Abstract
砷化物是已知的人類致癌物。它會誘發姊妹染色分體互換、染色體異 常以及微小核球 (micronucleus)，但其機制尚未明瞭。最近的研究顯示 細胞內的鈣離子濃度和含氧活性物(reactive oxygen species) 參與亞砷 酸鈉誘發微小核球，而一氧化氮(nitric oxide)也參與亞砷酸鈉誘發 poly(ADP-ribosylation)。我實驗的目的之一即是釐清上述分子那個是亞 砷酸鈉誘發微小核球的原因。為了研究亞砷酸鈉造成細胞內含氧活性物升 高的程度、鈣離子濃度及一氧化氮濃度的改變，我利用 dichlorofluorescin diacetate測量細胞內的氧化程度，用fluo3-AM測量 細胞內鈣離子的濃度，並使用diaminonaphthalene 測量培養過細胞的培 養液中的亞硝酸含量。這些結果顯示中國倉鼠卵巢細胞處理亞砷酸鈉四小 時超過5 uM時，氧化程度、一氧化氮和細胞內鈣離子濃度均會隨著亞砷酸 鈉處理的濃度增高而增高。亞砷酸鈉增加之細胞內氧化程度可被一氧化氮 合成脢抑制劑，S-methyl-L-thiocitrulline和 Nw-nitro-L-arginine methyl ester，以及鈣離子的螯合劑ethylene glycol-bis (beta- aminoethyl ether)-N,N,N',N'-tetraacetic acid, 和2-[(2-bis- [carboxy-methyl]-amino-5-methylphenoxy)-methyl]-6-methoxy-8- bis[carboxy-methyl]-amino-quinoline 等所抑低，但catalase 之抑制 劑，3-aminotriazole，對亞砷酸鈉所增加之細胞內氧化程度，卻無增高 作用。亞砷酸鈉誘發之一氧化氮也能被一氧化氮合成脢抑制劑與鈣離子螯 合劑所抑低。然而，亞砷酸鈉誘發之細胞內鈣離子濃度增高，雖會被鈣離 子螯合劑所抑低，但卻不會被一氧化氮合成脢抑制劑所抑低。當處理四小 時超過10 uM時，亞砷酸鈉誘發之微小核球也會隨著亞砷酸鈉處理的濃度 增高而增高。亞砷酸鈉誘發之微小核球，會被一氧化氮合成脢抑制劑、鈣 離子螯合劑、超氧歧化脢 (superoxide dismutase)以及尿酸 (uric acid) 所降低。這些結果顯示過氧亞硝酸 (peroxynitrite)參與亞砷酸鈉 誘發微小核球之產生。根據上述之結果，我推測干擾一氧化氮之生成會造 成心臟與末梢血管疾病，而過氧亞硝酸造成之DNA 損傷可能在受砷暴露的 人口中造成遺傳物質的不穩定因此而誘發癌症。 細胞經DNA損傷劑處 理後，通常可偵測到細胞週期的遲滯。此遲滯或許是為了DNA修補。因為 氧化物 (oxidants) 被發現參與中國倉鼠卵巢細胞中誘發姐妹染色分體互 換以及細胞凋亡，我乃研究含氧活性物參與亞砷酸鈉誘發細胞遲滯的可能 原因。以周期同步化的 (synchronized) 中國倉鼠卵巢細胞的實驗顯示， G1期與DNA合成期晚期 (late-S) 細胞對亞砷酸鈉與過氧化氫誘發之細胞 周期遲滯有相似的敏感度，但比合成期早期 (early-S) 敏感得多。 Catalase 前處理有效地去除過氧化氫所誘發之G1期細胞停滯，但只些許 減輕亞砷酸鈉誘發之G1期細胞停滯。Catalase抑制劑，3-aminotriazole ，顯著地增加過氧化氫誘發之G1和G2期細胞停滯，但它在亞砷酸鈉誘發之 G1和G2期細胞停滯並無明顯的作用。Mercaptosuccinic acid，是 glutathione 過氧化脢 (peroxidase) 的抑制劑，不論單獨或與3- aminotriazole共同處理對亞砷酸鈉誘發之G1及G2細胞周期停滯均無影響 。此外砷酸鈉所誘發之G1期細胞停滯亦不受neocuproine, Trolox, dimethylsulfoxide, 維他命C和維他命E的影響。因此，目前的結果顯示 含氧活性物並未參與亞砷酸鈉誘發之G1細胞周期停滯。此結論亦受過氧化 氫會增加亞砷酸鈉誘發之G1及G2細胞周期停滯的結果所支持。一氧化氮、 鈣離子以及DNA傷害可能也未參與亞砷酸鈉誘發之G1及G2細胞周期停滯。 因為亞砷酸鈉誘發之G1細胞周期停滯不會受一氧化氮合成脢抑制劑(Nw- nitro-L-arginine methyl ester, S-methyl-L-thiocitrulline)、鈣離 子螯合劑 (quin2, ethylene glycol-bis (beta-aminoethyl ether)-N, N,N',N'-tetraacetic acid) 以及DNA修補抑制劑(hydroxyurea, cytosine-b-D-arabinofuranoside, 3-aminobenzamide, caffeine) 之影 響。 於實驗中，我也注意到中國倉鼠卵巢細胞受亞砷酸鈉處理於G2期 會誘發濃縮不完全的染色體 (poorly condensed chromosomes) 以及染色 分體斷裂。經亞砷酸鈉處理G2期細胞，當再進入下一個細胞間期 (interphase) 時會出現微小核球。此現象亦證實染色分體斷裂的產生。 若將G2期以人為方式大略分為四段，處理亞砷酸鈉的時間越接近G2早期 (或G2/S交界)，所誘發的染色分體斷裂越多。除了誘發染色分體斷裂之外 ，亞砷酸鈉處理也很嚴重地阻礙了分裂期細胞 (mitotic cells) 重新近 入下一個細胞間期。藉由重新將亞砷酸鈉處理過的G2 細胞種殖於無藥的 培養液並加以培養，我觀察到一群DNA含量介於雙倍到四倍之間的細胞出 現。在下一個有絲分裂也出現了含有接近四倍體的染色體數目的分裂期細 胞。由於染色體的改變被認為跟惡性腫瘤化的轉型 (malignant transformation) 相關聯，且細胞染色體套數改變與遺傳物質的不穩定性 相關聯，此二現象在砷化物致癌性可能是重要的。 最近三氧化二砷被 報告對急性前黑色素細胞白血球過多症 (acute promyelocytic leukemia) 的病人有緩解的療效。此作用可能因為三氧化二砷會誘發變異 細胞之凋亡，其可能機制為三氧化二砷經由與鄰近的巰基結合而改變蛋白 質的磷酸化的結果。為了驗證此假說，我發現phenylarsine oxide，此乃 一巰基強結合劑，並不能在NB4細胞中產生細胞凋亡，而三氧化二砷及 dimethylarsinic acid則會。然而，phenylarsine oxide抑制去氫脢活性 的能力則比三氧化二砷強。Dithiothreitol (DTT) 也有效地降低 phenylarsine oxide抑制去氫脢活性的能力，但DTT、 dimercaptosuccinic aci、2,3-dimercaptopropanol、b- mercaptoethanol和glutathione則不會抑制三氧化二砷誘發之細胞凋亡。 相反地，DTT反而增高三氧化二砷在NB4細胞誘發之細胞凋亡。然而，人類 臍帶靜脈內皮細胞比NB4細胞對於三氧化二砷或三氧化二砷加DTT的細胞毒 性更有抗性。而且不論是三氧化二砷或是三氧化二砷加DTT在人類臍帶靜 脈內皮細胞細胞中均不會誘發細胞凋亡。在NB4細胞中三氧化二砷以及三 氧化二砷加DTT所誘發之細胞凋亡均可被pyruvate、selenite和catalase 所抑制，而被diethyldithiocarbamate、mercaptosuccinic acid和3- aminotriazole所增高。目前的結果顯示活性含氧活性物是三氧化二砷誘 發NB4細胞凋亡的機制，而非經由與鄰近的巰基結合而改變蛋白質的磷酸 化。本研究發現DTT增高三氧化二砷誘發NB4細胞凋亡，而不會增加正常細 胞的細胞凋亡的作用可能有治療上的價值。 