The N-butyl-N-4-hydroxybutyl Nitrosamine Mouse Urinary Bladder Cancer Model

  • Paula A. OliveiraEmail author
  • Cármen Vasconcelos-Nóbrega
  • Rui M. Gil da Costa
  • Regina Arantes-Rodrigues
Part of the Methods in Molecular Biology book series (MIMB, volume 1655)


Urinary bladder cancer (UBC) is a common and complex malignancy, with a multifactorial etiology, like environmental factors, such as cigarette smoking, occupational exposure, and genetic factors.

UBC exhibits considerable genotypic and phenotypic heterogeneity. Among all UBC lesions, urothelial carcinoma is the most frequently observed histological type. Despite all the developments made in urologic oncology field, therapeutic options remain inadequate. There is urgency for the identification and development of new antineoplastic drugs to replace or improve current protocols and in vivo models have been proven to be essential for this step. There are different animal models of UBC: Spontaneous and experimentally induced models (genetically engineered, transplantable-xenograft and syngeneic animals- and chemically induced models). N-butyl-N(4-hydroxybutil)nitrosamine (BBN) is the most suitable reagent to generate chemically induced in vivo models of UBC and to study bladder carcinogenesis. BBN has proven, over the years, to be very realistic and reliable. It is bladder specific, and induces high tumor incidence.

Key words

Bladder cancer Chemical carcinogenesis Arylamines Animal models 


  1. 1.
    Siegel R, Naishadham D, Jemal A (2012) Cancer statistics, 2012. CA Cancer J Clin 62(1):10–29CrossRefPubMedGoogle Scholar
  2. 2.
    Parkin DM (2008) The global burden of urinary bladder cancer. Scand J Urol Nephrol Suppl 218:12–20CrossRefGoogle Scholar
  3. 3.
    Bellmunt J, Mottet N, De Santis M (2016) Urothelial carcinoma management in elderly or unfit patients. EJC Suppl 14(1):1–20CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Antoni S, Ferlay J, Soerjomataram I, Znaor A, Jemal A, Bray F (2016) Bladder cancer incidence and mortality: a global overview and recent trends. Eur Urol 71(1):96–108. doi: 10.1016/j.eururo.2016.06.010 CrossRefPubMedGoogle Scholar
  5. 5.
    Mahdavifar N, Ghoncheh M, Pakzad R, Momenimovahed Z, Salehiniya H (2016) Epidemiology, incidence and mortality of bladder cancer and their relationship with the development index in the world. Asian Pac J Cancer Prev 17(1):381–386CrossRefPubMedGoogle Scholar
  6. 6.
    Kiriluk KJ, Prasad SM, Patel AR, Steinberg GD, Smith ND (2012) Bladder cancer risk from occupational and environmental exposures. Urol Oncol 30(2):199–211CrossRefPubMedGoogle Scholar
  7. 7.
    Chu H, Wang M, Zhang Z (2013) Bladder cancer epidemiology and genetic susceptibility. J Biomed Res 27:170–178PubMedPubMedCentralGoogle Scholar
  8. 8.
    Santos J, Chaves J, Videira M, Botelho M, Costa J, Oliveira J, Santos L (2012) Schistosomiasis haematobium and bladder cancer: retrospective analysis of 145 patients admitted to the urology. Department at the Américo Boavida Hospital, Luanda. Acta Urol 1:15–20Google Scholar
  9. 9.
    Kader KA (2011) Bladder cancer. Sci World J 11:2565–2566CrossRefGoogle Scholar
  10. 10.
    Oliveira PA, Arantes-Rodrigues R, Vasconcelos-Nóbrega C (2014) Animal models of urinary bladder cancer and their application to novel drug discovery. Expert Opin Drug Discov 9(5):485–503CrossRefPubMedGoogle Scholar
  11. 11.
    Kamat AM, Hahn NM, Efstathiou JA, Lerner SP, Malmström PU, Choi W, Guo CC, Lotan Y, Kassouf W (2016) Bladder cancer. Lancet 388(10061):2796–2810. doi: 10.1016/S0140-6736(16)30512-8 CrossRefPubMedGoogle Scholar
  12. 12.
    Chalasani V, Chin JL, Izawa JI (2009) Histologic variants of urothelial bladder cancer and non urothelial histology in bladder cancer. Can Urol Assoc J 3(6Suppl4):S193–S198PubMedPubMedCentralGoogle Scholar
  13. 13.
