Depletion of total non-protein sulphydryl groups in mouse tissues after administration of d-amphetamine

IOXICOiOGY FI SFVIER S('IENTIFI( PUItl [SHt RS IRt L \ N I )

Toxicology 83 (1993) 31-40

Depletion of total non-protein sulphydryl groups in mouse tissues after administration of d-amphetamine F61ix Dias Carvalho

~a

, Maria de Lurdes John A. Timbrell b

Bastos a,

aLaboratory of Toxicology, Faculty of Pharmao,, University of Oporto, 4000 Oporto, Portugal hToxicology Department, School of Pharmao', London, UK (Received 27 July 1992; accepted 14 April 1993)

Abstract

A study of total non-protein sulphydryl groups (TNPSH) depletion was performed in male Charles River CD1 mice (25-30 g) isolated or housed in pairs. The mice were injected intraperitoneally with d-amphetamine (5, 20 and 80 mg/kg), kept at 20 + I°C, sacrificed at different times (between 0 and 120 min) and TNPSH in brain, lungs, heart, liver, kidney and spleen was quantified, d-Amphetamine significantly reduced the TNPSH levels in the liver and kidney, the reduction being more pronounced in the latter organ. In the other organs assayed no depletion was observed. Housing in pairs increased depletion of TNPSH both in the liver and in the kidney. The dose of 20 mg/kg caused a higher TNPSH depletion in paired mice than the dose of 80 mg/kg did in isolated mice. Rectal temperatures (RT) were also determined in the different times for the same doses of d-amphetamine. For the two lowest doses temperature values were similar in isolated and paired mice and were inversely proportional to TNPSH depletion in kidney. The administration of 80 mg/kg induced the highest TNPSH depletion in kidney and liver, but the hyperthermic effect was lower than that elicited by 20 mg/kg. No proportion was observed between RT and TNPSH depletion in liver. These results suggest that d-amphetamine changes TNPSH homeostasis in mouse liver by a temperature independent mechanism. In kidney, the hyperthermic effect may have some influence. Housing mice in pairs increases TNPSH depletion, but has no influence in the hyperthermic effect of d-amphetamine, with this experimental protocol.

Key words." d-Amphetamine; Total non-protein sulphydryl groups; Glutathione; Housing in pairs; Mice; Hyperthermia * Corresponding author.

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F.D. Carvalho el al. / Toxicology 83 (1993) 3 1 - 4 0

I. Introduction

There has been some evidence that changes in cellular thiol status may be involved in the neurotoxicity of some amphetamines (Miller et al., 1986; G i b b e t al., 1990; Hiramatsu et al., 1990). This may also contribute to the toxicity detected in the peripheral organs where amphetamines have been associated with acute renal failure (Foley et al., 1984; Terada et al., 1988; Henry et al., 1992), cardiomyopathy (Zalis et al., 1967) and hepatotoxicity (Zalis et al., 1967; Foley et al., 1984; Henry et al., 1992). Also, amphetamine can induce hyperthermia (Zalis et al., 1967; Seale et al., 1985; Nowak, 1988; Terada et al., 1988; Henry et al., 1992) and it is known that hyperthermia may cause oxidative stress with a decrease of GSH content in liver (Skibba et al., 1991). Cellular non-protein thiols like glutathione and cysteine participate in the antioxidant defense systems preventing or mitigating the deleterious effects of oxidative stress. Any depletion of TNPSH can increase cell susceptibility to possible harmful agents for which GSH is a detoxicant, for instance a free radical generating substance. Thus the aim of the present study was to evaluate the extent of TNPSH depletion in various organs of mice and the effect in rectal temperature caused by the intraperitoneal injection of damphetamine. Since it is known that housing animals in the same cage modifies the toxicity of amphetamines (Swinyard et al., 1961; Moore, 1963; Duterteboucher et al., 1992), this study was performed upon isolated mice and results compared with mice housed in pairs. 2. Materials and Methods 2.1. Chemicals

d-Amphetamine sulphate, [5,5'-dithiobis-(2-nitrobenzoic acid)] (DTNB), glutathione (GSH) and sulphosalycylic acid (SSA) were purchased from Sigma Chemical Company (St. Louis, MO, USA). All other reagents were of analytical grade from Merck (D-6100 Darmstadt, Germany). 2.2. Animals

Adult male Charles River CD1 mice, weighing 25-30 g were used. The animals were housed five per cage lined with wood shavings, with food and water ad libitum for at least 1 week prior to experimentation, up to the time of the experiments. The animal quarters were maintained at an ambient temperature of 20 ± I°C, relative humidity between 40 and 60%, with a 12:12h light/dark cycle.

