Effect of smokeless tobacco products on human oral bacteria growth and viability

Abstract

To evaluate the toxicity of smokeless tobacco products (STPs) on oral bacteria, seven smokeless tobacco aqueous extracts (STAEs) from major brands of STPs and three tobacco-specific N-nitrosamines (TSNAs) were used in a growth and viability test against 38 oral bacterial species or subspecies. All seven STAEs showed concentration-dependent effects on the growth and viability of tested oral bacteria under anaerobic culture conditions, although there were strain-to-strain variations. In the presence of 1 mg/ml STAEs, the growth of 4 strains decreased over 0.32-2.14 log₁₀ fold, while 14 strains demonstrated enhanced growth of 0.3-1.76 log₁₀ fold, and the growth of 21 strains was not significantly affected. In the presence of 10 mg/ml STAEs, the growth of 17 strains was inhibited 0.3-2.11 log₁₀ fold, 18 strains showed enhanced growth of 0.3-0.97 log₁₀ fold, and 4 strains were not significantly affected. In the presence of 50 mg/ml STAEs, the growth of 32 strains was inhibited 0.3-2.96 log₁₀ fold, 8 strains showed enhanced growth of 0.3-1.0 log₁₀ fold, and 2 strains were not significantly affected. All seven STAEs could promote the growth of 4 bacterial strains, including Eubacterium nodatum, Peptostreptococcus micros, Streptococcus anginosus, and Streptococcus constellatus. Exposure to STAEs modulated the viability of some bacterial strains, with 21.1-66.5% decrease for 4 strains at 1 mg/ml, 20.3-85.7% decrease for 10 strains at 10 mg/ml, 20.0-93.3% decrease for 27 strains at 50 mg/ml, and no significant effect for 11 strains at up to 50 mg/ml. STAEs from snuffs inhibited more tested bacterial strains than those from snus indicating that the snuffs may be more toxic to the oral bacteria than snus. For TSNAs, cell growth and viability of 34 tested strains were not significantly affected at up to 100 μg/ml; while the growth of P. micros was enhanced 0.31-0.54 log₁₀ fold; the growth of Veillonella parvula was repressed 0.33-0.36 log₁₀ fold; and the cell viabilities of 2 strains decreased 56.6-69.9%. The results demonstrate that STAEs affected the growth of some types of oral bacteria, which may affect the healthy ecological balance of oral bacteria in humans. On the other hand, TSNAs did not significantly affect the growth of the oral bacteria.

Keywords: Cell viability; Oral bacteria; Smokeless tobacco; Tobacco-specific N-nitrosamines; Toxicology.

Fig. 1. The cell growth of oral bacteria incubated with STAEs from different STP brands for 18 or 24 h

The cell number change = Cell Number _{in presence of STAE}/Cell Number _{in absence of STAE}. When cell number change ≥0.3 log₁₀, the change is considered as significant. A: incubated with STAE from Snus 1 (Camel Snus Large Robust); B: incubated with STAE from Snus 2 (Marlboro Snus Mint); C: incubated with STAE from Snuff 3 (Copenhagen Snuff Original Fine Cut); D: incubated with STAE from Snuff 4 (Copenhagen Long Cut Wintergreen); E: incubated with STAE from Snuff 5 (Grizzly Long Cut Premium Wintergreen); F: incubated with STAE from Snuff 6 (Skoal Long Cut Classic Wintergreen); G: incubated with STAE from Snuff 7 (Skoal Banditis Wintergreen). Statistically significant values were presented here, the rest dada were shown in Table S1. * Strains were incubated for 18 h, while other strains were incubated for 24 h.

Fig. 2. The cell growth changes of oral bacteria incubated with STAEs from different STP brands for 36 or 48 h

The cell number change = Cell Number _{in presence of STAE}/Cell Number _{in absence of STAE}. When cell number change ≥0.3 log₁₀, the change is considered as significant. A: incubated with STAE from Snus 1 (Camel Snus Large Robust); B: incubated with STAE from Snus 2 (Marlboro Snus Mint); C: incubated with STAE from Snuff 3 (Copenhagen Snuff Original Fine Cut); D: incubated with STAE from Snuff 4 (Copenhagen Long Cut Wintergreen); E: incubated with STAE from Snuff 5 (Grizzly Long Cut Premium Wintergreen); F: incubated with STAE from Snuff 6 (Skoal Long Cut Classic Wintergreen); G: incubated with STAE from Snuff 7 (Skoal Banditis Wintergreen). Statistically significant values were presented here, the rest dada were shown in Table S1. * Strains were incubated for 36 h, while other strains were incubated for 48 h.

Fig. 3. The cell viability changes of oral bacteria incubated with STAE from different STP brands

Cell Viability Changes = Cell Viability _{in presence of STAE} – Cell Viability _{in absence of STAE}. When cell viability change ≥20%, the change is considered as significant. A: incubated with STAE from Snus 1 (Camel Snus Large Robust); B: incubated with STAE from Snus 2 (Marlboro Snus Mint); C: incubated with STAE from Snuff 3 (Copenhagen Snuff Original Fine Cut); D: incubated with STAE from Snuff 4 (Copenhagen Long Cut Wintergreen); E: incubated with STAE from Snuff 5 (Grizzly Long Cut Premium Wintergreen); F: incubated with STAE from Snuff 6 (Skoal Long Cut Classic Wintergreen); G: incubated with STAE from Snuff 7 (Skoal Banditis Wintergreen). * Strains were incubated for 18 h, while other strains were incubated for 24 h.

Fig. 4. The cell growth changes of oral bacteria incubated with TSNAs

The cell number change = Cell Number _{in presence of STAE}/Cell Number _{in absence of STAE}. When cell number change ≥0.3 log₁₀, the change is considered as significant. A: incubated with NNN for 24 h; B: incubated with NNAL for 24 h; C: incubated with NNK for 24 h; D: incubated with NNN for 48 h; E: incubated with NNAL for 48 h; F: incubated with NNK for 48 h * Strains were incubated for 18 h; y strains were incubated for 36 h.

Fig. 5. The cell viability changes of oral bacteria incubated with TSNAs

Cell Viability Changes = Cell Viability _{in presence of STAE} – Cell Viability _{in absence of STAE}. A: incubated with NNN for 24 h; B: incubated with NNAL for 24 h; C: incubated with NNK for 24 h * Strains were incubated for 18 h.