The response of mouse skin to hyperthermia combined with fast neutrons or X-rays

The effects of hyperthermia combined with fast neutrons (mean energy approximately 7.5 MeV) or X-rays (250 kVp) were studied in the skin of the mouse ear and foot. Hyperthermia was achieved by immersion in water at temperatures of 41.5-43.0 degrees C for 1 hour. The heat treatments used caused no observable tissue injury other than transient erythema but they enhanced the response to both neutrons and X-rays. The enhancement of neutron damage increased as the heating temperature was increased, as is well known for X-rays. When heat was given after irradiation the thermal enhancement ratio (t.e.r.) for neutrons was similar to that for X-rays. When heat was given before irradiation the neutron t.e.r. was less than that for X-rays. Consequently, the relative biological effectiveness of fast neutrons compared with X-rays was not altered by giving heat after irradiation but it was reduced by giving heat before irradiation.     

The differential response of the skin in young and old rats to a combination of X-rays and 'wet' or 'dry' hyperthermia

The left hind feet of groups of female rats aged 7, 14 and 52 weeks were irradiated at three dose levels of X-rays (20, 25 or 30 Gy). Hyperthermia (42.5 degrees C for 1 h) was carried out immediately following irradiation using either 'wet' or 'dry' heat, achieved by immersion in either water or fluorocarbon liquid. The results demonstrated that 'wet' heat produced a consistently greater enhancement of the irradiation damage than 'dry' heat. The thermal enhancement ratio for irradiation plus 'wet' heat was approximately 1.5 and for irradiation plus 'dry' heat it was in the range 1.17 to 1.39. Immersion of the feet in fluorocarbon liquid at 37 degrees C did not significantly modify the irradiation response of the skin. The lower thermal enhancement ratios obtained using immersion in fluorocarbon liquid at 42.5 degrees C are close to those obtained in large animal studies and also similar to the limited amount of data from clinical studies where microwave or ultrasound heating techniques were used. It has been demonstrated that there are large age-related differences in the response of the rat foot skin to irradiation alone. It has also been shown in the present study, using rats of the same age, that the response to irradiation plus hyperthermia was less age dependent. This finding may reflect the differing methods by which damage occurs in tissue after irradiation or hyperthermia.     

Increased levels of comet-detected spermatozoa DNA damage following in vivo isotopic- or X-irradiation of spermatogonia.

To investigate whether DNA damage arising in spermatogenic germ cells can be detected in resultant sperm, we have irradiated murine testis and collected spermatozoa from the vas deferens 45 days later. These cells were derived from spermatogonia present at the time of irradiation. Two forms of irradiation were used, external X-rays (4Gy) and internal auger electrons from contamination of the male mouse with the isotope Indium-114m (1.85MBq), which was localised in the testis. Both forms of irradiation produced a profound fall in vas deferens sperm count and testis weight, Indium-114m being more effective. Using the neutral Comet assay for double strand break detection, significant increases in sperm comet tail length and moment were observed. The levels of damage were similar for both treatments. Care had to be taken during the assay to distinguish between sperm and somatic cells as the proportion of the latter increased after irradiation. We conclude that the comet assay can detect DNA damage in spermatozoa after the in vivo exposure of male germ cells to a known testicular genotoxic agent. The assay may be useful for the assessment of sperm DNA damage (double stranded) associated with male infertility and post-fertilization developmental abnormalities in the offspring.     

An unexpected effect of hyperthermia on the expression of X-ray damage in mouse skin

The interaction between hyperthermia and X irradiation in the expression of injury to skin was investigated in the tail of adult mice. The X-ray treatments when given alone resulted in skin reactions which ranged in severity from "no observable gross injury" to "moist desquamation over most of the tail," the peak reaction occurring at approximately 20 days. When hyperthermia was given alone, the maximal reaction observed was "foci of moist desquamation, accompanied by severe erythema and edema" which, in contrast to the radiation response, peaked 1 to 2 days after treatment. For the combined treatments, hyperthermia at a temperature between 43.0 and 44.5 degrees C for 30 min was given either 3, 6, 9, or 10 days after X irradiation. When the interval was 3 days, there appeared to be no interaction between the treatments. As the interval was lengthened, so that hyperthermia was given 6 or more days after irradiation, i.e., within 7 days of the time of appearance of gross radiation injury, the severity of the observed skin reaction was greater than the individual responses following either treatment given alone. Using a 9-day interval, it could be seen that both the thermal and radiation reactions were enhanced in a dose-dependent manner. The peak times for each reaction were not significantly altered by the additional treatment. The results are discussed with reference to possible modes of interaction between X irradiation and hyperthermia in an in vivo system.     

