M.E. Watts

Structure-activity relationships in the development of hypoxic cell radiosensitizers. I. Sensitization efficiency.

The efficiency of 35 nitroaromatic and nitroheterocyclic compounds in radiosensitizing hypoxic Chinese Hamster cells in vitro was determined. The concentration C of the compound required to achieve an enhancement ratio of 1.6 was measured, and the redox and partition properties were quantified as the one-electron reduction potential at pH 7, E, and the octanol: water partition coefficient, P, respectively. Most of the compounds studied were 2-nitroimidazoles, but some 4- and 5-nitromidazoles, 5-nitrofurans and nitrobenzenes were investigated for comparison. Together with data for nine nitroimidazoles previously reported, the results were fitted to a structure-activity relationship of the form -log C = b0 + b1E + b2 log P + b3 (log P)2 using multiple linear regression analysis. Statistical tests showed that the coefficients b2 and b3 were not significantly different from zero and the simpler equation, obtained by omitting the terms in log P, explained 85 per cent of the variance in log C. Earlier reports that the radiosensitization efficiency of nitro compounds in vitro largely depends on the reduction potential were confirmed. The conclusive demonstration that P is unimportant in vitro is valuable in interpreting the results of experiments in vivo, where P is expected to have a much greater influence on biological response.

Mammalian cell toxicity of nitro compounds: Dependence upon reduction potentiakl

Summary The toxicity of several classes of nitro-aromatic and -heterocyclic compounds towards W79 mammalian cells in vitro has been determined. Cells with varying concentrations of drugs were incubated in air at 37°C for up to 14 days in order to form colonies. It was found that the concentration of nitro compound required to reduce cell colony-forming ability by 50% was a function of the one-electron reduction potential of the compound, more cytotoxic compounds having more positive potentials.

Isolation and characterization of microvessel endothelial cells from human mammary adipose tissue

SummaryA method for the isolation and long-term culture of human microvessel endothelial cells from mammary adipose tissue (HuMMEC) obtained at breast reduction surgery has been developed. Pure cultures of HuMMEC were isolated by sequential digestion of the fat with collagenase and trypsin followed by specific selection of microvessel fragments withUlex europaeus agglutinin-1 coated magnetic beads (Dynabeads). The resulting cells formed contact-inhibited monolayers on gelatin and fibronectin substrates and capillary-like “tubes” on Matrigel; they also expressed von Willebrand factor, angiotensin-converting enzyme, and accumulated acetylated low density lipoprotein. Further immunofluorescence characterization revealed the presence of antigens for the endothelial cell specific monoclonal antibodies EN4 and H4-7/33. In addition, the origin of these cells was confirmed by the demonstration of the cell adhesion molecules, platelet endothelial cell adhesion molecule-1 (CD31), and endothelial leukocyte adhesion molecule-1 (ELAM-1/E-selectin) upon stimulation with tumor necrosis factor (TNF)α. HuMMEC were found to express-1 ELAM-1 at lower levels of TNFα (<10 ng/ml) than required by human umbilical vein endothelial cells. These cells should provide a useful in vitro model for studying various aspects of microvascular biology and pathology.

Radiosensitization of Chinese Hamster Cells by Oxygen and Misonidazole at Low X-ray Doses

The radiosensitization of Chinese hamster V79 cells in vitro by air and misonidazole at low X-ray doses (0.2-6.0 Gy) had been studied. These survival data, together with high-dose data, were fitted to the linear quadratic model ln S = -(alpha D + beta D2), deriving estimates of alpha and beta by six different methods to illustrate the influence of the statistical treatment on the values so derived. This in vitro study clearly demonstrated that the survival parameters alpha and beta are dependent to some degree on the method of analysis of the raw survival data; however, their ratios, the values of oxygen enhancement ratios (OERs) and radiosensitizer enhancement ratios (SERs) derived from the different methods, are similar. All methods of analysis give reduced OERs at low radiation doses for combined low- and high-dose X-ray data. However, the OERs are still appreciably high, ranging from 2.45 to 2.50 for an oxic dose of 2 Gy. All methods of analysis gave reduced SERs at low doses for combined low and high X-ray dose data for hypoxic cells irradiated in 1 mmol dm-3 misonidazole. At survival levels corresponding to doses of 2 Gy in the presence of 1 mmol dm-3 misonidazole and SERs ranged from 1.2 to 1.5.

