Sub-toxic events induced by truck speed-facilitated PM2.5 and its counteraction by epigallocatechin-3-gallate in A549 human lung cells

To distinguish the influences of fuel type and truck speed on chemical composition and sub-toxic effects of particulates (PM2.5) from engine emissions, biomarkers—interleukin-6 (IL-6), cytochrome P450 (CYP) 1A1, heme oxygenase (HO)-1, and NADPH-quinone oxidoreductase (NQO)-1—were studied in A549 human lung cells. Fuel type and truck speed preferentially affected the quantity and ion/polycyclic aromatic hydrocarbon (PAH) composition of PM2.5, respectively. Under idling operation, phenanthrene was the most abundant PAH. At high speed, more than 50% of the PAHs had high molecular weight (HMW), of which benzo[a]pyrene (B[a]P), benzo[ghi]perylene (B[ghi]P), and indeno[1,2,3-cd]pyrene (I[cd]P) were the main PAHs. B[a]P, B[ghi]P, and I[cd]P caused potent induction of IL-6, CYP1A1, and NQO-1, whereas phenanthrene mildly induced CYP1A1. Based on the PAH-mediated induction, the predicted increases in biomarkers were positively correlated with the measured increases. HMW-PAHs contribute to the biomarker induction by PM2.5, at high speed, which was reduced by co-exposure to epigallocatechin-3-gallate.

Quantitation of PAHs in the dichloromethane extract of PM 2.5 indicated that phenanthrene ( Fig. 1) was the most abundant PAH in diesel-idle and gasoline-idle exhausts (Fig. 3B). Under idling operation, the order of the relative contents of PAHs was LMW-PAHs (45-73%) > MMW-PAHs (21-41%) > HMW-PAHs (7-14%) in the PM 2.5 emitted from both diesel and gasoline engines. At high speed, the emissions contained mainly HMW-PAHs (> 54%). Among the HMW-PAHs, B[a]P, B[ghi]P, and I[cd]P were the top three constituents in the diesel-high exhaust. According to the total amount of PAHs in the particulates, the amounts of B[a]P, B[ghi]P, and I[cd] P in the diesel-high exhaust were greater than those in the gasoline-high exhaust. Thus, phenanthrene, B[a]P, B[ghi]P, and I[cd]P were further studied to reveal their contribution to the differential effects of exhausts on the expression of biomarkers IL-6, CYP1A1, HO-1, and NQO-1.

Effects of the aqueous and dichloromethane extracts of PM 2.5 on cell viability.
For 48 h, A549 cells were exposed to the extracts at concentrations equivalent to PM 2.5 exposure as indicated in Fig. 4. After exposure to the aqueous extracts, a significant decrease (> 20%) in cell viability was not observed (Fig. 4A). Among the dichloromethane extracts, exposure to 20 μg/mL of PM 2.5 , in the diesel-high exhaust caused a 24% decrease in cell viability (Fig. 4B), while the other exhausts did not cause a significant decrease. Thus, to prevent the influence of cell death, the cells were exposed to PM 2.5 extracts at sub-toxic concentrations of aqueous and dichloromethane extracts at 10 μg PM 2.5 /mL, under which cell survival remained greater than 85%.

Effects of fuel type and truck speed on biomarker induction by fuel emissions. After 48-h expo-
sure to the aqueous and dichloromethane extracts of PM 2.5 (10 μg PM 2.5 /mL), compared to the vehicle control, the aqueous extract of gasoline-idle exhaust and the dichloromethane extract of diesel-high exhaust, signifi-   5A,B). Among the biomarkers, cellular CYP1A1 displayed the highest inducibility. The CYP1A1 transcript level significantly increased after exposure to the aqueous extract of diesel-high exhaust (5.0-fold, p < 0.05) (Fig. 5A), and the dichloromethane extracts of diesel-high (5.3-fold, p < 0.05) and gasoline-high exhausts (14.5-fold, p < 0.001) (Fig. 5B). Among the aqueous extracts, only the gasoline-idle exhaust increased HO-1 levels and none of the exhausts affected NQO-1 expression, compared to the vehicle control. The dichloromethane extracts of both gasoline-high and diesel-high www.nature.com/scientificreports/ exhausts caused significant increases in HO-1 expression, compared to vehicle control, and the induced HO-1 levels were significantly higher than those of idling operation. Among the dichloromethane extracts, only dieselhigh exhaust significantly increased NQO-1 transcript levels. In addition, the cells were exposed to a mixture of aqueous and dichloromethane extracts of the diesel-high exhaust at 10 μg PM 2.5 /mL. The mixed extracts significantly induced the mRNA levels of IL-6, CYP1A1, HO-1, and NQO-1 (Fig. 5C). These results revealed that CYP1A1 is a very sensitive biomarker for fuel emissions, and the PAH-rich fraction of PM 2.5 emitted from engines under high speed resulted in a greater increase in CYP1A1 and HO-1 than that under idle condition. The PAH-rich fraction from diesel-high exhaust, as well as a reconstituted mixture of the aqueous and dichloromethane extracts, stimulate biomarker expression in A549 cells.
