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Lipid peroxidation and changes in cytochrome c oxidase and xanthine oxidase activity in organophosphorus anticholinesterase induced myopathy
J Physiology (Paris) (1998) 92, 157-161 © Elsevier, Pads
Lipid peroxidation and changes in cytochrome c oxidase and xanthine oxidase activity in organophosphorus anticholinesterase induced myopathy Zhen R Yang, Wolf-D. Dettbarn* Department of Pharmacology and Neurology; Vanderbilt Universit3, School of Medicine, Nashville, TN 37212, USA
Abstract - - A possible role of radical oxygen species (ROS) initiated lipid peroxidation in diisopropylphosphorofluoridate (DFP)-induced muscle necrosis was investigated by quantifying muscle changes in F2-isoprostanes, novel and extremely accurate markers of lipid peroxidation in vivo. A significant increase in F2-isoprostanesof 56% was found in the diaphragm of rats 60 min after DFP-induced fasciculations. As possible source of ROS initiating lipid peroxidation, the cytocrome-c oxidase (Cyt-ox) and xanthine dehydrogenasexanthine oxidase (XD-XO) systems were investigated. Within 30 rain of onset of fasciculations Cyt-ox activity was reduced by 50% from 0.526 to 0.263 gmol/mg prot/min and XO activity increased from 0.242 to 0.541 gmol/mg prot/min. Total XD-XO activity was unchanged, indicating a conversion from XD into XO. In rats pretreatment with the neuromuscular blocking agent d-tubocurarine, prevented DFP-induced fasciculations, increases in F2-isoprostanes and changes in Cyt-ox or XD-XO. The decrease in Cyt-ox and increase in XO suggest that ROS are produced during DFP induced muscle fasciculations initiating lipid peroxidation and subsequent myopathy. (©Elsevier, Paris)
R4sum4 - - Peroxydation des lipides "et modification de la cytochrome C oxydase et de la xanthine oxydase dans la myopathie induite par les inhibiteurs anticholinest6rasiques organophosphor~s. Nous avons examin6 le r61e possible de la peroxydation de lipides par des r4sidug libres de I'oxyg~ne (ROS) dans la n4crose musculaire induite par le diisopropylphosphorofluoridate (DFP), en quantifiant le niveau d'isoprostane F2 musculaire, qui constitue un nouveau marqueur, extr~mement pr6cis, de la peroxydation lipidique in vivo. Nous avons trouv6 des augmentations significatives d'isoprostane F2 (56%) dans le diaphragme de rat, I heure apr~s I'induction de fasciculations par le DFP. La cytochrome C oxydase (Cyt-ox) et la xanthine d6shydrog6nase-xanthineoxydase (XD-XO) peuvent produire des radicaux libres (ROS) entralnant la peroxydation des lipides. En 30 minutes apr~:sle d6but des fasciculations, I'activit6 de la Cyt-ox ~tait r~duite de 50% (de 0,526 [t 0,263 gmole/mg de prot~ine/min) et l'activit6 de la XO 6tait augment~e de 0,242 ~ 0,541 I.tmole/mg de prot6ine/min. L'activit6 XD-XO totale n'6tait pas modifi6e, ce qui indique une conversion de XD en XO. Chez les rats, un pr6-traitement par la d-tubocurarine bloque l'activit6 musculaire et les fasciculations induites par le DFP, et emp~che I'augmentation d'isoprostane F2 et les changements de I'activit6 Cyt-Ox et du syst~:me XD-XO. Nous sugg6rons que l'hyperactivit6 cholinergique induite par le DFP provoque l'apparition de radicaux libres et la peroxydation des lipides. La diminution de Cyt-Ox et l'augmentation de XO sugg~rent que les ROS sont produits pendant les fasciculations et que la peroxydation des lipides qui en r6sulte est responsable de la myopathie induite par le DFP. (©Elsevier, Paris) lipid peroxidation / free radicals / myopathy / muscle hyperactivity
1. I n t r o d u c t i o n
Systemic injection of non-lethal concentrations of organophosphorus anticholinesterases (OP-antiChE) causes neuromuscular injury consequent to induced sustained muscle fasciculations [10]. The chain of events linking prolonged acetylcholine (ACh) receptor activation to neuromuscular pathology is complex and not fully understood. R e c e n t e v i d e n c e supports the a s s u m p t i o n that o v e r - p r o d u c t i o n of reactive oxygen species (ROS) and lipid peroxidation may have an important role in initiation of the neuromuscular lesions [20, 21]. In skeletal muscle, several sources of ROS have been identified, such as mitochondrial electron trans-
* Correspondence and reprints
port, oxidases in sarcoplasmic reticulum and sarcolemma and cytosolic xanthine oxidase. During normal conditions ROS, such as superoxide anion, hydrogen peroxide and hydroxyl radicals, are generated at a low rate by cytochrome c oxidase (Cyt-ox) and can be taken care of by the scavenger and antioxidant systems. However, a reduced capacity of this enzyme can lead to incomplete reduction of oxygen and thus increased ROS production [18], which may exceed the capacity of the cellular defence system. Another source of free radicals is xanthine oxidase (XO). This enzyme exist as NAD ÷ dependent dehydrogenase (XD, ECI. 1.1.2.0.4) and is converted into xanthine oxidase (EC1.1.3.2.2). Xanthine oxidase directly transfers electrons from the oxidation of h y p o x a n t h i n e to molecular oxygen, producing ROS such as superoxide and hydrogen peroxide; both of these are very toxic and can interact with
158
Z.P. Yang, W.D. Dettbarn
free fatty acids such as arachidonic acid which via prostaglandin synthesis generates more ROS [4, 6]. Conversion from XD to XO has been shown to occur during muscle ischemia [6], seizure activity [2] and muscle activity [17]. Previous studies have shown that a decrease in the endogenous antioxidant glutathione by buthionine sulfoxime (BSO) increased the DFP-induced lipid peroxidation and the severity of the myopathy [20]. The present investigation was undertaken to determine through appropriate measurements whether Cyt-ox, XD and XO activities are changed in muscle taken from DFP-treated rats. To rule out direct effects of DFP on these enzymes, the neuromuscular blocking a g e n t ' d - t u b o c u r a r i n e was used in another series of experiments to prevent DFP-induced muscle hyperactivity.
