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Evaluation of the genotoxicity of PM2.5 collected by a high-volume air sampler with impactor

Evaluation of the genotoxicity of PM2.5 collected by a high-volume air sampler with impactor Background: The harmful effects of fine particles with an aerodynamic diameter less than 2.5 μm (PM2.5) on respiratory organs are emphasized in pollution studies because PM2.5 have high deposition rates in the respiratory organs and contain various hazardous compounds. In this study, a sampling method combining a high-volume air sampler (HV) with a PM2.5 impactor was developed for collecting large quantities of PM2.5. The concentrations of elemental carbon (EC), organic carbon (OC), inorganic ions, and polycyclic aromatic hydrocarbons (PAHs) were measured in PM2.5 collected by the high-and low-volume air samplers (LV). Results: Similar results were obtained from the HV and LV methods, with respect to inorganic carbon, organic carbon, sodium ions, ammonium ions, and PAHs with more than four rings. Because of the much larger amount of PM2.5 could be collected by the HV method, the trace constituents, that were difficult to detect by the conventional LV method, were readily detected by the HV method. Furthermore, when the microsuspension method that was modified more sensitive Ames mutagenicity test, was used to test the PM2.5 samples at four sites, mutagenic activities were detected by strains TA100 and TA98. Most of the mutagenic activity was associated with the PM2.5 fraction and mutagenic activity in winter was greater than that in summer. Conclusions: The HV method produced results similar to those from the conventional LV method with respect to the PM2.5 components present in the atmosphere in relatively high concentrations, but its 40-fold greater flow rate enabled the detection of mutagenic compounds present in only trace concentrations. Keywords: PM2.5, High-volume air sampler, Impactor, Mutagenic activity, Microsuspension, TA98, TA100, Dibenzo [a,l]pyrene Background al. 2016). PM2.5 are widely studied because of their harm- Recent reports in Japan have indicated decreases in the ful influence on human health, such as cardiovascular dis- emission of suspended particulate matter (SPM) [40], ease and respiratory disorders, as a result of the high polycyclic aromatic hydrocarbons (PAHs) [7] and dioxins deposition rate in the respiratory organs [28]. Various epi- into the atmosphere, by emission control on car engines demiological and toxicological studies have shown strong and improved management of waste incineration. On the positive correlations between PM2.5 concentrations in the other hand, it has been reported that the Japanese atmos- atmosphere and the death rate by respiratory system dis- phere is influenced by sulfur oxides from coal combustion ease [8], and a direct influence on collective human mor- from China [30], so there is considerable interest in Japan tality [4, 26, 27]. Furthermore, PAHs, such as benzo [a] regarding air pollution by PM2.5 from China ([39], Ling et pyrene (BaP, [1, 3, 22, 29]), nitro-PAHs, and dioxins [28], which are related compounds with carcinogenic activity, have been detected in PM2.5. PAHs derived from atmos- * Correspondence: sugita@azabu-u.ac.jp pheric suspended particulates were shown to be retained Azabu University, 1-17-71, Fuchinobe, Chuou-ku, Sagamihara, Kanagawa in the lungs of a dog [32]. The World Health Organization 252-5201, Japan Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sugita et al. Genes and Environment (2019) 41:7 Page 2 of 11 (WHO) has set guidelines for PM2.5 in the atmosphere at phosphate potassium, sodium hydroxide, D-glucose (The 10 μg/m for the annual average concentration and at reagent best quality, FUJIFILM Wako Pure Chemical 25 μg/m for the daily average. The continuous monitor- Corporation, Oosaka, Japan) and agar (Nacalai Tesque Inc., ing of PM2.5 in Japan is essential, and an environmental Kyoto, Japan) were used for a mutation assay. standard of PM2.5 in the atmosphere was set in 2009 at S. typhimurium strains TA98 and TA100 were distrib- 3 3 15 μg/m for the annual average and at 35 μg/m for the uted from National Institute of Public Health. daily average, and continuous monitoring is carried out in all parts of Japan. PM2.5 is collected by LV as part of the PM2.5 sampling monitoring, but chemical analysis of the samples collected The PM2.5 samples were collected at four locations in is limited because only small samples can be collected by Japan, namely Maebashi (East longitude 36.4045°, North this method, which may be below the detection limit of latitude 139.0961°), Tsukuba (East longitude 36.0498°, some pollutants. Therefore, equipment collecting larger North latitude 140.1175°), Nagoya (East longitude 35.0990°, amounts of PM2.5 is needed. North latitude 136.9156°) and Fukuoka (East longitude It is currently impossible to measure all the potentially 33.5948°, North latitude 130.3646°). At Nagoya, parallel carcinogenic substances contained in PM2.5 samples be- sampling by the LV and HV methods was carried out to cause of the large number of compounds present at very measure the concentrations of EC, C and some ion compo- low concentrations. Therefore, to achieve a comprehen- nents in the spring and summer 2012 and winter 2013 sive evaluation of the harmful effects of substances associ- (21 samples) and to measure the concentrations of PAHs in ated with PM2.5, a mutagenic assay, such as the Ames the summer 2013 and winter 2014 (15 samples). At the test, is needed. On the other hand, the conventional Ames other sampling points, sampling by the HV method was mutagenicity test uses Salmonella typhimurium strains carried out to measure the mutagenic activities in the sum- TA98 (in which frameshift mutations are detected) and mer 2012 and winter 2013 (64 samples including 14 sam- TA100 (in which base substitution mutations are detected) ples in Nagoya). which cannot detect mutagenic activity because the sensi- Samples of PM2.5 were collected on 47 mmφ quartz-fiber tivity is not high enough, and the amount of PM2.5 col- filters (PALLFLEX 2500QAT-UP, PALL corporation, New lected is limited. Therefore, modified Ames tests have been York, USA) for a 24 h period by the LV method and on developed in which the mutagenicity of PM2.5 samples has 20.3 × 25.4 cm quartz-fiber filters (PALLFLEX 2500QAT-UP, been increased using TA98 and a TA98-derivative strain, PALL corporation, New York, USA) by the HV method. A YG1024, with elevated O-acetyltransferase levels. The low-volume air sampler, Partisol 2000-FRM Air Sampler YG1024 strain shows hyper-responsiveness to nitro-PAHs (Thermo Fisher Scientific, Massachusetts, USA) was used and amino-PAHs [6, 36], but there is the risk of introdu- with a flow rate of 16.7 L/min as LV method. In 2000-FRM, cing a bias toward these compounds when this modified PM2.5 were collected by back-up filter, and PM > 2.5 were Ames test is used. eliminated by impactor that consist of filter and pump oil In the current study, parallel sampling of PM2.5 was [25]. This PM 2.5 collection method is certified by the carried out using the HV and LV methods, with concen- Federal Reference Law of the United States and is consistent trations of EC, OC, some ion components, and PAHs be- with the manual of the Japanese Ministry of the Environ- ing compared in PM2.5 collected by the two sampling ment. A high-volume air sampler, HV-700R (SIBATA methods, to evaluate the validity of collecting PM2.5 using Scientific Technology Ltd., Saitama, Japan) was used with a the new HV method. Subsequently, PM2.5 and the par- flow rate of 700 L/min as HV method. In the HV method, ticle bigger than PM2.5 (PM > 2.5) were collected using an impactor for PM2.5 (Custom-made based on a HV-100 the HV method, and the carcinogenic potential was evalu- 2.5 impactor, SIBATA Scientific Technology Ltd., Saitama, ated using the TA98 and TA100 strains, these being the Japan) was mounted on HV and PM2.5 was collected on strains used for the conventional Ames test, to achieve a pre-treated back-up quartz-fiber filters, and PM > 2.5 was comprehensive evaluation of the carcinogenesis-related collected on slit quartz-fiber filters (2500QAT-UP, PALL substances in PM2.5 and PM > 2.5 collected by the HV corporation, New York, USA) referring Fig. 1. Each method. quartz-fiber filter was heat-treated at 450 °C for 2 h in a muffle furnace to remove organic pollutants and then Methods equilibrated in a desiccator at a constant 50% humidity Materials and weighed. Acetone (HPLC grade; FUJIFILM Wako Pure Chemical Corporation, Oosaka, Japan) and dimethyl sulfoxide Analysis of carbonaceous and ion components (DMSO, Dojindo Molecular Technologies, Inc., Kumamoto, A 1.0 cm sample of the quartz-fiber filter with aerosol Japan) were used for extraction. MgSO � (7H O), citric acid particles, collected using both the LV and HV, was 4 2 hydrate, dihydrogen phosphate ammonium, dihydrogen placed in an oven. Then, OC and EC were analyzed with Sugita et al. Genes and Environment (2019) 41:7 Page 3 of 11 a laboratory OC-EC aerosol analyzer (Sunset Laboratory Inc., Oregon, USA) following the Interagency Monitor- ing of Protected Visual Environments (IMPROVE) ther- mal/optical reflectance protocol [2]. 