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The antioxidant property of tetrahydrobiopterin
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AntoxidantCompounds
2.45 CELLULAR DISTRIBUTION OF GLUTATHIONE IN THE
2.47
NERVOUS SYSTEM. M.A. Philbert, D.K. Waters, H.E. Lowndes. Neurotoxicology Laboratories, College of Pharmacy, Rutgers University, Piscataway, NJ 08854, U.S.A.
PROTECTION OF CULTURED LENS EPITHELIAL CELLS FROM H202 INSULT BY SOD MIMICS J.R. Reddan, M.D. Sevilla, F.J. Giblin, D.C. Dziedzic and J.L. Peters. Oakland University, Rochester, MI 48309-4401, U.S.A.
Determining the cellular distribution of glutathione (GSH) is important to the understanding of selective neurotoxicity of chemicals. Eight or 5D-micron sections from peripheral nerve, spinal cord, dorsal root ganglia, brainstem, cerebellum, forebrain and cortex were stained with mercury orange (Brain Res 409, 1987) or o-phthaldialdehyde (Histochem J 18, 1986). GSH was localized by fluorescence microscopy. Strong fluorescence was principally associated with white matter and neuropil. Neuronal somata exhibited l i t t l e or no fluorescence, with the exceptions of cerebellar Purkinje and granule cells, dorsal root ganglion cells and granular and mitral cells of the olfactory bulbs. Complete concordance in distribution of fluorescence was obtained with the two methods; diethyl maleate pretreatment markedly reduced fluorescence obtained with either stain. Hence GSH appears localized primarily in neuropil, white matter and nerve endings; with a few exceptions, neuronal somata did not appear to contain appreciable GSH. The heterogeneous distribution of GSH may underlie selective vulnerability to neurotoxicants. Supported by NS-23325 and ES-05022.
SOD mimics protect Chinese hamster cells from H202 damage (Mitchell et al., Biochemistry, 29, 1990). H202 is a constituent of the aqueous humor (0.03-0.05mM), is elevated in the aqueous of humans with cataracts, and may contribute to cataract formation. Lens epithelial cells (LEC) play a major role in H202 detoxification. Here we report that TEMPO and TEMPOL protect LEC from H202 insult. 50,000 cells were cultured in MEM or in MEM containing imM TEMPO or 5mM TEMPOL and 0.05mM H202. EPR spectra indicated that both nitroxides were stable in MEM with H202. The concentration of H202 in MEM was unaffected by the nitroxides. Cells treated with H202 exhibited blebs, cell loss, and later displayed minimal growth in a serum-containing medium. The nitroxides which accumulated in the cells prevented all of these deleterious effects. Untreated cells or cells treated with TEMPO or TEMPOL showed a 30-fold increase in number after seven days in culture. Cells treated with 0.05mM H202 alone showed only a two-fold increase, whereas nitroxide-H202 treated cells increased 25-fold. Nitroxides may be useful in delineating the mechanism of H202-induced insult and may have therapeutic applications.
E. C O L I S T R A I N S A N D O X I D A N T S T R E S S : DIFFERENCES IN G R O W T H , LETHALITY, AND GSH. Mary Jo Romero 1, James A. Imlay, Andrew T. Canada 1. Departments of Anesthesiology 1 and Biochemistry, Duke University Medical Center, Durham, NC 27710, U.S.A
THE ANTIOXIDANTPROPERTY OF TETRAHYDROBIOPTERIN Rong-sen Shen and Yixian Zhang Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, TX 77550, U.S.A.
The effect of various oxidants and free radical generating processes on gluthathione (GSH) has been widely studied in bacteria, particulary E. coli. This is because E. coli is unique among procaryotes in containing an amount of GSH comparable to mammalian cells. Attempts to determine the importance of G S H to coli have usually employed the K12 strain and have relied on the use of G S H deficient or overproducing K12 mutants to accomplish this goal with mixed results. There is increasing evidence that the K12 strain is less sensitive to oxidant/free radical stress than E. coli B. Exposure of both wild-type strains to either paraquat (PQ) or t-butylhydroperoxide (TBH) resulted in a difference in both growth and lethality effects with the B strain the more sensitive of the two. Coincidentally, the GSH in the K12 strain was minimally affected by the oxidants at 5raM while a 80-90% reduction occurred with the B strain. A K12 GSH-deficient strain resembled the wild B strain in effect of TBH on lethality while neither K12 strain, parent or GSH-deficient, showed any difference in response to PQ. A B strain (RCI-1) deficient in G S H was constructed and resembled the K12 deficient strain in response to a wide variety of thiol-reacting compounds. This strain contained 2x SOD activity as the wild B and was more resistant to PQ effects on growth. This experiment show a correlation between the effects of organic peroxide stress but not paraquat on growth, and lethality, and GSH of E. coli B.
