Cytotoxicity Guangren Qian* School of Environmental and

Cytotoxicity Comparison between Fine Particles Emitted from the Combustion of Municipal Solid Waste and BiomassYu Shang, Meiying Wu, Jizhi Zhou, Xing Zhang, Yufang Zhong, Jing An, Guangren Qian*School of Environmental and Chemical Engineering, Shanghai University, 99 Shanghai Road, Shanghai 200444, China*Corresponding author:Guangren Qian, School of Environmental and Chemical Engineering, Shanghai University, Shanghai, China; Tel: 021-66137758; e-mail: [email protected] of interests: none.

Abstract:Fine particles (PM2.5) emitted from municipal solid waste incineration (MSWI) contain high amounts of toxic compounds and are posing a serious threat to human health. In this study, entire particles and extracted water soluble and insoluble parts of PM2.

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5 collected from MSWI and biomass incineration (BMI) were subjected to physiochemical characterization and cytotoxic tests in A549 and BEAS-2B cells. MSWI PM2.5 showed higher content of heavy metals (including Pb, Zn and Cu) and dioxin (PCDD/Fs) when compared with BMI PM2.5. The metals were enriched in water insoluble part, as measured with ICP-AES.

BMI PM2.5 presented a higher content of endotoxin, which is also enriched in insoluble part. Compared with BMI, MSWI PM2.5 caused more serious cell injuries, as indicated by the lower viability, higher ROS generation and DNA damage. BMI PM2.

5 presented increased pro-inflammatory potential, as indicated by increased mRNA induction of interleukin 6. Toxic effects of MSWI and BMI PM2.5 were mostly attributable to their water-insoluble fractions. Our results indicate a relationship existed between the heterogeneity in MSWI and BMI PM2.

5 compositions and their cytotoxicity. Normal human cell line BEAS-2B provided a higher sensitivity for testing viability, DNA damage, ROS response, and cytokine mRNA expression than did A549 cells.Key Words: Municipal solid waste incineration; Biomass incineration; PM2.

5; Cytotoxicity; Oxidative stress 1. IntroductionIncineration is applied throughout the world as one of the main municipal solid waste disposal methods 1. Associations between municipal solid waste incineration (MSWI) emissions and extensive adverse health outcomes (including respiratory diseases, cancer and birth defects) have been observed in epidemiological studies 2, 3.

Fine particulate matter (PM2.5) emitted from MSWI adsorbs plenty of hazardous substances, including sulfur dioxide (SO2), nitrogen oxides (NOx), hydrogen chloride (HCl), carbon monoxide (CO), volatile organic compounds (VOCs), heavy metals (such as Pb, Zn, Cu, Cd, Cr, etc.), and persistent organic pollutants (POPs) (such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs), and polychlorinated biphenyls (PCBs)) 4, 5. Recently, MSWI PM2.5 has been reported to pose a serious threat to human health and is becoming a highly pursued research field 4, 6.

Until now, regular urban PM2.5 has been well characterized, and its health effects and associated mechanisms are also well studied 7, 8. Epidemiological and toxicological studies reveal that adverse health effects of PM2.5 are at least partially attributable to their chemical compositions, including black carbon, metals, organic compounds and biogenic species 9, 10. As an important source for urban ambient particulate pollution, MSWI PM2.

5 is reported to contain higher amounts of metals, organic compounds, and water soluble salts 11, 12. The concentrations of toxic metals, especially Pb and Zn in MSWI PM2.5, are more than thousands of milligrams per kilogram 11, while their concentrations in the PM2.5 of mega cities (like Beijing and Shanghai) in China are always in the range of hundreds of milligrams per kilogram 13, 14. The levels of PCDD/Fs are also higher in MSWI PM2.5 12 than are those detected in common urban atmospheric PM2.

5 15, 16. Because of the completely different physiochemical characterizations, when compared to urban PM2.5, MSWI PM2.

5 could probably cause more serious health endpoints or activate different biological mechanisms.Silvia et al. reported that total MSWI particles (with diameter less than 20 ?m) could promote reactive oxygen species (ROS) generation and could increase the total cellular glutathione (tGSH) content in BEAS-2B human epithelial cells. This particle-induced oxidative stress level was correlated with the induction of the anti-oxidant enzyme heme oxygenase-1 (HO-1) and with the increase in the redox-sensitive transcription factor Nrf2 17.

Cao et al. have investigated the cytotoxic effects caused by MSWI particles with different sizes. They found that the ultrafine and fine fractions (PM0.2 and PM0.2-2.

