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Published in final edited form as: Free Radic Biol Med. 2018 Nov 2;130:343–347. doi: 10.1016/j.freeradbiomed.2018.10.454

Effects of oxygen challenging to tissue redox and pO2 status

Ken-ichiro Matsumoto a,*, James B Mitchell b, Murali C Krishna b
PMCID: PMC8202967  NIHMSID: NIHMS1701887  PMID: 30391676

Abstract

Nitroxide free radicals can serve as redox-sensitive MRI contrast agents useful to image the redox status of tissue of interest. In this study, the effect of oxygen content in the inspired gas on the kinetics of metabolism of three nitroxides has been evaluated in the muscle and tumor in mice.

SCC tumors (approximate size of 1.0 cm3) on the right hind leg of female C3H/Hen MTV− mice were prepared. Three nitroxides, 3-carboxy-2,2,5,5-tetramethylpyrrolidine-N-oxyl (CxP), 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (CmP), and 4-hydroxy-tetramethylpiperidine-N-oxyl (TEMPOL), having different lipophilicities were compared using MR redox imaging. T1-mapping of the tissues was obtained using a multi-slice multi-echo (MSME) sequence with several TRs.

The three nitroxides showed differences in accumulation and metabolism/clearance in muscle and tumor. The cell impermeable nitroxide CxP displayed kinetic patterns of slow enhancement followed by a slow decline typical of clearance rather than metabolism. The cell permeable CmP on the other hand showed a relatively faster uptake and metabolism with a modestly higher rate of metabolism in the tumor than muscle. The TEMPOL on the other hand displayed a rapid uptake and reduction with a trend of significantly rapid decay rate in tumor tissue, while slightly higher maximum signal intensity and slower decay rate was observed in normal muscle. The reduction rate of TEMPOL in the tumor was significantly enhanced when the breathing gas had 100%-oxygen while it was not significantly different in the muscle. EPR oximetry studies monitoring the oxygen dependent linewidth of TEMPOL showed that the pO2 in the healthy tissue during carbogen breathing significantly increased normal tissue pO2 compared to air breathing whereas breathing 100%-oxygen made normal tissue slight hypoxic. Since TEMPOL is a radioprotector, our studies show that a combination of 100%-oxygen breathing and TEMPOL has a potential to enhance radioprotective effects to normal tissue.

Keywords: Tumor oxygenation, Nitroxide, Redox sensitive contrast agent, MR redox imaging, T1-relaxivity

1. Introduction

Sensitizing tumor to ionizing radiation and protecting normal tissue in treatment fields is a desirable feature in radiotherapy [1–3]. Oxygen is a potent radiation sensitizer and tumors which are hypoxic display radiation resistance [4,5]. Similarly, radiation protectors which can selectively protect normal tissues while having no tumor radiation modification effects are desirable. Stable nitroxide radicals have been shown to selectively protect normal tissues but not tumors [6]. The selective protective capabilities of nitroxides to normal tissues but not tumors have been attributed to their relatively rapid conversion to the non-radioprotective hydroxylamine, a process kinetically favored in hypoxic tumors [7].

Nitroxide radicals are also used as a redox sensitive T1-weighted MR contrast agent [8,9]. The in vivo reduction rates of nitroxides are accelerated under hypoxic conditions [10,11]. In contrast, in vivo decay constants of nitroxides are slower due to re-oxidation of the probe in oxygenated tissue [12,13]. The difference of tissue pO2 in normal and tumor tissues may determine the radical/hydroxylamine ratio of 4-hydroxy-tetramethylpiperidine-N-oxyl (TEMPOL) in those tissues, which can confer radioprotection effects to TEMPOL.

Prior studies showed that cell-permeable nitroxides are metabolized to the corresponding hydroxylamines and piperidine nitroxides are metabolized faster than the pyrrolidine nitroxides [14–17]. The 3-carboxy-2,2,5,5-tetramethylpyrrolidine-N-oxyl, (CxP), is membrane impermeable molecule [18,19] whereas 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (CmP) is hydrophilic and has slight membrane permeability and is used as a redox sensitive contrast agent in EPR [20–25]. TEMPOL which has been used as a normal-tissue specific radioprotector [26,27], is an amphiphilic molecule and membrane permeable. Thus nitroxides can be used as tissue specific radioprotectors which can also be studied for their uptake and metabolism by MRI to probe their use as normal tissue specific radioprotectors.