Effects of the fuel type on IL-6 secretion by the dichloromethane extracts of PM 2.5 emitted under high speed. To further confirm the impact of increased IL-6 mRNA levels by the dichloromethane extract of PM 2.5 at high speed, the secretion of IL-6 in the medium was determined. Consistent with the induction of IL-6 transcript (Fig. 5C), IL-6 secretion was significantly stimulated by 1.3-and 1.4-fold, after exposure to the extract of diesel-high exhaust for 6 h and 48 h, respectively (Fig. 5D). The stimulation of IL-6 secretion was not observed with the gasoline-high exhaust, which did not induce IL-6 expression in A549 lung cells (Fig. 5C).
Protective effects of EGCG against the sub-toxic responses to the dichloromethane extract of diesel-high exhaust. Effects of EGCG on cell growth was first studied to determine the exposure concentration of EGCG, at which cell growth was not affected in A549 cells. With 48-h of exposure to EGCG, cell viability decreased by 200 μM (86% survival) (Fig. 6A). Thus, cells were exposed to EGCG at a concentration ≤ 100 µM in the following studies. The 6-h exposure to hydrogen peroxide and t-BHP resulted in an increase (> five fold) in cellular ROS levels. In contrast, EGCG (100 μM) exposure decreased the basal cellular ROS levels by 40% (Fig. 6B). The 6-h exposure to the aqueous extracts of gasoline-and diesel-engine exhausts did not increase the generation of cellular ROS (data not shown). Similarly, exposure to the dichloromethane extract of the dieselidle exhaust did not stimulate ROS generation. However, the dichloromethane extract of the diesel-high exhaust caused a twofold increase in ROS levels. This stimulation was diminished by the co-exposure to EGCG. Due to the greater induction of IL-6 by the PAH-rich dichloromethane extract of the diesel-high exhaust, the effect of EGCG (100 µM) on the diesel-high exhaust-mediated induction of the biomarkers was further studied. EGCG exposure caused time-dependent bi-functional effects on IL-6 and HO-1 transcript levels and NQO-1 transcript increased after 48-h EGCG exposure (Fig. 6C). After 6-h of exposure, the extract of diesel-high exhaust stimulated CYP1A1, HO-1, and NQO-1 expression without affecting IL-6 expression (Fig. 6C). However, the  After seeding, cells were cultured for 24 h and then exposed to the extracts of particulates for 48 h. Cell viability was monitored using MTT reduction assay as described in the Methods. Data represent the means ± SD of three separate experiments with triplicate determinations in each experiment. *p < 0.05 with a decrease > 20%. www.nature.com/scientificreports/ diesel-high-induced increases in HO-1 and NQO-1 could be reduced by EGCG co-exposure, while EGCG could not affect the potent CYP1A1 induction diesel-high exhaust after 6-h short time exposure. By prolonging the exposure to 48 h, the diesel-high exhaust extract caused a 1.7-fold increase in IL-6 expression, and EGCG coexposure significantly reduced IL-6 induction to a level comparable to the vehicle-control exposure. The elevation of transcript levels of CYP1A1 and NQO-1 by the extract also decreased after the cells were co-exposed to EGCG. Results showed the induction of the metabolic enzymes (CYP1A1, HO-1 and NQO-1) occurs shortly after the diesel-high exhaust exposure and the IL-6 stimulation required a longer time-period of exposure. Following the EGCG-decreased exhaust-mediated HO-1 and NQO-1 induction, EGCG ameliorated CYP1A1 and IL-6 induction by the exhaust extract. , cells were exposed to the aqueous extract (AE) and dichloromethane extract (DE) of PM 2.5 particulates at a concentration as indicated for 48 h. After exposure, the mRNA levels of biomarkers were analyzed using real-time PCR analysis as described in the Methods. In panel (E), cells were exposed to the DE of dieselhigh emission for 6 and 48 h as indicated. Data represent the means ± SD of three different experiments with duplicate determinations in each experiment. The lines between pairs of bars indicate that there are significant differences (p < 0.05) between two exposures in the one-way-ANOVA analysis with post-hoc Tukey's test for multiple comparison. *p < 0.05 (compared to the control using Student's t-test).  