Atomization method for verifying size effects of ...

STOTEN-21454; No of Pages 9 Science of the Total Environment xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Short Communication

Atomization method for verifying size effects of inhalable particles on lung damage of mice Chen Tao, Yue Tang, Lan Zhang, Yonggang Tian, Yingmei Zhang ⁎ Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000, China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• The new two-stage atomization device is used for in vivo inhalation experiment. • Aqueous aerosol containing cadmium was studied as a model. • The new device could control the particle size distribution of the aqueous aerosol. • Correlation coefficient between PM2.5 and lung damage was higher than that of PM10.

a r t i c l e

i n f o

Article history: Received 24 September 2016 Received in revised form 18 November 2016 Accepted 21 November 2016 Available online xxxx Editor: D. Barcelo Keywords: Pneumatic atomization Ultrasonic atomization Cadmium Inhalation Endoplasmic reticulum stress Inflammatory response

a b s t r a c t To explore the size effects of inhalable particles on lung damage, aqueous aerosol containing cadmium was studied as a model to design a new type of two-stage atomization device that was composed of two adjustable parts with electronic ultrasonic atomization and pneumatic atomization. The working parameters and effectiveness of this device were tested with H2O atomization and CdCl2 inhalation, respectively. By gravimetrically detecting the mass concentrations of PM2.5 and PM10 and analysing the particle size with a laser sensor, we confirmed the particle size distribution of the aqueous aerosol produced by the new device under different working conditions. Then, we conducted experiments in male Kunming mice that inhaled CdCl2 to determine the size effects of inhalable particles on lung damage and to confirm the effectiveness of the device. The new device could effectively control the particle size in the aqueous aerosol. The inhaled CdCl2 entered and injured the lungs of the mice by causing tissue damage, oxidative stress, increasing endoplasmic reticulum stress and triggering an inflammatory response, which might be related to where the particles deposited. The smaller particles in the aqueous aerosol atomized by the new two-stage atomization device deposited deeper into lung causing more damage. This device could provide a new method for animal experiments involving inhalation with water-soluble toxins. © 2016 Published by Elsevier B.V.

1. Introduction ⁎ Corresponding author. E-mail addresses: [email protected] (C. Tao), [email protected] (Y. Tang), [email protected] (L. Zhang), [email protected] (Y. Tian), [email protected] (Y. Zhang).

Aqueous aerosol treatment is one application of liquid atomization technology in inhalation toxicology studies (Oakes et al., 2014; Oakes et al., 2013). Currently, liquid atomization can be summarized as three

http://dx.doi.org/10.1016/j.scitotenv.2016.11.150 0048-9697/© 2016 Published by Elsevier B.V.

Please cite this article as: Tao, C., et al., Atomization method for verifying size effects of inhalable particles on lung damage of mice, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.150

2

C. Tao et al. / Science of the Total Environment xxx (2016) xxx–xxx

Fig. 1. The new type of two-stage atomization device developed in this study. Radiator (A), Electronic ultrasonic atomizer (B), Liquid level switch (C), Liquid inlet (D), Air entry (E), Compressed air inlet (F), Nozzle (G), Nylon net (H), Collecting tank (I), Shell (J).

types: mechanical atomization, pneumatic atomization (Mayer, 1961), and ultrasonic atomization (Patil, 1999). In mechanical atomization, liquid pressurization jets work at high speed to produce atomization without adding the additional energy of an atomizing agent. As a result, the average particle size after atomization is too large to enter into the lungs of humans or other animals. Pneumatic atomization uses pressurized gas as an energy carrier to impact or tear liquid in various ways for atomization, but the particle size distribution is fixed and uncontrolled while maintaining a certain atomization amount due to the structure of a single spray nozzle (Sovani et al., 2001). Ultrasonic atomization uses ultrasound to strengthen decomposition of liquid for atomization, which includes two types of power sources. One is electron acoustic energy transfer in which a transducer converts electrical energy into ultrasonic energy and the atomization amount is adjusted through changes in voltage (Lang, 1962). The other is the hydrodynamic type that uses fluid at a high velocity as a power source while simultaneously inducing ultrasonic energy transfer (Sun, 2004). Although electronic ultrasonic atomization and pneumatic atomization have been used for clinical respiratory medicine (Ibrahim et al., 2015), particles in the aqueous aerosol produced by electronic ultrasonic atomization are larger than particles produced by pneumatic atomization (Rau, 2002). Therefore, pneumatic atomization is more widely used in inhalation toxicology studies (Szkudlarek et al., 2004; Ge et al., 2016). Inhalation toxicology studies present some unique issues because the dosing method differs from oral or injection administration (Wolff, 2015). The particle size is an important characteristic for inhalation delivery. Particle size distribution also determines the mass concentration of particles of specific diameters, such as PM2.5 or PM10. Treatment time and drug concentration in an atomized liquid, rather than particle size distribution of aqueous aerosol (Szkudlarek et al., 2004; Ge et al., 2016), are common variables in inhalation exposure experiments because of limitations based on the uncontrollability of traditional pneumatic atomization (Sovani et al., 2001). Therefore, the development of a new type of atomization device is necessary for simultaneously controlling inhalable particle sizes and mass concentrations. Normally, both PM2.5 and PM10 could be inhaled into the lungs, but the inhalation of PM2.5 is easier than PM10. Consequently, more attention should be given to the association between PM2.5 and cardiopneumatic diseases (Miller et al., 2007). Cadmium (Cd) is a widespread heavy metal in natural environments (Flora et al., 2008) and is often found in the atmosphere, which could cause concentration-dependent acute or chronic poisoning in organisms through inhalation (Anetor, 2012). In industries involved with Cd mining and refining or processing of used batteries, particles containing Cd could be inhaled into the lungs. Due to its long-term biological halflife, Cd could also accumulate in the body (Ercal et al., 2001; Waisberg et al., 2003; Chen et al., 2013). The main toxicity of inhalable particles containing Cd is caused by the induction of oxidative stress that leads to

