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ABSTRACT: To evaluate a radioprotective effect of sodium n-propyl thiosulfate (NPTS) and sodium 2-propenyl thiosulfate (2PTS) derived from onions and garlic, respectively, rat hepatoma H4IIE cells and mouse lymphoma L5178Y cells were preincubated with each of these compounds for 48 hours at 37°C before receiving 10 Gy of X-ray irradiation. Cell damage caused by the irradiation was quantified as comet tail moment, which represents the degree of DNA damage. X-ray-induced DNA damage was significantly decreased in both H4IIE and L5178Y cells by micromolar concentrations of NPTS and 2PTS compared with the control without the compounds. The protective effect was more potent with 2PTS than NPTS. Onions and garlic have antiradiation potential.
TheScientificWorldJOURNAL 01/2012; 2012:846750. · 1.66 Impact Factor
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ABSTRACT: 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 O₂⁻-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 O₂⁻-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.
Journal of Veterinary Medical Science 03/2011; 73(3):299-304. · 0.85 Impact Factor
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ABSTRACT: We have reported that treatment with trientine, Cu-chelating agent, inhibits tumor growth in a murine transplantation model using fibrosarcoma and induces apoptosis in tumor cells in vivo and in vitro. When fibrosarcoma cells were treated with 10 mM trientine, the activities of p38 MAPK in treated cells were approximately 3-4 times higher than those in untreated cells. Proportions of cells in which apoptosis was induced by trientine increased in an incubation time-dependent manner from days 2 to 6. The proportions of apoptotic cells in the cells treated with trientine and SB203580, an inhibitor of p38 MAPK, were approximately 50% in those of cells treated with trientine alone. The present results showed that the p38 MAPK pathway may play an important role in induction of apoptosis in fibrosarcoma cells by trientine.
Journal of Veterinary Medical Science 11/2009; 71(11):1541-4. · 0.85 Impact Factor
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ABSTRACT: The carrier rates of Complex Vertebral Malformation (CVM) in 9 Holstein dairy herds in Hokkaido, number of usages of CVM carrier semen for breeding and gene frequencies of CVM carriers were measured. The mean CVM carrier rates of 140 cows from 4 herds in 1994 and 315 cows from 5 herds in 2003 were 10.8%(range 4.7-30.0%) and 5.1%(range 0.0-6.1%), respectively. The rate of use of CVM carrier semen in the Hokkaido district was 5.6% in 2002. The gene frequencies calculated from CVM carriers among the 315 cows and number of CVM carrier semen samples used were 0.032 and 0.028, and the occurrence of homozygous CVM in 2003 was estimated to be 0.1% in the local districts of Hokkaido, Japan.
Journal of Veterinary Medical Science 07/2009; 71(6):793-5. · 0.85 Impact Factor
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ABSTRACT: Combined effects of treatment with trientine, a copper-chelating agent, and X-irradiation on development of fibrosarcoma using a murine transplantation model in vivo and on cellular survival in vitro were examined. Copper contents in the tumors and serum of trientine-treated mice were significantly lower than those of untreated mice. The tumor volumes of mouse fibrosarcoma QRsp-11 cells increased more slowly in the trientine-treated and the X-irradiated mice than in the control mice from 10 to 24 days postinoculation. The extent of inhibition of tumor growth by X-irradiation at 3 Gy was similar to that obtained by treatment with trientine. A combination of trientine and X-irradiation at 3 Gy showed inhibitory effects on tumor growth similar to those obtained by X-irradiation at 6 Gy. The results showed that trientine and X-irradiation interacted additively in inhibition of tumor growth. When QRsp-11 cells and mouse and bovine endothelial cells were treated with trientine after X-irradiation, the surviving fractions of the cells with combined treatments were essentially consistent with the products of the surviving fractions of trientine-treated cells and those of X-irradiated cells. When the cells were pretreated with trientine and X-irradiated, the surviving fractions of the pretreated cells were lower than those of cells without treatment.
