Redox Monitoring in Nuclear Medical Imaging

Abstract

Significance:

The imbalance in redox homeostasis is known as oxidative stress, which is relevant to many diseases such as cancer, arteriosclerosis, and neurodegenerative disorders. Overproduction of reactive oxygen species (ROS) is one of the factors that trigger the redox state imbalance in vivo. The ROS have high reactivity and impair biomolecules, whereas antioxidants and antioxidant enzymes, such as ascorbate and glutathione, reduce the overproduction of ROS to rectify the redox imbalance. Owing to this, redox monitoring tools have been developed to understand the redox fluctuations in oxidative stress-related diseases.

Recent Advances:

In an attempt to monitor redox substances, including ROS and radical species, versatile modalities have been developed, such as electron spin resonance, chemiluminescence, and fluorescence. In particular, many fluorescent probes have been developed that are selective for ROS. This has significantly contributed to understanding the relevance of ROS in disease onset and progression.

Critical Issues:

To date, the dynamics of ROS and radical fluctuation in in vivo redox states remain unclear, and there are a few methods for the in vivo detection of redox fluctuations.

Future Directions:

In this review, we summarize the development of radiolabeled probes for monitoring redox-relevant species by nuclear medical imaging that is applicable in vivo. In the future, translational research is likely to be advanced through the development of highly sensitive and in vivo applicable detection methods, such as nuclear medical imaging, to clarify the underlying dynamics of ROS, radicals, and redox substances in many diseases. Antioxid. Redox Signal. 36, 797–810.

Introduction

The balance between oxidation and reduction (redox balance) is generally maintained in living organisms; however, when the redox balance is disrupted and oxidation is accelerated, irreversible oxidative degeneration occurs, leading to functional abnormalities. This imbalance in the redox state is called oxidative stress. Reactive oxygen species (ROS) are major contributors of oxidative stress. ROS, such as superoxide anion radical, hydroxyl radical, and hydrogen peroxide (H₂O₂), are highly reactive chemical species that oxidize biomolecules, including lipids, proteins, and DNA, causing them to lose their original functionality (39).

On the other hand, antioxidants and antioxidant enzymes, such as ascorbate, glutathione, and superoxide dismutase, are ROS scavengers and thus protect biomolecules from oxidative stress (5). Various studies have shown that ROS are involved in the onset and progression of many diseases such as cancer (40), stroke (19), arteriosclerosis (7), neurodegenerative disorders (38), inflammatory conditions (23), and aging (12). Therefore, the detection of ROS and other molecules that are relevant to redox imbalances is postulated to contribute to the early detection of oxidative stress-related diseases, elucidation of their mechanisms, and development of therapeutic methods.

To date, various detection methods have been developed, such as chemiluminescence, fluorescence, and electron spin resonance (ESR) (55). Owing to these methods, the involvement of oxidative stress in the pathophysiology of various diseases has been clarified by using cultured cells and animal models. To apply the results of the aforementioned laboratory-based research to clinical practice, it is necessary to understand the dynamics of ROS and radicals in vivo. Therefore, the development of detection techniques applicable to clinical use is necessary.

Nuclear Medical Imaging

In nuclear medical imaging, extremely small amounts of imaging probes labeled with radioisotopes are administered into the body, and the biodistribution and kinetics of the radioactivity are noninvasively measured by single-photon emission computed tomography (SPECT) and positron emission tomography (PET). Because of its high sensitivity (10⁻¹⁰ to 10⁻¹² mol/L) and quantitative performance, the nuclear medical imaging technique involving the use of molecular-targeted imaging probes enables spatiotemporal visualization of biological processes at the cellular and molecular levels in vivo for biochemical, biological, or diagnostic applications (37, 43).

These characteristics make it possible to understand the mechanisms underlying disease initiation and progression, detect disease at an early treatable phase, and identify individuals at risk for disease. The spatial resolution achieved by SPECT and PET techniques is relatively low in sub-millimeter, though the optical imaging with the fluorescence and chemiluminescence has quite high resolution; however, the amount of probe required for SPECT and PET is quite small, 10⁻¹⁰ to 10⁻¹² mol/L or even less, as almost same as optical imaging offering high sensitivity, whereas ESR imaging needs the probe concentration as high as 10⁻³ to 10⁻⁵ mol/L.

The quantitativity of SPECT and PET is superior to other imaging modalities, because the emitted radiation shows quite low absorption and scattering by biological tissues. In addition, high depth penetration of gamma rays is advantageous whereas the skin depth of a visible light for optical imaging is less than 1 cm, which is hard to measure in a deeper region of the body.

With the development of radiolabeled imaging probes for noninvasive diagnosis, gamma-ray emitters [e.g., iodine-123 (¹²³I) and technetium-99m (^99mTc)] and positron emitters [e.g., carbon-11 (¹¹C), fluoride-18 (¹⁸F), copper-64 (⁶⁴Cu), and gallium-68 (⁶⁸Ga)] have been commonly utilized for SPECT and PET, respectively, taking into account their decay type, photon energy, and half-lives.

¹¹C and ¹⁸F are positron-emitting radionuclides (annihilation photon energy: 511 keV), and their half-lives are ∼20 and 110 min, respectively. Various PET probes labeled with ¹¹C and ¹⁸F are available for clinical use. Since fluorine, unlike carbon, is a nonconstituent element of biologically active substances, careful drug design is needed for the development of ¹⁸F-labeled probes. In contrast to ¹¹C, ¹⁸F, and ⁶⁴Cu (half-life: 13 h) produced by cyclotron, a ⁶⁸Ga radiometal has recently received a lot of attention as a positron emitter because it shows suitable radiophysical properties (half-life: 68 min, photon energy: 511 keV) for PET and can easily be eluted onsite from germanium-68 (⁶⁸Ge)/⁶⁸Ga generators.

¹²³I is one of the most widely used radioisotopes suitable for SPECT because of its superior radiation properties (photon energy: 159 keV, half-life: 13 h). ^99mTc is also commonly employed for SPECT (photon energy: 141 keV, half-life: 6.01 h), because it is produced from molybdenum-99 (⁹⁹Mo, half-life: 65.9 h) by commercially available generator systems.

During small molecule-labeling applications, ¹²³I can form a covalent bond with carbon; however, the van der Waal's radius of iodine is relatively larger, making it is possible to induce problematic steric perturbations. On the other hand, ^99mTc-labeling requires a chelating agent to produce a metal complex; therefore, it is possible to compromise the binding affinity of probes for target molecules by introducing chelating agents to key compounds.

The expression levels of target molecules related to redox (e.g., ROS, free radicals, and some redox substances) are considered extremely low; thus, redox imaging using highly sensitive nuclear medical imaging techniques has been broadly investigated. In the past two decades, several nuclear medical imaging probes have been reported (Table 1). The strategy of the probe design is very different from that of fluorescent or ESR probes, in which the signal is switched off and on depending on the reaction.

Table 1.