elevated malondialdehyde (MDA) (Donaldson and Stone, 2003) and reactive oxygen species levels (Wei et al., 2014), as well as by the induction of endoplasmic reticulum (ER) stress (Chen et al., 2016), and a link-coupled inflammatory response (Ribeiro et al., 2005). Water mist in a natural environment could combine with Cd in the atmosphere, which comes from pollution events through wind action that forms aqueous aerosol containing Cd. For this study, droplets containing Cd were used to simulate inhalable toxic particles in the atmosphere to elicit lung damage in an animal model. However, because of the lack of control over particle size distribution of aqueous aerosol, few systematic studies have reported the size effects of inhaled particles in aqueous aerosol containing Cd or other toxins. Therefore, it was necessary to develop an instrument that could produce various size distributions of particles in aqueous aerosol for Cd or other environment pollutants. Some research studies of computational fluid dynamics (CFD) of lung airway models in vitro indicate that particle size can influence particle deposition in lung airways (Zhang et al., 2002; Longest and Xi, 2007; Hofmann et al., 2005), and epidemiological surveys have shown that cardiopneumatic diseases are closely related to PM2.5 (Nordberg, 2009; Nemmar et al., 2002; Rogers and Dunlop, 2006); however, those studies were analogue computations isolated from in vivo experiments. To detect the biological effects of particles in the aqueous aerosol containing Cd with different particle sizes inhaled via autonomous breathing, we have developed a new type of two-stage atomization device and have used it on male Kunming mice to demonstrate the size effects of inhaled aqueous aerosol particles containing Cd in vivo. 2. Materials and methods 2.1. Generation and detection of aqueous aerosol containing CdCl2 2.1.1. Devices used in CdCl2 disposal experiments The new two-stage atomization device (Fig. 1) was composed of electronic ultrasonic atomization (Fig. 1A–E) and pneumatic

Table 1 Primers sequences used for Real-Time PCR reactions. Genes

Primer pairs

ATF-6

Upper:5′-GAGCAAGAATCCCGAAGAGT-3′ Lower:5′-CAGGGGTTGACATGGAGGTG-3′ Upper:5′-TCCCTGCCTTTCACCTTG-3′ Lower:5′-CGTTCTCCTGCTCCTTCTC-3′ Upper:5′-CAACGGCATGGATCTCAAAGAC-3′ Lower:5′-AGATAGCAAATCGGCTGACGGT-3′ Upper:5′-ATCCAGTTGCCTTCTTGGGACTGA-3′ Lower:5′-TAAGCCTCCGACTTGTGAAGTGGT-3′ Upper:5′-AGTATGATGACATCAAGAAGG-3′ Lower:5′-ATGGTATTCAAGAGAGTAGGG-3′

Chop TNF-α IL-6 GAPDH

Please cite this article as: Tao, C., et al., Atomization method for verifying size effects of inhalable particles on lung damage of mice, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.150

C. Tao et al. / Science of the Total Environment xxx (2016) xxx–xxx

3

Two-stage atomization was processed with different air velocities and input voltages of an electronic ultrasonic atomizer. Both the pneumatic atomization part in the new device and the traditional single pneumatic atomization were operated with the same medical air compressor QW (WBL Medical, Chengdu, China) (Supplemental Fig. 1) to ensure comparability. Traditional single pneumatic atomization is widely used in clinical procedures and was used as a positive control for the new two-stage atomization. The air velocity of QW was consistently maintained at 5 L min−1 in traditional single pneumatic atomization for proper functioning but could be adjusted in two-stage atomization with a gas flow switch.

Fig. 2. Air background including number and mass concentration of particles. In air, small particles are more numerous than big particles. PM2.5 includes particles whose sizes are less than or equal to 2.5 μm and PM10 includes particles whose sizes are less than or equal to 10 μm.

atomization (Fig. 1F and G) parts. A radiator (Fig. 1A) refrigerated the electronic ultrasonic atomizer (Fig. 1B), which had power or input voltage adjusted by an electronic ultrasonic power controller with a rated input voltage of 12 V. The resonant frequency of the electronic ultrasonic atomizer was 1.7 MHz. A peristaltic pump controlled by a liquid level switch (Fig. 1C) was used to deliver liquid into the electronic ultrasonic atomization part through a liquid inlet (Fig. 1D). A blower received air through an air entry (Fig. 1E) and was used to deliver aqueous aerosol produced by the electronic ultrasonic atomization part into the pneumatic atomization part. Compressed air through the inlet (Fig. 1F) was used for pneumatic atomization through a nozzle (Fig. 1G) with a hole diameter of 0.8 mm. Room-temperature aqueous aerosol was filtered and collected by a mipor nylon net (Fig. 1H) with a hole diameter of 1.0 μm and was stored by the tank (Fig. 1I). The last aerosol was adsorbed by an allochroic silica gel microballoon in a shell (Fig. 1J). The volume of exposure chamber is 2.5 L.

