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The interaction of RPE melanin and ascorbic acid with the photooxidation of linoleic acid
Session 8: Photooxidative Stress 8:9
THE INTERACTIONOF RPE MELANIN AND ASCORBIC ACID WITH THE PHOTOOXIDATION OF LINOLEIC ACID Randolph D. Glickman and Kwok-Wai Lam, Department of Ophthalmology, The Univ. of Texas Health Science Center, San Antonio, TX, 78284-6230, USA. We have demonstrated that light-activated melanin rapidly oxidized ascorbic acid (AA), but had negligible reaction with glutathione, atocopherol, trolox, taurine, or 3-aminotymsine. We have now evaluated the ability of light-induced melanin radicals to oxidize linoleic acid (LA), a model cellular lipid. Retinal pigment epithelium (RPE) melanin granules were prepared from freshly obtained bovine eyes as in our earlier work (Glickman & Lam, Photochem. & Photobiol., 55:191-196, 1992). Photooxidation of LA was studied in several experiments: LA alone; LA with AA; LA with melanin granules; and LA with AA and melanin. In all these experiments, the light source was the broadband output of a 150 W xenon arc lamp, producing -215 mW/cm’ at the sample. LA was diluted in methanol, dispersed with 1 pl lubrol, and combined with the reagent under test in a total reaction volume of 25 fl. Following light exposure, a 5 fl aliquot was analyzed on a Delta PAK C,, HPLC column, eluted by 85% acetonitrile. The UV absorption of the eluant was recorded at 210 nm (LA), 232 nm (hydroperoxide of LA), and 265 nm (AA). In the absence of melanin granules, a presumed hydroperoxide peak appeared, dependent on light exposure time and initial LA concentration. Addition of AA completely prevented the photooxidation of LA. Melanin granules inhibited the oxidation of LA: 50% inhibition was achieved at a concentration of 4.48 x lo9 granules/ml. With AA and melanin present in the reaction mixture, there was essentially no appearance of the lipid hydroperoxide, but more AA was oxidized as the number of melanin granules was increased. These results do not support a direct interaction between activated melanin and LA, yet both are able to oxidize ascorbic acid during photooxidative stress. Interestingly. in this in vitro model, ascorbic acid, a hydrophilic antioxidant, is able to protect LA against photooxidative attack. Supported by grant AFOSR-91-0208, and a development grant from Research to Prevent Blindness, Inc.
8:ll
cDNA SEQUENCES OF Mn-SOD AND Fe-SOD IN CHLAMYDOMONAS REINHARDTII AND THEIR
OXIDATIVE
529 DAMAGE
TO
DNA
IN
in vitro.
DECOMPOSITION OF PHENYL N-fert-BUTYLNITRONE Walee Chamulitrat’,
MUTANTS Kaoru Kitayama and Robert K. Togas&i,
Sandra J. Jordan*, Ronald P. Mason*,
Kieko Saito# and Richard G. Cutler#
Department of Biology, Indiana University, Bloomington,
*National
Indiana, 47405 USA.
Institute
of Environmental
Health
Research Triangle Park, National Institutes Plant cells exsert defense responses against oxygen toxicity under highly oxidative stress during active photosynthesis. We are interested in tbe regulation of such mechanisms and trying to evaluate the role of SOD in photooxidative stress responses. An unicellular green algae, -r&&&ii, is used as a simple model syslem for photosynthetic eukaryote. c. &&&i has at least three forms of
Mn-SOD and one Fe-SOD which is localized in chloroplast. A cDNA clone coding for Mn-SOD was isolated from c. reinhu using an oligomer designed from a highly conserved region among all known Mn and Fe-SOD sequences. The 1.4kb cDNA sequence covers an entire coding sequence of the mature protein with the molecular weight of 23900 from the deduced amino acid sequence. The clone has no apparent transit peptide sequence, indicating its cytosolic localization. Mu-SOD of c. reinhardtii shows higher homology to bacterial h4nSOD sequences than to sequences from eukaryotes. Partial sequence of a Fe-SOD was obtained by F’CR amplification. The sequence covers three fourth of the mature protein sequence and shows high homology to Fe-SOD sequences from &&&Q&W and Nicotiana
olumbanlnlfolla. In order to assess an involvement of SOD in a of paraquat
resistance
in s.
paraquat
resistant mutants were isolated by gene disruption method. SMies on transcriptional and translational expression of h&SOD and Fe-SOD in these mutants are currently undenvay.