2. Materials and methods 2.1. Animals Male Sprague-Dawley rats weighing 200-250 g were maintained on a 12 h light/dark cycle with food and tap water available ad libitum. For biochemical studies rats were decapitated 30-120 min following the DFP administration (0.5-1.7 mg/kg, s.c.), d-Tubocurarine (0.07 mg/kg) was given i.p. 30 min prior to the DFP injection and !mmediately after the DFP injection. Buthionine sulfoxime (BSO) 900 mg/kg, i.p. was given 3.5 and 1.5 h before DFP. For histochemistry rats were decapitated 24 h after the start of the experiment. 2.2. Enzyme determination Cyt-ox activity was determined by measuring spectrophotometrically the oxidation rate of ferrocytochrome c at the absorbance of it's or-band, 550 nm, as described by Wharton and Tzagoloff [19]. 0.1 M 2-(N-morpholine) ethanesulfonic acid (MES, pH 6.0) with 10 glVl EDTA was used as buffer. XO or XD activity was determined spectrophotometricaily based on the production of uric acid at 295 nm [15]. XO activity was assayed in the absence of NAD ÷, and the XD-XO was measured in the presence of NAD ÷.
2.3. Lipid peroxidation A series of prostaglandin F2-1ike compounds termed F2isoprostanes are formed in vivo from the free radical-catalyzed peroxidation of arachidonic acid and are useful markers of oxidant stress [13]. We have used these F2-isoprostanes as an index of lipid peroxidation. 2.4. Statistical evaluation Student's t-test was used to establish significance at P < 0.05.
3. R e s u l t s 3.1. DFP induced lipid peroxidation and muscle necrosis Rats treated with DFP (1.5-1.7 mg/kg, s.c.) developed fasciculations within 10 min lasting for 4--8 h. Histochemical studies revealed necrotic lesions involving endplates. The number of necrotic lesions was reduced significantly when rats were pretreated with d-tubocurarine in order to prevent fasciculations (table 1). Glutathione (GSH), an endogenous antioxidant, protects against oxidative stress in vivo, its depletion should increase lipid peroxidation and the number of necrotic fibers in our experiments. In rats when pretreated with BSO 3.5 and 1 h before DFP, the n u m b e r o f n e c r o t i c f i b e r s was s i g n i f i c a n t l y higher than seen with DFP only (table 1). The combined treatment o f BSO and DFP reduced G S H from 1.36 + 0.20 (100%) to 0.39 + 0.11 (28%) t.tmol/g muscle. Significant increases of F2-isoprostanes were detected following exposure of rats to 1.7 mg/kg over 30, 60 and 120 min. The largest increase o f 56% from control 1.66 + 0.20 ng/g tissue in F2-isoprostanes was observed in rats treated with 1.7 mg/kg DFP following 60 min o f exposure (P < 0.01). Fasciculations also were detected under these conditions. Lower doses of DFP, such as 1 mg/kg, which did not induce fasciculations, produced no increases in
Table I. Modification of DFP myopathy by pretreatment with d-tubocurarine'~r BSO. Treatmenta
Fasciculations Number of necrotic fibers/1000 muscle fibers
Control
DFP
d-Tubocurarine + DFP
BSO + DFP
0 2 + 2 (0.2%)
++++ 479 + 45 b (47.9%)
0 73 + 19ce' d
++++ 655 + 67 b' c (65.5%)
a Treatment: DFP (1.7 mg/kg), d-tubocurarine (0.07 mg/kg), BSO (900 mg/kg). All drugs were given i.p. with the exception of DFP which was given s.c. Rats were killed 24 h after DFP treatment for histochemistry. Values are the mean + S.E.M. of seven muscles.bp < 0.001 and ep < 0.01, between control and treated rats; dp < 0.01 and ep < 0.05 between DFP-only-treated rats and the rats with drug pretreatments.
