Ohyama et al. Environmental Sciences Europe
https://doi.org/10.1186/s12302-022-00693-1(2022) 34:112

Open Access

RESEARCH

Role of nitrous acid in the association
between nitrogen dioxide and asthma
symptoms: effect of nitrous acid exposure
on specific airway resistance in guinea pigs
Masayuki Ohyama1*, Kenichi Azuma2, Chika Minejima3, Norimichi Takenaka4 and Shuichi Adachi5

Abstract
Epidemiological evidence regarding the effects of nitrogen dioxide ­(NO2) on asthma and chronic obstructive pulmonary disease (COPD) is inconclusive because ­NO2 assays measure nitrous acid (HONO) as ­NO2. Epidemiologicalstudy using separate measurements of indoor HONO and ­NO2 suggests that indoor HONO levels are associated withdecrease in lung function, and HONO exposure experiment in rats suggests that HONO adversely affects respiratory
function more than ­NO2. Effects of ­NO2 on respiratory tract resistance have not been observed in rats but have been
observed in guinea pigs, including baseline specific airway resistance (sRaw). The present study aimed to investigate
the effects of HONO exposure on baseline sRaw in guinea pigs. Eighteen male Hartley guinea pigs were divided into
Groups C, M, and H (n = 6 per group) and exposed to HONO at three concentrations, respectively, 24 h/d for 7 weeks.
Double-flow plethysmography was used to measure the sRaw once a week and grade respiratory waveforms,
indicating increased airway resistance. The experiment was performed twice. Mean HONO concentrations of two
experiments in Groups C, M, and H were 0.02, 0.66, and 3.43 parts per million (ppm). The sRaw increased significantly
in Groups H and M compared with sRaw in Group C, and in a concentration-dependent manner after the 4th week
of HONO exposure. Increased airway resistance was observed in 50% of animals in Group M and 100% of animals inGroup H in some weeks. However, in some animals from Group M, increased airway resistance was observed earlier than the airway resistance increases observed in all animals in Group H. These sRaw results suggest that HONO
affects asthma symptoms or COPD, at a lowest observed adverse effect level (LOAEL) of < 0.66 ppm. HONO may affectbaseline sRaw after shorter exposure periods and at lower concentrations compared with the reported effects of N
­ O 2.
The present results suggest that HONO is the most important nitrogen oxide affecting asthma and COPD indoors, andthat HONO may be the causative agent in the association between N­ O2 and asthma symptoms in epidemiologicalstudies.
Keywords: Nitrous acid, Specific airway resistance, Asthma, Chronic obstructive pulmonary disease, Nitrogen dioxide,
Guinea pig*Correspondence: ohyama@iph.osaka.jp
1
Department of Environmental Health, Osaka Institute of Public Health,
1‑3‑69, Nakamichi, Higashinari‑Ku, Osaka 537‑0025, Japan
Full list of author information is available at the end of the article

Background
Numerous epidemiological studies have shown a relationship between ­NO2 and alterations in respiratory
function or asthma symptoms [1–3]. However, conventional nitrogen dioxide ­(NO2) assays measure both
nitrous acid (HONO) and N­ O2 as N­ O2 [4]. HONO contamination in N
­ O2 measurements causes uncertainty in© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/.

