APPROACHES TO IDENTIFY LESS HARMFUL TOBACCO AND TOBACCO PRODUCTS
Aspects of the invention concern methods for detecting, identifying and evaluating tobacco and tobacco products to determine the potential that these compositions have to contribute to a tobacco-related disease. It is based, at least in part; on the discovery that exposure of pulmonary cells to smoke or smoke condensate obtained from tobacco or tobacco products induces double stranded breaks in cellular DNA, which were efficiently detected using assays that measure the presence, absence, or amount of phosphorylation of the histone, H2AX.
1 . A method of analyzing whole smoke from a cigarette for the ability to induce a double-strand DNA break in a human cell comprising:
contacting a human cell with whole smoke obtained from a cigarette; and
performing an assay that detects a double strand DNA break in said human cell.
2 . The method of claim 1 , wherein the assay that detects a double strand DNA break in said human cell is selected from the group consisting of a Comet assay; a TUNEL assay; a sister chromatid exchange assay; an assay that detects a phosphorylation of histone H2AX; and an assay that detects a chromosomal translocation.
3 . The method of claim 1 , wherein said cigarette comprises a filter.
4 . The method of claim 3 , wherein said filter comprises an antioxidant or a radical scavenger.
5 . The method of claim 3 , wherein said filter comprises a charcoal.
6 . The method of claim 1 , wherein said cigarette comprises a Burley tobacco.
7 . A method of making a cigarette comprising:
contacting a first population of human cells with whole smoke obtained from a first tobacco;
performing an assay that detects double strand DNA breaks in said first population of human cells;
determining whether the whole smoke obtained from said first tobacco produces fewer double strand DNA breaks in said first population of human cells than the amount of double strand DNA breaks produced in a second population of human cells that were contacted with whole smoke from a second tobacco; and
incorporating said first tobacco into a cigarette when said whole smoke from said first tobacco produces fewer double strand DNA breaks in said first population of human cells than the amount of double strand DNA breaks produced in a second population of human cells that were contacted with whole smoke from a second tobacco.
8 . The method of claim 7 , wherein the assay that detects double strand DNA breaks in said first population of human cells is selected from the group consisting of a Comet assay; a TUNEL assay; a sister chromatid exchange assay; an assay that detects a phosphorylation of histone H2AX; and an assay that detects a chromosomal translocation.
9 . The method of claim 7 , wherein said first tobacco is provided in a first cigarette and said second tobacco is provided in a second cigarette and said whole smoke is obtained from said first and second cigarettes.
10 . The method of claim 9 , wherein said first and second cigarettes comprise a filter.
11 . The method of claim 10 , wherein said filter comprises an antioxidant or a radical scavenger.
12 . The method of claim 10 , wherein said filter comprises a charcoal.
13 . A method of making a cigarette comprising:
contacting a first population of human cells with whole smoke obtained from a first tobacco;
performing an assay that detects the amount of apoptosis in said first population of human cells;
determining whether the whole smoke obtained from said first tobacco produces more apoptosis in said first population of human cells compared to the amount of apoptosis produced in a second population of human cells, which were contacted with whole smoke from a second tobacco; and
incorporating said first tobacco into a cigarette when said whole smoke obtained from said first tobacco produces more apoptosis in said first population of human cells compared to the amount of apoptosis produced in said second population of human cells, which were contacted with whole smoke from the second tobacco.
14 . The method of claim 13 , wherein the assay that detects the amount of apoptosis in said first population of human cells is selected from the group consisting of detection of caspase activation; detection of cleavage of the protein poly(ADP-ribose) polymerase; detection of annexin V binding to the cell membrane; detection of chromatin condensation; detection of an increase sensitivity of chromatin in cells to acid or heat-induced denaturation; detection of fractional DNA content; and TUNEL assay.
15 . The method of claim 13 , wherein said first tobacco is provided in a first cigarette and said second tobacco is provided in a second cigarette and said whole smoke is obtained from said first and second cigarettes.
16 . The method of claim 15 , wherein said first and second cigarettes comprise a filter.
17 . The method of claim 16 , wherein said filter comprises an antioxidant or a radical scavenger.
18 . The method of claim 16 , wherein said filter comprises a charcoal.
19 . The method of claim 13 , wherein said tobacco is a Burley tobacco.