Cytotoxic Effects of Water-Soluble Extracts of Coarse and Fine Atmospheric Particulate Matter on Mast Cell Lines

Abstract

Fine particulate matter (PM_2.5) pollution causes serious health disorders, because PM_2.5 becomes deposited in the tracheobronchial and alveoli regions. In the extrathoracic region, there are more deposits of coarse particulate matter than fine particulates. As adverse health issues caused by coarse particulates have not been well investigated, this study examined the cytotoxicity of water-soluble extracts of both fine (0.05–3 µm, PM_0.05–3) and coarse (> 3 µm, PM_>3) particulates collected from April 2016 to March 2019 in Fukuoka, Japan. Also evaluated were concentrations of NH₄⁺ and SO₄²⁻, multi-components of well-known secondary generation substances. The findings revealed that PM_>3 showed stronger cytotoxic effects on mast cell lines than PM_0.05–3. Cytotoxic effects were observed at concentrations of over 15 mM of (NH₄)₂SO₄ and over 30 mM of NH₄Cl. In contrast, Na₂SO₄ caused few cytotoxic effects up to a concentration of 50 mM. The causative substances for this cytotoxicity may not have been NH₄⁺ and SO₄²⁻ because their PM_>3 concentrations indicating the largest cytotoxic effects were 1 and 0.4 mM, respectively. The cytotoxicities of PM_>3 and PM_0.05–3 were the highest in the first half of FY2016. These cytotoxicities seem to be due to cross-border pollution, although this pollution has been declining in recent years. An increasing trend of cytotoxicity was observed in the second half of FY2018. This study showed that cytotoxicity and particulate concentrations are not always correlated. Thus, we should focus not only on the quantity of atmospheric particulate matter, but also on its quality.

INTRODUCTION

Fine particulate matter (PM_2.5) refers to fine particulate matter of less than 2.5 µm in diameter in the ambient air. PM_2.5 pollution is the source of serious social issues, causing health disorders, such as cardiovascular and respiratory diseases. High levels of PM_2.5 have been associated with the risk of cardiopulmonary diseases and lung cancer mortality by epidemiologic studies.^1–3) Exposure to PM_2.5 has also been associated with increases in the risk for mental illness⁴⁾ and postpartum depression symptoms.⁵⁾ Atmospheric particulates are classified into two types: primary particulates directly emitted from anthropogenic and natural sources and secondary particulates (e.g. (NH₄)₂SO₄ and NH₄NO₃) formed by conversion from air pollutant gases (e.g. SO₂, NOx, and NH₃). Measurements of PM_2.5 components in several countries have shown that the concentrations of total water-soluble components such as sulfate, nitrate, and ammonium ion are the highest and that their proportions display slight seasonal changes.^6–12)

Water-soluble components of the fine particulates in the atmosphere have been reported to cause adverse biological effects such as mast cell degranulation,¹³⁾ cytotoxicity and cell proliferation,^14,15) biotoxicity,¹⁶⁾ serum metabolite profile,¹⁷⁾ and genotoxicty.¹⁸⁾ However, the relationship between water-soluble particulates and their biological effects is not yet fully understood. We previously studied the effects of multi-component ammonium sulfate in PM_2.5 on the degranulation of a mast cell line (C57 cells).¹⁹⁾ Ammonium sulfate (initial concentration: 1 mM) significantly caused degranulation of C57 cells and significantly enhanced the degranulation induced by a stimulant (thapsigargin). Furthermore, we found that the ammonium ion in ammonium sulfate contributed to the degranulation of C57 cells.¹⁹⁾ The extrathoracic region, such as the pharynx and the posterior nasal passage, does not show much deposition of particulates less than around 3 µm, but does of coarse particulates (>3 µm).²⁰⁾

