Table 5.

Cytotoxicity assays for cigarette smoke (CSC/TPM), WS, and gas vapor phase smoke

AssayEnd point measuredCell studiedSmoke conditionsCigarette typeTest substance*Cytotoxicity reported in studyReference
Acid phosphatase activityCell membrane acid phosphatase activityCHO WBL strainFTC1R4FTPM (Cambridge method)Cytotoxic effect observedPutnam, KP 2002 (44)
Alamar blue assayMitochondrial dehydrogenase activityHuman bronchial epithelial cell line HBE135-50 mL puffs within 2 minN/AGVP (Exposure chamber)Little effect at 1 h, but increased response with greater dose at 24 hSpencer, JP 1995 (93)
ATP bioluminescence assayIntracellular ATP content3PC keratinocytes and J774A.1 macrophagesFTC1R4F,ECLIPSECSC (Cold trap)Concentration-dependent response observed, eclipse CSC exhibited reduced cytotoxicityCurtin, GM 2004 (272)
3H-adenine releaseMembrane integrityRat Alveolar type II cellsN/A1R1 cigaretteWS (Solvent trap)Cigarette smoke treated medium showed no cytotoxicityWirtz, HR 1996 (74)
Cell viability assayCount of live and dead cells by automated counterHuman fetal bronchial epithelial cellsFTCResearch cigarette type A and BWS (exposure chamber), smoke diluted with synthetic airDifferent effects by air dilution and cigarette typeRitter, D 2003 (85)
CytoTox 96 cytotoxicity assayLDH release into mediumHuman aortic endothelial cellsN/A1R4FTPM (Cambridge method)Minimal cytotoxicityNordskog, BK 2003 (136)
Dye-exclusion assay ViaCountFlow cytometryHuman airway epithelial cells, primary and transformedMDPHMajor U.S. brand of filtered, “full-flavor” cigaretteWS (Jet impaction) and GVP (Solvent trap)Concentration-dependent response differed by cell typesHays, LE 2008 (162)
LDH releaseAmount of LDH released as a result of membrane damageGuinea pig and human AMsFTCCommercial cigarette, brown tobacco, without filterGVP (exposure chamber)The LDH release from both cells exposed to smoke and to purified air were not significantly differentDubar, V 1993 (91)
Pig pulmonary artery endothelial cellsN/ACommercial Marlboro cigaretteWS (Solvent trap)No cytotoxic effect was observedSu,Y 1998 (71)
Human foreskin, bronchial epithelial, coronary endothelial, coronary smooth muscle, and rat liver epithelial cellsFTC1R4F, 1R5F, and a cigarette that primarily heats tobacco (TOB-HT)TPM (Cambridge method)1R4F and 1R5F increased cytoxicity. TOB-HT did not show effect in any of the cell types tested.McKarns, SC 2000 (50)
Beas-2B cellsN/AKentucky reference 1R3GVP (exposure chamber)Gas phase smoke-induced cytotoxicityArora, A 2001 (90)
CHO WBL strainFTC1R4FTPM (Cambridge method)LDH release was most sensitive assay after 1 h of exposurePutnam, KP 2002 (44)
Mouse lung epithelial cell line LA-4 cellsISOCigarettes with different filtersGVP (exposure chamber)Cytotoxicity differed by filter typePouli, AE 2003 (94)
Lung epithelial cellsISOA major international brandGVP (exposure chamber)Dose- and time-dependent response observedPiperi, C 2003 (92)
Murine 3T3 fibroblastsISO and an intense smoking conditionDark, American, and Virginian with standard and modified cellulose acetate filtersCS (Solvent trap) and GPCS (Solvent trap)Time-dependent response observed, CS and GPCS induced different cytotoxicitiesCulcasi, mol/L 2006 (88)
Normal human bronchial epithelial cellsFTC2R4F,Quest low nicotine and nicotine freeTPM (Cambridge method)Dose response observed but no significant difference among cigarettesChen, J 2008 (146)
Human gingival fibroblastsN/AN/ACSCDose-dependent increase in cytotoxicity observedZhang,W 2009 (151)
MTTReduction of tetrazolium salts to formazan dyes by cellular enzymesHuman normal breast epithelial cell line MCF10AFTC1R4FTPM (Cambridge method)Concentration-dependent response observed.Narayan, S 2004 (21)
Human epidermal keratinocytes and oral carcinoma cellsFTC1R3FCSC (Cambridge method)Concentration- and time-dependent response observedNagaraj, NS 2006 (48)
Human lung epithelial cell line A549N/AAn Indian cigarette with filterCSC (Solvent trap)Concentration response observedKaushik, G 2008 (76)
Human pulmonary artery endothelial cellsN/ALong Life cigarettesCSE (Cambridge method)Dose-dependent increase observedHsu, CL 2009 (159)
Human lung epithelial cells A549N/ACommercial filtered cigaretteAECS (Solvent trap)Dose- and time-dependent increase observedDas, A 2009 (152)