2.1.2. Detection of the mass concentrations of PM2.5 and PM10 Mass concentrations of PM2.5 and PM10 after atomization were detected by DT-9881M (CEM, Shenzhen, China). Aqueous aerosol passed through a polystyrene mipor filter membrane and the change of filter mass was gravimetrically detected to meet particular criteria (HJ 6182011, China), which described how to measure the mass concentration of PM2.5 and PM10 in the air. 2.1.3. Detection of particle size distribution Particles in the aqueous aerosol were counted in six channels (0.3, 0.5, 1, 2.5, 5, 10 μm) by DT-9881M to analyze particle size distribution. The particles went through a laser device, diffraction light signals were collected by a sensor and the particle numbers per litre for the different diameters were recorded with a microcomputer. All processes of measurement conformed to particular criteria (GB/T 19077-2016, China), which described how to analyze particle size by laser diffraction. 2.2. Materials and animals CdCl2 (99% purity) was purchased from Tianjin Chemical Reagent Plant (Tianjin, China). RNAiso Plus (9108), the RT reagent Kit with gDNA Eraser (RR047A) and SYBR green master mix (RR820A) were purchased from Takara Biotechnology (Dalian, China). All other chemicals

Fig. 3. The mass concentrations of PM2.5 and PM10 in different operating conditions (A) and simultaneous particle size distribution in aqueous aerosol (B). The mass concentrations and size distribution of particles were all detected in the nozzle (Fig. 2G) and they could be adjusted in two-stage atomization. The operations (B) are also applied in the next inhalation exposure to mice. The different single-letter labels such as ‘a’ and ‘b’ mean significant differences between groups (p b 0.05) while same-letter labels mean no significant differences between groups such as ‘A’ and ‘AD’ (p N 0.05).

Please cite this article as: Tao, C., et al., Atomization method for verifying size effects of inhalable particles on lung damage of mice, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.150

4

C. Tao et al. / Science of the Total Environment xxx (2016) xxx–xxx

were analytical grade. Male Kunming mice (6–8 weeks old) were purchased from the Experimental Animal Centre of Lanzhou University (Lanzhou, China). All mice were acclimated in the laboratory for one week before the experiments and were kept at 22–24 °C with an alternating 12 h light/dark cycle. Food and water were available ad libitum. 2.3. Inhalation exposure In a preliminary experiment, the mice inhaled different concentrations of CdCl2 (0, 0.5, 1, 2.5, 5, 10, 25, or 50 mM 2 h/day) for 6 days using the devices (Fig. 1). More than half of the mice inhaling CdCl2 (≥25 mM) died, and their bodies developed a red colour. None of the mice inhaling CdCl2 (≤ 5 mM) died, so 5 mM CdCl2 was selected for the following experiments. Mice were randomly divided into six groups, and each group contained eight mice. Five aqueous aerosols with particles of different diameters were inhaled by the mice from the five respective groups. Control group was treated by inhalation exposure with air only. Following inhalation exposure, the mice were sacrificed after heart perfusion, and their lungs were harvested. All the experiments with animals were carried out according to the guidelines of the Institutional Animal Ethical Committee (IAEC). The protocol was

approved by the Committee on the Ethics of Animal Experiments of Lanzhou University. 2.4. Measurement of lung damage in mice 2.4.1. Measurement of the lung coefficient The fasted pretreated mice that inhaled CdCl2 were weighed. The lungs were dissected from the mice after they were treated with an anaesthetic. Blood was removed from the surface of the dissected lungs with neutral filter paper and the lungs were weighed. The lung coefficient was calculated by taking the lung wet weight and dividing it by the wet weight of the mice who received different Cd treatments. The lung coefficient can reflect the degree of lung damage (Zhao et al., 2016). 2.4.2. Measurement of Cd concentration in the lungs of mice The lungs of the mice weighed 0.5 g before digested with 10 mL mixed acid digestion (Nitrate, Perchlorate = 4:1 (v/v)) in a DK20 digestion furnace (VELP, Milan, Italy). Then, Cd concentration in the sample was detected using a ZEEnit-700P atomic absorption

Fig. 4. Particle numbers in certain sizes which included 0.3, 0.5, 1, 2.5, 5, 10 μm changed with time and were detected in chamber (while operation was 12 V, 5 L min−1). These data are used to reflect mobility and stability of aqueous aerosol in airtight exposure chamber (Volume = 2.5 L). The different single-letter labels such as ‘a’ and ‘b’ mean significant differences between groups (p b 0.05) while same-letter labels mean no significant differences between groups such as ‘a’ and ‘ab’ (p N 0.05).

