Biological effects of adipocytes in sulfur mustard induced toxicity

Accepted Manuscript Title: Biological Effects of Adipocytes in Sulfur Mustard Induced Toxicity Authors: Hua Xu, Zhongcai Gao, Peng Wang, Bin Xu, Yajiao Zhang, Long Long, Cheng Zong, Lei Guo, Weijian Jiang, Qinong Ye, Lili Wang, Jianwei Xie PII: DOI: Reference:

S0300-483X(17)30341-4 https://doi.org/10.1016/j.tox.2017.11.011 TOX 51979

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Toxicology

Received date: Revised date: Accepted date:

18-8-2017 14-10-2017 7-11-2017

Please cite this article as: Xu, Hua, Gao, Zhongcai, Wang, Peng, Xu, Bin, Zhang, Yajiao, Long, Long, Zong, Cheng, Guo, Lei, Jiang, Weijian, Ye, Qinong, Wang, Lili, Xie, Jianwei, Biological Effects of Adipocytes in Sulfur Mustard Induced Toxicity.Toxicology https://doi.org/10.1016/j.tox.2017.11.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

Biological Effects of Adipocytes in Sulfur Mustard Induced Toxicity Hua Xua,1, Zhongcai Gaoa,b,1, Peng Wanga, Bin Xua, Yajiao Zhanga, Long Longc,

a

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Cheng Zonga, Lei Guoa, Weijian Jiangb, Qinong Yed, Lili Wangc,*, and Jianwei Xie a,*

State Key Laboratory of Toxicology and Medical Countermeasures, and Laboratory of Toxicant

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Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing, China

The Rocket Force General Hospital, PLA, No. 16, Xinjiekouwai Street, Xicheng District,

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State Key Laboratory of Toxicology and Medical Countermeasures, Institute of Pharmacology and

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Beijing 100088, China

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Toxicology, Academy of Military Medical Sciences, Beijing, China d

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Department of Medical Molecular Biology, Institute of Biotechnology, Academy of Military

Medical Sciences, Beijing, China

Corresponding authors.

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*

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E-mail addresses: [email protected] (J. Xie), [email protected] (L. Wang).

These authors made equal contributions to this work.

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Highlights Biological effects caused by SM-accumulated adipocytes were studied.

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Cytotoxicity was compared between SM exposed adipocytes and nonadipocytes.

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Pathological changes of adipose tissues in exposed rats were examined.

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Adipokines induced local and systemic inflammation response.

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Adipose tissue is not only a target but also a modulator in the SM toxicity.

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Abstract

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Sulphur mustard (2,2’-dichloroethyl sulfide; SM) is a vesicant chemical warfare agent

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whose mechanism of acute or chronic action is not known with any certainty and to

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date there is no effective antidote. SM accumulation in adipose tissue (AT) has been

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originally verified in our previous study. To evaluate the biological effect caused by

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the presence of abundant SM in adipocyte and assess the biological role of AT in SM

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poisoning, in vitro and in vivo experiments were performed. High content analysis revealed multi-cytotoxicity in SM exposed cells in a time and dose dependent manner,

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and adipocytes showed a relative moderate damage compared with non-adipocytes.

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Cell co-culture model was established and revealed the adverse effect of SM-exposed adipocyte supernatant on the growth of co-cultured cells. The pathological changes in

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AT from 10 mg/kg SM percutaneously exposed rats were checked and inflammation phenomena were observed. The mRNA and protein levels of inflammation-related adipokines secreted from AT in rats exposed to 1, 3 and 10 mg/kg doses of SM were determined by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assays. The expressions of proinflammatory and anti-inflammatory 2

adipokines together promoted the inflammation development in the body. The positive correlations between AT and serum adipokine levels were explored, which demonstrated a substantial role of AT in systemic inflammation responding to SM

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exposure. Thus, AT is not only a target of SM but also a modulator in the SM toxicity.

Abbreviations: SM, sulfur mustard; AT, adipose tissue; RT-PCR, reverse transcriptase-

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polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; GC-MS, gas

chromatography-mass spectrometry; LC-MS/MS, liquid chromatography-tandem mass spectrometry; POPs, persistent organic pollutants; MnSOD, manganese superoxide

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dismutase; pH2AX, phosphorylated gamma H2AX; MMP, mitochondrial membrane

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potential; NMP, nuclear membrane permeability; CCK-8, cell counting kit-8; PBS,

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phosphate-buffered saline; HCA, high-content analysis; IL-6, interleukin-6; TNF-a, tumor

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necrosis factor-a; PPAR, proliferator-activated receptor; COX-2, cyclooxygenase 2

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Keywords: Sulfur mustard; Adipocyte; Adipose tissue; Serum; Cytotoxicity;

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Adipokine

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1. Introduction Sulfur mustard (2,2’-dichloroethyl sulfide; SM), the highly reactive alkylating agent, was used as a chemical warfare agent in World War I and the 1980s Iran-Iraq conflict and more recently in Syria conflict (Ghabili et al., 2011; Kehe et al., 2008;

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Sawyer et al., 2017; Thiermann et al., 2013). Due to the ease of production and storage, SM is a potential threat to both military and civilian targets. Exposure to SM

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may cause ocular, respiratory, and cutaneous damage that is palpable only several

hours after the exposure. Dermal contact can produce severe skin blisters that last for

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weeks to months, and inhaled SM can cause severe lung damage. In addition to acute

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SM poising, chronic diseases induced by SM have been reported including asthma,

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bronchiectasis, pulmonary fibrosis, immunohematological and psychological

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disorders (Panahi et al., 2016; Razavi et al., 2014; Smith et al., 1995), which greatly

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reduced the life quality of sufferers.

Despite decades of research, the mechanism of SM induced injury and toxicity has

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not been fully clarified. As a strong vesicant and a bi-functional alkylating agent, SM

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mainly interacts with nucleic acids, enzymes, proteins, amino acids and other biomacromolecules when it penetrates into the body, causing extensive damage. The

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consequent alkylation products and metabolites have been considered to be associated with the lesions and used as biomarkers to verify SM exposure (Barr et al., 2008; Benschop et al., 1997; Black et al., 1997a; Black et al., 1992; Black et al., 1997b; Fidder et al., 1996; Li et al., 2013; Nie et al., 2014; Noort et al., 2008; Xu et al., 2014; Yue et al., 2014; Zhang et al., 2014). DNA damage, glutathione (GSH) reduction and 5

inflammation are believed to be the crucial factors in initiating SM-induced toxicity. But these theories cannot well interpret the occurrence and causes of long-term toxic effects after a single SM exposure, given the 30-60 min short half-life of SM in the blood (Xu et al., 2017). It is therefore essential to further elucidate the toxic

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mechanisms of SM, which will allow for better healthcare planning in the event of an exposure and aid in the development of a therapeutic strategy for acute symptoms and

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long-term complications.

