Development of Novel Fluorescence Probes That Can Reliably Detect Reactive Oxygen Species and Distinguish Specific Species

We designed and synthesized 2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2- [6-(4′-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF) as novel fluorescence probes to detect selectively highly reactive oxygen species (hROS) such as hydroxyl radical (⋅OH) and reactive intermediates of peroxidase. Although HPF and APF themselves scarcely fluoresced, APF selectively and dose-dependently afforded a strongly fluorescent compound, fluorescein, upon reaction with hROS and hypochlorite (−OCl), but not other reactive oxygen species (ROS). HPF similarly afforded fluorescein upon reaction with hROS only. Therefore, not only can hROS be differentiated from hydrogen peroxide (H2O2), nitric oxide (NO), and superoxide (O2⨪) by using HPF or APF alone, but −OCl can also be specifically detected by using HPF and APF together. Furthermore, we applied HPF and APF to living cells and found that HPF and APF were resistant to light-induced autoxidation, unlike 2′,7′-dichlorodihydrofluorescein, and for the first time we could visualize −OCl generated in stimulated neutrophils. HPF and APF should be useful as tools to study the roles of hROS and−OCl in many biological and chemical applications. We designed and synthesized 2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2- [6-(4′-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF) as novel fluorescence probes to detect selectively highly reactive oxygen species (hROS) such as hydroxyl radical (⋅OH) and reactive intermediates of peroxidase. Although HPF and APF themselves scarcely fluoresced, APF selectively and dose-dependently afforded a strongly fluorescent compound, fluorescein, upon reaction with hROS and hypochlorite (−OCl), but not other reactive oxygen species (ROS). HPF similarly afforded fluorescein upon reaction with hROS only. Therefore, not only can hROS be differentiated from hydrogen peroxide (H2O2), nitric oxide (NO), and superoxide (O2⨪) by using HPF or APF alone, but −OCl can also be specifically detected by using HPF and APF together. Furthermore, we applied HPF and APF to living cells and found that HPF and APF were resistant to light-induced autoxidation, unlike 2′,7′-dichlorodihydrofluorescein, and for the first time we could visualize −OCl generated in stimulated neutrophils. HPF and APF should be useful as tools to study the roles of hROS and−OCl in many biological and chemical applications. reactive oxygen species 2′,7′-dichlorodihydrofluorescein 2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid 2-[6-(4′-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid horseradish peroxidase 4β-phorbol-12-myristate-13-acetate 2′,7′-dichlorodihydrofluorescein diacetate 10-acetyl-3,7-dihydroxyphenoxazine 3-(4-hydroxyphenyl)propionic acid myeloperoxidase N,N-dimethylformamide human hepatocellular carcinoma cell line charge coupled device highly reactive oxygen species alkylperoxyl radical nitric oxide superoxide radical Reactive oxygen species (ROS)1 play key roles in many pathogenic processes, including carcinogenesis (1Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Google Scholar), inflammation (2McCord J.M. Science. 1974; 185: 529-531Google Scholar), ischemia-reperfusion injury (3Dobashi K. Ghosh B. Orak J.K. Singh I. Singh A.K. Mol. Cell. Biochem. 2000; 205: 1-11Google Scholar), and signal transduction (4Schmidt K.N. Amstad P. Cerutti P. Baeuerle P.A. Chem. Biol. 1995; 2: 13-22Google Scholar, 5Yermolaieva O. Brot N. Weissbach H. Heinemann S.H. Hoshi T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 448-453Google Scholar, 6Frohlich K.U. Madeo F. FEBS Lett. 2000; 473: 6-9Google Scholar, 7Nishida M. Maruyama Y. Tanaka R. Kontani K. Nagao T. Kurose H. Nature. 2000; 408: 492-495Google Scholar). Several methods, including electron spin resonance (8Kuppusamy P. Chzhan M. Vij K. Shteynbuk M. Lefer D.J. Giannella E. Zweier J.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3388-3392Google Scholar) and chemiluminescence (9Yasui H. Sakurai H. Biochem. Biophys. Res. Commun. 