In vivo enzyme control through a strong stationary magnetic field--the case of thymidine kinase in mouse bone marrow cells

The influence of a strong homogeneous and stationary magnetic field (SMF) on the activity of the enzyme thymidine kinase (TdR-K) in bone marrow cells, and as a consequence of this on the incorporation of 125I-labelled 5-iodo-2-deoxyuridine (125IUdR) into DNA of mice and into isolated bone marrow cells in vitro, was assayed after exposure of immobilized mice. No effect could be elicited in moving mice, in cells in suspension or in enzyme in solution. The response depended on the body temperature during exposure: at 27 degrees C and 29 degrees C there was an increase and at 37 degrees C and a depression of enzyme activity. The TdR-K activity at low temperature increased with the field strength ranging from 0.2 to 1.4T. Thirty minutes were required for full expression of the effect at 1.4T; 5-10 min were needed after exposure for a return to base-line levels. Mice were given total-body irradiation at a dose of 0.1 Gy 137Cs gamma rays and then exposed immediately to a magnetic field at 1.4T for 30 min at a body temperature of 27 degrees C; gamma irradiation no longer inhibited the enzyme. Exposure to the magnetic field further removed from the time of gamma irradiation, did not negate the inhibitory effect of gamma irradiation. The observed responses to given challenges in this complex system support the hypothesis that the magnetic field affects TdR-K activity by way of a mediating structure, such as a membrane.     

Long-term effects of low-level 239Pu contamination on murine bone-marrow stem cells and their progeny

The effects of long-term internal contamination with 13.3 kBq kg-1 239Pu injected intravenously were studied in 10-week-old ICR (SPF) female mice. Radiosensitivity of spleen colony-forming units (CFU-S) and 125IUdR incorporating into proliferating cells of vertebral bone marrow and spleens were determined in plutonium-treated and control animals one year after nuclide injection. The CFU-S in 239Pu-treated mice were more sensitive to X-rays (D0 = 0.52 +/- 0.01 Gy) than in controls (D0 = 0.84 +/- 0.02 Gy). 125IUdR incorporation into bone marrow and spleen cells was reduced after plutonium contamination. At one year following plutonium injection, the occurrence of chromosome aberrations was evaluated in metaphase figures of femoral bone marrow cells. The frequency of aberrations increased early after plutonium treatment, at later intervals it tended to decrease but not below the control level. While the relative numbers of vertebral marrow CFU-S decreased significantly, but only to 86 per cent of normal, cellularity of vertebral bone marrow, peripheral blood counts and survival of 239Pu-treated mice did not differ from the control data.     

Uptake of 125I-iododeoxyuridine in mouse organs during deuteration of body water: Evidence for a thymus-specific effect

The effects of body water deuteration on mammalian DNA synthesis $\text{in vivo}$ during the deuterium equilibration period in the body were studied. Young adult mice were given 15% or 30% D₂O in the drinking water for 4, 10 or 21 days. Control mice were given distilled water. Eighteen hours prior to sacrifice, ¹²⁵IUdR, a conveniently monitored synthetic analogue of the DNA precursor thymidine, was injected intravenously. Although neither radioiodine activity of the total body nor body weight varied significantly among the three groups, thymic radioactivity per g tissue was significantly lower in mice given 30% D₂O and, to a lesser extent, in mice given 15% D₂O than in the control group. In contrast, intestine and hemopoietic bone marrow displayed minor changes in ¹²⁵IUdR incorporation. This reduction of ¹²⁵IUdR incorporation is discussed in relation to the particular importance of thymidine reutilization in the thymus.     

Fundamentals, trends and our experiences with total body irradiation (TBI) before bone marrow transplantation (BMT)

At the end of the sixties and to beginning of the seventies years the total body irradiation (TBI) was introduced in the concept of bone marrow transplantation (BMT). The aim is the destruction of leukaemic or normal stem cells surviving the chemotherapy or the overcoming of the immunological defense. From March 1980 to January 1987 we have treated 84 patients with single exposure of 8.5 to 10.5 Gy midline dose for body and lung in cases of leukaemia and of 6 to 7 Gy for patients with aplastic anaemia. We used a dose rate of about 5.5 cGy/min delivered by a linear accelerator. The results were comparable with other centres but a further indicator for the effectiveness of a irradiation technique is also the idiopathic interstitial pneumonitis (IIP). Our incidence of IIP was 10.7 per cent and the mortality was 2.4 per cent. Additional we have had 8.3 per cent interstitial pneumonitis (IP) caused by an infection. All patients with a combination of IP and GVHD had a fatal prognosis. In present time a tendency is to see to fractionation techniques in total body irradiation for decreasing of the pneumonitis rate, the reduction of severe acute and delayed side effects, for a better homogenisation of the dose in the whole body and for using of synchronizing effects on the stem cells.     