2 2 The quartz-fiber filter of 4 cm in HV method and 5 cm in LV method was subjects to ultrasonic treatment with 10 ml of pure water to extract ion species respectively. After extraction, filter debris and suspended insoluble par- ticles were removed using a Teflon filter (DISMIC-25HP, Advantec Toyo Kaisha, Ltd., Tokyo, Japan). Cation species + + + 2+ 2+ such as Na ,NH ,K ,Mg and Ca and anion species − − 2− such as Cl ,NO and SO were measured by ion chro- 3 4 matograph (Dionex ICS-1000, Thermo Fisher Scientific, Massachusetts, USA). Analysis of polycyclic aromatic hydrocarbons One-half of the quartz-fiber filter collected by the LV method and one-fourth of the quartz-fiber filter collected by theHVmethodwereusedfor PAHmeasurements. The Fig. 1 Schematic of PM2.5 impactor for high-volume air sampler targeted PAHs (including dibenzo [a,l] pyrene (DBalP)), for (HV-1000 PM 2.5 impactor).① Base of impactor, ②; filter with slits, gas chromatography-mass spectrometry (GC/MS) analysis ③; Nozzle, ④; Washer, ⑤; Nut are shown in Table 1. A 16 PAHs mixture (representing the U.S. EPA priority PAHs) (each 20 ng) labeled with deuterium was added to the filter sample as an internal standard, followed by Soxhlet extraction with acetone. The Table 1 Information on the PAHs measured in this study PAHs Abbreviation Number of benzene Vapor pressure* (Pa at Target ion mass Retention time IARC Mutagenicity** rings 25 °C) (m/z) (min) classify Naphthalene Nap 2 10.4 128 9.6 2B – –1 Acenaphthylene Acy 3 8.9 × 10 152 12.6 – (?) –1 Acenaphthene Ace 3 2.9 × 10 153 12.8 – (?) –2 Fluorene Flu 3 9.0 × 10 166 13.9 3 – –2 Phenanthrene Phe 3 1.6 × 10 178 16.5 3 (?) –4 Anthracene Ant 3 8.0 × 10 178 16.6 3 – –3 Fluoranthene FLN 4 1.2 × 10 202 20.0 3 + –4 Pyrene Pyr 4 6.0 × 10 202 20.8 3 (?) –5 Benzo BaA 4 2.8 × 10 228 24.5 2A + [a]anthracene –5 Chrysene Chy 4 8.4 × 10 (at 20 °C) 228 24.8 3 + –5 Benzo BbF 5 6.7 × 10 252 27.8 2B + [b]fluoranthene –8 Benzo BkF 5 1.3 × 10 (at 20 °C) 252 27.9 2B + [k]fluoranthene –7 Benzo [a]pyrene BaP 5 7.3 × 10 252 29.0 2A + –8 Dibenzo dBahA 5 1.3 × 10 278 33.3 2A + [a,h]anthracene –8 Indeno[1,2,3- IndP 6 1.3 × 10 276 33.3 2B + c,d]pyrene –8 Benzo [ghi]perylene BghiP 6 1.4 × 10 276 35.0 3 + *: International programme on chemical safety environmental health criteria 202 selected non-heterocyclic policyclic aromatic hydrocarbons Table 4 [37] **: International programme on chemical safety environmental health criteria 202 selected non-heterocyclic policyclic aromatic hydrocarbons Table 2 [37] +: Positive, −: Negative, (?): Questionable Sugita et al. Genes and Environment (2019) 41:7 Page 4 of 11 extract was concentrated to a few ml using a rotary evapor- The microsuspension method, using the sensitized Ames ator set at a pressure of 39.9 kPa and a temperature of test, was used for the mutagenicity assay. It was carried out 40 °C, then a solvent was removed under the nitrogen flow with S9 mix addition and non-addition conditions gently, and a 0.5 ml of toluene was added to extract finally. using S. typhimurium strains TA98 and TA100 follow- One-fifth of the concentrated extract was measured and ing the protocols used in previous studies [15, 16, 31, re-dissolved in 30 mL hexane and once again concentrated 33]. When the dose-response relationship was ob- to ca. 3 ml with a rotary evaporator set at a pressure of tained, the case of the colony by which the number 23.9 kPa and a temperature of 40 °C. Then, a 3 ml whole of obtained return mutation colonies is beyond 2 volume of the hexane solution was added to a precondi- times of the control value was defined as positiveness tioned silica gel cartridge and eluted with 6 mL of and 1.5-two times, as weak positiveness and less than dichloromethane-hexane (8:2 (v/v)). An aliquot (100 μL) of 1.5 were difined as negative. n-nonane was added to the eluent, and the resulting solu- tion was then concentrated to 100 μL under nitrogen flow Results and discussion gently. Finally, the solution was transferred to an insert vial. Carbonaceous and inorganic chemicals in PM2.5 PAHs were analyzed by GC/MS (Model 5973 N, Agilent The EC concentration was 1.4 ± 0.7 μg/m , and the OC Technology, California, USA) in the selected ion monitor- concentration was 3.7 ± 1.8 μg/m in PM2.5 collected by ing mode (injection port temperature 300 °C, injected vol- the HV method. The atmospheric EC and OC concen- ume 1 μL, and GC temperature profile as follows: initial trations measured in this study were at a level similar to temperature: 50 °C for 5 min; the temperature was raised those in other reports of Japanese atmospheric EC and by 15 °C/min until it reached 185 °C; from 185 °C, the OC [9, 11, 12, 24]. The ion concentrations in PM2.5 + + + 2+ temperature was raised by 8 °C/min until it reached 320 °C, collected by the HV method for Na ,NH ,K ,Mg , 2+ − − 2− at which it was maintained for 22 min. Ionization was in Ca ,Cl ,NO and SO were 0.14 ± 0.10, 2.47 ± 1.88, 3 4 the EI mode. PAHs were determined by the internal stand- 0.10 ± 0.07, 0.02 ± 0.02, 0.07 ± 0.16, 0.20 ± 0.58, 1.37 ± ard method using PAHs deutriumed. 2.04, and 4.87 ± 4.22 μg/m , respectively. The atmos- pheric ion concentrations measured in this study were Mutagenicity test by the microsuspension method similar to those in other reports on Japanese atmos- One-fourth of both quartz-fiber filters for PM2.5 and PM > pheric ion species [20, 30]. The results from the HV and 2.5 of HV method were used for bioassay by the microsus- LV methods were compared in Figs. 2 and 3. Figure 2 pension method. The organic fractions were extracted by showed that there was a close agreement between con- ultrasonication (Ultra Sonic Cleaner AU-501CO; AIWA centrations of carbonaceous components in PM2.5 from Medical Industrial Corporation, Tokyo, Japan) with acet- the HV method and LV method. Furthermore, the slopes one twice. The two extracts were combined and then con- of the regression lines were in the range of 1.06 to 1.07, centrated to a few mL using a rotary evaporator. The very close to 1. In Fig. 3, a close agreement was shown + + + 2− solvent was removed under a nitrogen flow gently, and the for Na ,NH ,K , and SO between the two methods, 4 4 2+ 2+ − − dried samples were stored at − 80 °C until assayed. but the concentrations of Mg ,Ca ,Cl , and NO After defrosting at room temperature [23], an extracted were not similar between the two methods. The concen- 2+ 2+ − − sample was dissolved in DMSO and tested for mutagenicity. trations of Mg ,Ca ,Cl ,and NO by the HV (a) (b) (c) Fig. 2 Comparison of (a) elemental carbon (EC), (b) organic carbon (OC), and (c) total carbon (TC) concentrations measured with HV and LV methods. The solid line presents the regression line by at least two multiplication. The broken line presents the line equivalent in HV method and LV method Sugita et al. Genes and Environment (2019) 41:7 Page 5 of 11 (a) (b) (c) (d) (e) (f) (g) (h) + + + 2+ 2+ − − 2− Fig. 3 Comparison of (a)Na ,(b)NH ,(c)K ,(d)Mg ,(e)Ca ,(f)Cl ,(g)NO , and (h)SO concentrations measured with HV and LV 4 3 4 methods. The solid line presents the regression line by at least two multiplication. The broken line presents the line equivalent in HV method and LV method method were about 1.4, 2.6, 1.4, and 1.4 times higher, by the LV and HV methods. In contrast, for PAHs, the − 4 respectively, than those by the LV method. Uchiyama vapor pressure of which was greater than 10 Pa, the reported that the ion concentrations in SPM differed concentrations