Establishment, characterization, and toxicological application of a spontaneous immortalized cell line from the striped field mouse, Apodemus agrarius

  • Ji Min Lee
  • Byoung-Hee Lee
  • Seo-Na Chang
  • Hanseul Oh
  • Bokyeong Ryu
  • Ukjin Kim
  • Jae-Hak ParkEmail author


It is important to secure various biological resources in situations of diminishing wildlife genetic diversity. Cultured cells are useful bioresources because they can stably store genetic information for a long time and can be expanded efficiently. Here, we established fibroblast cell lines from Apodemus agrarius as a new living resource. A. agrarius is an important sub-predator species in ecosystem food chains and for the study of infection epidemiology. Established cell lines were characterized by chromosome and mitochondrial gene analysis, the observation of cell morphology, and their anchorage-dependent growth pattern. We also examined susceptibility to endocrine disruptors (EDCs), which threaten biodiversity, using these established cell lines. Nonylphenol (NP) is a well-known EDC that threatens wildlife; however, its impact is poorly understood. Sensitivity to NP was confirmed based on two cell viability assays, namely MTT and lactate dehydrogenase. Cells exposed to NP were analyzed for abnormalities in cell growth and mitochondrial function by evaluating the expression of genes (specifically, those encoding growth hormone receptor and cytochrome C oxidase). This newly established cell line represents a valuable tool for the evaluation of toxic substances such as EDCs and this cell was biobanked for study about relationship between various environmental pollution and decreasing biodiversity.


Bioresource Apodemus agrarius Immortalized cell line Endocrine disruptors Nonylphenol 


Funding information

This work was supported by the National Institute of Biological Resources (NIBR) [grant numbers NIBR201526201], funded by the Ministry of Environment (MOE), Republic of Korea, and the Research Institute for Veterinary Science and the BK21 PLUS Program for Creative Veterinary Science Research, College of Veterinary Medicine, Seoul National University, Seoul, Korea.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