SM was assumed to quickly and completely metabolize shortly after penetrating

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the skin, due to its highly reactivity and low stability in biological fluids. However, a

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secondary peak of the hydrolysis metabolite thiodiglycol (TDG) and the persistent

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increasing N-terminal valine adduct to hemoglobin during the first several days were

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observed (Nie et al., 2011), which confused the researchers. One study did report

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unexpectedly high level of SM in tissues, especially in the subcutaneous AT from a dead Iranian victim 7 days postexposure, as detected by GC-MS (Drasch et al., 1987).

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Thus people postulate that a SM depot perhaps exists in AT in vivo (Drasch et al.,

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1987; Xu et al., 2014), considering the lipophilic nature of SM, but suffered from the lack of appropriate method for monitoring SM in biological matrices. Recently, we

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successfully established a novel isotope-dilution LC-MS/MS derivatization method to conveniently and accurately quantitate and profile the toxicokinetics and tissue distribution of intact SM in vivo and initially confirmed that SM significantly accumulated in AT where the concentrations of SM were at least 15 times greater than those in non-adipose tissues in cutaneous exposed rats (Xu et al., 2017). We also 6

studied the fate of DNA adducts in exposed AT and cultured adipose cells (Wang et al., 2015). The adduct persistence behavior in AT was observed, which in turn supported the fact that SM tends to persistently accumulate in lipid-rich environment. In view of SM accumulation in AT has historically been overlooked and to the

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best of our knowledge, no research has described the subsequent biological effect, the

studies on the role of adipocyte in the pathogenesis of the chemical injury are urgently

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needed. Previous researches indicate that AT serves as more than just an energy depot for the body. Its physiological function has been appreciably assessed and its

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metabolic and endocrine functions have been evidenced (Mullerova and Kopecky

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2007; Myre and Imbeault 2014). Adipocytes secrete multiple endocrine factors such

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as resistin and adiponectin to regulate metabolic as well as inflammatory functions.

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AT is now recognized as an endocrine-immune organ which play a crucial role in the

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kinetics and the toxicity of persistent organic pollutants (POPs) (Barrett 2013; La Merrill et al., 2013). Due to the lipophilic properties, POPs, including certain

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organochlorine pesticides and numerous industrial chemicals, are sequestered in AT.

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Studies on how AT both modulates and serves as a target of POP toxicity have been carried out and attracted attention (Lee et al., 2017; Myre and Imbeault 2014). Based

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on these facts, exploring the role of AT in SM toxicity arouses our research interest. This present study aimed to evaluate the biological effect caused by SM

accumulation in adipocytes and adipose tissues, by detecting the cytotoxic or pathological changes in cultured adipocytes and adipose tissues in rats percutaneously exposed to SM. At mRNA and protein levels, the expressions of adipokines in rat 7

model were determined by RT-PCR and ELISAs respectively. The correlations between AT and serum adipokine levels were further explored to demonstrate the role of these molecules secreted from AT involved in system inflammation and immune response to SM exposure.

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2. Materials and methods 2.1. Caution

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SM is a reactive alkylating and cytotoxic agent. Handling of the agent should be

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stringent protective measures should be adopted.

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carried out in well-ventilated fume-cupboard by experienced personnel. Gloves and

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2.2. Chemical and reagents

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All reagents were of analytical reagent grade. SM was supplied by the Institute of Chemical Defense of the Chinese People’s Liberation Army, with purity higher than

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95%. Propidiumiodide (PI), bovine serum albumin (BSA) and Trizol reagent were

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purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluorescence probes (including MitoTracker Red CMXRos, LysoTracker Red, TOTO-3 iodide, Cell Mask

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Deep Red, Hoechst 33342, primary antibodies to manganese superoxide dismutase [MnSOD] and phosphorylated gamma H2AX [pH2AX]) as well as all fluorescent-

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labeled secondary antibodies were purchased from Life Technologies (Carlsbad, CA, USA). SYBR green was the product of TOYOBO Co. (Osaka, Japan). Cell counting kit-8 (CCK-8) was the product of Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). ELISA kits for detection resistin, IL-6 and TNF-α were all obtained from

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Cloud-clone Corp. (Houston, USA). Ultrapure water was produced by a Milli-Q A10 purification system from EMD Millipore Co. (MA, USA). 2.3. Cell lines and cell culture The HPA-s cell line that was derived from human subcutaneous fat tissue was

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purchased from Sciencell Research Laboratories (CA, USA) and cultured in

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preadipocyte medium (PAM) supplemented with 5% fetal bovine serum (FBS, Gibco, NY, USA). The mature adipocytes, HA-s, were differentiated from HPA-s with the previously established method (Wang et al., 2015). Oil-red-O staining was performed

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to monitor the differentiation of HPA-s according to the protocol provided by

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Sciencell Research Laboratories. The human keratinocyte cell line HaCaT, the human

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hepatocyte cell line L02, and the human lung fibroblasts cell line HLF were provided by the Shanghai Cell Bank of the Chinese Academy of Sciences and cultured in

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Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose supplemented

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with 10% FBS. All cultures were maintained at 37 °C in a humidified incubator in 5% CO2. Cells were passaged when they reached 70% to 80% confluency, and all

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experiments were performed below passage number 20. 2.4. Multi-parametric cytotoxicity assay for SM treated cells

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Before SM exposure, HPA-s and HA-s cells were individually plated at 8000

cells/well in poly-L-lysine-coated 96-well plates to grow overnight. SM was initially diluted with DMSO and subsequently diluted to a working concentration with medium, in which the final concentration of DMSO was restricted to less than 0.2%. In concentration-dependent and time-course experiments, 0, 200, and 400 µM SM 9

were used to expose the cells for different duration of 3, 6, 24, and 48 h. Each test was performed in five replicates. After SM treatments for the preset times, multiparametric assays were performed according to the assay panel shown in Table 1. The

For cytotoxicity assay 1, a 50 μL cell culture medium containing 1 μM

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procedures for each assay were as follows.

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MitoTracker Red, 4 μM Hoechst 33342, and 0.4 μM TOTO-3 iodide was added 30

min before the end of SM treatment. Then the cells were fixed in phosphate-buffered saline (PBS) containing 4% formaldehyde for 20 min and then permeabilized using

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0.1% Triton X-100 for 20 min at room temperature (RT). After blocked in PBS

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containing 5% BSA for 30 min at RT, the cells were incubated with a 1:500 diluted

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MnSOD primary antibody at 37 °C for 1 h. After washes with PBS, Alexa Fluor 488

30 min at RT.

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donkey anti-mouse secondary antibody were used at a dilution of 1:500 for staining

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For cytotoxicity assay 2, a 50 μL aliquot of cell culture medium containing 200 nM LysoTracker Red and 4 μM Hoechst 33342 was added to each well 30 min before the

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end of SM treatment. After that, the cells were fixed and permeabilized, and a standard immunofluorescence procedure was conducted. Mouse anti-pH2AX primary

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antibody and Alexa Fluor 488 donkey anti-mouse secondary antibody were individually used at dilutions of 1:1000 and 1:500. Finally, the cells were washed three times, and incubated with PBS containing 100 ng/mL Cell Mask DeepRed at RT for 1 h to label the cytoplasm in order to display the whole-cell shape and texture.