Another social issue arising in recent years is that of microplastic pollution of oceans and rivers caused by plastic waste, automobile tire wear, and chemical microfibers. Microplastic fragments of fibers or films are transported over great distances by the wind and deposited in remote areas. About half the microplastic fragments are less than 25 µm, with the remainder being 25–150 µm, and in rare cases exceeding 150 µm. Many of the fibrous materials are reported to be 100–300 µm long and up to 750 µm long.²¹⁾ Issues of the atmospheric transport of microplastics have been reported.^22,23) We consider this to be not only a water pollution problem but also an air pollution problem. Various substances that can be health hazards may become adsorbed on coarse atmospheric particulate matter. Fine and coarse atmospheric particulate matter, i.e., atmospheric particulate matter between 0.05 to 3 µm in diameter (abbreviated as PM_0.05–3) and atmospheric particulate matter of more than 3 µm in diameter (abbreviated as PM_>3), were collected approximately twice a month over three years in Fukuoka, Japan from April 2016 to March 2019. The average concentration of PM_2.5 in Fukuoka was about 3 µg/m³ higher than the average value in Japan.²⁴⁾ The purpose of this study was to examine the cytotoxic effects on C57 cells of water-soluble components of both coarse particles (PM_>3) and fine particles (PM_0.05–3). To identify the factors responsible for cytotoxic effects of particulate matter samples, we focused on ammonium and sulfate ions, multi-components of well-known secondary generation substances. We assayed the concentrations of ammonium and sulfate ions in particulate matter samples and the cytotoxic effects on C57 cells of ammonium and sulfate ions at several concentrations.

MATERIALS AND METHODS

Sample Preparation

Particulate matter samples were collected approximately twice a month on the campus of Daiichi University of Pharmacy (Fukuoka, Japan) from April 2016 to March 2019, avoiding rainy days. These samplings were carried out in the atrium of the second floor which is 5 m above the ground. Two filter holders with membrane filters with pore sizes of 3 and 0.05 µm (SSWP04700 and WMWP04700, Merck Millipore Ltd., Ireland) were connected in series and attached to a diaphragm pump. The atmospheric particulates were collected for 34.56 m³ air per 24 h. Water-soluble samples of PM_>3 and PM_0.05–3 were prepared as follows. The atmospheric particulate matter trapped on the 3 µm filter (PM_>3) and on the 0.05 µm filter (PM_0.05–3) were placed in polypropylene tubes and then ultrapure water (≥ 18.0 MΩ·cm, 2 mL) was added. These polypropylene tubes were vortexed for 1 min and sonicated at 10 min. then centrifuged. The supernatants were collected and filtered to remove traces of membrane filter debris with 0.8 µm membrane filters (DISMIC^®-25CS080AN, Toyo Roshi Kaisha Ltd., Japan).

Reagents

RPMI 1640 (RPMI + GlutaMAX™, [+] 25 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES)) medium, and Antibiotic-Antimycotic™ (100 ×; mixture of 10000 IU/mL penicillin, 10000 µg/mL streptomycin, 25 µg/mL amphotericin B, and 0.85% sodium chloride solution) were purchased from GIBCO^®-Life Technologies (Grand Island, NY, U.S.A.). Fetal bovine serum (FBS) was purchased from Biowest (Nuaille, France), and heat-inactivated at 56 °C for 30 min prior to use. Triton X-100 and albumin (bovine, F-V, pH 5.2), ammonium sulfate (biotechnology grade), and sodium sulfate (for pesticide residue analysis) were obtained from Nacalai Tesque Inc. (Kyoto, Japan). Ammonium chloride (99.995%) was purchased from Merck KGaA (Darmstadt, Germany).

Cell Culture

Mouse mast cell line C1.MC/C57.1 (C57 cell)²⁵⁾ was cultured in RPMI 1640 containing 10% FBS and Antibiotic-Antimycotic™ as mentioned above. Cells were maintained at 37 °C in a 5% CO₂ incubator. Confluent cultures were passed every 2–3 d.

