In vivo measurement of tumor redox environment using ... - Springer Link

Molecular and Cellular Biochemistry 234/235: 393-398, 2002. © 2002 Kluwer Academic Publishers.

In vivo measurement of tumor redox environment using EPR spectroscopy Govindasamy Ilangovan, Haiquan Li, Jay L. Zweier and Periannan Kuppusamy The EPR Center and Division of Cardiology, Department ofMedicine, Johns Hopkins University, School ofMedicine, Baltimore, MD, USA

Abstract Solid tumors are characterized by a number ofphysiological properties such as occurrence of significant hypoxia, large amounts of cellular reducing equivalents, compromised blood-flow and low pH, all of which are distinctly different from normal tissues. Tumor therapeutic regimens such as radiation or chemotherapy attempt to exploit these physiological differences between normal and malignant tissue. Thus, methods that can detect these subtle differences would greatly aid in devising appropriate treatment strategies. Low-frequency in vivo electron paramagnetic resonance (EPR) spectroscopy is capable of providing non-invasive measurements ofthese parameters in tumors. This requires the use of appropriate exogenously injected free radical reporter molecules (probes), such as nitroxides. In the present study we performed measurements of nitroxide metabolism in RIF-I murine tumors, in vivo, and demonstrated that the rate of nitroxide decay correlated with the tumor redox environment. The results showed the existence of significantly higher reducing environment in the tumor tissue compared to normal tissue. The dependence of the tumor redox status on the intracellular GSH levels and tissue oxygenation was investigated. The measurement ofredox status and its manipulation may have important implications in the understanding of tumor growth and therapy. (Mol Cell Biochem 234/235: 393-398, 2002)

Key words: EPR spectroscopy, RIF-I tumor, nitroxide, glutathione, redox status Abbreviations: 3-CP - 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl, or 3-carbamoyl-proxyl; RIF - radiation induced fibrosarcoma; EPR - electron paramagnetic resonance; GSH - glutathione; BSO - L-buthionine-s,R-sulfoximine

Introduction Solid tumors are known to have altered redox status, which may determine the response ofa tumor to certain chemotherapeutic agents [I], radiation [2], and bioreductive hypoxic cell cytotoxins [3-5]. A variety of intracellular molecules may contribute to the overall redox status (or redox environment) in tissues including glutathione (GSH), thioredoxins, NADPH, flavins, ascorbate, and others. Collectively, the reducing species may have an impact on how a particular tissue responds to oxidative stress induced by a particular drug or treatment modality and, therefore, a non-invasive means of determining the redox status of a tissue should be a useful adjunct for clinical oncology.

Small molecular weight stable nitroxide free radicals have been used as biophysical and biochemical probes in a variety of biological experiments [6-8], in which electron paramagnetic resonance (EPR) spectroscopy is conveniently used for detection. The nitroxides exist in biological systems as a redox pair (Fig. I), namely the nitroxide free radical form and the diamagnetic hydroxylamine, which is the one-electron reduction product of the nitroxide free radical [9, 10]. Prior studies indicate that nitroxides are reduced to the corresponding hydroxylamine in cellular incubations as well as in vivo and that the rate of nitroxide reduction is significantly enhanced under hypoxic conditions [11-15]. Presumably the nitroxide accepts cellular reducing equivalents that would have gone to reduction ofmolecular oxygen. Hence, the phar-

Address for offPrints: P. Kuppusamy, Johns Hopkins University, School of Medicine, 550 I Hopkins Bayview Circle, Baltimore, MD 21224, USA

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single cell suspension ofRIF-1 (radiation-induced fibrosarcoma) tumor cells (10 6 cells in 0.1 ml) in PBS. Tumors were allowed to grow to a size of about 8-10 mm in the greatest dimension at the time of experimentation.

