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Properties, Inducibility, and an Improved Method of Analysis of Aryl Hydrocarbon Hydroxylase in Cultured Human Lymphocytes¹

Department of Experimental Therapeutics, Grace Cancer Drug Center [H. L. G., N. B.], and Departments of Molecular Biology [N. B.] and Immunology [J. M.], Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14203

The properties and inducibility of aryl hydrocarbon hydroxylase (AHH) in cultured human lymphocytes were studied, and a sensitive method of analysis has been developed. In agreement with other reports, peripheral blood lymphocytes per se had no activity and required pretreatment in culture with a mitogen for conversion to lymphoblasts to possess AHH activity. This activity had an absolute requirement for reduced nicotinamide adenine dinucleotide phosphate. Under our conditions of incubation, reduced nicotinamide adenine dinucleotide (1.3 mM) or ethylenediaminetetraacetic acid disodium salt (1 mM), when added to reduced nicotinamide adenine dinucleotide phosphate-fortified incubation mixture, caused about 30% increase in activity and MnCl₂ caused an appreciable inhibition. The reaction was linear with the number of cells, with up to 16.8 x 10⁶ cells contained in the incubation mixture, and had a pH optimum of 8.5. The enzyme was fairly stable at -70° and retained about 90% of the original activity for 15 days.

The enzyme activity in the mitogen-stimulated lymphocyte cultures appeared slowly and reached a maximum at 48 to 72 hr of incubation at 37°, after which it considerably decayed with time. Viability of the cells in culture also decayed considerably after 72 hr and reached about 50% level at 120 hr. The enzyme was inducible with 3-methylcholanthrene and dibenz(a,h)anthracene. Dibenz(a,h)anthracene was a much more potent inducer than 3-methylcholanthrene and evoked a response in cells that would be considered noninducible with 3-methylcholanthrene.

By using purified lymphocytes and Roswell Park Memorial Institute Medium 1640, providing larger surface area during culture, harvesting the cells at the time of minimum cell death and maximum AHH activity, and assaying AHH at its pH optimum of 8.5 instead of 7.6, it was possible to enhance the sensitivity of the assay an average of 17-fold.

¹ This investigation was supported in part by Core Program Grant CA-13038, Erie County United Fund of New York, and an Institute allocation from Dr. G. M. Murphy, Director, Roswell Park Memorial Institute, Buffalo, New York 14203.

² To whom reprint requests should be addressed.

Received 10/29/74. Accepted 1/27/75.

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