In vitro exposure of human lung cells to emissions of several indoor ...

101

In vitro exposure of human lung cells to emissions of several indoor air sources created in a climate chamber

1,2, 2 2Philomena M. Bluyssen, * Marcel J. Alblas and Ilse L. Tuinman

1Department of Architectural Engineering and TechnologyDelft University of TechnologyDelft 2600 GA, the Netherlands

2TNO Technical SciencesDelft 2600 AA, the Netherlands

Key Words: Ultrafine dust, indoor pollution sources, in vitro method, lung cell exposure, health effects

*Corresponding authorEmail: [email protected]

ABSTRACT

INTRODUCTION

In the last decade, studies on indoor air pollution suggest a link between exposure to indoor particulate matter and compounds, in particular ultrafine particles and secondary organic aerosols, and several health effects. The mechanisms of how those complex mixtures relate to health effects are still not fully understood. In vitro testing, i.e., performing a given procedure in a controlled environment outside of a living organism, provides an additional source of information next to the exposure of persons or animals to controlled environmental conditions in a laboratory environment. Possible end-points that can be determined with such a system are oxidative stress, inflammation and cell-death. The applicability of an in vitro system with human lung cells as an instrument to evaluate possible biological effects of emissions of several indoor air sources (scented candles, hair and water resistant spray) created in a climate chamber was studied. Results demonstrate that the procedure for testing the emissions of scented candles and two sprays resulted in reproducible test conditions and reproducible toxicological results. In vitro testing seems to have potential as a meansto get more and better understanding of the mechanisms and causes for health effects of 'real-life' complex mixtures caused by sources such as burning candles and using spray cans. More tests with different indicators and endpoints, different concentrations and exposure time are required. .

For over 30 yr, research has demonstrated associa-tions between ambient particulate matter (PM) and increased human health outcomes (e.g., [1,2]). In the last decade studies on indoor air pollution also suggest a possible link between exposure to indoor PM and several health effects [3,4]. PM exposure has been associated with cardio-vascular diseases, atherosclero-sis, and problems in the regions of the respiratory tract, such as lung cancer, asthma, and bronchitis [5]. Particles may be also transported to extra-pulmonary organs, such as the liver, kidney and brain potentially causing neurological effects [6-8]. Particles generated during combustion processes such as (gas) cooking have been specifically associated with respiratory problems [9,10] and lung cancer [11-13]. .

Toxicological evidence provides an indication that aspects of PM other than mass alone determine toxicity [1,14]. Particles of different sizes, from differ-ent sources and with different composition should be considered [14]. Many compounds generated indoors are semi volatiles such as phthalates, flame retardants, polycyclic aromatic hydrocarbons (PAHs), chlorophe-nols, pesticides, organotins and metals which may adsorb to PM present in the indoor air and to house dust. These particles may be inhaled or ingested, de-pending on their size. Particulate air pollutants thus have very diverse chemical compositions that are highly dependent on their source. Additionally, most pollution sources produce gaseous compounds re-sulting in a complex mixture of particles and com-pounds. Semi volatile organic compounds (SVOCs), VOCs and inorganic compounds have all been associ-ated with negative impacts on human health [15,16].

Sustain. Environ. Res., 23(2), 101-112 (2013)

102

Products from indoor chemical reactions (e.g., ozone-terpene reactions producing secondary organic aerosols (SOA), a type of ultrafine particles) have also been found to contribute to adverse effects on human health [17,18]. With respect to the size of the particles, there is a need to identify the role of the ultrafine particle (UFP) fraction (the concentration of particles with an aerodynamic diameter smaller than 0.1 µm or PM ) 0.1