    Shanks JH, Iczkowski KA (2009) Divergent differentiation in urothelial carcinoma and other bladder cancer subtypes with selected mimics. Histopathology 54(7):885–900CrossRefPubMedGoogle Scholar
  14. 14.
    Anastasiadis A, de Reijke TM (2012) Best practice in the treatment of nonmuscle invasive bladder cancer. Ther Adv Urol 4(1):13–32CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Pasin E, Josephson DY, Mitra AP, Cote RJ, Stein JP (2008) Superficial bladder cancer: an update on etiology, molecular development, classification, and natural history. Rev Urol 10(1):31–43PubMedPubMedCentralGoogle Scholar
  16. 16.
    Grignon DJ (2009) The current classification of urothelial neoplasms. Mod Pathol 22:S60–S69CrossRefPubMedGoogle Scholar
  17. 17.
    Zuiverloon TC, van der Aa MN, van der Kwast TH, Steyerberg EW, Lingsma HF, Bangma CH, Zwarthoff EC (2010) Fibroblast growth factor receptor 3 mutation analysis on voided urine for surveillance of patients with low-grade non-muscle-invasive bladder cancer. Clin Cancer Res 16(11):3011–3018CrossRefPubMedGoogle Scholar
  18. 18.
    Knowles MA (2007) Role of FGFR3 in urothelial cell carcinoma: biomarker and potential therapeutic target. World J Urol 25:581–593CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Noël N, Couteau J, Maillet G, Gobet F, d'Aloisio F, Minier C, Pfister C (2013) Preliminary study of p53 and FGFR3 gene mutations in the urine for bladder tumors. Prog Urol 23(1):29–35CrossRefPubMedGoogle Scholar
  20. 20.
    Van Rhijn BW, Montironi R, Zwarthoff EC, Jöbsis AC, van der Kwast TH (2002) Frequent FGFR3 mutations in urothelial papilloma. J Pathol 198(2):245–251CrossRefPubMedGoogle Scholar
  21. 21.
    Memon AA, Sorensen BS, Melgard P, Fokdal L, Thykjaer T, Nexo E (2004) Expression of HER3, HER4 and their ligand heregulin-4 is associated with better survival in bladder cancer patients. Br J Cancer 91:2034–2041CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Jarow J, Maher VE, Tang S, Ibrahim A, Kim G, Sridhara R, Pazdur R (2015) Development of systemic and topical drugs to treat non-muscle invasive bladder cancer. Bl Cancer 1(2):133–136CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Palou J, Sylvester RJ, Faba OR, Parada R, Pena JA, Algaba F, Villavicencio H (2012) Female gender and carcinoma in situ in the prostatic urethra are prognostic factors for recurrence, progression, and disease-specific mortality in T1G3 bladder cancer patients treated with bacillus Calmette-Guerin. Eur Urol 62(1):118–125CrossRefPubMedGoogle Scholar
  24. 24.
    Garcia del Muro X, Torregrosa A, Muñoz J, Castellsagué X, Condom E, Vigués F, Arance A, Fabra A, Germà JR (2000) Prognostic value of the expression of E-cadherin and beta-catenin in bladder cancer. Eur J Cancer 36(3):357–362CrossRefPubMedGoogle Scholar
  25. 25.
    Wu XR (2005) Urothelial tumorigenesis: a tale of divergent pathways. Nat Rev Cancer 5(9):713–725CrossRefPubMedGoogle Scholar
  26. 26.
    Shariat SF, Tokunaga H, Zhou J, Kim J, Ayala GE, Benedict WF, Lerner SP (2004) p53, p21, pRB, and p16 expression predict clinical outcome in cystectomy with bladder cancer. J Clin Oncol 22(6):1014–1024CrossRefPubMedGoogle Scholar
  27. 27.
    Cordon-Cardo C, Zhang ZF, Dalbagni G, Drobnjak M, Charytonowicz E, Hu SX, Xu HJ, Reuter VE, Benedict WF (1997) Cooperative effects of p53 and pRB alterations in primary superficial bladder tumors. Cancer Res 57(7):1217–1221PubMedGoogle Scholar
  28. 28.
