Infectivity and Immunogenicity of Irradiated Babesia rodhaini*

SYNOPSIS. Babesia rodhaini-parasitized mouse blood exposed to varied doses of γ radiation up to 30 kRad was inoculated into mice. Mice inoculated with nonirradiated B. rodhaini developed progressive infections and died 7–11 days postinoculation. Mice infected with B. rodhaini-parasitized blood exposed to doses up to and including 22 kRad developed progressive parasitemias which were delayed in comparison to mice inoculated with non-irradiated B. rodhaini. Some mice receiving parasitized blood irradiated at 26 kRad did not develop progressive parasitemias. Progressive infections were prevented by exposure to irradiation at 30 kRad. The results of 2 separate experiments revealed that one inoculation of parasitized blood exposed to 30 kRad or higher apparently stimulated a resistance to a challenge infection with nonirradiated parasitized blood. While 20 of 20 control mice died as a result of challenging infections, 9 of 28 mice previously exposed to irradiated parasitized blood survived. The injection of irradiated nonparasitized blood did not produce a discernible acquired resistance to B. rodhaini. Presumably the irradiated parasitized blood was responsible for the development of acquired resistance to B. rodhaini.     

On the Wholesomeness of γ-Irradiated Potatoes

Fattening pigs and pigs for breeding have been fed γ-irradiated and non-irradiated control potatoes. The irradiation dose was 14–15 kilorad at the rates 175 r/sec. and 625 r/hr. Rats (Wistar) were also fed irradiated and control potatoes, but in this case the tubers were given a dose of about 200 kilorad. No unfavourable effects have been observed from the feeding with irradiated potatoes. All the facts indicate that the nutritional adequacy of the irradiated and the control tubers is equal. In some cases, the animals have even shown an improvement after feeding on irradiated potatoes which was not apparent after feeding on non-irradiated potatoes; for example there was a more rapid growth, somewhat higher fertility, and increased haemoglobin values. However, current experiments with larger groups of animals will provide more definite evidence in these questions.     

Frequency of micronuclei in 4–8 cell mouse embryos generated after maternal gamma-irradiation in the presence and in the absence of vitamin C

The objective of this investigation was to evaluate the frequency of chromosomal aberrations expressed as micronuclei (MN) in 4–8 cell embryos generated by gamma-irradiation of female mice in the absence and in the presence of vitamin C. Female NMRI mice were whole body exposed to 4 Gy gamma-irradiation after intraperitoneal (i.p.) injection of pregnant mare’s serum gonadotrophin (PMSG) followed by injection of human chorionic gonadotrophin (HCG) and mating with non-irradiated NMRI male mice. Pregnant animals were sacrificed and embryos flushed from the oviducts and fixed on slides. Cells were treated for MN observation using standard method. To investigate the protective effect of vitamin C (ascorbic acid) on the frequency of MN, 100 mg/kg vitamin C was i.p. injected 1 h before irradiation. Results show that the frequency of MN generated in the embryos of irradiated mother compared to those of control in the non-irradiated group increased dramatically (P < 0.001). Frequency of MN in embryos generated in irradiated female mice treated with vitamin C dramatically and statistically decreased relative to the frequency observed in the irradiation only group (P < 0.001). This decrease returned the combined treatment group to a level that was not statistically different from the controls (P > 0.05). Thus, irradiation of preovulatory stage oocytes leads to stable chromosome abnormalities expressed as micronuclei in successive preimplantation embryos. Vitamin C reduces these clastogenic effects of radiation in preovulatory oocytes and thus the reduced frequency of MN in embryos is probably due to its antioxidation and radical scavenging properties.     