ED. Carvalho et a L / Toxicology 83 (1993) 31-40

33

2.3. Experiments All experiments were performed between 0900 h and 1200 h. NaC1 0.9% or d-amphetamine sulphate (5, 20 and 80 mg/kg in NaC1 0.9%) were administered intraperitoneally using a 0.1 ml volume solution/10 g body weight. Then the mice were placed in polyethylene cages, lined with wood shavings, with wire mesh at the top, two per cage in the paired series and one per cage in the isolated series. The volume of the cage was 250 ml in the isolated series and 500 ml in the aggregated series. During the experiments the ambient temperature was maintained at 20 ± I°C. The mice remained in the cages for periods between 30 and 120 min without food or water. The animals were sacrificed by cervical dislocation, 30, 60 and 120 min after d-amphetamine administration. For each experiment, an equal number of saline injected animals was sacrificed at the same time as those injected with d-amphetamine, to serve as controls. A group of five animals were sacrificed without prior handling to serve as control at time zero. 2.4. Non-protein thiol determination The brain, heart, kidney, liver, lungs and spleen were quickly removed, rinsed in ice-cold saline, blotted dry, minced with scissors in a 1:4.5 w/v dilution of ice-cold 0.1 M phosphate-1 mM EDTA buffer (pH 7.4) and then homogenized using a Polytron homogenizer. To the homogenate an equal volume of sulphosalicylic acid (4%) was added, mixed and centrifuged at 3000 rev./min for 10 min. The supernatant was used for subsequent quantitation of total non-protein sulphydryl groups (TNPSH) by the spectrophotometric method of Ellman (1959), compared with a glutathione standard curve and then determined as micromoles of TNPSH/g of tissue. 2.5. Measurement oJ rectal temperature (RT) A similar experimental protocol was used for measurement of RT in mice treated with the same doses of d-amphetamine in all studied times but only once in each experiment. RT was measured by holding the animals by their tails while their forepaws were on a steel grid floor, and inserting a thermistor probe type PRC-A (Ellab, Denmark), which has been dipped in liquid silicon, into the rectum. The temperature was read on an Ellab thermometer type DU 3S when it reached steady state within 10 s. 2.6. Statistical evaluations Results are presented in the graphs as rectal temperature in °C and as the absolute amount of TNPSH in ttmol/g of tissue, 30, 60 and 120 min after administration of d-amphetamine, plotted against control tissues and are represented as means ± S.E.M. Differences between the TNPSH of control

F.D. Carvalho et al. / Toxicology 83 (1993) 31-40

34

and treated tissues were compared by Student's t-test for unpaired data and those with P-values of 0.05 or less were considered significant. 3.

Results

Administration of d-amphetamine (5, 20 and 80 mg/kg, i.p.) resulted in stereotyped behaviour in agreement with the literature (Weiss et al., 1960; Swinyard et al., 1961), namely periods of extreme excitement, rare fighting in paired mice and rhythmic wagging of the head. The chest and the neck usually became wet and the fur piloerected. Extensive motor activity was not possible in the cages used. For the organs studied, the control T N P S H values measured in paired and isolated mice, were similar. Thus, the control values for each time were added in order to give single control data for 0, 30, 60, or 120 min. With mice housed in pairs, the dose of 80 mg/kg was lethal in 100% of the mice (the deaths occurred within the first 10 rain after administration of the drug). In this particular case, T N P S H content in all organs assayed, just after death was not significantly different from the control (data not shown). Paired mice treated with 5 or 20 mg/kg and isolated mice treated with 5, 20 or 80 mg/kg survived during the experiments. In the kidneys (Figs. 1A,B), the level of T N P S H in controls was about 6.5 #mol/g of tissue at time 0 and the control values for 30, 60 and 120 min were similar, d-amphetamine caused a dose- and time-dependent depletion of TNPSH in isolated and paired mice. After 120 min the dose of 20 mg/kg

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Fig. 1. TNPSH content of kidney in adult mice (means ± S.E.M.) at 0, 30, 60 and 120 min in controls or mice treated with d-amphetamine after 5, 20 and 80 mg/kg in isolated mice (A) and 5 and 20 mg/kg in paired mice (B). n = 5, *P < 0.05, ** P < 0.01 (between treated and control).

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F.D. Carvalho et al. / Toxicology 83 (1993) 31-40

resulted in a TNPSH depletion 2.57 times higher in paired mice, than that obtained with the dose of 80 mg/kg in isolated mice. In the liver (Fig. 2A,B), the level of TNPSH control was about 9/~mol/g of tissue at time 0 and the control values for 30, 60 and 120 min were similar. In isolated and paired mice the dose of 5 mg/kg had no effect. In isolated mice, after a dose of 20 mg/kg, depletion was only significant at 120 min (13%) and with the dose of 80 mg/kg was significant at 60 min (21%) and recovered to 9% at 120 min. TNPSH level in the liver of paired mice treated with 20 mg/kg was similar to the control at 30 min but at 60 min was depleted by 36% and this depletion remained until 120 min (35%). At 60 and 120 min, the dose of 20 mg/kg caused a more pronounced depletion in the liver of paired mice than that obtained with the dose of 80 mg/kg in isolated mice, 1.73 and 3.93 times greater, respectively. The extent of TNPSH depletion was more severe in the kidney than in the liver for the same doses. Also, for the lowest dose (5 mg/kg) the kidney was the only organ where depletion of TNPSH was observed. The content of TNPSH was not affected by amphetamine administration in the brain, heart, lungs and spleen, for which the control content of TNPSH was, respectively 2.1, 1.6, 1.8 and 4.1/~mol/g of tissue in zero time. In accordance with the liver and kidney, the control values for these organs at 30, 60 and 120 min were not significantly different from those found at time 0. The effect of d-amphetamine on rectal temperature (RT) was dose dependent for 5 and 20 mg/kg in isolated mice (Fig. 3) and paired mice (Fig. 4).

