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and(b)oneexposedbyftregas
A549 cel ls were cultured in 75 cm2 culture flasks (Coming， Coming，NY)inEaglemedium(Nisui680541)suplemented with10%fetalcalfserum(CCB171012)，0.5%penicilin小島討i Seika)，5%L-glutamine(Wako2707)叩 d 1.5%Meylon(Nipon Yakyoku)at37oCwith5%CO2 inahurnidiftedatmosphere After48hoursofincubation，celswerereleased企omflaskby brief(1rnin)treatmentwith0.1%trypsinanddistributedinto standard35m mPetridishescontaining3x10コ cels/rn1each Thedisheswerethenplacedinat37oCwith5%CO2 for48h and血enobservedforaceptablebacterialcolonygrowthfor thiss印 dy.
2.2 Firegascolectionandexposureapparatus
The apara旬 s for the colection of gas evolved from combustion products is shown in Fig.3.The sampleswere placedinthealuminumdishandignitedbypilotflame.The combustiongaseswereproducedasthematerialsbum，thenthe gasesweredrawnintothealurninumcylindricalcontainerof 20mm in diameterby vacuum pump れ凡VAC DAM-OI0) Combustion byproducts were futered to eliminate the particulatema仕er，andthenacumulatedinanaluminumgas pack(GLScienceInc.l00LAAP8)
The cel-exposure device is shown in Fig. 4 and Fig. 5.Tworectangular plastic exposure chambers (SUGIYA.Miι GENA-l0)withseparategasentryandexitportswereplaced inaconventiona1incubator(SANSYO，Sfi-35)at37oC
Figure1.HumanA549lungcels
Thre e di百erent incubation gases were investigated: 95% a江 with5%CO2 (controlcelcultureatmosphericcondition)，air (79%nitrogenand21% oxygen)andthecombustiongas.The 95% air with 5% C02was delivered 企om CO2 incubator (ThermoScientificBB-15)byanairpump(Nisoα150).The combustiongaswassuplied企omthealuminumgaspackand then delivered into the test chanlber presurizing by 出e aspirator(HarvardAparatus，Inc.687MouseVentilator)ata constant flow rate of 10.0 liter per rninute (L/min) for al l experiments.Each ofthe exposure chambers contained two standard35m mPetridishes.Thesterilewatercontainerwas placedateachoftheexposurechamberstohumidi命 thecel
2.3Analysisandmeasurements
For A549cel ls， visual count 企 o m each plate was used as a measureofcelviability.Celcountswereexpresedasrelative percentageofcfuinthetreatedgroupsincomparisontothe controls.Priortoanalysis，theoldmediawasremovedandcels werewashedwithPBStwiceandreleasedfromdishbybrief treatmentwith0.1%trypsin.Afterstainingwi白 t可panblue， thenumberoflivecelswascountedbyafulautomaticcel counter(BIORAD，TCI0白1automaticcelcounter)
Forgasanalysis，agaschromatograph(SHIMAZUGC目 2014B) equipedwithflarnethermionicdetector(FTD)andthena1 conductivity detector (TCD) was used.Gas detector tube systems(GastecGV-I00S)wereusedwithshort-termquick- measuringdetectortubesforcarbonmonoxide，carbondioxide， hydrogencyanide，aldehydesandammonia
3 Resultsanddiscusion
3.1 Gasanalysis
The gas composition measured by the gas chromatograph equipped with FTD and TCD is shown in Table l.The HCNproductionwasexpresedbytherelativevalueduetolack ofstandardcalibrationgas.ThefirstrowinTable2showsthe relative values for H C N production for four materials. ThefolowingisHCNgenerationorderasmeasuredformthe m0
incuhltiun Fircglscxpusurc Figure2.TypicalshapeofHumanA549lungcelsthat(a)
culturedwithCO2gasand(b)exposedbyfiregas
5~も C0
1
Figure3Firegascolectionapparatus
Figure4Firegasexposureapparatus