Available online at www.sciencedirect.com

www.elsevier.com/locate/toxinvit

Toxicology in Vitro 21 (2007) 1341–1347

Comparative in vitro cytotoxicity assessment of selectedgaseous compounds in human alveolar epithelial cells

S. Bakand a,b, C. Winder a, A. Hayes a,*

a Chemical Safety and Applied Toxicology (CSAT) Laboratories, School of Safety Science, The University of New South Wales,

UNSW Sydney 2052, Australiab Department of Occupational Health, School of Public Health, Iran University of Medical Sciences, Tehran, Iran

Received 11 October 2006; accepted 23 April 2007Available online 5 May 2007

Abstract

Exposure to airborne contaminants is significantly associated with human health risks, ranging from bronchial reactivity to morbidityand mortality due to acute intense or long term low level repeated exposures. However, the precise mechanisms that derive such effectsare not always understood. Although inhalation studies are technologically complicated, correct hazard characterisation is essential forcomparable risk assessment of inhaled materials. The aim of this study was to investigate the comparative in vitro cytotoxicity of selectedgaseous contaminants in human lung cells. The cytotoxicity of nitrogen dioxide (NO2), sulphur dioxide (SO2) and ammonia (NH3) wasinvestigated in A549- human pulmonary type II-like epithelial cell lines cultured on porous membranes in Snapwell inserts. A dynamicdirect exposure method was established by utilizing the horizontal diffusion chamber system (Harvard Apparatus Inc, USA) for deliveryof test atmospheres. Test atmospheres were generated using a dynamic direct dilution method and the concentration monitored byappropriate analytical methods. A diversified battery of in vitro assays including the MTS (tetrazolium salt; Promega), NRU (neutralred uptake; Sigma) and ATP (adenosine triphosphate; Promega) assays was implemented. Airborne IC50 (50% inhibitory concentration)values were calculated based on the most sensitive assay for each test gas including NO2 (IC50 = 11 ± 3.54 ppm; NRU) > SO2

(IC50 = 48 ± 2.83 ppm; ATP)> and NH3 (IC50 = 199 ± 1.41 ppm; MTS). However, all in vitro assays revealed similar toxicity rankingfor selected gaseous contaminants. Identical toxicity ranking was achieved using both in vitro and published in vivo data. This compar-ison suggests that results of in vitro methods are comparable to in vivo data and may provide greater sensitivity for respiratory toxicitystudies of gaseous contaminants.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Adenosine triphosphate; Gaseous contaminants; In vitro cytotoxicity; Neutral red uptake; Tetrazolium salt

0887-2333/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.tiv.2007.04.013

Abbreviations: ACGIH, American Conference of Governmental Industrial Hygienists; ATP, adenosine triphosphate; A549, human pulmonary type II-like epithelial cell lines; ATCC, American type culture collection; C, ceiling; CAS, chemical abstracts service; CSAT, chemical safety and appliedtoxicology laboratories; DMEM/F12, Dulbecco’s modified eagle medium: Ham’s F-12 nutrient mixture; DPBS, Dulbecco’s phosphate buffered saline;EDTA, ethylene diamine tetra acetic acid; FCS, fetal calf serum; GSH, glutathione; HBSS, Hank’s balanced salt solution; HEPES, N-(2-hydroxyethyl)-piperazine-N0-2-ethanesulfonic acid; IC50, 50% inhibitory concentration; LC50, 50% lethal concentration; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carbo-xymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt; NIOSH, National Institute for Occupational Safety and Health; NRU, neutral red uptake;OSHA, Occupational Safety and Health Administration; PMS, phenazine methosulfate; TLV, threshold limit value; UNSW, University of New SouthWales.

* Corresponding author. Tel.: +61 2 9385 4200; fax: +61 2 9385 6190.E-mail address: a.hayes@unsw.edu.au (A. Hayes).

