Abstract
六價鉻化物是一個致癌物，它藉由細胞膜上陰離子通道進入細胞後，再被代謝還原產生五價、四價、三價鉻及一些自由基，這些物質都可能會傷害核酸。維生素C是細胞中代謝六價鉻的一個重要還原劑。本研究利用穿梭質體系統，以未處理或六價鉻/維生素C在Hepes緩衝溶液中共同處理pSP189質體，經一個對核酸雙股連結及氧化性傷害修補有缺陷的法科尼氏貧血細胞 (Fanconi's anemia; FA) 複製，再將子代質體萃取、純化後轉殖到指示型大腸桿菌，分析質體supF基因的突變性，並與經293細胞複製後的突變性做比較 (Tsou et al., 1998)。未處理藥物的質體經FA細胞複製後自發性突變頻率比293細胞高2.6倍。由FA細胞產生的87個自發性supF突變株，鹼基取代佔47％，大段的核酸片段缺失及基因重組佔46％。相對地，在293細胞產生的自發性突變以鹼基取代 (71％) 為主。FA細胞中可能含有較高的氧化自由基，它們會攻擊穿梭質體，而造成較多的核酸斷裂和重組。以六價鉻/維生素C處理質體轉殖到FA細胞中產生的突變頻率顯著地高於在293細胞產生的突變頻率 (P＜0.03)。核酸序列分析六價鉻/維生素C誘發的113個突變株，其中75﹪為單一鹼基取代，12％為單一鹼基缺失，12％為核酸片段缺失。鹼基取代大多發生在G• C 配對上 (94％)，且以G• C → T•A (62﹪) 為主，此結果與在293細胞中誘發的突變型式類似。雖然在這兩個細胞中，六價鉻/維生素C誘發的鹼基取代大多發生在5'GG及5'GG的位置，但所誘發的鹼基取代高頻區卻各有特性。六價鉻/維生素C在Hepes緩衝溶液中會誘發8-hydroxydeoxyguanine (8-OHdG)，以及核酸雙股連結。8-OHdG核酸鍵結物在核酸複製時會造成G．C → T•A突變，相反地，質體含有核酸雙股連結則無法進行複製。以氫氧自由基清除劑mannitol處理FA細胞，可以顯著抑制質體在FA細胞產生的自發性突變頻率 (P＜0.02) 和六價鉻/維生素C所誘發的突變頻率 (P＜0.02)。由本研究推測氫氧自由基促使8-OHdG形成，是六價鉻/維生素C誘發突變的主因，FA產生較高的突變頻率可能是由於該細胞氧化自由基含量較高，而核酸雙股連結在FA細胞並非突變源.The carcinogenic Cr(VI) is transported into cells through theanion transport system and is reduced intracellularly,generating Cr(V), Cr(IV), Cr(III), and reactive oxygen species.All the Cr(VI) metabolites may be potent agents that damage DNA.Ascorbic acid (AsA) is a major cellular reductant for Cr(VI)metabolism. In this study, we have adopted a shuttle vectorsystem to determine the molecular mutagenicity of untreated andCr(VI)/AsA-treated shuttle vector pSP189 during replication inFanconi's anemia (FA) cells that have defects in repairing DNAcross-links and oxidative damage.The pSP189 vector, containing abacterial suppressor tRNA gene, supF, was transfected into FAcells for DNA replication to take place. The progeny plasmidswere then rescued, purified, and introduced into an indicator E.coli that had an amber mutation in the β-galactosidase gene.The mutations generated during replication in FA cells werecompared with that obtained in human 293 cells (Tsou et al.,1998).The supF mutation frequency obtained in untreated plasmidsreplication in FA cells was 2.6-fold higher than that obtainedin 293 cells. DNA sequencing analysis of 87 spontaneous supFmutants derived from FA cells showed that 47% were basesubstitutions, and 46% were large deletions and rearrangements.In contrast, the majority of spontaneous supF mutations obtainedin 293 cells were base substitutions (71%). FA cells may havehigh level of reactive oxygen species that attack DNA andthereby generating high frequency of large deletions andrearrangements. The supF mutation frequency obtained in Cr(VI)/AsA-treated plasmids replication in FA cells was significantlyhigher than that obtained in 293 cells (p < 0.02). DNAsequencing analysis of 113 Cr(VI)/AsA-induced mutants showedthat 75% were base substitutions, 12% were single-basedeletions, and 12% were deletions. Almost all (94%) the basesubstitutions occurred at G•C base pairs, and the major basesubstitutions were G•C → T•A transversions (62%). Most ofthe base substitutions were located at 5'GG and 5'GG sites.Thetypes of mutations induced by Cr(VI)/AsA-treated plasmidsreplication in FA cells were similar to that obtained in 293cells, however, different hot spots were induced in these twocell lines. 8-Hydroxydeoxyguanosine (8-OHdG) adducts and DNA-interstrand cross-links were induced by Cr(VI)/AsA in a Hepesbuffer. During DNA replication, 8-OHdG adducts could result inG•C → T•A transversions, whereas DNA-interstrand cross-linkscould block DNA synthesis. Administrating mannitol to FA cellssignificantly reduced the mutation frequency occurredspontaneously (p < 0.02) or induced by Cr(VI)/AsA (p <0.02).Taken together, these results suggest that 8-OHdG adducts,resulting from hydroxyl radicals, may be the mutagenic lesionsin the Cr(VI)/AsA system, and that the high mutation frequencyobserved in FA cells may due to high amounts of intracellularreactive oxygen species, and not derived from DNA-interstrandcross-links.