Table 10.

Apoptosis testing for cigarette smoke

AssayEnd point measuredCell studiedSmoke conditionsCigarette typeTest Substance*Cytotoxicity reported in study?Reference
Annexin V and PI measured by FACSMeasure apoptotic and necrotic cellsHuman fetal lung fibroblasts (HFL-1 lung, diploid, human)N/AUnfiltered cigarettesCSE (Solvent trap)Dose-dependent increaseCarnevali, S 2003 (157)
Human umbilical vein endothelial cellsN/ATwo commercial filtered cigarettesCSE (Solvent trap)Increase in cell death observed compared with controlBernhard, D 2004 (277)
Human lung epithelial cells A549N/ACommercial filtered cigaretteAECS (Solvent trap)Dose-dependent increaseDas, A 2009 (152)
Annexin V assay kitAnnexin V binds to apoptotic cellsPrimary airway epithelial cells, some transformed with SV40MDPHCommercial brandsCSC (Jet impaction), GVP (Solvent trap)Dose-dependent increaseHays, LE 2008 (162)
Annexin V/7-AAD flow cytometric assayAnnexin/7-AAD binds to apoptotic cellsImmortalized human oral keratinocytes cell line OKF6/TERT1FTC2R4FCSE (Solvent trap)Reduced cell death observed in chronic (6 mo in culture) CSE treatmentChang, SS 2009 (158)
Caspase 3 Activation AssayMeasure active caspase 3Immortalized human oral keratinocytes cell line OKF6/TERT1FTC2R4FCSE (Solvent trap)Cells treated for extended time periods were observed to have chronic Casepase-3 activationChang, SS 2009 (158)
Human pulmonary artery endothelial cellsN/ALong Life cigarettesCSE (Cambridge method)Dose-dependent increaseHsu, CL 2009 (159)
Human bronchial epithelial cell line BEAS-2BFTC2R4FCSC (Cambridge method)Dose-dependent increaseCarter, CA 2009 (160)
Elisa kit for nucleosome detectionDetection of free nucleosomes due to apoptosisNormal human bronchial epithelial cellsFTCQuest low nicotine, nicotine free, and 2R4FCSC (Cambridge method)Increased with higher dose for Quest nicotine-free but not low-nicotine or reference cigarette.Chen, J 2008 (146)
FLICA Caspase detection kitStain-activated caspase 3 and 7Mouse embryonic fibroblastsN/A2R1, 1R4F and four commercial cigarettesMS (Solvent trap)Dose-dependent increaseLin, S 2009 (161)
Microscopy — Giemsa and Acridine orangeMorphologic changes associated with apoptosisRat, mice, human, and murine AMs, and human blood monocyte– derived macrophagesN/ACommercial cigaretteCSE (Solvent trap), GVP (Solvent trap)AMs exposed to CSE showed morphologic changes including cellular shrinkage and chromatin condensationAoshiba, K 2001 (156)
PI stainingPI stains necrotic cellsHuman lung epithelial type-II cells A549N/AIndian cigarette with filterCSC (Solvent trap)Dose-dependent increaseKaushik, G 2008 (76)
TUNEL assay dUTP labelingMeasure DNA fragments due to apoptosisRat, mice, human and murine AMs and human blood-monocyte derived macrophagesN/ACommercial cigaretteCSE (Solvent trap), GVP (Solvent trap)Dose- and time-dependent increase observed. GVP caused less apoptosis than CSE. All cells behaved similarly.Aoshiba, K 2001 (156)
TUNEL stainingMeasure DNA fragments due to apoptosisPrimary airway epithelial cells, some transformed with SV40MDPHCommercial brandsCSC (Jet impaction), GVP (Solvent trap)Dose-dependent increaseHays, LE 2008 (162)
Rat-1 FibroblastsBAT protocol (ISO)N/ATPM (Glass fiber filter)Dose-dependent increasePalozza, P 2005 (153)
  • *TPM or CSC indicated as reported in publication, but actual method shown in parenthesis.