Skip to main content

Involvement of Multidrug Resistance-Associated Protein 1 in Intestinal Toxicity of Methotrexate

Pharmaceutical Research volume 26, pages 1467–1476 (2009)Cite this article

Abstract

Purpose

Methotrexate (MTX) causes dose-limiting gastrointestinal toxicity due to exposure of intestinal tissues, and is a substrate of the multidrug resistance-associated protein (MRP) 1. Here we examine the involvement of MRP1, which is reported to be highly expressed in the proliferative crypt compartment of the small intestine, in the gastrointestinal toxicity of MTX.

Methods

MTX was intraperitonealy administered to mrp1 gene knockout (mrp1 (−/−)) and wild-type (mrp1 (+/+)) mice. Body weight, food and water intake were monitored, intestinal histological studies and pharmacokinetics of MTX were examined.

Results

mrp1 (−/−) mice more severely decreased body weight, food and water intake than mrp1 (+/+) mice. Almost complete loss of villi throughout the small intestine in mrp1 (−/−) mice was observed, whereas the damage was only partial in mrp1 (+/+) mice. Plasma concentration and biliary excretion profiles of MTX were similar in mrp1 (−/−) and mrp1 (+/+) mice, though accumulation of MTX in immature proliferative cells isolated from mrp1 (−/−) mice was much higher compared to mrp1 (+/+) mice. Immunostaining revealed localization of Mrp1 in plasma membrane of the intestinal crypt compartment in mrp1 (+/+) mice, but not in mrp1 (−/−) mice.

Conclusion

Mrp1 determines the exposure of proliferative cells in the small intestine to MTX, followed by gastrointestinal toxicity.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. K. Welte, M. A. Bonilla, A. P. Gillio, T. C. Boone, G. K. Potter, J. L. Gabrilove, M. A. Moore, R. J. O'Reilly, and L. M. Souza. Recombinant human granulocyte colony-stimulating factor. Effects on hematopoiesis in normal and cyclophosphamide-treated primates. J. Exp. Med. 165:941–948 (1987) doi:10.1084/jem.165.4.941.

    PubMed  Article  CAS  Google Scholar 

  2. S. E. Steinberg, C. L. Campbell, W. A. Bleyer, and R. S. Hillman. Enterohepatic circulation of methotrexate in rats in vivo. Cancer Res. 42:1279–1282 (1982).

    PubMed  CAS  Google Scholar 

  3. D. Griffin, and H. M. Said. The enterohepatic circulation of methotrexate in vivo: inhibition by bile salt. Cancer Chemother. Pharmacol. 19:40–41 (1987) doi:10.1007/BF00296253.

    PubMed  Article  CAS  Google Scholar 

  4. M. Masuda, Y. I’izuka, M. Yamazaki, R. Nishigaki, Y. Kato, K. Ni'inuma, H. Suzuki, and Y. Sugiyama. Methotrexate is excreted into the bile by canalicular multispecific organic anion transporter in rats. Cancer Res. 57:3506–3510 (1997).

    PubMed  CAS  Google Scholar 

  5. P. Breedveld, N. Zelcer, D. Pluim, O. Sönmezer, M. M. Tibben, J. H. Beijnen, A. H Schinkel, O. van Tellingen, P. Borst, and J. H. Schellens. Mechanism of the pharmacokinetic interaction between methotrexate and benzimidazoles: potential role for breast cancer resistance protein in clinical drug-drug interactions. Cancer Res. 64:5804–5811 (2004) doi:10.1158/0008-5472.CAN-03-4062.

    PubMed  Article  CAS  Google Scholar 

  6. M. Takeda, S. Khamdang, S. Narikawa, H. Kimura, M. Hosoyamada, S. H. Cha, T. Sekine, and H. Endou. Characterization of methotrexate transport and its drug interactions with human organic anion transporters. J. Pharmacol. Exp. Ther. 302:666–671 (2002) doi:10.1124/jpet.102.034330.

    PubMed  Article  CAS  Google Scholar 

  7. K. Miyamoto, T. Shiraga, K. Morita, H. Yamamoto, H. Haga, Y. Taketani, I. Tamai, Y. Sai, A. Tsuji, and E. Takeda. Sequence, tissue distribution and developmental change in rat intestinal oligopeptide transporter. Biochim. Biophys. Acta. 1305:34–38 (1996).