1342 S. Bakand et al. / Toxicology in Vitro 21 (2007) 1341–1347

1. Introduction

Occupational and environmental exposure to airbornecontaminants is significantly associated with human healthrisks ranging from bronchial reactivity to morbidity andmortality due to acute intense or long term low levelrepeated exposures (Klaassen, 2001; Chauhan and John-ston, 2003; Greenberg et al., 2003; Winder and Stacey,2004). However, the precise mechanisms by which air con-taminants produce such effects are not always fully under-stood. Conventional animal toxicity tests do not provide asatisfactory basis for risk assessment of human chemicalexposures due to scientific, economic and ethical concerns(O’Hare and Atterwill, 1995; Gad, 2000; Blaauboer,2002; Goldberg, 2004; Bakand et al., 2005a; Hayes et al.,2007). Although inhalation toxicity studies are technologi-cally more complicated, proper hazard characterization isessential for comparable risk assessment of inhaled materi-als. Recent studies demonstrate that in vitro methods mayhave a significant potential for toxicity testing of airbornecontaminants (Chen et al., 1993; Tu et al., 1995; Knebelet al., 1998; Muckter et al., 1998; Aufderheide et al.,2003; Bakand et al., 2005b, 2006a,b).

The aim of this study was to investigate the comparativecytotoxicity of selected gaseous contaminants in humanlung cells using a dynamic direct exposure method (Bakandet al., 2006b). Test gaseous airborne contaminantsincluded: nitrogen dioxide (NO2), sulphur dioxide (SO2)and ammonia (NH3). Cytotoxicity was investigated usingthe MTS, NRU and ATP in vitro assays. The multiple cyto-toxicity endpoints were selected to represent the vital bio-logical functions of the cellular system and to provide ageneral mechanism of toxicity.

2. Materials and methods

2.1. Test chemicals

Nitrogen dioxide (NO2, CAS No. 10102-44-0, 50 ppm),sulphur dioxide (SO2, CAS No. 7446-09-5, 200 ppm) andammonia (NH3, CAS No. 7664-41-7, 200 ppm) balancedin synthetic air were purchased from Linde Gas Pty Ltd,Australia. Synthetic air was also purchased from LindeGas Pty Ltd, Australia.

2.2. Cell types and culture conditions

Human pulmonary type II-like epithelial cell lines(A549, ATCC No. CCL-185) were cultured in sterile,vented 75-cm2 cell culture flasks with Dulbecco’s modifiedeagle medium: Ham’s F12 nutrient mixture (DMEM/F12;Gibco, USA) supplemented with 5% (v/v) fetal calf serum(FCS); JS Bioscience, Australia), and 1% (v/v) of an antibi-otic solution (Sigma, USA) containing: L-glutamine(2 mM), penicillin (100 U/ml) and streptomycin (0.1 mg/ml). Cultured cells were kept at 37 �C in a humidified 5%CO2 incubator.

For cytotoxicity experiments, newly confluent cell layerswere enzymatically removed, resuspended in culture med-ium and viable cell number determined (Bakand et al.,2005b). Human cells were grown on porous membranes(0.4 lm) in Snapwell inserts (Bakand et al., 2006a,b). TheSnapwell insert is a modified Transwell culture insert witha 12 mm diameter providing a growth area of 1.12 cm2

(clear polyester SnapwellTM insert, 3801, Corning), sup-ported by a detachable ring that was placed in a six wellculture plate. Culture media supplemented with HEPESbuffer (0.01 M) was added and the Snapwell inserts wereincubated at 37 �C for 1 h as an initial equilibrium timeto improve cell attachment. Culture media from the topwas replaced with fresh media (0.5 ml) containing25 · 104 cells, supplemented with HEPES buffer (0.01 M).Cell cultures were incubated at 37 �C in a humidified incu-bator for 24 h. Before exposure, cell confluence (75–80%)and attachment was observed using the light microscope,the medium was removed from newly confluent cells andmembranes washed with Hank’s balanced salt solution(HBSS; Gibco, USA). Cells were exposed to airborne con-centrations of test gases on their apical side while beingnourished from their basolateral side, using the dynamicdirect exposure method.

2.3. Dynamic generation of test atmospheres

Different concentrations of selected test gases were gen-erated using a dynamic direct dilution method (Bakandet al., 2006b). The system comprised of a metered test gassource, a metered clean-air source and mixer to dilute thetest gas to the desired concentration. Accurate flow rateswere continuously monitored and directed to the dilutionchamber in order to produce the final desired concentra-tions. For each gaseous contaminant, workplace recom-mended threshold limit values were considered for theinitial concentration of test atmospheres (ACGIH, 2006a;ACGIH, 2006b).