Tissue glutathione assay

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macology of nitroxides in an EPR experiment should report on the differences that exist in tissues having different redox and oxygen status. Previously we performed non-invasive in vivo EPR spectroscopic measurements ofnitroxide reduction profiles in RIF-I tumor-bearing mice and reported that the tumor tissues were significantly hypoxic and highly reducing compared to the normal tissues [16]. The purpose of this study was to develop a non-invasive method to measure redox status in tissues and to examine the effect of intracellular glutathione and hyperoxygenation on the tumor redox status. Experiments were performed using low-frequency (1.3 GHz) in vivo EPR spectroscopy with nitroxide probes. The results show the existence of significantly enhanced levels of reducing equivalents in tumor compared to normal tissue. Further, there is differential depletion of the reducing equivalents in the tumor compared to normal tissue in mice treated with carbogen or inhibitors of glutathione synthesis.

Mice were injected (i.p.) with 2.25 mmol/kg (body wt) of buthionine sulfoximine (BSO) in 100 J.lL saline. Six hours later the animals were sacrificed and about 0.2-0.5 g oftumor tissue on the right leg and normal muscle tissue on the left leg were carefully excised and weighed. The tissues were placed in glass dounce homogenizer (5 mL) and homogenized with I mL of200 mM methanesulfonic acid containing 5 mM diethylenetriaminepentaacetic acid (DTPA) (pH < 2.0). This homogenate was centrifuged for 30 min at 14,000 rpm at 4°C. The resultant supematantwas diluted I: I with mobile phase and stored frozen at -70°C for GSH assay. The GSH assays were performed by high-performance liquid chromatography - electrochemical method [17]. Samples were separated on a Polaris 5 11m, 0.4 x 20 cm C-18 column eluted with a mobile phase of 50 mM NaHl04, 0.05 mM octanesulfonic acid, and 2% acetonitrile adjusted to pH 2.7 with phosphoric acid and a flow rate of I mL/min. An ESA CoulArray detector was used with the guard cell set at +950 mV, electrode I at +400 mV, and electrode 2 at +880 m V. Full-scale output was set at 100 IlA and peak areas were analyzed using the CoulArray software (ESA, Chelmsford, MA, USA). A standard curve was obtained using 10 IlM-I mM solution of GSH, from which concentrations of GSH were determined. The GSH values were expressed as J.lmol/g of tissue wet weight.

Materials and methods

L-band EPR spectrometer

Nitroxide

Hydroxylamine

Fig. 1. Structure of 3-CP and its tissue metabolite. The paramagnetic 3CP (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl, or 3-carbamoylproxyl) nitroxide probe undergoes one-electron reduction in tissues to diamagetic hydroxylamine form.

Chemicals The nitroxide probe 3-CP (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl, or 3-carbamoyl-proxyl) was purchased from Aldrich (Milwaukee, WI, USA). Solutions of the nitroxide were freshly prepared at a stock concentration of 300 mM in saline. Glutathione (L-y-glutamyl-L-cysteinyl-glycine) and L-buthionine-s,R-sulfoximine were purchased from Sigma.

EPR measurements were performed using an L-band (1.32 GHz) EPR spectrometer with a custom-built surface resonator and personal computer-based data acquisition software [16, 18-20]. The resonator was capable of sampling a cylindrical volume measuring 10 mm in diameter and 5 mm deep. The open structure of this resonator was ideal for localized measurements on large objects and thus was not limited by the size of the object.

In vivo EPR measurements

Mice and RIF-l tumor growth Female C3H mice (weight: 25 ± 5 g) were used. The mice were injected subcutaneously in their right hind leg with a

Mice were anesthetized by breathing air containing 1% isoflurane delivered through a nose cone. The tail vein was cannulated with a heparin-filled 30-gauge catheter for infu-

395 sion of the nitroxide probes. Either the tumor in the right leg or the nonnal tissue (muscle) on the left leg was prepared for measurements. The animal was placed on a bedplate with a circular slot (20 mm diameter) and placed on top of the resonator such that the tumor or the nonnal muscle was in direct contact with the active surface of the resonator. An infrared lamp was used to maintain nonnal body temperature, which was measured using a rectal thennistor probe.