Modification of the effects of hyperthermia and neutron radiation on the activity of acid phosphatase in CaNT tumors.

Acid phosphatase activity was measured in implanted murine CaNT tumors of varying volumes. There is a clear monotonically increasing relation between acid phosphatase activity and tumor volume. Also the tumors were subjected to either induced artificial hypoxia or hyperthermia (41.0 degrees C) alone, or combined with neutron irradiation (3.8 Gy). Changes in the activity of this enzyme following radiation damage could reflect tissue damage associated with metabolic disturbances. The effect on enzyme activity after sequential hyperthermia and neutron irradiation is not synergistic, as is shown in the quantitative experimental data. This implies that the mechanisms of heat damage differ from that of neutron beam damage, as reflected by acid phosphatase activity. The CaNT tumor was also shown to be thermosensitive after administration of mitoxantrone. Finally, the role of exogenous ATP was shown to provide heat protection by modification of those thermal effects resulting in the activity of acid phosphatase. The augmentation of this hydrolytic enzyme probably represents initial metabolic damage in the tumor after different modalities of radiation alone, or combined with mitoxantrone and exogenous ATP.     

The effect of hyperthermia on radiation-induced carcinogenesis

Ten groups of mice were exposed to either a single (30 Gy) or multiple (six fractions of 6 Gy) X-ray doses to the leg. Eight of these groups had the irradiated leg made hyperthermic for 45 min immediately following the X irradiation to temperatures of 37 to 43 degrees C. Eight control groups had their legs made hyperthermic with a single exposure or six exposures to heat as the only treatment. In mice exposed to radiation only, the postexposure subcutaneous temperature was 36.0 +/- 1.1 degrees C. Hyperthermia alone was not carcinogenic. At none of the hyperthermic temperatures was the incidence of tumors in the treated leg different from that induced by X rays alone. The incidence of tumors developing in anatomic sites other than the treated leg was decreased in mice where the leg was exposed to hyperthermia compared to mice where the leg was irradiated. A systemic effect of local hyperthermia is suggested to account for this observation. In mice given single X-ray doses and hyperthermia, temperatures of 37, 39, or 41 degrees C did not influence radiation damage as measured by the acute skin reactions. A hyperthermic temperature of 43 degrees C potentiated the acute radiation reaction (thermal enhancement factor 1.1). In the group subjected to hyperthermic temperatures of 37 or 39 degrees C and X rays given in six fractions, the skin reaction was no different from that of the group receiving X rays alone. Hyperthermic temperatures of 41 and 43 degrees C resulted in a thermal enhancement of 1.16 and 1.36 for the acute skin reactions. From Day 50 to Day 600 after treatment, the skin reactions showed regular fluctuations with a 150-day periodicity. Following a fractionated schedule of combined hyperthermia and X rays, late damage to the leg was less than that following X irradiation alone. Mice subjected to X rays and hyperthermic temperatures of 41 and 43 degrees C had a lower median survival time than the mice treated with hyperthermia alone. This effect was not associated with tumor incidence.     

The individual and combined effects of X-irradiation and hyperthermia on early somite mouse embryos in culture.

