Chem. Res. Toxicol. 1996, 9, 1031-1036
1031
d-Amphetamine Interaction with Glutathione in Freshly Isolated Rat Hepatocytes Fe´lix Carvalho,*,† Fernando Remia˜o,† Francisco Amado,‡ Pedro Domingues,‡ A. J. Ferrer Correia,‡ and Maria Lourdes Bastos† CEQUP, Laborato´ rio de Toxicologia, Faculdade de Farma´ cia da Universidade do Porto, Rua Anibal Cunha, 164, 4050 Porto, Portugal, and Departamento de Quı´mica da Universidade de Aveiro, 3800 Aveiro, Portugal Received October 12, 1995X
Hepatocellular damage has been reported as a consequence of amphetamine intake for which little is known about the respective biological mechanisms involved. To give a better insight of cellular d-amphetamine effects, the present study was performed to evaluate d-amphetamine effects on glutathione homeostasis, in vitro, using freshly isolated rat hepatocytes. 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 glutathione depletion which was observed at all times (1, 2, and 3 h of incubation). After 3 h of incubation, cellular GSH decreased to 85%, 78%, 71%, and 47% of control levels for the referred concentrations, respectively. At the third hour of incubation, GSSG levels were only slightly increased for the three higher concentrations of d-amphetamine. The mass spectral study of the methanolic supernatants obtained from hepatocytes incubated with all d-amphetamine concentrations revealed the presence of the p-hydroxyamphetamine glutathione adduct (glutathion-S-yl)-p-hydroxyamphetamine. Pretreatment of hepatocytes with the P450 inhibitors metyrapone (1 mM) and iprindole (10 µM) significantly prevented the glutathione depletion induced by d-amphetamine. This inhibition was more effective for iprindole than for metyrapone. Incubation of isolated hepatocytes with p-hydroxyamphetamine (0.10 mM) for 3 h did not result in any modification of cell viability or GSH or GSSG levels. Also, in the mass spectrum study performed on these samples, the characteristic adduct obtained for damphetamine incubations was not detected. The above data suggest that the observed glutathione depletion induced by d-amphetamine is at least in part due to the conversion of d-amphetamine into (glutathion-S-yl)-p-hydroxyamphetamine and that P450 2D seems to have an important role in this metabolism. In spite of the results obtained, showing glutathione homeostasis alterations, incubation of freshly isolated rat hepatocytes with d-amphetamine did not result in any modification of cell viability or lipid redox status.
Introduction Hepatocellular damage as a consequence of amphetamine administration has been reported since the beginning of its therapeutical use in the 1930s. In fact, after the first observations of liver necrosis were described as a consequence of amphetamine sulfate ingestion by different species of experimental animals (1) and man (2, 3), many other reports appeared in the literature concerning amphetamines and this toxicological outcome (4-13). In spite of these reports concerning amphetamineinduced liver toxicity, little is known about the respective biological mechanisms involved. Recently, it was shown that in vivo d-amphetamine depletes nonprotein thiol groups in mouse liver and kidney in a dose dependent manner (14). There are some possible causes for these homeostatic alterations verified in vivo which include hyperpyrexia (14, 15) or catecholamine-induced glutathione depletion (16, 17). Another possibility could be the metabolic conversion of d-amphetamine to a reactive intermediate capable of forming adducts with glutathione (GSH).1 * Author to whom correspondence should be addressed. † Universidade do Porto. ‡ Universidade de Aveiro. X Abstract published in Advance ACS Abstracts, August 1, 1996.
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The aim of the present study was to get a better insight of d-amphetamine biochemical activity in freshly isolated rat hepatocytes, by evaluating d-amphetamine effects in glutathione homeostasis. For this purpose, the glutathione oxidation and/or adduct formation after damphetamine incubation with freshly isolated rat hepatocytes was determined. Some evidence for the mechanism involved in d-amphetamine reactivity is provided by the use of P450 inhibitors and by the evaluation of p-hydroxyamphetamine direct interaction with hepatocyte glutathione. Cell viability and lipid peroxidation were also determined.
Materials and Methods Chemicals. All reagents were of analytical grade. Water for HPLC was deionized (µs/cm e 0.055) (Seral Water Purification Systems, D-800 Munich, Germany). Collagenase (grade II), d-amphetamine sulfate, bovine serum albumin (fraction V), 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), glu1 Abbreviations: glutathione (GSH); disulfide form of glutathione (GSSG); total glutathione (GSH + GSSG); 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA); (reduced) nicotinamide adenine dinucleotide phosphate [NADP(H)]; (reduced) nicotinamide adenine dinucleotide [NAD(H)]; thiobarbituric acid reactive substances (TBARS); massresolved ion kinetic energy scanning (MIKES); collision induced dissociation (CID); no observed adverse effect level (NOAEL); (methylenedioxy)methamphetamine (MDMA).
