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Biomedical subjects

M E Watts

Publications and source records attributed to M E Watts.

At least 19 recordsLinked to original sources
PubMed1992

Flavone acetic acid as a modifier of endothelial cell function.

Flavone acetic acid (FAA) causes significant regression of larger established tumors in murine systems in vivo, but is only slightly toxic in vitro. This in vivo effect is thought to be indirect, or immunological, rather than a direct cytotoxic effect on tumor cells. Using the WHFIB fibrosarcoma, which grows both in vivo and in vitro, and the murine endothelial cell line B10, we have studied the effect of FAA on the survival of tumor and endothelial cells in vitro. The times taken for 1 mg ml-1 FAA to reduce survival to 0.1 surviving fraction were 63 hr for B10 and greater than 85 hr for WHFIB in vitro. WHFIB tumors in vivo were more sensitive than tumor cells in vitro, a single dose of 150 mg kg-1 FAA inducing a tumor growth delay of 10 days at treatment size + 2 mm. As FAA is more toxic to tumor-bearing animals than to those which are non-tumor bearing the effect of tumor conditioned medium on the cytotoxicity of FAA toward B10 cells was studied; no enhanced effect was seen. As FAA is only weakly cytotoxic in vitro to endothelial cells, and even less so to tumor cells, sublethal effects of FAA on endothelial cell function in vitro were studied. The permeability of monolayers of human unbilical vein endothelial cells (HUVEC) in vitro is transiently increased by FAA. Also, procoagulant activity of HUVEC is induced by FAA and this activity is further enhanced in the presence of a factor isolated from Meth-A tumor cells.

Animals↗
PubMed1992

Flavone acetic acid induced changes in human endothelial permeability: potentiation by tumour-conditioned medium.

Flavone acetic acid (FAA) causes significant regression of larger established tumours in murine in vivo systems. This in vivo effect of FAA has been shown to include a vascular component. In an effort to elucidate the mechanism of action of FAA, we have studied the effects of FAA on the permeability of human endothelium in vitro. Monolayers of human umbilical vein endothelial cells (HUVEC) grown on polycarbonate filters were incubated in 1 mg/ml FAA for 120 min at 37 degrees C. During the first 60 min, there was a 6-8-fold increase in permeability; this was followed by a return to control levels even in the continued presence of FAA. In contrast, in the presence of tumour conditioned medium, FAA caused a rapid 6-fold increase in permeability which did not subsequently return to control levels. The permeability changes which occurred under the latter conditions were accompanied by a rapid contraction of the cytoskeleton. The permeability of monolayers of human melanoma cells was unaffected by FAA.

Antineoplastic Agents↗
PubMed1990

Radiosensitization by misonidazole, pimonidazole and azomycin and intracellular uptake in human tumour cell lines.

The radiosensitization of two human tumour in vitro cell lines, HT-1080 and LoVo, has been compared with that of the Chinese hamster cell line V79-379A. Although the two human tumour cell lines were more radiosensitive than the V79 cell line sensitizer, enhancement ratios for misonidazole, pimonidazole and azomycin were similar (relative to extracellular concentration) for all three cell lines. Average intracellular concentrations of radiosensitizer were measured by high-performance liquid chromatography. In all three cell lines the uptake of misonidazole and azomycin was extremely rapid whereas that of pimonidazole was initially much slower before reaching a plateau. The ratios of intracellular concentration of radiosensitizer to extracellular concentration (Ci to Ce) for misonidazole were 0.8 (HT-1080) and 0.7 (LoVo and V79); for azomycin 0.9 (HT-1080 and LoVo) and 0.8 (V79). In contrast Ci/Ce for pimonidazole varied with cell line, the values being 1.8 (LoVo), 2.6 (HT-1080) and 3.3 (V79). Intracellular amounts of non-protein sulphydryl (NPSH) varied between cell lines by about a factor of three. However, when the average cell volume was taken into consideration the concentrations of NPSH were very similar, being 4.2 (HT-1080), 5.6 (LoVo) and 5.7 (V79) mmol dm-3. NPSH levels expressed as nmol per mg protein were also similar.

