[CANCER RESEARCH 37, 460-463, February 1977] SUMMARY With a direct fluorescence assay, the levels of mixed function oxidase activity were determined in mitogen-acti vated human lymphocytes. The O-deethylation of ethoxyre sorufin to resorufin was used to quantitate mixed-function axidase activity. Ethoxyresarufin O-deethylase activity was low to nondetectable in noninduced, mitogen-activated cells, but it was readily detected in 3-methylcholanthrene, mitogen-activated lymphocytes. The activity was: (a) de pendent on assay time and number of lymphocytes; (b) dependent on the presence of reduced nicotinamide ade nine dinucleatide phosphate; (c) stable to freezing at —80° for at beast 2 weeks; (d) reproducibly detected in duplicate samples of blood from one individual when cultured and assayed at the same time; but (e) quite variable in samples of blood from one individual at different times. Since in hepatic and pulmonary tissue of model animal systems ethaxyresorufin is a specific substrate for cytochnome P 448-associated monooxygenases, the use of this chemical could proffer an assay that specifically measures human cytachnome P-448-assaciated activity. INTRODUCTION It has been suggested that a correlation exists (11) in man between susceptibility to bronchogenic cancer (cigarette smoke associated) and the inducibility by chemical cancina gens of monooxygenase enzymes in cultured blood lym phacytes that hydroxylate such carcinogens (2, 6, 8—1 1, 13, 26). This observation seems to be analogous to the situation in model systems using inbred strains of mice in which the activity of a specific monooxygenase, AHH3 is (a) host-gene regulated (7, 17, 19, 20, 24, 25), (b) genetically linked to cancer susceptibility caused by MC and benzo(a)pyrene (12, 15, 16, 18), and (c) specifically associated with the presence of cytochrame P-448 (see review, Refs. 19 and 20). Ethoxyresarufin can serve as a substrate far mixed-func tian axidases in mat(3-5), mouse,4 or hamster (5)tissue, and I This work was supported by National Cancer Institute Contracts N01-CP 33362, N01-CP-43309, and NO1-CP-55605and by contracts from the Council for Tobacco Research. The mention herein of a proprietary product does not constitute an endorsement by the United States Department of Agriculture. 2 Present address: Forensic Science Department, Karolinska Institutet, S-10401 Stockholm, Sweden. 3 The abbreviations used are: AHH, aryl hydrocarbon hydroxylase; MC, 3-methylcholanthrene. 4 R. E. Kouri, L. M. Schechtman, T. Rude, C. E. McKinney, K. Nayar, and M. D. Burke, manuscript in preparation. Received January 19, 1976; accepted November 9, 1976. its metabolism seems to be intimately associated with the level of cytachrome P-448 (4, 5). Recent evidence4 suggests that the metabolism of ethoxyresomufin in inbred strains of mice is under the same genetic control as is the metabolism of benza(a)pyrene via the AHH enzyme complex. Since the assay for O-deethylation of ethaxymesomufin (3) is direct (requiring no extraction steps), sensitive, and amenable to automation and since ethaxyresarufin may be specifically metabolized by only cytochrome P-448, this chemical may serve as an effective alternate substrate [rather than benza(a)pyrene] for determination of mixed-function oxi dase activity in human tissue. This report describes such an assay. MATERIALS AND METHODS Ethoxyresarufin and resarufin were prepared as previ ously described (3). Lymphocytes were isolated from appar ently healthy human volunteers (ages 24 to 39) who were under no current drug regimen. All were nonsmokers ex cept for Subject T. R. Lymphocytes were isolated in Ficoll Hypaque gradients (specific gravity, 1.080) (Microbiological Associates, Bethesda, Md.), washed once with Hanks' bal anced salt solution, and cultured in 25-sq cm plastic flasks in 8.0-mb aliquots at a density of 0.5 x 10@cells/mb. The culture medium was Roswell Park Memorial Institute Me dium 1640 supplemented with 17% fetal calf serum, 0.025 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfanic acid buffer, 1% phytohemagglutinin M (Bumroughs-Wellcame, Greenville, N. C.), 50 j.@gstreptomycin per ml, and 50 units penicillin pen ml. Mitogen stimulation is a prerequisite far the development of manoaxygenase activity in lymphocytes (6, 26). After 72 hr of exposure to the mitagens, lymphocyte cultures were treated with either 1.5 nmales MC pen ml medium or an equivalent amount of the solvent acetone. Acetone concentration was 0.2%. Cells were collected 24 hr later and either assayed immediately amstoned as frozen pellets in 0.2 ml 0.1 M Tnis-HCI, pH 8.5, at —80° for up to 4 weeks. Approximately 4 x 10@cells were frozen pentube. For assay of frozen material , each cell pellet was thawed to 4°in the presence of 0.2 ml of 0.01 mM ethaxymesomufin (dissolved in 0.1 M Tnis-HCI, pH 8.5), sonically disrupted (three 2-sec bursts), and transferred to a 0.5- x 0.5-cm microfluorametem cuvet. The fluorescence of the suspen sian was measured at an emission wavelength of 586 nm with an excitation wavelength of 520 nm, using an Aminco Bowman spectrophatofluarometer with a sample compart ment thermostatically regulated to 25°.After the fluanes 460 CANCER RESEARCH VOL. 37 3-Methylcholanthrene-'induced Monooxygenase (0-Deethylation) Activity of Human Lymphocytes1 M. Danny Burke,2 Richard T. Mayer, and Richard E. Kourl Biochemistry Department, University of Texas Health Science Center, Dallas, Texas 75235 fM. D. B.); Veterinary Toxicology and Entomology, Research Laboratory, Agricultural Research Service, United States Department of Agriculture, College Station, Texas 77840 (R. T. M.J; and Department of Biochemical Oncology, Microbiological Associates, Bethesda, Maryland 20014 (R. E. K.) Research. on December 10, 2021. © 1977 American Association for Cancer cancerres.aacrjournals.org Downloaded from