Repair of DNA breaks after irradiation in misonidazole or oxygen.

Biphasic kinetics (half-times 7' and 250') adequately describe the aerobic repair of DNA breaks after irradiation of mammalian cells. The proportions repaired by the two components are affected by conditions prior to, during and after irradiation. DNA of cells irradiated in hypoxia have approximately twice as much of the damage which is repaired by the slow component as cells irradiated in air. If, instead of oxygen, misonidazole is present during hypoxic radiation, the repair resembles the repair of oxic damage more closely than repair of hypoxic damage. The biphasic nature of the repair curves is interpreted to be due to two classes of initial damage, proportions of which can be altered by sensitizers such as O2 and misonidazole.

Effect of BSO on the radiation response at low (0-4 Gy) doses.

Depletion of thiols by various agents has been shown to radiosensitize hypoxic cells. The agent BSO is used to specifically inhibit GSH synthesis, and the effect of this treatment on radiation response, particularly in the region of clinical interest, was studied using automated microscopic identification of cells for measurement of high survival levels. In both CHO and V79 cell lines, the enhancement rations were greater at low doses (80% S) than at high doses (2% S) in cells irradiated in hypoxia. GSH depletion also affected the aerobic response in both dose regions, with again higher ER's observed at low doses. These results support previous studies which suggest that GSH levels affect the shoulder portion of the survival curve. However, as with untreated cells, the OER's remain higher at high doses than at low doses, in BSO treated cells of both lines. These studies complement an earlier low-dose study on diamide by Skarsgard and co-workers, and were carried out to increase our understanding of the factors which affect radiation sensitivity in the range of doses used in fractionated regimens in the clinic.

Enhanced radiation-sensitivity by preincubation with nitroimidazoles: effect of glutathione depletion.

PURPOSE: The mechanism of enhanced radiosensitization by nitroheterocyclics after a preincubation period under hypoxic conditions was investigated. The hypothesis that this phenomenon was caused by glutathione depletion was tested. METHODS AND MATERIALS: The phenomenon of enhanced radiosensitization by nitroheterocyclics after a preincubation period under hypoxic conditions is potentially of importance therapeutically because essentially nonlethal preradiation exposures to the electron affinic drugs cause a much larger radiation sensitization than would otherwise be expected. We have investigated this interesting property of several 2-nitroimidazoles to determine its possible cause and to test various hypotheses about maximizing its possible therapeutic benefit. In view of many observations that thiols are depleted by incubation of cells with nitroimidazoles under hypoxic conditions, we have specifically investigated this aspect of the preincubation effect. Depletion of glutathione was either enhanced by an overnight incubation with buthionine sulfoximine or minimized by preincubation with a 2-nitroimidazole which is sterically inhibited from causing thiol depletion. RESULTS: When conditions were chosen which minimized variations in cellular glutathione content during the preincubation period, no preincubation effect was observed. At low, therapeutically relevant radiation doses, where 2-nitroimidazoles are less efficient sensitizers, the preincubation effect may be even more important, but thiol depletion still minimizes its impact in this region of the dose-response curve. CONCLUSION: These results suggest that the preincubation effect is caused by a "self-sensitization" involving the known enhancement of radiation sensitization by thiol depletion.

Low concentrations of nitroimidazoles: effective radiosensitizers at low doses.

