in β diversity (Fig. S7). This may also be related to the

19＜

sensitivity of the rats to DEHP exposure.

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3.6 DEHP exposure led to significant differential effects on the abundance and function of intestinal microbiota in

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rodents

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At phylum level, in the feces, only SD rats showed a significant increase in the abundance of Proteobacteria

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caused by DEHP exposure (Fig. 5A). In the cecal contents, the high-dose DEHP exposed SD rats showed a

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significant increase in Firmicutes, whilst the Proteobacteria and Actinobacteria in C57LB/6J mice showed a

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significant downward trend with the increase of exposure concentration (Fig. 6A). Tenericutes showed only a

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significant increase in the cecal contents of C57LB/6J mice exposed to median-dose DEHP (Fig. 6A). The values of

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Firmicutes/Bacteroidetes ratio showed that only the high-dose DEHP exposed SD rats showed a significant increase

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of the ratio in cecal contents (Fig. 6B), which was associated with the body weight gain exhibited by SD rats.

20＜

At the genus level, changes in gut microbiota in all four rodents showed significant differences. There were

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limited changes in the fecal microbiome in mice (Fig. 5C; S7). In the BALB/c mice exposed to DEHP, only

208

Bacteroides showed a decrease, while Runimococcaceae and Rikenellaceae showed significant increase. In

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C57LB/6J mice, the abundances of Prevotella, Lachnospiraceae, and Desulfovibrio showed significant decrease. In

210

contrast, the microbiota in rat feces showed multiple genus changes with DEHP exposure. In the feces of SD rats,

211

Oscillospira, Peptostreptococcaceae, Mycoplasma, Roseburia, Clostridiaceae, Sutterella, Clostridiales, RF32,

212

Christensenellaceae, Blautia, rc4-4 showed increase in abundance caused by DEHP exposure. Only Prevotella

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showed decrease in the feces of SD rats. In the feces of Wistar rats, except for the increasing abundance of

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Roseburia similar to that of SD rats, other changes were different from SD rats, including decreasing Coprococcus,

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Dehalobacteiaceae, and increasing Adlercreutzia, Eubacateriaceae. Changes of microbiota in the ceacal contents

21＜

were significantly different from those in the feces (Fig. 6C; S8). Microbiota in the cecal content of C57LB/6J mice

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exhibited the most abundant changes caused by DEHP exposure, these included a significant decrease in

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Actinobacteria,

Desulfovibrio,

Allobaculum,

Bifidobacterium,

Lactobacillus,

Prevotella,

Adlercreutzia,

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Desulfovibrionaceae, Clostridiaceae, and a significant increase in the abundance of Ruminococcus. In contrast,

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BALB/c showed only a decrease in S24-7 and an increase in Rikenellaceae. Changes in abundance of cecal

221

contents in both rats also showed completely different trends. In SD rats, Actinomyces, Arthrobacter and

222

Porphyromonas showed a significant increase, and Bacteroides showed a significant decrease. While in the Wistar

223

rats, only Desulfovibrionaceae and Ruminococcus showed a significant decline, and other genera did not change

224

significantly. The changes in functional pathways of the microbiota in the four rodents also indicated the

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susceptibility of SD rats to DEHP exposure (Fig. S10). All above differences indicated that they may be responsible

22＜

for differences in metabolic changes and differences in tissue damage caused by DEHP in the different rodents. The

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susceptibility of SD rats to DEHP may be related to the significant gut microbiota dysbiosis.

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4. Discussion

229

Di-(2-ethylhexyl) phthalate (DEHP) belongs to endocrine-disruptor chemicals, causing a series of chronic

230

diseases, and host genetics appeared to play important roles in susceptibility to this chemical (Lim and Ghosh,

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2005). The toxic kinetics of DEHP is related to the type and age of the subject (Botelho et al., 2009; Ito et al., 2005).

