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Abstract

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.

Gene Expression Changes and Signal Transduction Pathway Alterations in Primary Human Oral Epithelial Cells Exposed to Smokeless Tobacco Extracts

“…We were unable to see any apoptotic induction with the treatment before 24 h at the protein level. This finding is in line with a report that showed that induction of apoptosis with the same product (1S3) started from 48 h and remained as so until 96 h suggesting STE induced apoptosis is a late event [18].…”

“…Mechanistically, the induction of apoptosis as a result of STE exposure was suggested to be associated with the modulation of nitric oxide [18], oxidative stress [16], and osmotic stress [37]. Components in STE are implicated in the activation of Cytochrome-c, an activator of the intrinsic pathway of apoptosis, with the production of superoxide anions [16].…”

“…Smokeless tobacco (moist snuff) extract (STE) was prepared by using research grade reference 1S3 moist snuff (a kind gift from Dr. David Danehower, North Carolina State University) as described in the literature [18]. Briefly, 10 g of smokeless tobacco was mixed with 100 ml of basal medium (CNT-32, ZenBio, NC).…”

“…Previous reports indicated an increase in the induction of apoptosis by STE in a variety of cell types [15] [16]. Interestingly, STE induced toxicity leading to cell death is attributed to apoptosis on various oral cell types, including oral keratinocytes, hamster cheek pouch cells, and EBV-infected B cells [16]- [18]. However, an understanding of the molecular mechanism (s) leading to STE-induced apoptosis at the transcription level is minimal.…”

Smokeless tobacco induced biophysical and biochemical alterations in the plasma, erythrocytes, and platelets of panmasala users: Subsequent biological effects

“…Mangipudy and Vishwanatha [ 53 ] reported that smokeless tobacco extract causes a dose-dependent activation of apoptosis through the immediate actions of nitric oxide. We examined the participation of anti-apoptotic and pro-apoptotic markers, which have been described as importantly involved in the process of apoptosis.…”