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Image acquisition were performed with an IN Cell Analyzer 2000 (GE Healthcare, USA) using a 20 × lens, 50 ms exposure and 15 fields per well. Excitation and emission filters were selected specially according to the specific wavelength of each fluorescent dye. The acquired images were analyzed using an INCell Analyzer

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Workstation 3.5 and the Multi Target Analysis Module (GE Health Care, USA).

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2.5. Cell cultivation with the culture supernatant of SM-exposed HA-s

Mature adipose cells HA-s were exposed to 0.1, 0.5 and 1 mM SM diluted in FBSfree culture medium for 1 h at 37 °C. The supernatant was removed and the cells were

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washed three times with medium. Subsequently, cells were cultured in fresh complete

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medium at 37 °C in the presence of 5% CO2. After 24 h, the supernatants of SM-

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exposed adipocytes were individually collected. The supernatants of 0.1 mM SMexposed HLF cells were harvested in the same way. The culture supernatants from

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exposed HA-s or HLF were used to co-culture with SM exposed cells of HacaT, HLF and L02, respectively. The influence of co-culture treatment was evaluated by CCK-8

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method according to the manufacturer’s instruction, to evaluate the cell proliferation

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under the SM exposure, with or without the co-culture supernatant. Briefly, HacaT, HLF and L02 cells were separately seeded in 96-well plates at a density of 1×104

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cells/well and grown overnight. Then the cells were exposed to 0, 25, 50, 100, 200, 250, 300, 350 and 400 µM SM respectively for 1 h, and then the media were removed and cells were washed with FBS-free culture medium. After that, cells were cultured in normal medium or co-cultured with the harvested culture supernatant from SM exposed HA-s or HLF for another 48 h. Viable cells were quantified by a CCK-8 11

assay in which the absorbance of the samples was measured at 450 nm. IC50 was calculated according to the cell growth curves. Experiments were repeated at least three times. 2.6. Animals

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Adult male, specific pathogen free (SPF), Sprague-Dawley rats (180-200 g) were

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purchased from the Laboratory Animal Center of Beijing. After acclimatization for 3 days, 65 rats were randomly allocated into 13 experimental groups (n=5 per group)

including a control group and four groups per each dosage level. Treatment procedure

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was the same as that described previously (Xu et al., 2017). After the back hair of rat

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was depilated by 8% Na2S, a 10 μL fresh dilution of SM in dimethyl sulfoxide was

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uniformly pipetted onto the bare skin with a square area of approximately 1 cm2. And the treatment site of the skin was immediately covered by plastic wrap. SM was

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administrated as a single dose of 1, 3 and 10 mg/kg body weight. The rats were

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euthanized with urethane by intraperitoneal injection at 1, 3, 9 and 12 h after exposure, and subcutaneous adipose tissues were immediately isolated. Specimens

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were stored at -70 °C prior to biological analysis. To observe the pathological changes of adipose tissues in SM exposed rats, animals

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were dermally exposed to SM at a dosage of 10 mg/kg. The rats were euthanized at 24, 48, 72 and 96 h after exposure (n=5), four types of adipose tissues including epididymal, perirenal, subcutaneous and brown fat were isolated and weighed. Tissue coefficients of four adipose tissues were monitored by the ratios of wet weights of adipose vs. the body weights. Partial specimens from rats exposed for 96 h were fixed 12

in formalin and embedded in paraffin for H&E histological examination. Control rats were also included in this experiment. For high-dose exposure group (10 mg/kg), another 20 rats were divided into four groups and individually euthanized after a cutaneous treatment of 0.5, 24, 48 and 72

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h. Blood samples were obtained from the inferior vena cava, allowed to clot at 4 °C

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and then sera were obtained after centrifugation. Subcutaneous adipose tissues were also quickly dissected and frozen in liquid nitrogen for adipokine analyses.

During treatment, the rats were housed in individual cages under constant

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temperature and humidity. Food and water were available ad libitum under a 12 h

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light/ 12 h dark cycle. All animal experiments were in compliance with the guidelines

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of the Association for Assessment and Accreditation of Laboratory Animal Care

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International. 2.7. Quantitative RT-PCR

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Total RNA was isolated from rat subcutaneous adipose tissues using Trizol

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reagent and the concentration was calculated on spectrophotometer. For RT-PCR, 1 µg of total RNA was reverse-transcribed in a 20 µl reaction volume using cDNA Synthesis kit for RT-qPCR (Shenggong Co., Shanghai, China). The cDNA was

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diluted to 50 µL of which 1 µL was used as templates in subsequent PCR assays. PCRs were performed using the primers listed in Table 2 at a final concentration of 200 nM, by qTOWER 2.0 Real-Time PCR System (Analytik Jena AG, Jena,

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Germany). Relative transcript abundance of a gene was quantitated using the formula of 2-ΔΔCT. Gapdh mRNA levels were used for normalization. 2.8. Extraction of total proteins in adipose tissues Frozen subcutaneous adipose tissues were placed on ice, and 200 mg excisions

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were taken from these frozen samples. The excised tissues were dissolved in 1 mL

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precooled PBS (0.01 mol/L, pH 7.4) and homogenized using a TissueLyser (Scientz,

Ningbo, China) with 7 mm stainless steel beads. The parameters were set at 70 Hz for 60 sec and the adapter sets were precooled at -80 ºC. After two freeze-thaw cycles,

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homogenates were centrifuged at 14,000 g for 10 min. The middle layer was the

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protein-required liquid.

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2.9. ELISA

Concentrations of resistin, IL-6, and TNF-α in both subcutaneous adipose tissues

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and sera were measured with ELISA kits. The detection of adipokines was performed

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according to the manufacturer’s instruction. For tissue-extracted proteins, after being diluted to 1 µg/ µL with PBS, protein isolates were aspirated (100 µL per sample) to

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detect the three adipokines. For sera, a 1:10 dilution of specimen was employed for detection of IL-6 or resistin, and a 1:100 dilution solution was used for detection of

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TNF-α.

2.10. Statistical analysis All values were reported as the means ± standard deviation (SD). Data were analyzed with SPSS version 19.0 using single factor analysis of variance (ANOVA). 14

Comparison analyses among the means of groups were performed by Fisher’s least significant difference (LSD) method, in which differences were considered statistically significant at p < 0.05. Spearman linear correlation analysis between AT

3. Results and discussion

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3.1. Cytotoxic effect caused by SM in HPA-s and HA-s

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and serum adipokine levels was also applied.

Cytotoxicity is a complex process, associated with a variety of molecules and pathways. High-content analysis (HCA) is an image-based methodology for the

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simultaneous detection of multiple cellular parameters in live or fixed cells.

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Multichannel fluorescent signals can be quantitatively measured at the single-cell

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level, increasing the effectiveness and authenticity of the analysis. In recent years it has been widely used in cytotoxicity analysis (Nierode et al., 2016; Rausch 2006).

