Direct determination of functional activity of cytochrome p‐4501A1 and nadph DT‐diaphorase in hepatoma cell lines using noninvasive scanning laser cytometry

Abstract

Mammalian organisms possess a variety of enzymes that catalyze the biotransformation of numerous chemicals with diverse structure. The gene superfamily comprising the cytochrome P‐450 monooxygenases (P‐450) are key participants in these reactions, and certain P‐450 genes are highly inducible upon xenobiotic exposure. Many of the standard techniques used in the study of these systems rely on the disruption of tissues and cells, together with the preparation of subcellular particles. We have adopted a sensitive new technique, scanning laser cytometry, to monitor P‐450‐mediated O‐dealkylation activities directly in cultured cells. Metabolism in single cells was quantified by fluorescence detection of resorufin, the P‐450‐mediated O‐dealkylation product of alkoxyresorufin ether substrate probes. Functional activities associated with P‐4501A1 and NADPH DT‐diaphorase were compared among a human hepatoma (Hep G2) cell line and cells derived from mouse (Hepa 1clc7 wt) and rat (H4‐II‐E) hepatomas. Pretreating cells with the polyaromat/c hydrocarbon indvcer β‐naphthoflavone resulted in 50‐ to 100‐fold increases in single cell rates of O‐dealkylation of ethoxyresorufin (EROD activity). The use of scanning laser cytometry enabled in situ analysis of both constitutive and inducible biotransformation activities without disruption of cells or intracellular processes that determine the toxi‐cologic fate of exogenous chemicals in vivo.