To effectively treat a hypoxic tumor, increasing tumor oxygenation or delivering higher radiation depending to hypoxic region is necessary. Oxygenation of tumor tissue has been investigated to improve the efficacy of radiation- and/or chemo-therapy [28–30]. Increasing tissue oxygenation by breathing Carbogen, 100%-oxygen, or hyperbaric oxygen (HBO) was reported in several studies [31–34].

In this study the effect of oxygenation challenges on the in vivo decay rates of three different nitroxides, CxP, CmP, and TEMPOL, in tumor and normal muscle was compared based on MR redox imaging. The effects of several gas-breathing challenges on the tissue oxygenation levels were also investigated. The purpose of this study was to determine an appropriate oxygenation regimen and a nitroxide to selectively achieve tumor radiosensitization and normal tissue radioprotection.

2. Methods

2.1. Chemical

CxP, CmP, and TEMPOL were purchased from Sigma-Aldrich Chem. Co. (St. Louis, MO). CxP was prepared as 150 mM, while CmP and TEMPOL were as 300 mM solution, which were adjusted to isotonic and neutral pH.

2.2. Animals

C3H/Hen MTV− female mice supplied by the National Cancer Institute Animal Production Facility (Frederick, MD) were used. Details of animal and tumor conditions used were described in previous paper [33]. Experiments were carried out in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), National Research Council, and approved by the National Cancer Institute Animal Care and Use Committee.

2.3. MR redox imaging

MRI measurements were performed at 4.7 T controlled with ParaVision® 3.0.1 (Bruker BioSpin MRI GmbH, Rheinstetten, Germany). Details of animal handling and scan operation were described in previous paper [8]. After a mouse was set in the magnet and the body temperature reached to 36 °C, the breathing gas was switched to Carbogen or 100%-oxygen, or stayed on air. A T2-weighted image was obtained using a multi-slice multi-echo (MSME) sequence. The dynamic scan using spoiled gradientrecalled echo (SPGR) was started 20 min after switching breathing gas. The 1.5 μmol/g b.w. of a nitroxide was i.v. injected 5 min after starting the SPGR scan.

2.4. Tissue oxygenation

After a mouse was set in the MRI magnet, data for T1- and T2-mappings were simultaneously obtained using a MSME sequence. The breathing gas was switched to Carbogen or 100%-oxygen, and then T1- and T2-mappings were again obtained 15 min after switching gas.

2.5. EPR oximetry

The animal setting and EPR conditions were described elsewhere [35]. After a mouse was set in the magnet and the body temperature reached to 36 °C, the breathing gas was switched to 100%-oxygen or Carbogen. The CmP or TEMPOL solution (1.5 μmol/g b.w.) was i.v. infused through the tail vein 15 min after starting the gas challenge and EPR spectra (only center peak) were measured repeatedly. A Gaussian lineshape was fitted on the acquired EPR spectra, and linewidth of the fitted Gaussian line was measured. An averaged linewidth from 2 min after administration for 5 min was obtained.

2.6. MR image analyses

The image data were analyzed using the ImageJ software package (http://rsb.info.nih.gov/ij/). T1- and T2-mappings were calculated using a plug-in (MRI analysis calculator, Karl Schmidt, HypX Laboratory, Brigham and Women's Hospital) available in ImageJ. Supplemental Fig. 1 shows a typical T2-map of an axial slice including both the SCC tumor implanted and the contralateral normal legs, which was used as the scout image for ROI selection.

2.7. Statistics

Statistical differences were estimated when the p-value was less than 0.05 using TTEST function in the Microsoft Excel 2010.

3. Results

Fig. 1A shows typical semi-logarithmic plot of T1-weighted MRI image intensity kinetic profiles of CxP in the tumor and the normal muscle ROIs when breathing room air. CxP took a relatively long time to reach maximal intensity level, then gradually decayed linearly. Fig. 1B and C shows semi-logarithmic decay profiles of CxP under Carbogen breathing and 100%-oxygen breathing, respectively and summarized in Supplemental Table 1. The decay profiles in both tumor and normal tissue of CxP under Carbogen breathing were similar to the air breathing ones, but the decay rates of CxP in the tumor under 100%-oxygen challenge differed from that of air and Carbogen breathing conditions. These results show that CxP decay constants are characteristic of tissue type but do not vary significantly with the oxygen content in the inspired gas. However, a slight increase in tumor decay constant was observed when the breathing gas was 100%-oxygen. Based on previous studies, the relatively slow decay rates are consistent with the cell impermeable nature of CxP.

Fig. 1.

Fig. 1.

