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the oxidative stress, histopathological changes, inflammatory responses, etc. Mice. 22 were divided into 3 groups and exposed to intermittent concentr...
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Differential health effects of constant versus intermittent exposure to formaldehyde in mice: implications for building ventilation strategies Xu Zhang, Yun Zhao, Jing Song, Xu Yang, Junfeng (Jim) Zhang, Yinping Zhang, and Rui Li Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05015 • Publication Date (Web): 02 Jan 2018 Downloaded from http://pubs.acs.org on January 3, 2018

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Differential Health Effects of Constant versus Intermittent Exposure

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to Formaldehyde in Mice: Implications for Building Ventilation

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Strategies

4 5 6

Xu Zhang1,2#, Yun Zhao3#, Jing Song3, Xu Yang3, Junfeng (Jim) Zhang4,5, Yinping Zhang1,2 *, Rui Li3*

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1

8

2

9

100084, China;

Department of Building Science, Tsinghua University, Beijing 100084, China; Beijing Key Laboratory of Indoor Air Quality Evaluation and Control, Beijing

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3

11

Life Sciences, Central China Normal University, Wuhan 430079, Hubei, China;

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4

13

Durham, NC, USA.

14

5

Duke Kunshan University, Kunshan, Jiangsu Province, China

15

#

These authors contributed equally to this work.

Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of

Global Health Institute and Nicholas School of the Environment, Duke University,

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Abstract

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Formaldehyde, an air pollutant in the indoor environment, may have severe

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effects on human health. The aim of this study is to compare the health effects caused

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by intermittent exposure to formaldehyde (based on real monitoring) to those caused

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by exposures at constant concentration. Health effects explored in this study including

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the oxidative stress, histopathological changes, inflammatory responses, etc. Mice

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were divided into 3 groups and exposed to intermittent concentration formaldehyde

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(0.8ppm for 12 hours and 0 ppm for another 12 hours), or constant concentration

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formaldehyde (0.4 ppm for 24 hours) or zero concentration formaldehyde (reference)

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per day for 7, 14 and 28 days. Following these exposures, bronchoalveolar lavage

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fluid (BALF), lung tissue and lung tissue homogenate were prepared to measure 1

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biomarkers of oxidative stress (ROS, MDA, GSH), histopathological changes,

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inflammatory responses (EOS, NEU, LYM, IL-4, IL-5, IL-13, IL-6, IL-17A, NF-κB,

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IL-1β) and apoptosis (caspase-3). Compared to the constant exposure, intermittent

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exposure to fluctuating formaldehyde concentrations resulted in more profound

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increases in numbers of inflammatory cells in the BALF, greater biological alterations

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including apoptosis. The findings imply that with the same average indoor

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formaldehyde concentrations over the same time, a ventilation strategy to avoid

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higher peak concentrations would lead to lower health risks.

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1. Introduction

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Formaldehyde is a ubiquitous pollutant in indoor environments.1 Exposure to

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formaldehyde may lead to severe effects on human health.2-5 Formaldehyde

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concentrations in many residential buildings in China have been found to be much

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higher than the WHO threshold value of 0.08mg/m3 averaged over 0.5h.6-9 The lung

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cancer risk attributable to formaldehyde is the greatest among that attributable to other

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VOCs in China,10, 11 and is the 4th highest among disease burdens from all indoor air

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pollutants in the US.12 Many countries have set indoor air quality standards for

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formaldehyde. China’s standard is 0.1mg/m3 for a 1-hour average13; in France the

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standard is 0.05mg/m3 for 8-h average14; and in the US (California) it is 0.094mg/m3

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for 1-h average and 0.003mg/m3 for an annual average.15, 16

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Different fresh air intakes and air purifying approaches in the indoor

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environment tend to correspond to variations in the form of indoor formaldehyde

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concentrations.17 For example, constant fresh air intake may correspond to a constant

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indoor formaldehyde concentration because the emission rate of formaldehyde from

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indoor sources (e.g., furniture) changes little for a given indoor air temperature and

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humidity18-20, especially when the indoor climate is controlled by air conditioning or

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space heating. In contrast, natural ventilation tends to correspond to fluctuating indoor

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formaldehyde concentrations because the fresh air intake varies.21 To understand the 2

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health effects and the mechanisms behind them is important to: (1) more accurately

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evaluate the health risk or damage by considering the formaldehyde concentration

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variations; (2) optimize the control strategies for indoor air pollutants by considering

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the health effects, energy consumption and air purification costs.