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Previous studies have reported a relatively higher sensitive to DEHP-induced injury of juvenile versus adult rodents

233

(Sjöberg et al., 2010; Sjöberg et al., 1986; Tonk et al., 2012). This study demonstrated that damages to different

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tissues caused by short term exposure to DEHP during prepuberty are dosage and duration of exposure dependent,

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and also related to the species/strain of experimental animals. Considering the differences in the effects of doses of

23＜

DEHP exposure on gut microbiota in different rodents, we believe that the differential effects of DEHP on gut

237

microbiota may be an important cause of the different susceptibility of rodents to DEHP.

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DEHP belongs to the peroxisome proliferator class of non-genotoxic carcinogens, which can cause liver

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enlargement and peroxisome proliferation (Ashby et al., 1994; Lake et al., 1975; Moody et al., 1991). As a

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peroxisome proliferator, DEHP also activates the peroxide-activated receptor (PPARα) to induce oxidative stress

241

(Ito and Nakajima, 2008; Lapinskas et al., 2005; Wang et al., 2016). In addition, it was reported that MEHP can

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significantly increase level of TNF-α, MCP-1 (Manteiga and Lee, 2017). The most severe tissue damages, strongest

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liver toxicity, most significant induction of liver oxidative stress and strongest sensitivity to reproductive toxicity

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and immunotoxicity were observed in DEHP exposure SD rats in this study, indicates that DEHP-induced toxicity

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not only is dose-dependent but also species/strain dependence. Interestingly, the most significant body weight gain

24＜

and lipid metabolic disorder in SD rats in this study are in agreement with earlier suggestions that DEHP and

247

MEHP disturb lipid metabolism, promote preadipocytes differentiation and induce obesity via activation of PPARγ

248

(Chiang et al., 2017; Grün and Blumberg, 2006). However, our data showed that DEHP exposure caused increase

249

of both PPAR-α and PPAR-β levels, but not PPAP-γ levels in SD rats’ liver. These are in agreement with previous

250

reports that DEHP promoted accumulation of lipids by regulating the PPAR-α signaling pathway in hepatocytes

251

(Zhang et al., 2017), and the activation of PPAR-α could cause oxidative damage to liver (Lapinskas et al., 2005).

252

Meanwhile, PPAR-β showed an essential anti-inflammatory role in the liver except for the regulation on hepatic

253

glucose utilization and lipoprotein metabolism (Poulsen et al., 2012). This seems to be related to the significant

254

inflammatory response in SD rats, as increasing evidence suggests that metabolic disorders such as obesity are

255

accompanied by low levels of inflammation (Das, 2001; Sideleva et al., 2012). In addition, the different trends of

25＜

leptin and insulin changes in the serum of SD and Wistar rats corresponded to the body weight gain, lipid

257

metabolisc disorder, and significant decrease in testosterone levels of SD rats, which are in agreement with other

258

previous studies (Boguszewski et al., 2010; Mantzoros et al., 2011; Thanakun et al., 2017; Zhao et al., 2014).

259

Lyche had demonstrated that rats are more sensitive to phthalates negative effects than mice (Lyche et al., 2009).

2＜0

This difference in resistance to environmental pollutants may be related to its genetic background. However, there

2＜1

is now more evidences to suggest that changes in gut microbiota are closely related to the occurrence of various

2＜2

diseases. For example, metabolic diseases such as obesity and type 2 diabetes may be associated with low levels of

2＜3

inflammation due to disorders of the intestinal microbiota (Collins et al., 2015; Qin et al., 2012; Shen et al., 2013).

2＜4

Many reports indicate that exposure to endocrine disruptors in the environment (such as BPA, DEP, MPB, TCS)

2＜5

can have a significant impact on the gut microbiota (Hu et al., 2016; Javurek et al., 2016; Koestel et al., 2017; Lai

2＜＜

et al., 2016). Therefore, we have good reason to suspect that the physiological damage caused by DEHP exposure is

2＜7

likely to be related to the changes in gut microbiota caused by DEHP. The susceptibility of SD rats to DEHP may

2＜8

be due to changes in their gut microbiota. Our study, for the first time, found that exposure to DEHP caused

2＜9

significant changes in the gut microbiota of rodents, and these changes are host species and strain dependent. More

270

interestingly, the difference in gut microbiota variation in different species/strain of host is also related to the

271

physiological damage caused by DEHP in different species/strain of rodents.