Effects of irradiated medium with or without cells on bystander cell responses

Recent studies have indicated that extranuclear or extracellular targets are important in mediating the bystander genotoxic effects of alpha-particles. In the present study, human-hamster hybrid (A(L)) cells were plated on either one or both sides of double-mylar dishes 2-4 days before irradiation, depending on the density requirement of experiments. One side (with or without cells) was irradiated with alpha-particles (from 0.1 to 100 Gy) using the track segment mode of a 4 MeV Van de Graaff accelerator. After irradiation, cells were kept in the dishes for either 1 or 48 h. The non-irradiated cells were then collected and assayed for both survival and mutation. When one side with cells was irradiated by alpha-particles (1, 10 and 100 Gy), the surviving fraction among the non-irradiated cells was significantly lower than that of control after 48 h co-culture. However, such a change was not detected after 1h co-culture or when medium alone was irradiated. Furthermore, co-cultivation with irradiated cells had no significant effect on the spontaneous mutagenic yield of non-irradiated cells collected from the other half of the double-mylar dishes. These results suggested that irradiated cells released certain cytotoxic factor(s) into the culture medium that killed the non-irradiated cells. However, such factor(s) had little effect on mutation induction. Our results suggest that different bystander end points may involve different mechanisms with different cell types.     

Enhancement of transformation rates in higher plants by low-dose irradiation: Are DNA repair systems involved in the incorporation of exogenous DNA into the plant genome?

Irradiation (X-ray; 5–15 Gy) of protoplasts treated with plasmid-DNA and PEG yielded higher transformation rates in comparison to non-irradiated protoplasts transformed by the same method. This could be demonstrated for four plant species. The irradiation doses used did not affect the total number of colonies regenerated without selection pressure, but resulted in 3–6-fold enhancement of hygromycin- or kanamycin-resistant colonies. Plant regeneration frequencies of transformed colonies derived from irradiated and non-irradiated protoplasts were similar in tobacco as well as in Petunia. Higher integration rates of foreign DNA as a consequence of an increased recombination machinery in irradiated cells may be responsible for the enhancement of the number of stably transformed colonies.     

Bystander normal human fibroblasts reduce damage response in radiation targeted cancer cells through intercellular ROS level modulation

The radiation-induced bystander effect is a well-established phenomenon which results in damage in non-irradiated cells in response to signaling from irradiated cells. Since communication between irradiated and bystander cells could be reciprocal, we examined the mutual bystander response between irradiated cells and co-cultured with them non-irradiated recipients. Using a transwell culture system, irradiated human melanoma (Me45) cells were co-cultured with non-irradiated Me45 cells or normal human dermal fibroblasts (NHDF) and vice versa. The frequency of micronuclei and of apoptosis, ROS level, and mitochondrial membrane potential were used as the endpoints. Irradiated Me45 and NHDF cells induced conventional bystander effects detected as modest increases of the frequency of micronuclei and apoptosis in both recipient neighbors; the increase of apoptosis was especially high in NHDF cells co-cultured with irradiated Me45 cells. However, the frequencies of micronuclei and apoptosis in irradiated Me45 cells co-cultured with NHDF cells were significantly reduced in comparison with those cultured alone. This protective effect was not observed when irradiated melanomas were co-cultured with non-irradiated cells of the same line, or when irradiated NHDF fibroblasts were co-cultured with bystander melanomas. The increase of micronuclei and apoptosis in irradiated Me45 cells was paralleled by an increase in the level of intracellular reactive oxygen species (ROS), which was reduced significantly when they were co-cultured for 24h with NHDF cells. A small but significant elevation of ROS level in NHDF cells shortly after irradiation was also reduced by co-culture with non-irradiated NHDF cells. We propose that in response to signals from irradiated cells, non-irradiated NHDF cells trigger rescue signals, whose nature remains to be elucidated, which modify the redox status in irradiated cells. This inverse bystander effect may potentially have implications in clinical radiotherapy.     