Radiolabeled Probes for the Detection and Monitoring of Reactive Oxygen Species, Radicals, and Redox Status

Compound number	Compound name	Main target	Studied animal disease model	References
1	[¹⁸F]FDMT or [¹⁸F]DHMT	O₂˙⁻	DOX-induced cardiac inflammation	(6, 11, 54)
2	[¹⁸F]ROStrace	O₂˙⁻	LPS-induced neuroinflammation	(15, 44)
3	[¹⁸F]FDHM	O₂˙⁻	Brain ROS production induced by microinfusion of SNP	(13)
4	[³H]Hydromethidine	O₂˙⁻	Brain ROS production induced by microinfusion of SNP, ischemia-reperfusion model, Cisplatin-induced nephrotoxicity	(1, 2, 36)
5	[¹¹C]HM	O₂˙⁻	Brain ROS production induced by microinfusion of SNP	(45)
6	¹⁸F-labeled hydrocyanine dye	O₂˙⁻, HO˙	―	(3)
7	^125/131I-PISO	O₂˙⁻	LPS-induced inflammation, hind paw inflammation	(16)
8	PC-[¹⁸F]FLT-1	H₂O₂	―	(8)
9	[⁶⁸Ga]Galuminox	O₂˙⁻, H₂O₂, ONOO⁻	LPS-induced lung inflammation	(21, 32)
10	¹²⁵I-labeled edaravone	HO˙	―	(28)
11	¹²⁵I-HIPBN	Oxygen radicals	―	(41)
12	¹²⁵I-IPBN	Oxygen radicals	―	(14)
13	¹²⁵I-labeled nitroxide derivative	Lipid carbon radicals	―	(50)
14	¹²⁵I-labeled nitroxide derivative	Lipid carbon radicals	Carbon tetrachloride-treated acute liver inflammation	(4)
15	[¹⁸F]DFA	redox substances (e.g., GSH)	Diethyl maleate-induced glutathione-deficient model	(47 –49)
16/17	[¹¹C]VitC/[¹¹C]DHA	ROS and GSH	―	(9)
18/19	[¹¹C]DHQ/[¹¹C]Q1⁺	Redox status	Apocynin-treated brain redox alteration	(25)
20	[¹⁸F]FASu	System x_c ⁻	Tumor xenograft model	(42, 53)

DFA, 6-deoxy-6-fluoro-L-ascorbic acid; DHA, dehydroascorbic acid; DHMT, 6-(4-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-methyl-5,6-dihydrophenanthridine-3,8-diamine; DOX, doxorubicin; FASu, 5-fluoroaminosuberic acid; FDHM, 6-(4-fluorophenyl)-5-methyl-5,6-dihydrophenanthridine-3,8-diamine; FDMT, 6-(4-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-methyl-5,6-dihydrophenanthridine-3,8-diamine; LPS, lipopolysaccharide; ROS, reactive oxygen species; SNP, sodium nitroprusside.

On the other hand, a precise molecular design for radiolabeled probes is required because the radiation from these probes is emitted at all times, affording that the signal is always on. Therefore, in nuclear medical imaging, a sufficient amount of time is required for the clearance of the unreacted probe before attempting to obtain images to achieve a good signal-to-noise ratio. In this review, we outline the detection probes of redox imbalances, including ROS and other chemical species, utilizing nuclear medical imaging techniques.

Superoxide and Hydroxyl Radicals

Dihydroethidium analogues

Dihydroethidium (DHE) is the reduced form of ethidium bromide (EtBr) (Fig. 1A) used for the detection of nucleic acids. In contrast to EtBr, DHE is a neutral molecule with a non-planar phenanthridine moiety. When DHE is oxidized, it becomes positively charged and planar in orientation, allowing it to intercalate into DNA and remain in the cell. From the mechanism cited earlier, DHE has been well used as a “turn-on” type fluorescent probe by the reaction with superoxide, but it has been pointed out that its fluorescence spectrum was slightly differed from that of EtBr (56). In 2006, Kalyanaraman and colleagues reported the formation of 2-OH ethidium form (2-OH-E⁺) after the reaction of DHE with superoxide (Fig. 1A) (57).

FIG. 1.

Radiolabeled dihydroethidium analogues for superoxide detection. (A) Dihydroethidium, ethidium bromide, reaction with superoxide and side-reaction products; (B) radiolabeled derivatives.

In the presence of other one-electron oxidants such as ferricytochrome c (even in the absence of superoxide), DHE has been reported to produce ethidium without a hydroxyl group (E⁺), or a dimer (E⁺−E⁺) (Fig. 1A) (58). These products have similar structural characteristics with 2-OH-E⁺ and EtBr and would show the accumulation in the cell.

On the basis of the reaction of DHE with superoxide and other one-electron oxidants including hydroxyl radical mentioned earlier, the radiolabeled DHE derivatives 1–5 has been reported. In 2014, Chu et al. reported a probe 1 (Fig. 1B), which was synthesized from a Boc-protected DHE derivative and ¹⁸F-labeled azidoethane using a click reaction of copper(I)-assisted 1,3-dipolar cycloaddition (11). The probe showed a high retention time in the heart during microPET when administered to mice treated with doxorubicin (DOX), which is known to contribute to the production of superoxide, which leads to cardiotoxicity in vivo.

In 2016, Zhang et al. reported an automated synthetic system for 1, which was given another name as [¹⁸F]DHMT (Fig. 1B) (54). Further, in 2018, Boutagy et al. found that 1 accumulated in the myocardium before the onset of cardiotoxicity (a reduction in left ventricular ejection fraction [LVEF]) caused by the administration of DOX, concluding that the detection of ROS production could be used to identify signs of cardiotoxicity before a reduction in LVEF (6). In 2018, Hou et al., who first developed 1, reported another ¹⁸F-labeled DHE analogue 2 ([¹⁸F]ROStrace) (Fig. 1B) (15).

Probe 1 has a larger substituent consisting of a triazole group in the para position of the phenyl ring because of a click reaction that is used to introduce ¹⁸F, whereas ¹⁸F in 2 is introduced via nucleophilic substitution and thus has a smaller substituent due to the absence of a triazole linker, probably leading to an increase in lipophilicity and a decrease of molecular size. Owing to this modification, compound 2 has been shown to cross the blood–brain barrier (BBB), whereas 1 failed to show BBB permeability.

The radiolabeled probe 2 has been tested in a mouse model of lipopolysaccharide (LPS)-induced neuroinflammation. In the study, the degree of accumulation of the compound in the central nervous system almost correlated with the condition score stratified by visual observation of distress signs. In 2019, using the same LPS mouse model, unlabeled 2 (ROStrace) was shown to localize in microglia/macrophages and neurons, and serve as a PET probe for imaging the pro-inflammatory form of neuroinflammation (44).

Further, in 2020, Egami et al. synthesized another ¹⁸F-labeled dihydromethidine analogue 3 ([¹⁸F]FDHM), which has a fluorine atom directly attached to the para-position of the benzene ring (13) (Fig. 1B). This compound also showed BBB permeability and significantly accumulated in brain regions where sodium nitroprusside (SNP), which produces ROS by reacting with intracellular oxygen, was microinfused.

In the same year that 1 was first reported, Abe et al. reported tritium (³H)-labeled dihydromethidine (4, [³H]hydromethidine), which is an isotopologue of dihydromethidine without any further chemical modification (Fig. 1B) (1). In this study, ³H-labeling was used as a prototype since it is feasible to synthesize ¹¹C-labeled analogue (5) by N-methylation. This compound showed rapid penetration into the brain, and a significant accumulation of radioactivity was detected in the ipsilateral side of the SNP-microinfused oxidative stress mouse model in comparison to the contralateral side.

