Dog and rat hepatocytes were treated with phospholipogenics to identify the more sensitive species and to determine whether lysosomal or mitochondrial changes were the primary cause of cytotoxicity. Endpoints included cell death, lysosome membrane integrity, mitochondrial membrane polarization, and fluorescent phospholipid (NBD-PE). Dog cells exhibited lower survival IC50 values than did rat cells with all phospholipogenic treatments and exhibited a lower capacity to accumulate NBD-PE in 4 of 5 phospholipogenic test conditions. The lysosomal modulator Bafilomycin A1 (Baf) rescued dog cells from cytotoxicity caused by 3 phospholipogenic 5HT1b antagonists and hydroxychloroquine, but not fluoxetine, and rescued rat cells from hydroxychloroquine and NMTMB, a 5HT1b antagonist. Following NMTMB treatment, rat mitochondrial membrane hyperpolarization was observed at modestly cytotoxic concentrations and depolarization at the highest concentration. At the highest test concentration, lysosomal loss of acridine orange occurred by 30？min, mitochondrial polarity changes by 1？hr, and NBD-PE accumulation by 2？hr, respectively. Baf shifted mitochondrial polarity from a depolarized state to a hyperpolarized state. These data demonstrate that (a) dog hepatocytes were generally less capable of mounting an adaptive, protective phospholipidotic response than rat hepatocytes, (b) effects on mitochondria and survival were preventable by lysosomal protection, and (c) destabilizing changes in both organelles are involved causally in cytotoxicity. 1. Introduction Cationic amphiphilic drugs have chemical structures composed of hydrophilic and lipophilic regions, combined with a cationic amine group. Many cationic amphiphilic drugs are phospholipogenic (here defined as a compound that causes phospholipidosis (PLD)). A phospholipidotic response is characterized by the accumulation of phospholipids, lamellar bodies, and drug in cellular lysosomes and results from inhibited lysosomal phospholipase activity, either by binding of drug to phospholipids or by direct inhibition of phospholipases, and through alkalization of the normally acidic lysosomal mileu [1]. Lysosomotropic properties and accumulation potential of phospholipogenics are underscored by studies employing lysosomal modulators. The ionophore monensin, ammonium chloride (which causes direct lysosomal alkalinization), and the specific vacuolar sodium-potassium proton pump inhibitor Bafilomycin A1 (Baf) have been shown to abolish the pH gradient from cytoplasm (near neutral) to lysosome (low internal pH) and preclude
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