Journal of Veterinary Medical Science 11/2007; 69(10):1039-45. · 0.85 Impact Factor
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ABSTRACT: Anti-copper treatments have been investigated to determine whether they suppress angiogenesis and tumor development since Cu is widely accepted as being required for angiogenesis. We examined the effects of treatment with trientine, a copper-chelating agent, on tumor development in a murine xenograft model using fibrosarcoma-derived transplantable QRsp-11 cells and C57BL/6 mice and induction of apoptosis in tumor cells and endothelial cells in vivo and in vitro. The tumor volumes increased more slowly in trientine-treated mice than in untreated mice. Tumor volumes in the treated mice were significantly smaller than those in the untreated mice at 24 days postinoculation (d.p.i.) of tumor cells. A cluster of pyknotic tumor cells and morphological abnormalities in capillary endothelial cells were observed in the tumors of trientine-treated mice but not in the tumors of untreated mice. The proportions of apoptotic and necrotic cells in the tumors of treated mice were approximately 3.5-fold higher than those in the tumors of untreated mice at 14 d.p.i. When the cells were treated with trientine in vitro, mouse endothelial cells and bovine primary endothelial cells showed an approximately 10-fold higher sensitivity to trientine than QRsp-11 cells in terms of D37. However, the proportion of apoptotic cells in endothelial cells was significantly lower than that in QRsp-11 cells after treatment with trientine. These results show that apoptosis was induced in tumor cells by treatment with trientine in vivo and in vitro.
Journal of Veterinary Medical Science 03/2007; 69(2):137-42. · 0.85 Impact Factor
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ABSTRACT: Copper accumulation and induction of DNA strand breaks were investigated in the brain of Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson disease that is a heritable disease of copper accumulation and copper toxicity in the liver, kidney and brain. Copper contents in the brain of LEC rats increased from 20 weeks of age and were approximately 3.5 to 6 folds higher than those in the brain of WKAH rats at 24 weeks of age. Hepatic copper contents in LEC rats increased from 4 to 12 weeks of age in an age-dependent manner, and then decreased from 16 to 20 weeks of age. Thus, we consider that copper accumulated in the liver was released from severely damaged hepatocytes and deposited in the brain, although copper contents in the brain were 1/20-fold lower than those in the liver. We also evaluated the amounts of DNA single-strand breaks (SSBs) in the brain by comet analysis. The proportions of nuclei in the cerebrum and cerebellum without DNA damage decreased, and nuclei with severe DNA damage appeared in LEC rats at 24 weeks of age. The comet scores of cerebrum and cerebellum cells significantly increased in LEC rats and were significantly higher than those in WKAH rats at 24 weeks of age. The results show that SSBs in LEC rat brain cells are induced at a lower concentration of copper than are SSBs in hepatic cells.
Experimental Animals 11/2006; 55(5):419-26. · 0.92 Impact Factor
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ABSTRACT: To determine whether changes in expression level of the phosphatase and tensin homolog deleted on the chromosome 10 (PTEN) gene are associated with malignant transformation in mammary gland tumors in dogs.
Specimens of 5 benign and 8 malignant mammary gland tumors and 2 unaffected mammary glands from dogs.
The open reading frame (ORF) sequence of PTEN gene in each specimen was analyzed via a direct-sequencing method; expression levels of PTEN gene were quantified via a competitive reverse transcription (RT)-PCR method.
Compared with findings in clinically normal samples, amounts of PTEN mRNA were increased 2- to 4-fold in 4 of the 5 benign mammary gland tumor samples. In contrast, PTEN expression was remarkably low in 4 of the 8 malignant tumor samples (approx 12% to 37% of the level in unaffected mammary gland specimens). Gene amplification via the RT-PCR method with total RNA prepared from malignant tumor samples as a template yielded 3 bands that were smaller than the full-length ORF product of PTEN gene; in 2 of those 3 RT-PCR products, exons 6 and 7 or exons 3 to 8 were absent. No mutation was detected in the full-length ORF product of PTEN gene.
Results suggested that a decreased level of PTEN gene expression (compared with unaffected mammary gland tissue) is associated with malignancy in canine mammary tumors. Analysis of PTENgene expression level in dogs with mammary gland tumors may provide useful prognostic information.
American Journal of Veterinary Research 02/2006; 67(1):127-33. · 1.27 Impact Factor
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ABSTRACT: The effects of treatment with trientine, a specific copper-chelating agent, on the accumulation of copper and induction of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson's disease. Copper accumulated in the kidneys of LEC rats in an age-dependent manner from 12 to 18 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, renal copper contents did not increase and were maintained at the same levels as those in 4-week-old LEC rats. Estimation of the amounts of DNA single-strand breaks (SSBs) by comet assay showed that SSBs of DNA were induced in a substantial population of LEC rat renal cortex cells around 12 weeks of age and that the amounts of SSBs increased in an age-dependent manner from 12 to 18 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, the observed number of cells with DNA damage decreased, suggesting that induction of SSBs of DNA was inhibited and/or SSBs were repaired during the period of treatment with trientine. The results show that SSBs of DNA in LEC rat kidney cells are induced prior to occurrence of clinical signs of hepatic injury and that treatment of LEC rats with trientine decreases the number of DNA strand breaks.
