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首页> 外文期刊>Cellular Physiology and Biochemistry >Comparative Evaluation of Different Cell Lysis and Extraction Methods for Studying Benzo(a)pyrene Metabolism in HT-29 Colon Cancer Cell Cultures
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Comparative Evaluation of Different Cell Lysis and Extraction Methods for Studying Benzo(a)pyrene Metabolism in HT-29 Colon Cancer Cell Cultures

机译：HT-29结肠癌细胞培养物中苯并（a）py代谢的不同细胞裂解和提取方法的比较评价

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摘要

Lysis and extraction of cells are essential sample processing steps for investigations pertaining to metabolism of xenobiotics in cell culture studies. Of particular importance to these procedures are maintaining high lysis efficiency and analyte integrity as they influence the qualitative and quantitative distribution of drug and toxicant metabolites in the intra- and extracellular milieus. In this study we have compared the efficiency of different procedures viz. homogenization, sonication, bead beating, and molecular grinding resin treatment for disruption of HT-29 colon cells exposed to benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) compound and a suspected colon carcinogen. Also, we have evaluated the efficiency of various procedures for extracting BaP parent compound/metabolites from colon cells and culture media prior to High Performance Liquid Chromatography (HPLC) analyses. The extraction procedures include solid phase extraction, solid-supported liquid- liquid extraction, liquid-liquid extraction, and homogeneous liquid- liquid extraction. Our findings showed that bead-beating in combination with detergent treatment of cell pellet coupled with liquid-liquid extraction yielded greater concentrations of BaP metabolites compared to the other methods employed. Our method optimization strategy revealed that disruption of HT-29 colon cells by a combination of mechanical and chemical lysis followed by liquid-liquid extraction is efficient and robust enough for analyzing BaP metabolites from cell culture studies.
机译：细胞裂解和提取是细胞培养研究中有关异源生物代谢研究的基本样品处理步骤。这些程序特别重要的是保持高裂解效率和分析物完整性，因为它们会影响细胞内和细胞外环境中药物和有毒代谢产物的定性和定量分布。在这项研究中，我们比较了不同程序的效率。均质化，超声处理，微珠打浆和分子研磨树脂处理，以破坏暴露于苯并（a）re（BaP），多环芳烃（PAH）化合物和怀疑的结肠致癌物的HT-29结肠细胞。同样，我们评估了在高效液相色谱（HPLC）分析之前从结肠细胞和培养基中提取BaP母体化合物/代谢物的各种程序的效率。萃取程序包括固相萃取，固相支持的液-液萃取，液-液萃取和均相液-液萃取。我们的研究结果表明，与其他采用的方法相比，将珠子打浆与细胞沉淀的去污剂处理以及液-液萃取相结合可产生更高浓度的BaP代谢产物。我们的方法优化策略表明，通过机械和化学裂解以及液-液萃取相结合的方法破坏HT-29结肠细胞对于分析细胞培养研究中的BaP代谢物是足够有效和强大的。

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