Benzo[a]pyrene Induces Autophagic and Pyroptotic Death

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Benzo[a]pyrene induces autophagic and pyroptotic death simultaneously in HL-7702 human normal liver cells Li Yuan, junyi liu, Hong Deng, and chunxia gao J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 9, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

Autophagic vacuoles Beclin-1 LC-3I

LC-3II

LC-3II

Autophagy

BaP

Pyroptosis Pro-Caspase-1

Active Caspase-1

Inflammatory cytokines

HL-7702 liver cell

ACS Paragon Plus Environment


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Benzo[a]pyrene induces autophagic and pyroptotic death simultaneously in

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HL-7702 human normal liver cells

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Li Yuan1*, Junyi Liu1, Hong Deng, Chunxia Gao

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Key Laboratory of Ministry of Education for Medicinal Resource and Natural

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Pharmaceutical Chemistry, College of Food Engineering and Nutritional Science,

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Shaanxi Normal University, Xi’an, 710119, China

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*Corresponding author: Li Yuan, Ph. D., Xian, Shaanxi, 710119, China. Tel. /fax: 029-85310517; email: [email protected]

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These authors contributed equally to this work.

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Abbreviations: BaP , Benzo(α)pyrene; HL-7702 cells, HL-7702 human normal liver

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cells; MDC, monodansyl cadaverine; AO, acridine orang; ROS, reactive oxygen

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species; NO, nitric oxide; LDH, lactate dehydrogenase; 3-MA, 3-methyladenine;

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polycyclic aromatic hydrocarbon (PAHs).

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Abstract As a common polycyclic aromatic hydrocarbon compound, benzo(α)pyrene (BaP)

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is readily produced in processing of oil and fatty foods. It is not only a strong

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carcinogen, but also a substance with strong immunotoxicity and reproduction toxicity.

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Autophagy and pyroptosis are two types of programmed cell death. Whether or not

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BaP damages body tissues via autophagy or pyroptosis remains unknown. The present

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study investigated the effects of BaP on autophagy and pyroptosis in HL-7702 cells.

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The results showed that BaP induced cell death in HL-7702 cells, enhanced the

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intracellular levels of ROS, arrested cell cycle at the S phase. Additionally, BaP

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resulted in cell death through autophagy and pyroptosis. Compared with BaP group,

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the autophagy inhibitor 3-MA significantly (p< 0.01) inhibited the release of LDH by

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70.53% ± 0.46 and NO by 50.36% ± 0.80 , the increase of electrical conductivity by

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12.08% ± 0.55, and the expressions of pyroptotic marker proteins (Caspase-1, Cox-2,

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IL-1β, IL-18). The pyroptosis inhibitor Ac-YVAD-CM also notably (p< 0.01) blocked

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BaP-induced autophagic cell death characterized by the increase of autophagic

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vacuoles and overexpression of Beclin-1 and LC3-II. In conclusion, BaP led to injury

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by inducing autophagy and pyroptosis simultaneously, the two of which coexisted and

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promoted each other in HL-7702 cells.

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KEYWORDS:

benzo(α)pyrene,

autophagy,

pyroptosis,


crosstalk,

injury


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INTRODUCTION

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As a well-known carcinogen, mutagen and immuno toxicant, Benzo[a]pyrene

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(BaP) is a major constituent of polycyclic aromatic hydrocarbons (PAHs) which are

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persistent and potential environmental pollutants. BaP can enter the body through

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tobacco smoke, polluted water, air and soil, as well as daily foods primarily including

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charcoal-broiled, grilled, baked, and smoked meats and fishes or poorly cleaned

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vegetables.1,2 BaP is widely present in hot dog, fried eggs, hamburger, steak,

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pepperoni pizza, yogurt, edible vegetable oil and so on. 1 It has reported that BaP

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produced in burnt food is 20 times more than that in ordinary food, and it can be

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spread through air and bioaccumulated through food chain, finally leading to harmful

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effects on humans.3 It is demonstrated that BaP may act through various toxic

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mechanisms including forming DNA adducts, altering DNA methylation,4 changing

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histone acetylation,5 disordering the cell cycle and signal transduction,6 inducing

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oxidative stress and apoptotic cell death in human normal cell lines.7-9

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The liver has a much higher ability to metabolize BaP than stomach, jejunum,

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and colon. 10 As the main metabolic organ for ingested BaP, hepatotoxicity of BaP is

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also stronger than other organ toxicity.72h after oral administration of 40 µmol/kg of

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BaP in male Sprague-Dawley rats, only 14.07% of BaP was detected in feces, about

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85% of BaP consumption is metabolize by liver.