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  1. Borowicz S, Van Scoyk M, Avasarala S, Rathinam MKK, Tauler J, Bikkavilli RK, Winn RA (2014) The soft agar colony formation assay. J Visual ExpGoogle Scholar
  2. Caride A, Fernández-Pérez B, Cabaleiro T, Tarasco M, Esquifino AI, Lafuente A (2010) Cadmium chronotoxicity at pituitary level: effects on plasma acth, gh, and tsh daily pattern. J Physiol Biochem 66:213–220CrossRefGoogle Scholar
  3. Clarke A (2009) The frozen ark project: the role of zoos and aquariums in preserving the genetic material of threatened animals. International zoo yearbook 43:222–230CrossRefGoogle Scholar
  4. Dang VH, Choi K-C, Jeung E-B (2009) Estrogen receptors are involved in xenoestrogen induction of growth hormone in the rat pituitary gland. J Reprod Dev 55:206–213CrossRefGoogle Scholar
  5. García L, Ortiz S, Osorio G, Torrico MC, Torrico F, Solari A (2012) Phylogenetic analysis of bolivian bat trypanosomes of the subgenus schizotrypanum based on cytochrome b sequence and minicircle analyses. PLoS One 7:e36578CrossRefGoogle Scholar
  6. Gong Y, Wu J, Huang Y, Shen S, Han X (2009) Nonylphenol induces apoptosis in rat testicular sertoli cells via endoplasmic reticulum stress. Toxicol Lett 186:84–95CrossRefGoogle Scholar
  7. Gortat T, Rutkowski R, Gryczynska-Siemiatkowska A, Kozakiewicz A, Kozakiewicz M (2013) Genetic structure in urban and rural populations of apodemus agrarius in Poland. Mamm Biol-Zeitschrift für Säugetierkunde 78:171–177CrossRefGoogle Scholar
  8. Hanson AM, Ickstadt AT, Marquart DJ, Kittilson JD, Sheridan MA (2017) Environmental estrogens inhibit mrna and functional expression of growth hormone receptors as well as growth hormone signaling pathways in vitro in rainbow trout (oncorhynchus mykiss). Gen Comp Endocrinol 246:120–128CrossRefGoogle Scholar
  9. Hescot S, Slama A, Lombes A, Paci A, Remy H, Leboulleux S, Chadarevian R, Trabado S, Amazit L, Young J (2013) Mitotane alters mitochondrial respiratory chain activity by inducing cytochrome c oxidase defect in human adrenocortical cells. Endocr Relat Cancer 20:371–381CrossRefGoogle Scholar
  10. Hull KL, Harvey S (2000) Growth hormone: a reproductive endocrine-paracrine regulator? Rev Reprod 5:175–182CrossRefGoogle Scholar
  11. Irwin RW, Yao J, Hamilton RT, Cadenas E, Brinton RD, Nilsen J (2008) Progesterone and estrogen regulate oxidative metabolism in brain mitochondria. Endocrinology 149:3167–3175CrossRefGoogle Scholar
  12. Ishibashi H, Hirano M, Matsumura N, Watanabe N, Takao Y, Arizono K (2006) Reproductive effects and bioconcentration of 4-nonylphenol in medaka fish (oryzias latipes). Chemosphere 65:1019–1026CrossRefGoogle Scholar
  13. Ishiniwa H, Sakai M, Tohma S, Matsuki H, Takahashi Y, Kajiwara H, Sekijima T (2013) Dioxin pollution disrupts reproduction in male japanese field mice. Ecotoxicology 22:1335–1347CrossRefGoogle Scholar
  14. Jiao B, Cheng CH (2010) Disrupting actions of bisphenol A and malachite green on growth hormone receptor gene expression and signal transduction in seabream. Fish Physiol Biochem 36:251–261CrossRefGoogle Scholar
  15. Jones TT, Brewer GJ (2009) Critical age-related loss of cofactors of neuron cytochrome c oxidase reversed by estrogen. Exp Neurol 215:212–219CrossRefGoogle Scholar
  16. Kim J-H, Yoon M-H, Ueda Y, Honda K, Min B-Y (2006) Relationships between the abnormalities of the male reproductive organs and the accumulation of phenol compounds in the striped field mouse, apodemus agrarius, inhabiting Korea. Environ Pollut 144:716–725CrossRefGoogle Scholar
  17. Kim HC, Klein TA, Kang HJ, Gu SH, Moon SS, Baek LJ, Chong ST, O'Guinn ML, Lee JS, Turell MJ (2011) Ecological surveillance of small mammals at dagmar north training area, gyeonggi province, Republic of Korea, 2001–2005. J Vector Ecol 36:42–54CrossRefGoogle Scholar
  18. Lamche G, Burkhardt-Holm P (2000) Changes in apoptotic rate and cell viability in three fish epidermis cultures after exposure to nonylphenol and to a wastewater sample containing low concentrations of nonylphenol. Biomarkers 5:205–218CrossRefGoogle Scholar
  19. Lee PC, Arndt P, Nickels KC (1999) Testicular abnormalities in male rats after lactational exposure to nonylphenols. Endocrine 11:61–68CrossRefGoogle Scholar