5) could cause more cell death and could induce more ROS formation than coarse particles (PM2.5-10) 18. Until now, in most toxicological investigations, MSWI particles were always collected and characterized in their entirety (from ultrafine to coarse fractions). Several studies have tried to differentiate the toxic contributions from the water soluble and insoluble parts of ambient particles; however, this mechanism is still far from clear 19, 20.

Thus, further studies are needed that are focused on the fine fraction of MSWI (MSWI PM2.5) and their toxic data that are separately attributable to the water-insoluble and soluble parts.Biomass, mainly including straw, cornstalk, and wood chips, is reported to be one of the components of MSW. In addition, biomass combustion is being increasingly applied as a domestic and regenerative energy source, especially in developed countries 21. Thus, the adverse health effects of PM2.

5 from biomass incineration (BMI) are also becoming an issue of growing concern. BMI PM2.5 could up_regulate the expressions of inflammatory genes in BEAS-2B cells 22. Studies have also found that ultrafine particles from BMI exhibited lower anti-oxidant responses than ultrafine particles from diesel exhaust in BEAS-2B cells 23. BMI PM2.

5 presents lower chlorine, sulfur and metals when compared with MSWI PM2.5 24. Thus, when compared with MSWI PM2.5, BMI PM2.5 could probably result in different toxic endpoints.The present study aims to compare the toxic effects caused by MSWI and BMI PM2.

5 and to differentiate the contributions from the soluble and insoluble parts. To do this, the fine fractions of particles emitted from the electrostatic precipitators of municipal solid waste and biomass incinerator were collected. The entire particles and the extracted water soluble and insoluble parts of MSWI and BMI PM2.5 were subjected to physiochemical characterization, including morphology and typical toxic chemical and biological compositions (metals, dioxins and endotoxin).

The cytotoxic tests were conducted in two in vitro models, human carcinomatous lung epithelial (A549) and normal human bronchial epithelial cells (BEAS-2B). The cell viability, oxidative stress responses and inflammatory potentials of MSWI and BMI PM2.5 were explored and compared.2.

Materials and methods2.1 Particle collectionThe MSWI particles were collected from a bag dust collection in the Shanghai YQ MSWI plant. This plant has a treatment capacity of 2×500 tons per day, with two grate furnaces.

The biomass particles were also collected from bag dust collection in a biomass incineration company in Changsha (China). The treatment capacity of this biomass incinerator is 300 tons per day with a circulating fluidized bed furnace. Usually, the smoke control units of solid waste incinerators are composed of multiple stages, including coarse particle removal, acid gas removal and fine particle removal.

The bag dust collection system is the most effective method to catch the fine particles from incinerator smoke emission. The fine particles captured by bag dust collection are similar to those emitted into the atmosphere both in physical and chemical characterizations.The particles obtained from bag dust collection were size-fractionated into different fractions by a fluidization classifier. The fine particulate fractions with a diameter less than 2.5 ?m, defined as MSWI PM2.

5 and BMI PM2.5, respectively, are used in the following experiment in this study. The particle sizes were found to be less than 2.5 ?m, as shown in Fig.

s1 and Fig. s2. One half of MSWI and BMI PM2.

5 was applied for physiochemical characterizations. The other half were suspended with sterile ultrapure water to 10 mg/mL and stored at -20 °C for cell treatment.Soluble and insoluble extracts of MSWI and BMI PM2.5:A total of 10 g of MSWI or BMI PM2.

5 was mixed with 40 mL sterile ultrapure water under a magnetic stirring of 25 rpm at room temperature for 30 min. The mixture was then centrifuged (3000 rpm, 10 min), and the aqueous supernatant was collected and filtered (0.22 ?m) as the water-soluble extracts. The precipitate was the water-insoluble extracts. A portion of these extracts were used for chemical analysis. Another portion was freeze-dried, weighed and diluted to a 10 mg/mL stock solution using sterile ultrapure water and stored in -20 °C for cell treatment.

2.2 Physicochemical CharacterizationsScanning electron microscopy (SEM, JSM-7500F, Japan) combined with energy dispersive X-ray (EDX) spectroscopy were used to analyze morphological properties and associated qualitative chemical compositions. Before the SEM/EDX analysis, a gold film with 10 nm thickness on average was coated on the samples in a vacuum coating machine.

Energy-dispersive X-ray fluorescence spectrometer (XRF-1800, Shimadzu Limited, Japan) was further used to determine the elemental compositions.Inductively coupled plasma-atomic emission spectrometry (ICP-AES, Leeman, USA) was applied to determine the concentrations of heavy metals (including Pb, Zn, Cu, Cr, Cd, and Ni). Prior to the ICP analysis, approximately 0.