Please cite this article as: Tao, C., et al., Atomization method for verifying size effects of inhalable particles on lung damage of mice, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.150

C. Tao et al. / Science of the Total Environment xxx (2016) xxx–xxx

5

Fig. 5. Mass concentrations of PM2.5 and PM10 changed with time in chamber and came to be stabilized after 5 min (while operation was 12 V, 5 L min−1) (A). Collection of aqueous aerosol by the device (Fig. 2H, I and J) (B). The different single-letter labels such as ‘a’ and ‘b’ mean significant differences between groups (p b 0.05) while same-letter labels mean no significant differences between groups such as ‘a’ and ‘ab’ (p N 0.05).

spectrophotometer (Analytic Jena AG, Jena, Germany). All measurement processes conformed to particular criteria (GB/T5009. 15-2003, China)

groups (p b 0.05) while same-letter labels mean no significant differences between groups such as ‘a’ and ‘ab’ (p N 0.05). 3. Results

2.4.3. Measurement of MDA The rest of the excised lungs were used for extraction at a low temperature with an animal tissue lysis kit (E061; Nanjing Jiancheng, Nanjing, China). The lungs that weighed 0.1 g beforehand were treated with 1 mL extraction buffer. The extract was used to measure MDA with a detection kit (S0131; Beyotime, Shanghai, China). The 100 μL of the extract was added to 200 μL MDA detecting solution and the reaction mixture was added to a boiling water bath for 15 min. Then, the mixture was centrifuged at 1000g for 10 min and the absorbance at 532 nm was determined. 2.4.4. Histological and morphological analyses After heart perfusion, the mouse lungs were excised and fixed overnight in 10% neutral formaldehyde at room temperature. Fixed tissues were processed for paraffin embedding, and serial 5 μm thick sections were prepared and stained with haematoxylin/eosin (HE; Nanjing Jiancheng, Nanjing, China) to assess pulmonary alveoli histology and the morphology of the lungs from the mice. The histology and morphology of the lungs were scored using a Carl-Zeiss Axiovert 200 microscope (Carl-Zeiss, Jena, Germany) and a computer-assisted image analysis program (AxioVision Ver. 4.0; Carl-Zeiss) at 200× magnification.

3.1. The mass concentrations of PM2.5 and PM10 under different operating conditions and simultaneous particle size distribution in the aqueous aerosol The air background included the number and mass concentration of particles (Fig. 2). In air, small particles are more numerous than big particles. PM2.5 includes particles whose sizes are less than or equal to 2.5 μm and PM10 includes particles whose sizes are less than or equal to 10 μm. With the constant input voltage of electronic ultrasonic atomization in the new device, the mass concentrations of PM10 significantly increased (p b 0.05) with a decrease in the air velocity of pneumatic atomization of the new device. Meanwhile, the decrease of mass concentration for PM2.5 was significant (p b 0.05) as well (Fig. 3A). With the constant air velocity of pneumatic atomization in the new device and the decrease of the input voltage of electronic ultrasonic atomization in the new device, mass concentrations of PM10 and PM2.5 significantly decreased (p b 0.05) (Fig. 3A). In some cases of concomitant air velocity and input voltages (Fig. 3), the mass concentration of

2.4.5. Real-Time PCR The frozen lung tissue samples were homogenized using 1 mL of RNAiso Plus reagent per 100 mg of sample. Total RNA was extracted from the tissue according to the protocol of the manufacturer. RNA pellets were dissolved in 70 μL diethylpyrocarbonate (DEPC)-treated water, and the total RNA concentration was determined by spectrophotometric analysis at 260 nm. Total RNA was converted into cDNA using the RT reagent Kit with gDNA Eraser, and cDNA was amplified with the SYBR green master mix using a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, USA). The 2− ΔCT method was used (Livak and Schmittgen, 2001). The PCR primers for IL-6, TNF-a, ATF-6, and Chop were designed online with Primer-Blast from the National Centre of Biotechnology Information (NCBI, USA). The primer pairs are shown in Table 1. 2.5. Statistical analysis Data were expressed as the mean ± SD. Statistical comparisons were made using a one-way analysis of variance (ANOVA) with the FisherLSD test. Pearson's correlation analysis was used. All statistical analyses were performed using Originpro 8.0 (OriginLab, USA). The different single-letter labels such as ‘a’ and ‘b’ mean significant differences between

Fig. 6. Mass concentrations of Cd in lungs of mice after exposure for 6 days. Control group was treated by inhalation exposure with air only. QW group meant inhalation treatment with traditional single pneumatic atomization, other exposures were accomplished by the new device in different operations (Fig. 3B) such as 12 V, 5 L min−1 et al. The different single-letter labels such as ‘a’ and ‘b’ mean significant differences between groups (p b 0.05) while same-letter labels mean no significant differences between groups such as ‘a’ and ‘ab’ (p N 0.05).

Please cite this article as: Tao, C., et al., Atomization method for verifying size effects of inhalable particles on lung damage of mice, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.150

6

C. Tao et al. / Science of the Total Environment xxx (2016) xxx–xxx

Fig. 7. Lung coefficient of mice (A) and MDA level in lungs of mice (B). Control group was treated by inhalation exposure with air only. QW group meant inhalation treatment with traditional single pneumatic atomization, other exposures were accomplished by the new device in different operations (Fig. 3B) such as 12 V, 5 L min−1 et al. The different singleletter labels such as ‘a’ and ‘b’ mean significant differences between groups (p b 0.05) while same-letter labels mean no significant differences between groups such as ‘a’ and ‘ab’ (p N 0.05).