Alkaline and acid phosphatase activity in murine femoral bone marrow following X-irradiation, or X-irradiation and repopulation with syngenic or allogeneic bone marrow or marrow stroma cells

The activity of alkaline and acid phosphatases in the bone marrow from the femoral cavity was investigated in the following groups of mice: (1) normal (non-irradiated); (2) irradiated with 600 R; (3) irradiated and repopulated with syngeneic bone marrow; (4) irradiated and repopulated with syngeneic marrow stroma; (5) non-irradiated, infused with allogeneic bone marrow (host versus graft reaction, HvG); (6) irradiated and repopulated with allogeneic bone marrow (graft versus host reaction, GvH). In addition, the activity of alkaline and acid phosphatases was examined in bone marrow stromal cultures. In irradiated animals the activity of both enzymes was lower than in non-irradiated ones, repopulation with syngeneic bone marrow restoring it to normal. Repopulation with allogeneic marrow (GvH) resulted in a very deep reduction of alkaline, but not acid, phosphatase. It is postulated that the decrease in bone marrow alkaline phosphatase activity can be a sensitive test for the early GvH reaction, preceding such parameters as splenomegaly. Marrow stroma cultured in vitro also showed very low alkaline phosphatase activity.     

Enhancement of the Tolerance to Oxidative Stress in Cucumber (Cucumis sativus L.) Seedlings by UV-B Irradiation: Possible Involvement of Phenolic Compounds and Antioxidative Enzymes

Cucumis sativus L.) seedlings were irradiated or not irradiated with UV-B for several days in environment-controlled growth chambers. The first leaves irradiated with UV-B were retarded in growth but simultaneously acquired a remarkably high tolerance to oxidative stress, as induced by paraquat treatment, compared with the non-irradiated leaves. This enhanced tolerance was observed within 1d after the start of UV-B irradiation and was maintained during the 12 d period of UV-B treatment. The effects of UV-B on several antioxidative enzymes were examined, and activities of superoxide dismutase, ascorbate peroxidase and guaiacol peroxidase, but not of glutathione reductase, were found to be enhanced. However, activation of these enzymes occurred only from 6 d after the start of irradiation. In contrast, accumulation of phenolic compounds was observed within 1d after the start of UV-B irradiation. HPLC analysis of phenolic compounds showed the distinct enhancement of a substance, which may have antioxidative properties in cucumber seedlings irradiated with UV-B. On the basis of these results, we conclude that not only antioxidative enzymes but also other factors in cucumber seedlings irradiated with UV-B, such as phenolic compounds, may participate in the enhanced tolerance to oxidative stress. Received 10 June 2000/ Accepted in revised form 1 July 2000     

Lack of effect on the chromosomal non-disjunction in aged female mice after low dose X-irradiation.

Karyotypes were determined in 1064 embryos of aged C57/BL mothers. The virgin female mice were irradiated with 0, 4, 8 or 16 R of X-rays, respectively, and placed with young untreated males 5 days after irradiation. 10.5-days old embryos were recovered from the uterus. Aneuploid embryos classified as alive (heart beats observed at the dissection) were 1 monosomic in the control group (496 embryos) and 2 trisomics in the irradiated group (568 embryos). The number of aneuploid embryos classified as dead was 4 trisomic cases in the control group and 3 trisomics in the irradiated group. The data indicate that trisomic embryos are not uncommon in the mouse but are eliminated in post-implantation death. In contrast to the results of Yamamoto et al. the present data do not demonstrate an increased frequency of chromosome abnormalities in embryos of aged mice X-irradiated before mating as compared to non-irradiated ones.     

The effect of erythropoietin on the growth and development of spleen colony-forming cells

The progressive growth and development of spleen colonies was studied in heavily irradiated host mice in which erythropoiesis was modified by various procedures. Erythropoietic activity in non-polycythemic hosts bearing spleen colonies was not increased by injections of exogenous erythropoietin. Detectable levels of erythropoietin were found in the heavily irradiated host mice suggesting that the failure of exogenous erythropoietin to modify erythropoiesis was because the host mice were already maximally stimulated by the high endogenous erythropoietin levels. Spleen colonies do not become erythroid in polycythemic mice. The injection of exogenous erythropoietin into heavily irradiated polycythemic hosts did not decrease the total number of spleen colonies produced by a given bone marrow transplant, as would be expected if erythropoietin acted directly on the colony-forming cells. Comparison of growth curves for colony-forming cells in the spleens of polycythemic hosts either receiving or not receiving erythropoietin indicated that the overall doubling time of colony-forming cells during the first ten days after transplantation was not changed by the daily injection of erythropoietin. These experiments are consistent with the concept that erythropoietin is necessary for the development of erythroid colonies. Erythropoietin acts upon some progeny of the colony-forming cell rather than the colony-forming cell itself.     