2.4. Dynamic exposure protocol

The Navicyte horizontal diffusion chamber system wasadapted for dynamic delivery and exposure of airbornecontaminants to human cells (Bakand et al., 2006b). Thechamber created an environment in which the apical sur-face of the cell monolayer was exposed to airborne contam-inants while the basolateral surface was perfused withculture medium (Fig. 1). Human cells grown on mem-branes were exposed to selected gaseous contaminantsdirectly at the air/liquid interface (Bakand et al., 2006b).Briefly, after washing the cell monolayer with HBSS, mem-branes were placed in the horizontal diffusion chamberscontaining serum free culture media, supplemented withHEPES buffer. The upper compartment of the chamberwas closed and test atmospheres were delivered throughthe chambers (25 ml/min) for 1 h at 37 �C. The volume ofculture media in the basolateral compartment was sufficient

Fig. 1. Dynamic direct exposure of human cells at the air/liquid interface.

S. Bakand et al. / Toxicology in Vitro 21 (2007) 1341–1347 1343

to keep the cells hydrated. At the same time a humidifiedatmosphere was supported inside the chamber by standar-dising exposure temperature at 37 �C. After exposure,membranes were replaced in six well plates. Fresh culturemedia were added to the bottom part of the membranesand in vitro assays of MTS, NRU and ATP wereperformed.

2.5. Cytotoxicity endpoints

2.5.1. MTS-tetrazolium salt assay

The Promega CellTiter 96� AQueous Non-RadioactiveCell Proliferation Assay was used to measure the cytotox-icity of test gases by determining the number of viable cellsin culture (Promega, 2001). The MTS (3-(4,5-dimethylthia-zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium) assay is based on the ability of viable cellsto convert a soluble tetrazolium salt to a formazan prod-uct. After exposure, the MTS/PMS reagent was addedand cell cultures were incubated at 37 �C for 1 h (Bakandet al., 2006a,b). At the end of incubation period, absor-bance was recorded at 492 nm (Multiskan Ascent, ThermoLabsystems, Finland).

2.5.2. NRU – neutral red uptake

The Neutral red (3-amino-7-dimethyl-amino-2-methyl-phenazine hydrochloride) uptake (NRU; Sigma) assay isa cell survival/viability technique based on the ability ofviable cells to incorporate and bind supravital neutral reddye (Babich and Borenfreund, 1992). The NRU assaywas adapted for measuring the cytotoxicity of airbornechemicals (Bakand et al., 2006a,b). After exposure, theNRU solution was added and cells were incubated for3 h at 37 �C. The medium was then removed and cells fixedwith fixative solution. Membranes were rinsed with HBSSand assay solubilisation solution was added. The platewas shaken for 10 min and absorbance was recorded at540 nm (Multiskan MS, Labsystems, Finland).

2.5.3. ATP – adenosine triphosphate

ATP content was measured using the CellTiter-Glo�

Luminescent Cell Viability Assay (Promega, 2004). In thisassay the addition of CellTiter-Glo� Reagent induces cell

lysis and generation of a luminescent signal proportionalto the amount of cellular ATP content. After exposure,Cell-Titer-Glo� reagent (0.25 ml) was added and the lumi-nescence level was recorded (Berthold Detection Systems,Germany) as previously described (Bakand et al., 2006b).

2.5.4. ControlsFor all in vitro experiments, three controls were set up

including: an IC100 (100% inhibitory concentration; mediaonly), an IC0 (0% inhibitory concentration; cells only)and a synthetic air control exposed to a dynamic flow ofair during the exposure time to consider any cell viabilityreduction induced by the dynamic air flow (Bakand et al.,2006b). The synthetic air control was used as a referencefor percentage cell viability calculations of exposed cells.

2.6. Calibration of test atmospheres

The concentration of generated test atmospheres wascalculated based on the flow dilution ratios. To verify fur-ther, generated concentrations of gaseous airborne con-taminants were also monitored by appropriate samplingand analytical techniques.

The measurement of NO2 was carried out by the 6014NIOSH method (NIOSH, 1994) as previously described(Bakand et al., 2006b). In summary, sorbent tubes contain-ing triethanolamine-impregnated molecular sieve (TEA-IMS; SKC, USA) were utilised for air sampling. Air sam-ples were collected from sampling outlets of the dilutionchamber and were analysed using a colorimetric method.Calibration of NO2 test atmospheres indicated a high cor-relation between calculated and measured NO2 test concen-trations (R2 = 0.99).

