Hypoxic Toxicity Research Articles

Decreased hypoxic toxicity and binding of misonidazole by low glucose concentration

The modulation of the hypoxic toxicity and binding of Misonidazole (MISO) by glucose and lactate was studied by exposing exponential EMT6/Ro cells to 5 mM MISO under hypoxic conditions. The concentrations of glucose used were 0.015 mM and 5 mM, and the concentrations of lactate were 0, 3 and 10 mM. There was no significant hypoxic toxicity due to MISO in the absence of glucose. However, with 5 mM glucose, after a latent period of 0.5 hours, there was a rapid decrease in cell survival to less than 0.1% at 2.5 hours incubation in 5 mM MISO. The binding of MISO was also increased by glucose. The amount of MISO bound to cells in 0.015 mM glucose leveled off at 2 nmoles MISO/7 × 105 cells at 1 hour, whereas the binding in 5 mM glucose continued to increase to more than 5 nmoles MISO/7 × 105 cells after 3 hours incubation. There was no detectable effect of lactate on the binding of MISO to the cells either in 0.015 mM or 5 mM glucose. The high affinity of this binding was indicated by the lack of exchange of radioactive MISO with non-radioactive MISO even after 2 hours of incubation. These data showed that glucose concentrations could modify the toxicity and binding of MISO to hypoxic cells.

Studies on the toxicity of RSU-1069

RSU-1069 combines an aziridine function with a 2-nitroimidazole and has been reported to exhibit extraordinary radiosensitization both in vitro and in vivo. Such sensitization appears to be at variance with the electron affinity of the compound. In addition, recent experiments suggest that the compound is highly toxic to hypoxic tumor cells in vivo. On the assumption that the observed radiosensitizing ability may be a manifestation of toxicity and because of the high in vivo toxicity, we have investigated aerobic and hypoxic toxicity, both in wild type CHO cells and in mutants sensitive to a variety of DNA damaging agents. With wild type cells under aerobic conditions, the compound is approximately 50 times as toxic as misonidazole and under hypoxic conditions, approximately 250 times as toxic. The ratio of hypoxic to aerobic toxicity is approximately 80 times. Under aerobic conditions, repair-deficient mutants are 10 times as sensitive to RSU-1069 as wild type cells and approximately 100 times as sensitive under hypoxic conditions. The ratio of hypoxic to aerobic toxicity for the mutant cells is approximately 900. Based on these observations, we suggest that under aerobic conditions the aziridine function is primarily responsible for toxicity, whereas, under hypoxic conditions, the aziridine moiety combined with a reduced 2-nitroimidazole moiety produces a bifunctional agent.

The differential cytotoxicity of RSU 1069: Cell survival studies indicating interaction with DNA as a possible mode of action

The hypoxic cell radiosensitizer RSU 1069 (1-(2-nitro-1-imidazolyl)-3-(1-aziridinyl)-2-propanol) shows, on a concentration basis, a 100-fold greater toxicity towards hypoxic relative to aerobic cells. This toxicity is substantially greater than that of misonidazole, a compound of similar electron affinity. Reductive processes are important for hypoxic toxicity; this is demonstrated by the fact that misonidazole, in excess, can protect against the hypoxic but not aerobic toxicity of RSU 1069. The importance of the interaction of RSU 1069 with DNA, suggested initially by molecular studies, is supported by the fact that cells containing 5-bromodeoxyuridine (5-BUdR) incorporated into their DNA show greater sensitivity towards the lethal effects of RSU 1069 both in air and nitrogen, compared to cells not treated with 5-BUdR. Experiments with RSU 1069 and 3-aminobenzamide (3-AB) show the latter compound to potentiate aerobic toxicity, consistent with monofunctional alkylation by RSU 1069. In contrast, 3-AB has no effect on the hypoxic cytotoxicity of RSU 1069, which would be predicted if RSU 1069 is functioning as a bifunctional agent under these conditions. It is our contention that in air, RSU 1069 functions as a typical monofunctional alkylating agent, presumably due to the presence of the aziridine group whereas, in hypoxia, reduction of the nitro group provides an additional alkylating species, converting the compound into a bifunctional agent.

