USE OF 7-ALKOXYPHENOXAZONES, 7-ALKOXYCOUMARINS AND 7-ALKOXYQUINOLINES AS FLUORESCENT SUBSTRATES FOR RAINBOW-TROUT HEPATIC MICROSOMES AFTER TREATMENT WITH VARIOUS INDUCERS
USE OF 7-ALKOXYPHENOXAZONES, 7-ALKOXYCOUMARINS AND 7-ALKOXYQUINOLINES AS FLUORESCENT SUBSTRATES FOR RAINBOW-TROUT HEPATIC MICROSOMES AFTER TREATMENT WITH VARIOUS INDUCERS
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DOI：
10.1016/0006-2952(94)90490-1
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发表时间：
1994-03-02
影响因子：
5.8
通讯作者：
LECH, JJ
中科院分区：
文献类型：
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作者：
HAASCH, ML;GRAF, WK;LECH, JJ
在鱼类和哺乳动物中，不同的荧光底物被用作不同细胞色素P450同工酶的诱导或活性的特异性指示物。为了确定更多用于鱼类的确定的荧光底物，我们研究了一系列7-烷氧基苯恶酮、7-烷氧基香豆素和7-烷氧基喹啉作为底物，用虹鱼(Oncorhynchus MykISS)的肝微粒体进行O-脱烷基测定。用β-萘黄酮(β-NF)、孕烯醇酮-16α-碳腈(PCN)、苯巴比妥(PB)、异黄樟素(ISF)或地塞米松(DEX)处理48h后制备微粒体。获得了总的P450光谱，并进行了光谱结合研究。7-甲氧基、7-乙氧基、7-丙氧基和7-苯氧基苯恶宗经ISF处理后，微粒体O-脱烷基速率高于β-NF处理后，而7-丁氧基苯恶宗则不能。地塞米松处理导致戊氧基苯恶宗代谢显著增加(约144倍)，与β-核因子(11倍)和ISF(37倍)诱导的微生物体相比。经ISF处理的微粒子对烷氧基苯恶唑酮的脱烷基速率依次为：甲氧基和GT；乙氧基和GT；丙氧基和GT；苯甲氧基和GT；丁氧基和GT；己氧基。7-烷氧基香豆素类化合物的代谢顺序为：甲氧基-GT；乙氧基-GT；丙氧基-GT；丁氧基-GT；苄氧基；己氧基；7-烷氧基香豆素的代谢顺序为：乙氧基远大于丁氧基>；丙氧基-甲氧基>；其他处理均未显著提高任何烷氧基香豆素的代谢率。使用β-核因子治疗并不能显著提高任何烷氧基喹啉的代谢率。地塞米松处理显著提高了苄氧基、乙氧基和丁氧基的代谢率，依次接近己氧基和丙氧基喹啉。ISF处理显著提高了苯氧基喹啉、甲氧基喹啉和丁氧基喹啉的代谢速率。这些结果表明，除了常用的乙氧基苯恶酮和乙氧基香豆素外，其中一些新的荧光底物还可以用来表征ISF和DEX对虹鱼肝微粒体单加氧酶活性的诱导，以表征β-NF或其他3-甲基胆蒽类P450诱导剂的诱导作用。用7-甲氧基香豆素作为底物来区分虹鱼肝微粒体中ISF型和β-NF型诱导剂可能是最好的。使用7-甲氧基苯恶宗作为底物，可以最好地区分ISF型和DEX型诱导剂以及β-NF型和DEX型诱导剂。在β-NF诱导下，7-甲氧基香豆素、ISF诱导下的7-甲氧基苯恶宗和DEX诱导下的7-乙氧基喹啉的代谢最大。
Various fluorescent substrates have been used as specific indicators of induction or activity of different cytochrome P450 isozymes in both fish and mammalian species. In an attempt to identify additional definitive fluorescent substrates for use in fish, we examined a series of 7-alkoxyphenoxazones, 7-alkoxycoumarins and 7-alkoxyquinolines as substrates in O-dealkylation assays with hepatic microsomes from rainbow trout (Oncorhynchus mykiss). Microsomes were prepared after 48 hr of treatment with beta-naphthoflavone (beta-NF), pregnenolone-16 alpha-carbonitrile (PCN), phenobarbital (PB), isosafrole (ISF), or dexamethasone (DEX). Total P450 spectra were obtained, and spectral binding studies were performed. Microsomal O-dealkylation rates were greater after ISF treatment than after beta-NF treatment for 7-methoxy-, 7-ethoxy-, 7-propoxy- and 7-benzyloxyphenoxazones but not for 7-butoxyphenoxazone. DEX treatment resulted in a significant elevation of pentoxyphenoxazone metabolism (about a 144-fold increase) compared with microsomes induced by beta-NF (11-fold) and ISF (37-fold). The rates of dealkylation of the alkoxyphenoxazones by ISF-treated microsomes occurred in the following order: methoxy > ethoxy > propoxy > benzxyloxy > butoxy > pentoxy. When beta-NF-treated microsomes were used, the 7-alkoxyphenoxazones were metabolized as follows: methoxy > ethoxy > propoxy > butoxy > benzyloxy approximate to pentoxy, while the order of metabolism of the 7-alkoxycoumarins was: ethoxy much greater than butoxy > propoxy approximate to methoxy > benzyloxy > pentoxy. None of the other treatments significantly increased the rate of metabolism of any of the alkoxycoumarins. Treatment with beta-NF did not significantly elevate the rate of metabolism of any of the alkoxyquinolines. DEX treatment produced significant elevations in the rate of metabolism of benzyloxy-, ethoxy-, and butoxy- approximate to pentoxy- approximate to propoxyquinoline, in that order. ISF treatment significantly elevated the rate of metabolism of benzyloxy-, methoxy- and butoxyquinoline, in that order. These results suggest that some of these new fluorescent substrates can be used to characterize induction of rainbow trout hepatic microsomal monooxygenase activity by ISF and DEX, in addition to the commonly used ethoxyphenoxazone and ethoxycoumarin for the characterization of induction by beta-NF or other 3-methylcholanthrene-type P450 inducers. Distinction between ISF-type and beta-NF-type inducers in rainbow trout hepatic microsomes may best be made using 7-methoxycoumarin as a substrate. Distinction between ISF-type and DEX-type inducers and between beta-NF-type and DEX-type inducers may best be made using 7-methoxyphenoxazone as a substrate. With beta-NF induction 7-methoxycoumarin, with ISF induction 7-methoxy- phenoxazone, and with DEX induction 7-ethoxyquinoline were metabolized to the greatest extent compared with controls and all other substrates tested.