2000; 269: 131-136Google Scholar), have been developed to detect ROS, but fluorescence detection is superior in terms of high sensitivity and experimental convenience. Experimental studies on Ca2+-dependent signal transduction in cells were greatly facilitated by the development of fluorescent indicators for cytosolic Ca2+ (10Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Google Scholar, 11Minta A. Kao J.P.Y. Tsien R.Y. J. Biol. Chem. 1989; 264: 8171-8178Google Scholar). Several fluorescence probes to detect ROS, such as 2′,7′-dichlorodihydrofluorescein (DCFH) and dihydrorhodamine 123, have also been developed. However, as Hempel and co-workers (12Hempel S.L. Buettner G.R. O'Malley Y.Q. Wessels D.A. Flaherty D.M. Free Radical Biol. Med. 1999; 27: 146-159Google Scholar) pointed out, DCFH and dihydrorhodamine 123 can react with various ROS and oxidizing species (superoxide (O2⨪), hydrogen peroxide (H2O2), nitric oxide (NO), ferrous ion, and others), and in addition, DCFH is easily autoxidized, resulting in a spontaneous increase in fluorescence upon exposure to light. Therefore, it is not appropriate to think of these probes as detecting a specific oxidizing species in cells, such as H2O2 or NO, but rather they should be considered as detecting a broad range of oxidizing reactions that may be increased during intracellular oxidative stress (12Hempel S.L. Buettner G.R. O'Malley Y.Q. Wessels D.A. Flaherty D.M. Free Radical Biol. Med. 1999; 27: 146-159Google Scholar).There are many species of ROS, as mentioned above, but they tend to be considered collectively as “oxidative stress” when their effects in living cells are discussed. However, we believe that each species of ROS is likely to have a specific role in living cells. There is some evidence for this view. For example, H2O2 is an endothelium-derived hyperpolarizing factor in human and mice (13Matoba T. Shimokawa H. Nakashima M. Hirakawa Y. Mukai Y. Hirano K. Kanaide H. Takeshita A. J. Clin. Invest. 2000; 106: 1521-1530Google Scholar), p38 mitogen-activated protein kinase mediates caspase-3 activation during apoptosis induced by singlet oxygen (1O2) but not by H2O2 (14Zhuang S.G. Demirs J.T. Kochevar I.E. J. Biol. Chem. 2000; 275: 25939-25948Google Scholar), and hydroxyl radical (⋅OH) plays an important role as a second messenger in T cell activation (15Tatla S. Woodhead V. Foreman J.C. Chain B.M. Free Radical Biol. Med. 1999; 26: 14-24Google Scholar). In addition, each species of ROS has a characteristic chemical reactivity; for example, 1O2 reacts with anthracenes to yield endoperoxides in the Diels-Alder mode (16Umezawa N. Tanaka K. Urano Y. Kikuchi K. Higuchi T. Nagano T. Angew. Chem. Int. Ed. 1999; 38: 2899-2901Google Scholar), whereas ⋅OH can react directly with aromatic rings to yield hydroxylated products (17Coudray C. Favier A. Free Radical Biol. Med. 2000; 29: 1064-1070Google Scholar), and NO reacts with guanine to yield the deaminated compound (18Grishko V.I. Druzhyna N. LeDoux S.P. Wilson G.L. Nucleic Acids Res. 1999; 27: 4510-4516Google Scholar). However, because of the problems,i.e. lack of selectivity among species and autoxidation (12Hempel S.L. Buettner G.R. O'Malley Y.Q. Wessels D.A. Flaherty D.M. Free Radical Biol. Med. 1999; 27: 146-159Google Scholar,19Rota C. Fann Y.C. Mason R.P. J. Biol. Chem. 1999; 274: 28161-28168Google Scholar, 20Rota C. Chignell C.F. Mason R.P. Free Radical Biol. Med. 1999; 27: 873-881Google Scholar), the roles of an individual species of ROS in living cells remain uncertain. Therefore, we believe that it is very important to be able to detect each species of ROS selectively. If novel fluorescence probes that overcome the above problems were available, they would contribute greatly to the elucidation of the roles of individual ROS in living cells, because we would be able to “see” the generation of specific ROS with high resolution in time and space.It is known that ⋅OH participates in various biological processes. For example, HeLa, MW451, and HL-60 cells are induced to undergo apoptosis by ⋅OH (21Ren J.-G. Xia H.-L. Just T. Dai Y.-R. FEBS Lett. 2001; 488: 123-132Google Scholar). ⋅OH can damage DNA bases (1Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Google Scholar) and mediates redox alteration of cell-membrane Ca2+channels (22Az-ma T. Saeki N. Yuge O. Br. J. Pharmacol. 