Histochemistry and Cell Biology

, Volume 130, Issue 5, pp 967–977 | Cite as

A long-term study on female mice fed on a genetically modified soybean: effects on liver ageing

  • Manuela MalatestaEmail author
  • Federica Boraldi
  • Giulia Annovi
  • Beatrice Baldelli
  • Serafina Battistelli
  • Marco Biggiogera
  • Daniela Quaglino
Original Paper


Liver represents a suitable model for monitoring the effects of a diet, due to its key role in controlling the whole metabolism. Although no direct evidence has been reported so far that genetically modified (GM) food may affect health, previous studies on hepatocytes from young female mice fed on GM soybean demonstrated nuclear modifications involving transcription and splicing pathways. In this study, the effects of this diet were studied on liver of old female mice in order to elucidate possible interference with ageing. The morpho-functional characteristics of the liver of 24-month-old mice, fed from weaning on control or GM soybean, were investigated by combining a proteomic approach with ultrastructural, morphometrical and immunoelectron microscopical analyses. Several proteins belonging to hepatocyte metabolism, stress response, calcium signalling and mitochondria were differentially expressed in GM-fed mice, indicating a more marked expression of senescence markers in comparison to controls. Moreover, hepatocytes of GM-fed mice showed mitochondrial and nuclear modifications indicative of reduced metabolic rate. This study demonstrates that GM soybean intake can influence some liver features during ageing and, although the mechanisms remain unknown, underlines the importance to investigate the long-term consequences of GM-diets and the potential synergistic effects with ageing, xenobiotics and/or stress conditions.


Ageing Cell nucleus Genetically modified soybean Liver Mitochondria 



We thank M. Storaci of the Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche for his assistance in mouse breeding. This work was supported by the Agenzia Servizi Settore Agroalimentare delle Marche, Italy.


  1. Anantharaju A, Feller A, Chedid A (2002) Aging liver. Gerontology 48:343–353PubMedCrossRefGoogle Scholar
  2. Bernhard W (1969) A new staining procedure for electron microscopical cytology. J Ultrastruct Res 27:250–265PubMedCrossRefGoogle Scholar
  3. Bjellqvist B, Pasquali C, Ravier F, Sanchez JC, Hochstrasser D (1993) A nonlinear wide-range immobilized pH gradient for two-dimensional electrophoresis and its definition in a relevant pH scale. Electrophoresis 14:1357–1365PubMedCrossRefGoogle Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  5. Carreras MC, Poderoso JJ (2007) Mitochondrial nitric oxide in the signaling of cell integrated responses. Am J Physiol Cell Physiol 292:C1569–1580PubMedCrossRefGoogle Scholar
  6. Dai G, He L, Chou N, Wan YJ (2006) Acetaminophen metabolism does not contribute to gender difference in its hepatotoxicity in mouse. Toxicol Sci 92:33–41PubMedCrossRefGoogle Scholar
  7. Doull J, Gaylor D, Greim HA, Lovell DP, Lynch B, Munro IC (2007) Report of an Expert Panel on the reanalysis by Séralini of a 90-day study conducted by Monsanto in support of the safety of a genetically modified corn variety (MON 863). Food Chem Toxicol 45:2073–2085PubMedCrossRefGoogle Scholar