PURPOSE: There have been various reports that nitroimidazole radiosensitizers are less effective modifiers of radiation response in the clinically relevant x-ray dose regions (0-4 Gy) than they are at doses used in classical in vitro experiments. Our studies at low concentrations of etanidazole led us to question this generalization, and our purpose was to further investigate low concentrations at low doses, using the microscopic location of cells to facilitate these experiments. The observations are compared with data on these drugs in the literature using other methods and systems. METHODS AND MATERIALS: Survival of V79 cells after irradiation in hypoxia +/- drug at various concentrations was assessed using the Cell Analyser (DMIPS) for low doses, in comparison with the standard clonogenic assay (higher doses). Enhancement ratios (ERs) were calculated at 80% and 2% survival, respectively. RESULTS: Etanidazole (SR-2508) consistently gave higher ERs at low doses (measured at 80% survival) than at high doses (survival 2%), when cells were exposed to drug concentrations below approximately 2 mM (e.g., at 1 mM, ER80% = 2.2, ER2% = 1.8 in CHO cells after 1 h preincubation at 37 degrees C). Preincubation of cells for 1 h or 15 min at 37 degrees C with etanidazole prior to irradiation increased the ERs at high and low doses but did not change the "cross-over" behavior (ER80% > ER2% at low concentrations, ER2% > ER80% above 2 mM, regardless of pretreatment at 37 degrees C or cell line, CHO and V79 cells), whereas the 5-nitroimidazole nimorazole consistently gave the same ERs whether determined at high or low radiation doses (e.g., at 1 mM, 1 h preincubation at 37 degrees C ER80% = ER2% = 1.3). This crossover behavior also occurred after irradiation/preincubation at 0 degrees C. The 2-nitroimidazole predecessor, misonidazole, shows the same cross-over behavior. CONCLUSION: Two nitroimidazoles at low concentrations appear to be as effective sensitizers (or better) at low doses (80% S); at high doses (2% S).

The interaction of trans-DDP [PtCl2(NH3)2] with low doses of radiation in mammalian cells.

Trans-DDP, a less toxic isomer of cisplatin, was examined for its radiosensitizing activity at high and low doses of ionizing radiation. Cells were exposed to the drug to produce low toxicity before exposure to X rays. A sensitive assay using the DMIPS Cell Analyzer was employed to measure cell survival response in low dose region (0-4 Gy) and conventional assay was used for high doses (4-25 Gy). The results show that trans-DDP is a much more effective radiosensitizer at low doses than at high doses, whether or not cytotoxicity was pronounced. This is in contrast to any other sensitizer studied to date, including oxygen, misonidazole, SR-2508 and Ro-03-8799, regardless of prior incubation and/or cytotoxicity.

Energy and misonidazole toxicity: the effects of 5-thio-D-glucose.

Cell inactivation and DNA damage (single-strand breaks) were used to study the effects of inhibitors of anaerobic glucose oxidation on the toxicity of misonidazole to hypoxic Chinese hamster cells. Citrate and 2-deoxyglucose produced no effects on the toxicity. 5-thio-D-glucose (5-TG) protected cells of the CH2B2 line to some extent (SSB decreased by about 30%). In the CHO lines used (wild, and ethylmethanesulfonate-sensitive mutants), 5-TG had varied effects. Non-protein sulfhydryl (NPSH) levels were measured in all lines. Cells with lower NPSH levels are more sensitive to misonidazole; these are the cells which are protected by 5-TG. Cell line variations must be considered when studying interactions between a drug and other forms of treatment as possible treatments of cancer.

A new ruthenium radiosensitizer: RuCl2(DMSO)2(4-nitroimidazole)2.

Earlier studies on Ru complexes, such as, cis-RuCl2(DMSO)4, as antineoplastic agents, have suggested a DNA binding mechanism similar to that of the clinically successful platinum complex, cis-diammine(dichloro)platinum(II), (cis-DDP). As part of our study on metal-radiosensitizer complexes, cis-RuCl2(DMSO)4 is used as a precursor for synthesis of Ru(II)-nitroimidazole complexes. These complexes were identified and characterized and their toxicity and radiosensitizing abilities were examined in vitro. In the series of Ru(II)-nitroimidazole complexes synthesized, RuCl2(DMSO)2(4-nitroimidazole)2, "Ru-(4-nitro)," was the most effective radiosensitizer. At 200 microM, Ru-(4-nitro) produced a sensitizer enhancement ratio (SER) of 1.6 in hypoxic Chinese hamster ovary (CHO) cells, but did not sensitize oxic CHO cells. Other Ru-nitroimidazole complexes gave SER values of 1.2-1.4 at 200 microM. These Ru(II)-nitroimidazole complexes also showed lower toxicity than the free nitroimidazoles alone at equimolar concentration. The enhanced radiosensitizing effect of Ru-(4-nitro) may be due to the metal's ability to target the sensitizer to DNA, or may be related to changes in reduction potential: from -685 mV for the free ligand to -355 mV and -615 mV for the complex. This complex did not deplete non-protein sulfhydryls (NPSH) at the time intervals and concentrations used. DNA binding was studied using inhibition of restriction enzyme activity on plasmid DNA.