1999; 126: 1462-1470Google Scholar). However, because of the lack of effective direct detection methods for ⋅OH, its participation in these events has been established only indirectly by using inhibitors such as dithioethanol, glutathione, and desferrioxamine (1Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Google Scholar, 15Tatla S. Woodhead V. Foreman J.C. Chain B.M. Free Radical Biol. Med. 1999; 26: 14-24Google Scholar, 21Ren J.-G. Xia H.-L. Just T. Dai Y.-R. FEBS Lett. 2001; 488: 123-132Google Scholar, 22Az-ma T. Saeki N. Yuge O. Br. J. Pharmacol. 1999; 126: 1462-1470Google Scholar). Therefore, we wished to develop novel fluorescence probes for highly reactive oxygen species (hROS). Here, we use the term hROS to indicate reactive oxygen species with strong oxidizing power sufficient to directly hydroxylate aromatic rings (for example, ⋅OH or reactive intermediates of peroxidase).We report herein the development of novel fluorescence probes for ROS, 2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2-[6-(4′-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF), which can specifically detect certain species of ROS in terms of an increase of fluorescence and exhibit complete resistance to autoxidation both in vitro and in vivo. We also describe the visualization of hypochlorite (−OCl) in stimulated neutrophils.DISCUSSIONWe have succeeded in developing novel autoxidation-resistant fluorescence probes, HPF and APF, that can reliably detect hROS and/or−OCl selectively. Because it is likely that individual ROS have distinct roles in biological systems, the availability of selective fluorescence probes will be extremely useful. For example, by using HPF or APF, we can distinguish ⋅OH from NO. This is very important, because DCFH reacts with both ⋅OH and NO and so cannot be used reliably to study the biological role of ⋅OH. In addition, the mere production of H2O2 is completely different in terms of cell damage from the situation in which H2O2 is converted into hROS in the presence of low-valent metal ions. We feel our probes are useful here, because they can distinguish these two situations. Furthermore, we can also distinguish ONOO− from NO or O2⨪. It has been reported that ONOO− can be generated from NO and O2⨪ in vitro and in vivo (46Blough N.V. Zafiriou O.C. Inorg. Chem. 1985; 24: 3502-3504Google Scholar, 47Xia Y. Dawson V.L. Dawson T.M. Snyder S.H. Zweier J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6770-6774Google Scholar), and therefore we will be able to visualize the production of ONOO− with a clear distinction from that of NO or O2⨪, and this will allow a reliable evaluation of the role of ONOO− in various processes. Furthermore, we could detect−OCl selectively by using HPF and APF together, because HPF shows no fluorescence increase with −OCl, whereas APF shows a dose-dependent increase. The ability to selectively detect individual species of ROS represents a major advance.As shown in Table I and Fig. 4, the currently used fluorescence probe DCFH is easily autoxidized by light irradiation. This means that precautions must be taken to exclude light during incubation to load DCFH-DA into cells, and it is necessary to change the visual field often during observations. However, HPF and APF are not autoxidized at all, as shown in Table I and Fig. 4. Therefore, we believe HPF and APF will contribute greatly to the elucidation of the roles of ROS in living cells by making it possible to see the generation of specific ROS with high resolution in time and space. Although the sensitivity of HPF and APF is inferior to that of DCFH (Table I), lability to autoxidation and selectivity among ROS, rather than sensitivity, are considered to be critical for fluorescence probes for ROS.The question arises, why are HPF and APF selective for hROS, unlike DCFH? DCFH is nonfluorescent, and HPF and APF possess low fluorescence quantum efficiency, and all of them are converted to strongly fluorescent compounds, dichlorofluorescein or fluorescein, by oxidation. However, DCFH is converted to dichlorofluorescein, initially via abstraction of the hydrogen atom at the 9′-position, whereas HPF and APF are converted to fluorescein, initially via abstraction of the hydrogen atom of the phenolic hydroxy group or abstraction of one electron from the nitrogen atom. The hydrogen atom at the 9′-position of DCFH is readily abstracted because this hydrogen atom can be considered as being located at the