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Lipoic acid prevents endoneurial ischemia and improves experimental diabetic neuropathy
INTERACTIONS OF HEAVY METAL COMPOUNDS WITH THE GLUTATHIONE SYSTEM IN 06 GLIOMA CELLS. M_~R. Cookson and V.W. Pentreath University of Salford, Salford M5 4WT, UK.
CYTOTOXICITY OF PIPERIDINE NITROXIDES. E. Monti 1, M.B. Gariboldit, R. Supino2, F. Piccinini' ~laarmacology, V.~Vanvitelli, 32-20129 Milan; 2Ist. Nazionale Tumori, V.Venezian, 1-20133, Milan, Italy.
One of the functions of glial cells in the nervous system is a protective one" they contain high levels of antioxidants and enzymes of the glutathione pathway. W e have examined the interactions of toxic heavy metals with the C6 glioma cell line. Cytotoxic effects were assessed by the neutral red uptake assay. The order of potency was triethyltin (TET) > trimethyltin (TMT) > CdCI2 > methylmercury (MeHg)> HgCI 2. The addition of reduced glutathione (GSH) to the culture media protected the cells, in that the EC50 values for neutral red were shifted 22-fold: for MeHg, 8-fold for CdCI 2 and 14-fold for HgCI 2. N o effects of GSH on the toxicity of TET or TMT were seen. Pretreatment of the cells with the glutathione synthesis inhibitor L-o-buthionine sulfoximine increased the sensitivity t ° CdCI 2 28-fold and to HgCI 2114-fold but had no effect on the EC50 values for TET, TMT or MeHg. Intracellular GSH : levels were also estimated using the fluorescent probe chlorobimane. With sub-cytotoxic concentrations of HgCI=, there was a depletion of GSH over 4h with a subsequent rise (to 150% of control) after 24h. Similar effects were seen with CdCI 2 and MeHg although the time' course of these was more protracted. A rise in GSH was also seen with the organotin compounds. These results show a direct interaction of heavy metal compounds with the glutathione system in gila, which m a y have important consequences for brain function after low level exposure.
Piperidine nitroxide free radicals are currently under investigation for their potential role as antioxidants, due to their ability to participate in one-electron reactions. While such compounds are generally reported as non cytotoxic upon short term exposure (10 rain-2 hr), their long term effects on cell survival and proliferation have not been investigated. We examined the effects of three piperidine nitroxide spin labels, 2,2,6,6-tetramethylpiperidine-N-oxyl (Tempo) and its 4-hydroxy- and 4-amino-derivatives (Tempol and Tempamine, respectively), against a panel of neoplastic and nonneoplastic cell lines. Our results show that all three nitroxides display varying degrees of cytotoxicity against the cell lines tested, with IC50 values between 0.1 to 1 mM after 4-day exposure. In particular, Tempol was consistently found to be more cytotoxic against neoplastic than against non-neoplastic cells of similar lineage. Cell cycle studies indicate that cell death does not occur by apoptosis. The mechanism of the cytotoxic action of Tempol seems to involve free radical-mediated reactions, as indicated by the significant protection achieved in a human adenocarcinoma cell line (MCF-7) by simultaneous exposure to 5-10 mM N-acetylcysteine. This hypothesis is further supported by the observation that cells with higher levels of intracellular glutathione (GSH) and/or GSH-related enzyme activity were less sensitive to the cytotoxic action of Tempol. In addition, partially antagonistic effects between Tempol and adriamycin observed in MCF-7 cells suggest a mutual inactivation of the free radical species corresponding to the active forms of the two drugs. Therapeutic exploitation of the antiproliferative effects of Tempol could be worth pursuing if the observed selectivity against neoplastic versus non-neoplastic cells, should be confirmed by further in vivo studies. On the other hand, the possibility of citotoxic side effects should not be neglected when planning antioxidant treatments with these compounds.
ENZYMATICALLY REDUCED LIPOIC ACID MAY R E G E N E R A T E G L U T A T H I O N E AND V I T A M I N C
LIPOIC ACID PREVENTS ENDONEURIAL ISCHEMIA AND IMPROVES EXPERIMENTAL DIABETIC NEUROPATHY M. Nagamatsu ~, K.K. Nickander~, J.D. Schmelzer~, A. Raya*, D,A. Rohwer ~, H. Tritschler H', P.A. Low PAa a, Mayo Foundation, Rochester, MN, 55905, USA; *, Department of Physiology, University of Valencia, Spain; ~', Asta Medica, Dresden, Germany.