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An extensive literature exists reporting the effects of average concentrations or

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the effects of peak exposures.22 These studies have been used for supporting

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formaldehyde exposure limits.23-25 However, these studies may not represent the

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real-world exposure conditions, for example, indoor formaldehyde concentrations in

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residences may exhibit a cyclic pattern driving by ventilation differences between the

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daytime (with windows open) and nighttime (with windows closed). It is also rare for

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a home to have an emission source generating an unusually high indoor concentration

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for a short period. A limited number of animal studies have compared the health

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effects of fluctuating formaldehyde concentrations with those of constant (average)

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formaldehyde concentrations but have generated insistent results.26-29 Such studies

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have yielded results helpful in understanding the toxicology mechanisms of

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formaldehyde. However, these studies seem to be inconclusive for guiding

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formaldehyde control in indoor environment. Firstly, although the majority of these

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studies concluded that fluctuating exposures were more harmful26-28, the

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Swiecichowski et al. study concluded the opposite29. Secondly, the exposure settings

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of these studies failed to reflect the fluctuations in actual indoor environments. The

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time pattern is different. For example, in Wilmer’s studies27, 28, the concentration of

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interval groups changes so frequently (every 30-min), which implies that residents

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would have to change and close windows every half hour. This situation differs from

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our monitoring results and the survey of window-opening habits.30 Also, since people

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typically spend most of their time indoors31, they are exposed to indoor air

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formaldehyde most of the day as opposed to a brief period of peak concentration.

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However, the previous studies did not attempt to address exposures representative to

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real-world situations. In addition, these studies only examined respiratory effects of

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formaldehyde. 3

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Based on diurnal variations in formaldehyde concentrations in actual residences,

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we measured a suite of biomarkers related to not only the respiratory system, but also

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the immune system. The present study was designed to: (1) investigate the impact of

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constant versus fluctuating indoor formaldehyde concentrations on respiratory toxicity

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and immunotoxicity when the average concentration is the same for both exposure

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conditions; (2) provide a new perspective for assessment of the operating effect of

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different strategies of indoor air pollution control.

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2. Methods

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2.1 Fluctuating formaldehyde concentration schedule

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To examine the temporal variability in formaldehyde concentration in a real

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apartment, we selected a typical residence in Beijing to monitor the indoor

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formaldehyde concentration for 7 days during the space-heating season (February

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13-20, 2016). The apartment was occupied by two elderly people and an infant for

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almost the entire time during the monitoring period. The formaldehyde concentration

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was measured and recorded every 5 minutes using a PPM monitor (PPM technology,

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HTV-M). The monitor was placed in a bedroom at 10cm higher than the bed. The

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PPM was calibrated prior to monitoring using the DNPH (dinitrophenylhydrazone)

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method with High Performance Liquid Chromatography (HPLC) (Agilent 1260). As

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shown in Figure S1 in Supporting Information, the indoor formaldehyde

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concentration showed a clear diurnal variation approximating a square “wave” with

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12 hours of higher concentration (nighttime) and 12 hours of lower concentration

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(daytime). This was due to the occupants opening the windows during the day and

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closing them at night.

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As for the exposure settings, we have chosen the time patterns based on the

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monitoring results. For exposure concentration, the equivalent exposure concentration

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for mice was about 3 times of the formaldehyde concentration for humans (discussed

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in Section S1 of Supporting Information). Considering our limited experiment time (4 4

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weeks) compared to exposure duration for humans (years), we chose a higher

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formaldehyde concentration which has been reported in literature6, 7, 0.27ppm for

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peak exposure and about 0.13ppm for average intensity. These concentrations are

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about 3 and 1.5 times higher than the WHO recommended values. After concentration

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conversion for mice, the peak and average concentrations were chosen at 0.8ppm and

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0.4ppm, respectively. Thus, the exposure conditions for our toxicological experiments

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as follows. The intermittent exposure condition consisted of 12 hours with a

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formaldehyde concentration of 0.8ppm, followed by 12 hours with the concentration

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at 0 ppm. The monitoring results showed the high concentration period to be from

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9:00pm to the 9:00am, which corresponds to most of the sleeping time for the

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occupants. However, given that the sleeping time and habits of mice differ from that

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of humans, it is not necessary to mimic human sleep patterns in our mouse

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experiments. We set the high concentration period from 12:00 AM to 12:00 PM and

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the low concentration period from 12:00 PM to 12:00 AM. The constant concentration

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exposure condition was set at 0.4ppm. These two exposure conditions had the same

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24-hour average exposure concentrations (see Figure S2 in Supporting Material).

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2.2 Experimental system and monitoring

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Formaldehyde was produced by a generator developed in our laboratory

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sketch of the generator system is shown in Figure S3 in Supporting Material. Before

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the exposure experiment, this system was operated for 7 days in the exposure setting.

