中藥中毒—探尋台灣泌尿上皮癌之特殊危險因子

Herbs intoxication –Identification of a novel risk factor for urothelial carcinoma in Taiwan

陳忠信(Chung-Hsin Chen)

指導教授 ： 蒲永孝

共同指導教授 ： 孫家棟

國立臺灣大學/醫學院/臨床醫學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.02064

摘要

泌尿上皮癌(urothelial carcinoma)是台灣地區第九大常見的惡性腫瘤。泌尿上皮癌可發生在任何泌尿上皮(urothelium)存在之處，包括腎盂、輸尿管、膀胱和尿道。其中，腎盂與輸尿管合稱為上泌尿道。而台灣與巴爾幹半島地區同為全世界上泌尿道泌尿上皮癌(upper tract urothelium carcinoma)發生率最高的地區，目前的發生率約為每十萬人每年4.2人,為歐美等國的四至八倍。依據台灣癌症登記小組從1979年至2007年的登記結果顯示，上泌尿道泌尿上皮癌的發生率竟成長了八倍，同期間膀胱泌尿上皮癌卻僅成長兩倍；這結果顯示上泌尿道泌尿上皮癌在最近十數年來快速增加，但直到本文前，其原因卻仍未明。此外，從統計資料內也發現膀胱泌尿上皮癌的病人，男性居多(70%)；但是上泌尿泌尿上皮癌的病人中，卻是女性較多(55%)。因此，上泌尿道泌尿上皮癌的發生原因可能不同於膀胱泌尿上皮癌，需要我們進一步去探究。
目前研究上泌尿道泌尿上皮癌的成因包括：砷中毒、吸菸、苯環類中毒、及馬兜鈴酸。台灣流行病學家發現烏腳病流行的台灣西南沿海，膀胱泌尿上皮癌及上泌尿道泌尿上皮癌的發生率約為非烏腳病地區的十至二十倍。因此，造成烏腳病的致病主因—砷，曾被當成解釋台灣上泌尿道泌尿上皮癌流行的一個主要成因。然而，國內烏腳病地區(嘉義縣義竹鄉、布袋鎮，台南市北門區、學甲區)的人口僅佔全國約兩百分之一，且污染區域的衛生條件也明顯改善，多數區域均改飲用自來水而非地下水。排除了砷流行區，其他台灣地區的上泌尿道泌尿上皮癌發生率，依然是歐美國家的四到八倍。所以砷並無法解釋台灣上泌尿道泌尿上皮癌的增加趨勢。另外，歐美國家的上泌尿道泌尿上皮癌均可歸因於吸菸，但台灣地區的女性病人僅有百分之四以下有吸菸史；而且近年來，台灣男性人口的吸菸比率也大幅下降(<38%)，所以吸菸這一個危險因子只能解釋一小部份病人的成因，對於大多數女性病人並不適用。
西元1992年起，從比利時到巴爾幹半島的腎病變，均被證實導因於馬兜鈴酸。馬兜鈴酸也被發現，不僅僅是一個腎毒物，也是一個致癌物質。馬兜鈴酸是目前已知中藥中，會造成腎臟損傷及致癌的一種成分，其存在於馬兜鈴屬植物之藥劑中；在國內常用的中藥如關木通、廣防己、青木香、天仙藤、馬兜鈴、朱砂蓮、尋骨風、黃木香、南木香、管南香、漢中防己、淮通、白金古欖、白金果欖、通城虎、金獅藤、籮蔔防己等至少十七種藥材都含有馬兜鈴酸。而這些中藥藥材在過去未被發現其副作用前，均曾廣泛使用於國人的處方中。
因此我們建立一個假說：台灣上泌尿道泌尿上皮癌的增加，一部分導因於砷汙染，另一部分導因於馬兜鈴酸暴露。並據此進行四部分研究，以證實我們的假說。
