CHOP THERAPY INDUCED MITOCHONDRIAL REDOX STATE ALTERATION IN NON-HODGKIN'S LYMPHOMA XENOGRAFTS.

We are interested in investigating whether cancer therapy may alter the mitochondrial redox state in cancer cells to inhibit their growth and survival. The redox state can be imaged by the redox scanner that collects the fluorescence signals from both the oxidized-flavoproteins (Fp) and the reduced form of nicotinamide adenine dinucleotide (NADH) in snap-frozen tissues and has been previously employed to study tumor aggressiveness and treatment responses. Here, with the redox scanner we investigated the effects of chemotherapy on mouse xenografts of a human diffuse large B-cell lymphoma cell line (DLCL2). The mice were treated with CHOP therapy, i.e., cyclophosphamide (C) + hydroxydoxorubicin (H) + Oncovin (O) + prednisone (P) with CHO administration on day 1 and prednisone administration on days 1-5. The Fp content of the treated group was significantly decreased (p = 0.033) on day 5, and the mitochondrial redox state of the treated group was slightly more reduced than that of the control group (p = 0.048). The decrease of the Fp heterogeneity (measured by the mean standard deviation) had a border-line statistical significance (p = 0.071). The result suggests that the mitochondrial metabolism of lymphoma cells was slightly suppressed and the lymphomas became less aggressive after the CHOP therapy.

Metabolic network analysis of DB1 melanoma cells: how much energy is derived from aerobic glycolysis?

A network model has been developed for analysis of tumor glucose metabolism from (13)C MRS isotope exchange kinetic data. Data were obtained from DB1 melanoma cells grown on polystyrene microcarrier beads contained in a 20-mm diameter perfusion chamber in a 9.4 T Varian NMR spectrometer; the cells were perfused with 26 mM [1,6-(13)C(2)]glucose under normoxic conditions and 37°C and monitored by (13)C NMR spectroscopy for 6 h. The model consists of ∼150 differential equations in the cumomer formalism describing glucose and lactate transport, glycolysis, TCA cycle, pyruvate cycling, the pentose shunt, lactate dehydrogenase, the malate-aspartate and glycerophosphate shuttles, and various anaplerotic pathways. The rate of oxygen consumption (CMRO(2)) was measured polarographically by monitoring differences in pO(2). The model was validated by excellent agreement between model predicted and experimentally measured values of CMRO(2) and glutamate pool size. Assuming a P/O ratio of 2.5 for NADH and 1.5 for FADH2, ATP production was estimated as 46% glycolytic and 54% mitochondrial based on average values of CMRO(2) and glycolytic flux (two experiments).

Lactate detection in inducible and orthotopic Her2/neu mammary gland tumours in mouse models.

This study compared the steady state concentration of lactate in an inducible Her2/nue transgenic breast cancer mouse model and in tumours from the same Her2/neu model grown orthotopically. In vivo lactate was detected by MRS using the Hadamard encoded selective multiple quantum coherence pulse sequence (HadSelMQC) recently developed by our laboratory. A lower lactate signal was observed in the inducible tumours compared to orthotopic tumours in vivo, while ex vivo analysis of perchloric acid extracts revealed similar amounts of this metabolite in both models. Histological staining of mammary tumour specimens showed a much higher level of fat tissue in inducible tumours compared to the orthotopic model. Phantom studies with [3-(13) C] lactate indicated that a lipid environment could significantly reduce the T2 of lactate and impede its detection. The transgenic inducible model for breast cancer not only better recapitulated the biological aspects of the human disease but also provided additional characteristics related to in vivo detection of lactate that are not available in orthotopic or xenograft models. This study suggests that the level of lactate measured by the HadSelMQC pulse sequence may be underestimated in human patients in the presence of high lipid levels that are typically encountered in the breast.

In vivo and ex vivo MR imaging of slowly cycling melanoma cells.