Ohyama et al. Environmental Sciences Europe(2022) 34:112

epidemiological studies regarding whether ­NO2 exposure has an independent effect on asthma development.
According to the US Environmental Protection Agency(EPA) [5], this uncertainty has been partly reduced byexperimental animal studies by Kobayashi & Miura
characterizing the potential mode of action of ­NO2 inasthma development [5]. Kobayashi & Miura (1995) conducted an ­NO2 exposure experiment in guinea pigs using
baseline specific airway resistance (sRaw) as an index
of asthma and chronic obstructive pulmonary disease(COPD); exposure of guinea pigs to N
­ O2 at 2 and 4 parts
per million (ppm) for 12 weeks induced a concentrationdependent increase in sRaw compared with a control
group [6]. However, no increase in sRaw was observed at
12 weeks of exposure to ­NO2 at 0.06, 0.5, and 1.0 ppm, orat 6 weeks of N
­ O2 exposure at up to 4 ppm [6].
We previously reported that HONO exposure at
5.8 ppm with secondary products of 0.7 ppm ­NO2 and
2.3 ppm NO in rats significantly increased the baseline
pulmonary resistance (RLung) and Muc5ac mRNA levelsin the lung but had no effects on dynamic lung compliance (Cdyn) and pulmonary fibrosis [7]. RLung is used
as an indicator of asthma symptoms and COPD in rats.
Until the publication of our HONO exposure experiment, sulfur dioxide ­(SO2) was the only known air pollutant that increased RLung in rats. 250 ppm ­SO2 causes not
only increased RLung, but also pulmonary fibrosis andreduced Cdyn in rats [8]. However, pulmonary fibrosis
is not always observed in asthmatics. The HONO exposure experiment in rats suggested that HONO has more
adverse effects on respiratory function than ­NO2 or ­SO2
[7].
The World Health Organization (WHO) [9] reported
that the extent of health effects that can be attributed to­NO2 itself, or rather to primary and secondary combustion-related products (i.e., organic carbon and HONO)
is unclear in epidemiological studies [9]. For instance,
Jarvis et al. conducted an epidemiological study using
separate measurements of indoor HONO and ­NO2 andfound that indoor HONO levels, but not indoor ­NO2levels, are associated with a decrease in lung function[10]. Additionally, in an epidemiological pilot study,
Ohyama et al. found that indoor HONO and nitric oxide(NO), which are mainly produced by the combustion ofcertain substances, were significantly associated withasthma attacks, as revealed via statistical analysis using
the Mann–Whitney U-test [11]. However, WHO revised
the annual air quality guidelines (AQG) for ­NO2 from40 µg/m3 (approximately 0.020 ppm) in 2005 to 10 µg/m3(approximately 0.005 ppm) in 2021 based on the effectsof long-term N­ O2 on all-cause mortality and respiratory
mortality according to epidemiological studies without HONO measurements [12]. One of the reports that

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contributed to this revision in the AQG demonstrated
that the evidence for ­NO2 effects on COPD mortality was
the most reliable [13].
Although atmospheric ­NO2 levels has been decreasing in recent years [2], numerous epidemiological studies
have demonstrated an association between indoor N­ O2
and asthma [14–18]. Hansel et al. [14] suggested thatinterventions that lower N­ O2 concentrations in homes
will reduce asthma morbidity in preschool children [14].
According to Belanger et al. (2013), asthmatic children
exposed to indoor ­NO2 levels below the US EPA outdoor
standard (53 ppb) are at risk for increased asthma morbidity [16]. However, these reports did not consider the
involvement of HONO. Lee et al. reported correlations
between indoor HONO and indoor ­NO2, with a correlation coefficient of 0.51, and between indoor HONO andoutdoor ­NO2, with a correlation coefficient of 0.35, but
not between outdoor HONO and indoor and outdoor­NO2 [19]. The involvement of HONO in indoor N­ O2
effects should be considered even more than the outdoor effects, as the ratio of HONO to ­NO2 is about 1:3–4
indoors [10] and 1:20–60 outdoors [20].
Further animal studies are needed to determine
whether HONO or ­NO2 has stronger effects on asthma
and COPD. The present study aimed to investigate theeffects of HONO exposure on baseline sRaw in guinea
pigs over time. This experiment will help us compared
the effects of HONO versus ­NO2 on asthma and COPD.

