Ischemia reperfusion injury in tumors: the role of oxygen radicals and nitric oxide.

Oxidative stress is a key process involved in the action of several therapeutic modalities used in cancer treatment. Ischemia reperfusion insult provides a model system for investigating the processes involved in determining the sensitivity of tumor tissue to oxidative stress. We have investigated the response of the murine CaNT tumor to ischemia reperfusion injury and the role that oxygen radicals and nitric oxide may play in this phenomenon. Our results show that little or no cell kill is detected in tumors exposed to up to 3 h of ischemia if the tumors are excised immediately before reperfusion. However, if reperfusion is permitted, then extensive cell kill is evident 24 h later. i.v. administration of superoxide dismutase or catalase, at the time when vascular reperfusion occurred, resulted in a significant protection against tumor cell kill, suggesting that the damage was mediated by oxygen radicals. Conversely, administration of an inhibitor of nitric oxide synthase, N omega-nitro-L-arginine, resulted in potentiation of tumor cell damage. Administration of a nitric oxide (NO) donor, diethylamine NO, at the time when vascular reperfusion occurred resulted in significant protection against tumor damage. These results suggest that nitric oxide is a potent mediator in determining tumor damage after ischemia reperfusion injury. The role of intrinsic NO production by murine tumors was investigated by measuring the accumulation of nitrate in the medium of tumor explants cultured in vitro in two tumors with differing sensitivity to ischemia reperfusion damage. The clamp-insensitive tumor SaS showed a greater nitrate accumulation than the clamp-sensitive tumor CaNT, which may confer a greater capacity for preventing tumor and endothelial cell damage after oxidative stress.

Cell proliferation in human tumour xenografts: measurement using antibody labelling against bromodeoxyuridine and Ki-67.

Cell proliferation was investigated in human tumour xenografts using bromodeoxyuridine (BrdUrd) labelling, evaluated either by flow cytometry or in tissue sections, and also using the proliferation marker Ki-67. BrdUrd labelling was found to increase when cryostat tumour sections were digested with an enzymic solution. This yielded a labelling index up to four times higher than that obtained using the flow cytometer. Ki-67 indices were found to be higher than those reported for human tumour biopsies, as may be expected due to the enhanced growth rate of the xenografts. Significant heterogeneity was observed in the results for cervix, breast and bladder tumours, and the results of the three methods were poorly correlated. However, three of the four tumour types showed that the tumour with the lowest Ki-67 index also had the longest potential doubling time. Since the measurement of Ki-67 index was found technically easier to perform, and also adequately reflects relative tumour cell proliferation, it is preferred over the other techniques.

Tumour blood flow changes induced by application of electric pulses.

The effect of electric pulses on tumour blood flow was investigated in the murine fibrosarcoma SA-1. After the application of short intense electric pulses, relative tumour perfusion was measured using an 86RbC1 extraction technique. A significant reduction of tumour perfusion (approximately 30% of control) was observed within 1 h following the application of eight electric pulses to the tumour. Thereafter, tumour blood flow slowly recovered, almost reaching the pretreatment level by 24 h. No change in perfusion was induced in the untreated contralateral normal leg muscle. A similar pattern of blood flow reduction was induced when a second set of electric pulses was applied to the tumour following a 24 h interval. The degree of tumour blood flow reduction was dependent upon the number of electric pulses applied, at 1040 V, and less effect was observed if less than eight pulses were applied. A modification of the amplitude of the electric pulses resulted in changes in the direction of tumour blood flow response. Tumour blood flow increased following pulses in the range between 80 and 560 V and decreased at amplitudes higher than 640 V. These results demonstrate that the local application of electric pulses to solid tumours can modify tumour blood flow. Pulses of increased amplitude resulted in the progressive reduction of tumour blood flow with a corresponding increase in tumour cytotoxicity as measured by growth delay. Tumour blood flow reduction by electric pulses could have potential in exploiting modalities mediated by tumour hypoxia, e.g. activation of bioreductive agents.

Is there a loss of repair capacity in mouse lungs with increasing numbers of dose fractions?

The capability of mouse lung to repair sublethal damage after up to 10 fractions of X rays was assessed by an in situ breathing rate assay and lethality. The whole thorax of mice was irradiated "daily" with 4, 7 or 10 fractions of 2.75 or 3.0 Gy of X rays followed at 24 hours by graded "test" doses of X rays or neutrons. Repair capability was measured by determining the difference in test dose between 4 and 7 fractions or 7 and 10 fractions at a given isoeffect. Damage was assessed monthly up to 76 weeks after irradiation, during pneumonitis and chronic fibrosis. The data from both assays for the pneumonitis phase suggested that there may be some loss of repair between 7 and 10 fractions, although it was not large enough in only 10 fractions to be clearly demonstrated. In contrast, there was no suggestion of loss of repair for late damage after up to 10 fractions of X rays using either assay.