Slowly cycling cells are believed to play a critical role in tumor progression and metastatic dissemination. The goal of this study was to develop a method for in vivo detection of slowly cycling cells. To distinguish these cells from more rapidly proliferating cells that constitute the vast majority of cells in tumors, we used the well-known effect of label dilution due to division of cells with normal cycle and retention of contrast agent in slowly dividing cells. To detect slowly cycling cells, melanoma cells were labeled with iron oxide particles. After labeling, we observed dilution of contrast agent in parallel with cell proliferation in the vast majority of normally cycling cells. A small and distinct subpopulation of iron-retaining cells was detected by flow cytometry after 20 days of in vitro proliferation. These iron-retaining cells exhibited high expression of a biological marker of slowly cycling cells, JARID1B. After implantation of labeled cells as xenografts into immunocompromised mice, iron-retaining cells were detected in vivo and ex vivo by magnetic resonance imaging that was confirmed by Prussian Blue staining. Magnetic resonance imaging detects not only iron retaining melanoma cells but also iron positive macrophages. Proposed method opens up opportunities to image subpopulation of melanoma cells, which is critical for continuous tumor growth.

In vivo 31P MR spectral patterns and reproducibility in cancer patients studied in a multi-institutional trial.

The standardization and reproducibility of techniques required to acquire anatomically localized 31P MR spectra non-invasively while studying tumors in cancer patients in a multi-institutional group at 1.5 T are reported. This initial group of patients was studied from 1995 to 2000 to test the feasibility of acquiring in vivo localized 31P MRS in clinical MR spectrometers. The cancers tested were non-Hodgkin's lymphomas, sarcomas of soft tissue and bone, breast carcinomas and head and neck carcinomas. The best accrual and spectral quality were achieved with the non-Hodgkin's lymphomas. The initial analysis of the spectral values of the sum of phosphoethanolamine plus phosphocholine normalized by the content of nucleotide triphosphates in a homogeneous sample of 32 NHL patients studied by in vivo (31)P MRS showed good reproducibility among different institutions. No statistical differences were found between the institution with the largest number of cases accrued and the rest of the multi-institutional NHL data (2.28 +/- 0.64, mean +/- standard error; n = 17, vs 2.08 +/- 0.14, n = 15). The preliminary data reported demonstrate that the institutions involved in this trial are obtaining reproducible 31P MR spectroscopic data non-invasively from human tumors. This is a fundamental prerequisite for the international cooperative group to be able to demonstrate the clinical value of the normalized determination of phosphoethanolamine plus phosphocholine by 31P MRS as predictor for treatment response in cancer patients.

Quantitative assessment of regional myocardial function in a rat model of myocardial infarction using tagged MRI.

We characterized global and regional left ventricular (LV) function during post myocardium infarction (MI) remodeling in rats, which has been incompletely described by previous MRI studies. To assess regional wall motion, four groups of infarcted animals corresponding to 1-2, 3-4, 6-8 and 9-12 weeks post-MI respectively were imaged using a fast gradient echo sequence with a 2D spatial modulation of magnetization (SPAMM) tagging preparation. An additional group was serially imaged (1-2 and 6-7 weeks post-MI) to assess the global function. Regional and global functional parameters of infarcted rats were compared to non-infarcted normal rats. Compared to normal rats, a decrease in ejection fraction (70 +/-7 vs. 40 +/- 8%, p<0.05) was observed in rats with MI. Maximal and minimal principal stretches (lambda1, lambda2) and strains (E1, E2), principal angle (beta) and displacement varied regionally in normal rats but deviated significantly from the normal values in rats with MI particularly in the infarcted and adjacent zones. Not only was strain magnitude reduced segmentally post-MI, but strain direction became more circumferentially oriented, particularly in rats with larger infarctions. We report the first regional myocardial strain values in normal and infarcted rats. These results parallel findings in humans, and provide a unique tool to examine regional mechanical influences on the remodeling process.

Methodological standardization for a multi-institutional in vivo trial of localized 31P MR spectroscopy in human cancer research. In vitro and normal volunteer studies.

