以體外及體內模式探討 5-去甲基川陳皮素與川陳皮素抑制苯駢芘誘導 DNA 損傷之分子機制

Abstract

不完全燃燒產生的多環芳香烴 (polycyclic aromatic hydrocarbons, PAHs) 已被證明會引起基因突變。PAHs 中的苯駢芘 (benzo[a]pyrene, B[a]P) 已被廣泛證明具有強烈基因毒性，而飲食攝取 B[a]P 作為主要暴露途徑之一，將嚴重影響腸上皮細胞，增加罹患腸癌風險。B[a]P 會與芳香烴受體 (aryl hydrocarbon receptor, AhR) 結合從而上調細胞色素 P450 家族 1 酵素 (cytochrome P450 family 1, CYP1s) 將其代謝為 B[a]P-7,8-dihydrodiol-9,10-epoxide (B[a]P-7,8-dihydrodiol-9,10-epoxide, BPDE)。該環氧化物可與 DNA 結合形成 DNA 加合物，從而導致 DNA 損傷。值得注意的是，許多研究表明，類黃酮化合物不僅可以作為 AhR 配體調控 AhR/CYP1s 路徑，還可以作為?抑制劑降低 CYP1s 的活性。先前文獻亦表明，多甲氧基黃酮類具有透過調節 CYP1s 為抗癌的良好潛力，尤其是川陳皮素 (nobiletin, NBT) 和 5-去甲基川陳皮素 (5-demethylnobiletin, DMNB)。然而 NBT 和 DMNB 在 B[a]P 代謝途徑中之作用尚未釐清，因此本研究旨在闡明 NBT 和 DMNB 對 B[a]P 誘導 DNA 損傷之影響。體外實驗結果顯示 DMNB 和 NBT 可能透過調節 NCM460 細胞中 AhR/CYP1s 信號途徑潛在地抑制 B[a]P 之代謝轉化，從而使 DMNB 與 NBT 減輕 DNA 損傷並抑制 BPDE-DNA 加合物之形成以防止 NCM460 細胞受到 B[a]P 引發之細胞毒性。另一方面，在 BALB/c 小鼠的實驗結果，發現 DMNB 與 NBT 對 B[a]P 誘導 DNA 損傷具有保護效果。DMNB 與 NBT 減緩 B[a]P 所造成的肝損傷及減少尿液中 8-羥基-2-脫氧鳥? (8-hydroxy-2’-deoxyguanosin, 8-OHdG)；此外，更可以藉由抑制 BPDE-DNA 加合物和 DNA 受損反應以改善肝臟和結腸之 DNA 損傷。在 B[a]P 代謝的干預作用中，DMNB 和 NBT分別具有抑制?活性和調節 AhR/CYP1s 路徑之能力。綜合上述，DMNB 和 NBT 可以透過調控生物轉化機制減緩 B[a]P 誘導 DNA 損傷，對 B[a]P 具有化學預防作用且在阻斷癌症發展方面有良好應用價值，有助於未來發展以 DMNB 與 NBT 作為結腸直腸癌化學預防試劑之策略，更可提供農業副產物於保健食品應用之依據。

Polycyclic aromatic hydrocarbons (PAHs) generated from incomplete combustion have been proved that they cause gene mutations. Benzo[a]pyrene (B[a]P) of PAHs has been widely evidenced by its strong genotoxicities. Oral consumption as the primary route of B[a]P exposure will severely affect intestinal epithelial cells and rise the risk of colorectal cancer. B[a]P will be metabolized into B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE) by cytochrome P450 family 1 enzymes (CYP1s) that are upregulated through aryl hydrocarbon receptor (AhR). The epoxide can bind with DNA to form DNA adducts, leading to DNA injuries. Notably, many studies have indicated that flavonoids not only mediate the AhR/CYP1s pathway as AhR ligands, but also inhibit CYP1s activity as enzyme inhibitors. Previous studies also showed polymethoxyflavones possess high potential for anti-cancer effects through regulating CYP1s, especially nobiletin (NBT) and 5-demethylnobiletin (DMNB). However, the roles of NBT and DMNB in B[a]P metabolism remain unclear. Therefore, this study aimed to clarify the effect of NBT and DMNB on B[a]P-induced DNA damage in vitro and in vivo. The in vitro results showed DMNB and NBT might potentially abate the metabolic transformation of B[a]P via regulating AhR/CYP1s signaling pathway in NCM460 cells. Therefore, DMNB and NBT prevented NCM460 cells from B[a]P-induced cytotoxicity via alleviating DNA damage and inhibiting the formation of BPDE-DNA adduct. On the other hand, a protective effect of DMNB and NBT against B[a]P-induced DNA damage was found in BALB/c mice. DMNB and NBT mitigated liver damage and reduced urinary 8-hydroxy-2’-deoxyguanosine (8-OHdG) which were brought by B[a]P. Furthermore, DMNB and NBT ameliorated hepatic and colonic DNA injury via inhibition of BPDE-DNA adduct and DNA damage response. In B[a]P metabolism of the intervening roles, DMNB and NBT possess the capacity of inhibiting enzyme activity and regulating the AhR/CYP1s pathway, respectively. In conclusion, these results showed that DMNB and NBT attenuated B[a]P-induced DNA damage via mediating biotransformation, indicating their chemopreventive effects against B[a]P and high potential of application in blocking carcinogenesis. It will be a promising strategy using DMNB and NBT as colorectal cancer chemopreventive agents in the future, and also provide a basis for the application of agricultural by-products in dietary supplements.