Myles Astor

Biochemistry of reduction of nitro heterocycles.

Misonidazole is a metabolically active drug. Its addition to cells causes an immediate alteration in cellular electron transfer pathways. Under aerobic conditions the metabolic alterations can result in futile cycling with electron transfer to oxygen and production of peroxide. Thiol levels are extremely important in protecting the cell against the peroxide formation and potentially hazardous conditions for hydroxyl radical production. Nevertheless such electron shunting out of cellular metabolism will result in alterations in pentose cycle, glycolysis and cellular capacity to reduce metabolites to essential intermediates needed in DNA metabolism (i.e. deoxyribonucleotides). Glutathione must be depleted to very low levels before toxic effects of misonidazole and other nitro compounds are manifested in cell death via peroxidative damage. Under hypoxic conditions misonidazole also diverts the pentose cycle via its own reduction; however, unlike the aerobic conditions, there are a number of reductive intermediates produced that react with non-protein thiols such as GSH as well as protein thiols. The reaction with protein thiols results in the inhibition of glycolysis and other as yet undetermined enzyme systems. The consequences of the hypoxic pretreatment of cells with nitro compounds are increased vulnerability to radiation and chemotherapeutic drugs such as L-PAM, cis-platinum and bleomycin. The role that altered enzyme activity has in the cellular response to misonidazole and chemotherapeutic agents remains to be determined. It is also clear that the GSH depleted state not only makes cells more vulnerable to oxidative stress but also to hypoxic intermediates produced by the reduction of misonidazole beyond the one electron stage. The relevancy of the present work to the proposed use of thiol depletion in vivo to enhance the radiation or chemotherapeutic response of tumor tissue lies with the following considerations. Apparently, spontaneous peroxidative damage to normal tissue such as liver can occur with GSH depletion to 10-20% of control and with other normal tissue when GSH reaches 50% of control. This situation can obviously become more critical if peroxide producing drugs are administered. The only advantage to such combined drug treatments would lie in the possibility that tumors vary in their catalase and peroxidase activity and consequently may be more vulnerable to oxidative stress (cf. review by Meister. Our tumor model, the A549 human lung carcinoma cell in vitro, appears to be an exception because it has catalase, peroxidase and a high content of GSH.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytotoxicity of Ro-07-0582; enhancement by hyperthermia and protection by cysteamine.

The selective cytotoxicity which Ro-07-0582 exhibits towards hypoxic cells is strongly temperature-dependent. This cytotoxicity is reduced by the radical scavenger cysteamine, suggesting that nitro radicals or nitroso intermediates are involved in cell killing by the drug. Chromosome aberrations are not induced by Ro-07-0582 even when the surviving fraction is reduced to 0-01.

Non-protein thiols and cellular response to drugs and radiation

Abstract Our results have enabled us to make a number of observations concerning the reactivity of nitrocompounds and non-nitrocompounds with glutathione (GSH) and cellular nonprotein thiols (NPSH), which may be as much as 90 % GSH. We have summarized our observations as follows with respect to the different types of reactions responsible for part or all of the cellular NPSH depletion by hypoxic cell radiosensitizing drugs. (A) Some nitrocompounds, such as 4-nitroimidazoles containing a 5-sulfonamide group, react spontaneously with GSH; (B) A number of hypoxic cell radiosensitizing drugs such as chlorodinitrobenzene (CDNB), dimethylfumarate (DMF) and diethylmaleate (DEM) are substrates for the enzyme glutathione-S-transferase and form covalent bonds with GSH; (C) NPSH may be oxidized by diamide; (D) form covalent bonds with N-ethylmaleimide; (E) or be converted by thiol-reactive drug intermediates formed under anaerobic conditions. The latter reaction occurs with misonidazole, Ro-05–9963, SR 2508 and SR 255:5; its mechanism is still unknown. It is obvious from the above that there are a variety of means by which radiosensitiizing drugs can alter cellular metabolism as reflected by changes in the NPSH. It remains to be determined whether a relationship exists between altered NPSH, metabolism and the radiosensitizing capacity of nitrocompounds when used alone or in combination with other drugs. Our studies strongly suggest that potential new sensitizers be routinely examined for their capacity to react spontaneously with GSH or to remove cellular NPSH under aerobic as well as anaerobic conditions. This is especially true for radiosensitizing drugs showing anomalous behavior, i.e., better sensitization than predicted by their one-electron reduction potentials. Such screening would pay dividends insofar as drugs that are too reactive could be excluded from further in vivo study.