[16], because of their greater number concentration, rendering them potentially more biologically active with greater potential for transcytosis across epithelial barriers, thus reaching target tissues beyond the lungs [19]. 50-80% of indoor UFP is produced indoors [3] by home cooking and heating appliances, tobacco smoke, burning candles, vacuuming, natural gas clothes dryers and other household activities [20,21]. In office buildings, laser printers are a major source of UFP [22,23]. There is a need for a procedure or method with which health effects of different indoor air source emissions including UFPs as encountered indoors can be assessed. To estimate human health effects of indoor air pollutants several techniques are available. One can test exposed persons in an epidemiological or cohort study, in which questionnaires and health data are used whether or not in combination with biomarker sample collection (e.g., blood, urine). Health data are combined with information on living environment and lifestyle in order to find correlations (e.g., [4,9]). Interrelated risk factors, such as psychological stress can however affect the outcome that is being studied. It has been hypothesised that stress, which can in-fluence the immune function and susceptibility, may potentiate effects of air pollution in respiratory disease onset and exacerbation [24]. Persons exposed to con-trolled environmental conditions in a laboratory environment is the most powerful method for testing hypotheses related to mechanisms by which specific air pollutants might cause effects [25,26]. However, these studies are limited for ethical and financial reasons. Instead of using humans as exposure subjects, several animals like monkeys or rodents (mice, rats) are therefore used for exposure test (in vivo testing) [27,28] Besides the ethical issues, the challenges with animal studies in particular involve appropriate extrapolation of doses and biological responses to humans [29]. In vitro testing, which refers to the technique of performing a given procedure in a controlled environ-ment outside of a living organism, provides an addi-tional source of information. It has the major ad-vantage that there are very limited ethical issues and human cells being used, so species-to-species extrapo-lations can be avoided [30]. The disadvantages are two folds: the poor definition of dose (making extrapolation from in vitro to in vivo difficult) and the lack of the complex physiological organisation of tissues and organs limiting the studies to direct responses at the

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cellular level [30]. Several in vitro testing methods are available. These methods differ in the cell types used (human or other, primary cells or transformed cell lines, epithelial or other), the exposure method (suspension of collected materials, exposure of con-ventional cultures to aerosols, or air-liquid interface exposures), and the toxicological endpoints assessed (cell viability (cell death), cytokine production, oxidant stress, cell type-specific function) [29]. With

®the commercially available CULTEX system [31], it is possible to expose cells cultivated on porous transwell membrane inserts to a dynamic flow of test atmospheres without medium interference, emulating in vivo exposure.

® The CULTEX system was developed for the investigation of toxic gaseous mixtures, such as cigarette smoke and diesel exhaust [31]. The use of

®human lung epithelium cells and the CULTEX system [32] for the assessment of biological effects seems to be promising. The sensitivity of the system has been shown to be high enough for studying relevant con-centrations of single components such as NO , benzene 2

and complex mixtures, such as diesel exhaust and cigarette smoke [29,34,35]. The main advantage is that the system permits direct assessment of the toxicity of complex test atmospheres that have undergone minimal or no changes to their physicochemical properties. In previous studies, polluted air was either first stored into Teflon bags or produced next to the

®CULTEX system (for example by a tobacco machine) and then introduced to the lung cells via short tubes [29,31,34]. In the underlying study, polluted air was produced in a 'real life' climate chamber and led into

®the CULTEX system. The reproducibility of the test conditions and outcome were investigated. In the underlying study the goal was to test whether this system has potential to study the biological effects of indoor air sources, as an essential step in the ultimate goal to estimate health effects of emissions from indoor air sources.

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MATERIALS AND METHODS

1. Test Procedure

The emissions of different sources, 25 scented candles of one brand type (non-burning (Ncandles) n = 3, burning (Candles) n = 5) and two types of nano particles producing sprays (hairspray n = 5; water re-pellent spray (spray) n = 4), were tested several times (see Table 1). The emissions were produced in an over-pressurized climate chamber (4 x 4 x 2.5 m). The internal wall was covered with aluminium foil to prevent emissions of construction materials, which might interfere with the experiments. The air supply (via a F7 and a F11 filter) was located in one of the walls at a height of 2 m high next to the chamber door

.