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36

F.D. Carvalho et al. / Toxicology 83 (1993) 31-40

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Nevertheless, despite the differences in TNPSH depletion between isolated and paired mice, there were no differences between these two groups in the effects of d-amphetamine on RT (Fig. 5). Also, the control temperature of isolated and paired mice was similar (37.5 + 0.2°C) and maintained for the duration of the experiment.

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F.D. Carvalho et al. / Toxicology 83 (1993) 31-40

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4. Discussion

GSH is an important protective agent against xenobiotic toxicity, namely participating in metabolism and in the maintainance of the redox state of various organs, including the kidney and the liver (Bakke, 1990; Lew and Quintanilha, 1991; Sies, 1991). The results presented in this study demonstrate that in the liver and the kidney of the mouse, acute administration of d-amphetamine changes the level of TNPSH, (which mostly comprises GSH). This action may decrease the capability of those organs to counteract the possible toxic effects of amphetamines and their metabolites and other drugs taken together in polypharmacy. TNPSH depletion in the kidney was caused by a lower dose of damphetamine (5 mg/kg) and was greater than that in the liver for all the doses administered. Our results show that d-amphetamine does not alter TNPSH content in brain, heart, lungs and spleen. However, care should be taken in the interpretation of these results because the percentage of GSH in the total nonprotein thiol content in these organs is not as high as in liver and kidney (Simmons et al., 1990). Despite of this observation, the toxic effects of amphetamines are nevertheless observed also in some of these organs (Zalis et al., 1967; Christopher, 1986; Commins et al., 1986; Miller et al., 1986), which means that GSH may not be involved in the toxicity to these organs. A possible explanation for the exclusive depletion of TNPSH in kidney and liver of mice could be a rise in the rectal temperature (RT). In fact, amphetamine in high doses has a thermogenic action in mice (Seale et al., 1985;

38

I~D. Carvalho et al./ Toxicology 83 (1993) 31-40

Nowak, 1988), and it is known that hyperthermia causes oxidative stress with a decrease of GSH content in liver (Skibba et al., 1991). In this study, there was no direct proportionality between RT and TNPSH depletion in liver, since in isolated and paired mice the dose of 5 mg/kg increased RT but had no effect on TNPSH levels; the dose of 20 mg/kg induced the highest increase in RT. However, in isolated mice it was the dose of 80 mg/kg that induced the highest depletion in TNPSH. The kidney was the organ in which a stricter correlation was observed between RT and TNPSH depletion, because the two effects were both dosedependent in the lowest two doses. Nevertheless, in this organ, TNPSH depletion after administration of 20 mg/kg and 80 mg/kg in isolated mice was similar for these two doses but the RT increase during the experiments was similar at 30 min and significantly higher for 20 mg/kg at 60 and 120 min. Despite of the differences in TNPSH depletion between isolated and paired mice, there were no differences in the effects of d-amphetamine on RT between these two groups (Fig. 5). Endogenous catecholamines may have a role in the observed thiol depletion in the kidney and liver of mice induced by amphetamines, since these drugs exert some of their pharmacological and toxicological actions through the release of endogenous catecholamines (Burn and Rand, 1958; Trendelenburg et al., 1962; Heffner and Seiden, 1979; Parker and Cubeddu, 1986). In fact, both adrenaline, noradrenaline and other adrenergic agonists have already been shown to suppress hepatic glutathione in mice and this effect was counteracted by adrenergic antagonists (Register and Bartlett, 1954; James et al., 1987; Harbison et al., 1991). Nevertheless, this adrenergic effect in the kidney is understudied. Furthermore, housing saline-treated mice together does not result in any depletion of catecholamine stores, but enhances the catecholamine depleting action of d-amphetamine (Moore, 1963). In conclusion, the present results demonstrate that d-amphetamine changes TNPSH homeostasis in mouse liver and kidney in a dose-dependent manner, the effect being more pronounced in the kidney. The hyperthermic effect is also observed in a dose-dependent manner for 5 and 20 mg/kg. A proportionality with TNPSH depletion for these two doses is observed for the kidney, but not for the liver. Housing mice in pairs does not increase rectal temperature, but enhances lethality and TNPSH depletion in kidney and liver of the mouse induced by non-lethal doses of d-amphetamine, which means that the aggregation mediated effect is not due to hyperthermia.

5. Acknowledgments This work was supported by Instituto Nacional de Investigag~o Cientifica (INIC) Grant 89/SAD/4, and by Associag~.o Nacional das Farm~cias (ANF).

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