The measurement of SO2 concentration was carried outby the ID-104 OSHA analytical method with a detectionlimit of 0.01 ppm in 60-L air (OSHA, 1989). A calibratedpersonal sampling pump was used to transfer a known vol-ume of air through a midget impinger containing 10 ml ofhydrogen peroxide (0.3 N). Collected air samples were fil-tered (0.45 lm; Millipore) and analysed as total sulphate(SO4) by an ion chromatograph equipped with a conductiv-ity detector (Waters IC PAK-A Column; model 430 Con-ductivity and 484 Absorbance Detectors). Calibration ofSO2 test atmospheres indicated a high correlation betweencalculated and measured SO2 test concentrations(R2 = 0.99).

The measurement of ammonia (NH3) was carried out bythe 6015 NIOSH recommended sampling method (NIOSH,1994). Solid sorbent tubes containing sulphuric acid-trea-ted silica gel were utilised. Each tube was a two-sectiontube packed with a 200-mg front and a 100-mg back-upsection (SKC, Cat No 226-10-06). Air samples were col-lected from sampling outlets of the dilution chamber at0.1 L/min using a calibrated sampling pump (SKC,USA). Each section was extracted separately in ammoniafree deionised water (5 ml) and pH of samples wasadjusted to 5.0–6.5. Collected samples were analysed using

MTSNRUATP

0

25

50

75

100

125

10 20 40 80 200

Concentration (ppm)

Cel

l Via

bili

ty (

pp

m)

Fig. 3. Cytotoxic effects of SO2 in A549 cells with three in vitro assays.

Fig. 4. Cytotoxic effects of NH3 in A549 cells with three in vitro assays.

1344 S. Bakand et al. / Toxicology in Vitro 21 (2007) 1341–1347

a reflectometric method (Merck, Germany). In this analyt-ical method ammonium ions ðNHþ4 Þ were reacted with achlorinating agent to form monochloramine that in returnreacts with a phenol compound to form indophenol bluewhich is proportional to the ammonia concentration.Calibration of NH3 test atmospheres also indicated a highcorrelation between calculated and measured test concen-trations (R2 = 0.99).

2.7. Statistical analysis

Statistical analyses were performed using MicrosoftExcel 2002 and SPSS (version 12.0) Software. Experimentalresults were expressed as mean ± standard deviation(m ± SD) for three different replicates at each test concen-tration. For all in vitro assays the percentage of cell viabil-ity at each test concentration was calculated (Bakand et al.,2006a). After testing the homogeneity of variances usingthe F test, the Student t-test was used to compare the aver-age cell viability of exposed cells and control cells. Differ-ences were considered as statistically significant at p < 0.05.

3. Results

3.1. Cytotoxic effects of nitrogen dioxide

Cytotoxic effects of NO2 in human A549 lung derivedcell lines with the MTS, NRU and ATP assays are pre-sented in Fig. 2. Cell viability was significantly reduced ina concentration dependent manner after exposure ofhuman A549 lung derived cells to test concentrations ofNO2 ranging from 2.5 to 15 ppm (p < 0.05).

3.2. Cytotoxic effects of sulphur dioxide

Cytotoxic effects of SO2 in human A549 lung derivedcell lines with the MTS, NRU and ATP assays is presentedin Fig. 3. Cell viability was reduced in a concentrationdependent manner after exposure of human A549 lungderived cells to test concentrations of SO2 ranging from10 to 200 ppm.

0

25

50

75

100

125

2.5 5 10 15Concentration (ppm)

Cel

l Via

bili

ty %

MTSNRUATP

Fig. 2. Cytotoxic effects of NO2 in A549 cells with three in vitro assays.

3.3. Cytotoxic effects of ammonia

Cytotoxic effects of NH3 in human A549 lung derivedcell lines with the MTS, NRU and ATP assay is presentedin Fig. 4. Cell viability was reduced in a concentrationdependent manner after exposure of human A549 lungderived cells to test concentrations of NH3 ranging from40 to 200 ppm.

3.4. Comparative toxicity assessment

Toxicity ranking of selected gaseous compounds wasdetermined using both in vitro and in vivo published data.The airborne IC50 values were derived for selected gaseousairborne contaminants considering both linear and expo-nential concentration-effect relationships of each test gas(Table 1). Airborne IC50 values were calculated based onthe most sensitive in vitro assays for each test gas. However,all three in vitro assays revealed similar toxicity ranking forselected gaseous contaminants. The 50% lethal concentra-tion (LC50) values were reported for all selected gaseouscontaminants by the NIOSH Registry of Toxic Effects ofChemical Substances (RTECS) in rats (NIOSH, 2004).Identical toxicity ranking of selected gaseous contaminants

Table 1Toxicity ranking of gaseous compounds using in vitro and in vivo data

Test gas In vitro toxicity data In vivo toxicity data

IC50 (A549) (m ± SD) In vitro assay LC50 (rat) Reference

NO2 11 ± 3.54 ppm NRU 117 ppm/1 h RTECSSO2 48 ± 2.83 ppm ATP 2520 ppm/1 h RTECSNH3 199 ± 1.41 ppm MTS 2000 ppm/4 h RTECS

S. Bakand et al. / Toxicology in Vitro 21 (2007) 1341–1347 1345

were achieved using both in vitro and in vivo publishedinhalation toxicity data.