Bluyssen et al., Sustain. Environ. Res., 23(2), 101-112 (2013)

103

(see Fig. 1). The air was supplied with a controllable ventilator, set to create an overpressure of 5 to 10 Pa. To minimize the distance between the source and the measurements, sources were placed close to the wall behind which the measurement equipments, including

®the CULTEX system (Fig. 2), were located. This resulted in a travelling distance of 3 m through tubing of 5 mm inner diameter. In Fig. 1 the test set-up is presented schematically. To prevent pollutants from outside entering the chamber when the door is opened, an extra opening with built-in gloves was created. Via these gloves it was possible to light the candles or to spray without having to enter the chamber (see Figs. 3 and 4). The candles were placed on a table close to the wall with the gloves and lighted with an electrical lighter (Fig. 3). While the (burning) candles repre-sented a continuous source, both sprays were sprayed directly into the hood every 10 min for 1 min (Fig. 4). . The sprays and scented candles were selected as sources because their emissions consist of complex mixtures of pollutants such as UFP, SVOC, VOC, and possibly SOA. Per type of source several repetitions of the test conditions were performed during a time

Table 1. Summary of tests

No.

1

2

3

4

5

6

7

8

9

Name

Blank

Ncandles-1

Ncandles-2

Ncandles-3

Candles-1

Candles-2

Candles-3

Candles-4

Candles-5

No.

10

11

12

13

14

15

16

17

Name

Hairspray-1

Hairspray-2

Hairspray-3

Hairspray-4

Spray-1

Spray-2

Spray-3

Spray-4

Fig. 1. Horizontal cross-section of the experimental set-up: Measurement points for respectively fine dust, lung cells, SO + NO , (S) VOC + PAH, Temperature (T) + Relative humidity (RH), O , ventilation + pressure, are noted by 2 2 3

numbers 1 to 7. Measurement points 1, 2 and 4 are located next to the wall of chamber. Measurement points 3 and 6 are connected with tubes to continuous measurement equipment outside the chamber. Measurement 5 is performed inside the chamber. F11 and F7 represent two different air filters through which the incoming air is filtered. .

1 = fine dust2 = lung cells3 = SO + NO2 x

4 = (S)VOC, PAH5 = T + RH6 = O3

7 = ventilation + pressure

outdoor air supply fan orifice plate: 7

Test chamber

5 at 1.5 m gloves

Door

1

2

3

4

6

F11 F7

hood table

36

period of 1.5 h for candles and 1 h for both sprays(because it was expected that the sprays will cause strong immediate effects). Lung cells can dry out when they are exposed too long with polluted air that is not humidified to approximately the same humidity lung cells are normally exposed to in the lungs. Based on


Table 2. Overview of measurements and equipment

(Ultra) fine particles

Toxicity

Gasses

Other

Component

Fine particles (10-523 nm)

Toxicity human lung cells

Nitrogen oxides (NO )x

SO2

O3

Temperature (T) [°C]/

Relative humidity (RH) [%]3 -1Ventilation rate [m h ]

Methods

Scanning mobility particle sizer (SMPS)®CULTEX : LDH, Alamar blue, RC-DC and IL-8

Chemilumescence NO/NO -analyzerx

Fluorescence SO -analyzer2

Photometric O -analyzer3

Thermometer/air humidity meter

Air pressure measurement

104

Ritter et al. [33] an exposure time of 90 min is con-sidered to be on the safe side. Additional one blank situation was tested (empty chamber) and three situations with the candles in the chamber but not burning (Ncandles).

2. Measurements and Analysis

During the experiments the air flow, temperature, relative humidity (RH), several gases (NO, NO , SO , 2 2

and O ) and fine particles and UFPs were monitored or 3

collected. Table 2 gives an overview of the measure-ments and applied equipment. The outdoor airflow was calculated from the pressure difference over the orifice plate (see Fig. 1).

The concentration measurements for fine particles and UFPs were continuously conducted by a TSI(Minnesota, USA) Scanning Mobility Particle Sizer or SMPS (TSI 3936) in the configuration long DMA,

-1model 3081 (sheath gas flow rate 3 L min ; aerosol -1flow rate 0.30 L min ) in combination with a TSI

Condensation Particle Counter 3786. The used pre-impactor had a cut-off of 0.0508 cm. The SMPS Scan Up Time was set to 150 s, and the retrace period between two scans was 30 s. The SMPS measurement

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.

.

.

® Fig. 2. CULTEX system.

Fig. 3. Lighting of the candles with an electrical lighter.

Fig. 4. Spraying.


