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=J Till: COL'Nf"II. Fo1t TOBACCO Rf:SlSAKCII-U.S.A., INC. January 7, 1977 MEMORANDUM 9IiC2228028 T0: Drs. Hockett, Huebner, Meier, Sommers, Stone and Wattenberg FROM: W. U. Gardner SUBJECTi Application 1120. Elroy T. Cantrell, Ph.D., Departmer.t of Pharmacology, Texas College of Osteopathic Medicine, North Texas State University, Denton. Aryl Hydrocarbon Hydroxylase in single cells and subpopulations of human lymphocytes and other ce11s." In the past Wattenberg ard Kouri had attempted to measure the concentrations of PAH metabolites using estimations of the fluorescence of tissue sections or isolated cells. These methods were technically difficult and not too quantitative. The present application is a collaborative effort between Dr. Tyrer at Becton Dickenson Research Institute and Cantrell and Busbee at North Texas State, the instrumentation being done at the former and the biological aspects at the latter. A Fluorescence Activated Cell Sorter (FACS) has been developed which will separate cells according to size and specific fluorescence. Enough work haz been done to show that the method is sensitive, specific and chemically simpler than other methods. The end pbint is the quantitation BaPOH in cells of different sizes. The research proposal is described in a general way. A vital stain will be used to attempt to quantitate DNA (H 33258) using the FACS-II. SuafructAons will then be stained with mithramycin and rerun in the FACS, and DNA content per cell determined using the diphenylamine reaction. Attempts will be made to determine whether cells in different stayes of the mitotic cycle show differences in uptake of DPOH. Several modifications of the handling of the cultures and of the estimation of AI:H will be studied on selected populations of persons to check the reproducibility of the method and what might cause the day- to-day differences that are observed by the conventional method. In addition to lymphocytes the method will be applied to PAMs and to skin. The applicants are experienced in the area and have adequate space and equipment. They do have some support -- approximately $33,000 per year terminating September 1977 for a similar proposal. They are asking 115% overhead for the Becton-Dickinson part and 42.8% for the Texas part. They have a source for PAMs at both sources. They have already noted differences in PaBOH levels ih cells of different sizes. Their methods can detect lOd molecules per cell. W.U.G. WUG:ek 1