WR-2721 protection of pneumonitis and fibrosis in mouse lung after single doses of x rays.

The radioprotective effect of WR-2721 has been studied in mouse lung after single doses of radiation. Using the breathing rate assay and lethality, radioprotection was assessed at monthly intervals between 3 and 18 months after irradiation during both pneumonitis and chronic fibrosis. The degree of radioprotection was greater for fibrosis than for pneumonitis using both assays. In replicate experiments, dose modifying factors (DMF's) ranging from 1.2 to 1.4 were obtained for pneumonitis and 1.5 and 1.6 for fibrosis. The differences in DMF's for the two phases of lung damage were significant. A difference in the time course of expression of damage was seen in both the breathing rate and lethality assays between mice irradiated with and without WR-2721: the damage ended sooner in the drug-treated mice. This difference is best explained by protection of all damage after 5 months by WR-2721. No evidence of drug toxicity was found. We conclude that WR-2721 protects against chronic lung fibrosis caused by radiation at least as well as against the earlier appearing pneumonitis after single doses of radiation. Thus, if WR-2721 is dose modifying and if late tissue complications are dose limiting in clinical radiotherapy, then a therapeutic benefit would be obtained by the use of this drug in clinical radiotherapy, provided that the radioprotection of tumors did not exceed a factor of 1.5-1.6.

Ischaemia induced cell death in tumors: importance of temperature and pH.

PURPOSE: Increasing attention has focussed on the therapeutic potential of agents which can reduce tumor blood flow and induce ischaemia for long enough to result in tumor cell death. A confounding factor in this approach is the fact that the core temperature of superficial tumors reduces when the supplying blood flow is occluded and therefore protects the tumor cells from "metabolic" death. Consequently, we have tested the importance of tumor temperature on the relationship between vascular occlusion and cell death. METHODS AND MATERIALS: The murine tumor CaNT used in this study was implanted subcutaneously in the dorsum. Total vascular occlusion was achieved by physically occluding the blood supply to the tumors for periods between 1 and 20 h. The mouse temperature was controlled by placing the whole body in a thermostatically controlled incubator maintained at 35 degrees C. Tumor cell survival was assessed using an excision assay and by measuring the delay in growth of treated tumors. Measurement of tumor pH was achieved using microelectrodes. RESULTS: The core temperature of unclamped tumors was approximately 33 degrees C, but fell by about 5 degrees C during vascular occlusion at room temperature. Tumor cell survival was decreased with increasing periods of vascular occlusion at room temperature, but a greater reduction in cell survival and correspondingly increased regrowth delay was observed when the tumor temperature was prevented from cooling below preocclusion values. The extracellular pH (pHe) fell during vascular occlusion and this reduction was greater when the tumor temperature was maintained at preocclusion values. This extracellular acidosis is expected to partly explain the observation of greater tumor cell death in those tumors whose temperature does not reduce during occlusion. CONCLUSION: The temperature of superficial tumors reduces in response to vascular occlusion. This may result in an underestimation of the cytotoxicity of agents which reduce tumor blood flow as the tumor cooling protects the cells from the acidosis that accumulates within the occluded tumor.

Early and late effects in mouse lung and rectum.

No higher RBE's were found for late than for early damage in mouse rectum up to 70 weeks or mouse lungs up to 48 weeks after irradiation with 3.0 MeV neutrons (4 MeV deuterons on Be). The smallest neutron doses per fraction were 1.5 and 0.6 Gy for rectal and lung irradiation, respectively. There was a suggestion of higher late RBE's for small doses per fraction in the lung. Slow repair after 2 neutron doses split by 31 days was unequivocally demonstrated in the lung, of magnitude about 1 Gy, possibly decreasing after about 40 weeks.

Collagen synthesis in CBA mouse heart after total thoracic irradiation.

CBA mice were irradiated to the whole thorax with single doses of 240 kVp X-rays in the dose range 8-16 Gy. Collagen and total protein synthesis rates in the heart were measured at 2-monthly intervals using a radio-isotope incorporation technique. Doses of 10 Gy or greater caused a slight increase in collagen synthesis, followed by significantly reduced collagen synthesis by 16 weeks or longer after treatment. The depression in synthesis appeared correspondingly earlier with increasing dose. Total protein synthesis in heart followed similar patterns although changes were not statistically significant, indicating that the changes reflected alterations to collagen synthesis specifically, and not protein synthesis in general. Total hydroxyproline measurements showed no significant changes in heart collagen at any time as a result of X-irradiation.

