| 摘要: | 無機砷廣泛存在土壤及水中之類金屬化合物，並在工業上大量的使用。使得環境長期受到砷污染，因此常可見到無機砷高暴露之人類族群。在流行病學的研究報告中充分顯示無機砷為人類致癌物，長期無機砷攝取除主要誘引皮膚癌外也會有膀胱癌、肝癌和腎癌的發生。然而動物實驗尚未直接證實砷化物的致癌性。為進一步了解無機砷的致癌性，因此利用0.5或1 mM亞砷酸鈉處理不朽但無致癌性的人類皮膚細胞株( HaCaT )大約六個月，乃探討暴露在長時間低濃度無機砷下的HaCaT 細胞之生長和基因表現的情形。結果顯示HaCaT 細胞經數月培養在含無毒性的亞砷酸鈉(0.5或1 mM)之培養液中，再以皮下注射方式將細胞打入裸鼠背部，則可見到腫瘤的形成。此外，HaCaT細胞仍有下列幾項的變化: (1) 於凝膠培養皿有較高的細胞群落; (2) 於培養皿內細胞長滿時有較高的細胞密度; (3) 每一代之平均分裂次數較高及(4) 細胞內GSH含量增加。以西方點墨法分析0.5或1 mM的亞砷酸鈉處理的細胞中c-fos, c-jun, keratins, catalase, heme oxygenase-1 and galectin-1蛋白質的含量，發現這些蛋白質都被抑制。以cDNA基因微陣分析長時間無毒性的1 mM亞砷酸鈉處理HaCaT 細胞的基因表現，發現細胞內dihydrodiol dehydrogenase, ferritin, thioredoxin peroxidase, oxidation resistance-1 and glutamate-cysteine ligase這些基因是增強表現。另一方面也發現1 mM亞砷酸鈉處理的細胞內transmembrane protein(63 kD), catenin a2, gelsolin, caveolins and keratin 14這些基因是減少表現。綜之，本研究結果顯示經長時間、低劑量的處理下，無致癌性的HaCaT細胞會改變其基因表達的形式及於裸鼠身上形成腫瘤。為了更進一步了解低濃度無機砷在in vivo狀況下致癌的機制，因此先將無致癌性的人類皮膚細胞株( HaCaT ) 以皮下注射方式將細胞打入SCID mice背部。持續五個月內喝含不同濃度亞砷酸鈉的水(50, 100, 200和500 ppb)，發現若餵食含亞砷酸鈉的水會造成SCID mice身上無致癌性的HaCaT細胞轉形成腫瘤，此研究模式將有助於進一步探討無機砷致癌的分子機制。 Arsenic is a naturally occurring constituent of soil and water, and arsenic metalloid is widely used in industry. The combination of lengthy environmental persistence with widespread arsenic contamination contributes to a high exposure incidence among human populations. Arsenic is a human carcinogen. With ingestion of inorganic forms of this metalloid causes primarily skin cancer, but also cancers of the bladder, liver, and kidney. However inorganic arsenic fails to induce tumors in most laboratory animals. To explore the mechanisms of arsenic carcinogenesis, we have investigated the growth property and gene expression profile in spontaneously immortalized human skin keratinocytes (HaCaT), that were continuously exposed to non-toxic doses of arsenite (0.5 and 1 mM) for approximately 6 months. Through a continuous exposure of HaCaT to non-toxic doses of arsenite(0.5 and 1 mM) for several months, HaCaT cells became apparently tumorigenic in nude mice. In addition, we have observed the following changes caused by long term exposure to arsenite: (1) higher colony forming efficiency on soft agar; (2) higher cell density at confluency; (3) more population doublings in each passage; and (4) higher levels of GSH. By Western blotting analysis, c-fos, c-jun, keratins, catalase, heme oxygenase-1 and adhesion molecular galectin-1 were inhibited in arsenite-exposured cultures. By using a colorimetric cDNA microarray analysis, we found that the expression of dihydrodiol dehydrogenase, ferritin, thioredoxin peroxidase, oxidation resistance-1 and glutamate-cysteine ligase were enhanced in 1 mM arsenite-exposured cultures. We also found that the transmembrane protein(63kD) ,catenin a2, gelsolin, caveolins and keratin 14 were decreased in 1 mM arsenite-exposured cultures. These results suggest that after long-term and low dose exposure of HaCaT cells to arsenite leads to alterations in the expression profile and tumor formation in nude mice. We have investigated the carcinogenesis in low concentration arsenite in vivo in non-tumorigenic HaCaT cells, which were subcutaneously injected into SCID mice. Through a continuous exposure of low dose arsenite (50, 100, 200 and 500 ppb) to SCID mice for approximately 5 months, HaCaT cells became apparently tumorigenic in SCID mice. This animal model may be valuable for further investigation in molecular mechanisms of arsenic carcinogenicity. |
