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Microbial Populations and the Development of Tobacco Specific Nitrosamines in Moist Snuff Products

Principal Investigator: Steve Foley and Colleen Rogers

Funding Mechanism:  Internal FDA

ID number: C13061/E07568.01

Award Date: 3/12/2015

Institution: National Center for Toxicological Research (NCTR)

Certain bacteria and fungi in tobacco may contribute to the development of tobacco-specific nitrosamines (TSNAs), which are cancer-causing substances in smokeless tobacco and other tobacco products. The goal of this study is to evaluate potential additional ways that TSNAs can be limited in smokeless tobacco products. Study aims are: (1) to determine whether there is a difference in the types of bacteria and fungi found in products purchased at different times of the year; (2) to compare the TSNA levels with the types of microorganisms present; and (3) to examine the impact of storage time and temperature on microbial populations and TSNA levels. Researchers will count cultured bacteria and fungi from moist snuff, identify unique strains using DNA sequencing, and evaluate these strains for their ability to reduce nitrate and nitrite, which are associated with TSNA development. Researchers will also analyze the total microbial DNA in snuff to determine the relative proportions of microorganisms present in the samples, and will screen them by polymerase chain reaction (PCR) for nitrate and nitrite reductase genes. Some bacteria and fungi can convert the nitrate in tobacco to nitrite, which then reacts with chemicals such as nicotine to yield TSNAs; thus, strains able to reduce nitrate and/or nitrite will be spiked into snuff to determine whether they affect TSNA development. Nitrosamine content in snuff samples will be measured by ultra-performance liquid chromatography and tandem mass spectrometry-based methods. Finally, researchers will evaluate the impact of storage conditions to determine the effects of time and temperature on microbial changes and TSNA production. The results should clarify the additional factors that contribute to TSNA development and identify additional conditions that can minimize the impact of TSNAs on user health, and thus may inform regulatory activities related to smokeless tobacco.

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