Lactate Dehydrogenase (LDH) Cytotoxicity Assay

LDH release by C57 cells was assayed using a commercially available LDH Cytotoxicity Detection kit (TaKaRa Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. Briefly, C57 cells were seeded at a density of 1 × 10⁵ cells/mL (100 µL) in a 96-well round bottom plate adding samples (100 µL). After 4 h incubation at 37 °C in 5% CO₂, the plate was centrifuged at 250 × g for 10 min, and 100 µL of supernatant from each well was collected and transferred to another 96-well flat bottom plate, to which 100 µL of the LDH assay reagent was added. Absorbance was then measured at 490 nm and evaluated by subtraction of the absorbance at 620 nm as a reference. The percentage of LDH release was calculated by using the following formula: percentage of release = 100 × (sample LDH release − control LDH release − spontaneous LDH release)/(maximal LDH release − spontaneous LDH release). To determine the maximal LDH release, C57 cells were treated with 2% Triton X-100. The ultrapure water present in the wells was confirmed to not affect this cytotoxicity assay protocol.

Ammonium Ion Concentration

The concentration of ammonium ion was analyzed using ammonia high range reagent kit (HANNA^® Instruments, Chiba, Japan) according to the manufacturer’s instructions with modifications for microscale analyses. Briefly, the sample (30 µL) was added to a 96-well flat bottom plate. Next, a mixture of reagents HI93733A-0 (10 µL) and HI93733B-0 (90 µL) was added to each sample solution. Absorbance was then measured at 415 nm. The concentration of ammonium ion was determined using the calibration curve of ammonium chloride. The minimum limit of determination was 1.0 mg/L. The data were determined from the means of two independent experiments.

Sulfate Ion Concentration

The concentration of sulfate ion was analyzed using sulfate reagent (HANNA^® Instruments) according to the manufacturer’s instructions with modifications for microscale analyses. Briefly, a sample (80 µL) was added to a 96-well flat bottom plate. Next, a reagent solution (20 µL) in which one pack of sulfate reagent (HI93751-0) had been dissolved in ultrapure water (2 mL), was added to each sample solution. Absorbance was then measured at 415 nm. The concentration of sulfate ion was determined using the calibration curve of sodium sulfate. The minimum limit of determination was 4.0 mg/L. The data were determined from the means of two independent experiments.

RESULTS AND DISCUSSION

We assayed the cytotoxicity of water-soluble samples of PM_>3 and PM_0.05–3 collected from April 2016 to March 2019 in Fukuoka (Fig. 1). The concentration of 40 mM ammonium sulfate was used as a positive control. The values of the negative controls were obtained by adding ultrapure water to an unused membrane filter and treating it in the same way as the sampling filter (No cytotoxicity effects were observed for the 3 µm filter and 0.05 µm filter extracts). The 2016 samples showed a relatively high trend of cytotoxicity from PM_>3 (white squares) and PM_0.05–3 (gray squares). In addition, the cytotoxicity of PM_>3 was generally higher than that of PM_0.05–3. In particular, clear cytotoxicity (approximately 20%) was observed for the PM_>3 sample collected on May 27, 2016 (C-160527) indicated by arrows. In the case of PM_0.05–3 particulates, the highest cytotoxicity was observed for the sample collected on April 14, 2016 (F-160414) and the second highest for the sample collected on May 27, 2016 (F-160527). For FY2016, the cytotoxicities of PM_>3 and PM_0.05–3 samples were high from spring to autumn. The days when yellow sand blown in from the continent was observed in Fukuoka are indicated with the $ mark (April 14, 2016 and May 8, 2017) based on Japan Meteorological Agency data.²⁶⁾ The April 14, 2016 (F-160414) sample showed the highest cytotoxicity among all the PM_0.05–3 particulate samples, but the samples collected on May 8, 2017 (PM_>3 and PM_0.05–3, C-170508 and F-170508, respectively) did not display cytotoxicity. Thus, the yellow sand may have some influence on cytotoxicity, but not always.