Effect of the balanced inhibition of DNA and protein synthesis on the in vitro and in vivo proliferative activity of the lymphocytes]

Lymphocytes isolated from the rabbit peripheral blood were irradiated in vitro with 200, 400 and 1000 r doses and cultivated with phytohemagglutinin (PHA) doses and cultivated with phytohemagglutinin (PHA) at 37 degrees C during 48 hours. In several experiments cycloheximide, inhibiting protein synthesis, was added to the cells 60 minutes before and 30 minutes after irradiation. There was no apparent difference in the viability of irradiated cells with or without cycloheximide. The ability of lymphocytes of the popliteal lymph nodes for proliferation after PHA injection into one of the hind foot-pads of the irradiated mice was studied, as well. The injection of cycloheximide or puromycin into one of the hind foot-pads immediately after irradiation of the animals augmented the proliferation of lymphocytes in this extremity in comparison with contralateral one, 1.5-2 times. Cytosine arabinoside, inhibiting DNA synthesis, was not effective under these conditions.     

Keratinocytes control the proliferation and differentiation of cultured epidermal melanocytes from ultraviolet radiation B-induced pigmented spots in the dorsal skin of hairless mice

Long-term exposure of ultraviolet radiation B (UVB)-induced pigmented spots in the dorsal skin of hairless mice of Hos:(HR-1 X HR//De) F1. Previous study showed that the proliferative and differentiative activities of cultured epidermal melanoblasts/melanocytes from UVB-induced pigmented spots increased with the development of the pigmented spots. To determine whether the increase in the proliferative and differentiative activities of epidermal melanoblasts/melanocytes was brought about by direct changes in melanocytes, or by indirect changes in surrounding keratinocytes, pure cultured melanoblasts/melanocytes and keratinocytes were prepared and co-cultured in combination with control and irradiated mice in a serum-free culture medium. Keratinocytes from irradiated mice stimulated the proliferation and differentiation of both neonatal and adult non-irradiated melanoblasts/melanocytes more greatly than those from non-irradiated mice. In contrast, both non-irradiated and irradiated adult melanocytes proliferated and differentiated similarly when they were co-cultured with irradiated adult keratinocytes. These results suggest that the increased proliferative and differentiative activities of mouse epidermal melanocytes from UVB-induced pigmented spots are regulated by keratinocytes, rather than melanocytes.     

The effect of a mammalian repair endonuclease on x-irradiated DNA.

DNA was extracted from rat liver of non-irradiated animals, and was irradiated in vitro, and from animals which received whole body doses of X-radiation. Sedimentation on neutral and alkaline sucrose gradients as well as measurements of 32P release after sequential treatment with endonuclease and alkaline phosphatase and determination of triphosphate incorporation after the sequential treatment with endonuclease, alkaline phosphatase and DNA polymerase indicated that DNA irradiated in vivo and in vitro were effective substrates for the mammalian repair endonuclease. The experiments suggest that in addition to strand breaks, X-radiation causes base damage and they have provided a plausible explanation for the formation of double strand breaks in DNA irradiated in vivo.     

Biochemical states of zinc incorporated in the liver of mice treated with a radioelement after irradiation with a sublethal dose of gamma-rays

Radioresistance has been observed in mice which have received an inorganic zinc 24 hours prior to gamma irradiation with a sublethal dose. In order to elucidate the mechanism of radioresistance, the biochemical states of the metal in filtrate extracted from the homogenated liver of the animal which have received an injection of zinc under the form of 65Zn as tracer 24 hours post irradiation, were analyzed and compared with those of non-irradiated (control) mice. Two peaks of radioactivity: P-1 and P-2, having molecular weight of about 45,000 and 20,000-25,000 respectively, were revealed by Sephadex G-75 separation. The radioactivity of P-2 (39%) from the irradiated mice is lower than that from the controls (46%). Chromatography of P-2 from control animals through DEAE-Sephadex A-50 revealed the existence of a large radioactive peak (P-U). Chromatography of P-2 from irradiated animals did not show any P-U peak. This P-U peak is composed principally from 2 to 3 proteins with a molecular weight of 12,000 to 26,000. Proteins of the P-U peak provoke a protection effect against the lethal effect of gamma-rays when they are injected into mice after irradiation.     