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CxP-induced MR T1-weighted signal decay profiles obtained in tumor tissue and normal muscle tissue, when mice were breathed (A) air, (B) carbogen, and (C) 100%-oxygen. A set of 50 T1-weighted SPGR time course images were scanned for CxP (total scan time was 25 min). T2-map of the corresponding slice was used as the scout image for ROI selection. Time courses of averaged T1-weighted signal intensity in either the tumor and the normal muscle ROIs were observed. Marks and error bars indicate average ± SD of 3 or 4 mice. The intensity decay constants of CxP were calculated from the slope of the time window 7–15 min (closed marks). The lines are slopes estimated by least square method.

Fig. 2 shows a representative semi-logarithmic plot of kinetic profiles of CmP, obtained in the tumor and normal muscle. CmP reached maximum level relatively faster compared to CxP, and gradually decayed linearly. Decay slopes of CmP in tumor were significantly steeper than that of CxP under air breathing. Both oxygen challenges attenuated the decay constants of CmP in normal muscle, though not significant. The decay constants in tumor when breathing Carbogen or 100%-oxygen were significantly faster than that in muscle. The enhanced reduction in tumors of CmP can be attributed to bio-reduction rather than clearance based on previous studies.

Fig. 2.

Fig. 2.

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CmP-induced MR T1-weighted signal decay profiles obtained in tumor tissue and normal muscle tissue, when mice were breathed (A) air, (B) carbogen, and (C) 100%-oxygen. A set of 50 T1-weighted SPGR time course images were scanned for CmP (total scan time was 25 min). T2-map of the corresponding slice was used as the scout image for ROI selection. Time courses of averaged T1-weighted signal intensity in either the tumor and the normal muscle ROIs were observed. Marks and error bars indicate average ± SD of 3 or 4 mice. The kinetic decay constants of CmP were calculated from the linear slope of the time window 3–10 min (closed marks). The lines are slopes estimated by least square method.

Fig. 3A shows a typical semi-logarithmic plot of intensity decay profiles of TEMPOL obtained in the tumor and normal muscle and the corresponding kinetic constants shown in Supplemental Table 1. TEMPOL reached maximum level quickly and then rapidly decayed to undetectable levels within 4 min in the tumor and within 10 min in the normal muscle. Both oxygen challenges had no effect on the decay slopes of TEMPOL in the normal muscle, though a faster decay was noticed in the tumor tissue (Fig. 3B and C). The enhanced decay constants noticed in the tumor compared to normal tissue can be attributed to enhanced bioreduction in the tumor based on prior studies. The decay constant in tumor when breathing 100%-oxygen was significantly greater than that in normal tissue and eliminating TEMPOL withn 2 min in tumor, while enough detectable TEMPOL was retained in normal muscle.

Fig. 3.

Fig. 3.

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TEMPOL-induced MR T1-weighted signal decay profiles obtained in tumor tissue and normal muscle tissue, when mice were breathed (A) air, (B) carbogen, and (C) 100%-oxygen. A set of 30 T1-weighted SPGR time course images were scanned for TEMPOL (total scan time was 15 min). T2-map of the corresponding slice was used as the scout image for ROI selection. Time courses of averaged T1-weighted signal intensity in either the tumor and the normal muscle ROIs were observed. Marks and error bars indicate average ± SD of 3 or 4 mice. The decay constants of TEMPOL were estimated from the time window 0.8–1.8 min for tumor and 1.3–5.9 min for normal tissue (closed marks). The lines are slopes estimated by least square method.

Molecular oxygen with two unpaired electrons can also provide image intensity enhancement in a T1-weighted MRI scan. The results of R1 (= 1/T1) measurements under air Carbogen, or 100%-oxygen breathing are summarized in Supplemental Table 2. Carbogen challenge showed significant increase of R1 in the tumor and normal tissue compare to the air breathing group. The 100%-oxygen challenge displayed minimal change in R1 in both normal and tumor tissues compared to air breathing group. These results show that while the magnitude in R1 changes (0.86% down) as a result of breathing higher content oxygen was smaller than the nitroxide decay constants (95% up), these changes can be directly attributable to intracellular oxygen changes. The nitroxide decay constants on the other hand depend on changes in pO2 among other factors.

