Angiogenesis, hypoxia and VEGF expression during tumour growth in a human xenograft tumour model.

Tumour growth and spread of tumour cells requires angiogenesis. Incipient angiogenesis is not induced by tumour cell hypoxia but probably by proangiogenic factors. During growth tumours depend on a further induction of vascular development for adequate oxygen and nutrient supply. If the oxygen supply is insufficient, the resulting hypoxia stimulates angiogenesis through upregulation of HIF-1 alpha and VEGF. VEGF upregulation is associated with a poor response to treatment and poor prognosis. The aim of the study was to analyze the interrelationship between hypoxia and angiogenesis during tumour growth. Therefore the tumour vasculature architecture and functional properties of the vessels were studied during subsequent phases of tumour growth in relation to hypoxia and VEGF-expression. Tumours from the human glioblastoma multiforme tumour line E106 were transplanted in athymic mice. Tumours were harvested at 2 days after transplantation and when tumours reached a mean size of 2, 4, 6, 8 and 10 mm. VEGF was present early in the onset of angiogenesis independent of HIF-1 alpha. During tumour growth VEGF increased from 0.94 to 7.27 ng/mg assessed by ELISA. However, there was increasing intratumoural heterogeneity in the architecture of the tumours, even in the largest tumours small well oxygenated areas were detected resembling the relatively well organized architecture of the smallest tumours. The observation that tumour vasculature develops in early phases under normoxic and at later phases under hypoxic conditions with the presence of both conditions in the larger tumours, suggested that anti-angiogenic therapy should be directed towards HIF-1 alpha dependent and HIF 1-alpha independent pathways.

The hypoxic tumour microenvironment, patient selection and hypoxia-modifying treatments.

Tumour hypoxia has been found to be a characteristic feature in many solid tumours. It has been shown to decrease the therapeutic efficacy of radiation treatment, surgery and some forms of chemotherapy. Successful approaches have been developed to counteract this resistance mechanism, although usually at the cost of increased short- and long-term side-effects. New methods for qualitative and quantitative assessment of tumour oxygenation have made it possible to establish the prognostic significance of tumour hypoxia. The ability to determine the degree and extent of hypoxia in solid tumours is not only important prognostically, but also in the selection of patients for hypoxia-modifying treatments. To provide the best attainable quality of life for individual patients it is of increasing importance that tools be developed that allow a better selection of patients for these intensified treatment strategies. Several genes and proteins involved in the response to hypoxia have been identified as potential candidates for future use in predictive assays. Although some markers and combinations have shown potential benefit and are associated with treatment outcome, their clinical usefulness needs to be validated in prospective trials. A review of published studies was carried out, focusing on the assessment of tumour hypoxia, patient selection and the possibilities to overcome hypoxia during treatment.

Transcriptional response to hypoxia in human tumors.

BACKGROUND: The presence of hypoxic regions within solid tumors is associated with a more malignant tumor phenotype and worse prognosis. To obtain a blood supply and protect against cellular damage and death, oxygen-deprived cells in tumors alter gene expression, resulting in resistance to therapy. To investigate the mechanisms by which cancer cells adapt to hypoxia, we looked for novel hypoxia-induced genes. METHODS: The transcriptional response to hypoxia in human glioblastoma cells was quantified with the use of serial analysis of gene expression. The time course of gene expression in response to hypoxia in a panel of various human tumor cell lines was measured by real-time polymerase chain reaction. Hypoxic regions of human carcinomas were chemically marked with pimonidazole. Immunohistochemistry and in situ hybridization were used to examine gene expression in the tumor's hypoxic regions. RESULTS: From the 24 504 unique transcripts expressed, 10 new hypoxia-regulated genes were detected-all induced, to a greater extent than vascular endothelial growth factor, a hypoxia-induced mitogen that promotes blood vessel growth. These genes also responded to hypoxia in breast and colon cancer cells and were activated by hypoxia-inducible factor 1, a key regulator of hypoxic responses. In tumors, gene expression was limited to hypoxic regions. Induced genes included hexabrachion (an extracellular matrix glycoprotein), stanniocalcin 1 (a calcium homeostasis protein), and an angiopoietin-related gene. CONCLUSIONS: We have identified the genes that are transcriptionally activated within hypoxic malignant cells, a crucial first step in understanding the complex interactions driving hypoxia response. Within our catalogue of hypoxia-responsive genes are novel candidates for hypoxia-driven angiogenesis.