Irradiated stem cells and ageing of the haematopoietic system

In the work presented here, changes in haematopoiesis of mice (B6129SF2/J) were studied 1 year after their whole-body exposure to a dose of 7 Gy (72% of mice survived). The irradiated mice were compared with non-irradiated younger (4 months of age) and older (16 months of age) mice. There was a significant increase in the relative abundance of primitive stem cells with long-term capability of the haematopoiesis recovery lin⁻/Sca-1⁺/CD117⁺/CD34⁻ in the bone marrow of mice aged 16 months (irradiated and non-irradiated) compared with those aged 4 months. In terms of the ability to respond to further whole-body irradiation at a dose of 1 Gy, the presence of γH2A.X foci was studied in lin⁻ bone marrow cells. There was a considerable number of persisting foci in lin⁻ stem cells isolated from the bone marrow of the older irradiated mice. In the blood count from the peripheral blood of the older mice (both non-irradiated and irradiated at 7 Gy), there was a significant increase in granulocytes. In the group exposed to 7 Gy, the numbers of thrombocytes significantly increased, and on the contrary, the numbers of erythrocytes, the amount of haemoglobin, and haematocrit significantly decreased.     

Exosomes Derived from Squamous Head and Neck Cancer Promote Cell Survival after Ionizing Radiation

Exosomes are nanometer-sized extracellular vesicles that are believed to function as intercellular communicators. Here, we report that exosomes are able to modify the radiation response of the head and neck cancer cell lines BHY and FaDu. Exosomes were isolated from the conditioned medium of irradiated as well as non-irradiated head and neck cancer cells by serial centrifugation. Quantification using NanoSight technology indicated an increased exosome release from irradiated compared to non-irradiated cells 24 hours after treatment. To test whether the released exosomes influence the radiation response of other cells the exosomes were transferred to non-irradiated and irradiated recipient cells. We found an enhanced uptake of exosomes isolated from both irradiated and non-irradiated cells by irradiated recipient cells compared to non-irradiated recipient cells. Functional analyses by exosome transfer indicated that all exosomes (from non-irradiated and irradiated donor cells) increase the proliferation of non-irradiated recipient cells and the survival of irradiated recipient cells. The survival-promoting effects are more pronounced when exosomes isolated from irradiated compared to non-irradiated donor cells are transferred. A possible mechanism for the increased survival after irradiation could be the increase in DNA double-strand break repair monitored at 6, 8 and 10 h after the transfer of exosomes isolated from irradiated cells. This is abrogated by the destabilization of the exosomes. Our results demonstrate that radiation influences both the abundance and action of exosomes on recipient cells. Exosomes transmit prosurvival effects by promoting the proliferation and radioresistance of head and neck cancer cells. Taken together, this study indicates a functional role of exosomes in the response of tumor cells to radiation exposure within a therapeutic dose range and encourages that exosomes are useful objects of study for a better understanding of tumor radiation response.     

Radiation causes increased production and decreased utilization of IL-2 in human mononuclear cells