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The formaldehyde concentration was calibrated with a gas analyzer (Innova 1312,

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Denmark) using the dinitrophenylhydrazone (DNPH) method with High Performance

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Liquid Chromatography (HPLC) (Agilent 1260, USA).

32

. A

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The mat in the exposure chamber adsorbs formaldehyde and decreases the

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accuracy of the formaldehyde concentration measurement. To reduce mat adsorption,

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the air intake was via horizontal intake ports, as shown in Figure S4. Horizontal air

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flow has a lower mass transfer coefficient and the multi-intake port decreases the air

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flow speed which also contributes to a lower mass transfer coefficient 33. 5

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During the exposure period, flowmeters were calibrated daily using a soap film

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flow meter (GL-100, Zhongkezhonghuan, Inc., Beijing, China). The formaldehyde

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concentration in the exposure chambers was monitored hourly using PPM (PPM

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technology,

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dinitrophenylhydrazone (DNPH) method with HPLC (Agilent 1260, USA). The

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temperature in the chamber was controlled with air-conditioning, and the humidity

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was controlled by the formaldehyde generator system.

HTV-M).

The

PPM

was

calibrated

weekly

using

the

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2.3 Animals

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SPF male Balb/c mice (5-6 weeks, approximately 20 g) were purchased from

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Wuhan Institute of Biological Products Co., Ltd. (Wuhan, China) and Hubei Research

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Center of Laboratory Animals (Wuhan, China). Mice were kept in cages and raised in

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a pathogen-free animal laboratory room at 55%-75% humidity and between

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20 °C - 25 °C for a week prior to the study. All animal experiments were conducted

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following the guidelines provided by the Institutional Animal Care and Use

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Committee

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CCNU-IACUC-2012-011). The experimental project ethics ratification is shown in

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Figure S5 in the Supporting Information.

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2.4 Reagents, antibodies and kits

of

Central

China

Normal

University

(Ratification

ID:

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Formaldehyde was purchased from Sigma-Aldrich (St Louis, MO, USA). All

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other reagents were analytically pure. Rabbit anti-NF-κB p65 (phosphor S536)

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(ab86299, Abcam, MA, USA), mouse anti-IL-6 (ab9324, Abcam, MA, USA), rabbit

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anti-IL-4 (BA0980-1, Boster, Wuhan, China), rabbit anti-IL-13 (BA1208-1, Boster,

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Wuhan, China), rabbit anti-IL-5 (BA0985, Boster, Wuhan, China), rabbit anti-IL-17A

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(A0688, ABclonal, MA, USA), IL-1β, Caspase 3. The GSH kit was purchased from

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Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The protein was

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determined using a Folin-phenol kit from

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Biotechnology Co., Ltd. (Beijing, China)

Beijing Dingguo

6

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2.5 Animal exposure

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Mice were randomly divided into three groups (n=6/group), comprising (1)

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control group; (2) constant exposure group; (3) intermittent exposure group. The three

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groups were exposed to air containing different levels of formaldehyde: the control

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group was exposed to pure air for 24h; the constant exposure group was exposed to a

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constant formaldehyde concentration at 0.4 (±20%) ppm for 24h; and the intermittent

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exposure group was exposed to 0.8 (±20%) ppm formaldehyde for 12h followed by

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pure air for 12h. All three exposure chambers were maintained at 23±3 °C, 60%±20%

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humidity, 3L/min gas flux. Three-rounds of experiments were performed for 7, 14 and

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28 consecutive days, respectively.

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2.6 Preparation of bronchoalveolar lavage fluid (BALF) and lung tissue homogenate

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On day 8 (first round), 15 (second round) and 29 (third round), all mice were

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anesthetized with 100mg/kg (body weight) pentobarbital sodium via intraperitoneal

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injection. Following anesthetization, 1mL saline was introduced into the lung of each

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mouse through a tracheal cannula, and rinsed and lavaged for about 1min. Then a

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syringe combined with the cannula was used to withdraw the liquid. This process was

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repeated three times so as to collect enough bronchoalveolar lavage fluid (BALF).

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More than 90% of the mice recovered from this procedure.

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After the mice were sacrificed by cervical dislocation, whole lung tissues were

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removed using medical scissors and rinsed in ice-cold phosphate buffer saline (PBS).

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The lung tissue was then homogenized with ice-cold PBS (10 mL/g, pH=7.5) in a

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glass homogenizer on ice. This homogenate was centrifuged (10,000 rpm, 4 °C, 10

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min) to collect the supernatant, which was then stored at -80 °C.