Fig. 1. Cytotoxic Effects of Coarse and Fine Particulates

In all LDH cytotoxicity assays, the concentration of 40 mM ammonium sulfate was used as the positive control and the cytotoxic effects were shown as the % of this control. The data were determined from the means of two independent experiments. $: Day when yellow sand was observed in Fukuoka (according to Japan Meteorological Agency data, <http://www.data.jma.go.jp/gmd/env/kosahp/59chiten/807.html>).

Ammonium (diamonds) and sulfate (circles) ion concentrations in the PM_>3 and PM_0.05–3 samples are shown in Fig. 2. The values of the negative controls were obtained by adding ultrapure water to an unused membrane filter and treating it in the same way as the sampling filter (the concentrations of ammonium ion in a 3 µm filter extract, of ammonium ion in a 0.05 µm filter extract, of sulfate ion in a 3 µm filter extract, and of sulfate ion in a 0.05 µm filter extract were <1.0, 1.3, <4.0, and 4.0 mg/L, respectively.) These ion concentrations were higher in PM_>3 than in PM_0.05–3 samples. In particular, higher concentrations were noted for ammonium and sulfate ions in C-160527 and in the PM_>3 samples collected on June 1, 2017 (C-170601), July 31, 2017 (C-170731), August 8, 2017 (C-170808), March 29, 2018 (C-180329), and May 1, 2018 (C-180501). Furthermore, the concentration of these ions tended to be high from spring to summer.

Fig. 2. Concentrations of Ammonium and Sulfate Ions (mg/L) of Coarse and Fine Particulates

The data were determined from the means of two independent experiments. Values below the minimum limit of determination of ammonium ion (<1.0 mg/L) and sulfate ion (<4.0 mg/L) are shown as zero in this figure. $: Day when yellow sand was observed in Fukuoka (according to Japan Meteorological Agency data, <http://www.data.jma.go.jp/gmd/env/kosahp/59chiten/807.html>).

Next, we examined the correlation between the cytotoxic effects of PM_>3 and PM_0.05–3 particulate samples and the concentration of suspended particulate matter (SPM), PM_2.5, ammonium ion and sulfate ion (Fig. 3). The averages of SPM and PM_2.5 corresponding to the collection time of the atmospheric particulates were calculated from the data of the Ohashi Motor Vehicle Exhaust Monitoring Station on the website of Fukuoka city.²⁷⁾ C-160527 having the highest cytotoxicity had the highest concentration of ammonium and sulfate ions, and the SPM concentration for the collection times was also higher. Furthermore, the concentration of PM_2.5 was the highest at the collection times of F-160527 which had the second highest cytotoxicity of all the PM_0.05–3 particulate samples. The average of SPM and PM_2.5 for the particulate samples (C-170508 and F-170508) were the highest in each group, and yellow sand was also observed on the collection day. However, these samples did not exhibit a cytotoxic effect. Altogether these data showed no correlation between the cytotoxic effects of PM_>3 and PM_0.05–3 and the concentrations of SPM, PM_2.5, ammonium ion and sulfate ion and also the observation day of yellow sand.

Fig. 3. No Correlation between Cytotoxic Effects of Coarse (A) and Fine (B) Particulates and Concentration of SPM (A-(a)), PM_2.5 (B-(a)), Ammonium Ion (b) and Sulfate Ion (c), Yellow Sand ($ Mark)

The concentrations of particulates (triangles), ammonium ion (diamonds), and sulfate ion (circles) are plotted against cytotoxicity. (A) and (B) indicate coarse and fine particulates, respectively. No correlation was observed between the cytotoxicity and the concentrations of these particles or ions. $ mark in parentheses represents day when yellow sand was observed in Fukuoka (according to Japan Meteorological Agency data, <http://www.data.jma.go.jp/gmd/env/kosahp/59chiten/807.html>).