Methods
Animals

Eighteen male Hartley guinea pigs (5 weeks of age, specific-pathogen free) were purchased from SLC (Shizuoka, Japan). Guinea pigs were divided randomly into
three groups of 6 animals each (control group, Group C;
middle concentration exposure group, Group M; highconcentration exposure group, Group H). The animals
in each group were housed preliminarily in flat-flooredhanging 3 cages for 4 weeks in each exposure chamber,as described previously [7, 21, 22]. Briefly, the exposure
chambers were custom-made out of acrylic, and had volume of 0.2 ­m3 and flow air volume of 15 L/min. Room
air was filtered using about 5 kg of charcoal-activatedgranules and 15 sheets of American air filters for vinyl
isolators (Clea Japan, Inc., Tokyo, Japan), air compressors (0.4LE-8S; Hitachi Industrial Equipment Systems
Co., Ltd., Tokyo, Japan), dehumidifiers (RAX3F; Orion
Machinery Co., Ltd., Nagano, Japan), high-pressure regulator valves (with the largest supply pressure of about
0.078 MPa; model No. 44–2263-241; Kojima Instruments
Inc., Kyoto, Japan), and mass flow controllers to control
air flow (model 8350MC-0-1-1; Kojima Instruments Inc.,
Kyoto, Japan). The internal pressure in the chambers wasOhyama et al. Environmental Sciences Europe(2022) 34:112

adjusted to about + 1 mm ­H2O relative to atmosphericpressure. Food and water were available freely during all
experimental periods. The animal room was maintained
under a dynamic temperature of 24 ± 3 °C to stabilize thechamber temperature and humidity. The room lighting
was turned on/off by staff at 9:00 and 17:30.
HONO exposure

A HONO generation system (as described previously)
was used with the exposure chambers described above[23, 24]. Briefly, the HONO generation system sprayed a
mixture of aqueous sodium nitrite (> 98.5% pure sodium
nitrite; Wako Pure Chemical Industries, Ltd. Osaka,
Japan) with aqueous acid (85–92% lactic acid; Wako
Pure Chemical Industries, Ltd. Osaka, Japan) solution
in a porous polytetrafluoroethylene (PTFE) tube (TB1008, approximately 15 cm length; Sumitomo ElectricFine Polymer, INC., Osaka, Japan) with filtrated room
air through an atomizer-nozzle (BN90s-IS[V], SUS316L,
1/8PT, M14; Atomax Co., Shizuoka, Japan). Air outside
the porous PTFE tube, including that containing HONO,
was continuously supplied to the exposure chamberwith secondary products of ­NO2 and NO for 7 weeks.
The concentration of aqueous sodium nitrite was 200
and 80 mmol/L in Groups H and M, respectively. The
concentration of lactic acid was 250 and 100 mmol/L inGroups H and M, respectively. The flow velocity of eachsolution was maintained at approximately 0.4 mL/min,and the flow velocity of clean air was maintained at 15 L/
min. HONO exposure was stopped for approximately 1 hevery week during exchange of cages and cleaning of thechambers.
Measurement of nitrogen oxides in chamber air

Within the same sampling period, we used a non-continuous method to measure HONO levels and a continuous
method for measuring ­NO2 and NO with a NOx analyzer (Model 42 i, Thermo Fisher Scientific., Yokohama,Japan). In measurement of HONO, we used a Harvard
EPA Annular Denuder System consisting of two annular denuders (URG-2000-30 × 150-3CSS; URG Corporation, NC) coated with sodium carbonate (> 99.5% pure,
Wako Pure Chemical Industries, Ltd. Osaka, Japan) andglycerol (> 99% pure, Wako Pure Chemical Industries,
Ltd. Osaka, Japan) [25, 26]. Chamber air was aerated tothe denuder by a NOx analyzer equipped with a device
that switched six paths hourly. The amount of HONO in
the denuder extracts was measured by ion chromatography (700 series; Metrohm Japan LTD., Tokyo, Japan). The
concentrations of HONO were calculated by the aeration
volume data of the NOx analyzer and the ion chromatography data. The concentrations of N
­ O2 and NO were
measured in the air passing through the denuder. Only

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stable data collected from 20 min after the switching ofsampling routes were used.
Measurement of sRaw and observation of respiratory
waveform