Xth International Symposium on Cholinergic Mechanisms
formation of F2-isoprostanes when compared to control animals. Pretreatment with the neuromuscular blocking agent d-tubocurarine prevented the muscle hyperactivity and increases in F2-isoprostanes formation otherwise elevated by DFP treatment.
3.2. Sources for ROS production-cytocrome c oxidase c reduction In diaphragm, following DFP, Cyt-ox was transiently reduced to 51% of control activity after 15 min (P < 0 . 0 0 1 ) and to 77% within 30 min (P < 0.01). By 60 rain, the Cyt-ox activity had completely recovered (figure 1). The 0.5 mg/kg DFP treatment did neither cause muscle hyperactivity such as fasciculations, nor any significant Cyt-ox reduction (figure 1), myopathic lesions or increases in lipid peroxidation. As shown infigure 2, following pretreatment with d-tubocurarine (0.07 mg/kg), only a 7.6% decrease in Cyt-ox activity was seen within 15 min following DFP (1.7 mg/kg). This is a significant protection of Cyt-ox activity when compared to the 51% reduction seen after DFP alone, Under these conditions, muscle fasciculations, increases in F2-isoprostanes as well as the muscle necrosis were prevented.
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DFP 1.7 mg/kg, s.c. and d-tubocurarine
30
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DFP exposure time (min) Figure 2. Effect of d-tubocurarine pretreatment on the cytocrome c oxidase reduction in rat diaphragm following various in vivo DFP treatments, d-Tubocurarine (0.07. mg/kg, i.p.) was given twice: 30 rain before DFP injection and immediately after DFP injection. Control activity of cytochrome c oxidase was: 0.526 _+0.093 lamol/min/mg protein. *P < 0.05; ** P < 0.01; *** P < 0.001.
3.3. Xanthine dexydrogenase/xanthine oxidase conversion Within 30 min of DFP-induced fasciculations, an increase of XO activity was seen. During DFP-induced fasciculations, however, XO activity rose to 70% of the total XD + XO activity (table II). This increase in XO activity appears to have resulted from conversion of XD to XO, since the total activity of XD + XO remained constant throughout the 60 rain observation period. Prevention of muscle hyperactivity through pretreatment with d-tubocurarine inhibited the conversion of XD to XO otherwise seen with DFP-induced muscle fasciculations (table I1). These findings rule out a direct effect of DFP on Cyt-ox or XD-XO.
0 15
30
60
4. Discussion
DFP exposure time (min) Figure 1. Reduction (%) of cytochrome c oxidase in rat diaphragm following various in vivo DFP treatm[nts. Control activity of cytochrome c oxidase: 0.526 + 0.093 p.mol/mirdmg protein. *P < 0.01; ** P < 0.001.
Radical oxygen species are considered to be active mediators in the hyperactivity-induced skeletal muscle damage and inflammation, and in various muscle diseases [17]. While the potential sources of
160
Z.P. Yang, W.D. Dettbarn
Table II. Conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO) in rat diaphragm following in vivo DFP treatments, with or without the d-tubocurarine pretreatmenta.
XOb XD+XOc XO/XD Increase of XO/XD from control (%)
Control
D I P 30 min
DFP 60 min
DFP 30 min + d-tubocurarine
DFP 60 rain + d-tubocurarine
0.242 + 0.05 0.794 + 0.18 0.44 -
0.541 + 0.13a 0.768 + 0.20 2.39a 443
0.553 + 0.09a 0.803 + 0.14 2.21d 402
0.264 + 0.07 0.771 5:0.22 0.52 18
0.236 + 0.07 0.825 + 0.21 0.40
aDFP was given 1.7 mg/kg, s.c. Rats were killed after 30 or 60 min of exposure to DFP. The drug pretreatment was same as in tdable I. bxo, t.tmol/min/mg protein. CXO + XD:/.tmol/min/mg protein. Values were mean + S.D. of five rats. P < 0.001 between control and treated animals.