8:lO
The photobiological activities of psoralens have been thought to involve their photobinding to, and crosslinking of, DNA However, there is recent evidence that psoralens can also photosensitize the formation of reactive oxygen species. Guanosine hydroxylation was used as a marker for assessing photooxidation of both isolated and cellular DNA by psoralens. The monofunctional psoralen, 3-carbethoxypsoralen (3-CP), in the presence of ultraviolet light, efficiently sensitized the formation of 8-hydroxyguanosine (S-OHdG) in isolated DNA. Up to 0.4% of guanosine residues were converted to 8-OHdG. The bifunctional photosensitizer, psoralen (Ps), showed low but detectable photooxidizing activity. The phototoxicities of Ps and 3-CP were determined in an in vitro assay using skin fibroblasts irradiated with UVA (320-400 run). Ps was found to be significantly more phototoxic (IC,,: 4 PM, 0.82 J/cm’) than 3-CP (IQ,: 40 FM, 0.82 J/cm* UVA). To determine the role of DNA oxidation in the phototoxicity of Ps and 3-CP, levels of 8-OHdG in cellular DNA were measured following treatment of skin fibroblasts with Ps or 3-CP and UVA yielding 80% cytotoxicity. Neither Ps nor 3-CP treatments resulted in elevated levels of 8-OHdG in cellular DNA. These data suggest that, although psoralen analogues can effectively photosensitize oxidation of isolated DNA, oxidation of cellular DNA is not mechanistically important in psoralen phototoxicity
NITRIC OXIDE FORMATION DURING LIGHT-INDUCED
EXPRESSIONAL REGULATION IN PARAQUAT RESISTANT
mechanism
PSORALEN
PHOTOSENSITIZED CYTOTOXICITY Wayne G Warner, Rong Wei, William C. Timmer and Andrija Komhauser, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Washington, DC, 20204
27709 USA, #National
Sciences,
of Health, NC,
Institute of Aging, National Institutes
of Health, MD, 21224 USA. Phenyl N-tert-butylnitrone (PBN) is a spin trap commonly employed in free radical research, which has been shown to have both adverse and beneficial effects on various biological systems. We report here evidence that photolysis decomposes PBN to nitric oxide in aqueous solutions. Non-heme and heme proteins have been employed to form nitrosyl complexes which were detected using EPR spectroscopy. Concomitantly, nitrite formation was detected after light-induced decomposition of PBN. In addition, we found that tertnitrosobutane and decomposed PBN caused an activation of guanylate cyclase. We propose a mechanism where PBN is decomposed by light to tert-nitrosobutane. The latter compound is in turn decomposed to nitric oxide. This study suggests the possibility that PBN or PBN radical adducts may be sources of nitric oxide in biological environments. When utilizing PBN as a spin trap in biological samples, not only is the trapping of reactive free radicals operative, but nitric oxide produced from PBN decomposition may play an important role in altering biological functions.
a:12
THE INTERACTIONOF RPE MELANIN AND ASCORBIC ACID WITH THE PHOTOOXIDATION OF LINOLEIC ACID Randolph D. Glickman and Kwok-Wai Lam, Department of Ophthalmology, The Univ. of Texas Health Science Center, San Antonio, TX, 78284-6230, USA. We have demonstrated that light-activated melanin rapidly oxidized ascorbic acid (AA), but had negligible reaction with glutathione, atocopherol, trolox, taurine, or 3-aminotymsine. We have now evaluated the ability of light-induced melanin radicals to oxidize linoleic acid (LA), a model cellular lipid. Retinal pigment epithelium (RPE) melanin granules were prepared from freshly obtained bovine eyes as in our earlier work (Glickman & Lam, Photochem. & Photobiol., 55:191-196, 1992). Photooxidation of LA was studied in several experiments: LA alone; LA with AA; LA with melanin granules; and LA with AA and melanin. In all these experiments, the light source was the broadband output of a 150 W xenon arc lamp, producing -215 mW/cm’ at the sample. LA was diluted in methanol, dispersed with 1 pl lubrol, and combined with the reagent under test in a total reaction volume of 25 fl. Following light exposure, a 5 fl aliquot was analyzed on a Delta PAK C,, HPLC column, eluted by 85% acetonitrile. The UV absorption of the eluant was recorded at 210 nm (LA), 232 nm (hydroperoxide of LA), and 265 nm (AA). In the absence of melanin granules, a presumed hydroperoxide peak appeared, dependent on light exposure time and initial LA concentration. Addition of AA completely prevented the photooxidation of LA. Melanin granules inhibited the oxidation of LA: 50% inhibition was achieved at a concentration of 4.48 x lo9 granules/ml. With AA and melanin present in the reaction mixture, there was essentially no appearance of the lipid hydroperoxide, but more AA was oxidized as the number of melanin granules was increased. These results do not support a direct interaction between activated melanin and LA, yet both are able to oxidize ascorbic acid during photooxidative stress. Interestingly. in this in vitro model, ascorbic acid, a hydrophilic antioxidant, is able to protect LA against photooxidative attack. Supported by grant AFOSR-91-0208, and a development grant from Research to Prevent Blindness, Inc.
