Table 6.

Cytotoxicity assays used for ST extracts

AssayEnd point measuredCell studiedExtraction solutionST productCytotoxicity reported in studyReference
Cell survival assayCounting of mutant cells resistant to purine analogue 6TGHuman lymphoblasts cell lines AHH-1 and TK-6H2OTwo American moist snuffsDose-dependent response. Both cell lines showed similar sensitivityShirname-More, L 1991 (98)
DAPI stainingNumber of cells that incorporate fluorescent labelHuman skin fibroblastsDMEMCommercial chewing tobaccoDose- and time-dependent response observed, increase at low concentrations, cytotoxicity observed at higher concentrationsCoppe, JP 2008 (105)
LDH releaseAmount of LDH released as a result of membrane damageMacrophage J774A.1 cellsPhosphate bufferKentucky moist snuffConcentration- and time-dependent increase in cytotoxicity observedBagchi, D 1995 (244)
Golden Syrian hamster oral epidermoid carcinoma cell lineDMEM1S3No effect observedMangipudy, RS 1999 (103)
CHO cellsH2OKentucky Reference chewing tobaccoNo effect observedYildiz, D 1999 (99)
Neutral redMeasure the amount of dye incorporated into lysosomesCHO cellsDMSO, DCM, artificial saliva11 ST productsConcentration-dependent increase in cytotoxicity observed in DMSO extracted STE, Artificial saliva, and DCM showed no dose responseRickert, WS 2009 (118)
MTSReduction of tetrazolium salts to formazan dyes by cellular enzymesGolden Syrian hamster oral epidermoid carcinoma cell lineDMEM1S3Concentration-dependent response observed, differs by exposure timeMangipudy, RS 1999 (103)
MTTReduction of tetrazolium salts to formazan dyes by cellular enzymesNormal human oral keratinocytesPBSKentucky moist snuffConcentration- and time-dependent responseBagchi, M 2001 (144)
Oral leukoplakia cells (AMOL-III)H2OCommercial khainiCytotoxicity was observed at higher concentrationsRohatgi, N 2005 (97)
Trypan blue dye exclusionCounting the number of cells that incorporate dyeGolden Syrian hamster oral epidermoid carcinoma cell lineDMEMReference Kentucky 1S3No effect observedMuns, G 1994 (106)
Macrophage J774A.1 cellsPhosphate bufferKentucky moist snuffConcentration- and time-dependent increase in cytotoxicity observedBagchi, D 1995 (244)
HT-29 human colon adenocarcinoma epithelial cellsSalineReference Kentucky moist snuffConcentration- and time-dependent response observedGregory, RL 1996 (117)
Spleen cells, T cells and oral epithelial cells from ratDMEMCommercial Swedish moist snuffConcentration- dependent response observed.Hasseus, B 1997 (107)
Normal human oral keratinocytesPBSKentucky moist snuffConcentration- and time-dependent responseBagchi, M 2001 (144)

Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.