三亞螨藥劑對大鼠體內睪固酮及雌素二醇代謝之調控作用

三亞蟎為一防治果樹蟎類及牛、羊蜱類等用途之甲咪類(formamidine)農業用藥。本研究主要目的，在探討三亞蟎調控大鼠睪固酮及雌素二醇的代謝作用，及血清中睪固酮(testosterone)及雌素二醇(17β-estradiol)含量所扮演的角色。內分泌干擾物的定義為，外來的化學物質具有干擾內生性荷爾蒙在生合成，分泌、傳輸、結合及生理功用，體內的代謝、排泄等方面的機制者稱之。因此本研究假設三亞蟎為一內分泌干擾物質，具調控睪固酮及雌素二醇在大鼠的代謝及血清中的含量。首先本研究進行睪固酮、雌素二醇及代謝產物在大鼠血清中，及微粒體分析方法的建立。分別以液相層析附串聯式質譜儀(liquid chromatography- tandem mass spectrometry, LC/MS)，對睪固酮代謝產物及睪固酮的鑑定與分析，以及以氣相層析附質譜(gas chromatography-mass spectrometry, GC/MS)及電子捕獲式檢出器(gas chromatography-electron capture detector, GC-ECD)，對雌素二醇代謝產物及雌素二醇在血清中的鑑定與分析。接著為瞭解三亞蟎對大鼠肝臟P450代謝酵素活性的影響，雄性及雌性大鼠以腹腔注射的方式，分別處理25及50 mg/kg三亞蟎藥劑持續五及七天。切除卵巢雌性大鼠，除處理相同劑量三亞蟎外，另以腹腔注射處理0.5 mg/kg雌素二醇。大鼠在經處理後，分別製備肝臟微粒體(microsome)，以進行實驗。在P450含量及erythromycin N-demethylase活性部分，雄性大鼠在以50 mg/kg處理後，活性分別增加39及68 %。而雌性大鼠則在處理50 mg/kg後，分別增加51及81%。切除卵巢雌性大鼠，在共同處理0.5 mg/kg雌素二醇及50 mg/kg三亞蟎後，活性分別增加178及71%。本研究再以相同劑量處理雄、雌性大鼠，觀察對phaseⅡ酵素活性及蛋白表現的影響。三亞蟎對葡萄糖醛酸基轉移酶(UDP-Glucuronosyltransferase)在雄及雌性大鼠均呈劑量關係，同時雄性大鼠分別增加39%及0.8倍，雌性大鼠則分別增加57及113%。麩胱甘肽轉移酶(glutathione-S-transferase)在雄性大鼠的活性，隨處理劑量的增加而增加，在以50 mg/kg處理時活性增加一倍，在雌性大鼠於25 mg/kg處理時降低0.8倍，但在50 mg/kg處理時則增加40%。對氮-乙醯基轉移酶(N-acetyltransferase)雄性大鼠活性在50 mg/kg處理後，顯著增加2.0倍，雌性大鼠則分別增加72及86%。對兒茶酚-O-甲基轉移酶( catechol O-methyltransferase)在雄及雌性大鼠均不會產生影響。對硫基轉移酶(sulfo-transferase)在雄性大鼠呈顯著增加的趨勢，在以25 mg/kg處理時，活性增加46%，以50 mg/kg處理後，活性則增加1.13倍。在雌性大鼠則有降低活性的趨勢，以25 mg/kg處理時，活性下降至0.73倍，而50 mg/kg處理後，活性降低0.3倍呈顯著性的降低。為證實三亞蟎對SULT活性影響的性別差異，本研究進行三亞蟎對SULT1E2及SULT2A1此兩種SULT isoform蛋白表現的測試。在以西方墨點分析SULT1E2的蛋白結果，以雄性大鼠的表現較雌性大鼠為顯著，但三亞蟎對此蛋白的影響，雄性大鼠則隨處理劑量的增加，而降低蛋白表現的趨勢，雌性大鼠則是隨處理劑量的增加，而增加蛋白的表現。至於在對SULT2A1的蛋白表現，以雌性大鼠的表現較雄性大鼠為顯著。但三亞蟎對此蛋白的影響，雄性大鼠隨處理劑量的增加，而增加蛋白表現，雌性大鼠則是隨處理劑量的增加，而呈現降低蛋白表現的趨勢。在睪固酮代謝部分，液相層析分析結果顯示，以50 mg/kg三亞蟎處理後，雄性大鼠肝臟微粒體代謝產生睪固酮6β-, 16α-以及2β-羥基代謝產物的生成，分別增加2.0，1.4及2.8倍。而在雌性大鼠則分別增加1.5，2.9以及3.0倍。同時三亞蟎亦增加雌性大鼠8.6倍在16β-羥基睪固酮，以及另一代謝產物androstenedione的代謝生成。16β-羥基睪固酮以及androstenedione的代謝生成，在雄性大鼠則顯示分別增加1.7及2.6倍。三亞蟎在對切除卵巢及外加雌素二醇後，則只對睪固酮6β-羥基代謝產物的生成，顯示1.5倍的增加，但對其他的羥基代謝產物則無影響。在雌素二醇代謝方面，以氣相層析分別結果顯示，三亞蟎對肝臟微粒體代謝雌素二醇，在雄性大鼠分別增加1.7及3.6倍2-羥基雌素二醇及estrone的產生，而對雌性大鼠則增加1.9及3.1倍，但三亞蟎對切除卵巢雌性大鼠的肝臟微粒體，代謝雌素二醇的能力則無影響。除了以肝臟微粒體，探討三亞蟎對睪固酮及雌素二醇代謝的能力外，本研究另進行以三亞蟎處理後，大鼠血清中睪固酮、雌素二醇以及代謝產物濃度的測定。雄性大鼠在經三亞蟎處理後，血清中睪固酮及代謝產物2β-羥基睪固酮的濃度，分別增加12及20倍。而切除卵巢雌性大鼠在外加雌素二醇且經三亞蟎處理後，顯示會降低6.7倍血清中雌素二醇的濃度，但代謝產物2-羥基雌素二醇在血清中的濃度則反而增加。由上述各實驗結果顯示，三亞蟎能誘導大鼠肝臟對睪固酮及雌素二醇代謝的能力，且能因此而影響血清中睪固酮及雌素二醇的濃度，研究結果呈現，三亞蟎藥劑具內分泌干擾作用。

