, Volume 24, Issue 6, pp 1123–1131 | Cite as

Effect of diquat-induced oxidative stress on iron metabolism in male Fischer-344 rats

  • Masashi Higuchi
  • Yasunaga Yoshikawa
  • Koichi Orino
  • Kiyotaka Watanabe


Diquat toxicity causes iron-mediated oxidative stress; however, it remains unclear how diquat affects iron metabolism. Here, we examined the effect of diquat-induced oxidative stress on iron metabolism in male Fischer-344 rats, with particular focus on gene expression. Hepatic nonheme iron content was unchanged until 20 h after diquat treatment. Hepatic free iron levels increased markedly in the early stages following treatment and remained elevated for at least 6 h, resulting in severe hepatotoxicity, until returning to control levels at 20 h. The level of hepatic ferritin, especially the H-subunit, increased 20 h after diquat treatment due to elevated hepatic ferritin-H mRNA expression. These results indicate that early elevated levels of free iron in the liver of diquat-treated rats cause hepatotoxicity, and that this free iron is subsequently sequestered by ferritin synthesized under conditions of oxidative stress, thus limiting the pro-oxidant challenge of iron. The plasma iron concentration decreased at 6 and 20 h after diquat treatment, whereas the level of plasma interleukin-6 increased markedly at 3 h and remained high until 20 h. In the liver of diquat-treated rats, expression of hepcidin mRNA was markedly upregulated at 3 and 6 h, whereas ferroportin mRNA expression was downregulated slightly at 20 h. Transferrin receptor 1 mRNA expression was significantly upregulated at 3, 6, and 20 h. These results indicate that inhibition of iron release from iron-storage tissues, through stimulation of the interleukin-6-hepcidin-ferroportin axis, and enhanced iron uptake into hepatocytes, mediated by transferrin receptor 1, cause hypoferremia.