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ranged from 11.1 to 523 nm. Data collection and analysis were performed by the Aerosol Instrument Manager software (TSI, AIM version 8.0.0). All data were corrected for diffusion losses and potential multi-ple charges by the software. The candle soot measure-ments were also corrected for aggregate formation (Primary Particle Diameter 10.0 nm). NO , SO and O were continuously measured with x 2 3

Erespectively a NO/NO analyzer (Teledyne type 200 ), xESO analyzer (Teledyne type 100 ) and an O analyzer 2 3

(Advanced Pollution Instrumentation, type 400).

®. CULTEX System

Several toxicological indicators were investigated ®on human lung cells (A549) in the CULTEX system

[31,34]. Originating from the alveolar epithelial lining, A549 cells are widely used to assess the toxicity of airborne toxicants reaching the lungs [35]. The human lung Epithelial Cell (EC) line A549 is derived from human lung non-small cell adenocarcinoma [36], which has been described to exhibit a closely matched type II alveolar cell phenotype and to share many properties with human primary alveolar ECs, including cytokeratin expression and growth parameters [35]. The A549 cells were cultivated in Dulbecco's modified eagle medium (DMEM, Gibco, Invitrogen, Breda, The Netherlands) supplemented with 10% foetal calf serum (PAA Laboratories GmbH, Pasching, Austria) and

-1100 g mL penicillin and streptomycin (Sigma, Zwijndrecht, The Netherlands) termed as total medi-um - at 37 °C under humidified conditions containing 5% CO . 2

® Exposure in the CULTEX system took place with 2cells on Transwell (4.7 cm , Falcon BD Biosciences,

Erembodegem, Belgium). These inserts were first cultivated for a day in total medium, 3 mL under and 2 mL above the inserts (6-wells plate). After that the cells were seeded and cultivated for 3 or 4 d before they were seeded in a 1% foetal calf serum (induction medium) and transferred to a new 6-wells plate with 3 mL of induction medium below the inserts (air-lifted) for 12 h. For the exposure, the inserts were transferred

®to the exposure unit of the CULTEX system. ® The CULTEX system contains two exposure units

in which each three vitro-cell inserts can be placed. Cells are fed and moistened from the basal side through the porous membrane with 1% foetal calf serum and exposed to air at their apical side. The airflow rate of the exposed air amounts normally 8 mL

-1min , but for these experiments, the flow was set at 5 -1 ®L min . In the CULTEX system the lung cells are

-1 exposed to a piston flow of 8 mL min with the air blown in the direction of the cells. The process of drying out can be influenced in this way. The flow rate above the cells during exposure influences cell viabil-ity. Based on values found in literature [29,31,34],

-1a flow of 5 L min seems to be realistic for the

.

.

.

.

.

3

ì

®CULTEX system (leading to an air velocity of ap--1proximately 0.17 mm s above the cells). One ex-

posure unit was exposed to medical air (Air liquid ALM007-B0HA) and the other to the 'polluted' air to be tested.

® After exposure in the CULTEX system, exposed cells were cultivated in total medium up to 24 h after the experiment on a conventional 6-wells plate at 37 °C and 5% CO . After 24 h 400 µL medium was 2

frozen (for determination of lactate dehydrogenase (LDH) an

to Measure Health Effects

Based on literature, previous studies and lab capabilities, it was chosen to measure the health effects with two possible end-points: (1) Inflammation: in-crease of IL-8; and (2) Cell viability: increase of LDH, reduction of Alamar Blue and reduction of RC-DC (as an additional indicator). IL-8 release was determined in the cell media with a PeliKine compact™ human IL-8 kit (Sanquin, Amsterdam, The Netherlands). The optical densities of the IL-8 assay were measured at 450 nm in a plate reader uQuant (Bio-Tek, Germany, Bad Friedrichshall, Germany). From each vitro-cell insert 1 medium sample was taken, resulting in 3 samples per control and 3 samples per exposed unit per test. LDH release was measured with an LDH assay (colorimetric determination) according to manufac-turer's instructions (Roche, Mannheim, Germany). The optical density was measured at a wavelength of 450 nm with a micro plate reader uQuant (BioTek Germany, Bad Friedrichshall, Germany). From each vitro-cell insert 3 medium samples were taken, re-sulting in 9 samples per control and 9 samples per exposed unit per test. Alamar Blue (10 vol%) was added for 1 h to the cell layer and medium. The reduction of Alamar Blue was measured with a fluorescence reader Cytofluor II (Perceptive Biosystems, Framingham, USA) at an excitation wavelength of 560 nm and an emission wavelength of 595 nm. Viability was calculated for both assays as percentage of the control cells. From each vitro-cell insert 4 medium samples were taken, resulting in 12 samples per control and 12 samples per exposed unit per test. The RC-DC Protein Assay (Bio-Rad, Veenendaal, The Netherlands) is a colorimetric assay for protein quantification, which is a modified Lowry procedure. It is a measure of the number of cells in the sample and