    PubMed  Google Scholar 

  8. I. Tamai, T. Nakanishi, K. Hayashi, T. Terao, Y. Sai, T. Shiraga, K. Miyamoto, E. Takeda, H. Higashida, and A. Tsuji. The predominant contribution of oligopeptide transporter PepT1 to intestinal absorption of ß-lactam antibiotics in the rat small intestine. J. Pharm. Pharmacol. 49:796–801 (1997).

    PubMed  CAS  Google Scholar 

  9. Y. Wang, R. Zhao, R. G. Russell, and I. D. Goldman. Localization of murine reduced folate carrier as assessed by immuhistochemical analysis. Biochim. Biophys. Acta. 1513:49–54 (2001) doi:10.1016/S0005-2736(01)00340-6.

    PubMed  Article  CAS  Google Scholar 

  10. M. Shayeghi, G. O. Latunde-Dada, J. S. Oakhill, A. H. Laftah, K. Takeuchi, N. Halliday, Y. Khan, A. Warley, F. E. McCann, R. C. Hider, D. M. Frazer, G. J. Anderson, C. D. Vulpe, R. J. Simpson, and A. T. McKie. Identification of an intestinal heme transporter. Cell. 122:789–801 (2005) doi:10.1016/j.cell.2005.06.025.

    PubMed  Article  CAS  Google Scholar 

  11. Y. Wang, A. Rajgopal, I. D. Goldman, and R. Zhao. Preservation of folate transport activity with a low-pH optimum in rat IEC-6 intestinal epithelial cell lines that lack reduced folate carrier function. Am. J. Physiol. 288:C65–C71 (2005) doi:10.1152/ajpcell.00533.2004.

    Article  CAS  Google Scholar 

  12. A. Qiu, M. Jansen, A. Sakaris, S. H. Min, S. Chattopadhyay, E. Tsai, C. Sandoval, R. Zhao, M. H. Akabas, and I. D. Goldman. Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption. Cell. 127:917–928 (2006) doi:10.1016/j.cell.2006.09.041.

    PubMed  Article  CAS  Google Scholar 

  13. T. Terao, E. Hisanaga, Y. Sai, I. Tamai, and A. Tsuji. Active secretion of drugs from the small intestinal epithelium in rats by P-glycoprotein functioning as an absorption barrier. J. Pharm. Pharmacol. 48:1083–1089 (1996).

    PubMed  CAS  Google Scholar 

  14. K. Naruhashi, I. Tamai, N. Inoue, H. Muraoka, Y. Sai, N. Suzuki, and A. Tsuji. Involvement of multidrug resistance-associated protein 2 in intestinal secretion of grepafloxacin in rats. Antimicrob. Agents Chemother. 46:344–349 (2002) doi:10.1128/AAC.46.2.344-349.2002.

    PubMed  Article  CAS  Google Scholar 

  15. D. Rost, S. Mahner, Y. Sugiyama, and W. Stremmel. Expression and localization of the multidrug resistance-associated protein 3 in rat small and large intestine. Am. J. Physiol. 28:G720–G726 (2002).

    Google Scholar 

  16. H. Zeng, Z. S. Chen, M. G. Belinsky, P. A. Rea, and G. D. Kruh. Transport of methotrexate (MTX) and folates by multidrug resistance protein (MRP) 3 and MRP1: effect of polyglutamylation on MTX transport. Cancer Res. 61:7225–7232 (2001).

    PubMed  CAS  Google Scholar 

  17. K. C. Peng, F. Cluzeaud, M. Bens, J. P. Van Huyen, M. A. Wioland, R. Lacave, and A. Vandewalle. Tissue and cell distribution of the multidrug resistance associated protein (MRP) in mouse intestine and kidney. J. Histochem. Cytochem. 47:757–767 (1999).

    PubMed  CAS  Google Scholar 

  18. J. Wijnholds, R. Evers, M. R. van Leusden, C. A. Mol, G. J. Zaman, U. Mayer, J. H. Beijnen, M. van der Valk, P. Krimpenfort, and P. Borst. Increased sensitivity to anticancer drugs and decreased inflammatory response in mice lacking the multidrug resistance-associated protein. Nat. Med. 3:1275–1279 (1997) doi:10.1038/nm1197-1275.

    PubMed  Article  CAS  Google Scholar 

  19. A. Kurita, S. Kado, N. Kaneda, M. Onoue, S. Hashimoto, and T. Yokokura. Modified irinotecan hydrochloride (CPT-11) administration schedule improves induction of delayed-onset diarrhea in rats. Cancer Chemother. Pharmacol. 46:211–220 (2000) doi:10.1007/s002800000151.