8:ll
cDNA SEQUENCES OF Mn-SOD AND Fe-SOD IN CHLAMYDOMONAS REINHARDTII AND THEIR
OXIDATIVE
529 DAMAGE
TO
DNA
IN
in vitro.
DECOMPOSITION OF PHENYL N-fert-BUTYLNITRONE Walee Chamulitrat’,
MUTANTS Kaoru Kitayama and Robert K. Togas&i,
Sandra J. Jordan*, Ronald P. Mason*,
Kieko Saito# and Richard G. Cutler#
Department of Biology, Indiana University, Bloomington,
*National
Indiana, 47405 USA.
Institute
of Environmental
Health
Research Triangle Park, National Institutes Plant cells exsert defense responses against oxygen toxicity under highly oxidative stress during active photosynthesis. We are interested in tbe regulation of such mechanisms and trying to evaluate the role of SOD in photooxidative stress responses. An unicellular green algae, -r&&&ii, is used as a simple model syslem for photosynthetic eukaryote. c. &&&i has at least three forms of
Mn-SOD and one Fe-SOD which is localized in chloroplast. A cDNA clone coding for Mn-SOD was isolated from c. reinhu using an oligomer designed from a highly conserved region among all known Mn and Fe-SOD sequences. The 1.4kb cDNA sequence covers an entire coding sequence of the mature protein with the molecular weight of 23900 from the deduced amino acid sequence. The clone has no apparent transit peptide sequence, indicating its cytosolic localization. Mu-SOD of c. reinhardtii shows higher homology to bacterial h4nSOD sequences than to sequences from eukaryotes. Partial sequence of a Fe-SOD was obtained by F’CR amplification. The sequence covers three fourth of the mature protein sequence and shows high homology to Fe-SOD sequences from &&&Q&W and Nicotiana
olumbanlnlfolla. In order to assess an involvement of SOD in a of paraquat
resistance
in s.
paraquat
resistant mutants were isolated by gene disruption method. SMies on transcriptional and translational expression of h&SOD and Fe-SOD in these mutants are currently undenvay.
8:lO
The photobiological activities of psoralens have been thought to involve their photobinding to, and crosslinking of, DNA However, there is recent evidence that psoralens can also photosensitize the formation of reactive oxygen species. Guanosine hydroxylation was used as a marker for assessing photooxidation of both isolated and cellular DNA by psoralens. The monofunctional psoralen, 3-carbethoxypsoralen (3-CP), in the presence of ultraviolet light, efficiently sensitized the formation of 8-hydroxyguanosine (S-OHdG) in isolated DNA. Up to 0.4% of guanosine residues were converted to 8-OHdG. The bifunctional photosensitizer, psoralen (Ps), showed low but detectable photooxidizing activity. The phototoxicities of Ps and 3-CP were determined in an in vitro assay using skin fibroblasts irradiated with UVA (320-400 run). Ps was found to be significantly more phototoxic (IC,,: 4 PM, 0.82 J/cm’) than 3-CP (IQ,: 40 FM, 0.82 J/cm* UVA). To determine the role of DNA oxidation in the phototoxicity of Ps and 3-CP, levels of 8-OHdG in cellular DNA were measured following treatment of skin fibroblasts with Ps or 3-CP and UVA yielding 80% cytotoxicity. Neither Ps nor 3-CP treatments resulted in elevated levels of 8-OHdG in cellular DNA. These data suggest that, although psoralen analogues can effectively photosensitize oxidation of isolated DNA, oxidation of cellular DNA is not mechanistically important in psoralen phototoxicity
NITRIC OXIDE FORMATION DURING LIGHT-INDUCED
EXPRESSIONAL REGULATION IN PARAQUAT RESISTANT
mechanism
PSORALEN
PHOTOSENSITIZED CYTOTOXICITY Wayne G Warner, Rong Wei, William C. Timmer and Andrija Komhauser, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Washington, DC, 20204
27709 USA, #National
Sciences,
of Health, NC,
Institute of Aging, National Institutes
of Health, MD, 21224 USA. Phenyl N-tert-butylnitrone (PBN) is a spin trap commonly employed in free radical research, which has been shown to have both adverse and beneficial effects on various biological systems. We report here evidence that photolysis decomposes PBN to nitric oxide in aqueous solutions. Non-heme and heme proteins have been employed to form nitrosyl complexes which were detected using EPR spectroscopy. Concomitantly, nitrite formation was detected after light-induced decomposition of PBN. In addition, we found that tertnitrosobutane and decomposed PBN caused an activation of guanylate cyclase. We propose a mechanism where PBN is decomposed by light to tert-nitrosobutane. The latter compound is in turn decomposed to nitric oxide. This study suggests the possibility that PBN or PBN radical adducts may be sources of nitric oxide in biological environments. When utilizing PBN as a spin trap in biological samples, not only is the trapping of reactive free radicals operative, but nitric oxide produced from PBN decomposition may play an important role in altering biological functions.
a:12