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Incubator Ctrl Media AVG Incubator Ctrl Dry AVG Ctrls AIR AVG 0.03 ppm Benzene AVG 0.1 ppm Benzene AVG 0.3 ppm Benzene AVG

Mean Tail Intensities(%)

Figure 2: Mean tail intensity (%) of the control and exposed cells. Errorbars represent the standard deviation of the means. (***) indicates astatistically significant difference between the exposed sample and theirrelative controls. The figure includes 3 independent replicates for thecontrols, and a single independent replicate for the exposed cells. Eachindependent replicate consisted of 3 technical replicates.

N(Cells)

Mean MedianStandard Deviation

VarianceH

(Variance/Mean)

Incubator Controls (Media) 1550 4.52 0.74 9.40 70.98 20.61

Incubator Controls (Dry) 1376 6.73 3.40 11.80 135.26 18.98

Controls 1028 7.40 3.15 11.60 147.82 20.77

0.03 ppm benzene 314 11.49 3.21 15.58 243.83 21.26

0.1 ppm benzene 318 16.02* 5.53* 21.28 456.49 28.37

0.3 ppm benzene 375 12.27* 5.13* 16.90 303.75 23.29

In vitro exposure of A549 cells to benzene using an air-liquid interface exposure system: DNA damage and ROS production

Massimiliano Mascelloni1*, Juana Maria Delgado-Saborit1, Nikolas Hodges2, Roy M. Harrison1

1School of Geography, Earth and Environmental Sciences, University of Birmingham, B15 2TT, United Kingdom2School of Biosciences, University of Birmingham, B15 2TT, United Kingdom

*Now at Department of Environmental Health Sciences, University of Massachusetts, Amherst, MA 01003, USA

Figure 1: Example of results from the Comet assayA: control cells exposed to synthetic air for 2h;B: Cells exposed to 0.1ppm benzene for 2h.

Metabolically competent

cellular model

Metabolomic analyses

Proteomic analyses

Mechanistic endpoint analyses

PollutantPollutant-

specific metabolic pathway

Introduction

Exposure to volatile compounds that have adverse effects isoften monitored through their metabolites or biomarkers. Thefield of biomarker discovery for such compounds is still nascent.Extensive studies which are both costly and time consuming areneeded to identify the targets that can later be used forbiomonitoring. The potential of the presented device, togetherwith the increasing availability and lower costs of 3D printers andCNC machines enables the creation of a high numbers ofreproducible samples with a relatively small investment. The highnumber of samples can facilitate further analyses and methoddevelopment towards biomarker discovery.

The authors wish to thank Shrikant Jondhale for providing the A549 cells. Authors wish to thank CEFIC LRI 2010 Long Range Initiative Innovative Science Award for funding this study.

Reference paper:

Materials and methods

• An exposure chamber was designed around Transwell inserts(Corning Inc., UK), in order to expose A549 cells to differentgas mixtures.

• The cells were grown on a monolayer and the apical mediawas removed for the exposure, obtaining a physiologicallysignificant model of exposure.

• Cells were exposed for 2 hours to synthetic air and to threeconcentrations of benzene to study short the term effects oflow level exposure (0.03; 0.1; 0.3ppm).

• ROS production was analysed via DCFH-DA (10 µM) assay• DNA damage was measured by Comet assay (Singh et al.,

1988)

Exposure vessel and model construction

• The exposure vessel was designed to have three wells, allowing to perform eachexperiment in triplicate

• The temperature was kept at 37°C by keeping the exposure vessel in a GC oven• The gases (synthetic air and 1ppm benzene in nitrogen) were delivered using

two mass flow controllers• The air was humidified by bubbling through deionized water• The two gases were mixed in a heated glass mixing chamber

Results

• Exposure to air in the exposure vessel did not show significantdifferences with the incubator control in both comet assay andDCFH-DA fluorescence assay

• DCFH-DA assay in benzene exposed cells, showed significantdifferences between the pre- and post-exposure only for0.3ppm

• Comet assay showed increased DNA breaks in cells exposed tobenzene, with a peak on 0.1ppm

• Replicates were found to be reproducible and themethodology could be applied to different environments fortoxicological studies

Conclusions

• Short term exposure to benzene proved to cause DNA damagein a concentration-dependent fashion

• High concentration of benzene was found to cause ROSproduction

• Negative control (air exposure) did not show significantdifferences with incubator controls

• Results were consistent and reproducible, making theprocedure a good proof of concept for future applications

Future directions

• CNC technology and 3D printing are becoming easily available, editing and modifyingdesigns via Computer Aided Design (CAD) programs is a reality

• In-house printing of devices and ad-hoc modification can provide cheap and purpose-oriented exposure devices for toxicological studies of different atmospheres andpollutants

• Upscaling the exposure vessel can allow production of large samples that can be usedfor metabolomics, proteomics and toxicological endpoint analyses, provided that thecell model is suitable

Graphic representation of comet results: Y represents % tail intensity

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Incubator Ctrl Dry Incubator Ctrl Media Air exposure 0.03 ppm Benzene 0.1 ppm Benzene 0.3 ppm Benzene

Pre/Post Exposure Fluorescence(Fluorescence Units)

Pre Exposure Post Exposure

Figure 3: Summary of the DCF fluorescence measurements before andafter exposure. Error bars represent standard deviation. (***) indicates astatistically significant difference between the pre and the post exposure.

Table 1: Descriptive statistics of the Comet assay data (% tail intensity). * indicates astatistically significant difference with the control.

Contact information:mmascelloni@umass.edu

Figure 4: Schematics of the exposure vessel assembly.

Figure 5: Schematic with gas flow representation of the assembled exposure vessel. Onthe right is reported a picture of the experimental setup in working condition, inside a gaschromatography oven.