Development and Application of a Novel Bioassay System for Dioxin

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Development and application of a novel bioassay system for dioxin determination and AhR activation evaluation in ambient-air samples SONGYAN ZHANG, Shuaizhang Li, Zhiguang Zhou, Hualing Fu, Li Xu, Heidi Qunhui Xie, and Bin Zhao Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06376 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 15, 2018

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Environmental Science & Technology

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Research article Development and application of a novel bioassay system for dioxin determination and AhR activation evaluation in ambient-air samples †,‡,# Songyan Zhang , Shuaizhang Li†,‡,#, Zhiguang Zhou§,#, Hualing Fu†,‡, Li Xu †,‡, Heidi Qunhui Xie *,†,‡, Bin Zhao *,†,‡

†

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China ‡ University of Chinese Academy of Sciences, Beijing, China § State Environmental Protection Key Laboratory of Dioxin Pollution Control, National Research Center for Environmental Analysis and Measurement, Beijing, China *Address correspondence to Dr. Heidi Qunhui Xie, Research Center for Eco-Environmental Sciences (RCEES), Chinese Academy of Sciences, Beijing 100085, China; Telephone: (86) 010-62842865; Email: [email protected]; or Dr. Bin Zhao, Research Center for Eco-Environmental Sciences (RCEES), Chinese Academy of Sciences, Beijing 100085, China; Telephone: (86) 010-62842867; Email: [email protected] # These authors contributed equally to this study.

ABSTRACT

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Airborne persistent toxic substances are associated with health impacts resulting from air pollution,

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e.g. dioxins, dioxin-like polychlorinated biphenyls and certain polycyclic aromatic hydrocarbons

25

(PAHs), which activate aryl hydrocarbon receptor (AhR) and thereby produce the adverse

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outcomes. Thus, a bioassay for evaluating AhR activation is required for the risk assessment of

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ambient-air samples, and for this purpose, we developed a new and sensitive recombinant mouse

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hepatoma cell line, CBG2.8D, in which a novel luciferase-reporter plasmid containing 2 copies of

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a newly designed dioxin-responsive domain and a minimal promoter derived from a native gene

30

were integrated. The minimal detection limit for 2,3,7,8-tetrachlorodibenzo-p-dioxin with this

31

assay system was 0.1 pM. We used CBG2.8D to determine dioxin levels in 45 ambient-air

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samples collected in Beijing. The measured bioanalytical equivalent (BEQ) values were closely

33

correlated with the toxic equivalent values obtained from chemical analysis. In haze ambient-air

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samples, the total activation of AhR (TAA) was considerably higher than the BEQ of dioxin-rich

35

fractions according to the results of the cell-based bioassay. Notably, the haze samples contained

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abundant amounts of PAHs, whose relative toxicity equivalent was correlated with the TAA; this

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finding suggests that PAHs critically contribute to the AhR-related biological impacts of haze

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ambient-air samples.

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Key words: aryl hydrocarbon receptor, dioxins, polycyclic aromatic hydrocarbon, bioassay

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ACS Paragon Plus Environment


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Table of Content (TOC art)

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INTRODUCTION

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Ambient air pollution has recently become a major public health concern in China. Exposure to

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ambient particulate matters (PMs), particular fine PMs such as PM2.5 and PM10, is regarded as

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one of the main causes of the adverse health effects resulting from air pollution.1 Industry

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emissions, coal combustion, diesel and gasoline exhaust, and biomass burning represent potential

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sources of ambient PMs.2, 3 The organic carbon (OC) fraction of the ambient PM, which contains

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thousands of organic compounds, is one of the critical PM constituents examined in toxicity

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studies.4 Among these organic compounds, persistent toxic substances (PTSs) are particularly

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indispensable for the monitoring and risk assessment of air pollution.5-8

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Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs; dioxins) are present in

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ambient-air samples and are mostly bound to ambient PMs.9, 10 Although airborne dioxins are not

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the most abundant PTSs in the air, their bioavailability, persistence in the environment and

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organisms, and close association with multiple diseases make dioxins crucial for toxicity analysis

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and risk assessments. In animals and humans, dioxins produce diverse toxicological effects,11

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which are predominately mediated by aryl hydrocarbon receptor (AhR), a ligand-dependent

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transcription factor.12 AhR is involved in regulating the expression of cytochrome P-450 enzyme

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family members and other genes related to numerous critical physiological processes, such as cell

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cycle and proliferation and the immune response.13-15 The AhR-dependent signaling pathway is

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regarded as a key mediator linking dioxin exposure to diverse pathological conditions, such as

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chloracne, immune disorders16, 17 and cardiopulmonary diseases.18, 19, which could occur after

