Low-dose effects of X-rays and negative pions on the pronuclear zygote stage of mouse embryos

Summary Mouse embryos in the pronuclear-zygote stage (day 0 of gestation) were irradiated with 13.5 rad of 140 kV X-rays or negative pions. On day 13 the fetuses were examined for developmental anomalies such as intrauterine death, growth retardation and malformations. Significant decreases in the percentage of normal implantations were obtained with peak pions (high LET) and X-rays, whereas the effect of plateau pions was less obvious. Irradiation with peak pions was more effective than with X-rays by a factor of about 1.7.     

Induction of congenital anomalies in offspring of female mice exposed to varying doses of X-rays

Female mice were exposed to varying absorbed doses (108–504 rad) of X-rays and mated at different intervals after irradiation (1–7, 8–14, 15–21 and 22–28 days). Uterine contents were examined at late pregnancy in order to detect early fetal deaths (dominant lethality) and malformations in the live fetuses.Two trends were apparent from data on abnormal fetuses. At each weekly interval, the incidence of abnormalities tended to rise with increase in dose, and, at any given dose, the incidence tended to increase with time after irradiation. Dwarfism and exencephaly were the two most common malformations found.The changes in incidence of dominant lethality and of abnormal fetuses with time and with dose follow each other closely, the highest incidence for both being reached in week 3 (59±4.7% for dominant lethals and 12.5±3.1% for abnormal fetuses, after 504 rad) indicating increased radiosensitivity of less mature oocytes. These results parallel those obtained from known genetic effects reported by other workers and suggest that testing for incidence of congenital malformations among offspring of treated animals may prove a useful means of assessing genetic hazards of radiation of chemicals.     

Effect of negative pi mesons on vascular permeability of brain in neonatal rats

Summary Neonatal rats and their radiation-induced cerebral petechial hemorrhages were used as an experimental system for evaluating the effects of negative pions on healthy tissue and especially on the microvasculature. Dose response curves for peak and plateau pions (dose range 150–250 rad and 100–400 rad, respectively) were obtained and compared with those of 200 kV X-rays of corresponding dose rates. The RBE of the peak pions was 1.1, that of the plateau pions 0.6 resulting in a peak/plateau ratio of 1.8. Implications were made as to the importance of this favorable peak/plateau relationship since the response of the capillary endothelium to pion-irradiation might be one of the limiting factors in radiotherapy.     

Chromatid breaks induced in Chinese hamster cells by low doses (12–100 rad) ofπ −-mesons

Summary Monolayer cultures of the fibroblast-like Chinese hamster cell-line 19/1 were irradiated in the G₂-phase of the cell cycle by ^–-mesons (6 rad/min peak-pion dose rate). Frequencies of induced single- and isochromatid breaks, acentric fragments and interchanges were compared with data obtained from 140 kV X-rays.The RBE-values were for the pion dose peak between 0.8–1.2 and for the pion dose plateau 0.5–0.9. Whereas for single chromatid breaks there was no significant difference between X-rays and peak pions for identical physical doses, the isochromatid breaks alone showed a significantly higher frequency for 100 rad peak pions.     

Preclinical Radiobiology with Pions: RBE — Values for Mouse Skin Damage as a Function of Single Doses of Pions

Summary The macroscopic reaction of the mouse skin was used to derive RBE values for negative-Mesons. Hind limbs of mice were irradiated with pions or X-rays. The pions were produced by the 590 MeV accelerator of the Schweizerisches Institut für Nuklearforschung (SIN). Early skin reaction was assessed over a period of 6–30 days after irradiation with single doses (20–45 Gy). The radiation damage was scored using an arbitrary scale of effect. The time pattern of development of the skin reaction and the subsequent healing after exposure both to pions and X-rays were similar, indicating that depletion and repopulation of the basal cells of the skin were comparable, both after pions and X-rays. RBE values as a function of pion doses at the peak (dose maximum), plateau and at the postpeak (12 mm downstream of the dose maximum) were computed with nonparametric statistical methods. The RBE at the peak and at the plateau relative to X-rays of the same dose rate was 1.15–1.25 and 0.85, respectively. The RBE of peak pions manifested a marked dependence on dose, when plateau pions were chosen as reference radiation. In this experiment there was no significant difference in RBE between peak and postpeak. The importance of some experimental condition (dose rate, irradiation volume) is discussed.Supported by the Swiss National Science Foundation (grant no. 3.682-0.75)     

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.     