The effects of radiation on the kinetics of Interleukin-2 (IL-2) production and utilization by mononuclear cells (MNCs) were studied. Mononuclear cells from normal, healthy individuals were subjected to various doses of radiation ranging from 0 to 2,000 rad and cultured in the presence of PHA. Supernatants from these cultures were harvested at various periods and their IL-2 contents determined by both the standard bioassay and ELISA. A radiation dose of 800 rad and higher had a marked effect on both IL-2 production and consumption. Although the supernatants from both the irradiated and non-irradiated MNCs contained maximal concentrations of IL-2 between 8 and 24 h of culture, the former had three times as much IL-2 as the latter. An increase in IL-2-mRNA levels was also noticed in irradiated, PHA-stimulated cells. Moreover, the supernatants from irradiated MNCs collected as late as 72 h after the initiation of culture contained more than 30% of the total IL-2 produced compared to less than 8% in supernatants from non-irradiated cells. Supernatants from non-irradiated cells incubated further with irradiated cells contained relatively higher quantities of IL-2 than those incubated continuously with non-irradiated cells. Supernatants from co-cultures of irradiated and non-irradiated MNCs contained less than expected amounts of IL-2 in two of the three subjects. Despite a difference in both the production and consumption of IL-2 between the irradiated and non-irradiated cells, there was no difference in their ability to generate IL-2 receptors. The results indicate that inactivation of radiosensitive suppressor T cells is associated with superinduction of IL-2 mRNA, increased production and decreased consumption of IL-2 by MNCs, thereby resulting in increased accumulation of IL-2.     

Stimulation of alkalothermophilic Aspergillus terreusxylanase by low-intensity laser radiation

In this study, Aspergillus terreus was irradiated by a 7.3 mW He–Ne laser in the presence of crystal violet, toluidine blue O and hematoporphyrin as photosensitizers. Xylanases recovered from non-irradiated and irradiated fungi were purified and characterized. The maximum production of xylanase (42.2 U/ml) was obtained after 5 min of laser irradiation in the absence of the photosensitizer. The irradiation of the sensitized fungus diminished the production of xylanase. On purification using G-100, the specific activity of xylanase recovered from the irradiated fungus was 292 U/mg protein representing a 37-fold purification over the crude extract compared with 95.6 U/mg protein representing the 12.8-fold for the enzyme recovered from the non-irradiated fungus. The enzyme recovered from the irradiated fungus had lower molecular weight as compared with that recovered from the non-irradiated one. Characterization of the purified enzymes revealed that the enzyme recovered from the irradiated fungus was more thermostable and had a wider range of optimum reaction temperature (60–70°C) and pH (4.0–12.0), compared to the non-irradiated one.     

Catalytic properties of testicular hyaluronidase after gamma-irradiation

The effect of gamma-irradiation on ovine testicular hyaluronidase was studied in aqueous solution. Following irradiation, hyaluronidase is inhibited, and the kinetics of inhibition follow a pattern in which Km and Vmax decline as radiation dose is increased. It was indicated that the binding affinity of the residual activity of hyaluronidase with substrate is enhanced and depends upon radiation damage. Effects of various agents such as pH, salts, PCMB and glutathione on irradiated hyaluronidase have been compared with non-irradiated enzyme. The irradiated hyaluronidase was more sensitive to inhibition by CuSO4 than the non-irradiated enzyme. The residual activity after irradiation is less refractory to FeCl3 inhibition and less sensitive to NaCl stimulation compared to non-irradiated hyaluronidase. pH response curves of ovine testicular hyaluronidase show two maxima which become more evident after irradiation.     

A comparison of chromatin degradation in irradiated normal and tumorous lymphoid cells

Irradiation of mice with doses of 2 and 4 Gy induced extensive chromatin degradation in the thymocytes within 6 hours accompanied by an increase in polydeoxynucleotide (PDN) content (36 and 42 times, respectively). Fifteen hours after irradiation the PDN level was considerably lower, however, still being 4.7 and 14 times the control values after doses of 2 and 4 Gy. The PDN content in control LS/BL lymphosarcoma cells was similar as that in the thymocytes of non-irradiated mice. Unlike in the thymocytes, irradiation of lymphosarcoma cells did induce no statistically significant increase in the PDN level 6 and 15 hours after the irradiation, respectively. It has been reported previously (Matyásová et al. 1973) that chromatin of LS/BL cells degraded similarly as that in the irradiated thymocytes. The results of the present experiments thus provide additional evidence for changes of LS/BL cell properties due to long term cultivation. These cells, however, are still able to react by chromatin fragmentation to nitrogen mustard treatment.