ROS in muscle are numerous, such as mitochondrial electron transport, oxidases in sarcoplasmatic reticulum and sarcolemma and cytosolic xanthine oxidase, the mechanisms that control the reactive oxidant production and the sites at which oxidative stress causes • dysfunction remain little understood. The present data demonstrate that DFP-induced muscle hyperactivity results in a rapid, significant reduction in the activity of Cyt-ox (figure 1) and an increased conversion of XD to XO (table 113, suggesting the generation of ROS. This agrees with previous observations of increased levels of ROS following exercise-induced muscle damage [1, 5, 9, 14, 161. Cytochrome c oxidase is the terminal complex in the mitochondria respiratory chain, which generates ,ATP by oxidative phosphorylation. During intensive muscle activity, the activity of Cyt-ox is reduced [7, 18], leading to an increase in the electron pressure within the electron transport chain and to the ROS production. The significant increase in the ratio XO/XD following the onset of DFP-induced muscle fasciculations appear to be due to a conversion from XD to XO as no significant changes in the total activity (XO + XD) were observed. The shift of XD to XO begins with the onset of fasciculations and is still present after 60 min. As previously observed, DFPinduced fasciculations caused a rapid fall in ATP [8], leading to increased accumulation of Ca 2÷ [11]. The increased calcium may initiate the conversion of XD to XO through a calcium-dependent protease [3]. The loss of ATP is followed by an increase in ADP, which is further metabolized to adenosine, inosine and hypoxanthine, a substrate for XO [12]. Xanthine oxidase through the oxidation of hypoxanthine produces ROS as superoxide and hydrogen peroxide leading to lipid peroxidation and membrane damage. - A greatly increased rate of free radical production may exceed the capacity of the cellular defence sys-
tem such as antioxidants and permit a substantial attack of free radicals on lipids in the cell membrane causing lipid peroxidation leading to cell injury. This suggestion is supported by findings that BSO induced loss of GHS increased lipid peroxidation and the severity of the myopathy due to DFP [20]. The prevention of the DFP-induced muscle hyperactivity through the neuromuscular blocking agent d-tubocurarine attenuated the increase of F2isoprostanes [20, 21], prevented the change in Cytox (figures 1, 2) and the conversion of XD into XO (table 113. These data suggest that free radical generation during DFP-induced fasciculation is associated with AChE inhibition by DFP, and is not due to direct effects of DFP on Cyt-ox and XD/XO. No studies to date have reported a relationship between OP-antiChE-induced muscle hyperactivity, lipid peroxidation, and muscle lesions. From the present studies, however, it is concluded that the accumulation of oxygen free radicals and the related increases in lipid peroxidation are caused by the DFP-induced muscle hyperactivity and that lipid peroxidation is a contributing initial factor in the mechanism of OP-antiChE-induced cell injury. In summary, the results of this study demonstrated: 1) that the OP-antiChE, DFP, caused fasciculations leading to lipid peroxidation and muscle necrosis; 2) prevention of DFP-induced muscle hyperactivity-by d-tubocurarine inhibits lipid peroxidation and necrosis and rules out a direct effect of DFP on Cyt-ox and XD-XO and thus support the hypothesis that OP-antiChE-induced muscle hyperactivity stimulates ROS production, through changes in cytochrome c oxidase and xanthine oxidase activity.
Acknowledgment This work was supported by NIH grant RO 1 ES04597.
Xth International Symposium on Cholinergic Mechanisms
References [ I]
Alessio H.M., Goldfarb A.H., Cutler R.G., MDA content increases in fast and slow twitch skeletal muscle with high intensity of exercise in rat, Am. J. Physiol. 255 (1988) C874-C877. [21 Battelli M.G., Buonamici L., Abbondanza A., Virgili M., Contestabile A., Stirpe E, Excitotoxic increase of xanthine dehydrogenase and xanthine oxidase in rat olfactory cortex, Dev. Brain Res. 86 (1995) 340-344. [3] Bindoli A., Cavallini L., Rigobello M.P., Coassin M.G., Dilisa E, Modification of the xanthine-converting enzyme of perfused rat heart during ischemia and oxidative stress, Free Rad. Biol. Med. 4 (1988) 163-167. [41 Bratell S., Folmerz P., Hansson R., Jonsson O., Lundstam S., Pettersson B.R., Schersten T., Effects of oxygen free radical scavengers, xanthine oxidase inhibition and calcium entry blockers on leaking of albumin after ischemia: an experimental study in rabbit kidneys, Acta Physiol. Scand. 134 (1988) 35--41. [5] Davies K.J.A., Quintanilha A.T., Brooks G.A., Packer L., Free radicals and tissue damage produced by exercise, Biochem. Biophys. Res. Commun. 107 (1982) 1198-1205. [6] Downey J.M., Hearse D.J., Yellon-D.M., The role of xanthine oxidase during myocardia ischemia in several species including man, J. Mol. Cell Cardiol. 20 (1988) 55-63. [7] Gollnick P.D., Bertocci L.A., Kelso T.B., Witt E.H., Hodgson D.R., The effect of high intensity exercise on the respiratory capacity of skeletal muscle, Pfliigers Arch. (Eur. J. Physiol.) 415 (1990) 407--413. [8] Gupta R.C., Dettbarn W.-D., Alterations of high-energy phosphate compounds in the skeletal muscles of rats intoxicated with diisopropylphosphorofluoridate (DFP) and soman, Fund. Appl. Toxicol. 8 (1987) 400-407. [9] Jenkins R.R., Free radical chemistry: relationship to exercise, Sport Med. 5 (1988) 156-170. [10] Laskowski M.B., Olson W.H., Dettbarn W.-D., Initial ultrastructural abnormalities at the motor endplate produced by a eholinesterase inhibitor, Exp. Neurol. 57 (1977) 94-100.