4. Discussion

Comparative cytotoxicity of selected gaseous contami-nants was studied in vitro using a dynamic direct exposuremethod in which human A549 lung derived cells weregrown on porous membranes and exposed to gaseous con-taminants directly at the air/liquid interface using a hori-zontal diffusion chamber system (Bakand et al., 2006b).Test atmospheres of selected gaseous contaminants wereproduced using a dynamic direct dilution method and ver-ified via appropriate analytical methods. Test gases wereselected from known environmental or occupational gas-eous contaminants and included: NO2, SO2 and NH3.

Nitrogen dioxide (NO2) is a well known indoor and out-door oxidant gas. Motor vehicles are the major mobilesource of ambient NO2, whereas industrial processes suchas electric power generation, arc and gas welding, electro-plating, nitric acid production and storage of silage in agri-cultural operations are the main stationary sources(Schlesinger et al., 2000). NO2 is a pulmonary toxicantinducing irritation, acute inflammation, pulmonaryoedema and pneumonia (Winder, 2004). Due to both lowwater solubility and high reactivity, NO2 may react withpulmonary cells causing direct cytotoxicity (Tu et al.,1995).

In this study, concentration-dependent effects wereobserved in human lung cells immediately after 1 h expo-sure to NO2 concentrations from 2.5–15 ppm (Fig. 2)which compared favourably to other published studies(Tu et al., 1995; Ritter et al., 2001). Ritter et al. (2001)investigated cellular responses of human lung fibroblasts(LK004) and human bronchial epithelial (HFBE-21) cellsto NO2 under environmentally relevant concentrations.Cytotoxic effects of NO2 were investigated in humanA549 lung derived cell lines, IC-21 mouse macrophage celllines and HUVE human umbilical cord vein endothelialcells at concentrations between 2–20 ppm (Tu et al.,1995). One hour exposure to 5 ppm NO2 increased lactatedehydrogenase (LDH) release of HUVE cells (7.9–21.6%),IC-21 macrophages (5.7–10.9%) and A549 cells (2.0–3.4%).At 10 ppm NO2 the depletion of intracellular glutathione(GSH) was significant in all cell types (p < 0.05). However,considerable resistance of A549 cells to the acute toxicity ofNO2 has been reported and may be due to significantlyhigher level of glutathione in this cell line (Tu et al., 1995).

At the lowest test concentration, 2.5 ppm, NO2 exposureresulted in a significant decrease in cell viability of humanA549 cells with the MTS and NRU in vitro assays(p < 0.05). However, no reduction was observed in cellularATP content after exposure of A549 cells to 2.5 ppm NO2

for 60 min (Fig. 2). An increased cellular ATP/ADP ratiohas also been reported in human bronchial epithelial cellsHFBE-21 after exposure to NO2 concentrations lower than1 ppm, probably due to enhanced metabolic activity of thecells (Ritter et al., 2001). In contrast to in vivo, cultured pri-mary cells or the A549 cell line may have a predominantlyglycolytic metabolism, which is well known for renal andhepatic epithelial in vitro systems. Therefore, higher con-centrations of test gas were perhaps necessary to affectATP or ATP/ADP ratio than proliferation of cells. Toinvestigate this, assessment of mitochondrial membranepotential using rhodamine (Rh 123) or carbocyanine (JC-1) fluorescent dyes could be used (Chen, 1988; Cossarizzaet al., 1993). However, NO2 concentration at 5 ppminduced a significant reduction in cellular ATP content ofA549 cells when compared to control cells (p < 0.01).

Sulphur dioxide, an indoor and outdoor air pollutant,can be oxidised to acid aerosols during its atmospheric life.SO2 is generated as a by-product from diverse industrialprocesses such as smelting of sulphide ores, electrical gen-eration, iron and steel mills, paper manufacturing andpetroleum refining (McDow and Tollerud, 2003). Theharmful effects of SO2 and particulates have been docu-mented in several severe air pollution episodes in Londonduring the 1950s (McDow and Tollerud, 2003). Upon inha-lation, SO2 rapidly dissolves into the upper airway liningfluids and dissociates into bisulphite and sulphite ions.Bisulphite is known as a bronchial constrictor (McDowand Tollerud, 2003).