A murine model of lip epidermal/mucosal reactions to X-irradiation.

A new radiobiological test system has been developed for lip epidermal/mucosal reactions in mice. This is intended for use in investigations of the effect of non-standard fractionation and of modifying drugs on oral radiation reactions in human cancer patients. An arbitrary scale of scores was devised, with separate scores for oedema of the lips and for erythema or exudation. After single doses of 13-20 Gy, the mouse lip epidermal reactions began at 5 days, reached a peak about 10-13 days, and had fallen to low values, but not to zero, by 21 days. Several different periods for averaging the reaction scores were tested for relative steepness and variability, the most useful being 10-12 days inclusive or the 12th day score alone. The use of longer periods of averaging led to apparent saturation of the scores. It was found that large doses of X-rays repeated at 21-23 day intervals did not lead to escalating waves of reactions unless each dose was greater than 17 Gy. With these larger doses, escalation of reactions occurred even if the intervals were extended.

Evaluation of cell attachment matrix (CAM) coated plates for primary culture of human tumour biopsies.

Baker et al. [Cancer Res. 46: 1263-1274, 1986] developed an adhesive tumour cell culture system (ATCCS) using a culture surface coated with a cell attachment matrix (CAM). The ability of CAM-coated plates to support the growth of cells from human tumour biopsies has been evaluated. Successful growth was obtained in 9/22 samples (41%), but fibroblasts, rather than tumour cells, grew in the majority. Comparison of CAM with other surfaces showed that CAM was no better for establishing tumour cell growth than the presence of feeder cells or an alternative attachment factor vitronectin.

Collagen metabolism in mouse lung after X irradiation.

Collagen and total protein synthesis rates have been determined in the lungs of CBA mice irradiated with single doses of X rays between 8 and 16 Gy. Mice were injected with [3H]proline accompanied by a large dose of unlabeled proline, and synthesis rates were measured at 2-month intervals from 8 to 31 weeks after irradiation. At 2 months after radiation treatment, collagen and total protein synthesis rates were significantly depressed but they had recovered by 4 months. By 6 months collagen synthesis rates had increased above control in a dose-dependent manner, so that in the 14-Gy dose group the fractional synthesis rate for collagen was 4.6 times higher than in control mice as measured by incorporation of [3H]proline. However, a significant net accumulation of collagen was seen only in the lungs of the highest dose group at 31 weeks, as indicated by total hydroxyproline measurements. There was a slight increase in the ratio of types I and III collagen. Late radiation damage in the CBA mouse lung is characterized by increased collagen metabolism, which may or may not lead to a net accumulation of collagen.

Repair in mouse lung between multiple small doses of X rays.

Multiple fraction experiments have been carried out to determine the response of mouse lung to repeated small doses of 240 kV X rays down to 150 rad/fraction using breathing rate and lethality to assess damage. Two experimental approaches were used to measure the effect of small doses in vivo: (1) multiple equal doses and (2) multiple priming doses followed by a large test dose. Analysis was performed using the multitarget two-component model and the linear quadratic model of cell survival. The amount of repair was calculated as a function of either dose per fraction (FR) or total dose (Frec). Both FR and Frec increased with decreasing dose per fraction but the change in FR was small. The advantage of Frec was that it varied more rapidly with dose per fraction than FR, so that possible differences between tissue repair capabilities are more visible on plots of repair as a function of dose per fraction. FR and Frec both decreased with the level of single-dose isoeffect injury; thus neither parameter is acceptable for comparing repair capability of different normal tissues with widely differing single-dose end point levels. Beta/alpha values were calculated and found to be a more acceptable index of repair capability than either FR or Frec because unlike those two parameters, beta/alpha varied little with level of damage. Beta/alpha values of 1.7 to 4.2 krad-1 were obtained for both lung death and increased breathing rate and are clearly intermediate between the lower beta/alpha ratios for acute reactions, i.e., skin and intestine, and the higher values for late reactions in kidney and spinal cord.

Involvement of oxygen free radicals in ischaemia-reperfusion injury to murine tumours: role of nitric oxide.