Statistical analysis All data are expressed as mean ± S.E.M. Comparisons among groups were perfonned by student's t-test or one-way ANOVA, with Student-Newman-Keuls tests. The significance level was defined as p < 0.05.

Results Phannacokinetics of nitroxide uptake and clearance from nonnal and tumor tissues ofRIF-l tumor-bearing mice were measured in vivo using L-band EPR spectroscopy. 3-CP was used as a probe at a dose of 185 mg/kg. Mice were infused (bolus dose), via tail vein catheter, with a saline solution of 3-CP and EPR spectra were measured continuously from the tumor on right leg or nonnal muscle on the left leg. Figure 2 shows a typical EPR spectrum measured from the tumor leg.

The spectrum is characterized by a triplet, arising due to hyperfine splitting from the '4N nucleus, with coupling constants 15.78 and 16.30 G. The unequal coupling constants, which are usually observed at low-frequency measurements, are due to breakdown ofthe high-field approximation, known as the 'Breit-Rabi' effect. Each triplet of the spectrum is also characterized by a unique peak-to-peak width and amplitude. While the coupling constants and width ofthe spectra remain more or less constant, the amplitude varies with time reflecting changes in the nitroxide concentration in the tissue. The area under the peak, measured as double integral ofthe spectrum was used as a quantitative measure of the nitroxide concentration, in arbitrary units. Figure 3 shows a semi-logarithmic plot of time course of the signal intensity of 3-CP in the nonnal muscle and tumor tissues. The data, representative of measurements in each group, was measured after the nitroxide intensity peaked, usually in about 3-4 min. It is observed that the nitroxide concentrations (in logarithmic units) in the tumor as well as nonnal tissues decrease linearly with time, suggesting that the clearance phannacokinetics can be modeled by a pseudo-first order rate equation. The solid lines through the data points are linear fit to the respective data set suggesting compliance with a pseudo first-order rate law. The bar graph in Fig. 4 shows the measured pseudo first-order rate

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Magnetic Field Fig. 2. EPR spectrum of3-CP nitroxide in a RIF-I tumor. A tumor-bearing mouse under anesthesia was infused (i.v.) with a saline solution of 3CP (185 mglkg). The EPR spectrum of the nitroxide in the tumor tissue was measured in vivo using L-band (1.3 GHz) EPR spectrometer at 4 min after infusion. The triplet signal, arising due to hyperfine splitting from the 14N nucleus, is characterized with coupling constants 15.78 and 16.30 G and peak-to-peak width 1.50 G. Measurement parameters: microwave power 8 mW; modulation amplitude, 1.0 G; modulation frequency, 100 kHz; scan time 15 sec.

Time (min) Fig. 3. Pharmacokinetics of nitroxide in normal muscle and tumor tissues. Time course of the EPR signal intensity of3-CP in the normal leg muscle and tumor tissue of RIF-I tumor-bearing mice, infused (i.v.) with a saline solution of 3-CP, were obtained by double integration of the spectra. The semi-log plot shows the clearance of the nitroxide (in arbitrary units) as a function of time in the normal muscle and tumor tissue of untreated, airbreathing tumor-bearing mice and in the tumor tissue of carbogen-breathing mice and mice treated (i.p.) with L-buthionine-s,R-sulfoximine (880, 2.25 mmol/kg) 6 h before the measurements. The solid lines through the data points are linear fit to the respective data set suggesting compliance with a pseudo first-order rate law.