The effects of 1) X-irradiation and 2) hyperthermia at a temperature of 43 degrees C individually and in combination have been investigated using cultured 8-day mouse embryos. B6C3F1 embryos were exposed to 0.3-2.0 Gy of X-rays, 5-20 min of heating, or 5 min of heating and irradiation at 0.3, 0.6, and 0.9 Gy. Irradiation alone at 0.3 Gy showed no apparent effect on embryonic development, but irradiation at 0.6-2.0 Gy caused a dose-dependent increase in malformed embryos. Heating alone for 5 min produced no malformed embryos, while heating for 10-20 min caused malformations as a function of heating time. Combined treatments produced higher frequencies (22.2-100%) of malformations than those of the sum of the separate treatments (0-41.7%). Malformations observed were primarily microphthalmia, microcephaly, and open neural tubes. The results indicate that in cultured mouse embryos irradiation combined with a "nonteratogenic dose" of hyperthermia directly exerts an additive effect on formation of the malformed embryos. In addition, a single occurrence of left-sided tail was produced by hyperthermia alone, while four occurrences were produced in combination with radiation.     

The response of mouse intestine to combined hyperthermia and radiation: the contribution of direct thermal damage in assessment of the thermal enhancement ratio

The thermal enhancement of X-ray damage to mouse jejunum has been assessed when heating was achieved by immersion of an exteriorized loop of intestine in Krebs-Ringer solution. The results have been compared with those previously obtained following heating in situ. The primary effect of 1 hour of mild hyperthermia was to reduce the should of the crypt survival curve obtained following X-rays given alone. Thermal enhancement ratio (TER) values increased with increasing temperature, up to 42.3 degrees C, and were within the range reported for other normal tissues. However, when hyperthermia itself caused crypt loss and the contribution of hyperthermal killing to the overall tissue response was taken into account, there was little enhancement of radiation damage. There was no evidence of a large increase in TER at high temperatures, as is seen in some tumours and has been reported by Merino, Peters, Mason and Withers (1978) for intestine. It is possible that very high TER values which have previously been reported mainly reflect the heat-alone component of damage. Some of the implications of these results are discussed in relation to the combination of heat and radiation in therapy.     

Effects of hyperthermia and X-irradiation on mouse stromal tissue

The sensitivity of normal stroma to heat, irradiation and heat combined with irradiation has been studied using the tumour bed effect (TBE) assay. Irradiation before implantation led to a TBE. This TBE was dose dependent below 15 Gy, the TBE remaining relatively constant above 15 Gy. The interval (0-90 days) between irradiation and tumour implantation did not influence the magnitude of the TBE. Hyperthermia with large heat doses (45-60 min at 44 degrees C) before implantation may lead to a TBE. The interval between hyperthermia and tumour implantation proved to be very important. Our results show that the recovery from heat-induced stromal damage is very rapid. When the interval between hyperthermia and tumour implantation is 10 days or longer, no TBE could be observed. Irradiation combined with large heat doses (30-60 min at 44 degrees C) decreased the radiation-induced TBE. However, the combination of irradiation with mild heat treatments (15 min at 44 degrees C) could lead to a larger TBE than after irradiation alone. When hyperthermia was given prior to irradiation, the interval between heat and irradiation proved to be very important. With large intervals (21 days or longer) the TBE values were about the same as with irradiation alone. When heat was given after irradiation it always reduced the irradiation-induced TBE.     

Variations in epidermal cytochrome oxidase activity after local irradiation

Summary Cytochrome oxidase activity was evaluated histochemically as an index of mitochondrial damage after local irradiation with X-rays. It was determined by microphotometry on the tail skin of newly born Wistar rats four days after irradiation with doses ranging from 2 to 16 krad.The enzyme activity of the whole epidermis increased after irradiation, the increases being related to the increase in thickness of the epithelium which was observed as a response to irradiation injury. Within the dose range tested, the enzyme concentration (expressed per unit volume of tissue) decreased in relation to the dose applied.At the electron microscopy level, the cytochemical demonstration of cytochrome oxidase revealed an irregular reaction over the cristae, intramitochondrial vacuolization and partial homogenization of the matrix. Positive membrane fragments were seen around lipid droplets. This reaction confirms the mitochondrial origin of these previously observed radiation-induced vacuoles.     

