- 學位論文
過量酒精攝取對乳腺腫瘤大鼠之影響
Effects of Excess Alcohol Intake on the Rats with Breast Tumor
摘要
目的：酒精一直被認為是致癌因子之一，主要因素與攝取酒精後其代謝產物-乙醛 (acetaldehyde)以及代謝過程中活性氧的生成 (reactive oxygen species, ROS)有關，進一步造成細胞抗氧化狀態失衡導致疾病的發生。女性乳癌一直是國人不可忽視的重要疾病之一，然而酒精在乳癌發展過程中扮演何種角色，以及影響體內哪些生物指標的表現，為探討之主要目標。因此本研究利用乳腺腫瘤大鼠給予酒精攝取，評估對於腫瘤增生與轉移因子、氧化及抗氧化能力與細胞凋亡之影響。 材料與方法：本研究以雌性大鼠為實驗對象，利用7,12-dimethylbenz(a)anthracene (DMBA)作為誘發乳腺腫瘤之模式，同時合併酒精攝取之因素，於犧牲後取出血液、肝臟和腫瘤組織，進行肝功能、脂質、全血球計數、免疫球蛋白、血管增生與轉移因子、氧化與抗氧化指標、細胞凋亡相關因子及穀胱甘肽轉移酶等測定分析。 結果與討論：本實驗所取出之腫瘤組織經由蘇木紫伊紅 (Hematoxylin Eosin, HE)染色確認為乳腺腫瘤，單獨給予DMBA條件下誘發率達到80%，顯示DMBA模式能成功誘發乳腺腫瘤形成。此外無論有無乳腺腫瘤因素下，攝取酒精的確造成Triglycerides (TG)表現量上升，推測大鼠確實受到酒精刺激。有攝取酒精或乳腺腫瘤的條件下，Matrix Metalloproteinase-2 (MMP2)與Vascular Endothelial Growth Factor (VEGF)的表現量均有上升的情況，然而當兩因素合併的條件下，MMP2與VEGF的表現量反而有下降的趨勢，顯示酒精具有拮抗DMBA誘發乳腺腫瘤的能力。與腫瘤惡化相關之Transforming Growth Factor-beta (TGF-beta)方面，若乳腺腫瘤大鼠攝取酒精時，則有顯著上升的現象，顯示若DMBA成功誘發乳腺腫瘤後，酒精確實扮演著不良因子之一。還原型榖胱甘肽(glutathione, GSH)方面，同樣於攝取酒精或乳腺腫瘤的條件下，血液中的GSH含量較高，我們推測應體內氧化力較高所造成之代償因素，然而當乳腺腫瘤大鼠攝取酒精後，GSH含量降低，同樣推測酒精具有拮抗DMBA的作用。細胞凋亡方面，於代謝器官肝臟中，攝取酒精會導致促進細胞凋亡之Bax蛋白表現量增加，而乳腺腫瘤大鼠肝臟中抑制細胞凋亡之Bcl-2蛋白則有下降的現象，然而兩者合併的情況下，Bax和Bcl-2的表現情況皆有恢復之情形。穀胱甘肽轉移酶 (pi calss of glutathione S-transferase, GSTP)方面，乳腺腫瘤大鼠肝臟中的GSTP表現量有顯著上升的現象；乳腺腫瘤同時攝取酒精之大鼠GSTP表現量雖然比Control組來的高，然而相較無攝取酒精之乳腺腫瘤大鼠，GSTP則有下降的趨勢，顯示DMBA在代謝過程中被酒精抑制毒性。由於DMBA代謝過程中會藉由生物轉換酵素系統 I 中的P450s與mEH (microsomal epoxide hydrolase)形成更具毒性之代謝產物，進一步刺激生物轉換酵素系統 II 中的GSTP表現量上升，然而慢性酒精攝取會誘發CYP2E1的表現，同時負向調控下游之mEH，導致DMBA無法形成具癌化能力之代謝產物。在腫瘤組織中，攝取酒精之乳腺腫瘤大鼠GSTP表現量顯著高於未攝取酒精之組別，顯示若成功誘發乳腺腫瘤後，酒精會促使腫瘤抗氧化能力提升，使腫瘤更易生長。 結論：酒精攝取會影響DMBA誘發大鼠形成乳腺腫瘤之能力；然而，若DMBA成功誘發出乳腺腫瘤時，酒精在此階段則扮演不良因子，促使腫瘤更加惡化的角色。
並列摘要
Objectives: Alcohol has been considered one of the carcinogenic factors, the main causes of which are acetaldehyde, the metabolic product, and reactive oxygen species generation (ROS) produced in the metabolic process; they further lead to unbalanced cell antioxidation which results in the development of disease. Female breast cancer is one of the major cancers in Taiwan. The role alcohol plays in the process of breast cancer development as well as the effects of biological indicators is the main objective of this study. Therefore, in this study, we used rats with breast tumors which also received intake of alcohol to assess the effects of tumor proliferation and migration, apoptosis, oxidant and antioxidant statuses. Materials and Methods: In this study, we used female rats, which were treated with 7,12-dimethylbenz(a)anthracene (DMBA) to induce breast tumors and with the concomitant of alcohol intake, as the experimental models. We analyzed the liver function tests, lipid profiles, complete blood counts, immunoglobulins, vascular proliferation and migration factors, oxidants and antioxidants, apoptosis-related factors and the glutathione S-transferase. Results and Discussion: The Hematoxylin Eosin stain was used to confirm breast cancer tumors. When DMBA was administered alone, the induction rate of breast tumors was induced to 80%, which indicated that the DMBA model