DNA turnover and thymidine re-utilization in mouse tissues.

Mice were injected intravenously with tritiated thymidine (TdR) and 125I-labeled iododeoxyuridine (IUdR) in low doses to provide a simultaneous labeling of tissue DNA with non-toxic amounts of these two precursors. The total activity per organ and the ratio of the two isotopes was measured in the DNA at various times between 1 and 15 days after the injection. Since TdR from dying cells is re-utilized more effeciently than IUdR from the same cells, more labeled TdR than IUdR was retained in the tissue DNA in these experiments. From the slopes of the regression lines, the true rated of turnover of replicating tissue DNA and the per cent re-utilization of TdR were calculated. Re-utilization of TdR varied from 37 to 60% in the six tissues examined.     

Radiotoxicity of 5-[125I]iodo-2'-deoxyuridine in mammalian cells following treatment with 5-fluoro-2'-deoxyuridine.

We have enhanced the uptake of 5-[125I]iodo-2'-deoxyuridine (125IUdR) in Chinese hamster V79 cells with 5-fluoro-2'-deoxyuridine (FUdR) and have examined the combined toxicity of these agents. Although the uptake of 125IUdR increases approximately 3.2 +/- 0.5-fold in the presence of 1 microM FUdR, when cell survival fraction is plotted as a function of intranuclear 125IUdR content, the biphasic curve obtained reaches a plateau at a higher survival fraction than with control cells not exposed to FUdR. The results suggest that a greater number of cells were prevented from entering the S phase and consequently from incorporating 125IUdR. An FUdR- 125IUdR combination, therefore, does not seem to enhance the therapeutic potential of 125IUdR. Such observations are also of importance when FUdR and other inhibitors are used to enhance cold IUdR uptake in an effort to obtain an increase in radiosensitization effects.     

DNA TURNOVER AND THYMIDINE RE-UTILIZATION IN MOUSE TISSUES

Mice were injected intravenously with tritiated thymidine (TdR) and ¹²⁵I-labeled iododeoxyuridine (IUdR) in low doses to provide a simultaneous labeling of tissue DNA with non-toxic amounts of these two precursors. The total activity per organ and the ratio of the two isotopes was measured in the DNA at various times between 1 and 15 days after the injection. Since TdR from dying cells is re-utilized more efficiently than IUdR from the same cells, more labeled TdR than IUdR was retained in the tissue DNA in these experiments. From the slopes of the regression lines, the true rate of turnover of replicating tissue DNA and the per cent re-utilization of TdR were calculated. Re-utilization of TdR varied from 37 to 60 % in the six tissues examined.     

Large granular lymphocytic (LGL) leukemia in rats exposed to intermittent 60 Hz magnetic fields

An animal model for large granular lymphocytic (LGL) leukemia in male Fischer 344 rats was utilized to determine whether magnetic field exposure can be shown to influence the progression of leukemia. We previously reported that exposure to continuous 60 Hz, 1 mT magnetic fields did not significantly alter the clinical progression of LGL leukemia in young male rats following injection of spleen cells from donor leukemic rats. Results presented here extend those studies with the following objectives: (a) to replicate the previous study of continuous 60 Hz magnetic field exposures, but using fewer LGL cells in the inoculum, and (b) to determine if intermittent 60 Hz magnetic fields can alter the clinical progression of leukemia. Rats were randomly assigned to four treatment groups (18/group) as follows: (1) 1 mT (10 G) continuous field, (2) 1 mT intermittent field (off/on at 3 min intervals), (3) ambient controls ( < 0.1 microT), and (4) positive control (5 Gy whole body irradiation from cobalt-60 four days prior to initiation of exposure). All rats were injected intraperitoneally with 2.2 x 10(6) fresh, viable LGL leukemic spleen cells at the beginning of the study. The fields were activated for 20 h per day, 7 days per week, and all exposure conditions were superimposed over the natural ambient magnetic field. The rats were weighed and palpated for splenomegaly weekly. Splenomegaly developed 9-11 weeks after transplantation of the leukemia cells. Hematological evaluations were performed at 6, 8, 10, 12, 14, and 16 weeks of exposure. Peripheral blood hemoglobin concentration, red blood cells, and packed cell volume declined, and total white blood cells and LGL cells increased dramatically in all treatment groups after onset of leukemia. Although the positive control group showed different body weight curves and developed signs of leukemia earlier than other groups, differences were not detected between exposure groups and ambient controls. Furthermore, there were no overall effects of magnetic fields on splenomegaly or survival in exposed animals. In addition, no significant and/or consistent differences were detected in hematological parameters between the magnetic field exposed and the ambient control groups.     