obtained by the HV method were less than + 2− + depending on particle size, with the NH ,SO ,and K those obtained by the LV method. The linear velocity of 4 4 concentrations in the fine particles being about 10, 4, and filter surface by HV method is 28.7 cm/sec, is 1.23 times 2 times higher than those in coarse particles, in the other higher than that of LV method. A little difference was rec- 2+ 2+ + hand, Ca ,Mg ,and Na concentrations in coarse parti- ognized between HV method and LV method. And, be- cles were about 10, 5, and 3 times higher than those in cause of the shape of the suspended particulate has a fine particles [34]. Because the impactor couldn’tbe di- distorted shape, not globular, the particles were separated vided among the particle diameter perfectly for 50% by an aerodynamic diameter, could not separate correctly cut-off value, it was suggested that a possibility with some by particle size. Therefore, it is consider that the coarse properties with HV method tends to undergo influence of particle near the PM2.5 influence PM2.5 fraction. The a particle large compared with LV method. factor for the difference is not clear, so cause investiga- tion is need urgently. But, HV method could collect − 4 Polycyclic aromatic hydrocarbons (PAHs) in PM2.5 low-vapor-pressure PAHs (less than 10 Pa) select- The PAH concentration per cubic meter of total sam- ively, This means that the HV method is suitable for − 4 pling flow, collected as particulate components using the low-vapor-pressure PAHs (less than 10 Pa), which are LV and HV methods, was measured and compared. mutagenic or carcinogenic [13]. These results are shown in Table 2. The atmospheric PAH concentrations measured in this study are at a level Highly carcinogenic PAHs in PM2.5 similar to those in other reports on Japanese atmos- In this study, DBalP, which is highly carcinogenic and has pheric PAHs [22, 38]. The comparison of the PAH con- six benzene rings, was also measured by GC/MS. DBalP is centrations of PM2.5 between the HV and LV methods not included in the U.S. Environmental Protection Agency was shown in Table 2 in which the results were classified (U.S EPA) priority 16 PAHs; however, it is notable that the by the vapor pressure of PAHs. The results demonstrated relative potency factor (RPF) [35] of DBalP is 30 [19]. significant correlation with significant level < 0.01 (R = DBalP could be detected in the PM2.5 samples by the HV 0.795 0.985) for low-vapor-pressure PAHs (less than 10 method, but it was not detected in PM2.5 by the LV Pa) between the concentrations associated with PM2.5 method. The atmospheric concentration of DBalP ranged Sugita et al. Genes and Environment (2019) 41:7 Page 6 of 11 Table 2 The atmospheric PAH concentration levels as determined by the LV and HV methods Abbreviation of Vapor pressure (Pa at Atmosphereic concentration by LV method ave. Atmosphereic concentration by HV method R 3 3 PAH 25 °C) ± SD; ng/m ) (ave. ± SD; ng/m ) value Nap 10.4 1.76 ± 0.73 0.70 ± 0.42 −0.369 –1 Acy 8.9 × 10 40.1 ± 20.9 0.47 ± 0.46 −0.058 –1 Ace 2.9 × 10 0.24 ± 0.07 0.04 ± 0.03 −0.144 –2 Flu 9.0 × 10 0.25 ± 0.17 0.05 ± 0.02 0.344 –2 Phe 1.6 × 10 3.79 ± 0.81 0.45 ± 0.21 0.649 –4 Ant 8.0 × 10 0.11 ± 0.08 0.03 ± 0.02 0.077 –3 FLN 1.2 × 10 3.32 ± 0.85 0.49 ± 0.25 0.265 –4 Pyr 6.0 × 10 1.71 ± 0.96 0.29 ± 0.15 0.068 –5 BaA 2.8 × 10 0.16 ± 0.12 0.13 ± 0.12 0.795 Chy 8.4 × 10–5(at 20 °C) 0.54 ± 0.34 0.44 ± 0.35 0.906 –5 BbF 6.7 × 10 0.31 ± 0.29 0.40 ± 0.29 0.951 –8 BkF 1.3 × 10 (at 20 °C) 0.17 ± 0.12 0.16 ± 0.13 0.928 –7 BaP 7.3 × 10 0.25 ± 0.14 0.19 ± 0.15 0.954 –8 dBahA 1.3 × 10 0.03 ± 0.02 0.03 ± 0.02 0.944 –8 IndP 1.3 × 10 0.22 ± 0.19 0.25 ± 0.20 0.985 –8 BghiP 1.4 × 10 0.33 ± 0.27 0.28 ± 0.22 0.964 R means the coefficient of correlation from 0.014 to 0.078 ng/m , and this concentration was samples than did the TA98 strain [10, 21]. But it was about one-tenth that of BaP. DBalP concentrations in considered that mutagenic evaluation with YG1024 had present study were one-tenth of lower than that in China an in-built TA1537 (or TA97 and TA97a), TA98, TA100 (Wei [17]) by three days sampling, and were similar to and WP2uvrA (or WP2uvrA (pKM101) or TA100) have those in Chiba prefecture [14] by one week sampling. The been used for mutagenic assay (OECD 1997). The measurement of low concentration PAHs by HV method YG1024 strain is not selected for standard mutagenicity could be shortened sampling time to 24 h from a few days assays. So, in this study, two types of strain were used, one successfully. Then, the comparison of mutagenic potency is TA98 strain that detected mutagenicity of the frame- of DBalP and BaP calculated by their concentration and shift type, another is TA100 that detected mutagenicity of PRF showed that the atmospheric carcinogenic potency of the base substitution type. Furthermore, the measure- DBalP is three times higher than that of BaP. It is a recog- ments of the mutagenic activity in PM2.5 samples were nized that the HV method is suitable for the quantification tried here using the microsuspension method [15]that ex- of one of the most-highly carcinogenic PAHs. The major hibits greater sensitivity than does the original Ames test. advantage of the HV method is the extreme microanalysis In previous studies, the sample of mutagenic assay for of PM2.5 composition which is possible given the large environmental atmosphere using TA98 and TA100 used amount of PM2.5 available from the HV method, a to be collected by HV method for 24 h. The sample of 1/3 process which is impossible to detect with the small to 1/2 was used for mutagenic assay, that equivalent to air 3 3 amount of PM2.5 collected by the LV method. volume 400 m to 600 m [5]. And, Matsumoto et al. car- ried out sampling for mutagenic assay for atmosphere by Mutagenicity of PM2.5 LV method for 3 days and collected air volume120 m to A LV method was used for conventional PM2.5 collec- 240 m [18]. Because of these air volume were equivalent tion and analysis of airborne particles, but the sample to sampling volume for 5 days to 10 days by LV method, it size achieved by this method was very small. Because was impossible to elucidate diversification of a testing nitro-PAHs were reported as mutagen which could be condition and fluctuation during the day. present in airborne particles, the S. typhimurium strain, In this study, the mutagenic evaluation for environ- YG1024, developed to exhibit very high sensitivity to mental atmosphere using TA98 and TA100 strain was nitro-PAHs, was used to assay for mutagenicity in such attempt by combining microsuspention sensitized Ames small airborne particle samples. YG1024 strain was re- test and large volume sampling method. ported to exhibit approximately 10 times greater sensi- The air volume necessary to detection of the positive tivity to mutagenesis from food and environmental and weak positive of mutation are indicated in Table 3. Sugita et al. Genes and Environment (2019) 41:7 Page 7 of 11 Table 3 Comparison of the air volume (m ) necessary to false positive in present study (in the case of single dose) (unit: m ) TA100 - S9mix TA100 + S9mix TA98 – S9mix TA98 + S9mix Total average 7.85 4.65 1.12 4.45 18.07 sd 5.98 4.07 1.31 4.40 12.72 50% of detection parcentage 6.36 3.40 0.99 3.06 14.09 90% of detection parcentage 14.95 7.96 1.74 12.57 35.59 95% of detection parcentage 18.70 12.54 2.05 14.15 50.22 All 32.28 26.59 10.67 17.88 59.19 In the case of actual assay, the necessary air volume was mutagenicity were positive in all four conditions. But, four times of value in Table 3, because mutation assay mutagenicity were negative in the conditions of TA100 need assay with different 5 concentrations and duplicate without S9mix (TA100 S9mix-) and TA98 with S9mix measurement. Its air volume was different depending on (TA98 S9mix+), was weak positive in the condition of samples by the percentage of the mutagenic substance TA100 with S9mix (TA100 S9mix+) and was positive in 3 3 included in a sample, 14 m (actually, 56 m ) was needed the condition of TA98 without S9mix (TA98 S9mix-). by 4 conditions by the detection rate of the 50% in present An outline of the detection rate of mutagenic activity in study. This air volume was equivalent to the sample for PM2.5 is shown in Table 4. The positive rate, including 48 h by LV method. Additionally, the air volume necessary weak positives, was 98.4% by