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[11] Leonard J.E, Salpeter M.M., Agonist-induced myopathy at the neuromuscular junction is mediated by calcium, Cell Biol. 82 (1979) 811-819. [121 McCord J.M., Free radicals and myocardial ischemia overview and outlook, Free Rad. Biol. Med. 4 (1988) 9-19. [13] Morrow J.D., Hill K.E., Burk R.E, Nammour T.M., Badr K.F., Roberts L.J., A series of F2-1ike compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism, Proc. Natl. Acad. Sci. USA 87 (1990) 9383-9387. [141 Packer L., Vitamin E, physical exercise and tissue damage in animals, Med. Biol. 62 (1984) 105-109. [15] Parks D.A., Williams T.K., Beckman J.S., conversion of xanthine dehydrogenase to oxidase in ischemic rat intestine: a reevaluation, Am. J. Physiol. 254 (Gastroint. Liver Physiol. 17) (1988) G768-774. [16] Radak Z., Asano K., Inoue M., Kizaki T., Oh-lshi S., Suzuki K., Taniguchi N., Ihno H., Superoxide dismutase derivative reduces oxidative damage in skeletal muscle of rats during exhaustive exercise, J. Appl. Physiol. 79 (1995) 129-135. [17] Sjodin B., Westing Y.H., Apple F.S., Biochemical mechanisms for oxygen free radical formation during exercise, Sports Med. 10 (1990) 236-254. [18] Soussi B., Idstrom J.-P., Schersten T., Bylund-Fellcnius A.C., Cytochrome c oxidase and cardiolipin alterations in response to skeletal muscle ischemia and reperfusion, Acta Physiol. Scand. 138 (1990) 107-114. [19] Wharton D.C., Tzagoloff A., Cytochrome oxidase from beef heart mitochondria, Methods Enzymol. 10 (1967) 245-250. [20] Yang Z.P., Dettbarn W.-D., Diisopropylphosphorofluoridateinduced cholinergic hyperactivity and lipid peroxidation, Toxicol. Appl. Pharmacol. 138 (1996) 48-53. [21] Yang Z.P., Morrow J.D., Wu A., Roberts L.J. II, Dettbarn W.-D., Diisopropylphosphorofluoridate-induced muscle hyperactivity associated with enhanced lipid peroxidation in vivo, Biochem. Pharmacol. 52 (1996) 357-361.
Lipid peroxidation and changes in cytochrome c oxidase and xanthine oxidase activity in organophosphorus anticholinesterase induced myopathy Zhen R Yang, Wolf-D. Dettbarn* Department of Pharmacology and Neurology; Vanderbilt Universit3, School of Medicine, Nashville, TN 37212, USA
Abstract - - A possible role of radical oxygen species (ROS) initiated lipid peroxidation in diisopropylphosphorofluoridate (DFP)-induced muscle necrosis was investigated by quantifying muscle changes in F2-isoprostanes, novel and extremely accurate markers of lipid peroxidation in vivo. A significant increase in F2-isoprostanesof 56% was found in the diaphragm of rats 60 min after DFP-induced fasciculations. As possible source of ROS initiating lipid peroxidation, the cytocrome-c oxidase (Cyt-ox) and xanthine dehydrogenasexanthine oxidase (XD-XO) systems were investigated. Within 30 rain of onset of fasciculations Cyt-ox activity was reduced by 50% from 0.526 to 0.263 gmol/mg prot/min and XO activity increased from 0.242 to 0.541 gmol/mg prot/min. Total XD-XO activity was unchanged, indicating a conversion from XD into XO. In rats pretreatment with the neuromuscular blocking agent d-tubocurarine, prevented DFP-induced fasciculations, increases in F2-isoprostanes and changes in Cyt-ox or XD-XO. The decrease in Cyt-ox and increase in XO suggest that ROS are produced during DFP induced muscle fasciculations initiating lipid peroxidation and subsequent myopathy. (©Elsevier, Paris)
R4sum4 - - Peroxydation des lipides "et modification de la cytochrome C oxydase et de la xanthine oxydase dans la myopathie induite par les inhibiteurs anticholinest6rasiques organophosphor~s. Nous avons examin6 le r61e possible de la peroxydation de lipides par des r4sidug libres de I'oxyg~ne (ROS) dans la n4crose musculaire induite par le diisopropylphosphorofluoridate (DFP), en quantifiant le niveau d'isoprostane F2 musculaire, qui constitue un nouveau marqueur, extr~mement pr6cis, de la peroxydation lipidique in vivo. Nous avons trouv6 des augmentations significatives d'isoprostane F2 (56%) dans le diaphragme de rat, I heure apr~s I'induction de fasciculations par le DFP. La cytochrome C oxydase (Cyt-ox) et la xanthine d6shydrog6nase-xanthineoxydase (XD-XO) peuvent produire des radicaux libres (ROS) entralnant la peroxydation des lipides. En 30 minutes apr~:sle d6but des fasciculations, I'activit6 de la Cyt-ox ~tait r~duite de 50% (de 0,526 [t 0,263 gmole/mg de prot~ine/min) et