A multi-institutional group has been created to demonstrate the utility of in vivo 31P magnetic resonance spectroscopy (31P-MRS) to study human cancers in vivo. This review is concerned with the novel problems concerning quality control in this large multinational trial of 31P MRS. Our results show that the careful and systematic performance of the quality control tests depicted here (standardized dual 1H/31P tuned radiofrequency probe, quality control procedures, routine use of 1H irradiation while acquiring 31P MR signals) has ensured comparable results between the different institutions. In studies made in vitro, the root-mean-square error was 3.6 %, and in muscle of healthy volunteers in vivo the coefficients of variance for the ratios phosphocreatine/nucleotide-triphosphates, phosphocreatine/noise and nucleotide-triphosphate/noise were 12.2, 7.0 and 10.8 %, respectively. The standardization of the acquisition protocol for in vivo-localized 31P MR spectroscopy across the different institutions has resulted in comparable in vivo data, decreasing the possible problems related to a research study carried out under a multi-institutional setting.

Real-time detection of 13C NMR labeling kinetics in perfused EMT6 mouse mammary tumor cells and betaHC9 mouse insulinomas.

A method was developed for obtaining high signal-to-noise 13C NMR spectra of intracellular compounds in metabolically active cultured cells. The method allows TCA cycle labeling kinetics to be determined in real time without significant oxygen transport limitations. Cells were immobilized on the surface of nonporous microcarriers that were either uncoated or coated with polypeptides and used in a 12-cm3 packed bed. The methods were tested with two EMT6 mouse mammary tumor cell lines, one strongly adherent and the other moderately adherent, and a weakly adherent mouse insulinoma line (betaHC9). For both EMT6 lines, NTP and oxygen consumption measurements indicated that the number of cells in the spectrometer ranged from 6 x 10(8) to 1 x 10(9). During infusion of [1-13C]glucose, labeling in C-4 glutamate (indicative of flux into the first half of the TCA cycle) could be detected with 15-min resolution. However, labeling for C-3 and C-2 glutamate (indicative of complete TCA cycle activity) was fivefold lower and difficult to quantify. To increase TCA cycle labeling, cells were infused with medium containing [1,6-13C2]glucose. A 2.5-fold increase was observed in C-4 glutamate labeling and C-3 and C-2 glutamate labeling could be monitored with 30-min resolution. Citrate synthase activity was indirectly detected in real time, as [3,4-13C2]glutamate was formed from [2-13C]oxaloacetate and [2-13C]acetate (of acetyl-CoA). Cell mass levels observed with betaHC9 cells were somewhat lower. However, the 13C S/N was sufficient to allow real-time monitoring of the response of intracellular metabolite labeling to a step change in glucose and a combined glutamine/serum pulse.

Effects of hyperglycemia on oxygenation, radiosensitivity and bioenergetic status of subcutaneous RIF-1 tumors.

Since tissue oxygen tension is a balance between delivery and consumption of oxygen, considerable effort has been directed at increasing the former and/or decreasing the latter. Techniques to decrease the rate of cellular oxygen consumption (increasing the distance oxygen can diffuse into tissues) include increasing glycolysis by administering supra-physiologic levels of glucose. We have examined the effect of hyperglycemia produced by intravenous glucose infusion on the tissue oxygenation and radiation response of subcutaneously implanted murine radiation induced fibrosarcomas (RIF-1). A 0.3 M glucose solution was delivered via tail vein injection according to a protocol that maintained glucose at a plasma concentration of 17+/-1 mM. The effect of this treatment on radiation response (clonogenic and growth delay studies), tumor oxygenation (needle electrode pO2 and 2-[2-nitro-1H-imidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide (EF5) binding), and tumor bioenergetics and pH (31P NMR spectroscopy) was examined. Systemic measurements included hematocrit and blood glucose and lactate concentrations. The results of these studies suggest that these subcutaneously implanted RIF-1 tumors are both radiobiologically and metabolically hypoxic and that intravenous glucose infusion is not an effective method of modifying this metabolic state.

Quantitative T1rho magnetic resonance imaging of RIF-1 tumors in vivo: detection of early response to cyclophosphamide therapy.

This study compares two potential magnetic resonance imaging (MRI) indices for noninvasive early detection of tumor response to chemotherapy: the spin-lattice relaxation in the rotating frame (T1rho) and the transverse relaxation time (T2). Measurements of these relaxation parameters were performed on a s.c. murine radiation-induced fibrosarcoma (RIF-1) model before and after cyclophosphamide treatment. The number of pixels exhibiting T1rho values longer than controls in viable regions of the tumor increased significantly as early as 18 h after drug administration and remained elevated up to 36 h after treatment (P < 0.005). Although a trend of increasing T2s relative to controls was noted in viable regions of the tumor 36 h after treatment, the changes were not statistically significant. Histological examination indicated a decrease in mitotic index that paralleled the changes in T1rho. We conclude that T1rho measurements may be useful for noninvasive monitoring of early response of tumors to chemotherapy.