Recovery capacity of glial progenitors after in vivo fission-neutron or X irradiation: age dependence, fractionation and low-dose-rate irradiations.

Previous experiments on the radiosensitivity of O-2A glial progenitors determined for single-dose fission-neutron and X irradiation showed log-linear survival curves, suggesting a lack of accumulation of recovery of sublethal damage. In the present study, we addressed this question and further characterized the radiobiological properties of these glial stem cells by investigating the recovery capacity of glial stem cells using either fractionated or protracted whole-body irradiation. Irradiations were performed on newborn, 2-week-old or 12-week-old rats. Fractionated irradiations (four fractions) were performed with 24-h intervals, followed by cell isolations 16- 24 h after the last irradiation. Single-dose irradiations were followed by cell isolation 16-24 h after irradiation or delayed cell isolation (4 days after irradiation) of the O-2A progenitor cells from either spinal cord (newborns) or optic nerve (2- and 12-week-old rats). Results for neonatal progenitor cell survival show effect ratios for both fractionated fission-neutron and X irradiation of the order of 1.8 when compared with single-dose irradiation. A similar ratio was found after single-dose irradiation combined with delayed plating. Comparable results were observed for juvenile and adult optic nerve progenitors, with effect ratios of the order of 1.2. The present investigation clearly shows that fractionated irradiation regimens using X rays or fission neutrons and CNS tissue from rats of various ages results in an increase in O-2A progenitor cell survival while repair is virtually absent. This recovery of the progenitor pool after irradiation can be observed at all ages but is greatest in the neonatal spinal cord and can probably be attributed to repopulation.

Modulation of hypoxia in murine liver metastases of colon carcinoma by nicotinamide and carbogen.

There is increasing evidence that modulation of tumor hypoxia may improve therapy outcome. However, most preclinical data are derived from subcutaneous rather than orthotopic tumor models. We investigated the effect of the hypoxia-modulating agents nicotinamide and carbogen on tumor hypoxia, tumor blood perfusion, and proliferative activity in liver metastases of the murine colon carcinoma line C26a. In untreated C26a liver metastases, we observed a considerable amount of hypoxia, similar to the amount in liver metastases of patients with colorectal cancer. Compared to untreated mice, we observed a significantly smaller hypoxic fraction in the liver metastases of mice treated with nicotinamide and carbogen breathing as single treatments or in combination. In the group of mice that underwent carbogen breathing, perfusion was significantly lower than in the untreated group, but the decrease was only marginal. The proliferative activity was similar in all groups. In C26a subcutaneous tumors, a similar effect on hypoxia has been observed that was, however, combined with a decrease in proliferative activity. The different effects of nicotinamide and carbogen on parameters of the tumor microenvironment in liver metastases and subcutaneous tumors suggest that the host tissue influences the mechanism by which nicotinamide and carbogen exert their effects. Since tumor hypoxia may be a clinical problem in colorectal liver metastases, our results open possibilities for further research on the effect of hypoxia modifiers on colorectal liver metastases to improve therapy outcome.

Noninvasive assessment of the functional neovasculature in 9L-glioma growing in rat brain by dynamic 1H magnetic resonance imaging of gadolinium uptake.

Pathophysiologic parameters of the functional neovasculature and the blood-brain barrier of 9L-glioma in rat brain were measured noninvasively by dynamic 1H magnetic resonance imaging studies of gadolinium (Gd)-DTPA uptake. Changes of apparent [Gd-DTPA] uptake in time (CT[t]) were analyzed in a slice through the center of 10 9L-gliomas using fast T1 measurements. The distribution of the contrast agent was spatially correlated with the distribution of perfused microvessels as determined by immunohistochemical analysis. This method permits a distinction between perfused and nonperfused microvessels with a disrupted blood-brain barrier. In transverse slices of the whole tumor, a spatial correlation was observed between CT maps and the two-dimensional distribution of perfused microvessels. In the next step, Gd-DTPA uptake rates were spatially related to the perfused microvessel density (Np) or vascular surface area (Sp). In tumor voxels with perfused microvessels, a linear correlation was found between Gd-DTPA uptake rate constants (k values) and Np or Sp. No correlation was observed between k values and the total microvessel density. These are the first data that show a relation between Gd-DTPA uptake rates and parameters of the functional neovasculature in 9L-glioma growing in rat brain. Now that Gd-DTPA uptake studies can be related to parameters of the functional neovasculature, they may be used more efficiently as a prognostic tool before or during therapy.