The method for measurement of sRaw by double-flow
plethysmography has been reported previously [27–29].
sRaw was assessed weekly in nonanesthetized spontaneously breathing guinea pigs using the PULMOS-I system(MIPS Co. Ltd., Osaka, Japan) from the stage of preliminary housing to the end of HONO exposure. The object
animal range of the PULMOS-I system was fixed at therange for “guinea pig”. The data of 100 respirations following the 10th respiration after the start of measurement were used for analysis. sRaw values were calculated
as the mean of these data. Any data obtained while theguinea pigs struggled were excluded. Raw is used as anindicator of asthma symptoms or COPD in guinea pig.
Respiratory waveforms can be observed while airway
resistance is being measured using the PULMOS-I system. Under normal conditions in guinea pigs, expiratory flow in the first half of expiration is greater than the
expiratory flow in the latter half of expiration. However,when airway resistance is increased, expiratory flow inthe latter half of expiration is greater than expiratory flowin the first half of expiration. We classified the respiratory
waveforms of each measurement based on these characteristics into three grades: normal, larger flow in thefirst half of expiration than in the latter half of expiration;affected, larger flow in the latter half of expiration thanin the first half of expiration; and strongly affected, clear
and large flow in the latter half of expiration than in thefirst half of expiration.
Measurement of tidal volume (TV) and peak expiratory
flow (PEF)

TV and PEF were measured by the PULMOS-I system in
100 respirations, as for sRaw.
Experiment to confirm reproducibility

A same above mentioned experiment, as describedabove, was conducted again to confirm reproducibility.
Therefore, the total animal number of animals in eachgroup was 12.
Statistical analysis

The mean ± standard deviation values of sRaw, TV, andPEF were calculated for 12 animals in each group. Statistical analyses were performed for twice experiments datausing analysis of variance followed by Dunnett’s multiple
comparison tests. p < 0.05 was considered significant. Alldata analyses were performed using SPSS statistical software, version 26 (IBM Corp, Armonk, NY, USA).

Ohyama et al. Environmental Sciences Europe(2022) 34:112

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Table 1 Concentrations of nitrogen oxides in each camber

Results

Group

Concentrations of nitrogen oxides in chamber air

HONO (ppm)

NO2 (ppm)

NO (ppm)

Experiment 1
Group C

0.005

Group M

0.775

Group H

4.179

Experiment 2
Group C

0.030

Group M

0.542

Group H

2.670

AveragebGroup CGroup M
Group Ha0.044 ± 0.063

0.027 ± 0.062

1.374 ± 1.175

3.997 ± 1.626

0.174 ± 0.291

1.032 ± 0.495− 0.011 ± 0.0 ­35a

0.094 ± 0.131

0.385 ± 0.212

2.639 ± 0.569

0.000 ± 0.042

0.721 ± 0.219

0.018 ± 0.013

0.017 ± 0.028

0.061 ± 0.0335

3.425 ± 0.755

0.880 ± 0.495

3.318 ± 0.679

0.659 ± 0.117

0.087 ± 0.087

0.877 ± 0.156

Negative value due to inadequate of calibration of the NOx analyzerbData are presented as the mean value ± SD based on the average value of 2
experiments

Table 1 shows the concentrations of HONO, ­NO2, andNO, in chamber air for each group. Although the nitrogen oxide concentrations were lower in Groups H and M
in Experiment 2 than in Experiment 1, the concentration
ratios of Groups H/M were similar for HONO and NO in
Experiment 1 and 2 (Groups H/M for HONO and NO:
5.4 and 3.9 in Experiment 1, and 4.9 and 3.7 in Experiment 2, respectively). The concentration level of ­NO2 waslower those of HONO and NO in both Experiment 1 and
2. Although a slight drift of NOx analyzer was observedin the measurement of Experiment 2, the N­ O2 concentration in Group M was similar to that in Group C.
Respiratory function

Figure 1 schematically depicts the plan for measuringrespiratory functions during the HONO exposure experiment. Arrows indicate the measurement of respiratory
functions. The average values of three measurements of
respiratory functions obtained during the preliminary
housing period were considered zero-week data.
Respiratory waveforms of the three grades

Fig. 1 An outline of the measurements of respiratory functions in
the HONO exposure experiment. The timing of respiratory functions
is indicated using arrows. All measurements of respiratory functions
during the preliminary housing period were considered zero-weekdata

Figure 2 (A, B, and C) shows examples of respiratory waveforms for the normal, affected, and strongly
affected respiratory waveform grades. In each subfigure, the upper two waveforms were obtained in Experiment 1, and the lower two waveforms were obtained inExperiment 2. All examples of the normal grade were
respiratory waveforms of Group C, and the examples of
the affected and strongly affected grades were respiratory waveforms of Groups M and H in each experiment.