www.nature.com/scientificreports/ HO-1, and NQO-1 were studied (Fig. 7A). According to the reported exposure concentration range of B[a] P for evoking biomarker induction and genotoxic potential [25][26][27] , the cells were exposed to these four PAHs at the same concentration of 2 μM for the purpose of comparison. P to the differential effects of fuel emission under idling operation and high speed, relative increases in the biomarkers were predicted. The predicted and measured increases in transcript levels of IL-6, CYP1A1, HO-1, and NQO-1 (16 sets of values) showed a positive correlation with a Spearman's correlation coefficient (r) of 0.691 (95% confidence interval: 0.283-0.888, p = 0.003). While the data were assumed to be sampled from Gaussian populations, the results of Pearson's test showed no significant correlation with Pearson's r of 0.238 (95% confidence interval: -0.292-0.657, p = 0.374), potentially due to two outliers. Following the removal of the two outliers, the predicted and measured increases in transcript levels of IL-6, CYP1A1, HO-1, and NQO-1 (14 sets of values) showed a positive correlation with a Spearman's r of 0.582 (95% confidence interval: 0.057 -0.855; p = 0.0289) and Pearson's r of 0.904 (95% confidence interval: 0.719 -0.970; p < 0.0001) (Fig. 7B). The results suggested that phenanthrene, B[a]P, B[ghi]P, and I[cd]P represent the principal PAHs for the differential responses of the biomarkers in lung cells after exposure to various exhausts.

Discussion
In our previous study of PM 2.5 in northern Taiwan, the results showed that traffic activity generates higher PM 2.5 concentration (mg/m 3 ) than long-range transport events and outdoor cooking (night market) 28 . The potential factors influencing the PM 2.5 composition in vehicle emissions include fuel type, engine age, running distance, vehicle displacement (c.c.), wind speed, temperature, and humidity while collecting the exhaust 15,16,29,30 . In the gasoline exhausts of 15 cars (1328 ~ 2378 c.c.), the concentrations of PM 2.5 in the emission were found to be 0.36-1.02 mg/m 315 . In another study of the diesel exhausts from 15 heavy trucks (4009-11,149 c.c.), the PM 2.5 levels in the emission were 3.88-16.50 mg/m 316 . From those results, diesel emissions seemed to contain greater PM 2.5 particulates than gasoline emissions. However, there was fourfold difference in the mean engine displacement of vehicles between these two reports. Our study is the first to demonstrate that a light diesel-truck (2835 c.c.) emitted a PM 2.5 level of more than eightfold that of emission from a gasoline-truck (2000 c.c.). The emissions from one diesel truck at high speed (80 km/h) had a 94% increase in the particulate exhaust rate (mg/min) than that at low speeds (20 km/h) 31 . Our findings further indicated that engines under high speed emitted a PM 2.5 level of 35%-108% higher than the engines under idling operation. Compared to speed, both our present findings, and that from previous studies, revealed that fuel type had a greater influence on the absolute amounts of PM 2.5 , emitted from vehicles.
Analysis of the ionic and metallic (Suppl. Fig. 1) composition of the emitted PM 2.5 , revealed that, among the truck exhausts, the PM 2.5 , in gasoline-idle exhaust, had the highest levels of total cation/anion and metals. When the gasoline and diesel engines changed from idling operation to high speed, the most abundant cations and anions changed from Na + to Ca 2+ and from NO 3 or PO 4 3− to NO 2 − , respectively. The increased speed changed the most abundant metals in PM 2.5 , from sodium to aluminum (Suppl. Fig. 2). The relative abundance of several metals increased with the increased speed, e.g. abundance of zinc increased 23-26 fold. In this study, used trucks were studied and the same truck was used for the PM 2.5 collections under idle and high-speed conditions. The components of PM 2.5 could be from fuel combustion, crustal dust and other absorbed substances. The actual causes for the changes in the relative abundance of ions, such as Na + , and metals, such as Zn, in the PM 2.5 remained unclear. Potential causes may include the differential engine-temperature and the relative contribution of crustal dust and fuel combustion to the PM 2.5 emitted from the engines under different speed conditions.