Effects of fixation method on image cytometric measurement of DNA content and distribution in cells stained for fluorescence with propidium iodide.

We performed image cytometric measurements of DNA content and distribution on cycling human HCT-8 cells stained for fluorescence with propidium iodide (PI). Seven different fixation protocols were evaluated for stoichiometry of PI staining and for their ability to preserve in vivo chromatin structure. Bimodal integrated optical intensity (IOI) histograms were obtained with all fixation protocols. Increased accessibility of DNA to the dye was evident in increased values of the IOI at the GI peak. The fixatives studied, in order of increasing accessibility to DNA, were Regaud's Boehm-Sprenger, Carnoy's, air-drying, methanol, ethanol, and acetone/methanol. In general, the coefficient of variation of the IOI within the G1 peak was higher for fixatives where DNA is less accessible. Features describing the spatial distribution of stain exhibited dramatic changes for Boehm-Sprenger fixation, which were consistent with the observation that in vivo conformation of chromatin is best preserved with this method.

Modification of radiation response by metal complexes: a review with emphasis of nonplatinum studies.

There is a need to develop compounds which alter the effects of radiation, particularly in the hypoxic radioresistant cell, following the limited success to date of the electron-affinic nitroimidazoles. The chemistry of transition metals is briefly outlined to point out certain aspects which might be exploited in the design of radiosensitizers. The best known clinical example of a metal complex which enhances the effect of radiation in hypoxic cells is the successful antineoplastic cisplatin. Past studies on enhancement of radiation damage by complexes of metals other than platinum, mainly in bacterial spores and bacterial and mammalian cells, have been summarized according to the metal used. The many mechanisms by which metal complexes could interact with radiation are outlined, and examples are given where possible. This survey emphasizes the need for a systematic study of the effect of metal/ligand variation on radiosensitization with regard to mechanisms of action to assess the potential of these compounds as radiosensitizers. Metal complexes offer many advantages, both for the study of mechanisms by which radiation kills cells and for drug development.

The contribution of hydroxyl radical to radiosensitization: a study of DNA damage.

Using the radioprotector dimethylsulfoxide, DMSO, as a scavenger of hydroxyl radicals, the proportions of DNA damage caused by OH. were determined in mammalian cells irradiated in hypoxia with or without the radiosensitizers misonidazole and TAN or in air. Yields of both single-strand breaks (SSB) and base/sugar damage (MLS for Micrococcus luteus sensitive sites) were measured for each situation. Most of the damage enhanced by the sensitizers was found to be OH. dependent, for both MLS and SSB classes of damage: most breaks (greater than 80%) enhanced by oxygen and about two-thirds of the breaks enhanced by misonidazole (hypoxia) occur at OH.-damaged sites; most if not all base/sugar damage enhanced by the sensitizers misonidazole and TAN (in hypoxia) occurs only in the presence of OH., whereas in air, some (about one-quarter) of the enhanced MLS damage does not require OH.. The sensitizer enhancement ratios in the presence of scavenger and the degree of protection afforded by the scavenger determined for total (MLS + SSB) damage agree well with those derived from corresponding survival experiments.

Molecular, cellular, and genetic basis of radiosensitivity at low doses: a case of inducible repair?

