Peggy L. Olive

Evaluation of Nitroheterocyclic Radiosensitizers Using Spheroids

Publisher Summary This chapter focuses on the studies regarding in vitro testing of sensitizers largely using multicell spheroids, and presents the optimistic and pessimistic views as to the future of hypoxic cell sensitizers in the clinical setting. Some nitroheterocycles show promise for enhancing the response of selected tumors to radiation. Use of the drugs in humans is predicated on the belief that the presence of hypoxic cells within some tumors limits the success of conventional radiotherapy; however, in many tumors, conclusive evidence supporting this prejudice is lacking. The development of new drugs may be self-limiting as nitroheterocycles that are more effective as sensitizers are generally more reactive at a biochemical level. The effects of the nitroheterocycles can be observed under controlled and quantifiable conditions, and the roles of growth inhibition, cytotoxicity, and radiosensitization can be separated. The correlations between chemical and biological activities of nitroheterocycles suggest that at least with compounds that are effective as sensitizers only through the property of nitro-group reactivity, sensitization and toxicity are likely to be inseparable. As with most predictions, this may or may not be accurate. Combinations of radiation and hypoxic cell sensitizers with other modalities such as hyperthermia and selected chemotherapeutic agents such as alkylating agents, and even radioprotectors may improve the therapeutic ratio.

Evidence suggesting that the mechanism for aerobic and hypoxic cytotoxicity of nitroheterocycles is the same

The fluorescence of three nitroheterocycles (AF-2, trans-5-amino-3-(2-(5-nitro-2furyl) vinyl)-1,2,4-oxadiazole and 4-NQO) was used to quantitate cellular uptake and binding using a fluorescence-activated cell sorter. Mean cellular fluorescence, proportional to the amount of bound drug, allowed accurate prediction of the amount of cell killing. At equitoxic concentrations, the same amount of drug was bound under either aerobic or hypoxic conditions. In addition, 5 mM glutathione was equally effective at inhibiting aerobic and hypoxic cell killing by AF-2. These results suggest that the mechanism for cell killing may be similar under aerobic and hypoxic conditions, and the presence of oxygen may influence the rate of drug uptake rather than the nature of the toxic species. The nitro anion radical, formed in the presence and absence of oxygen, seems a likely candidate for the toxic species.

Correlation between the half-wave reduction potentials of nitroheterocycles and their mutagenicity in Chinese hamster V79 spheroids.

Toxicity and DNA damage by nitroheterocycles has previously been correlated with their redox potentials. Resistance to 6-thioguanine was measured using Chinese hamster V79 cells grown in suspension culture as three-dimensional cell clusters of spheroids. Since diffusion gradients of oxygen and other nutrients are largely responsible for the growth properties of spheroids, cells grown as spheroids might better simulate cells exposed to mutagens in vivo. The log of the concentration inducing 10 mutants/plate or 1.6 X 10(5) clonogenic cells from spheroids (equivalent to about 300 rad), was correlated with the half-wave reduction potential of a series of nitroheterocycles. FANFT and 4NQO were more mutagenic than predicted on the basis of redox potential.

Cellular metabolism of fluorescent nitroheterocycles

Since nitroheterocycles are preferentially metabolized and bound in hypoxic cells, we have examined more than 2 dozen nitroheterocycles as potential fluorescent probes for hypoxia. Using flow cytometry, several patterns of cellular fluorescence (CF) have been observed; for most nitroheterocycles, CF was several fold higher for anoxic than for aerobic cells (which was not predicted based on comparison of the fluorescence spectra of parent drug and reduced products). CF gradually increased when cells were exposed to 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) or to 4-nitroquinoline-1-oxide (4-NQO), and cells remained fluorescent when the drug was washed off. In contrast, cells exposed to trans-5-amino-3-[5-nitro-2-furyl)vinyl-1,2,4-oxadiazole (NFVO) lost fluorescence with a half-time of 60 minutes. Cells exposed to nitrofurazone (NF-7) reached maximum fluorescence within 30 minutes and then lost fluorescence, even in the presence of the drug. Finally, cells exposed to 3-nitropyrene (NP-3) were initially more fluorescent when incubated under aerobic conditions than anoxic conditions; however, after 2 hours in the presence of NP-3, anoxic cells continued to increase in fluorescence while aerobic cells lost fluorescence. Differences in the patterns of cellular accumulation of fluorescent nitroheterocycles were accompanied by differences in the toxicity and metabolism of these drugs. Therefore, chemical studies alone do not allow us to predict the potential of a compound as a hypoxic probe; studies at the cellular level are also essential.