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2.7 Cell counts

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Pellets of BALF were collected after centrifugation (1500 rpm, 4 °C, 10 min)

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and resuspended with 0.5 mL saline. Then differential cell count of eosinophils,

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neutrophils lymphocytes and total cells was carried out using a Blood Cell Analysis 7

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System (MTN-21, Matee3nu Technology Corp., Jinan, China).

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2.8 Measurement of reactive oxygen species (ROS), malondialdehyde (MDA) and

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glutathione (GSH) content

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ROS

fluorescence

intensity

was

measured

using

the

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2′,7′-Dichlorodihydrofluorescein

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previously.34 Briefly, DCFH, a product of hydrolysis of DCFH-DA by cell esterases,

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is oxidized by peroxides to DCF, whose fluorescence intensity reflects ROS levels.

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The supernatant of lung tissue homogenate was diluted ten times with PBS (pH = 7.5),

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100µL supernatant was then added into a 96-well microplate, followed by the addition

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of 100µL DCFH-DA (10 µmol/L). The mixture was kept from light for 30 min at

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37 °C, and the fluorescence intensity was measured after this incubation time (488 nm

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excitation wavelength, 525 nm emission wavelength) by a fluorescence microplate

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reader (FLx800, BioTek Instruments, Vinooski, VT, USA). The bone marrow cell

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sample level was adjusted to 106/mL. 300µL of this was added to DCFH-DA to arrive

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at 10 µmol/L. The mixture was incubated for 30min at 37 °C in the dark, following

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which the cells were washed and resuspended with PBS. 200µL of the suspension was

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then added to a 96-well microplate and the fluorescence intensity determined as

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described above.

diacetate

(DCFH-

DA)

method

described

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Malondialdehyde (MDA) was measured from the chromophoric production of its

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reaction with 2-Thiobarbituric acid (TBA).35 The mixture of 0.5 mL supernatant with

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2 mL 0.6% TBA solution (dissolved in 10% trichloroacetic acid) was incubated in

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boiling water for 15 minutes to form the pink pigments and protein precipitates. The

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mixture was cooled and centrifuged (10,000 rpm, 5min, 4 °C) and the supernatant

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collected. The absorbance of the supernatant was 532, 600, and 450 nm. MDA

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concentration was calculated as:

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MDA (µmol/L) = 6.45 × (OD532 – OD600) – 0.56 × OD450.

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The GSH contents of the lung homogenate and bone marrow cells were determined

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using a GSH test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) 8

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according to the manufacturer’s instructions.

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2.9 Histological examination

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Half of the lung tissue was removed and fixed in 10% polyformaldehyde for 24h

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at room temperature and later stained with hematoxylin and eosin (H&E). The stained

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lung tissues were embedded in paraffin, and then sectioned for histopathological

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observation using a DM4000B Microscope (Leica Microsystems GmbH, Wetzlar,

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Germany).

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2.10 Immunohistochemical assay

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The lung tissue sections were incubated with 0.3% H2O2 to quench the

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endogenous peroxides after deparaffinization, rehydration and antigen retrieval, and

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then blocked by 5% BSA in PBS for 30 min (room temperature). Primary antibodies,

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rabbit anti-NF-κB p65 (phosphor S536) (ab86299, Abcam, MA, USA), mouse

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anti-IL-6 (ab9324, Abcam, MA, USA), rabbit anti-IL-4 (BA0980-1, Boster, Wuhan,

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China), rabbit anti-IL-13 (BA1208-1, Boster, Wuhan, China), rabbit anti-IL-5

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(BA0985, Boster, Wuhan, China), rabbit anti-IL-17A (A0688, ABclonal, MA, USA),

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IL-1β, Caspase 3, were used for immunohistochemical detection of NF-kB p65, IL-1β,

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IL-6, IL-4, IL-5, IL-17, IL-13, and Caspase 3 respectively. After overnight incubation

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at 4 °C with primary antibodies, the sections were incubated with secondary

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antibodies (anti-rabbit IgG) for 30min (37 °C). 3% H2O2 and diaminobenzidine

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tetrahydrochloride (5mg/10mL) were used to incubate such sections after being

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detected with an avidinbiotin peroxidase complex. A DM 4000B Microscope (Leica)

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was used to view the immunostained slides and the average optical densities of such

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proteins were determined using Image-Pro Plus 6.0 software.

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2.11 Statistical analysis

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Data are presented as the mean ± SEM and statistical graphs were generated

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using GraphPad Prism version 5.01 (GraphPad Software Inc., San Diego, CA, USA).

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Differences between groups were determined using one-way analysis of variance 9

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(ANOVA). Statistical analyses were performed using SPSS software version 23.0

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(SPSS Inc., Chicago, IL, USA). p