Magnetic field exposure of marrow donor mice can increase the number of spleen colonies (CFU-S 7d) in marrow recipient mice

Transplantation of bone marrow cells of magnetic-field-exposed mice led to increased numbers of spleen colonies (CFU-S 7d) in conditioned recipient mice (Peterson et al. 1986). Here we report on the dependence of this phenomenon on body temperature, field strength and exposure time. It was found that the effect can only be seen when the body temperature is 27 degrees C, the field strength not less than 1.4 T and the exposure time at least 15 min. It is suggested that the magnetic field increases the number of spleen colonies either directly by affecting membrane components (receptors) responsible for the seeding of the transplanted stem cells to the recipient spleens or indirectly affecting radical/redox-systems that may have a regulatory function in the stem cells.     

Nonthermal 60 Hz sinusoidal magnetic-field exposure enhances 45Ca2+ uptake in rat thymocytes: dependence on mitogen activation

The effect of a 60 Hz sinusoidal magnetic field of nonthermal intensity on Ca2+ metabolism in rat thymic lymphocytes (thymocytes) was assessed in resting cells and in cells activated with the mitogen Concanavalin A (Con A). A 60 min exposure at 37 degrees C to an induced electric field of 1.0 mV/cm produced an average 2.7-fold increase in Con A-dependent 45Ca2(+)-uptake compared to non-exposed, isothermal control cells. In contrast, 45Ca2+ uptake remained unaltered during exposure of resting thymocytes. It was also found that thymocytes with a diminished ability to mobilize Ca2+ in response to Con A were most sensitive to the 60 Hz magnetic field. Although the precise mechanism of field interaction is at present unknown, modulation of Ca2+ metabolism during cell activation may represent a common pathway for field coupling to cellular systems.     

Radioprotective effect of acute non-specific inflammation in mice

A full protection against a 8,5 Gy gamma irradiation is observed in Mice bearing a one day old granuloma induced by polyacrylamide microbeads, when these Mice are injected, 1 hr before irradiation, with the eluate obtained from a 1 day old granuloma. In these Mice, a striking increase of the uptake of 125IUdR in bone marrow is observed.     

Biochemical and morphological modifications in rabbit Achilles tendon during maturation and ageing.

1. The plasma clearance of intravenously injected 125I-labelled mitochondrial malate dehydrogenase (half-life 7 min) was not influenced by previous injection of suramin and/or leupeptin (inhibitors of intralysosomal proteolysis). 2. Pretreatment with both inhibitors considerably delayed degradation of endocytosed enzyme in liver, spleen, bone marrow and kidneys. 3. The tissue distribution of radioactivity was determined at 30 min after injection, when only 3% of the dose was left in plasma. All injected radioactivity was still present in the carcass. The major part of the injected dose was found in liver (49%), spleen (5%), kidneys (13%) and bone, including marrow (11%). 4. Liver cells were isolated 15 min after injection of labelled enzyme. We found that Kupffer cells and parenchymal cells had endocytosed the enzyme at rates corresponding to 9530 and 156 ml of plasma/day per g of cell protein respectively. Endothelial cells do not significantly contribute to uptake of the enzyme. 5. Uptake by Kupffer cells was saturable, whereas uptake by parenchymal cells was not. This suggests that these cell types endocytose the enzyme via different receptors. 6. Previous injection of carbon particles greatly decreased uptake of the enzyme by liver, spleen and bone marrow.     

Endocytosis of lactate dehydrogenase isoenzyme M4 in rats in vivo. Experiments with enzyme labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose.