  8. Durante W, Johnson FK, Johnson RA (2007) Arginase: a critical regulator of nitric oxide synthesis and vascular function. Clin Exp Pharmacol Physiol 34:906–911PubMedCrossRefGoogle Scholar
  9. Ewen SW, Pustzai A (1999) Effects of diets containing genetically modified potatoes expressing Galanthus nivalis lecitin on rat small intestine. Lancet 354:1353–1354PubMedCrossRefGoogle Scholar
  10. Fakan S (2004) Ultrastructural cytochemical analyses of nuclear functional architecture. Eur J Histochem 48:5–14PubMedGoogle Scholar
  11. Frasca D, Nguyen D, Riley RL, Blomberg BB (2003) Decreased E12 and/or E47 transcription factor activity in the bone marrow as well as in the spleen of aged mice. J Immunol 170:719–726PubMedGoogle Scholar
  12. Fujita T (1999) Senescence marker protein-30 (SMP30): structure and biological function. Biochem Biophys Res Commun 254:1–4PubMedCrossRefGoogle Scholar
  13. Fukaya Y, Yamaguchi M (2004) Regucalcin increases superoxide dismutase activity in rat liver cytosol. Biol Pharm Bull 27:1444–1446PubMedCrossRefGoogle Scholar
  14. Galy V, Olivo-Marin JC, Scherthan H, Doye V, Rascalou N, Nehrbass U (2000) Nuclear pore complexes in the organization of silent telomeric chromatin. Nature 403:108–112PubMedCrossRefGoogle Scholar
  15. Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM (1999) Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 20:601–605PubMedCrossRefGoogle Scholar
  16. Gilca M, Stoian I, Atanasiu V, Virgolici B (2007) The oxidative hypothesis of senescence. J Postgrad Med 53:207–213PubMedCrossRefGoogle Scholar
  17. Granby K, Johannesen S, Vahl M (2003) Analysis of glyphosate residues in cereals using liquid chromatography–mass spectrometry. Food Addit Contam 20:692–698PubMedCrossRefGoogle Scholar
  18. Hallen A (2002) Accumulation of insoluble protein and ageing. Biogerontology 3:307–315PubMedCrossRefGoogle Scholar
  19. Hildebrand R (1980) Nuclear volume and cellular metabolism. Adv Anat Embryol Cell Biol 60:1–54PubMedGoogle Scholar
  20. Hochstrasser DF, Patchornik A, Merril CR (1988) Development of polyacrylamide gels that improve the separation of proteins and their detection by silver staining. Anal Biochem 173:412–423PubMedCrossRefGoogle Scholar
  21. Honore B, Ostergaard M, Vorum H (2004) Functional genomics studied by proteomics. Bioessays 26:901–915PubMedCrossRefGoogle Scholar
  22. Jameson CW (2004) Towards a unified and interdisciplinary model of ageing. Med Hypotheses 63:83–86PubMedCrossRefGoogle Scholar
  23. Justo R, Boada J, Frontera M, Oliver J, Bermúdez J, Gianotti M (2005) Gender dimorphism in rat liver mitochondrial oxidative metabolism and biogenesis. Am J Physiol Cell Physiol 289:C372–378PubMedCrossRefGoogle Scholar
  24. Kato K, Giulivi C (2006) Critical overview of mitochondrial nitric-oxide synthase. Front Biosci 11:2725–2738PubMedCrossRefGoogle Scholar
  25. Kuiper HA, König A, Kleter GA, Hammes WP, Knudsen I, European Network on Safety Assessment of Genetically Modified Food Crops (ENTRANSFOOD) (2004) Safety assessment, detection and traceability, and societal aspects of genetically modified foods. European Network on Safety Assessment of Genetically Modified Food Crops (ENTRANSFOOD). Food Chem Toxicol 42:1195–1202PubMedCrossRefGoogle Scholar
  26. Kumar AN, Kalyankar GD (1984) Effect of steroid hormones on age dependent changes in rat arginase isoenzymes. Exp Gerontol 19:191–198PubMedCrossRefGoogle Scholar