central carbon of a triphenylmethane. It is therefore vulnerable even to a weakly oxidizing species, and this is the reason why DCFH lacks the selectivity among ROS. However, a strongly oxidizing species is required for theipso-substitution reaction of HPF and APF. Therefore, we conclude that the difference of oxidizing power required for oxidation reaction used for detection causes the difference of selectivity among ROS. Furthermore, the fact that HPF shows no fluorescence increase with−OCl, whereas APF does (Fig. 3 and Table I), reflects the difference in lability to oxidation between an aryloxyphenol and an aryloxyaniline.HPF and APF could detect hROS generated in the HRP/H2O2 system (Fig. 5). HRP is often used as an enzyme label in immunohistochemical studies, 3,3-diaminobenzidine is commonly used as a substrate for measurement of the peroxidase activity. However, 3,3-diaminobenzidine can be detected only by absorbance measurement and is easily autoxidized by light irradiation. Because HPF and APF permit fluorescence detection, which has higher sensitivity than absorbance detection, and they are not autoxidized by light irradiation at all, they are likely to be more effective reagents for immunohistochemistry using peroxidase than 3,3-diaminobenzidine and related compounds.We also used HPF and APF to visualize the production of−OCl from neutrophils (Fig. 8). Dye-loaded neutrophils weakly fluoresced before the stimulation with PMA, because the dyes were taken up by pinocytosis and MPO was slightly released into pinocytic vacuoles. Nevertheless, the fluorescence intensity of APF-loaded neutrophils markedly increased, in contrast to little fluorescence increase of HPF-loaded cells upon stimulation with PMA.−OCl is believed to play important roles not only in bacterial killing bacteria by neutrophils but also in injury to the venular endothelial surface in platelet-activating factor-induced microvascular damage (48Suematsu M. Kurose I. Asako H. Miura S. Tsuchiya M. J. Biochem. 1989; 106: 355-360Google Scholar). However, it has been difficult to draw firm conclusions concerning direct participation of −OCl because a completely selective detection method for −OCl has never been developed. Therefore, our finding that we could detect−OCl selectively by using HPF and APF together will make it possible for the first time to elucidate reliably the roles of−OCl in biological systems such as neutrophils.In summary, we have developed novel fluorescence probes, HPF and APF, that can selectively and dose dependently detect certain species among ROS and that are highly resistant to autoxidation. They can be used in enzymatic and cellular systems. They are greatly superior to the existing fluorescence probes for ROS, and are expected to have many chemical and biological applications. Reactive oxygen species (ROS)1 play key roles in many pathogenic processes, including carcinogenesis (1Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Google Scholar), inflammation (2McCord J.M. Science. 1974; 185: 529-531Google Scholar), ischemia-reperfusion injury (3Dobashi K. Ghosh B. Orak J.K. Singh I. Singh A.K. Mol. Cell. Biochem. 2000; 205: 1-11Google Scholar), and signal transduction (4Schmidt K.N. Amstad P. Cerutti P. Baeuerle P.A. Chem. Biol. 1995; 2: 13-22Google Scholar, 5Yermolaieva O. Brot N. Weissbach H. Heinemann S.H. Hoshi T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 448-453Google Scholar, 6Frohlich K.U. Madeo F. FEBS Lett. 2000; 473: 6-9Google Scholar, 7Nishida M. Maruyama Y. Tanaka R. Kontani K. Nagao T. Kurose H. Nature. 2000; 408: 492-495Google Scholar). Several methods, including electron spin resonance (8Kuppusamy P. Chzhan M. Vij K. Shteynbuk M. Lefer D.J. Giannella E. Zweier J.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3388-3392Google Scholar) and chemiluminescence (9Yasui H. Sakurai H. Biochem. Biophys. Res. Commun. 