Potentially lethal radiation damage repair and its inhibition by hyperthermia in normal hamster cells, mouse cells, and transformed mouse cells

The capacity of plateau-phase Chinese hamster V79 and normal and transformed C3H-10T1/2 cells for repair of potentially lethal radiation damage (PLD) was evaluated for cells irradiated alone or given combined treatments of heat and radiation. The data show that all cell lines tested could repair PLD and that transformation to the tumorigenic state may reduce the capacity to repair PLD, especially if cells are evaluated at equal survival levels. Hyperthermia treatments before irradiation produced less sensitization than treatments after irradiation. In addition, hyperthermia treatment led to the inhibition of cellular capacity to repair PLD. This effect was the greatest for cells heated after irradiation, and repair of PLD could be completely eliminated. Several temperature isodose heat treatments were evaluated, and the lower temperature heat treatments were more effective in the inhibition of PLD than the higher temperature heat treatments; this is consistent with earlier results indicating temperature dependence in thermal radiosensitization (S. A. Sapareto et al., Int. J. Radiat. Oncol. Biol. Phys. 5, 343-347 (1979)).     

Dramatic Increase in Oxidative Stress in Carbon-Irradiated Normal Human Skin Fibroblasts

Skin complications were recently reported after carbon-ion (C-ion) radiation therapy. Oxidative stress is considered an important pathway in the appearance of late skin reactions. We evaluated oxidative stress in normal human skin fibroblasts after carbon-ion vs. X-ray irradiation. Survival curves and radiobiological parameters were calculated. DNA damage was quantified, as were lipid peroxidation (LPO), protein carbonylation and antioxidant enzyme activities. Reduced and oxidized glutathione ratios (GSH/GSSG) were determined. Proinflammatory cytokine secretion in culture supernatants was evaluated. The relative biological effectiveness (RBE) of C-ions vs. X-rays was 4.8 at D₀ (irradiation dose corresponding to a surviving fraction of 37%). Surviving fraction at 2 Gy (SF2) was 71.8% and 7.6% for X-rays and C-ions, respectively. Compared with X-rays, immediate DNA damage was increased less after C-ions, but a late increase was observed at D_10% (irradiation dose corresponding to a surviving fraction of 10%). LPO products and protein carbonyls were only increased 24 hours after C-ions. After X-rays, superoxide dismutase (SOD) activity was strongly increased immediately and on day 14 at D_0% (irradiation dose corresponding to a surviving fraction of around 0%), catalase activity was unchanged and glutathione peroxidase (GPx) activity was increased only on day 14. These activities were decreased after C-ions compared with X-rays. GSH/GSSG was unchanged after X-rays but was decreased immediately after C-ion irradiation before an increase from day 7. Secretion of IL-6 was increased at late times after X-ray irradiation. After C-ion irradiation, IL-6 concentration was increased on day 7 but was lower compared with X-rays at later times. C-ion effects on normal human skin fibroblasts seemed to be harmful in comparison with X-rays as they produce late DNA damage, LPO products and protein carbonyls, and as they decrease antioxidant defences. Mechanisms leading to this discrepancy between the two types of radiation should be investigated.     

Effects of X-irradiation on the kinetics of abnormal sperm production and sperm loss in the mouse

Exposure of male mice to 6 Gy of X-rays resulted in a very rapid and extensive sloughing of the germinal epithelium as shown by the accumulation of non-sperm cells within the lumen of the epididymis. These cells were identified as stage 1 and 2 round spermatids. After accumulating in the caput, they progressed through the epididymis over the weeks of sampling and, by Week 9 after irradiation, they had completely disappeared from the organ. It is suggested that the precocious loss of round spermatids is responsible for the induction of oligospermy within the testis and the caput epididymidis. Similar sperm losses from the cauda epididymidis were not observed. Radiation also enhanced the frequency of misshapen spermatozoa normally found in this strain. From kinetic considerations, it is suggested that the generation of abnormal spermatozoa may be biphasic with an early component comprising maturing spermatids and a late contingent composed of affected spermatocytes. Return to the pre-irradiation level of abnormal frequency was not observed within the time frame of this study (10 weeks), perhaps indicating residual damage. The synchrony that existed among the various organs in terms of both sperm loss and the generation of abnormal spermatozoa may be the result of a rapid dispersion of gametes from the testis and not due to local responses as would be expected if sperm flow were affected by the irradiation. The distribution of abnormal sperm types was different in the testis from that in the epididymis, presumably because of a testicular spermatophagic mechanism specific for the removal of certain deformities. It is concluded that the kinetics of spermatogenesis, of spermiogenesis, and of sperm transport in the mouse is not affected by exposure to 6 Gy of X-rays.     