L-erythro-5,6,7,8-Tetrahydrobiopterin (BH4), the obligatory cofactor for aromatic amino acid hydroxylases (AAA-H), is ubiquitous in nature. When BH4 is oxidized to quinonoid dihydrobiopterin by AAA-H, it is regenerated by dihydropteridine reductase (DHPR) using NADH as the cofactor. High levels of BH4 and DHPR, however, are found in non-neural organs, such as bone marrow, spleen, thymus and blood cells. The physiological function ofBH 4 and DHPR in these tissues, which are devoid of AK&-H, is unclear. This study showed that BH4, in the presence of NADH and DHPR, acted as an antioxidant against catecholamine autoxidation in Dulbecco's phosphate buffered saline, by increasing the lag periods and by decreasing the rates of aminochrome formation. In the presence of a fourth component, peroxidase (PO), this BH4-mediated antioxidant system also protected rat pheochromocytoma PC 12 and mouse macrophage J774A.1 cells against 02 toxicity caused by 1-1202, nitrofurantoin (NF), and xanthine/xanthine oxidase (X/XO) reaction. The BH4 antioxidant system, evaluated by the [3H]-thymidine incorporation and the MTT colorimetric assays, was more effective than catalase and PO in protecting cells against H202 and NF toxicity, and was as effective as catalase and ascorbic acid in protecting cells against X/XO-induced toxicity. Since BH4, PO, NADH, and DHPR are widely distributed in major organs and blood cells, they may constitute an as of yet little known antioxidant system against reactive 02 species in mammalian cells. (Sponsored by John Sealy Memorial Endowment Fund).
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2.48
AntoxidantCompounds
2.45 CELLULAR DISTRIBUTION OF GLUTATHIONE IN THE
2.47
NERVOUS SYSTEM. M.A. Philbert, D.K. Waters, H.E. Lowndes. Neurotoxicology Laboratories, College of Pharmacy, Rutgers University, Piscataway, NJ 08854, U.S.A.
PROTECTION OF CULTURED LENS EPITHELIAL CELLS FROM H202 INSULT BY SOD MIMICS J.R. Reddan, M.D. Sevilla, F.J. Giblin, D.C. Dziedzic and J.L. Peters. Oakland University, Rochester, MI 48309-4401, U.S.A.
Determining the cellular distribution of glutathione (GSH) is important to the understanding of selective neurotoxicity of chemicals. Eight or 5D-micron sections from peripheral nerve, spinal cord, dorsal root ganglia, brainstem, cerebellum, forebrain and cortex were stained with mercury orange (Brain Res 409, 1987) or o-phthaldialdehyde (Histochem J 18, 1986). GSH was localized by fluorescence microscopy. Strong fluorescence was principally associated with white matter and neuropil. Neuronal somata exhibited l i t t l e or no fluorescence, with the exceptions of cerebellar Purkinje and granule cells, dorsal root ganglion cells and granular and mitral cells of the olfactory bulbs. Complete concordance in distribution of fluorescence was obtained with the two methods; diethyl maleate pretreatment markedly reduced fluorescence obtained with either stain. Hence GSH appears localized primarily in neuropil, white matter and nerve endings; with a few exceptions, neuronal somata did not appear to contain appreciable GSH. The heterogeneous distribution of GSH may underlie selective vulnerability to neurotoxicants. Supported by NS-23325 and ES-05022.