Arsenic, a human carcinogen is known to induce sister- chromatid exchanges, chromosome aberrations and micronuclei, but its mechanisms remain unknown. Recently independent studies have suggested that intracellular calcium and reactive oxygen species are involved in arsenite-induced micronuclei (MN), and nitric oxide (NO) is involved in arsenite-induced poly(ADP- ribosylation). One of the aims of my research is to clarify which of the above molecules is responsible for the MN induction by arsenite. In order to study the perturbation of arsenite treatment on the cellular oxidant, calcium, and nitric oxide level, I have used dichlorofluorescin diacetate to measure intracellular oxidant level, fluo3-AM to measure intracellular calcium level, and diaminonaphthalene to measure the nitrite in cell culture medium. The results show that a 4-h treatment with arsenite above 5 uM, caused a dose-dependent increase of oxidant, NO, as well as intracellular calcium level Chinese hamster ovary (CHO) cells. The arsenite-increased intracellular oxidant level was inhibited by NO synthase inhibitors, S-methyl- L-thiocitrulline and Nw-nitro-L-arginine methyl ester and calcium chelators, ethylene glycol-bis (beta-aminoethyl ether)- N,N,N',N'-tetraacetic acid, and 2-[(2-bis-[carboxymethyl]- amino-5-methylphenoxy)-methyl]-6-methoxy-8-bis[carboxy- methyl]aminoquinoline, but catalase inhibitor, 3-aminotriazole did not increase arsenite-increased intracellular oxidant level. The arsenite-increased NO could also be suppressed by NO synthase inhibitors and calcium chelator. However, the arsenite-increased intracellular calcium level was inhibited by calcium chelators, but not by NO synthase inhibitors. A 4-h treatment with arsenite above 10 uM, also induced MN dose- dependently. The arsenite-increased MN could be reduced by NO synthase inhibitors, calcium chelators, as well as superoxide dismutase and uric acid. These results suggest the involvement of peroxynitrite in arsenite-induced MN. I surmise that the disturbance of NO production may cause cardio/peripheral vascular disorders, and the peroxynitrite-mediated DNA damages may cause genetic instability and hence cancers in arsenic- exposed humans. When cells are challenged with a DNA-damaging agent, a delay of cell cycle progression is usually detected. This is presumably for the purpose of repair. Since oxidants have been shown to involve in the induction of sister-chromatid exchanges and apoptosis in CHO cells, I have investigated the possible involvement of hydrogen peroxide in arsenite-induced cell-cycle arrest. Experiments with synchronized CHO cells, indicate that the G1 and late-S cells were similar sensitive, but were more sensitive than the early-S cells to the arsenite- and H2O2-induced cell-cycle delay. Catalase pretreatment effectively removed the H2O2-induced G1 arrest, but only slightly reduced the arsenite-induced G1 arrest. Catalase inhibitor, 3-aminotriazole, markedly enhanced the H2O2-induced G1 and G2 arrest, but it had no apparent effect on the arsenite- induced G1 or G2 arrest. Neither, mercaptosuccinic acid, an inhibitor of glutathione peroxidase, alone or in combination with 3-aminotriazole, had an enhancing effect on arsenite- induced G1 or G2 arrest. The arsenite-induced G1 arrest was not be affected by neocuproine, Trolox, dimethylsulfoxide, vitamin C and vitamin E. Therefore, the present results demonstrate that reactive oxygen species are not involved in arsenite-induced G1 arrest. This