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exposures to airborn dioxins. Besides dioxins, AhR can sense, respond to and defend against other

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PTSs, such as dioxin-like polychlorinated biphenyls (DL-PCBs) and certain polycyclic aromatic

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hydrocarbons (PAHs).20-22 Notably, with rapid industrial development and the consequent massive

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consumption of fossil fuels, PAHs have become one of the predominant classes of PTSs in the air

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in China.23 It has been reported that exposure to airborne PAHs might lead to respiratory and

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cardiovascular diseases.24,

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environmental health effects produced by air pollution. Because activation of the AhR-dependent

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pathway is the common mechanism through which these compounds exert their biological effects,

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measuring the potency of AhR activation by these compounds in ambient-air samples might

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represent a suitable bioassay for evaluating the overall potential health impacts of the pollutants.

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Accordingly, AhR-based bioassays have been used as reliable methods for monitoring both

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dioxins and PAHs26,

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analytical methods (such as high-resolution gas chromatography/high-resolution mass

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spectrometry (HRGC-HRMS) for chemical analysis of dioxins), particularly for rapid screening

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and risk assessment in the case of a large number of samples.28

27

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Thus, the airborne PAHs are also key contributors to the

and these assays could also serve as a supplement for instrumental

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One of the AhR-based bioassay methods that has been developed involves using the

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AhR-mediated reporter-gene assay system. A representative of this type of methods is the

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chemically activated luciferase expression (CALUX) method, which is considered to be highly

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sensitive and has been applied in USA, Japan, and European Union for diverse environmental

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samples.29-31 Anezaki et al. employed a similar method and successfully determined the

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international-toxic equivalent (I-TEQ) of PCDD/Fs and DL-PCBs (0.0029-0.53 pg I-TEQ m-3) in

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ambient-air samples by using DR-EcoScreen cells.32 AhR activity was evaluated to deduce dioxin

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concentrations in the ambient-air samples, and the bioanalytical equivalent (BEQ) values (11-1860

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fg BEQ m-3) from the DR-EcoScreen cell assay were found to correlate well with the toxic

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equivalent (TEQ) values obtained from HRGC-HRMS analysis; this suggested that the bioassay

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was suitable for evaluating dioxins in ambient-air samples.30

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Given the importance of airborne dioxins, DL-PCBs, and PAHs in the risk assessment of air

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pollution in China, urgent demand exists for a bioanalytical method that can fulfil the

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requirements of dioxin screening and evaluation of the total potential biological impacts of all

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these compounds in ambient-air samples. Here, we developed a new sensitive AhR-mediated

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reporter-gene

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luciferase-reporter plasmid. The novel detection plasmid contained relative less number of DREs

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and a native minimal promoter, which is different from that of other similar bioassay systems with

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comparable sensitivity to dioxin. We evaluated the suitability of this bioassay for dioxin

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measurement in ambient-air samples by comparing with the data obtained from HRGC-HRMS

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analysis. By using this bioassay, the total potencies of AhR activation (TAA) by crude extracts of

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haze ambient-air samples were determined. These results together with those of chemical analysis

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bioassay

based

on

a

novel

dioxin-responsive

domain

(DRD)-driven

suggested that the airborne PAHs compounds contributed substantially to the TAA.

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MATERIALS AND METHODS

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Chemicals and reagents

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toluene, and dichloromethane, J. T. Baker, Co., Ltd. (Center Valley, PA); dimethyl sulfoxide

105

(DMSO), Sigma (St. Louis, MO); calibration standard solutions and isotope labeled standards for

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PCDD/Fs, polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs), DL-PCBs and PAHs,

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Wellington Laboratories Inc. (Guelph, ON, Canada);

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chromatography, Wako Pure Chemicals Inc., Ltd. (Osaka, Japan).

These chemicals were from commercial sources: acetone, n-hexane,

and silica gel for multilayer column

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Fetal bovine serum (FBS), α-modified Eagle’s minimum essential medium (α-MEM),

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penicillin-streptomycin (antibiotics) solutions, 0.25% trypsin/0.02% ethylene diamine tetra-acetic

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acid (EDTA) disodium salt solution, and Lipofectamine LTX transient transfection system were

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obtained from Invitrogen (Carlsbad, CA); geneticin (G418) was from Solarbio (Beijing, China);

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luciferase substrate and reporter vector, pGL3-Basic, were from Promega (Madison, WI); and

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High-fidelity Polymerase was from the GeneStar Company (Beijing, China).

