Free Radical Research, September 2014; 48(9): 990–995 © 2014 Informa UK, Ltd. ISSN 1071-5762 print/ISSN 1029-2470 online DOI: 10.3109/10715762.2014.898844
ORIGINAL ARTICLE
Histological detection of catalytic ferrous iron with the selective turn-on fluorescent probe RhoNox-1 in a Fenton reaction-based rat renal carcinogenesis model* T. Mukaide1, Y. Hattori1,2, N. Misawa1, S. Funahashi1, L. Jiang1, T. Hirayama3, H. Nagasawa3 & S. Toyokuni1 1Department
of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Japan, of Obstetrics /and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan, and 3Laboratory of Pharmaceutical and Medicinal Chemistry, Gifu Pharmaceutical University, Gifu, Japan
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2Department
Abstract Iron overload of a chronic nature has been associated with a wide variety of human diseases, including infection, carcinogenesis, and atherosclerosis. Recently, a highly specific turn-on fluorescent probe (RhoNox-1) specific to labile ferrous iron [Fe(II)], but not to labile ferric iron [Fe(III)], was developed. The evaluation of Fe(II) is more important than Fe(III) in vivo in that Fe(II) is an initiating component of the Fenton reaction. In this study, we applied this probe to frozen sections of an established Fenton reaction-based rat renal carcinogenesis model with an iron chelate, ferric nitrilotriacetate (Fe-NTA), in which catalytic iron induces the Fenton reaction specifically in the renal proximal tubules, presumably after iron reduction. Notably, this probe reacted with Fe(II) but with neither Fe(II)-NTA, Fe(III) nor Fe(III)NTA in vitro. Prominent red fluorescent color was explicitly observed in and around the lumina of renal proximal tubules 1 h after an intraperitoneal injection of 10–35 mg iron/kg Fe-NTA, which was dose-dependent, according to semiquantitative analysis. The RhoNox-1 signal colocalized with the generation of hydroxyl radicals, as detected by hydroxyphenyl fluorescein (HPF). The results demonstrate the transformation of Fe(III)-NTA to Fe(II) in vivo in the Fe-NTA-induced renal carcinogenesis model. Therefore, this probe would be useful for localizing catalytic Fe(II) in studies using tissues. Keywords: catalytic ferrous iron, fluorescent probe, kidney, oxidative stress, morphometry
Introduction Iron is an essential element in all living organisms on earth and is the most abundant heavy metal in humans. Human adults hold approximately 4 g of iron in total. Hemoglobin in red blood cells maintains 60% of this iron as the heme prosthetic group for oxygen binding. The remaining portions of iron are present in either the cells or extracellular space, including serum. Iron is a cofactor for various enzymes, is tightly bound to transferrin in serum, forms an iron reserve as ferritin or may transform into insoluble hemosiderin when overloaded [1]. Iron both has benefits and poses risks. Whereas iron deficiency causes anemia and muscle weakness, iron overload or even iron misdistribution that leads to localized chronic iron overload is associated with and is a risk for various diseases, including infection, cancer, atherosclerosis, and autoimmune diseases [2,3]. An iron importing transporter, DMT1 (Nramp2; natural resistance-associated macrophage protein), and a hepatic peptide hormone, hepcidin, were previously discovered in association with a risk for infection [4,5]. There are a plethora of reports of an association between iron overload and carcinogenesis in both human and animal studies [6–8]. Iron accumulation in an atheroma that results from hemorrhage appears
to be associated with its rupture, which is a direct cause of infarction in small arteries [9]. It is established that the synovial fluid in rheumatoid arthritis patients contains catalytic iron [10]. It is generally accepted that the Fenton reaction, which leads to the generation of hydroxyl radicals, causes all of the pathologies described above [11]. Therefore, the localization of ferrous iron [Fe(II)] has always been a subject of interest as an initiator of the Fenton reaction [12]. However, thus far, no histological methodology has been established to detect labile or catalytic Fe(II). Recently, a highly specific probe, called RhoNox-1, for labile Fe(II) was developed [13]. In this study, we describe for the first time the application of this probe to frozen sections of kidney after in vivo oxidative injury through ferric nitrilotriacetate [Fe(III)-NTA], which is an established model of Fenton reaction-based renal carcinogenesis in rats [14–19].
Methods Materials RhoNox-1 was a kind gift from Prof. Hideko Nagasawa (Gifu Pharmaceutical University, Gifu, Japan) [13].
*Special section for SFRR Asia 2013 in Taiwan. Correspondence: Shinya Toyokuni, MD, PhD, Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Tel: ⫹ 81-52-744-2086. Fax: ⫹ 81-52-744-2091. E-mail: toyokuni@med. nagoya-u.ac.jp (Received date: 21 January 2014; Accepted date: 23 February 2014; Published online: 21 March 2014)
Localizing catalytic Fe(II) in tissue
Hydroxyphenyl fluorescein (HPF) was obtained from Sekisui Medical (Tokyo, Japan). Fe(NO3)3 9H2O was obtained from Wako (Osaka, Japan), and nitrilotriacetate disodium salt was obtained from Nakalai Tesque (Kyoto, Japan). Ferric nitrilotriacetate [Fe(III)-NTA] was produced by mixing 300 mM ferric nitrate solution and 600 mM nitrilotriacetate solution, followed by pH adjustment to 7.4 with sodium hydrogen bicarbonate, as previously described [15], and was used within 30 min. All other agents were of analytical grade.