TA100 (-S9mix), 95.3% by to detection of 90% was approximately 140 m ,thisvol- TA100 (+S9mix), 100% by TA98 (-S9mix) and 92.2% by ume was equal to the suction amount of the previous TA98 (+S9mix). The average mutagenic activities per study. unit volume at each location are shown in Fig. 6.In spite Samples of PM2.5, taken at four locations from the of PM > 2.5 particles accounting for about 20% of the Kanto area to the Kyushu area, were collected by the particle weight of the sample, mutagenic activity in HV method. Then, the mutagenic activities of these PM > 2.5 was very weak (Fig. 6). It was considered that PM2.5 samples were tested using the microsuspension most of mutagenic compounds were associated with method with TA98 and TA100 strains. Typical results of the fine particles like PM2.5 in the atmosphere (Fig. 5). the mutagenicity assay are shown in Fig. 4. In Fig. 4 a, Significant differences in mutagenic activity of PM2.5 (a) (b) Fig. 4 Typical dose–response curves of mutation assay for PM2.5 in Tsukuba in (a) 30th July, 2012 and (b) 8th August, 2012.a: Results of typical positive mutagenicity. b: Results of negative, false-positive and positive mutagenicity. TA100 S9mix- and TA98 S9mix + were negative, TA100 S9mix + was false-positive and TA98 S9mix- was positive Sugita et al. Genes and Environment (2019) 41:7 Page 8 of 11 Table 4 Detection rates of mutagenic activity by the microsuspension method Tsukuba Maebashi Nagoya Fukuoka number parcentage(%) number parcentage(%) number parcentage(%) number parcentage(%) Negative 3 (4.7) 1 (1.6) 2 (3.1) 3 (4.7) week positive 13 (20.3) 18 (28.1) 13 (20.3) 11 (17.2) Positive 48 (75.0) 45 (70.3) 49 (76.6) 50 (78.1) sum 64 (100.0) 64 (100.0) 64 (100.0) 64 (100.0) between summer and winter were recognized in other hand, it was found that mutagenic activity dif- Nagoya and Fukuoka in spite of it being recognized fered significantly, in spite of the similar PM2.5 con- that there were no significant differences in particle centrations being reported from the different sites over concentrations between summer and winter. The aver- the two seasons. From the relationship between PM2.5 age (mean ± SD) concentration of PM2.5 was 24.0 ± concentration and mutagenicity, it is apparent that 3 3 2.9 μg/m in summer and 24.6 ± 9.3 μg/m in winter at more data need to be collected and the distribution an- Tsukuba, 28.7 ± 2.8 μg/m in summer and 26.8 ± alyzed to identify management concentrations. 3 3 13.8 μg/m in winter at Maebashi, 31.2 ± 5.1 μg/m in The concentration of PM2.5 in the atmosphere is regu- summer and 28.2 ± 13.9 μg/m in winter at Nagoya, lated globally because of the detrimental impact of these 3 3 and 25.8 ± 5.3 μg/m in summer and 18.2 ± 15.0 μg/m particles on human health. On the other hand, since the in winter at Fukuoka. Furthermore, the relationship be- composition of the chemical substances associated with tween atmospheric PM2.5 concentration and mutage- PM2.5 differs according to the sources, it can also be used nicity is showninFig. 6. The correlation coefficients in source analysis by the two commonly used receptor between PM2.5 concentration and mutagenic activity models, the chemical mass balance (CMB) model and the were significant (p <0.05, n = 64) for each assay, namely positive matrix factorization (PMF) model. In this study, 0.568 for TA100-S9mix, 0.525 of TA100 + S9mix, 0.445 the HV method could detect significant differences in mu- of TA98-S9mix, and 0.391 of TA98 + S9mix. On the tagenic activity between summer and winter samples, Fig. 5 Comparison of mutation activity in PM2.5 and PM > 2.5 per unit air volume at different sites and seasons. *: Results with a common letter present significant differences (p < 0.05) Sugita et al. Genes and Environment (2019) 41:7 Page 9 of 11 Fig. 6 Relationship between mutagenicity and PM2.5 concentration The solid line presents the regression line, the both broken lines present the 95% prediction interval although there was no significant difference between sea- (HV method) using high-volume air sampler with an sons in the concentration of PM2.5 at the same location. impactor and compared and evaluated it using the These results suggest that the components of particle-based Low-volume air sampler based authentication method pollutants related to carcinogenesis in PM2.5 could change (LV method). The results showed that the HV method depending on the season. Therefore, it is considered that had the same performance with LV method in terms of the human health risk posed by PM2.5 would also vary, de- measuring the inorganic components (OC, EC, Na , + + 2− pending on the composition of the substances, but these NH ,K and SO ) and low-volatile PAHs that are 4 4 effects have not yet been clarified in detail. The HV method mutagenic or carcinogenic. Because of the air volume of fine particle collection can evaluate the risk of PM2.5 collected by HV method was forty times higher than the precisely. Since the toxic effects of PM2.5 extend to cardio- LV method, the high carcinogen and mutagen like DBalP vascular and respiratory diseases, the measurement of that could be not detected by LV method were detected chemical substances related to such diseases and the deter- at 0.014 0.078 ng/m . Furthermore, HV method with mination of the end-points of these compounds are ur- microsuspension method could evaluate the mutagenic gently required. It is considered that the HV method activity of PM2.5 by TA98 and TA100 strain. The HV proposed in this study is a very useful tool for the compre- method for PM2.5 proposed in present study is very hensive evaluation of the health effects of carcinogens by useful for detecting targeted micropollutants present at the mutation assay and by precise measurement of trace very low concentrations in the air. On the other hand, for chemicals in the fine particles. low molecular weight PAH and some inorganic ionic spe- 2+ 2+ − − cies (Mg ,Ca ,Cl and NO ), the HV method tended Conclusions to show higher concentration than the LV method. Atten- A strong harmful effect is attributed to a fine particle tion should be paid according to the application. (PM2.5) in the atmosphere because PM2.5 has a high Abbreviations deposition rate in the respiratory organs. In present BaP: Benzo [a]pyrene; CMB: The chemical mass balance; DBalP: Dibenzo study, we would propose a new PM2.5 sampling method [a,l]pyrene; DMSO: Dimethyl sulfoxide; EC: Elemental carbon; GC/MS: Gas Sugita et al. Genes and Environment (2019) 41:7 Page 10 of 11 chromatography–mass spectrometry; IMPROVE: The Interagency Monitoring K.: Mutagenicity of airborne particles, river waters and soils in Japan from of Protected Visual Environments; LV: Low-volume air sampler; OC: Organic 1996 to 2003. Environ Mutagen Res, Vol.26, pp9–22 (2004). carbon; PAHs: Polycyclic aromatic hydrocarbons; PM > 2.5: Particle bigger 6. Endo, O., Sugita, K., Goto, S., Amagai, T. and Matsushita H.: Mutagenicity of than PM2.5; PM2.5: Fine particles with an aerodynamic diameter less than size-fractionated airborne particles collected with Andersen low pressure 2.5 μm; PMF: The positive matrix factorization; RPF: Related potency factor; impactor, L Health Sci Vol.49, No.1, pp22–27 (2003). SPM: Suspended particulate matter; WHO: The World Health Organization 7. Ezoe, Y., Goto, S., Tanabe, K., Endo, O., Koyano, M., Watanabe, I. and Matsushita, H.: Polycyclic aromatic hydrocarbon concentrations of airborne Acknowledgements particles in urban air over the past twenty years. Polycyclic Aromatic We thank Mrs. Yamagami of Nagoya City Institute for Environmental Comounds, Vol.24, pp635–646 (2004). Sciences for providing assistance with PM2.5 sampling. 8. Fu J, Jiang D, Lin G, Liu K, Wang Q. An ecological analysis of PM2.5 concentrations and lung cancer mortality rates in China. BMJ Open. 2017;5: e009452. Funding 9. Fujikawa,K., Shigekazu,Y.,Shiro,T.,Hisao,C., Okihiro, O.and Shinji I.: The Not applicable. behavior of carbon compounds (EC,OC) in aerosols, and its relationships with other compounds. - analysis of daily data. Annual Reports of Fukuoka Institute of Availability of data and materials Health and Environmental Sciences, Vol.35, pp93–97 (2008). All data analyzed during this study are included in this published article. 10. Gabbani G, Nardini B, Bordin A, Pavanello S, Janni L, Celotti L, Clonfero E. Urinary mutagenicity on TA98 and YG1024 Salmonella typhimurium strains Authors’ contributions after a hamburger meal: influence of GSTM1 and NAT2 genotypes. KS and DN was the two major contributor in designing this study and KS Mutagenesis. 1998;13(2):187–91. wrote this manuscript mainly. MY collected PM2.5 and performed the 11. Hashimoto, T.: Investigation of PM2.5 measurements in the environmental analysis of PAHs. FI collected PM2.5, and performed analysis of Carbonaceous atmosphere of Kagawa prefecture (II). Annual Report of Kagawa Prefectural and inorganic chemicals. YK and YT performed the Ames test. KK, TO, MK Research Institute for Environmental Sciences and Public Health, Vol.12, and KN collected PM2.5. All authors read and approved the final manuscript. pp45–55 (2013). 12. 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Evaluation of the genotoxicity of PM2.5 collected by a high-volume air sampler with impactor

