Journal of Clinical Immunology

, Volume 31, Issue 1, pp 22–29 | Cite as

Immunochemical Studies on Catechol-Estrogen Modified Plasmid: Possible Role in Rheumatoid Arthritis

  • Wahid Ali KhanEmail author
  • Moinuddin
  • Abdullah S. Assiri



Increased concentrations of estrogen metabolites (catecholestrogens) have been found in rheumatoid arthritis (RA) but the exact patho-etiology remains elusive.


The binding of antibodies from the sera of RA patients and control subjects to native and modified DNA was studied by direct binding and inhibition ELISA, quantitative precipitin titration. Experimentally induced antibodies were also checked to detect oxidative lesions in the DNA as well as for the estimation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels in different fluids of RA.


Anti-DNA IgG from RA sera, exhibited increased recognition of modified DNA than native DNA (nDNA; P < 0.001). The relative affinity of anti-DNA antibodies for modified and nDNA was in the order of 1.85 × 10−7, 1.23 × 10−7, and 1.2 × 10−6. Samples of DNA from RA patients showed a significant inhibition in the induced antibody activity in comparison to DNA isolates from controls (P < 0.001). The concentration of 8-OHdG evaluated by induced antibody in RA patients was found to be significantly higher than controls ((P < 0.0001, P < 0.01, P < 0.05).


High binding of modified DNA with the IgG from RA patient might explain possible antigenic role of 4-OHE2-modified DNA in the production of anti-DNA antibodies. In addition, the induced antibodies have been shown to represent an alternative immunochemical probe to detect oxidative lesions in DNA as well as for the estimation of 8-OHdG levels in different body fluid of RA patients, which may be used as marker in the diagnosis of the disease.


4-Hydroxyestradiol 8-OHdG antibodies rheumatoid arthritis ELISA 



The authors thank Dr. H S Chabbra for his help and support. We are greatly thankful to him for providing lab facility and constructive discussions during this work.