  27. Lafarga M, Berciano MT, Andres MA (1993) Protein-synthesis inhibition induces perichromatin granule accumulation and intranuclear rodlet formation in osmotically stimulated supraoptic neurons. Anat Embryol 187:363–369PubMedCrossRefGoogle Scholar
  28. Lin G, Cui YY, Liu XQ (2003) Gender differences in microsomal metabolic activation of hepatotoxic clivorine in rat. Chem Res Toxicol 16:768–774PubMedCrossRefGoogle Scholar
  29. Malatesta M, Bertoni-Freddari C, Fattoretti P, Baldelli B, Fakan S, Gazzanelli G (2004) Aging and vitamin E deficiency are responsible for altered RNA pathways. Ann N Y Acad Sci 1019:379–382PubMedCrossRefGoogle Scholar
  30. Malatesta M, Bertoni-Freddari C, Fattoretti P, Caporaloni C, Fakan S, Gazzanelli G (2003a) Altered RNA structural constituents in aging and vitamin E deficiency. Mech Ageing Dev 124:175–181PubMedCrossRefGoogle Scholar
  31. Malatesta M, Biggiogera M, Manuali E, Rocchi MBL, Baldelli B, Gazzanelli G (2003b) Fine structural analyses of pancreatic acinar cell nuclei from mice fed on GM soybean. Eur J Histochem 47:385–388PubMedGoogle Scholar
  32. Malatesta M, Caporaloni C, Gavaudan S, Rocchi MBL, Tiberi C, Gazzanelli G (2002a) Ultrastructural morphometrical and immunocytochemical analyses of hepatocyte nuclei from mice fed on genetically modified soybean. Cell Struct Funct 27:173–180PubMedCrossRefGoogle Scholar
  33. Malatesta M, Caporaloni C, Rossi L, Battistelli S, Rocchi MBL, Tonucci F, Gazzanelli G (2002b) Ultrastructural analysis of pancreatic acinar cells from mice fed on genetically modified soybean. J Anat 201:409–416PubMedCrossRefGoogle Scholar
  34. Malatesta M, Mannello F, Sebastiani M, Cardinali A, Marcheggiani F, Renò F, Gazzanelli G (1998) Ultrastructural characterization and biochemical profile of human gross cystic breast disease. Breast Cancer Res Treat 48:211–219PubMedCrossRefGoogle Scholar
  35. Malatesta M, Tiberi C, Baldelli B, Battistelli S, Manuali E, Biggiogera B (2005) Reversibility of hepatocyte nuclear modifications in mice fed on genetically modified soybean. Eur J Histochem 49:237–242PubMedGoogle Scholar
  36. Marc J, Le Breton M, Cormier P, Morales J, Belle R, Mulner-Lorillon O (2005) A glyphosate-based pesticide impinges on transcription. Toxicol Appl Pharmacol 203:1–8PubMedCrossRefGoogle Scholar
  37. Molloy MP, Brzezinski EE, Hang J, McDowell MT, VanBogelen RA (2003) Overcoming technical variation and biological variation in quantitative proteomics. Proteomics 3:1912–1919PubMedCrossRefGoogle Scholar
  38. Morris ME, Lee HJ, Predko LM (2003) Gender differences in the membrane transport of endogenous and exogenous compounds. Pharmacol Rev 55:229–240PubMedCrossRefGoogle Scholar
  39. Multhoff G (2006) Heat shock proteins in immunity. Handb Exp Pharmacol 172:279–304PubMedCrossRefGoogle Scholar
  40. Numao N, Masuda H, Sakai Y, Okada Y, Kihara K, Azuma H (2007) Roles of attenuated neuronal nitric-oxide synthase protein expression and accelerated arginase activity in impairing neurogenic relaxation of corpus cavernosum in aged rabbits. BJU Int 99:1495–1499PubMedCrossRefGoogle Scholar
  41. Padgette SR, Kolacz KH, Delannay X, Re DB, LaVallee BJ, Tinius CN, Rhodes WK, Otero YI, Barry GF, Eichholtz DA, Peschke VM, Nida DL, Taylor NB, Kishore GM (1995) Development, identification and characterization of a glyphosate-tolerant soybean line. Crop Sci 35:1451–1461Google Scholar
