James Margaret O, Stuchal Leah D, Nyagode Beatrice A
Department of Medicinal Chemistry, University of Florida, Gainesville, FL 32610-0485, United States.
Aquat Toxicol. 2008 Jan 31;86(2):227-38. doi: 10.1016/j.aquatox.2007.11.003. Epub 2007 Nov 9.
The organochlorine pesticide, methoxychlor (MXC), is metabolized in animals to phenolic mono- and bis-demethylated metabolites (OH-MXC and HPTE, respectively) that interact with estrogen receptors and may be endocrine disruptors. The phase II detoxication of these compounds will influence the duration of action of the estrogenic metabolites, but has not been investigated extensively. In this study, the glucuronidation and sulfonation of OH-MXC and HPTE were investigated in subcellular fractions of liver and intestine from untreated, MXC-treated and 3-methylcholanthrene (3-MC)-treated channel catfish, Ictalurus punctatus. MXC-treated fish were given i.p. injections of 2mg MXC/kg daily for 6 days and sacrificed 24h after the last dose. The 3-MC treatment was a single 10mg/kg i.p. dose 5 days prior to sacrifice. In hepatic microsomes from control fish, the V(max) value (mean+/-S.D., n=4) for glucuronidation of OH-MXC was 270+/-50pmol/min/mg protein, higher than found for HPTE (110+/-20pmol/min/mg protein). For each substrate, the V(max) values observed in intestinal microsomes were approximately twice those found in the liver. The K(m) values for OH-MXC and HPTE glucuronidation in control liver were not significantly different and were 0.32+/-0.04mM for OH-MXC and 0.26+/-0.06mM for HPTE. The K(m) for the co-substrate, UDPGA, was higher in liver (0.28+/-0.09mM) than intestine (0.04+/-0.02mM). Treatment with 3-MC but not MXC increased the V(max) for glucuronidation in liver and intestine. Glucuronidation was a more efficient pathway than sulfonation for both substrates, in both tissues. The V(max) values for sulfonation of OH-MXC and HPTE, respectively, in liver cytosol were 7+/-3 and 17+/-4pmol/min/mg protein and in intestinal cytosol were 13+/-3 and 30+/-5pmol/min/mg protein. Treatment with 3-MC but not MXC increased rates of sulfonation of OH-MXC and HPTE and the model substrate, 3-hydroxy-benzo(a)pyrene in both intestine and liver. Comparison of the kinetics of the conjugation pathways with those published for the demethylation of MXC showed that formation of the endocrine-active metabolites was more efficient than either conjugation pathway. Residues of OH-MXC and HPTE were detected in extracts of liver microsomes from MXC-treated fish. This work showed that although OH-MXC and HPTE could be eliminated by glucuronidation and sulfonation, the phase II pathways were less efficient than the phase I pathway leading to formation of these endocrine-active metabolites.
有机氯农药甲氧滴滴涕（MXC）在动物体内代谢为酚类单脱甲基和双脱甲基代谢物（分别为OH-MXC和HPTE），它们与雌激素受体相互作用，可能是内分泌干扰物。这些化合物的II相解毒作用会影响雌激素代谢物的作用持续时间，但尚未得到广泛研究。在本研究中，对未处理、经MXC处理和经3-甲基胆蒽（3-MC）处理的斑点叉尾鮰肝脏和肠道亚细胞组分中OH-MXC和HPTE的葡萄糖醛酸化和磺化作用进行了研究。经MXC处理的鱼每天腹腔注射2mg MXC/kg，持续6天，并在最后一剂后24小时处死。3-MC处理是在处死前5天单次腹腔注射10mg/kg剂量。在对照鱼的肝微粒体中，OH-MXC葡萄糖醛酸化的V(max)值（平均值±标准差，n = 4）为270±50pmol/min/mg蛋白质，高于HPTE（110±20pmol/min/mg蛋白质）。对于每种底物，在肠道微粒体中观察到的V(max)值约为肝脏中的两倍。对照肝脏中OH-MXC和HPTE葡萄糖醛酸化的K(m)值无显著差异，OH-MXC为0.32±0.04mM，HPTE为0.26±0.06mM。共底物UDPGA的K(m)值在肝脏中（0.28±0.09mM）高于肠道中（0.04±0.02mM）。用3-MC而非MXC处理可增加肝脏和肠道中葡萄糖醛酸化的V(max)。对于两种组织中的两种底物，葡萄糖醛酸化都是比磺化更有效的途径。OH-MXC和HPTE在肝脏胞质溶胶中的磺化V(max)值分别为7±3和17±4pmol/min/mg蛋白质，在肠道胞质溶胶中分别为13±3和30±5pmol/min/mg蛋白质。用3-MC而非MXC处理可增加OH-MXC、HPTE以及模型底物3-羟基苯并(a)芘在肠道和肝脏中的磺化速率。将结合途径的动力学与已发表的MXC脱甲基化动力学进行比较表明，内分泌活性代谢物的形成比任何一种结合途径都更有效。在经MXC处理的鱼的肝微粒体提取物中检测到了OH-MXC和HPTE的残留量。这项工作表明，尽管OH-MXC和HPTE可通过葡萄糖醛酸化和磺化作用消除，但II相途径比导致这些内分泌活性代谢物形成的I相途径效率更低。