272

Many studies have shown that the rise of Firmicutes/Bacteroidetes ratio is often accompanied by the

273

occurrence of various diseases such as obesity and diabetes (Cani et al., 2008; Turnbaugh et al., 2006). In the

274

present study, the increase in Firmicutes/Bacteroidetes ration only occurred in the cecal contents of SD rats with a

275

significant increase in body weight. Meanwhile, Proteobacteria, which may cause obesity and diabetes, also

27＜

showed an increase in abundance only in the feces of SD rats. This suggests that DEHP may affect the body at

277

metabolic level by affecting Firmicutes/Bacteroidetes ratio. This result is consistent with the results of a previous

278

study in which DEP and MPB reduce Firmicutes/Bacteroidetes ratio, which led to body weight loss in adolescent

279

rats (Hu et al., 2016). Currently, several mechanisms involving gut microbiota have been proposed to explain the

280

development of obesity (Indiani et al., 2018). The first one relates to their ability to digest non-digestible

281

polysaccharides releasing an extra source of calories to the host. The second mechanism refers to a low grade

282

systemic chronic inflammation caused by lipopolysaccharide (LPS) from intestinal bacteria. The third one relies on

283

the ability of gut microbiota in regulating host genes associated with energy metabolism (Khan et al., 2016;

284

Tsukumo et al., 2015). Increased Firmicutes/Bacteroidetes ratio was believed to be associated with increased

285

production of SCFAs and energy harvest from fermentation in colon (Fernandes et al., 2014; Turnbaugh et al.,

28＜

2006). Firmicutes are enriched with genes related to nutrient transporters which could extract more energy from the

287

diet (Tilg and Kaser, 2011; Turnbaugh et al., 2009). Furthermore, Bacteroidetes own fewer genes for enzymes

288

partake in lipid and carbohydrate metabolism than Firmicutes (Kallus and Brandt, 2012). Therefore, an increase in

289

Firmicutes/Bacteroidetes ratio has been found in obese individuals. Furthermore, LPS of the Gram-negative

290

Proteobacteria has been reported to associate with host inflammation (Finegold et al., 2010). Therefore, the

291

Firmicutes/Bacteroidetes ratio and abundance of Proteobacteria only increased in the gut of SD rats with body

292

weight gain, which confirms the first two hypotheses above. In obese individuals, the abundance of Actinobacteria

293

was found to increase and the abundance of Tenericutes decrease (Delzenne and Reid, 2009; Panasevich et al.,

294

2018), although no corresponding changes were found in SD rats in this study, the opposite trend was observed in

295

the cecal contents of C57LB/6J mice. This may also be related to the stability of C57LB/6J mice body weight

29＜

despite of exposure to DEHP.

297

In addition, the significant decrease in Actinobacteria in C57LB/6J mice was mainly due to the decrease in the

298

abundance of Bifidobacterium of the same phylum. Bifidobacterium plays an important role in maintaining

299

intestinal immune homeostasis (Dong et al., 2010; Menard et al., 2008) . A decrease in Bifidobacterium leads to an

300

increase in intestinal permeability and an increase in the concentration of LPS in the blood (Duca et al., 2013),

301

leading to the development of chronic inflammation leading to diabetes, liver damage, etc (Scarpellini and Tack,

302

2012). The intake of Bifidobacterium can reduce the levels of inflammatory cytokines and chemokines, especially

303

IL-6 and MCP-1, elevated by a high-fat diet (Cano et al., 2013). Considering the changes of cytokines level in the

304

four rodents, the decrease in the level of Actinobacteria (Bifidobacterium) in C57LB/6J mice induced by DEHP

305

may be related to the elevated levels of IL-6 and MCP-1 in the serum of this mice.