l'activit6 de la XO 6tait augment~e de 0,242 ~ 0,541 I.tmole/mg de prot6ine/min. L'activit6 XD-XO totale n'6tait pas modifi6e, ce qui indique une conversion de XD en XO. Chez les rats, un pr6-traitement par la d-tubocurarine bloque l'activit6 musculaire et les fasciculations induites par le DFP, et emp~che I'augmentation d'isoprostane F2 et les changements de I'activit6 Cyt-Ox et du syst~:me XD-XO. Nous sugg6rons que l'hyperactivit6 cholinergique induite par le DFP provoque l'apparition de radicaux libres et la peroxydation des lipides. La diminution de Cyt-Ox et l'augmentation de XO sugg~rent que les ROS sont produits pendant les fasciculations et que la peroxydation des lipides qui en r6sulte est responsable de la myopathie induite par le DFP. (©Elsevier, Paris) lipid peroxidation / free radicals / myopathy / muscle hyperactivity
1. I n t r o d u c t i o n
Systemic injection of non-lethal concentrations of organophosphorus anticholinesterases (OP-antiChE) causes neuromuscular injury consequent to induced sustained muscle fasciculations [10]. The chain of events linking prolonged acetylcholine (ACh) receptor activation to neuromuscular pathology is complex and not fully understood. R e c e n t e v i d e n c e supports the a s s u m p t i o n that o v e r - p r o d u c t i o n of reactive oxygen species (ROS) and lipid peroxidation may have an important role in initiation of the neuromuscular lesions [20, 21]. In skeletal muscle, several sources of ROS have been identified, such as mitochondrial electron trans-
* Correspondence and reprints
port, oxidases in sarcoplasmic reticulum and sarcolemma and cytosolic xanthine oxidase. During normal conditions ROS, such as superoxide anion, hydrogen peroxide and hydroxyl radicals, are generated at a low rate by cytochrome c oxidase (Cyt-ox) and can be taken care of by the scavenger and antioxidant systems. However, a reduced capacity of this enzyme can lead to incomplete reduction of oxygen and thus increased ROS production [18], which may exceed the capacity of the cellular defence system. Another source of free radicals is xanthine oxidase (XO). This enzyme exist as NAD ÷ dependent dehydrogenase (XD, ECI. 1.1.2.0.4) and is converted into xanthine oxidase (EC1.1.3.2.2). Xanthine oxidase directly transfers electrons from the oxidation of h y p o x a n t h i n e to molecular oxygen, producing ROS such as superoxide and hydrogen peroxide; both of these are very toxic and can interact with
158
Z.P. Yang, W.D. Dettbarn
free fatty acids such as arachidonic acid which via prostaglandin synthesis generates more ROS [4, 6]. Conversion from XD to XO has been shown to occur during muscle ischemia [6], seizure activity [2] and muscle activity [17]. Previous studies have shown that a decrease in the endogenous antioxidant glutathione by buthionine sulfoxime (BSO) increased the DFP-induced lipid peroxidation and the severity of the myopathy [20]. The present investigation was undertaken to determine through appropriate measurements whether Cyt-ox, XD and XO activities are changed in muscle taken from DFP-treated rats. To rule out direct effects of DFP on these enzymes, the neuromuscular blocking a g e n t ' d - t u b o c u r a r i n e was used in another series of experiments to prevent DFP-induced muscle hyperactivity.
2. Materials and methods 2.1. Animals Male Sprague-Dawley rats weighing 200-250 g were maintained on a 12 h light/dark cycle with food and tap water available ad libitum. For biochemical studies rats were decapitated 30-120 min following the DFP administration (0.5-1.7 mg/kg, s.c.), d-Tubocurarine (0.07 mg/kg) was given i.p. 30 min prior to the DFP injection and !mmediately after the DFP injection. Buthionine sulfoxime (BSO) 900 mg/kg, i.p. was given 3.5 and 1.5 h before DFP. For histochemistry rats were decapitated 24 h after the start of the experiment. 2.2. Enzyme determination Cyt-ox activity was determined by measuring spectrophotometrically the oxidation rate of ferrocytochrome c at the absorbance of it's or-band, 550 nm, as described by Wharton and Tzagoloff [19]. 0.1 M 2-(N-morpholine) ethanesulfonic acid (MES, pH 6.0) with 10 glVl EDTA was used as buffer. XO or XD activity was determined spectrophotometricaily based on the production of uric acid at 295 nm [15]. XO activity was assayed in the absence of NAD ÷, and the XD-XO was measured in the presence of NAD ÷.