In addition, the same group reported the accumulation of radioactivity derived from 4 in brain regions of mice induced with transient middle cerebral artery occlusion, which is a model of ischemia-reperfusion that is known to produce ROS such as superoxide and hydroxyl radicals (2).

The accumulation of radioactivity was decreased after treatment with dimethylthiourea (DMTU), a hydroxyl radical scavenger, indicating that the ROS produced by ischemia-reperfusion could be detected. Moreover, the same group of researchers investigated the accumulation of 4 in the corticomedullary junction by autoradiography in a mouse model of cisplatin-induced nephrotoxicity, which is also known to be involved in hydroxyl radical generation (36). This ³H–labeled compound 4 demonstrated the usefulness of this chemical structure as a probe for detecting superoxide and hydroxyl radicals.

The ¹¹C-labeled analogue 5 ([¹¹C]HM, Fig. 1B) was reported by Wilson et al. in 2017 (45). Compound 5 is an isotopologue of HM (Fig. 1A) that is substituted with ¹¹CH₃. The probe 5 showed good uptake as well as a rapid washout from the brain. LPS-treated rats showed a significant increase in retention of radioactivity in the brain. Ex vivo autoradiography and PET imaging of rats treated with SNP-containing microinjections showed increased retention of radioactivity in the treated region of the brain.

With regard to synthesis and probe preparation, the authors mentioned some difficulties, namely direct methylation of phenanthridine (oxidized form of 5,6-dihydrophenanthridine) by nucleophilic substitution was not feasible due to the low nucleophilicity of the attached nitrogen atom with a preference for methylating the amine groups at the 3,8-positions. Boc protection of amine groups is necessary for direct methylation; however, the authors opted out of this method, citing stability concerns of the reduced form and thus making it unsuitable as a precursor in radiosynthesis. Moreover, because of the high susceptibility of 5 to the oxidants, the authors recommended the coexistence of ascorbic acid in all purification, preparation, and storage processes.

In addition to the concerns mentioned earlier, Kalyanaraman and colleagues have highlighted other shortcomings of DHE derivatives, including instability, complexity of reaction products, and potential interference with heme enzymes (58). The compounds are light sensitive and are prone to autoxidation. When DHE derivatives react with superoxide, they only give off 2-OH-E⁺ (Fig. 1A). In other words, to prove the presence of superoxide, 2-OH-E⁺ or 2-OH-methidium (2-OH-M⁺) must be detected.

However, in the presence of other one-electron oxidants (even in the absence of superoxide), these derivatives produce E⁺, methidium without a hydroxyl group (M⁺), or a dimer (E⁺−E⁺ or M⁺−M⁺) (Fig. 1A), which have similar fluorescence characteristics to 2-OH-E⁺ and 2-OH-M⁺. This also applies to radiolabeled DHE derivatives 1–5, because the emission signals from these chemical analogues cannot be distinguished. When the involvement of superoxide is considered during the use of DHE derivative probes, it is necessary to carefully examine the chemical form of the superoxide, that is, whether it is detectable or not, and whether it is nonspecific to the accumulation of other oxidants, for example, by comparison with standard compounds using high-performance liquid chromatography (HPLC) analysis.

Recently, the reaction mechanism and kinetics of DHE derivatives with superoxide and other biological radicals were reported (22). Radical cations were reported to be initially formed by one-electron oxidation, and the spin density distribution was localized at the 2-position of 5,6-dihydrophenanthridine based on density functional theory calculations. Superoxide then reacted with atoms at the aforementioned position to form a 2-OH form or intramolecular dimerization.

In the same study, methylation of the amine moiety was found to prevent the formation of dimers, but not the prevention of the reaction between the radical cation and the superoxide. This chemical modification may be useful as a new scaffold for the sensitive detection of superoxide.

Hydrocyanine derivative

Hydrocyanine is a reduced form of cyanine dye that typically has a polymethine structure and nitrogen-containing heterocyclic rings at both ends. Cyanine dyes have a positive charge on nitrogen atoms and emit strong fluorescence, whereas hydrocyanine is a neutral molecule with less fluorescent emission properties. Hydrocyanine is a “turn-on” type probe for oxygen radicals such as superoxide and hydroxyl radical, which attract hydrogen atoms from hydrocyanine, triggering oxidation to form cyanine dye.

Next, electronically neutral hydrocyanine is converted to cationic cyanine, which is expected to be trapped in the cell after the reaction with ROS. The ¹⁸F-labeled hydrocyanine probe 6 (Fig. 2) was reported in 2017 as an ROS probe (3). ¹⁸F-Labeling was achieved by direct fluorination of hydrocyanine (reduced form) with a decay-corrected radiochemical yield of 62%.

FIG. 2.

Radiolabeled hydrocyanine derivative for superoxide and hydroxyl radical detection.

During stability studies of 6 in 10% ethanol/saline solution, a peak that appeared to be of the oxidized form of 6 was observed during HPLC analysis at 120 min after preparation; however, the addition of ascorbic acid (0.5 mg/mL) was found to prevent oxidation of 6. PET/computed tomography (CT) imaging in healthy mice showed that the distribution of 6 was quite different from that of its oxidized form at 2 h post-injection. Nonetheless, an evaluation of the oxidative stress model is required to assess the usefulness of this ROS probe.

Fluorescein derivative

As a superoxide reacting probe, fluorescein-based radiolabeled probe 7 (^125/131I-PISO, Fig. 3) was reported in 2018 by Huang et al. (16). Fluorescein in a form of a non-fluorescent but ring-opening spirolactam structure, under physiological conditions, gives rise to strong fluorescence emission. When the hydroxyl groups of the xanthene moiety are protected, fluorescein forms a spirolactam structure, resulting in no fluorescence.

FIG. 3.

Radiolabeled fluorescein derivative for superoxide detection.

When this protection is offered by a chemical moiety that is selectively cleaved by target molecules, fluorescein works as a sensor by reacting with targets, thus offering high fluorescence capabilities. Huang et al. used trifluoromethanesulfonyl as a superoxide reactive group and the protective group of the hydroxyl group in the xanthene backbone and labeled the benzene moiety with radioiodine.

The log p value of the resultant compound was −1.62 (log p = 2.46 before the reaction), and it was thus postulated to accumulate intracellularly after reacting with intracellular superoxide. In an LPS-induced inflammation mouse model, SPECT/CT imaging showed that the accumulation of 7 was focused at the target site, indicating the potential of this probe in aiding the visual distribution of superoxide in living organisms.

Hydrogen peroxide

Thymidine analogue

H₂O₂ is an ROS and is considered to be an important messenger molecule because of its relatively long half-life and extensive diffusion distance compared with other ROS. PC-[¹⁸F]FLT-1 (8, Fig. 4) was developed by Carroll et al. (8) as a PET probe for H₂O₂. [¹⁸F]FLT (Fig. 4) was used as a PET probe in clinical settings for cancer diagnosis. It is a thymidine analogue that is phosphorylated by thymidine kinase and incorporated into DNA when taken up by cells, mimicking endogenous thymidine. Owing to this, 3′-deoxy-3′-fluorothymidine (FLT) accumulates in cancer cells because of their high rate of proliferation.

FIG. 4.

Radiolabeled thymidine analogue for H₂O₂ detection. H₂O₂, hydrogen peroxide.