In the PAH content of vehicle emissions, large variations exist based on different reports 30 . In previous studies on the determination of 21 PAHs, the mean concentrations of total PAHs (Σ 21 PAHs) were 206 ng/m 3 and 5310 ng/m 3 in the gasoline exhaust of cars (< 2378 c.c.) and diesel exhausts from heavy trucks (> 4009 c.c.), respectively 15,16 . Compared to the reported PAH contents in gasoline and diesel exhausts, our determinations of the total PAHs in PM 2.5 emitted from light trucks were relatively low. Our findings revealed that the PM 2.5 from diesel emissions contained greater total PAHs compared to gasoline emissions under idle or high-speed condition, and the diesel-idle exhaust generated the highest total PAH content in PM 2.5 (135 ng/mg PM 2.5 ). Our study is the first to show that under idling operation, LMW-PAHs was the most abundant type of PAH (37-45% of total PAHs), emitted from trucks with either diesel (with a catalyzed diesel particulate filter) or gasoline engine. Consistent with a previous report that indicated that the abundance of heavier PAHs increased while the speed of a heavy-duty diesel truck increased 18 , our findings further showed that HMW-PAHs became the most abundant type of PAH (54-87% of total PAHs) in the PM 2.5 of not only diesel but also gasoline exhaust when the speed of light trucks increased to 70 km/h. Under idle condition, the temperature of gas flow would be lower than that under high-speed condition. Temperature of gas flow could affect the partition of LMW-PAHs between particle and gas phases, which may lead to the relatively lower phenanthrene abundance in high-speed exhausts 32 . Temperature of gas flow could be one of the factors for the differential PAH composition of exhausts under idle and high speeds. Vehicle speed appeared to be a determining factor for the ion/metal/PAH composition of the PM 2.5 emitted from trucks. The TEF values used for calculating B[a]P toxicity equivalents were estimated based on the tumor incidence or carcinogenicity of PAHs 33 . In the early stages of cancer development after PAH exposure, CYP1A1, HO-1, NQO-1, and IL-6 were identified as exposure biomarkers 34,35 . Among the aqueous extract of exhausts, gasolineidle exhaust caused the highest increases in IL-6 and HO-1 expression. Compared to the biomarker induction caused by the PAH-rich dichloromethane extract of PM 2.5 emissions, the aqueous extract-mediated increase was weak to moderate. Among the exhausts, PM 2.5 in the diesel-idle exhaust had the highest levels of total PAHs (ng/ mg PM 2.5 ) and B[a]Peq (ng/mg PM 2.5 ). However, under sub-toxic acute exposure to the exhaust extracts, the PAH-rich dichloromethane extract of diesel-high exhaust effected the highest increase in IL-6 transcript level. Exposure to diesel-high exhaust extract resulted in a greater increase in HO-1 compared to effect of exposure to the diesel-idle exhaust. CYP1A1 was significantly induced by the diesel-high exhaust extract, but not by the diesel-idle exhaust extract. The dichloromethane extract of gasoline-high exhaust also significantly induced CYP1A1 and HO-1, compared with gasoline-idle. The PAH-rich fraction of PM 2.5 , emitted from trucks under high speed, resulted in greater biomarker induction compared to trucks under idling operation. Compared to the most abundant LMW-PAHs in the diesel-idle exhaust, phenanthrene, the HMW-PAHs, B[a]P, B[ghi]P, and I[cd]P effected greater increases in IL-6, CYP1A1, and NQO-1 levels. Our estimates of the relative increases in biomarkers correlated well with the measured increases after the exposure of A549 cells to PAH-rich extracts. This indicates that the PAH-mediated biomarker induction by exhausts is primarily attributed to these HMW-PAHs, whose abundance increased when truck ran at high speed. The lack of HO-1 induction by the 3 main HMW-PAHs suggested the presence of other types of inducers in the dichloromethane extract of diesel-high exhaust. In addition to PAHs, nitro-PAHs and other organic compounds as the persistent organic pollutants (POPs), dibenzo-p-dioxins, dibenzofurans, and polychlorinated biphenyls (PCBs), are other classes of stimulators 27 .