Experimental Animals 11/2005; 54(5):403-12. · 0.92 Impact Factor
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ABSTRACT: Complex vertebral malformation (CVM), a hereditary lethal disease in Holstein calves, is characterized by complex anomalies of the vertebral column and limbs in an aborted fetus and in prematurely born, stillborn, and neonatal calves. The mode of inheritance of CVM is autosomal recessive, and CVM is caused by a point mutation from G to T at nucleotide position 559 of the bovine solute carrier family 35 member 3 (SLC35A3) gene. Although an allele-specific polymerase chain reaction (AS-PCR) is a useful method for diagnosis of CVM, the AS-PCR requires selected DNA polymerases and strictly controlled reaction conditions to obtain reliable results. Therefore, an alternative screening method for the CVM gene would be useful. Polymerase chain reaction-primer introduced restriction analysis (PCR-PIRA) is a method that can be used for detecting a single nucleotide mutation in any gene without a restriction site around the mutation site. In this study, primers were designed to introduce PstI or EcoT22 sites into PCR products from the wild-type and CVM alleles, respectively. The wild-type allele, a heterozygote, and a homozygote of the CVM allele could be discriminated by restriction fragment length polymorphism analysis. Specific introduction of restriction sites into PCR products depending on the change in a single nucleotide of template was shown using a variety of DNA polymerases and PCR machines. Therefore, the PCR-PIRA technique using primers designed in this study might provide a more useful method for extensive screening of CVM.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 06/2005; 17(3):258-62. · 1.21 Impact Factor
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ABSTRACT: A method for the isolation of genomic fragments of RNA virus based on cDNA representational difference analysis (cDNA RDA) was developed. cDNA RDA has been applied for the subtraction of poly(A)(+) RNAs but not for poly(A)(-) RNAs, such as RNA virus genomes, owing to the vast quantity of ribosomal RNAs. We constructed primers for inefficient reverse transcription of ribosomal sequences based on the distribution analysis of hexanucleotide patterns in ribosomal RNA. The analysis revealed that distributions of hexanucleotide patterns in ribosomal RNA and virus genome were different. We constructed 96 hexanucleotides (non-ribosomal hexanucleotides) and used them as mixed primers for reverse transcription of cDNA RDA. A synchronous analysis of hexanucleotide patterns in known viral sequences showed that all the known genomic-size viral sequences include non-ribosomal hexanucleotides. In a model experiment, when non-ribosomal hexanucleotides were used as primers, in vitro transcribed plasmid RNA was efficiently reverse transcribed when compared with ribosomal RNA of rat cells. Using non-ribosomal primers, the cDNA fragments of severe acute respiratory syndrome coronavirus and bovine parainfluenza virus 3 were efficiently amplified by subtracting the cDNA amplicons derived from uninfected cells from those that were derived from virus-infected cells. The results suggest that cDNA RDA with non-ribosomal primers can be used for species-independent detection of viruses, including new viruses.
Nucleic Acids Research 02/2005; 33(6):e65. · 8.03 Impact Factor
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ABSTRACT: 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.
Journal of Veterinary Medical Science 11/2004; 66(10):1177-82. · 0.85 Impact Factor
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ABSTRACT: Effects of treatment with trientine, a specific copper-chelating agent, on accumulation of copper and induction of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson's disease. Copper accumulated in the livers of LEC rats in an age-dependent manner from 4 to 13 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, hepatic copper contents did not increase and were maintained at the same levels as those in 10-week-old LEC rats. When the amounts of DNA single-strand breaks (SSBs) were estimated by a comet assay, SSBs of DNA were induced in a substantial population of LEC rat hepatic cells around 8 weeks of age and the amounts of SSBs increased in an age-dependent manner from 8 to 15 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, the observed number of cells with DNA damage decreased dramatically, suggesting that induction of SSBs of DNA was inhibited and/or SSBs were repaired during the period of treatment with trientine. The results show that treatment of LEC rats with trientine decreases the number of DNA strand breaks observed, although copper contents remain high in the liver.