The effect of intraspinal cytosine arabinoside on the re-irradiation tolerance of the cervical spinal cord of young and adult rats.

Intrathecal treatment with cytosine arabinoside (ara-C) in combination with radiation has been used as prophylactic treatment in children with acute lymphatic leukaemia. Animal experiments have shown that ara-C enhances the effect of radiation on the spinal cord when administered shortly before irradiation, and that the long-term recovery after a combined treatment may be impaired. In the present experiments immature, 3-week-old rats, were treated with ara-C and radiation on the cervical spinal cord, and the long-term recovery was examined by reirradiation after different intervals. The endpoint of the study was paresis due to radiation myelopathy. The results showed a clear enhancement of the radiation effect with a dose-modifying factor of 1.2, when ara-C was administered before irradiation. However, no indications for impaired long-term recovery were observed. Additional experiments in adult rats with ara-C treatments during a 6-month interval between two radiation doses also did not suggest any interference between ara-C treatment and long-term recovery of radiation induced injury. It is concluded that for both the adult and immature nervous tissue, only when ara-C is administered intraspinally shortly before irradiation, interaction between ara-C and radiation results in a significant reduction of the isoeffective radiation dose by a factor of 1.2 (1.13-1.37, 95% confidence interval).

Radiation response of the rat cervical spinal cord after irradiation at different ages: tolerance, latency and pathology.

PURPOSE: The investigation of the age dependent single-dose radiation tolerance, latency to radiation myelopathy, and the histopathological changes after irradiation of the rat cervical spinal cord. METHODS AND MATERIALS: Rats, ages 1-18 weeks, were irradiated with graded single doses of 4 MV photons to the cervical spinal cord. When the rats showed definite signs of paresis of the forelegs, they were killed and processed for histological examination. RESULTS: The radiation dose in paresis due to white matter damage in 50% of the animals (ED50) after single dose irradiation was about 21.5 Gy at all ages > or = 2 weeks (mean 21.4 (mean 21.4 Gy; 95% CI 21.0, 21.7 Gy). Only the ED50 at 1 week was significantly lower (19.5 Gy; 18.7, 20.3 Gy). The latency to the development of paresis clearly changed with the age at irradiation, from about 2 weeks after irradiation at 1 week to 6-8 months after irradiation at age > or = 8 weeks. The white matter damage was similar in all symptomatic animals studied. The most prominent were areas with diffuse demyelination and swollen axons, often with focal necrosis, accompanied by glial reaction. This was observed in all symptomatic animals, irrespective of the age at irradiation. Expression of vascular damage appeared to depend on the age at irradiation. No vascular damage was observed in the rats irradiated at 1 week, clearly altered blood vessels were seen in animals symptomatic 10 weeks after irradiation at > or = 3 weeks, and vascular necrosis occurred after > or = 6 months in some rats irradiated at > or = 8 weeks. CONCLUSION: Although the latency to myelopathy is clearly age dependent, single dose tolerance is not age dependent at age > or = 2 weeks in the rat cervical spinal cord. The white matter damage is similar in all symptomatic animals studied, but the vasculopathies appear to be influenced by the age at irradiation. It is concluded that white matter damage and vascular damage are separate phenomena contributing to the development of radiation myelopathy, expression of which may depend on the radiation dose applied and the age at irradiation.

Radiation tolerance and fractionation sensitivity of the developing rat cervical spinal cord.

