Jones T W, Gerdes R G, Ormstad K, Orrenius S
Chem Biol Interact. 1985 Dec 31;56(2-3):251-67. doi: 10.1016/0009-2797(85)90010-9.
The nephrotoxicity of hexachloro-1,3-butadiene (HCBD) appears to depend on the initial formation of a glutathione (GSH) conjugate in the liver. In the present study we have examined the hepatic metabolism of HCBD using isolated hepatocytes and following in vivo administration. Exposure of isolated hepatocytes to HCBD resulted in a dose-dependent depletion of GSH. HPLC analysis of the incubation medium demonstrated the formation of two products. When isolated hepatocytes containing [3H]GSH were exposed to [14C]HCBD, coincident elution of 3H and 14C corresponding to the previously recognized HPLC peaks was observed. Both products were sensitive to treatment with gamma-glutamyl transpeptidase (gamma-GT), providing additional support for their identification as GSH conjugates. The ratio of 3H to 14C in the two peaks indicated the formation of both a mono- and a bis-substituted GSH conjugate of HCBD. The identification of the mono- and bis-GSH conjugates was further confirmed by the preparation of synthetic standards which displayed retention times by HPLC identical to the biological products. The production of the total and individual GSH conjugates displayed both dose and time dependence. The production of the total as well as the ratio of mono- to bis-conjugate was found to depend on the availability of GSH. At low HCBD exposure levels the bis-substituted conjugate accounted for more than 20% of the total conjugate produced by isolated hepatocytes. This value decreased at higher HCBD concentrations. Analysis of bile collected from rats following intraportal administration of [14C]HCBD revealed the presence of both the mono- and bis-substituted GSH conjugates of HCBD as well as additional 14C-containing metabolites. The results of the present study clearly demonstrate the production of both a mono- and a bis-substituted GSH conjugate of HCBD. The potential importance of this finding in terms of the nephrotoxicity of HCBD is discussed.
六氯-1,3-丁二烯（HCBD）的肾毒性似乎取决于其在肝脏中最初形成谷胱甘肽（GSH）结合物。在本研究中，我们使用分离的肝细胞并在体内给药后检测了HCBD的肝脏代谢情况。将分离的肝细胞暴露于HCBD会导致GSH呈剂量依赖性消耗。对孵育培养基进行HPLC分析表明形成了两种产物。当将含有[³H]GSH的分离肝细胞暴露于[¹⁴C]HCBD时，观察到与先前识别的HPLC峰相对应的³H和¹⁴C的同时洗脱。两种产物对γ-谷氨酰转肽酶（γ-GT）处理敏感，这为它们被鉴定为GSH结合物提供了额外支持。两个峰中³H与¹⁴C的比例表明形成了HCBD的单取代和双取代GSH结合物。通过制备合成标准品进一步证实了单取代和双取代GSH结合物的鉴定，这些标准品通过HPLC显示出与生物产物相同的保留时间。总GSH结合物和单个GSH结合物的产生均显示出剂量和时间依赖性。发现总结合物的产生以及单结合物与双结合物的比例取决于GSH的可用性。在低HCBD暴露水平下，双取代结合物占分离肝细胞产生的总结合物的20%以上。在较高的HCBD浓度下，该值会降低。对经门静脉注射[¹⁴C]HCBD后的大鼠胆汁进行分析，发现存在HCBD的单取代和双取代GSH结合物以及其他含¹⁴C的代谢产物。本研究结果清楚地证明了HCBD单取代和双取代GSH结合物的产生。讨论了这一发现对于HCBD肾毒性的潜在重要性。