  20. Lei F, Zu-guo W, Yao Y, Xiang-ming X, Pin-qiang G (2015) Population change of farmland rodent and the influences of climate and cultivation factors in fengxian district of Shanghai, China. Yingyong Shengtai Xuebao 26Google Scholar
  21. Lepretti M, Paolella G, Giordano D, Marabotti A, Gay F, Capaldo A, Esposito C, Caputo I (2015) 4-nonylphenol reduces cell viability and induces apoptosis and er-stress in a human epithelial intestinal cell line. Toxicol in Vitro 29:1436–1444CrossRefGoogle Scholar
  22. Lermen D, Bloemeke B, Browne R, Clarke A, Dyce PW, Fixemer T, Fuhr GR, Holt WV, Jewgenow K, Lloyd RE (2009) Cryobanking of viable biomaterials: implementation of new strategies for conservation purposes. Mol Ecol 18:1030–1033CrossRefGoogle Scholar
  23. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative pcr and the 2−δδct method. Methods 25:402–408CrossRefGoogle Scholar
  24. MacKenzie KL, Franco S, Naiyer AJ, May C, Sadelain M, Rafii S, Moore MA (2002) Multiple stages of malignant transformation of human endothelial cells modelled by co-expression of telomerase reverse transcriptase, sv40 t antigen and oncogenic n-ras. Oncogene 21:4200–4211CrossRefGoogle Scholar
  25. Maruyama M, Kobayashi N, Westerman KA, Sakaguchi M, Allain JE, Totsugawa T, Okitsu T, Fukazawa T, Weber A, Stolz DB (2004) Establishment of a highly differentiated immortalized human cholangiocyte cell line with sv40t and htert. Transplantation 77:446–451CrossRefGoogle Scholar
  26. Nooteboom M, Johnson R, Taylor RW, Wright NA, Lightowlers RN, Kirkwood TB, Mathers JC, Turnbull DM, Greaves LC (2010) Age-associated mitochondrial DNA mutations lead to small but significant changes in cell proliferation and apoptosis in human colonic crypts. Aging Cell 9:96–99CrossRefGoogle Scholar
  27. Qi Y, Zhang Y, Liu Y, Zhang W (2013) Nonylphenol decreases viability and arrests cell cycle via reactive oxygen species in raji cells. Exp Toxicol Pathol 65:69–72CrossRefGoogle Scholar
  28. Shen X-J, Si I, Mizutani M, Yamamoto Y (2002) Phylogenetic analysis in chicken breeds inferred from complete cytochrome b gene information. Biochem Genet 40:129–141CrossRefGoogle Scholar
  29. Sicheritz-Pontén T, Kurland CG, Andersson SG (1998) A phylogenetic analysis of the cytochrome b and cytochrome c oxidase i genes supports an origin of mitochondria from within the rickettsiaceae1. Biochim Biophys Acta (BBA)-Bioenergetics 1365:545–551CrossRefGoogle Scholar
  30. Soares A, Guieysse B, Jefferson B, Cartmell E, Lester J (2008) Nonylphenol in the environment: a critical review on occurrence, fate, toxicity and treatment in wastewaters. Environ Int 34:1033–1049CrossRefGoogle Scholar
  31. Strutz F, Okada H, Lo CW, Danoff T, Carone RL, Tomaszewski JE, Neilson EG (1995) Identification and characterization of a fibroblast marker: Fsp1. J Cell Biol 130:393–405CrossRefGoogle Scholar
  32. Suzuki H, Filippucci MG, Chelomina GN, Sato JJ, Serizawa K, Nevo E (2008) A biogeographic view of apodemus in asia and europe inferred from nuclear and mitochondrial gene sequences. Biochem Genet 46:329–346CrossRefGoogle Scholar
  33. Todaro GJ, Green H (1963) Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol 17:299–313CrossRefGoogle Scholar
  34. Tyler C, Jobling S, Sumpter J (1998) Endocrine disruption in wildlife: a critical review of the evidence. Crit Rev Toxicol 28:319–361CrossRefGoogle Scholar
  35. Vapalahti O, Mustonen J, Lundkvist Å, Henttonen H, Plyusnin A, Vaheri A (2003) Hantavirus infections in Europe. Lancet Infect Dis 3:653–661CrossRefGoogle Scholar
  36. Vos JG, Dybing E, Greim HA, Ladefoged O, Lambré C, Tarazona JV, Brandt I, Vethaak AD (2000) Health effects of endocrine-disrupting chemicals on wildlife, with special reference to the european situation. Crit Rev Toxicol 30:71–133CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2018

Authors and Affiliations

  • Ji Min Lee
    • 1
  • Byoung-Hee Lee
    • 2
  • Seo-Na Chang
    • 1
  • Hanseul Oh
    • 1
  • Bokyeong Ryu
    • 1
  • Ukjin Kim
    • 1
  • Jae-Hak Park
    • 1
    Email author
  1. 1.Department of Laboratory Animal Medicine, College of Veterinary MedicineSeoul National UniversitySeoulRepublic of Korea
  2. 2.Microorganism Resources DivisionNational Institute of Biological ResourcesIncheonRepublic of Korea

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