G. Ph. Biewenga, G.R.M.M. Haenen and A. Bast Leiden/Amsterdam Center for Drug Research, Vrije Universiteit, Department of Pharmacochemistry, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
Experimental diabetic neuropathy (SDN) induced by streptozotocin results in a reduction in nerve blood flow (NBF), endoneurial hypoxia, increased lipid peroxidation, and impaired nerve conduction. Experimental depletion of nerve tx-tocopherol will not only aggravate SDN but will cause neuropathy in otherwise normal nerves. We evaluated the effect of supplementation of lipoic acid (LA), a powerful lipophilie antioxidant, on normal and ot-tocopheroI-depleted control and diabetic peripheral nerves of Sprague-Dawley male rats. Lipoie acid, in doses of 20, 50, and 100 mg/kg, was administered intrapedtoneally, five times per week, after onset of diabetes. Measurements of NBF, electrophysiology, and indices of oxidative stress were studied at one month. NBF in SDN was reduced by 50%; LA improved NBF of SDN in a dose-dependent manner. After one month of treatment, LA supplemented rats (100 mg/kg) had normal NBF. The most sensitive and reliable indicator of oxidative stress is a reduction in reduced glutathione (GSH), which is signifteantly reduced in SDN and o~-toeopherol deficient nerves. LA treatment resulted in a dose-dependent improvement in GSH in SDN, resulting in normal values for the higher doses in LA-supplemented rats. The conduction velocity of digital nerve was reduced in SDN and was significantly improved by LA. These studies suggest that LA improves SDN, in significant part by reducing the effects of oxidative stress. The drug may have potential in the treatment of human diabetic neurupathy. The improvement in distal and sensory fibers is of particular interest, suggesting a role in the treatment of distal painful diabetic neuropathy.
The therapeutic value of lipoic acid (LA) is found in pathologies in which free radicals are involved. Therefore, we study the antioxidant profile of LA. In vivo LA is also present in its reduced form viz. dihydrolipoic acid (DHI_,A). In the protection against lipid peroxidation it was found that DHLA pOtentiates the protection provided by glutathione (GSH). It keeps GSH in the reduced, active form. During oxidative stress vitamin C is converted into a semi-dehydroascorbate radical and subsequently into dehydroascorbate. Our experiments show that DHLA regenerates vitamin C much faster than GSH. We propose that cooperation of DHLA with other anti-oxidants as GSH and vitamin C is an important mechanism by which LA shows anti-oxidant activity. Therefore we studied lipoamide dehydrogenase (LipDH) which can reduce LA. It was shown that LipDH reduces the Renantiomer of LA 30 times faster than S-LA. The initial reduction rate of racemic LA shows exactly half the rate of R-LA. When LipDH is the main reducing enzyme in vivo R-LA wilt be reduced preferentially. This makes R-LA a more potent anti-oxidant than S-
LA.
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CYTOTOXICITY OF PIPERIDINE NITROXIDES. E. Monti 1, M.B. Gariboldit, R. Supino2, F. Piccinini' ~laarmacology, V.~Vanvitelli, 32-20129 Milan; 2Ist. Nazionale Tumori, V.Venezian, 1-20133, Milan, Italy.
One of the functions of glial cells in the nervous system is a protective one" they contain high levels of antioxidants and enzymes of the glutathione pathway. W e have examined the interactions of toxic heavy metals with the C6 glioma cell line. Cytotoxic effects were assessed by the neutral red uptake assay. The order of potency was triethyltin (TET) > trimethyltin (TMT) > CdCI2 > methylmercury (MeHg)> HgCI 2. The addition of reduced glutathione (GSH) to the culture media protected the cells, in that the EC50 values for neutral red were shifted 22-fold: for MeHg, 8-fold for CdCI 2 and 14-fold for HgCI 2. N o effects of GSH on the toxicity of TET or TMT were seen. Pretreatment of the cells with the glutathione synthesis inhibitor L-o-buthionine sulfoximine increased the sensitivity t ° CdCI 2 28-fold and to HgCI 2114-fold but had no effect on the EC50 values for TET, TMT or MeHg. Intracellular GSH : levels were also estimated using the fluorescent probe chlorobimane. With sub-cytotoxic concentrations of HgCI=, there was a depletion of GSH over 4h with a subsequent rise (to 150% of control) after 24h. Similar effects were seen with CdCI 2 and MeHg although the time' course of these was more protracted. A rise in GSH was also seen with the organotin compounds. These results show a direct interaction of heavy metal compounds with the glutathione system in gila, which m a y have important consequences for brain function after low level exposure.
