Postradiation disorders in the adenine-phosphoribosyltransferase pathway of AMP biosynthesis

A study was made of the activity of adenine-phosphoribosyltransferase (EC 2.4.2.7), which catalizes the biosynthesis of AMP from adenine and 5-phospho-alpha-D-riboso-1-diphosphate, in extracts from thymus and liver during 24 h following irradiation of mice in a dose of 245.1 mC/kg (dose-rate of 9.16 X 10(-4)-9.0 X 10(-4) A/kg). Adenine-phosphoribosyltransferase activity of liver remains normal during the entire period of observation. In thymus extracts, the activity of the enzyme decreases down to 70-80% of the control level 3 h after irradiation, it is normalized after 6 h and increases up to 150-160% of the control level by the end of the first day of radiation sickness.     

Glutathione concentration and peptidase activity in the lens after exposure to microwaves

This study was undertaken for observation of early changes in glutathione concentration and the activity of carboxypeptidase A and aminopeptidase in the cortex and core of the lens as well as for determination of the cumulating effect of microwave energy after repeated exposures to microwaves. Experiments were carried out on New Zealand rabbits. The control group was compared to experimental groups exposed every day for 5 minutes to microwave irradiation of the eyeballs at power densities of 5 X 10(-3) W/cm2 and 10 X 10(-3) W/cm2 during 10, 20 and 30 days. Differences were found between the control group and the groups of animals exposed to microwaves in which the glutathione concentration in the cortex and core of the lens was decreasing with time in proportion to the number of exposures. Parallelly to the number of days of exposure to microwaves the enzymatic activity of carboxypeptidase A and aminopeptidase increased in the cortex of the lens. The observed changes demonstrate cumulation of the absorbed microwave energy leading to changes in the permeability of the capsule and membranes of lenticular fibres which lead to secondary metabolic disturbances in the lens of the eye.     

Rapid loss of bone mass and strength in mice after abdominal irradiation

Localized irradiation is a common treatment modality for malignancies in the pelvic-abdominal cavity. We report here on the changes in bone mass and strength in mice 7-14 days after abdominal irradiation. Male C57BL/6 mice of 10-12 weeks of age were given a single-dose (0, 5, 10, 15 or 20 Gy) or fractionated (3 Gy × 2 per day × 7.5 days) X rays to the abdomen and monitored daily for up to 14 days. A decrease in the serum bone formation marker and ex vivo osteoblast differentiation was detected 7 days after a single dose of radiation, with little change in the serum bone resorption marker and ex vivo osteoclast formation. A single dose of radiation elicited a loss of bone mineral density (BMD) within 14 days of irradiation. The BMD loss was up to 4.1% in the whole skeleton, 7.3% in tibia, and 7.7% in the femur. Fractionated abdominal irradiation induced similar extents of BMD loss 10 days after the last fraction: 6.2% in the whole skeleton, 5.1% in tibia, and 13.8% in the femur. The loss of BMD was dependent on radiation dose and was more profound in the trabecula-rich regions of the long bones. Moreover, BMD loss in the total skeleton and the femurs progressed with time. Peak load and stiffness in the mid-shaft tibia from irradiated mice were 11.2-14.2% and 11.5-25.0% lower, respectively, than sham controls tested 7 days after a single-dose abdominal irradiation. Our data demonstrate that abdominal irradiation induces a rapid loss of BMD in the mouse skeleton. These effects are bone type- and region-specific but are independent of radiation fractionation. The radiation-induced abscopal damage to the skeleton is manifested by the deterioration of biomechanical properties of the affected bone.     

Sex behavior of male rats after gamma-irradiation: various radiobiological characteristics

The authors showed a pronounced and stable decrease in sexual motivation of male rats immediately after gamma-irradiation of the head with a dose of 2.58 C/kg. Exposure of the body to 1.29-2.58 C/kg radiation also inhibited sexual behaviour but only by the 45th-55th minute following irradiation: with higher doses some increase in sexual activity was observed immediately after irradiation.     

Dose-rate effects of 8-methoxypsoralen plus 365-NM irradiation on cell killing in Saccharomyces cerevisiae.