1. Pig lactate dehydrogenase isoenzyme M4 was labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose and injected intravenously into rats. Previous work has shown that this label does not influence the clearance of the enzyme (half-life about 26 min) and that it is retained within the lysosomes for several hours after endocytosis and breakdown of the protein [De Jong, Bouma & Gruber (1981) Biochem. J. 198, 45--51]. 2. The distribution of the radioactivity over a large number of tissues was determined 2 h after injection. A high percentage of the injected dose was found in liver (41%), spleen (10%) and bone including marrow (21%). 3. Autoradiography indicated uptake of the enzyme mainly by Kupffer cells of the liver, by spleen macrophages and by bone marrow macrophages. 4. Liver cells were isolated 1 h after injection of the enzyme. Kupffer cells, endothelial cells and parenchymal cells were found to endocytose the enzyme at rates corresponding to 4230, 35 and 25 ml of plasma/day per g of cell protein, respectively. 5. Previous injection of carbon particles greatly reduced the uptake of the enzyme by liver and spleen, but the uptake by bone marrow was not significantly changed.     

Man's sensitivity to bone marrow failure following whole body exposure to low LET ionising radiation: inferences to be drawn from animal experiments

Evidence concerning the sensitivity of man to bone marrow failure following exposure to brief but substantial doses of ionising radiation is sparse. There is, however, a relatively substantial body of information on such effects in large animals. Reported experiments on six species where exposure to low LET radiation was uniform to the whole body and of brief duration (exposure times of the order of one hour or less) have been reanalysed both in terms of exposure and of midline tissue dose. The results indicate a marked lack of homogeneity among values for LD50 within species thus questioning the applicability of LD50 as a species dependent constant. It is, however, suggested that on a purely empirical basis these large animal data suggest that the dose killing 'most' (where 'most' is between 90 and 95 per cent) is about twice that killing 'few' (where 'few' is between 5 and 10 per cent). For man, where there is evidence that the dose killing few is unlikely to be less than 3 Gy, this relationship might indicate a gradient of mortality with dose between 3 and 6 Gy.     

A STATIC MAGNETIC FIELD OF 125 mT STIMULATES THE INITIAL GROWTH STAGES OF BARLEY (Hordeum vulgare L.)

In this paper the influence of a stationary magnetic field on the initial stages of barley plant development was evaluated. A stationary magnetic field has a stimulating effect on the first stages of growth of barley seeds for all exposure times studied. When germinating barley seeds were subjected to a magnetic field of 125 mT for different times (1, 10, 20, and 60 min, 24 h, and chronic exposure), increases in length and weight were observed. Maximum increases in the measured parameters were obtained when the time of exposure to magnetic field was long (24 h and chronic); however, the exposure for a short time (1 min) had a similar effect on growth.     

Effect of prolonged exposure to a magnetic field on the haematopoietic stem cell.

The authors studied the effect of prolonged exposure (3, 4 and 5 months) to the action of a magnetic field of 180-200 gauss formed by the poles of a rotating permanent magnet on the haematopoietic stem cells of mouse bone marrow donors. The effect of the field was evaluated from the ability of the donors' bone marrow cells to form haematopoietic colonies in the spleen of lethally irradiated mice. It was found that the number of stem cells was not reduced by the action of the above magnetic field and that proliferative capacity was likewide unimpaired.     

DIFFERENCES IN REUTILIZATION OF THYMIDINE IN HEMOPOIETIC AND LYMPHOPOIETIC TISSUES OF THE NORMAL MOUSE

Swiss Albino mice received a single i.v. injection of ³H-thymidine (TdR) or of ¹²⁵I-deoxyuridine (IUdR). Bone marrow, thymus, spleen and mesenteric lymph node were examined for the efficiency of precursor incorporation into DNA, and for DNA renewal from day 1 to day 8.
TdR is 5–8 times more efficiently incorporated by the different organs in vivo and in vitro than is IUdR. This indicates that the discrimination against IUdR occurs at the level of DNA synthesizing cells.
A diminished DNA turnover rate measured with ³H-TdR in comparison with ¹²⁵I-UdR is interpreted to indicate reutilization of TdR.
TdR reutilization was observed in bone marrow and spleen from at least day 1 on, and in the thymus from day 3 on, following pulse labeling of DNA synthesizing cells. The degree of TdR reutilization appears higher in the thymus (67%) than the bone marrow (43%) and spleen (38%). The mesenteric lymph node indicates either no, or a very low efficiency of TdR reutilization. The data are also consistent with a reutilization equally efficient for TdR and IUdR.
It is suggested that the TdR salvage pathway in hemopoietic tissues is largely localized to single organs which have immediate access to TdR made available by catabolism of DNA. The contribution of TdR from systemic reutilization to the organs studied falls within the limits of error of measurements. Moreover, the TdR salvage pathway especially in the lymph node may involve other DNA breakdown products than nucleosides.     