PM2.5 was significantly different (p b 0.05) from each other because the numbers of particles in 1 and 2.5 μm diameters were significantly different (p b 0.05) (Fig. 3B). The traditional single pneumatic atomization and the partial pneumatic atomization of the new device were accomplished using the same medical air compressor QW (Supplemental Fig. 1). In the process of inhalation exposure, the mass concentration and the number of particles in the chamber (Fig. 1) were detected to mirror mobility and stability of aqueous aerosol in the new device. The operation (12 V, 5 L min−1) was representative because the induced mass concentration of PM2.5 was significantly higher (p b 0.05), making collision and combination of particles more difficult. Particle size distribution affected mass concentration of PM2.5 and PM10 and the results (Fig. 4) showed that particle numbers with certain sizes, which included 0.3, 0.5, 1, 2.5, 5, and 10 μm, changed over time gradually stabilizing (Time ≥ 5 min). The mass concentration of PM2.5 and PM10 stabilized as well (Fig. 5A). This data showed that mobility and stability of aqueous aerosol in the new device were suitable for inhalation exposure. Collection of the aqueous aerosol containing Cd was accomplished with a new device (Fig. 1H, I and J) designed to reduce hazards to the environment. The mass concentration of the aqueous aerosol significantly decreased (p b 0.05) after the collection treatment (Fig. 5B).

3.2. Lung coefficient variation of mice induced by the aqueous aerosol containing Cd with rising MDA accumulation The aqueous aerosol containing Cd atomized by the new device could be inhaled into the lungs. Mass concentrations of Cd in the lungs of mice were significantly different (p b 0.05) with different settings for the new device or exposure from traditional single pneumatic atomization (Fig. 6) with increasing concentrations over time (Supplemental Fig. 2). The lung coefficients of the mice increased with the rising mass concentration of Cd in the lungs (Fig. 7A). The redness and roughness of the pulmonary surface observed in dissection increased as well (Supplemental Fig. 3). The MDA levels in the lungs of mice also increased with increasing mass concentrations of Cd in the lungs (Fig. 7B).

3.3. Histological and morphological damage in the lungs of mice induced by the aqueous aerosol containing Cd Aqueous aerosol containing Cd atomized by the new device could induce increased pulmonary parenchymas in mice. The pulmonary parenchyma in mice exposed to either traditional single pneumatic

Fig. 8. Histological and morphological damages in lungs of mice. (A) Control treated by inhalation exposure with air only, (B) Group treated under QW, (C) Group treated under 12 V, 5 L min−1, (D) Group treated under 12 V, 4 L min−1, (E) Group treated under 10 V, 4 L min−1, (F) Group treated under 6 V, 3 L min−1. Arrows indicated increasing pulmonary parenchymas in mice. Inhalation exposures in different operations induced different levels of damages in lungs of mice.

Please cite this article as: Tao, C., et al., Atomization method for verifying size effects of inhalable particles on lung damage of mice, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.150

C. Tao et al. / Science of the Total Environment xxx (2016) xxx–xxx

7

Fig. 9. Detection of pulmonary ATF-6 (A), Chop (B), TNF-α (C) and IL-6 mRNAs (D). Control group was treated by inhalation exposure with air only. QW group meant inhalation treatment with traditional single pneumatic atomization, other exposures were accomplished by the new device in different operations (Fig. 3B) such as 12 V, 5 L min−1 et al. The different singleletter labels such as ‘a’ and ‘b’ mean significant differences between groups (p b 0.05) while same-letter labels mean no significant differences between groups such as ‘a’ and ‘ab’ (p N 0.05).

atomization powered by only QW or exposed to different operations with the new device were different. The rise in pulmonary parenchyma induced a decrease in the effective volume of pulmonary alveoli, which was adverse to the lungs of the mice (Fig. 8). 3.4. Detection of pulmonary ATF-6, Chop, TNF-α and IL-6 mRNAs Activating transcription factor 6 (ATF-6) and C/EBP-homologous protein (CHOP) are positively related to ER stress in cells (Yoshida et al., 2001). Tumor necrosis factor-α (TNF-α) and interleukin-6

(IL-6) are positively related to the inflammatory response (Lü, 2016). The aqueous aerosol containing Cd atomized by the new device could induce an increase in pulmonary ATF-6, Chop, TNF-α, and IL6 mRNAs in mice. The levels of these pulmonary related mRNAs in mice exposed to either traditional single pneumatic atomization powered by only QW or treated with different operations with the new device were different. The rise in pulmonary ATF-6 and Chop mRNAs reflected an increase in ER stress. In addition, the rise in pulmonary TNF-α and IL-6 mRNAs also reflected an inflammatory response (Fig. 9).

Fig. 10. Pearson correlation analysis between aqueous aerosol containing Cd and lung damages of mice. The deeper the blue, the lower the Pearson correlation. The deeper the black, the higher the significance. Pearson correlations were considered significant (p b 0.05) such as data between ‘PM2.5’ and ‘IL-6 mRNA level’ (the number is gray). Pearson correlations were considered highly significant (p b 0.01) such as data between ‘PM2.5’ and ‘Cd concentration’ which was detected in lungs of mice rather than the inhalation solution (the number is black). Pearson correlations were not considered significant (p N 0.05) such as data between ‘PM10’ and ‘MDA level’ (the number is white). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Tao, C., et al., Atomization method for verifying size effects of inhalable particles on lung damage of mice, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.150