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Herein, we used HCA-based multi-parametric toxicity assay to access the dynamic

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cytotoxic profiles of SM in preadipocyte HPA-s and adipocyte HA-s. SM was found to produce concentration and time-dependent cellular responses in both cells,

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including decreased cell counts, increased membrane permeability, oxidative stress, DNA damage, and lysosome impairment (Fig. 1; Fig. S1).

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The number of HPA-s or HA-s cells labeled with Hoechst 33342 decreased with

the increase in exposure dose and time, but the change degree of HA-s was less. The nuclear area of both cell lines increased with the exposure dose and time, while HA-s demonstrated a relatively moderate change (Data not shown). These results suggested that SM caused significant damage to nucleus and affected the survival of exposure 15

cells. On the other hand, the nucleus damage caused by SM in HA-s is less than in HPA-s. This might be related to the lipid rich environment of adipocyte which may to some extent inhibit the reaction activity of SM. Cell manganese superoxide dismutase (MnSOD) content, mitochondrial membrane potential (MMP), and nuclear membrane

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permeability (NMP) were measured at 3, 6, 24, and 48 h after 400 μM SM treatment. Significant increases in MnSOD content and the changes of NMP and MMP levels

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were observed at 48 h, and there were more severe oxidative stress and mitochondrial damage caused by SM in HPA-s than in HA-s. MnSOD, one of the major scavengers

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of endogenous reactive oxygen species (ROS), is believed to be an indicator of

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adaptive response against oxidative stresses and a major factor in oxidant toxicity

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(Long et al., 2016; Lustgarten et al., 2011). MnSOD alterations have preceded the

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changes in MMP and NMP, which are considered to participate in the induction of

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apoptosis. These results were consistent with the observed apoptosis response in

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HPA-s and HA-s, in which the response of HPA-s was more pronounced. In Cytotoxicity assay panel 2, LysoTracker Red- stained lysosomes and Alexa

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Fluor 488-labeled pH2AX were assayed at 3, 6, 24, and 48 h after SM treatment. The lysosome intensity increased with the dose and exposure time in both cells. The rising

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trend in HPA-s was more obvious than in HA-s, indicating a stronger lysosomal injury in HPA-s and a further cell damage may be caused by the loss of function of intracellular proteins (Maejima et al., 2013). The expression of pH2AX, an indicator of DNA damage, increased in both cells especially in HPA-s. The fluorescence became stronger with the prolonged exposure time and significant differences were 16

observed at each time point between HPA-s and HA-s. H2AX is a variant of H2A and its conserved SQ motif (SQRY) is phosphorylated in response to DNA-damaging agents that can introduce double-strand breaks (DSBs). pH2AX is now recognized as a marker for DNA damage induced by oxidative bases, DNA adducts, single strand

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breaks (SSBs), and crosslinks, and also by the repair of this damage (Ibuki and Toyooka 2015; Rios-Doria et al., 2006; Scully and Xie 2013). Results showed that

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SM induced a time and concentration-dependent DNA damage in both cells and the

fluorescence intensity of pH2AX foci in exposed cells was significantly stronger than

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other cytotoxicity parameters, indicating DNA was the main target of SM. A

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relatively modest DNA damage appeared in HA-s, demonstrated by the staining

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pattern of pH2AX foci, reflecting to some degree a fat-rich environment in HA-s

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might protect DNA from SM rapidly attack, which is also supported by the fact that

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concentrations of DNA adducts were low in AT (Wang et al., 2015). On the other hand, pH2AX is known to be involved in caspase-dependent apoptosis pathway and

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its expression may become high in preapoptotic cells (Firsanov et al., 2011). A higher

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pH2AX staining signal was observed in HPA-s than in HA-s, probably suggesting more preapoptosis occurred in HPA-s, which was consistent with the Hoechst 33342 staining results that SM caused more apoptosis in HPA-s cells (Fig. S1). Taken

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together, the lesion degree of the exposed HA-s is not as severe as HPA-s evidenced by this multi-parametric cytotoxicity assay. 3.2. Co-culture effect of exposed adipocyte supernatants on SM-exposed cells

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In order to study whether SM-sequestered adipocytes have biological effects on other cells, we established a co-culture model. The culture supernatants from exposed adipocytes HA-s or lung fibroblasts HLF were collected and employed to co-culture with SM exposed cells of HacaT, HLF and L02, respectively. As shown in Fig. 2,

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HLF supernatant co-culture led to higher IC50 values of SM exposed cells, while HAs supernatant resulted in decreased IC50 values of the three cells. This showed that

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exposed-HLF supernatant had a protective effect on cells in vitro, suggesting that its secretion of cytokines may promote the repair and survival of damage cells. On the

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contrary, the supernatant of exposed mature adipocyte HA-s further inhibited the

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growth activity of SM-treated cells. The negative effect may be due to the cytokine

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secretion from exposed HA-s aggravated cell injury via promoting the apoptosis and

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necrosis of treated cells. Furthermore, the sequestered SM in adipocytes may release

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to the supernatant after the cell death and consequently induce the cytotoxicity in other cells. The survival inhibition in all three cells exacerbated by HA-s supernatants

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was dose-dependent, for example, the IC50 value of SM-exposed HLF was 73 μM

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without co-culture HA-s supernatant and decreased by 8%, 16% and 29% when cells were co-cultured with 0.1, 0.5 and 1 mM SM treated HA-s supernatant respectively. Pathological changes of adipose tissues in rats with percutaneous exposure

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3.3.

to SM In order to systematically evaluate the biological effect of SM accumulation in AT, we established a cutaneous intoxication model with SD male rats (Xu et al., 2017). The rats were treated with 10 mg/kg SM, and the body weight was weighed before 18

sacrifice. The four major adipose tissues of subcutaneous fat, perirenal fat, epididymal fat and brown fat were weighed and recorded. Fat coefficient was calculated by the percentage of the wet weight of AT versus body weight, which reflects the content of body fat within rat. As shown in Fig. S2, the weight of untreated rats increased during

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the observation period while treated rats lost their weight with time. The poisoned rats gradually reduced the intake of food and water, became lethargic and curled up in the

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corner of the cage. Urinary and fecal incontinence were observed in these injured rats and continuous death of the animals occurred during the observation period.