    PubMed  Article  CAS  Google Scholar 

  20. O. C. Trifan, W. F. Durham, V. S. Salazar, J. Horton, B. D. Levine, B. S. Zweifel, T. W. Davis, and J. L. Masferrer. Cyclooxygenase-2 inhibition with celecoxib enhances antitumor efficacy and reduces diarrhea side effect of CPT-11. Cancer Res. 62:5778–5784 (2002).

    PubMed  CAS  Google Scholar 

  21. F. M. Sirotnak, D. M. Moccio, and C. H. Yang. Similar characteristics of folate analogue transport in vitro in contrast to varying dihydrofolate reductase levels in epithelial cells at different stages of maturation in mouse small intestine. Cancer Res. 44:5204–5211 (1984).

    PubMed  CAS  Google Scholar 

  22. M. M. Weiser. Intestinal epithelial cell surface membrane glycoprotein synthesis. I. An indicator of cellular differentiation. J. Biol. Chem. 248:2536–2541 (1973).

    PubMed  CAS  Google Scholar 

  23. K. Ueda, Y. Kato, K. Komatsu, and Y. Sugiyama. Inhibition of biliary excretion of methotrexate by probenecid in rats: quantitative prediction of interaction from in vitro data. J. Pharmacol. Exp. Ther. 297:1036–1043 (2001).

    PubMed  CAS  Google Scholar 

  24. E. S. Henderson, R. H. Adamson, C. Denham, and V. T. Oliverio. The metabolic fate of tritiated methotrexate 1. Absorption, excretion, and distribution in mice, rats, dogs and monkeys. Cancer Res. 25:1008–1017 (1965).

    PubMed  CAS  Google Scholar 

  25. A. Lorico, G. Rappa, R. A. Finch, D. Yang, R. A. Flavell, and A. C. Sartorelli. Disruption of the murine MRP (multidrug resistance protein) gene leads to increased sensitivity to etoposide (VP-16) and increased levels of glutathione. Cancer Res. 57:5238–5242 (1997).

    PubMed  CAS  Google Scholar 

  26. L. J. Bain, and R. A. Feldman. Altered expression of sulfotransferases, glucuronosyltransferases and mrp transporters in FVB/mrp1-/- mice. Xenobiotica. 33:1173–1183 (2003) doi:10.1080/00498250310001609138.

    PubMed  Article  CAS  Google Scholar 

  27. H. Cheng, and C. P. Leblond. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian Theory of the origin of the four epithelial cell types. Am. J. Anat. 141:537–561 (1974) doi:10.1002/aja.1001410407.

    PubMed  Article  CAS  Google Scholar 

  28. C. A. Loehry, D. N. Croft, A. K. Singh, and B. Creamer. Cell turnover in the rat small intestinal mucosa: an appraisal of cell loss. Gut. 10:13–16 (1969) doi:10.1136/gut.10.1.13.

    PubMed  Article  CAS  Google Scholar 

  29. I. B. Renes, M. Verburg, N. P. Bulsing, S. Ferdinandusse, H. A. Büller, J. Dekker, and A. W. Einerhand. Protection of the Peyer's patch-associated crypt and villus epithelium against methotrexate-induced damage is based on its distinct regulation of proliferation. J. Pathol. 198:60–68 (2002) doi:10.1002/path.1183.

    PubMed  Article  Google Scholar 

  30. Y. Miyazono, F. Gao, and T. Horie. Oxidative stress contributes to methotrexate-induced small intestinal toxicity in rats. Scand. J. Gastroenterol. 39:1119–1127 (2004) doi:10.1080/00365520410003605.

    PubMed  Article  CAS  Google Scholar 

  31. B. A. de Koning, J. M. van Dieren, D. J. Lindenbergh-Kortleve, M. van der Sluis, T. Matsumoto, K. Yamaguchi, A. W. Einerhand, J. N. Samsom, R. Pieters, and E. E. Nieuwenhuis. Contributions of mucosal immune cells to methotrexate-induced mucositis. 1. Int. Immunol. 18:941–949 (2006) doi:10.1093/intimm/dxl030.

    PubMed  Article  Google Scholar 

  32. B. A. de Koning, M. Sluis, D. J. Lindenbergh-Kortleve, A. Velcich, R. Pieters, H. A. Büller, A. W. Einerhand, and I. B. Renes. Methotrexate-induced mucositis in mucin 2-deficient mice. J. Cell. Physiol. 210:144–152 (2007) doi:10.1002/jcp.20822.