Neutral redMeasure the amount of dye incorporated into lysosomesCHO-WBLFTCCommercial and novel carbon filter cigarettesTPM (Cambridge method) and WS (Exposure chamber)Dose-response reported for CSC but no difference between cigarettes. WS of novel carbon cigarettes was less cytotoxic.Bombick, DW 1997 (59)
CHO WBL strainFTC1R4F, 1R5F, and a cigarette that primarily heats tobacco (TOB-HT)WS (Exposure chamber)WS from the 1R4F cigarette was significantly more cytotoxic on a per cigarette basis than the smokes from the 1R5F and TOB-HT cigarettesBombick, DW 1998 (86)
CHO WBL strainFTCKentucky 1R4F and 1R5F, a cigarette primarily heats tobacco (TOB-HT)TPM (Cambridge method)Dose-dependent response observed for reference cigarettes, but not for TOB-HTBombick, BR 1997 (60)
CHO WBL strainFTC1R4FTPM (Cambridge method)Concentration-dependent response observedPutnam, KP 1999 (43)
Mouse embryo BALB/c 3T3 cellsISO1R4F, cigarettes with different flavoring and tobaccoTPM (Cambridge method) and GVP (Solvent trap)No differences in GVP cytotoxicity, but there were some differences for TPM among the cigarettesRoemer, E 2002 (72)
CHO WBL strainFTC1R4FTPM (Cambridge method)Cytotoxic effect observed. Most sensitive following 24 h of exposure.Putnam, KP 2002 (44)
Mouse embryo BALB/c 3T3 cellsFTC1R4F,an EHC(EHC)TPM (Cambridge method) and GVP (Solvent trap)TPM and GVP from EHC were less cytotoxic than 1R4F on a per cigarette basisTewes, FJ 2003 (89)
Human lung epithelial cell line A549ISO2R4F, 2R4F with modified acetate or charcoal filterWS and GVP (Exposure chamber)GVP less cytotoxic than WS. Charcoal filter decreased the cytotoxicity of GVPFukano, Y 2004 (83)
Mouse embryo BALB/c 3T3 cellsFTC/ISO, MDPHEight commercial cigarettes, three reference cigarettes, EHCTPM (Cambridge method) and GVP (Solvent trap)MDPH showed higher cytotoxicity on a per cigarette basis. The opposite was found on a per milligram basis, although the results may not be stat. significant. EHC less cytotoxic than commercial cigarettes. GVP was more or less cytotoxic than TPM depending on the reporting method and product.Roemer, E 2004 (139)
CHO-WBL cellsFTC, HC, MDPHUltralights and EclipseTPM (Cambridge method)Ultralights were cytotoxic, whereas Eclipse was notFoy, JW 2004 (61)
BALB/c 3T3ISO1R4F, test cigarette containing typical commercial tobacco blend no additives with cellulose acetate filtersTPM (Cambridge method) and GVP (Solvent trap)No difference seen between licorice treated and control cigarettesCarmines, EL 2005 (132)
BALB/c 3T3ISO1R4F, test cigarette containing typical commercial tobacco blend no additives with cellulose acetate filtersTPM (Cambridge method) and GVP (Solvent trap)No difference seen between glycerin treated and control cigarettesCarmines, EL 2005 (131)
Normal human bronchial epithelial cellsFTC1R4F, EclipseCSC (Cambridge method)Concentration dependent decrease observed with 1R4F, no decrease observed with EclipseFields, WR 2005 (155)
Mouse embryo BALB/c 3T3 cellsFTC1R4F and 2R4FTPM (Cambridge method) and GVP (Solvent trap)There were no statistically significant differences between the cigarettesCounts, ME 2006 (130)
CHO K1ISODark, American, and Virginian with standard and modified cellulose acetate filtersCS (Solvent trap) and GPCS (Solvent trap)Modified cellulose acetate filters which included charcoal showed significantly lower EC50 values than modified cellulose acetate filters without charcoal in GPCSCulcasi, mol/L 2006 (88)
CHOFTCCigarettes with different levels of fructose corn syrup and reference cigarettesCSC (Cambridge method), WS (Exposure chamber)No significant differences for CSC and WS of cigarettes testedStavanja, MS 2006 (140)
CHOISO, HCKentucky reference 1R4F and 2R5F,Canadian MonitorTPM (Cambridge method) and GVP (Solvent trap)TPM for HC less cytotoxic on a per milligram basis. GVP more cytotoxic for HCRickert, WS 2007 (141)
CHOFTCStandard American tobacco blend, three test cigarettes with banded cigarette paper technologies and control cigaretteCSC (Cambridge method) and WS (Exposure chamber)No significant difference in cytotoxicity observed between control and test cigarettes in both CSC and WSTheophilus, EH 2007 (68)
BALB/c 3T3ISOTypical American blend with and without vanillinTPM (Glass filter) and GVP (Solvent trap)No statistically significant differences seen between test and control cigaretteLemus, R 2007 (134)