396 constants ofnitroxide clearance in the tissues. The data represents mean ± S.E.M. ofmeasurements on 3-5 mice per group. In order to study the effect of oxygenation on the redox status oftumor tissue, we performed the above measurements under hyperoxic conditions. Tumor-bearing animals were allowed to breathe carbogen (a mixture of 95% 0z and 5% COz) for 20 min. After 20 min, 3-CP was infused via tail vein and the intensity of the nitroxide signal at the tumor was followed. The pharmacokinetic data obtained from the tumor at the hyperoxygenation condition are presented in the Fig. 3. The tumor pOz was measured independently using locally implanted lithium phthalocyanine (LiPc) particulate oximetry probe during the experiments. The tumor tissue pOz values were 4 ± 3 mmHg in air-breathing mice and 40105 mmHg in carbogen-breathing mice. It was observed that the nitroxide depletion rate considerably reduced when the tumor was hyperoxygenated, as can be seen from Fig. 3. Thiols such as intracellular glutathione and protein thiols are key to maintaining the redox status oftissues. Chemically, nitroxides do not react with thiols. This was verified using EPR measurements on nitroxide in presence of up to 7.5 mM ofGSH in saline (data not shown). However, in microsomal preparations, thiol-containing biomolecules have been shown to playa significant role in the bioreduction ofnitroxides [21]. Since thiol/disulfide status generally reflects the redox state of the tissue, altering the concentration of the thiol or changing the ratio ofredox pairs should have an impact on the clearance of the nitroxide and thus provide a means to study

the importance of this class of compounds as a function ofbioreduction. For example, depleting the GSH levels and monitoring the pharmacokinetics ofredox sensitive nitroxide probes in both normal as well as tumor tissue should delineate the role ofthiols in modulating tissue redox state. In order to investigate the effect of glutathione depletion on the rate of clearance of tissue nitroxide, the pharmacokinetic experiments were performed on tumor-bearing animals treated with L-buthionine-s,R-sulfoximine (BSO), a specific inhibitor ofy-glutamylcysteine synthetase enzyme that is responsible for GSH synthesis [22]. Mice were injected (i.p.) with 2.25 mmollkg BSO in saline and the EPR measurements were made 6 h later. The redox data (Figs 3 and 4) show that the BSO treatment significantly decreased the rate constant of nitroxide reduction in the tumor tissue. The effect ofBSO treatment on the redox constants obtained in the present experiments thus can be attributed to alterations in the tissue glutathione levels. In order to investigate the correlation between the observed rate constants and tissue glutathione, the tissue levels ofGSH were measured in the tissues of normal (control) and BSOtreated (same dose as for the EPR measurements) mice. The GSH assay data are shown in Fig. 5. The BSO-treated tissue data show a significant reduction in the GSH levels in the tumor tissue compared to untreated control. On the other hand, no significant differences were observed between treated and untreated normal tissues. The measured GSH levels correlate well with the nitroxide reduction rate constants in different tissues. Thus it is evident that BSO causes a differential depletion among normal and tumor tissues.

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Fig. 4. Bar-graph showing the measured pseudo first-order rate constants of nitroxide reduction in the tissues. The data represents mean ± S.E.M. of measurements on 3-5 mice per group. The rate constants were: untreated normal muscle, 0.037 ± 0.005 min"; untreated (air-breathing) tumor, 0.063 ± 0.008 min"; carbogen-breathing tumor, 0.047 ± 0.007 min"; BSO-treated tumor, 0.052 ± 0.006 min· l . *Significantly different from untreated (airbreathing) RIF-I tissue.

The present data obtained using in vivo EPR techniques and a nitroxide spin probe in RIF-I tumor implanted in a C3H mouse model shows the differential physiology between normal and tumor tissue. The nitroxide pharmacokinetic data indicate that the nitroxide is reduced more rapidly in the tumor tissue compared to normal muscle tissue in the tumor-bearing mice and that the reduction rate constant in the tumor of carbogenbreathing or BSO-treated mice is decreased. The in vivo stability of nitroxi des depends primarily on the ease of the oxidation ofthe nitroxide to the oxoammonium cation [23] or the reduction to the hydroxylamine [24]. Consistent with this hypothesis are observations that the in vivo stability of the nitroxides is inversely related to the ease of reduction of the nitroxide. Further, in vivo reduction of nitroxides is dependent on the oxygen content as well as levels of endogenous reducing agents, such as glutathione. The enhanced reduction of the nitroxide in tumor compared to normal tissue, thus, may be due to lower oxygen concentration as well as to the significantly enhanced GSH levels in the tumor.

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Fig. 5. Glutathione (GSH) levels in normal muscle and RIF-I tumors of untreated and BSO-treated mice. The normal muscle tissue and tumor tissue levels ofGSH were measured in untreated RIF-I tumor-bearing mice (N = 7) and in mice 6-h post-treatment of 2.25 mmol/kg of BSO (N = 7). ·Significantly different from the untreated group.

In the pharmacokinetic experiments we have followed changes in the nitroxide signal intensity in the tumor, in vivo, as a function of post-infusion period. The measured rate of decrease of nitroxide signal intensity at the tumor site may depend on the following dynamic processes: (i) infusion of the probe into the tumor, (ii) changes in the probe concentration due to bioreduction/oxidation in the tumor tissue, and (iii) elimination of the probe out of the tumor volume by blood flow. It has been reported by Gallez et al. [25] that bioreduction at the local site is the major factor that contributes to the overall decrease. Thus the observed decrease in the nitroxide signal is attributed mainly to the bioreduction. This is also evident from the present work since different rate constants of clearance are observed between the tumor tissue and muscle on the non-tumor-bearing leg. If infusion of nitroxide in to the tissue is the major contributor then any local measurement should reflect on the systemic levels of the probe, and thus the rate constants would have been identical between the two tissues. Thus, the rate constant observed by this localized measurement can be used as a measure of bioreduction of the nitroxide probe in the tissue and hence is correlated to its redox status. Several studies have shown that tissue oxygenation is an important factor that will affect the tissue redox status and hence the reduction rate of the nitroxide. Particularly, tissue hypoxia, which is known to be present in tumors, has been shown to enhance the bioreduction of nitroxides [16]. While this is evident from the enhanced rate of reduction from the tumor as compared to muscle tissue, which is relatively well

oxygenated, the involvement of other factors such as altered redox state, and pH may also be important. The carbogenbreathing study shows that the nitroxide reduction in tumor is dependent on the tumor oxygenation. Carbogen-breathing is in general expected to result in increased tumor oxygenation, though not without exceptions. GSH is present in significantly large quantities inside cells and it is a versatile protector. Some of the protective roles of the glutathione include radical scavenging, restoration of damaged molecules by hydrogen donation, reduction of peroxides and maintenance of protein thiols in the reduced state. Of these roles, hydrogen atom donation to DNA radicals is probably the most important [26]. Since competing reactions are very rapid, particularly with oxygen in welloxygenated normal tissues, high concentrations of GSH are required for protection. Thus, moderate depletion of GSH in normal cells may no effect on the radiosensitivity while under hypoxic conditions, such as that occurs in tumor tissues, GSH may playa dominant protective role. Conversely, the depletion of GSH in the hypoxic tumor tissue may have beneficial effects on the treatment of tumors.

Conclusions Non-invasive measurements of tumor redox status in tumorbearing mice were performed using low-frequency electron paramagnetic resonance spectroscopy. Implanted RIF-I tumor in the right leg was studied, while the normal tissue on the left leg was used as control. Nitroxide pharmacokinetics showed overall higher magnitude of reducing equivalents in the tumor tissue compared to normal tissue. It was also observed that tumor hyperoxygenation induced by carbogenbreathing significantly decreased the tumor tissue redox state. Mice treated with BSO, a glutathione depleting agent, showed differential reduction ofGSH and redox status in tumors. This differential reduction of reducing equivalents between the normal and malignant tissue might be useful for selective killing of tumor.

Acknowledgement We acknowledge the financial support from the NIH grant CA 78886.

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