  42. Pastorelli R, Carpi D, Campagna R, Airoldi L, Pohjanvirta R, Viluksela M, Hakansson H, Boutros PC, Moffat ID, Okey AB, Fanelli R (2006) Differential expression profiling of the hepatic proteome in a rat model of dioxin resistance: correlation with genomic and transcriptomic analyses. Mol Cell Proteomics 5:882–894PubMedCrossRefGoogle Scholar
  43. Patel NN, Crincoli CM, Kennedy EL, Frederick DM, Tchao R, Harvison PJ (2008) Effect of gender, dose, and time on 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT)-induced hepatotoxicity in Fischer 344 rats. Xenobiotica 38:435–449PubMedCrossRefGoogle Scholar
  44. Peixoto F (2005) Comparative effects of the Roundup and glyphosate on mitochondrial oxidative phosphorylation. Chemosphere 61:1115–1122PubMedCrossRefGoogle Scholar
  45. Pujol G, Soderqvist H, Radu A (2002) Age-associated reduction of nuclear protein import in human fibroblast. Biochem Biophys Res Comm 274:354–358CrossRefGoogle Scholar
  46. Rea IM, McNerlan S, Pockley AG (2001) Serum heat shock protein and anti-heat shock protein antibody levels in aging. Exp Gerontol 36:341–352PubMedCrossRefGoogle Scholar
  47. Richard S, Moslemi S, Sipahutar H, Benachour N, Seralini GE (2005) Differential effects of glyphosate and roundup on human placental cells and aromatase. Environ Health Perspect 113:716–720PubMedCrossRefGoogle Scholar
  48. Roijkind M, Dominguez-Rosales JA, Nieto N, Greenweld P (2002) Role of hydrogen peroxide and oxidative stress in healing responses. Cell Mol Life Sci 59:1872–1891CrossRefGoogle Scholar
  49. Sanvido O, Romeis J, Bigler F (2007) Ecological impacts of genetically modified crops: ten years of field research and commercial cultivation. Adv Biochem Eng Biotechnol 107:235–278PubMedGoogle Scholar
  50. Schmitt E, Gehrmann M, Brunet M, Multhoff G, Garrido C (2007) Intracellular and extracellular functions of heat shock proteins: repercussions in cancer therapy. J Leukoc Biol 81:15–27PubMedCrossRefGoogle Scholar
  51. Schmucker DL (1990) Hepatocyte fine structure during maturation and senescence. J Electron Microsc Tech 14:106–125PubMedCrossRefGoogle Scholar
  52. Schwarzacher HG, Wachtler F (1993) The nucleolus. Anat Embryol 188:515–536PubMedCrossRefGoogle Scholar
  53. Séralini GE, Cellier D, Spiroux de Vendomois J (2007) New analysis of a rat feeding study with a genetically modified maize reveals signs of hepatorenal toxicity. Arch Environ Contam Toxicol 52:596–602PubMedCrossRefGoogle Scholar
  54. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850–858PubMedCrossRefGoogle Scholar
  55. Sverko V, Sobocanec S, Balog T, Marotti T (2004) Age and gender differences in antioxidant enzyme activity: potential relationship to liver carcinogenesis in male mice. Biogerontology 5:235–242PubMedCrossRefGoogle Scholar
  56. Szarek J, Siwicki A, Andrzejewska A, Terech-Majewska E, Banaszkiewicz T (2000) Effects of the herbicide Roundup on the ultrastructural pattern of hepatocytes in carp (Cyprinus carpio). Mar Environ Res 50:263–266PubMedCrossRefGoogle Scholar
  57. Tobisawa M, Tsurusaki Y, Yamaguchi M (2003) Decrease in regucalcin level and enhancement of protein tyrosine phosphatase activity in rat brain microsomes with increasing age. Int J Mol Med 12:577–580PubMedGoogle Scholar
  58. Tong BC, Barbul A (2004) Cellular and physiological effects of arginine. Mini Rev Med Chem 4:823–832PubMedGoogle Scholar
