Identification of genes responsive to gamma radiation in rat hepatocytes and rat liver by cDNA array gene expression analysis

The mechanisms underlying hepatocellular damage after irradiation are obscure. We identified genes induced by radiation in isolated rat hepatocytes in vitro by cDNA array gene expression analysis and then screened in vivo experiments with those same genes using real-time PCR and Western blotting. Hepatocytes were irradiated and cDNA array analyses were performed 6 h after irradiation. The mRNA of differentially expressed genes was quantitatively analyzed by real-time PCR. cDNA array analyses showed an up-regulation of 10 genes in hepatocytes 6 h after irradiation; this was confirmed by real-time PCR. In vivo, rat livers were irradiated selectively. Treated and sham-irradiated controls were killed humanely 1, 3, 6, 12, 24 and 48 h after irradiation. Liver RNA was analyzed by real-time PCR; expression of in vivo altered genes was also analyzed at the protein level by Western blotting. Up-regulation was confirmed for three of the in vitro altered genes (multidrug resistance protein, proteasome component C3, eukaryotic translation initiation factor 2). Histologically, livers from irradiated animals were characterized by steatosis of hepatocytes. Thus we identified genes that may be involved in liver steatosis after irradiation. The methods shown in this work should help to further clarify the consequences of radiation exposure in the liver.     

Biosynthesis and degradation of collagen in X-irradiated mouse lung

Fibrosis, characterized by accumulation of collagen, is a delayed result of radiation injury in many tissues, including lung. To investigate its development, synthesis and degradation of collagen were measured in lungs of mice after X irradiation of the whole thorax. The ratio of type I (coarse fibered) to type III (meshwork) collagen was also determined. Synthesis of procollagen, measured as the activities of prolyl-4-hydroxylase and protein disulfide isomerase in lung tissue, was increased at 2 months after X-ray doses of 5, 7.5, and 9 Gy. Maximal increases were observed 6 to 7 months after doses of 9 Gy and persisted up to 15 months after exposure. Increases after 5 and 7.5 Gy were more gradual, but by 1 year after irradiation they had reached levels similar to those after 9 Gy. X irradiation had no effect on the degradation of collagen as assessed by collagenase activity in lung. The ratio of type I to type III collagen, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of collagen-derived cyanogen bromide peptides, was the same in irradiated lungs as in age-matched controls. Therefore, increased synthesis of procollagen, rather than decreased degradation of collagen or changes in collagen type, is an important factor in the accumulation of collagen in irradiated lung.     

Time-dependent neurosphere-forming ability of adult rat spinal cord after irradiation

To determine whether there was evidence for long-term time-dependent changes in neurosphere-forming ability of rat spinal cord after irradiation, a 15-mm length of spinal cord (C2-T2) of 10-week-old female rats was irradiated with a single dose of 2, 5, 10 or 19 Gy. Cells were isolated from the central 10-mm segment of the irradiated spinal cord immediately or at 0.5, 1, 2 or 5 months to form neurospheres. The number and sizes of neurospheres were determined at day 10, 12, 14 and 16 in vitro. The multipotential properties of neurosphere cells were assessed by immunocytochemistry using lineage-specific markers for neurons and glia. In nonirradiated controls, the number and size of the neurospheres decreased with increasing age of the animals. Regardless of the time after irradiation, there was a dose-dependent decrease in the number and size of neurospheres obtained from the irradiated cord compared to age-matched controls. Using three-way ANOVA, the number of neurospheres was dependent on radiation dose (P < 0.0001), time after irradiation (P < 0.0001), and day of counting in vitro (P < 0.0001). Compared to cells cultured immediately after irradiation, there was an increase in the relative plating efficiency of neurospheres cultured 1 month after irradiation. However, no further increase was apparent up to 5 months after irradiation. The multipotential properties of neurosphere cells in vitro remained unchanged with increasing time after irradiation. These results may suggest a time-dependent recovery of radiation damage using neurosphere-forming ability as the end point and agree with data that show time-dependent recovery of radiation damage in spinal cord using histological or functional end points.     

Effects of acute X-irradiation on pre-implantation embryos and on the implantation reaction in the mouse

Summary Pregnant mice were treated on the 1st, 2nd, and 3rd day of pregnancy by a single dose of 300 R X-rays. Uterine dissections at day 6 p.c. topographically revealed decrease of the implantation sites from 9.67 per female in the controls to 8.00 in females irradiated on day 1, to 6.63 in females irradiated on day 2, and to 7.00 in females irradiated on day 3 p.c. Among a number of 22 implantations after irradiation on day 1, 19 after irradiation on day 2 and 11 after irradiation on day 3, however no living embryo could be detected on histological examination. The degree of damage as indicated by the total resorptions was highest (94,7%) after irradiation on day 2 p.c., and lowest (31,8%) after irradiation on day 1 p.c. Since the decidual cell reaction was either unaffected or only slightly reduced after irradiation on day 2 p.c. as indicated by cytomorphological criteria and the alkaline phosphatase reaction, not maternal effects but direct effects only of the irradiation on the embryo must account for embryonic deaths.     

Effects of split fast neutron doses on the liver cells of albino Swiss mice

The effect of neutron doses from a D-T compact neutron generator on the liver cells of adult male and female albino Swiss mice was investigated. Fast neutrons (14.5 MeV) were delivered to the whole body in a single dose or in two, four, six or eight equal doses separated by 3-day intervals. The lowest dose, 100 rem, was given for an exposure time of 6 hours and was then steadily raised to 912 rem for an exposure time of 48 hours. During exposure the neutron flux was controlled by the activation foil technique. Animals were killed for testing after each irradiation. Histological examination of the hepatocytes in the light microscope showed marked degenerative changes only after the longer irradiation periods (24, 36 and 48 h). Electron microscopy showed cytological (cytoplasmic and nuclear) changes in the hepatocytes after only 12 hours' irradiation. Densitometric scans of electron micrographs of control and 12 h-irradiated livers indicated that the control hepatocyte interphase nucleus contains approximately 72% heterochromatin, while the irradiated nucleus contains only 64% heterochromatin.     

Decreased weight, DNA, RNA and protein content of the brain after neutron irradiation of the 18-day mouse embryo

Pregnant mice were irradiated with 0.5 Gy fission neutrons on the eighteenth day of their gestation. The average litter size at birth was unchanged but mortality increased 5-6 fold in the first 3 days. The irradiated mice were the same weight as control mice at birth but showed a progressively increasing weight deficiency up to at least 36 days as compared to controls. Brain weight was 37, 45 and 25 per cent less in 2-, 3- and 52-week old irradiated animals, respectively, and the ratio of brain weight to body weight was 25, 27 and 13 per cent less. The concentrations of DNA, RNA and protein (mg/g wet tissue) were the same in irradiated and control mice in both brain and liver at all three ages. Total DNA, RNA and protein contents of whole brain after irradiation were 56-75 per cent of the control levels. No definite decrease was observed in liver. Histological study at 6 hours after irradiation showed nuclear pyknosis in the central nervous system from definite to very severe according to the part examined. It is concluded that damage to the central nervous system of the 18-day mouse foetus after neutron irradiation is mainly due to killing and/or inhibition of the differentiation of neuroblasts.     

Lack of relationship of transplantation immunogenicity in C3H/He mammary carcinomas,with lymphoid hyperplasia and radiation-induced growth enhancement

Summary The therapeutic use of (a) radiation-inactivated tumor cells, (b) Bacillus Calmette-Guérin (BCG), and (c) heparinized plasma from normal mice to reduce radiation-induced impairment of existing antitumor resistance was investigated in female C3H/He hosts of syngeneic mammary carcinoma implants. The mice, which had been moderately presensitized 50 days before challenge, were given 300 rad whole-body irradiation at various times up to the day of challenge and 3 days after. Irradiated presensitized and irradiated unsensitized animals were maximally immunodepressed 1–2 weeks after exposure. The levels of resistance seen in unirradiated presensitized and in unirradiated unsensitized controls were recovered by irradiated presensitized and by irradiated unsensitized mice in about 3 and 4 weeks, respectively. Repeated injections of radiation-inactivated tumor cells were most effective in supporting the immune status of irradiated mice and in promoting an early recovery. Injections of BCG had only an insignificant effect. Injections of normal plasma was effective in reducing the immune suppression but did not promote an earlier recovery.     

Long-term effects of low-dose mouse liver irradiation involve ultrastructural and biochemical changes in hepatocytes that depend on lipid metabolism

The aim of the study was to investigate long-term effects of radiation on the (ultra)structure and function of the liver in mice. The experiments were conducted on wild-type C57BL/6J and apolipoprotein E knock-out (ApoE^?/?) male mice which received a single dose (2 or 8 Gy) of X-rays to the heart with simultaneous exposure of liver to low doses (no more than 30 and 120 mGy, respectively). Livers were collected for analysis 60 weeks after irradiation and used for morphological, ultrastructural, and biochemical studies. The results show increased damage to mitochondrial ultrastructure and lipid deposition in hepatocytes of irradiated animals as compared to non-irradiated controls. Stronger radiation-related effects were noted in ApoE^?/? mice than wild-type animals. In contrast, radiation-related changes in the activity of lysosomal hydrolases, including acid phosphatase, β-glucuronidase, N-acetyl-β-d-hexosaminidase, β-galactosidase, and α-glucosidase, were observed in wild type but not in ApoE-deficient mice, which together with ultrastructural picture suggests a higher activity of autophagy in ApoE-proficient animals. Irradiation caused a reduction of plasma markers of liver damage in wild-type mice, while an increased level of hepatic lipase was observed in plasma of ApoE-deficient mice, which collectively indicates a higher resistance of hepatocytes from ApoE-proficient animals to radiation-mediated damage. In conclusion, liver dysfunctions were observed as late effects of irradiation with an apparent association with malfunction of lipid metabolism.     

Lasting effects of an impairment of Th1-like immune response in γ-irradiated mice: A resemblance between irradiated mice and aged mice

Although one of the several chronic effects of ionizing radiation is aging, there are no experimental data on radiation-induced immunological aging. The most interesting change in aging was a helper T (Th) 1/Th2 imbalance. We investigated chronic effect on immune responses after ionizing radiation and its effects in irradiated mice were compared with those of aged mice. The 2-month-old mice received a whole-body irradiation of 5 Gy. At 6 months after irradiation, we compared the immune functions of the irradiated mice with those of normal mice of the same age and with those of older. Interferon (IFN)-γ and antigen-specific immunoglobulin (Ig)G2a level were lower in the irradiated mice than in normal mice of same age, showing similar levels to those of old normal mice. In contrast, interleukin (IL)-4 and IL-5 and antigen-specific IgG1 level were increased in irradiated mice when compared with the same aged-normal mice. Next, we investigated the low expression of IL-12p70, IL-12 receptors and IL-18 receptors in irradiated and old mice. Also, the decrease of natural killer cell activity was intensified in the irradiated mice, showing lower than values to those of old mice. Interestingly, in irradiated mice, the absolute numbers and the percentages of natural killer (NK) cells was extremely decreased. But the absolute numbers of Th cells and cytotoxic T (Tc) cells in old mice were significantly decreased. In conclusion, an immunological imbalance by the whole-body irradiation of 5 Gy induces to persist in the long term, resulting in the similar results with aging. Our results suggest that the downregulation of the Th1-like immune response shown in old mice rapidly occurred through exposure of ionizing radiation.     

Total body 4.5 Gy gamma irradiation-induced early delayed learning and memory dysfunction in the rat.

In an attempt to determine the consequences of total body radiation damage on learning and memory in the rat, twenty-eight male Wistar rats aged 4 months received 4.5 Gy total body gamma-irradiation (TBI) while 28 rats received sham irradiation. Sequential behavioral studies of negative reinforcement including a/ one- and b/ two-way avoidance tasks were undertaken. a/ One-way avoidance test: this test was performed before and after TBI. Prior to irradiation both groups were similar. At 20 days (D) and at 3 months post-TBI, irradiated rats had a significantly lower percentage of avoidance than controls but no statistical difference was found at 5 months post-TBI. b/ Two-way avoidance test: this test was performed only after TBI. At days 21, 22, 23, 24, (leaming) and at 4 or 6 months (recalls) post-TBI the mean percentage of avoidance was significantly lower in irradiated than in control rats. This study demonstrates that total-body exposure to 4.5 Gy gamma-irradiation induces behavioral dysfunction affecting learning and transitorily memory. These results suggest that a relatively low dose of total body irradiation can induce neurological complications, which persist 4-6 months later.     

Radiation injury in rat lung. I. Prostacyclin (PGI2) production, arterial perfusion, and ultrastructure

Pulmonary prostacyclin (PGI2) production, arterial perfusion, and ultrastructure were correlated in rats sacrificed from 1 day to 6 months after a single exposure of 25 Gy of gamma rays to the right hemithorax. PGI2 production by the irradiated lung decreased to approximately half the normal value 1 day after irradiation (P less than 0.05), then increased steadily throughout the study. By 6 months postirradiation, the right lung produced two to three times as much PGI2 as did either shielded left lung or sham-irradiated lungs (P less than 0.05). Perfusion scans revealed hyperemia of the right lung from 1 to 14 days after irradiation. From its peak at 14 days postirradiation, however, perfusion of the irradiated lung decreased steadily, then reached a plateau from 3 to 6 months at less than half that in the shielded left lung. Electron micrographs of the right lung revealed perivascular edema from 1 to 30 days after irradiation. The right lung then exhibited changes typical of radiation pneumonitis followed by progressive interstitial fibrosis. Platelet aggregates were not observed at any time. Thus, decreased PGI2 production is an immediate but transient response of the lung to radiation injury. Then from 2 to 6 months after irradiation, the fibrotic, hypoperfused lung produces increasing amounts of the potent vasodilator and antithrombotic agent, PGI2. Pulmonary PGI2 production and arterial perfusion are inversely correlated for at least 6 months after hemithoracic irradiation.     

Radiation injury in rat lung. IV. Modification by D-penicillamine

To determine whether D-penicillamine, known to reduce fibrosis in irradiated rat lung (W. F. Ward, A. Shih - Hoellwarth , and R. D. Tuttle , Radiology 146, 533-537, 1983), also ameliorates radiation injury in the pulmonary endothelium, we measured angiotensin-converting enzyme (ACE) activity, plasminogen activator (PLA) activity, and prostacyclin (PGI2) production in the lungs of penicillamine-treated (10 mg/day, po, continuous after irradiation) and untreated rats from 2 weeks to 6 months after a single dose of 25 Gy of 60Co gamma rays to the right hemithorax. Both ACE and PLA activity in the irradiated right lung of untreated rats decreased dramatically between the 1st and 2nd months after exposure, then reached a plateau through 6 months at approximately 25 and 50% of the normal level, respectively. For the first 2 months after irradiation, penicillamine-treated animals exhibited significantly (P less than 0.05) higher activities of both ACE and PLA than did untreated rats. From 3 to 6 months after irradiation, however, the only significant drug effect on these enzymes was a 25% increase in PLA activity at 6 months. PGI2 production by the irradiated lung of untreated rats increased continuously, and at 6 months was approximately 10 times higher than normal. Penicillamine significantly (P less than 0.05) reduced this hypersecretion, and at 6 months after irradiation, PGI2 production by the lungs of drug-treated rats was only half that of untreated animals. In contrast, the drug had no significant effect on enzyme activities in the lungs of sham-irradiated rats. Thus the antifibrotic agent D-penicillamine delays the onset of radiation-induced enzyme dysfunction in the pulmonary endothelium. In addition at 6 months after irradiation, the lungs of penicillamine-treated rats exhibit 25% more PLA activity and only half as severe a hypersecretion of PGI2 as do the lungs of untreated animals. The drug is most effective in ameliorating endothelial damage during the first 2 months after irradiation, preceding the development of interstitial fibrosis. However, the effect of this penicillamine regimen on pulmonary endothelial function is not as large as its effect on collagen accumulation in irradiated rat lung.     

Acceleration of regeneration of mucosa in small intestine damaged by ionizing radiation using anabolic steroids

Damage to intestine is a serious problem after accidental radiation exposure. To examine substances to ameliorate damage by postirradiation administration, we focused on the regeneration process after irradiation of the intestine. Using experimental systems, the effects of clinically used sex hormones on regeneration were compared. An anabolic steroid, nandrolone (19-nortestosterone), stimulated proliferation in IEC-6 epithelial cells. A single injection of 19-nortestosterone ester with prolonged action into mice 24 h after abdominal irradiation at a lethal dose of 15.7 Gy showed significant life-saving effects. Regeneration indicators such as microcolonies of BrdU-incorporated cells at day 5 and c-myb mRNA expression levels at day 4 were enhanced by 19-nortestosterone administration. In contrast, high concentrations of estradiol inhibited growth of IEC-6 cells. Treatment of abdominally irradiated mice with estradiol ester decreased levels of regeneration indicators and survival. These results suggest the effectiveness of the anabolic steroid as well as the importance of manipulation of steroid receptors in the recovery of mucosa damaged by radiation.     

Cellular mechanisms of cirrhotic rat liver regeneration. II. Proliferation, polyploidization and hypertrophy after partial hepatectomy

Using cytofluorimetry and absorptional cytophotometry, hepatocyte DNA and total protein contents were measured in intact and cirrhotic rats in 1, 3 and 6 months after partial hepatectomy (PH). It has been found that within one month of intact rat liver regeneration the level of hepatocyte ploidy rised by 25% to remain elevated for the next 6 months. This was due mainly to reducing the number of cells with diploid nuclei (2c 2-fold, 2c x 2 - 6.6-fold) and to rising the number of octaploid hepatocytes. In cirrhotic animals the ploidy level in hepatocytes increased in 3 months after PH, and decreased by 15% in 6 months. The number of hepatocytes with diploid nuclei (2c and 2c x 2) increased within 3-6 months in both control and cirrhotic rats. The protein content per diploid hepatocyte rised by 30% within 3-6 months of liver regeneration after PH. Special calculations have shown that within 3 months after PH the increase in the liver mass of control and cirrhotic rats was due completely to hepatocyte DNA synthesis, i. e. proliferation and polyploidization. Within the next 3 months of liver regeneration after PH, the contribution of polyploidization to liver mass increase was negative because of depolyploidization of liver parenchyma cell population. At this time hypertrophy was the main process determining the liver mass increase.     

Deficits in T helper cells after total lymphoid irradiation (TLI): reduced IL-2 secretion and normal IL-2 receptor expression in the mixed leukocyte reaction (MLR)

Spleen cells from BALB/c mice treated with total lymphoid irradiation (TLI) and from normal, unirradiated mice were compared in the mixed leukocyte reaction (MLR). Although the percentage of CD4+ cells in the spleen was close to normal, 4 to 6 weeks after TLI, the MLR of unfractionated spleen cells from irradiated mice was more than 10-fold lower than controls. A similar reduction was observed when purified CD4+ cells were used as responders in the MLR. Secretion of IL-2 by cells from irradiated mice was also about 10-fold lower than controls. However, the percentage of CD4+ and CD8+ cells which expressed IL-2 surface receptors during the MLR was similar using spleen cells from irradiated and control mice. Addition of an exogenous source of IL-2 restored the proliferative capacity of the irradiated cells and suggests that the lack of IL-2 secretion is the likely explanation of the marked deficit in the MLR of CD4+ spleen cells after TLI.     

Role of radiation-induced granulocyte colony-stimulating factor in recovery from whole body gamma-irradiation

The purpose of this study was to further elucidate the radioprotective role of granulocyte colony-stimulating factor (G-CSF) induced in response to irradiation. The induction of G-CSF and interleukin-6 (IL-6) in response to radiation exposure was evaluated in mice. The level of cytokine in serum was determined by multiplex Luminex. The role of G-CSF on survival and tissue injury after total body gamma-irradiation was evaluated by administration of neutralizing antibody to G-CSF before radiation exposure. An isotype control was used for comparison and survival was monitored for 30 d after irradiation. Jejunum samples were used for immunohistochemistry. Ionizing radiation exposure induced significant levels of the hematopoietic cytokines G-CSF and IL-6, in mice receiving 9.2 Gy radiation. Maximal levels of G-CSF were observed in peripheral blood of mice 8h after irradiation. IL-6 levels were maximum at 12h after irradiation. Administration of G-CSF antibody significantly enhanced mortality in irradiated mice. G-CSF antibody-treated mice had higher numbers of CD68(+) cells and apoptotic cells in intestinal villi. Our results confirm that radiation exposure induces elevations of circulating G-CSF and IL-6. Neutralizing antibody to G-CSF exacerbates the deleterious effects of radiation, indicating that G-CSF induced in response to irradiation plays an important role in recovery.     

Topical tocopherol acetate reduces post-UVB, sunburn-associated erythema, edema, and skin sensitivity in hairless mice.

Exposure of the skin of the back of skh-1 hairless mice to UVB (310 nm peak) irradiation at doses of 0.115-0.23 J/cm2 results after 24-48 h in an erythema which can be quantified using an erythema meter, providing a useful model of sunburn. Application of pure d-alpha-tocopherol acetate, a thick oil, to the skin immediately following the exposure to UVB significantly reduces the increase in erythema index, by 40-55%. At the lower dose (0.115 J/cm2), skin thickness (associated with edematous swelling of the sunburned skin) was measured by a novel non-invasive technique not previously reported for this purpose--magnetic resonance imaging (MRI). In two experiments the UVB-induced increase in skin thickness was significantly reduced at 24 hr by 29 and 54%, and at 48 hr by 26 and 61%. After 8 days the untreated irradiated mouse skin still showed a significant increase in thickness (24%) compared to the untreated unirradiated control, while the treated irradiated control was not significantly thicker than the unexposed control. Skin sensitivity was tested using a modification of the technique of esthesiometry, by observing rapid avoidance responses of the mouse to a pressure of 0.96 g/cm2 exerted by applying to the skin the tip of a nylon esthesiometer fiber extended to 60 mm in length. The untreated irradiated mice were more sensitive (p less than 0.07, Wilcoxon test) than the treated irradiated mice, and also significantly different from the untreated unirradiated control mice (p less than 0.04, Wilcoxon test), but the treated irradiated mice were not significantly differently sensitive when compared to the unirradiated controls (p less than 0.32). Taken together these data indicate that the erythema, edema, and skin sensitivity commonly associated with UVB-induced sunburn are significantly reduced by topical application of tocopherol acetate even after the exposure has occurred. This observation suggests that treatment of sunburn may be possible even after the irradiation has stopped, by a derivative of d-alpha-tocopherol which is stable to autooxidation.     

Polyploidization of rat hepatocytes due to cell fusion caused by radiations of different LET

The method of flow cytometry was used to study polyploidization of hepatocytes following X-, gamma-, and neutron-irradiation. Ionizing radiation was shown to induce cell polyploidization by two different ways: (1) cells and nuclei fusion, and (2) restriction of mitosis after DNA replication. RBE of 14 MeV neutrons with respect to fusion was about 5.10(3). With neutron irradiation, the sensitivity of cells by fusion was not lower than that by chromosome mutations.     

Cellular mechanisms of rat liver regeneration after experimental myocardial infarction

Morphological changes and regeneration activity of rat liver after experimental myocardial infarction (MI) caused by a permanent left coronary artery occlusion were investigated. It was shown that, 6 months after MI, considerable changes were observed in the rat liver circulatory system: the vessel amount per unit area increased by 118%, the thickness of their walls increased by 19%, and the average area of vessel lumens increased by 159%. The contribution of connective tissue 6 months after MI increased by more than one- and-a-half times in comparison with control. Inflammatory and necrotic changes in rat liver remained for 6 months after MI. The liver injury caused by MI leads to activation of regeneration processes in its parenchyma. Six months after MI, the number of 4c-hepatocytes decreased by 12% in comparison with control and the number of 4c×2- and 8c-hepatocytes increased by 45 and 71%, respectively. Six months after MI, the hepatocyte ploidy increased by 11%. In this period, the dry mass of rat hepatocytes increased by 19%. Thus, liver regeneration after MI is stipulated by hepatocyte hypertrophy rather than their polyploidization.     

Treatment of mice with a herbal preparation (Liv. 52) reduces the frequency of radiation-induced chromosome damage in bone marrow

Induction of chromosomal aberrations was studied from 1/4 to 14 days post irradiation in the bone marrow of mice treated or not with Liv. 52, a herbal preparation, prior to 4.5 Gy exposure. The frequency of chromatid and chromosomal aberrations started increasing at day 1/4 in the irradiation and Liv. 52 + irradiated groups. The highest frequency of aberrations was recorded at day 1/2 post exposure which declined after day 1 in both groups. The frequency of both types of aberrations was significantly lower in the Liv. 52 + irradiated group than in the irradiated group.