Radiobiological Studies on a Series of Human Cell Lines of Varying Glutathione Content

The effect of decreased levels of intracellular glutathione (GSH) on the radiosensitivity of aerated and hypoxic cells was studied using human skin fibroblasts obtained from patients affected with the inborn error of metabolism, 5-oxoprolinuria. The oxygen enhancement ratios (OER) were determined for four cell lines obtained from a single family, SR and SUR (heterozygous parents) and GM3877 and GM3878 (affected homozygous siblings). Glutathione values ranged from 7.4 to 130% of control values. Only GM3877 with a GSH value 7.4% of control exhibited a reduced OER of 1.9 compared with a control value of 3. These results suggest that a reduction in OER is observed only when GSH levels reach extremely low values.

The enhanced sensitivity of mammalian cells to killing by X rays after prolonged exposure to several nitroimidazoles

Electron affinic compounds, such as misonidazole, preferentially sensitize hypoxic cells to killing by X rays, and are also preferentially cytotoxic to cells deficient in oxygen. Prolonged exposure of cells to misonidazole prior to irradiation results in an increased radiosensitization. This is expressed as the Extra Enhancement Ratio (EER), defined as the ratio of the doses delivered immediately after the addition of the sensitizer or after prolonged incubation, that produce a given biological effect. Chinese hamster V79 cells have been used to investigate this prolonged incubation effect for a variety of 2-nitroimidazoles including misonidazole, desmethylmisonidazole and SR-2508 and as well as two ortho-substituted-4-nitroimidazoles with a bromine or sulfonamide group substituted in the 5-position. A considerable variation was observed in the magnitude of the Extra Enhancement Ratio (EER) produced by pre-incubation with different compounds at concentrations that produce the same sensitizing effect. There is a good correlation between the EER and the measured rate at which the various sensitizers deplete cells of non-protein sulfhydryl compounds. There is also a good correlation between the EER and the fraction of cells killed by the pre-incubation period in the drug.

Response of cells of human origin, normal and malignant, to acute and low dose rate irradiation

Dose response curves were obtained for normal human fibroblasts and for several cell lines derived from human tumors, including melanomas and an osteosarcoma. Most of the tumor lines are similar in radiosensitivity to the normal fibroblasts, except for the melanoma lines, which are significantly more resistant. The two melanoma lines differ, one being much more radioresistant than the other. Potentially lethal damage repair (PLDR) has been studied in these cell lines as well. The extent of PLDR does not appear to correlate with radioresistance; for example, the most resistant melanoma line shows very little repair of PLD. In addition, the normal fibroblasts repair PLD at least as well as any of the tumor derived lines, which casts doubts on the wisdom of introducing into clinical practice inhibitors of PLD until a clear differential between normal tissues and tumors has been demonstrated in vivo. Low dose-rate studies with normal human fibroblasts indicate a smaller dose-rate effect than for most established cell lines of rodent origin. Indeed, in the human cells studied, the effect of sublethal damage repair is quantitatively similar to the repair of potentially lethal damage. Dose response curves for acute and protracted exposures have been obtained for cells derived from patients with cancer-prone syndromes including ataxia telangiectasia (AT) and Blooms syndrome. Both cell lines are much more radiosensitive than normal human fibroblasts; the AT cells show a dose-rate effect, while Blooms syndrome cells do not.