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three different wavelengths, was used for analyses. To obtain quantitative data, the exposure condition was recorded for each image. For the quantification of labile Fe(II), each image was divided into RGB elements, and only the red component was used for the analysis using the built-in software (BZ-II analyzer) or ImageJ version 1.47 software (http://www.rsb.info.nih.gov/ij/). Green component was used for HPF and blue component was used for nucleus. The number of nuclei was determined, and the final value was the integration of the red color (RhoNox-1) in tissue divided by the number of nuclei included in the analyzed area with a 40 ⫻ objective lens. Eight random areas in the proximal tubules were used for the analysis of each rat.
The animal experiment committee of the Nagoya University Graduate School of Medicine approved the following animal experiments. In total, 42 male Wistar rats (8-week old; Shizuoka Laboratory Animal Center, Shizuoka, Japan) were purchased. These rats were divided into time-course and dose-dependency groups. The time-course group was evaluated 0, 1, 2, 4, 6, 24, and 48 h after a single injection of 10 mg iron/kg body weight of Fe(III)-NTA; the dosedependency group was evaluated following the administration of 0, 10, 15, 20, 25, 30, and 35 mg iron/kg of Fe(III)-NTA (N ⫽ 3). Fe-NTA was intraperitoneally injected, and the animals were euthanized at the indicated time. The kidneys were immediately removed. Some portions of the kidney were fixed with 10 mM phosphate-buffered 10% formalin and were processed for routine paraffin embedding and sectioning at 3 μm, which was followed by hematoxylin and eosin staining to examine the histology. Some portions were embedded in optimum cutting temperature compound (Sankyo Miles, Tokyo) for frozen sections.
A black 96-well microplate (#MS-8096K, Sumitomo Bakelite Co., Osaka, Japan) and RhoNox-1 (1 μM final concentration in 10 mM phosphate buffer) were used for this analysis. Fe(II) solution was prepared from FeSO4 7H2O (Wako, Osaka, Japan). Fe(II)-NTA was produced in a manner similar to the preparation of Fe(III)-NTA. The pH was adjusted to 7.4 and immediately used. Each solution containing iron (100 μl) was mixed with 1 μM RhoNox-1 (100 μl), which was incubated for 1 h at room temperature. Then, RhoNox-1-specific fluorescence was measured using Powerscan 4 (DS Pharma Biomedical, Osaka, Japan; excitation, 530 nm; emission, 575 nm; gain 100). The data are shown as ([Sample fluorescence value][Background])/[Background].
Histological detection of labile Fe(II)
Statistical analysis
RhoNox-1 was preserved in a deep freezer at ⫺80°C and dissolved in dimethyl sulfoxide to produce a 1 mM solution, which was further diluted (1:200) with 10 mM phosphate-buffered saline (pH 7.4) before use (final concentration 5 μM). This diluted solution was used within a single day. Frozen sections of 8 μm thickness were prepared with a cryostat on MAS-GP type A grass slides (Matsunami, Osaka, Japan), air dried for 3 min, fixed in 10 mM phosphate-buffered 20% formalin in methanol for 1 min, and washed in deionized water for 5 min. Then, 200 μl of 5 μM RhoNox-1 was placed on those specimens and incubated for 30 min at 37°C in a dark chamber. Unfixed frozen sections were also used in some experiments. Thereafter, the specimen was counterstained with 4′,6-diaminido-2-phenylindole, dihydrochloride (DAPI) and observed as described below. Some of the specimens were further incubated with HPF after three washes with PBS for 30 min at 37°C in a dark room. We were able to preserve the frozen sections at ⫺80°C after cutting at least for a week.
The data are shown as the mean ⫾ SD. Unpaired t-test, Cochran-Armitage trend test and Pearson correlation coefficient were used where appropriate with SPSS 13.0 (SPSS Inc., Chicago, IL). P ⬍ 0.05 was considered statistically significant.
Imaging analysis A fluorescence microscope (BZ-9000, Keyence, Osaka, Japan), which allows simultaneous data acquisition of
Reactivity of the iron solution and iron chelates with RhoNox-1
Results Reactivity of Fe(II)-NTA, Fe(III), and Fe(III)-NTA with RhoNox-1 RhoNox-1 dose-dependently reacted with Fe(II) in a range of 0–10 μM as previously described [13]. However, RhoNox-1 reacted with neither Fe(II)-NTA, Fe(III), nor Fe(III)-NTA (Figure 1). Fe-NTA-induced renal carcinogenesis model Oxidative stress, as indicated by lipid peroxidation and DNA modification, is reported to reach its maximum 30 min to 3 h after an intraperitoneal injection of Fe-NTA [20]. First, we used unfixed frozen sections to locate Fe(II) 1 h after 10 mg iron/kg Fe(III)-NTA administration, and
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Figure 1. Reactivity of different forms of iron with RhoNox-1. RhoNox-1 is specific to Fe(II) among Fe(II), Fe(III), Fe(II)-NTA, and Fe(III)NTA. Each solution was incubated with RhoNox-1 for 1 h, and emission at 575 nm after excitation at 540 nm was measured. Refer to the text for details. NTA, nitrilotriacetate.
found strong positivity not only in the renal proximal tubules but also in their lumina. However, the image was blurred in the absence of fixation (Figure 2). Because these were not optimal for the morphometric analyses, we tried light fixation as described in the methods section. Light fixation provided acceptable results both in morphology and sensitivity, and the results were in good parallel with those of unfixed specimens. Then, we performed a time-course study. We noted some levels of background fluorescence in the normal kidney but found that RhoNox1-specific fluorescence significantly increased 1 h after the injection, then further increased and was maintained up to at least 6 h. The fluorescence decreased 24 and 48 h after the injection, when proximal tubular necrosis was dominant. The increase and decrease in HPF-specific fluorescence were consistent with RhoNox-1-specific fluorescence. Nucleus-specific fluorescence (DAPI) gradually decreased after Fe(III)-NTA injection as renal proximal tubular cells degenerated and necrotized (Figure 3).