Piperidine nitroxide free radicals are currently under investigation for their potential role as antioxidants, due to their ability to participate in one-electron reactions. While such compounds are generally reported as non cytotoxic upon short term exposure (10 rain-2 hr), their long term effects on cell survival and proliferation have not been investigated. We examined the effects of three piperidine nitroxide spin labels, 2,2,6,6-tetramethylpiperidine-N-oxyl (Tempo) and its 4-hydroxy- and 4-amino-derivatives (Tempol and Tempamine, respectively), against a panel of neoplastic and nonneoplastic cell lines. Our results show that all three nitroxides display varying degrees of cytotoxicity against the cell lines tested, with IC50 values between 0.1 to 1 mM after 4-day exposure. In particular, Tempol was consistently found to be more cytotoxic against neoplastic than against non-neoplastic cells of similar lineage. Cell cycle studies indicate that cell death does not occur by apoptosis. The mechanism of the cytotoxic action of Tempol seems to involve free radical-mediated reactions, as indicated by the significant protection achieved in a human adenocarcinoma cell line (MCF-7) by simultaneous exposure to 5-10 mM N-acetylcysteine. This hypothesis is further supported by the observation that cells with higher levels of intracellular glutathione (GSH) and/or GSH-related enzyme activity were less sensitive to the cytotoxic action of Tempol. In addition, partially antagonistic effects between Tempol and adriamycin observed in MCF-7 cells suggest a mutual inactivation of the free radical species corresponding to the active forms of the two drugs. Therapeutic exploitation of the antiproliferative effects of Tempol could be worth pursuing if the observed selectivity against neoplastic versus non-neoplastic cells, should be confirmed by further in vivo studies. On the other hand, the possibility of citotoxic side effects should not be neglected when planning antioxidant treatments with these compounds.
ENZYMATICALLY REDUCED LIPOIC ACID MAY R E G E N E R A T E G L U T A T H I O N E AND V I T A M I N C
LIPOIC ACID PREVENTS ENDONEURIAL ISCHEMIA AND IMPROVES EXPERIMENTAL DIABETIC NEUROPATHY M. Nagamatsu ~, K.K. Nickander~, J.D. Schmelzer~, A. Raya*, D,A. Rohwer ~, H. Tritschler H', P.A. Low PAa a, Mayo Foundation, Rochester, MN, 55905, USA; *, Department of Physiology, University of Valencia, Spain; ~', Asta Medica, Dresden, Germany.
G. Ph. Biewenga, G.R.M.M. Haenen and A. Bast Leiden/Amsterdam Center for Drug Research, Vrije Universiteit, Department of Pharmacochemistry, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
Experimental diabetic neuropathy (SDN) induced by streptozotocin results in a reduction in nerve blood flow (NBF), endoneurial hypoxia, increased lipid peroxidation, and impaired nerve conduction. Experimental depletion of nerve tx-tocopherol will not only aggravate SDN but will cause neuropathy in otherwise normal nerves. We evaluated the effect of supplementation of lipoic acid (LA), a powerful lipophilie antioxidant, on normal and ot-tocopheroI-depleted control and diabetic peripheral nerves of Sprague-Dawley male rats. Lipoie acid, in doses of 20, 50, and 100 mg/kg, was administered intrapedtoneally, five times per week, after onset of diabetes. Measurements of NBF, electrophysiology, and indices of oxidative stress were studied at one month. NBF in SDN was reduced by 50%; LA improved NBF of SDN in a dose-dependent manner. After one month of treatment, LA supplemented rats (100 mg/kg) had normal NBF. The most sensitive and reliable indicator of oxidative stress is a reduction in reduced glutathione (GSH), which is signifteantly reduced in SDN and o~-toeopherol deficient nerves. LA treatment resulted in a dose-dependent improvement in GSH in SDN, resulting in normal values for the higher doses in LA-supplemented rats. The conduction velocity of digital nerve was reduced in SDN and was significantly improved by LA. These studies suggest that LA improves SDN, in significant part by reducing the effects of oxidative stress. The drug may have potential in the treatment of human diabetic neurupathy. The improvement in distal and sensory fibers is of particular interest, suggesting a role in the treatment of distal painful diabetic neuropathy.
The therapeutic value of lipoic acid (LA) is found in pathologies in which free radicals are involved. Therefore, we study the antioxidant profile of LA. In vivo LA is also present in its reduced form viz. dihydrolipoic acid (DHI_,A). In the protection against lipid peroxidation it was found that DHLA pOtentiates the protection provided by glutathione (GSH). It keeps GSH in the reduced, active form. During oxidative stress vitamin C is converted into a semi-dehydroascorbate radical and subsequently into dehydroascorbate. Our experiments show that DHLA regenerates vitamin C much faster than GSH. We propose that cooperation of DHLA with other anti-oxidants as GSH and vitamin C is an important mechanism by which LA shows anti-oxidant activity. Therefore we studied lipoamide dehydrogenase (LipDH) which can reduce LA. It was shown that LipDH reduces the Renantiomer of LA 30 times faster than S-LA. The initial reduction rate of racemic LA shows exactly half the rate of R-LA. When LipDH is the main reducing enzyme in vivo R-LA wilt be reduced preferentially. This makes R-LA a more potent anti-oxidant than S-
LA.
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163
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