    Braasch MR, Bohle A, O’Donnell MA (2009) Intravesical instillation treatment of non–muscle-invasive bladder cancer. Eur Urol Suppl 8:549–555CrossRefGoogle Scholar
  29. 29.
    Rosenberg JE, Hahn WC (2009) Bladder cancer: modeling and translation. Genes Dev 23:655–659CrossRefPubMedGoogle Scholar
  30. 30.
    Zips D, Thames HD, Baumann M (2005) New anticancer agents: in vitro and in vivo evaluation. In Vivo 19(1):1–7PubMedGoogle Scholar
  31. 31.
    Van Dam D, De Deyn PP (2006) Drug discovery in dementia: the role of rodent models. Nat Rev Drug Discov 5:956–970CrossRefPubMedGoogle Scholar
  32. 32.
    Oliveira PA, Gil da Costa RM, Vasconcelos-Nóbrega C, Arantes-Rodrigues R, Pinto-Leite R (2016) Challenges with in vitro and in vivo experimental models of urinary bladder cancer for novel drug discovery. Expert Opin Drug Discov 11(6):599–607CrossRefPubMedGoogle Scholar
  33. 33.
    Gil da Costa RM, Oliveira PA, Vilanova M, Bastos MM, Lopes CC, Lopes C (2011) Ptaquiloside-induced B-cell lymphoproliferative and early-stage urothelial lesions in mice. Toxicon 58:543–549CrossRefPubMedGoogle Scholar
  34. 34.
    Gil da Costa RM, Bastos MMSM, Oliveira PA, Lopes C (2012) Bracken-associated human and animal health hazards: chemical, biological and pathological evidence. J Hazard Mater 203-204:1–12CrossRefPubMedGoogle Scholar
  35. 35.
    Gil da Costa RM, Oliveira PA, Bastos MMSM, Lopes CC, Lopes C (2014) Ptaquiloside-induced early-stage urothelial lesions: increased cell proliferation and intact β-catenin and E-cadherin expression. Environ Toxicol 29:763–769CrossRefPubMedGoogle Scholar
  36. 36.
    Knapp DW, Ramos-Vara JA, Moore GE et al (2014) Urinary bladder cancer in dogs, a naturally occurring model for cancer biology and drug development. ILAR J 55(1):100–118CrossRefPubMedGoogle Scholar
  37. 37.
    Knapp DW, Waters DJ (1997) Naturally occurring cancer in pet dogs: important models for developing improved cancer therapy for humans. Mol Med Today 3(1):8–11CrossRefPubMedGoogle Scholar
  38. 38.
    Lairmore MD, Khanna C (2014) Naturally occurring diseases in animals: contributions to translational medicine. ILAR J 55(1):1–3CrossRefPubMedGoogle Scholar
  39. 39.
    Arentsen HC, Hendricksen K, Oosterwijk E, Witjes JA (2009) Experimental rat bladder urothelial cell carcinoma models. World J Urol 27(3):313–317CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Ding J, Xu D, Pan C, Ye M, Kang J, Bai Q, Qi J (2014) Current animal models of bladder cancer: awareness of translatability (review). Exp Ther Med 8(3):691–699CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Arantes-Rodrigues R, Colaço A, Pinto-Leite R, Oliveira PA (2013) In vitro and in vivo experimental models as tools to investigate the efficacy of antineoplastic drugs on urinary bladder cancer. Anticancer Res 33(4):1273–1296PubMedGoogle Scholar
  42. 42.
    Rosenbert MP, Bortner D (1998) Why transgenic and knockout animal models should be used (for drug efficacy studies in cancer). Cancer Metastasis Rev 17:295–299CrossRefGoogle Scholar
  43. 43.
    Russell PJ, Raghavan D, Gregory P et al (1986) Bladder cancer xenografts: a model of tumor cell heterogeneity. Cancer Res 46:2035–2040PubMedGoogle Scholar
  44. 44.
    Günther JH, Jurczok A, Wulf T et al (1999) Optimizing syngeneic orthotopic murine bladder cancer (MB49). Cancer Res 59:2834–2837PubMedGoogle Scholar
  45. 45.
    Chan ESY, Patel AR, Smith AK et al (2009) Optimizing orthotopic bladder tumor implantation in a syngeneic mouse model. J Urol 183(6):2926–2931CrossRefGoogle Scholar
  46. 46.
