鎳與鉻暴露以及基質金屬蛋白酶之相關性對口腔癌的影響

Abstract

在過去的二十年間，坐落於中台灣的彰化縣被發現到有著土壤金屬汙染的情況，而當地的金屬相關工業所排放之廢水更被懷疑是造成土壤金屬汙染的主要原因。此外，一般已知的口腔癌危險因子並不能有效地解釋彰化縣居高不下的口腔癌盛行率而且此地區亦存有不少口腔癌的特異性，而過去的職業性及生態學研究則顯示了金屬暴露與口腔癌發展之間存在著可能的相關性。基質金屬蛋白酶(MMPs)這一系列的金屬依賴性蛋白分解酵素與許多癌症的侵犯及轉移有所關聯，其中基質金屬蛋白酶-2(MMP-2)與基質金屬蛋白酶-9(MMP-9)的基因表現以及酵素活性則被發現與口腔癌的發展有關。再者，細胞實驗與流行病學研究都表示金屬暴露可能會增加基質金屬蛋白酶-2與基質金屬蛋白酶-9的表現量與活性。 本研究之目的在於釐清金屬暴露與口腔癌發展之間的關係，並了解口腔癌病人中金屬暴露與基質金屬蛋白酶-2及基質金屬蛋白酶-9的相關性，以及探討金屬暴露與基質金屬蛋白酶-2及基質金屬蛋白酶-9之間相關性對於口腔癌之影響為何。 本計劃之研究設計為一個醫院型的個案對照研究，在彰化基督教醫院收集了101位口腔癌病人，以及104位沒有口腔癌病史的對照組病人。在受試者結束定期回診檢查後進行問卷訪談與血樣採集的動作。受訓過之面訪者以問卷調查受試者的人口學資料、生活型態、抽菸、喝酒與吃檳榔習慣、環境與職業暴露史，以及家庭癌症史等相關資料。同時，利用感應耦合電漿質譜儀(ICP-MS)量測血液樣本中鎳(Ni)、鉻(Cr)、砷(As)、鎘(Cd)、汞(Hg)、鉛(Pb)、銅(Cu)、鋅(Zn)等八種重金屬含量。而血漿中之基質金屬蛋白酶-2與基質金屬蛋白酶-9的含量則是使用酵素免疫法(ELISA)來進行定量，至於基質金屬蛋白酶-2的啟動子基因上-1306 C>T與基質金屬蛋白酶-9的啟動子基因上-1562 C>T這兩個點位的基因多型性則是由聚合酶連鎖反應(PCR)進行分析。 第一部分的研究結果顯示，病例組中血液鎳、鉻、銅與鋅的濃度皆高於對照組(P’s < 0.01)，而利用居住地區還有抽菸、喝酒與吃檳榔的習慣將受試者進行分層之後，則發現到血液中鎳與鉻的濃度差異比例在病例組與對照組之間是最為明顯的。此外，在控制了可能的干擾因子後，口腔癌與嚼食檳榔(OR = 1.10, 95% CI: 1.06-1.13)、高濃度的血中鎳(OR = 16.2, 95% CI: 6.59-39.8)以及高濃度的血中鉻(OR = 6.80, 95% CI: 2.84-16.3)有顯著的相關性。 第二部分的研究結果發現病例組只有在血漿中基質金屬蛋白酶-9含量上顯著高於對照組(P < 0.0001)，而在基質金屬蛋白酶-2含量的部分兩組並無明顯差異。在控制了干擾因子的影響後，觀察到血漿中高含量的基質金屬蛋白酶-9(OR = 1.01, 95% CI: 1.00-1.02)以及嚼食檳榔(OR = 1.08, 95% CI: 1.05-1.11)與口腔癌有顯著的相關。再者，口腔癌病人的血液中鉻濃度與血漿中基質金屬蛋白酶-9含量呈現正相關(P < 0.05)。而且在調整了已知的口腔癌危險因子後，亦可在口腔癌病人中觀察到血中各濃度與血漿中基質金屬蛋白酶-9含量的顯著相關性(P < 0.05)。 第三部分的研究結果則顯示，病例組在基質金屬蛋白酶-2的-1306 C>T點位帶有C/C這種基因型的比例較對照組來的高(P < 0.05)，而病例組在基質金屬蛋白酶-9的-1562 C>T點位帶有T這種對偶基因的比例則是顯著的高於對照組(P < 0.05)。不論是在病例組或是對照組，血漿中基質金屬蛋白酶-2的含量都會受到基質金屬蛋白酶-2之-1306 C>T點位的基因多型性所調控(P’s < 0.05)；而血中砷與汞的濃度亦會調控到血漿中基質金屬蛋白酶-2的含量(P’s < 0.01)。另一方面，病例組之血漿基質金屬蛋白酶-9的含量會受到基質金屬蛋白酶-9之-1562 C>T點位的基因多型性所調控(P < 0.001)；而血中鉻與鋅的濃度亦會調控到血漿中基質金屬蛋白酶-9的含量(P’s < 0.01)。在控制了可能的危險因子之影響後，嚼食檳榔(OR = 18.7, 95% CI: 6.69-52.3)、高濃度的血中鎳(OR = 18.8, 95% CI: 6.93-51.0)、高濃度的血中鉻(OR = 7.47, 95% CI: 2.88-19.4)、高濃度的血漿中基質金屬蛋白酶-9(OR = 3.35, 95% CI: 1.30-8.66)、以及基質金屬蛋白酶-9的-1562 C>T點位為T對偶基因(OR = 3.21, 95% CI: 1.17-8.80)等因素，皆與口腔癌有顯著的相關性。 總結，本研究提供了直接的流行病學證據去加強金屬暴露與口腔癌發展之間可能的相關性，並且支持了血漿中基質金屬蛋白酶-9的含量以及基質金屬蛋白酶-9的-1562 C>T點位上的T對偶基因在口腔癌發展機制中的重要性。此外，目前的結果更顯示，鉻暴露調控血漿中基質金屬蛋白酶-9含量的能力是其與口腔癌相關聯的一種可能解釋方向。整體而言，本研究提供了環境金屬污染與基質金屬蛋白酶之間對於口腔癌發展之影響的證據，有助於未來進一步了解口腔癌的成因，進而改善口腔癌的健康風險。

