Abstract
8-methoxypsoralen（8-MOP）加上一次長波長紫外光（UVA）處 理細胞（PUVA-I）主要產生的核酸損傷為單鍵結物。若在 PUVA-I 處理之 後洗去未鍵結的 8-MOP 並照以第二次較高劑量的 UVA （PUVA-II）會將 大部分的單鍵結物轉變為雙股連結物。單鍵結物和雙股連結物這兩種核酸 損傷都必須靠核酸切除修補途徑來去除。為了探究這兩類核酸損傷在突變 誘發所扮演的重要性﹐以及核酸切除修補對其突變誘發的影響﹐我們以核 酸修補能力正常的人類成纖細胞（HFW 細胞）和缺乏此能力的著色性皮膚 乾萎症 A 族病人的成纖細胞（XP-A 細胞）為比較對象﹐觀察 PUVA-I 以及 PUVA-II 對它們造成的細胞毒性和突變特異性。對於 HFW 細胞﹐ PUVA-II 有顯著的細胞毒性﹐並且能誘發 數目高於背景值 10 倍的抗 6-thioguanine 突變株﹐然而 PUVA-I 對於 HFW 細胞僅有輕微的毒性﹐ 但也能誘發 14 倍數目的突變株﹐此結果顯示核酸單鍵結物比雙股連結物 較不具細胞毒性但是一樣有致變性。以聚合酵素鏈反應以及核酸定序分析 PUVA-I 以及PUVA-II 在 HFW 細胞的 hprt 基因所造成的突變﹐發現兩者 主要均造成位於 5'TA 或 5'AT 位置上的 T:A 鹼基置換﹐且大部分發生 突變的 thymine 是位於非轉錄股。但不 同的是﹐PUVA-I 造成的 T:A 鹼 基置換都是 T -> A transversions﹐而 PUVA-II 產生的鹼基突變會受到 thymine 之 5' 鹼基種類的影響。另一方面﹐XP-A 細胞比 HFW 細 胞對 PUVA-I 以及 PUVA-II 的毒性較敏感。其中﹐PUVA-II 的細胞毒性遠較 PUVA-I 強﹐但對 XP-A 細胞卻不能像對 HFW 細胞一樣誘發明顯高於背 景值的突變﹐此結果顯 示核酸切除修補途徑可能參與雙股連結物造成突 變的過程。相反的﹐PUVA-I 可以誘發 XP-A 細胞高於背景值 6 倍的hprt 基因突變率﹐且這些突變主要是 T -> A transversions。因為 PUVA-I 在 HFW 與 XP-A 兩種細胞造成的突變主要都是 T -> A transversions﹐ 所以推測此類突變的形成與核酸切除修補途徑較無關。再者﹐PUVA-I 在 XP-A 細胞所誘發的突變中﹐發生突變的 thymines 有 1/3 是位於轉錄股 上﹐此結 果吻合核酸切除修補與基因轉錄過程偶合的理 論。 Treatment of cells with 8-methoxypsoralen (8-MOP) plus a single dose of UVA (365 nm) radiation (PUVA-I) generates mostly monoadducts on DNA. Further irradiation of cells to another high dose of UVA (PUVA-II) after PUVA-I treatment and wash of the unbound 8-MOP converts most monoadducts to crosslinks. Both monoadducts and crosslinks are removed by nucleotide excision repair (NER). To investigate the mutagenic effects of monoadducts and crosslinks as well as the role of NER on the mutagenesis induced by these lesions, a comparison of cytotoxicity and mutagenicity was made between normal human fibroblasts and NER- deficient xeroderma pigmentosum group A (XP-A) fibroblasts treated with PUVA-I and PUVA-II. In normal cells, PUVA- II treatment markedly reduced cell viability and induced 6-thioguanine resistant mutants 10 folds higher than UVA- treated populations. Whereas, PUVA-I only slightly affected cell survival and induced 14 folds mutant frequency at subcytotoxic doses of 8-MOP. These results indicated that 8- MOP monoadducts were less cytotoxic but as mutagenic as crosslinks. Molecular analysis of hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene in mutants derived from PUVA-I and PUVA-II treated normal cells have shown that both treatment induced mainly T:A base substitutions at 5'TA or 5'AT sites, and most of the mutated thymines were located on the non- transcribed strands. However, the mutations derived from PUVA-I-treated normal cells were predominantly T -> A transversions, whereas those derived from PUVA-II-treated ones were a specific kind of base changes related to the 5'flanking base of thymines. On the other hand, XP-A cells were more sensitiveto cytotoxic effects of both PUVA- I and PUVA-II than normal cells. PUVA-II was much more cytotoxic than PUV-I, but did not induce significant hprt mutations in XP-A cells as it did in normal cells, suggesting that NER is involved in the mutagenic process of 8-MOP crosslinks. In contrast, PUVA-I treatment in XP-A cells enhanced hprt mutation frequency 6-fold higher than control populations, and most of these mutations were T -> A transversions. The results that PUVA-I induced mainly T ->A transversions in both normal and XP-A cells indicated that this kind of mutations was not affected by NER deficiency. In addition, 1/3 of the T:A base substitutions of PUVA-I-induced XP-A mutants had thymines on the transcribed strand, while those mutations in normal cells were almost located on non-transcribed strand. These results were in consistent with the fact that NER is coupled with transcription.