Tow types of dose-rate effect that alter the survival response of haploid yeast cells to 8-methoxypsoralen (8-MOP) plus treatment with irradiation at 365 nm were studied. (1) When the concentration of 8-MOP was varied between 9.2 X 10(-5) and 2.3 X 10(-8) M and the dose rate of 365-nm irradiation kept constant, the efficiency of the irradiation for killing increased relatively to that of 8-MOP whe the concentration of 8-MOP decreased. This indicated that there was no strict reciprocity between radiation dose and concentration of drug. (2) When the dose rate of radiation was varied between 0.66 X 10(3) and 108 X 10(3) J m-2 h-1 and the concentration of 8-MOP was kept constant, the survival of wild-type cells increased strikingly at low dose rates of radiation as compared with high dose rates. Cells responded more to changes at low dose rates than to equal changes a high dose rates. The high resistance of wild-type cells to 8-MOP plus radiation delivered at low dose rates absent from rad 1-3 cells defective in excision-repair. This suggests that the dose-rate effect seen in wild-type cells depended at least in part on an active excision-repair function. At low dose rates of radiation, the shoulder of the survival curve for rad1-3 cells, i.e. the ability to accumulate sub-lethal damage, was increased by a factor of about 2 when compared with that seen at a high dose rate. Thus it is likely that at low dose rates a repair function other than excision-resynthesis may operate in rad1-3 cells.     

In vivo postirradiation protection by a vitamin E analog, alpha-TMG

The water-soluble vitamin E derivative alpha-TMG is an excellent radical scavenger. A dose of 600 mg/kg TMG significantly reduced radiation clastogenicity in mouse bone marrow when administered after irradiation. The present study was aimed at investigating the radioprotective effect of postirradiation treatment with alpha-TMG against a range of whole-body lethal (8.5-12 Gy) and sublethal (1-5 Gy) doses of radiation in adult Swiss albino mice. Protection against lethal irradiation was evaluated from 30-day mouse survival and against sublethal doses was assessed from micronuclei and chromosomal aberrations in the bone marrow 24 h after irradiation. An intraperitoneal injection of 600 mg/kg TMG within 10 min of lethal irradiation increased survival, giving a dose modification factor (DMF) of 1.09. TMG at doses of 400 mg/kg and 600 mg/kg significantly reduced the percentage of aberrant metaphases, the different types of aberrations, and the number of micronucleated erythrocytes. DMFs of 1.22 and 1.48 for percentage aberrant metaphases and 1.6 and 1.98 for micronuclei were obtained for 400 mg/kg and 600 mg/kg TMG, respectively. No drug toxicity was observed at these doses. The effectiveness of TMG when administered postirradiation suggests its possible utility for protection against unplanned radiation exposures.     

Metallothionein induction as a potent means of radiation protection in mice

A striking resistance to lethal damage from a single dose of 6-8 Gy of X rays has been found in mice which had received various pretreatments to induce metallothionein (MT) synthesis in the liver prior to irradiation. Mice were injected with manganese (10 mg Mn/kg) or cadmium (3 mg Cd/kg) salt subcutaneously, or a patch of dorsal skin (2 X 2 cm2) was excised 1 or 2 days prior to irradiation. The increased tolerance of these mice to radiation was established by a marked decrease of mortality rate, an increase of mean survival time, a reduction of weight loss, and a smaller decrease in the number of leukocytes as compared with the control group. The LD50/30 for control mice was 6.3 Gy, while the corresponding values for the groups pretreated with Mn, Cd, and skin excision were 7.5, 7.7, and 7.9 Gy, respectively. The normal level of MT in mouse liver was approximately 25 micrograms/g tissue. This level increased 2.5- to 3-fold 24 h after 6.3 Gy irradiation. The MT levels of mice pretreated with Cd, Mn, and skin excision were increased 8-, 5-, and 7-fold, respectively, prior to irradiation as compared with the preirradiation control. These results indicate that the induction of MT in mouse liver is a significant factor in the mechanism of protection against radiation.     