It was demonstrated that AhR induction is linked to Nrf2 activated downstream targets, HO-1 and NQO-1 36 . Induction of CYP1A1 and NQO-1 is crucial for the degradation of harmful toxicants, including PAHs and quinones 37 . However, persistent AhR activation and subsequent oncogene activation can also lead to the increase of carcinogenic metabolite-induced DNA damage and the unbalanced promotion of cell proliferation 1,7,10 . EGCG exposure was reported to cause inconsistent regulation of Nrf2, HO-1, and NQO-1 expression in different cell types 38,39 . In consistence with that EGCG (100 μM EGCG, 24 h) increased the expression of HO-1 in A549 cells and A549 xenografts 40 , our findings revealed that EGCG decreased HO-1 expression after 6-h exposure and then induced HO-1 in a dose-dependent manner after 48-h exposure. The 6-h exposure to EGCG alone decreased the basal level of ROS and IL-6 expression. Co-exposure to EGCG reduced the ROS, HO-1 and NQO-1 level increased by PAH-rich dichloromethane extract of diesel-high-emitted PM 2.5 . Although both EGCG and the PAH-rich diesel-high exhaust extract induced the expression of IL-6 and NOQ-1 after 48-h exposure, coexposure to EGCG reduced the stimulation by the diesel-high exhaust extract. The 6 and 48-h exposure to a high concentration of EGCG alone did not affect the basal CYP1A1 transcript level, but 48-h co-exposure to EGCG significantly decreased the diesel-high exhaust-mediated CYP1A1 induction due to its potential inhibitive effect on the AhR-signaling 19,20 . The time-dependent influence of EGCG on fuel-emission-induced biomarker responses was first reported in this study. EGCG decreased the diesel-high extract-and the HMW-PAH-stimulated IL-6, indicating the benefit of EGCG in ameliorating the IL-6 stimulation by diesel-high-emitted PM 2.5 .
In conclusion, our findings reveal that truck speed is a determining factor of the ionic, PAH and metallic composition of PM 2.5 . B[a]P, B[ghi]P and I[cd]P were identified to be the main PAHs facilitated by diesel-truck speed and caused potent induction of biomarkers in lung cells. The diesel-high exhaust-stimulated ROS and relevant HO-1 and NQO-1 levels were first attenuated by EGCG and then the elevated CYP1A1 and IL-6 levels decreased. www.nature.com/scientificreports/ gal, India) with the precision of ± 3%. One sixteenth of the filter was cut using a pair of Nanocera scissors (Hola China, Shanghai, China), immersed in 10 mL deionized water and sonicated in a water bath at room temperature for 1 h. After centrifugation, the aqueous extract was then collected and lyophilized, while the filter was air-dried and placed in a Thimble filter (item no. 84, 31 mm (i.d.) × 35 mm × 120 mm, Advantec Toyo Kaisha, Ltd., Tokyo, Japan) for further preparation of dichloromethane extract. All apparatuses were then washed using acetone followed by dichloromethane. Soxhlet-extraction was performed using dichloromethane for 6 h without light. The extracts were concentrated using a rotor evaporator and dried under a stream of nitrogen gas at room temperature. The filter residues were used for further metallic determination (Suppl. Methods 1.1 and Results) and the detection rates were listed in Suppl. Table 1. A clean filter was extracted and analyzed as a blank sample for the chemical analyses. All extracts were stored at -20 °C in the dark.