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Springer Journals
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Copyright © 2019 by The Author(s)
Subject
Biomedicine; Human Genetics; Life Sciences, general
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1880-7062
DOI
10.1186/s41021-019-0120-0
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Abstract

Background: The harmful effects of fine particles with an aerodynamic diameter less than 2.5 μm (PM2.5) on respiratory organs are emphasized in pollution studies because PM2.5 have high deposition rates in the respiratory organs and contain various hazardous compounds. In this study, a sampling method combining a high-volume air sampler (HV) with a PM2.5 impactor was developed for collecting large quantities of PM2.5. The concentrations of elemental carbon (EC), organic carbon (OC), inorganic ions, and polycyclic aromatic hydrocarbons (PAHs) were measured in PM2.5 collected by the high-and low-volume air samplers (LV). Results: Similar results were obtained from the HV and LV methods, with respect to inorganic carbon, organic carbon, sodium ions, ammonium ions, and PAHs with more than four rings. Because of the much larger amount of PM2.5 could be collected by the HV method, the trace constituents, that were difficult to detect by the conventional LV method, were readily detected by the HV method. Furthermore, when the microsuspension method that was modified more sensitive Ames mutagenicity test, was used to test the PM2.5 samples at four sites, mutagenic activities were detected by strains TA100 and TA98. Most of the mutagenic activity was associated with the PM2.5 fraction and mutagenic activity in winter was greater than that in summer. Conclusions: The HV method produced results similar to those from the conventional LV method with respect to the PM2.5 components present in the atmosphere in relatively high concentrations, but its 40-fold greater flow rate enabled the detection of mutagenic compounds present in only trace concentrations. Keywords: PM2.5, High-volume air sampler, Impactor, Mutagenic activity, Microsuspension, TA98, TA100, Dibenzo [a,l]pyrene Background al. 2016). PM2.5 are widely studied because of their harm- Recent reports in Japan have indicated decreases in the ful influence on human health, such as cardiovascular dis- emission of suspended particulate matter (SPM) [40], ease and respiratory disorders, as a result of the high polycyclic aromatic hydrocarbons (PAHs) [7] and dioxins deposition rate in the respiratory organs [28]. Various epi- into the atmosphere, by emission control on car engines demiological and toxicological studies have shown strong and improved management of waste incineration. On the positive correlations between PM2.5 concentrations in the other hand, it has been reported that the Japanese atmos- atmosphere and the death rate by respiratory system dis- phere is influenced by sulfur oxides from coal combustion ease [8], and a direct influence on collective human mor- from China [30], so there is considerable interest in Japan tality [4, 26, 27]. Furthermore, PAHs, such as benzo [a] regarding air pollution by PM2.5 from China ([39], Ling et pyrene (BaP, [1, 3, 22, 29]), nitro-PAHs, and dioxins [28], which are related compounds with carcinogenic activity, have been detected in PM2.5. PAHs derived from atmos- * Correspondence: sugita@azabu-u.ac.jp pheric suspended particulates were shown to be retained Azabu University, 1-17-71, Fuchinobe, Chuou-ku, Sagamihara, Kanagawa in the lungs of a dog [32]. The World Health Organization 252-5201, Japan Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sugita et al. Genes and Environment (2019) 41:7 Page 2 of 11 (WHO) has set guidelines for PM2.5 in the atmosphere at phosphate potassium, sodium hydroxide, D-glucose (The 10 μg/m for the annual average concentration and at reagent best quality, FUJIFILM Wako Pure Chemical 25 μg/m for the daily average. The continuous monitor- Corporation, Oosaka, Japan) and agar (Nacalai Tesque Inc., ing of PM2.5 in Japan is essential, and an environmental Kyoto, Japan) were used for a mutation assay. standard of PM2.5 in the atmosphere was set in 2009 at S. typhimurium strains TA98 and TA100 were distrib- 3 3 15 μg/m for the annual average and at 35 μg/m for the uted from National Institute of Public Health. daily average, and continuous monitoring is carried out in all parts of Japan. PM2.5 is collected by LV as part of the PM2.5 sampling monitoring, but chemical analysis of the samples collected The PM2.5 samples were collected at four locations in is limited because only small samples can be collected by Japan, namely Maebashi (East longitude 36.4045°, North this method, which may be below the detection limit of latitude 139.0961°), Tsukuba (East longitude 36.0498°, some pollutants. Therefore, equipment collecting larger North latitude 140.1175°), Nagoya (East longitude 35.0990°, amounts of PM2.5 is needed. North latitude 136.9156°) and Fukuoka (East longitude It is currently impossible to measure all the potentially 33.5948°, North latitude 130.3646°). At Nagoya, parallel carcinogenic substances contained in PM2.5 samples be- sampling by the LV and HV methods was carried out to cause of the large number of compounds present at very measure the concentrations of EC, C and some ion compo- low concentrations. Therefore, to achieve a comprehen- nents in the spring and summer 2012 and winter 2013 sive evaluation of the harmful effects of substances associ- (21 samples) and to measure the concentrations of PAHs in ated with PM2.5, a mutagenic assay, such as the Ames the summer 2013 and winter 2014 (15 samples). At the test, is needed. On the other hand, the conventional Ames other sampling points, sampling by the HV method was mutagenicity test uses Salmonella typhimurium strains carried out to measure the mutagenic activities in the sum- TA98 (in which frameshift mutations are detected) and mer 2012 and winter 2013 (64 samples including 14 sam- TA100 (in which base substitution mutations are detected) ples in Nagoya). which cannot detect mutagenic activity because the sensi- Samples of PM2.5 were collected on 47 mmφ quartz-fiber tivity is not high enough, and the amount of PM2.5 col- filters (PALLFLEX 2500QAT-UP, PALL corporation, New lected is limited. Therefore, modified Ames tests have been York, USA) for a 24 h period by the LV method and on developed in which the mutagenicity of PM2.5 samples has 20.3 × 25.4 cm quartz-fiber filters (PALLFLEX 2500QAT-UP, been increased using TA98 and a TA98-derivative strain, PALL corporation, New York, USA) by the HV method. A YG1024, with elevated O-acetyltransferase levels. The low-volume air sampler, Partisol 2000-FRM Air Sampler YG1024 strain shows hyper-responsiveness to nitro-PAHs (Thermo Fisher Scientific, Massachusetts, USA) was used and amino-PAHs [6, 36], but there is the risk of introdu- with a flow rate of 16.7 L/min as LV method. In 2000-FRM, cing a bias toward these compounds when this modified PM2.5 were collected by back-up filter, and PM > 2.5 were Ames test is used. eliminated by impactor that consist of filter and pump oil In the current study, parallel sampling of PM2.5 was [25]. This PM 2.5 collection method is certified by the carried out using the HV and LV methods, with concen- Federal Reference Law of the United States and is consistent trations of EC, OC, some ion components, and PAHs be- with the manual of the Japanese Ministry of the