Many proteins are induced by ionizing radiation, and genes are activated. We still do not know which, if any, are responsible for IRR, or what leads to the adaptive response seen at still lower doses. Are these the same responses? Are they related to apoptosis, repair of potentially lethal damage and other responses? Does the cell have a whole battery of responses, depending on the dose? I suspect this is the case. Can the responses be explained more simply, as effects on regulators of cell cycle or induction of fidelity, or is there induction of repair? Are there still other explanations for the apparent protection? The initial slope of the survival curve which was addressed earlier (1) must take on new meaning given the hyperradiosensitive portion. Similarly, we may have to change our thinking with respect to the LQ description of survival data. It is not surprising that this workshop, held at such an early stage primarily to address the phenomenon of increased radioresistance, produced more questions than answers. Single-strand breaks may trigger resistance, but additional lesions or classes of damage may be relevant. Some physicists expect the damage caused to be linear with dose; the biologists suggest that the response is nonlinear (e.g. saturation of an enzyme, induction of repair, cell cycle effects) and there is room for biochemistry which could also vary with dose (e.g. consumption of a protector or a sensitizer). Some biophysicists would argue that the observed structures in survival curves might be explained by change in the target cross section such as a large change in DNA conformation caused by a very low dose. There is some reluctance in the radiobiology community to accept that cells may respond to ionizing radiation by inducing or activating protective mechanisms, although the cell exhibits defensive responses to many other detrimental stimuli. If "the heart of the matter is in the shape of the survival curve" as suggested by Dr. Elkind in his summary of the 1974 "low doses" conference (p. 385 in ref. 1), then we are fortunate indeed that there are now additional methods to attack the question directly of what is turned on or activated. It is anticipated that there will be many further developments within the year, to be presented at related sessions at larger meetings, and at a closely related meeting to be held in June 1994 in Montreal, entitled "Gene Induction and Adaptive Responses in Irradiated Cells: Mechanisms and Clinical Implications."

Comparisons of cellular radiation response using absolute rather than relative parameters.

One important goal of radiobiology is to describe the response to radiation damage in quantitative terms. Because the dose response is nonlinear, this typically involves the comparison of several dose-dependent parameters. Past attempts at simplifying this process have often involved manipulations of these dose-dependent parameters to derive a single comparative number. Unfortunately, the advantages of a single comparative number, often a ratio or even a ratio of ratios, can be outweighed by the loss of significant biological information. Examples are given in four areas of research: (1) definition of radiation response at clinically relevant radiation doses, (2) modification of radiation sensitivity by oxygen, (3) effects of combined radiation modifiers (e.g., sensitizers and protectors), and (4) comparisons of radiation modifiers in different dose/response regions. In each area, to define and compare dose response, we propose the use of consistent, simple, and absolute radiation response parameters: the inverse dose (or dose) required to produce a given effect. The use of absolute sensitivity (or resistance) avoids the use of many other parameters, ratios, and definitions and permits a uniform and unambiguous description of "radiation response."

Small doses of high-linear energy transfer radiation increase the radioresistance of Chinese hamster V79 cells to subsequent X irradiation.

Chinese hamster V79 cells show a complex X-ray survival response which is characterized by hypersensitivity followed by increased resistance as the dose increases to 1 Gy. This hypersensitivity can be eliminated by pretreating cells with X rays or hydrogen peroxide. Accordingly, the protective effect that results from the priming treatments could be considered analogous to the "adaptive response" induced by low-linear energy transfer (LET) radiation and some chemical agents in human lymphocytes. Indeed, no hyper-radiosensitive response after single treatments in V79 cells or adaptive response in human lymphocytes has been reported after exposure to high-LET radiation. To investigate this further, we measured the survival after X irradiation of V79-379A cells previously irradiated with small priming doses of high-LET radiation. After a 0.2-Gy priming dose of neutrons followed by a 1-Gy 250 kVp X-ray dose given 4 h later, survival was 1.08 +/- 0.04 compared to 0.73 +/- 0.03 when the doses were given concurrently. Increases in survival were also observed from 0.80 +/- 0.03 to 0.96 +/- 0.05 after a 0.2-Gy priming treatment with 250 kVp X rays and from 0.78 +/- 0.03 to 0.84 +/- 0.03 with a priming dose of Bragg-peak negative pi mesons. The results indicate that a protective effect, as measured by an increase in radioresistance, is induced by high-LET neutrons, as well as by Bragg-peak pi mesons and X rays, and that a threshold level of damage is required for adaptation to occur.

Inactivation of hypoxic cells by cisplatin and radiation at clinically relevant doses.