Histopathology, electrodiagnostic testing, and magnetic resonance imaging show significant peripheral and central nervous system myelin abnormalities in the cat model of alpha-mannosidosis.

Alpha-mannosidosis is a disease caused by the deficient activity of alpha-mannosidase, a lysosomal hydrolase involved in the degradation of glycoproteins. The disease is characterized by the accumulation of mannose-rich oligosaccharides within lysosomes. The purpose of this study was to characterize the peripheral nervous system (PNS) and central nervous system (CNS) myelin abnormalities in cats from a breeding colony with a uniform mutation in the gene encoding alpha-mannosidase. Three affected cats and 3 normal cats from 2 litters were examined weekly from 4 to 18 wk of age. Progressively worsening neurological signs developed in affected cats that included tremors, loss of balance, and nystagmus. In the PNS, affected cats showed slow motor nerve conduction velocity and increased F-wave latency. Single nerve fiber teasing revealed significant demyelination/remyelination in affected cats. Mean G-ratios of nerves showed a significant increase in affected cats compared to normal cats. Magnetic resonance imaging of the CNS revealed diffuse white matter signal abnormalities throughout the brain of affected cats. Quantitative magnetization transfer imaging showed a 8%-16% decrease in the magnetization transfer ratio in brain white matter of affected cats compared to normal cats, consistent with myelin abnormalities. Histology confirmed myelin loss throughout the cerebrum and cerebellum. Thus, histology, electrodiagnostic testing, and magnetic resonance imaging identified significant myelination abnormalities in both the PNS and CNS that have not been described previously in alpha-mannosidosis.

Enhancement of hyperglycemia-induced acidification of human melanoma xenografts with inhibitors of respiration and ion transport.

RATIONALE AND OBJECTIVES: The authors performed this study to evaluate the selective acidification of a human melanoma xenograft in mice with severe combined immunodeficiency with the induction of hyperglycemia (mean blood glucose level +/- standard error of the mean, 26 mmol/L +/- 1) and the intraperitoneal administration of metaiodobenzylguanidine (MIBG, 30 mg/kg), alpha-cyano-4-hydroxycinnamate (CNCn, 300 mg/kg), lonidamine (100 mg/kg), cariporide (HOE642, 160 mg/kg), or 4.4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS, 50 mg/kg). MATERIALS AND METHODS: The intra- and extracellular pH levels of tumor were estimated from the chemical shifts of inorganic phosphate and 3-aminopropylphosphonate, respectively, with phosphorus-31 nuclear magnetic resonance (MR) spectroscopy. The relative level of steady-state lactate was monitored with hydrogen-1 MR spectroscopy. RESULTS: In small tumors (< or = 8.0 mm), hyperglycemia decreased the intra- and extracellular pH levels by less than 0.2. The combination of hyperglycemia and MIBG decreased the intra- and extracellular pH levels by approximately 0.4 and 0.6, respectively, and lowered the beta-nucleoside triphosphate (NTP)/inorganic phosphate (Pi) ratio of tumor and liver by about 60% and 25%, respectively. The combination of hyperglycemia, MIBG, and CNCn produced a transient decrease in the intracellular pH of about 0.6. The combination of hyperglycemia and lonidamine produced a sustained (>3 hours) 0.8-unit decrease in intracellular pH and an 83% and 100% decrease in PCr/P1 and beta-NTP/P1 ratios, respectively. The combination of hyperglycemia. MIBG, cariporide, and DIDS produced a gradual decrease in intra- and extracellular pH by 1.1 and 1.0, respectively. The relative level of steady-state lactate concentration in tumors increased 10% with hyperglycemia alone, about 20% with MIBG plus hyperglycemia, and increased more than twofold when hyperglycemia was combined with MIBG and CNCn administration. CONCLUSION: These preliminary data suggest that hyperglycemia and combinations of respiratory and ion transport inhibitors can be used to selectively acidify tumors and, thereby, sensitize them to hyperthermnia or other pH-sensitive therapeutic modalities.

In vivo detection of gene expression in liver by 31P nuclear magnetic resonance spectroscopy employing creatine kinase as a marker gene.

In vivo assessment of gene expression is desirable to obtain information on the extent and duration of transduction of tissue after gene delivery. We have developed an in vivo, potentially noninvasive, method for detecting virally mediated gene transfer to the liver. The method employs an adenoviral vector carrying the gene for the brain isozyme of murine creatine kinase (CK-B), an ATP-buffering enzyme expressed mainly in muscle and brain but absent from liver, kidney, and pancreas. Gene expression was monitored by (31)P magnetic resonance spectroscopy (MRS) using the product of the CK enzymatic reaction, phosphocreatine, as an indicator of transfection. The vector was administered into nude mice by tail vein injection, and exogenous creatine was administered in the drinking water and by i.p. injection of 2% creatine solution before (31)P MRS examination, which was performed on surgically exposed livers. A phosphocreatine resonance was detected in livers of mice injected with the vector and was absent from livers of control animals. CK expression was confirmed in the injected animals by Western blot analysis, enzymatic assays, and immunofluorescence measurements. We conclude that the syngeneic enzyme CK can be used as a marker gene for in vivo monitoring of gene expression after virally mediated gene transfer to the liver.

T1rho imaging of murine brain tumors at 4 T.

RATIONALE AND OBJECTIVES: The goal of this study was to evaluate the utility of T1rho weighting in magnetic resonance imaging of murine brain tumors. MATERIALS AND METHODS: S91 Cloudman melanoma was implanted in mouse brains (n = 4). A T2-weighted spin-echo (SE) and a T1rho-weighted fast SE-based sequence were performed on a 4-T clinical imager. T2 and T1rho maps were computed. The tumor-to-normal-tissue contrast was compared between T2-weighted, T1rho-weighted, proton-density-weighted, and pre- and postcontrast T1-weighted SE images. RESULTS: The tumor-tissue contrast of the T1rho-weighted images was similar to that of the T2-weighted images but less than that of the postcontrast T1-weighted images. The T1rho-weighted images provided better definition of tumor boundaries than T2-weighted images. At spin-locking powers of 0.5 and 1.5 kHz, the T1rho of the tumor was 64.0 msec +/- 0.46 and 68.65 msec +/- 0.59, respectively. There was no significant inter- or intra-animal variation in T1rho for tumor or normal brain cortex. CONCLUSION: T1rho-weighted imaging performed at low spin-lock strengths qualitatively depicted tumor borders better than proton-density or T2-weighted imaging and could be useful in treatment planning when combined with other imaging sequences.

Metabolism of alternative substrates and the bioenergetic status of EMT6 tumor cell spheroids.

In order to evaluate the ability of EMT6/Ro multicellular spheroids to utilize various pathways of energy production, (13)C and (31)P MRS have been employed to monitor the metabolism of glucose, glutamine, acetate and propionate. EMT6/Ro spheroids perfused with culture medium containing 5.5 mM glucose maintain stable levels of nucleotide triphosphates (NTP) and phosphocreatine (PCr) for up to 48 h, even in the absence of glutamine. The metabolism of 1-(13)C-glucose was almost entirely to 3-(13)C-lactate (88 +/- 12%, n = 7), even though the perfusion medium was equilibrated with 95% O(2). Labeling was also observed in other glycolytic metabolites, primarily alanine and alpha-glycerolphosphate. A low level of (13)C labeling in glutamate, indicative of mitochondrial oxidative metabolism (TCA cycle), was consistently detected when spheroids were perfused with 1-(13)C-glucose, almost exclusively in the C4 position of glutamate. Labeling of glutamate C2 and C3 was always less than 20% of the labeling in C4 and was usually undetectable. No evidence of adjacent carbon labeling in individual glutamate molecules (indicative of multiple cycles of label incorporation) was found, even in high-resolution (13)C NMR spectra of extracts from cells or spheroids. Despite the predominantly glycolytic metabolism of glucose, the mitochondrial substrate glutamine (2 mM, in the presence of < or =0.5 mM glucose from fetal bovine serum), supported stable levels of NTP and PCr in the tumor cells for up to 12 h. In the presence of 2.5 mM acetate, the bioenergetic status of cells in EMT6 spheroids declined slowly but measurably, and no incorporation of label from 2-(13)C-acetate into other metabolites was detected either in intact perfused spheroids or in high-resolution spectra of extracts. In contrast, when the anaplerotic TCA cycle substrate 3-(13)C-propionate replaced acetate, the high-energy phosphate levels in EMT6/Ro spheroids were somewhat reduced, but stabilized at a new lower level. Incubation of spheroids with 3-(13)C-propionate (with natural abundance glucose and glutamine) resulted in label detectable in the C2 and C3 of glutamate, but the primary labeled compound was methylmalonate, an intermediate in propionate metabolism. Addition of vitamin B(12), a cofactor for methylmalonyl CoA reductase, to the growth medium 24 h prior to perfusion with propionate resulted in the elimination of the methylmalonate resonance. A variety of 2- and 3-labeled metabolites were detected, including succinate, malate and glutamate. Labeling of C2 and C3 of lactate implicated cytoplasmic malic enzyme activity.

Intracellular acidification of human melanoma xenografts by the respiratory inhibitor m-iodobenzylguanidine plus hyperglycemia: a 31P magnetic resonance spectroscopy study.

In vivo 31P magnetic resonance spectroscopy demonstrates that human melanoma xenografts can be significantly acidified by induction of hyperglycemia combined with administration of m-iodobenzylguanidine (MIBG), an inhibitor of mitochondrial respiration. In melanoma xenografts (< or =8 mm diameter), intracellular pH (pHi, measured by the chemical shift of the Pi resonance) and extracellular pH (pHe, measured with 3-aminopropylphosphonate) was reduced by less than 0.2 unit during i.v. infusion of glucose for 40 min. Administration of MIBG (30 mg/kg) under hyperglycemic conditions (26 mM) reduced tumor pHi and pHe by approximately 0.4 (P < 0.001) and approximately 0.6 (P < 0.001) unit, respectively; coincidentally, the nucleoside triphosphates:Pi ratio decreased approximately 60% (P < 0.004) relative to the baseline level. Minimal changes in pHi and pHe and a small decrease in nucleoside triphosphates:Pi ratio (26%, P = 0.2) were observed in liver in response to MIBG plus hyperglycemia. These results suggest that under normoglycemic and hyperglycemic conditions, small human melanoma xenografts (< or =8 mm) may exhibit a relatively high level of oxidative phosphorylation that may be blocked by MIBG. The acidification may result from increased lactate production as a direct effect of MIBG inhibition of respiration in mitochondria of tumor cells, or through indirect systemic effects, which remain to be identified. The synergetic effects of MIBG and hyperglycemia result in significant acidification of the tumor and a decrease in tumor bioenergetic status, and the effects are largely selective for tumors in comparison with normal tissues.

Lactate editing and lipid suppression by continuous wavelet transform analysis: application to simulated and (1)H MRS brain tumor time-domain data.

Determination of lactate concentrations in vivo is required in the noninvasive diagnosis, staging, and therapeutic monitoring of diseases such as cancer, heart disease, and stroke. An iterative filtering process based on the continuous wavelet transform (CWT) method in the time domain is proposed to isolate the lactate doublet signal from overlapping lipid resonances and estimate the magnetic resonance spectroscopy (MRS) parameters of the lactate methyl signal (signal amplitude, chemical shift, J-coupling and apparent transverse relaxation time (T*(2))). This method offers a number of advantages over the multiple quantum (MQ) and difference spectroscopy approaches, including: 1) full recovery of the lactate methyl signal, whereas the MQ methods usually detect 50% of the signal intensity; 2) in contrast to MQ methods, the lipid signal is retained together with J-coupling data on the lactate peak; 3) the CWT method is much less sensitive to motion artifacts than difference spectroscopy. Application of the method to simulated and real (1)H MRS data collected from human blood plasma and brain tumors demonstrated that this filter provides accurate estimates of the MRS parameters of the lactate doublet and efficiently removes lipid contributions.