30＜

Consistent with the most significant Th1 response in SD rats, we found a decrease in the level of butyrate in

307

the intestine of SD rats. Butyrate, as the energy source for the colon epithelial cells, contributes to the normal

308

function of the intestinal barrier (Gao et al., 2009). Many studies have suggested that obesity is accompanied by a

309

decrease in the level of SCFAs, especially a decrease in the level of butyrate, which indicates a low level of

310

inflammation in the intestine (McNabney and Henagan, 2017). In addition, in SD rats, an increase in the abundance

311

of some pathogenic bacteria, such as Actinomyces, Mycoplasma, Porphyromonas were found. Bacteria which are

312

known to associate with intestinal and liver diseases such as Blautia, Peptostreptococcaceae, Sutterella were also

313

significantly increased in the feces of SD rats (Jiang et al., 2015; Labus et al., 2017; Matsushita et al., 2016;

314

Santoru et al., 2017). It has been reported that the level of DEHP in children with autism is higher than in healthy

315

children (Kardas et al., 2016; Testa et al., 2012). The decrease in Prevotella (reducing Bifidobacterium, increasing

31＜

Sutterella and Firmicutes) is also a significant phenomenon in children with autism (Bourassa et al., 2016).

317

Although no social disorders such as autism was observed in this study, a significant reduction in Prevotella

318

abundance was found in the feces of SD rats and in the feces and cecal contents of C57LB/6J mice. This suggests

319

that DEHP may affect the functional integrity of the nervous system by affecting gut microbiota, and the effect of

320

DEHP on the mother may cause autism in the offspring. Interestingly, the increase in Eubacteriaceae in Wistar rats

321

predicts that Wistar rats have some resistance to IBD, autism, and type 1 diabetes caused by DEHP (De Angelis et

322

al., 2013; Omori et al., 2017). Although other rodents did not show an increase in body weight as SD rats, some of

323

the microbiota associated with obesity and diabetes, such as Allobaculum, Dehalobacteiaceae, Rikenellaceae,

324

Ruminococcus were also found. This indicates that metabolic disorder caused by exposure to DEHP can be

325

predicted from changes in gut microbiota profile before the relevant metabolic indicators are fully expressed. In

32＜

addition, the functional pathways of the microbiota in SD rats was most affected by DEHP exposure. The

327

significantly increased abundance of microbial genes associated with pathways such as carbohydrate digestion and

328

absorption, starch and sucrose metabolism, glycolysis/gluconeogenesis in the fecal and cecal contents of SD rats,

329

and a significant increase in microbial gene abundance associated with colorectal cancer, Parkinson's, and type 2

330

diabetes in feces of SD rats, suggested that the most significant structural and physiological damage caused by

331

DEHP exposure in SD rats is likely to be related to the changes in gut microbiota.

332

However, it must be clarified that the genetic background of the host itself is also an important reason for the

333

characteristic changes of the gut microbiota during DEHP exposure. Previous studies have shown that

334

Firmicutes/Bacteroidetes ratio changes significantly from infants, adults and elderly individuals, which is directly

335

related to the diet and intestinal environment (Mariat et al., 2009). In this study, the abundance of Firmicutes in SD

33＜

rats was higher than that in other rodents, suggesting that the intestinal environment of SD rats might be more

337

conducive to the proliferation of Firmicutes. Besides, the original abundance of Protebacteria in the four rodents

338

were also quite different. SD rats had the lowest Protebacteria abundance and was increased after DEHP exposure.

339

While in the other strains with high abundance of Protebacteria, especially C57LB/6J and Wistar, Protebacteria

340

abundance showed a downward trend after DEHP exposure. In addition, differences in the metabolism of DEHP by

341

the host could cause differences in the chemicals available to the bacteria in the intestine, which might lead to

342

differences in the abundance of gut microorganisms. The changes in gut microbiota, host physiological and

343

biochemical, and diseases are interlocked.

344

5. Conclusions

345

All animals, including humans, are exposed daily to a variety of environmental chemicals that affect the gut

34＜

microbiota. Exposure to these chemicals may result in downstream systemic effects secondary to gut microbiota

347

dysbiosis. The intestinal microbial composition of the host may directly affect the effects of the chemicals to the

348

host. Analysis of sensitivity of different prepubertal male rodents to different doses of DEHP exposure suggested

349

that the susceptibility to DEHP exposure may be related to the gut microbiota. Despite the lack of clear evidence of

350

how DEHP-induced changes in the gut microbiota subsequently induce pathological responses in the host, it may

351

open new therapeutic strategies in animals (including humans) exposed to such chemicals through modulation of

352

gut microbiota dysbiosis.

