Investigation of co-genotoxic effects of radiofrequency electromagnetic fields in vivo.

We investigated the possible combined genotoxic effects of radiofrequency (RF) electromagnetic fields (900 MHz, amplitude modulated at 217 Hz, mobile phone signal) with the drinking water mutagen and carcinogen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Female rats were exposed to RF fields for a period of 2 years for 2 h per day, 5 days per week at average whole-body specific absorption rates of 0.3 or 0.9 W/kg. MX was given in the drinking water at a concentration of 19 microg/ml. Blood samples were taken at 3, 6 and 24 months of exposure and brain and liver samples were taken at the end of the study (24 months). DNA damage was assessed in all samples using the alkaline comet assay, and micronuclei were determined in erythrocytes. We did not find significant genotoxic activity of MX in blood and liver cells. However, MX induced DNA damage in rat brain. Co-exposures to MX and RF radiation did not significantly increase the response of blood, liver and brain cells compared to MX exposure only. In conclusion, this 2-year animal study involving long-term exposures to RF radiation and MX did not provide any evidence for enhanced genotoxicity in rats exposed to RF radiation.

Long-term effects on tumour incidence and survival from 241Am exposure of the BALB/c mouse in utero and during adulthood.

BALB/c mice were given 100, 500 or 1500 Bq/g 241Am at day 14 of pregnancy. The offspring were separated from the mothers at birth and followed until death. In addition, adult females and one group of males were also studied for the effects of 241Am following treatment with 45-213 Bq/g. Adults treated with 241Am showed significantly shortened survival and increased incidence of osteosarcoma (to 40 - 50%). The data also suggest that the female mouse is more susceptible to induction of osteosarcoma than the male. There was also a significant increase in osteosarcoma, all bone tumours, all sarcomas, and all leukaemias in the offspring from the contaminated mothers, although this appeared to occur independently of dose. Calculations of the number of osteosarcomas induced per Gy varied for contamination of adult mice between 0.2 and 0.01 and for the offspring between 6 and 0.6. Thus, offspring seemed to be about 10 times more at risk if osteosarcomas induced per mouse Gy are compared. Surprisingly, offspring from mothers treated with 241Am displayed a longer survival time than controls, possibly due to fewer deterministic lung diseases appearing early in life.

Haemopoietic and osteogenic toxicity testing in vitro using murine bone marrow cultures.

Bone marrow and the surrounding bone with its high storage capacity for inorganic compounds may accumulate various lipophilic and electrophilic substances that enter the bloodstream. In bone marrow a few stem cells are responsible for the continuous production of blood cells and bone cells during the entire life of the organism. Damage to these cells may result in haemopoietic failure, blood disorders or bone diseases. Therefore bone marrow needs to be considered as one of the major targets of chemicals that enter the circulation. A battery of different in vitro bone marrow assays is established in which interference of chemicals with proliferation and differentiation of marrow cells with haemopoietic, stromal or bone forming marrow commitment may be screened routinely. Stromal cells form the network of extracellular matrix and growth factors that is needed by the haemopoietic cells to proliferate and differentiate. If stromal marrow cells are cultured in the presence of ascorbic acid and beta-glycerophosphate, bone-specific proteins and an extracellular matrix are produced, which calcifies within 3 wk. To evaluate the specificity of the effects on marrow cells, a general cytotoxicity assay is included using 3T3-fibroblasts. Various concentrations of xenobiotics were added over the course of 3 days to the different asssays. Lead nitrate inhibited proliferation of stromal stem cells and their calcification in the bone-forming assay at much lower concentrations than those which were inhibitory to the proliferation of 3T3 cells. The benzene metabolite hydroquinone was equally inhibitory in all the marrow assays, but 3T3 cells needed 10 times more hydroquinone to reach the same degree of inhibition. Catechol, which is another benzene metabolite, was highly toxic but was equally effective in all the assays and showed no specific effects on the marrow cells. As in vivo, benzene itself and phenol showed hardly any effect in the in vitro assays. Not only pollutants but also cytokines may be screened with these assays. Differential effects on marrow cells could be demonstrated for interleukin 10.

Effects of in vitro alpha-particle irradiation on osteogenic bone marrow cultures.

Murine bone marrow contains osteogenic precursor cells that undergo differentiation during in vitro cultivation. In vitro these cells are potential target cells for alpha-irradiation-induced bone tumour formation. Under defined tissue culture conditions these differentiating cells were directly exposed to alpha-particle irradiation from the radon daughter 210Po. Po deposits in soft tissue and it was shown to be associated with marrow cells and with the extracellular marrow tissue formed in vitro. These differentiating marrow cultures showed high sensitivity to alpha-irradiation. Cell death was observed at 210Po concentrations in tissue culture medium (TCM) > 7 Bq 210Po/ml. At lower concentrations (between 1 and 5 Bq 210Po per ml TCM) proliferation was enhanced as measured by uptake of 3H-thymidine, also differentiation was stimulated as measured by alkaline phosphatase activity and incorporation of 3H-proline in newly synthesized collagen. At several times of culture, the association of 210Po with the extracellular matrix and cells was measured. These retention data enabled us to calculate the daily alpha-particle fluence. At 1 Bq 210Po present per ml tissue culture, a daily alpha-particle fluence as low as 3-6 per 1000 cells seemed very efficient in changing the expression of osteogenic differentiation of marrow cells.