Diquat Iron metabolism Oxidative stress Rat 


  1. Abe T, Kinda T, Takano Y, Chikazawa S, Higuchi M, Kawasaki N, Orino K, Watanabe K (2006) Relationship between body iron stores and diquat toxicity in male Fischer-344 rats. Biometals 19:651–657PubMedCrossRefGoogle Scholar
  2. Anderson GJ, Powell LW (2002) HFE and non-HFE hemochromatosis. Int J Hematol 76:203–207PubMedCrossRefGoogle Scholar
  3. Andrews SC, Arosio P, Bottke W, Briat JF, von Darl M, Harrison PM, Laulhere JP, Levi S, Lobreaux S, Yewdall SJ (1992) Structure, function, and evolution of ferritins. J Inorg Biochem 47:161–174PubMedCrossRefGoogle Scholar
  4. Canonne-Hergaux F, Donovan A, Delaby C, Wang HJ, Gros P (2006) Comparative studies of duodenal and macrophage ferroportin proteins. Am J Physiol Gastrointest Liver Physiol 290:156–163CrossRefGoogle Scholar
  5. Collawn JF, Stangel M, Kuhn LA, Esekogwu V, Jing S, Trowbridge IS, Tainer JA (1990) Transferrin receptor internalization sequence YXRF implicates a tight turn as the structural recognition motif for endocytosis. Cell 63:1061–1072PubMedCrossRefGoogle Scholar
  6. Donovan A, Brownlie A, Zhou Y, Shepard J, Pratt SJ, Moynihan J, Paw BH, Drejer A, Barut B, Zapata A, Law TC, Brugnara C, Lux SE, Pinkus GS, Pinks JL, Kingsley PD, Palis J, Fleming MD, Andrews NC, Zon LI (2000) Positional cloning of zebrafish ferroportin 1 identifies a conserved vertebrate iron exporter. Nature 403:776–781PubMedCrossRefGoogle Scholar
  7. Esposito BP, Epsztejn S, Breuer W, Cabantchik ZI (2002) A review of fluorescence methods for assessing labile iron in cells and biological fluids. Anal Biochem 304:1–18PubMedCrossRefGoogle Scholar
  8. Ford GC, Harrison PM, Rice DW, Smith JMA, Treffry A, White JL, Yariv J (1984) Ferritin: design and formation of an iron-storage molecule. Philos Trans R Soc Lond B Biol Sci 304:551–565PubMedCrossRefGoogle Scholar
  9. Frazer DM, Wilkins SJ, Becker EM, Murphy TL, Vulpe CD, Mckie AT, Anderson GJ (2003) A rapid decrease in the expression of DMT1 and Dcytb but not Ireg1 or hephaestin explains the mucosal block phenomenon of iron absorption. Gut 52:340–346PubMedCrossRefGoogle Scholar
  10. Ganz T (2003) Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation. Blood 102:783–788PubMedCrossRefGoogle Scholar
  11. Ganz T (2005) Hepcidin: a regulator of intestinal iron absorption and iron recycling by macrophages. Best Pract Res Clin Haematol 18:171–182PubMedCrossRefGoogle Scholar
  12. Harrison PM, Arosio P (1996) The ferritins: molecular properties, iron storage function and cellular regulation. Biochim Biophys Acta 1275:161–203PubMedCrossRefGoogle Scholar
  13. Hentze MW, Kuhn LC (1996) Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc Natl Acad Sci USA 93:8175–8182PubMedCrossRefGoogle Scholar
  14. Hentze MW, Muckenthaler MU, Andrews NC (2004) Balancing acts: molecular control of mammalian iron metabolism. Cell 117:285–297PubMedCrossRefGoogle Scholar
  15. Hershko C, Graham G, Bates GW, Rachmilewitz EA (1978) Non-specific serum iron in thalassaemia: an abnormal serum iron fraction of potential toxicity. Br J Haematol 40:255–263PubMedCrossRefGoogle Scholar
  16. Higuchi M, Kobayashi S, Kawasaki N, Hamaoka K, Watabiki S, Orino K, Watanabe K (2007) Protective effects of wheat bran against diquat-induced oxidative stress in male Fischer-344 rats. Biosci Biotechnol Biochem 71:1621–1625PubMedCrossRefGoogle Scholar
  17. Higuchi M, Oshida J, Orino K, Watanabe K (2011) Wheat bran protects Fischer-344 rats from diquat-induced oxidative stress by activating antioxidant system: selenium as an antioxidant. Biosci Biotechnol Biochem 75:496–499PubMedCrossRefGoogle Scholar
  18. Jones GM, Vale JA (2000) Mechanism of toxicity, clinical features, and management of diquat poisoning: a review. Clin Toxicol 38:123–128CrossRefGoogle Scholar
  19. Kawabata H, Yang R, Hirama T, Vuong PT, Kawano S, Gombart AF, Koeffler HP (1999) Molecular cloning of transferrin receptor 2 A new member of the transferrin receptor-like family. J Biol Chem 274:20826–20832PubMedCrossRefGoogle Scholar
  20. Kawabata H, Germain RS, Vuong PT, Nakamaki T, Said JW, Koeffler HP (2000) Transferrin receptor 2-α supports cell growth both in iron-chelated cultured cells and in vivo. J Biol Chem 275:16618–16625PubMedCrossRefGoogle Scholar
  21. Kemna EH, Tjalsma H, Willems HL, Swinkels DW (2008) Hepcidin: from discovery to differential diagnosis. Haematologica 93:90–97PubMedCrossRefGoogle Scholar
  22. Kobune M, Kohgo Y, Kato J, Miyazaki E, Niitsu Y (1994) Interleukin-6 enhances hepatic transferrin uptake and ferritin expression in rats. Hepatology 19:1468–1475PubMedCrossRefGoogle Scholar
  23. Kozlov AV, Bini A, Gallesi D, Giovannini F, Iannone A, Masini A, Meletti E, Tomasi A (1996) ‘Free’ iron, as detected by electron paramagnetic resonance spectroscopy, increases unequally in different tissues during dietary iron overload in the rat. Biometals 9:98–103PubMedCrossRefGoogle Scholar
  24. Krause A, Neitz S, Magert H, Shulx A, Forssmann W, Shulz-Knappe P, Adermann K (2000) LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett 480:147–150PubMedCrossRefGoogle Scholar
  25. Levi S, Luzzago A, Cesareni G, Cozzi A, Franceshinelli F, Albertini A, Arosio P (1988) Mechanism of ferritin iron uptake: activity of the H-chain and deletion mapping of the ferro-oxidase site A study of iron uptake and ferro-oxidase activity of human liver recombinant H-chain ferritins, and of two H-chain deletion mutants. J Biol Chem 263:18086–18092PubMedGoogle Scholar