Cytogenetic effects of X-rays and fission neutrons in female mice

The induction by X-rays of chromosomal damage in oocytes was studied, while the genetic consequences of X- and neutron-induced damage in female mice were looked for by testing offspring for dominant lethality and semi-sterility. None out of 386 sons of hybrid females given 300 rad X-rays showed evidence of semi-sterility or translocation heterozygosity, but 9 out of 294 daughters were diagnosed as semi-sterile. At least 3 and probably 4 of these (1.4%) carried reciprocal translocations, 2 of which caused male sterility. Complete or partial loss of the X-chromosome may have been responsible for some of the other sermi-steriles. Examination of oocytes at metaphase-I during the first and third weeks after X-irradiation with 100 or 400 rad revealed both multivalents (some of the ring quadrivalent type) and fragments (mainly double). These were thought to arise mainly from chromatid intercchanges (both symmetrical and asymmetrical) and isochromatid intrachanges respectively. Since neither the proportion of asymmetrical interchanges nor the amount of hidden damage was known it was not thought possible to predict the magnitude of F₁ effects from metaphase-I findings. The aberration frequency in oocytes rose with dose and (at the 400 rad level only) with time after irradiation, reaching a maximum of 10% multivalents and 22% fragments in the third week after 400 rad. The frequency of univalents showed no consistent trend, but chiasma counts decreased in the first week after 400 rad. The increase in levels of chromosomal damage with dose and time after irradiation was reflected in dominant lethal frequencies after the same radiation-conception intervals and doses of 0–400 rad. Induced post-implantation lethality was over twice as high in the third week after 200–400 rad than in the first. Pre-implantation loss also greatly increased in the third week after 300 or 400 rad; this was associated with increased non-fertilization of ova. No evidence for the induction of translocations in oogonia or resting oocytes by fast neutron irradiation was obtained, although there was evidence for X-chromosomal loss after 200 rad to oocytes. The relative biological effectiveness (RBE) for fission neutrons vs. X-rays with respect to dominant lethal induction in oocytes was found to vary with dose, but seamed to be around 1 at lower levels.     

Combined genotoxic effects of radiation and a tobacco-specific nitrosamine in the lung of gpt delta transgenic mice

It is important to evaluate the health effects of low-dose-rate or low-dose radiation in combination with chemicals as humans are exposed to a variety of chemical agents. Here, we examined combined genotoxic effects of low-dose-rate radiation and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the most carcinogenic tobacco-specific nitrosamine, in the lung of gpt delta transgenic mice. In this mouse model, base substitutions and deletions can be separately analyzed by gpt and Spi- selections, respectively. Female gpt delta mice were either treated with gamma-irradiation alone at a dose rate of 0.5, 1.0 or 1.5 mGy/h for 22 h/day for 31 days or combined with NNK treatments at a dose of 2 mg/mouse/day, i.p. for four consecutive days in the middle course of irradiation. In the gpt selection, the NNK treatments enhanced the mutation frequencies (MFs) significantly, but no obvious combined effects of gamma-irradiation were observable at any given radiation dose. In contrast, NNK treatments appeared to suppress the Spi- large deletions. In the Spi- selection, the MFs of deletions more than 1 kb in size increased in a dose-dependent manner. When NNK treatments were combined, the dose-response curve became bell-shaped where the MF at the highest radiation dose decreased substantially. These results suggest that NNK treatments may elicit an adaptive response that eliminates cells bearing radiation-induced double-strand breaks in DNA. Possible mechanisms underlying the combined genotoxicity of radiation and NNK are discussed, and the importance of evaluation of combined genotoxicity of more than one agent is emphasized.     

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.     

Effects of topically-applied olive oil on the response of hamster skin to single or multiple doses of 230kV X-rays.

