Smokeless tobacco, oxidative stress, apoptosis, and antioxidants in human oral keratinocytes

We have investigated the effects of a smokeless tobacco extract (STE) on li pid peroxidation, cytochrome c reduction, DNA fragmentation and apoptotic c ell death in normal human oral keratinocyte cells, and assessed the protect ive abilities of selected antioxidants. The cells, isolated and cultured fr om human oral tissues, were treated with STE (0-300 &mu l;g/ml) for 24 h. S uperoxide anion production was determined by cytochrome c reductase. Oxidat ive tissue damage was determined by lipid peroxidation and DNA fragmentatio n, whereas apoptotic cell death was assessed by flow cytometry. STE-induced fragmentation of genomic DNA was also determined by gel electrophoresis. T he comparative protective abilities of vitamin C (75 mu M), vitamin E (75 m u M), a combination of vitamins C & E (75 mu M each), and a novel grape see d proanthocyanidin (IH636) extract (GSPE) (100 mu g/ml) against STE induced oxidative stress and tissue damage were also determined. Following treatme nt of the cells with 300 mu g STE/ml 1.5-7.6-fold increases in lipid peroxi dation, cytochrome c reduction and DNA fragmentation were observed. The add ition of the antioxidants to cells treated with STE provided 10-54% decreas es in these parameters. Approximately 9, 29, and 35% increases in apoptotic cell death were observed following treatment with 100, 200, and 300 mu g S TE/ml, respectively, and 51-85% decreases in apoptotic cell death were obse rved with the antioxidants. The results demonstrate that STE produces oxida tive tissue damage and apoptosis, which can be attenuated by antioxidants i ncluding vitamin C, vitamin E, a combination of vitamins C plus E and GSPE. GSPE exhibited better protection against STE than vitamins C and E, singly and in combination. (C) 1999 Elsevier Science Inc.