2 g of each MSWI or BMI PM2.5 was digested using HNO3, HF and HClO4 in a heat-dispelling furnace. Digested samples were filtered and diluted with 5% HNO3 to a final volume of 50 mL.Dioxins analyses of the MSWI and BMI PM2.5 were delegated to CCIC physical and chemical testing company (CCICLAB).Endotoxins of the MSWI and BMI PM2.

5 was detected with chromogenic end-point tachypleus amebocyte lysate (CE TAL, Xiamen, China) according to the manufacturer’s instructions.2.3 Cell culture and treatmentA human adenocarcinoma alveolar basal epithelial cell line (A549) and a bronchial epithelial cell line (BEAS-2B) were chosen for the cytotoxic evaluation. The cells were cultured in DMEM modified with high glucose supplemented with L-Glutamine and pyruvate (Life Technologies, Karlsruhe, Germany), 10% (v/v) fetal bovine serum (FBS) (Gibco, UK) and 100 U/mL penicillin/streptomycin (Invitrogen, Darmstadt, Germany) at 37 °C, 5% CO2 (v/v) in humidified air.

In each cell treatment, the stock solution (10 mg/mL) of the total, insoluble and soluble fractions of MSWI and BMI PM2.5 were unfrozen, sonicated to disperse equally and diluted into different concentrations (0, 25, 50, 100, 200 ?g/mL) with the culture medium.2.4 Cytotoxicity testsCell viability was analyzed using the MTT method; the oxidative stress was evaluated by the ROS generation and detected using 2,7-dichlorofluorescein-diacetate (H2DCFH-DA) fluorescent probe (Beyotime, China) according to previous studies 25, 26. DNA damage in A549 and BEAS-2B cells were measured with the Comet assay.

The %tail DNA was selected as the parameter to estimate DNA damage 27, 28. Cell apoptosis was measured using the flow cytometry method with the Annexin V-FITC and Propidium Iodide probes according to the manufacturer’s instructions (Beyotime, China). Gene expressions were analyzed with the reverse transcription – quantitative polymerase chain reaction (RT-qPCR). The detailed information for these methods were described in the Supplementary Materials (SM).

2.5 Statistical analysisAll the data were presented as the mean ± standard error of the mean. A t-test was conducted using Microsoft Excel 2007 to examine the differences between various groups. Differences were considered statistically significant if the p value was less than 0.05.3.

Results3.1 Physicochemical characterizationsThe morphology of MSWI and BMI PM2.5 were measured with the SEM method, and the images are shown in Fig. 1.

MSWI and BMI PM2.5 were observed to be both irregular and conglobatus. Some rods were found in MSWI PM2.5 but not in BMI PM2.5. The EDX spectra (Fig.

1 c and f) mainly showed the energy reflection of Cl, Zn, Pb, Ca, Fe, Al, S, and O. After washing with pure water, the percentages of water-soluble fractions in MSWI and BMI PM2.5 were approximately 29.

8% and 3.7% (m/m), respectively (Table 1). Lima et al. (2008) presented a dissolution of nearly 24% in MSW fly ash 29. The water washing process can effectively remove soluble chlorides such as NaCl, KCl and CaCl2 from MSWI fly ash 30.The chemical and biological compositions of the entire, water soluble and insoluble fractions of MSWI and BMI PM2.

5 are shown in Table 1. The concentrations of Pb, Zn, Cu and Cd in MSWI PM2.5 were respectively 4.4, 6.

3, 23.9 and 1.6 times higher than those in BMI PM2.5, and these metals were found to be mainly enriched in the water-insoluble fractions. Meanwhile, there were higher levels of Pb, Zn, Cu and Cd in the water-insoluble fractions of MSWI PM2.5 compared with those of BMI PM2.

5. Meanwhile, in water-soluble fraction of MSWI PM2.5, Pb and Zn were also higher than those in water-soluble fraction of BMI PM2.5. The observed results were similar to those obtained when measuring with the XRF method, as shown in the SM, Table s2.The organic and biogenic compounds were also measured in MSWI and BMI PM2.

5. The toxic equivalent quantity (TEQ) of the total dioxins in MSWI PM2.5 was 7.6 times higher than those in BMI PM2.

5 (Table 1). In contrast, it showed that the endotoxin level in BMI PM2.5 was slightly higher than that in MSWI PM2.5. In addition, the endotoxin was also enriched in the water-insoluble part.3.2 Biological responses3.2.1 Cytotoxicity comparison between MSWI and BMI PM2.5The cell proliferation, apoptosis rate, and DNA damage were measured to evaluate the cytotoxic effects induced by MSWI and BMI PM2.5. Fig. 2a shows that a significantly dose-dependent viability decrease was observed in A549 and BEAS-2B cells after treatment with both MSWI and BMI PM2.5 (ANOVA test, p


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