The cytotoxic effects of various concentrations of ammonium sulfate, the major water-soluble component of secondary particulates on C57 cells, were examined. As shown in Fig. 4 (circles), the cytotoxic effects increased in a concentration-dependent manner for ammonium sulfate concentrations over 15 mM (ammonium ion, 30 mM; sulfate ion, 15 mM).

Fig. 4. Cytotoxic Effects of Ammonium Sulfate, Ammonium Chloride and Sodium Sulfate

Data are represented as the mean ± standard error (S.E.). (n = 8). ** p < 0.01 versus ammonium sulfate.

We next investigated whether ammonium or sulfate ion affected this cytotoxicity (Fig. 4). We found that the cytotoxic effect of ammonium chloride (squares) increased in a concentration-dependent manner over the concentration range of 30 to 80 mM (ammonium ion, 30–80 mM; sulfate ion, not included), similarly to ammonium sulfate. On the other hand, in the case of sodium sulfate (triangles), a slight increasing tendency of cytotoxicity was observed at 30 mM (ammonium ion, not included; sulfate ion, 30 mM) or more, but almost no cytotoxicity was exhibited. Sodium sulfate concentrations over 20 mM (ammonium ion, not included; sulfate ion, 20 mM) showed significant differences in the cytotoxicity compared with each corresponding concentration of ammonium sulfate (ammonium ion, over 40 mM; sulfate ion, over 20 mM, ** p < 0.01). Moreover, a significant difference (** p < 0.01) was observed between 50 mM ammonium sulfate (ammonium ion, 100 mM; sulfate ion, 50 mM) and 100 mM ammonium chloride (ammonium ion, 100 mM; sulfate ion, not included). Taken together, these results suggested that the cytotoxicity of ammonium sulfate is involved in the effect of ammonium ion, but not of sulfate ion. This result is consistent with our previous findings¹⁹⁾ that the ion affecting degranulation of C57 cells was ammonium ion. Interestingly, the cytotoxic effect of ammonium sulfate was concentration-dependent, whereas that of ammonium chloride reached a plateau at around 80 mM. Thus, although the ammonium ion concentrations (100 mM) were the same for 50 mM ammonium sulfate and 100 mM ammonium chloride, a clear difference in their cytotoxicity was observed. We suspected the effect of pH as one of the causes of differences in cytotoxicity due to differences in ammonium salts. The theoretical pH of 50 mM ammonium sulfate and 100 mM ammonium chloride are the same (pH 5.12). Also, the theoretical pH of 40 mM ammonium sulfate and 80 mM ammonium chloride are the same (pH 5.17). These were estimated from the dissociation constant (ammonium ion, K_a = 5.6 × 10⁻¹⁰ M).²⁸⁾ Therefore, these results did not support the idea of the difference in cytotoxicity between 50 mM ammonium sulfate and 100 mM ammonium chloride being affected by pH. The mechanisms of these cytotoxicities by ammonium ion remain to be elucidated, but the present findings indicated that the ammonium ion has a role in the cytotoxicity on C57 cells.

We next considered whether ammonium ion and sulfate ion in the C-160527 sample contributed to the cytotoxicity to C57 cells. The results in Figs. 1, 2 and 4 suggested that the ammonium ion concentration (approximately 18 mg/L = approx. 1 mM) and the sulfate ion concentration (approximately 38 mg/L = approx. 0.4 mM) in the C-160527 sample were below the concentrations leading to cytotoxic effects. Therefore, the water-soluble extract of C-160527 sample may contain cytotoxicity-inducing components other than ammonium and sulfate ion.

