Aryl hydrocarbon hydroxylase in a stable human B-lymphocyte cell line, RPMI-1788, cultured in the absence of mitogen.

Abstract

A stable, immunologically defined human B-lymphocyte cell line (RPMI-1788) in a long-term culture has shown high basal and inducible aryl hydrocarbon hydroxylase (AHH) activity in the absence of mitogenic prestimulation, making it an excellent and a prolific source of human tissue for studies on human mixed-function oxygenases. Various parameters influencing the enzyme activity and inducibility were studied and optimized. This allowed attainment of enzyme levels and inducibility ratios comparable to those previously reported for fresh, mitogen-activated human lymphocytes. Dibenz( a,h )anthracene, at an optimal concentration of 0.3 µm and an optimal exposure time of 24 hr, was 30 times more potent an inducer than 1,2-benzanthracene. Maximal basal and induced enzyme activities and cell viability occurred at 48 hr of culture while the cell viability frequently declined by 72 hr of culture. The mitogens, phytohemagglutinin M (200 µg/ml) and concanavalin A (40 µg/ml), which were not obligatory for AHH expression, enhanced AHH activity while, at the same time, decreasing the number of viable cells per culture. However, combinations of phytohemagglutinin and concanavalin A or those of either of these with pokeweed mitogen (itself ineffective) did not potentiate AHH activity over that obtained with phytohemagglutinin or concanavalin A used alone; however, various mitogen combinations were more cytotoxic than individual mitogens. Lipopolysaccharide B did not affect either cell growth or AHH activity. Using cells cultured under optimal conditions and induced optimally with dibenz( a,h )anthracene, metabolism of benzo( a )pyrene was analyzed by high-pressure liquid chromatography. The metabolite profile produced by these cells had several similarities with those produced by mitogen-stimulated, short-term cultured human lymphocytes derived from fresh blood and by rodent liver microsomes.