Radiotoxicity of 125I in mammalian cells

The radiotoxicity of 125I in Chinese hamster V79 lung fibroblasts has been studied following extracellular (Na125I), cytoplasmic [125I]iododihydrorhodamine (125I-DR), and nuclear (125IUdR) localization of the radionuclide. Exposure of the cells for 18 h to Na125I (less than or equal to 7.4 MBq/ml) had no effect on survival. A similar exposure to 125I-DR produced a survival curve with a distinct shoulder and with a mean lethal dose (D37) of 4.62 Gy to the nucleus. While this value compares well with the 5.80 Gy X-ray D37 dose, it is in contrast to the survival curve obtained with DNA-bound 125IUdR which is of the high LET type and has a D37 of 0.80 Gy to the nucleus. Furthermore, when the uptake of 125I into DNA is reduced by the addition of nonradioactive IUdR or TdR to the medium and the survival fraction is determined as a function of 125I contained in the DNA, a corresponding increase in survival is observed. This work demonstrates the relative inefficiency of the Auger electron emitter 125I when located in the cytoplasm or outside the cell. It indicates that a high dose deposited within the cytoplasm contributes minimally to radiation-induced cell death and that radiotoxicity depends not upon the specific activity of IUdR but upon the absolute amount of 125I that is associated with nuclear DNA.     

Hemopoietic progenitor cells with limited potential for differentiation: erythropoietic function of mouse marrow "lymphocytes"

Filtration of mouse marrow cell suspensions over columns of glass wool increased the frequency of small and medium-sized lymphocytes (SML) and of erythropoietic progenitor units (EPU) by about the same factor. Identical results were obtained when erythropoiesis was assayed by isotope uptake (⁵⁹FeCl₃ and ¹²⁵IUdR) or by the spleen-colony techniques. Transfusion of prospective donor mice with erythrocytes virtually eliminated morphologically recognizable erythroid cells from marrow without affecting the frequency of EPU. Injection of prospective donors with cortisol decreased the frequency of SML in marrow but not that of EPU or erythropoietin-sensitive cells. However, glass wool filtration of lymphocyte-poor marrow taken from mice pretreated with cortisol resulted in a similar increase in frequency of residual SML and of EPU. Therefore, it appears that a subpopulation of marrow SML are EPU. Whereas glass wool filtration increased the frequency of erythropoietic progenitor and colony-forming units, the filtration failed to change the frequency of leukopoietic progenitor or colony-forming units (assayed in mice hypertransfused with erythrocytes to suppress erythropoiesis). It follows that separate progenitor cells for erythropoiesis and leukopoiesis are present in bone marrow of adult mice, in addition to pluripotent stem cells.     

磁场作用对小鼠抗应激能力的影响

目的 :探讨磁场作用对小鼠抗应激能力的影响。方法 :对磁场处理 30分钟和 1 5分钟的两组实验组与非磁场处理的正常对照组进行游泳耐疲劳运动时间的比较。结果 :游泳耐疲劳运动实验表明 30分钟磁场处理组与正常对照组比较 ,动物游泳耐疲劳运动时间延长 ,且具有显著性差异 (p <0 .0 5 ) ;1 5分钟磁场处理组与正常对照组比较 ,动物游泳耐疲劳运动时间无明显差异 (p >0 .0 5 ) ;30分钟磁场处理组与 1 5分钟磁场处理组比较 ,动物游泳耐疲劳运动时间延长 ,且具有显著性差异 (p <0 .0 5 )。结论 :在一段时间( 30天 )内 ,每天给予小鼠 30分钟的磁场处理明显提高了小鼠的抗应激能力 ,而磁场处理 1 5分钟对小鼠的抗应激能力不产生影响。