The authors designed and synthesized a molecule consisting of boronic acid, which is often used as an H₂O₂ reactive substituent, attached to the 5′-OH of FLT by a linker and labeled with ¹⁸F at the 3′ position. Before uncaging by H₂O₂, compound 8 could not be incorporated into DNA and was thus washed out. However, once the boronic acid and linker were cleaved off by H₂O₂ to form FLT, it could then be phosphorylated by thymidine kinase and incorporated into DNA.

In other words, it accumulated in cells where both H₂O₂ and thymidine kinase activity were elevated. The accumulation of radioactivity in UOK262 renal carcinoma cells was increased in a concentration-dependent manner after the addition of H₂O₂, and a significant increase in cellular uptake was observed in UOK262 cells treated with paraquat, an ROS inducer, from 4 h after treatment compared with the untreated group. There are potential limitations to the uncaging of ¹⁸F by ROS and cleaving of the linker because of its short half-life.

Boronic acids or boronate esters are the only caging moieties for H₂O₂; however, the reaction rate between boronic acid and H₂O₂ is relatively slow (∼1 M⁻¹ s⁻¹) (31). Therefore, it is desirable to develop a more reactive caging moiety for H₂O₂ detection.

Total ROS and other radicals

Luminol derivative

In 2020, ⁶⁸Ga-labeled probe 9 ([⁶⁸Ga]Galuminox, Fig. 5) was developed for imaging oxidative stress-induced inflammation in the lungs, by reacting with superoxide and its downstream molecules such as H₂O₂ and peroxynitrite (32). Galuminox comprised luminol and a chelator (NOTA), which are tethered with a linker. In an LPS-induced inflammation A549 lung epithelial cell model, non-radiolabeled Galuminox showed colocalization with the mitochondrial superoxide probe MitoSOX, indicating that Galuminox measures mitochondrial production of ROS.

FIG. 5.

Radiolabeled luminol derivative for ROS detection. ROS, reactive oxygen species.

To assess the potential ROS imaging ability of 9, PET imaging was conducted in mice with LPS-induced lung inflammation. LPS is known to initiate pro-inflammatory responses in mice and cause acute lung injury (21). A high accumulation of radioactivity was observed in the lungs of the animals 24 h after treatment with LPS, with a standard uptake value that was 5-fold higher than that of the saline-treated control mice group.

Then, this probe has a potential to monitor the ROS activity in acute lung injury. On the other hand, the biodistribution data in control mice at 60 min showed that most of the probe were accumulated in the bladder, next in the lung, and then in the kidney. This supposed that the probe was readily reacted in the blood, as luminol is a famous reagent for blood detection and excreted immediately into the urine. Then, the intravenous administration may lead to the loss of probe in the blood before reaching the target site in the unreacted form. Therefore, the result of accumulation needs to be precisely considered whether the probe reacted with the target molecule onsite, or the product reacted elsewhere was just distributed to the target site.

Edaravone derivative

There has been a report on the use of 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone, Fig. 6A), which is a potent free radical scavenger, for detecting free radicals such as hydroxyl radical (28). Edaravone has been developed and used as a medicine to protect the brain from damage after acute cerebral infarction. When edaravone, a lipophilic molecule, scavenges free radicals in the brain, it converts to a carboxylic acid [2-oxo-3-(2-phenylhydrazono)butanoic acid (OPB)] (Fig. 6A), which is a hydrophilic compound, leading to retention in the brain.

FIG. 6.

Radiolabeled edaravone derivative for free radical detection. (A) Reaction of edaravone and its product; (B) ¹²⁵I-labeled edaravone.

For this purpose, 1-(3′-[¹²⁵I]iodophenyl)-3-methy-2-pyrazolin-5-one 10 (¹²⁵I-labeled edaravone) (Fig. 6B) was developed by using two labeling methods via isotopic exchange and interhalogen exchange under solvent-free conditions, in which iodo- and bromo-derivatives were used as labeling precursors, respectively. The specific activities were lower for the former labeling method, whereas the latter gave higher specific radioactivity.

The radiochemical purity of 10 with low specific activity decreased to 80% after 1 h of storage at 37°C and to 40% after 3 h of storage at the same temperature. For the high specific activity compound, its radiochemical purity dropped to between 60% and 70% during an attempt to prepare an injectable formulation. Next, the distribution was assessed by using the labeled compound with the low specific activity, and it was observed to be uniformly distributed in all organs of the body, and mostly disappeared after 24 h.

The high specific activity compound was not stable and has not yet been investigated in vivo. Nevertheless, the applicability of this probe needs further investigation in a brain oxidative stress model.

Derivatives of spin trapping reagents

A radiolabeled spin-trapping reagent for the detection of free radicals in vivo has been reported by Fujibayashi et al. (14, 41). A spin-trapping reagent is a nitrone or a nitroso compound that reacts with radicals to form stable radical adducts that can be detected by ESR. In their study, one of the spin-trapping reagents, α-phenyl-N-tert-butylnitrone (PBN) (Fig. 7A), was labeled with radioiodine. Initially, in 1994, nitrone 11 ([¹²⁵I]HIPBN) (Fig. 7B), which has a hydroxy group at the para position and ¹²⁵I at the meta position, had been reported (41).

FIG. 7.

Radiolabeled derivatives of spin trapping reagents for free radical detection. (A) Reaction of PBN with free radicals and its product; (B) ¹²⁵I-labeled PBN derivatives. PBN, α-phenyl-N-tert-butylnitrone.

In 1997, another nitrone 12 ([¹²⁵I]IPBN) (Fig. 7B) was reported to have an ¹²⁵I at the para position (14). [¹²⁵I]IPBN showed higher stability in cell culture media than 11, and the blood retention of 12 in ddY mice remained high and did not decrease even after 60 min. The authors postulated the long retention to the high protein binding of 12 in the blood. To date, a study in an oxidative stress model has not been performed with this probe, and further investigations are still required to determine its usefulness.

Nitroxide derivatives

Nitroxides, which are also called aminoxyls, are radical compounds whose spin density localizes on N–O atoms and are relatively stable as they can be handled at room temperature and normal pressure. They have potential reactivity toward one-electron oxidation and reduction as well as carbon radicals to form covalent bonds (26). Using these reactivities, nitroxides have been used for various applications such as catalysts in alcohol oxidation (30), antioxidants (33), and agents for nitroxide-mediated radical polymerization (29).

The reactivities of nitroxides vary according to the basic structure of the compounds, such as ring- or chain-type, and by electronic and steric substituent effects. It has been reported that six-membered ring nitroxides have various reactivities toward ascorbate, depending on the substituents proximal to the radical moiety (52). In addition, an inhibition effect toward lipid peroxidation was shown by using a series of nitroxides with alkyl groups as substituents (51).

This effect was enhanced by the increase in lipophilicity due to the alkyl substituent, which facilitated the trapping of carbon radicals in the lipid fraction. One fluorescent probe application involving lipid carbon radical detection through taking advantage of the off/on quenching of fluorescence before and after the reaction, which is a quenching effect derived from the diamagnetism of the radical moiety, has been reported to date (46).

Based on the reactivity of this nitroxide compound for lipid radical detection, we developed ¹²⁵I-labeled nitroxide derivatives 13 and 14 (Fig. 8) for in vivo detection, assuming that the adduct of nitroxide with lipid radicals might be trapped in membranes by increasing their lipophilicity (4, 50). In these studies, the problematic reactivity toward bioreduction by ascorbate was reduced and the selectivity to lipid radicals was increased after the replacement of the methyl group (13) by the alkyl substituent (14).

