ABSTRACT
The effects of chlorpromazine (CPZ) on the lipid accumulation induced by the cationic lipophilic compound tetraphenylphosphonium chloride (TPP) were examined using proton nuclear magnetic resonance spectroscopy (NMR), lipid extraction and thin layer chromatography (TLC), and electron microscopy (EM). Chlorpromazine at concentrations of 12 or 25 microM significantly reduced the NMR-visible lipid accumulation induced by a 48-h treatment with 6.25 microM TPP in the human breast cell line, HBL-100, without affecting cell viability. TPP caused threefold increases in whole-cell triglyceride levels that were attenuated by the addition of CPZ. Electron micrographs of TPP-treated HBL-100 cells showed that the destruction of mitochondria was accompanied by the accumulation of lipid droplets and myelinoid bodies. The addition of CPZ to TPP-treated cells reduced the occurrence of lipid droplets but not of mitochondrial destruction. Treatment with CPZ, in the presence or absence of TPP, resulted in large cytoplasmic inclusions indicating the inhibition of lysosomal metabolism. The induction and attenuation of NMR-visible lipids in conjunction with concomitant changes in both intracellular lipid droplets and whole-cell triglyceride levels provides evidence that NMR-visible lipids arise from cytoplasmic neutral lipid droplets. The observation that CPZ, a known inhibitor of lysosomal and cytosolic lipid metabolism, attenuates the formation of neutral triglycerides indicates that lysosomal processing may be a major step in the accumulation of NMR-visible lipid in breast cell lines.
Subject(s)
Breast/drug effects , Breast/metabolism , Chlorpromazine/pharmacology , Lipid Metabolism , Onium Compounds/pharmacology , Organophosphorus Compounds/pharmacology , Breast/cytology , Cell Line , Chlorpromazine/administration & dosage , Dose-Response Relationship, Drug , Drug Interactions , Female , Humans , Lysosomes/drug effects , Lysosomes/metabolism , Lysosomes/ultrastructure , Magnetic Resonance Spectroscopy , Microscopy, Electron , Onium Compounds/administration & dosage , Organophosphorus Compounds/administration & dosage , Triglycerides/metabolismABSTRACT
Proton nuclear magnetic resonance (NMR) spectroscopy, histological lipid staining, and electron microscopy were used to assess the biochemical and structural changes induced by treating the cultured human breast cell line HBL-100 with the cationic lipophilic phosphonium salts p-(triphenylphosphoniummethyl) benzaldehyde chloride (drug A) and [4-(hydrazinocarboxy)-1-butyl] tris-(4-dimethylaminophenyl) phosphonium chloride (drug B). The major biochemical change detected by (1)H NMR in drug-treated cells was a significant time- and concentration-dependent increase in lipid acyl chain resonances arising from mobile lipids. The amount of NMR-visible lipid strongly correlated with morphometric measurements of oil red O-staining lipid detected in the cytoplasm by light microscopy. Ultrastructural investigations revealed substantial damage to mitochondria and the progressive development of lipid droplets accompanied by end-stage autophagic vacuoles, in the form of densely staining myelinoid bodies, after treatment of HBL-100 cells with drug B at the IC(50). No apparent increase in acid phosphatase activity was observed using electron microscopy, indicating that the accumulation of phospholipids in myelinoid bodies may result from substrate inundation of the lysosome, rather than increased lysosomal activity. These results indicate a potential role for lysosomal lipid catabolism in the formation of NMR-visible lipids in models of cytotoxic insult.