Monitoring pharmacokinetics of anticancer drugs: non-invasive investigation using magnetic resonance spectroscopy.

Magnetic resonance spectroscopy (MRS) offers the unique advantage of detecting, identifying and quantifying chemicals deep within the living body in a totally non-invasive manner. In studies on pharmacology and toxicology of anticancer drugs, MRS and the closely related technique magnetic resonance imaging (MRI) have many uses. MRS in particular, despite its low sensitivity, offers unique insights into pharmacokinetics (the changing concentration of the drug at its site of action) which can be monitored, and metabolism (both activation and detoxification) can be detected in real time. This review considers some recent work on (19)F, (31)P, (1)H and (13)C MRS of anticancer drugs. Future possibilities for (13)C MRS and (1)H MRS studies of drugs and their metabolites are considered in detail.

MIBG inhibits respiration: potential for radio- and hyperthermic sensitization.

INTRODUCTION: Meta-iodobenzylguanidine (MIBG) in its 131I-labeled form is clinically used as a tumor-targeted radiopharmaceutical in the diagnosis and treatment of adrenergic tumors. This well established drug may have additional clinical applications as a radiosensitizer or hyperthermic agent, ie., MIBG reportedly inhibits mitochondrial respiration in vitro. The mechanism for MIBG inhibition of cellular oxygen consumption is uncertain. Moreover, MIBG reportedly stimulates glycolysis both in vitro and in vivo. Our studies show the effect of MIBG on 9L glioma oxygen consumption and redox status with tumors cells in vitro and in vivo. MATERIALS AND METHODS: The effects on electron transfer were determined by following oxygen consumption with a Clark oxygen electrode. Fluorescence measurements were used to determine effects of MIBG on intracellular electron acceptors, NADPH and flavoproteins, in vitro and in vivo. 31P-NMR was used to determine alterations in tumor cell pH in vivo. RESULTS: Our results show the inhibition of oxygen utilization with MIBG for cell suspensions in vitro. The same results were demonstrated for tumor cell suspensions rapidly isolated from tumors grown in rats. Moreover, NAD(P)H and flavoprotein (Fp) fluorescence changes were observed to rapidly occur following MIBG addition in vitro. Changes in intracellular pH measured with 31P-NMR, in vivo, precede the changes in fluorescence of NAD(P)H and Fp obtained with frozen sections of tumor. CONCLUSIONS: We conclude that 31P-NMR measurements and fluorescence changes, following MIBG injection, can be used as criterion for selecting the proper time to treat tumors with ionizing radiation or hyperthermia.

Estimations of intra- and extracellular volume and pH by 31P magnetic resonance spectroscopy: effect of therapy on RIF-1 tumours.

Quantification of metabolite or drug concentrations in living tissues requires determination of intra- and extracellular volumes. This study demonstrates how this can be achieved non-invasively by 31P magnetic resonance spectroscopy (MRS) employing dimethyl methylphosphonate (DMMP) as a marker of total water space, 3-aminopropylphosphonate (3-APP) as a marker of extracellular space and P and 3-APP as markers of intracellular pH (pH) and extracellular pH (pHe) respectively. The MRS measurements of the tumour volumes were validated by classic radiolabelling methods using 3H2O and [14C]inulin as markers of total and extracellular space respectively. The extracellular volume fraction measured by radiolabelling of RIF-1 tumours was 23 +/- 0.83% (mean +/- s.e.m. n = 9), not significantly different (P > 0.1) from that found by MRS (27 +/- 2.9%, n = 9, London, and 35 +/- 6.7, n = 14, Baltimore). In untreated RIF-1 tumours, pH was about 0.2 units higher than pHe (P < 0.01). 5-Fluorouracil (5FU) treatment (165 mg kg(-1)) caused no significant changes in either pHe or per cent extracellular volume. However significant increases in pH, 48 h after treatment (P < 0.01) correlated with decreased tumour size and improved bioenergetic status [NTP/inorganic phosphate (Pi) ratio]. This study shows the feasibility of an MR method (verified by a 'gold standard') for studying the effects of drug treatment on intra- and extracellular spaces and pH in solid tumours in vivo.