2000; 269: 131-136Google Scholar), have been developed to detect ROS, but fluorescence detection is superior in terms of high sensitivity and experimental convenience. Experimental studies on Ca2+-dependent signal transduction in cells were greatly facilitated by the development of fluorescent indicators for cytosolic Ca2+ (10Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Google Scholar, 11Minta A. Kao J.P.Y. Tsien R.Y. J. Biol. Chem. 1989; 264: 8171-8178Google Scholar). Several fluorescence probes to detect ROS, such as 2′,7′-dichlorodihydrofluorescein (DCFH) and dihydrorhodamine 123, have also been developed. However, as Hempel and co-workers (12Hempel S.L. Buettner G.R. O'Malley Y.Q. Wessels D.A. Flaherty D.M. Free Radical Biol. Med. 1999; 27: 146-159Google Scholar) pointed out, DCFH and dihydrorhodamine 123 can react with various ROS and oxidizing species (superoxide (O2⨪), hydrogen peroxide (H2O2), nitric oxide (NO), ferrous ion, and others), and in addition, DCFH is easily autoxidized, resulting in a spontaneous increase in fluorescence upon exposure to light. Therefore, it is not appropriate to think of these probes as detecting a specific oxidizing species in cells, such as H2O2 or NO, but rather they should be considered as detecting a broad range of oxidizing reactions that may be increased during intracellular oxidative stress (12Hempel S.L. Buettner G.R. O'Malley Y.Q. Wessels D.A. Flaherty D.M. Free Radical Biol. Med. 1999; 27: 146-159Google Scholar). There are many species of ROS, as mentioned above, but they tend to be considered collectively as “oxidative stress” when their effects in living cells are discussed. However, we believe that each species of ROS is likely to have a specific role in living cells. There is some evidence for this view. For example, H2O2 is an endothelium-derived hyperpolarizing factor in human and mice (13Matoba T. Shimokawa H. Nakashima M. Hirakawa Y. Mukai Y. Hirano K. Kanaide H. Takeshita A. J. Clin. Invest. 2000; 106: 1521-1530Google Scholar), p38 mitogen-activated protein kinase mediates caspase-3 activation during apoptosis induced by singlet oxygen (1O2) but not by H2O2 (14Zhuang S.G. Demirs J.T. Kochevar I.E. J. Biol. Chem. 2000; 275: 25939-25948Google Scholar), and hydroxyl radical (⋅OH) plays an important role as a second messenger in T cell activation (15Tatla S. Woodhead V. Foreman J.C. Chain B.M. Free Radical Biol. Med. 1999; 26: 14-24Google Scholar). In addition, each species of ROS has a characteristic chemical reactivity; for example, 1O2 reacts with anthracenes to yield endoperoxides in the Diels-Alder mode (16Umezawa N. Tanaka K. Urano Y. Kikuchi K. Higuchi T. Nagano T. Angew. Chem. Int. Ed. 1999; 38: 2899-2901Google Scholar), whereas ⋅OH can react directly with aromatic rings to yield hydroxylated products (17Coudray C. Favier A. Free Radical Biol. Med. 2000; 29: 1064-1070Google Scholar), and NO reacts with guanine to yield the deaminated compound (18Grishko V.I. Druzhyna N. LeDoux S.P. Wilson G.L. Nucleic Acids Res. 1999; 27: 4510-4516Google Scholar). However, because of the problems,i.e. lack of selectivity among species and autoxidation (12Hempel S.L. Buettner G.R. O'Malley Y.Q. Wessels D.A. Flaherty D.M. Free Radical Biol. Med. 1999; 27: 146-159Google Scholar,19Rota C. Fann Y.C. Mason R.P. J. Biol. Chem. 1999; 274: 28161-28168Google Scholar, 20Rota C. Chignell C.F. Mason R.P. Free Radical Biol. Med. 1999; 27: 873-881Google Scholar), the roles of an individual species of ROS in living cells remain uncertain. Therefore, we believe that it is very important to be able to detect each species of ROS selectively. If novel fluorescence probes that overcome the above problems were available, they would contribute greatly to the elucidation of the roles of individual ROS in living cells, because we would be able to “see” the generation of specific ROS with high resolution in time and space. It is known that ⋅OH participates in various biological processes. For example, HeLa, MW451, and HL-60 cells are induced to undergo apoptosis by ⋅OH (21Ren J.-G. Xia H.-L. Just T. Dai Y.-R. FEBS Lett. 2001; 488: 123-132Google Scholar). ⋅OH can damage DNA bases (1Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Google Scholar) and mediates redox alteration of cell-membrane Ca2+channels (22Az-ma T. Saeki N. Yuge O. Br. J. Pharmacol. 1999; 126: 1462-1470Google Scholar). However, because of the lack of effective direct detection methods for ⋅OH, its participation in these events has been established only indirectly by using inhibitors such as dithioethanol, glutathione, and desferrioxamine (1Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Google Scholar, 15Tatla S. Woodhead V. Foreman J.C. Chain B.M. Free Radical Biol. Med. 1999; 26: 14-24Google Scholar, 21Ren J.