Role of nitric oxide in the induction of apoptosis by smokeless tobacco extract.
Smokeless tobacco usage is a growing public health concern in the United States. Lesions of the oral cavity have been clearly linked to smokeless tobacco use. The objective of this study was to determine the biochemical effects of smokeless tobacco extract (STE) exposure upon hamster cheek pouch cell (HCPC-1) cultures. HCPC-1 cells were exposed to a 5 -fold dose-range of STE (0.5, 1.0 and 2.5%) over a time-course of 24-96 h. Following each exposure we measured various biochemical parameters of cell proliferation and cell death. Cell viability, cell cycle progression and S-phase DNA synthesis were measured as markers of cell proliferation. We measured lactate dehydrogenase leakage as a marker of cell membrane damage and cell death due to necrosis. No significant alterations were observed in cell cycle progression and cell proliferation as a result of exposure to STE. LDH measured colorimetrically indicated no significant effect with the lower doses(0.5, 1.0 and 2.5% STE). Apoptosis measured as the A0 peak and by the TUNEL procedure revealed that STE caused significant rates of apoptosis. Maximal apoptosis was noted between 48-96 h. In order to probe the mechanism further we measured the levels of nitrites as an indicator of nitric oxide (NO) in the media. NO levels were significantly elevated at the doses that caused an induction of apoptosis. The results from this study indicate that STE causes a dose-dependent induction of apoptosis and that this is mediated by nitric oxide.
Mangipudy
R S
RS
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha 68198-4525, USA.
Vishwanatha
J K
JK
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology*; D002460:Cell Line; D006224:Cricetinae; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008565:Membrane Proteins; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D010936:Plant Extracts / Q000737:chemistry / Q000633:toxicity; D010947:Plants, Toxic; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D014030:Tobacco, Smokeless / Q000737:chemistry* / Q000633:toxicity*
D007527:Isoenzymes; D008565:Membrane Proteins; D010936:Plant Extracts; D009569:Nitric Oxide; D007770:L-Lactate Dehydrogenase; D019001:Nitric Oxide Synthase; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone