Factors Influencing the Measurement and the Reproducibility of Aryl Hydrocarbon Hydroxylase Activity in Cultured Human Lymphocytes2

Abstract

Several factors that could influence the activity of aryl hydrocarbon hydroxylase (AHH) in cultured human lymphocytes were examined in an effort to determine optimal conditions for the measurement and reproducibility of this enzyme activity. The following observations were made: a) Under the culture conditions defined here, optimal AHH activity in cultures in Falcon T-30 flasks occurred after 72 hours of incubation; b) AHH-inducibility ratio was affected by the starting cell density; c) storage of blood for 24 hours resulted in the deterioration of the activity, but storage of purified lymphocytes in RPMI-1640 at room temperature for 24 hours resulted in a minimal loss of the activity; d) DNA determinations performed subsequent to the enzyme assays were more reliable than cell counts for the calculation of specific AHH activity; e) a good correlation existed between AHH activity, cellular DNA content, and blastogenesis measured by [³H]thymidine and [¹⁴C]amino acid incorporation when phytohemagglutinin (PHA) or concanavalin A was used as mitogen but not when pokeweed or bacterial lipopolysaccharide was used as mitogen; and f) different lots of fetal calf serum significantly affected AHH activity. When culture conditions were strictly controlled and lymphocytes were cultured in the presence of a mitogen combination (1:100 dilution each of PHA and pokeweed mitogen) and 3-methylcholanthrene was used as the inducer, AHH-inducibility ratio in repeat determinations on the same individual was much less variable than the corresponding basal and induced AHH activities, as well as the degree of mitogenic activation (blastogenesis) measured by the incorporation of [³H]thymidine and [¹⁴C]amino acids.