To reveal putative bioactivation pathways of diclofenac, in vitro human liver materials such as microsomal fractions and hepatocytes were used to confirm metabolic activation of diclofenac by ³⁵S-cysteine trapping assay and covalent binding assay. Candidate human liver proteins possibly targeted by ¹⁴C-diclofenac via bioactivation were investigated using two-dimensional gel electrophoresis followed by detection of remaining radioactivity on the modified proteins with bio-imaging analyzer.

In the ³⁵S-cysteine trapping assay, three and two adducts with ³⁵S-cysteine were observed in NADPH-fortified and UDPGA-fortified human liver microsomes, respectively. In the covalent binding assay using ¹⁴C-diclofenac in human hepatocytes, the extent of covalent binding of diclofenac to human hepatic proteins increased time-dependently. Addition of glutathione attenuated the extent of covalent binding of ¹⁴C-diclofenac to human liver microsomal proteins.

Fifty-nine proteins from human hepatocytes were proposed as the candidate proteins targeted by reactive metabolites of diclofenac. Proteins modified by cytochrome P450-mediated reactive metabolites were identified by using a cytochrome P450 inhibitor, 1-aminobenzyltriazole and seven of the nine radioactive protein spots were removed by 1-aminobenzyltriazole treatment.

In contrast, the remaining two radioactive protein spots, mainly containing human serum albumin and heat shock proteins, were not affected by the addition of 1-aminobenzyltriazole, which suggested the involvement of the acyl glucuronide of diclofenac, formed via uridine diphosphate-glucuronosyl transferases, in the covalent modifications induced by diclofenac.

为了揭示双氯芬酸的假定生物活化途径，通过³⁵ S-半胱氨酸捕获测定法和共价结合测定法，使用体外人肝材料（如微粒体级分和肝细胞）来确认双氯芬酸的代谢活化。使用二维凝胶电泳研究了可能被¹⁴ C-双氯芬酸通过生物激活作用靶向的人肝蛋白质候选物，然后使用生物成像分析仪检测了修饰蛋白质上的剩余放射性。

在³⁵ S-半胱氨酸捕获分析中，分别在NADPH强化和UDPGA强化的人肝微粒体中观察到3个和³⁵ S-半胱氨酸的加合物。在人肝细胞中使用¹⁴ C-双氯芬酸的共价结合测定中，双氯芬酸与人肝蛋白的共价结合程度随时间增加。谷胱甘肽的添加减弱了¹⁴ C-双氯芬酸与人肝微粒体蛋白的共价结合程度。

有人提议将人肝细胞中的59种蛋白质作为双氯芬酸反应性代谢产物靶向的候选蛋白质。通过使用细胞色素P450抑制剂鉴定出被细胞色素P450介导的反应性代谢产物修饰的蛋白质，使用1-氨基苄基三唑，并通过1-氨基苄基三唑处理去除了九个放射性蛋白质斑点中的七个。

相反，剩余的两个放射性蛋白斑点（主要包含人血清白蛋白和热休克蛋白）不受1-氨基苄基三唑添加的影响，这表明双氯芬酸酰基葡糖醛酸苷是通过尿苷二磷酸-葡糖醛酸糖基转移酶形成的，双氯芬酸诱导的共价修饰。