    Oliveira PA, Colaço A, De la Cruz Palomino LF, Lopes C (2006) Experimental bladder carcinogenesis–rodent models. Exp Oncol 28:2–11PubMedGoogle Scholar
  47. 47.
    Gil da Costa RM, Oliveira PA, Vasconcelos-Nóbrega C, Arantes-Rodrigues R, Pinto-Leite R, Colaço A, de la Cruz PLF, Lopes C (2015) Altered expression of CKs 14/20 is an early event in a rat model of multistep bladder carcinogenesis. Int J Exp Pathol 96(5):319–325. doi: 10.1111/iep.12145 CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Cohen SM (1983) Promotion in urinary bladder carcinogenesis. Environ Health Perspect 50:51–59CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Johansson SL, Cohen SM (1997) Epidemiology and etiology of bladder cancer. Semin Surg Oncol 13:291–298CrossRefPubMedGoogle Scholar
  50. 50.
    Cohen SM (2008) Tresholds in genotoxicity and carcinogenicity: urinary bladder carcinogenesis. Genes Environ 30(4):132–138CrossRefGoogle Scholar
  51. 51.
    Jiang T, Lui T, Li L, Yang Z, Bai Y, Liu D, Kong C (2016) Knockout of phospholipase Cε attenuates N-butyl-N-(4-hydroxybutyl) nitrosamine-induced bladder tumorigenesis. Mol Med Rep 13:2039–2045CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Bonfanti M, Magagnotti C, Bonati M, Fanelli R, Airoldi L (1988) Pharmacokinetic profile and metabolism of N-nitrosobutyl-(4-hydroxybutyl)amine in rats. Cancer Res 48:3666–3669PubMedGoogle Scholar
  53. 53.
    Mirvish SS (1995) Role of N-nitroso compounds (NOC) and N-nitrosation in etiology of gastric, esophageal, nasopharyngeal and bladder cancer and contribution to cancer of known exposures to NOC. Cancer Lett 93:17–48CrossRefPubMedGoogle Scholar
  54. 54.
    Iida K, Itoh K, Maher JM, Kumagai Y, Oyasu R, Mori Y, Shimazui T, Akaza H, Yamamoto M (2007) Nrf2 and p53 cooperatively protect against BBN-induced urinary bladder carcinogenesis. Carcinogenesis 28(11):2398–2403CrossRefPubMedGoogle Scholar
  55. 55.
    Suzuki E, Okada M (1980) Metabolic fate of N-butyl-N-(4-hydroxybutyl)nitrosamine in the rat. Gann 71(6):856–862PubMedGoogle Scholar
  56. 56.
    Mochizuki M, Suzuki E, Okada M (1997) Structure and metabolic fate of N-nitrosodialkylamines in relation to their organotropic carcinogenicity with special reference to induction of urinary bladder tumors. Yakugaku Zasshi 117(10-11):884–894CrossRefPubMedGoogle Scholar
  57. 57.
    Cohen SM (1998) Urinary bladder carcinogenesis. Toxicol Pathol 26:121–127CrossRefPubMedGoogle Scholar
  58. 58.
    Cohen SM, Ohnishi T, Clark NM, He J, Arnold LL (2007) Investigations of rodent urinary bladder carcinogens: collection, processing, and evaluation of urine and bladders. Toxicol Pathol 35(3):337–347CrossRefPubMedGoogle Scholar
  59. 59.
    Okada M, Suzuki E, Hashimoto Y (1976) Carcinogenicity of N-nitrosamines related to N-butyl-N-(4-Hydroxybutyl)nitrosamine and N, N-Dibutylnitrosamine in ACI/N rats. Gann 67:825–834PubMedGoogle Scholar
  60. 60.
    Airoldi L, Magagnotti C, Bonfanti M, Fanelli R (1987) Development of an experimental model for studying bladder carcinogen metabolism using the isolated rat urinary bladder. Cancer Res 47:3697–3700PubMedGoogle Scholar
  61. 61.
    Yamamoto K, Nakata D, Tada M, Tonoki H, Nishida T, Hirai A, Ba Y, Aoyama T, Hamada J, Furuuchi K, Harada H, Hirai K, Shibahara N, Katsuoka Y, Moriuchi T (1999) A functional and quantitative mutational analysis of p53 mutations in yeast indicates strand biases and different roles of mutations in DMBA- and BBN-induced tumors in rats. Int J Cancer 83:700–705CrossRefPubMedGoogle Scholar
  62. 62.