FIG. 8.

Radiolabeled nitroxide derivatives for free radical detection.

In a carbon tetrachloride-treated mouse model that showed enhanced lipid radicals from metabolic processes, radiolabeled nitroxide 14 accumulated largely in the liver, kidney, and lung tissues, where lipid peroxidation was promoted, whereas the methoxyamine derivative, which lost reactivity to lipid radicals, showed less accumulation compared with 14.

Nitroxides are often used for in vivo redox monitoring probe (17, 35). It is known to be reduced mainly by ascorbate that exists in a relatively high concentration, even if its reaction rate constant is low in the order of 10⁰–10¹ M⁻¹s⁻¹ (18, 20). On the other hand, nitroxides have a high reaction rate constant in order of ∼10⁹ M⁻¹s⁻¹ for the coupling reaction with carbon radicals (10) although the amount of lipid carbon radicals generated in vivo is assumed to be quite low.

In addition, lipid peroxidation has been associated with a variety of diseases such as cancer, ischemia/reperfusion injury, and neurodegenerative disorders. And ferroptosis, a new type of cell death characterized by iron-dependent lipid peroxidation (34), is receiving a great deal of attention in recent years. To elucidate the mechanism of these disease generation and progression, it would be useful to clarify the localization of lipid radicals in vivo.

From the viewpoint of these requirements for sensitivity and imaging, the use of nuclear medical imaging is suitable for the development of the in vivo detection method for lipid radicals. Therefore, radiolabeled nitroxide 14 would be one of the candidate probes for in vivo lipid radicals.

Redox state

Ascorbic acid derivatives

Ascorbic acid, also known as vitamin C, is a water-soluble antioxidant that is oxidized to dehydroascorbic acid (DHA) by extracellular ROS (24). In the process of H₂O₂ elimination, it is recycled via the glutathione-ascorbate cycle. In addition, ascorbic acid regenerates vitamin E, which is a lipophilic antioxidant. Therefore, the dynamics of ascorbic acid are believed to reflect the in vivo redox state.

One of the radiolabeled ascorbic acids, 6-deoxy-6-[¹⁸F]fluoro-L-ascorbic acid 15 ([¹⁸F]DFA, Fig. 9A) was synthesized by Yamamoto et al. in 1992 (48). This compound accumulated in the brain of a global cerebral ischemia-reperfusion rat model, inducing superoxide production and redox alterations (49). The relationship between the accumulation of 15 and glutathione content was investigated in glutathione-deficient rats treated with diethyl maleate, a glutathione-depleting agent (47).

FIG. 9.

Radiolabeled ascorbic acid derivatives for redox state detection. (A) ¹⁸F-Labeled ascorbic acid; (B) ¹¹C-labeled ascorbic acid and its redox couples.

A lower accumulation of radioactivity was observed as glutathione content decreased. This suggested that cellular uptake of 15 competed with endogenous ascorbic acid due to the de novo synthesis of ascorbic acid triggered by glutathione depletion. As seen in these results, compound 15 demonstrated the ability to monitor redox status. On the other hand, DHA exists predominantly in the bicyclic form in an aqueous solution, is a substrate for glucose transporters (GLUT), and is taken up into cells.

Carroll et al. proposed that this principle could provide for the design of a probe that reflects extracellular ROS. They synthesized ¹¹C-labeled ascorbic acid 16 ([¹¹C]VitC, Fig. 9B) (9). When the labeling reaction was performed under carrier-free conditions, the radiochemical yield was ∼14%. This was lower than that observed for carrier-added conditions (∼45%). These results indicated that the produced ascorbic acid was immediately oxidized to DHA in situ, which was probably due to the generation of ROS by radiolysis.

To evaluate the cellular transport of this probe, DHA labeled with ¹¹C (17) ([¹¹C]DHA, Fig. 9B) was also prepared by oxidation of 16. The uptake of 17 was about 10-fold higher in a U87 glioblastoma cell line than that of 16, indicating that 16 is mainly taken up in an oxidation-dependent manner. In vivo microPET imaging of rat brains showed that 17 had a higher accumulation than 16, because DHA permeates the BBB via GLUT1.

Further, to investigate endogenous ROS-dependent accumulation in cells, the radioactivity accumulation of 16 was evaluated by using HL60 human promyelocytic leukemia cells and isolated human neutrophils that were stimulated with phorbol 12-myristate 13-acetate (PMA), which is an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activator that causes the production of ROS, and is blocked by using cytochalasin B, a potent inhibitor of GLUT transport.

In both cells, the radioactivity increased after the stimulation of PMA, and its accumulation was significantly decreased by cytochalasin B. This indicated that 16 was taken up by cells via oxidation by endogenously produced ROS.

As seen earlier, radiolabeled probe derivatized from ascorbic acid can monitor the in vivo redox status through the redox reaction. Especially, as seen in a probe 16, the extracellular oxidation leads to the intracellular accumulation that utilizes the fast transport of DHA into the cell and the following reduction to the ascorbic acid, which is hard to diffuse out of the cell.

However, since this probe is oxidized extracellularly and trapped in the cell via GLUT transporter, not only the ROS production but also the expression level of GLUT would affect the results of probe accumulation. Therefore, the precise consideration would be required for the relationship of the ROS production and GLUT expression in each of the disease models.

Dihydroquinoline derivatives (NAD⁺/NADH)

The NAD⁺/NADH is a redox substance that is involved in redox reactions in vivo. Similar to NADH, its derivatives, 1,4-dihydropyridine and 1,4-dihydroquinoline, become quaternary ammonium salts when oxidized. In other words, it may be possible to detect the redox balance when the reduced form passes through the cellular membrane with relatively high hydrophobicity and accumulates in the cell after oxidation, depending on the redox state.

Okamura et al. reported ¹¹C-labeled 1,4-dihydroquinoline 18 ([¹¹C]DHQ1; Fig. 10) in 2015 for brain redox status monitoring, because unlabeled DHQ1 is more chemically stable to oxidation than NAD or 1,4-dihydropyridine (25). However, they found that 18 was not stable and changed to its oxidized form after purification for HPLC analysis.

FIG. 10.

Radiolabeled dihydroquinoline derivatives for redox state detection

The radiochemical purity immediately after purification was less than 90%, even with the addition of ascorbate into the collecting flask. It was found that oxidation could be inhibited by adding ascorbate to the mobile phase during purification. This mixture was stable in phosphate-buffered saline at 37°C, whereas the fraction of 18 disappeared in brain homogenate of mice, with the initial rate of the disappearance increasing with an increase in the concentration of the homogenate.

These results suggest that 18 was oxidized by some enzymes to its oxidized form 19 ([¹¹C]Q1⁺, Fig. 10). To examine BBB permeability of the reduced and oxidized forms, quinolinium cation 19 was also prepared. PET imaging showed that the brain radioactivity of cationic 19 was much lower than that of 18. Dihydroquinoline 18 was enzymatically oxidized and accumulated in the brain of mice.

Further, to investigate whether 18 could respond to the redox status of the brain, apocynin was pretreated as a redox modulator, which is an NADPH oxidase inhibitor that reduces the generation of ROS. In apocynin-pretreated mice, the radioactivity rapidly disappeared compared with the vehicle group, indicating that the clearance of 18 from the brain occurred because of its inhibition to form the hydrophilic 19 moiety by apocynin. Although the possibility of reacting with ROS cannot be excluded, the oxidation of 18 was concluded to proceed enzymatically and was determined to be affected by the redox status.