Animals↗
PubMed1989

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.

Animals↗
PubMed1989

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.

Animals↗
PubMed1987

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.

Animals↗
PubMed1987

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.

Animals↗
PubMed1986

Glutathione-reactive nitro compounds as radiosensitizers: mechanistic and therapeutic implications.

The "extra" radiosensitization seen with GSH-reactive nitro compounds is too large to be accounted for by GSH-depletion acting independently--there must be competition. The GS-conjugate leaks out of cells slowly and is trapped at high concentrations. Its properties, such as concentration trapped and reduction potential, must be considered. Limited therapeutic exploitation of the glutathione conjugate trapping and concomitant GSH depletion may be possible if intratumor injection is permitted.

Animals↗
PubMed1986

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.

Animals↗
PubMed1985

Cellular uptake of misonidazole and analogues with acidic or basic functions.

Average intracellular concentrations of five radiosensitizers in hamster fibroblast-like V79-379A cells in vitro were measured by high performance liquid chromatography, varying the extracellular pH (pHe) and estimating the apparent intracellular pH from the distribution of 5,5-dimethyloxazolidine-2,4-dione. The intracellular: extracellular concentration ratio for the 2-nitroimidazole, misonidazole was constant at about 0.7 for pHe = 6.6-7.6, whereas the weak base, Ro 03-8799 (1-(2-nitro-1-imidazolyl)-3-N-piperidino-2-propanol) was concentrated intracellularly at pHe = 7.3-7.4 by a factor of 3.3, the factor increasing from about 0.8 at pHe = 6.0, to 7.5 at pHe = 7.85. The weak acid, azomycin (2-nitroimidazole) showed approximately constant uptake (factor 1.1) between pHe = 6.0-7.0, decreasing to 0.8 at pHe = 7.3 and 0.4 at pHe = 7.8. Measurements of intracellular uptake of Ro 31-0052 (the more hydrophilic and less basic 3'-hydroxypiperidino analogue of Ro 03-8799) and of Ro 31-0258 (3-(2-nitro-1-imidazolyl)propionic acid, a stronger acid than azomycin) were made for comparison. The results were compared with theoretical calculations of pH-induced concentration gradients; the time dependence of the uptake of the bases is not at present clearly understood. These measurements of uptake are broadly consistent with the distribution of misonidazole and Ro 03-8799 in human and animal tissues and provide a useful insight into the likely intracellular concentrations in the clinical use of Ro 03-8799 or other basic radiosensitizers. The measurements also resolve the apparent discrepancy in radiosensitizer efficiency for weak bases in vitro and in vivo which has been previously noted.

Animals↗
PubMed1985

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.

Animals↗
PubMed1979

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.

Animals↗
PubMed1979

Structure-activity relationships in the development of hypoxic cell radiosensitizers. II. Cytotoxicity and therapeutic ratio.

This paper describes measurements of the aerobic cytotoxicity of 42 nitroaromatic and nitroheterocyclic compounds towards Chinese Hamster cells in vitro. The results of acute and chronic exposure were quantified, and the concentration C required to achieve a standard response estimated. Fitting the data to an equation of the form - log C = b0 + b1E, where E is the one-electron reduction potential, explained 47 and 71 per cent of the variance in the acute and chronic aerobic cytotoxicity respectively. The addition of further terms to the equation, quantifying partition properties, was not statistically significant. The coefficient b1 was similar for both acute and chronic exposure; the dependence of both cytotoxicity and radiosensitization efficiency on reduction potential was also similar. A therapeutic ratio derived from these in vitro measurements showed no dependence on redox or partition properties. The insensitivity of cytotoxicity and radiosensitization properties to variations in molecular structure, other than those which influence redox behaviour, offers exceptional flexibility in developing compounds of improved therapeutic ratio.

Animals↗