Environ- ing compared in PM2.5 collected by the two sampling ment. A high-volume air sampler, HV-700R (SIBATA methods, to evaluate the validity of collecting PM2.5 using Scientific Technology Ltd., Saitama, Japan) was used with a the new HV method. Subsequently, PM2.5 and the par- flow rate of 700 L/min as HV method. In the HV method, ticle bigger than PM2.5 (PM > 2.5) were collected using an impactor for PM2.5 (Custom-made based on a HV-100 the HV method, and the carcinogenic potential was evalu- 2.5 impactor, SIBATA Scientific Technology Ltd., Saitama, ated using the TA98 and TA100 strains, these being the Japan) was mounted on HV and PM2.5 was collected on strains used for the conventional Ames test, to achieve a pre-treated back-up quartz-fiber filters, and PM > 2.5 was comprehensive evaluation of the carcinogenesis-related collected on slit quartz-fiber filters (2500QAT-UP, PALL substances in PM2.5 and PM > 2.5 collected by the HV corporation, New York, USA) referring Fig. 1. Each method. quartz-fiber filter was heat-treated at 450 °C for 2 h in a muffle furnace to remove organic pollutants and then Methods equilibrated in a desiccator at a constant 50% humidity Materials and weighed. Acetone (HPLC grade; FUJIFILM Wako Pure Chemical Corporation, Oosaka, Japan) and dimethyl sulfoxide Analysis of carbonaceous and ion components (DMSO, Dojindo Molecular Technologies, Inc., Kumamoto, A 1.0 cm sample of the quartz-fiber filter with aerosol Japan) were used for extraction. MgSO � (7H O), citric acid particles, collected using both the LV and HV, was 4 2 hydrate, dihydrogen phosphate ammonium, dihydrogen placed in an oven. Then, OC and EC were analyzed with Sugita et al. Genes and Environment (2019) 41:7 Page 3 of 11 a laboratory OC-EC aerosol analyzer (Sunset Laboratory Inc., Oregon, USA) following the Interagency Monitor- ing of Protected Visual Environments (IMPROVE) ther- mal/optical reflectance protocol [2]. 2 2 The quartz-fiber filter of 4 cm in HV method and 5 cm in LV method was subjects to ultrasonic treatment with 10 ml of pure water to extract ion species respectively. After extraction, filter debris and suspended insoluble par- ticles were removed using a Teflon filter (DISMIC-25HP, Advantec Toyo Kaisha, Ltd., Tokyo, Japan). Cation species + + + 2+ 2+ such as Na ,NH ,K ,Mg and Ca and anion species − − 2− such as Cl ,NO and SO were measured by ion chro- 3 4 matograph (Dionex ICS-1000, Thermo Fisher Scientific, Massachusetts, USA). Analysis of polycyclic aromatic hydrocarbons One-half of the quartz-fiber filter collected by the LV method and one-fourth of the quartz-fiber filter collected by theHVmethodwereusedfor PAHmeasurements. The Fig. 1 Schematic of PM2.5 impactor for high-volume air sampler targeted PAHs (including dibenzo [a,l] pyrene (DBalP)), for (HV-1000 PM 2.5 impactor).① Base of impactor, ②; filter with slits, gas chromatography-mass spectrometry (GC/MS) analysis ③; Nozzle, ④; Washer, ⑤; Nut are shown in Table 1. A 16 PAHs mixture (representing the U.S. EPA priority PAHs) (each 20 ng) labeled with deuterium was added to the filter sample as an internal standard, followed by Soxhlet extraction with acetone. The Table 1 Information on the PAHs measured in this study PAHs Abbreviation Number of benzene Vapor pressure* (Pa at Target ion mass Retention time IARC Mutagenicity** rings 25 °C) (m/z) (min) classify Naphthalene Nap 2 10.4 128 9.6 2B – –1 Acenaphthylene Acy 3 8.9 × 10 152 12.6 – (?) –1 Acenaphthene Ace 3 2.9 × 10 153 12.8 – (?) –2 Fluorene Flu 3 9.0 × 10 166 13.9 3 – –2 Phenanthrene Phe 3 1.6 × 10 178 16.5 3 (?) –4 Anthracene Ant 3 8.0 × 10 178 16.6 3 – –3 Fluoranthene FLN 4 1.2 × 10 202 20.0 3 + –4 Pyrene Pyr 4 6.0 × 10 202 20.8 3 (?) –5 Benzo BaA 4 2.8 × 10 228 24.5 2A + [a]anthracene –5 Chrysene Chy 4 8.4 × 10 (at 20 °C) 228 24.8 3 + –5 Benzo BbF 5 6.7 × 10 252 27.8 2B + [b]fluoranthene –8 Benzo BkF 5 1.3 × 10 (at 20 °C) 252 27.9 2B + [k]fluoranthene –7 Benzo [a]pyrene BaP 5 7.3 × 10 252 29.0 2A + –8 Dibenzo dBahA 5 1.3 × 10 278 33.3 2A + [a,h]anthracene –8 Indeno[1,2,3- IndP 6 1.3 × 10 276 33.3 2B + c,d]pyrene –8 Benzo [ghi]perylene BghiP 6 1.4 × 10 276 35.0 3 + *: International programme on chemical safety environmental health criteria 202 selected non-heterocyclic policyclic aromatic hydrocarbons Table 4 [37] **: International programme on chemical safety environmental health criteria 202 selected non-heterocyclic policyclic aromatic hydrocarbons Table 2 [37] +: Positive, −: Negative, (?): Questionable Sugita et al. Genes and Environment (2019) 41:7 Page 4 of 11 extract was concentrated to a few ml using a rotary evapor- The microsuspension method, using the sensitized Ames ator set at a pressure of 39.9 kPa and a temperature of test, was used for the mutagenicity assay. It was carried out 40 °C, then a solvent was removed under the nitrogen flow with S9 mix addition and non-addition conditions gently, and a 0.5 ml of toluene was added to extract finally. using S. typhimurium strains TA98 and TA100 follow- One-fifth of the concentrated extract was measured and ing the protocols used in previous studies [15, 16, 31, re-dissolved in 30 mL hexane and once again concentrated 33]. When the dose-response relationship was ob- to ca. 3 ml with a rotary evaporator set at a pressure of tained, the case of the colony by which the number 23.9 kPa and a temperature of 40 °C. Then, a 3 ml whole of obtained return mutation colonies is beyond 2 volume of the hexane solution was added to a precondi- times of the control value was defined as positiveness tioned silica gel cartridge and eluted with 6 mL of and 1.5-two times, as weak positiveness and less than dichloromethane-hexane (8:2 (v/v)). An aliquot (100 μL) of 1.5 were difined as negative. n-nonane was added to the eluent, and the resulting solu- tion was then concentrated to 100 μL under nitrogen flow Results and discussion gently. Finally, the solution was transferred to an insert vial. Carbonaceous and inorganic chemicals in PM2.5 PAHs were analyzed by GC/MS (Model 5973 N, Agilent The EC concentration was 1.4 ± 0.7 μg/m , and the OC Technology, California, USA) in the selected ion monitor- concentration was 3.7 ± 1.8 μg/m in PM2.5 collected by ing mode (injection port temperature 300 °C, injected vol- the HV method. The atmospheric EC and OC concen- ume 1 μL, and GC temperature profile as follows: initial trations measured in this study were at a level similar to temperature: 50 °C for 5 min; the temperature was raised those in other reports of Japanese atmospheric EC and by 15 °C/min until it reached 185 °C; from 185 °C, the OC [9, 11, 12, 24]. The ion concentrations in PM2.5 + + + 2+ temperature was raised by 8 °C/min until it reached 320 °C, collected by the HV method for Na ,NH ,K ,Mg , 2+ − − 2− at which it was maintained for 22 min. Ionization was in Ca ,Cl ,NO and SO were 0.14 ± 0.10, 2.47 ± 1.88, 3 4 the EI mode. PAHs were determined by the internal stand- 0.10 ± 0.07, 0.02 ± 0.02, 0.07 ± 0.16, 0.20 ± 0.58, 1.37 ± ard method using PAHs deutriumed. 2.04, and 4.87 ± 4.22 μg/m , respectively. The atmos- pheric ion concentrations measured in this study were Mutagenicity test by the microsuspension method similar to those in other reports on Japanese atmos- One-fourth of both quartz-fiber filters for PM2.5 and PM > pheric ion species [20, 30]. The results from the HV and 2.5 of HV method were used for bioassay by the microsus- LV methods were compared in Figs. 2 and 3. Figure 2 pension method. The organic fractions were extracted by showed that there was a close agreement between con- ultrasonication (Ultra Sonic Cleaner AU-501CO; AIWA centrations of carbonaceous components in PM2.5 from Medical Industrial Corporation, Tokyo, Japan) with acet- the HV method and LV method. Furthermore, the slopes one twice. The two extracts were combined and then con- of the regression lines were in the range of 1.06 to 1.07, centrated to a few mL using a rotary evaporator. The very close to 1. In Fig. 3, a close agreement was shown + + + 2− solvent was removed under a nitrogen flow gently, and the for Na ,NH ,K , and SO between the two methods, 4 4 2+ 2+ − − dried samples were stored at − 80 °C until assayed. but the concentrations of Mg ,Ca ,Cl , and NO After defrosting at room temperature [23], an extracted were not similar between the two methods. The concen- 2+ 2+ − − sample was dissolved in DMSO and tested for