To investigate the influence of age at irradiation on single dose radiation tolerance and fractionation sensitivity, the cervical spinal cord of rats was irradiated at the age of 1 week and at 15-18 weeks (adult). While the main histological lesions seem to be comparable after irradiation at the two ages, differences were found in single dose tolerance, latency to paresis due to white matter lesions, and fractionation sensitivity. The 50% effect dose (ED50) for single dose irradiation at one week was 19.5 Gy, which is only 10%, but significantly (p < 0.05), lower than the ED50 of about 21.5 Gy at 3 weeks and above. The latency to paresis was clearly influenced by the age at irradiation. The latency in the rats irradiated at 1 week was about 2 weeks, while for adult rats a latency of about 8 months was observed. The fractionation sensitivity for irradiation at 1 week was lower than the fractionation sensitivity of the adult rats; the alpha/beta value at 1 week was estimated to be 4.5 Gy, while for the adult rats an alpha/beta value of 1.8 Gy was found. As a consequence, the observed small difference in tolerance to single doses between 1 week-old and adult rats is further enhanced after fractionated irradiation. During prolonged irradiation treatments this decreased tolerance may be compensated by a higher proliferation rate in the immature central nervous system. The results of the present experiments indicate that, for a single tissue and endpoint, paresis due to white matter lesions in the rat cervical spinal cord, the latency to expression of damage and the fractionation sensitivity clearly change with age at irradiation.

Effect of X rays and neutrons on repair and regeneration in the rat spinal cord.

Clinical and experimental results of neutron irradiation have shown higher RBE values for the central nervous system (CNS) than for most other normal tissues. This is because of a considerable impairment of the large capacity of the CNS to repair subeffective damage induced by low LET radiation. Decreasing the dose per fraction of X rays increases the CNS tolerance significantly; this has no effect for neutrons. In the cervical spinal cord and the brain, two types of delayed damage can be described, so-called early and late. Different target cells are assumed to be involved, oligodendroglial cells in the early, and vascular endothelium in the late type. In the lumbar cord, the main lesion is nerve root necrosis, with the Schwann cell as the most probable target. These target cells show differences in response to X rays and neutrons, resulting in different RBE values. The highest RBE is obtained for cervical white matter necrosis. In addition to cellular repair of subeffective damage, long-term tissue regeneration is observed in the spinal cord, beginning at different times for the various types of damage. With neutrons, the rate of long-term regeneration is at least similar, or even more pronounced than for X rays.

Accelerated fractionation radiotherapy for laryngeal cancer, acute, and late toxicity.

The acute toxicity of an accelerated radiotherapy scheme was compared with a conventional schedule. Eighteen patients with squamous cell carcinoma of the larynx were treated with accelerated fractionation radiotherapy. An average reduction of overall treatment time of 11 days was accomplished by giving 2 fractions a day during the last part of the treatment. Total dose and fraction dose were left unchanged. Acute reactions of skin and mucosa in these patients were compared with those in 40 patients treated with a conventional fractionation scheme, that is, 2 Gy per fraction, 5 fractions per week, to a total dose of 64-70 Gy. Acute reactions were maximal between 5 and 7 weeks after the start of treatment. Complete healing occurred within 3 months in all patients. Mucosal reactions and, as a consequence, dysphagia were clearly increased in those patients treated with accelerated fractionation. For confluent mucositis an ED50 of 66 Gy was calculated compared to 69 Gy for conventional fractionation. To a lesser degree, skin toxicity was also enhanced in the patients treated with the accelerated schedule. Severe edema of the laryngeal mucosa occurred only in patients treated to a total dose of 68 or 70 Gy and was somewhat more frequent with accelerated fractionation (4/10) than with conventional fractionation (4/24). One patient in the accelerated fractionation group underwent laryngectomy for persistent edema and laryngeal necrosis. No severe late skin reactions were observed. It can be concluded that the fractionation schedule tested in this study is feasible. Further shortening of overall treatment time without reduction of total dose will likely lead to unacceptable acute and, possibly, also late toxicity.

Clinical implications of incomplete repair parameters for rat spinal cord: the feasibility of large doses per fraction in PDR and HDR brachytherapy.