8

C. Tao et al. / Science of the Total Environment xxx (2016) xxx–xxx

3.5. Correlation analysis between the particle size of the aqueous aerosol containing Cd and lung damage of mice Particles in the air could be divided into total suspended particulates (TSP), inhalable particulates (namely thoracic particulates, PM10) and respirable particulates (PM2.5). By definition, PM2.5 and PM10 in the aqueous aerosol could be simultaneously inhaled because PM2.5 is a portion of PM10, but the two had different correlations in the results related to lung damage in mice for Cd concentrations (Fig. 6), lung coefficients (Fig. 7A), MDA levels (Fig. 7B) and related mRNA levels (Fig. 9). Additionally, correlation coefficients between PM2.5 and the results related to the lung damage of mice were significantly higher (p b 0.05) than the correlation coefficients of PM10 (Fig. 10). 4. Discussion The electronic ultrasonic atomizer in this new type of two-stage atomization device could be used to separately adjust the mass concentration of aqueous aerosol according to the input voltage, which was positively related to the atomization rate. The resonant frequency of the atomizer was inversely related to particle sizes in the aerosol (Lang, 1962), but the resonant frequency was immutable after atomizer was manufactured. Pneumatic atomization part in this new type of twostage atomization device could separately adjust particle size distribution according to the principle of fluid dynamics, i.e., the air velocity increased with a decrease in the cross-sectional area under the same volume flow (Christophe, 2008), and the compressed air could tear droplets produced by electronic ultrasonic atomization in the narrow nozzle of the pneumatic atomization part in this new type of twostage atomization device, as well as change the mass concentrations of particles in the specific size distribution. Therefore, the particles produced by the new type of two-stage atomization device could conform to the process in single pneumatic atomization or electronic ultrasonic atomization. Consistent with this outcome, the mass concentrations of the aqueous aerosol produced by the new device were closely related to the input voltage or air velocity. For maintaining the stability and durability of the electronic ultrasonic atomizer in the new device, we detected the particle size and tested mice below 12 V, which was the rated input voltage of the electronic ultrasonic atomizer. The particle number decreased when the particle size increased. However, this observation was reversed from 0.3 to 0.5 μm, and especially from 5 to 10 μm (Fig. 3B). This opposite trend might be caused during the generation process of aqueous aerosol. In this experiment, particles in aqueous aerosol were from two-stage atomization namely primary atomization (electronic ultrasonic atomization) and secondary atomization (pneumatic atomization). The big droplets from electronic ultrasonic atomization could be crushed for generating small droplets by compressed air in pneumatic atomization. However, the limited crushing capacity of compressed air at a certain air velocity could not afford to crush particularly small particles. This might cause the 0.5 μm sized particles to be more numerous than 0.3 μm sized particles. Meanwhile, the generated droplets could also collide and combine with each other in the air or the narrow nozzle of pneumatic atomization (Fig. 1G). The particles in 5 and 10 μm were minorities in total particles but partial particles in 5 μm could form partial particles in 10 μm through collision and combination, so, the number of particles in 10 μm was more than that of particles in 5 μm. Compared with unadjustable traditional single pneumatic atomization (Robichaud et al., 2015; Guillon et al., 2015), the new device was more adaptable and had obvious advantages for the control of mass concentration or particle size distribution of the aqueous aerosol. Meanwhile, particles accumulated in the PM2.5 range were easier to inhale (Heidi, 2000), and our detection experiments showed that the new device could produce the aqueous aerosol for inhalation with more control than traditional single pneumatic atomization. Previous reports showed that environmental exposure of Cd was carcinogenic to human beings

(Waalkes, 2003; Satarug et al., 2003), and could induce oxidative stress in humans and mice (Odewumi et al., 2015; Surolia et al., 2015), elevate ER stress (Chen et al., 2016), as well as increase the inflammatory response (Nordberg, 2009). We used these phenomena to show the size effects of particles in the aqueous aerosol containing Cd and tested the effectiveness of the new device. For our inhalation exposures, only male mice were used as the model species because cadmium had estrogenic-like effect (Garcia-Morales et al., 1994). The results in the lungs of the mice were consistent with the previous studies mentioned. These results suggested that our new device was effective. PM2.5 had a higher correlation with the lung damage of mice treated with the aqueous aerosol containing Cd, indicating PM2.5 deposited in the lungs of mice more easily than PM10. ATF-6 had a highly significant correlation with Chop (Pearson Corr. = 1, p b 0.05) because ATF-6 was located upstream of Chop in the molecular signalling pathways of ER stress (Oyadomari and Mori, 2004). TNF-α also had a highly significant correlation with IL-6 (Pearson Corr. = 0.99796, p b 0.05) because TNF-α was located upstream of IL-6 in the molecular signalling pathways of the inflammatory response (Zimmermann et al., 2015). Combined with the genes' relationship between upstream and downstream in molecular signalling pathways, the correlation analysis showed that ER stress and inflammatory response happened. We measured the concentration of Cd in the lungs of mice to determine whether Cd could be inhaled. The concentration of Cd in the lungs dramatically increased over time compared to the control, but the differences among the groups did not correspond well to the treatment time of the aqueous aerosol containing Cd. This is probably because partial Cd was transported into other tissues through pulmonary capillaries, as shown in the Supplementary material (Supplemental Fig. 2) and in a previous study (Schöpfer et al., 2010). MDA as a lipid peroxide could be naturally removed by an antioxidant system in organisms, while its level could increase with the increase of ROS. Therefore, MDA is used as a marker for oxidative stress (Noll et al., 1987). Our results for the lung coefficient and MDA levels in the lungs of the mice coincided with previous research (Odewumi et al., 2015; Surolia et al., 2015), which suggests that the new device is reliable. CFD in the in vitro lung airway model (Hofmann et al., 2005; Nordberg, 2009; Nemmar et al., 2002), and in epidemiological surveys indicated that particle size influenced particle deposition in the lung airways (Rogers and Dunlop, 2006; Zhao et al., 2016; Livak and Schmittgen, 2001). We showed this effect in vivo with new two-stage atomization technology. The results and new method in this paper could provide a new prospect for a better understanding of the formation mechanism of lung diseases induced by the aqueous aerosol containing Cd.