2.3. Lipid peroxidation A series of prostaglandin F2-1ike compounds termed F2isoprostanes are formed in vivo from the free radical-catalyzed peroxidation of arachidonic acid and are useful markers of oxidant stress [13]. We have used these F2-isoprostanes as an index of lipid peroxidation. 2.4. Statistical evaluation Student's t-test was used to establish significance at P < 0.05.
3. R e s u l t s 3.1. DFP induced lipid peroxidation and muscle necrosis Rats treated with DFP (1.5-1.7 mg/kg, s.c.) developed fasciculations within 10 min lasting for 4--8 h. Histochemical studies revealed necrotic lesions involving endplates. The number of necrotic lesions was reduced significantly when rats were pretreated with d-tubocurarine in order to prevent fasciculations (table 1). Glutathione (GSH), an endogenous antioxidant, protects against oxidative stress in vivo, its depletion should increase lipid peroxidation and the number of necrotic fibers in our experiments. In rats when pretreated with BSO 3.5 and 1 h before DFP, the n u m b e r o f n e c r o t i c f i b e r s was s i g n i f i c a n t l y higher than seen with DFP only (table 1). The combined treatment o f BSO and DFP reduced G S H from 1.36 + 0.20 (100%) to 0.39 + 0.11 (28%) t.tmol/g muscle. Significant increases of F2-isoprostanes were detected following exposure of rats to 1.7 mg/kg over 30, 60 and 120 min. The largest increase o f 56% from control 1.66 + 0.20 ng/g tissue in F2-isoprostanes was observed in rats treated with 1.7 mg/kg DFP following 60 min o f exposure (P < 0.01). Fasciculations also were detected under these conditions. Lower doses of DFP, such as 1 mg/kg, which did not induce fasciculations, produced no increases in
Table I. Modification of DFP myopathy by pretreatment with d-tubocurarine'~r BSO. Treatmenta
Fasciculations Number of necrotic fibers/1000 muscle fibers
Control
DFP
d-Tubocurarine + DFP
BSO + DFP
0 2 + 2 (0.2%)
++++ 479 + 45 b (47.9%)
0 73 + 19ce' d
++++ 655 + 67 b' c (65.5%)
a Treatment: DFP (1.7 mg/kg), d-tubocurarine (0.07 mg/kg), BSO (900 mg/kg). All drugs were given i.p. with the exception of DFP which was given s.c. Rats were killed 24 h after DFP treatment for histochemistry. Values are the mean + S.E.M. of seven muscles.bp < 0.001 and ep < 0.01, between control and treated rats; dp < 0.01 and ep < 0.05 between DFP-only-treated rats and the rats with drug pretreatments.
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formation of F2-isoprostanes when compared to control animals. Pretreatment with the neuromuscular blocking agent d-tubocurarine prevented the muscle hyperactivity and increases in F2-isoprostanes formation otherwise elevated by DFP treatment.
3.2. Sources for ROS production-cytocrome c oxidase c reduction In diaphragm, following DFP, Cyt-ox was transiently reduced to 51% of control activity after 15 min (P < 0 . 0 0 1 ) and to 77% within 30 min (P < 0.01). By 60 rain, the Cyt-ox activity had completely recovered (figure 1). The 0.5 mg/kg DFP treatment did neither cause muscle hyperactivity such as fasciculations, nor any significant Cyt-ox reduction (figure 1), myopathic lesions or increases in lipid peroxidation. As shown infigure 2, following pretreatment with d-tubocurarine (0.07 mg/kg), only a 7.6% decrease in Cyt-ox activity was seen within 15 min following DFP (1.7 mg/kg). This is a significant protection of Cyt-ox activity when compared to the 51% reduction seen after DFP alone, Under these conditions, muscle fasciculations, increases in F2-isoprostanes as well as the muscle necrosis were prevented.
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DFP exposure time (min) Figure 2. Effect of d-tubocurarine pretreatment on the cytocrome c oxidase reduction in rat diaphragm following various in vivo DFP treatments, d-Tubocurarine (0.07. mg/kg, i.p.) was given twice: 30 rain before DFP injection and immediately after DFP injection. Control activity of cytochrome c oxidase was: 0.526 _+0.093 lamol/min/mg protein. *P < 0.05; ** P < 0.01; *** P < 0.001.
3.3. Xanthine dexydrogenase/xanthine oxidase conversion Within 30 min of DFP-induced fasciculations, an increase of XO activity was seen. During DFP-induced fasciculations, however, XO activity rose to 70% of the total XD + XO activity (table II). This increase in XO activity appears to have resulted from conversion of XD to XO, since the total activity of XD + XO remained constant throughout the 60 rain observation period. Prevention of muscle hyperactivity through pretreatment with d-tubocurarine inhibited the conversion of XD to XO otherwise seen with DFP-induced muscle fasciculations (table I1). These findings rule out a direct effect of DFP on Cyt-ox or XD-XO.