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Construction of plasmid pCL-CR2

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sequence

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5ʹ-CACAGAGCTCGTGGTGACCCCAACCTTTAT-3ʹ;

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5ʹ-CACAAAGCTTTAGGGAGGATCGGGGAAGCT-3ʹ. SacI and HindIII restriction sites were

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added at the 5ʹ ends of the sense and antisense primers, respectively. Subsequently, this sequence

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was inserted into a pGL3-Basic vector for use as the minimal promoter of the luciferase-encoding

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gene. We selected a dioxin-responsive element (DRE)-enriched region, located from -1099 to -802

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upstream of the transcription start site of the mouse CYP1A1 gene, as the DRD, and we cloned

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this 297-bp-DRD and doubly inserted it into the vector mentioned above. The sense and antisense

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primers

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5ʹ-GATAGGTACCCTTTAAGAGCCTCACCCAC-3ʹ

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5ʹ-GATACCATGGAGGGTGGAGGAAGGATCCA-3ʹ (restriction sites, KpnI and NcoI); and for

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the second 297-bp sequence: F: 5ʹ-GATACCATGGCTTTAAGAGCCTCACCCAC-3ʹ; and R:

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5ʹ-GATAGAGCTCAGGGTGGAGGAAGGATCCA-3ʹ (restriction sites, NcoI and SacI). All

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inserted sequences were verified through DNA sequencing. An overview of the plasmid

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construction is shown in Figure 1A.

containing

were

the

a

From the mouse CYP1A1 promoter, we cloned a 260-bp

TATA

following

box

by

(respectively):

using

for

this

primer

pair:

and

the

6


first

297-bp and

F: R:

sequence:

F R

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Mouse hepatoma (Hepa1c1c7)

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Cell culture, transient transfection, and chemical treatment

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cells were seeded in 100-mm dishes, grown in MEM supplemented with 10% FBS and 100 U/mL

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penicillin, and incubated at 37 ℃ in 95% air/5% CO2. For transient transfections, cells were plated

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in 24-well-plates at 50%–80% confluence one day before transfection, and pCL-CR2 and

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pCDNA3 were co-transfected into the Hepa1c1c7 cells by using LTX reagent according to the

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manufacturer’s instructions. After 24 h incubation, the transfected cells were treated with DMSO

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(1%, final solvent concentration) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD 1 nM) for

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another 24 h, after which the medium was removed and the cells were washed gently with 100 µL

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of PBS. Next, 100 µL of cell-lysis buffer was added into each test-well and the plates were shaken

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at room temperature for 15 min, and after incubating with the Luciferase Reporter Assay System

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kit reagent, luciferase activity was measured using a microplate luminometer (GLOMAX MULTI

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PLUS, Promega, Madison, WI), with the optical signal collected as relative light unit (RLU)

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values.

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Stable transfection and chemical treatment

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transiently transfected cells in each well of the plates were incubated for 24 h, after which the cells

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were trypsinized and resuspended in culture medium containing 600 µg/mL G418 and then

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divided equally to plate in two 100-mm petri dishes. The culture medium was replaced every 4

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days until colonies could be isolated (at roughly 4 weeks), and thus a stably transfected cell line

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was obtained, which we named CBG2.8D. We plated these cells in white clear-bottom 96-well

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cell-culture plates at various densities (1–8 ×104 cells/well) and incubated them for distinct times

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(4–48 h) after chemical treatment to identify the optimal treatment condition. A calibration

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standard curve for 2,3,7,8-TCDD was generated using concentrations between 0.01 and 1000 pM

For selecting stably transfected clones, the

7



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per well to identify the minimal detection limit (MDL), described as the luciferase activity that

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was significantly higher than background.22 Each plate contained a control solution of 12.3 pM for

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verification of stability, and the analysis was based on the difference from the mean value.

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Standard substance dilutions of other dioxin congeners and DL-PCBs were used for EC50 and

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relative potency (REP) calculation.

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Cell-based bioassay developed using CBG2.8D cell line

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104 cells/well in a volume of 100 µL/well. After culturing for 24 h, 100 µL of growth medium

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containing the test sample (dissolved in DMSO) was added to the cells in triplicate (final DMSO

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concentration: 1%).29 In parallel, a calibration standard curve for 2,3,7,8-TCDD was generated

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using 9 concentrations between 0.15 and 1000 pM/well, besides including a control solution. After

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exposure for 24 h, the RLUs were measured by performing the treatment described above. The

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dioxin concentrations in the test samples were calculated as BEQ values by using the standard

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curve for 2,3,7,8-TCDD. The calculations of luciferase activity data were processed using the Hill

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equation.