can successfully induce breast tumors. In addition, there was increased expression of triglycerides in the group with alcohol intake, which confirmed the rats were indeed affected by alcohol intake. The matrix metalloproteinase-2 (MMP2) and vascular endothelial growth factor (VEGF) levels were higher in the groups with alcohol intake and breast tumors than in the control group; however, there was a downward trend in the expressions of MMP2 and VEGF in the breast tumor group with alcohol intake, which indicates the ability of DMBA to induce breast tumors was suppressed by alcohol intake. transforming growth factor-beta (TGF-beta), which was involved in the progression of breast tumors, was significantly increased in the rats with breast tumors when they were given alcohol intake, meaning that alcohol intake played one of the negative roles after DMBA had induced mammary tumors. There were high levels of glutathione (GSH) in the groups with alcohol intake and breast tumor compared with the control group. We believe it was the compensative effect of the oxidative response. In addition, when the rats in the breast tumors group with alcohol intake, their GSH levels were reduced, which confirmed again that alcohol intake suppressed the ability of DMBA. In the liver tissues, alcohol intake can lead to the promotion of the proapoptotic protein Bax expression. The antiapoptotic protein Bcl-2 was decreased in the group with breast tumors; however, when the breast tumors group with alcohol intake, the expressions of the Bax and Bcl-2 protein were restored. The expression of pi calss of glutathione S-transferase (GSTP) was significantly increased in the rats with breast tumors in their liver tissues. Although it was higher in the breast tumors group with alcohol intake than in the control group, there was a downward trend in the group with breast tumors which did not receive any alcohol intake, the result of which indicates the metabolic process of DMBA could be restrained by alcohol intake. DMBA is metabolized through the P450s enzyme in the phase I of the biotransformation enzyme system and microsomal epoxide hydrolase (mEH) to form a more toxic metabolite which further stimulates the increased expression of GSTP in the phase II of the biotransformation enzyme system. However, chronic alcohol intake can induce the expression of CYP2E1 while negatively regulate the mEH downstream, and finally inhibit the ability of DMBA to induce breast tumors. In the tumor tissues, the GSTP expression in the breast tumors group with alcohol intake was significantly higher than that in the group with breast tumors which received no alcohol intake, the result of which showed that alcohol intake would enhance the antioxidant capacity of the tumors after breast tumors had been successfully induced by DMBA. Conclusion: Alcohol might inhibit the ability of DMBA to induce breast tumors. However, if DMBA successfully induces breast tumors, alcohol plays a poor role in the tumor progression.