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Just Accepted by Free Radical Research

Histological detection of catalytic ferrous iron with the selective turn-on fl uorescent probe RhoNox-1 in a Fenton reaction-based rat renal carcinogenesis model Takahiro Mukaide , Yuka Hattori , Nobuaki Misawa , Satomi Funahashi , Li Jiang , Tasuku Hirayama , Hideko Nagasawa and Shinya Toyokuni

doi: 10.3109/10715762.2014.898844

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 specifi c turn-on fl uorescent probe (RhoNox-1) specifi c 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 carcino-genesis model with an iron chelate, ferric nitrilotriacetate (Fe-NTA), in which catalytic iron induces the Fenton reaction specifi cally 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 fl uorescent color was explicitly observed in and around the lumina of renal proximal tubules one hour 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 fl uorescein (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 ferrous iron in studies using tissues.

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Mukaide T et al. 1

Histological detection of catalytic ferrous iron with the selective

turn-on fluorescent probe RhoNox-1 in a Fenton reaction-based

rat renal carcinogenesis model

Takahiro Mukaide1, Yuka Hattori1,2, Nobuaki Misawa1, Satomi Funahashi1, Li Jiang1,

Tasuku Hirayama3, Hideko Nagasawa3 and Shinya Toyokuni1

1Department of Pathology and Biological Responses, and 2Department of Obstetrics and

Gynecology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan

3Laboratory of Pharmaceutical and Medicinal Chemistry, Gifu Parmaceutical University,

Gifu 501-1196, Japan

Correspondence: Shinya Toyokuni, M.D., Ph.D.; 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

Short title: Localizing catalytic Fe(II) in tissue

Abstract

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Mukaide T et al. 2

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 one hour

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 ferrous iron in studies

using tissues.

Keywords: catalytic ferrous iron, fluorescent probe, kidney, oxidative stress,

morphometry

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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 grams 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 has always been a subject of interest as an initiator

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of the Fenton reaction [12]. However, thus far, no histological methodology has

been established to detect labile or catalytic ferrous iron.

Recently, a highly specific probe, called RhoNox-1, for labile ferrous iron

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 using 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]. Hydroxyphenyl fluorescin (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.

Animal experiments

The  animal  experiment committee of the Nagoya University Graduate

School of Medicine approved the following animal experiments. In total, 42 male

Wistar rats (8 weeks old; Shizuoka Laboratory Anima Center, Shizuoka, Japan)

were purchased. These rats were divided into time-course and dose-dependency

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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 dose-dependency

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 m, 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.

Histological detection of labile ferrous iron

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

M). This diluted solution was used within a single day. Frozen sections of 8 m

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 l of 5 M 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.

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Imaging analysis

A fluorescence microscope (BZ-9000, Keyence, Osaka, Japan), which

allows simultaneous data acquisition of three different wavelengths, was used for

analyses. To obtain quantitative data, the exposure condition was recorded for

each image. For the quantification of labile ferrous iron, 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 40x objective lens. Eight

random areas in the proximal tubules were used for the analysis of each rat.

Reactivity of the iron solution and iron chelates with RhoNox-1

A black 96-well microplate (#MS-8096K, Sumitomo Bakelite Co., Osaka,

Japan) and RhoNox-1 (1 M final concentration in 10 mM phosphate buffer) were

used for this analysis. Ferrous iron [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 l) was mixed with 1 M RhoNox-1 (100 l),

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].

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Statistical analysis

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.

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 M as

previously described [13]. However, RhoNox-1 reacted with neither Fe(II)-NTA,

Fe(III) nor Fe(III)-NTA (Figure 1).

Ferric nitrilotriacetate (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 found strong positivity not only in the

renal proximal tubules but also in their lumina. In addition, 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 RhoNox-1-specific fluorescence

significantly increased 1 h after the injection, then further increased and was

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Mukaide T et al. 8

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 HPF-specific 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 ferrous

iron 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.

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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, acrolein, etc.)

[18,26]. Eventually, repeated reactions of this nature cause renal cell carcinoma

[17], 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 ferrous iron was principally intraluminal (unfixed) and 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

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Mukaide T et al. 10

labile ferrous iron 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 -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 4-hydroxy-2-nonenal (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 ferrous iron 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 ferrous iron and is

a novel marker for evaluating numerous oxidative stress-associated diseases.

Nevertheless, further studies are necessary to determine the followings: 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

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discrepancies mean (We suspect that labile ferrous iron 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 ferrous iron 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.

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Acknowledgments

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.

Conflicts of interest

The authors declare that they have no conflicts of interest.

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4-hydroxy-2-nonenal histidine adduct. FEBS Lett

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Figure Legends

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.

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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 m).

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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. **, P<0.01; ***, P<0.001; ####, P<0.0001

vs time=0; unpaired t-test.

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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 m). 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. Corresponding data on

integrated fluorescence intensity (IFI) of RhoNox-1 and HPF was compared,

which were proportional. AU, arbitrary unit; HPF, hydroxyphenyl fluorescein.

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