Studies on drug-induced lipidosis: subcellular localization of phospholipid and cholesterol in the liver of rats treated with chloroquine or 4,4'-bis (diethylaminoethoxy)@-diethyldiphenylethanel

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 i n liver. T o 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 chloroquineor 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(monoacy1g1ycero)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 chloroquineor DH-treated liver can be accounted for by the drug-induced multilarnellar bodies. Triton WR1339-induced lysosomes, which were isolated for comparison, also contain bis(monoacylglycero)phosphate and bis(n1onoacylglycero)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( monoacylg1ycero)phosphate since it has been identified as an acyl donor in bis(monoacy1glycero)phosphate synthesis.-Matsuzawa, Y., and K. Y. Hostetler. Studies on drug-induced lipidosis: subcellular localization of phospholipid and cholesterol in the liver of rats treated with chloroquine or 4,4'-his(diethylaminoethoxy)cu,P-diethyldiphenylethane.,]. Lipzd Res. 1980. 21: 202-214. Supplementary key words bisphosphatitlic acid . Triton WR-I 339 diethylaminoethoxyhexestrol . lysoThe administration of certain cationic ainphiphilic drugs can cause phospholipid storage in body tissues. In one of the first examples of this form of drug toxicity, the chronic administration of 4,4'-bis(Pdiethy1aminoethoxy)qp-diethyldiphenylethane (DH) 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). The tissue concentration of all phospholipids was increased in liver, spleen and other tissues, but the tissue content of bis(monoacy1glycero)phosphate (also known as lysobisphosphatidic acid) was increased many fold more than other phosphoglycerides (1 , 2). The concentration of acidic phospholipids in liver correlated with the degree of accumulation of the drug and its metabolites (3). Administration of DH to rats or monkeys caused an increase in the tissue content of phospholipids and his( monoacylg1ycero)phosphate and was accompanied by the formation of multilamellar bodies in the cytoplasm (4). Chloroquine administration to rats also causes a substantial increase in liver phospholipid content and bis(monoacy1glycero)phosphate accumulation is particularly marked (5). It is Abbreviations: DH, 4,4'-bis(diethylaminoethyoxy)a$-diethyldiphenylethane. These studies were presented in part at the 63rd Annual Meeting of the Federation of American Societies for Experimental Biology and Medicine, Dallas, TX, April, 1979. To whom correspondence should be addressed. 202 Journal of Lipid Research Volume 21, 1980 by gest, on N ovem er 6, 2017 w w w .j.org D ow nladed fom known to inhibit the degradation of 1251-labeled low density lipoprotein (LDL) in cultured skin fibroblasts but has no effect on the binding of LDL to the cell surface receptors (6). Chloroquine also inhibits the in vitro degradation of radiolabeled LDL and VLDL by a liver postnuclear supernatant at pH 4.4 (7), and also inhibits various other lysosomal acid hydrolases presumably by raising the intralysosomal pH (8,9). However, the metabolic basis for the marked accumulation of bis(monoacylg1ycero)phosphate and other phospholipids in the liver of chloroquinetreated rats is unknown. In liver, bis(monoacylg1ycero)phosphate is found only in lysosomes as first demonstrated by Wherret and Huterer (10). The studies of Renkonen and coworkers with BHK-21 cells (1 1, 12) and of Mason, Stossel, and Vaughan (13) with alveolar macrophages also indicate a lysosomal localization of bis(monoacylg1ycero)phosphate. Electron microscopic study of liver tissue from humans (14, 15) or rats (16, 17) treated with DH or chloroquine indicates that a prominent feature of the lipidosis is the presence in the cytoplasm of numerous autophagic vacuoles and multilamellar bodies (also called myelin figures). These multilamellar bodies are of lysosomal origin based on the histochemical demonstration of acid phosphatase activity (1517) and they are thought to be rich in phospholipid, based on their histochemical properties (14, 16, 17). Numerous other drugs with widely differing pharmacologic effects also cause the accumulation of phospholipids in body tissues. The other agents include chlorophenteramine, fenfluramine, triparanol, trans1,4-bis(2-chlorobenzylaminomethyl)cyclohexane, azacosterol, 1 -chloroamitryptyline, iprindol, and chlorcyclizine (18). The drugs which cause systemic phospholipidosis have certain structural features in common. An uncharged hydrophobic region is present (usually an aromatic ring structure) and an amine group is found at varying distances from the hydrophobic region. I t is believed that the ability of these drugs to cause phospholipidosis is related to their structural similarities rather than to any common pharmacologic action (19, 20). Lysosomes isolated from the liver of rats treated with chloroquine or DH have been shown to be enriched in bis(monoacylg1ycero)phosphate (2 1, 22). In these studies, no increase in liver total phospholipid was found with DH while a slight increase (10%) was noted with chloroquine; total cholesterol was not increased with chloroquine treatment but no data are presented for DH (21). The intracellular sites of phospholipid accumulation in chloroquine-treated liver cannot be determined in these studies since the other membrane pools of phospholipid were not analyzed; marker enzyme determinations were not presented to indicate the degree of purity of the lysosomal fractions (2 1, 22). In this publication, 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. Mitochondria, microsomes, and highly-purified multilamellar bodies (lysosomes) have been isolated and the purity of these fractions has been assessed by electron microscopic analysis and by marker enzyme determinations. The lipid composition of the normal subcellular fractions has been compared with that of the rats treated with Triton WR1339, chloroquine, and DH. Our results indicate that the increased hepatic content of phospholipid caused by chloroquine and DH can be accounted for by the presence of these multilamellar lysosomes which also contain the respective drugs in high concentrations. These lysosomes contain large amounts of bis(monoacylg1ycero)phosphate and the enzyme required for its synthesis.