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. 2000 Feb 1;345(Pt 3):665–672.

Dehydroascorbic acid uptake in a human keratinocyte cell line (HaCaT) is glutathione-independent.

I Savini 1, S Duflot 1, L Avigliano 1
PMCID: PMC1220802  PMID: 10642526

Abstract

Vitamin C plays an important role in neutralizing toxic free radicals formed during oxidative metabolism or UV exposure of human skin. This study was performed to investigate the mechanisms that regulate the homoeostasis of vitamin C in HaCaT cells by identifying the events involved in the transport and in the reduction of dehydroascorbic acid. Dehydroascorbic acid accumulated to a greater extent and faster compared with ascorbic acid; its transport appeared to be mediated by hexose transporters and was entirely distinct from ascorbic acid transport. Dehydroascorbate reductase activity was unaffected by glutathione depletion, although it was sensitive to thiol protein reagents. These observations, as well as the subcellular distribution of this enzymic activity and the cofactor specificity, indicate that thioredoxin reductase and lipoamide dehydrogenase play an important role in this reduction process. HaCaT cells were able to enhance their dehydroascorbic acid reductase activity in response to oxidative stress.

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Selected References

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  1. Anderson M. E. Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol. 1985;113:548–555. doi: 10.1016/s0076-6879(85)13073-9. [DOI] [PubMed] [Google Scholar]
  2. Bode A. M., Yavarow C. R., Fry D. A., Vargas T. Enzymatic basis for altered ascorbic acid and dehydroascorbic acid levels in diabetes. Biochem Biophys Res Commun. 1993 Mar 31;191(3):1347–1353. doi: 10.1006/bbrc.1993.1365. [DOI] [PubMed] [Google Scholar]
  3. Boukamp P., Petrussevska R. T., Breitkreutz D., Hornung J., Markham A., Fusenig N. E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol. 1988 Mar;106(3):761–771. doi: 10.1083/jcb.106.3.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Del Bello B., Maellaro E., Sugherini L., Santucci A., Comporti M., Casini A. F. Purification of NADPH-dependent dehydroascorbate reductase from rat liver and its identification with 3 alpha-hydroxysteroid dehydrogenase. Biochem J. 1994 Dec 1;304(Pt 2):385–390. doi: 10.1042/bj3040385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dreher F., Denig N., Gabard B., Schwindt D. A., Maibach H. I. Effect of topical antioxidants on UV-induced erythema formation when administered after exposure. Dermatology. 1999;198(1):52–55. doi: 10.1159/000018064. [DOI] [PubMed] [Google Scholar]
  7. Griffith O. W., Meister A. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J Biol Chem. 1979 Aug 25;254(16):7558–7560. [PubMed] [Google Scholar]
  8. Guaiquil V. H., Farber C. M., Golde D. W., Vera J. C. Efficient transport and accumulation of vitamin C in HL-60 cells depleted of glutathione. J Biol Chem. 1997 Apr 11;272(15):9915–9921. doi: 10.1074/jbc.272.15.9915. [DOI] [PubMed] [Google Scholar]
  9. Kuo S. M., Morehouse H. F., Jr, Lin C. P. Effect of antiproliferative flavonoids on ascorbic acid accumulation in human colon adenocarcinoma cells. Cancer Lett. 1997 Jun 24;116(2):131–137. doi: 10.1016/s0304-3835(97)00183-3. [DOI] [PubMed] [Google Scholar]
  10. Lykkesfeldt J., Hagen T. M., Vinarsky V., Ames B. N. Age-associated decline in ascorbic acid concentration, recycling, and biosynthesis in rat hepatocytes--reversal with (R)-alpha-lipoic acid supplementation. FASEB J. 1998 Sep;12(12):1183–1189. doi: 10.1096/fasebj.12.12.1183. [DOI] [PubMed] [Google Scholar]
  11. May J. M., Qu Z. C., Whitesell R. R., Cobb C. E. Ascorbate recycling in human erythrocytes: role of GSH in reducing dehydroascorbate. Free Radic Biol Med. 1996;20(4):543–551. doi: 10.1016/0891-5849(95)02130-2. [DOI] [PubMed] [Google Scholar]
  12. Mendiratta S., Qu Z. C., May J. M. Enzyme-dependent ascorbate recycling in human erythrocytes: role of thioredoxin reductase. Free Radic Biol Med. 1998 Jul 15;25(2):221–228. doi: 10.1016/s0891-5849(98)00060-4. [DOI] [PubMed] [Google Scholar]
  13. Meredith M. J., Reed D. J. Status of the mitochondrial pool of glutathione in the isolated hepatocyte. J Biol Chem. 1982 Apr 10;257(7):3747–3753. [PubMed] [Google Scholar]
  14. Mitchell J. B., Russo A., Biaglow J. E., McPherson S. Cellular glutathione depletion by diethyl maleate or buthionine sulfoximine: no effect of glutathione depletion on the oxygen enhancement ratio. Radiat Res. 1983 Nov;96(2):422–428. [PubMed] [Google Scholar]