d interleukin-8 (IL-8), the metabolic activity was measured (Alamar Blue), the inserts were washed with 2 mL phosphate buffered solution and lysed for 10 min with 0.4 mL lysis buffer on ice of the GSH-kit (GSH + RC-DC (reducing agent and detergent com-patible)). 50 ìL lysate was frozen separately (for RC-DC measurement) and the rest of the lysate was deproteinized and frozen.

4. Toxicological Endpoints

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was taken as an additional indicator. One measurement per vitro-cell insert was performed.

RESULTS AND DISCUSSION

1. Reproducibility of Test Conditions

Figure 5 presents the average airflow and standard deviations per test. Test 1 represents the 'blank' situa-tion, tests 2, 3 and 4 represent the non-burning candle test situation; tests 5 to 9 represent the burning candles test situation; tests 10-13 the hairspray test situation; and test 14-17 the water repellent spray (spray) test situation.

Table 3 presents the average values (for tempera-ture, RH and gas concentrations (SO , O )) and stand-2 3

ard deviations per type of test conditions (non-burning candles, candles, hairspray and water repellent spray (spray)). For temperature, RH and O , the repro-3

ducibility is good for all tested situations. For the tests with candles the SO concentrations are reproducible. 2

For the other test situations, the concentrations are so low that the presented numbers are difficult to inter-pret. For NO , only the burning candle situation was x

measured. The concentrations were reproducible.

.

.

. Fig. 5. Average airflow and standard deviation per test.

3-1

Air

flow

rat

e (m

h)

Test number

Table 3. Average concentrations (AV), standard deviations (SD) and reproducibility (R) per type of test. R is defined as the standard deviation divided by the average

T [°C] AV

T [°C] SD

R

RH [%] AV

RH [%] SD

R

SO [ppb] AV2

SO [ppb] SD2

R

O [ppb] AV3

O [ppb] SD3

R4 -3Particles [10 cm ] AV4 -3Particles [10 cm ] AV

R

Geo. mean [nm]

SD [nm]

R

Ncandles (n = 3)

22.6

0.7

0.03

42.5

12.6

0.29

0

0

-

12.4

2.2

0.18

0.04

0.04

0.96

51

25

0.5

Candles

(n = 5)

31.3

0.2

0.01

31.2

0.5

0.02

9.2

0.46

0.05

5.01

0.34

0.07

13.7

3.3

0.24

29

2

0.08

Hairspray

(n = 4)

21.7

0.3

0.01

62.4

2.6

0.04

0.32

0.40

1.24

2.50

0.08

0.03

156

13

0.09

56

4

0.07

Spray

(n = 4)

22.8

0.6

0.02

52.8

1.5

0.03

2.29

1.37

0.6

4.37

2.31

0.53

7.44

2.0

0.27

61

1

0.02

Figure 6 shows the average particle size distributions for the tests with the candles, the hairspray and the water repellent spray. The standard deviations show that the reproducibility of the test conditions with re-spect to number of particles and particle size is good for all three test situations (Table 3). In the non-burning candle situation, the number of particles was so low that nothing can be reported on that.

. Concentrations of Gasses and Particles

For the tests with burning candles, the average

.