The cytotoxicities of particulate matter samples were summarized every six months (Fig. 5). The cytotoxicities of PM_>3 and PM_0.05–3 in the first half of FY2016 were significantly higher than all and several other periods, respectively. Water-soluble components of atmospheric particulates affecting cytotoxicity showed a tendency to decline with the passage of time while a slight increasing trend was observed in the second half of FY2018. The PM_2.5 concentrations in Fukuoka have been reported to be influenced by cross-border pollution from China.²⁹⁾ As the concentration of PM_2.5 in China decreased, the concentration in Fukuoka was found to decrease from 2013 to 2016²⁹⁾ and from 2014 to 2019.³⁰⁾ Therefore, one of the causes of the high cytotoxicity levels in FY2016 may have been due to the effect of cross-border pollution from China.

Fig. 5. Semiannual Variations in Cytotoxic Effects of Coarse (A) and Fine (B) Particulates

The graphs were expressed as box plots (percentile: 90, 75, 50, 25, 10) semiannually. The data were reconstituted from Fig. 1. ** p < 0.01 and * p < 0.05 indicate the statistical significance compared with all other periods.

At a clean remote island in Nagasaki, the aerosol composition ratios in 2019 changed from SO₄²⁻ to NO₃⁻ in PM_2.5 and concentration of NO₃⁻ in 2019 increased by almost four-fold compared to the 2012–2014 period.³⁰⁾ Although the concentration of PM_2.5 in China has been decreasing in recent years, we have observed an increasing trend of cytotoxicity in the second half of FY2018. This increase of the cytotoxicity may be related to changes in the aerosol composition.

Pavagadhi et al.³¹⁾ reported that a decrease in cell viability or an increase in cell death could be related to the cytotoxic action exerted by water-soluble metals and polycyclic aromatic hydrocarbons (PAHs) contained in PM_2.5 samples collected from high-level periods of haze aerosols. Furthermore, the cytotoxicity of PM_2.5 was closely associated with heavy metals and organic pollutants but less related to water-soluble ions.¹⁴⁾ Several metals are carried by microplastics,³²⁾ and microplastic fragments are transported over great distances by the wind and deposited in remote areas.^21–23) Also, the low-molecular-weight organic matter generated by oxidation of volatile organic compounds (VOCs) are known as water-soluble secondary atmospheric particulate matter.^33,34) Although the causative substance inducing the cytotoxicity on C57 cells in the C-160527 sample remains unknown, candidate substances were presumed to be water-soluble metals or low-molecular-weight organic compounds.

In sum, our results showed that (1) the cytotoxicities of 2016 samples of PM_>3 from April to September were significantly higher than for other periods, (2) the C-160527 sample having the highest cytotoxicity had the highest concentration of ammonium and sulfate ions, and the SPM concentration for the collection times was also higher, (3) the C-160527 sample may contain cytotoxicity-inducing components other than ammonium and sulfate ions, and (4) high SPM or PM_2.5 levels and the arrival of yellow sand from the continent were not always linked to high cytotoxicity.

Taken together, although further studies are required to elucidate the relationship between atmospheric particulate matter and their cytotoxicities, our findings indicate that several types of coarse atmospheric particulate matter had cytotoxic effects which were higher than those of fine atmospheric particulates. This study showed that cytotoxicity and atmospheric particulate matter (PM_2.5 and SPM) concentrations are not always correlated. It also indicated that we should focus not only on the quantity of atmospheric particulate matter, but also on its quality, such has its cytotoxicity.

Statistical Analysis

The data for cytotoxicities of ammonium sulfate, ammonium chloride and sodium sulfate were analyzed for statistical significance using the Tukey–Kramer multiple comparisons test. Semiannual variation data in the cytotoxic effects of coarse and fine particulates were analyzed for statistical significance using the Steel–Dwass test. BellCurve for Excel (Social Survey Research Information Co., Ltd., Ver3.20) software was used. p Values less than 0.05 were considered significant. In the figures, ** and * indicate statistical significance at p values of 0.01 and 0.05, respectively.

Conflict of Interest

The authors declare no conflict of interest.

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