We have examined the effects of exposure to cisplatin (cis-diamminedichloroplatinum(II] on the response of exponentially growing V79 cells to low (0-4 Gy) and high (up to 30 Gy) doses of X rays under hypoxic and aerobic conditions. Survival in both dose regions was assessed by clonogenic assays; the low-dose studies were facilitated by a Cell Analyser (B. Palcic and B. Jaggi, Int. J. Radiat. Biol. 50, 345-352 (1986]. The results show that cisplatin, like its isomer trans-DDP, exhibits greater interaction with low than with high radiation doses in hypoxic cells. This increased interaction could be seen even with subtoxic exposures to cisplatin as low as 1 mumol dm-3. In contrast, with cells irradiated in air in the presence of either complex, the interaction seen with high doses of radiation is completely lost or greatly diminished in the low radiation dose region. Further experiments showed that enhanced interaction of hypoxic cells with low doses of radiation could be equally effective with cisplatin pretreatments in air or in hypoxia, even if the cells are exposed to cisplatin only after irradiation. In experiments with nonproliferating plateau-phase cultures, the same enhanced interaction was observed in the low-dose region. These results, for example enhancement ratios of 2.3 and 1.2 at low- and high-dose regions, respectively, for 5 mumol dm-3 cisplatin, are contrasted with those for nitroimidazoles which are better sensitizers in the high-dose region.

Platinum complexes with one radiosensitizing ligand [PtCl2(NH3) (sensitizer)]: radiosensitization and toxicity studies in vitro.

Complexes of general formula [PtCl2(NH3)L] with one radiosensitizing ligand per platinum are compared with ligand L alone, complexes with two radiosensitizers per platinum [PtCl2L2], and their analogs with NH3 ligands, with respect to radiosensitizing properties and toxicity in CHO cells. Radiosensitizing ligands, L, were misonidazole, metronidazole, 4(5)-nitroimidazole, and 2-amino-5-nitrothiazole, and the ammine analogs were cis- and trans-DDP [diamminedichloroplatinum(II)] and the monoammine, K[PtCl3(NH3)]. Results are related to a previous study on plasmid DNA binding by these series. The toxicity of the mono series [PtCl2(NH3)L], attributable to DNA binding, is much higher than the corresponding bis complexes, [PtCl2L2]. For L = misonidazole, toxicity is similar to the monoammine, but higher in hypoxic than in aerobic cells. trans-[PtCl2(NH3)-(misonidazole)] is more toxic than the cis isomer. Except for L = 4(5)-nitroimidazole, the complexes [PtCl2(NH3)L] are more toxic than L in air and hypoxia. Hypoxic radiosensitization by the mono complexes is comparable to the monoammine and is not better than free sensitizers, again except for L = 4(5)-nitroimidazole. Significantly lower sensitization is observed in oxic cells. The bis complexes [PtCl2L2], which do not bind to DNA as well as the mono complexes, are less effective radiosensitizers and less toxic than the [PtCl2(NH3)L] series.

The effect of radiosensitizers on the survival response of hypoxic mammalian cells: the low X-ray dose region, hypersensitivity and induced radioresistance.

It has been shown previously that the extent of chemical modification of the hypoxic radiation response is dependent on dose. Some types of sensitizer are more effective at low doses (to 4 Gy) than at higher doses. Since such drugs are possible adjuvants to radiotherapy, the mechanisms responsible for the variable response at clinical doses are of interest. Existing reports on sensitization at low doses are summarized, and the effects of cisplatin and buthionine sulfoximine on the purported induced response to radiation in hypoxic cells are presented. Cisplatin at a low, nontoxic concentration (1 microM) appears to abolish the increased radioresistant portion of the survival response. A role for high-mobility-group protein binding by platinum drugs is hypothesized to explain their interaction with radiation, and conversely, it is suggested that the heretofore unexplained different behavior of certain hypoxic sensitizers at low doses could be, at least in part, an effect on the induction of resistance.

The effect of oxygen on low-dose hypersensitivity and increased radioresistance in Chinese hamster V79-379A cells.

Chinese hamster V79 cells irradiated in air are hypersensitive to X-ray doses less than 0.5 Gy and show an increased radioresistance over the dose range 0.5-1 Gy (Marples and Joiner, Radiat. Res. 133, 41-51, 1993). Of considerable interest from both a mechanistic and clinical viewpoint is the response of hypoxic cells over this dose range. The data presented here indicate that hypoxic cells are also hypersensitive to low X-ray doses and exhibit an increased radioresistant response, albeit triggered at a somewhat higher dose (0.69 Gy, SEM +/- 0.18 Gy) than observed in oxygenated cells (0.5 Gy, SEM +/- 0.21 Gy). These data indicate that the triggering event for increased radioresistance may be independent of oxygen. As reported by others previously, the oxygen enhancement ratio was found to decrease with a decreasing X-ray dose.

