一氧化氮供給劑及內毒素對RAW264.7巨噬細胞之細胞-遺傳毒性的研究。

中文摘要 (Chinese Abstract) 近年來隨著工商業發達，人們生活水準也隨之提高，但車輛數量與工廠密度偏高，導致長期空氣品質不佳，容易影響到人類的健康。例如空氣污染物之一的NOx (氮氧化物) 被呼吸進入人體肺的深部，可引起支氣管炎或肺氣腫，也會增加呼吸道阻力及氣喘發作比例。另外，在大氣中的N2O氧化變成硝酸，是造成酸雨的原因之一。 由於NO對免疫系統的細胞毒性及遺傳毒性很少被研究，所以本研究以RAW 264.7 macrophages為實驗標本，加入四種不同的NO donors ，3-morpholinosydnonimine (SIN-1) ，S-nitroso-N-acetyl-DL-penicillamine (SNAP)，S-nitrosoglutathione (GSNO)，和sodium nitroprusside (SNP)，或lipopolysaccharide (LPS) 來探討外生性NO和內生性NO對RAW 264.7 macrophages的細胞-遺傳毒性之影響，及觀察對細胞形態的改變 。 實驗方法包括有: 微小核法 (遺傳毒性的指標) ，細胞核分裂係數 (NDI) 法，MTT分析法 (細胞毒性的指標) ，亞硝酸鹽 (Nitrite) 測量法，CPP32 測量法 -- 等方法 。 並進一步以自由基捕捉劑 (Catalase和Superoxide dismutase) 和Cytarabine (Ara - C) 研究NO造成遺傳毒性之機轉，是否和自由基 (ROS) 或DNA修復系統 (DNA repair enzyme system) 有關 ? 另外，也以Lewis lung carcinoma (LLC) 為實驗標本，探討SNP是否和adriamycin一樣可經由細胞凋亡 (apoptosis) 方式毒殺LLC癌細胞 ? 實驗結果顯示RAW 264.7 macrophages經四種NO donors (外生性NO) 處理後，均具統計意義地增加微小核頻率和降低細胞存活率，四種NO donors在加藥12小時所產生之遺傳毒性最強，亦均會降低RAW 264.7 macrophages之NDI值，同時可測到培養液中nitrite的釋放具統計意義地增加。這些結果顯示外生性NO donors可經由NO產生細胞-遺傳毒性。此外，SOD和Catalase會降低SNP， SIN-1和SNAP之遺傳毒性，卻不影響GSNO，暗示SNP，SIN-1和SNAP增加微小核頻率的機轉，可能和superoxide，hydrogen peroxide有關，而GSNO可能和superoxide，hydrogen peroxide無關，但尚需進一步的研究 。 四種NO donors分別和Ara - C相加並不會進一步增加微小核頻率，暗示NO donors不是經由excision - repairable DNA lesions增加微小核頻率。 另外，SNP，SIN-1和SNAP分別與Ara-C相加，亦不增加細胞毒性，但GSNO具統計意義地對抗Ara - C對RAW 264.7 macrophages的細胞毒性，可能和GSNO可解離釋出GSH有某種程度上的關連 。 而在RAW 264.7 macrophages給與LPS的實驗中，觀察到0.3和1μg/ml LPS有統計意義的增加微小核，而LPS處理2小時所產生之遺傳毒性最強。LPS需在30μg/ml之高濃度才具統計意義的降低細胞存活率，而LPS之細胞毒性可分別被SOD，Catalase或L-NAME有統計意義的降低。LPS亦具統計意義的增加nitrite之釋放，此作用亦顯著的被L - NAME降低，顯示LPS經由iNOS途徑產生NO。根據這些結果可推論LPS的細胞毒性和內生性NO，superoxide和hydrogen peroxide有關 。 此外，SNP和LPS均會明顯改變細胞形態。 為更進一步證實NO有抗癌的作用，所以使用 SNP和ADM研究其在LLC的細胞毒性: SNP和ADM皆具統計意義的降低LLC之細胞存活率。SNP和ADM均會增加CPP32的活性，此增加活性之作用會被CPP32抑制劑 (DEVD-CHO) 所降低，所以SNP和ADM均以細胞凋亡方式殺死LLC癌細胞。 結論是四種NO donors均能增加RAW 264.7 macrophages之微小核頻率，降低其存活率和核分裂係數，又同時均增加NO2-之形成，所以此四種NO donors對RAW 264.7 macrophages均透過NO而產生細胞-遺傳毒性。由於其遺傳毒性不被Ara-C所加強，但被自由基捕捉劑 (Catalase和Superoxide dismutase) 所減弱 (GSNO例外)，所以其造成遺傳毒性之機轉和excision-repairable DNA lesion無關，而是經由含氧自由基 (ROS) 造成DNA之斷裂或影響紡綞體功能而增加微小核頻率。LPS在RAW 264.7 macrophages造成細胞-遺傳毒性亦至少部分和NO之生成有關。

英文摘要 (English Abstract) There are limited data on NO-induced cytogenotoxicity in immune cells. In the present study, micronucleus assay, nuclear division index (NDI), MTT assay and cell morphology were assayed to evaluate cytogenetic toxicities of different NO donors, 3-morpholinosydnonimine (SIN-1), S-nitroso-N-acetyl-DL-penicillamine (SNAP), S-nitrosoglutathione (GSNO), and sodium nitroprusside (SNP), or lipopolysaccharide (LPS) in RAW 264.7 macrophages. On the other hand, NO release was determined spectrophotomertrically by measuring the accumulation of nitrite in the culture medium. In order to approach the mechanisms of NO-induced genotoxicity, the genotoxic interactions of NO donors with cytarabine (Ara-C) or free radical scavengers (Catalase and Superoxide dismutase) were conducted in RAW 264.7 macrophages；the cytotoxic interactions of LPS with L-NAME, Catalase and Superoxide dismutase were also studies.We also assayed CPP32 activity of Lewis lung carcinoma (LLC) to clarify SNP-induced apoptosis in LLC as positive control, adriamycin. In the present study, all