  59. Torres AM (1996) Gender-differential liver plasma membrane affinities in hepatic tetrabromosulfonephthalein (TBS) uptake. Biochem Pharmacol 51:1117–1122PubMedCrossRefGoogle Scholar
  60. Trabalza-Marinucci M, Brandi G, Rondini C, Avellini L, Giammarini C, Costarelli S, Acuti G, Orlandi C, Filippini G, Chiaradia E, Malatesta M, Crotti S, Antonini C, Amagliani G, Manuali E, Mastrogiacomo AR, Moscati L, Haouet MN, Gaiti A, Magnani M (2008) A three year longitudinal study on the effects of a diet containing genetically modified Bt176 maize on the health status and performance on sheep. Livestock Sci 113:178–190CrossRefGoogle Scholar
  61. Tipton KD (2001) Gender differences in protein metabolism. Curr Opin Clin Nutr Metab Care 4:493–498PubMedCrossRefGoogle Scholar
  62. Tudisco R, Lombardi P, Bovera F, d’Angelo D, Cutrignelli MI, Mastellone V, Terzi V, Avallone L, Infascelli F (2006) Genetically modified soya bean in rabbit feeding: detection of DNA fragments and evaluation of metabolic effects by enzymatic analysis. Anim Sci 82:193–199CrossRefGoogle Scholar
  63. Valdez LB, Zaobornyj T, Alvarez S, Bustamante J, Costa LE, Boveris A (2004) Heart mitochondrial nitric oxide synthase. Effects of hypoxia and aging. Mol Aspects Med 25:49–59PubMedCrossRefGoogle Scholar
  64. Vecchio L, Cisterna B, Malatesta M, Martin TE, Biggiogera M (2004) Ultrastructural analysis of testes from mice fed on genetically modified soybean. Eur J Histochem 48:449–453Google Scholar
  65. Voss MR, Stallone JN, Li M, Cornelussen RN, Knuefermann P, Knowlton AA (2003) Gender differences in the expression of heat shock proteins: the effect of estrogen. Am J Physiol Heart Circ Physiol 285:H687–H692PubMedGoogle Scholar
  66. Wei Y, Chan LL, Wang D, Zhang H, Wang J, Dai J (2008) Proteomic analysis of hepatic protein profiles in rare minnow (Gobiocypris rarus) exposed to perfluorooctanoic acid. J Proteome Res 7:1729–1739PubMedCrossRefGoogle Scholar
  67. Witte MB, Barbul A (2003) Arginine physiology and its implication for wound healing. Wound Repair Regen 11:419–423PubMedCrossRefGoogle Scholar
  68. Yamaguchi M (2005) Role of regucalcin in maintaining cell homeostasis and function. Int J Mol Med 15:371–389PubMedGoogle Scholar
  69. Yamaguchi M, Tsurusaki Y, Misawa H, Inagaki S, Ma ZJ, Takahashi H (2002) Potential role of regucalcin as a specific biochemical marker of chronic liver injury with carbon tetrachloride administration in rats. Mol Cell Biochem 241:61–67PubMedCrossRefGoogle Scholar
  70. Zhang HJ, Drake VJ, Morrison JP, Oberley LW, Kregel KC (2002) Selected contribution: differential expression of stress-related genes with aging and hyperthermia. J Appl Physiol 92:1762–1769PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Manuela Malatesta
    • 1
    Email author
  • Federica Boraldi
    • 2
  • Giulia Annovi
    • 2
  • Beatrice Baldelli
    • 3
  • Serafina Battistelli
    • 3
  • Marco Biggiogera
    • 4
  • Daniela Quaglino
    • 2
  1. 1.Dipartimento di Scienze Morfologico-Biomediche, Sezione di Anatomia e IstologiaUniversity of VeronaVeronaItaly
  2. 2.Department of Biomedical SciencesUniversity of Modena e Reggio EmiliaModenaItaly
  3. 3.Istituto di Istologia e Analisi di LaboratorioUniversity of UrbinoUrbino (PU)Italy
  4. 4.Dipartimento di Biologia Animale, Laboratorio di Biologia Cellulare e NeurobiologiaUniversity of PaviaPaviaItaly

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