Fig. 2 Examples of respiratory waveform for each of the three grades A, B and C. Horizontal lines are zero flow level. Expiratory waves are displayed
above the zero flow level, and inspiratory waves are displayed below the zero flow level. The blue lines indicate thoracic flow, and red lines indicate
nasal flow. The upper two waveforms were obtained in Experiment 1, and the lower two waveforms were obtained in Experiment 2

Ohyama et al. Environmental Sciences Europe(2022) 34:112

Although breathing speed varied among the animals,
no effects of breathing speed on the respiratory waveform grades were observed. Respiratory waveforms likethose in Fig. 2A were classified as normal. The expiratory
flows in the first half of expiration were slightly greaterthan the expiratory flows in the latter half of expiration inmost respiratory waveforms. Respiratory waveforms likethose in Fig. 2B were classified as affected. The expiratory
flows in the latter half of expiration were slightly greaterthan the expiratory flows in the first half of expiration insome respiratory waveforms, and some respiratory waveforms corresponded to normal. Respiratory waveforms
like those in Fig. 2C were classified as strongly affected.
The expiratory flows in the latter half of expiration wereclearly greater than the expiratory flows in the first half of
expiration in most respiratory waveforms. In each group,similar waveforms were observed in the two experiments.
Effects on sRaw and the respiratory waveform grades

Figure 3 shows changes in sRaw and the respiratory
waveform grade for each animal over time. Although thesRaw for each animal was not very stable over time, allrespiratory waveforms in Group C were graded as normal. In the respiratory waveforms of Groups M and H,
waveforms were often graded as affected or strongly
affected when sRaws were greater than 2 cm ­H2O × mL/(mL/s), and waveforms were sometimes graded asaffected when sRaws were less than 2 cm ­H2O × mL/(mL/s). Animals with sRaw above 3 cm ­H2O × mL/(mL/s)
in some week tended to have similar sRaw in otherweekly measurements. In both Experiments 1 and 2, thestrongly affected or affected grades were observed ineither week six animals in Group H and three animals inGroup M. Although the HONO concentration of GroupM in Experiment 2 was lower than the concentrationin Experiment 1, the strongly affected weeks earlier inExperiment 2 than in Experiment 1. Moreover, strongly
affected or affected grades were observed in five animals
in Group M and one animal in Group H within 3 weeks
of exposure.
Table 2 shows the results of the statistical analysis ofchanges in sRaw in each group for combined Experiments 1 and 2. Significant increases in sRaw wereobserved after the 4th week of HONO exposure inGroups H and M and in the 1st week of HONO exposurein Group M compared with the sRaw in Group C, and in
a concentration-dependent manner after the 4th week of
HONO exposure. Changes in sRaw for Groups M and H
were similar after the 2nd week of HONO exposure.
Effects of HONO exposure on TV and PEF

Figure 4 shows changes in the average TV over time foreach group for the combined Experiments 1 and 2. The

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TV for each group ranged from approximately 3–4 mL.
No significant changes over time were observed, and TVs
in Groups H and M were not significantly different fromTV in Group C.
Figure 5 shows changes in the average PEF over timefor each group for the combined Experiments 1 and2. The PEF for each group ranged from approximately
14–19 mL/s. No significant changes over time wereobserved, and PEFs in Groups H and M were not significantly different from PEF in Group C.