mutagenicity. trations of Mg ,Ca ,Cl ,and NO by the HV (a) (b) (c) Fig. 2 Comparison of (a) elemental carbon (EC), (b) organic carbon (OC), and (c) total carbon (TC) concentrations measured with HV and LV methods. The solid line presents the regression line by at least two multiplication. The broken line presents the line equivalent in HV method and LV method Sugita et al. Genes and Environment (2019) 41:7 Page 5 of 11 (a) (b) (c) (d) (e) (f) (g) (h) + + + 2+ 2+ − − 2− Fig. 3 Comparison of (a)Na ,(b)NH ,(c)K ,(d)Mg ,(e)Ca ,(f)Cl ,(g)NO , and (h)SO concentrations measured with HV and LV 4 3 4 methods. The solid line presents the regression line by at least two multiplication. The broken line presents the line equivalent in HV method and LV method method were about 1.4, 2.6, 1.4, and 1.4 times higher, by the LV and HV methods. In contrast, for PAHs, the − 4 respectively, than those by the LV method. Uchiyama vapor pressure of which was greater than 10 Pa, the reported that the ion concentrations in SPM differed concentrations obtained by the HV method were less than + 2− + depending on particle size, with the NH ,SO ,and K those obtained by the LV method. The linear velocity of 4 4 concentrations in the fine particles being about 10, 4, and filter surface by HV method is 28.7 cm/sec, is 1.23 times 2 times higher than those in coarse particles, in the other higher than that of LV method. A little difference was rec- 2+ 2+ + hand, Ca ,Mg ,and Na concentrations in coarse parti- ognized between HV method and LV method. And, be- cles were about 10, 5, and 3 times higher than those in cause of the shape of the suspended particulate has a fine particles [34]. Because the impactor couldn’tbe di- distorted shape, not globular, the particles were separated vided among the particle diameter perfectly for 50% by an aerodynamic diameter, could not separate correctly cut-off value, it was suggested that a possibility with some by particle size. Therefore, it is consider that the coarse properties with HV method tends to undergo influence of particle near the PM2.5 influence PM2.5 fraction. The a particle large compared with LV method. factor for the difference is not clear, so cause investiga- tion is need urgently. But, HV method could collect − 4 Polycyclic aromatic hydrocarbons (PAHs) in PM2.5 low-vapor-pressure PAHs (less than 10 Pa) select- The PAH concentration per cubic meter of total sam- ively, This means that the HV method is suitable for − 4 pling flow, collected as particulate components using the low-vapor-pressure PAHs (less than 10 Pa), which are LV and HV methods, was measured and compared. mutagenic or carcinogenic [13]. These results are shown in Table 2. The atmospheric PAH concentrations measured in this study are at a level Highly carcinogenic PAHs in PM2.5 similar to those in other reports on Japanese atmos- In this study, DBalP, which is highly carcinogenic and has pheric PAHs [22, 38]. The comparison of the PAH con- six benzene rings, was also measured by GC/MS. DBalP is centrations of PM2.5 between the HV and LV methods not included in the U.S. Environmental Protection Agency was shown in Table 2 in which the results were classified (U.S EPA) priority 16 PAHs; however, it is notable that the by the vapor pressure of PAHs. The results demonstrated relative potency factor (RPF) [35] of DBalP is 30 [19]. significant correlation with significant level < 0.01 (R = DBalP could be detected in the PM2.5 samples by the HV 0.795 0.985) for low-vapor-pressure PAHs (less than 10 method, but it was not detected in PM2.5 by the LV Pa) between the concentrations associated with PM2.5 method. The atmospheric concentration of DBalP ranged Sugita et al. Genes and Environment (2019) 41:7 Page 6 of 11 Table 2 The atmospheric PAH concentration levels as determined by the LV and HV methods Abbreviation of Vapor pressure (Pa at Atmosphereic concentration by LV method ave. Atmosphereic concentration by HV method R 3 3 PAH 25 °C) ± SD; ng/m ) (ave. ± SD; ng/m ) value Nap 10.4 1.76 ± 0.73 0.70 ± 0.42 −0.369 –1 Acy 8.9 × 10 40.1 ± 20.9 0.47 ± 0.46 −0.058 –1 Ace 2.9 × 10 0.24 ± 0.07 0.04 ± 0.03 −0.144 –2 Flu 9.0 × 10 0.25 ± 0.17 0.05 ± 0.02 0.344 –2 Phe 1.6 × 10 3.79 ± 0.81 0.45 ± 0.21 0.649 –4 Ant 8.0 × 10 0.11 ± 0.08 0.03 ± 0.02 0.077 –3 FLN 1.2 × 10 3.32 ± 0.85 0.49 ± 0.25 0.265 –4 Pyr 6.0 × 10 1.71 ± 0.96 0.29 ± 0.15 0.068 –5 BaA 2.8 × 10 0.16 ± 0.12 0.13 ± 0.12 0.795 Chy 8.4 × 10–5(at 20 °C) 0.54 ± 0.34 0.44 ± 0.35 0.906 –5 BbF 6.7 × 10 0.31 ± 0.29 0.40 ± 0.29 0.951 –8 BkF 1.3 × 10 (at 20 °C) 0.17 ± 0.12 0.16 ± 0.13 0.928 –7 BaP 7.3 × 10 0.25 ± 0.14 0.19 ± 0.15 0.954 –8 dBahA 1.3 × 10 0.03 ± 0.02 0.03 ± 0.02 0.944 –8 IndP 1.3 × 10 0.22 ± 0.19 0.25 ± 0.20 0.985 –8 BghiP 1.4 × 10 0.33 ± 0.27 0.28 ± 0.22 0.964 R means the coefficient of correlation from 0.014 to 0.078 ng/m , and this concentration was samples than did the TA98 strain [10, 21]. But it was about one-tenth that of BaP. DBalP concentrations in considered that mutagenic evaluation with YG1024 had present study were one-tenth of lower than that in China an in-built TA1537 (or TA97 and TA97a), TA98, TA100 (Wei [17]) by three days sampling, and were similar to and WP2uvrA (or WP2uvrA (pKM101) or TA100) have those in Chiba prefecture [14] by one week sampling. The been used for mutagenic assay (OECD 1997). The measurement of low concentration PAHs by HV method YG1024 strain is not selected for standard mutagenicity could be shortened sampling time to 24 h from a few days assays. So, in this study, two types of strain were used, one successfully. Then, the comparison of mutagenic potency is TA98 strain that detected mutagenicity of the frame- of DBalP and BaP calculated by their concentration and shift type, another is TA100 that detected mutagenicity of PRF showed that the atmospheric carcinogenic potency of the base substitution type. Furthermore, the measure- DBalP is three times higher than that of BaP. It is a recog- ments of the mutagenic activity in PM2.5 samples were nized that the HV method is suitable for the quantification tried here using the microsuspension method [15]that ex- of one of the most-highly carcinogenic PAHs. The major hibits greater sensitivity than does the original Ames test. advantage of the HV method is the extreme microanalysis In previous studies, the sample of mutagenic assay for of PM2.5 composition which is possible given the large environmental atmosphere using TA98 and TA100 used amount of PM2.5 available from the HV method, a to be collected by HV method for 24 h. The sample of 1/3 process which is impossible to detect with the small to 1/2 was used for mutagenic assay, that equivalent to air 3 3 amount of PM2.5 collected by the LV method. volume 400 m to 600 m [5]. And, Matsumoto et al. car- ried out sampling for mutagenic assay for atmosphere by Mutagenicity of PM2.5 LV method for 3 days and collected air volume120 m to A LV method was used for conventional PM2.5 collec- 240 m [18]. Because of these air volume were equivalent tion and analysis of airborne particles, but the sample to sampling volume for 5 days to 10 days by LV method, it size achieved by this method was very small. Because was impossible to elucidate diversification of a testing nitro-PAHs were reported as mutagen which could be condition and fluctuation during the day. present in airborne particles, the S. typhimurium strain, In this study, the mutagenic evaluation for environ- YG1024, developed to exhibit very high sensitivity to mental atmosphere using TA98 and TA100 strain was nitro-PAHs, was used to assay for mutagenicity in such attempt by combining microsuspention sensitized Ames small airborne particle samples. YG1024 strain was re- test and large volume sampling method. ported to exhibit approximately 10 times greater sensi- The air volume necessary to detection of the positive tivity to mutagenesis from food and environmental and weak positive of mutation are indicated in Table 3. Sugita et al. Genes and Environment (2019) 41:7 Page 7 of 11 Table 3 Comparison of the air volume (m ) necessary to false positive in present study (in the case of single dose) (unit: m ) TA100 - S9mix TA100 + S9mix TA98 – S9mix TA98 + S9mix Total average 7.85 4.65 1.12 4.45 18.07 sd 5.98 4.07 1.31 4.40 12.72 50% of detection parcentage 6.36 3.40 0.99 3.06 14.09 90% of detection parcentage 14.95 7.96 1.74 12.57 35.59 95% of detection