第一部分、是否砷汙染仍是造成台灣泌尿上皮癌的主因，及砷汙染區泌尿上皮癌的特色? 流性病學分析發現砷汙染嚴重程度確實與泌尿上皮癌的發生率，有顯著相關；但是，近年來非砷汙染區的病例數快速增加，佔全國病例數的比率也達到近80%，所以顯然還有其他的因素會導致泌尿上皮癌發生率的增加。臨床世代研究也發現，砷污染核心區的泌尿上皮癌病人的臨床表現，異於非砷汙染區。砷污染核心區的病人表現出較差的腫瘤分化、較高的腫瘤期別、較差的預後狀況；這代表著非砷汙染區的泌尿上皮癌的病因，可能不同於砷汙染區，而是導因於其他致癌物質。
第二部分、台灣泌尿上皮癌之馬兜鈴酸暴露研究。 我們藉由直接檢驗上泌尿道泌尿上皮癌病人的腎臟皮質的馬兜鈴酸去氧核醣核酸加合物之有無(直接證據)，來決定病人是否有馬兜鈴酸暴露。結果發現女性上泌尿道泌尿上皮癌病人，有65.7%可檢得馬兜鈴酸去氧核醣核酸加合物；男性上泌尿道泌尿上皮癌病人，也有54.2%可檢得馬兜鈴酸去氧核醣核酸加合物。不過，對照組的腎細胞癌病人，也有57.9%被發現腎實質中有兜鈴酸去氧核醣核酸加合物。因此，我們雖然證實了多數上泌尿道泌尿上皮癌病人，確實暴露過馬兜鈴酸。但是，馬兜鈴酸真的是致病因嗎?
第三部分、台灣上泌尿道泌尿上皮癌的基因突變譜，與馬兜鈴酸的關係。由於細胞、動物、與人體檢體發現，基因上的A:T to T:A突變可以當作是一個馬兜鈴酸效應的生物指標。我們檢測台灣上泌尿道泌尿上皮癌是否擁有這些特異性突變，並與巴爾幹半島馬兜鈴酸暴露病人及世界腫瘤基因突變資料庫內的基因突變譜比較。結果顯示台灣上泌尿道泌尿上皮癌的TP53突變譜，以A:T to T:A transversions為主，佔全部突變數的53.1%。根據第十一版IARC的報導，這種突變在全部人類的腫瘤中，僅佔5.1%；而在泌尿腫瘤內，也僅有4.8%；在上泌尿道(腎盂與輸尿管)腫瘤中，更僅有1.4%會有A:T to T:A transversion。比對巴爾幹半島的資料發現，台灣與巴爾幹半島病人的腫瘤突變分布與位置，非常相像。據此，我們已證實了三分之一的台灣上泌尿道泌尿上皮癌，確實導因於馬兜鈴酸的暴露。
第四部分、馬兜鈴酸引起的上泌尿道泌尿上皮癌的臨床表現特徵。同時利用暴露指標(馬兜鈴酸去氧核醣核酸加合物)，與效應指標(A:T to T:A transversion)，馬兜鈴酸引起的上泌尿道泌尿上皮癌可以被確認，其臨床表現也可用來與非馬兜鈴酸相關腫瘤比較。結果發現不同致病因的腫瘤，在腫瘤診斷時表現類似(分布位置、多發性、腫瘤期別、腫瘤分化)；但是，馬兜鈴酸引起的上泌尿道泌尿上皮癌病人，有較不好的腎功能、較多的女性病人、較少的吸菸史、較多的中藥服用史；更重要的是，馬兜鈴酸引起的腫瘤容易有對側上泌尿道復發的特色。所以，我們應於診斷初期找出何人有馬兜鈴酸相關的腫瘤，擬定特別的治療與追蹤對策，以改善病人的預後。
台灣的泌尿上皮癌的致癌物質，不僅僅是砷、吸菸，也包括了馬兜鈴酸。特別是近年快速增加的上泌尿道泌尿上皮癌，更多是導因於馬兜鈴酸的暴露。由於馬兜鈴酸引起的上泌尿道泌尿上皮癌，有容易對側上泌尿道復發的特色，目前可藉由兩種生物指標，腎實質馬兜鈴酸去氧核醣核酸加合物與腫瘤特徵性TP53突變，確認馬兜鈴酸相關的腫瘤，並據此擬定特別的治療與追蹤策略。