Cytotoxic effects of SO2 were studied in human A549lung derived cells immediately after 1 h exposure to 10–200 ppm (Fig. 3). Cell viability reduced in a concentra-tion-dependent manner, however, considering the compar-atively low cell viability reductions, and need to assess thecomplete concentration-response relationship, SO2 testconcentrations were increased to 200 ppm. No furtherreduction in cell viability was observed after 24 h post incu-bation (data are not shown) which compared favourably toKnorst et al., who studied the toxic effects of SO2 in humanalveolar macrophages (Knorst et al., 1996). The ability ofalveolar macrophages to release tumour necrosis factor-a(TNF-a) and interleukin-1b (IL-1b) was impaired signifi-cantly following a 30-min SO2 exposure (up to 5 ppm),

1346 S. Bakand et al. / Toxicology in Vitro 21 (2007) 1341–1347

whereas their capacity to produce IL-6 and transforminggrowth factor-b (TGF-b) was not affected. Up to 9.5%reduction in cell viability was measured immediately after30-min exposure of human alveolar macrophages to SO2

and no further reduction was reported after 24 h post incu-bation (Knorst et al., 1996). In this study, cellular ATPcontent appeared to be the most sensitive test for cytotox-icity of SO2 in human A549 cells among selected in vitro

cytotoxicity endpoint assays. Cell viability of humanA549 cells reduced to approximately 50% (53.82 ±12.57%) immediately following 1 h exposure to 40 ppmSO2 using the ATP assay.

Ammonia is a colourless gas with a pungent smell and ismainly an industrial indoor air pollutant. NH3 is involvedin the production of fertilisers, nitric acid, plastics, explo-sives, household cleaners and refrigerants (Weisskopfet al., 2003; Winder, 2004). The hazards of NH3 are asso-ciated to its high solubility that forms a strong alkalinesolution capable of inducing extreme irritation of the upperrespiratory tract. Depending on the concentration, ammo-nia exposure can cause different consequences ranging frommild irritation to inflammatory responses of the entirerespiratory tract, pulmonary oedema and bronchopneumo-nia (Winder, 2004).

Cytotoxic effects of NH3 were investigated in humanA549 cells after 1 h exposure to 40–200 ppm (Fig. 4). Cellviability of human A549 cells was reduced in a concentra-tion-dependent manner after exposure to ammonia gas. Cellviability of human A549 cells reduced to less than 50%(49.51 ± 1.22%) immediately after 1 h exposure to200 ppm NH3. While increasing the incubation time to24 h did not significantly reduced cell viability of A549 cellsusing the ATP assay, cell viability was significantly reducedusing the MTS (15.0 ± 1.2%) and NRU (9.0 ± 1.1%)assays. A diversified battery of in vitro test methods measur-ing different cytotoxic endpoints may potentially provide abetter understanding of mechanisms involved in toxicity oftest chemicals. Different results of biological endpoints mayprovide an indication of the possible mechanisms responsi-ble for the cytotoxic effects (Knebel et al., 1998).

Extrapolation of an accurate IC50 value for a test chem-ical requires a complete dose-response relationship repre-senting the response at diverse range of concentrations.Nevertheless, to quantify the cytotoxicity of selected testgases for comparative and ranking purposes, airborneIC50 values were derived for selected airborne contaminantsconsidering both linear and exponential concentration-effect relationships of each test gas (Table 1). AirborneIC50 values were calculated based on the most sensitivein vitro assays for each test gas including NO2

(IC50 = 11 ± 3.54 ppm; NRU) > SO2 (IC50 = 48 ± 2.83ppm; ATP)> and NH3 (IC50 = 199 ± 1.41 ppm; MTS).However, all three in vitro assays revealed similar toxicityranking for selected gaseous contaminants. The inhala-tional LC50 values were reported for all selected gaseouscontaminants by RTECS for the rat including: NO2;117 ppm/1 h > SO2; 2520 ppm/1 h > and NH3; 2000 ppm/

4 h (NIOSH, 2004). Considering both concentration andexposure time, identical toxicity ranking of selected gases(NO2 > SO2 > NH3) was achieved using both in vitro andpublished in vivo inhalational toxicity data (Table 1).Although in vitro data is not a substitute for whole animalstudies, this comparison suggests that results of in vitro testmethods are comparable to in vivo data with more sensitiv-ity for respiratory toxicity of selected gaseous contaminants.