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Sampling procedure, extraction, and cleanup

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sites covering different functional zones in Beijing from February 2011 to March 2012

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(Supporting Information, Figure S1), and 8 samples were collected from February to June 2014

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(Supporting Information, Table S1). All samplings were performed using a SIBATA HV-1000F

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sampler, equipped with a quartz fiber filter (QFF) and followed by a glass cartridge containing 2

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polyurethane foam (PUF) plugs. Roughly 2160 m3 of ambient-air samples were collected during a

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72-h period, using at a flow rate of 0.50 m3 min-1. For the haze ambient-air samples, a 24-h

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sampling time was used and 720 m3 of ambient-air was collected. Briefly, for extraction, the QFFs

Cells were seeded at a density of 4 ×

Here, 45 samples were collected at 6 sampling

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and PUF plugs of all 53 samples were extracted through accelerated solvent extraction (ASE 200,

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Dionex, Sunnyvale, CA) with toluene and n-hexane:dichloromethane (1:1), respectively. The 45

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extracts (collected in 2011–2012) were separated into 2 sets and used for HRGC-HRMS analysis

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(a 13C12-labeled standard mixture was spiked into this set) and the bioassay. The solvents of these

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extracts were completely evaporated and then dissolved in n-hexane. The 8 samples collected in

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2014 were separated into 4 sets: 2 sets were treated using the same procedure as described above,

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and the other 2 sets were prepared for method validation and total AhR-activity analysis by using

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CBG2.8D cells (the ASE extracts were evaporated and solvent-exchanged in 300 µL of DMSO

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under a gentle stream of nitrogen gas).

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Cleanup procedure for chemical analysis: For PAHs analysis, the extracts were cleaned up on

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a neutral silica-gel column. The PAHs fraction was obtained by eluting with 50 mL of a

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hexane/dichloromethane mixture (9:1, v/v), with the first 10 mL being discarded. The eluates were

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evaporated and then reconstituted in hexane for GC-MS analysis. For analyses of DL-PCBs,

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PBDD/Fs and PCDD/Fs, the extracts were cleaned up using a multilayered silica-gel

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activated-carbon column and then analyzed using HRGC-HRMS. All data were subject to

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stringent quality-control procedures to meet EPA requirements for field research. The detailed

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instrumental conditions and the method evaluation were as described previously.33

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Cleanup for the bioassay: The ASE extract was cleaned up using an acid silica-gel column,

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which consisted mainly of 44% (w/w) sulfuric acid silica gel and anhydrous sodium sulfate. The

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cleaned extract was concentrated and subsequently fractionated using the activated-carbon column,

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which was packed with 0.4 g activated carbon and celite 545 (1:99, w/w). The sample extract was

196

loaded onto the carbon column and washed with 10 mL of n-hexane, and the extract was lastly

9



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eluted with 20 mL of toluene to obtain the dioxin-rich fraction, which was then solvent-exchanged

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in 200 µL of DMSO.

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Chemical analysis

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PBDD/Fs, DL-PCBs and followed the method of HJ 77.2-2008 (Environmental Protection

201

Standard of China). The system used was an Agilent 6890 N gas chromatograph connected with a

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Waters AutoSpec Ultima NT high-resolution mass spectrometer (Waters Micromass, Manchester,

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UK). Samples were injected in the split-less mode at an injector temperature of 270 °C. The PAHs

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analysis was performed by GC-MS (Agilent 6890-5975). The TEQ values for each test compound

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(including the 17 dioxin congeners and 12 DL-PCBs) were calculated by multiplying the mass

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concentration with the corresponding WHO TEF (Toxic Equivalent Factor).34 described as I-TEQ.

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The REPs of the 7 PAHs were used to calculate the toxicity equivalent of PAHs according to a

208

previous study.35

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Statistics analysis

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figure plotting. EC50 values were derived from the sigmoidal ﬁt equation, and REPs were obtained

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by dividing the values with the EC50 of 2,3,7,8-TCDD. Data are presented as means ± sem.

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Differences between groups were examined using Student’s t test.

HRGC-HRMS analyses were performed to identify and quantify PCDD/Fs,

Graph Pad Prism software (version 5) was used for statistical analysis and

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RESULTS AND DISCUSSION

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New AhR-mediated reporter-gene assay established using CBG2.8D cell line

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constructed a novel DRD-driven luciferase-reporter plasmid, pCL-CR2, containing 2 copies of a

217

newly designed DRD harboring a total of 8 DREs (Figure 1A); each 297-bp-long DRD contained

218

4 DREs from the mouse CYP1A1 gene promoter (Figure 1A). This DRD includes a highly

10


We

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conserved fragment containing a DRE (-821 -5‘ CACGC 3’- -817) and its adjacent sequences,

220

whose transcriptional activity is relative higher than the others based on our previous work,36 and