Then, we performed a dose-dependence study at 1 h. Most of the animals were dying at a dose of 35 mg iron/ kg at 1 h. The kidneys were swollen with edema and showed significantly increased weight at 30 and 35 mg iron/kg. We observed dose-dependent RhoNox-1-specific fluorescence in the renal proximal tubular cells, which was inversely associated with the number of viable cells as seen by DAPI-positivity. Furthermore, RhoNox-1 and HPF coexisted (Figure 4A–C). Thus, the intensity of HPFspecific fluorescence was in parallel with RhoNox-1, and the correlation coefficient was r ⫽ 0.912 (Figure 5). Discussion RhoNox-1, a highly specific turn-on probe for labile ferrous ion, was recently established. The effect of the turn on for red fluorescence was immense. In that paper, the application of RhoNox-1 in cultured cells loaded with
Figure 2. RhoNox-1-specific fluorescence in rat renal proximal tubules 1 h after intraperitoneal injection of 10 mg iron/kg Fe(III)-NTA with unfixed specimens. Note that both renal proximal tubules and their lumina are strongly stained (bar ⫽ 40 μm).
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Localizing catalytic Fe(II) in tissue
Figure 3. Time-course study of RhoNox-1-specific integrated fluorescence intensity in rat renal proximal tubules after intraperitoneal injection of 10 mg iron/kg Fe(III)-NTA with lightly fixed specimens. RhoNox-1 intensity and HPF increased up to 6 h, whereas nuclear fluorescence (DAPI; 4′, 6-diaminido-2-phenylindole, dihydrochloride) decreased with degeneration and necrosis. Analyses were through BZ-II. AU, arbitrary unit; NTA, nitrilotriacetate; HPF, hydroxyphenyl fluorescein. HPF: **p ⬍ 0.01; ***p ⬍ 0.001; RhoNox-1: ####p ⬍ 0.0001 vs time ⫽ 0; unpaired t-test.
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Fe(II) was successful [13]. In this study, we applied this technique for the first time to frozen sections of rat tissue and found that this technique works well for systemic studies in animals and could be easily extended to samples from humans as well as other species. We could use unfixed and lightly fixed specimens. We used an established rat model of the Fenton reaction in vivo. Fe(III)-NTA is an iron chelate that is soluble at a neutral pH and still retains 3–4 free catalytic ligands. Thus, this molecule is thought to be the most potent iron catalyst for the Fenton reaction after reduction [21–23]. An intraperitoneal injection of Fe-NTA causes the Fenton reaction in the lumina of renal proximal tubules after it is absorbed into the systemic blood flow, which is followed by filtration through the glomeruli of the kidney [24], where it is believed that Fe(III)-NTA is reduced to Fe(II)NTA due to the presence of L-cysteine from the glutathione cycle [25]. There are many reports on the generation of hydroxyl radical-modified molecules in situ in this model, such as oxidative DNA base modifications [16] and various aldehydes [malonaldehyde, 4-hydroxy-2-nonenal (HNE), acrolein, etc.] [18,26]. Eventually, repeated reactions of this nature cause renal cell carcinoma [17],
Figure 4. Dose-dependence study of RhoNox-1-specific integrated fluorescence intensity in rat renal proximal tubules 1 h after intraperitoneal Fe(III)-NTA injection with lightly fixed specimens. A: Representative pictures obtained from a single frozen section with multi-color analysis. Colocalization of RhoNox-1 and HPF is evident. DAPI, 4′, 6-diaminido-2-phenylindole, dihydrochloride; HPF, hydroxyphenyl fluorescein; NTA, nitrilotriacetate (bar ⫽ 40 μm). B: Quantification of A for RhoNox-1 and HPF. AU, arbitrary unit. C: Integrated fluorescence intensity per cell; trends p ⬍ 0.001 with Cochran–Armitage test. Analyses were through ImageJ; the results through BZ-II and ImageJ were proportional (HPF: ***p ⬍ 0.001 vs dose ⫽ 0; ****p ⬍ 0.0001 vs dose ⫽ 0; RhoNox-1: ###p ⬍ 0.001 vs dose ⫽ 0; ####p ⬍ 0.0001 vs dose ⫽ 0; unpaired t-test).
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Figure 5. The association of RhoNox-1 and HPF fluorescence. Corresponding data on integrated fluorescence intensity (IFI) of RhoNox-1 and HPF were compared, which were proportional. AU, arbitrary unit; HPF, hydroxyphenyl fluorescein.
