There is a growing awareness of the role of habits and diet in the induction of human cancer. The identification of mutagens in ingested material should yield important information in formulating a healthy diet and life-style. As a model for complex ingested mixtures, we will examine snuff and betel quid and its ingredients. The general popularity of snuff dipping in the United States, especially among young people, is a relatively recent trend. A number of retrospective studies have linked oral cancer with chronic snuff use. This excess risk of oral cancer among snuff dippers in U.S. resembles that observed among betel quid chewers in India and other Asian countries. One of the problems in the resolution of complex mutagenic mixtures is that the standard mutagenicity assay systems require large amounts of test material. The Microscreen assay, developed in this laboratory, requires only a small fraction of the material needed in an assay such as the Ames test. The original assay used strain E. coli WP2s(lambda) and measured toxicity, prophage induction and Trp+ revertants - all from the same exposed cell population in the microtitre plates (V=250Mul). We have demonstrated that this one strain will detect mutagens of all classes. Recently, we have introduced an additional marker for forward mutation - induction of resistance to T5 phage. The Microscreen assay has also been adapted to include the Ames tester strains, since it is possible that some of the strains may be advantageous in detecting certain classes of mutagens. In this proposal, we will separate and identify mutagens in these complex mixtures (snuff and betel quid) based on high performance liquid chromatography (HPLC) coupled with the genotoxicity assay, Microscreen. The proposal involves: 1) Comparison of the different end points: toxicity, Lambda prophage induction, Trp+ reversion, T5 resistance and his+ reversion in the Microscreen assay with E. coli WP2s(lambda) and S. typhimurium strains TA100, TA1535, TA1538, TA98, TA97, TA102 and TA104, using a variety of known mutagens found in snuff and betel quid. 2) The extraction and fractionation via HPLC of our model mixtures, snuff and betel quid. The Microscreen assay will be used to monitor the mutagenicity of HPLC fractions. Purified material will be chemically characterized. 3) Time permitting, the extension of this approach to other complex mixtures such as saliva and urine of snuff dippers and betel quid chewers.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Unknown (R23)
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Oral Biology and Medicine Study Section (OBM)
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Massachusetts Institute of Technology
Schools of Arts and Sciences
United States
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Shirname-More, L (1991) Smokeless tobacco extracts mutate human cells. Carcinogenesis 12:927-30
Shirname-More, L (1991) Forward mutation of S. typhimurium by smokeless tobacco extracts. Mutat Res 259:37-42