A Rapidin VitroAssay for Evaluation of Metabolism-Dependent Cytotoxicity of Antiepileptic Drugs on Isolated Human Lymphocytes

Abstract

In vitroassessment of human lymphocyte viability by trypan blue dye exclusion in the presence of an external metabolizing system (microsomes plus NADPH) has been shown to be a useful method in assessing predisposition to idiopathic toxicity in response to various anticonvulsant drugs. The trypan blue method, however, is labor intensive, is time consuming, is prone to human error, is not suitable for high-volume toxicity screening, and excludes autolysed cells. The objective of this study was to develop a rapid, high-capacity, objective, and easyin vitrocytotoxicity method for the detection of metabolism-dependent cytotoxicity of a test chemical. Thein vitrosystem uses an external metabolizing system (rabbit microsomes) in conjunction with isolated human lymphocytes as the target cells. Cellular toxicity was determined by assessing plasma membrane integrity using 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), or phenobarbital (62.5 μm) showed net increases in percentage cell death of 31 ± 5, 11 ± 4, 0 ± 3, and 2 ± 3, respectively. A metabolism-dependent concentration–response was observed for valproic acid-induced cytotoxicity, which approached a plateau at a concentration of 4000 μg/ml with a net percentage cell death of 31 ± 4. This technique resolves various technical difficulties inherent in viability determinations by the trypan blue exclusion method. The YO-PRO-1 method also may be useful in a clinical setting for the assessment of patients with a genetically determined susceptibility to certain drugs and for identifying the responsible drug in patients with idiopathic toxicity undergoing multiple-drug therapy.