and it was recently shown that the genomic alterations in these cancers are quite similar to those alterations in human counterparts [19]. The detection of labile Fe(II) was principally intraluminal (unfixed) and in the surrounding cells (both unfixed and lightly fixed) in the renal proximal tubules. We found a clear dose-dependence in the quantification of the signals. The results demonstrate that this probe can be successfully applied to frozen sections obtained from tissues. We fixed a part of the specimen with neutral buffered formalin followed by paraffin embedding, and performed Perls’ iron staining. However, we did not obtain positive staining. It is thought that Perls’ iron staining detects hemosiderin and a part of ferritin; it is unknown whether those compounds are actually damaging to cellular molecules in vivo. We believe that a portion of these compounds would be solubilized to a catalytic form. In this sense, the detection of labile Fe(II) is more direct. Furthermore, our data clearly demonstrated that Fe(II) is produced in vivo from Fe(III)-NTA. We suspect that the Fe(II)-NTA generated via reduction, presumably through L-cysteine derived from glutathione through γ-glutamyl transferase and dipeptidase, in the lumina of renal proximal tubules [25] is absorbed from the villous luminal membrane as Fe(II) via DMT1 [27] after de-chelation at a mild acidic pH. This hypothesis requires further investigation. There are already many markers for oxidative stress. Those markers include molecules modified by the reaction of hydroxyl radicals, such as 8-hydroxy-2′-deoxyguanosine (8-OHdG) [28] and HNE [29]. Previously, we developed monoclonal antibodies against 8-hydroxy-2′-deoxyguanosine [30] and 4-hydroxy-2-nonenal-modified proteins [31]. We could successfully apply these antibodies to this model in formalin-fixed paraffin-embedded sections. We believe that there is a conceptual difference between these monoclonal antibodies and the present probe, i.e., the presence of labile Fe(II) constitutes the precise risk for the Fenton reaction, whereas modified products are the sum of the production and the repair/
removal of the modifications. Furthermore, we demonstrated the coexistence of RhoNox-1 and HPF. This result indicates that RhoNox-1-positive foci can indeed initiate the Fenton reaction in situ. Therefore, RhoNox-1 detects catalytic Fe(II) and is a novel marker for evaluating numerous oxidative stress-associated diseases. Nevertheless, further studies are necessary to determine the following: 1) whether this method is applicable to formalin-fixed paraffin-embedded sections; 2) whether there are any discrepancies between the data regarding this probe and other modified products in various models, and, if so, what those discrepancies mean (We suspect that labile Fe(II) may be unexpectedly stable in the absence of hydrogen peroxide in situ); 3) whether this method is applicable to time-lapse studies using animals; and 4) what kind of efforts would be necessary to decrease the background fluorescence of RhoNox-1. In conclusion, in the present study, we developed a novel strategy to localize catalytic Fe(II) in frozen sections of tissue. We recommend to use both unfixed and lightly fixed specimens for the initial evaluation. This strategy would be helpful for analyzing various iron-associated pathologies and physiologies, including neurodegenerative diseases and iron absorption through the duodenum. This probe may open up novel research areas for various oxidative stress-associated diseases. Acknowledgements The authors thank all members of Toyokuni laboratory for useful discussion.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. This work was supported in part by a grant-in-aid for research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (24390094; 221S0001-04; 24108001).
References [1] Wriggleworth JM, Baum H. The biochemical function of iron. In: Jacobs A, Worwood M (eds.). Iron in biochemistry and medicine, II. London: Academic Press; 1980. pp. 29–86. [2] Weinberg ED. The hazards of iron loading. Metallomics 2010;2:732–740. [3] Toyokuni S. Iron as a target of chemoprevention for longevity in humans. Free Radic Res 2011;45:906–917. [4] Weinberg ED. Iron availability and infection. Biochim Biophys Acta 2009;1790:600–605. [5] Hentze MW, Muckenthaler MU, Galy B, Camaschella C. Two to tango: regulation of Mammalian iron metabolism. Cell 2010;142:24–38. [6] Weinberg ED. The role of iron in cancer. Eur J Cancer Prev 1996;5:19–36. [7] Toyokuni S. Iron-induced carcinogenesis: the role of redox regulation. Free Radic Biol Med 1996;20:553–566.
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Localizing catalytic Fe(II) in tissue [8] Toyokuni S. Role of iron in carcinogenesis: cancer as a ferrotoxic disease. Cancer Sci 2009;100:9–16. [9] Yuan XM. Apoptotic macrophage-derived foam cells of human atheromas are rich in iron and ferritin, suggesting ironcatalysed reactions to be involved in apoptosis. Free Radic Res 1999;30:221–231. [10] Rowley D, Gutteridge JM, Blake D, Farr M, Halliwell B. Lipid peroxidation in rheumatoid arthritis: thiobarbituric acid-reactive material and catalytic iron salts in synovial fluid from rheumatoid patients. Clin Sci (Lond) 1984;66:691–695. [11] Halliwell B, Gutteridge JMC. Free radicals in biology and medicine. New York: Oxford University Press; 2007. [12] Fenton HJH. Oxidation of tartaric acid in presence of iron. J Chem Soc 1894;65:899–910. [13] Hirayama T, Okuda K, Nagasawa H. A highly selective turn-on fluorescent probe fro iron(II) to visualize labile iron in living cells. Chem Sci 2013;4:1250–1256. [14] Ebina Y, Okada S, Hamazaki S, Ogino F, Li JL, Midorikawa O. Nephrotoxicity and renal cell carcinoma after use of ironand aluminum-nitrilotriacetate complexes in rats. J Natl Cancer Inst 1986;76:107–113. [15] Toyokuni S, Uchida K, Okamoto K, Hattori-Nakakuki Y, Hiai H, Stadtman ER. Formation of 4-hydroxy-2-nonenalmodified proteins in the renal proximal tubules of rats treated with a renal carcinogen, ferric nitrilotriacetate. Proc Natl Acad Sci USA 1994;91:2616–2620. [16] Toyokuni S, Mori T, Dizdaroglu M. DNA base modifications in renal chromatin of Wistar rats treated with a renal carcinogen, ferric nitrilotriacetate. Int J Cancer 1994;57:123–128. [17] Nishiyama Y, Suwa H, Okamoto K, Fukumoto M, Hiai H, Toyokuni S. Low incidence of point mutations in H-, Kand N-ras oncogenes and p53 tumor suppressor gene in renal cell carcinoma and peritoneal mesothelioma of Wistar rats induced by ferric nitrilotriacetate. Jpn J Cancer Res 1995; 86:1150–1158. [18] Toyokuni S, Luo XP, Tanaka T, Uchida K, Hiai H, Lehotay DC. Induction of a wide range of C2–12 aldehydes and C7–12 acyloins in the kidney of Wistar rats after treatment with a renal carcinogen, ferric nitrilotriacetate. Free Radic Biol Med 1997;22:1019–1027. [19] Akatsuka S, Yamashita Y, Ohara H, Liu YT, Izumiya M, Abe K, et al. Fenton reaction induced cancer in wild type rats recapitulates genomic alterations observed in human cancer. PLoS ONE 2012;7:e43403.