BALB/c 3T3ISOTypical American blend with and without potassium sorbate, 2R4FTPM (Glass filter) and GVP (Solvent trap)No statistically significant differences seen between test and control cigaretteGaworski, CL 2008 (133)
Human lung carcinoma cell line NCI-H292 and A549ISO2R4FTPM (Cambridge method)Similar cytotoxicity was observed for both cell lines at highest test doseNewland, N 2008 (273)
BALB/c 3T3 clone A31ISO1R4F, three commercial cigarettesTPM (Cambridge method) and GVP (Solvent trap)TPM from two and GVP from one of the commercial cigarettes were observed to be significantly different from the 1R4F when reported on a per cigarette basisPatskan, GJ 2008 (127)
Mouse embryo BALB/c 3T3 cellsFTC/ISO, MDPH, human puff profile (HPP)Two EHC prototypes, eight commercial cigarettes, and K1R4FTPM (Cambridge filters)Cytotoxicity from EHC was 50-75% lower per milligram of TPM, less than 50% per milligram of nicotine, and 95% lower on a per cigarette basis compared with commercial cigarettesRoemer, E 2008 (224)
Mouse embryo BALB/c 3T3 cellsISO, HPPsTwo Marlboro brands, reference cigarette, EHCTPM (Cambridge method) and GVP (Solvent trap)GVPs generated under ISO conditions showed less cytotoxicity than those by HPP on a per cigarette basis. EHC GVP and TPM less cytotoxic.Werley, MS 2008 (129)
Human bronchial epithelial cell line BEAS-2BFTC2R4FCSC (Cambridge method)Time- and concentration-dependent decrease observedCarter, CA 2009 (160)
CHOFTCReference cigarette, commercial tobacco blend, with and without cast sheetsTPM (Cambridge Method) and WS (Exposure chamber)Concentration-dependent increase observed with both CSC and WS.Potts, RJ 2009 (84)
Mouse embryo BALB/c 3T3 cellsFTC with modifications for tar production1R5F, 2R4F, 2R1FTPM (Glass fiber filter) CSC (impaction trap)Concentration-dependent decreases in viability observed, cigarettes smoked to the same TPM yield showed different cytoxicitiesRoemer, E 2009 (274)
Nigrosin stainingDye enters damaged cellsPolymorphonuclear blood cellsFTCBritish cigarette high tar, middle tar, and low tarCSCControl cells from heavy smokers without respiratory disability were more resistant to cytotoxicity than those from heavy smokers with severe diseaseHopkin, JM 1981 (275)
Resazurin bindingMetabolic activity of living cellsCHO WBL strainFTC1R4FTPM (Cambridge method)Cytotoxic effect observedPutnam, KP 2002 (44)
Sulforhodamine B bindingAmount of dye removed estimates number of viable cellsCHO WBL strainFTC1R4FTPM (Cambridge method)Cytotoxic effect observedPutnam, KP 2002 (44)
trypan blue dye exclusionDye enters damaged cells, healthy cells stay white.Mouse L-929 cells, a fibroblast-like cell linePuff number and puff volume variedDifferent filter typesWS (Exposure chamber)Cytotoxicity decreased with increasing smoke age and increased with puff numbers and increasing amounts of charcoal in filterSonnenfeld, G 1985 (135)
Human lung epithelial cell line A549FTC2R4FTPM (Cambridge method)Dose-dependent response observedFukano, Y 2006 (55)
WST-1Reduction of tetrazolium salts to formazan dyes by cellular enzymesLung epithelial cellsISOA major international brandGVP (Exposure chamber)Dose-dependent response observedPiperi, C 2003 (92)
Mouse lung epithelial cell line LA-4 cellsISOCigarettes with different filtersGVP (Exposure Chamber)Cytotoxicity differed by filter type, dose- and time-dependent response observedPouli, AE 2003 (94)
Human fetal bronchial epithelial cellsFTCResearch cigarette type A and BWS (Exposure chamber), smoke diluted with synthetic airDifferent effects by air dilution and cigarette typeRitter, D 2003 (85)
Human lung epithelial cell line A549ISO2R4F, 2R4F with modified acetate or charcoal filterWS and GVPGVP less cytotoxic than WS, charcoal filter decreased the cytotoxicity of vapor phase.Fukano, Y 2004 (83)
Human gingival fibroblastsN/AN/ACSCCytotoxicity increased in a dose-dependent mannerZhang, W 2009 (151)
WST-8Reduction of tetrazolium salts to formazan dyes by cellular enzymesNormal human bronchial epithelial cellsFTC2R4F,Quest low nicotine and nicotine freeTPM (Cambridge method)Dose-response observed, differences in cytotoxicity existed at some concentrationsChen,J 2008 (146)
XTT assayActivity of mitochondrial dehydrogenasesCHO WBL strainFTC1R4FTPM (Cambridge method)Cytotoxic effect observed after 12-24 h of exposurePutnam, KP 2002 (44)
  • *TPM and CSC as reported in publication, with the actual method indicated in parenthesis.