Discussion
Most ­NO2 epidemiological studies discuss the effects of­NO2 without measuring HONO, even though conventional ­NO2 assays measure HONO as ­NO2 [4]. Moreover,
HONO is in equilibrium with NO, ­NO2, and ­H2O [30]:2HONO ⇄ ­NO2 + NO + ­H2O. Therefore, some ­NO2 may
have changed to HONO in animal experiments focusedon ­NO2 exposure. Even in the present HONO exposure
experiment, the secondary products of ­NO2 and NO
could not be eliminated. However, since HONO and N­ O2
were both measured, we can discuss whether the effects
were due to HONO or ­NO2.
The ­NO2 concentration in Group M from Experiment
1 was 0.17 ppm, and the N­ O2 concentration in Group M
from Experiment 2 was similar to the ­NO2 concentrationin Group C from Experiment 2. However, the effects on
respiratory waveforms in Group M were similar between
Experiments 1 and 2. This result suggests that 0.17 ppm­NO2 levels in HONO exposure is not important in respiratory resistance. Although the highest concentrationof ­NO2 in this experiment was approximately 1.4 ppm inGroup H of Experiment 1, Kobayashi & Miura reported
no effect on sRaw even at 4 ppm N­ O2 6 weeks exposure[6]. Moreover, the sRaws of Groups M and H in thisexperiment were similar. In addition, we observed thepulmonary emphysema-like alterations in guinea pigs
exposed to 1.7 ppm HONO with secondary productsof 0.0 ppm N­ O2 and 0.2 ppm NO [22]. Based on theseresults and knowledges, the increases in sRaw in Groups
M and H are not likely to be due to the secondary product ­NO2. Although the concentrations of NO are similar
to the concentrations of HONO in each group, NO is
not very toxic and is used for treating respiratory insufficiency in certain conditions [31]. Overall, our results
suggest that the significant increase in sRaw was due to
HONO and the LOAEL was less than 0.66 ppm.
Although nitrogen oxide concentrations in Groups H
and M were lower in Experiment 2 than in Experiment
1, the experimental conditions, such as reagent concentrations, remained the same, and the reason for this finding is unknown. According to the results of Experiment
1 and 2, there was no air leakage based on the identicalOhyama et al. Environmental Sciences Europe(2022) 34:112

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Fig. 3 Change in sRaw and grade of respiratory waveform for each animal over time. Data for 0 weeks of exposure are averages of sRaw of the
3-week preliminary housing period. Grade of respiratory waveform is indicated as follows: circle, normal; triangle, affected; square, strongly affected.

Ohyama et al. Environmental Sciences Europe(2022) 34:112

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Table 2 Change in sRaw for each group over time after exposure to HONO
Weeks ofexposureGroup C
na

1

12

2

12

3

12

4

12

5

12

6

12

7

12

p ­valueb

Group M
Mean ± SD
1.24 ± 0.37

1.44 ± 0.37

1.42 ± 0.37

1.36 ± 0.28

1.44 ± 0.32

1.70 ± 0.21

1.63 ± 0.22

na
12
12
12
12
12
12
12

na

Mean ± SD
1.79 ± 0.58

1.65 ± 0.51

2.16 ± 1.19

2.30 ± 1.04

∗ 0.034
0.395

12
12

0.197

12

†0.00612

2.44 ± 0.87

†0.002

12

0.067

12

2.27 ± 0.83

∗ 0.02812

2.47 ± 1.22

p ­valueb

Group Hp ­valuec

Mean ± SD
1.44 ± 0.64

1.78 ± 0.36

2.14 ± 1.45

2.20 ± 0.66

2.46 ± 0.76

2.59 ± 0.85

2.19 ± 0.63

ANOVA

0.565

0.056

0.103

0.157

0.213

0.196

∗ 0.015†0.006†0.002†0.001∗ 0.031∗ 0.0350.058∗ 0.030

Data are presented as the mean ± SD ­(cmH2O × mL/(mL/s)) for Experiments 1 and 2. Dunnett’s multiple comparison was applied for comparison with the control
following ANOVAa

Number of animalsb

P value for comparison with the controlc

P value for ANOVA*

p < 0.05†

p < 0.01

Fig. 4 Change in TV for each group over time after exposure to
HONO. Data are presented as the mean ± SD for Experiments 1
and 2. Dunnett’s multiple comparison was applied for comparison
with the control following ANOVA TVs in Groups H and M were notsignificantly different from TV in Group C