The effects of topically-applied olive oil on the response of hamster skin to single or multiple doses of X-rays has been studied. The olive oil was applied either 15 min or 1 hour before the radiation exposures. The treatment did not alter the temporal pattern of development and recovery from the radiation injury. For single exposers, olive oil did not alter the 1- to 30-day average skin response. However, when it was administered at each treatment when three radiation fractions were given over a 4-day interval (3 fractions/4 days), a significant increase in the amount of dose recovered was found compared with control irradiated animals. For controls, the average amount of dose recovered per fractionation interval, (Dn-D1)/(n-1), was about 505 rad. For animals treated with olive oil 15 min before irradiation, it was about 720 rad; and for those treated 1 hour before irradiation, it was 782 rad. The data indicate a definite radioprotective effect of topical administration of olive oil, but at present the mechanism is not known.     

The response of mouse skin to combined hyperthermia and X-rays.

The effects of combined hyperthermia and X-irradiation were studied in the skin of the mouse ear. Ears were heated for 1 hour by immersion in a waterbath at temperatures ranging from 37 degrees C--43 degrees C. These heat treatments had little visible effect alone, but when combined with X-rays, enhanced the radiation response. Enhancement depended on the degree of heating. When heat was given immediately after X-rays, the radiation dose to cause a given skin reaction had to be reduced by about 10 per cent for 37 degrees C and about 40 per cent of 43 degrees C. The timing and sequence of the two treatments were important. Heat after X-rays was less effective than heat before X-rays. When heat followed X-rays, the enhancing effect was lost completely if the interval exceeded 4 hours. When heat preceded X-rays, the effect was lost more slowly, depending on temperature. The implications of this for the treatment of cancer by combined therapy are discussed.     

The antiangiogenic agents SU5416 and SU6668 increase the antitumor effects of fractionated irradiation.

Angiogenesis is critical for tumor development, growth and metastasis. The vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) and their tyrosine kinase receptors are major regulators of angiogenesis. Radiation induces the production of VEGF, FGF and PDGF in many tumor cells. We hypothesized that inhibition of the function of these growth factors could inhibit tumor angiogenesis and thereby enhance the efficacy of radiation therapy. To test this hypothesis, we used the small molecule inhibitors SU5416 (an inhibitor for Vegf receptor) and SU6668 (an inhibitor for Vegf, Fgf and Pdgf receptors) alone and in combination with fractionated irradiation to treat C3H mice bearing SCC VII carcinomas. The SCC VII tumors express Vegf, Fgf2 (also known as bFGF), Pdgf and their associated receptors. Animals were given either SU5416 or SU6668 daily before or after irradiation (2 Gy per fraction per day for 5 days). The results from these experiments demonstrate that administration of either SU5416 or SU6668 without radiation delayed tumor growth. Administration of SU5416 at a dose of 25 mg/kg per day (the maximum tolerated effective dose) inhibited tumor growth by 17.9% on day 7 (P < 0.05 compared to untreated control mice) and produced an average tumor growth delay time of 0.5-2.0 days. When combined with fractionated irradiation, administration of SU5416 increased the inhibition of tumor growth to 50-53% on day 7 and the tumor growth delay time to 5.7-6.5 days (P < 0.001 compared with SU5416 alone; P < or = 0.05 compared with radiation alone). SU6668 alone inhibited tumor growth in a dose-dependent manner. Administration of SU6668 at a dose of 75 mg/kg per day (a suboptimal dose) inhibited tumor growth by 36% on day 7 and produced an average tumor growth delay time of 3.3 +/- 1.4 days. The combination of SU6668 with fractionated radiation increased inhibition of tumor growth to 66-70% and the tumor growth delay time from 3.3 days to 11.9 days (P < or = 0.001 compared with either radiation alone or SU6668 alone). Administration of these agents before or after irradiation produced similar results (P = 0.40 for SU5416; P = 0.98 for SU6668). SU5416 or SU6668 alone or in combination with radiation was very well tolerated with little or no toxicity. These results suggest that inhibition of Vegf, Fgf and Pdgf receptor function by SU5416 and SU6668 can enhance the efficacy of irradiation. The targeting of multiple tyrosine kinase receptors by SU6668 is more effective than inhibition of the Vegf receptor alone by SU5416 for the enhancement of tumor cell killing by fractionated irradiation.     