Protective effects of 5,6,7,8-tetrahydroneopterin against X-ray radiation injury in mice

The protective effects of 5,6,7,8-tetrahydroneopterin (NH4) against radiation injury in mice were studied. (C57BL/6xA/J)F1 (B6A) mice received a single whole-body irradiation dose of 200, 400, 700 or 800 cGy of X-rays. NH4 (30 mg/kg body weight) or phosphate-buffered saline (PBS) was injected intraperitoneally into irradiated mice 10 min before and after the irradiation and again after 6 h. All mice which received the 800 cGy radiation+PBS died between 8 and 11 days after the treatment. In contrast, those which also received NH4 demonstrated a significantly prolonged survival time and 40% lived more than 5 months. Total numbers of thymocytes and spleen cells on day 5 post-irradiation were dramatically reduced in line with the radiation dose. The survival was significantly enhanced by NH4 in treated mice. The proliferation of spleen cells in mice stimulated by concanavalin A (Con A) or lipopolysaccharide (LPS) was also greater in NH4 treated mice. The immune response of survivors 5 months after 800 cGy+NH4 treatments, against Con A, LPS, allogenic mouse, and sheep red blood cells had essentially recovered to the levels of normal mice. These results indicate that NH4 had an important role in modifying radiation injury.     

The metabolism of neuropeptides. Phase separation of synaptic membrane preparations with Triton X-114 reveals the presence of aminopeptidase N.

The property of solutions of Triton X-114 to separate into detergent-rich and detergent-poor phases at 30 degrees C has been exploited to investigate the identities of the aminopeptidases in synaptic membrane preparations from pig striatum. When titrated with an antiserum to aminopeptidase N (EC 3.4.11.2), synaptic membranes solubilized with Triton X-100 revealed that this enzyme apparently comprises no more than 5% of the activity releasing tyrosine from [Leu]enkephalin. When assayed in the presence of puromycin, this proportion increased to 20%. Three integral membrane proteins were fractionated by phase separation in Triton X-114. Aminopeptidase activity, endopeptidase-24.11 and peptidyl dipeptidase A partitioned predominantly into the detergent-rich phase when kidney microvillar membranes were so treated. However, only 5.5% of synaptic membrane aminopeptidase activity partitioned into this phase, although the other peptidases behaved predictably. About half of the aminopeptidase activity in the detergent-rich phase could now be titrated with the antiserum, showing that aminopeptidase N is an integral membrane protein of this preparation. Three aminopeptidase inhibitors were investigated for their ability to discriminate between the different activities revealed by these experiments. Although amastatin was the most potent (IC50 = 5 X 10(-7) M) it failed to discriminate between pure kidney aminopeptidase N, the total activity of solubilized synaptic membranes and that in the Triton X-114-rich phase. Bestatin was slightly more potent for total activity (IC50 = 6.3 X 10(-6) M) than for the other two forms (IC50 = 1.6 X 10(-5) M). Puromycin was a weak inhibitor, but was more selective. The activity of solubilized membranes was more sensitive (IC50 = 1.6 X 10(-5) M) than that of the pure enzyme or the Triton X-114-rich phase (IC50 = 4 X 10(-4) M). We suggest that the puromycin-sensitive aminopeptidase activity that predominates in crude synaptic membrane preparations may be a cytosolic contaminant or peripheral membrane protein rather than an integral membrane component. Aminopeptidase N may contribute to the extracellular metabolism of enkephalin and other susceptible neuropeptides in the brain.     

Chromosome translocations in the sex cells of mice subjected to chronic low-dose gamma-irradiation

Translocation induction in mouse spermatogonia by continuous whole-body gamma irradiation (radium 226) was studied. Total doses, delivered at a rate of 13.0 +/- 1.3 X 10(-4) rad/min for various time intervals, were 97, 195, 294 and 442 rad. Cytological examination within 3 to 4 months after irradiation indicated the presence of translocations in 0.16, 0.30, 0.75 and 1.29 percent respectively, of primary spermatocytes at diakinesis metaphase I. Data on translocation induction (Y) as related to total irradiation dose (D) were best fitted to a second power parabola equation (Y=5.1 X 10(-6)D2 + 7.32 X 10(-4) X D). The results obtained confirm that chronic gamma irradiation is of low genetic efficiency, and support the suggestion that there exists a dose-rate threshold under which no more changes in exposure efficiency will occur.     

Angiotensin III: A Potent Inhibitor of Enkephalin-Degrading Enzymes and an Analgesic Agent

Various angiotensins, bradykinins, and related peptides were examined for their inhibitory activity against several enkephalin-degrading enzymes, including an aminopeptidase and a dipeptidyl aminopeptidase, purified from a membrane-bound fraction of monkey brain, and an endopeptidase, purified from the rabbit kidney membrane fraction. Angiotensin derivatives having a basic or neutral amino acid at the N-terminus showed strong inhibition of the aminopeptidase. Dipeptidyl aminopeptidase was inhibited by angiotensins II and III and their derivatives, whereas the endopeptidase was inhibited by angiotensin I and its derivatives. The most potent inhibitor of aminopeptidase and dipeptidyl aminopeptidase was angiotensin III, which completely inhibited the degradation of enkephalin by enzymes in monkey brain or human CSF. The Ki values for angiotensin III against aminopeptidase, dipeptidyl aminopeptidase, endopeptidase, and angiotensin-converting enzyme, which degraded enkephalin, were 0.66 X 10(-6), 1.03 X 10(-6), 2.3 X 10(-4), and 1.65 X 10(-6) M, respectively. Angiotensin III potentiated the analgesic activity of Met-enkephalin after intracerebroventricular coadministration to mice in the hot plate test. Angiotensin III itself also displayed analgesic activity in that test. These actions were blocked by the specific opiate antagonist naloxone.     

(ADP-ribose)n-transferase activity of enterocyte nuclei during radiation injury of small intestine mucosa in rats

The (ADP-ribose)n transferase activity of enterocyte nuclei of small intestine is studied in rats in normal and under radiation injury of intestine mucosa. It is shown that kinetics of [14C]-NAD incorporation into the acid-insoluble fraction of enterocyte nuclei is of a two-step character when the medium contains 1-15 microM of NAD. The X-ray irradiation of animals (a dose of 0.21 cells per kg) evokes changes in the (ADP-ribose)n transferase activity in nuclei of enterocytes isolated 1-72 h after irradiation. The irradiation results in distortion of the two-step kinetics of (ADP-ribose) synthesis in nuclei. The most pronounced activation of this biopolymer synthesis occurs 1,24 and 36 h after irradiation. In other periods a decrease in the level of (ADP-ribose)n synthesis is observed. Changes in the (ADP-ribose)n transferase activity in nuclei of enterocytes reflect the phase character of radiation sickness course in the small intestine. Activation of these processes is supposed to be a result of intensification of molecular mechanisms of DNA molecule reparation.     

Alterations in monoamine oxidase activity of the mouse brain and liver after mixed neutron-gamma irradiation

Monoamine oxidase (MAO) plays an important role in the metabolism of neuro-transmitter biogenic amines. Its activity was determined in mouse brain and liver after exposure to different kinds of ionizing radiation and after pretreatment with a radioprotective agent. After a lethal dose of mixed neutron-gamma irradiation the MAO activity decreased in the brain and increased in the liver. In contrast, after a lethal dose of 60Co-gamma irradiation enzyme activity was considerably increased in the brain while in the liver it increased like after mixed neutron-gamma irradiation. AET (S2-aminoethyl-isothiuronium-Br X HBr), when administered in a radio-protective dose, inhibited MAO activity in the brain, while it increased in the liver. Even more marked changes of enzyme activity were observed in both brain and liver after AET pretreatment and mixed neutron-gamma irradiation. On the basis of the results it is suggested that different kinds of ionizing radiation lead to different types of lipid peroxidation in the lipid environment surrounding MAO, an event leading to altered enzyme activity. AET itself inhibited MAO in the brain and increased the activity in the liver but did not prevent the alterations caused by ionizing radiation in enzyme activity.     

Assessment of the genetic risk of radiation by irradiation data from laboratory mammals

An attempt has been made to assess quantitatively genetic risk of radiation for man based on mammalian (mostly mouse) data and using the direct method proposed by UNSCEAR. The parameter employed was induction of reciprocal translocations. Two assumptions were made: human radiosensitivity equals that of the mouse; and dose-response is linear. From observations with acute gamma irradiation the estimate of risk per 10(-2) Gy was as follows: 39 translocation heterozygotes are expected among one million F1 conceptions, 5 cases of multiple congenital anomalies, 25 abortions recorded and 49 unrecorded. Chronic gamma irradiation at dose rates of 1.3 X 10(-5), 1.7 X 10(-4) and 1.0 X 10(-4) Gy/min was 3 to 10 times less effective. Exposure to 4.2 GeV deuterons proved inferior in effectiveness to gamma irradiation. Chronic exposure to 4.1 MeV neutrons delivered at 8 X 10(-4) Gy/min showed 7 times the effectiveness of chronic gamma irradiation. Administration of tritiated water (from 37 to 37 X 10(2) kBq/g b.w.) to rats entailed a risk of the same order of magnitude as external chronic gamma irradiation. Reduction of genetic risk was achieved by pretreatment with either AFT-, ATP-serotonin mixtures or the molecular combinations, Adeturon and Cytriphos. Study of interspecies differences in genetic radiosensitivity showed decline in the following order: rat-rabbit-mouse-Syrian hamster. A dose-rate effect was most clearly seen in the rat, and least clearly in the rabbit. In female mice, examination of oocyte depletion indicated primary follicles to be highly susceptible to acute gamma irradiation; decrease in sensitivity was observed beginning with stage 4. Chronic gamma irradiation was found to be less effective.     

Protective effect of corticosteroids on radiation pneumonitis in mice

We explored the protective effect of corticosteroids on the mortality of mice that received thoracic irradiation. Methylprednisolone, 100 mg/kg/week, given from 11 weeks after gamma irradiation of the thorax resulted in an increase in the LD50 (11-26 weeks) from 14.3 +/- 0.3 (mean +/- SE) Gy to 17.6 +/- 0.4 Gy, P less than 0.001, a protection factor of 1.2. Withdrawal of steroids at various times during the period of radiation pneumonitis resulted in accelerated mortality in the next 2-4 weeks, so that the cumulative mortality "caught up" with that of control animals by 4 weeks after steroid withdrawal. However, after the end of the usual period of pneumonitis withdrawal of steroids did not result in accelerated mortality, suggesting that the time when steroids are protective corresponds to the duration of pneumonitis. A smaller dose of steroids, 25 mg/kg/week, was found to be as protective as the larger dose used in the above experiments. The possibility that corticosteroids reduce mortality, even when given many weeks after radiation, may have important practical and theoretical implications.     

The effects of ionizing radiation on the pulmonary vasculature of intact rats and isolated pulmonary endothelium

We studied the effects of ionizing radiation on the morphology of the pulmonary circulation using an in vivo rat model and an in vitro pulmonary artery endothelial cell model. Gamma radiation was given as either an acute (30 Gy) or fractionated (5 X 6 Gy) dose to one hemithorax of rats. An acute 30-Gy dose delivered resulted in a 70% decrease in pulmonary arterial perfusion, using technetium-99m microaggregated albumin (99mTc-MAA), in the irradiated lung by 2-3 weeks after irradiation. Pulmonary microradiographs, using a barium sulfate perfusion method, obtained 2-3 weeks after irradiation demonstrated widespread loss of capillary filling and segmentation of the vessels. Histologic examination demonstrated intact capillaries, suggesting that the alterations in pulmonary perfusion were at the precapillary level. Similar abnormalities in lung perfusion and morphology were found after delivery of fractionated doses of radiation, but the onset of the changes was delayed, occurring 4-6 weeks postirradiation. Using cultured pulmonary endothelial cell monolayers, cell sloughing and retraction from the surface substrate were observed within 24 h after in vitro delivery of 30 Gy. Similar findings occurred in monolayers given fractionated doses (5 X 6 Gy) of radiation 2-3 days after the final dose. The in vivo animal and in vitro endothelial cell models offer a useful means of examining the morphologic alterations involved in radiation lung vascular damage.     

Effect of subtherapeutic doses of docetaxel (taxotere) on the efficacy of radiotherapy and pro-oxidant-antioxidant balance in rats with Guerin's carcinoma

In the experiments at Wistar male rats the effect of subtherapeutic doses of docetaxel (5 and 10 mg/kg) on the radiotherapy efficacy (20 Gy of single-dose X-rays) namely growth rate of Guerin's tumor and prooxidant-antioxidant balance in liver and blood of animals bearing tumors was investigated. It has been demonstrated that docetaxel at dose 5 mg/kg given 18 hours before irradiation resulted in significant tumor growth delay (2.3-2.7-fold) in comparison with group of rats that received only irradiation. After application of higher dose of docetaxel there was no statistically significant change of tumor size along the whole experiment (14 and 21 days after tumor implantation). Content of lipid peroxidation products was revealed to be considerably increased after chemotherapy and concurrent irradiation when docetaxel was used in a dose of 10 mg/kg. At the same time glutatione peroxidase activity and antioxidative activity of blood plasma were reduced. In the rat liver chemoradiotherapy led to decrease of glutathion peroxidase and glutathione-S-transferase activity to greater degree at docetaxel dose of 10 mg/kg. The obtained results allow to conclude that higher dose of docetaxel and concurrent irradiation resulted in the most effective Guerin's carcinoma growth delay and considerable deviation of antioxidant-prooxidant balance of tissues in the direction of the last.     

Effect of ionizing radiation on the properties of rat liver phosphoprotein phosphatase

Phosphoproteidphosphatase (3.1.3.16) of high specificity for lysil-tRNA-synthetase (6.1.1.6) and proteins of high-molecular-weight multienzyme complex of aminoacyl-tRNA-synthetases (6.1.1.) was isolated from rat liver. Irradiation of animals with an absolutely lethal dose of 0.21 C/kg decreased phosphoproteidphosphatase activity: a 3-4-fold decrease was noted 1 hr following irradiation. The activity of the enzyme isolated 24 hr after irradiation increased but did not reach the control level.     

Soluble and immobilized clostridial aminopeptidase and aminopeptidase P as metal-requiring enzymes

The dependence of enzymatic activity on Co2+ concentration was found to be bell-shaped for the soluble and immobilized clostridial aminopeptidase (alpha-aminoacyl-peptide hydrolase, EC 3.4.11.13) and aminopeptidase P (aminoacylpropyl-peptide hydrolase, EC 3.4.11.9), with maxima in the 3-18 microM range of Co2+ concentration. The Co2+-enzyme association constants derived from the activation of soluble, glass- and cellulose-bound clostridial aminopeptidase by Co2+ were KE-Co = 5.2 X 10(5), 4.5 X 10(6) and 2.0 X 10(5) M-1, respectively; for soluble and glass-bound aminopeptidase P, the KE-Co were 1.5 X 10(5) and 8.2 X 10(5) M-1, respectively. Kinetic measurements indicate the involvement of Co2+ in the enzyme-substrate binding. Cobalt-citrate (Co-cit) acted as a useful metallobuffer and protected both enzymes against inhibition by high concentrations of CoSO4. For association of citrate with Co2+ under the assay conditions, KCo-cit was determined as (5.3 +/- 1.4) X 10(3) M-1 by anodic stripping polarography. In contrast to the rapid association of Co2+ with soluble and glass-bound clostridial aminopeptidase (less than 1 min at 4 degrees C), the dissociation process was very slow (hours to days), being slower for the glass-bound than for the soluble and cellulose-bound enzyme. For aminopeptidase P, both processes were rapid. All the interactions were shown to be reversible.     

Evaluation of Systemic Antifungal Agents in X-Irradiated Mice

The effect of X irradiation on the survival time of animals experimentally infected with pathogenic fungi was studied, and the activity of antifungal agents in pre-irradiated hosts was evaluated. A 24-hr preinfection dose of X irradiation decreased the survival time of mice infected with Cryptococcus neoformans and Histoplasma capsulatum to a greater extent than Candida albicans or Blastomyces dermatitidis infections. Exposure to 400 r caused a significant reduction in the variation (S(2)) survival time of C. albicans or H. capsulatum mouse infections. A single 100-mg/kg dose of 5-fluorocytosine or amphotericin B administered within 24 hr postinfection significantly extended the survival time of mice infected with C. albicans. Delayed treatment with amphotericin B was effective against C. neoformans infections. Four 50-mg/kg doses of 5-fluorocytosine were more effective than a single 200-mg/kg dose against C. neoformans infections. A single dose of amphotericin B provided significant protection when administered 48 hr postinfection against B. dermatitidis in preirradiated mice. A single dose of saramycetin 48 hr postinfection was highly effective against H. capsulatum mouse infections. A 100-mg/kg dose of amphotericin B was only effective against this fungal pathogen when administered within 8 hr postinfection. In vivo activity of the antifungal agents studied was detected within 8 to 14 days. The relative in vivo activity of several antifungal agents indicated the importance of considering their individual pharmacological properties for optimum effectiveness. The experimental model used in this study should be useful for the detection and for the preclinical evaluation of new antifungal agents.