of four NO donors (exogenous NO) significantly increased MN frequency and decreased both cell viability in RAW 264.7 macrophages and NDI. At the same time, all of the four NO donors increased Nitrite concentration in cultureed medium in a concentration-dependent manner. In addition, the just incubation time of NO donors-induced genotoxicity in RAW 264.7 macrophages was 12 hr. These results indicated that NO donors induced cytogenotoxicity RAW 264.7 macrophages via NO pathway. The data of genotoxic interactions showed that both SOD and Catalase attenuated the genotoxic effects of SNP, SIN-1 and SNAP, but did not affect GSNO-induced genotoxicity. We suggested that the mechanism of increased MN frequency of SNP, SIN-1 and SNAP may involve with superoxide and hydrogen peroxide generation, but GSNO-induced genotoxicity might be not with ROS, it needs to be further study. All of the four NO donors co-culture with Ara-C did not further increase MN frequency, suggested that NO donors did not cause excision - repairable DNA lesions. Ara-C also did not affect the cytotoxicity of SNP, SIN-1 and SNAP. However, GSNO significantly decreased the cytotoxicity of Ara-C in RAW 264.7 macrophages, it might be related to GSH was dissociated from GSNO. LPS at concentrations of 0.3 and 0.1μg/ml LPS significantly increased MN frequency in RAW 264.7 macrophages, and the just culture time of LPS-induced genotoxicity was 2hrs. However, LPS only at high concentration of 30μg/ml significantly decreased cell viability. In addition SOD, Catalase or L-NAME significantly decreased LPS-induced cytotoxicity, respectively. LPS also significantly increased nitrite concentration in medium, this effect of LPS was markedly decreased by L-NAME, which suggested that LPS produced NO through iNOS pathway. According to these results, we suggested that LPS-induced involved with generation endogenous NO, superoxide and hydrogen peroxide. In the present study, we observed SNP and LPS markedly changed the morphology of RAW 264.7 macrophages. In order to further confirm the antitumor effect of NO, the cytotoxic effect of SNP and ADM in LLC was conducted using MTT assay. Both SNP and ADM significantly decreased viability of LLC. In addition, both SNP and ADM increased CPP32 activity, and this effect decreased by CPP32 inhibitor (DEVD-CHO). It appears that SNP induced apoptosis of LLC as ADM. In conclusion, we have shown that all of the four NO donors significantly increased MN frequency, decreased cell viability and NDI, even SNP changed cell morphology of RAW 264.7 macrophages. All of four NO donors also increased of nitrite concentration in medium at the same time. Accordingly, all of four NO donors induced cytogenotoxicities via NO formation pathway in raw 264.7 macrophages. However, NO donors-induced genotoxicity did not potentiate by Ara-C, but attenuated (except GSNO) by free radical scavengers (Catalase and SOD). It appears that the mechanism of NO donors-induced genotoxicity did not involve with excision-repairable DNA lesion, they induced genotoxicity via production of oxygen free radicals. In addition, LPS induced cytogenotoxicities in RAW 264.7 macrophages, which is, at least in part, due to NO and oxygen free radicals generation.