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is distinct from other DRDs used in DR-EcoScreen or different generations of CALUX

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systems.37-39, 27 In terms of the minimal promoter of the luciferase-reporter plasmid, we inserted a

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260-bp fragment derived from the native mouse CYP1A1 promoter, which might facilitate

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improving reliability of the risk assessments using the bioassay system. While in the other reporter

225

bioassay systems, a viral promoter (mouse mammary tumor virus) or a minimal heat shock

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promoter from Drosophila has been employed.39, 40 Moreover, the total length of the inserted

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fragment, containing 2 DRDs and the minimal promoter, was less than that of other such inserted

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fragments in DR-EcoScreen or third-generation CALUX system.37, 39 This relatively shorter insert

229

were used for avoiding nonspecific signals, although this might have limited the extent of

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sensitivity enhancement. Hepa1c1c7 is a mouse hepatoma cell line and sensitive to dioxins and

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other AhR agonists, which has been wildly used in toxicological study of dioxins.41 It can

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moderately response to dioxins in AhR-mediated reporter gene assays.29, 38 Thus, we further tested

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the 2,3,7,8-TCDD responsiveness of this luciferase-reporter plasmid after transiently transfecting

234

it into Hepa1c1c7 cells.. The MDL for 2,3,7,8-TCDD was 0.1 pM in the transfected cells

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(Supporting Information, Figure S2). Next, we stably transfected the plasmid into the mouse

236

hepatoma cells and selected the recombinant clone that showed the highest 2,3,7,8-TCDD

237

responsiveness: CBG2.8D. For the CBG2.8D cell-based bioassay, we investigated the optimal

238

number of cells to grow on culture plates and the dilution factor of the solvent. The results showed

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that 4 × 104 cells/well plated in 96-well plates yielded the highest induction of luciferase activity

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after 24-h treatment with 2,3,7,8-TCDD at 1000 pM as compared to the solvent control

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(Supporting Information, Figure S3). Under this condition, we further examined the time course

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and the concentration-dependent effect of 2,3,7,8-TCDD treatment in CBG2.8D cells. The cells

243

were incubated with increasing concentrations of 2,3,7,8-TCDD and the luciferase activity was

244

measured after treatment for 4–48 h. All the obtained concentration-effect curves presented an “S”

245

shape and fit the Hill function. The highest 2,3,7,8-TCDD responsiveness with both of the

246

maximal fold-induction and the lowest 50% of maximal response was obtained with the 24-h

247

treatment, and this condition was used for further characterizations (Figure 1B).

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Critical parameters for analyzing 2,3,7,8-TCDD standard in CBG2.8D cells

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calibration curves, each containing 9 points, were obtained upon treating CBG2.8D cells with

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2,3,7,8-TCDD at 0.15–1000 pM (Figure 2A). The RLU values obtained were in a range of 103–

251

105, which are within the detection capability of regular luminometers. The MDL and EC50

252

determined were 0.1 pM (p < 0.05) and 14.94 ± 0.30 pM, respectively. This MDL value for

253

2,3,7,8-TCDD is comparable to those obtained from other reporter-gene assays used for dioxin

254

analysis in ambient-air samples, including DR-EcoScreen cell and the third-generation mouse

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CALUX cell-line (H1L7.5c3) bioassays, which are integrated with 7 and 20 DREs, respectively.29,

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37

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Thus, apart from DRE number, optimization of DRE-rich region could be another critical factor

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for sensitivity improvement. The repeatability of the 2,3,7,8-TCDD responsiveness in the

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CBG2.8D cell assay was revealed through luciferase induction at the 12.3 pM calibration point,

260

which is close to the EC50, in independent assays performed on 17 different days: most of the

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collected data showed 100-fold of control in CBG2.8D bioassay.

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point could be used as the point for the quality-control monitoring of the CBG2.8D cell assay.

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Relative responsiveness of CBG2.8D to other dioxin congeners

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other dioxin congeners can also activate AhR and its downstream gene expression, but with

266

distinct potencies.32 Thus, we examined the responsiveness of CBG2.8D cells to 17 congeners of

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dioxins and 4 selected DL-PCBs featuring high TEFs, which are listed in the TEF system

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(WHO-2005).32 Cells were treated for 24 h with a concentration series of the 17 dioxins and 4

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DL-PCBs, and the luciferase-induction curves were obtained and the REPs of the distinct

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compounds relative to 2,3,7,8-TCDD were calculated by comparison of the EC50 values: The

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ratios of the measured REPs to the corresponding TEFs (WHO-2005) for all tested congeners are

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listed in Table 1. The REPs were generally higher than the corresponding TEFs for most dioxins,

273

which is similar to results from another study.32 For the most toxic congeners of PCDDs and