Biochimica et Biophysica Acta 11/2004; 1674(3):312-8. · 4.66 Impact Factor
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ABSTRACT: Amounts of DNA strand breaks were estimated by the proportion of cells without tails (PCWT) and the average lengths of tail momentum (ALTM) in comet images of tissue cells of senescence-accelerated prone (SAMP1) mouse and senescence-accelerated resistant (SAMR1) mouse. The PCWT and ALTM of brain cells from SAMR1 were unchanged from 4 to 15 months of age. In the case of SAMP1 brain cells, the PCWT decreased and the ALTM increased in an age-related manner from 8 to 15 months of age. In the cases of liver and kidney, the PCWT and the ALTM of both SAMP1 and SAMR1 cells showed constant values from 4 to 15 months of ages.
Experimental Animals 08/2003; 52(4):353-7. · 0.92 Impact Factor
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ABSTRACT: 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.
Journal of Veterinary Medical Science 03/2003; 65(2):249-54. · 0.85 Impact Factor
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ABSTRACT: 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.
Journal of Veterinary Medical Science 10/2002; 64(9):761-5. · 0.85 Impact Factor
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ABSTRACT: 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.
Journal of Veterinary Medical Science 08/2002; 64(7):597-601. · 0.85 Impact Factor
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ABSTRACT: The DNA-dependent protein kinase (DNA-PK) complex has been implicated in the repair of DNA double-strand breaks (DSBs). DNA-PK is a heterotrimeric protein complex comprised of two components: a large catalytic subunit, Prkdc, with serine/threonine kinase activity and a DNA-targeting component, G22p1 and Xrcc5. In previous report, we showed that approximately 80% of the G22p1 and Xrcc5 proteins were observed in the cytoplasm of rat fibroblasts, and that nuclear translocation of the proteins from the cytoplasm is important for the repair of DNA DSBs. In the present study, we showed that nuclear accumulation of the G22p1 and Xrcc5 proteins was not observed in fibroblasts from a mutant strain of Long-Evans Cinnamon (LEC) rat that has an enhanced radiosensitivity and a reduced level of repair of DSBs after X irradiation. Nuclear translocation of the proteins was observed in both LEC rat cells and control rat cells with normal radiosensitivity at 5 min after X irradiation. Although high levels of G22p1 and Xrcc5 proteins were observed in the nuclei of control rat cells until 60 min postirradiation, the amounts of the proteins decreased rapidly in the nuclei of LEC rat cells in the first 10 min after X irradiation. These findings suggest that there are some defects in maintaining the levels of G22p1 and Xrcc5 proteins in the nuclei of LEC rat cells. An analysis of fibroblasts from backcross rats showed that the deficiency in nuclear accumulation of G22p1 and Xrcc5 proteins is genetically linked to enhanced radiosensitivity. Since the nucleotide sequences of the G22p1 and Xrcc5 genes of the LEC rats coincided with those of the control rats, the deficiency in nuclear accumulation may not be caused by mutations of the G22p1 and Xrcc5 proteins.
Radiation Research 06/2002; 157(5):553-61. · 2.68 Impact Factor
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ABSTRACT: To demonstrate whether protein synthesis is required for ionizing radiation-induced apoptosis through activation of caspases in human leukaemia cell line MOLT-4, the effects of a protein synthesis inhibitor, cycloheximide, on the apoptotic signalling pathway including the activation of caspase family and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and the expression of Fas/CD95/APO-1 (Fas) were examined in X-irradiated MOLT-4 cells.
MOLT-4 cells pretreated with 0.5 microg/ml cycloheximide for 1h were exposed to 7.5Gy of X-rays. The appearance of apoptosis, expression of Fas, activation of caspases-3, -8, -9, SAPK/JNK and AP-1, the release of mitochondrial cytochrome-C and the formation of death-induced signalling complex (DISC) between Fas and the Fas-associated death domain (FADD) were measured by fluorescence microscopy, Western blotting, flow cytometry, gel shift assay and immunoprecipitation, respectively.
Nuclear fragmentation and chromatin condensation were observed at 6 h after X-irradiation and gradually increased up to 12 h. These phenomena were significantly attenuated by cycloheximide. Cycloheximide also inhibited the activation of caspases and AP-1, the expression of Fas, the formation of DISC and the release of cytochrome-C, but not the activation of SAPK/JNK in X-irradiated MOLT-4 cells.
These results indicate that apoptosis of X-ray-induced MOLT-4 cells is dependent on the activation of caspases regulated by de novo protein synthesis through SAPK/JNK activation.
International Journal of Radiation Biology 03/2002; 78(2):115-24. · 2.28 Impact Factor