關鍵字

馬兜鈴酸 ； 泌尿上皮癌 ； 砷 ； p53 ； 去氧核醣核酸加合物

並列摘要

In Taiwan, the incidence of upper tract urothelial carcinomas (UUC) is the highest among all countries in the world. Also in Taiwan, there is a strong association between UUC and end-stage renal disease (ESRD). Although arsenic contamination of drinking water has been found to be associated with UUC, exposure to this carcinogen in Taiwan occurs only in very few areas. Cigarette smoking is also a proven risk factor for UUC; however, smoking does not account for the high incidence rate of UUC in Taiwanese women as the smoking rate among Taiwanese women is less than 5%. Recently, aristolochic acid (AA), a proven nephrotoxin and human carcinogen, was shown to be a cause of UUC in Balkan countries.

This prospective, case-controlled, molecular epidemiologic study was undertaken to explore the proposition that AA, an intrinsic component of all Aristolochia herbal remedies, contributes significantly to the high incidence of UUC in Taiwan. Subjects of this study included 151 patients (82 men) with UUC and 25 patients (18 men) with renal cell carcinoma (RCC). TP53 mutational spectra and aristolactam (AL)-DNA adducts were utilized as biomarkers to establish the contribution of AA to the prevalence of UUC in Taiwan. A total of 113 TP53 mutations were detected in 84 of 151 (56%) patients with UUC. All but one mutation consisted of single-base substitutions in exons 2-11. The TP53 mutational spectra for patients with UUC was dominated by A:T to T:A transversions (53.1% of the total), a class of mutations found in only 4.8% of all urothelial carcinomas and 1.4% of all UUC in the world. Remarkably, the positions of TP53 A:T to T:A transversions in the Taiwanese tumor samples were almost identical to those observed for residents of endemic regions of Bosnia, Croatia and Serbia where AA has been linked to the development of the so-called Balkan Endemic Nephropathy (BEN). Three of the 25 patients with RCC displayed mutations in TP53. Not a single A:T base pair was mutated in this control group.

Among patients with UUC, 60% of women and 50% of men with TP53 mutations displayed the characteristic A:T to T:A transversions. Thirty-seven per cent of all A:T to T:A transversions occurred at splice sites. All but one of 60 mutated adenines were located on the non-transcribed strand of DNA. We identified mutational “hotspots” for A:T to T:A transversions at introns 6, 7 and 8 and at codons 131, 209 and 249, respectively.

The major adduct found in renal cortex DNA of UUC patients with the signature TP53 mutation was identified by mass spectrometric analysis as dA-AL-I. Adduct levels ranged between 1.4 and 234 adducts per 108 nucleotides, as estimated by 32P-postlabeling assays. dG-AL adducts were not observed. Eighty-four % of patients with A:T to T:A transversions in TP53 were adduct positive, underscoring the close association between exposure to AA and its carcinogenic effect. Evidence for AA exposure, indicated by the presence of AL-DNA adducts in the renal cortical DNA and/or A to T transversions in tumor DNA, was present in 66 % of all UUC cases. Of males with no evidence of exposure, 47% were smokers, an established risk factor for UUC. None of the females who tested negative for AA exposure smoked.

Defining aristolochic acid-induced UUC (AA-UUC) as having both AL-DNA adducts in the renal cortex and A to T transversions in tumors, we found that AA-UUC patients were younger, predominately female, had more end-stage renal disease, and were infrequent smokers compared to possible-AA-UUC and non-AA-UUC patients. All 14 patients who developed contralateral UUC had aristolactam-DNA adducts; ten of these also had signature mutations. The contralateral UUC-free survival period was shorter in AA-UUC compared to possible- or non-AA-UUC, whereas no differences among groups were observed for bladder cancer recurrence.

In conclusion, results of this study provide compelling evidence for the role of AA in the etiology of UUC in Taiwan. Due to lifelong persistence of mutagenic DNA-AL-I adducts in target tissues, persons treated with Aristolochia herbal preparations at any time in their life are at significant risk of developing UUC. AA-UUC patients tend to be younger and female, and have more advanced renal disease. Notably, AA exposure was associated with an increased risk for developing synchronous bilateral and metachronous contralateral UUC.

並列關鍵字

aristolochic acid ； urothelial carcinoma ； arsenic ； p53 ； aristolactum-DNA adduct

參考文獻

1. anonymous, 1860. 吉林地方誌.

2. anonymous, 200. 神農本草經.

3. Arlt,V.M., Schmeiser,H.H., and Pfeifer,G.P., 2001a. Sequence-specific detection of aristolochic acid-DNA adducts in the human p53 gene by terminal transferase-dependent PCR. Carcinogenesis, 22(1): 133-140.

4. Arlt,V.M., Schmeiser,H.H., and Pfeifer,G.P., 2001b. Sequence-specific detection of aristolochic acid-DNA adducts in the human p53 gene by terminal transferase-dependent PCR. Carcinogenesis, 22(1): 133-140.

5. Arlt,V.M., Stiborova,M., and Schmeiser,H.H., 2002. Aristolochic acid as a probable human cancer hazard in herbal remedies: a review. Mutagenesis, 17(4): 265-277.