This project will attempt to identify mutagenic compounds in smokeless tobaccos. As the use of smokeless tobacco is strongly associated with cancer of the oral cavity, identification of genotoxic agents could be an important step in the identification of carcinogens in smokeless tobacco. A number of smokeless tobaccos will be purchased in local shops and these will serve as the tobacco samples for this project. The tobaccos will be extracted in a neutral buffer and subsequently with organic solvents to yield a neutral-aqueous, and a neutral-organic layer. The aqueous layer will be further fractionated in acidic and basic organic and aqueous fractions. Each fraction will be assayed for mutagenic activity in 3 bacterial strains in a modified Ames Salmonella assay. In addition a forward modification of the Ames assay will be used. All extracts will be tested under several different conditions. Active extracts will subjected to HPLC and collected fractions will be assayed for mutagenic activity. Active fractions will be rechromotographed on a different type of column. Fractions which appear pure will be analyzed by mass-spectral and/or other means in an attempt to identify the active compounds. Several tobacco samples will be compared with mutagenicity results from other extractions. In addition saliva will be obtained from a volunteer who will """"""""chew"""""""" one or more of the smokeless tobaccos. The saliva will be extracted and fractionated as above, and mutagenic activity in the fractions will be compared with mutagenic activity obtained under the previous conditions.
