Xenobiotica ( IF 1.3 ) Pub Date : 2021-09-08 , DOI: 10.1080/00498254.2021.1975060 Minghan Yong 1 , Kathryn Elisa Burns 1 , Janak de Zoysa 2, 3 , Nuala Ann Helsby 1
Abstract
1.The conversion of the cyclophosphamide intermediate metabolite 4-hydroxycyclophosphamide (4-OHCP) to the final cytotoxic metabolite phosphoramide mustard (PAM) is classically assumed to occur via chemical hydrolysis of the phospho-ester bond. Whilst it has been suggested previously that this reaction could be enzyme-catalysed, there was only indirect evidence for this (i.e. formation of the by-product acrolein).
2. Using an assay to detect formation of DNA-alkylating adducts which block PCR amplification (QPCR-block assay), we have demonstrated that 4-OHCP can be activated by peripheral blood mononuclear cells (PBMC). The DNA-alkylating potency of 4-OHCP in PBMC increased >18-fold compared to the intrinsic reactivity of 4-OHCP for purified gDNA.
3. We also found that immortalised T-cells (Jurkat) had a similar ability to activate 4-OHCP into a DNA alkylating agent, whereas there was no appreciable activation in epithelial derived (Caco-2) cells. This suggests the possibility of tissue-specific enzyme expression.
4. Of the candidate enzymes tested only recombinant human cAMP-phosphodiesterase-PDE4B and snake-venom phosphodiesterase (PDE-I) could catalyse this activation into a DNA-alkylating agent.
5. This enzymatic catalysis of the phospho-ester bond (P-O-C) is a hitherto unrecognised feature of this important immunomodulatory drug and should be investigated further.
中文翻译:
人白细胞中 4-羟基环磷酰胺在细胞内活化成 DNA 烷化剂
摘要
1. 环磷酰胺中间代谢物 4-羟基环磷酰胺 (4-OHCP) 向最终细胞毒性代谢物磷酰胺芥 (PAM) 的转化被认为是通过磷酸酯键的化学水解发生的。虽然之前已经提出该反应可以是酶催化的,但仅存在间接证据(即副产物丙烯醛的形成)。
2. 使用一种检测方法来检测阻止 PCR 扩增的 DNA 烷基化加合物的形成(QPCR 阻断检测),我们已经证明 4-OHCP 可以被外周血单核细胞 (PBMC) 激活。与 4-OHCP 对纯化 gDNA 的内在反应性相比,PBMC 中 4-OHCP 的 DNA 烷基化效力增加了 > 18 倍。
3. 我们还发现永生化 T 细胞 (Jurkat) 具有将 4-OHCP 激活为 DNA 烷化剂的类似能力,而在上皮衍生细胞 (Caco-2) 中没有明显的激活。这表明组织特异性酶表达的可能性。
4. 在测试的候选酶中,只有重组人 cAMP-磷酸二酯酶-PDE4B 和蛇毒磷酸二酯酶 (PDE-I) 可以将这种活化催化为 DNA 烷化剂。
5. 磷酸酯键 (POC) 的这种酶促催化作用是这种重要的免疫调节药物迄今为止未被认识的特征,应进一步研究。