Imaging Mass Spectrometry Revealed the Accumulation Characteristics of the 2-Nitroimidazole-Based Agent "Pimonidazole" in Hypoxia

PLoS One. 2016 Aug 31;11(8):e0161639. doi: 10.1371/journal.pone.0161639. eCollection 2016.

Abstract

Hypoxia, or low oxygen concentration, is a key factor promoting tumor progression and angiogenesis and resistance of cancer to radiotherapy and chemotherapy. 2-Nitroimidazole-based agents have been widely used in pathological and nuclear medicine examinations to detect hypoxic regions in tumors; in particular, pimonidazole is used for histochemical staining of hypoxic regions. It is considered to accumulate in hypoxic cells via covalent binding with macromolecules or by forming reductive metabolites after reduction of its nitro group. However, the detailed mechanism of its accumulation remains unknown. In this study, we investigated the accumulation mechanism of pimonidazole in hypoxic tumor tissues in a mouse model by mass spectrometric analyses including imaging mass spectrometry (IMS). Pimonidazole and its reductive metabolites were observed in the tumor tissues. However, their locations in the tumor sections were not similar to the positively stained areas in pimonidazole-immunohistochemistry, an area considered hypoxic. The glutathione conjugate of reduced pimonidazole, a low-molecular-weight metabolite of pimonidazole, was found in tumor tissues by LC-MS analysis, and our IMS study determined that the intratumor localization of the glutathione conjugate was consistent with the area positively immunostained for pimonidazole. We also found complementary localization of the glutathione conjugate and reduced glutathione (GSH), implying that formation of the glutathione conjugate occurred in the tumor tissue. These results suggest that in hypoxic tumor cells, pimonidazole is reduced at its nitro group, followed by conjugation with GSH.

MeSH terms

  • Animals
  • Cell Hypoxia / drug effects
  • Glutathione / metabolism*
  • Humans
  • Male
  • Mass Spectrometry*
  • Mice
  • Mice, Inbred BALB C
  • Mice, Nude
  • Neoplasms, Experimental* / diagnostic imaging
  • Neoplasms, Experimental* / drug therapy
  • Neoplasms, Experimental* / metabolism
  • Nitroimidazoles* / pharmacokinetics
  • Nitroimidazoles* / pharmacology

Substances

  • Nitroimidazoles
  • pimonidazole
  • Glutathione

Grants and funding

This study was supported by the Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program, Ministry of Education, Culture, Sports, Science and Technology, Japan (http://www.jst.go.jp/shincho/sentanyugo/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This study was also funded by Shionogi & Co., Ltd. (http://www.shionogi.co.jp/index.html). The funder provided support in the form of salaries for authors [YM TY KH YN], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.