A Rapid in Vitro Assay for Evaluation of Metabolism-Dependent Cytotoxicity of Antiepileptic Drugs on Isolated Human Lymphocytes

In vitro assessment of human lymphocyte viability by trypanblue dye exclusion in the presence of an external metabolizingsystem (microsomes plus NADPH) has been shown to be a usefulmethod in assessing predisposition to idiopathic toxicity inresponse to various anticonvulsant drugs. The trypan blue method,however, is labor intensive, is time consuming, is prone tohuman error, is not suitable for high-volume toxicity screening,and excludes autolysed cells. The objective of this study wasto develop a rapid, high-capacity, objective, and easy in vitrocytotoxicity method for the detection of metabolism-dependentcytotoxicity of a test chemical. The in vitro system uses anexternal metabolizing system (rabbit microsomes) in conjunctionwith isolated human lymphocytes as the target cells. Cellulartoxicity was determined by assessing plasma membrane integrityusing a membrane-impermeant fluorescent nucleic acid dye (YO-PRO-1)and a multiwell plate scanner for fluorescence. Using this system,cells incubated with either acetaminophen (1500 µg/ml),carbamazepine (62.5 µM), phenytoin (62.5 µm), orphenobarbital (62.5 µM) showed net increases in percentagecell death of 31 ± 5, 11 ± 4, 0 ± 3, and2 ± 3, respectively. A metabolism-dependent concentration-responsewas observed for valproic acid-induced cytotoxicity, which approacheda plateau at a concentration of 4000 µ/ml with a net percentagecell death of 31 ± 4. This technique resolves varioustechnical difficulties inherent in viability determinationsby the trypan blue exclusion method. The YO-PRO-1 method alsomay be useful in a clinical setting for the assessment of patientswith a genetically determined susceptibility to certain drugsand for identifying the responsible drug in patients with idiopathictoxicity undergoing multiple-drug therapy.