-G. Xia H.-L. Just T. Dai Y.-R. FEBS Lett. 2001; 488: 123-132Google Scholar, 22Az-ma T. Saeki N. Yuge O. Br. J. Pharmacol. 1999; 126: 1462-1470Google Scholar). Therefore, we wished to develop novel fluorescence probes for highly reactive oxygen species (hROS). Here, we use the term hROS to indicate reactive oxygen species with strong oxidizing power sufficient to directly hydroxylate aromatic rings (for example, ⋅OH or reactive intermediates of peroxidase). We report herein the development of novel fluorescence probes for ROS, 2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2-[6-(4′-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF), which can specifically detect certain species of ROS in terms of an increase of fluorescence and exhibit complete resistance to autoxidation both in vitro and in vivo. We also describe the visualization of hypochlorite (−OCl) in stimulated neutrophils. DISCUSSIONWe have succeeded in developing novel autoxidation-resistant fluorescence probes, HPF and APF, that can reliably detect hROS and/or−OCl selectively. Because it is likely that individual ROS have distinct roles in biological systems, the availability of selective fluorescence probes will be extremely useful. For example, by using HPF or APF, we can distinguish ⋅OH from NO. This is very important, because DCFH reacts with both ⋅OH and NO and so cannot be used reliably to study the biological role of ⋅OH. In addition, the mere production of H2O2 is completely different in terms of cell damage from the situation in which H2O2 is converted into hROS in the presence of low-valent metal ions. We feel our probes are useful here, because they can distinguish these two situations. Furthermore, we can also distinguish ONOO− from NO or O2⨪. It has been reported that ONOO− can be generated from NO and O2⨪ in vitro and in vivo (46Blough N.V. Zafiriou O.C. Inorg. Chem. 1985; 24: 3502-3504Google Scholar, 47Xia Y. Dawson V.L. Dawson T.M. Snyder S.H. Zweier J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6770-6774Google Scholar), and therefore we will be able to visualize the production of ONOO− with a clear distinction from that of NO or O2⨪, and this will allow a reliable evaluation of the role of ONOO− in various processes. Furthermore, we could detect−OCl selectively by using HPF and APF together, because HPF shows no fluorescence increase with −OCl, whereas APF shows a dose-dependent increase. The ability to selectively detect individual species of ROS represents a major advance.As shown in Table I and Fig. 4, the currently used fluorescence probe DCFH is easily autoxidized by light irradiation. This means that precautions must be taken to exclude light during incubation to load DCFH-DA into cells, and it is necessary to change the visual field often during observations. However, HPF and APF are not autoxidized at all, as shown in Table I and Fig. 4. Therefore, we believe HPF and APF will contribute greatly to the elucidation of the roles of ROS in living cells by making it possible to see the generation of specific ROS with high resolution in time and space. Although the sensitivity of HPF and APF is inferior to that of DCFH (Table I), lability to autoxidation and selectivity among ROS, rather than sensitivity, are considered to be critical for fluorescence probes for ROS.The question arises, why are HPF and APF selective for hROS, unlike DCFH? DCFH is nonfluorescent, and HPF and APF possess low fluorescence quantum efficiency, and all of them are converted to strongly fluorescent compounds, dichlorofluorescein or fluorescein, by oxidation. However, DCFH is converted to dichlorofluorescein, initially via abstraction of the hydrogen atom at the 9′-position, whereas HPF and APF are converted to fluorescein, initially via abstraction of the hydrogen atom of the phenolic hydroxy group or abstraction of one electron from the nitrogen atom. The hydrogen atom at the 9′-position of DCFH is readily abstracted because this hydrogen atom can be considered as being located at the central carbon of a triphenylmethane. It is therefore vulnerable even to a weakly oxidizing species, and this is the reason why DCFH lacks the selectivity among ROS. However, a strongly oxidizing species is required for theipso-substitution