Abstract
Administration of chloroquine or 4,4'-bis(diethylaminoethoxy)alpha, beta-diethyldiphenylethane (DH) to rats in oral doses of 100 mg/kg for 7 days causes phospholipid and cholesteryl ester accumulation in liver. To further characterize this drug-induced lipidosis, we have isolated and characterized the lipids of subcellular fractions from control rats and rats treated with chloroquine, DH, and Triton WR-1339. The phospholipid content of liver is increased 1.5-fold by chloroquine or DH treatment but is unaffected by Triton WR-1339. Acid phosphatase is increased by treatment with these three agents. Chloroquine and DH cause a shift of acid phosphatase from the light mitochondrial fraction (L) to the heavy mitochondrial fraction (M). Multilamellar bodies, an ultrastructural hallmark of chloroquine and DH-induced lipidosis, were isolated in a highly-purified form from the M fraction of chloroquine- or DH-treated rats. They are highly enriched in acid phosphatase indicating their lysosomal origin. In addition, they contain large amounts of phospholipid, cholesterol, and cholesteryl ester and are the sole site of bis(monoacylglycero)phosphate and the enzyme which catalyzes its synthesis from phosphatidylglycerol. Analysis of the phospholipid content of the respective control and drug-treated liver fractions shows that the entire excess phospholipid content of chloroquine- or DH-treated liver can be accounted for by the drug-induced multilamellar bodies. Triton WR-1339-induced lysosomes, which were isolated for comparison, also contain bis(monoacyglycero)phosphate and bis(monoacyglycero)phosphate synthetase. However, they differ from the drug-induced lysosomes in that their sphingomyelin content is much higher and their total phospholipid and phosphatidylinositol content much lower. The multilamellar bodies are the principal intracellular site of accumulation of chloroquine and DH, respectively. Increased delivery of phospholipid to lysosomes and decreased lysosomal catabolism of phospholipid are the factors which are thought to cause this experimental lipidosis. High levels of phosphatidylinositol in the multilamellar body may be in part responsible for the increased content of bis(monoacyglycero)phosphate since it has been identified as an acyl donor in bis(monoacylglycero)phosphate synthesis.
Highlights
Administration of chloroquine o r 4,4'-bis(diethylaminoethoxy)a,P-diethyldiphenylethane(DH) to rats in oral doses of 100 mg/kg for 7 days causes phospholipid and cholesteryl ester accumulation in liver
In one of the first examples of this form of d r u g toxicity, the chronic administration of 4,4'-bis(Pdiethy1aminoethoxy)qp-diethyldiphenylethane( D H ) to patients was found to cause a phospholipidosis characterized by the accumulation of intracellular multilamellar bodies and by the appearance in the bone marrow of foamy histiocytes which resemble those found in Niemann-Pick disease [1, 2]
We report studies of the subcellular distribution of phospholipids and cholesterol in normal rat liver and in the liver of rats treated with Triton WR-1339, chloroquine, or DH
Summary
Male Sprague-Dawley rats were given chloroquine phosphate (Aralen@o) r 4,4’-bis(diethy1aminoethoxy)a#-diethyldiphenylethane (DH) for seven days by stomach tube (100 mg/kg). T h e following marker enzymes were determined in the respective subcellular fractions: succinate dehydrogenase (mitochondria) was determined by the method of Green, Mii, and Kohout [24]; /3-glycerol phosphatase (lysosomes) as described by Trouet [23] and rotenone-insensitive NADPH-. Analyzed by thin-layer chromatography on 0.25 mm plates of silica gel G prepared with 0.4 M boric acid and developed in chloroform-methanol-concentrated ammonia-water 70:30:3:2 (by volume) as previously described [26]. T h e total lipid extracts of homogenates and the respective subcellular fractions were prepared and washed with 0.73% NaCl as described by Folch et al. In chloroquine- or DH-treated determined by fluorescence using an Amicon spectro- livers, the heavy mitochondrial fraction (M) had the photofluorimeter (285 nm, excitation and 397 nm, highest relative specific activity of acid phosphatase emission).
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