While in vitro methods have been increasingly imple-mented in the multiple disciplines of toxicology, the appli-cation of in vitro models in inhalation toxicology is stilllimited. However, results of this study confirm thatin vitro models with appropriate exposure techniques thatmore closely reflect inhalation exposure in vivo have a sig-nificant potential to be applied widely for toxicity testing ofairborne contaminants. These comparable in vitro exposuretechniques have the potential to substantially reduce theuse of animals for future risk assessment of human air-borne chemical exposures.

Acknowledgements

The authors thank Dr. Christian Khalil for his technicalassistance and Dr. Zhanhe Wu (Westmead Hospital, Syd-ney) for supplying the human cells. The authors would alsolike to thank Dr. Paul Thomas (Department of Medicine,Prince of Wales Clinical School) and Dr. Maria Kavallaris(Experimental Therapeutics Program Children’s CancerInstitute Australia for Medical Research) for initially pro-viding the A549 cell lines.

References

ACGIH, 2006a. 2006 TLVs and BEIs; Based on the documentation ofthreshold limit values for chemical substances and physical agents andbiological exposure indices. In: American Conference of GovernmentalIndustrial Hygienists, ACGIH Worldwide, Cincinnati, USA.

ACGIH, 2006b. 2006 Guide to occupational exposure values. In:American Conference of Governmental Industrial Hygienists, ACGIHWorldwide, Cincinnati, USA.

Aufderheide, M., Knebel, J.W., Ritter, D., 2003. Novel approaches forstudying pulmonary toxicity in vitro. Toxicology Letters, 205–211.

Babich, H., Borenfreund, E., 1992. Neutral red assay for toxicologyin vitro. In: Watson, R.R. (Ed.), In Vitro Methods of Toxicology. CRCPress, Inc, Boca Raton, pp. 237–252.

Bakand, S., Winder, C., Khalil, C., Hayes, A., 2005a. Toxicity assessmentof industrial chemicals and airborne contaminants: transition fromin vivo to in vitro test methods: a review. Inhalation Toxicology 17,775–787.

Bakand, S., Hayes, A., Winder, C., Khalil, C., Markovic, B., 2005b. In

vitro cytotoxicity testing of airborne formaldehyde collected in serum-free culture media. Toxicology and Industrial Health 21, 147–154.

Bakand, S., Winder, C., Khalil, C., Hayes, A., 2006a. A novel in vitro

exposure technique for toxicity testing of selected volatile organiccompounds. Journal of Environmental Monitoring 8 (1), 100–105.

Bakand, S., Winder, C., Khalil, C., Hayes, A., 2006b. An experimentalin vitro model for dynamic direct exposure of human cells to airbornecontaminants. Toxicology Letters 165 (1), 1–10.

Blaauboer, B.J., 2002. The applicability of in vitro-derived data in hazardidentification and characterisation of chemicals. Environmental Tox-icology and Pharmacology 11 (3–4), 213–225.

S. Bakand et al. / Toxicology in Vitro 21 (2007) 1341–1347 1347

Chauhan, A.J., Johnston, S.L., 2003. Air pollution and infection inrespiratory illness. British Medical Bulletin 68 (1), 95–112.

Chen, L.B., 1988. Mitochondrial membrane potential in living cells.Annual Review of Cell Biology 4, 155–181.

Chen, L.C., Fang, C.P., Qu, Q.S., Fine, J.M., Schlesinger, R.B., 1993. Anovel system for the in vitro exposure of pulmonary cells to acidsulphate Aerosols. Fundamental and Applied Toxicology 20 (2), 170–176.

Cossarizza, A., Baccaranicontri, M., Kalashnikova, G., Franceschi, C.,1993. A new method for the cytofluorometric analysis of mitochon-drial membrane potential using the J-aggregate forming lipophiliccation 5,5 0,6,6 0-tetrachloro-1,1 0,3,3 0-tetraethylbenzimidazolcarbocya-nine iodide (JC-1). Biochemical and Biophysical Research Communi-cations 197 (1), 40–45.

Gad, S.C., 2000. In Vitro Toxicology. Taylor and Francis, New York.Goldberg, A.M., 2004. Animals and alternatives: social expectations and

scientific need. Alternatives to Laboratory Animals 32 (6), 545–551.

