• Journal of radiological science and technology
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• v.26 no.1
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• pp.91-97
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• 2003

The purpose of this study is to investigate the mechanism of inhibition of chondrogenic differentiation by X-irradiation. Cultures of chick limb bud mesenchymal cells were exposed to various dose of X-ray and chondrogenesis was examined. X-irradiation inhibited accumulation of proteoglycan based on the observation of alcian blue staining and expression of chondorcyte specific-type II collagen. X-irradiation also inhibited expression of protein kinase $C{\alpha}$ while expression of $PKC{\lambda}({\iota}),\;{\varepsilon}$ was not altered. Expression of Erk-1 was not changed by X-irradiation but phosphorylation of Erk-1 was increased. In addition, inhibition of Erk-1 phosphorylation by PD98059 overcame inhibitory effect of X-irradiation on the chondrogenic differentiation. PNA staining data showed that X-irradiation inhibited cellular aggregation. Taken together, these results suggest that X-irradiation inhibits chondrogenic differentiation by inhibiting cellular aggregation and suppressing expression of $PKC{\alpha}$ and promoting phosphorylation of Erk-1. In addition to above pathway, our results also suggest that X-irradiation may exerts its inhibitory effect by another signaling pathways.

• Toxicological Research
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• v.20 no.2
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• pp.117-122
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• 2004

The present study was carried out to establish a short-term teratogenicity study for detecting developmental toxic potential induced by gamma radiation in ICR mice. Pregnant mice were exposed at dose levels of 0, 0.5, 1, 2, or 4 Gy on gestational day 8.5. All dams were subjected to caesarean section on gestational day 10.5 and their embryos were examined for growth, differentiation, and morphological abnormalities. An increase in the number of resorption was found at 4 Gy in a dose-dependent manner. Dose-dependent decreases in the developmental score of yolk sac circulation and olfactory system at above 1 Gy, in the number of somite pairs and developmental score of allantois, optic system, and maxillary process at above 2 Gy, and in the all growth and developmental parameters examined at 4 Gy were found. Various types of morphological abnormalities were seen at dose levels of 0.5 Gy or greater. Characteristic malformations induced by gamma radiation were abnormal axial rotation, hematoma, craniofacial hypoplasia, open neuropore, shortened prosencephalon, kinked somites, irregular somites, swelling, hydropericardium, absent branchial bar, and absent limb bud. Morphological alterations such as hematoma, craniofacial hypoplasia, open neuropore, and kinked somites were noted even in the lowest dose (0.5 Gy). These results indicated that the short-term teratogenicity study established in this study can be a useful tool for not only detecting the developmental toxic potential induced by gamma radiation, but also screening radio-protective agents in ICR mice.

• Animal cells and systems
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• v.2 no.2
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• pp.229-232
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• 1998

Lysophosphatidylcholine (LPC) has been reported to be responsible for the sustained activation of protein kinase C (PKC). As chondroqenesis is known to be regulated by PKC, this study was performed to investigate the effects of LPC on chondrogenesis of chick limb bud mesenchymes in vitro. LPC treatment of mesenchymes during micromass culture significantly enhanced chondrogenic differentiation. The most effective time of LPC on the stimulation of chondrogenesis was the first day of micromass culture. Analysis of LPC effects on the expression of PKC isoforms revealed that LPC treatment increased expression of PKCa, among the multiple PKC isoforms, in the membrane fraction on day one of culture. The stimulatory effect of LPC on chondrogenesis was abolished if PKCa was down regulated by the prolonged treatment of cells with phorbol ester. The results suqqest that LPC promotes chondrogenesis through the activation of PKCa at the early stage of chondrogenic differentiation.

• Animal cells and systems
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• v.4 no.4
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• pp.361-366
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• 2000

In order to investigate the role of protein kinase C (PKC) in chondrogenic differentiation, we examined the localization of PKC isoforms in a limb bud micromass culture system. PKC$\alpha$ is specifically localized in the regions which would become cartilage nodules, while PKC$\lambda/l$ and $\zeta$ display widespread distribution in the whole culture. Distribution of PKC$\alpha$ change along with promotion or inhibition of chondrogenesis by lysophosphatidylcholine or phorbol 12-myristate 13-acetate. On the other hand, localization of PKC$\lambda/l$ or $\zeta$ a was not changed by the modulation of chondrogenesis. Peanut agglutinin binding protein which is associated with cell aggregation during chondrogenesis was present in the cell condensation regions and its expression in those regions was influenced by PKC activity. Expression of fibronectin and N-cadherin in the cell condensing area were also affected by modulation of PKC activity. These results suggest involvement of PKC$\alpha$ in the cell condensation, possibly through regulating expression of fibronectin and N-cadherin.

• The Korean Journal of Zoology
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• v.33 no.3
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• pp.310-321
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• 1990

To establish the in vitro culture system and quantitation for chondrogenesis, and to investigate the relationship between cell aggregation and chondrogenesis, chick limb bud mesenchymal cells of Hamburger-Hamilton stage 23/24 were micromass cultured in various cell densities. The chondrogenesis was assayed based on checking the alcian blue-stained nodule numbers, the amount of alcian blue extraded, the change in cell numbers, the rate of [35 S] sulfate incorporation and expression of type II collagen. Mesenchymal cells plated with an initial density of high (1 x 107 cells/ml)- and intermediates (5. $\times$ 106 cells/ml)-density were differentiated into cartilage. On the other hand, the cells of low density (2 x 106 cells/mi, 5 $\times$ 105 cells/ml) of stage 23/24 cells and the stage 18/19 cells in three kinds of cell density did not differentiate into cartilage even though the cells formed an aggregated core at the center of cultured mass. From these results and others obtained in this study, it can be stated that the stage 23/24 mesenchymal cells are likely to pass over the aggregation step and have the potentiality to differentiate into chondrocytes. Thus chondrogenesis in vitro can be observed when mesenchymal cells are plated over the threshold density of 5 $\times$ 106 cells/ml. Hyaluronidase (HAase) activity was relatively constant throughout the culture, suggesting that the role of HAase may not be important for the cells of stage 23/24.

