Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Fluorescence probes to detect lipid-derived radicals

Nature Chemical Biology volume 12, pages 608–613 (2016)Cite this article

Subjects

Abstract

Lipids and their metabolites are easily oxidized in chain reactions initiated by lipid radicals, forming lipid peroxidation products that include the electrophiles 4-hydroxynonenal and malondialdehyde. These markers can bind cellular macromolecules, causing inflammation, apoptosis and other damage. Methods to detect and neutralize the initiating radicals would provide insights into disease mechanisms and new therapeutic approaches. We describe the first high-sensitivity, specific fluorescence probe for lipid radicals, 2,2,6-trimethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)-6-pentylpiperidine-1-oxyl (NBD-Pen). NBD-Pen directly detected lipid radicals in living cells by turn-on fluorescence. In a rat model of hepatic carcinoma induced by diethylnitrosamine (DEN), NBD-Pen detected lipid radical generation within 1 h of DEN administration. The lipid radical scavenging moiety of NBD-Pen decreased inflammation, apoptosis and oxidative stress markers at 24 h after DEN, and liver tumor development at 12 weeks. Thus, we have developed a novel fluorescence probe that provides imaging information about lipid radical generation and potential therapeutic benefits in vivo.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Spectroscopic responses and selectivity for lipid radicals.
Figure 2: Liver cell fluorescence imaging and in vivo detection of lipid radicals produced during DEN metabolism.
Figure 3: OH-Pen, but not OH-Pen-NOMe (NOMe), suppresses DEN-induced HCC.
Figure 4: Influence of OH-Pen and OH-Pen-NOMe (NOMe) on hepatic injury and compensatory cell proliferation.
Figure 5

References

  1. Yin, H., Xu, L. & Porter, N.A. Free radical lipid peroxidation: mechanisms and analysis. Chem. Rev. 111, 5944–5972 (2011).

    Article  CAS  Google Scholar 

  2. Niki, E. Biomarkers of lipid peroxidation in clinical material. Biochim. Biophys. Acta 1840, 809–817 (2014).

    Article  CAS  Google Scholar 

  3. West, X.Z. et al. Oxidative stress induces angiogenesis by activating TLR2 with novel endogenous ligands. Nature 467, 972–976 (2010).

    Article  CAS  Google Scholar 

  4. Rudolph, T.K. & Freeman, B.A. Transduction of redox signaling by electrophile-protein reactions. Sci. Signal. 2, re7 (2009).

    Article  Google Scholar 

  5. Buettner, G.R. Spin trapping: ESR parameters of spin adducts. Free Radic. Biol. Med. 3, 259–303 (1987).

    Article  CAS  Google Scholar 

  6. Bagryanskaya, E.G. & Marque, S.R. Scavenging of organic C-centered radicals by nitroxides. Chem. Rev. 114, 5011–5056 (2014).

    Article  CAS  Google Scholar 

  7. Blinco, J.P., Fairfull-Smith, K.E., Morrow, B.J. & Bottle, S.E. Profluorescent nitroxides as sensitive probes of oxidative change and free radical reactions. Aust. J. Chem. 64, 373–389 (2011).

    Article  CAS  Google Scholar 

  8. Jia, M., Tang, Y., Lam, Y.F., Green, S.A. & Blough, N.V. Prefluorescent nitroxide probe for the highly sensitive determination of peroxyl and other radical oxidants. Anal. Chem. 81, 8033–8040 (2009).

    Article  CAS  Google Scholar 

  9. Mito, F. et al. Oxygen concentration dependence of lipid peroxidation and lipid-derived radical generation: application of profluorescent nitroxide switch. Free Radic. Res. 45, 1103–1110 (2011).

    Article  CAS  Google Scholar 

  10. Abbas, K. et al. Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps. Free Radic. Biol. Med. 71, 281–290 (2014).

    Article  CAS  Google Scholar 

  11. Bartsch, H., Hietanen, E. & Malaveille, C. Carcinogenic nitrosamines: free radical aspects of their action. Free Radic. Biol. Med. 7, 637–644 (1989).

    Article  CAS  Google Scholar 

  12. Hebels, D.G., Briedé, J.J., Khampang, R., Kleinjans, J.C. & de Kok, T.M. Radical mechanisms in nitrosamine- and nitrosamide-induced whole-genome gene expression modulations in Caco-2 cells. Toxicol. Sci. 116, 194–205 (2010).

    Article  CAS  Google Scholar 

  13. Yamada, K., Yamamiya, I. & Utsumi, H. In vivo detection of free radicals induced by diethylnitrosamine in rat liver tissue. Free Radic. Biol. Med. 40, 2040–2046 (2006).