5. Conclusions Our results show that this new device can adjust the particle size of the aqueous aerosol. The input voltage of the electronic ultrasonic atomizer and the air velocity of pneumatic atomization in the new device influenced particle size. Upon the inhalation exposure of mice to the aqueous aerosol containing Cd with certain input voltages and air velocities, oxidative stress, ER stress, lung damage and inflammation were closely correlated with the particle size of the aqueous aerosol, specifically PM2.5, which was easily deposited in the lungs. In conclusion, this new type of two-stage atomization device is potentially valuable for treating animals and offers a new method to be used for experimental lung research.

Funding The study was funded by the National Natural Science Foundation of China (No. 31572216).

Please cite this article as: Tao, C., et al., Atomization method for verifying size effects of inhalable particles on lung damage of mice, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.150

C. Tao et al. / Science of the Total Environment xxx (2016) xxx–xxx

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Acknowledgements Thank for the help from American Journal Experts to improve the writing level of our manuscript (Editorial Certificate Verification Key: EBA5-125D-7498-B1DA-4AA4). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2016.11.150. References Anetor, J.I., 2012. Rising environmental cadmium levels in developing countries: threat to genome stability and health. J. Environ. Anal. Toxicol. 27, 103–115. Chen, S., Ren, Q., Zhang, J., Ye, Y., Zhang, Z., Xu, Y., et al., 2013. N-acetyl-L-cysteine protects against cadmium-induced neuronal apoptosis by inhibiting ROS-dependent activation of Akt/mTOR pathway in mouse brain. Neuropathol. Appl. Neurobiol. 40, 759–777. Chen, R.C., Sun, G.B., Yang, L.P., Wang, J., Sun, X.B., 2016. Salvianolic acid B protects against doxorubicin induced cardiac dysfunction via inhibition of ER stress mediated cardiomyocyte apoptosis. Toxicol. Res. http://dx.doi.org/10.1039/c6tx00111d. Christophe, D., 2008. On the experimental investigation on primary atomization of liquid streams. Exp. Fluids 45, 371–422. Donaldson, K., Stone, V., 2003. Current hypotheses on the mechanisms of toxicity of ultrafine particles. Ann. Ist. Super. Sanita 39, 405–410. Ercal, N., Gurer Orhan, H., Aykin, B.N., 2001. Toxic metals and oxidative stress part I: mechanisms involved in metal induced oxidative damage. Curr. Top. Med. Chem. 1, 529–539. Flora, S.J., Mittal, M., Mehta, A., 2008. Heavy metal induced oxidative stress & its possible reversal by chelation therapy. Indian J. Med. Res. 128, 501–523. Garcia-Morales, P., Saceda, M., Kenney, N., Kim, N., Salomon, D.S., Gottardis, M.M., et al., 1994. Effect of cadmium on estrogen receptor levels and estrogen-induced responses in human breast cancer cells. J. Biol. Chem. 269, 16896–16901. Ge, L.T., Liu, Y.N., Lin, X.X., Shen, H.J., Jia, Y.L., Dong, X.W., et al., 2016. Inhalation of ambroxol inhibits cigarette smoke-induced acute lung injury in a mouse model by inhibiting the Erk pathway. Int. Immunopharmacol. 33, 90–98. Guillon, A., Mercier, E., Lanotte, P., Haguenoer, E., Darrouzain, F., Barc, C., et al., 2015. Aerosol route to administer teicoplanin in mechanical ventilation: in vitro study, lung deposition and pharmacokinetic analyses in pigs. J. Aerosol Med. Pulm. Drug Deliv. 28, 290–298. Heidi, O., 2000. Suspended particulate matter in indoor air: adjuvants and allergen carriers. Toxicology 152, 53–68. Hofmann, W., Bolt, L., Sturm, R., Fleming, J.S., Conway, J.H., 2005. Simulation of threedimensional particle deposition patterns in human lungs and comparison with experimental SPECT data. Aerosol Sci. Technol. 39, 771–781. Ibrahim, M., Verma, R., Garcia Contreras, L., 2015. Inhalation drug delivery devices: technology update. Med. Devices (Auckl.) 8, 131–139. Lang, R., 1962. Ultrasonic atomization of liquids. J. Acoust. Soc. Am. 34, 6–8. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25, 402–408. Longest, W., Xi, J.X., 2007. Computational investigation of particle inertia effects on submicron aerosol deposition in the respiratory tract. J. Aerosol Sci. 38, 111–130. Lü, P., 2016. Inhibitory effects of hyperoside on lung cancer by inducing apoptosis and suppressing inflammatory response via caspase-3 and NF-κB signaling pathway. Biomed Pharmacother. 82, 216–225. Mayer, E., 1961. Theory of liquid atomization in high velocity gad streams. ARSJ 31, 1738–1785. Miller, K.A., Siscovick, D.S., Sheppard, L., Shepherd, K., Sullivan, J.H., Anderson, G.L., et al., 2007. Long-term exposure to air pollution and incidence of cardiovascular events in women. N. Engl. J. Med. 356, 447–458.

