ABSTRACT. The relationship between deoxyribonucleic acid (DNA) damage and the cell death induced by γ-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. KEY WORDS: comet assay, DNA strand break, γ-irradiation, predictive assay, radiosensitivity.J. Vet. Med. Sci. 65(4): 471-477, 2003 Different types of cell lines show different radiosensitivities. In addition, even a tumor of the same origin and pathology, can show radiosensitivity over quite a wide range [4], and therefore, understanding the radiosensitivity of an individual tumor is of great importance in the clinical situation. Knowledge of the intrinsic radiosensitivities of cells from a tumor biopsy can help produce an optimized radiotherapy protocol for individual patients.Reproductive death measured by a clonogenic assay is an important index for the cell death induced by radiation, and the cellular radiosensitivities of fibroblasts and tumor cells have been evaluated by a clonogenic assay [13,17]. A good correlation was found between the reproductive death of fibroblasts measured by the clonogenic assay and the severity of late normal tissue effects, such as fibrosis and erythema, after radiotherapy [7,14]. However, this clonogenic assay consumes a lot of time, and the cells must be proliferated in vitro [4,14]. Hence this assay is not optimal for routine clinical use, and thus there is now much interest in exploiting a more rapid and simple assay to evaluate cellular radiosensitivity [8,25].It is said that the cell death caused by radiation is due to deoxyribonucleic acid (DNA) damage [29]. DNA double strand breaks (dsbs) are considered to be closely related to cell death because it is suggested that they can lead directly to chromosome aberration and the loss of genetic material [22]. To date, the correlation between radiose...

ABSTRACT. The electrophoresis pattern of apoptotic cells detected by the comet assay has a characteristic small head and spread tail. Thi s image has been referred to as an apoptotic comet, but it has not been previously proven to be apoptotic cells by any direct method. In order to identify this image obtained by the comet assay as corresponding to an apoptotic cell, the frequency of appearance of apoptosis was examined using CHO-K1 and L5178Y cells which were exposed to gamma irradiation. As a method for detecting apoptosis, the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay was used. When the frequency of appearance of apoptotic cells following gamma irradiation was observed over a period of time, there was a significant increase in appearanc e of apoptosis when using the TUNEL assay. However, there was only a slight increase when using the comet assay. In order to verify th e low frequency of appearance of apoptosis when using the comet assay, we attempted to use the TUNEL assay to stain the apoptotic comets detected in the comet assay. The apoptotic comets were TUNEL positive and the normal comets were TUNEL negative. This indicate s that the apoptotic comets were formed from DNA fragments with 3'-hydroxy ends that are generated as cells undergo apoptosis. Th erefore, it was understood that the characteristic pattern of apoptotic comets detected by the comet assay corresponds to cells undergoing apoptosis.

ABSTRACT. Gastrointestinal stromal tumor (GIST), a mesenchymal neoplasm affecting the gastrointestinal tract, shows a variety of clinical behaviors from inactive benign to aggressive malignant in dogs. In this study, the feasibility of using clinically significant ultrasonographic features to predict the metastatic potential of canine GIST was investigated through comparison with actual metastatic incidence and findings of malignancy obtained by postoperative pathological examination. Ultrasonographic features, including large tumor size, irregular margin and heterogeneous internal echogenicity with large hypoechoic areas, related closely with the presence of metastasis as well as a high-risk ranking by the human classification system according to pathological findings. Based on these ultrasonographic features, the potential of metastasis in canine GIST could be preoperatively predicted.

Ferritin utilizes ferroxidase activity to incorporate iron. Iron uptake kinetics of bovine spleen apoferritin (H: L = 1 : 1.1) were compared with those of recombinant H chain ferritin and L chain ferritin homopolymers. H chain ferritin homopolymer showed an iron uptake rate identical to bovine spleen apoferritin (0.19 and 0.21 mmol/min/micromol of protein, respectively), and both showed iron concentration-dependent uptake. In contrast, the L chain homopolymer, which lacks ferroxidase, did not incorporate iron and showed the same level of iron autoxidation in the absence of ferritin. Bovine spleen apoferritin was shown to have two iron concentration-dependent uptake pathways over a range of 0.02-0.25 mM ferrous ammonium sulfate (FAS) by an Eadie-Scatchard plot (v/[FAS] versus v), whereas the H chain ferritin homopolymer was found to have only one pathway. Of the two Km values found in bovine spleen apoferritin, the lower mean Km value was 9.0 microM, while that of the H chain homopolymer was 11.0 microM. H chain ferritin homopolymer reached a saturating iron uptake rate at 0.1 mM FAS, while bovine spleen apoferritin incorporated more iron even at 0.25 mM FAS. These results suggest that the intrinsic ferroxidase of ferritin plays a significant role in iron uptake, and the L chain cooperates with the H chain to increase iron uptake.

Concentrations of pig plasma folates were determined by a high-performance liquid chromatography with an electrochemical detector (HPLC-ECD). Tetrahydrofolic acid (THF) and 5-methyltetrahydrofolic acid (5-MF) were found to be the plasma folates. The concentration of THF (10.3 +/- 4.3 ng/ml) was significantly higher than that of 5-MF (3.5 +/- 1.0 ng/ml). The sum of the THF and 5-MF concentrations determined by the HPLC-ECD analysis in Göttingen miniature pigs was comparable to the total folate concentration of the same plasma sample determined by a radioligand assay. No or only trace amounts of THF were found in the plasma of the rats, mice, rabbits, dogs, cows, horses, or humans. Although 5-MF is generally recognized as the only principal plasma folate in many species, THF may be the principal congener with 5-MF as a secondary congener in the pig.