Cystine-glutamate transporter substrate

During oxidative stress, the ubiquitin-ligase protein KEAP1 is oxidatively modified to form disulfide bonds, which inhibit the degradation of transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and promote the transcription of xCT by binding to the antioxidant response element promoter region. xCT is a subunit of the cystine-glutamate transporter (system x_c ⁻), which imports cystine from the extracellular space to the cytosol and releases glutamate simultaneously.

The xCT subunit maintains low basal expression in normal tissues but is universally upregulated in tumors in response to oxidative stress. Therefore, cellular responses to oxidative stress induce xCT expression and promote cystine uptake for glutathione synthesis in tumors. 5-[¹⁸F]Fluoroaminosuberic acid 20 ([¹⁸F]FASu) (Fig. 11) has been reported as a system x_c ^- substrate for PET imaging (42).

FIG. 11.

Radiolabeled cystine derivative for redox state detection.

Cellular uptake in human ovarian carcinoma cell SKOV-3 was inhibited by sulfasalazine, an inhibitor of system x_c ^-. In the SKOV-3 cancer transplantation model, 20 showed a fivefold higher accumulation than [¹⁸F]FDG. In a breast cancer transplantation model, an uptake of 20 was increased when MDA-MB-231 cells were treated with diethyl maleate, which increases xCT expression as well as oxidative stress (53).

In addition, in three breast cancer models, MDA-MB-231, which has the highest xCT messenger RNA level, had the highest uptake of 20. These results showed that 20 could be useful for monitoring oxidative stress through the activity of system x_c ^-.

Conclusions

Nuclear medical imaging probes that target ROS, free radicals, and redox substances related to redox fluctuations have been outlined in this review. Since these substances are highly reactive and in very small quantities, the high sensitivity of measurement in nuclear medical imaging is very vital. On the other hand, since radioactive material constantly emits a signal, it is difficult to distinguish between probes that have reacted with the target molecules and those that have not.

Therefore, it is necessary to design probes in which only the reacted probes remain where they are reacted, and the unreacted probes are quickly eliminated. In fact, as in the case of most of the probes presented in this review, the strategy of molecular design is to (1) increase cell membrane permeability by decreasing the molecular weight and increasing the lipophilicity of the probes before the intracellular reaction and (2) reduce permeability by making ionic compounds of probes on a reaction with the target molecule so that they remain in the cell.

Regarding to these requirements, fluorescein derivative 7 is currently best suited for superoxide in terms of specificity and reactivity among the superoxide targeted probes 1–7. And for H₂O₂, the boronic acid 8 is relatively specific to H₂O₂. However, the boronic acid is also known to be reactive to peroxynitrite and hypochlorous acid with the reaction rate in the order of ∼10⁶ and ∼10² M⁻¹s⁻¹, respectively, whereas the rate for H₂O₂ is around 1 M⁻¹s⁻¹ (27).

And for probes targeting other ROS such as hydroxyl and alkoxyl radicals, the specificity is not sufficient at this time. To elucidate the role of these species in pathology, the improvements of specificity or discovery of novel reacting groups are indispensable. In addition to the strategy mentioned earlier, another trapping method is useful in molecular design, for example, which is trapped in DNA by intercalation or in membrane by trapping lipid-derived radicals and trapped inside cells by utilizing the transporter.

At the same time, the ideal scaffold needs the abilities to be easily introduced in a reacting group combined with the trapping mechanism, and to be easily labeled by radioisotope with high radiochemical yield and radiochemical purity. In addition, if a probe is targeted to the brain disease, the BBB permeability is needed for a probe design. Namely, the scaffold should be chosen considering the molecular weight/size and moderate lipophilicity. Since these properties should be altered with the types of the reacting group, they need to be investigated precisely one by one for the target molecule and target disease.

As we have seen with DHE derivatives, as well as in nuclear medical imaging probes, it should be carefully considered what the probe reacts with and what reflects the produced result(s). This is because the unique problem with radiolabeled probes, especially ROS-targeting probes, is that the radiation from the radioisotope is known to cause radiolysis of water and generate ROS, which not only degrades the probe itself and reduces its purity before it is administered into the body, but may also reflect results other than the reaction with the targeted endogenous ROS.

Nonetheless, the addition of carriers or radical scavengers is an effective solution for degradation before administration into the body. To examine whether the reaction with endogenous ROS can be detected, it is necessary to confirm the estimated reaction products by comparison with standards or identification by liquid chromatography–mass spectrometry analysis or by utilizing specific inhibitors for target molecules.

Thus, although radiolabeled probes require precise molecular design that controls internal behavior and may cause problems specific to them, they have many advantages over existing imaging modality probes, such as superior sensitivity and capability of noninvasive measurements, including deep regions. Also, it would be possible to measure from cells to the living body with one common structure that has fluorescent/chemiluminescent moiety and radioisotope such as fluorescein (6) and luminol (9) derivatives.

The use of such radiolabeled probes is expected to promote translational research that can verify the contribution of redox fluctuations to diseases at the molecular level and enhance drug discovery as well as the development of diagnostic technologies based on the elucidation of redox alterations.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

A part of this work was supported by JSPS KAKENHI [Grant Nos. 19K17283 (T.Y.), 19H03607 (T.M.)] and Hyogo Science and Technology Association (T.Y.).

Abbreviations Used

References

Abe

, Takai

, Fukumoto

, Imamoto

, Tonomura

, Ito

, Kanegawa

, Sakai

, Morimoto

, Todoroki

, and Inoue

. In vivo imaging of reactive oxygen species in mouse brain by using [³H]Hydromethidine as a potential radical trapping radiotracer. J Cereb Blood Flow Metab, 34: 1907–1913, 2014.

Abe

, Tonomura

, Ito

, Takai

, Imamoto

, Rokugawa

, Momosaki

, Fukumoto

, Morimoto

, and Inoue

. Imaging of reactive oxygen species in focal ischemic mouse brain using a radical trapping tracer [³H]hydromethidine. EJNMMI Res, 5: 1–9, 2015.

Al-Karmi

, Albu

, Vito

, Janzen

, Czorny

, Banevicius

, Nanao

, Zubieta

, Capretta

, and Valliant

. Preparation of an¹⁸F-labeled hydrocyanine dye as a multimodal probe for reactive oxygen species. Chem A Eur J, 23: 254–258, 2017.

Azuma

, Yamasaki

, Sano

, Munekane

, Matsuoka

, Yamada

, and Mukai

. A radioiodinated nitroxide probe with improved stability against bioreduction for in vivo detection of lipid radicals. Free Radic Biol Med, 163: 297–305, 2021.

Birben

, Sahiner

, Sackesen

, Erzurum

, and Kalayci

. Oxidative stress and antioxidant defense. World Allergy Organ J, 5: 9–19, 2012.

Boutagy

, Wu

, Cai

, Zhang

, Booth

, Kyriakides

, Pfau

, Mulnix

, Liu

, Miller

, Young

, Carson

, Huang

, Liu

, and Sinusas

. In vivo reactive oxygen species detection with a novel positron emission tomography tracer, ¹⁸F-DHMT, allows for early detection of anthracycline-induced cardiotoxicity in rodents. JACC Basic Transl Sci, 3: 378–390, 2018.