Ischaemia-reperfusion (I/R) injury is a model system of oxidative stress and a potential anti-cancer therapy. Tumour cytotoxicity follows oxygen radical damage to the vasculature which is modulated by tumour production of the vasoactive agent, nitric oxide (NO.). In vivo hydroxylation of salicylate, to 2,3- and 2,5-dihydroxybenzoate (DHBs), was used to measure the generation of hydroxyl radicals (OH.) following temporary vascular occlusion in two murine tumours (with widely differing capacity to produce NO.) and normal skin. Significantly greater OH. generation followed I/R of murine adenocarcinoma CaNT tumours (low NO. production) compared to round cell sarcoma SaS tumours (high NO. production) and normal skin. These data suggest that tumour production of NO. confers resistance to I/R injury, in part by reducing production of oxygen radicals and oxidative stress to the vasculature. Inhibition of NO synthase (NOS), during vascular reperfusion, significantly increased OH. generation in both tumour types, but not skin. This increase in cytotoxicity suggests oxidative injury may be attenuation by tumour production of NO.. Hydroxyl radical generation following I/R injury correlated with vascular damage and response of tumours in vivo, but not skin, which indicates a potential therapeutic benefit from this approach.

Induction of systemic neutrophil response in mice by photodynamic therapy of solid tumors.

Photodynamic therapy (PDT) of solid tumors elicits a strong, acute inflammatory response characterized by a rapid and massive infiltration of activated neutrophils into the tumor. The present study investigated the impact of PDT on the systemic and local (treatment site) kinetics of neutrophil trafficking and activity in mouse SCCVII and EMT6 tumor models. Differential leukocyte counts in the peripheral blood of treated mice revealed a pronounced neutrophilia developing rapidly after Photofrin porfimer sodium (Photofrin)- or tetra(m-tetrahydroxyphenyl)chlorin (mTHPC)-based PDT. Significant neutrophilia was also observed upon PDT treatment of normal dorsal skin but not on the footpad of tumor-free mice. The changes in circulating neutrophil numbers were accompanied by an efflux of these cells from the bone marrow. An increased proportion of cells with high L-selectin (CD62L antigen) expression was found among bone-marrow-residing neutrophils 6-24 h after PDT, and in neutrophils in the peripheral circulation and treated tumors 24 h after therapy. Complement inhibition completely prevented the development of PDT-induced neutrophilia. The results of the present study demonstrate that treatment of solid tumors by PDT induces a strong and protracted increase in systemic neutrophil numbers mediated by complement activation. This reaction reflects rapid and massive mobilization and activation of neutrophils for the destruction of PDT-treated tumor tissue.

The role of nitric oxide in cancer. Improved methods for measurement of nitrite and nitrate by high-performance ion chromatography.

The short lifetime of nitric oxide (NO) in vivo impedes its quantitation directly; however, the determination of nitrite and nitrate ions as the end-products of NO oxidation has proven a more practical approach. High-performance ion chromatographic analysis of nitrite in biological fluids is hampered by the large amount of chloride ion (up to approximately 100 mmol/l) which results in insufficient peak resolution when utilizing conductimetric detection. Analysis of both anions in small sample volumes is also constrained by the need to minimise sample handling to avoid contamination by environmental nitrate. We report a means to remove Cl- ions from small sample volumes using Ag+ resin which facilitates quantitation of either nitrite and nitrate anions in biological samples, using silica or polymer based ion-exchange resins with conductimetric or electrochemical and spectrophotometric detection. Including a reversed-phase guard column before the anion-exchange guard and analytical column also greatly extends column lifetime.

Analysis of the acidic microenvironment in murine tumours by high-performance ion chromatography.

High-performance ion chromatography (HPIC) has been utilised to probe the biochemistry associated with changes in tumour pH following total vascular occlusion. Samples from the tumour extracellular compartment were obtained by insertion of a microdialysis probe and analysed by HPIC with conductivity detection. Separations were carried out by ion-exclusion chromatography using an IonPac ICE AS1 weak-acid column. The eluent (0.5 mM octanesulphonic acid) was chemically suppressed with 5 mM tetrabutylammonium hydroxide through a micromembrane suppressor. After complete vascular occlusion induced by a clamp, lactate levels increased in the extracellular compartment.

Enhancement of mitomycin-C cytotoxicity by combination with flavone acetic acid in a murine tumour.

The antitumour activity of a combination of Mitomycin C (MMC), a prototypical bioreductive agent, with the vascular occluding agent, flavone acetic acid (FAA), was examined in a murine tumour. Flavone acetic acid induced a long lived reduction of blood flow in the tumour thereby inducing hypoxia and cell death by ischaemia. Dose response curves for tumour cell survival and regrowth delay were constructed after each agent administered alone or when combined. The greatest antitumour effect was measured when the two agents were administered simultaneously. The reduced tumour perfusion induced by the combination was not lower than that for FAA alone indicating that the greater in vivo effect was not due to enhancement of the vascular occluding effect of the FAA. The role of the tumour microenvironment in determining the MMC cytotoxicity was examined using in vitro incubations of MMC under controlled oxygenation and pH. MMC cytotoxicity was increased in hypoxia (enhancement ratio = 2.0) and also when extracellular pH was reduced from 7.4 to 6.4 (enhancement ratio = 1.5). Since the microenvironmental consequences of induced ischaemia are reductions in both oxygenation and pH these parameters are probably responsible in large part for the enhancement of cytotoxicity observed when MMC and FAA are combined.