parcentage 18.70 12.54 2.05 14.15 50.22 All 32.28 26.59 10.67 17.88 59.19 In the case of actual assay, the necessary air volume was mutagenicity were positive in all four conditions. But, four times of value in Table 3, because mutation assay mutagenicity were negative in the conditions of TA100 need assay with different 5 concentrations and duplicate without S9mix (TA100 S9mix-) and TA98 with S9mix measurement. Its air volume was different depending on (TA98 S9mix+), was weak positive in the condition of samples by the percentage of the mutagenic substance TA100 with S9mix (TA100 S9mix+) and was positive in 3 3 included in a sample, 14 m (actually, 56 m ) was needed the condition of TA98 without S9mix (TA98 S9mix-). by 4 conditions by the detection rate of the 50% in present An outline of the detection rate of mutagenic activity in study. This air volume was equivalent to the sample for PM2.5 is shown in Table 4. The positive rate, including 48 h by LV method. Additionally, the air volume necessary weak positives, was 98.4% by TA100 (-S9mix), 95.3% by to detection of 90% was approximately 140 m ,thisvol- TA100 (+S9mix), 100% by TA98 (-S9mix) and 92.2% by ume was equal to the suction amount of the previous TA98 (+S9mix). The average mutagenic activities per study. unit volume at each location are shown in Fig. 6.In spite Samples of PM2.5, taken at four locations from the of PM > 2.5 particles accounting for about 20% of the Kanto area to the Kyushu area, were collected by the particle weight of the sample, mutagenic activity in HV method. Then, the mutagenic activities of these PM > 2.5 was very weak (Fig. 6). It was considered that PM2.5 samples were tested using the microsuspension most of mutagenic compounds were associated with method with TA98 and TA100 strains. Typical results of the fine particles like PM2.5 in the atmosphere (Fig. 5). the mutagenicity assay are shown in Fig. 4. In Fig. 4 a, Significant differences in mutagenic activity of PM2.5 (a) (b) Fig. 4 Typical dose–response curves of mutation assay for PM2.5 in Tsukuba in (a) 30th July, 2012 and (b) 8th August, 2012.a: Results of typical positive mutagenicity. b: Results of negative, false-positive and positive mutagenicity. TA100 S9mix- and TA98 S9mix + were negative, TA100 S9mix + was false-positive and TA98 S9mix- was positive Sugita et al. Genes and Environment (2019) 41:7 Page 8 of 11 Table 4 Detection rates of mutagenic activity by the microsuspension method Tsukuba Maebashi Nagoya Fukuoka number parcentage(%) number parcentage(%) number parcentage(%) number parcentage(%) Negative 3 (4.7) 1 (1.6) 2 (3.1) 3 (4.7) week positive 13 (20.3) 18 (28.1) 13 (20.3) 11 (17.2) Positive 48 (75.0) 45 (70.3) 49 (76.6) 50 (78.1) sum 64 (100.0) 64 (100.0) 64 (100.0) 64 (100.0) between summer and winter were recognized in other hand, it was found that mutagenic activity dif- Nagoya and Fukuoka in spite of it being recognized fered significantly, in spite of the similar PM2.5 con- that there were no significant differences in particle centrations being reported from the different sites over concentrations between summer and winter. The aver- the two seasons. From the relationship between PM2.5 age (mean ± SD) concentration of PM2.5 was 24.0 ± concentration and mutagenicity, it is apparent that 3 3 2.9 μg/m in summer and 24.6 ± 9.3 μg/m in winter at more data need to be collected and the distribution an- Tsukuba, 28.7 ± 2.8 μg/m in summer and 26.8 ± alyzed to identify management concentrations. 3 3 13.8 μg/m in winter at Maebashi, 31.2 ± 5.1 μg/m in The concentration of PM2.5 in the atmosphere is regu- summer and 28.2 ± 13.9 μg/m in winter at Nagoya, lated globally because of the detrimental impact of these 3 3 and 25.8 ± 5.3 μg/m in summer and 18.2 ± 15.0 μg/m particles on human health. On the other hand, since the in winter at Fukuoka. Furthermore, the relationship be- composition of the chemical substances associated with tween atmospheric PM2.5 concentration and mutage- PM2.5 differs according to the sources, it can also be used nicity is showninFig. 6. The correlation coefficients in source analysis by the two commonly used receptor between PM2.5 concentration and mutagenic activity models, the chemical mass balance (CMB) model and the were significant (p <0.05, n = 64) for each assay, namely positive matrix factorization (PMF) model. In this study, 0.568 for TA100-S9mix, 0.525 of TA100 + S9mix, 0.445 the HV method could detect significant differences in mu- of TA98-S9mix, and 0.391 of TA98 + S9mix. On the tagenic activity between summer and winter samples, Fig. 5 Comparison of mutation activity in PM2.5 and PM > 2.5 per unit air volume at different sites and seasons. *: Results with a common letter present significant differences (p < 0.05) Sugita et al. Genes and Environment (2019) 41:7 Page 9 of 11 Fig. 6 Relationship between mutagenicity and PM2.5 concentration The solid line presents the regression line, the both broken lines present the 95% prediction interval although there was no significant difference between sea- (HV method) using high-volume air sampler with an sons in the concentration of PM2.5 at the same location. impactor and compared and evaluated it using the These results suggest that the components of particle-based Low-volume air sampler based authentication method pollutants related to carcinogenesis in PM2.5 could change (LV method). The results showed that the HV method depending on the season. Therefore, it is considered that had the same performance with LV method in terms of the human health risk posed by PM2.5 would also vary, de- measuring the inorganic components (OC, EC, Na , + + 2− pending on the composition of the substances, but these NH ,K and SO ) and low-volatile PAHs that are 4 4 effects have not yet been clarified in detail. The HV method mutagenic or carcinogenic. Because of the air volume of fine particle collection can evaluate the risk of PM2.5 collected by HV method was forty times higher than the precisely. Since the toxic effects of PM2.5 extend to cardio- LV method, the high carcinogen and mutagen like DBalP vascular and respiratory diseases, the measurement of that could be not detected by LV method were detected chemical substances related to such diseases and the deter- at 0.014 0.078 ng/m . Furthermore, HV method with mination of the end-points of these compounds are ur- microsuspension method could evaluate the mutagenic gently required. It is considered that the HV method activity of PM2.5 by TA98 and TA100 strain. The HV proposed in this study is a very useful tool for the compre- method for PM2.5 proposed in present study is very hensive evaluation of the health effects of carcinogens by useful for detecting targeted micropollutants present at the mutation assay and by precise measurement of trace very low concentrations in the air. On the other hand, for chemicals in the fine particles. low molecular weight PAH and some inorganic ionic spe- 2+ 2+ − − cies (Mg ,Ca ,Cl and NO ), the HV method tended Conclusions to show higher concentration than the LV method. Atten- A strong harmful effect is attributed to a fine particle tion should be paid according to the application. (PM2.5) in the atmosphere because PM2.5 has a high Abbreviations deposition rate in the respiratory organs. In present BaP: Benzo [a]pyrene; CMB: The chemical mass balance; DBalP: Dibenzo study, we would propose a new PM2.5 sampling method [a,l]pyrene; DMSO: Dimethyl sulfoxide; EC: Elemental carbon; GC/MS: Gas Sugita et al. Genes and Environment (2019) 41:7 Page 10 of 11 chromatography–mass spectrometry; IMPROVE: The Interagency Monitoring K.: Mutagenicity of airborne particles, river waters and soils in Japan from of Protected Visual Environments; LV: Low-volume air sampler; OC: Organic 1996 to 2003. Environ Mutagen Res, Vol.26, pp9–22 (2004). carbon; PAHs: Polycyclic aromatic hydrocarbons; PM > 2.5: Particle bigger 6. Endo, O., Sugita, K., Goto, S., Amagai, T. and Matsushita H.: Mutagenicity of than PM2.5; PM2.5: Fine particles with an aerodynamic diameter less than size-fractionated airborne particles collected with Andersen low pressure 2.5 μm; PMF: The positive matrix factorization; RPF: Related potency factor; impactor, L Health Sci Vol.49, No.1, pp22–27 (2003). 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Published: Feb 28, 2019

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