Greenberg, M.I., Hamilton, R.J., Phillips, S.D., McCluskey, G.J.(Eds.), 2003. Occupational, Industrial, and Environmental Toxicol-ogy, second ed. Mosby, Inc, Philadelphia, Pennsylvania.

Hayes, A., Bakand, S., Winder, C., 2007. Novel in vitro exposuretechniques for toxicity testing and biomonitoring of airborne contam-inants. In: Marx, U., Sandig, V. (Eds.), Drug Testing In Vitro-Breakthroughs and Trends in Cell Culture Techniques. Wiley-VCH,Berlin, pp. 103–124.

Klaassen, C.D. (Ed.), 2001. Casarett and Doull’s Toxicology: The BasicScience of Poisons, sixth ed. McGraw-Hill, New York.

Knebel, J.W., Ritter, D., Aufderheide, M., 1998. Development of anin vitro system for studying effects of native and photochemicallytransformed gaseous compounds using an air/liquid culture technique.Toxicology Letters, 1–11.

Knorst, M.M., Kienast, K., Muller-Quernheim, J., 1996. Effect of sulfurdioxide on cytotoxine production of human alveolar macrophagesin vitro. Archives of Environmental Health 51 (2), 150–156.

McDow, S.R., Tollerud, D.J., 2003. Outdoor air pollution. In: Greenberg,M.I., Hamilton, R.J., Phillips, S.D., McCluskey, G.J. (Eds.), Occupa-tional, Industrial, and Environmental Toxicology, second ed. Mosby,Inc, Philadelphia, Pennsylvania, pp. 622–635.

Muckter, H., Zwing, M., Bader, S., Marx, T., Doklea, E., Liebl, B., Fichtl,B., Georgieff, M., 1998. A novel apparatus for the exposure of culturedcells to volatile agents. Journal of Pharmacological and ToxicologicalMethods 40, 63–69.

NIOSH, 1994. NIOSH Manual of Analytical Methods (NMAM), fourthed., http://www.cdc.gov/niosh/nmam/nmammenu.html.

NIOSH, 2004. The Registry of Toxic Effects of Chemical Substances. USNational Institue for Occupational Safety and Health. http://www.cdc.gov/niosh/rtecs/.

O’Hare, S., Atterwill, C.K., 1995. Methods in molecular biology. In vitro

toxicity testing protocols. Humana Press, New Jersey, USA.OSHA, U., 1989. Sulfur Dioxide in Workplace Atmospheres, Method

Number: ID-104. US Occupational Safety & Health Administration.http://www.osha.gov/dts/sltc/methods/toc_s.html.

Promega, 2001. CellTiter 96� Aqueous Non-Radioactive Cell Prolifera-tion Assay: Technical Bulletin No 169. Promega Corporation, Mad-ison, USA.

Promega, 2004. CellTiter-GIO Luminescent Cell Viability Assay. Techni-cal Bulletin No. 288. Promega Corporation, Madison, USA.

Ritter, D., Knebel, J.W., Aufderheide, M., 2001. In vitro exposure ofisolated cells to native gaseous compounds – development andvalidation of an optimized system for human lung cells. Experimentaland Toxicologic Pathology 53, 373–386.

Schlesinger, R.B., Chen, L.C., Zelikoff, J.T., 2000. Sulfur and nitrogenoxides. In: Cohen, M.D., Zelikoff, J.T., Schlesinger, R.B. (Eds.),Pulmonary Immunotoxicology. Kluwer Academic Publishers, Boston,pp. 337–352.

Tu, B., Wallin, A., Moldeus, P., Cotgreave, I.A., 1995. Cytotoxicity ofNO2 gas to cultured human and murine cells in an inverted monolayerexposure system. Toxicology 96 (1), 7–18.

Weisskopf, M.G., Drew, J.M., Hanrahan, L.P., Anderson, H.A., Haugh,G.S., 2003. Hazardous ammonia releases: public health consequencesand risk factors for evacuation and injury, United States, 1993–1998.Journal of Occupational and Environmental Medicine 45 (2), 197–204.

Winder, C., 2004. Toxicology of gases, vapours and particulates. In:Winder, C., Stacey, N.H. (Eds.), Occupational Toxicology, second ed.CRC Press, Boca Raton, pp. 399–424.

Winder, C., Stacey, N.H. (Eds.), 2004. Occupational Toxicology, seconded. CRC Press, Boca Raton.