• Toxicological Research
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• v.14 no.3
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• pp.357-363
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• 1998

The teratogenic potential of the anticonvulsant drug phenytoin (PHT) has been well documented both in the human and in the experimental animals. However there are few reports on the effects of PHT on embryonic development in rats in vitro. The present study was performed to evaluate the teratogenic effects of PHT using whole-embryo culture system in rats. Sprague-Dawley rat embryos were explanted on gestational day (GD) 9.5 and cultured for 48 hrs in the immediately centrifuged and heat-inactivated rat serum containing 0,25,50, or $100{\mu}g$ PHT/mL. At the end of culture period the embryos were scored for morphological development according to the procedure of Van Maele-Fabry, and their total protein contents were determined. At 100 ${\mu}$g/mL of culture medium. PHT caused significant reduction in developmental score and protein content of embryos and a high incidence morphological abnormalities (100%). Characteristic malformations included altered yolk and embryonic circulation, craniofacial hypoplasia, neural tube schisis, branchial arch defects, abnormal ratation, and limb bud hypoplasia, among others. There were no adverse effects on embryonic growth and development at concentrations of 25 and 50 ${\mu}$g /mL of culture medium. The results indicated that the dysmorphogenic effect of PHT on cultured embryos is due to a direct interference with embryonic development.

• Journal of radiological science and technology
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• v.25 no.2
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• pp.77-82
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• 2002

It is well known that X-irradiation affects on maturing process of differentiated chondrocytes. Nevertheless, It has been remained elusively whether X-irradiation affects the process of differentiation of mesenchymal cells which differentiate into chondrocyte, fibroblast, or muscle cells. In this study, we examined the effect of X-irradiation (with 1 to 10 Gy) on chondrogenesis using the mesenchymal cells of chick limb bud. Our results show that X-irradiation dose-dependently inhibited chondrogenesis. This result suggests that immature chondroblast-like mesenchymal cells are sensitive to X-irradiation. Moreover, X-irradiation affects not only maturing process of chondrocytes, but also inhibits the chondrogenesis. Taken together, we demonstrate that the whole process of differentiation of mature chondrocytes from mesenchymal cells is affected by X-irradiation and undifferentiated cells were more affected by X-irradiation than mature cells.

• Toxicological Research
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• v.26 no.3
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• pp.209-216
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• 2010

Limb bud (LB) and central nerve system (CNS) cells were prepared from 12.5 day old pregnant female Crj:CD (SD) rats and treated with olaquindox and vitamin A. Cytotoxicity and inhibition on differentiation were measured in each cell. Three doses of olaquindox (4, 21 and 100 mgkg), and 0.2 and 75 mg/kg of vitamin A were administered to pregnant rat for 11 days from $6^{th}$ to $16^{th}$ of pregnancy. $IC_{50}$ values of olaquindox for proliferation and differentiation in CNS cell were 22.74 and $28.32\;{\mu}g/ml$ and 79.34 and $23.29\;{\mu}g/ml$ in LB cell and those values of vitamin A were 8.13 and $5.94\;{\mu}g/ml$ in CNS cell and 0.81 and $0.05\;{\mu}g/ml$ in LB cell, respectively. Mean body weights of pregnant rats were decreased at high dose of olaquindox (110 mg/kg) but relative ovary weight, number of corpus lutea, and number of implantation were not changed. Resorption and dead fetus were increased at high dose of olaquindox, and relative ovary weight, the number of corpus lutea and implantation, and sex ratio of male to female were not significantly changed in all dose of olaquindox. Mean fetal and placenta weights were significantly (p < 0.01) decreased in rats of high group. Seven fetuses out of 103 showed external anomaly like bent tail, and 10 out of 114 fetuses showed visceral anomalies at high group. The ossification of sternebrae and metacarpals were significantly (p < 0.01) increased by low and middle dose of olaquindox but it was significantly (p < 0.01) prohibited by high dose of olaquindox. In rats treated with vitamin A, the resorption and dead fetus were increased by high dose. Mean fetal weights were significantly (p < 0.01) increased by low dose but significantly (p < 0.01) decreased by high dose. Thirty four fetuses out of 52 showed external anomaly; bent tail (1), cranioarchschisis (14), exencephaly (14), dome shaped head (22), anophthalmia (15), brcahynathia (10) and others (19). Forty five fetuses out of 52 showed soft tissue anomaly; cleft palate (42/52) and anophthalmia (22/52) by high dose of vitamin A. Sixty one fetuses out of 61 (85.2%) showed skull anomaly; defect of frontal, partial and occipital bone (21/61), defect of palatine bone (52/61) and others (50/61). In summary, we support that vitamin A is strong teratogen based on our micromass and in vivo data, and olaquindox has a weak teratogenic potential in LB cell but not in CNS cell. We provide the in vivo evidence that a high dose of olaquindox could have weak embryotoxic potential in rats.