Radiosensitization, Pharmacokinetics, and Toxicity of a 2-Nitroimidazole Nucleoside (RA-263)

A 2-nitroimidazole nucleoside, 1-(2',3'-dideoxy-alpha-D-erythro-hex-2'-enopyranosyl)-2-nitroimida zole (RA-263), has been investigated for its radiosensitization, pharmacokinetics, and toxicity properties. The in vitro radiosensitization tests against hypoxic Chinese hamster (V-79) cells demonstrated that RA-263 was a more potent radiosensitizer than misonidazole and at 2 mM concentration approached the oxic curve. Significant in vitro radiosensitization activity was also observed in EMT6 mammary tumor cells. The in vitro cytotoxicity data suggested that RA-263 is considerably more toxic to hypoxic cells than misonidazole. The increased cytotoxicity may be related to its higher depletion of nonprotein thiols (NPSH) than misonidazole. The combined effects of radiosensitization and hypoxic cell toxicity were measured by preincubation of the V-79 cells for 4 h under hypoxic conditions before irradiation. The results demonstrated a synergistic response by causing a significant decrease in the extrapolation number with loss of shoulder of the radiation survival curves. The in vivo radiosensitization experiments conducted by the in vivo-in vitro cloning assay with the EMT6 mammary tumor indicate that RA-263 is an effective sensitizer. Pharmacokinetic data suggested that RA-263 was eliminated from plasma by a rapid alpha phase and a slower beta phase with T 1/2 of 36 and 72 min, respectively. The concentration in the brain was approximately one-sixth of tumor concentration, suggesting that RA-263 is excluded from the CNS. Moreover, RA-263 was two times less toxic than misonidazole on equimolar basis by acute LD50 tests. This agent was also significantly less mutagenic than misonidazole in a strain of Escherichia coli.

Effects of glutathione depletion using buthionine sulphoximine on the cytotoxicity of nitroaromatic compounds in mammalian cells in vitro

The inhibitor of glutathione biosynthesis, buthionine sulphoximine (BSO) has been used to deplete endogenous thiols in mammalian cells in vitro. The effect of such depletion on the toxicity of nitroaromatic compounds has been investigated. Substantial enhancement of both aerobic and hypoxic toxicity of the 2-nitroimidazole, misonidazole is observed in thiol-depleted cells; the hypoxic toxicities of metronidazole, nitrofurantoin and nimorazole are also increased by thiol depletion. These data of significance for the potential combined use of BSO with nitroaromatic radiosensitizers to increase their radiosensitizing efficiency in radiotherapy, and as a potential method for enhancing the efficiency of anti-protozoal nitroaromatic drugs.

Combined Effects of Misonidazole Radiosensitization and Hypoxic Cell Toxicity in Mammalian Cells

It has been established that the hypoxic cell radiosensitizer misonidazole (Ro-07-0582) is also a toxic agent to hyposic cells in the absence of radiation. In this study, the combined effects of radiation sensitization and toxicity were examined in hypoxic CHO cells. Complete radiation survival curves were measured for cells in the presence of 15 mM misonidazole prior to or following incubation with the drug for various times in hypoxia at 37/sup 0/C. The latter case is the condition for the preincubation effect observed previously for misonidazole. The cells were irradiated as a monolayer in stoppered glass flasks with 280-kVp x rays; survival was assayed by colony formation. The survival data obtained in these experiments were analyzed by comparing the experimentally observed survival curves with those theoretically predicted based on the concepts of independent and additive interaction of the two effects of hypoxic cell toxicity and radiation sensitization. For incubation times of 1 to 3 hr, for which the cytotoxic effect is moderate, the combined response appears to be of an additive rather than an independent nature. The data in fact suggest a somewhat greater than additive response, and also a slight change in the slope as well as the shouldermore » of the survival curve, when compared with the predicted curves. The results are discussed with regard to both in vitro and in vivo studies.« less

Toxicity of Nitro Compounds Toward Hypoxic Mammalian Cells In Vitro: Dependence on Reduction Potential

Fifteen nitroaromatic and nitroheterocyclic compounds that can act as "radiosensitizers" were tested for their cytotoxicity toward hypoxic Chinese hamster V79 cells in vitro. The cytotoxicity increased markedly as the electron affinity, measured as a one-electron reduction potential, increased. Non-nitro-containing compounds of similar electron affinities (such as quinones) that also act as radiosensitizers did not exhibit this specific toxicity toward hypoxic cells. The implications of the presence of the nitro group as a prerequisite for the hypoxic cell toxicity were discussed, and the mechanism of the cytotoxicity was compared with that of hypoxic cell radiosensitization.