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an object in this paper.

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Therefor, liver was researched as

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Autophagy is known as type II programmed cell death, it can maintain cell

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homeostasis by eliminating superfluous, damaged or aged cells, organelles and cell


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wastes and providing the building blocks and energy for cells.12 However, excessive

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autophagy results in the loss of homeostasis, and subsequently leads to cell death. It

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has reported that the Particulate Matter2.5 (PM2.5) could trigger autophagy in A549

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human lung epithelial cells. 13 As a major and strongest toxic component of PM2.5, the

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autophagic injury effect of BaP on HL-7702 human normal liver cells remains

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unknown.

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Furthermore, pyroptosis is a newly discovered programmed cell death which is

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uniquely dependent on caspases-1 activation and inflammation. Some research show

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that BaP is able to promote the release of pro-inflammatory cytokines including

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TNF-α, IL-1β as well as Cox-2 and enhance the protein expression of iNOS in mice

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colon,

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BaP can cause inflammation. Whether or not inflammation induced by BaP is

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mediated by pyroptosis, and the relationship between autophagy and pyroptosis

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remain unknown.

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lungs,15, 16 airway,17 intestine,18 and zebrafish embryos.19 In other words,

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In the present study, we investigated the effects of BaP on autophagy and

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pyroptosis in HL-7702 human normal liver cells, and demonstrated the interplay

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between autophagy and pyroptosis induced by BaP in HL-7702 cells.

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MATERIAL AND METHODS

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Chemicals and reagents

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Benzo[a]pyrene (BaP, purity of ≥ 96%), 3-(4,5-dimethylthiazol-2-yl)-2,5-

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diphenyltetrazolium bromide (methyl tetrazolium, MTT) , dimethylsulfoxide (DMSO),

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trypan blue, and monodansyl cadaverine (MDC) were purchased from Sigma (St.



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Louis, MO, USA). 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and

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acridine orange (AO) were purchased from Beyotime biotechnology company

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(Jiangsu, China). The RPMI-1640 medium modified and penicillin-streptomycin

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solution were obtained from Thermo Fisher (Shanghai, China).

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Cell culture and treatment

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The immortalized normal human fetal hepatocyte cell line HL-7702 was

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purchased from the Type Culture Collection of the Chinese Academy of Sciences

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(Shanghai, China). HL-7702 cells were grown in RPMI-1640 medium with 10% fetal

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bovine serum and 1% penicillin-streptomycin, at 37°C under a 5% CO2 atmosphere.

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HL-7702 cells were treated 24 h following seeding (5×103 cell/cm2) with different

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concentration BaP and/or the different inhibitors.

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Cell viability by MTT assay

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HL-7702 cells were seeded in 96-well plates at a density of 1×106 cells/mL.

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After 18 h, cells were treated with 100 µL different doses of BaP for 24 h or 48 h at

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37°C in 5% CO2 atmosphere. Subsequently, 100 µL of 0.5% (w/v) MTT was added to

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each well and incubated for 4 h. Then, removed MTT, and added DMSO to dissolve

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the formazan crystals. Absorbance at 490 nm was detected using a microplate reader

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(Thermo Fisher, US.). Cell viability was expressed as a percentage of the untreated

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cells.

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Trypan Blue, MDC and AO staining assay

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The cell mortality rate was analysed by trypan blue exclusion assay (0.04%,

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10min). And the level of autophagy was surveyed by MDC (50 mol/L MDC at 37 °C


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for 1 h.) and AO (1µg/mL AO at 37 °C for 15 min) fluorescence staining. After

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incubation, trypan blue, MDC and AO were removed and cells were examined under

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fluorescence microscopy (Olympus Optical Co., Ltd., Tokyo, Japan).