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Anatomical experiments found that exposed rats suffered severe intestinal damage

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and the intestinal wall turned almost transparent due to the edema. The observation

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indicated that high dose of SM seriously affected the normal diet and behavior of

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animal. The fat coefficients of four ATs declined with the exposure time, which is

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consistent with the anatomical results that the AT volume significantly decreased with exposure time in exposed rats. Compared with the weight loss, the decreases of fat

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coefficients in the subcutaneous, perirenal, epididymal and brown fat tissues were

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more obvious which proved that adipose tissues were consumed after SM exposure. This also suggests that the high level of SM retention in AT is likely to undergo secondary release with the large consumption of AT, and it is meaningful to clear the

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SM in AT to avoid the subsequent poisoning. In order to explore the pathological damage in AT, sections from ATs in exposed rats or control rats were stained with H&E. The blood vessel dilatation, vessel wall thickness and inflammatory cell infiltration were observed in subcutaneous AT. 19

Newborn adipocytes were visible in perirenal AT, as marked in the red box in Fig. 3. Capillary congestion was also observed in epididymal AT. For brown AT, at the 4th day postexposure, there were obvious pathological changes that poisoned cells turned unexpectedly larger compared to normal brown AT cells and became white

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adipocyte-like. The appearance of “whitening” in brown AT indicated the adipocyte differentiation was seriously affected and a significant increase in body energy

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expenditure. Brown adipocytes contain multiple lipid droplets that are packed with

mitochondria, different from white adipocytes which mainly consist of a single large

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cytoplasmic lipid droplet and possess only a few mitochondria, leading to a high

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efficiency in energy mobilization. Recently, brown adipocyte-like i.e. “browning”

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cells were identified in white AT depots in response to cold and hormonal stimuli

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(Elattar and Satyanarayana 2015; Harms and Seale 2013; Qin et al., 2016). In our

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experiment, we were surprised to find the opposite case that whitening phenomena occurred in brown AT, which has not been reported previously. The mechanism and

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the biological significance associated with the increased white fat mass in brown AT

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is worthy of the further study. 3.4. SM accumulation regulates the mRNA expression of adipokines in AT

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In above mentioned experiments, we found that infiltrating immune cells presented

in adipose tissues and adipocytes had a relative resistance to SM exposure. AT has been found to contain diverse cell types, including adipocytes, preadipocytes and immune cells. AT especially white adipose tissue (WAT) is now regarded as an active contributor to whole-body homeostasis rather than just a fat depot with the discovery 20

of more than 50 cytokines (Lago et al., 2007; Shibata et al., 2017). These adipokines engage, through endocrine, paracrine, autocrine or juxtacrine mechanisms of action, in a wide variety of physiological or pathological processes, including immunity and inflammation. Most adipokines such as cyclooxygenase 2 (COX-2), leptin, visfatin,

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resistin, TNF-a and IL-6 display proinflammatory properties and are harmful in the setting of diseases. In contrast, a few adipokines including adiponectin and

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peroxisome proliferator-activated receptor (PPAR) α exert protective effects in

metabolic, inflammatory and rheumatic diseases. The imbalanced production of

U

adipokines was proven to contribute to the pathogenesis of obesity-linked metabolic

N

and cardiovascular complications (Jung and Choi 2014; Shibata et al., 2017).

A

However, no study focuses on the adipokine production after SM intoxication.

M

Therefore, herein we aimed to assess the impact of adipokines produced in AT as

ED

inflammation regulation cytokines responding to SM poisoning.

PT

On the whole, the expressions of adipokines after SM accumulated in AT were obviously dose and time dependent. As demonstrated in Fig. 4, adiponectin mRNA

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level showed a first-drop and then-rise trend, especially in 3 mg/kg and 10 mg/kg exposure groups. As an anti-inflammatory factor (Lago et al., 2007), an early decline

A

of adiponectin gene expression suggested an up-regulation of AT inflammation, and the later risen level may be involved in the subsequent systemic inflammatory response. The mRNA level of COX-2 dramatically increased rapidly after exposure, reached its peak at 1 h, and then decreased. This is consistent with the characteristics of COX-2 which is the product of an “immediate-early” gene that is rapidly inducible 21

and tightly regulated during inflammation (Crofford 1997). The subsequent down regulation of COX-2 may be due to the negative feedback of its downstream products. It is worth noting that the overall level of COX-2 mRNA in AT of exposed rats is high, indicating that COX-2 plays an important role in the development of AT

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inflammation. As for IL-6, an essential inflammation-related cytokine although its pro-inflammatory role in chronic inflammation diseases or its anti-inflammatory role

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in acute inflammatory responses remains to be elucidated (Kaplanski et al., 2003), its mRNA level showed a gradual increase tendency with the increased exposure dose

U

and time, suggesting its essential role in the AT inflammation. PPAR-α as an anti-

N

inflammatory factor (Lo Verme et al., 2005), the mRNA level decreased rapidly after

A

exposure, and maintained at a significantly low level after 3 h postexposure. This

M

proved a persistence of inflammation in AT, and meanwhile suggested that PPAR-α

ED

was not greatly affected by other feedback within 12 h. PPAR-γ, a proinflammatory cytokine (Cipolletta et al., 2012), exhibited different mRNA expression changes in

PT

different dose groups. The mRNA level of PPAR-γ increased with the exposure time

CC E

in high-dose group, while decreased in low-dose group, reflecting the inflammation state of AT responded to different SM exposure level. In addition, PPAR-γ has been identified as a “master-regulator” of adipocyte differentiation (Rosen et al., 1999), the

A

fluctuation of its mRNA level may meanwhile reflect the adipocyte differentiation affected by SM exposure and the exposure dosage, as hinted by the “whitening” of brown AT. As for leptin, in middle and high dose groups, its mRNA level has an overall increase with the exposure time. As one of the main peripheral endocrine 22

signals involved in the regulation of appetite and body weight (Lago et al., 2007) expect for its pro-inflammation function, the up-regulated mRNA was consistent with the observation of decreased food intake in exposed rats. Resistin level showed a marked rise in three groups, especially at 9 h postexposure in high-dose group, which

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elevated by 90% compared to the control group. Resistin is known to engage in inflammatory conditions in humans by means of its secretion in substantial quantities

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by mononuclear cells. And there was evidence that resistin increased the expression

of cell adhesion molecules to regulate the inflammation progress (Lago et al., 2007).

U

Therefore, high mRNA level of resistin indicated its key role in inducing

N

inflammatory factors and the recruitment of inflammatory cells in exposed AT. The

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mRNA level of visfatin increased to maximum at about 3 h postexposure and then

M

gradually returned to the normal level. Visfatin was reported to highly express in

ED

visceral adipose tissue of both humans and rodents and mimic the effects of insulin by lowering plasma glucose levels (Lago et al., 2007), so its increased expression may be

PT

related to energy metabolism under the intoxication stress. TNF-α, as an important

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immune effector, its mRNA level quickly increased postexposure and increased with the exposure dose. The results demonstrated that TNF-α had a major role in the early

A

stage of AT inflammation. 3.5.

Effects of SM accumulation on levels of adipokines in adipose tissues and

sera of rats The effects of SM accumulation in AT on serum resistin, IL-6 and TNF-α levels were assessed by ELISA. The concentration- time profile of adipokines exhibited a 23

similar fluctuation tendency between subcutaneous adipose tissue and serum. As mentioned above, the inflammatory factor resistin showed a significant dose and timedependent increase in the mRNA level postexposure, so its protein levels both in AT and sera were emphatically discussed here. The expression of resistin raised

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significantly at 0.5 h and 24 h in exposed AT compared with the control group (Fig. 5). There was a consequent decline in resistin concentration at 48 h and 72 h. The

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profile of sera resistin level showed a similar tendency, which slightly increased at first and then significantly decreased at 48 h and 72 h compared with the control

U

group. For the inflammation effectors IL-6 and TNF-α, both of them have been

N

reported to show a rapid increase level in sera after SM exposure, here we also

A

observed their sera levels increased and achieved the highest at 24 h (P< 0.05) and

M

then gradually decreased. The correlation between subcutaneous adipose tissue and

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serum adipokine levels were further assessed. As shown in Table 3, a positive correlation between AT and serum was found for IL-6 and TNF-α. The results

PT

suggested that AT contributed to circulating levels of adipokines and played a critical

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role in the systemic inflammation and metabolic disorders after SM exposure and accumulation in AT.