© 1996 American Chemical Society
1032 Chem. Res. Toxicol., Vol. 9, No. 6, 1996 tathione (GSH), glutathione reductase (EC 1.6.4.2), (reduced) nicotinamide adenine dinucleotide phosphate [NADP(H)], (reduced) nicotinamide adenine dinucleotide [NAD(H)], pyruvic acid, tris(hydroxymethyl)aminomethane (Tris), [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), 2-thiobarbituric acid, metyrapone, and iprindole were purchased from Sigma Chemical Co. (St. Louis, MO). Chloroacetic acid, methanol (gradient grade), and perchloric acid, and all other chemicals, were obtained from Merck (D-6100 Darmstadt, Germany). p-Hydroxyamphetamine was a generous gift from the United Nations Drug Control Program (Vienna). (Glutathion-S-yl)-p-hydroxyamphetamine was synthesized for mass spectra comparison with the in vitro formed adduct. The synthesis of this suspected glutathione conjugate was performed by incubating GSH and p-hydroxyamphetamine (equimolar concentrations) in water at pH 9, in a water bath at 37 °C for 4 h (18). No further treatment was performed. Animals. Adult male Wistar rats, weighing 200-250 g, were used. The rats remained in polyethylene cages, lined with wood shavings, with wire mesh at the top, at an ambient temperature of 20 ( 1 °C, humidity between 40% and 60%, and 12/12 h light/ dark cycle in our animal house at least 2 weeks before sacrifice. Surgical procedures, which were performed under diethyl ether anesthesia, occurred always between 9.00 a.m. and 10.00 a.m. Hepatocyte Isolation and Incubation. Hepatocyte isolation was performed by collagenase perfusion as previously described (19). Cell viability at the beginning of the experiments was between 85% and 95%. Incubations were performed in a shaking water bath (90 oscillations/min) at 37 °C, using 106 cells/mL in Krebs-Henseleit buffer supplemented with 25 mM HEPES (pH 7.4), and gassed continuously with an air stream of humidified carbogen. In a first set of experiments, isolated hepatocytes were preincubated for 60 min and then incubated with d-amphetamine sulfate (0, 0.04, 0.10, 0.20, and 1.00 mM, which correspond to 0, 0.08, 0.20, 0.40, and 2.00 mM, respectively, of d-amphetamine free base). Samples were taken at 60 min intervals for 3 h for evaluation of cell viability, lipid peroxidation, and glutathione status. After 180 min, sample aliquots were taken for detection and characterization of glutathione adducts. In a second set of experiments, isolated hepatocytes were preincubated for 50 min and then for 10 min with metyrapone (1 mM) or with iprindole (10 µM) (dissolved in Krebs-Henseleit buffer), or just Krebs-Henseleit buffer. d-Amphetamine (0, 0.08, 0.20, 0.40, and 2.00 mM) was then added to these three different groups. Samples were taken at 60 min intervals for 3 h for evaluation of cell viability and glutathione status. In a third set of experiments, isolated hepatocytes were preincubated for 60 min and then incubated with p-hydroxyamphetamine (100 µM) for 3 h. At the end of this time, samples were taken for evaluation of cell viability, glutathione status, and detection of glutathione adducts. Analytical Techniques. Cell viability was determined after isolation by the trypan blue exclusion test. During the course of the experiments cell viability was determined by the cell LDH leakage method which was randomly confirmed by the trypan blue exclusion test. GSH and the disulfide form of glutathione (GSSG) measurement was performed as previously described (20), with modifications. Briefly, for GSH measurement, cell suspension aliquots (500 µL) were precipitated with 500 µL of perchloric acid (5% final acid concentration) and centrifuged 10 min at 6000 rpm, the supernatant was filtered through a 0.45 µm filter, and 20 µL of each sample was injected into an HPLC system with electrochemical detection. For GSSG determination, 500 µL aliquots of cell suspensions were sonicated (Vibracell 375 W sonicator, Sonics & Materials Danbury-USA) for 12 s at medium intensity in order to disrupt the cells. These aliquots were then incubated with glutathione reductase (6 UI) and NADPH (0.2 mM) during 2 min for reduction of GSSG to GSH. This incubation was stopped by addition of perchloric acid (5% final concentration) and total glutathione (GSH + GSSG) measured
Carvalho et al.
Figure 1. Effect of d-amphetamine (0.08, 0.20, 0.40, and 2.00 mM) on GSH content of freshly isolated rat hepatocytes for 3 h of exposure. *p