PURPOSE: To evaluate the clinical implications of the repair parameters determined experimentally in rat spinal cord and to test the feasibility of large doses per fraction or pulses in daytime high-dose-rate (HDR) or pulsed-dose-rate (PDR) brachytherapy treatment schedules as an alternative to continuous low-dose-rate (CLDR) brachytherapy. METHODS AND MATERIALS: BED calculations with the incomplete repair LQ-model were performed for a primary CLDR-brachytherapy treatment of 70 Gy in 140 h or a typical boost protocol of 25 Gy in 50 h after 46-Gy conventional external beam irradiation (ERT) at 2 Gy per fraction each day. Assuming biphasic repair kinetics and a variable dose rate for the iridium-192- (192Ir) stepping source, the LQ-model parameters for rat spinal cord as derived in three different experimental studies were used: (a) two repair processes with an alpha/beta ratio = 2.47 Gy and repair half-times of 0.2 h (12 min) and 2.2 h (Pop et. al.); (b) two repair processes with an alpha/beta ratio = 2.0 Gy and repair half-times of 0.7 h (42 min) and 3.8 h (Ang et al.); and (c) two repair processes with an alpha/beta ratio = 2.0 Gy and repair half-times of 0.25 h (15 min) and 6.4 h (Landuyt et al.). For tumor tissue, an alpha/beta ratio of 10 Gy and a monoexponential repair half time of 0.5 h was assumed. The calculated BED values were compared with the biologic effect of a clinical reference dose of conventional ERT with 2 Gy/day and complete repair between the fractions. Subsequently, assuming a two-catheter implant similar to that used in our experimental study and with the repair parameters derived in our rat model, BED calculations were performed for alternative PDR- and HDR-brachytherapy treatment schedules, in which the irradiation was delivered only during daytime. RESULTS: If the repair parameters of the study of Pop et al., Ang et al., or Landuyt et al. are used, for a CLDR-treatment of 70 Gy in 140 h, the calculated BED values were 117, 193, or 216 Gy(sc) (Gy(sc) was used to express the BED value for the spinal cord), respectively. These BED values correspond with total doses of conventional ERT of 65, 96, or 104 Gy. The latter two are unrealistic high values and illustrate the danger of a straightforward comparison of BED values if repair parameters are used in situations quite different from those in which they were derived. For a brachytherapy boost protocol, the impact of the different repair parameters is less, due to the fact that the percentage increase in total BED value by the brachytherapy boost is less than 50%. If a primary treatment with CLDR brachytherapy delivering 70 Gy in 140 h has to be replaced, high doses per fraction or pulses (> 1 Gy) during daytime can only be used if the overall treatment time is prolonged with 3-4 days. The dose rate during the fraction or pulse should not exceed 6 Gy/h. For a typical brachytherapy boost protocol after 46 Gy ERT, it seems to be safe to replace CLDR delivering a total dose of 25 Gy in 50 h by a total dose of 24 Gy in 4 days with HDR or PDR brachytherapy during daytime only. Total dose per day should be limited to 6 Gy, and the largest time interval as possible between each fraction or pulse should be used. CONCLUSION: Extrapolations based on longer repair half-times in a CLDR reference scheme may lead to the calculation of unrealistically high BED values and dangerously high doses for alternative HDR and PDR treatment schedules. Based on theoretical calculations with the IR model and using the repair parameters derived in our rat spinal cord model, it is estimated that with certain restrictions, large doses per fraction or pulses can be used during daytime schedules of HDR or PDR brachytherapy as an alternative to CLDR brachytherapy, especially for those treatment conditions in which brachytherapy is used after ERT for only less than 50% of the total dose.

Lack of evidence for increased tolerance of rat spinal cord with decreasing fraction doses below 2 Gy.

The radiation tolerance of the spinal cord, both in man and in rats, has been shown to depend strongly on the size of the dose per fraction. With fraction doses down to about 2 Gy, the spinal cord tolerance can be predicted by a modified Ellis formula: D approximately N0.43. More recently alternative isoeffect formulas were based on the linear-quadratic (LQ) model of cell survival where the effect of dose fractionation is characterized by the ratio alpha/beta which varies from tissue to tissue. For the spinal cord, as well as for other late responding tissues, the ratio alpha/beta is small, in contrast to most acutely responding tissues. Both the Ellis-type formula, and to a lesser extent the LQ-model, predict a continuously increasing tolerance dose with decreasing fraction size. From the LQ model, the concept of "flexure dose" has been derived, which proposes the limit of effective fractionation to be about 0.1 alpha/beta. At this dose per fraction no significant further gain in tolerance would be detected. From previous experiments on the rat cervical spinal cord with doses per fraction down to about 2 Gy, the ratio alpha/beta was determined to be 1.7 Gy, and the LQ-model would predict a rise in tolerance with a reduction in fraction size to far below 2 Gy. Based on these predictions clinical studies have been initiated assuming a significantly increased tolerance by reduction of fraction size to about 1 Gy. However, in the present experiments no evidence was found for such an increase in tolerance with fraction sizes below 2 Gy.