  15. Ngkeekwong F. C., Ng L. L. Two distinct uptake mechanisms for ascorbate and dehydroascorbate in human lymphoblasts and their interaction with glucose. Biochem J. 1997 May 15;324(Pt 1):225–230. doi: 10.1042/bj3240225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. PENNINGTON R. J. Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem J. 1961 Sep;80:649–654. doi: 10.1042/bj0800649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Park J. B., Levine M. Purification, cloning and expression of dehydroascorbic acid-reducing activity from human neutrophils: identification as glutaredoxin. Biochem J. 1996 May 1;315(Pt 3):931–938. doi: 10.1042/bj3150931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rafferty T. S., McKenzie R. C., Hunter J. A., Howie A. F., Arthur J. R., Nicol F., Beckett G. J. Differential expression of selenoproteins by human skin cells and protection by selenium from UVB-radiation-induced cell death. Biochem J. 1998 May 15;332(Pt 1):231–236. doi: 10.1042/bj3320231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sachi Y., Hirota K., Masutani H., Toda K., Okamoto T., Takigawa M., Yodoi J. Induction of ADF/TRX by oxidative stress in keratinocytes and lymphoid cells. Immunol Lett. 1995 Jan;44(2-3):189–193. doi: 10.1016/0165-2478(95)00213-o. [DOI] [PubMed] [Google Scholar]
  20. Salvemini F., Franzé A., Iervolino A., Filosa S., Salzano S., Ursini M. V. Enhanced glutathione levels and oxidoresistance mediated by increased glucose-6-phosphate dehydrogenase expression. J Biol Chem. 1999 Jan 29;274(5):2750–2757. doi: 10.1074/jbc.274.5.2750. [DOI] [PubMed] [Google Scholar]
  21. Savini I., D'Angelo I., Ranalli M., Melino G., Avigliano L. Ascorbic acid maintenance in HaCaT cells prevents radical formation and apoptosis by UV-B. Free Radic Biol Med. 1999 May;26(9-10):1172–1180. doi: 10.1016/s0891-5849(98)00311-6. [DOI] [PubMed] [Google Scholar]
  22. Schallreuter K. U., Pittelkow M. R., Wood J. M. Free radical reduction by thioredoxin reductase at the surface of normal and vitiliginous human keratinocytes. J Invest Dermatol. 1986 Dec;87(6):728–732. doi: 10.1111/1523-1747.ep12456848. [DOI] [PubMed] [Google Scholar]
  23. Siushansian R., Tao L., Dixon S. J., Wilson J. X. Cerebral astrocytes transport ascorbic acid and dehydroascorbic acid through distinct mechanisms regulated by cyclic AMP. J Neurochem. 1997 Jun;68(6):2378–2385. doi: 10.1046/j.1471-4159.1997.68062378.x. [DOI] [PubMed] [Google Scholar]
  24. Thomas D. R. Specific nutritional factors in wound healing. Adv Wound Care. 1997 Jul-Aug;10(4):40–43. [PubMed] [Google Scholar]
  25. Tsukaguchi H., Tokui T., Mackenzie B., Berger U. V., Chen X. Z., Wang Y., Brubaker R. F., Hediger M. A. A family of mammalian Na+-dependent L-ascorbic acid transporters. Nature. 1999 May 6;399(6731):70–75. doi: 10.1038/19986. [DOI] [PubMed] [Google Scholar]
  26. Vera J. C., Rivas C. I., Fischbarg J., Golde D. W. Mammalian facilitative hexose transporters mediate the transport of dehydroascorbic acid. Nature. 1993 Jul 1;364(6432):79–82. doi: 10.1038/364079a0. [DOI] [PubMed] [Google Scholar]
  27. Vera J. C., Rivas C. I., Velásquez F. V., Zhang R. H., Concha I. I., Golde D. W. Resolution of the facilitated transport of dehydroascorbic acid from its intracellular accumulation as ascorbic acid. J Biol Chem. 1995 Oct 6;270(40):23706–23712. doi: 10.1074/jbc.270.40.23706. [DOI] [PubMed] [Google Scholar]
  28. Washburn M. P., Wells W. W. Identification of the dehydroascorbic acid reductase and thioltransferase (Glutaredoxin) activities of bovine erythrocyte glutathione peroxidase. Biochem Biophys Res Commun. 1999 Apr 13;257(2):567–571. doi: 10.1006/bbrc.1999.0508. [DOI] [PubMed] [Google Scholar]
  29. Watt F. M., Jordan P. W., O'Neill C. H. Cell shape controls terminal differentiation of human epidermal keratinocytes. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5576–5580. doi: 10.1073/pnas.85.15.5576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wells W. W., Xu D. P., Yang Y. F., Rocque P. A. Mammalian thioltransferase (glutaredoxin) and protein disulfide isomerase have dehydroascorbate reductase activity. J Biol Chem. 1990 Sep 15;265(26):15361–15364. [PubMed] [Google Scholar]
  31. Will J. C., Ford E. S., Bowman B. A. Serum vitamin C concentrations and diabetes: findings from the Third National Health and Nutrition Examination Survey, 1988-1994. Am J Clin Nutr. 1999 Jul;70(1):49–52. doi: 10.1093/ajcn/70.1.49. [DOI] [PubMed] [Google Scholar]
  32. Winkler B. S., Orselli S. M., Rex T. S. The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective. Free Radic Biol Med. 1994 Oct;17(4):333–349. doi: 10.1016/0891-5849(94)90019-1. [DOI] [PubMed] [Google Scholar]
  33. Xu D. P., Wells W. W. alpha-Lipoic acid dependent regeneration of ascorbic acid from dehydroascorbic acid in rat liver mitochondria. J Bioenerg Biomembr. 1996 Feb;28(1):77–85. [PubMed] [Google Scholar]

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