Fig. 5 Change in PEF for each group over time after exposure to
HONO. Data are presented as the mean ± SD for Experiments 1 and2. Dunnett’s multiple comparison was applied for comparison withthe control following ANOVA PEFs in Groups H and M were notsignificantly different from PEF in Group Cconcentration ratios of Groups H/M for HONO and NO.
A possible reason for the lower N­ O2 concentrations inExperiment 2 could be that the humidity in Experiment
2 was higher than that in Experiment 1, which could
have resulted in the formation of HONO from ­NO2 inequilibrium. HONO is water soluble, but ­NO2 and NO
are insolubule in water. Water soluble HONO is more
readily absorbed by animals than non water soluble­NO2 and NO, which may have resulted in a reduction
in all nitrogen oxide concentrations. Thus, Group M in
Experiment 2 would have produced more HONO thanGroup M in Experiment 1, despite the HONO values

being lower. Generally, in animal exposure experiments,
the HONO concentration is measured after the animals
have absorbed the exposed HONO through respiration
and body hair. Consequently, it is likely that the animals
in Group M in Experiment 2 inhaled more HONO thanthose in Group M in Experiment 1, and that the effecton respiratory function was observed from the first week
of HONO exposure. In the general environment, theweather conditions under which equilibrium reactions
produce HONO from N­ O2 may be an important factor
associated with asthma symptoms.

Ohyama et al. Environmental Sciences Europe(2022) 34:112

Our previous experiments on rats [7] and guinea
pigs [21] utilized the highest possible concentrations of
HONO to indicate clear biological effects at a single time
point observation. The N­ O2 ranged from 0.3 to 0.7 ppm
in the HONO exposure groups in those experiments, andthere was no basis for ignoring the ­NO2 effects. Therefore, in those reports we discussed that HONO withsecondary products of ­NO2 and NO adversely affects respiratory function and pulmonary emphysema-like alterations without inflammation. We believe that the previous
discussion stating that “HONO with secondary productsof ­NO2 and NO adversely affects the lungs” should be
modified to “HONO adversely affects the lungs.”
The respiratory waveforms indicative of increased airway resistance were reproducible in the two experiments,
with some animals in Group M exhibiting such respiratory waveforms earlier than in Group H. The respiratory
waveform results in Group M suggest that some animals
experience short-term effects of airway resistance at low
concentrations of HONO. The respiratory waveforms
in Group H suggest that high concentrations of HONO
affect airway resistance in most guinea pigs. However, thereason for the later effects on the respiratory waveforms
in Group H compared to the waveforms in Group M isunclear. Whether NO can delay the effects of HONO on
the respiratory waveforms requires further investigation.
HONO significantly increased sRaw, but no significant
effects on TV and PEF were detected. These results are
consistent with the results of previous HONO exposure rat experiment [7] showing increased LRung but noeffects on Cdyn. Our results suggest that HONO specifically enhances respiratory resistance.
NO2 is often considered as the most important nitrogen oxide affecting respiratory function. However,according to the results of Kobayashi & Miura, 12 weeks
of exposure to N
­ O2 above 2 ppm was required to produce
a significant increase in sRaw, whereas, in our experiment, exposure to HONO at 0.66 ppm increased sRaw
after only 4 weeks. It may be difficult to explain the relationship between nitrogen oxides and asthma by HONO
alone because of the discrepancy that Group M observed
the respiratory waveforms indicating increased airway
resistance earlier than Group H. However, we believethat HONO is an important nitrogen oxide that increases
airway resistance at low concentrations and short exposure periods without inflammatory alterations. Although
the concentration of HONO that affects airway resistance is lower than the effective concentration of ­NO2, the
HONO concentration in the environment is also lower
than the environmental concentration of N
­ O2 [10, 20].
The LOAEL ratios of HONO/NO2 are greater than theconcentration ratios of HONO/NO2 in outdoor [20], but