    Article  CAS  Google Scholar 

  14. Negre-Salvayre, A., Coatrieux, C., Ingueneau, C. & Salvayre, R. Advanced lipid peroxidation end products in oxidative damage to proteins. Potential role in diseases and therapeutic prospects for the inhibitors. Br. J. Pharmacol. 153, 6–20 (2008).

    Article  CAS  Google Scholar 

  15. Farazi, P.A. & DePinho, R.A. Hepatocellular carcinoma pathogenesis: from genes to environment. Nat. Rev. Cancer 6, 674–687 (2006).

    Article  CAS  Google Scholar 

  16. Naugler, W.E. et al. Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Science 317, 121–124 (2007).

    Article  CAS  Google Scholar 

  17. Nakae, D. et al. Involvement of 8-hydroxyguanine formation in the initiation of rat liver carcinogenesis by low dose levels of N-nitrosodiethylamine. Cancer Res. 57, 1281–1287 (1997).

    CAS  PubMed  Google Scholar 

  18. Sakurai, T., Maeda, S., Chang, L. & Karin, M. Loss of hepatic NF-kappa B activity enhances chemical hepatocarcinogenesis through sustained c-Jun N-terminal kinase 1 activation. Proc. Natl. Acad. Sci. USA 103, 10544–10551 (2006).

    Article  CAS  Google Scholar 

  19. Hui, L., Zatloukal, K., Scheuch, H., Stepniak, E. & Wagner, E.F. Proliferation of human HCC cells and chemically induced mouse liver cancers requires JNK1-dependent p21 downregulation. J. Clin. Invest. 118, 3943–3953 (2008).

    Article  CAS  Google Scholar 

  20. Uchida, K. et al. Activation of stress signaling pathways by the end product of lipid peroxidation. 4-hydroxy-2-nonenal is a potential inducer of intracellular peroxide production. J. Biol. Chem. 274, 2234–2242 (1999).

    Article  CAS  Google Scholar 

  21. Parola, M. et al. HNE interacts directly with JNK isoforms in human hepatic stellate cells. J. Clin. Invest. 102, 1942–1950 (1998).

    Article  CAS  Google Scholar 

  22. Bognár, B., Osz, E., Hideg, K. & Kalai, T. Synthesis of new double (spin and fluorescence) sensor reagents and labels. J. Heterocycl. Chem. 43, 81–86 (2006).

    Article  Google Scholar 

  23. Lin, S. & Struve, W.S. Time-resolved fluorescence of nitrobenzoxadiazole-aminohexanoic acid: effect of intermolecular hydrogen-bonding on non-radiative decay. Photochem. Photobiol. 54, 361–365 (1991).

    Article  CAS  Google Scholar 

  24. Yamasaki, T. et al. Structure-reactivity relationship of piperidine nitroxide: electrochemical, ESR and computational studies. J. Org. Chem. 76, 435–440 (2011).

    Article  CAS  Google Scholar 

  25. Yamasaki, T. et al. Structural concept of nitroxide as a lipid peroxidation inhibitor. J. Org. Chem. 76, 4144–4148 (2011).

    Article  CAS  Google Scholar 

  26. Blinco, J.P. et al. Experimental and theoretical studies of the redox potentials of cyclic nitroxides. J. Org. Chem. 73, 6763–6771 (2008).

    Article  CAS  Google Scholar 

  27. Yamasaki, T., Matsuoka, Y., Mito, F., Yamato, M. & Yamada, K. Redox potential of nitroxides is an index to evaluate superoxide dismutase mimic activity. Asian J. Org. Chem. 2, 388–391 (2013).

    Article  CAS  Google Scholar 

  28. Thaler, S. et al. Neuroprotective effects of Tempol acyl esters against retinal ganglion cell death in a rat partial optic nerve crush model. Acta Ophthalmol. 89, e555–e560 (2011).

    Article  CAS  Google Scholar 

  29. Wagner, B.A., Buettner, G.R. & Burns, C.P. Free radical-mediated lipid peroxidation in cells: oxidizability is a function of cell lipid bis-allylic hydrogen content. Biochemistry 33, 4449–4453 (1994).

    Article  CAS  Google Scholar 

  30. Krishna, M.C. et al. Do nitroxide antioxidants act as scavengers of O2−• or as SOD mimics? J. Biol. Chem. 271, 26026–26031 (1996).

    Article  CAS  Google Scholar 

  31. Lee, J.S. et al. Application of comparative functional genomics to identify best-fit mouse models to study human cancer. Nat. Genet. 36, 1306–1311 (2004).

    Article  CAS  Google Scholar 

  32. Maeda, S., Kamata, H., Luo, J.L., Leffert, H. & Karin, M. IKKβ couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis. Cell 121, 977–990 (2005).

    Article  CAS  Google Scholar 

  33. Factor, V.M. et al. Vitamin E reduces chromosomal damage and inhibits hepatic tumor formation in a transgenic mouse model. Proc. Natl. Acad. Sci. USA 97, 2196–2201 (2000).