Response of murine stromal bone marrow cultures to alpha-particle irradiation in vivo and in vitro at osteosarcomogenic alpha-radiation doses.

Stromal cells belonging to the bone marrow microenvironment are altered in mice at risk of bone tumour development after (241)Am injection. This can be observed with selective cell culture techniques long before the tumours become manifest. Colony forming assays in vitro showed increases in the number of stromal stem cells at lower dose levels but a decrease in cell number at higher dose levels. The in vitro osteogenic capacity of marrow, which is attributed to stromal cells of the osteogenic lineage, was significantly reduced. The changes were persistent until at least 1 yr after (241)Am injection. In vitro alpha-irradiation of bone marrow also reduced the osteogenic capacity of the bone marrow at dose levels that were comparable with those producing an effect in vivo. This suggests a direct effect of alpha-particle irradiation on stromal bone marrow cells. These data and previous results on the regulatory role of the stroma in haemopoiesis show that stromal bone marrow cells should be considered as sensitive targets for chronic low level alpha-particle irradiation.

High radiosensitivity of the mineralization capacity of adult murine bone marrow in vitro to continuous alpha-irradiation compared to acute X-irradiation.

Adult BALB/c mice, injected with osteosarcomogenic amounts of 241Am (between 40 and 500 Bq/g mouse) showed an impaired mineralization capacity of their femoral bone marrow. This effect persisted until at least 1 year after 241Am injection and was expressed after incubation of bone marrow cells in vitro in conditions allowing osteogenic differentiation. The mineralization capacity of marrow in vitro was evaluated by measurement of 85Sr uptake from the tissue culture medium. Two osteogenic assays were used: in marrow cultured as an intact organ (marrow organ cultures), reduced mineralization was observed in mice given 149 Bq 241Am/g mouse or more (skeletal dose rate of 25 mGy/day), in stromal marrow cells cultured from adherent cell layers and subsequently brought into a three-dimensional (3D) mineralizing condition (stromal 3D cultures), reduced 85Sr uptake was observed from the lowest dose level tested (42 Bq 241Am/g mouse, skeletal dose rate of 7 mGy/day). Taking into account that only a fraction of the skeletal alpha-dose reached the marrow of the femoral diaphyses, marrow organ cultures and stromal 3D cultures exhibited high radiosensitivity to alpha-irradiation in vivo. However, after acute X-irradiation of marrow in vivo or in vitro prior to initiation of the marrow organ cultures, X-ray doses of 4 Gy or higher were needed to significantly impair the mineralization capacity of marrow organ cultures in vitro. Our data demonstrated that the osteogenic cells from the bone marrow are subjected to long-term damage after low doses of continuous alpha-irradiation in vivo.

Establishment of an osteogenic cell line derived from adult mouse bone marrow stroma by use of a recombinant retrovirus.

In order to characterize fibroblastic colony-forming units (CFU-F) from murine bone marrow in relation to osteogenesis, adherent cells of 7-day-old BALB/c mouse bone marrow cultures were infected with a recombinant retrovirus (N2/ delta fosB) containing the bacterial neomycin resistance gene. One of the G418-resistant clones, MN7, was selected for further analysis on the basis of its high expression of the bone-specific alkaline phosphatase. The cells have now been in culture for more than 1 year and maintain a stable phenotype. The osteogenic nature of the immortalized clone MN7 was demonstrated as follows: (1) Mineralization was detected by 85Sr uptake and with the Von Kossa staining method only after in vitro cultivation on a collagen type I matrix. (2) Osteoblastic phenotype markers, including the synthesis of type I collagen, osteonectin, and the bone-specific isoenzyme of alkaline phosphatase were expressed in vitro. (3) MN7 cells responded to bone effectors such as parathyroid hormone and 1,25-dihydroxyvitamin D3. (4) Intraperitoneal injection of MN7 cells into 1-day-old BALB/c mice produced typical osteosarcomas in all animals. We conclude that MN7, derived entirely in vitro from a stromal CFU-F colony, represents a stable murine osteosarcoma cell line expressing the osteoblastic phenotype and provides the first direct evidence needed to establish adult mouse marrow-derived, nonhematopoietic stromal cells as osteoprogenitors.

