Abstract
鉛化物是環境中普遍存在的污染物質，會誘發鼠類細胞的轉型、基因之突變及動物腫瘤之形成。然而鉛化物對人類細胞之細胞毒性、基因突變、細胞轉型及突變分子機制，目前為止仍不清楚。論文首先探討醋酸鉛誘發人類成纖細胞之毒性、次黃嘌呤-鳥糞嘌呤磷酸核醣轉化酵素基因hypoxanthine guanosine phosphoribosyltransferase gene (hprt)基因突變及非固著性細胞群落生長 (Anchorage-independence; A.I.) 之能力。細胞以醋酸鉛0?2 mM，在37℃處理24小時，細胞之毒性隨著鉛濃度的增加而增加，0.8 mM鉛可以造成37% 之細胞存活率。此外，隨著鉛濃度之增加其誘發A.I.群落之能力也顯著的增加，大約造成10?30% 的細胞毒性的濃度之下，鉛誘發A.I.頻率與強致癌劑MNNG造成的大致相等。醋酸鉛並不會誘發人類成纖細胞hprt基因之突變頻率。為探討自由基是否參與鉛的細胞毒性、基因突變及細胞之轉型。細胞以過氧化氫酵素抑制劑3-aminotriazole (3-AT; 80 mM) 前處理1小時再與鉛共同處理24小時之後，並不會加強鉛之細胞毒性、細胞轉型及鉛之弱突變性。然而3-AT會明顯促進鉛之吸收與累積。由這些結果推測，鉛對人類成纖細胞可能扮演著一個腫瘤促進劑之角色。本論文接著探討鉛對核酸的傷害及誘發分子突變的機轉。在活體外，以0.5 mM鉛與0?5 mM維生素C在10 mM HEPES緩衝溶液(pH 6.8)，在37℃共同處理超螺旋質體核酸1小時，發現鉛會造成核酸斷裂，並且隨著維生素C濃度增加而顯著加強。以上述相同的處理條件處理小牛胸腺核酸，發現隨著維生素C濃度上升，鉛誘發八-氫氧化鳥糞嘌呤去氧核醣核酸鍵結物(8-hydroxy-2-deoxyguanine ; 8-OHdG) 也隨之遞增。鉛/維生素C造成的核酸斷裂及8-OHdG之形成皆能有效的被單線態氧清除劑NaN3及過氧化氫酵素抑制，但不受氫氧自由基清除劑D-mannitol的影響。推測單線態氧及過氧化氫參與鉛/維生素C造成核酸之傷害及8-OHdG之生成。此外，利用感應耦合電漿質譜分析儀 (ICP-MS) 發現維生素C會加強鉛與核酸的鍵結，單線態氧清除劑NaN3可以降低鉛/維生素C共同處理中鉛與核酸之鍵結。利用包含supF基因之穿梭質體系統 (shuttle vector)，研究鉛/維生素C處理的質體在人類胚胎腎臟細胞株; 293細胞複製,質體核酸經抽取、純化後轉入指示型大腸桿菌 (E. coli) 產生的突變分子機制。發現鉛誘發之突變頻率會隨著維生素C濃度增加而增加。當鉛與維生素C劑量分別為0.5 mM與5 mM時所誘發之突變頻率為控制組之5.8倍。質體核酸的相對轉殖能力只隨著維生素C濃度上升而稍微下降。分析鉛/維生素C誘發的132個突變株之核酸序列改變，包括鹼基取代 (59株)，鹼基缺失(40株)，鹼基插入 (3株)，重複一段鹼基序列 (3株)，鹼基序列重排(27株)。所誘發的鹼基取代有98% 之突變發生在G•C配對上，以G•C * T•A顛換突變佔全部鹼基取代的48%，其次是G•C * C•G顛換突變(26%)，G•C * A•T轉換突變(25%)。根據文獻報導，氫氧自由基及單線態氧皆會誘發核酸鍵結物8-OHdG之形成，造成G•C * T•A突變。此外，單線態氧也會誘發G•C * C•G突變。由這些結果推測核酸序列缺失及G•C鹼基取代可能是因為鉛在維生素C存在下，藉由自由基產生核酸斷裂及8-OHdG鍵結物之形成。而單線態氧及過氧化氫可能是鉛/維生素C誘發突變的主因。Lead compounds are ubiquitous environmental contaminantsthat have been shown to induce cellular transformations and genemutations in cultured rodent cells, as well as tumours in liveanimals. However, the mencahisms by which this metal causecellular transformations and mutations in human cells have notbeen explored. In this study, we investigated the abilities oflead to induce cytotoxicity, hypoxanthine guanosinephosphoribosyltransferase(hprt) gene mutations and anchorage-independent transformations in diploid human fibroblasts. Humanfibroblasts were exposed to lead acetate (0?2 mM) at 37 ℃for24 h. Lead significantly induced cytotoxicity in a dose-dependent manners. 0.8 mM of lead acetate was required to reducethe survival of treated cell to 37% (Do). The anchorage-independent colonies was shown to be dose-dependently induced bylead, and the frequencies of anchroage-independent coloniesinduced by lead is similar to those induced by strong carcinogenN-methyl-N*-nitro-N-nitrosoguanidine (MNNG) at approxi-matelyequal cytotoxic dose ranges (30?10 % survival). Lead did notinduced 6-thioguanine-resistant mutation frequnecy in humanfibroblasts. Additionally, the role of ROS on lead-inducedcytotoxicity, mutagenicity and transformation, cells wereincubated with 80 mM of 3-AT for 1 h before co-exposure to leadfor 24 h. In this dosage, lead-induced cytotoxicity, anchorage-independence and weak mutagenicity of lead