Bioluminescence imaging of metabolites in a human tumour xenograft after treatment with hyperthermia and/or the radiosensitizer pimonidazole.

The levels and distribution of ATP, glucose and lactate in human tumour xenografts following either single or combined treatment with hyperthermia (43 degrees C for 30 min) and/or pimonidazole (1 mg/g b.w) were determined by bioluminescence and compared with the mean 'global' levels obtained from the same tumours using conventional biochemical analysis. In general, the levels of ATP, glucose and lactate measured with both methods were in good agreement although the latter was consistently lower after bioluminescence determination. Compared with controls, neither the levels of ATP nor glucose were greatly affected in this tumour following treatment with the various modalities, whereas those of lactate were considerably increased as determined by both methods. The spatial distribution of ATP and glucose from controls and treated tumours was largely confined to the periphery and generally remained unchanged irrespective of treatment without any apparent alterations in the shape of the distribution curve. However, the increased lactate levels tended to accumulate towards the central region of the tumour after hyperthermia and/or sensitizer, showing an almost Gaussian-like distribution compared with controls. These results are in agreement with previous studies of global tumour metabolism showing an enhanced glycolytic activity with increased lactate levels after single or combined modality treatment.

Metabolic imaging in tumours by means of bioluminescence.

A bioluminescence technique involving single photon imaging was used to quantify the spatial distribution of the metabolites ATP, glucose and lactate in cryosections of various solid tumours and normal tissue. Each section was covered with an enzyme cocktail linking the metabolite in question to luciferase with light emission proportional to the metabolite concentration. The photons emitted are imaged directly through a microscope and an imaging photon counting system. In some cases, good agreement was observed between the distribution of relatively high concentrations of ATP and glucose in viable cell regions of the periphery, while the reverse was seen in more necrotic tumour centres with comparatively high lactate levels. In general, lactate was distributed more diffusely over the sections while ATP was more highly localised and glucose assumed an intermediate pattern. In contrast to the large degree of heterogeneity seen in tumours, distribution patterns of metabolites were much more homogeneous in normal tissue, such as heart muscle. Mean values for metabolite levels in cryosections using bioluminescence are in good agreement with those obtained from the same tumour by conventional methods.

Metabolic studies and neurotoxicity in tumors and brain of mice after hypoxic cell sensitizers.

PURPOSE: The effects of the radiosensitizers RK-28 and RP-170, both 2-nitroimidazole nucleoside analogues, and KU-2285, a fluorinated 2-nitroimidazole, as well as etanidazole (ETA) on glucose metabolism in mouse tumors and brain were studied to assess their degree of neurotoxicity. METHODS AND MATERIALS: Adult male C57Bl mice received differing doses of the above sensitizers IP. Blood, brain, and tumor samples were removed at various times and the levels of glycolytic metabolites determined. Glucose uptake and phosphorylation in brain was measured by the 2-deoxyglucose method of Sokoloff et al. (6). RESULTS: RP-170 showed neither signs of toxicity nor significant alterations in glucose metabolism in brain or tumor at doses up to 4 g/kg b.w. up to 4 h. By contrast, RK-28 was extremely neurotoxic at a dose of 1 g/kg b.w. with a high degree of lethality, resulting in a highly significant increase in the brain glucose level from 0.38 mumol/g to 2.20 mumol/g (p < 0.001) 2 h after administration, whereas that in the tumor was decreased. KU-2285 and ETA were significantly (p < 0.01) less toxic than RK-28 at this dose, as reflected in a lower increase in the brain glucose level (0.60 mumol/g), although KU-2285 approached that of RK-28 (1.43 mumol/g; p < 0.01) after 2 h following a dose of 2 g/kg b.w. However, in contrast to the other sensitizers, KU-2285 concomitantly also resulted in a highly significant continuous increase (p < 0.01) in tumor glucose levels. Labeled 3H-2-deoxyglucose studies showed that RP-170 neither markedly affected the uptake of total radioactivity into the brain nor its degree of phosphorylation whereas, KU-2285 (2 g/kg) and RK-28 (1 g/kg) decreased uptake by approximately 50% and phosphorylation approximately 3 and 4-fold, respectively. At doses of 1 g/kg, ETA and KU-2285 showed no significant changes in these parameters. This indicates a decreased level of neurotoxicity. CONCLUSION: Since the adult brain relies solely on glucose metabolism for its energy supply, interference to this pathway may be instrumental in the development of neurotoxicity, thus, underlining the need for such metabolic studies to assess the level of toxicity by radiosensitizers.

Evaluation of a new 2-nitroimidazole nucleoside analogue, RK-28 as a radiosensitizer for clinical use.

