Motorcycle exhaust and metabolic activation

機車廢氣是台灣城市主要空氣污染源 之一，含致癌物苯及benzo(a)pyrene 等許多 毒性化學物質，有潛在的健康危害性。細 胞色素P450 (CYP) 是藥物、致癌物、類固 醇荷爾蒙、環境汙染物的主要代謝酵素系 統。CYP 基因表現很容易受到外來物所調 控，因此是研究基因-環境交互作用的良好 工具。本研究發現在接近人類暴露狀況 下，呼吸暴露雄性大鼠機車廢氣四週後， 會造成睪丸、副睪重量下降，睪丸及副睪 尾精子濃度減少，副睪尾及輸精管精子活 動力下降。組織病理分析結果顯示機車廢 氣造成睪丸萎縮、副睪小管壞死。機車廢 氣造成睪丸及副睪P450 相關單氧酶活 性，包括7-ethoxycoumarin O-deethylation 下降及鐵基質氧化酶活性上昇。RT-PCR 分 析結果顯示CYP11A1 及CYP17 mRNA 在 機車廢氣暴露組睪丸及副睪的表現較對照 組之表現為低。由本研究成果得知，機車 廢氣對雄鼠具有生育毒性，其可能機制是 透由抑制P450 及膽固醇生合成酵素及誘 導氧化壓迫蛋白的途徑而造成毒性。研究 成果應有助於評估暴露機車廢氣的健康風 險。Motorcycle exhaust (ME) is a major source of air pollution in the urban areas and a potential health hazard in Taiwan. ME contains carcinogenic benzene and benzo(a)pyrene and many toxic chemicals. Cytochrome P450 (CYP) is the primary enzyme system responsible for the metabolism of drugs, carcinogens, steroid hormones, and environmental pollutants. CYP genes are markedly responsive to the stimulatory and inhibitory effects of xenobiotics and provide a powerful tool to investigate gene-environment interaction. The present studies showed that inhalation exposure of male rats to ME at environmental realistic conditions for 4 weeks decreased testis and epididymis weights, testis and cauda epididymis sperm concentrations, and cauda epididymis and vas deferens sperm mobility. The result of histological analysis showed ME produced testicular atrophy and epididymal tubule necrosis. ME exposure resulted in decreases of P450-dependent monooxygenases activities including 7-ethoxycoumarin O-deethylation in testis and epididymis microsomes. The exposure also caused decreases of heme oxygenase activity in testis and epididymis. The results of RT-PCR analysis suggested that CYP11A1 and CYP17 mRNA expression in testis and epididymis from ME-exposed rats were lower than their respective controls. The results of the present studies show that ME inhalation has the ability to cause developmental toxicity in male rats and the underlying mechanisms involve suppression of P450 and steroidogenesis enzymes and induction of oxidative stress proteins in testis and epididymis. These results may help to assess the health risk associated with ME exposure

Motorcycle exhaust and metabolic activation

本研究探討機車廢氣微粒 (motorcycle exhaust particulate，MEP) 有機萃取物對人類乳腺癌MCF-7 細胞及 幼雌鼠的內分泌干擾作用。處理MCF-7 細胞1、10、50 g/ml MEP 2 天及4 天 分別造成劑量相關胸苷結合及細胞生 長，對1 nM 雌二醇(17-estradiol，E2) 共同處理細胞亦然。MEP 取代雌激素受 器結合[3H] E2 能力與時間及濃度相 關。細胞色素P450 抑制劑及多環芳香烴 受器拮抗劑-naphthoflavone (ANF)，對 MEP 的抗雌激素活性有抑制作用。MEP 處理過MCF-7 細胞之微粒體對E2 羥化 作用有較高的代謝活性， 此活性會被 ANF 所抑制。共同處理MEP 及E2 會降 低E2 所誘升之子宮重量。MEP 的多環 芳香烴成分benzo(a)pyrene 對MCF-7 細胞及幼鼠產生類似MEP 的抗雌激素活 性。本研究結果顯示MEP 是一種體外、 體內的抗雌激素物質，細胞色素P450 的 誘升可能是MEP 抗雌激素活性的機制之 一。The emissions from 2- and 4-stroke motorcycles pollute the air of urban areas where motorcycle is a popular means of transportation. This study aimed to determine the endocrine-disrupting activity of motorcycle exhaust particulate (MEP) using MCF-7 human breast cancer cells and immature female Wistar rats treated with organic extracts of MEP. Treatments with 1, 10, and 50 μg/ml MEP extract for 2 and 4 days produced dose-dependent inhibition of thymidine incorporation and cell growth, respectively, in untreated and 1 nM 17-estradiol (E2)-treated cells. Treatments of MCF-7 cells with MEP extract replaced [3H]E2 from the estrogen receptor in a timeand concentration-dependent manner. The antiestrogenic effects and the receptor binding property of MEP extract were blocked by cotreatment of the cells with 2 μM -naphthoflavone, a cytochrome P450 inhibitor and aryl hydrocarbon receptor antagonist. E2 metabolism and HPLC analysis showed that treatment of MCF-7 cells with 50 μg/ml MEP extract for 24 h increased E2 2- and 4-hydroxylation in microsomes. The MEP-mediated increase of E2 2-hydroxylation was inhibited by addition of 1 μM -naphthoflavone to MCF-7 microsomes. Cotreatment of immature female rats with 10 μg/kg E2 and 10 mg/kg MEP extract intraperitoneally for 3 days decreased the E2-induced uterine weights. MEP extract alone showed no effect on rat uterine weight. The endocrine-disrupting activity of MEP extract was further confirmed in paralleled experiments using MCF-7 cells and immature female rats treated with benzo(a)pyrene, an MEP constituent compound. The present findings demonstrate that MEP extract is antiestrogenic in vitro and in vivo and cytochrome P450 induction is an underlying mechanism