The effect of sampling time on radiation-induced translocation yield in spermatogonial stem cells of male mice, differing in chromosomal constitution and sexual activity

We have investigated the frequency of reciprocal translocations in the first differentiating spermatogonia entering the first meiotic division after 2 x 2.5 Gy X-rays, given 24 h apart, as well as the development of this parameter in later stem-cell generations by studying multivalent configurations at the first meiotic division. Diakinesis-metaphase I cells were found for the first time between 30 and 40 days after irradiation. Subsequently, meiotic stages were sampled at 120, 180 and 280 days post irradiation. From day 40 post irradiation on, half of the males were allowed to impregnate females which enabled us to estimate the length of the post-irradiation sterile period, the development of litter size and the possible effect of sexual activity on the development of reciprocal translocation-containing stem cells. Half of the males were karyologically normal, the other half were homozygous for a reciprocal translocation (T/T) that affects testis weight and about halves sperm production. Irrespective of male karyotype, the first meiocytes had an induced translocation frequency of 9.00 +/- 2.56% (n = 8 males), followed by frequencies of 20.70 +/- 4.87% (n = 15) at 180 days and 20.20 +/- 4.30% (n = 20) at 280 days (males with and without mating behavior showing no difference). At 120 days post irradiation, +/+ males had a frequency of 14.59 +/- 2.97% irrespective of sexual activity. T/T males (120 days post irradiation) that had mated showed a frequency of 18.63 +/- 0.85% (n = 4) compared with 13.64 +/- 2.36% (n = 7) for those that had not. The observed rise of multivalent-carrying spermatocytes in time was highly significant. Notwithstanding the differences in testis weight and epididymal sperm count between the karyotypes, fertile matings occurred on average 72 days after irradiation, though with relatively wide margins. For the T/T karyotype, the first litter was statistically smaller than the subsequent litters. At 78 days post irradiation, testis weights were back in the subnormal range for both karyotypes and hardly improved in time. Restoration of fertility thus coincided with the period just prior to the return to subnormal testis weights. The first diakinesis-metaphase I cells precede those that are numerous enough to accomplish 'return to fertility' by about 2 weeks. Thus differentiation of stem-cell spermatogonia already follows a few days after irradiation. A pattern of spermatogonial cell divisions compatible with 'return to fertility' is only established some 2 weeks later.     

Interaction between radiation and cadmium or mercury in mouse embryos during organogenesis

The response of mouse embryos to different concentrations of cadmium or mercury with or without low LET radiation was measured in terms of gross morphological anomalies and cellular changes. Single doses of the heavy metals (2 mg/kg body weight) were injected i.p. on day 8 of gestation 30 min before whole-body irradiation. Combined exposures to CdCl2 and X-rays led to a significant reduction in the rate of exencephaly compared with the high frequency after cadmium alone. The hypothesis that metallothionein, a sulphur-rich and metal-binding protein, may be responsible for the antagonism observed could not be confirmed. Mercuric chloride alone induced a low rate of exencephaly and the data on combined treatment suggest additivity with 0.5 and 1.0 Gy X-rays. Regarding cellular criteria, cell death in the eye anlage on day 9 of gestation was significantly suppressed after 0.5 Gy and especially after CdCl2 plus 0.5 Gy compared to cadmium alone. It is assumed that the reduction of cell lethality is correlated with the low occurrence of exencephaly. Concerning the proliferation of neuroblasts, cadmium stimulated the mitotic activity whereas X-rays depressed the proliferation capacity. After the combined treatment a distinct antimitotic effect was established.     

