Kaphalia Bhupendra S, Mericle Kelly A, Ansari G A S
Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609, USA.
Toxicol Appl Pharmacol. 2004 Oct 1;200(1):7-15. doi: 10.1016/j.taap.2004.03.018.
Earlier, we have shown that rat hepatic and pancreatic fatty acid ethyl ester (FAEE) synthases are structurally and functionally similar to rat liver carboxylesterase (CE) and pancreatic cholesterol esterase (ChE), respectively. We have also reported that only hepatic FAEE synthase is inhibited by tri-o-tolylphosphate (TOTP) in vivo and in human hepatocellular carcinoma (HepG2) cells. The metabolism of TOTP is a prerequisite for the inhibition of hepatic FAEE synthase as well as esterase activity. To further elucidate the mechanism of such differential inhibition by inhibitors of serine esterases, we synthesized two metabolites of TOTP, 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphoran-2-one (CBDP; cyclic saligenin phosphate) and di-o-tolyl-o-( proportional, variant -hydroxy)tolylphosphate (HO-TOTP), and one ChE inhibitor, 3-benzyl-6-chloro-2-pyrone (3-BCP). The inhibitory effect of CBDP, HO-TOTP, and 3-BCP on FAEE synthase and esterase activity was studied using rat hepatic and pancreatic postnuclear (PN) fractions, commercial porcine hepatic CE and pancreatic ChE, and in HepG2 and rat pancreatic tumor (AR42J) cell lines. Only HO-TOTP and CBDP inhibited FAEE synthase as well as esterase activity of hepatic PN fraction and commercial CE and ChE in a concentration-dependent manner, and the inhibition was found to be irreversible. However, no inhibition was found in pancreatic PN fraction by both TOTP metabolites and 3-BCP. Although 3-BCP inhibited only the esterase activity of commercial ChE in a concentration-dependent manner, the activity was reversible within 30 min of incubation. Studies with HepG2 cells also showed a significant inhibition of FAEE synthase-esterase activity by CBDP and HO-TOTP within 15 min of incubation, while no inhibition was observed in AR42J cells. 3-BCP did not inhibit FAEE synthase-esterase activity either in HepG2 or AR42J cells. Such differential inhibitory effect of the TOTP metabolites on hepatic and pancreatic FAEE synthase-esterase is supported by our earlier in vivo and in vitro studies. Further investigations are needed to understand the biochemical mechanism(s) of inactivation of TOTP metabolites and 3-BCP in the pancreas and AR42J cells towards FAEE synthase-esterase activities.
此前，我们已经表明，大鼠肝脏和胰腺脂肪酸乙酯（FAEE）合酶在结构和功能上分别与大鼠肝脏羧酸酯酶（CE）和胰腺胆固醇酯酶（ChE）相似。我们还报道，在体内以及人肝癌（HepG2）细胞中，只有肝脏FAEE合酶会被磷酸三邻甲苯酯（TOTP）抑制。TOTP的代谢是抑制肝脏FAEE合酶以及酯酶活性的前提条件。为了进一步阐明丝氨酸酯酶抑制剂产生这种差异抑制作用的机制，我们合成了TOTP的两种代谢产物，2-（邻甲苯基）-4H-1:3:2-苯并二氧磷杂环己二烯-2-酮（CBDP；环亚水杨基磷酸酯）和二邻甲苯基-邻-（比例可变的、羟基）甲苯基磷酸酯（HO-TOTP），以及一种ChE抑制剂3-苄基-6-氯-2-吡喃酮（3-BCP）。使用大鼠肝脏和胰腺核后（PN）组分、市售猪肝脏CE和胰腺ChE以及HepG2和大鼠胰腺肿瘤（AR42J）细胞系，研究了CBDP、HO-TOTP和3-BCP对FAEE合酶和酯酶活性的抑制作用。只有HO-TOTP和CBDP以浓度依赖性方式抑制肝脏PN组分以及市售CE和ChE的FAEE合酶和酯酶活性，并且发现这种抑制作用是不可逆的。然而，两种TOTP代谢产物和3-BCP对胰腺PN组分均未产生抑制作用。尽管3-BCP仅以浓度依赖性方式抑制市售ChE的酯酶活性，但在孵育30分钟内该活性是可逆的。对HepG2细胞的研究还表明，在孵育15分钟内，CBDP和HO-TOTP对FAEE合酶-酯酶活性有显著抑制作用，而在AR42J细胞中未观察到抑制作用。3-BCP在HepG2细胞或AR42J细胞中均未抑制FAEE合酶-酯酶活性。TOTP代谢产物对肝脏和胰腺FAEE合酶-酯酶的这种差异抑制作用得到了我们早期体内和体外研究的支持。需要进一步研究以了解TOTP代谢产物和3-BCP在胰腺和AR42J细胞中对FAEE合酶-酯酶活性失活的生化机制。
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