.2

140

120

100

80

60

40

20

01 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17


107

concentrations of NO (379 ± 24 ppb) and SO (9 ± 3 2 2

ppb) were above the long term WHO guideline values -3 -3of 40 g m (221 ppb) (annual mean) and 20 g m (8

ppb) (24-h mean), respectively, but below the short -3term WHO guideline values of 200 g m (1106 ppb)

-3and 500 g m (191 ppb). These gas concentrations may cause some effects on the toxicological indicators. For the test with the water repellent spray the average con-centration of NO and SO was 43 ± 13 and 2 ± 1 ppb, 2 2

respectively, well below any guideline values. The average SO concentration for the hairspray was nearly 2

zero (NO was unfortunately not measured). As ex-2

pected, burning candles caused higher concentrations

Fig. 6. Average number of particles per diameter per (a) candle test (b) hairspray test (c) water repellent spray test. During tests 16 (spray-3) and 17 (spray-4) the spraying, hence concentration build-up and reduction, was not properly coordinated with the SMPS scans and due to the short spraying period time and 30 s downscan time averaging did not result in usable concentration profiles. .

(a)

(b)

(c)

hairspray 4hairspray 3hairspray 2hairspray 1

spray 1spray 2

X

Particle diameter (nm)

-3C

once

ntra

tion

(pa

rtic

les

cm)

-3C

once

ntra

tion

(pa

rtic

les

cm)

-3C

once

ntra

tion

(pa

rtic

les

cm)

10 100 1000

8000

6000

4000

2000

0

60000

50000

40000

30000

20000

10000

0

2500

2000

1500

1000

500

0

candles 1 candles 2 candles 3 candles 4 candles 5

X

of NO and SO than the sprays. x 2

The average O concentrations for the burning 3

candles, hairspray and the water repellent spray, re-spectively 5 ± 1, 3 ± 0 and 4 ± 2 ppb, were not signifi-cantly different from the blank situation (test 17: 3 ppb). The non burning candles tests (tests 2, 3 and 4) did show a slightly higher average O concentration 3

(12 ± 2 ppb), but still well below the WHO guideline -3value of 100 g m (196 ppb) for a daily maximum 8-h

mean. No effect of spraying on the O concentration 3

was observed. Because particle distributions are approximately log normal and measurements spread often over dec-ades, in general the geometric mean is used as a representative value. The geometric mean diameter in the candle burning tests was around 29 nm, in the hairspray tests around 56 nm and in the watertight spray tests around 61 nm. The diameter of 29 nm for the candle burning tests indicated a steady state burn (no smoke) with one peak distribution [37]. Sun et al. [37] found that with an unsteady burning, smoke occurs with a bimodal distribution, in which two peaks lie within the 10-500 nm range (high peak at ca 40 nm and a low peak at 250 nm). The total number concentration of particles during hairspray tests was 10 times higher than during candle burning and even 20-fold higher than during the water repellent spray tests. The average particle concentra-

4 4 4 -3tions were 13 x 10 , 160 x 10 and 7.4 x 10 cm for candles, hairspray and water repellent spray, respec-tively. The number of particles from burning scented candles was of the same order of magnitude as found by Afshari et al. [21]. They measured fine particles and UFPs in a chamber with different sources among which were pure wax candles and scented candles. Their outcome showed that the highest concentration

4of UFP came from wax candles (ca 24.1 x 10 particles -3cm ), twice as high as for scented candles.

. Reproducibility of Toxicological Endpoints

Figure 7 presents the difference in percentage between the polluted air exposure and the medical air exposure per test for each toxicological endpoint tested. For the blank situation (test 1) no difference with medical air was seen, indicating a 'clean' room. Average difference between the test exposure and the medical air exposure of the lung cells, the standard deviation and the reproducibility is presented in Table 4 per test situation (non-burning candles, candles, hairspray and water repellent spray).

The exposure results with candles, hairspray and water repellent spray showed a reproducibility be-tween 0.05 and 0.30 (where R = 0.01 is excellent with R > 1 very bad). The non-burning candle situation was not as reproducible as the other situations (range of 0.44 for Alamar Blue to 1.66 for LDH).

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.

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.

.

