Mechanism of bilirubin toxicity on tissue culture cells: Factors that affect toxicity, reversibility by albumin, and comparison with other respiratory poisons and surfactants

Abstract

Various factors that influence the cytotoxic effects of bilirubin, a known electron-transport poison, on mammalian cells in culture have been studied. The cellular uptake of free bilirubin is rapid, being complete in 10 minutes or less following its addition to a cell suspension. Increasing bilirubin concentration increased cell death; there is a lag phase at all concentrations tested, inversely proportional to the concentration of the bile pigment seen with this effect on viability. Cytotoxicity increased at pH 7.6 and below. Albumin-bound bilirubin was nontoxic to tissue culture cells. Albumin added to bilirubin-treated cells resulted in extraction of significant quantities of the cellular-bound bilirubin. However, even when the albumin was added as rapidly as 1 minute after the bilirubin to cells there was still some loss in cell viability. Uncouplers of oxidative phosphorylation and inhibitors of NADH oxidase activity, unlike bilirubin, neither decreased cell viability nor brought about the loss of soluble proteins from cells. Bilirubin more closely resembled agents such as deoxycholate that affect cell membranes in bringing about the loss of cellular proteins. The suggestion is made that the electron-transport lesion induced by bilirubin may not in itself bring about cell death, but that the primary lethal lesion may be on multiple cell membrane systems, perhaps brought about by some alteration of membrane lipids.