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comparable to the concentration ratio of HONO/NO2
in indoor [10]. We believe that both the exposure period
and concentration of HONO are important when comparing the effects of HONO to ­NO2. The present results
suggest that HONO is the most important nitrogen oxide
affecting asthma and COPD indoors.
HONO is readily decomposed to NO and the OH radical by sunlight at < 400 nm [32, 33]. Therefore, HONO
reach the maximum concentration during the night inan urban atmosphere. Moreover, in the indoor environment, HONO is produced directly by combustion processes such as gas appliances or kerosene heaters [34, 35].
Therefore, the effects of HONO might be considered as
an indoor pollution rather than air pollution. Thus, inthe homes of asthma and COPD patients, measures toreduce HONO indoors may help reduce symptoms.

Conclusion
The nitrogen oxide concentrations in Groups H and M in
Experiment 2 were lower than those in Groups H and M
in Experiment 1, although the experimental settings, suchas reagent concentrations, were the same in both experiments. However, the effects on respiratory function weresimilar in Experiments 1 and 2. These results suggest that
HONO has a more negative impact on respiratory function than ­NO2 and that the LOAEL of HONO was lessthan 0.66 ppm. Some animals in Group M showed respiratory waveforms indicative of increased respiratory
function earlier than in Group H. This finding indicatesthat some animals may experience short-term respiratory
effects from low HONO concentrations. These results
suggest that HONO affects asthma and COPD symptoms at a lower concentration and shorter exposure timethan ­NO2. We believe that HONO is the most significant
nitrogen oxide impacting asthma and COPD inside dueto the fact that HONO concentrations are frequently
higher indoors than outdoors.
Abbreviations
NO2: Nitrogen dioxide; COPD: Chronic obstructive pulmonary disease; HONO:
Nitrous acid; sRaw: Specific airway resistance; ppm: Parts per million; LOAEL:
Lowest observed adverse effect level; EPA: Environmental Protection Agency;
RLung: Pulmonary resistance; Cdyn: Dynamic lung compliance; SO2: Sulfur
dioxide; WHO: World Health Organization; AQG: Air quality guidelines; PTFE:
Polytetrafluoroethylene; NO: Nitric oxide; TV: Tidal volume; PEF: Peak expiratoryflow.
Acknowledgements
The authors would like to acknowledge Mr. Takahiro Matuyama and Mr.
Minoru Tsujii for their assistance in the animal cage washing. The authors
would like to thank Enago (www.​enago.​jp) for the English language review.
Author contributions
MO: conceptualization, funding acquisition, investigation, validation, writing—original draft. KA: formal analysis. CM: visualization. NT: methodology of HONO

Ohyama et al. Environmental Sciences Europe(2022) 34:112

generation system. SA: methodology of animal exposure system. All authors
read and approved the final manuscript.
Funding
This study is supported by a Grant-in-Aid for Scientific Research (B) from JSPS
KAKENHI Grant Number JP26281032.

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9.
10.
11.

Availability of data and materials
The datasets used and/or analyzed during the current study are available fromthe corresponding author on reasonable request.

12.

Declarations

13.

Ethics approval and consent to participate
The procedures were approved by the committee on animal care of the Osaka
Institute of Public Health, and all experiments were performed in accordancewith guidelines and regulations of the Osaka Institute of Public Health.
Consent for publication
Not applicable.

14.

15.

Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Environmental Health, Osaka Institute of Public Health,
1‑3‑69, Nakamichi, Higashinari‑Ku, Osaka 537‑0025, Japan. 2 Departmentof Environmental Medicine and Behavioural Science, Kindai University Facultyof Medicine, Osakasayama, Japan. 3 Department of Natural Sciences, Collegeof Liberal Arts, International Christian University, Mitaka, Japan. 4 Departmentof Environmental Materials Science, Graduate School of Sustainable System
Sciences, Osaka Metropolitan University, Sakai, Japan. 5 Department of Public
Health, Sagami Women’s University, Sagamihara, Japan.

16.
17.

18.

Received: 21 July 2022 Accepted: 12 November 2022
19.
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