EPR based oximetry provides quantitative assessment of tissue pO2 and associated changes in response to changes in oxygen content in breathing gas by monitoring the spectral linewidths of the nitroxides. EPR spectral linewidths of the cell permeable nitroxides CmP and TEMPOL immediately after administration under different breathing gas conditions in healthy normal femoral muscle of mice were monitored (Fig. 4). CmP showed similar pO2 levels under all conditions of the breathing gas presumably due to its slight lipophilic nature. This result agrees with values reported in a previous paper that measured oxygen levels using LiPc as the oxygen probe [31]. TEMPOL on the other hand displayed significant differences in pO2 with changes in breathing gas. While the pO2 values were similar when breathing air and 100%-oxygen, Carbogen breathing significantly enhanced pO2 values by > 4-fold compared to air-breathing and 100%-oxygen breathing. Amphiphilic TEMPOL may penetrate deeper into the tissues and may reflect intracellular pO2 levels. In both cases, carbogen breathing increased tissue pO2 level.

Fig. 4.

Fig. 4.

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Comparison of pO2 level in normal muscle tissue during breathing several different gases. The pO2 values were estimated by EPR oximetry using (A) CmP or (B) TEMPOL as an paramagnetic oxygen probe. EPR linewidth measured were translated to oxygen concentration using previously obtained calibration curve. Columns and error bars indicate average ± SD of 3 or 4 mice.

4. Discussion

Modulating the levels of radiosensitizers in tumor and radioprotectors in normal tissue will allow improved outcome in radiotherapy by protecting normal tissue and sensitizing tumors. Oxygen is one of the most potent radiosensitizers but is low in tumors. Its levels can be selectively increased by inhalation of breathing gases with higher O2 content compared to room air [33,36]. Nitroxides on the other hand are normal tissue radioprotectors with minimal protective effects in tumors. Oxygen and nitroxides, both being paramagnetic species allow the use of T1-weighted MRI and EPR techniques to monitor their distribution and changes with time. Further, the spectral linewidths of nitroxides can be used to quantify pO2 and changes in response to hyperoxic challenges. In this study hyperoxygenating challenges such as Carbogen or 100%-oxygen breathing on the metabolism of the radioprotective nitroxides were evaluated in muscle and tumors in tumor bearing mice. Similarly, oxygen levels were determined by T1-weighted MRI and EPR oximetry.

We find that CmP and TEMPOL were metabolized faster in tumors than in normal tissue when breathing air. Carbogen breathing resulted in enhanced metabolism of the nitroxides in both tumors and with minimal effects in muscle. However when breathing 100%-oxygen, TEMPOL metabolism was significantly enhanced in tumors even compared with Carbogen breathing with minimal effects in muscle. This creates a condition of significant differences in radioprotector levels in the muscle than in tumor allowing selective radioprotection. EPR assessment of pO2 based on linewidths show that pO2 levels are lower in the muscle when breathing 100%-oxygen than when breathing Carbogen. This result is consistent with earlier studies from our group using HBO [33] where we find a selective increase in tumor oxygenation associated with a temporally coincident decrease in muscle oxygenation levels.

Overall our studies show that breathing 100%-oxygen causes enhanced levels of the radioprotector TEMPOL in normal tissue with an associated decrease in pO2 while in tumors the significantly enhanced bioreduction of the radioprotector to an inactive form with an associated increase in pO2 creates a favorable condition of selective sensitization of the tumor and protection to the normal tissue. In our mouse tumor model, 2 min after TEMPOL injection under breathing 100%-oxygen may be an optimal window for radiation treatment.

5. Conclusion

Under breathing 100%-oxygen, TEMPOL in tumor tissue eliminated within 2 min, while 100%-oxygen tends to make the decay rate in the normal muscle 20% slower of air-breathing. A combination of 100%-oxygen can make large difference of TEMPOL concentration between normal and tumor tissue for 4–5 min without oxygenation of tissues. EPR oximetry showed that 100%-oxygen breathing did not affect to the normal tissue pO2. The result gives a potential to enhance the radioprotection effect selective to normal tissue.

Supplementary Material

Supple fig
Supple table

Acknowledgements

This research was supported by the Intramural Research Program, Center for Cancer Research, National Cancer Institute, NIH. The authors express heartfelt condolences for untimely passing of Dr. Marcelino Bernardo, who made a great contribution to implement our T1-weighted SPGR sequence running on the 4.7T MRI scanner at MRI Research Facility, National Institute of Neurological Disorder and Stroke, Bethesda, MD, USA. The authors also express heartfelt condolences for passing of Mr. Frank Harrington, who made our special mouse holder for MRI experiments.

Footnotes

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.freeradbiomed.2018.10.454.

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Associated Data

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Supplementary Materials

Supple fig
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Supple table
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