The Effect of Extracellular pH on Radiosensitization by Misonidazole and Acidic or Basic Analogues

The effect of extracellular pH (pHe) on the radiosensitization of hypoxic Chinese hamster V79 cells in vitro by the 2-nitroimidazole, misonidazole, and analogues substituted with basic or acid functions has been studied. Misonidazole (1 mmol dm-3) gave an enhancement ratio (e.r.) of 1.6 which remained unchanged over the pHe range of 3.8-9.5. Control hypoxic survival curves in the absence of sensitizer also remained essentially unchanged over this pHe range. These results contrast with those seen for 0.1 mmol dm-3 Ro 03-8799 (1-(2-nitro-1-imidazolyl)-3-N-piperidino-2-propanol), a base with pKa = 8.9): the ER increased from 1.4 to 2.1 as pHe increased from 5.6 to 8.4. However, with the weaker bases, Ro 03-8800 and nimorazole (morpholino derivatives with pKa = 6.3 and 5.2 respectively) the e.r. remained constant over a wide pHe range. Nitroimidazoles substituted with acidic functions gave decreasing sensitization with increasing pHe. For azomycin (pKa = 7.2) at 1 mmol dm-3 the e.r. decreased from 1.9 at pHe 4 to 1.0 at pHe 9. The effect of the proton conductor carbonyl cyanide-3-chlorophenylhydrazone (CCCP, 10 mumol dm-3) on radiosensitization by Ro 03-8799 (0.1 mmol dm-3) and misonidazole (1.0 mmol dm-3) was also studied. At pHe 6.67 the e.r. for Ro 03-8799 was increased from 1.36 to 1.76 by the presence of CCCP, whereas at pHe 7.33 the e.r. was unchanged. In contrast the e.r. for misonidazole was unchanged at pHe 6.65 and 7.33. These results are consistent with pH differentials across the cell membrane creating intracellular:extracellular concentrations gradients for radiosensitizers with acidic or basic functions.

Radiosensitizer-DNA interactions in relation to intracellular uptake

We have studied the intracellular uptake of a number of neutral, acidic, and basic radiosensitizers. For neutral sensitizers, we observed a correlation between the measured intracellular concentration and sensitization, but for bases, a large change in average intracellular concentration results in only a small change in sensitization. In addition, by modifying the intralysosomal pH, we have altered the measured average intracellular concentration of the weak base pimonidazole by a factor of two, although this had no detectable effect upon sensitization. Using spin filtration of solutions of sensitizers with naked calf thymus DNA or chromatin we have assessed the affinity of DNA for sensitizers with different prototropic and lipophilic properties. We have also shown that this anomalous behavior of the basic sensitizers could be partly explained on the basis of intracellular localization adjacent to the DNA due to ionic interactions. Thus, intracellular localization needs to be considered when interpreting average intracellular uptake data.

A comparison of colorimetric and clonogenic assays for hypoxic-specific toxins with hamster and human cells

The hypoxic cytotoxicities of misonidazole and pimonidazole (Ro 03-8799) towards the human tumor cell lines HT-1080 and LoVo have been compared with those seen with Chinese hamster V79-379A cells. Survival was assayed using two colorimetric assays, either a tetrazolium salt (MTT) or methylene blue, and by conventional colony scoring. The drugs were more cytotoxic towards HT-1080 and LoVo cells than V79 cells. The times taken for 10 mmol dm-3 misonidazole to reduce survival to 0.1 surviving fraction (SF) using colony formation as the end point were 2.6 hr for HT-1080, 2.4 hr for LoVo, and 3.5 hr for V79; using the MTT assay these times were 3.5 hr, 2.1 hr, and 2.9 hr, respectively. The times for 2 mmol dm-3 pimonidazole to reduce survival to 0.1 SF using colony formation as the end point were 2.0 hr for HT-1080, 1.7 hr for LoVo, and 3.7 hr for V79; using the MTT assay these times were 2.5 hr, 1.4 hr, and 2.5 hr, respectively.