Piotron pion irradiation of cerebral capillaries in neonatal rats

Summary The microvasculature of the neonatal rat brain and its radiation-induced petechial hemorrhages were used as an in vivo model for studying the effect of negative pions on healthy normal tissue. By means of dose response curves (range: 2–6 gray) the RBE of peak pions with respect to 220 kVp X-rays was calculated. Whereas earlier studies involved irradiations with single beam pions (E3) of low dose rate (0.06–0.11 Gy per min) the present work was performed using pions of a multiport applicator (Piotron) of higher dose rate (1 Gy per min). The results obtained by Piotron pions agreed well with those obtained byE3 pions resulting in a RBE of 1.1. The difference between pion and X-rays, however, was statistically not significant. 0-(beta-hydroxyethyl)-rutosides known for their protective effect on radiation damage induced by either X-rays or electrons were used for evaluating any radioprotective effect on pion-induced damage of the microvasculature. They were applied subcutaneously in doses of 450 mg/kg body weight 30 min prior to irradiation. It could be demonstrated that the rutosides decreased the pion-induced radiation response significantly by a factor of 1.3.     

Combined effect of misonidazole and glutathione depletion by buthionine sulphoximine on cellular radiation response

Chinese hamster cells (V79) and glutathione-proficient (GSH+/+) and glutathione-deficient (GSH-/-) human fibroblasts were treated with a glutathione (GSH)-depleting agent buthionine sulphoximine (BSO) and the hypoxic radiosensitizer misonidazole (MISO), separately or in combination. Subsequently, the cells were exposed to X-rays. Determination of the yield of single-strand DNA breaks (ssb) immediately after irradiation indicated no effect of BSO or MISO treatment when radiation exposure was made aerobically. Assuming that ssb determined immediately after irradiation reflects mainly the effect of radical processes, the results obtained with BSO and MISO, singly and in combination, agreed well with the predictions of a modified version of the 'competition model' using V79 and GSH+/+ cells. Some results obtained with GSH-/- cells could not be so explained.     

Serial analysis of chromosomal aberrations and proliferation kinetics of mouse spermatogonia after single or fractionated X-ray exposures

Dose-fractionation studies on translocation induction in stem-cell spermatogonia of mice, as measured by spermatocyte analysis many cell generations after irradiation, revealed that a small conditioning dose of X-rays sensitizes the stem cells to the induction of translocations by a second dose 24 h later (Van Buul and Léonard, 1974, 1980). To find out whether such sensitization effects also occur at other spermatogonial stages, a comparison was made of the effects of single (50, 100 and 150 rad) and fractionated (100 + 50 rad, with 24 h in between) doses of X-rays on the induction of chromosomal aberrations in spermatogonia by analysing spermatogonial metaphases shortly after irradiation at multiple sampling times (0–48 h; every 4 h). In addition, the kinetics of spermatogonial proliferation was studied by using, in vivo, a BrdU chromosome-labelling procedure. The recorded frequencies of chromosomal aberrations did not indicate any sensitization effect of dose fractionation. It is concluded that the sensitization effects, as observed for chromosomal aberrations in male premeiotic germ cells, are characteristic for the stem-cell spermatogonia and do not occur in the more differentiated spermatogonia.     

Combined effects of radiation and caffeine on embryonic development in mice

The combined effect of radiation and caffeine has been studied in mouse embryos. Radiation and/or caffeine were administered to ICR mice on Day 11 of gestation. Intrauterine death, gross malformation, and fetal body weight were selected as indicators of effects. Doses of whole-body gamma irradiation were 0.5 to 2.5 Gy and those of caffeine were 100 and 250 mg/kg maternal body wt. Intrauterine mortality increased with increasing radiation dose; this trend was more remarkable in combination with caffeine. Gross malformations such as cleft palate and defects of forelegs and hindlegs appeared frequently in the fetuses treated with both radiation and caffeine. Decreased fetal weight was observed even in mice treated with 0.5 Gy of radiation or 100 mg/kg caffeine. There was a linear relationship between dose and reduction of fetal weight. The fetal weight was a sensitive, precise, and easy-to-handle indicator for the effects of growth retardation. Intrauterine mortality and frequencies of cleft palate and defects of forelegs and hindlegs were higher than the sum of those induced by radiation and by caffeine separately. The results indicated that the combined action of radiation and caffeine on intrauterine death and malformations was synergistic.     