9

Nemmar, A., Hoet, P.H., Vanquickenborne, B., Dinsdale, D., Thomeer, M., Hoylaerts, M.F., et al., 2002. Passage of inhaled particles into the blood circulation in humans. Circulation 105, 411–414. Noll, T., de Groot, H., Sies, H., 1987. Distinct temporal relation among oxygen uptake, malondialdehyde formation, and low-level chemiluminescence during microsomal lipid peroxidation. Arch. Biochem. Biophys. 252, 284–291. Nordberg, G.F., 2009. Historical perspectives on cadmium toxicology. Toxicol. Appl. Pharmacol. 238, 192–200. Oakes, J.M., Scadeng, M., Breen, E.C., Prisk, G.K., Darquenne, C., 2013. Regional distribution of aerosol deposition in rat lungs using magnetic resonance imaging. Ann. Biomed. Eng. 41, 967–978. Oakes, J.M., Marsden, A.L., Grandmont, C., Shadden, S.C., Darquenne, C., Vignon Clementel, I.E., 2014. Airflow and particle deposition simulations in health and emphysema: from in vivo to in silico animal experiments. Ann. Biomed. Eng. 42, 899–914. Odewumi, C.O., Latinwo, L.M., Ruden, M.L., Badisa, V.L., Fils Aime, S., Badisa, R.B., 2015. Environ. Toxicol. http://dx.doi.org/10.1002/tox.22165. Oyadomari, S., Mori, M., 2004. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 11, 381–389. Patil, P.S., 1999. Versatility of chemical spray pyrolysis technique. Mater. Chem. Phys. 59, 185–198. Rau, J.L., 2002. Design principles of liquid nebulization devices currently in use. Respir. Care 47, 1257–1275. Ribeiro, O.G., Cabrera, W.H., Maria, D.A., De Franco, M., Massa, S., Di Pace, R.F., et al., 2005. Genetic selection for high acute inflammatory response confers resistance to lung carcinogenesis in the mouse. Exp. Lung Res. 31, 105–116. Robichaud, A., Fereydoonzad, L., Schuessler, T.F., 2015. Delivered dose estimate to standardize airway hyperresponsiveness assessment in mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 308, 837–846. Rogers, J.F., Dunlop, A.L., 2006. Air pollution and very low birth weight infants: a target population. Pediatrics 118, 156–164. Satarug, S., Baker, J.R., Urbenjapol, S., Haswell Elkins, M., Reilly, P.E., Williams, D.J., et al., 2003. A global perspective on cadmium pollution and toxicity in non-occupationally exposed population. Toxicol. Lett. 137, 65–83. Schöpfer, J., Drasch, G., Schrauzer, G.N., 2010. Selenium and cadmium levels and ratios in prostates, livers, and kidneys of nonsmokers and smokers. Biol. Trace Elem. Res. 134, 180–187. Sovani, S., Sojka, P., Lefebvre, A., 2001. Effervescent atomization. Prog. Energy Combust. Sci. 27, 483–521. Sun, X.X., 2004. Recent research advances of ultrasonic atomizer. Ind. Furnace 26, 19–25. Surolia, R., Karki, S., Kim, H., Yu, Z., Kulkarni, T., Mirov, S.B., et al., 2015. Heme oxygenase-1 mediated autophagy protects against pulmonary endothelial cell death and development of emphysema in cadmium treated mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 309, 280–292. Szkudlarek, U., Zdziechowski, A., Witkowski, K., Kasielski, M., Luczyńska, M., Luczyński, R., et al., 2004. Effect of inhaled N-acetylcysteine on hydrogen peroxide exhalation in healthy subjects. Pulm. Pharmacol. Ther. 17, 155–162. Waalkes, M.P., 2003. Cadmium carcinogenesis. Mutat. Res. 533, 107–120. Waisberg, M., Joseph, P., Hale, B., Beyersmann, D., 2003. Molecular and cellular mechanisms of cadmium carcinogenesis. Toxicology 192, 95–117. Wei, X., Qi, Y.M., Zhang, X.N., Qiu, Q., Gu, X.Y., Tao, C., et al., 2014. Cadmium induces mitophagy through ROS-mediated PINK1/Parkin pathway. Toxicol. Mech. Methods 24, 504–511. Wolff, R.K., 2015. Toxicology studies for inhaled and nasal delivery. Mol. Pharm. 12, 2688–2696. Yoshida, H., Matsui, T., Yamamoto, A., Okada, T., Mori, K., 2001. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107, 881–891. Zhang, Z., Kleinstreuer, C., Kim, C.S., 2002. Micro-particle transport and deposition in a human oral airway model. J. Aerosol Sci. 33, 1635–1652. Zhao, D., Qu, H., Guo, J., Zhao, H., Yang, Y., Zhang, P., et al., 2016. Protective effects of myrtol standardized against radiation-induced lung injury. Cell. Physiol. Biochem. 38, 619–634. Zimmermann, M., Aguilera, F.B., Castellucci, M., Rossato, M., Costa, S., Lunardi, C., Ostuni, R., Girolomoni, G., Natoli, G., Bazzoni, F., Tamassia, N., Cassatella, M.A., 2015. Chromatin remodelling and autocrine TNFα are required for optimal interleukin-6 expression in activated human neutrophils. Nat. Commun. 6, 6061.

Please cite this article as: Tao, C., et al., Atomization method for verifying size effects of inhalable particles on lung damage of mice, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.150