The experimental data previously reported on RK-28, a hypoxic cell sensitizer which is now being tested in a phase I clinical trial, are confusing. Some data indicate superiority of RK-28 over misonidazole (MISO), whereas others do not. This paper presents our experimental data on the efficacy, toxicity, and pharmacokinetics of RK-28, in comparison with those of MISO, and also summarizes the data of other investigators. In our experiments, RK-28 had a 1.5-2.5 times higher sensitizing activity in vitro on EMT6 and SCCVII cells than MISO, and the difference was larger when the pre-irradiation incubation time was longer. The latter was considered to be due to the time-dependent cellular uptake and reactivity of RK-28 with non-protein sulphydryls. In vivo, RK-28 was almost as efficient as or slightly inferior to MISO against SCCVII and EMT6 tumours when assayed with an in vivo/in vitro assay and a growth delay time assay. The LD50/7 by a single injection of RK-28 was half that of MISO, but when 60% of LD50/7 was injected into mice every day, the total dose that could be given was higher for RK-28 than for MISO. Pharmacokinetic studies using mice, rats, rabbits, and a dog showed that RK-28 was rapidly eliminated from the blood and various tissues. From our results it was concluded that the possible success of the clinical trial of RK-28 depends on its low cumulative toxicity.

Effects on intermediary metabolism in mouse tissues by Ro-03-8799.

Glucose and lipid metabolism in the brain, liver and in a transplanted tumour were found to be variously altered within 2 to 3 h of administering single doses of the radiosensitizer Ro-03-8799 to normal and tumour-bearing mice. Hepatic lactate and glycerol-3-phosphate (G3P) levels were decreased but those of the ketone body beta-hydroxybutyrate (beta-HOBu) were raised. However, in the tumour, these levels were all enhanced. The lactate levels in brain remained relatively constant but both beta-HOBu and G3P levels were altered in a manner similar to that in the liver. The levels of glucose were approximately doubled in blood, brain and tumour, but whereas tumour G6P levels increased, those in the brain were lowered to below the limits of detection. Hepatic glucose levels were significantly decreased after 1 h but G6P levels were not affected. These changes could neither be related to inhibitory effects on hepatic glucokinase or brain hexokinase activity nor to limiting amounts of ATP in both tissues. However, the activity of glucose-6-phosphatase (G6P'ase) was distinctly raised in the liver and the hepatic glycogen stores were also rapidly lowered. Overall, the results suggest that Ro-03-8799 exerts a stimulatory effect on glucose production in the liver. In both liver and brain the levels of free fatty acids and phospholipids were increased whereas those of esterified fatty acids were lowered. Most importantly, the changes in metabolite levels affect the cellular redox couples; those of the cytosol (lactate/pyruvate; G3P/dihydroxyacetone phosphate (DAP] are directed towards the oxidised state in the liver but to a more reduced state in the tumour. The mitochondrial couple (beta-HOBu/acetoacetate (AcAc)) in both tissues is shifted towards the reduced state. These metabolic changes may result in an increase in the degree of hypoxia in the tumour and may well play an important role in the development of neuropathies.

The effects of X irradiation, hyperthermia, and combined modality treatment on poly(ADPR)synthetase activity in human melanoma cells.

The effects of X irradiation and/or hyperthermia on both the intrinsic and total poly(ADPR)synthetase activity in permeabilized human melanoma cells were determined. All treatments were given in the early phase of exponential growth (early log phase). The intrinsic and total enzyme activities were significantly decreased immediately after heat treatment at either 42 degrees or 44 degrees C for 3 hr. These activities remained below the respective control levels over the whole period of culture, and some recovery was observed after 48 hr in both cases. X-ray doses of 3.76 to 11.28 Gy produced an increase in intrinsic activity 30 min postradiation (p.r.) but had no significant effect on the total activity. The increase may thus be associated with DNA repair. At later times after irradiation with 3.76 Gy no significant changes were observed. Combined treatment of X irradiation (3.76 Gy) followed by hyperthermia (42 degrees C for 3 hr) produced an enhanced decrease in intrinsic activity, equivalent to that after hyperthermia at 44 degrees C. The effects on cell proliferation were similar in both cases. However, the total activity was not modified to the same extent after combined treatment. The long-term decrease in total activity at 42 degrees C hyperthermia may be due to inhibitors produced during heat treatment since the enzyme activity was partially restored by changes of fresh medium. These results suggest that heat, X irradiation, and combined treatment act in a different manner on the chromatin-associated enzyme or at different sites on the enzyme complex.

The effects of radiosensitizers on intermediary metabolism in vivo.

In the liver, MISO has little effect on glycolytic intermediates but both the lactate and G3P contents are significantly decreased shortly after administration, whereas the level of ketone-bodies is raised. The changes in hepatic metabolite levels following treatment with SR-2508 are less marked. However, in an adenocarcinoma, both the lactate and ketone-body concentrations are enhanced with MISO. The redox equilibria states are shifted to the oxidized metabolites in the liver but instead to the reduced metabolites in tumor. These effects may have relevance for the radiotherapy in tumors.

N-methylnicotinamide as a possible prognostic indicator of recovery from leukaemia in patients treated with total-body irradiation and bone marrow transplants.

N-methylnicotinamide was determined in urine from patients with acute myelocytic leukaemia following total-body X-irradiation with 8.6 Gy and bone marrow transplantation. Patients that are alive and in excellent condition i.e. with acute leukaemia in full remission showed a distinct enhanced excretion of this metabolite about 20 days p.r. which returned to normal levels at about day 40 p.r. Patients that have died intercurrently of early leukaemic recurrences showed considerable fluctuations in N-methylnicotinamide excretion over the entire period and no "normalization" of levels in these patients was seen. In those cases where late leukaemic recurrence or infections were the cause of death, usually after discharge from the clinic, excretion patterns typical of those seen in disease-free patients were observed. We thus conclude that this metabolite appears to be a suitable tentative prognostic indicator for the overall state of recovery from leukaemia in the patients.

