갑상선세포에서 산화스트레스에 대한 여러 종류의 식이항산화제의 효과

Abstract

Potassium bromate (KBrO3)는 산화스트레스와 암을 유발하며, 수돗물과 생수의 오존화 과정에서 형성된다. 이 연구는 갑상선세포에서 KBrO3가 유발할 수 있는 산화독성에 대해 일상생활에서 섭취할 수 있는 여러 항산화물질들이 보호효과를 알아보고자 하였다. 흰쥐의 갑상선세포주인 FRTL-5 세포를 6H 배양액에 배양한 후 200 μM의 KBrO3를 4시간 동안 처리하여 산화독성을 유발하였고, KBrO3 독성에 대한 항산화제들의 보호효과를 관찰하기 위하여 N-acetylsysteine (NAC), melatonin, (-)-epigallocatechin-3 -gallate (EGCG), D-methionine, caffeine, estrogen과 비타민 A, C, D, E를 각각 4 ～ 24시간 전처치한 후 FRTL-5 세포의 생존율을 비교하였다. 배양액에 KBrO3를 처치한 후 FRTL-5 세포의 생존율은 100, 200, 300, 400, 500 μM KBrO3를 처치하였을 때 각각 97.4 ± 2.5, 61.5 ± 4.0, 35.0 ± 4.1, 21.4 ± 2.6, 13.7 ± 1.6%로 농도에 의존적으로 감소하였다. 200 μM KBrO3만을 처치하였을 때와 비교하여 0.05, 0.1, 0.2, 0.4, 1 nM NAC를 전처치하였을 때 세포생존율은 각각 85.6 ± 1.2, 92.9 ± 1.0, 92.8 ± 0.8, 95.0 ± 0.6, 96.1 ± 0.4%로 KBrO3의 독성을 거의 차단하였다. Estrogen, 비타민 C 및 E를 각각 전처치한 후 200 μM KBrO3를 투여하였을 때 세포생존율은 KBrO3만을 처치하였을 때와 비교해서 유의하게 증가하였다. EGCG, 비타민 A 및 D를 각각 전처치한 후 200 μM KBrO3를 투여하였을 때 세포생존율은 낮은 농도에서는 유의하게 증가하였으나 고농도에서는 생존율이 오히려 감소하였다. 그러나 melatonin, D-methionine, caffeine을 각각 전처치한 후 200 μM KBrO3를 투여하였을 때 세포생존율은 KBrO3만을 처치하였을 때와 비교해서 변화가 없었다. 이상의 결과에서 FRTL-5 갑상선세포에서 KBrO3에 의해 유발된 산화스트레스에 대하여 EGCG, estrogen, 비타민 A, C, D, E는 보호효과를 나타내었으나 고농도에서 EGCG, 비타민 A와 D는 오히려 세포독성을 유발할 가능성이 있어 항산화제의 섭취에 주의를 기울여야 할 것으로 생각된다. Potassium bromate (KBrO3) is a prooxidant and carcinogen that is frequently detected in tap and botteled water. The present study was designed to evaluate the protective effects of dietary antioxidants on oxidative stress induced by KBrO3 in FRTL-5 rat thyroid cells. FRTL-5 cells were primarily incubated in the absence or presence of KBrO3 ranging 100 ～ 500 μM concentrations for 4 hours to induce the cytotoxicity, which was measured by the viability assay using 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfophenyl) -2H-tetrazolium (MTS) reagent. The protective effect of dietary antioxidants, including melatonin, D-methionine, (-)-epigallocatechin-3 -gallate (EGCG), estrogen, or vitamins (A, C, D, E) on KBrO3-mediated cytotoxicity in FRTL-5 cells was evaluated by the MTS-based viability assay after pre-treating each antioxidant for 4 ～ 24 hours prior to the exposure of 200 μM of KBrO3. Treatment with 100, 200, 300, 400, and 500 μM of KBrO3 caused a concentration-dependent reduction of FRTL-5 cell viability with 97.4± 2.5, 61.5 ± 4.0, 35.0 ± 4.1, 21.4 ± 2.6, 13.7 ± 1.6%, respectively, compared with control. However, the reduction of FRTL-5 cell viability by 200 μM of KBrO3 was blocked by pre-treatment with 0.05-1 mM of N-acetylcysteine in a dose-dependent fashion. In particular, pre-treatment with NAC at 1 mM was found to almost completely block the reduction of FRTL-5 cell viability by 200 μM of KBrO3. Moreover, pre-treatments with vitamin C, vitamin E, and estrogen, respectively, showed a protective effect on FRTL-5 cells against the oxidative stress induced by 200 μM KBrO3. Notably, while pre-treatment with EGCG, vitamin A, or vitamin D in low concentrations effectively protected FRTL-5 cells against the oxidative stress induced by KBrO3, that with respective antioxidant in high concentrations further decreased the viability of FRTL-5 cells probably due to their prooxidant properties. On the other hand, pre-treatment with melatonin, D-methionine or caffeine had no protective effect against the KBrO3-induced oxidative stress on FRTL-5 cells. In conclusion, these results demonstrate that the dietary intake of EGCG, estrogen, vitamin A, C, D or E has protective effects against KBrO3-induced oxidative stress in thyroid cells. However, it is recommended to have a caution when to intake these antioxidants particularly in high concentrations because of their prooxidant effects.