Alterations in the antioxidant defense of peripheral nervous tissue ...

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92s Biochemical Society Transactions (1 993) 21. Alterations in the antioxidant defense of peripheral nervous tissue following acute ethanol administration.
92s Biochemical Society Transactions ( 1 993) 21 Alterations in the antioxidant defense of peripheral nervous tissue following acute ethanol administration. ANGEL RAYA, JOAQUfN ROMA, CARLOS HERMENEGILDO and FRANCISCO J. ROMERO Experimental Toxicology & Neurotoxicology Unit, Department of Physiology, School of Medicine & Dentistry, University of Valencia, E46010 Valencia, Spain. The exact mechanisms by which ethanol induces toxic effects on both central and peripheral nervous tissues remain undefined. The primary site of action of ethanol is thought to be the lipids of the neural membrane [I].On this regard, ethanol has been shown to disorder the lipid portions of neural membranes, resulting in an increase in membrane fluidity [2]. However, because the magnitude of this disordering is relatively small, it is probable that membranemediated biological responses would involve functional entities in membranes [3]. It is known that free radical reactions can initiate and/or propagate oxidative membrane damage [4].The implication of free radical mechanisms in the ethanol-induced disorders has been recently reviewed (see ref. [S]).Despite some contradictory data, i t has been reported that acute administration of ethanol induces glutathione (GSH) depletion and promotes lipid peroxidation in several tissues and organs, among others, liver and brain. Nevertheless, there is no available information on the effects of ethanol on the antioxidant defense of peripheral nervous tissue. Here we have determined the GSH content and GSHrelated enzymatic activities in peripheral nerve after acute administration of ethanol to the rat, once electrophysiological signs of neuromuscular disfunction appeared. Male Wistar rats (body weight 319.6f 17.6 g) were used in this study. All animals were maintained in uniform conditions at room temperature and given a standard diet and water ad libitum. Electrophysiological determinations were performed throughout the experiment on the rat's tail musculature, i n order to monitor the peripheral neuromuscular function. The ethanol-treated group (n = 4)was injected i.p. with 5 g/Kg body weight of ethanol. Ethanol was administered at the same time each day to avoid circadian effects. The control group (n = 3)received only the anesthetic agent used in the electrophysiological study (ketamine i.p., 100 mg/kg of body weight). Ethanol-treated rats showed a complete block of extracellular-recorded evoked muscle action potentials [6]. When this occurs (67.5 f 9.7 minutes after ethanol injection) the rats were killed by decapitation and both sciatic nerves were carefully exposed and a = 3 cm section of them removed and kept frozen until use. Fresh homogenates were prepared in 0.2 M potassium phosphate buffer pH 7.0.GSH content was measured in the acidic homogenate supernatant following the method of [7].GSHenzymatic activities were determined in the cytosolic fraction after a 100,000 g centrifugation for 1 hcur, according to the following methods: GSH S-transferases (GST) towards I-chlor-2,4-dinitrobenzene(CDNB) [8], GSH S-transferases towards 4-hydroxy-2,3-frans-nonenal (CHNE) [9],GSH peroxidase (GSH Px) towards hydrogen peroxide [lo], GSH peroxidase towards f -butyl hidroperoxide (t-BOOH) [lo] and GSSG reductase (GSSG R) [ 1 I]. Proteins were measured according to [ 121. Results are expressed as mean f standard error. Statistical differences between groups were determined by the one way ANOVA and Student's t-test with unpaired samples.

Table 1. GSH and related enzymatic activities i n sciatic nerve of control and ethanol-treated rats

*

Values are means standard error of three control and four ethanol-treated animals. GSH values are expressed as nmollg wet tissue, and enzymatic activities as nmol/mg prot. x min. * Significant at p < 0.05vs. control group. Control GSH GST (CDNB) GST (4-HNE) GSH Px (H202) GSH Px (t-BOOH) GSSG R

94.46k 11.71 34.35f 4.50 58.82f 6.37 33.33f 5.83 37.42 1.42 17.53f 0.95

*

Ethanol

% -

40.24f 8.40* 43 38.29f 4.52 115 42.45rt 5.73 72 36.36f 4.48 109 92 34.49f 6.17 80 13.94* 1.09*

Table 1 shows the GSH content, as well as GSH-related enzymatic activities of control and ethanol-treated rats (5 g/kg of body weight). A significant decrease was observed in ethanol-treated rats once the evoked muscle action potentials were blocked. Moreover, the GSSG reductase activity also showed a significant decrease. Whether these changes are related to the neuromuscular function impairment observed, is currently under investigation. This work was partially supported by grants No. PM92/0146 from the DGICYT (Spain) and No. 92/0403 from the FIS (Spain) to FJR. AR and CH are research fellows of the Conselleria de Cultura, Educacio i Ciencia of the Generalitat Valenciana, Valencia, Spain. 1. Little, H.J., Dolin, S.J. & Halsey, M.J. (1986)Life Sci.

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