    PubMed  Article  Google Scholar 

  33. J. Wijnholds, G. L. Scheffer, M. van der Valk, P. van der Valk, J. H. Beijnen, R. J. Scheper, and P. Borst. Multidrug resistance protein 1 protects the oropharyngeal mucosal layer and the testicular tubules against drug-induced damage. J. Exp. Med. 188:797–808 (1998) doi:10.1084/jem.188.5.797.

    PubMed  Article  CAS  Google Scholar 

  34. M. G. Belinsky, P. Guo, K. Lee, F. Zhou, E. Kotova, A. Grinberg, H. Westphal, I. Shchaveleva, A. Klein-Szanto, J. M. Gallo, and G. D. Kruh. Multidrug resistance protein 4 protects bone marrow, thymus, spleen, and intestine from nucleotide analogue-induced damage. Cancer Res. 67:262–268 (2007) doi:10.1158/0008-5472.CAN-06-2680.

    PubMed  Article  CAS  Google Scholar 

  35. Y. Mochida, K. Taguchi, S. Taniguchi, M. Tsuneyoshi, H. Kuwano, T. Tsuzuki, M. Kuwano, and M. Wada. The role of P-glycoprotein in intestinal tumorigenesis: disruption of mdr1a suppresses polyp formation in Apc(Min/+) mice. Carcinogenesis. 24:1219–1224 (2003) doi:10.1093/carcin/bgg073.

    PubMed  Article  CAS  Google Scholar 

  36. H. Wang, X. Wu, K. Hudkins, A. Mikheev, H. Zhang, A. Gupta, J. D. Unadkat, and Q. Mao. Expression of the breast cancer resistance protein (Bcrp1/Abcg2) in tissues from pregnant mice: effects of pregnancy and correlations with nuclear receptors. Am. J. Physiol. Endocrinol. Metab. 291:E1295–E1304 (2006) doi:10.1152/ajpendo.00193.2006.

    PubMed  Article  CAS  Google Scholar 

  37. H. Bleiberg. CPT-11 in gastrointestinal cancer. Eur. J. Cancer. 35:371–379 (1999) doi:10.1016/S0959-8049(98)00423-7.

    PubMed  Article  CAS  Google Scholar 

  38. J. T. Hartmann, and H. P. Lipp. Camptothecin and podophyllotoxin derivatives: inhibitors of topoisomerase I and II—mechanisms of action, pharmacokinetics and toxicity profile. Drug Saf. 29:209–230 (2006) doi:10.2165/00002018-200629030-00005.

    PubMed  Article  CAS  Google Scholar 

  39. Z. S. Chen, T. Furukawa, T. Sumizawa, K. Ono, K. Ueda, K. Seto, and S. I. Akiyama. ATP-Dependent efflux of CPT-11 and SN-38 by the multidrug resistance protein (MRP) and its inhibition by PAK-104P. Mol. Pharmacol. 55:921–928 (1999).

    PubMed  CAS  Google Scholar 

Download references

Acknowledgement

We thank Ms. Lica Ishida and Mr. Syuichi Yamazaki for technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research provided by the Ministry of Education, Science and Culture of Japan and funds from the Kanehara Grant Program 2004 of the Ichiro Kanehara Foundation (Tokyo, Japan).

Author information

Affiliations

  1. Division of Pharmaceutical Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa, 920–1192, Japan

    Sayaka Kato, Katsuaki Ito, Yukio Kato, Yoshiyuki Kubo & Akira Tsuji

  2. Department of Histology and Embryology, Graduate School of Medical Science, Kanazawa University, 13–1 Takara-machi, Kanazawa, Ishikawa, 920–8640, Japan

    Tomohiko Wakayama & Shoichi Iseki

Authors
  1. Sayaka Kato
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Katsuaki Ito
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yukio Kato
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tomohiko Wakayama
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yoshiyuki Kubo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shoichi Iseki
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Akira Tsuji
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Akira Tsuji.

Electronic supplementary material

Below is the image is a link to a high resolution version.

ESM 1
figure 5

(GIF 489 kb)

Full size image

ESM 1

(TIF 134 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kato, S., Ito, K., Kato, Y. et al. Involvement of Multidrug Resistance-Associated Protein 1 in Intestinal Toxicity of Methotrexate. Pharm Res 26, 1467–1476 (2009). https://doi.org/10.1007/s11095-009-9858-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-009-9858-6

KEY WORDS

  • intestinal crypt compartment cells
  • methotrexate
  • MRP1
  • toxicity
  • transporters