Distribution of 241Am in offspring from BALB/c mice injected with 241Am at 14 days of gestation: relation to calcium and iron metabolism and comparison with distribution of 241Am after injection of adults.

Pregnant mice were given intravenous injections of 241Am citrate at 14 days of gestation. The fetal skeleton had a higher or similar uptake of 241Am per gram of fresh tissue than the liver. In comparison, the liver in adults concentrated 5 to 20 times more 241Am per gram of fresh tissue than the bones. Measurement of changes in calcium and iron content and concentration with time, showed that in the developing mice intensive calcification of bones determined the uptake of 241Am. The 241Am uptake was related to the calcium concentration of the fetal bones, which was greater at 14 days of gestation in the anterior bones, the mandibles and calvaria, than in the ribs and femurs. Transfer of 241Am to pups via milk resulted in further accumulation of 241Am in the skeleton and liver. The incorporation in the skeleton persisted after weaning and contributed to the lifetime body burden. The 241Am concentration decreased rapidly with time after injection in relation to the growth of the organs. Radiation dose rates and cumulative radiation doses were calculated for liver and bones of contaminated offspring.

Effects of radioprotective glycans on the arrest of blood-borne lymphoma cells.

The mode of action of radioprotective glycans is not understood. In view of the known importance of cell migration in haematology, on the one hand, and of carbohydrates in homing processes, on the other, we have investigated the effect of several glycans on blood-borne arrest of lymphoma cells. Radioprotective glycans (but not heparin) modified the arrest of injected cells in the spleen. Altered recirculation and homing processes may play a role in the radioprotective properties of glycans.

Modification of blood-borne arrest properties of lymphoma cells by inhibitors of protein glycosylation suggests the existence of endogenous lectins.

The requirement for intact carbohydrates of glycoproteins at the cell surface was investigated after treatment of lymphoma cells with compounds which interfere at different steps in N-linked glycosylation: swainsonine and 1-deoxynojirimycin act at different levels during the processing, so that complex oligosaccharides cannot be formed; 2-deoxyglucose, beta-hydroxynorvaline, and tunicamycin completely prevent the formation of N-linked (high-mannose as well as complex) oligosaccharides. The role of sialic acid was investigated by treating the cells with neuraminidase. These treatments resulted in altered patterns of surface-labelled glycoproteins after SDS-polyacrylamide gel electrophoresis. Blood-borne arrest of lymphoma cells in the spleen was sensitive to neuraminidase and to treatments interfering with the processing of complex N-linked oligosaccharides. It is suggested that carbohydrates are signals for cellular interactions involved in the recirculation and homing behaviour of lymphoid cells and probably interact with endogenous lectins at their site of homing.

Detection and spontaneous alteration of lymphocyte antigens on slide smears.

Smears of cell suspensions from murine lymphoid organs were prepared on slides, air dried, and processed for detection of immunoglobulin (Ig) and theta (Thy-1) antigens by the unlabeled antibody peroxidase-antiperoxidase (PAP) technique. Satisfactory results were obtained for both antigens on recently made smears. However, slides kept at room temperature showed a progressive decrease in staining of the two antigens with time. Various fixatives and preservation procedures were tested to prevent this alteration. Good conservation of smears was obtained when slides were kept at -18 degrees C and/or air isolated before or after fixation with alcohols. A similar degradation of Ig and/or Thy-1 antigens occurred also in histological tissue sections or in serum spots dried on slides. The major cause for this degradation is thought to be contact with air, residual enzymatic hydrolysis playing a less important role.

Radioimmunoassay determination of two main cross-reacting viral antigens during radiation and Rauscher induced leukemias.

The two main proteins p 30 and gp 70 from the Rauscher leukemia virus (RLV) have been isolated, labeled with 125I and used in radioimmune competition assays to estimate the amount of cross-reacting antigens produced during the evolution of various leukemias. The Rauscher leukemia in Balb/c mice, the radiation induced leukemia (RadLV) in C57Bl mice and two types derived therefrom by serial passages in mice (RadLV-RS) and in rats (RadLV-rat) were studied. Whereas the p 30 from RLV or RadLV (rat) viruses showed complete identity, the cross-reaction of their gp 70 proteins wasonly partial. The main findings in the tissues were the following: 1. The concentration of p 30 and gp 70 antigens increased much more during the RadLV-rat leukemia than in irradiated or RadLV (RS) treated mice. 2. In the serum, the ratio p 30/gp 70 was in general higher than one in the Rauscher leukemic mice and less than one in all types of RadLV leukemias. 3. In the spleen, RLV and RadLV (RS) infected mice had higher levels of p 30 than of gp 70. The reverse occurred in irradiated mice and in leukemic rats. Finally, the catabolic degradation of labeled RLV gp 70 was similar in normal and leukemic mice.