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[20] Tanaka T, Nishiyama Y, Okada K, Hirota K, Matsui M, Yodoi J, et al. Induction and nuclear translocation of thioredoxin by oxidative damage in the mouse kidney: independence of tubular necrosis and sulfhydryl depletion. Lab Invest 1997;77:145–155. [21] Toyokuni S, Sagripanti JL. Iron-mediated DNA damage: sensitive detection of DNA strand breakage catalyzed by iron. J Inorg Biochem 1992;47:241–248. [22] Toyokuni S, Sagripanti J-L. DNA single- and double-strand breaks produced by ferric nitrilotriacetate in relation to renal tubular carcinogenesis. Carcinogenesis 1993;14:223–227. [23] Toyokuni S, Sagripanti JL. Iron chelators modulate the production of DNA strand breaks and 8- hydroxy-2’-deoxyguanosine. Free Radic Res 1999;31:123–128. [24] Toyokuni S, Okada S, Hamazaki S, Minamiyama Y, Yamada Y, Liang P, et al. Combined histochemical and biochemical analysis of sex hormone dependence of ferric nitrilotriacetate-induced renal lipid peroxidation in ddY mice. Cancer Res 1990;50:5574–5580. [25] Okada S, Minamiyama Y, Hamazaki S, Toyokuni S, Sotomatsu A. Glutathione cycle dependency of ferric nitrilotriacetate-induced lipid peroxidation in mouse proximal renal tubules. Arch Biochem Biophys 1993;301:138–142. [26] Kawai Y, Furuhata A, Toyokuni S, Aratani Y, Uchida K. Formation of acrolein-derived 2’-deoxyadenosine adduct in an iron-induced carcinogenesis model. J Biol Chem 2003; 278:50346–50354. [27] Veuthey T, Hoffman D, Vaidya VS, Wessling-Resnick M. Impaired renal function and development in Belgrade rats. Am J Physiol Renal Physiol 2014;306:F333–343. [28] Kasai H. Analysis of a form of oxidative DNA damage, 8hydroxy-2’-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis. Mutat Res 1997;387:147–163. [29] Uchida K. 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog Lipid Res 2003;42:318–343. [30] Toyokuni S, Tanaka T, Hattori Y, Nishiyama Y, Ochi H, Hiai H, et al. Quantitative immunohistochemical determination of 8-hydroxy-2’-deoxyguanosine by a monoclonal antibody N45.1: its application to ferric nitrilotriacetate-induced renal carcinogenesis model. Lab Invest 1997;76:365–374. [31] Toyokuni S, Miyake N, Hiai H, Hagiwara M, Kawakishi S, Osawa T, Uchida K. The monoclonal antibody specific for the 4-hydroxy-2-nonenal histidine adduct. FEBS Lett 1995; 359:189–191.
ORIGINAL ARTICLE
Histological detection of catalytic ferrous iron with the selective turn-on fluorescent probe RhoNox-1 in a Fenton reaction-based rat renal carcinogenesis model* T. Mukaide1, Y. Hattori1,2, N. Misawa1, S. Funahashi1, L. Jiang1, T. Hirayama3, H. Nagasawa3 & S. Toyokuni1 1Department
of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Japan, of Obstetrics /and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan, and 3Laboratory of Pharmaceutical and Medicinal Chemistry, Gifu Pharmaceutical University, Gifu, Japan
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2Department
Abstract Iron overload of a chronic nature has been associated with a wide variety of human diseases, including infection, carcinogenesis, and atherosclerosis. Recently, a highly specific turn-on fluorescent probe (RhoNox-1) specific to labile ferrous iron [Fe(II)], but not to labile ferric iron [Fe(III)], was developed. The evaluation of Fe(II) is more important than Fe(III) in vivo in that Fe(II) is an initiating component of the Fenton reaction. In this study, we applied this probe to frozen sections of an established Fenton reaction-based rat renal carcinogenesis model with an iron chelate, ferric nitrilotriacetate (Fe-NTA), in which catalytic iron induces the Fenton reaction specifically in the renal proximal tubules, presumably after iron reduction. Notably, this probe reacted with Fe(II) but with neither Fe(II)-NTA, Fe(III) nor Fe(III)NTA in vitro. Prominent red fluorescent color was explicitly observed in and around the lumina of renal proximal tubules 1 h after an intraperitoneal injection of 10–35 mg iron/kg Fe-NTA, which was dose-dependent, according to semiquantitative analysis. The RhoNox-1 signal colocalized with the generation of hydroxyl radicals, as detected by hydroxyphenyl fluorescein (HPF). The results demonstrate the transformation of Fe(III)-NTA to Fe(II) in vivo in the Fe-NTA-induced renal carcinogenesis model. Therefore, this probe would be useful for localizing catalytic Fe(II) in studies using tissues. Keywords: catalytic ferrous iron, fluorescent probe, kidney, oxidative stress, morphometry
Introduction Iron is an essential element in all living organisms on earth and is the most abundant heavy metal in humans. Human adults hold approximately 4 g of iron in total. Hemoglobin in red blood cells maintains 60% of this iron as the heme prosthetic group for oxygen binding. The remaining portions of iron are present in either the cells or extracellular space, including serum. Iron is a cofactor for various enzymes, is tightly bound to transferrin in serum, forms an iron reserve as ferritin or may transform into insoluble hemosiderin when overloaded [1]. Iron both has benefits and poses risks. Whereas iron deficiency causes anemia and muscle weakness, iron overload or even iron misdistribution that leads to localized chronic iron overload is associated with and is a risk for various diseases, including infection, cancer, atherosclerosis, and autoimmune diseases [2,3]. An iron importing transporter, DMT1 (Nramp2; natural resistance-associated macrophage protein), and a hepatic peptide hormone, hepcidin, were previously discovered in association with a risk for infection [4,5]. There are a plethora of reports of an association between iron overload and carcinogenesis in both human and animal studies [6–8]. Iron accumulation in an atheroma that results from hemorrhage appears
