Differential Health Effects of Constant versus Intermittent Exposure to

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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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Environmental Science & Technology

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Differential Health Effects of Constant versus Intermittent Exposure

2

to Formaldehyde in Mice: Implications for Building Ventilation

3

Strategies

4 5 6

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

7

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;

12

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,

16

17

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

ACS Paragon Plus Environment


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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

133

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


Changsheng

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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

213

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

235

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

236

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

242

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

243

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

244

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

248

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

251

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

Differential Health Effects of Constant versus Intermittent Exposure to

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