Hepatic nuclear estrogen receptor concentrations in the rat--influence of age, sex, gestation, lactation and estrous cycle.

Nuclear estrogen receptor concentrations in rat liver were determined by exchange assay. The concentration of estrogen receptors in nuclei from vehicle-treated male and female rats show age-dependent, but not sex-dependent variations in the course of life. Levels are highest during the perinatal period (approximately 1500 binding sites/nucleus), whereafter they decrease towards the onset of puberty (approximately 300 binding sites/nucleus) before rising again to reach the postpuberal maximum (approximately 800 binding sites/nucleus). Pregnancy further raised receptor concentrations in the last week of gestation when they reach approximately 1200 binding sites/nucleus. Studies with ethynylestradiol-treated rats demonstrated that virtually no translocation can be detected before the onset of puberty; thereafter the number of translocated receptors increases dramatically reaching a maximum (9000 binding sites/nucleus) between day 80 and 87 of life. The extractability of the nuclear receptors with 0.4 M KCl varies during the course of life. Extractability is very high (approximately 90%) up to about day 12 of life, but then decreases markedly (to approximately 70% in vehicle-treated and to approximately 50% in ethynylestradiol-treated rats) before the onset of puberty.

Nuclear oestrogen receptors in rat liver. Development of assay conditions, characterization and the translocation of oestrogens in vivo.

A method for the determination of specific oestrogen-receptor binding sites in rat liver nuclei is described. Nuclear receptors showed a high affinity for oestradiol (Kd approximately 3 x 10(-9)M), a low capacity, and a distinct specificity for substances with known oestrogenic and anti-oestrogenic activity. No sex differences were seen in the concentrations of nuclear receptors from either vehicle- or ethynyloestradiol-pretreated rats. Only a limited number of binding sites could be extracted with 0.4 M-KCl. The remaining sites, which were solubilized by sonication and treatment with deoxyribonuclease I, sedimented at 3-4 S. Of four oestrogens tested (oestradiol, ethynyloestradiol, diethylstilboestrol, tri-p-anisylchloroethylene), ethynyloestradiol was the most effective translocation agent in vivo, nuclear uptake occurring at doses below 1 microgram/rat; changes in salt extractability of nuclear receptors occurred at doses lower than those required to achieve absolute increases in nuclear receptor concentrations.

Misonidazole as a radiosensitizer in the radiotherapy of glioblastomas and oesophageal cancer. Pharmacokinetic and clinical studies.

Since May 1978 the hypoxic-cell radiosensitizer, misonidazole (MIS), has been under clinical investigation in a phase III trial with multiple doses of the drug in 11 patients with brain tumours (seven glioblastomas, four recurrent brain tumours) and three patients with oesophageal carcinoma. The doses of MIS administered were usually well tolerated but the principal toxicities observed were peripheral neuropathy as well as nausea and vomiting was completely reversible. The incidence of neuropathy was not related to the pharmacological parameters of plasma level or half-life. Pharmacological assessment by high-pressure liquid chromatography included assays of plasma, urine and cerebrospinal fluid. The demethylated product, Ro-05-9963, was detected as the major metabolite. Peak plasma levels were obtained one to four hours after administration of MIS, with a half-life of five to ten hours. Cerebrospinal fluid levels of MIS correlated well with those of the plasma. MIS was mainly excreted as the demethylated metabolite, but less than 40% of the given dose could be recovered. The results obtained suggest that the present MIS dosage for glioblastoma patients results in a low plasma level with no observable therapeutic effect.

Localization of oxidized nocotinamide--adenine dinucleotide glycohydrolase in the mouse liver nuclear envelope.

NAD+ glycohydrolase activity located in the nuclear envelope was maximally solubilized by treatment with 0.1--0.2% Triton X-100. The residual activity largely represents the chromatin-associated NAD+ glycohydrolase. Under these conditions the phospholipids were extensively solubilized (over 90%) while leaving the nuclei physically stable, although the nuclear membranes were removed, as shown by electron microscopy. After Triton X-100 treatment, deoxyribonuclease I did not significantly affect the residual NAD+ glycohydrolase activity, although the DNA was completely broken down. This enzyme activity can be released from the nuclear pellet by incubation with phospholipase C. For comparative studies, the glucose 6-phosphatase activity, known to be present in the nuclear envelope, was investigated. Treatment with 0.01% Triton X-100 released 10--20% of the phospholipids, but without solubilizing either glucose 6-phosphatase or NAD+ glycohydrolase. Higher Triton X-100 concentrations (0.1--1.0%) inhibited glucose 6-phosphatase, but not NAD+ glycohydrolase activity. NAD+ glycohydrolase is apparently present in a latent form in the nuclear envelope. Glucose 6-phosphatase, However, shows no such latency.