to be associated with its rupture, which is a direct cause of infarction in small arteries [9]. It is established that the synovial fluid in rheumatoid arthritis patients contains catalytic iron [10]. It is generally accepted that the Fenton reaction, which leads to the generation of hydroxyl radicals, causes all of the pathologies described above [11]. Therefore, the localization of ferrous iron [Fe(II)] has always been a subject of interest as an initiator of the Fenton reaction [12]. However, thus far, no histological methodology has been established to detect labile or catalytic Fe(II). Recently, a highly specific probe, called RhoNox-1, for labile Fe(II) was developed [13]. In this study, we describe for the first time the application of this probe to frozen sections of kidney after in vivo oxidative injury through ferric nitrilotriacetate [Fe(III)-NTA], which is an established model of Fenton reaction-based renal carcinogenesis in rats [14–19].
Methods Materials RhoNox-1 was a kind gift from Prof. Hideko Nagasawa (Gifu Pharmaceutical University, Gifu, Japan) [13].
*Special section for SFRR Asia 2013 in Taiwan. Correspondence: Shinya Toyokuni, MD, PhD, Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Tel: ⫹ 81-52-744-2086. Fax: ⫹ 81-52-744-2091. E-mail: toyokuni@med. nagoya-u.ac.jp (Received date: 21 January 2014; Accepted date: 23 February 2014; Published online: 21 March 2014)
Localizing catalytic Fe(II) in tissue
Hydroxyphenyl fluorescein (HPF) was obtained from Sekisui Medical (Tokyo, Japan). Fe(NO3)3 9H2O was obtained from Wako (Osaka, Japan), and nitrilotriacetate disodium salt was obtained from Nakalai Tesque (Kyoto, Japan). Ferric nitrilotriacetate [Fe(III)-NTA] was produced by mixing 300 mM ferric nitrate solution and 600 mM nitrilotriacetate solution, followed by pH adjustment to 7.4 with sodium hydrogen bicarbonate, as previously described [15], and was used within 30 min. All other agents were of analytical grade.
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three different wavelengths, was used for analyses. To obtain quantitative data, the exposure condition was recorded for each image. For the quantification of labile Fe(II), each image was divided into RGB elements, and only the red component was used for the analysis using the built-in software (BZ-II analyzer) or ImageJ version 1.47 software (http://www.rsb.info.nih.gov/ij/). Green component was used for HPF and blue component was used for nucleus. The number of nuclei was determined, and the final value was the integration of the red color (RhoNox-1) in tissue divided by the number of nuclei included in the analyzed area with a 40 ⫻ objective lens. Eight random areas in the proximal tubules were used for the analysis of each rat.
The animal experiment committee of the Nagoya University Graduate School of Medicine approved the following animal experiments. In total, 42 male Wistar rats (8-week old; Shizuoka Laboratory Animal Center, Shizuoka, Japan) were purchased. These rats were divided into time-course and dose-dependency groups. The time-course group was evaluated 0, 1, 2, 4, 6, 24, and 48 h after a single injection of 10 mg iron/kg body weight of Fe(III)-NTA; the dosedependency group was evaluated following the administration of 0, 10, 15, 20, 25, 30, and 35 mg iron/kg of Fe(III)-NTA (N ⫽ 3). Fe-NTA was intraperitoneally injected, and the animals were euthanized at the indicated time. The kidneys were immediately removed. Some portions of the kidney were fixed with 10 mM phosphate-buffered 10% formalin and were processed for routine paraffin embedding and sectioning at 3 μm, which was followed by hematoxylin and eosin staining to examine the histology. Some portions were embedded in optimum cutting temperature compound (Sankyo Miles, Tokyo) for frozen sections.
A black 96-well microplate (#MS-8096K, Sumitomo Bakelite Co., Osaka, Japan) and RhoNox-1 (1 μM final concentration in 10 mM phosphate buffer) were used for this analysis. Fe(II) solution was prepared from FeSO4 7H2O (Wako, Osaka, Japan). Fe(II)-NTA was produced in a manner similar to the preparation of Fe(III)-NTA. The pH was adjusted to 7.4 and immediately used. Each solution containing iron (100 μl) was mixed with 1 μM RhoNox-1 (100 μl), which was incubated for 1 h at room temperature. Then, RhoNox-1-specific fluorescence was measured using Powerscan 4 (DS Pharma Biomedical, Osaka, Japan; excitation, 530 nm; emission, 575 nm; gain 100). The data are shown as ([Sample fluorescence value][Background])/[Background].