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PCDFs, such as 1,2,3,7,8-PentaCDD and 2,3,7,8-TetraCDF, the REPs were same as the TEFs,

275

whereas the congeners featuring relative low TEFs showed clear differences between the TEFs

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and the REPs, as in the case of OctaCDD and OctaCDF (Table 1). For an AhR-based bioassay, the

277

total potency of AhR activation is derived based on measurements with different dioxin congeners,

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and this depends not only on the amount of each congener used in the assay, but also on the REPs

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of the related compounds. In standardized HRGC-HRMS analyses of dioxins, the TEF system

280

(WHO-2005) is used for TEQ calculations after determination of each congener.34 Thus, a

281

comparison of the TEFs and REPs could facilitate interpretations of the difference between the

282

TEQ obtained from HRGC-HRMS analysis and the BEQ from our cell-based bioassay.

283

Comparison of the TEQ from HRGC-HRMS analysis and BEQ from CBG2.8D cell assay for

284

ambient-air samples

Besides 2,3,7,8-TCDD,

Beijing is a typical megacity affected by air pollution.23, 42 We collected

13



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45 ambient-air samples from different functional urban zones in Beijing during 2011–2012; the

286

sites and periods of sample collection paralleled those from our previous work.33 HRGC-HRMS

287

analyses and CBG2.8D assays were performed using equivalent aliquots originating from the

288

same crude extract for each sample. The HRGC-HRMS analysis data revealed that the TEQs of

289

PCDD/PCDFs ranged from 55 to 747 fg I-TEQ m-3 (mean 254 fg I-TEQ m-3), in line with the

290

previously reported range.33 BEQs were obtained using the dioxin-rich fraction from the crude

291

extract of the ambient-air samples. The corresponding BEQs obtained using CBG2.8D cell assay

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ranged from 309 to 4684 fg BEQ m-3, which are higher than the respective TEQs from the

293

HRGC-HRMS analysis. This discrepancy might result from the difference between the REPs and

294

the TEFs measured for most of the dioxin congeners. Nevertheless, the BEQ and TEQ values were

295

found to be closely correlated (r2 = 0.97), with the slope of the correlation line being 5.81 (Figure

296

3). Thus, a conversion coefficient of 0.17, which is the reciprocal of the slope of the regression

297

line (5.81), could be used to predict the TEQ values of dioxins in general ambient-air samples.

298

Moreover, for most of the dioxin congeners, the REPs for the CBG2.8D cells used here were

299

higher than the respective DR-REPs for DR-EcoScreen cells, which might potentially explain the

300

higher slope of the regression line obtained here than that of the corresponding line from the

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DR-EcoScreen cell assay (3.66).32 Ambient PMs are key carriers of airborne dioxins.23 Recently,

302

Khedidji et al. used a highly sensitive CALUX cell line, the mouse hepatoma H1L7.5c1 cell line,

303

to measure both PCDD/Fs and DL-PCBs in PM10 in Algeria;43 in this ambient PM10 fraction, the

304

total concentrations of the dioxins were 0.016–0.15 pg BEQ m-3. Although parallel data from

305

HRGC-HRMS analysis were lacking, the study suggested that the method might be suitable for

306

dioxin determination in the ambient PM fraction. Because our CBG2.8D cell-based assay system

14


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307

showed a similar responsiveness to 2,3,7,8-TCDD as the H1L7.5c1 system did, it would be worth

308

investigating whether our system could also be used for dioxin determination in ambient PM

309

samples.

310

Contributions of PAHs present in haze ambient-air samples to total activation of AhR

311

AhR-associated biological impacts of ambient-air samples could be due to not only dioxins but

312

also PAHs and DL-PCBs, which have all raised serious health concerns.20, 22, 26 We evaluated the

313

contribution of these compounds to total AhR activation in 8 haze ambient-air samples collected in

314

Beijing. Generally, PAHs are the most abundant type of chemicals among airborne PTSs in China,

315

and PAHs featuring a molecular weight (MW) of >210 are mostly bound to ambient PMs and are

316

considered to adversely affect public health. Thus, for our study, we selected 7 PAHs from 16

317

EPA-PAHs that fit the required criteria: featuring MWs of >210, being capable of activating AhR

318

and having recorded REPs from CALUX cell assays, and belonging to Class 1, 2A, or 2B of

319

IARC classification.44, 45 The amounts and the relative toxicity equivalents of the selected PAHs

320

were determined through chemical analysis of the haze ambient-air samples. In line with the

321

literature, PAHs were the most abundant of all tested PTSs in the study (at the ng m-3 level;

322

Supporting Information, Table S2). The relative toxicity equivalents of the 7 PAHs were