  1. 1.
    Komi J, Lassila O. Nonsteroidal anti-estrogens inhibit the functional differentiation of human monocyte-derived dendritic cells. Blood. 2000;95:2875–82.PubMedGoogle Scholar
  2. 2.
    Whitacre CC. Sex differences in autoimmune disease. Nat Immunol. 2001;2:777–80.CrossRefPubMedGoogle Scholar
  3. 3.
    Han X, Lundberg P, Tanamachi B, Openshaw H, Longmate J, Cantin E. Gender influences herpes simplex virus type 1 infection in normal and gamma interferon-mutant mice. J Virol. 2001;75:3048–52.CrossRefPubMedGoogle Scholar
  4. 4.
    Kanda N, Tsuchida T, Tamaki K. Estrogen enhancement of anti-double-stranded DNA antibody and immunoglobulin G production in peripheral blood mononuclear cells from patients with systemic lupus erythematosus. Arthritis Rheum. 1999;42:328–37.CrossRefPubMedGoogle Scholar
  5. 5.
    Bauerova K, Bezek A. Role of reactive oxygen and nitrogen species in etiopathogenesis rheumatoid arthritis. Gen Physiol Biophys. 1999;18:15–20.PubMedGoogle Scholar
  6. 6.
    Wanchu A, Agnihotri N, Deodhar SD, Ganguly NK. Plasma reactive nitrogen intermediate levels in patients with clinically active rheumatoid arthritis. Indian J Med Res. 1996;104:263–8.PubMedGoogle Scholar
  7. 7.
    Sakurai H, Kohsaka H, Liu MF, Higashiyam H, Hirata Y, Kanno K, et al. Nitric oxide production and inducible nitric oxide synthase expression in inflammatory arthritis. J Clin Invest. 1995;96:2357–63.CrossRefPubMedGoogle Scholar
  8. 8.
    Weidler C, Harle P, Schedel J, Schmidt M, Scholmerich J, Straub RH. Patients with rheumatoid arthritis and systemic lupus erythematosus have increased renal excretion of mitogenic estrogen in relation to endogenous antiestrogens. J Rheumatol. 2004;31:489–94.PubMedGoogle Scholar
  9. 9.
    Castagnetta L, Cutolo M, Granata O, Di Falco M, Bellavia V, Carruba G. Endocrine end points in rheumatoid arthritis. Ann NY Acad Sci. 1999;876:180–92.CrossRefPubMedGoogle Scholar
  10. 10.
    Castagnetta L, Carruba G, Granata OM, Stefano R, Miele M, Schmidt M, et al. Increased estrogen formation and estrogen to androgen ratio in the SF of patients with rheumatoid arthritis. J Rheumatol. 2003;30:2597–605.PubMedGoogle Scholar
  11. 11.
    Khan WA, Moinuddin. Binding characteristics of SLE anti-DNA autoantibodies to catecholestrogen-modified DNA. Scand J Immunol. 2006;64:677–83.CrossRefPubMedGoogle Scholar
  12. 12.
    Khan WA, Alam K, Moinuddin. Catechol-estrogen modified DNA: a better antigen for cancer autoantibody. Arch Biochem Biophys. 2007;465:293–300.CrossRefPubMedGoogle Scholar
  13. 13.
    Khan WA, Moinuddin, Khan MWA, Chabbra HS. Catecholestrogen: possible role in systemic lupus erythematosus. Rheumatol (Oxford). 2009;48:1345–51.CrossRefGoogle Scholar
  14. 14.
    Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 1988;31:315–24.CrossRefPubMedGoogle Scholar
  15. 15.
    Bonvoisin B, Cordier G, Revillard JP, Lejeune E, Bouvier M. Increased DNA and/or RNA content of synovial fluid cells in rheumatoid arthritis: a flow cytometry study. Ann Rheum Dis. 1984;43:222–7.CrossRefPubMedGoogle Scholar
  16. 16.
    Ashok BT, Ali R. Binding of human anti-DNA autoantibodies to reactive oxygen species modified-DNA and probing oxidative DNA damage in cancer using monoclonal antibodies. Int J Cancer. 1998;78:404–9.CrossRefPubMedGoogle Scholar
  17. 17.
    Waris G, Alam K. Immunogenicity of superoxide modified-DNA: studies on induced antibodies and SLE anti-DNA antibodies. Life Sci. 2004;75:2633–42.CrossRefPubMedGoogle Scholar
  18. 18.
    Dixit K, Ali R. Role of nitric oxide modified DNA in the etiopathogenesis of systemic lupus erythematosus. Lupus. 2004;13:95–100.CrossRefPubMedGoogle Scholar
  19. 19.
    Khan WA, Habib S, Khan MWA, Alam K, Moinuddin. Enhanced binding of circulating SLE autoantibodies to catecholestrogen-modified DNA. Mol Cell Biochem. 2008;315:143–50.CrossRefPubMedGoogle Scholar
  20. 20.
    Burton K. A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956;62:315–23.PubMedGoogle Scholar
  21. 21.
    Bradford MM. A rapid and sensitive method for quantitation of micrograms quantity of protein utilizing the principle of protein dye binding. Anal Biochem. 1976;72:248–54.CrossRefPubMedGoogle Scholar