The response of Chinese hamster V79-379A cells exposed to negative pi-mesons: evidence that increased radioresistance is dependent on linear energy transfer.

Chinese hamster V79-379A cells exhibit low-dose hypersensitivity to 250 kVp X rays followed by an increased radioresistant response over the dose range 0.5-1 Gy. This phenomenon is not seen with neutrons (Marples and Joiner, Radiat. Res. 133, 41-51, 1993). It was therefore postulated the induction of radioresistance might develop as a response to a cellular event(s) which predominates after low- and not high-LET radiation. To test this hypothesis, we measured the survival response of V79-379A cells exposed to pions. Clonogenic survival was assessed for cells irradiated in the Bragg peak (35 keV/microns) and plateau region (10-20 keV/microns) of the beam, using an automated microscope (DMIPS cell analyzer). As expected, peak pions were found to be more effective per unit of dose at killing cells than plateau pions. The survival curve for cells irradiated in the plateau of the pion beam was found to incorporate a region of low-dose hypersensitivity and increased radioresistance, the effective D0 was dose-dependent, ranging from 3.5-5. This was not seen with peak pions, where the effective D0 was, on average, constant reflecting a single-exponential survival curve. Fitting the data with an induced repair model indicates that the phenomenon of increased radioresistance is almost certainly dependent on LET.

The effect of differentiation on photosensitizer uptake by HL60 cells.

The capability of human promyelocytic leukemia cells HL60 to be induced to differentiate to various stages along the monocytic or myelocytic pathway was exploited for investigation of the uptake of selected photosensitizers by diverse types of cells of the same origin. The results showed that there was no substantial difference in photofrin uptake between noninduced HL60 cells, immature monocytes, immature neutrophils and cells differentiated along the eosinophilic pathway. In contrast, HL60 cells differentiated into macrophages (HL60 phi) exhibited markedly increased photofrin uptake, which was further enhanced by their pretreatment with bacterial lipopolysaccharide. Similar results were obtained with other photosensitizers tested: di- and tetrasulfonated aluminum phthalocyanines (A1PcS2 and A1PcS4), tetrasulfonated zinc phthalocyanine (ZnPcS4), tetraphenylporphine tetrasulfonate (TPPS4) and benzoporphyrin derivative monoacid (BPD). Despite marked differences in the state of self-aggregation and other chemical properties of these compounds, the degree of their preferential uptake by HL60 phi cells showed very little variation. In a typical experiment, the uptake of these photosensitizers by HL60 phi cells was four to five times higher than the uptake by noninduced HL60 cells. In addition to the fluorometric assay employed in most of the experiments, cellular concentration of A1PcS4 was determined by measurement of elementary aluminum using atomic absorption spectroscopy.

Radiosensitization by metal complexes of 4(5)-nitroimidazole.

Four closely-related cis-platinum (Pt) complexes of 4(5)-nitroimidazole have been examined with respect to properties of radiobiological interest, to test the hypothesis that targeting a nitroimidazole (NO2Im) to DNA could enhance its radiosensitizing ability: I [PtCl2(5-NO2Im)2]; II [PtCl2(4-NO2Im)2]; III [PtCl2(NH3)(5-NO2Im)]; IV [PtCl2(NH3)(4-NO2Im)]. The reduction potential was affected to the same extent on metal binding in all of the complexes (delta E1/2 = +200 mV, cf. ligand measured polographically). Higher sensitization by 5-NO2 complexes I, III (cf. II, IV) was found. Only the mono complexes III and IV bind to DNA (in an assay using inhibition of restriction endonuclease activity); these radiosensitize as well as, or better than, free ligand in hypoxic CHO cells, and better than the bis complexes (I and II). The toxicity of the mono complexes is higher than ligand, and parallels the binding (III, IV, mono bis analogues). The complexes are compared with 4-nitroimidazole complexes of ruthenium, with respect to toxicity, binding and radiosensitization.