Influence of cell crowding on toxicity of nitroheterocycles.

Cell density (no. of cells per unit area or volume) during drug treatment may play a role of considerable importance in the interpretation of drug toxicity experiments performed in vitro. Chinese hamster V-79 and mouse L-929 cells exposed to nitroheterocycles under aerobic conditions are considerably more sensitive to the cytotoxic effects of these drugs when incubated at low cell density (10(2) cells/cm2 or 10(4) cells/ml) than at higher cell density (10(4) cells/cm2 or 10(6) cells/ml). This may be related to diffusion limitations when cells are in contact and to the ability of dense cell suspensions to inactivate drugs. In contrast, under anaerobic conditions, more toxicity is observed at high cell density than at low cell density, perhaps due to local effects of toxic metabolites. Toxicity appears to correlate with intracellular drug levels under both aerobic and hypoxic conditions.

Fluorescent probes for cellular hypoxia: Lack of transfer of fluorescence between cells in vitro

Fluorescent nitroheterocycles may be useful as probes for cellular hypoxia. Reductive metabolism of AF-2 (cis 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide) and NFVO (trans-5-amino-3((5-nitro-2-furyl)vinyl)1,2,4-oxadiazole) results in intracellular accumulation of fluorescent molecules; the mean cellular fluorescence has previously been shown to be related to the cellular oxygen content during incubation in vitro. However, factors in addition to oxygen and nitroreductive activity may also affect cellular accumulation of these drugs. The stability of cellular fluorescence and possible diffusion of metabolites were examined by flow cytometric analysis of mouse and hamster fibroblasts exposed to NFVO and AF-2. Incubation of cells with 14C-AF-2 allowed calibration of the flow cytometer for AF-2 fluorescence; 5 X 10(8) molecules/cell resulted in double the spontaneous cellular fluorescence. Cellular fluorescence was stable for days after exposure to AF-2, and no evidence of transfer between exposed and unexposed cells was observed. For concentrations resulting in less than 5 X 10(9) AF-2 adducts/cell, all of the metabolites could be accounted for intracellularly. Therefore, it is unlikely that transfer of reduced nitroheterocycles occurs between cells.

Influence of cellular environment on toxicity of nitroheterocycles.

The preferential sensitivity of hypoxic cells to nitroheteroxycles is thought to result from the actions of toxic intermediates of drug reduction produced under hypoxic conditions. However, a lack of oxygen also alters the biochemical state of the cell and may indirectly enhance the sensitivity, of hypoxic cells to these drugs. This hypothesis was tested by conditioning mouse L-929 cells in oxygen-free buffer, then exposing the cells to nitrofurazone under both aerobic and anaerobic conditions. After conditioning, the rate of cell inactivation by nitrofurazone was equal in air or nitrogen-equilibrated buffer. Pretreatment of cells in 1 muM rotenone or 0.5 mM 2,4-dinitrophenol for one hour under aerobic conditions increased the sensitivity of the cells to nitrofurazone under aerobic conditions. Similar rates of cell killing were obtained when mouse L-cells were heated in buffer for 30 min at 43 degrees before incubation with nitrofurazone in either air or nitrogen. Also, incubation of cells with nitrofurazone in the presence of 0.1% glucose, or at a cell density less than 10(5) cells/ml significantly enhanced cell killing, especially under aerobic conditions. Thus, the intracellular state of the cell, manipulated by altering the cellular environment, influenced the cellular sensitivity to nitrofurazone. Similar results were not, however, obtained with the nitroimidazoles, dimetronidazole and misonidazole; pretreatment for 2 h in buffer under anaerobic conditions did not increase the sensitivity of L cells to subsequent drug treatment in air-equilibrated buffer.

Recovery of thioguanine-resistant cells from Chinese hamster V79 spheroids

Multicell spheroids may prove useful in evaluating the interactions of mutagens with cells exposed in a tissue-like environment. However, direct comparisons among populations of Chinese hamster V79 spheroids of different sizes or with monolayers are complicated by the observation that as spheroids enlarge, the fraction of mutant cells resistant to 6-thioguanine (TGr) gradually decreases from about 5 in 10(5) to less than 1 in 10(5). There appear to be at least 2 explanations for these observations. First, TGr cells grow less well as spheroids than do 6-thioguanine-sensitive (TGS) cells. Second, the clonal nature of spheroid growth means that small samples of spheroids are likely to contain fewer pre-existing TGr cells.