關鍵字

三亞螨睪固酮；雌素二醇；代謝；內分泌干擾

並列摘要

The present study has investigated the ability of amitraz (N’-(2,4-dimethylphenyl)-N-[[2,4-dimethylphenyl]imino]methyl)-N-methanimidamide), a formamidine insecticide widely used to control eriophyid mites on pears and mange in animals, to modulate testosterone and 17β-estradiol (E2) metabolism and to alter the circulating levels of the hormones in rats. Endocrine-disrupting chemicals (EDCs) are exogenous agents that interfere with the production, release, transport, metabolism, binding, action, or elimination of the natural hormones in the body responsible for the maintenance of homeostasis and the regulation of developmental processes. The main hypothesis of this study was that amitraz is an EDC which has the ability to modulate testosterone and E2 metabolism and to alter the circulating levels of the hormones in rats. To test the hypothesis, the present study established analytical methods to detect and identify testosterone metabolites in rat serum and microsomes using high performance liquid chromatography-ultraviolet detector and tandem mass spectrometry and to detect E2 and metabolites using gas chromatography equipped with electron capture detector and mass spectrometry. To determine the effects of amitraz on liver cytochrome P450 content and erythromycin N-demethylase activity, amitraz was administered intraperitoneally at 25 and 50 mg/kg once daily for 5 and 7 days to male and female rats or to ovariectomized (OVX) female rats treated with 0.5 mg/kg E2. The results showed that in male rats, 50 mg/kg amitraz increased respective 39% and 68% of P450 and erythromycin N-demethylase activities. In female rats, 50 mg/kg of amitraz resulted in 51% and 81% increases of P450 content and erythromycin N-demethylase activity. In OVX female rats, cotreatment with E2 and 50 mg/kg of amitraz produced 178% and 71% increases of the aforementioned monooxygenase content or activity. The effects of amitraz on liver phase Ⅱ enzymes including uridine diphosphate-glucuronosyltransferase (UGT),glutathione S-transferase (GST), N-acetyltransferase (NAT), catechol O-methyltransferase (COMT), and sulfotransferase (SULT) activities were also studied. The results showed that in male rats, 25 mg/kg amitraz resulted in respective 39 and 46% increases of UGT and SULT activities. Amitraz at 50 mg/kg produced 0.8-, 1.0-, 2.0-, and 1.13-fold increases of UGT, GST, NAT and SULT enzymes activities, respectively. In female rats, 25 mg/kg amitraz produced 0.8- and 0.73-fold decreases of GST and SULT activities and 72% and 57% increases of NAT and UGT activities, respectively. Amitraz at 50 mg/kg produced 113%, 40%, and 86% increases of UGT, GST, and NAT activities, but decreased SULT by a 0.3-fold, respectively. The results of immunoblot analysis indicated that SULT 1E2 and 2A1 protein showed sex differences in their responses to amitraz treatment. Amitraz showed no effects on COMT activities of male and female rats. The results of testosterone metabolism studies of the liver of male and female rats showed that amitraz at the dose of 50 mg/kg produced respective 2.0-, 1.4-, and 2.8-fold increases of the ability of liver microsomes to metabolize testosterone to 6β-, 16α-, and 2β- hydroxylated products in male rats and 1.5-, 2.9-, and 3.0-fold increases in female rats. The insecticide resulted in an 8.6-fold increase of 16β-OH-testosterone formation in female rats and 2.6- and 1.7-fold increases of androstenedione formation in males and females, respectively. Amitraz increased testosterone 6β-hydroxylation by a 1.5-fold and had no effects on other metabolites formation in OVX and E2-treated female rats. The results of E2 metabolism on the liver of male and female rats showed that amitraz produced respective 1.7- and 3.6-fold increases of the capacity of liver microsomes to metabolize E2 to 2-OH-E2 and estrone in male rats and 1.9- and 3.1-fold increases in females. The formamidine did not have effects on E2 metabolism in OVX and E2-treated female rats. In male rat serum, amitraz increased testosterone and 2β-OH-testosterone concentrations by 12- and 20-fold, respectively. In OVX and E2-treated female rat serum, the insecticide produced a 6.7-fold decrease of E2 concentration and an increase of 2-OH-E2 concentration. The present findings show that amitraz induces testosterone and E2 metabolism in the liver and decreases the hormone concentrations in the serum of rats. These findings also demonstrate that amitraz is an endocrine disruptor.

並列關鍵字

amitraz ； testosterone ； estrogen ； metabolism ； endocrine disruption

參考文獻

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三亞螨藥劑對大鼠體內睪固酮及雌素二醇代謝之調控作用

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