323

determined based on literature.35, 46 In the haze ambient-air samples, the total relative toxicity

324

equivalents of PAHs ranged from 63 to 442 fg TEQ m-3 (average 267 fg TEQ m-3), which are

325

comparable to the respective TEQ of dioxins (117–599 fg I-TEQ m-3) (Supporting Information,

326

Table S2). Therefore, besides dioxins, PAHs might constitute another critical fraction in haze

327

ambient-air samples contributing to total AhR activation. Moreover, we selected DL-PCBs

328

featuring TEF values of ≥0.00001 as the target chemicals for revealing TEQ of DL-PCBs34

15


The


329

(Supporting Information, Table S2); the TEQ of DL-PCBs were within 7–36 fg I-TEQ m-3

330

(average 18 fg I-TEQ m-3), which were markedly lower than the relative toxicity equivalents of

331

PAHs in all tested haze ambient-air samples (Figure 4A). PBDD/Fs were also present in the tested

332

haze ambient-air samples, and their concentrations ranged from 0.47 to 3.65 pg m-3 and were

333

slightly lower than those of the DL-PCBs in different haze ambient-air samples (Supporting

334

Information, Table S2).

335

To evaluate the potency of TAA, one aliquot of the crude extract of each ambient-air sample

336

was directly used in the CBG2.8D assay, whereas another aliquot of an equivalent amount of the

337

crude extract was subject to cleanup procedures to obtain the dioxin-rich fraction before the

338

CBG2.8D assay. The TAA values in relation to 2,3,7,8-TCDD measured for the crude extracts

339

were within 41.65-72.18 pg BEQ m-3 (Supporting Information, Table S3); these values were

340

8.7-18.8-fold higher than those measured with the dioxin-rich fraction (Supporting Information,

341

Table S3). Such large differences have also been recorded previously using the DR-EcoScreen cell

342

assay.32 Because the PAHs and dioxins featured comparable TEQs in the test samples, we

343

speculate that the BEQs of the PAHs might be considerably higher than the TEQs, and the

344

difference might be more drastic than that of dioxins (~5.81-fold different in this study). Another

345

potential explanation for the difference could be the presence of AhR agonists other than the

346

well-documented DL-PCBs and PAHs in the crude extracts of the haze ambient-air samples.

347

Because DL-PCBs could be separated from PCDD/Fs by using the cleanup procedure35 they likely

348

contribute to the TAA potency in the crude extract but not in the dioxin-rich fraction; by contrast,

349

PBDD/Fs were not separated from PCDD/Fs after the cleanup procedure, and thus they could

350

contribute to the BEQ of the dioxin-rich fraction. Intriguingly, we found that the relative toxicity

16


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351

equivalents of the PAHs of the haze ambient-air samples were more closely correlated with the

352

TAA potencies than were TEQ of dioxins or DL-PCBs, which suggested that PAHs play critical

353

roles in AhR activation and are mostly independent from other AhR agonists (Figure 4B).

354

Furthermore, the TAA potency might potentially predict the TEQ of the PAHs in haze ambient-air

355

samples. Given the adverse effects of ambient PAHs on lung and cardiovascular system,

356

TAA might be useful in health risk assessment of haze ambient-air samples.

25

the

357 358

ASSOCIATED CONTENT:

359

Supporting information

360

Tables S1 shows the sampling information of 8 haze samples and Table S2 is a list of chemical

361

analysis data of 8 haze samples; Figure S1 is a draft city map of Beijing containing 6 sampling

362

sites, Figure S2 shows the determination of minimal detection limit and Figure S3 shows a density

363

gradient of 2.3.7.8-TCDD induced luciferase activity in CBG2.8D cell line.

364 365

ACKNOWLEDGMENTS:

366

This work was supported by the National Natural Science Foundation of China (Nos. 21525730,

367

21527901), the Special Scientific Research Funds for Environmental Protection Commonweal

368

Section (Grant No. 201309029) and the Strategic Priority Research Program of the Chinese

369

Academy of Sciences (Grant No. XDB14030401).

370

We thank Dr. Marjorie A. Philips from UC Davis for helpful comments during manuscript

371

preparation. All authors wrote and revised this paper. The authors declare no competing financial

372

interests.