    Weisburger JH, Williams GM (2000) The distinction between genotoxic and epigenetic carcinogens and implication for cancer risk. Toxicol Sci 57:4–5CrossRefPubMedGoogle Scholar
  63. 63.
    Drago JR (2004) The noble rat bladder cancer model–FANFT induced Tumours. Cancer 53:1093–1099CrossRefGoogle Scholar
  64. 64.
    Mann AM, Asamoto M, Chlapowski FJ, Masui T, Macatee TL, Cohen SM (1992) Ras involvement in cells transformed with 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) in vitro and with N-[4-(5-nitro-2-furyl)-2-thiazoyl]formamide in vivo. Carcinogenesis 13(9):1651–1655CrossRefPubMedGoogle Scholar
  65. 65.
    Reis LO, Pereira TC, Favaro WJ, Cagnon VH, Lopes-Cendes I, Ferreira U (2009) Experimental animal model and RNA interference: a promising association for bladder cancer research. World J Urol 27:353CrossRefPubMedGoogle Scholar
  66. 66.
    Spry LA, Rubinstein J, Rettke C, Zenser TV, Davis BB (1988) Renal metabolic/excretory coupling. Am J Physiol 254(1):F145–F152PubMedGoogle Scholar
  67. 67.
    Severs NJ, Barnes SH, Wright R, Hicks RM (1982) Induction of bladder cancer in rats by fractionated intravesicular doses of N-methyl-N-nitrosourea. Br J Cancer 45:337–351CrossRefPubMedPubMedCentralGoogle Scholar
  68. 68.
    Steinberg GD, Brendler CB, Ichikawa T, Squire RA, Isaacs JT (1990) Characterization of an N-methyl-N-nitrosourea-induced autochthonous rat bladder cancer model. Cancer Res 50(20):6668–6674PubMedGoogle Scholar
  69. 69.
    Vasconcelos-Nóbrega C, Colaço A, Lopes C, Oliveira PA (2012) Review: BBN as an urothelial carcinogen. In Vivo 26(4):727–739PubMedGoogle Scholar
  70. 70.
    Cohen SM, Friedell GH (1982) Neoplasms of the urinary system. In: The mouse in biomedical research. Academic Press, New York, pp 439–463Google Scholar
  71. 71.
    Ito N, Shirai T, Fukushima S, Hirose M (1984) Dose-response study of urinary bladder carcinogenesis in rats by N-butyl-N-(4-hydroxybutyl)nitrosamine. J Cancer Res Clin Oncol 108:169–173CrossRefPubMedGoogle Scholar
  72. 72.
    Ohtani M, Kakizoe T, Nishio Y, Sato S, Sugimura T, Fukushima S, Niijima T (1986) Sequential changes of mouse bladder epithelium during induction of invasive carcinomas by N-butyl-N-(4-hydroxybutyl)nitrosamine. Cancer Res 46:2011–2004Google Scholar
  73. 73.
    Wanibuchi H, Wei M, Salim EI, Kinoshita A, Morimura K, Sudo K, Fukushima S (2006) Inhibition of rat urinary bladder carcinogenesis by the antiangiogenic drug TNP-470. Asian Pac J Cancer Prev 7:101–107PubMedGoogle Scholar
  74. 74.
    Wang SC, Huang CC, Shen CH, Lin LC, Zhao PW, Chen SY, Deng YC, Liu YW (2016) Gene expression and DNA methylationstatus of glutathione-S-transferase Mu1 and Mu5 in urothelial carcinoma. PLoS One 11:e0159102CrossRefPubMedPubMedCentralGoogle Scholar
  75. 75.
    Oliveira PA, Palmeira C, Lourenço L, Lopes C (2005) Evaluation of DNA content in preneoplastic changes of mouse urinary bladder induced by N-butyl-N-(4-hydroxybutyl) nitrosamine. J Exp Clin Cancer Res 24:207–214Google Scholar
  76. 76.
    Arantes-Rodrigues R, Henriques A, Pinto-Leite R, Faustino-Rocha A, Pinho-Oliveira J, Teixeira-Guedes C, Seixas F, Gama A, Colaço B, Colaço A, Oliveira PA (2012) The effects of repeated oral gavage on the health of male CD-1 mice. Lab Anim (NY) 41(5):129–134CrossRefGoogle Scholar
  77. 77.