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Cell cycle analysis

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The cell cycle was analyzed with flow cytometry. After treatment, both the

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detached cells and the remaining adherent cells were collected and fixed with 70%

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ethanol at 4 °C overnight, washed and resuspended with 1.0 mL of PBS. Finally, cells

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were incubated with 1 mg/mL RNase (Sigma Aldrich) and 50 µg/mL PI (Sigma

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Aldrich) in the dark for 30 min at room temperature, and then examined by the

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GUAVA® easyCyte™ 8HT flow cytometry (Millipore Corporation, Billerica, MA,

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USA).

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Measurement of intracellular ROS

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ROS was detected using flow cytometer and fluorescence microscopy with

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H2DCFDA fluorescent probe. After treatment, cells were incubated with 10 µM

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H2DCFDA at 37 °C for 30 min. Subsequently, cells were washed twice with PBS, and

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the fluorescence of cells was observed under an inverted fluorescence microscope

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(Olympus Optical Co., Ltd., Tokyo, Japan) or CyFlow Cube flow cytometer (Partec

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GmbH, Münster, Germany).

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Cell ultrastructure

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After treatment by BaP, cells were fixed with 2.5% glutaraldehyde for 24h, then

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embedded in 2% agarose gel, and postfixed in 4% osmium tetroxide solution for 1 h.

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Next, the examples were dehydrated using a series of graded ethyl alcohols from 70 to



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100%, and embedded in epoxy resin at 30 °C for 24 h and 60 °C for 48 h, respectively.

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Finally, ultrathin sections were stained with uranyl acetate and lead citrate before

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examination using the transmission electron microscope (Hitachi, Japan).

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Western blot analysis

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After the indicated treatments, cells were collected and lysed with 100 µL cell

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lysis buffer and harvested the total protein. The homogenates of protein were

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incubated with SDS sample buffer at 100 °C for 10 min and then divided into

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centrifuge tube. After the 10 µL of protein sample were separated through the

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SDS-PAGE method and were electrotransferred onto a PVDF membrane (0.45µm,

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Millipore). Then, the membrane was blocked in 5% nonfat dry milk for 1 h, and was

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incubated with the primary antibodies (rabbit anti-Beclin-1, anti-LC3, anti- Caspase-1,

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anti-active-Caspase-1, anti-Cox-2, anti-IL-1β, anti-IL-18, anti-GAPDH) overnight at

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4 °C, respectively. Anti-rabbit IgG conjugated with horseradish peroxidase was used

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as a secondary antibody, and was incubated with the membrane at 25 °C for 2 h. The

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blots were detected by chemiluminescence using a Molecular Imager Chemidoc XRS

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System (UVP, Ltd., US.).

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LDH release assay

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Lactate dehydrogenase (LDH) was used to examine cellular permeability. After

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24h exposure to BaP, cell-free supernatant was separated and transferred to the

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96-well microtiter plates. Then incubated with LDH assay kit (Nanjing Jianchen

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Bioengineering Institute, China), and measured the absorbance at 450 nm. LDH was

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expressed as the percentage of the control cells.


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NO assay

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NO in culture supernatant liquid was determined by the Griess reagent. After

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treatment, culture supernatant was collected and incubated with the same volume of

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the Griess reagent (0.1%(w/v) N-(1-naphathyl)-ethylenediamine and 1%(w/v)

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sulfanilamide in 5% (v/v) phosphoric acid ) at 25°C for 15min. Then, measured

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absorbance of the reaction solution at 540nm using a Microplate Reader instrument

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(Thermo Fisher, US.). NO was calculated using a sodium nitrites standard curve.

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Relative electrical conductivity assay

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The relative electrical conductivity was determined by DDSJ-308A conductivity

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meter (Shanghai precision scientific instrument co., Ltd, China).After the indicated

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treatments, the culture supernatant liquid was collected and examined by conductivity

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meter. The tri-distilled was prepared to zero the conductivity meter.

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Statistical analysis

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All experiments were repeated three times and performed in triplicate and on the

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same set of cells, and the nine datas were presented as the means ± standard errors

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(SE) and were analyzed using the one-way factorial analysis of variance (ANOVA)

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and Duncan’s post hoc test (SPSS 16.0).

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RESULTS

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Cytotoxic effect of BaP on HL-7702 cells

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BaP induced HL-7702 cells death in a dose- and time-dependent manner in the

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concentration range from 0.5µM to 20µM. Treatments with 20 µM BaP for 24h and

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48h significantly (p

Benzo[a]pyrene Induces Autophagic and Pyroptotic Death

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