Reproductive and genetic effects of continuous prenatal irradiation in the pig

The stem germ cells of the prenatal pig are highly vulnerable to the cytotoxic effects of ionizing irradiation. This study was conducted to determine whether sensitivity to killing was also marked by a sensitivity to mutation and how prenatal depletion of the germ-cell population affects reproductive performance. Germ-cell populations were reduced by continuously irradiating sows at dose rates of either 0.25 or 1.0 rad/day for the first 108 days of gestation. The prenatally irradiated boars were tested for sperm-producing ability, sperm abnormalities, dominant lethality, reciprocal translocations, and fertility. Prenatally irradiated females were allowed to bear and nurture one litter, then tested for dominant lethality in a second litter; germ cell survival and follicular development were assessed in their serially sectioned ovaries. Sperm production was not significantly affected in the 0.25-rad boars, but boars irradiated with 1.0 rad per day produced sperm at only 17% of the control level. Incidence of defective sperm was 4.9% and 11.1% in the 0.25 and 1.0 groups, respectively. Four of the 1.0-rad boars were infertile, but prenatal irradiation apparently caused neither dominant lethality nor reciprocal translocations in fertile males. Number of oocytes was reduced to 66 +/- 7% of control in the 0.25-rad gilts, but reproductive performance was unaffected and no dominant lethality was observed. Only 7 +/- 1% of the oocytes survived in the 1.0-rad group. Reproductive performance was normal for the first litter, but four of the 23 sows tested were infertile at the second litter and a significant incidence of dominant lethality was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Quantitative changes in the arterial blood gases of mice following localized irradiation of the lungs

The arterial pH and partial pressures of oxygen (PaO2) and carbon dioxide (PaCO2) were evaluated in LAF 1 mice 15 and 38 weeks after localized irradiation of the animals' thoraxes. Graded radiation doses of 900 to 1200 rad were administered. These doses resulted in 0 to 100% lethality by 26 weeks (180 days) after irradiation. At 15 weeks after treatment mice receiving radiation doses which would subsequently result in lethality (by 180 days) exhibited significant reductions in their PaO2 and elevations in their PaCO2 values, respectively. However, there was no clear dose-response relationship between blood gas values and radiation dose, which may reflect the animals' ability to compensate for their poor blood gas exchange by an increased breathing frequency. At 38 weeks after irradiation the blood gas values were abnormal in mice from groups which had normal blood gas values at Week 15 (and no fatalities by Week 26) but in which animal deaths had occurred between Weeks 26 and 38. These data therefore indicated (i) that abnormal blood gas values occurred in the mice prior to fatalities resulting from the acute radiation pneumonitis syndrome and (ii) that mice surviving the initial radiation pneumonitis phase could still succumb to progressive pulmonary toxicity which was reflected by the increasing levels of animal lethality and altered blood gas tensions at the later times.     

Enhanced Post-Irradiation Recovery of the Haemopoietic System In Animals Pretreated With A Variety of Cytotoxic Agents

It is known that pretreatment of mice with bacterial endotoxin and certain stathmokinetic agents between 1 and 3 days prior to exposure to ionizing radiation reduce radiation lethality. In this communication it is shown that pretreatment with cytosine arabinoside, methotrexate, nortestosterone and chlorambucil reduces radiation (1000 rad) induced lethality. This reduction can be ascribed to enhanced regeneration of the haemopoietic system in pretreated animals and not to increased survival of colony-forming cells (CFU) in these animals. Regeneration of CFUs was underway within 24 hr after 900 rad in the pretreated mice but did not start until day 3 in mice treated with γ radiation only. Two agents, namely radiation itself (either 75 or 150 rad) and busulphan (10 mg/kg) did not reduce the lethal effects of subsequent γ irradiation nor enhance the regeneration of CFUs, even though radiation, like the protective cytosine arabinoside, induces early CFUs proliferation. The administration of nucleoside precursors of DNA enhanced regrowth of haemopoietic stem cells to an extent comparable with that of the most effective pretreatment, cytosine arabinoside. It is postulated that drugs like cytosine arabinoside operate by causing cell death, providing a source of DNA that can enhance the regrowth of surviving stem cells in the bone marrow.