was not enhanced by3-aminotriazole co-treatment. However, 3-aminotriazole didsignificantly enhance lead uptake and accumulation in humanfibroblasts. These results suggest that lead may act as tumourpromoter in diploid human fibroblasts. Subsequently, wehave adopted an in vitro system to investigate the DNA damageand mechanisms of mutations induced by lead. Lead acetate at 0.5mM could induce DNA strand breakage in 10 mM HEPES buffer, pH6.8, at 37℃for 1 h and lead-induced DNA breakage increasedlinearly with the increasing concentrations of ascorbic acid(AsA) at 0.5?5 mM as determined using the plasmid relaxationassay. Additionally, lead at 0.5 mM could cause a small amountof 8-hydroxydeoxyguanosine (8-OHdG) formation in calf thymusDNA, and dose-dependent induced the formation of this adduct inthe presence of increasing concentrctions of AsA using HPLC-UV-ECD method. Singlet oxygen scavenger NaN3 and hydrogen peroxidescaverger catalase markedly inhibited lead/AsA-induced DNAbreakage and 8-OHdG formation, but D-mannitol did not. Theseresults have indicated that singlet oxygen (1O2) and hydrogenperoxide (H2O2) are involved in the DNA strand breakage and8-OHdG formation induced by lead/AsA. AsA also enhanced lead andDNA association in a dose-dependent manner by increasing dosesof AsA using the ICP-MS analysis. Singlet oxygen scavenger NaN3markedly decreased lead/AsA-induced lead and DNA association. Wealso have adopted a shuttle vector system to determine themolecular mutagenicity of lead in the presence of AsA in humancells. Plasmid pSP189, containing a bacterial suppressor tRNAtarget gene, supF, was treated with lead/AsA and transfectedinto a human embryonic kidney cell line; 293, for DNAreplication to take place. The progeny plasmids were thenrescued, purified, and introduced into an indicator E.coli thathad an amber mutation in the b-galactosidase gene. The supFmutant frequency increased linearly with the increasingconcentrations of AsA. The highest supF mutant frequency inducedby lead/AsA was observed at 0.5 mM of lead and 5 mM of AsA, theinduced mutant frequincy was 5.8-fold of those observed in theuntreated plasmid. The relative transformation efficiency ofprogeny plasmids decreased with the incrasing concentrations ofAsA. SupF gene sequencing analysis of 132 of lead/AsA-inducedmutants showed that 59 mutants were base substitutions, 40 weredeletions, 3 were insertions, 3 were duplictions, 27 wererearrangements. Almost all (98%) the base substitutions occuredat G*C base pairs, and the major kinds of base substitutionswere G*C*T*A transversions (48%), G*C*C*G transversion (26%) andG*C*A*T transitions (25%). Taken together, the above resultsimplicate that the gene deletions and G*C base pair subsitutionsmay be derived from DNA strand breaks and 8-OHdG adducts thatwere induced by lead/AsA through reactive oxygen species.Singlet oxygen and hydrogen peroxide may be the major speciesfor lead/AsA mutagenesis.