373

17



374 375 376

TABLE TABLE 1. Comparison between WHO-TEF and REP values for dioxins and selected DL-PCBs

Dioxins TEFa REP Polychlorinated dibenzo-p-dioxins (PCDDs) 2,3,7,8-TetraCDD 1 1 1,2,3,7,8-PentaCDD 1 1 1,2,3,4,7,8-HexaCDD 0.1 0.48 1,2,3,6,7,8-HexaCDD 0.1 0.25 1,2,3,7,8,9-HexaCDD 0.1 0.18 1,2,3,4,6,7,8-HeptaCDD 0.01 0.06 OctaCDD 0.0003 0.002 Polychlorinated dibenzofurans (PCDFs) 2,3,7,8-TetraCDF 0.1 0.1 1,2,3,7,8-PentaCDF 0.03 0.1 2,3,4,7,8-PentaCDF 0.3 0.46 1,2,3,4,7,8-HexaCDF 0.1 0.41 1,2,3,6,7,8-HexaCDF 0.1 0.28 1,2,3,7,8,9-HexaCDF 0.1 0.4 2,3,4,6,7,8-HexaCDF 0.1 0.36 1,2,3,4,6,7,8-HeptaCDF 0.01 0.03 1,2,3,4,7,8,9-HeptaCDF 0.01 0.17 OctaCDF 0.0003 0.00433 Nonortho dioxin-like polychlorinated biphenyls (DL-PCBs) 3,3′,4,4′-TetraCB (77) 0.0001 0.00015 3,4,4′,5-TetraCB (81) 0.0003 0.00047 3,3′,4,4′,5-PentaCB (126) 0.1 0.017 3,3′,4,4′,5,5′-HexaCB (169) 0.03 0.1 377

Page 18 of 26

a: TEF (WHO-2005) values are cited from Ref. 34.

18


REP/TEF 1 1 4.8 2.5 1.8 6 6.67 1 3.33 1.53 4.1 2.8 4 3.6 3 17 14.43 1.5 1.57 1.7 3.33

Page 19 of 26


378

FIGURES AND FIGURE LEGENDS

379

FIGURE 1.

380 381

FIGURE 1. Overview of expression of luciferase-reporter plasmid pCL-CR2 and effect of

382

2,3,7,8-TCDD concentration and treatment time on the stably transfected bioassay cell line

383

CBG2.8D.

384

(A) A 297-bp fragment containing 4 DREs was doubly inserted into pGL3-Basic vector under

385

control of the 260-bp mouse CYP1A1 minimal promoter.

386

(B) To investigate the optimal time of 2,3,7,8-TCDD induction in this stable cell line, cells were

387

treated with increasing concentrations of 2,3,7,8-TCDD and luciferase activity (in RLU) was

388

determined at incubation times of 4,12,24 and 48 h. Values represent means ± sem of three

389

independent experiments.

19



390

FIGURE 2.

391 392

FIGURE 2. Stability and repeatability of inducible luciferase activity of CBG2.8D cells.

393

(A) Stability of CBG2.8D cell induction by 2,3,7,8-TCDD was derived from the statistics of 17

394

independent experiments. Cells were treated with 2,3,7,8-TCDD at a set of concentrations ranging

395

from 1.5 × 10-13 M to 10-9 M, and luciferase activity (in RLU) was measured after incubation for

396

24 h. Values represent means ± sem, n = 17.

397

(B) Repeatability was calculated by performing induction with one fixed concentration of

398

2,3,7,8-TCDD. The middle points in the standard curves from different experiments are collected

399

(n = 17). Values shown are the ratios of the average in 17 experiments.

400 401

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402

FIGURE 3.

403 404

FIGURE 3. Comparison between values obtained from CBG2.8D cell assays and HRGC-HRMS

405

analyses for determination of PCDD/Fs in 45 ambient-air samples. Forty-five ambient-air samples

406

were collected at 6 sampling sites in Beijing. Two aliquots of each sample extract were separately

407

subjected to cleanup method for HRGC-HRMS or for bioassay to obtain dioxin-rich fractions

408

which were then subjected to the chemical analysis or CBG2.8D cell assay for TEQ or BEQ

409

determination, respectively. The TEQ value of each dioxin congener was calculated as mentioned

410

in M&M. A sum of TEQ values of 17 dioxin congeners was then obtained and expressed as pg

411

I-TEQ/m3 for each sample. The BEQ value of each sample was obtained as mentioned in M&M,

412

which was expressed as pg BEQ/m3. The correlation between the TEQ and BEQ values of 45

413

samples was studied by scatter plot, correlation coefficient and regression analysis.

414

21



415

FIGURE 4.

416 417

FIGURE 4. (A) Comparison between TEQs of DL-PCB and relative toxicity equivalents of PAHs

418

in 8 haze ambient-air samples. Value were obtained from chemical analysis. (B) Correlation

419

between total activity values obtained from CBG2.8D cell assays and from GC-MS analyses for

420

PAHs.

421

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422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465

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Development and Application of a Novel Bioassay System for Dioxin

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