SOD mimics protect Chinese hamster cells from H202 damage (Mitchell et al., Biochemistry, 29, 1990). H202 is a constituent of the aqueous humor (0.03-0.05mM), is elevated in the aqueous of humans with cataracts, and may contribute to cataract formation. Lens epithelial cells (LEC) play a major role in H202 detoxification. Here we report that TEMPO and TEMPOL protect LEC from H202 insult. 50,000 cells were cultured in MEM or in MEM containing imM TEMPO or 5mM TEMPOL and 0.05mM H202. EPR spectra indicated that both nitroxides were stable in MEM with H202. The concentration of H202 in MEM was unaffected by the nitroxides. Cells treated with H202 exhibited blebs, cell loss, and later displayed minimal growth in a serum-containing medium. The nitroxides which accumulated in the cells prevented all of these deleterious effects. Untreated cells or cells treated with TEMPO or TEMPOL showed a 30-fold increase in number after seven days in culture. Cells treated with 0.05mM H202 alone showed only a two-fold increase, whereas nitroxide-H202 treated cells increased 25-fold. Nitroxides may be useful in delineating the mechanism of H202-induced insult and may have therapeutic applications.
E. C O L I S T R A I N S A N D O X I D A N T S T R E S S : DIFFERENCES IN G R O W T H , LETHALITY, AND GSH. Mary Jo Romero 1, James A. Imlay, Andrew T. Canada 1. Departments of Anesthesiology 1 and Biochemistry, Duke University Medical Center, Durham, NC 27710, U.S.A
THE ANTIOXIDANTPROPERTY OF TETRAHYDROBIOPTERIN Rong-sen Shen and Yixian Zhang Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, TX 77550, U.S.A.
The effect of various oxidants and free radical generating processes on gluthathione (GSH) has been widely studied in bacteria, particulary E. coli. This is because E. coli is unique among procaryotes in containing an amount of GSH comparable to mammalian cells. Attempts to determine the importance of G S H to coli have usually employed the K12 strain and have relied on the use of G S H deficient or overproducing K12 mutants to accomplish this goal with mixed results. There is increasing evidence that the K12 strain is less sensitive to oxidant/free radical stress than E. coli B. Exposure of both wild-type strains to either paraquat (PQ) or t-butylhydroperoxide (TBH) resulted in a difference in both growth and lethality effects with the B strain the more sensitive of the two. Coincidentally, the GSH in the K12 strain was minimally affected by the oxidants at 5raM while a 80-90% reduction occurred with the B strain. A K12 GSH-deficient strain resembled the wild B strain in effect of TBH on lethality while neither K12 strain, parent or GSH-deficient, showed any difference in response to PQ. A B strain (RCI-1) deficient in G S H was constructed and resembled the K12 deficient strain in response to a wide variety of thiol-reacting compounds. This strain contained 2x SOD activity as the wild B and was more resistant to PQ effects on growth. This experiment show a correlation between the effects of organic peroxide stress but not paraquat on growth, and lethality, and GSH of E. coli B.
L-erythro-5,6,7,8-Tetrahydrobiopterin (BH4), the obligatory cofactor for aromatic amino acid hydroxylases (AAA-H), is ubiquitous in nature. When BH4 is oxidized to quinonoid dihydrobiopterin by AAA-H, it is regenerated by dihydropteridine reductase (DHPR) using NADH as the cofactor. High levels of BH4 and DHPR, however, are found in non-neural organs, such as bone marrow, spleen, thymus and blood cells. The physiological function ofBH 4 and DHPR in these tissues, which are devoid of AK&-H, is unclear. This study showed that BH4, in the presence of NADH and DHPR, acted as an antioxidant against catecholamine autoxidation in Dulbecco's phosphate buffered saline, by increasing the lag periods and by decreasing the rates of aminochrome formation. In the presence of a fourth component, peroxidase (PO), this BH4-mediated antioxidant system also protected rat pheochromocytoma PC 12 and mouse macrophage J774A.1 cells against 02 toxicity caused by 1-1202, nitrofurantoin (NF), and xanthine/xanthine oxidase (X/XO) reaction. The BH4 antioxidant system, evaluated by the [3H]-thymidine incorporation and the MTT colorimetric assays, was more effective than catalase and PO in protecting cells against H202 and NF toxicity, and was as effective as catalase and ascorbic acid in protecting cells against X/XO-induced toxicity. Since BH4, PO, NADH, and DHPR are widely distributed in major organs and blood cells, they may constitute an as of yet little known antioxidant system against reactive 02 species in mammalian cells. (Sponsored by John Sealy Memorial Endowment Fund).
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