Burtenshaw

, Kitching

, Redmond

, Megson

, and Cahill

. Reactive oxygen species (ROS), intimal thickening, and subclinical atherosclerotic disease. Front Cardiovasc Med, 6: 1–18, 2019.

Carroll

, Michel

, Blecha

, Vanbrocklin

, Keshari

, Wilson

, and Chang

. A boronate-caged [¹⁸F]FLT probe for hydrogen peroxide detection using positron emission tomography. J Am Chem Soc, 136: 14742–14745, 2014.

Carroll

, Truillet

, Shen

, Flavell

, Shao

, Evans

, VanBrocklin

, Scott

PJH

, Chin

, and Wilson

. [¹¹C]Ascorbic and [¹¹C]dehydroascorbic acid, an endogenous redox pair for sensing reactive oxygen species using positron emission tomography. Chem Commun, 52: 4888–4890, 2016.

10.

Chateauneuf

, Lusztyk

, and Ingold

. Absolute rate constants for the reactions of some carbon-centered radicals with 2,2,6,6-tetramethyl-1-piperidinoxyl. J Org Chem, 53: 1629–1632, 1988.

11.

Chu

, Chepetan

, Zhou

, Shoghi

, Xu

, Dugan

, Gropler

, Mintun

, and Mach

. Development of a PET radiotracer for non-invasive imaging of the reactive oxygen species, superoxide, in vivo. Org Biomol Chem, 12: 4421–4431, 2014.

12.

Davalli

, Mitic

, Caporali

, Lauriola

, and D'Arca

. ROS, cell senescence, and novel molecular mechanisms in aging and age-related diseases. Oxid Med Cell Longev, 2016: 1–18, 2016.

13.

Egami

, Nakagawa

, Katsura

, Kanazawa

, Nishiyama

, Sakai

, Arano

, Tsukada

, Inoue

, Todoroki

, and Hamashima

. ¹⁸F-Labeled dihydromethidine: positron emission tomography radiotracer for imaging of reactive oxygen species in intact brain. Org Biomol Chem, 18: 2387–2391, 2020.

14.

Fujibayashi

, Tajima

, Wada

, Waki

, Sakahara

, Konishi

, and Yokoyama

. Radioiodinated α-p-iodophenyl-N-tert-butylnitrone as a radical detecting agent in vivo. Nucl Med Biol, 24: 399–403, 1997.

15.

Hou

, Hsieh

, Li

, Lee

, Graham

, Xu

, Weng

, Doot

, Chu

, Chakraborty

, Dugan

, Mintun

, and Mach

. Development of a positron emission tomography radiotracer for imaging elevated levels of superoxide in neuroinflammation. ACS Chem Neurosci, 9: 578–586, 2018.

16.

Huang

, Li

, Zhang

, Li

, Shi

, Zhang

, Su

, and Zhang

. Highly specific and sensitive radioiodinated agent for in vivo imaging of superoxide through superoxide-initiated retention. Anal Chem, 90: 12971–12978, 2018.

17.

Hyodo

, Murugesan

, Matsumoto

, Hyodo

, Subramanian

, Mitchell

, and Krishna

. Monitoring redox-sensitive paramagnetic contrast agent by EPRI, OMRI and MRI. J Magn Reson, 190: 105–112, 2008.

18.

Keana

and Van Nice

. Influence of structure on the reduction of nitroxide MRI contrast-enhancing agents by ascorbate. Physiol Chem Phys Med NMR, 16: 477–480, 1984.

19.

Komsiiska

Oxidative stress and stroke: a review of upstream and downstream antioxidant therapeutic options. Comp Clin Path, 28: 915–926, 2019.

20.

Lin

, Liu

, Ohno

, and Ogata

. Determination of ascorbate concentration in a raw leaf with electron spin resonance spectroscopy. Anal Sci, 15: 973–977, 1999.

21.

Matute-Bello

, Frevert

, and Martin

. Animal models of acute lung injury. Am J Physiol Cell Mol Physiol, 295: L379–L399, 2008.

22.

Michalski

, Thiebaut

, Michałowski

, Ayhan

, Hardy

, Ouari

, Rostkowski

, Smulik-Izydorczyk

, Artelska

, Marcinek

, Zielonka

, Kalyanaraman

, and Sikora

. Oxidation of ethidium-based probes by biological radicals: mechanism, kinetics and implications for the detection of superoxide. Sci Rep, 10: 18626, 2020.

23.

Mittal

, Siddiqui

, Tran

, Reddy

, and Malik

. Reactive oxygen species in inflammation and tissue injury. Antioxid Redox Signal, 20: 1126–1167, 2014.

24.

Njus

, Kelley

, Tu

Y-J

, and Schlegel

. Ascorbic acid: the chemistry underlying its antioxidant properties. Free Radic Biol Med, 159: 37–43, 2020.

25.

Okamura

, Okada

, Kikuchi

, Wakizaka

, and Zhang

. A ¹¹C-labeled 1,4-dihydroquinoline derivative as a potential PET tracer for imaging of redox status in mouse brain. J Cereb Blood Flow Metab, 35: 1930–1936, 2015.

26.

Prescott

and Bottle

. Biological relevance of free radicals and nitroxides. Cell Biochem Biophys, 75: 227–240, 2017.

27.

Rios

, Piacenza

, Trujillo

, Martínez

, Demicheli

, Prolo

, Álvarez

, López G

, and Radi

. Sensitive detection and estimation of cell-derived peroxynitrite fluxes using fluorescein-boronate. Free Radic Biol Med, 101: 284–295, 2016.

28.

Sano

, Motomura

, Yamamoto

, Fukuda

, Mukai

, and Maeda

. 1-(3′-[¹²⁵I]iodophenyl)-3-methy-2-pyrazolin-5-one: preparation, solution stability, and biodistribution in normal mice. Chem Pharm Bull, 58: 1020–1025, 2010.

29.

Sciannamea

, Jérôme

, and Detrembleur

. In-situ nitroxide-mediated radical polymerization (NMP) processes: their understanding and optimization. Chem Rev, 108: 1104–1126, 2008.

30.

Sheldon

, Arends

IWCE

, ten Brink

G-J

, and Dijksman

. Green, catalytic oxidations of alcohols. Acc Chem Res, 35: 774–781, 2002.

31.

Sikora

, Zielonka

, Lopez

, Joseph

, and Kalyanaraman

. Direct oxidation of boronates by peroxynitrite: mechanism and implications in fluorescence imaging of peroxynitrite. Free Radic Biol Med, 47: 1401–1407, 2009.

32.

Sivapackiam

, Liao

, Zhou

, Shoghi

, Gropler

, Gelman

, and Sharma

. Galuminox: preclinical validation of a novel PET tracer for non-invasive imaging of oxidative stress in vivo. Redox Biol, 37: 101690, 2020.

33.

Soule

, Hyodo

, Matsumoto

, Simone

, Cook

, Krishna

, and Mitchell

. The chemistry and biology of nitroxide compounds. Free Radic Biol Med, 42: 1632–1650, 2007.

34.