Factors involved in depletion of glutathione from A549 human lung carcinoma cells: Implications for radiotherapy☆

We have measured the rate of GSH resynthesis in plateau phase cultures of A549 human lung carcinoma cells subjected to a fresh medium change. Buthionine sulfoximine (BSO) blocks this resynthesis. Diethyl maleate (DEM) causes a decrease in accumulation of GSH. If DEM is added concurrently with BSO there is a rapid decline in GSH that is maximal in the presence of 0.5 mM DEM. GSH depletion rapidly occurs when BSO is added to log phase cultures which initially are higher in GSH content. Twenty-four hr treatment of A549 cells with BSO results in cells that are more radiosensitive in air and show a slight hypoxic radiation response. A 2 hr treatment with either 0.25 mM or 0.5 mM DEM results in some hypoxic sensitization and little increase in the aerobic radiation response. The 24 hr BSO + 2 hr DEM treatment sensitizes hypoxic cells to a greater degree than either agent alone but does not increase the aerobic response more than is seen with BSO alone. Cells treated simultaneously with BSO + DEM show little increase in the hypoxic radiation response, compared to DEM alone, but are more sensitive under aerobic conditions. Decreased cell survival for aerobically irradiated log phase A549 cells occurs within minutes after addition of a mixture of BSO + DEM. The decreased cell survival following aerobic irradiation, after prolonged treatment with BSO or acute exposure to BSO + DEM, may be in part due to inhibition of glutathione peroxidases. For example, glutathione-S-transferase, known to have glutathione peroxidase activity (non-selenium), is nearly completely inhibited by the BSO treatments. In addition, cellular capacity to react with peroxide (glutathione peroxidase, selenium containing) was also inhibited. We suggest that the enhanced aerobic radiation response is related to an inability of GSH depleted cells to inactivate either peroxy radicals or hydroperoxides that may be produced during irradiation of BSO treated cells. Furthermore, enhancement of the aerobic radiation response may be useful in vivo if normal tissue responses are not also increased.

Newly synthesized hypoxia-mediated drugs as radiosensitizers and cytotoxic agents

Chinese hamster cells in culture were used to compare the radiosensitizing efficiency and cytotoxicity of misonidazole with several 2, 4 and 5 substituted nitroimidazoles. The two substituted compounds (SR 2508 and SR 2555) are similar to misonidazole in radiosensitizing effectiveness, but are significantly less toxic to hypoxic cells. This reduced cytotoxicity may result from either slower drug penetration or slower removal of non-protein sulfhydryl compounds (NPSH). The compound MJL-1-191-VII (a 4-nitroimidazole) is a much more effective radiosensitizer than would be predicted from its electron affinity. It appears to sensitize by two mechanisms, the first resulting from its electron affinity and the second as consequence of its rapid removal of endogeneous cellular NPSH; which are naturally occurring radioprotective substances.

Mechanism of misonidazole-linked cytotoxicity and altered radiation response: role of cellular thiols

The effectiveness of misonidazole as a hypoxic radiosensitizer of mammalian cells is increased by prolonged exposure of hypoxic cells to the drug. Increased radiosensitization occurs even after the cells are washed free of excess Hall and Biaglow, 1977; Whitmore et al., 1978; Varnes et al., 1981). Hall et al. (1977) suggested and Biaglow et al. 1978) found that drug intermediates, produced during hypoxic incubation of cells with misonidazole, might react with endogenous non-protein thiols (NPSH). These thiols function to protect the cell against deleterious intermediates that could otherwise attack and modify critical macromolecules such as DNA, RNA and protein. This hypothesis is in agreement with the cytotoxic mechanisms proposed earlier for many different nitro compounds (Biaglow, 1981). With respect to the effects of preincubation with misonidazole on the radiation response, alterations in both NPSH and protein thiols (Varnes et al., 1981) would be expected to alter the shoulder of the radiation respo...

Radiobiological studies with therapeutic neutron beams generated by p+ → °Be or d+ → +°Be

Abstract Mammalian cells cultured in vitro were used to study the radiobiological characteristics of neutron beams generated by 43 MeV protons on beryllium or 25 MeV deutrons on beryllium. For an unfiltered beam of neutrons generated by 43 MeV p+→Be the relative biological effectiveness was found to be 8–12% higher at a depth of 2 cm than at a depth of 12 cm due to the presence of a large component of low-energy neutrons. The addition of a hydrogenous filter 4 cm thick preferentially removed the low-energy neutrons from the beam and, as a result, the neutron RBE was independent of depth. There was no significant difference in the oxygen enhancement ratio between the filtered neutrons produced by 43 Mev p+Be and neutrons produced by 25 MeV d+Be; for both beams the OER value was about 1.6.