3


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Table 4. Average difference (AV) between the test exposure and the medical air exposure of the lung cells, the standard deviation (SD) and the reproducibility (R) per test situation. 1: test 13 is not included, because the outcome was considered an outlier (see Fig. 7c) therefore n = 3

%Ncandles

(n = 3)

6.5

4.5

0.69

-7.1

3.1

0.44

6.2

10.3

1.66

-2.3

3.4

1.48

Candles

(n = 5)

24.1

6.7

0.28

-20.4

3.1

0.15

24.9

4.0

0.16

-21.5

4.3

0.20

Hairspray

(n = 4)126.0

4.6

0.18

-26.2

5.9

0.22

25.1

7.6

0.301-30.4

8.2

0.27

Spray

(n = 4)

13.6

2.4

0.18

-20.3

1.0

0.05

41.9

9.0

0.21

-21.3

4.8

0.22

IL-8 AV

SD

R

Alamar Blue AV

SD

R

LDH AV

SD

R

RC-DC AV

SD

R

Fig. 7. (a) Alamar blue reduction: Average and standard deviations per test (n = 12). (b) LDH increase: Average and standard deviations per test (n = 9). (c) RC-DC reduction: average and standard deviations per test (n = 3). (d) IL-8 increase: Average and standard deviations per test (n = 3). .

Standard deviations for the single tests performed (see Fig. 7) varied on average from 7.7% for tests with Alamar Blue, 12.1% for tests with LDH, 12.3% for test

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

40

30

20

10

0

-10

-20

Ala

mar

blu

e re

duct

ion

(%)

LD

H i

ncre

ase

(%)

RC

-DC

red

ucti

on (

%)

IL-8

inc

reas

e (%

)

Test number Test number

60

50

40

30

20

10

0

-10

-20

-30

60

50

40

30

20

10

0

-10

-20

-30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

70

60

50

40

30

20

10

0

-10

-20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17


(a) (c)

(b) (d)

with RC-DC and 15.4% for tests with IL-8. The lowest standard deviation was 2.6% for an RC-DC test on candles and the highest standard deviation was found

109

Average-3#Particles m

total #particles

Single tests-3#particles m

total #particles

Alamar blue

0.46

0.47

0.11

0.13

LDH

0.005

0.005

0.00003

0.0002

RC-DC

0.14

0.15

0.40

0.43

IL-8

0.45

0.48

0.17

0.19

Table 5. Correlation coefficients of particle concentrations and total number of particles for the single tests and for the average outcome of the exposure situation with Alamar blue, LDH, RC-DC and IL-8 results

for a RC-DC test of candles (27.7%). It has been shown in other studies that these standard deviations can be lower in precisely controlled environments. As for example in a study performed by Pariselli et al.

®[34], in which the cells in the CULTEX system were exposed to different concentrations of toluene and benzene. Average increases of LDH and standard deviations found for LDH in single test situations varied from 19.2 ± 3.6% 0.1 ppmv toluene in air and 27.0 ± 2.7% for 0.1 ppmv benzene in air to 34.6 ± 7.4% for 0.6 ppmv toluene in air and 39.6 ± 2.4% for 0.3 ppmv benzene in air. But it must be pointed out that in that study the test atmosphere was generated by mixing synthetic air with different concentrations of air pollutants in a mixing chamber, which was trans-ported directly via inert Teflon tubes into the device. No studies for comparison can be found in which tests with complex mixtures have been produced as in the present study.

4. UFP versus Toxicological End-points

It was investigated whether a correlation between Alamar Blue/LDH/RC-DC/IL-8 on the one hand and particles concentration on the other existed. Addi-tionally, it was investigated whether a correlation could be found between Alamar blue/LDH/RC-DC/IL-8 and total amount of particles to which the lung cells were exposed. In Table 5 those correlations are pre-sented for respectively the single tests performed and the average outcome of the different exposure situa-tions (Ncandles, candles, hairspray and water repellent spray). Alamar Blue and IL-8 gave the best correlation for the average outcome per exposure situation, for both the average concentration of particles as the total number of particles, the cells were exposed to. RC-DC is the only endpoint that gives a reasonable correlation with the single test comparison in which each test is taken independently. For the toxicological indicators (Alamar Blue, LDH, RC-DC and IL-8), it has been analysed how different the average outcomes for the different ex-

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Fig. 8. 95%-confidence interval for Alamar blue, LDH, RC-DC and IL-8 during tests with candles, hairspray and water repellent spray based on a students distribution.

posure situations (Ncandles, candles, hairspray and water repellent spray) are, using the 95% confidence interval for a t-distribution (see Fig. 8). The confidence intervals show a relevant difference between the re-duction in Alamar Blue and RC-DC for non-burning and burning candles, but not for LDH and IL-8. .

