The role of non-protein sulphydryls in determining the chemical repair rates of free radical precursors of DNA damage and cell killing in Chinese hamster V79 cells.

Chinese hamster V79 fibroblasts were irradiated in the gas explosion apparatus and the chemical repair rates of the oxygen-dependent free radical precursors of DNA double-strand breaks (dsb) and lethal lesions measured using filter elution (pH 9.6) and a clonogenic assay. Depletion of cellular GSH levels, from 4.16 fmol/cell to 0.05 fmol/cell, by treatment with buthionine sulphoximine (50 mumol dm-3; 18 h), led to sensitization as regards DNA dsb induction and cell killing. This was evident at all time settings but was particularly pronounced when the oxygen shot was given 1 ms after the irradiation pulse. A detailed analysis of the chemical repair kinetics showed that depletion of GSH led to a reduction in the first-order rate constant for dsb precursors from 385 s-1 to 144 s-1, and for lethal lesion precursors from 533 s-1 to 165 s-1. This is generally consistent with the role of GSH in the repair-fixation model of radiation damage at the critical DNA lesions. However, the reduction in chemical repair rate was not proportional to the severe thiol depletion (down to approximately 1% for GSH) and a residual repair capacity remained (approximately 30%). This was found not to be due to compartmentalization of residual GSH in the nucleus, as the repair rate for dsb precursors in isolated nuclei, washed virtually free of GSH, was identical to that found in GSH-depleted cells (144 s-1), also the OER remained substantially above unity. This suggests that other reducing agents may have a role to play in the chemical repair of oxygen-dependent damage. One possible candidate is the significant level of protein sulphydryls present in isolated nuclei.     

The response of spontaneous and transplantable murine tumors to vasoactive agents measured by 31P magnetic resonance spectroscopy.

31P magnetic resonance spectroscopy has been used to compare the effects of the vasoactive agents hydralazine and flunarizine on the oxygenation of the transplantable tumors, SCCVII/Ha and 16C, and a range of spontaneous mammary tumors arising in the breeding stock in the Genetics Division at the Radiobiology Unit. The vasodilator hydralazine, previously shown to increase the radiobiological hypoxic fraction of transplantable murine tumors, increased inorganic phosphate to total phosphate (Pi/total) in SCCVII/Ha and 16C tumors. However, only two spontaneous tumors responded to this agent (2/12). The calcium antagonist flunarizine, which sensitizes the SCCVII tumor to X rays, consistent with a reduction in hypoxic fraction, reduced Pi/total in this and the 16C tumor. Further, most spontaneous tumors tested (8/10) responded to this agent, as measured by a reduction in Pi/total. These results point to fundamental differences between transplantable and spontaneously arising tumors in mice in their response to vasoactive agents.     

Dual function nitroimidazoles less toxic than RSU 1069: selection of candidate drugs for clinical trial (RB 6145 and/or PD 130908.

Following the toxicity and synthetic difficulties encountered with the hypoxic cell radiosensitizer RSU 1069, efforts have focused on development of a superior analogue. Two compounds, RB 6145 and PD 130908, have emerged from this program which overcome the instability and synthetic problems associated with RSU 1069 while retaining favorable biological activity. Both agents show comparable radiosensitizing activity to RSU 1069 following oral or i.p. administration to mice bearing the KHT or RIF-1 tumors. Sensitizing efficiency is about 10 X greater than that observed for misonidazole or etanidazole. Toxicity toward hypoxic tumor cells in vivo is demonstrated by clamping tumors (for 60 min) following administration of PD 130908 or RB 6145. Both are effective hypoxic cytotoxins, but less potent than RSU 1069. Systemic toxicity is substantially reduced following oral drug administration. Further, doses achievable following fractionated drug treatments are sufficiently high to produce significant levels of radiosensitization.     

Pharmacokinetics of varying doses of nicotinamide and tumour radiosensitisation with carbogen and nicotinamide: clinical considerations.

Plasma concentrations, after administration of varying doses of nicotinamide, were measured in CBA male mice using a newly-developed high performance liquid chromatography assay. In all dose groups, peak levels were observed within the first 15 min after an i.p. administration of 0.1, 0.2, 0.3 or 0.5 mg g-1 of nicotinamide. There was a clear dose-dependent increase in plasma concentration with increasing dose, with almost a five-fold lower concentration (1.0 vs 4.9 mumol ml-1) achieved with a dose of 0.1 mg g-1 compared with 0.5 mg g-1, respectively. The half-life of nicotinamide increased from 1.4 h to 2.2 h over the dose range (P < 0.01). Comparisons with previous pharmacokinetic data in humans show that clinically-relevant oral doses of 6 and 9 g in humans give plasma levels slightly higher than those achieved at 1 h with doses of 0.1 to 0.2 mg g-1 in mice. Tumour radiosensitisation with carbogen alone, and with carbogen combined with varying doses of nicotinamide (0.05 to 0.5 mg g-1), was investigated using a 10-fraction in 5 days X-ray schedule. Relative to air-breathing mice, a statistically significant increase in sensitisation was observed with both a local tumour control and with an in vivo/in vitro excision assay (P < or = 0.007). With the local control assay, a trend was observed towards lower enhancement ratios (ERs) with decreasing nicotinamide dose (from 1.85 to 1.55); carbogen alone was almost as effective as when combined with 0.1 mg g-1 of nicotinamide. With the excision assay, ERs for carbogen combined with nicotinamide increased with decreased levels of cell survival. At a surviving fraction of 0.02, enhancement ratios of 1.39-1.48 were obtained for carbogen plus 0.1 to 0.3 mg g-1 of nicotinamide. These were lower than those seen with the two higher doses of 0.4 to 0.5 mg g-1 (ERs = 1.63-1.69).     

The effect of hypoxia on acquired drug resistance and response to epidermal growth factor in chinese hamster lung fibroblasts and human breast-cancer cells in vitro

Prolonged hypoxia induced transient drug resistance in Chinese hamster lung fibroblasts. Previously hypoxic cells were resistant to adriamycin and resistant to etoposide. Complete recovery of etoposide sensitivity was observed following re-aeration for 24 hr. A change in P-glycoprotein expression was unlikely to contribute to the resistance caused by hypoxia, since adriamycin resistance was not reversed by verapamil. However, alteration in the plasma membrane structure may be involved, since previously hypoxic cells were resistant to extracellular superoxide radical generated by the addition of xanthine/ xanthine oxidase. In contrast, adriamycin sensitivity was not altered by hypoxia in 3 human breast-cancer cell lines. MDA-468 and MCF-7/Adr differed in their response to EGF, independent of the presence of hypoxia. These results suggest that hypoxic-stress-induced drug resistance is not generalized.     

Determination of combretastatin A-4 and its phosphate ester pro-drug in plasma by high-performance liquid chromatography

High-performance liquid chromatography with both absorbance and fluorescence detection has been applied to the determination of the potential anti-tumour agent combretastatin A-4 and its phosphate ester in murine and human plasma. The presence of different interfering peaks in the two species makes absorbance detection at 295 nm the method of choice for the mouse, and fluorescence detection (295 nm/390 nm) for human plasma. The calibration was linear over the range studied (0.01-50 microM for combretastatin A-4, 0.02-200 microM for combretastatin A-4 phosphate), with quantitation limits of 0.05 microM for both drugs in the mouse, and 0.05 microM and 0.0125 microM for the phosphate ester and free drug, respectively, in human plasma. The method should be useful for pharmacokinetic studies in the forthcoming Phase I clinical trial of combretastatin A-4 phosphate.