reaction of HPF and APF. Therefore, we conclude that the difference of oxidizing power required for oxidation reaction used for detection causes the difference of selectivity among ROS. Furthermore, the fact that HPF shows no fluorescence increase with−OCl, whereas APF does (Fig. 3 and Table I), reflects the difference in lability to oxidation between an aryloxyphenol and an aryloxyaniline.HPF and APF could detect hROS generated in the HRP/H2O2 system (Fig. 5). HRP is often used as an enzyme label in immunohistochemical studies, 3,3-diaminobenzidine is commonly used as a substrate for measurement of the peroxidase activity. However, 3,3-diaminobenzidine can be detected only by absorbance measurement and is easily autoxidized by light irradiation. Because HPF and APF permit fluorescence detection, which has higher sensitivity than absorbance detection, and they are not autoxidized by light irradiation at all, they are likely to be more effective reagents for immunohistochemistry using peroxidase than 3,3-diaminobenzidine and related compounds.We also used HPF and APF to visualize the production of−OCl from neutrophils (Fig. 8). Dye-loaded neutrophils weakly fluoresced before the stimulation with PMA, because the dyes were taken up by pinocytosis and MPO was slightly released into pinocytic vacuoles. Nevertheless, the fluorescence intensity of APF-loaded neutrophils markedly increased, in contrast to little fluorescence increase of HPF-loaded cells upon stimulation with PMA.−OCl is believed to play important roles not only in bacterial killing bacteria by neutrophils but also in injury to the venular endothelial surface in platelet-activating factor-induced microvascular damage (48Suematsu M. Kurose I. Asako H. Miura S. Tsuchiya M. J. Biochem. 1989; 106: 355-360Google Scholar). However, it has been difficult to draw firm conclusions concerning direct participation of −OCl because a completely selective detection method for −OCl has never been developed. Therefore, our finding that we could detect−OCl selectively by using HPF and APF together will make it possible for the first time to elucidate reliably the roles of−OCl in biological systems such as neutrophils.In summary, we have developed novel fluorescence probes, HPF and APF, that can selectively and dose dependently detect certain species among ROS and that are highly resistant to autoxidation. They can be used in enzymatic and cellular systems. They are greatly superior to the existing fluorescence probes for ROS, and are expected to have many chemical and biological applications. We have succeeded in developing novel autoxidation-resistant fluorescence probes, HPF and APF, that can reliably detect hROS and/or−OCl selectively. Because it is likely that individual ROS have distinct roles in biological systems, the availability of selective fluorescence probes will be extremely useful. For example, by using HPF or APF, we can distinguish ⋅OH from NO. This is very important, because DCFH reacts with both ⋅OH and NO and so cannot be used reliably to study the biological role of ⋅OH. In addition, the mere production of H2O2 is completely different in terms of cell damage from the situation in which H2O2 is converted into hROS in the presence of low-valent metal ions. We feel our probes are useful here, because they can distinguish these two situations. Furthermore, we can also distinguish ONOO− from NO or O2⨪. It has been reported that ONOO− can be generated from NO and O2⨪ in vitro and in vivo (46Blough N.V. Zafiriou O.C. Inorg. Chem. 1985; 24: 3502-3504Google Scholar, 47Xia Y. Dawson V.L. Dawson T.M. Snyder S.H. Zweier J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6770-6774Google Scholar), and therefore we will be able to visualize the production of ONOO− with a clear distinction from that of NO or O2⨪, and this will allow a reliable evaluation of the role of ONOO− in various processes. Furthermore, we could detect−OCl selectively by using HPF and APF together, because HPF shows no fluorescence increase with −OCl, whereas APF shows a dose-dependent increase. The ability to selectively detect individual species of ROS represents a major advance. As shown in Table I and Fig. 4, the currently used fluorescence probe DCFH is easily autoxidized by light irradiation. This means that precautions