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CONCLUSIONS

From the results of the reported experiments per-®formed with the CULTEX system, it can be concluded

that the procedure for testing indoor air exposures re-sulted in reproducible test conditions and reproducible toxicological results. However, the discriminatory capabilities of the test in its current form were limited. It must be noted that the concentrations created during these experiments were higher than expected in real-life situations. Normally a dose-related response is assumed. However, especially at very high concen-trations as were applied in this study, the response to a high concentration for a short period can differ signifi-cantly from the response to a lower dose for a longer period due to a strong immediate response without a chance of clearance or recovery. These high exposures were chosen to guarantee a response from the cells during the relatively short exposure time of 1 h for the sprays and 90 min for the candles based on pilot studies. Whether it is possible to increase the exposure duration to for example 8 h needs to be explored. Other parameters such as airflow, air distribution over the cells (air velocity) and humidity of the exposed air are important to consider. It is not unlikely that a different “exposure” system is required. Other systems have been and are being developed, based on similar in vitro models and other models [38]. Even though the concentration and exposure time per source were not varied within this set of experi-ments, the toxicological results from the different test situations confirm earlier studies that UFP is probably an important indicator, but certainly not the only cause of the effects. The highest number of particles was produced during hairspray production (a factor of 10 higher than during the candles burning and even a factor of 20 higher than for the water repellent spray situation). However, this difference is not seen in the outcome of the toxicological indicators. It seems that other pollutants than UFP play a role in the observed effects. The reason for this can perhaps be found in the composition of particle-bound chemical pollutants or in the combined effect of gasses, vapours and aerosols. This needs to be investigated. Additionally, oxidative stress as a toxicological endpoint seems worthwhile investigating. The mech-anisms of PM related health effects are still not fully understood, but it is investigated whether many of the adverse health effects may be derived from oxidative stress, initiated by the formation of reactive oxygen species at the surface of and within the target cells [39]. Components of particles have the potential to generate free radicals in the lung environment and thereby cause oxidative stress, which is an important mechanism leading to inflammation [40]. In the latest discussions on the relevance of toxici-ty testing, the need for an integral approach is propa-gated. An integral approach comprises of (1) human

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exposure data to select doses for toxicity testing and facilitate development of environmental relevant hazard information, (2) biomonitoring data relating real-world human exposures with concentrations that perturb toxicity pathways to identify biologically relevant exposures, and (3) information on host susceptibility and background exposures to interpret and extrapolate in vitro test results for risk assessment [41]. To make them fully useful it will take more than the development and validation of these as individual tests [42]. Lung cells have been selected to determine the mechanisms of action of inhaled toxicants in terms of how they cause injury and repair in the respiratory system. Besides, different bacteria have been used to assess biological effects of pollutants, such as Salmonella typhimurium lines YG1024 [43] and TA98 [44]; other exposure routes are being identified that need to be taken into account in order to determine the full exposure risk. Besides the lungs and blood, another possible route for particles to reach the brain that is being investigated, is the olfactory neuronal pathway [45]. It has been shown that the olfactory neuronal pathway, the sensory nerve endings, repre-sents a significant exposure route of

CNS) tissue to inhale manganese oxide UFPs with rats and is a much more efficient pathway to the CNS than via the lungs and the blood circulation across the blood-brain barrier [46]. Still, in vitro testing seems to have potential as a means to get more and better understanding of the mechanisms and causes for health effects of 'real-life' complex mixtures caused by sources such as burning candles and using spray cans. More tests with different indicators and endpoints, different concentrations and exposure time are required. Combinations of different cells and toxicological end-points should result in more usable information. This is envisioned as the next step in this research.

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Nervous System System (

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Discussions of this paper may appear in the discus-sion section of a future issue. All discussions shouldbe submitted to the Editor-in-Chief within six monthsof publication. .

Manuscript Received: Revision Received:

and Accepted:

July 16, 2012October 27, 2012

December 14, 2012

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