0 15
30
60
4. Discussion
DFP exposure time (min) Figure 1. Reduction (%) of cytochrome c oxidase in rat diaphragm following various in vivo DFP treatm[nts. Control activity of cytochrome c oxidase: 0.526 + 0.093 p.mol/mirdmg protein. *P < 0.01; ** P < 0.001.
Radical oxygen species are considered to be active mediators in the hyperactivity-induced skeletal muscle damage and inflammation, and in various muscle diseases [17]. While the potential sources of
160
Z.P. Yang, W.D. Dettbarn
Table II. Conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO) in rat diaphragm following in vivo DFP treatments, with or without the d-tubocurarine pretreatmenta.
XOb XD+XOc XO/XD Increase of XO/XD from control (%)
Control
D I P 30 min
DFP 60 min
DFP 30 min + d-tubocurarine
DFP 60 rain + d-tubocurarine
0.242 + 0.05 0.794 + 0.18 0.44 -
0.541 + 0.13a 0.768 + 0.20 2.39a 443
0.553 + 0.09a 0.803 + 0.14 2.21d 402
0.264 + 0.07 0.771 5:0.22 0.52 18
0.236 + 0.07 0.825 + 0.21 0.40
aDFP was given 1.7 mg/kg, s.c. Rats were killed after 30 or 60 min of exposure to DFP. The drug pretreatment was same as in tdable I. bxo, t.tmol/min/mg protein. CXO + XD:/.tmol/min/mg protein. Values were mean + S.D. of five rats. P < 0.001 between control and treated animals.
ROS in muscle are numerous, such as mitochondrial electron transport, oxidases in sarcoplasmatic reticulum and sarcolemma and cytosolic xanthine oxidase, the mechanisms that control the reactive oxidant production and the sites at which oxidative stress causes • dysfunction remain little understood. The present data demonstrate that DFP-induced muscle hyperactivity results in a rapid, significant reduction in the activity of Cyt-ox (figure 1) and an increased conversion of XD to XO (table 113, suggesting the generation of ROS. This agrees with previous observations of increased levels of ROS following exercise-induced muscle damage [1, 5, 9, 14, 161. Cytochrome c oxidase is the terminal complex in the mitochondria respiratory chain, which generates ,ATP by oxidative phosphorylation. During intensive muscle activity, the activity of Cyt-ox is reduced [7, 18], leading to an increase in the electron pressure within the electron transport chain and to the ROS production. The significant increase in the ratio XO/XD following the onset of DFP-induced muscle fasciculations appear to be due to a conversion from XD to XO as no significant changes in the total activity (XO + XD) were observed. The shift of XD to XO begins with the onset of fasciculations and is still present after 60 min. As previously observed, DFPinduced fasciculations caused a rapid fall in ATP [8], leading to increased accumulation of Ca 2÷ [11]. The increased calcium may initiate the conversion of XD to XO through a calcium-dependent protease [3]. The loss of ATP is followed by an increase in ADP, which is further metabolized to adenosine, inosine and hypoxanthine, a substrate for XO [12]. Xanthine oxidase through the oxidation of hypoxanthine produces ROS as superoxide and hydrogen peroxide leading to lipid peroxidation and membrane damage. - A greatly increased rate of free radical production may exceed the capacity of the cellular defence sys-
tem such as antioxidants and permit a substantial attack of free radicals on lipids in the cell membrane causing lipid peroxidation leading to cell injury. This suggestion is supported by findings that BSO induced loss of GHS increased lipid peroxidation and the severity of the myopathy due to DFP [20]. The prevention of the DFP-induced muscle hyperactivity through the neuromuscular blocking agent d-tubocurarine attenuated the increase of F2isoprostanes [20, 21], prevented the change in Cytox (figures 1, 2) and the conversion of XD into XO (table 113. These data suggest that free radical generation during DFP-induced fasciculation is associated with AChE inhibition by DFP, and is not due to direct effects of DFP on Cyt-ox and XD/XO. No studies to date have reported a relationship between OP-antiChE-induced muscle hyperactivity, lipid peroxidation, and muscle lesions. From the present studies, however, it is concluded that the accumulation of oxygen free radicals and the related increases in lipid peroxidation are caused by the DFP-induced muscle hyperactivity and that lipid peroxidation is a contributing initial factor in the mechanism of OP-antiChE-induced cell injury. In summary, the results of this study demonstrated: 1) that the OP-antiChE, DFP, caused fasciculations leading to lipid peroxidation and muscle necrosis; 2) prevention of DFP-induced muscle hyperactivity-by d-tubocurarine inhibits lipid peroxidation and necrosis and rules out a direct effect of DFP on Cyt-ox and XD-XO and thus support the hypothesis that OP-antiChE-induced muscle hyperactivity stimulates ROS production, through changes in cytochrome c oxidase and xanthine oxidase activity.
Acknowledgment This work was supported by NIH grant RO 1 ES04597.
Xth International Symposium on Cholinergic Mechanisms
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