Methods
Analysis of chemicals. The aqueous extracts of PM 2.5 samples were filtered using a glass syringe and a 20 mm polytetrafluoroethylene (PTFE) filter (Advantec Toyo Kaisha, Ltd., Tokyo, Japan). The filtrate was subjected to ion chromatography using Dionex ICS-1000 (Thermo Fisher Scientific Inc., Waltham, MA, USA) to determine the ionic content (recovery range of 88-104%) 28 with a detection limit of 0.01-10.1 µg. For the quantitation of PAHs, we analyzed the contents of 27 PAHs in the dichloromethane extracts of PM 2.5 samples. Each PM 2.5 extract was spiked with internal standards (deuterated PAHs, 50 μl of 50 pg/μl for each sample) before reconstitution with dichloromethane (40 mL). The reconstituted samples were sonicated for 15 min and then incubated at room temperature in the dark for 5 min; this was repeated three times. The sample solutions were filtered as previously described and then concentrated using a rotary evaporator in a thermostatic bath (Eyela, Tokyo, Japan) at 33 °C. The final solution volume was reduced to 1.0 mL under a nitrogen stream. The PAHs were separated and quantitated by gas chromatography-mass spectrometry (GC-MS/MS) using the TRACE 1300 GC & TSQ 8000 Evo Triple Quadrupole system (Thermo Fisher Scientific Inc.) equipped with a DM-5MS column (60 m × 0.25 mm × 0.25 μm, DiKMA Technol. Inc., Foothill Ranch, CA, USA) (Suppl. Table 2) 41,42 . A mixture of PAH standards (deuterated PAHs) was added to the blank extract and analyzed as previously described. Acenaphthene-d 10 , pyrene-d 10 , and benzo[e]pyrene-d 12 were used as standards for the recovery determination. The recovery efficiency ranged from 72 to 95% and the limits of detection (LOD) ranged from 0.30 to 2.1 ng. To prevent underestimation, when the level of a PAH compound was lower than the LOD value, a LOD/2 value was assigned. The detection rates of ions and PAHs were shown in Suppl. Cell culture, exposure, and viability. A549 cells were purchased from the Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan. The characterization of the A549 cells was performed using short tandem repeat-polymerase chain reaction (PCR) using an AmpFLSTR Identifier PCR amplification kit (Applied Biosystems/Thermo Fisher, Foster City, CA, USA) and DNA profiling was conducted using an ABI 3730 Sequencer (Applied Biosystems Inc.). The cells (within 20 passages) were cultured in a F-12 K nutrient mixture (Kaighn's Modification, Gibco, Thermo-Fisher Scientific Inc.) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific Inc) in a humidified atmosphere at 37 °C with 5% CO 2 . The cells were cultured overnight and then washed with phosphate-buffered saline (PBS) containing 150 mM NaCl, 2.7 mM KCl, 1.3 mM KH 2 PO 4 , and 8.1 mM Na 2 HPO 4 (pH 7.4). They were then exposed to PAHs (in DMSO) or the PM 2.5 extracts (aqueous extract in PBS and dichloromethane extract in DMSO) in the culture medium 43 . The control cells were exposed to the same concentration of PBS and DMSO (0.1%) as in the exhaust extracts/EGCG exposed cells. Cell viability was monitored by measuring the cellular reduction activity toward 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium (MTT) 44 . After overnight culture of 4000 cells/well in a 96-well plate, cells were exposed to exhaust extracts/EGCG for 48 h before cell viability determination. Cell viability was expressed as the relative reduction activity compared to the vehicle control.
Real-time PCR analysis of the reverse-transcript of ribonucleic acid (RNA). Total RNA of cells (6-48-h exhaust extract/EGCG exposure of the overnight culture of 3 × 10 5 cells/well in a 6-well plate) was isolated using TRIzol reagent according to the manufacturer's protocol (Invitrogen Co., CA, USA). RNA samples (2 μg) were subjected to reverse transcription (RT) at 42 °C for 1 h and then at 70 °C for 5 min, using genetically modified M-MuLV reverse transcriptase from the RevertAidTM First Strand cDNA Synthesis kit (Fermantas Inc., MD, USA). Quantitative real-time PCR analysis was then performed using primer sets (Suppl. Table 4) and SYBR Green (Roche Molecular Biochemicals, Mannheim, Germany). The amplification products were analyzed using a LightCycler 480 II/96 (Roche Diagnostics Ltd., Rotkreuz, Switzerland). The amplification conditions were as follows: 2-min pre-incubation at 95 °C followed by 45 cycles of 5 s at 95 °C, 10 s at 60 °C, and 15 s at 72 °C. The quantity of target cDNA in each sample was determined using the fractional PCR threshold cycle number (Ct value). Relative mRNA expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression and the target cDNA was calculated using 2 −(Ct(target) -Ct(GAPDH)) .

IL-6 secretion and ROS determination.
For the determination of the release of IL-6, the medium was collected after exposure and centrifuged at 6000×g for 5 min at 4 °C. The supernatant was subjected to an ELISA using a commercially available ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA). Cellular ROS levels were determined after 6-h pollutant exposure using the Cellular ROS Assay Kit (ab113851; t-butylhydroperoxide License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.