    Article  CAS  Google Scholar 

  34. Park, E.J. et al. Dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing IL-6 and TNF expression. Cell 140, 197–208 (2010).

    Article  CAS  Google Scholar 

  35. Blair, I.A. DNA adducts with lipid peroxidation products. J. Biol. Chem. 283, 15545–15549 (2008).

    Article  CAS  Google Scholar 

  36. Marnett, L.J., Riggins, J.N. & West, J.D. Endogenous generation of reactive oxidants and electrophiles and their reactions with DNA and protein. J. Clin. Invest. 111, 583–593 (2003).

    Article  CAS  Google Scholar 

  37. Pollack, M., Yang, I.Y., Kim, H.Y., Blair, I.A. & Moriya, M. Translesion DNA Synthesis across the heptanone--etheno-2′-deoxycytidine adduct in cells. Chem. Res. Toxicol. 19, 1074–1079 (2006).

    Article  CAS  Google Scholar 

  38. Soresi, M. et al. Interleukin-6 and its soluble receptor in patients with liver cirrhosis and hepatocellular carcinoma. World J. Gastroenterol. 12, 2563–2568 (2006).

    Article  CAS  Google Scholar 

  39. Nagata, H. et al. Inhibition of c-Jun NH2-terminal kinase switches Smad3 signaling from oncogenesis to tumor- suppression in rat hepatocellular carcinoma. Hepatology 49, 1944–1953 (2009).

    Article  CAS  Google Scholar 

  40. Seki, E., Brenner, D.A. & Karin, M. A liver full of JNK: signaling in regulation of cell function and disease pathogenesis, and clinical approaches. Gastroenterology 143, 307–320 (2012).

    Article  CAS  Google Scholar 

  41. Gorrini, C., Harris, I.S. & Mak, T.W. Modulation of oxidative stress as an anticancer strategy. Nat. Rev. Drug Discov. 12, 931–947 (2013).

    Article  CAS  Google Scholar 

  42. Seglen, P.O. Preparation of isolated rat liver cells. Methods Cell Biol. 13, 29–83 (1976).

    Article  CAS  Google Scholar 

  43. Bligh, E.G. & Dyer, W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917 (1959).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported in part by JST PRESTO, by JSPS KAKENHI grant number 24390011 and 24659020, by the Platform Project for Supporting in Drug Discovery and Life Science Research from AMED, Japan, by the Suzuken Memorial Foundation, Nagoya, Japan, and by the Suntory Foundation for Life Sciences, Kyoto, Japan. We appreciate the technical support provided by the Research Support Center, Graduate School of Medical Sciences, Kyushu University.

Author information

Author notes

  1. Toshihide Yamasaki

    Present address: Current address: Aix-Marseille Université, Institut de Chimie Radicalaire, Campus St.-Jérôme, Marseille, France.,

Authors and Affiliations

  1. Physical Chemistry for Life Science Laboratory, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan

    Ken-ichi Yamada, Fumiya Mito, Yuta Matsuoka, Satsuki Ide, Kazushige Shikimachi, Ayano Fujiki, Daiki Kusakabe, Yuma Ishida, Masataka Enoki, Arisa Tada, Miyuki Ariyoshi & Toshihide Yamasaki

  2. Japan Science and Technology Agency, PRESTO, Saitama, Japan

    Ken-ichi Yamada

  3. Innovation Center for Medical Redox Navigation, Kyushu University, Fukuoka, Japan

    Mayumi Yamato

Authors
  1. Ken-ichi Yamada
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fumiya Mito
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuta Matsuoka
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Satsuki Ide
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kazushige Shikimachi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ayano Fujiki
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Daiki Kusakabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yuma Ishida
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Masataka Enoki
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Arisa Tada
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Miyuki Ariyoshi
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Toshihide Yamasaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Mayumi Yamato
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.Y. designed experiments and conceived and supervised the overall project. K.Y. also performed final data analysis and wrote the manuscript. F.M., Y.M., K.S. and Y.I. performed experiments and analyzed data for properties and mechanistic analysis of probes. S.I., A.F. and A.T. performed experiments and analyzed data for animal experiments. D.K. and M.E. performed experiments and analyzed data for cell experiments. M.A. performed experiments early in the project. T.Y. designed and performed chemical syntheses and wrote the synthetic methods for the manuscript. M.Y. designed experiments and performed final data analysis.

Corresponding author

Correspondence to Ken-ichi Yamada.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Table 1 and Supplementary Figures 1–16. (PDF 19711 kb)

Supplementary Note

Synthetic procedures (PDF 190 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yamada, Ki., Mito, F., Matsuoka, Y. et al. Fluorescence probes to detect lipid-derived radicals. Nat Chem Biol 12, 608–613 (2016). https://doi.org/10.1038/nchembio.2105

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2105

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer