Radiofrequency radiation at 40 kHz induces hepatic injury in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson disease

In the present study, we examined effects of radiofrequency (RF) radiation at 40 kHz on hepatic injury in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson disease, which is a heritable disease of copper metabolism in the liver. The activities of ALT and AST in serum of LEC rats exposed to RF radiation for 2 weeks were approximately 3.8-fold and 2-fold higher than those in serum of sham-exposed rats, respectively. Although there were no significant differences in hepatic copper contents between LEC rats exposed to RF radiation for 2 weeks and sham-exposed rats, copper contents in the kidney and serum of exposed LEC rats were approximately 4.2-fold and 12.9-fold higher than those in sham-exposed rats, respectively. Relative O??-scavenging activities in the S-100 fraction of the liver of LEC rats exposed to RF radiation for 2 weeks were 1.6-fold higher than those in sham-exposed rats. No significant differences were observed in activities of AST and ALT in serum and relative O??-scavenging activity in the S-100 fraction of the liver of normal control WKAH rats that were sham-exposed and exposed to RF radiation. No significant differences were observed in copper contents in the liver, kidney and serum of WKAH rats that were sham-exposed and exposed to RF radiation for 2 weeks. The results show that RF radiation at 40 kHz induced hepatic injury in LEC rats.     

Inhibition of replication induces non-apoptotic cell death in fibroblast cell lines derived from LEC rats

Hydroxyurea (HU), an anticancer drug, inhibits ribonucleoside diphosphate reductase and reduces pool sizes of deoxyribonucleoside triphosphate (dNTP). The reduction of dNTP results in inhibition of DNA replication. The cytotoxic effect of HU was investigated using fibroblast cell lines from LEC rats. LEC rat cells showed significantly higher sensitivity to HU than did cell lines from control WKAH rats. No significant differences were observed between the percentages of apoptotic cells in either LEC or WKAH rat cells that had been treated with HU and those that had not been treated with HU. LEC rat cells also showed significantly higher sensitivity to aphidicolin, which blocks DNA synthesis by inhibiting DNA polymerase alpha, than did WKAH rat cells. In both LEC and WKAH rat cells, intensified bands of p53 protein were observed immediately after treatment with HU. Although the high level of p53 protein persisted in WKAH rat cells until 6 hr post-incubation time after treatment with HU, the level of p53 protein had decreased at 6 hr post-incubation time in LEC rat cells. When the cells were X-irradiated in the absence or presence of HU, the ratio of the surviving fraction without HU to that with HU only slightly increased after X-irradiation in WKAH rat cells. In contrast, the ratio in LEC rat cells significantly increased after X-irradiation in a dose-dependent manner.     

Higher sensitivity of LEC strain rat in radiation-induced acute intestinal death.

LEC strain rats (LEC rats), which have been known to develop hereditarily spontaneous fulminant hepatitis 4-5 months after birth, were highly sensitive to whole-body X-irradiation as compared to WKAH strain rats (WKAH rats). Radiation-induced acute intestinal death occurred at doses higher than 6.5 Gy in LEC rats, and at doses higher than 12.8 Gy in WKAH rats, respectively. By the probit analysis of survival data, it was shown that the LD50/7 value of LEC rats was estimated to be 7.03 Gy which was significantly lower than that (12.99 Gy) of WKAH rats. Histopathological examinations of small intestines from LEC rats 2 days after irradiation at the dose of 8.5 Gy showed severe epithelial death together with edema, whereas little or no significant changes were noted in intestinal epithelium of 8.5 Gy-irradiated WKAH rats. These results suggest that the radiosensitivity of LEC rats to ionizing radiation appears to be higher than that of other strains of rats.     

High sensitivity of thymocytes of LEC strain rats to induction of apoptosis by X-irradiation

It is known that physical disruption of cell contacts induces apoptosis of thymocytes. When thymocytes from LEC and WKAH rats were incubated in vitro at 37 degrees C for 0-6 hr and then the proportion of apoptotic cells was determined using a flow cytometer, it was found that the percentages of apoptotic thymocytes from both LEC and WKAH rats increased with incubation time and that the proportion of apoptotic cells from LEC rats was significantly higher than that from WKAH rats at each incubation time. The fact that cycloheximide, an inhibitor of protein synthesis, did not show significant inhibitory effects on induction of apoptosis of thymocytes indicates that induction of apoptosis during in vitro cultivation did not require de novo protein synthesis. When thymocytes from LEC and WKAH rats were X-irradiated in vitro at 4 and 8 Gy, the percentages of radiation-induced apoptotic cells increased with post-incubation time after X-irradiation in both LEC and WKAH rat thymocytes and the proportions of apoptotic cells from LEC rats were significantly higher than those from WKAH rat cells at 2 and 4 hr post-incubation after X-irradiation. When thymocytes from LEC and WKAH rats were X-irradiated in the presence of cycloheximide, the induction of apoptosis was substantially inhibited, indicating that radiation-induced apoptosis of thymocytes from LEC and WKAH rats required de novo protein synthesis. The present results showed high sensitivities of thymocytes of LEC rats to induction of apoptosis during in vitro cultivation and by X-irradiation.     

High sensitivity of fibroblast cell lines derived from LEC rats to heat treatment

A fibroblast cell line derived from LEC rat was approximately twofold more sensitive to heat treatment at 45 degrees C than were that from WKAH rat in terms of heating time required to attain 50% loss of survival in a colony forming assay. The present study was carried out for understanding the mechanism underlying the higher sensitivity of LEC rat cells to heat treatment. Although apoptosis was not found in WKAH rat cells, the percentages of apoptotic cells in LEC rat cells significantly increased after heat treatment. LEC rat cells showed significantly lower sensitivity in induction of cell death and apoptosis to ceramide, a lipid signaling molecule that is associated with heat-induced apoptosis, than did WKAH rat cells. SP600125, an inhibitor of JNK suppressed the induction of cell death in both heated LEC and WKAH rat cells, but SB203580, an inhibitor of p38 mapk, did not. The relative surviving fractions of heated LEC and WKAH rat cells in the presence of both SB203580 and SP600125 were higher than those of cells in the presence of SP600125 alone. The amounts of hsp70 protein in WKAH rat cells increased from 4 to 12 hr after heat treatment, but did not in LEC rat cells. These results suggest that higher thermosensitivity in the fibroblast cell line from LEC rat is due to low inducibility of hsp70 protein after heat treatment.     

Higher sensitivity in induction of apoptosis in fibroblast cell lines derived from LEC strain rats to ultraviolet B radiation

When lung fibroblast cell lines from LEC and WKAH rats were irradiated with ultraviolet B (UVB) and assayed for colony formation, LEC rat cells showed a higher sensitivity than did WKAH rat cells. The LEC rat cells were approximately 1.5-fold more sensitive to UVB radiation than were the WKAH rat cells in terms of D37 values, which are the doses of UVB required to reduce cell survival to 37%. When the rat cells were irradiated with UVB in the presence of 0.5 M dimethyl sulfoxide (DMSO), which efficiently scavenges free radicals such as hydroxyl radicals, no significant difference was observed between the survival curves of either LEC or WKAH rat cells irradiated with UVB in the presence of 0.5 M DMSO and those irradiated with UVB in the absence of DMSO. Therefore, formation of free radicals may not be involved in cell death induced by UVB radiation. Flow cytometry showed that the percentage of apoptotic cells in the LEC rat cell population increased with post-incubation time after UVB radiation. The proportion of apoptotic cells in the UVB-irradiated LEC rat cell population increased as the dose of UVB was increased. In contrast, no significant proportion of apoptotic cells was observed in the UVB-irradiated WKAH rat cell population. These results showed a higher sensitivity in induction of apoptosis by UVB radiation in LEC rat cells than in WKAH rat cells.     

Wortmannin, a radiation sensitizer, enhances the radiosensitivity of WKAH rat cells but not that of LEC rat cells

No significant cytotoxic effect was observed in WKAH rat cells by the treatment of wortmannin, a radiation sensitizer, at concentrations lower than 30 microM for 24 hr. The relative surviving fractions of LEC rat cells were slightly, but significantly, lower than those of WKAH rat cells at each concentration of wortmannin. When the wortmannin-treated WKAH rat cells were X-irradiated, the relative surviving fractions decreased in a wortmannin concentration-dependent manner. On the contrary, no significant difference was observed between the survival curves of untreated and wortmannin-treated LEC rat cells after X-irradiation.     

Abnormal accumulation of G2/M-phase cells from LEC strain rats after X-irradiation at S phase.

The effect of X-irradiation on the progression of the cell cycle in cell lines from LEC and WKAH rats was investigated by a flow cytometer. When the cells were exposed to 5 Gy of X-rays at S phase, the proportion of S-phase cells in both cell populations decreased with incubation time and that of G2/M-phase cells was approximately 80% at 6 hr post-irradiation. At 12 hr post-irradiation, approximately 45% of the WKAH rat cells appeared in the G1 phase. However, 80-90% of LEC rat cells remained in the G2/M phase and less than 5% in the G1 phase during 6-12 hr post-irradiation. Thus, the LEC rat cells irradiated at S phase remained in the G2/M phase for at least 6 hr longer than did the WKAH rat cells.     

Analysis of the cell cycle of fibroblasts derived from the LEC rat after X-irradiation

The LEC rat is reported to exhibit hypersensitivity to X-irradiation, deficiency in DNA double-strand break repair, and radio-resistant DNA synthesis. This character of the LEC rat has been thought to be due to abnormal G1 arrest in cells after X-irradiation. In this report, we re-investigated the effect of X-irradiation on the cell cycle in primary-cultured fibroblasts. Primary-cultured fibroblasts derived from LEC and BN rats were exposed to 4 Gy of X-ray and their cell cycle analysis was performed with a flow cytometer. Fibroblasts derived from both rats showed normal response of the cell cycle, indicating the arrest at both G1--and G2/M-phase and no difference in the cell cycle population between fibroblasts derived from both rats. In contrast, when the same analysis was performed using the cell line, L7 and W8, which had been established from the lung fibroblasts of LEC and control WKAH rats, respectively, by immortalizing with SV40 T-antigen, L7 cells but not W8 cells showed impaired G1 arrest and abnormal cell cycle. These results suggest that fibroblasts derived from LEC rats possess the normal cell cycle response after X-irradiation, if they are kept naive as not immortalized with SV40 T-antigen.     

MR imaging of hepatic injury in the LEC rat under a high magnetic field (7.05 T).

Visualization of copper-induced hepatitis (CuH) in LEC rats was performed by using an MRI apparatus equipped with a magnet producing a high magnetic field of 7.05 T. When three groups of LEC rats (6-16 [pre-hepatitis], 15-26 [acute hepatitis] and 40-77 [chronic hepatitis] weeks old) were examined by MRI under T2-weighted imaging conditions which are suitable for the diagnosis of human hepatitis, hypointense MR images of the livers were, as a whole, obtained in all groups, suggesting that these conditions were not adequate for imaging of CuH of LEC rats. The shortening of the T1 and T2 relaxation times of livers due to an excess amount of paramagnetic irons under the high magnetic field was responsible for the lowering of MR signal intensities of the livers, especially those of 15 to 26-week old rats showing acute hepatitis. However, theoretical calculation of the MR signal intensities using the T1 and T2 relaxation times of the livers indicated that their imaging might be possible under proton density-weighted conditions even with a high magnetic field. Experimental results showed that hepatic injury was visualized as hyperintense regions in the MR image of the liver in the acute-phase rat.     

Restriction fragment length polymorphism for the Yc subunit gene of rat liver glutathione S-transferase

An altered expression of the Yc subunit gene of rat glutathione S-transferase (GST) in the liver of the LEC rat, which is a mutant strain with spontaneous hereditary hepatitis associated with severe jaundice, has been reported. To provide further information concerning the structure of the Yc subunit gene, we carried out the Southern blot hybridization analysis of DNA samples from rats of eight different inbred strains including LEC with cDNA complementary to mRNA specific for the Yc subunit of rat liver GST as a probe. The hybridization patterns of the DNA samples from rats belonging to the different inbred strains showed interstrain variation in the length of restriction fragments with four restriction endonucleases. Since the DNA samples prepared from several rats of one inbred strain gave an identical hybridization pattern, the restriction fragment patterns for the Yc gene could be used as markers for genetic monitoring of inbred rat strains. Although the altered expression of Yc-Yc activity of GST has been observed in the liver of the LEC rat, the characteristic changes in the gene structure of the Yc subunit of LEC rat were not detected in the present hybridization analysis.     

Copper-associated chronic hepatitis in Labrador Retrievers

This study summarizes the clinical and pathologic findings in 15 Labrador Retrievers with copper-associated chronic hepatitis (CACH). Our hypothesis was that this form of hepatitis is caused by a defect in hepatic copper metabolism, which most likely originates from a genetic defect. Affected Labradors consisted of 11 female and 4 male Labrador Retrievers. Eight family members of 2 of these patients were examined prospectively, as were 6 unrelated healthy Labrador Retrievers. All dogs were registered at the breed club. The average age at clinical presentation was 7 years (range, 2.5-10.5 years). All dogs were presented for anorexia, which was associated with vomiting in 8 patients. The diagnosis of CACH was based on histologic examination of liver biopsy specimens in all dogs, including semiquantitation of copper. A disproportionate increase in alanine aminotransferase (ALT) activity relative to alkaline phosphatase (ALP) activity, as well as the centrolobular localization of copper and the association of copper accumulation with hepatic lesions, suggested a primary copper storage disease rather than primary cholestatic liver disease causing copper accumulation. Mean hepatic copper concentration measured in related Labradors was 1,317 microg/g dry weight liver (range, 402-2,576 microg/g). Mean hepatic copper concentration of unrelated normal Labradors was 233 microg/g dry weight liver (range, 120-304 microg/g). Our findings support the hypothesis that a hereditary form of hepatitis occurs in Labrador retrievers and is caused by a defect in hepatic copper metabolism.     

Protective effect of a lignan-containing flaxseed extract against CCl(4)-induced hepatic injury

Carbon tetrachloride (CCl(4)) -induced hepatotoxicity is a commonly used model for investigating lipid peroxidation-related tissue injury. In the present study, the effect of flaxseed extract was observed on histological sections, glutathione-content and DNA strand breaks. Lignan-containing flaxseed extract (1.6 g/kg body weight/day) was daily administered with intragastric injection to rats for three days, on the fourth day, CCl(4) (2 g/kg) was intraperitoneally injected. Liver tissue was sampled at 24 hr after administering CCl(4). Liver-necrosis was observed in CCl(4)-injected rats without pretreatment of flaxseed extract. Pretreatment of flaxseed extract reduced extent of the necrosis found 24 hr after the intraperitoneal administration of CCl(4). Pretreatment of flaxseed extract protect against CCl(4)-induced decrease of reduced glutathione-content measured from reactions with 5,5'-dithiobis-(2-nitrobenzoic acid) and also protect against the elevation of DNA strand breaks in the liver cells measured by comet assay. Flaxseed-extract appears to protect liver cells against CCl(4)-induced necrosis.     

Estradiol inhibits hepatic stellate cell area and collagen synthesis in the chicken liver

Hepatic stellate cells (HSCs) are the main collagen‐producing cells in the liver. The HSC area and amount of collagen fibers are different between male and female chickens. This study was performed to confirm the effect of estradiol on collagen synthesis in the growing chicken liver. Blood estradiol levels in chicks were compared at 4 and 8 weeks of age, and the collagen fibril network in liver tissue was observed at 8 weeks by scanning electron microscopy. Intraperitoneal administrations of estradiol and tamoxifen to male and female chicks, respectively, were performed daily from 5 to 8 weeks of age. The areas of HSCs and collagen contents were measured in the liver tissue. The blood estradiol level was higher in females than in males, and the collagen fibril network was denser in males than in females at 8 weeks of age. Estradiol administration in males induced decreases in the HSC area and collagen content of the liver. Conversely, tamoxifen administration in females induced an increase in the HSC area but did not facilitate collagen synthesis. Based on these results, estradiol inhibits the area and collagen synthesis of HSCs in the growing chicken liver under normal physiological conditions.     

Falcon adenovirus infection in breeding Taita falcons (Falco fasciinucha).

Four female and 3 male Taita falcons (Falco fasciinucha) out of a breeding colony of 14 Taita falcons (7 pairs) died during the breeding season after showing lethargy and anorexia for 1 to 2 days. All animals were submitted for necropsy. Gross lesions in the female falcons were characterized by anemia secondary to marked hemorrhage into the ovary and oviduct, serofibrinous effusion into the cardioabdominal cavity and serosal petechiae. In addition, marked necrotizing splenitis and pulmonary hemorrhage were present. Histologically, the female falcons had mild necrotizing hepatitis with numerous intranuclear inclusion bodies and necrotizing splenitis with rare inclusion bodies. There were no gross lesions in the male falcons, and the histological lesions were characterized by urate deposition and rare intranuclear inclusion bodies in the renal tubular epithelial cells. Adenoviral particles were found by electron microscopy in the cloacal contents of the female Taita falcons but not in the male falcons. DNA in situ hybridization revealed widespread aviadenoviral nucleic acid within the nuclei of hepatocytes, renal tubular epithelial cells, and adrenal cells in the female falcons but no aviadenoviral nucleic acid in 1 male falcon and only a low quantity of adenoviral nucleic acid in the liver and kidney of another male Taita falcon. PCR amplified aviadenoviral DNA in the liver and intestine of all Taita falcons. The amplicons were sequenced, and the virus was identified as falcon adenovirus. The deaths of the female and male birds were attributed to the aviadenovirus infection.     

Age-Dependent Immunolocalization of Fibronectin and Histological Changes in the Thymus of Rats

Age-dependent variations in the immunolocalization of fibronectin (FN) in the thymus were investigated in 1-, 6-, 12- and 20-month-old male and female Swiss albino rats (Rattus rattus) at the light-microscopic level and the changes with ageing in the histological structure of the thymus were also studied. There were no significant differences in the age-dependent variations in the immunolocalization of fibronectin or in the histological structure of the thymus between male and female rats of the same age but there were increases with ageing in the fibronectin content of the thymic capsule, the connective tissue between the lobules, around blood vessels, and in the medulla and cortex of thymus. The connective-tissue content between lobules, fat cells, Hassall's corpuscles, the thickness of capsule and the ratio of the medulla to the cortex of the lobules showed age-dependent increases in the thymus of rats of both sexes. Decreases in the organ weight/body weight ratio were also observed with ageing.     

肉仔鸡饲粮中锌需要量的研究

取1日龄AA肉仔鸡600只,公、母各300只,随机分为10组,公、母各5组,每组4个重复,分别喂以含锌40mg/kg的基础日粮和含锌50、70、90和110mg/kg的添加锌日粮,以研究肉仔鸡对锌的需要量。试验期8周。结果表明,6周龄前日粮锌各水平对公、母鸡的体增重和饲料报酬无明显差异(P>0.05),40mg/kg锌可满足其生长需要。7周龄后,虽然公、母鸡分别在日粮含锌50和90mg/kg时可达到最大体增重,但差异不显著(P>0.05)。对于组织中的锌含量,不同组织、不同生长阶段对日粮补锌的反应不同。公、母鸡血液中锌含量并不随日粮锌含量的增加而升高;肾脏锌含量变化也不大;比较敏感的组织是胰和肝。组织锌含量依次为胰>肝>肾>血。血液中的碱性磷酸酶活性公、母鸡不同,但总的看来,随日龄增加而降低。铜锌超氧化物歧化酶(CuZnSOD)的活性不同组织、不同生长阶段和不同性别是不同的,母鸡比公鸡的酶活性高。根据各项指标的测定结果和经济效益综合分析,建议肉仔鸡对锌的需要量为40～50mg/kg。     

The relationship between cellular radiosensitivity and radiation-induced DNA damage measured by the comet assay

The relationship between deoxyribonucleic acid (DNA) damage and the cell death induced by gamma-irradiation was examined in three kinds of cells, Chinese hamster ovary fibroblast CHO-K1, human melanoma HMV-II and mouse leukemia L5178Y. Cell survival was determined by a clonogenic assay. The induction and rejoining of DNA strand breaks induced by radiation were measured by the alkaline and neutral comet assays. L5178Y cells were the most radiosensitive, while CHO-K1 cells and HMV-II cells were radioresistant. There was an inverse relationship between the survival fraction at 2 Gy (SF2) and the yield of initial DNA strand breaks per unit dose under the alkaline condition for the comet assay, and also a relationship between SF2 and the residual DNA strand breaks (for 4 hr after irradiation) under the neutral condition for the comet assay, the latter being generally considered to be relative to cellular radiosensitivity. In the present analysis, it was considered that the alkaline condition for the comet assay was optimal for evaluating the initial DNA strand breaks, while the neutral condition was optimal for evaluating the residual DNA strand breaks. Since the comet assay is simpler and more rapid than other methods for detecting radiation-induced DNA damage, this assay appears to be a useful predictive assay for evaluating cellular clonogenic radiosensitivity of tumor cells.     

The effect of age of host in resistance expressed at the gut level in rats infected with Fasciola hepatica

The development of oral and intraperitoneal infections with Fasciola hepatica in young and old rats showed that the gut was involved in the expression of age resistance. The role of the gut was assessed by comparison of the number of flukes recovered 4 and 10 weeks after oral or intraperitoneal infection of 2-, 6-, 15-, and 35-week-old male Wistar rats and 6- and 15-week-old male DA rats with encysted metacarcariae of F. hepatica.Rats of both strains behaved similarly in their response to F. hepatica infection. For both routes of infection the number of flukes recovered decreased as host age increased. In 22- and 6-week-old rats equal numbers of flukes were recovered at 4 and 10 weeks after oral or intraperitoneal infection. In 15-week-old rats, fluke burdens 4 weeks after infection were significantly greater following intraperitoneal infection than oral infection. A significant loss of flukes from the intraperitoneal infection of 15-week-old Wistar rats occured between 4 and 10 weeks after infection. In 35-week-old Wistar rats there was no significant effect for route or age of infection.As intraperitoneal infection (to by-pass rejection at the gut level) only partially eliminates the age response, additional age related mechanism(s), able to reject flukes at some time after they have entered the peritoneal cavity, must be operative in the peritoneal cavity and/or the liver.     

Influence of cholesterol administration and aging on the development of pulmonary foam cells in F344 rats

The influence of experimentally induced hyperlipidemia and aging on the development of pulmonary foam cells (PFCs) was examined in Fischer 344 rats. The male and female rats were administered orally with cholesterol at a dosage level of 1000 mg/kg/day for 30 days from 6 to 10 weeks or from 33 to 37 weeks of age. The control rats received the vehicle only in the same manner. Plasma levels of total cholesterol, phospholipid, triglyceride (TG), beta-lipoprotein (beta-LP) and calcium in both control and cholesterol-administered groups were higher at 37 weeks than at 10 weeks of age. Plasma beta-LP and TG levels in the treated groups were significantly higher or tended to be higher than those of the controls at 10 and 37 weeks of age. Males of the treated group showed the highest level of beta-LP at 37 weeks of age, positively correlated with the highest incidence of PFCs. PFCs developed singly or in a small cluster in peribronchial and subpleural regions. PFCs had an abundant cytoplasm filled with many fine vacuoles containing neutral lipid and cholesterol. PFCs stained with PAS and reacted immunohistochemically with both anti-rat monocytes/macrophages monoclonal antibody and anti-lysozyme antibody. Moderately swollen macrophages with a foamy appearance were detected in perivascular connective tissues of the lungs and they were considered to represent an initial stage of the development of PFCs. These observations suggest that hyperlipidemic conditions, particularly hyper beta-lipoproteinemia, resulting from cholesterol administration or aging may be involved in the development of PFCs in rats.