must be taken to exclude light during incubation to load DCFH-DA into cells, and it is necessary to change the visual field often during observations. However, HPF and APF are not autoxidized at all, as shown in Table I and Fig. 4. Therefore, we believe HPF and APF will contribute greatly to the elucidation of the roles of ROS in living cells by making it possible to see the generation of specific ROS with high resolution in time and space. Although the sensitivity of HPF and APF is inferior to that of DCFH (Table I), lability to autoxidation and selectivity among ROS, rather than sensitivity, are considered to be critical for fluorescence probes for ROS. The question arises, why are HPF and APF selective for hROS, unlike DCFH? DCFH is nonfluorescent, and HPF and APF possess low fluorescence quantum efficiency, and all of them are converted to strongly fluorescent compounds, dichlorofluorescein or fluorescein, by oxidation. However, DCFH is converted to dichlorofluorescein, initially via abstraction of the hydrogen atom at the 9′-position, whereas HPF and APF are converted to fluorescein, initially via abstraction of the hydrogen atom of the phenolic hydroxy group or abstraction of one electron from the nitrogen atom. The hydrogen atom at the 9′-position of DCFH is readily abstracted because this hydrogen atom can be considered as being located at the central carbon of a triphenylmethane. It is therefore vulnerable even to a weakly oxidizing species, and this is the reason why DCFH lacks the selectivity among ROS. However, a strongly oxidizing species is required for theipso-substitution reaction of HPF and APF. Therefore, we conclude that the difference of oxidizing power required for oxidation reaction used for detection causes the difference of selectivity among ROS. Furthermore, the fact that HPF shows no fluorescence increase with−OCl, whereas APF does (Fig. 3 and Table I), reflects the difference in lability to oxidation between an aryloxyphenol and an aryloxyaniline. HPF and APF could detect hROS generated in the HRP/H2O2 system (Fig. 5). HRP is often used as an enzyme label in immunohistochemical studies, 3,3-diaminobenzidine is commonly used as a substrate for measurement of the peroxidase activity. However, 3,3-diaminobenzidine can be detected only by absorbance measurement and is easily autoxidized by light irradiation. Because HPF and APF permit fluorescence detection, which has higher sensitivity than absorbance detection, and they are not autoxidized by light irradiation at all, they are likely to be more effective reagents for immunohistochemistry using peroxidase than 3,3-diaminobenzidine and related compounds. We also used HPF and APF to visualize the production of−OCl from neutrophils (Fig. 8). Dye-loaded neutrophils weakly fluoresced before the stimulation with PMA, because the dyes were taken up by pinocytosis and MPO was slightly released into pinocytic vacuoles. Nevertheless, the fluorescence intensity of APF-loaded neutrophils markedly increased, in contrast to little fluorescence increase of HPF-loaded cells upon stimulation with PMA.−OCl is believed to play important roles not only in bacterial killing bacteria by neutrophils but also in injury to the venular endothelial surface in platelet-activating factor-induced microvascular damage (48Suematsu M. Kurose I. Asako H. Miura S. Tsuchiya M. J. Biochem. 1989; 106: 355-360Google Scholar). However, it has been difficult to draw firm conclusions concerning direct participation of −OCl because a completely selective detection method for −OCl has never been developed. Therefore, our finding that we could detect−OCl selectively by using HPF and APF together will make it possible for the first time to elucidate reliably the roles of−OCl in biological systems such as neutrophils. In summary, we have developed novel fluorescence probes, HPF and APF, that can selectively and dose dependently detect certain species among ROS and that are highly resistant to autoxidation. They can be used in enzymatic and cellular systems. They are greatly superior to the existing fluorescence probes for ROS, and are expected to have many chemical and biological applications. We thank Dr. Hidehiko Nakagawa (National Institute of Radiological Sciences, Chiba, Japan) for providing peroxynitrite solution.