Inhalation anesthesia in experimental radiotherapy: a reliable and time-saving system for multifractionation studies in a clinical department.

An inhalation anesthesia system has been employed to overcome several of the limitations associated with the use of sodium pentobarbital and other i.p. administered anesthetics in experimental radiotherapy. The described method is reliable and time-saving. The depth and duration of anesthesia are easily controllable. Only 4 deaths have occurred with more than 6000 animal exposures. The use of polystyrene jigs is shown to provide adequate thermal isolation. Oxygen as a carrier of the anesthetic agent is expected to prevent a reduced tissue oxygenation and its radiobiological consequences. The whole system is constructed as a mobile unit in which up to 16 mice or rats can be anesthetized simultaneously and irradiated in a single field with clinical treatment equipment during short time intervals between patient irradiations. The described advantages of this method make it specially suited for experiments with protracted fractionation schedules.

The effect of small radiation doses on the rat spinal cord: the concept of partial tolerance.

To evaluate the tolerance of the rat spinal cord to small radiation doses per fraction, an increasing number of fractions is required for induction of paralysis. The assessment of doses of 1-2 Gy, as used in the clinic, would require that over 100 fractions be given. The validity of replacing part of a fractionated irradiation of the spinal cord by a single large dose has been tested. Fractionated irradiation doses with 18 MeV X rays were followed by a "top-up" dose of 15 Gy as a single treatment. This is the fraction size of a treatment with two irradiation doses leading to paralysis in 50% of the animals (ED 50). Fractionated treatments were carried out with 2, 5, 10 and 20 fractions followed by the top-up dose of 15 Gy. The isoeffect curve, as a function of the number of fractions, has the same slope as experiments performed without top-up dose. The results show that the quality and quantity of cellular repair is not modified when part of a multifractionated exposure is replaced by a larger top-up dose. An important consequence of this finding is, that in treatments with unequal fraction sizes, the partial tolerances can simply be added. Since a top-up dose can replace a sizable number of irradiation treatments, its application will allow investigations of the extent of sublethal damage repair for fraction sizes as low as 1 Gy.

Radiation nephropathy in young and adult rats.

The effects of bilateral kidney irradiation were compared in young and adult rats. During a 1 year period after a single dose of 0, 7.5, 10, 12.5, or 15 Gy on both kidneys, renal function (glomerular filtration rate and effective renal plasma flow), urine composition, and systolic blood pressure were measured periodically. The first changes after irradiation were observed in the glomerular filtration rate and urine osmolality. One month after 10, 12.5, and 15 Gy, glomerular filtration rate (GFR) and urine osmolality had declined below control values in the young rats. After this initial decline, renal function increased at control rate or even more during the third and fourth month after irradiation but decreased progressively thereafter. In the adult rats, GFR and urine osmolality started to decrease 3 months after 10, 12.5, and 15 Gy. A rise in systolic blood pressure and proteinuria started 2-3 months after 12.5 and 15 Gy in both age groups. Early changes in the glomerular filtration rate with a drop in urine osmolality in young rats, occurring during a period of rapid renal development indicated an irradiation-induced inhibition of glomerular and tubular development. Although renal function deteriorated at a later time in adult rats, dose-response relationships obtained in young and adult rats did not show significant differences.

Is the rate of repair of radiation-induced sublethal damage in rat spinal cord dependent on the size of dose per fraction?

