D-AMPHETAMINE INTERACTION WITH GLUTATHIONE IN FRESHLY ISOLATED RAT HEPATOCYTES

Hepatocellular damage has been reported as a consequence of amphetamin e intake for which little is known about the respective biological mec hanisms involved. To give a better insight of cellular d-amphetamine e ffects, the present study was performed to evaluate d-amphetamine effe cts on glutathione homeostasis, in vitro, using freshly isolated rat h epatocytes. Cell viability and lipid peroxidation were also evaluated. Incubation of freshly isolated rat hepatocytes with d-amphetamine (0. 08, 0.20, 0.40, and 2.00 mM) induced a concentration dependent glutath ione depletion which was observed at all times (1, 2, and 3 h of incub ation). After 3 h of incubation, cellular GSH decreased to 85%, 78%, 7 1%, and 47% of control levels for the referred concentrations, respect ively. At the third hour of incubation, GSSG levels were only slightly increased for the three higher concentrations of d-amphetamine. The m ass spectral study of the methanolic supernatants obtained from hepato cytes incubated with all d-amphetamine concentrations revealed the pre sence of the p-hydroxyamphetamine glutathione adduct (glutathion-S-yl) -p-hydroxyamphetamine. Pretreatment of hepatocytes with the P450 inhib itors metyrapone (1 mM) and iprindole (10 mu M) significantly prevente d the glutathione depletion induced by d-amphetamine. This inhibition was more effective for iprindole than for metyrapone. Incubation of is olated hepatocytes with p-hydroxyamphetamine (0.10 mM) for 3 h did not result in any modification of cell viability or GSH or GSSG levels. A lso, in the mass spectrum study performed on these samples, the charac teristic adduct obtained for d-amphetamine incubations was not detecte d. The above data suggest that-the observed glutathione depletion indu ced by d-amphetamine is at least in part due to the conversion of d-am phetamine into (glutathion-S-yl)-p-hydroxyamphetamine and that P450 2D seems to have an important role in this metabolism. In spite of the r esults obtained, showing glutathione homeostasis alterations, incubati on of freshly isolated rat hepatocytes with d-amphetamine did not resu lt in any modification of cell viability or lipid redox status.