A

In recent years, AT has been reported to constitute an internal source of stored

lipophilic xenobiotic chemical, in particular, POPs, and AT can protect other organs and tissues from POPs overload. But more recently, this protective function is proved to be a threat in the long run because the accumulated POPs are slowly release into the bloodstream leading to continuous exposure in body and AT can be a target for 24

the xenobiotic chemical that alters AT functions (Barrett 2013). Our findings, for the first time, confirmed the double-sided effects of AT in the toxic mechanism of lipophilic SM. Although AT showed a certain resistance and protection to SM exposure, accumulated intact SM in AT could induce adverse effects in vivo through

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increasing AT inflammation and modulate the differentiation of AT precursor cells by

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regulating the adipokine expression and release. 4. Conclusions

In this paper, in vitro and in vivo experiments were performed to reveal the role and

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biological effect of adipose tissues in SM intoxication, especially when SM

N

accumulated in these tissues. Adipocytes showed a multiple toxic response but a

M

A

relative resistance to SM compared to non-adipocytes, and co-culture adverse effects on other cultured cells were observed by the supernatant of SM-exposed adipocytes.

ED

The pathological changes and consumption of adipose tissues were initially discussed

PT

here. The disturbance of proinflammatory and anti-inflamamtory adipokine mRNA expression may lead to the local inflammation in adipose tissues and systemic

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inflammation. SM accumulation in AT affected adipokine levels in adipose tissues and sera in a time-dependent manner. The altered levels of adipokines in AT and sera

A

demonstrated a synchronization trend and even a positive correlation. These results improve our understanding of biological effects especially the inflammatory role of AT in a SM toxicity model. In addition to its buffering function, AT is also a target of SM and may mediate the metabolic and toxic effects by secretion of adipokines. Importantly, the identified double-sided properties of AT make it a novel and 25

interesting therapeutic target in SM intoxication. Development of lipophilic scavenger to clean the accumulated SM or targeting adipokines may be an efficacious approach to mitigating the inflammation effects of SM induced in humans.

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Conflict of interest The authors declare that there is no conflict of interest.

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Acknowledgments

This work was supported by the Chinese National Scientific Research Special-

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Purpose Project in Public Health Profession (Grant No. 2015SQ00192), and the Major

N

Projects of the PLA Medical Science and Technology (Grant No. AWS11C004

A

sub).References

A

CC E

PT

ED

M

Barr, J.R., Pierce, C.L., Smith, J.R., Capacio, B.R., Woolfitt, A.R., Solano, M.I., Wooten, J.V., Lemire, S.W., Thomas, J.D., Ash, D.H., Ashley, D.L., 2008. Analysis of urinary metabolites of sulfur mustard in two individuals after accidental exposure. J. Anal. Toxicol. 32,10-16. Barrett, J.R., 2013. POPs vs. fat: persistent organic pollutant toxicity targets and is modulated by adipose tissue. Environ. Health Perspect. 121, a61. Benschop, H.P., van der Schans, G.P., Noort, D., Fidder, A., Mars-Groenendijk, R.H., de Jong, L.P., 1997. Verification of exposure to sulfur mustard in two casualties of the Iran-Iraq conflict. J. Anal. Toxicol. 21, 249-251. Black, R.M., Clarke, R.J., Harrison, J.M., Read, R.W., 1997a. Biological fate of sulphur mustard: identification of valine and histidine adducts in haemoglobin from casualties of sulphur mustard poisoning. Xenobiotica 27, 499-512. Black, R.M., Hambrook, J.L., Howells, D.J., Read, R.W., 1992. Biological fate of sulfur mustard, 1,1'-thiobis(2-chloroethane). Urinary excretion profiles of hydrolysis products and beta-lyase metabolites of sulfur mustard after cutaneous application in rats. J. Anal. Toxicol. 16, 79-84. Black, R.M., Harrison, J.M., Read, R.W., 1997b. Biological fate of sulphur mustard: in vitro alkylation of human haemoglobin by sulphur mustard. Xenobiotica 27, 1132. Cipolletta, D., Feuerer, M., Li, A., Kamei, N., Lee, J., Shoelson, S.E., Benoist, C., Mathis, D., 2012. PPAR-gamma is a major driver of the accumulation and phenotype of adipose tissue Treg cells. Nature 486, 549-553. 26

A

CC E

PT

ED

M

A

N

U

SC R

IP T

Crofford, L.J., 1997. COX-1 and COX-2 tissue expression: implications and predictions. J. Rheumatol. Suppl. 49, 15-19. Drasch, G., Kretschmer, E., Kauert, G., von Meyer, L., 1987. Concentrations of mustard gas [bis(2-chloroethyl)sulfide] in the tissues of a victim of a vesicant exposure. J. Forensic. Sci. 32, 1788-1793. Elattar, S., Satyanarayana, A., 2015. Can Brown Fat Win the Battle Against White Fat? J. Cell Physiol. 230, 2311-2317. Firsanov, D. V., Solovjeva, L. V., Svetlova, M. P., 2011. H2AX phosphorylation at the sites of DNA double-strand breaks in cultivated mammalian cells and tissues. Clin. Epigenetics. 2, 283-297. Fidder, A., Noort, D., de Jong, A.L., Trap, H.C., de Jong, L.P., Benschop, H.P., 1996. Monitoring of in vitro and in vivo exposure to sulfur mustard by GC/MS determination of the N-terminal valine adduct in hemoglobin after a modified Edman degradation. Chem. Res. Toxicol. 9, 788-792. Ghabili, K., Agutter, P.S., Ghanei, M., Ansarin, K., Panahi, Y., Shoja, M.M., 2011. Sulfur mustard toxicity: history, chemistry, pharmacokinetics, and pharmacodynamics. Crit. Rev. Toxicol. 41, 384-403. Harms, M., Seale, P., 2013. Brown and beige fat: development, function and therapeutic potential. Nat. Med. 19, 1252-1263. Ibuki, Y., Toyooka, T., 2015. Evaluation of chemical phototoxicity, focusing on phosphorylated histone H2AX. J. Radiat. Res. 56, 220-228. Jung, U.J., Choi, M.S., 2014. Obesity and its metabolic complications: the role of adipokines and the relationship between obesity, inflammation, insulin resistance, dyslipidemia and nonalcoholic fatty liver disease. Int. J. Mol. Sci. 15, 6184-6223. Kaplanski, G., Marin, V., Montero-Julian, F., Mantovani, A., Farnarier, C., 2003. IL-6: a regulator of the transition from neutrophil to monocyte recruitment during inflammation. Trends Immunol. 24, 25-29. Kehe, K., Balszuweit, F., Emmler, J., Kreppel, H., Jochum, M., Thiermann, H., 2008. Sulfur mustard research--strategies for the development of improved medical therapy. Eplasty 8, e32. La Merrill, M., Emond, C., Kim, M.J., Antignac, J.P., Le Bizec, B., Clément, K., Birnbaum, L.S., Barouki, R., 2013. Toxicological function of adipose tissue: focus on persistent organic pollutants. Environ. Health Perspect. 121, 162-169. Lago, F., Dieguez, C., Gomez-Reino, J., Gualillo, O., 2007. Adipokines as emerging mediators of immune response and inflammation. Nat. Clin. Pract. Rheumatol. 3, 716-724. Lee, Y.M., Kim, K.S., Jacobs, D.R.Jr., Lee, D.H., 2017. Persistent organic pollutants in adipose tissue should be considered in obesity research. Obes. Rev. 18, 129139. Li, C., Chen, J., Liu, Q., Xie, J., Li, H., 2013. Simultaneous quantification of seven plasma metabolites of sulfur mustard by ultra high performance liquid chromatography-tandem mass spectrometry. J. Chromatogr. B. 917-918, 100107. 27