Histological detection of labile Fe(II)
Statistical analysis
RhoNox-1 was preserved in a deep freezer at ⫺80°C and dissolved in dimethyl sulfoxide to produce a 1 mM solution, which was further diluted (1:200) with 10 mM phosphate-buffered saline (pH 7.4) before use (final concentration 5 μM). This diluted solution was used within a single day. Frozen sections of 8 μm thickness were prepared with a cryostat on MAS-GP type A grass slides (Matsunami, Osaka, Japan), air dried for 3 min, fixed in 10 mM phosphate-buffered 20% formalin in methanol for 1 min, and washed in deionized water for 5 min. Then, 200 μl of 5 μM RhoNox-1 was placed on those specimens and incubated for 30 min at 37°C in a dark chamber. Unfixed frozen sections were also used in some experiments. Thereafter, the specimen was counterstained with 4′,6-diaminido-2-phenylindole, dihydrochloride (DAPI) and observed as described below. Some of the specimens were further incubated with HPF after three washes with PBS for 30 min at 37°C in a dark room. We were able to preserve the frozen sections at ⫺80°C after cutting at least for a week.
The data are shown as the mean ⫾ SD. Unpaired t-test, Cochran-Armitage trend test and Pearson correlation coefficient were used where appropriate with SPSS 13.0 (SPSS Inc., Chicago, IL). P ⬍ 0.05 was considered statistically significant.
Imaging analysis A fluorescence microscope (BZ-9000, Keyence, Osaka, Japan), which allows simultaneous data acquisition of
Reactivity of the iron solution and iron chelates with RhoNox-1
Results Reactivity of Fe(II)-NTA, Fe(III), and Fe(III)-NTA with RhoNox-1 RhoNox-1 dose-dependently reacted with Fe(II) in a range of 0–10 μM as previously described [13]. However, RhoNox-1 reacted with neither Fe(II)-NTA, Fe(III), nor Fe(III)-NTA (Figure 1). Fe-NTA-induced renal carcinogenesis model Oxidative stress, as indicated by lipid peroxidation and DNA modification, is reported to reach its maximum 30 min to 3 h after an intraperitoneal injection of Fe-NTA [20]. First, we used unfixed frozen sections to locate Fe(II) 1 h after 10 mg iron/kg Fe(III)-NTA administration, and
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Figure 1. Reactivity of different forms of iron with RhoNox-1. RhoNox-1 is specific to Fe(II) among Fe(II), Fe(III), Fe(II)-NTA, and Fe(III)NTA. Each solution was incubated with RhoNox-1 for 1 h, and emission at 575 nm after excitation at 540 nm was measured. Refer to the text for details. NTA, nitrilotriacetate.
found strong positivity not only in the renal proximal tubules but also in their lumina. However, the image was blurred in the absence of fixation (Figure 2). Because these were not optimal for the morphometric analyses, we tried light fixation as described in the methods section. Light fixation provided acceptable results both in morphology and sensitivity, and the results were in good parallel with those of unfixed specimens. Then, we performed a time-course study. We noted some levels of background fluorescence in the normal kidney but found that RhoNox1-specific fluorescence significantly increased 1 h after the injection, then further increased and was maintained up to at least 6 h. The fluorescence decreased 24 and 48 h after the injection, when proximal tubular necrosis was dominant. The increase and decrease in HPF-specific fluorescence were consistent with RhoNox-1-specific fluorescence. Nucleus-specific fluorescence (DAPI) gradually decreased after Fe(III)-NTA injection as renal proximal tubular cells degenerated and necrotized (Figure 3).
Then, we performed a dose-dependence study at 1 h. Most of the animals were dying at a dose of 35 mg iron/ kg at 1 h. The kidneys were swollen with edema and showed significantly increased weight at 30 and 35 mg iron/kg. We observed dose-dependent RhoNox-1-specific fluorescence in the renal proximal tubular cells, which was inversely associated with the number of viable cells as seen by DAPI-positivity. Furthermore, RhoNox-1 and HPF coexisted (Figure 4A–C). Thus, the intensity of HPFspecific fluorescence was in parallel with RhoNox-1, and the correlation coefficient was r ⫽ 0.912 (Figure 5). Discussion RhoNox-1, a highly specific turn-on probe for labile ferrous ion, was recently established. The effect of the turn on for red fluorescence was immense. In that paper, the application of RhoNox-1 in cultured cells loaded with
Figure 2. RhoNox-1-specific fluorescence in rat renal proximal tubules 1 h after intraperitoneal injection of 10 mg iron/kg Fe(III)-NTA with unfixed specimens. Note that both renal proximal tubules and their lumina are strongly stained (bar ⫽ 40 μm).
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Localizing catalytic Fe(II) in tissue
Figure 3. Time-course study of RhoNox-1-specific integrated fluorescence intensity in rat renal proximal tubules after intraperitoneal injection of 10 mg iron/kg Fe(III)-NTA with lightly fixed specimens. RhoNox-1 intensity and HPF increased up to 6 h, whereas nuclear fluorescence (DAPI; 4′, 6-diaminido-2-phenylindole, dihydrochloride) decreased with degeneration and necrosis. Analyses were through BZ-II. AU, arbitrary unit; NTA, nitrilotriacetate; HPF, hydroxyphenyl fluorescein. HPF: **p ⬍ 0.01; ***p ⬍ 0.001; RhoNox-1: ####p ⬍ 0.0001 vs time ⫽ 0; unpaired t-test.