353

Fundings

354

This work was supported by the National Natural Science Foundation of China (No. 31671839, 31972052), the

355

National Natural Science Foundation of China Key Program (No. 31530056), the Fundamental Research Funds for

35＜

the Central Universities (JUSRP51501), a project funded by the Priority Academic Program Development of

357

Jiangsu Higher Education Institutions, the national first-class discipline program of Food Science and Technology

358

(JUFSTR20180102), the Program of Collaborative Innovation Centre of Food Safety and Quality Control in

359

Jiangsu Province, the Natural Science Foundation of Jiangsu Province (BK20180613), the Project funded by China

3＜0

Postdoctoral Science Foundation (2018M642164) and the Postdoctoral Science Foundation of Jiangsu Province

3＜1

(2018K090C).

3＜2 3＜3

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Figure legends

578 579 580

Fig. 1. The effects of DEHP on the body damage in different species/strains of murine rodents. (A) Body weight gain. (B) Liver index. (C) Testis index. (D) Epididymis index. (E) Spleen index. One-way ANOVA with Tukey's

581

multiple comparisons test vs control group, values are mean ± SD calculated by SPSS, *P

582

***P<0.001.

0.05,**P< 0.01,

583 584 585

Fig. 2. Tissue damages caused by DEHP in different species/strains of murine rodents. (A) Liver enzymes (AST, ALT, ALP) activities in rodents. (B) Serum testosterone concentration in rodents. One-way ANOVA with Tukey's

58＜

multiple comparisons test vs control group, values are mean ± SD calculated by SPSS, *P

0.05, **P< 0.01.

587 588

Fig 3. Changes of serum cytokine levels in different species/strains of murine rodent exposed to DEHP. One-way

589

ANOVA with Tukey's multiple comparisons test vs control group (0mg/kg/day/day), values are mean ± SD

590

calculated by SPSS, *P

0.05, **P

0.01, ***P

0.001, ****P

0.0001.

591 592 593 594

Fig 4. Effects on SCFAs levels and gut microflora diversity in rodents by DEHP exposure. (A) Changes of acetic acid and butyric acid levels in feces. (B) α diversity in feces and cecal content indicated by Chao-1 index, Shannon index and Observed_OTUs. One-way ANOVA with Tukey's multiple comparisons test vs control group, values are

595

mean ± SD calculated by SPSS, *P

59＜ 597 598 599 ＜00

Fig 5. Changes in the faecal microbiota in different groups of rodents. (A) Relative abundance (percentage) of bacterial phyla in fecal samples from different rodents. (B) The values of Firmicutes/Bacteroidetes ratio in feces. (C) Variation of fecal bacteria abundance at the genus level in different rodents treated with different doses of DEHP. One-way ANOVA with Tukey's multiple comparisons test vs control group, values are mean ± SD calculated by

＜01

SPSS, *P

＜02 ＜03 ＜04 ＜05 ＜0＜

Fig 6. Changes in the cecal microbiota in different groups of rodents. (A) Relative abundance (percentage) of bacterial phyla in cecal samples from different rodents. (B) The values of Firmicutes/Bacteroidetes ratio in cecal contents. (C) Variation of cecal bacteria abundance at the genus level in different rodents treated with different doses of DEHP. One-way ANOVA with Tukey's multiple comparisons test vs control group, values are mean ± SD

＜07

calculated by SPSS, *P

＜08

0.05,**P< 0.01, ***P

0.05,**P< 0.01, ***P

0.001, ****P

0.05,**P< 0.01, ***P

0.001, ****P

0.0001.

0.0001.

0.001, ****P

0.0001.

Highlights 1. SD rats show most severe organ damage caused by DEHP in 4 species of rodents 2. Only SD rats show significant body weight gain cause by DEHP exposure 3. SD rats show gut microbiota changes corresponding to weight gain and body damage 4. Gut microbiota dysbiosis may be the cause of the susceptibility of SD rats to DEHP

The authors declare that all authors have no competing interests related to this manuscript.