  26. Levi S, Salfeld J, Franceshinelli F, Cozzi A, Dorner MH, Arosio P (1989) Expression and structural and functional properties of human ferritin L-chain from Escherichia coli. Biochemistry 28:5179–5184PubMedCrossRefGoogle Scholar
  27. Lieu PT, Heiskala M, Peterson PA, Yang Y (2001) The roles of iron in health and disease. Mol Aspect Med 22:1–87CrossRefGoogle Scholar
  28. McGrath H Jr, Rigby PG (2004) Hepcidin: inflammation’s iron curtain. Rheumatology 43:1323–1325PubMedCrossRefGoogle Scholar
  29. Means RT (2004) Hepcidin and cytokines in anemia. Hematology 9:357–362PubMedCrossRefGoogle Scholar
  30. Nemeth E, Rivera S, Gabayan V, Keller C, Taudorf S, Pedersen BK, Ganz T (2004a) IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J Clin Invest 113:1271–1276PubMedGoogle Scholar
  31. Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, Ganz T, Kaplan J (2004b) Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science 306:2090–2093PubMedCrossRefGoogle Scholar
  32. Nicolas G, Bennoun M, Devaux I, Beaumont C, Grandchamp B, Kahn A, Vaulont S (2001) Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice. Proc Natl Acad Sci USA 98:8780–8785PubMedCrossRefGoogle Scholar
  33. Orino K, Harada S, Natsuhori M, Takehara K, Watanabe K (2004) Kinetic analysis of bovine spleen apoferritin and recombinant H and L chain homopolymers: iron uptake suggests early stage H chain ferroxidase activity and second stage L chain cooperation. Biometals 17:129–134PubMedCrossRefGoogle Scholar
  34. Petry TW, Wolfgang GH, Jolly RA, Ochoa R, Donarski WJ (1992) Antioxidant-dependent inhibition of diquat-induced toxicity in vivo. Toxicology 74:33–43PubMedCrossRefGoogle Scholar
  35. Pigeon C, Ilynin G, Courselaud B, Leroyer P, Turlin B, Brissot P, Loreal O (2000) A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem 276:7811–7819PubMedCrossRefGoogle Scholar
  36. Ramey G, Deschemin JC, Durel B, Canonne-Hergaux F, Nicolas G (2010) Hepcidin targets ferroportin for degradation in hepatocytes. Haematologica 95:501–504PubMedCrossRefGoogle Scholar
  37. Reif DW, Beales ILP, Thomas CE, Aust SD (1988) Effect of diquat on the distribution of iron in rat liver. Toxicol Appl Pharmacol 93:506–510PubMedCrossRefGoogle Scholar
  38. Rikans LE, Ardinska V, Hornbrook KR (1997) Age-associated increase in ferritin content of male rat liver: implication for diquat-mediated oxidative injury. Arch Biochem Biophys 344:85–93PubMedCrossRefGoogle Scholar
  39. Rivera S, Nemeth E, Gabayan V, Lopez MA, Farshidi D, Ganz T (2005) Synthetic hepcidin causes rapid dose-dependent hypoferremia and is concentrated in ferroportin-containing organs. Blood 106:2196–2199PubMedCrossRefGoogle Scholar
  40. Rogers JT (1996) Ferritin translation by interleukin-1 and interleukin-6: the role of sequences upstream of the start codons of the heavy and light subunit genes. Blood 87:2525–2537PubMedGoogle Scholar
  41. Schägger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379PubMedCrossRefGoogle Scholar
  42. Smith CV (1987) Evidence for participation of lipid peroxidation and iron in diquat-induced hepatic necrosis in vivo. Mol Pharmacol 32:417–422PubMedGoogle Scholar
  43. Smith CV, Hughes H, Lauterburg BH, Mitchell JR (1985) Oxidant stress and hepatic necrosis in rats treated with diquat. J Pharmacol Exp Ther 235:172–177PubMedGoogle Scholar
  44. Stookey LL (1970) Ferrozine-a new spectrophotometric reagent for iron. Anal Chem 42:779–781CrossRefGoogle Scholar
  45. Theil EC (1987) Ferritin: structure, gene regulation, and cellular function in animals, plants, and microorganisms. Annu Rev Biochem 56:289–315PubMedCrossRefGoogle Scholar
  46. Thomas CE, Aust SD (1986) Reductive release of iron from ferritin by cation free radicals of paraquat and other bipyridyls. J Biol Chem 261:13064–13070PubMedGoogle Scholar
  47. Tomosugi N, Kawabata H, Wakatabe R, Higuchi M, Yamaya H, Umehara H, Ishikawa I (2006) Detection of serum hepcidin in renal failure and inflammation by using ProteinChip system. Blood 108:1381–1387PubMedCrossRefGoogle Scholar
  48. Tsuji Y, Ayaki H, Whitman SP, Morrow CS, Torti SV, Torti FM (2000) Coordinate transcriptional and translational regulation of ferritin in response to oxidative stress. Mol Cell Biol 20:5818–5827PubMedCrossRefGoogle Scholar
  49. Watanabe K, Hayashi K, Miyamoto T, Tanaka M, Okano S, Yamamoto S (2000) Characterization of ferritin and ferritin-binding proteins in canine serum. Biometals 13:57–63PubMedCrossRefGoogle Scholar
  50. Watt RK (2011) The many faces of the octahedral ferritin protein. Biometals 24:489–500PubMedCrossRefGoogle Scholar
  51. Weiss G (2005) Modification of iron regulation by the inflammatory response. Best Pract Res Clin Haematol 18:183–201PubMedCrossRefGoogle Scholar
  52. Wrighting DM, Andrews NC (2006) Interleukin-6 induces hepcidin expression through STAT3. Blood 108:3204–3209PubMedCrossRefGoogle Scholar
  53. Zhang D, Okada S, Kawabata T, Yasuda T (1995) An improved simple colorimetric method for quantitation of non-transferrin-bound iron in serum. Biochem Mol Biol Int 35:635–641PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Masashi Higuchi
    • 1
  • Yasunaga Yoshikawa
    • 1
  • Koichi Orino
    • 1
  • Kiyotaka Watanabe
    • 1
  1. 1.Laboratory of Veterinary Biochemistry, School of Veterinary MedicineKitasato UniversityTowadaJapan

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