A

CC E

PT

ED

M

A

N

U

SC R

IP T

Lo Verme, J., Fu, J., Astarita, G., La Rana, G., Russo, R., Calignano, A., Piomelli, D., 2005. The nuclear receptor peroxisome proliferator-activated receptor-alpha mediates the anti-inflammatory actions of palmitoylethanolamide. Mol. Pharmacol. 67, 15-19. Long, L., Li, W., Chen, W., Li, F.F., Li, H., Wang, L.L., 2016. Dynamic cytotoxic profiles of sulfur mustard in human dermal cells determined by multiparametric high-content analysis. Toxicol. Res. 5, 583-593. Lustgarten, M.S., Jang, Y.C., Liu, Y., Qi, W., Qin, Y., Dahia, P.L., Shi, Y., Bhattacharya, A., Muller, F.L., Shimizu, T., Shirasawa, T., Richardson, A., Van Remmen, H., 2011. MnSOD deficiency results in elevated oxidative stress and decreased mitochondrial function but does not lead to muscle atrophy during aging. Aging cell 10, 493-505. Maejima, I., Takahashi, A., Omori, H., Kimura, T., Takabatake, Y., Saitoh, T., Yamamoto, A., Hamasaki, M., Noda, T., Isaka, Y., Yoshimori, T., 2013. Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury. EMBO J. 32, 2336-2347. Mullerova, D., Kopecky, J., 2007. White adipose tissue: storage and effector site for environmental pollutants. Physiol. Res. 56, 375-381. Myre, M., Imbeault, P., 2014. Persistent organic pollutants meet adipose tissue hypoxia: does cross-talk contribute to inflammation during obesity? Obes. Rev. 15, 1928. Nie, Z, Zhang, Y., Dong, Y., Wu, B., Liu, Q., Feng, J., 2011. Determination of the Nterminal valine adduct in rabbit hemoglobin after skin exposure to sulfur mustard. Scientia Sinica 41, 965. Nie, Z., Zhang, Y., Chen, J., Lin, Y., Wu, B., Dong, Y., Feng, J., Liu, Q., Xie, J., 2014. Monitoring urinary metabolites resulting from sulfur mustard exposure in rabbits, using highly sensitive isotope-dilution gas chromatography-mass spectrometry. Anal. Bioanal. Chem. 406, 5203-5212. Nierode, G., Kwon, P.S., Dordick, J.S., Kwon, S.J., 2016. Cell-based assay design for high-content screening of drug candidates. J. Microbiol. Biotechnol. 26, 213225. Noort, D., Fidder, A., Degenhardt-Langelaan, C.E., Hulst, A.G., 2008. Retrospective detection of sulfur mustard exposure by mass spectrometric analysis of adducts to albumin and hemoglobin: an in vivo study. J. Anal. Toxicol. 32, 25-30. Panahi, Y., Jadidi-Niaragh, F., Jamalkandi, S.A., Ghanei, M., Pedone, C., Nikravanfard, N., Nikravesh, F., Sahebkar, A., 2016. Immunology of chronic obstructive pulmonary disease and sulfur mustard induced airway injuries: implications for immunotherapeutic interventions. Curr. Pharm. Des. 22, 2975-2996. Qin, X,, Park, H.G., Zhang, J.Y., Lawrence, P., Liu, G., Subramanian, N., Kothapalli, K.S., Brenna, J.T., 2016. Brown but not white adipose cells synthesize omega3 docosahexaenoic acid in culture. Prostaglandins Leukot. Essent. Fatty Acids. 104, 19-24. Rausch, O., 2006. High content cellular screening. Curr. Opin. Chem. Biol. 10, 316320. 28

A

CC E

PT

ED

M

A

N

U

SC R

IP T

Razavi, S.M., Negahban, Z., Pirhosseinloo, M., Razavi, M.S., Hadjati, G., Salamati, P., 2014. Sulfur mustard effects on mental health and quality-of-life: a review. Iran. J. Psychiatry Behav. Sci. 8, 11-21. Rios-Doria, J., Fay, A., Velkova, A., Monteiro, A.N., 2006. DNA damage response: determining the fate of phosphorylated histone H2AX. Cancer Biol. Ther. 5, 142-144. Rosen, E.D., Sarraf, P., Troy, A.E., Bradwin, G., Moore, K., Milstone, D.S., Spiegelman, B.M., Mortensen, R.M., 1999. PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro. Mol. cell 4, 611-617. Sawyer, T.W., McNeely, K., Louis, K., Lecavalier, P., Song, Y., Villanueva, M., Clewley, R., 2017. Comparative toxicity of mono- and bifunctional alkylating homologues of sulphur mustard in human skin keratinocytes. Toxicology 382, 36-46. Scully, R., Xie, A., 2013. Double strand break repair functions of histone H2AX. Mutat. Res. 750, 5-14. Shibata, R., Ouchi, N., Ohashi, K., Murohara, T., 2017. The role of adipokines in cardiovascular disease. J. cardiol. S0914-5087, 30060-30066. Smith, K.J., Hurst, C.G., Moeller, R.B., Skelton, H.G., Sidell, F.R., 1995. Sulfur mustard: its continuing threat as a chemical warfare agent, the cutaneous lesions induced, progress in understanding its mechanism of action, its long-term health effects, and new developments for protection and therapy. J. Am. Acad. Dermatol. 32, 765-776. Thiermann, H., Worek, F., Kehe, K., 2013. Limitations and challenges in treatment of acute chemical warfare agent poisoning. Chem. Biol. Interact. 206, 435-443. Wang, P., Zhang, Y., Chen, J., Guo, L., Xu, B., Wang, L., Xu, H., Xie, J., 2015. Analysis of different fates of DNA adducts in adipocytes post-sulfur mustard exposure in vitro and in vivo using a simultaneous UPLC-MS/MS quantification method. Chem. Res. Toxicol. 28, 1224-1233. Xu, B., Zong, C., Zhang, Y., Zhang, T., Wang, X., Qi, M., Wu, J., Guo, L., Wang, P., Chen, J., Liu, Q., Xu, H., Xie, J., Zhang, Z., 2017. Accumulation of intact sulfur mustard in adipose tissue and toxicokinetics by chemical conversion and isotope-dilution liquid chromatography-tandem mass spectrometry. Arch. Toxicol. 91, 735-747. Xu, H., Nie, Z., Zhang, Y., Li, C., Yue, L., Yang, W., Chen, J., Dong, Y., Liu, Q., Lin, Y., Wu, B., Feng, J., Li, H., Guo, L., Xie, J., 2014. Four sulfur mustard exposure cases: Overall analysis of four types of biomarkers in clinical samples provides positive implication for early diagnosis and treatment monitoring. Toxicol. Rep. 1, 533-543. Yue, L., Wei, Y., Chen, J., Shi, H., Liu, Q., Zhang, Y., He, J., Guo, L., Zhang, T., Xie, J., Peng, S., 2014. Abundance of four sulfur mustard-DNA adducts ex vivo and in vivo revealed by simultaneous quantification in stable isotope dilutionultrahigh performance liquid chromatography-tandem mass spectrometry. Chem. Res. Toxicol. 27, 490-500. Zhang, Y., Yue, L., Nie, Z., Chen, J., Guo, L., Wu, B., Feng, J., Liu, Q., Xie, J., 2014. 29