  22. 22.
    Langmuir I. The adsorption of gases on plane surface glass, mica and platinum. J Am Chem Soc. 1918;40:1361–403.CrossRefGoogle Scholar
  23. 23.
    Osawa T, Yoshida A, Kawakishi S, Yamashita K, Ochi H, Cutler RG. Protective role of dietary antioxidants in oxidative stress. In: Culter RG, Packer L, Bertram J, Mori A, editors. Oxidative stress and aging. Basel: Berkhauser; 1995. p. 367–77.Google Scholar
  24. 24.
    Toyokuni S, Tanaka T, Hattori Y, Nishiyama Y, Yashida A, Uchida K, et al. Quantitative immunohistochemical determination of 8-hydroxy-2′-deoxyguanosine by a monoclonal antibody N45.1: its application to ferric nitrilotriacetate-induced renal carcinogenesis model. Lab Invest. 1997;76:365–74.PubMedGoogle Scholar
  25. 25.
    Cutolo M, Villagio B, Seriolo B, Montagna P, Camopellino S, Straub RH, et al. Synovial fluid estrogens in rheumatoid arthritis. Autoimmun Rev. 2004;3:193–8.CrossRefPubMedGoogle Scholar
  26. 26.
    Cutolo M, Villaggio B, Craviotto C, Pizzorni C, Seriolo B, Sulli A. Sex hormones and rheumatoid arthritis. Autoimmun Rev. 2002;1:284–9.CrossRefPubMedGoogle Scholar
  27. 27.
    Straub RH, Cutolo M. Involvement of the hypothalamic-pituitary-adrenal/gonadal axis and the peripheral nervous system in rheumatoid arthritis: viewpoint based on a systemic pathogenetic role. Arthritis Rheum. 2001;44:493–507.CrossRefPubMedGoogle Scholar
  28. 28.
    Zutshi DW, Reading CA, Epstein WV, Ansell BM, Holborow EJ. FII haemagglutination test for serum antigammaglobulin factors in arthritides sero-positive and sero-negative by other tests. Ann Rheum Dis. 1969;28:289–99.CrossRefPubMedGoogle Scholar
  29. 29.
    Morgan K, Clague RB, Collins I, Ayad S, Phinn SD, Holt PJ. Incidence of antibodies to native and denatured cartilage collagens (types II, IX, and XI) and to type I collagen in rheumatoid arthritis. Ann Rheum Dis. 1987;46:902–7.CrossRefPubMedGoogle Scholar
  30. 30.
    George J, Shoenfeld Y. Natural autoantibodies. In: Peter JB, Shoenfeld Y, editors. autoantibodies. Amsterdam: Elsevier Scientific; 1996. p. 534–9.CrossRefGoogle Scholar
  31. 31.
    Yung RL, Richardson BC. Drug-induced lupus. Rheum Dis Clin North Am. 1994;20:61–86.PubMedGoogle Scholar
  32. 32.
    Graninger WB, Hassfeld W, Pesan BB, Machold KP, Zielinski LL, Smolen JS. Induction of systemic lupus erythematosus by interferon-γ in a patient with rheumatoid arthritis. J Rheumatol. 1991;18:1621–2.PubMedGoogle Scholar
  33. 33.
    Nutter LM, Wu YY, Ngo EO, Sierra EE, Abdul-Hajj YJ. An o-quinone form of estrogen produces free radicals in human breast cancer cells: correlation with DNA damage. Chem Res Toxicol. 1994;7:23–8.CrossRefPubMedGoogle Scholar
  34. 34.
    Zhu BT, Conney AH. Functional role of estrogen metabolism in target cells: review and perspectives. Carcinogenesis. 1998;19:1–27.CrossRefPubMedGoogle Scholar
  35. 35.
    Kamanli A, Naziroglu M, Aydile K, Hacievliyagil C. Plasma lipid peroxidation and antioxidant levels in patients with rheumatoid arthritis. Cell Biochem Funct. 2004;22:53–7.CrossRefPubMedGoogle Scholar
  36. 36.
    Bazzichi L, Ciompi ML, Betti L, Rossi A, Melchiorre D, Fiorini M, et al. Impaired glutathione reductase activity and level of collogenase and elastase in synovial fluid in rheumatoid arthritis. Clin Exp Rheumatol. 2002;20:761–6.PubMedGoogle Scholar
  37. 37.
    De Leo ME, Tranghese A, Passantino M, Mordente A, Lizzio MM, Galeotti T, et al. Manganese superoxide dismutase, glutathione peroxidase, and total radical trapping antioxidant capacity in active rheumatoid arthritis. J Rheumatol. 2002;29:2245–6.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Wahid Ali Khan
    • 1
    Email author
  • Moinuddin
    • 2
  • Abdullah S. Assiri
    • 3
  1. 1.Department of Clinical Biochemistry, College of MedicineKing Khalid UniversityAbhaKingdom of Saudi Arabia
  2. 2.Department of Biochemistry, Faculty of MedicineA.M.U.AligarhIndia
  3. 3.College of MedicineKing Khalid UniversityAbhaKingdom of Saudi Arabia

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