conclusion is also supported by the result that H2 O2 could enhance arsenite-induced G1 or G2 arrest. Nitric oxide, calcium, and DNA damages probably do not participate in the arsenite-induced G1 and G2 arrest either. This is because the arsenite-induced G1 arrest was not be affected by NO synthase inhibitors (Nw-nitro-L-arginine methyl ester, S-methyl- L-thiocitrulline), calcium chelators (quin2, ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid), DNA repair inhibitors (hydroxyurea plus cytosine-b-D-arabinofuranoside, 3-aminobenzamide, caffeine). On the other hand, H2O2 could enhance arsenite-induced G1. During the experiments, I have also noticed that treatment of CHO cells with arsenite during the G2 phase induced poorly condensed chromosomes and chromatid breaks. The induction of chromatid breaks was confirmed by the appearance of micronucleated cells after arsenite-treated G2 cells were allowed to re-enter interphase. When the duration of the G2 phase was artificially divided into 4 periods, more chromatid breaks were induced by treatment with arsenite during the very early G2 phase (or G2/S boundary). In addition to the induction of chromatid breaks, arsenite treatment also remarkably retarded the re-entry of mitotic cells into interphase. By replating and incubating arsenite-treated G2 cells in drug-free medium, I have observed the appearance of a population of cells whose DNA content was between 4C and 8C, and metaphase cells with near-tetraploid chromosome numbers in the next mitotic division. Since chromosomal alternations are known to be associated with the initiation of malignant transformation, and the changes in cell ploidy are associated with genetic instability, these two phenomena may be important in arsenic carcinogenesis. Recently arsenic trioxide (As2O3) has been reported to induce clinical remission in patients with acute promyelocytic leukemia, and modulation of protein phosphorylation by binding to the vicinal thiol groups has been suggested as a possible mechanism. In an attempt to test this hypothesis, I found that phenylarsine oxide, a strong vicinal thiol binding agent, did not induce apoptosis in NB4 cells, As2 O3 and dimethylarsinic acid did. However, phenylarsine oxide was much more potent in inhibiting the activity of cellular dehydrogenases than As2O3. Dithiothreitol (DTT) effectively suppressed the phenylarsine oxide-inhibited dehydrogenases activity, but DTT, dimercaptosuccinic acid, 2,3-dimercaptopropanol, b-mercaptoethanol and glutathione did not suppress As2O3-induced apoptosis. On the contrary, DTT enhanced the As2O3-induced apoptosis in NB4 cells. However, human umbilical vein endothelial cells (HUVEC) were relatively resistant to the cytotoxic effects of As2O3 or As2O3 plus DTT than NB4 cells, and neither As2O3 nor As2O3 plus DTT induced apoptosis in HUVEC. The As2O3- as well as As2O3 plus DTT- induced apoptosis in NB4 cells, could be reduced by sodium pyruvate, sodium selenite, and catalase and enhanced by diethyldithiocarbamate, mercaptosuccinic acid, and 3-aminotriazole. The results so far, suggest reactive oxygen species rather than modulation of protein phosphorylation by binding to vicinal thiol groups is the mechanism by which As2O3 induced apoptosis in NB4 cells. The enhancing effect of DTT on the As2O3-induced apoptosis in NB4 cells and not in normal cells may be of therapeutic value.