Soil metal contamination has been found over the past two decades in Chaughua County, which is located in central Taiwan. Wastewater from local metal-related industries was suspected to be the main source of soil metal pollution. Moreover, Changhua County has a high prevalence of oral cancer that could not be explained by the well-known potential risk factors of oral cancer, and some distinctive oral cancer features were observed in this area. Previous occupational and ecological studies implied that there was a possible association between metal exposure and oral cancer development. Matrix metalloproteinases (MMPs), a family of metal-dependent proteolytic enzymes, were associated with the potential invasion and metastasis of some tumors, and the gene expressions and enzyme activities of MMP-2 and MMP-9 were reported to be correlated with oral cancer development. In addition, cellular level experiments and epidemiological studies indicated that the metal exposure might increase the expression and activity of MMP-2 and MMP-9. The aim of this study was therefore to elucidate the relationship between metal exposure and oral cancer development, the correlations between metal exposure and MMP-2 and MMP-9 in oral cancer patients, and the effects of the associations between metal exposure and MMP-2 and MMP-9 on oral cancer development. A hospital-based case-control study was conducted with 101 oral cancer patients and 104 control patients without a previous history of oral cancer from Changhua Christian Hospital (CCH). Questionnaire administration and blood sample collection were implemented after the study subjects’ regular return clinic visits. A well-trained interviewer administered the questionnaire to collect study subjects’ demographic information, lifestyle information, smoking history, history of alcohol use, betel quid chewing history, environmental and occupational exposure history, and family history of cancer. Trace levels of several metals, including nickel (Ni), chromium (Cr), arsenic (As), cadmium (Cd), mercury (Hg), lead (Pb), copper (Cu), and zinc (Zn), in blood samples were determined by inductively coupled plasma mass spectrometry. The amounts of MMP-2 and MMP-9 in the plasma samples were quantified by enzyme-linked immunosorbent assays (ELISA), and the genetic polymorphisms of the MMP-2 promoter gene at -1306 C>T and the MMP-9 promoter gene at -1562 C>T were analyzed by polymerase chain reactions (PCR). The results of the first part of this study showed that the blood nickel, chromium, copper, and zinc concentrations were higher in the oral cancer cases than in the controls (all P’s < 0.01) and that the differences in concentration between the cases and controls were most significant for blood-Ni and blood-Cr levels after being stratified by participants’ residential locations and smoking, alcohol drinking, and betel quid chewing history. Additionally, oral cancer was significantly associated with betel quid chewing (OR = 1.10, 95% CI: 1.06-1.13), high levels of blood-Ni (OR = 16.2, 95% CI: 6.59-39.8), and high levels of blood-Cr (OR = 6.80, 95% CI: 2.84-16.3) after controlling for potential confounders. The results of the second part of this study indicated that only the plasma MMP-9 levels, and not MMP-2 levels, of the cases were significantly higher than those of controls (P < 0.0001) and that oral cancer was significantly associated with MMP-9 plasma levels (OR = 1.01, 95% CI: 1.00-1.02) and betel quid chewing (OR = 1.08, 95% CI: 1.05-1.11) after adjusting for the confounding factors. In addition, blood chromium level was positively correlated with plasma MMP-9 level in oral cancer patients (P < 0.05), and significant associations between blood chromium level and plasma MMP-9 level were observed in oral cancer patients after adjusting for the known risk factors for oral cancer (P < 0.05). The results of the third part of this study demonstrated that the C/C genotype frequency of MMP-2 -1306 C>T and the T allele frequency of MMP-9 -1562 C>T were higher in the oral cancer cases than in the controls (all P’s < 0.05). The plasma MMP-2 levels were modified not only by the polymorphisms of MMP-2 -1306 C>T in both the cases and controls (all P’s < 0.05) but also by the blood arsenic and mercury levels (all P’s < 0.01); the plasma MMP-9 levels were regulated not only by the polymorphisms of MMP-9 -1562 C>T in cases (P < 0.001) but also by the blood chromium and zinc levels (all P’s < 0.01). After controlling for potential risk factors, oral cancer was found to be significantly associated with betel quid chewing (OR = 18.7, 95% CI: 6.69-52.3), high levels of blood-Ni (OR = 18.8, 95% CI: 6.93-51.0), high levels of blood-Cr (OR = 7.47, 95% CI: 2.88-19.4), high levels of plasma-MMP-9 (OR = 3.35, 95% CI: 1.30-8.66), and the T allele of MMP-9 -1562 C>T (OR = 3.21, 95% CI: 1.17-8.80). In conclusion, this study provided direct epidemiological evidence that strengthens the possible association between metal exposure and oral cancer development and that underscores the importance of plasma MMP-9 levels and the T allele of MMP-9 -1562 C>T in the mechanism of oral cancer development. Moreover, the present results implied that chromium exposure, as a result of its regulation of plasma MMP-9 levels, has a possible role in the pathogenesis of oral cancer. On the whole, this study described the possible relationship between personal environmental metal exposure and MMPs levels in the development of oral cancer; these findings might warrant further study to reduce the risk of oral cancer in the future.