    Okada M, Ishidate M (1977) Metabolic fate of N-butyl-N-(4-hydroxybutyl)-nitrosamine and its analogues: selective induction of urinary bladder tumours in the rat. Xenobiotica 7(1):11–24CrossRefPubMedGoogle Scholar
  78. 78.
    Hashimoto Y, Suzuki K, Okada M (1974) Induction of urinary bladder tumors by intravesicular instillation of butyl(4-hydroxybutyl)nitrosoamine and its principal urinary metabolite, butyl(3-carboxypropyl)nitrosoamine in rats. Gann 65:69–73PubMedGoogle Scholar
  79. 79.
    Fuji K, Odashima S, Okada M (1977) Induction of tumours by administration of N-dibutylnitrosamine and derivatives to infant mice. Br J Cancer 35:610–614CrossRefGoogle Scholar
  80. 80.
    Tsuda H, Miyata Y, Hagiwara A, Hasegawa R, Shirai T, Ito N (1977) Damage and repair of DNA in urinary bladder epithelium of rats treated with N-butyl-N-(4-hydroxybutyl) nitrosamine. Gann 68:781–783PubMedGoogle Scholar
  81. 81.
    Oliveira PA, Pires MJ, Nóbrega C, Arantes-Rodrigues R, Calado AM, Carrola J, Ginja M, Colaço A (2009) Technical report: technique of bladder catheterization in female mice and rats for intravesical instillation in models of bladder cancer. Scand J Lab Anim Sci 36(1):5–9Google Scholar
  82. 82.
    Xu J, Wang Y, Hua X, Xu J, Tian Z, Jin H, Li J, Wu XR, Huang C (2016) Inhibition of PHLPP2/cyclin D1 protein translation contributes to the tumor suppressive effect of NFκB2 (p100). Oncotarget 7:34112–34130PubMedPubMedCentralGoogle Scholar
  83. 83.
    Suzuki E, Anjo T, Aoki J, Okada M (1983) Species variations in the metabolism of N-butyl-N-(4-hydroxybutyl)nitrosamine and related compounds in relation to urinary bladder carcinogenesis. Gann 74:60–68PubMedGoogle Scholar
  84. 84.
    Shin K, Lim A, Odegaard JI, Honeycutt JD, Kawano S, Hsieh MH, Beachy PA (2014) Cellular origin of bladder neoplasia and tissue dynamics of its progression to invasive carcinoma. Nat Cell Biol 16:469–478CrossRefPubMedPubMedCentralGoogle Scholar
  85. 85.
    Van Loo PLP, Kruitwagenb CLJJ, Koolhaasc JM, Van de Weerdd HA, Van Zutphena LFM, Baumansa V (2002) Influence of cage enrichment on aggressive behaviour and physiological parameters in male mice. Appl Anim Behav Sci 76:65–81CrossRefGoogle Scholar
  86. 86.
    Danneman PJ, Suckow MA, Brayton CF (2012) The laboratory mouse, 2nd edn. Taylor and Francis, LondonGoogle Scholar
  87. 87.
    Conn PM (2013) Animal models for the study of human disease, 1st edn. Elsevier, AmsterdamGoogle Scholar
  88. 88.
    Wolfer DP, Litvin O, Morf S, Nitsch RM, Lipp HP, Würbel H (2004) Laboratory animal welfare: cage enrichment and mouse behaviour. Nature 432:821–822CrossRefPubMedGoogle Scholar
  89. 89.
    Gouveia K, Hurst JL (2013) Reducing mouse anxiety during handling: effect of experience with handling tunnels. PLoS One 8:e66401CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Paula A. Oliveira
    • 1
    • 2
    Email author
  • Cármen Vasconcelos-Nóbrega
    • 3
  • Rui M. Gil da Costa
    • 1
    • 2
  • Regina Arantes-Rodrigues
    • 1
    • 2
  1. 1.Department of Veterinary SciencesUniversity of Trás-os-Montes and Alto Douro (UTAD)Vila RealPortugal
  2. 2.Center for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB)Vila RealPortugal
  3. 3.Centre for the Study of Education, Technologies and HealthPolythecnic Institute of ViseuViseuPortugal

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