Enhancement of chlorambucil cytotoxicity by combination with flavone acetic acid in a murine tumour.

Previous studies using murine tumours have shown enhanced action of certain chemotherapeutic compounds when combined with agents that reduce tumour blood flow. In the majority of cases the compounds used were cytotoxic to the induced hypoxic cells but in this study we have investigated the relative importance of changes in tumour pH following blood flow reductions. The role of tumour pH was investigated by using combinations of the cytotoxic alkylating agent Chlorambucil (CHL) with the vascular occluding agent Flavone Acetic Acid (FAA). Chlorambucil is a weak acid (pKa = 3.7) and is concentrated within cells exposed to culture media at low pH or to the acidic microenvironment in vivo. In vitro incubations showed that greater cytotoxicity was obtained when cells were incubated at low pH and that the cytotoxicity was independent of the level of oxygenation at the time of the drug incubation. Combination of both CHL and FAA in vivo resulted in greater reductions in cell survival and growth delay than when either agent was given alone. Simultaneous administration of the two agents were very effective, potentially due to two factors; firstly, that the pH of the tumour changes during ischaemia and secondly, that the reduced blood flow potentially alters the pharmacokinetic distribution of CHL resulting in 'drug-trapping' within the tumour. Since the action of CHL is independent of the induced hypoxia which results from blood flow reduction it is suggested that the increased in vivo action is due in part to the changes in tumour pH.

Cytotoxicity of bioreductive drug tirapazamine is increased by application of electric pulses in SA-1 tumours in mice.

The application of electrical pulses (electroporation) is a local tumour treatment resulting in the facilitated accumulation of non-permeant chemotherapeutic drugs (electrochemotherapy), as well as in the transient reduction of tumour blood flow. The aim of our study was to determine whether the application of electric pulses to the tumour increased the antitumour effectiveness of the bioreductive drug tirapazamine (TPZ). The survival of SA-1 fibrosarcoma cells was 150-fold lower after the exposure of cells for 1 h to TPZ under anoxic compared with normoxic conditions. The exposure of cells to electric pulses did not increase the cytotoxicity of TPZ. However, the in vivo treatment of subcutaneous tumours with a combination of TPZ (i.p. 25 mg/kg) injected 20 min before the application of electrical pulses significantly enhanced tumour response. Treatment with TPZ and electric pulses, repeated three times at 24-hour intervals resulted in tumour growth delay of 7.2 days. The results of our study showed that the observed antitumour effectiveness is unlikely to be due to increased cellular accumulation of TPZ by application of electric pulses, as indicated from in vitro experiments. The effect is more likely to be attributed to increased tumour hypoxia as a consequence of reduced tumour blood flow induced by application of electric pulses.

Radiation damage to glucose concentrating capacity and cell survival in kidney tubule cells: effects of fractionation.

The glucose concentrating capacity of cultured LLC-PK1 kidney epithelial cells has been measured after single and fractionated doses of X-rays. Steady-state glucose concentrating capacity (ratio of glucose concentration inside to outside cell) can be measured using radiolabelled analogues of glucose which are actively transported but not metabolized. These cells can be stimulated to increase their glucose concentrating capacity (up-regulation) by a reduction in the glucose concentration of the growth medium. However, after X-ray irradiation the cells have a reduced capacity to respond to up-regulation. This effect can be measured 7 days after irradiation and before radiation-induced cell killing affects the cell population. The previously reported radiosensitivity of this function to single doses of X-rays (in the range 1-16 Gy) was confirmed. Surprisingly, no significant sparing of this effect could be measured by fractionation of the X-ray dose into two or four fractions. However, the cells showed a significant fractionation effect if clonogenic survival was measured using the standard cell survival assay. These early effects have different fractionation response from the later phases of tissue damage, measured months to years after irradiation, which do show sparing due to fractionation and are thought to be mainly due to changes in cell survival. The lack of sparing by fractionation to the functional damage may suggest a different target from that which determines cell survival. These results support the hypothesis that radiation damages cellular functions, separately from cell replication.