Moderate hyperthermia and low dose rate irradiation

Moderate hyperthermia (4 h at 40 degrees C) enhances V-79 cell radiosensitivity at low irradiation dose rates with a maximum thermal enhancement ratio (TER) of 1.38. In comparison, the TER measured at acute dose rate is 1.13. Heat treatments given before and during irradiation are equally effective, and more so than postirradiation hyperthermia. Hyperthermia-induced inhibition of sublethal damage repair is a probable cause of the observed effect.     

Comparison of Three Inbred Mouse Strains with Respect to General and Genetic Radiosensitivity

The radiosensitivities of three mouse strains (BALB/cLacY, C3H/SnY, and 101/HY) have been compared using the following parameters: survival after irradiation at a dose of 6–7 Gy, chromosome aberration frequency in bone marrow cells after irradiation at a dose of 1.5 Gy, and the change in testis weight and frequency of abnormal sperm heads (ASHs) after irradiation at doses from 0.5 to 4 Gy. Strain BALB/c is the most radiosensitive with respect to the survival and chromosome aberration frequency in the bone marrow but the most resistant with respect to the change in testis weight and the frequency of abnormal sperm heads. Strain 101/HY was the most resistant with respect to survival and chromosome aberration frequency in bone marrow after irradiation but the most radiosensitive with respect to testis damage.     

Comparison of three inbred lines of mice by overall and genetic radiosensitivity

The radiosensitivities of three mouse strains (BALB/cLacY, C3H/SnY, and 101/HY) have been compared using the following parameters: survival after irradiation at a dose of 6-7 Gy, chromosome aberration frequency in bone marrow cells after irradiation at a dose of 1.5 Gy, and the change in testis weight and frequency of abnormal sperm heads (ASHs) after irradiation at doses from 0.5 to 4 Gy. Strain BALB/c is the most radiosensitive with respect to the survival and chromosome aberration frequency in the bone marrow but the most resistant with respect to the change in testis weight and the frequency of abnormal sperm heads. Strain 101/HY was the most resistant with respect to survival and chromosome aberration frequency in bone marrow after irradiation but the most radiosensitive with respect to testis damage.     

The response of previously irradiated skin to combinations of X radiation and ultrasound-induced hyperthermia

Areas of skin approximately 1.5 cm in diameter on the legs of mice were made hyperthermic (30 min at 42.7 degrees C) by exposure to an ultrasound beam (780 kHz), a single dose of X irradiation (2000 rad), or a combination of these treatments. After 35 days, when the acute reaction had reached a steady state, the same tissue was given a second treatment by either hyperthermia, irradiation, or a combination of hyperthermia and irradiation. When the first treatment was irradiation and the second treatment was either irradiation or a combination of hyperthermia and irradiation, the acute skin reactions were similar to those of skin not previously irradiated, indicating a large proportion of recovery from the first irradiation. When irradiation was the first treatment, a comparison of second treatments by hyperthermia plus irradiation with irradiation alone showed a thermal enhancement of 1.45. When the first treatment was hyperthermia plus irradiation, a comparison of second treatments by hyperthermia plus irradiation with irradiation also showed an enhancement factor of 1.45 for the combined treatment.     

Combined application of misonidazole and piotron pions on mouse embryos

Mouse embryos on day 8 of gestation were irradiated with negative pions (12.5-100 rad) or 200 kV X-rays (12.5-150 rad). Misonidazole (MISO), a hypoxic cell radiosensitizer, was applied 30 min before exposure. On day 13 the fetuses were examined for lethality, growth retardation and malformation. No significant embryolethal effects were observed after irradiation alone in the dose range of 12.5-100 rad (X-rays or pions). However, MISO alone and in combination with radiation led to high rates of lethality. The frequency of growth retardation was significantly increased at 100 rad and in combined treatments at low radiation doses. MISO and irradiation with 50 rad and more induced complex damages consisting of multiple and severe malformations and growth retardation. The relative biological effectiveness (RBE) for teratogenic effects was 1.6. In conclusion, the combined application of MISO and radiation of different LET revealed a strong enhancing action compared to single treatments. The extent of enhancement depends on both radiation quality and dose.