臺灣肺癌之基因體研究及臨床應用:著重於女性肺腺癌─分項計畫五：肺腺癌之環境因子及基因體毒理研究—機車排氣及肺部代謝之活化與癌症發生之關係（子計畫一）

機車是台灣主要交通工具之一，機車廢氣在都會區造成嚴重污染 問題。機車廢氣之微粒(motorcycle exhaust particulate, MEP)含有致癌 性的多環芳香烴化合物，包括苯駢芘(benzo(a)pyrene)， 苯甲 (benzanthracene) 及 benzo(g, h, i)perylene 等。本研究主要目的在探討 MEP 萃取物改變致癌物代謝基因、雌激素代謝基因、致癌基因、腫 瘤抑制基因表現的情形。本研究使用國人婦女肺腺癌CL5 細胞株， 用10，33 及100 ìg/ml MEP 萃取物處理 6 小時後，抽取RNA，再進 行9600 基因之微陣列分析。研究結果顯示MEP 萃取物造成許多基 因的改變，增加致癌物及雌激素代謝基因如細胞色素P450 (CYP) 2E，CYP2J，CYP27B，hydroxysteroid 17â 去氫酵素之表現，降低代 謝基因UDP-glucuronosyltransferase 及苯 氧化還原酵素表現，增加 K-ras 致癌基因表現，降低一假定腫瘤抑制基因表現。本年度初步研 究成果顯示機車廢氣微粒具有改變代謝活化及腫瘤生成標的基因表 現的能力。本研究成果應有助瞭解機車廢氣和基因交互作用在婦女肺 腺癌病因所扮演的角色。The emissions of motorcycle exhaust are a major source of environmental pollutants in urban areas where motorcycles are a major means of transportation in Taiwan. Motorcycle exhaust particulates (MEPs) contained carcinogenic polycyclic aromatic hydrocarbons including benzo(a)pyrene, benzanthracene, and bezno(g,h,i)perlyene. The major objective of this study is to determine the ability of MEP to alter the expression profiles of carcinogen- and estrogen-metabolizing genes as well as oncogenes and tumor suppressor genes in human lung cells treated with organic extracts of MEP. We have used cDNA microarrays to determine the expression level of 9600 genes in human female lung adenocarcinoma CL5 cells treated with 10 and 100 g/ml MEP extracts for 6 hr. Our recent data showed that MEP extract produced increases of the transcripts of many carcinogen- or estrogen-metabolism such as cytochrome P450 (CYP) 2E, CYP2J, CYP27B, and hydroxysteroid 17b dehydrogenase. MEP extract resulted in decreases of the expression of metabolism genes UDP-glucuronosyltransferase and quinone oxidoreductase. The extract induced the expression of a K-ras oncogene-associated gene and in contrast suppressed the expression of a human putative tumor suppressor gene. These recent data suggested that MEP has the ability to alter the expression profiles of some of the target genes related to metabolic activation and tumor promotion, which might lead to a better understanding of the role of MEP and gene interaction in the etiology of female lung carcinogenesis