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Simultaneous determination of four sulfur mustard-DNA adducts in rabbit urine after dermal exposure by isotope-dilution liquid chromatography-tandem mass spectrometry. J. Chromatogr. B 961, 29-35.

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Fig.1. Changes in cell count, NMP, MMP, lysosomes, MnSOD and pH2AX induced by SM in HPA-s and HA-s cells (n=5, means±SD). A: Dose-dependent effect of cytotoxic changes caused by different concentrations of SM for exposure 48 h. * means statistically significant at the same exposure dosage between HPA-s and HA-s. B: Time-dependent effect of

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cytotoxic changes caused by different exposure time of 3, 6, 24, 48 h at the same concentration of 400 μM SM. * means statistically significant at the same exposure

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timepoint between HPA-s and HA-s.

Fig. 2. Effects of SM-exposed HA-s or HLF cell supernatant on co-cultured cell growth.

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0.1, 0.5 or 1 mM SM was exposed to HA-s cells, and 0.1 mM SM was exposed to HLF

N

cells, the postexposure culture supernatant was collected and employed to co-culture

A

with SM exposed cells of HacaT, HLF and L02, respectively. The IC50 values were

M

obtained by CCK-8 method. ns means not statistically significant between the culture

ED

treatments for the same cell line. Other pair-wise comparisons in the same cell group

PT

were all statistically significant.

Fig. 3. Effect of SM on histopathology of adipose tissues. Subcutaneous (A), perirenal

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(B), epididymal (C), brown (D) adipose tissue sections, prepared 4 days after exposure of rats to 10 mg/kg SM or control were stained with H&E. SM-induced acute structural

A

changes include infiltration of inflammatory cells (arrow), thickened capillary wall, newborn adipocytes (red box) and “whitening” of brown adipocytes. Original magnification, × 200 (A, B, C panel) and × 400 (D panel). Representative sections from 5 rats per treatment group are shown.

31

Fig. 4. mRNA expression levels of adipokines in rat subcutaneous adipose tissues at 1, 3, 9, 12 h post cutaneous exposure to a low (1 mg/kg), middle (3 mg/kg) or high (10 mg/kg) dosage of SM. Results are expressed as the means ± SD (n = 5). * means statistically significant vs. control group at the same exposure dosage.

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Fig. 5. Adipokine levels of resistin, IL-6 and TNF-α in rat subcutaneous adipose

tissues and sera at 0.5, 24, 48, 72 h post cutaneous exposure to a high (10 mg/kg)

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dosage of SM. Results are expressed as the means ± SD (n = 5). * means statistically

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A

N

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significant vs. control group.

32

A ED

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N

A

M

Figr-1

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U

N

A

M

Figr-2

34

A ED

PT

CC E

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U

N

A

M

Figr-3

35

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PT

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N

A

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Figr-4

36

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Figr-5

37

Table 1 Multi-parametric cytotoxicity assay panel. Assay and parameters

Fluorescent probes

1. Cytotoxicity assay 1 Cell count



Hoechst 33342



MnSOD content



MnSOD Ab (Alexa 488)



Mitochondrial membrane potential and mass (MMP)



Mito Tracker Red CMXRos



Nuclear membrane permeability (NMP)



TOTO-3



Hoechst 33342



LysoTracker Red



pH2AX Ab (Alexa 647)

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IP T





Lysosome membrane potential and mass



Phosphohistone H2AX, Ser139 (DNA damage)

N

Cell count

A

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PT

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M

A



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2. Cytotoxicity assay 2

38

Table 2 Sequences of primers used for RT-PCR. Primer*

Primer sequence from 5’ to 3’

gapdh

F

gta ttg ggc gcc tgg tca cc

R

cgc tcc tgg aag atg gtg atg g

F

tcc tgg tca caa tgg gat acc

R

atc tcc tgg gtc acc ctt agg

F

ttt gtt gag tca ttc acc aga cag at

R

acg atg tgt aag gtt tca ggg aga ag

F

tgt ctc gag ccc acc agg aac gaa

R

agg gaa ggc agt ggc tgt caa ca

il-6

F

gac cag acc ctg gca gtc ta

N

leptin

SC R

cox-2

U

adipo

IP T

Gene name

F R F

ED

ppar-γ

CC E

PT

leptin

resistin

A

visfatin

Tnf-α

M

ppar-α

ctc agc att cag ggc taa gg

A

R

gaa ccc aag ttt gac ttc gc ccg atc tcc aca gca aat ta ctt tac cac ggt tga ttt ctc

R

cag gct cta ctt tga tcg ca

F

gac cag acc ctg gca gtc ta

R

ctc agc att cag ggc taa gg

F

cta cat tgc tgg tca gtc tcc

R

gct gtc cag tct atg ctt cc

F

ccc aga aaa gca agc aac ca

R

tgg tac tgt gct ctg ccg ct

F

ccc aga aaa gca agc aac ca

R

ccc aga aaa gca agc aac ca

* F, forward primer; R, reverse primer

39

Table 3 Spearman correlation between subcutaneous AT and serum adipokine levels

P

resistin

0.800

0.104

IL-6

0.900

0.037*

TNF-α

0.900

0.037*

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r

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of 10 mg/kg SM-exposed rats.

* P < 0.05 means significant correlation between adipose tissue and serum adipokine levels of

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PT

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M

A

N

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rats.

40