In the present study the possible dependency of the kinetics of repair of sublethal damage in rat spinal cord on the fraction size has been further investigated. A wide range of sizes of dose per fraction (1.7-17.5 Gy) has been given with interfraction intervals varying from 0.5 to 24 hr. A direct method for analysis of quantal response and an incomplete-repair (IR) model for survival after fractionated exposures with short intervals were used to interpret the data. The half time of repair (T1/2) was found to be 1.6, 1.6 and 1.9 hrs for fraction sizes of approximately 4, 9, and 14 Gy respectively. There appears to be no significant effect of fraction size on the rate of repair. A clinically relevant feature observed from these experimental data is that in this tissue it takes more than 4 hrs for repair of sublethal damage, induced by a dose of approximately 4 Gy, to approach completion (i.e., sparing beyond the limit of the experimental resolution). This has to be taken into account when several fractions are to be given each day. Another feature noted from the analysis of these results is that the alpha/beta determined from the complete repair data is considerably smaller than that estimated from the incomplete repair data (interval less than or equal to 4 hrs). The nature of the inconsistency is discussed.

Late effects of fractionated pi-mesons compared to X rays on mouse lung.

Early and late delayed effects of up to 20 fractions of pions and X rays were investigated in the mouse lung. The whole thorax of female CD-1 mice was irradiated under Ethrane/O2 anesthesia. Respiration rate was measured by whole body plethysmography at biweekly to monthly intervals. With signs of irreversible respiratory distress, animals were sacrificed and their lungs evaluated histologically. In addition to the effect of fractionation, the influence of dose-rate and anesthesia was studied as well. The degree of injury for the most predominant lesions (macrophage accumulation, fibrosis, vascular congestion) was scored, and the correlation with the relative change in respiratory rate and survival was analyzed. This analysis showed the primary lesion to be radiation pneumonitis at a median survival time of approximately 100 days. Focal fibrosis was observed to occur soon thereafter, and no evidence was obtained for an independent second wave of fibrotic injury. Fibrosis seemed primarily the result of pathological organization in areas with heavy concentration of macrophages. It was observed that the mice were unusually sensitive, with a single dose X ray ED50/180 of 8.8 Gy. A similar value was found for unanesthetized mice. This might have been the result of performing these studies at an altitude of 2100 m. The fractionation effect also seemed more pronounced, with alpha/beta values of 0.6 Gy for X rays and 4 Gy for pions, which is significantly lower compared to reported values. At the pion dose-rate of 0.25 Gy.min-1, RBE values for single doses were 0.9 when compared to high dose-rate X rays, and 1.36 at equivalent dose rates. This clearly shows that significant repair occurs during the relatively low dose-rate pion irradiations. With smaller doses per fraction, the dose-rate effect became less dominant, and for 20 fractions of pions the RBE was 1.4 compared to fractionated high dose-rate X rays. These RBE's are similar to values reported for acute effects in skin.

From 2 Gy to 1 Gy per fraction: sparing effect in rat spinal cord?

Recently published results, from this group, on rat cervical spinal cord, a late responding tissue, indicated no further sparing with lowering the fraction size from 2 to 1.8, 1.5, and 1.3 Gy. In the present experiments a small but probably significant rise in tolerance is suggested, when the dose per fraction was decreased from 2 Gy down to 1 Gy. This rise would however still be much less than what is predicted by the linear quadratic model, based on the experimental data obtained with fraction sizes larger than 2 Gy.

Effects of multifraction irradiation on the rat kidney.

Wag/Rij female rats were irradiated to the left kidney with single doses or 2, 4, 10, 20, or 40 equal dose fractions. The right kidney was removed 4 weeks after the last fraction. The kidney function was determined using three different parameters. The serum urea content indicated glomerular function. Urine osmolality and the total volume of urine excreted in 24 hr indicated tubular function. The onset as well as the rate of expression of radiation-induced kidney damage was dose-dependent. The kidney function decreased continuously. Differences in expression of damage between glomerular and tubular parameters were not observed. All parameters indicated marked sparing of the kidney by fractionation. In general, the data could be fitted to the linear-quadratic model, if the single dose data were not included in the analyses. However, the fit greatly improved when data obtained with high and low doses per fraction were analyzed separately. The Direct Analysis method was used to determine the alpha/beta ratios. No significant differences were observed between the alpha/beta ratios calculated for the different parameters. The ratios also did not change with increasing time after treatment. The alpha/beta for high doses per fraction was between 0.6 and 2.7 Gy, and that for low doses per fraction, with fractional doses in the clinical range, was between 0.5 and 3.8 Gy. The alpha/beta values for low doses per fraction were generally lower than those for high doses per fraction. These observations indicate a strong dependence of radiation-induced damage in the rat kidney on the size of the dose per fraction.