Reduction of nitroimidazoles in model chemical and biological systems

Some chemical and biological properties of intermediates obtained during reduction of nitroimidazoles are discussed. These include: rate data for the decay of the nitro radical-anion, stoichiometry and absorption spectra for reduction via the radical-anion or using dithionite, stoichiometry with other reducing agents, and rate of reduction by xanthine/xanthine oxidase. Increased radiosensitization by misonidazole is seen upon prolonged pre-irradiation incubation using E. coli, enabling demonstration that a freely-diffusable metabolite is responsible for this effect. Preliminary experiments designed to extend studies of the radiobiological properties of extracellularly-added metabolites to mammalian cells and the use of liver perfusion to generate metabolites are described.

A Comparison of the Intracellular Uptake and Radiosensitization Efficiency in Different Media of Uncharged 2-nitroimidazoles of Varying Lipophilicity

The effect of varying octanol: water partition coefficients, P, (range 0.026-260) on the uptake of uncharged 2-nitroimidazoles into Chinese hamster V79 379A cells has been studied. Average intracellular concentrations were measured by high performance liquid chromatography after centrifuging cells through oil or an aqueous medium. The ratio of intracellular concentration of radiosensitizer to extracellular concentration (Ci/Ce) for misonidazole (P = 0.43) was 0.85 for the oil method and 0.68 for the aqueous method. For values of P less than about 0.05 uptake was initially very slow and Ci was always less than Ce. When P greater than or equal to 0.1 uptake was rapid and then remained unchanged for times up to 3 h; for P greater than or equal to 10, Ci/Ce increased rapidly as P increased. Ro 31-1405 (P = 260) concentrated by a factor of 7 inside the cell. Although uptake was identical for cells suspended in full growth medium and PBS, radiosensitization was greater for cells in PBS: 1 mmol dm-3 misonidazole produced an enhancement ratio of 1.6 in full growth medium and 1.9 in PBS. This increase in radiosensitization could not be accounted for by protein binding. However, measurements on cellular non-protein sulphydryl (NPSH) demonstrated the levels to be reduced to about 60 per cent for cells in PBS. Similar reductions in NPSH levels have previously been shown not to increase the radiosensitivity of control cells but to increase greatly the effectiveness of nitroimidazole radiosensitizers.

Rapid-mixing Studies of Radiosensitivity with Thiol-depleted Mammalian Cells

A moderate reduction in the non-protein thiol content of V79 379A Chinese hamster cells, obtained by pretreatment with buthionine sulphoximine (BSO), diethyl maleate (DEM) or N-ethyl maleimide (NEM), increase both the absolute radiosensitivity of the cells in hypoxia and the radiosensitizing effect of adding oxygen 7 ms after irradiation. Combined pretreatment of cells with BSO and NEM removes most of the non-protein thiol and some of the protein thiol; such treatment further increases the radiosensitivity of hypoxic cells but there is no further effect of adding oxygen 7 ms after irradiation. Addition of 2-mercaptoethanol to cells 7 ms after irradiation gives protection factors that increase with increasing severity of thiol depletion. Substantial radioprotection can still be observed when 2-mercaptoethanol is added 70 ms after irradiation of cells pretreated with BSO and NEM; there is no effect of adding 2-mercaptoethanol to such cells 50s after irradiation. These observations support the repair-fixation model of radiation damage and suggest that, in addition to the established role of non-protein thiol in chemical repair of radiation damage, other endogenous reducing agents such as protein thiol may be important in determining cellular radiosensitivity. A relatively long-lived thiol-modifiable component of radiation damage has been observed within hypoxic thiol-depleted cells.