Stockwell

, Friedmann Angeli

, Bayir

, Bush

, Conrad

, Dixon

, Fulda

, Gascón

, Hatzios

, Kagan

, Noel

, Jiang

, Linkermann

, Murphy

, Overholtzer

, Oyagi

, Pagnussat

, Park

, Ran

, Rosenfeld

, Salnikow

, Tang

, Torti

S V.

, Toyokuni

, Woerpel

, and Zhang

DD.

Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease. Cell, 171: 273–285, 2017.

35.

Swartz

, Khan

, and Khramtsov

. Use of electron paramagnetic resonance spectroscopy to evaluate the redox state in vivo. Antioxid Redox Signal, 9: 1757–1772, 2007.

36.

Takai

, Abe

, Tonomura

, Imamoto

, Fukumoto

, Ito

, Momosaki

, Fujisawa

, Morimoto

, Takasu

, and Inoue

. Imaging of reactive oxygen species using [³H]hydromethidine in mice with cisplatin-induced nephrotoxicity. EJNMMI Res, 5: 38, 2015.

37.

Thakur

and Lentle

. Report of a summit on molecular imaging. Radiology, 236: 753–755, 2005.

38.

Uttara

, Singh

, Zamboni

, and Mahajan

. Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Curr Neuropharmacol, 7: 65–74, 2009.

39.

Valko

, Leibfritz

, Moncol

, Cronin

MTD

, Mazur

, and Telser

. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol, 39: 44–84, 2007.

40.

Valko

, Rhodes

, Moncol

, Izakovic

, and Mazur

. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact, 160: 1–40, 2006.

41.

Wada

, Fujibayashi

, Tajima

, and Yokoyama

. A new radioiodinated agent for detecting radicals in vivo: synthesis and preliminary evaluations. Nucl Med Biol, 21: 901–904, 1994.

42.

Webster

, Morton

, Johnson

, Yang

, Rishel

, Lee

, Miao

, Pabba

, Yapp

, and Schaffer

. Functional imaging of oxidative stress with a novel PET imaging agent, ¹⁸F-5-fluoro-L-aminosuberic acid. J Nucl Med, 55: 657–664, 2014.

43.

Weissleder

and Mahmood

. Molecular imaging. Radiology, 219: 316–333, 2001.

44.

Weng

, Carlin

, Hou

, Metz

, Li

, Lee

, Xu

, Makvandi

, and Mach

. Correlation analysis of [¹⁸F]ROStrace using ex vivo autoradiography and dihydroethidium fluorescent imaging in lipopolysaccharide-treated animals. Biochem Biophys Res Commun, 516: 397–401, 2019.

45.

Wilson

, Sadovski

, Nobrega

, Raymond

, Bambico

, Nashed

, Garcia

, Bloomfield

, Houle

, Mizrahi

, and Tong

. Evaluation of a novel radiotracer for positron emission tomography imaging of reactive oxygen species in the central nervous system. Nucl Med Biol, 53: 14–20, 2017.

46.

Yamada

, Mito

, Matsuoka

, Ide

, Shikimachi

, Fujiki

, Kusakabe

, Ishida

, Enoki

, Tada

, Ariyoshi

, Yamasaki

, and Yamato

. Fluorescence probes to detect lipid-derived radicals. Nat Chem Biol, 12: 608–613, 2016.

47.

Yamamoto

, Kaneshiro

, Kato

, Mukai

, Kuwabara

, Honda

, and Maeda

. Decreased tissue accumulation of 6-deoxy-6-[¹⁸F]fluoro-L-ascorbic acid in glutathione-deficient rats induced by administration of diethyl maleate. Biol Pharm Bull, 28: 1943–1947, 2005.

48.

Yamamoto

, Sasaki

, and Maeda

. Positron labeled antioxidants: synthesis and tissue biodistribution of 6-deoxy-6-[¹⁸F]fluoro-L-ascorbic acid. Int J Radiat Appl Instrumentation Part A Appl Radiat Isot, 43: 633–639, 1992.

49.

Yamamoto

, Shibata

, Watanabe

, Masuda

, and Maeda

. Positron-labeled antioxidant 6-deoxy-6-[¹⁸F]fluoro-L-ascorbic acid: increased uptake in transient global ischemic rat brain. Nucl Med Biol, 23: 479–486, 1996.

50.

Yamasaki

, Azuma

, Sano

, Munekane

, Matsuoka

, Yamada

, and Mukai

. Radioiodinated nitroxide derivative for the detection of lipid radicals. ACS Med Chem Lett, 11: 45–48, 2020.

51.

Yamasaki

, Ito

, Mito

, Kitagawa

, Matsuoka

, Yamato

, and Yamada

. Structural concept of nitroxide as a lipid peroxidation inhibitor. J Org Chem, 76: 4144–4148, 2011.

52.

Yamasaki

, Mito

, Ito

, Pandian

, Kinoshita

, Nakano

, Murugesan

, Sakai

, Utsumi

, and Yamada

. Structure-reactivity relationship of piperidine nitroxide: electrochemical, ESR and computational studies. J Org Chem, 76: 435–440, 2011.

53.

Yang

, Jenni

, Colovic

, Merkens

, Poleschuk

, Rodrigo

, Miao

, Johnson

, Rishel

, Sossi

, Webster

, Bénard

, and Schaffer

. ¹⁸F-5-Fluoroaminosuberic acid as a potential tracer to gauge oxidative stress in breast cancer models. J Nucl Med, 58: 367–373, 2017.

54.

Zhang

, Cai

, Li

, Ropchan

, Lim

, Boutagy

, Wu

, Stendahl

, Chu

, Gropler

, Sinusas

, Liu

, and Huang

. Optimized and automated radiosynthesis of [¹⁸F]DHMT for translational imaging of reactive oxygen species with positron emission tomography. Molecules, 21: 1696, 2016.

55.

Zhang

, Dai

, and Yuan

. Methods for the detection of reactive oxygen species. Anal Methods, 10: 4625–4638, 2018.

56.

Zhao

, Kalivendi

, Zhang

, Joseph

, Nithipatikom

, Vásquez-Vivar

, and Kalyanaraman

. Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic Biol Med, 34: 1359–1368, 2003.

57.

Zielonka

, Sarna

, Roberts

, Wishart

, and Kalyanaraman

. Pulse radiolysis and steady-state analyses of the reaction between hydroethidine and superoxide and other oxidants. Arch Biochem Biophys, 456: 39–47, 2006.

58.

Zielonka

, Srinivasan

, Hardy

, Ouari

, Lopez

, Vasquez-Vivar

, Avadhani

, and Kalyanaraman

. Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: identification of homo- and heterodimers. Free Radic Biol Med, 44: 835–846, 2008.

Redox Monitoring in Nuclear Medical Imaging

Abstract

Significance:

Recent Advances:

Critical Issues:

Future Directions:

Introduction

Nuclear Medical Imaging

Superoxide and Hydroxyl Radicals

Dihydroethidium analogues

Hydrocyanine derivative

Fluorescein derivative

Hydrogen peroxide

Thymidine analogue

Total ROS and other radicals

Luminol derivative

Edaravone derivative

Derivatives of spin trapping reagents

Nitroxide derivatives

Redox state

Ascorbic acid derivatives

Dihydroquinoline derivatives (NAD+/NADH)

Cystine-glutamate transporter substrate

Conclusions

Footnotes

Author Disclosure Statement

Funding Information

Abbreviations Used

References

Dihydroquinoline derivatives (NAD⁺/NADH)