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Effects of TCDD on micronucleus formation and cellular factors responsible for toxicity in human cell lines

Effects of TCDD on micronucleus formation and cellular factors responsible for toxicity in human cell lines
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대학원 화학과
이화여자대학교 대학원 화학과
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a powerful carcinogen for multiple species and is known to induce a wide spectrum of biological responses, including changes in gene expression, metabolism, cell growth, and differentiation. However, the mechanisms of TCDD-induced carcinogenesis are not completely understood. To enhance our understanding of TCDD carcinogenicity at the cellular or molecular level, we studied TCDD-induced responses in human cell lines. First, as an index of genetic toxicity, we investigated the effect of TCDD on micronucleus (MN) frequency. We also studied whether TCDD can transform human cells in vitro in skin keratinocyte HaCaT, Chang liver and breast MCF10A cells as nontumorigenic human cell lines. TCDD did not affect the cell viability of Chang liver, HaCaT, and MCF10A cells. The MN frequency was increased after TCDD treatment for 24 h in Chang liver and HaCaT cells, but was unchanged in the MCF10A cells. We observed putative transformed cells in Chang liver cells that were exposed to 1 μM TCDD for 2 weeks. The putative transformed cells were also observed in HaCaT cells that were initially exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and then to TCDD (0.1, 1, 10, and 100 nM) for 2 weeks; however, such cells were not observed in the MCF10A cells. Collectively, these results indicate that the ability of TCDD to induce micronuclei may lead to the cellular transformation of Chang liver and HaCaT cells. Our putative TCDD-transformed Chang liver and HaCaT cells are expected to provide a clue in elucidating the TCDD-induced transformation pathway. Second, we compared the TCDD-induced responses in two human liver cell lines - hepatoma HepG2 and nontumorigenic fetal hepatic WRL68. We found that the growth of HepG2 cells decreased by more than 50 % after treatment with TCDD at concentrations greater than 1 nM; however TCDD did not have any effect on WRL68 cell proliferation. Thus, it appears that the cancer cell line HepG2 is more sensitive to TCDD than the non-cancerous cell line WRL68 and that WRL68 cells are TCDD resistant. Further, in the HepG2 cells, the levels of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG), which is known to be a potential surrogate marker for oxidative DNA damage, increased in a time-dependent manner after exposure to 0.1 nM TCDD. In WRL68 cells, the 8-oxo-dG level increased after 2 h following treatment with 0.1 nM TCDD and then returned to the control level at 6 h. Therefore, it appears that TCDD can induce oxidative stress by increasing the 8-oxo-dG level in our experimental system. In addition, to determine whether stress and toxicity-related genes are induced or suppressed in response to the experimental conditions used in our study, we performed a cDNA “mini-array” analysis. Hsp90, which binds to the aromatic hydrocarbon receptor (AhR), was up-regulated 1.5 fold in HepG2 and WRL68 cells treated with 0.1 nM TCDD; AhR is a known TCDD receptor in the cytoplasm. Some marker genes of the p53 signaling pathway such as p21, gadd45, and mdm2 were down-regulated by approximately 2 fold in HepG2 cells. On the other hand, p21 and mdm2, which are known to be marker genes of the p53 signaling pathway, were up-regulated by more than 2 fold in WRL68 cells. Stress signal pathway-related genes such as p53, hsf1, hsp27, and hsp90, were upregulated in WRL68 cells but downregulated in HepG2 cells. Collectively, our results suggest that TCDD may induce cellular oxidative stress, and the genes related to stress may be responsible for TCDD-induced toxicity.;2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)는 가장 강력한 발암물질로서 유전자 발현, 대사과정, 세포 증식 및 분화 등 다양한 생물학적인 변화를 일으키는 것으로 알려져 있다. 그러나 TCDD에 의한 발암과정은 완전히 설명되고 있지 못하기 때문에, 본 연구에서는 세포 또는 분자수준에서 TCDD의 발암성에 대한 이해를 증진시키고자 사람유래세포를 이용하여 TCDD에 의한 영향을 조사하였다. 첫째, 사람유래 정상세포로 간주되는 skin keratinocyte HaCaT, Chang liver 및 breast MCF10A 세포를 이용하여 유전독성지표로서 소핵형성에 대한 TCDD의 영향 및 이들 세포에 대한 TCDD의 암화능력을 조사하였다. 먼저 Chang liver, HaCaT 및 MCF10A 세포 생존에 대한 TCDD의 영향을 조사한 결과, TCDD는 이들 세포의 성장에 영향을 주지 않았다. Chang liver 및 HaCaT 세포는 TCDD에 의해 소핵형성이 증가되었으나, MCF10A 세포에서는 TCDD가 소핵형성을 유발하지 않는 것으로 확인되었다. 또한, 1 μM TCDD를 2주간 처리하였을 때 Chang liver 및 HaCaT 세포에서 형질전환세포로 추정되는 foci가 관찰되었다. 이들 결과는 TCDD의 소핵유발 능력이 TCDD-induced transformation pathway를 설명하는 열쇠가 될 수 있음을 시사해준다. 둘째, 사람 유래 간세포중 hepatoma HepG2 및 nontumorigenic fetal hepatic WRL68 세포를 선택하여 TCDD에 의한 세포내 영향을 조사하였다. 먼저 HepG2 및 WRL68 세포의 세포증식에 대한 TCDD의 영향을 조사한 결과, 1 nM 이상의 TCDD 농도에서 HepG2 세포의 증식이 50%이상 감소되었으나, WRL68 세포의 증식에는 TCDD가 아무런 영향을 주지 않았다. 따라서, 이러한 결과들은 암세주인 HepG2 세포보다 정상세포 특성을 갖는 WRL68 세포가 TCDD에 대해 덜 민감하게 반응함으로써 정상세포의 TCDD에 대한 내성을 예측하게 해 준다. 또한 산화적 DNA 손상에 대한 지표인자로서 7,8-dihydro-8-oxo-2-deoxyguanosine (8-oxo-dG)를 분석한 결과, 0.1 nM TCDD를 처리한 HepG2 세포에서는 시간의존적으로 8-oxo-dG가 증가되었다. WRL68 세포에 있어서는 TCDD 처리 후 2시간째에 8-oxo-dG가 크게 증가였으며, 6시간 후에는 다소 감소되었다가 다시 증가되는 양상을 보였다. 본 시험조건에서 TCDD에 의해 8-oxo-dG 생성이 증가되는 결과로부터 TCDD가 세포내 산화적 스트레스를 유발하는 것으로 생각되어진다. 또한, 본 시험조건에서 스트레스 관련 유전자들의 변화를 관찰하기 위하여 0.1 nM TCDD 를 처리한 HepG2 및 WRL68세포에서 cDNA “mini-array” 시험을 실시하였다. 그 결과, HepG2 및 WRL68 세포 모두에서 TCDD의 수용체인 aromatic hydrocarbon receptor (AhR)과 결합하는 Hsp90 유전자가 1.5배 증가되는 것으로 나타났다. HepG2 세포에 있어서 p53 signal pathway에 관여하는 p21, gadd45, mdm2 유전자가 2배이상 감소하였으며, WRL68세포에서는 이들 유전자들이 2배이상 증가하였다. 이들 결과들을 종합해보면, 본 시험조건에서 TCDD는 세포내 산화적 스트레스를 유발하고, 스트레스 관련 유전자들이 TCDD의 독성작용에 관여할 수 있음을 시사해주고 있다.
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