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Fe(II) was successful [13]. In this study, we applied this technique for the first time to frozen sections of rat tissue and found that this technique works well for systemic studies in animals and could be easily extended to samples from humans as well as other species. We could use unfixed and lightly fixed specimens. We used an established rat model of the Fenton reaction in vivo. Fe(III)-NTA is an iron chelate that is soluble at a neutral pH and still retains 3–4 free catalytic ligands. Thus, this molecule is thought to be the most potent iron catalyst for the Fenton reaction after reduction [21–23]. An intraperitoneal injection of Fe-NTA causes the Fenton reaction in the lumina of renal proximal tubules after it is absorbed into the systemic blood flow, which is followed by filtration through the glomeruli of the kidney [24], where it is believed that Fe(III)-NTA is reduced to Fe(II)NTA due to the presence of L-cysteine from the glutathione cycle [25]. There are many reports on the generation of hydroxyl radical-modified molecules in situ in this model, such as oxidative DNA base modifications [16] and various aldehydes [malonaldehyde, 4-hydroxy-2-nonenal (HNE), acrolein, etc.] [18,26]. Eventually, repeated reactions of this nature cause renal cell carcinoma [17],
Figure 4. Dose-dependence study of RhoNox-1-specific integrated fluorescence intensity in rat renal proximal tubules 1 h after intraperitoneal Fe(III)-NTA injection with lightly fixed specimens. A: Representative pictures obtained from a single frozen section with multi-color analysis. Colocalization of RhoNox-1 and HPF is evident. DAPI, 4′, 6-diaminido-2-phenylindole, dihydrochloride; HPF, hydroxyphenyl fluorescein; NTA, nitrilotriacetate (bar ⫽ 40 μm). B: Quantification of A for RhoNox-1 and HPF. AU, arbitrary unit. C: Integrated fluorescence intensity per cell; trends p ⬍ 0.001 with Cochran–Armitage test. Analyses were through ImageJ; the results through BZ-II and ImageJ were proportional (HPF: ***p ⬍ 0.001 vs dose ⫽ 0; ****p ⬍ 0.0001 vs dose ⫽ 0; RhoNox-1: ###p ⬍ 0.001 vs dose ⫽ 0; ####p ⬍ 0.0001 vs dose ⫽ 0; unpaired t-test).
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Figure 5. The association of RhoNox-1 and HPF fluorescence. Corresponding data on integrated fluorescence intensity (IFI) of RhoNox-1 and HPF were compared, which were proportional. AU, arbitrary unit; HPF, hydroxyphenyl fluorescein.
and it was recently shown that the genomic alterations in these cancers are quite similar to those alterations in human counterparts [19]. The detection of labile Fe(II) was principally intraluminal (unfixed) and in the surrounding cells (both unfixed and lightly fixed) in the renal proximal tubules. We found a clear dose-dependence in the quantification of the signals. The results demonstrate that this probe can be successfully applied to frozen sections obtained from tissues. We fixed a part of the specimen with neutral buffered formalin followed by paraffin embedding, and performed Perls’ iron staining. However, we did not obtain positive staining. It is thought that Perls’ iron staining detects hemosiderin and a part of ferritin; it is unknown whether those compounds are actually damaging to cellular molecules in vivo. We believe that a portion of these compounds would be solubilized to a catalytic form. In this sense, the detection of labile Fe(II) is more direct. Furthermore, our data clearly demonstrated that Fe(II) is produced in vivo from Fe(III)-NTA. We suspect that the Fe(II)-NTA generated via reduction, presumably through L-cysteine derived from glutathione through γ-glutamyl transferase and dipeptidase, in the lumina of renal proximal tubules [25] is absorbed from the villous luminal membrane as Fe(II) via DMT1 [27] after de-chelation at a mild acidic pH. This hypothesis requires further investigation. There are already many markers for oxidative stress. Those markers include molecules modified by the reaction of hydroxyl radicals, such as 8-hydroxy-2′-deoxyguanosine (8-OHdG) [28] and HNE [29]. Previously, we developed monoclonal antibodies against 8-hydroxy-2′-deoxyguanosine [30] and 4-hydroxy-2-nonenal-modified proteins [31]. We could successfully apply these antibodies to this model in formalin-fixed paraffin-embedded sections. We believe that there is a conceptual difference between these monoclonal antibodies and the present probe, i.e., the presence of labile Fe(II) constitutes the precise risk for the Fenton reaction, whereas modified products are the sum of the production and the repair/
removal of the modifications. Furthermore, we demonstrated the coexistence of RhoNox-1 and HPF. This result indicates that RhoNox-1-positive foci can indeed initiate the Fenton reaction in situ. Therefore, RhoNox-1 detects catalytic Fe(II) and is a novel marker for evaluating numerous oxidative stress-associated diseases. Nevertheless, further studies are necessary to determine the following: 1) whether this method is applicable to formalin-fixed paraffin-embedded sections; 2) whether there are any discrepancies between the data regarding this probe and other modified products in various models, and, if so, what those discrepancies mean (We suspect that labile Fe(II) may be unexpectedly stable in the absence of hydrogen peroxide in situ); 3) whether this method is applicable to time-lapse studies using animals; and 4) what kind of efforts would be necessary to decrease the background fluorescence of RhoNox-1. In conclusion, in the present study, we developed a novel strategy to localize catalytic Fe(II) in frozen sections of tissue. We recommend to use both unfixed and lightly fixed specimens for the initial evaluation. This strategy would be helpful for analyzing various iron-associated pathologies and physiologies, including neurodegenerative diseases and iron absorption through the duodenum. This probe may open up novel research areas for various oxidative stress-associated diseases. Acknowledgements The authors thank all members of Toyokuni laboratory for useful discussion.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. This work was supported in part by a grant-in-aid for research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (24390094; 221S0001-04; 24108001).
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