Objective： To investigate whether CYP2E1 is responsible for the acrylamide metabolic activation in FIp-In CHO cell system. Methods： CYP2E1 cDNA was subcloned from the human liver full-length cDNA library and subsequ...Objective： To investigate whether CYP2E1 is responsible for the acrylamide metabolic activation in FIp-In CHO cell system. Methods： CYP2E1 cDNA was subcloned from the human liver full-length cDNA library and subsequently transfected into the FIp-In CHO cells to generate the stable transfectant of CYP2E1. The CYP2E1 mRNA expression was determined by RT-PCR. Acrylamide and its epoxide glycidamide induced cytotoxicity and cell cycle arrest in G2/M were conducted using MTS assay and flow cytometry, respectively. Results： In the CHO cell stably expressing CYP2E1 （CHO-2E1）, a -1.5 kb size of band was detected from the mRNA in the cells while no corresponding band in the CHO-vector cells, which indicated that CYP2E1 was successfully transfected in the CHO cells. Compared with the CHO-vector cells, acrylamide showed a concentration dependent loss of viability in the CHO-2E1 cells but no significant change of G2/M arrest was found. As expected, glycidamide induced similar profile of cytotoxicity in both of the cells, and G2/M arrest presented a concentration-dependent increased in the CHO-2E1 cells. Conclusion： The result suggested that CYP2E1 might be responsible for the acrylamide metabolism, and its metabolite glycidamide was a direct cytotoxic and genotoxic agent. It should be further considered whether acrylamide-induced toxicity is through its epoxide glycidamide in the presence of CYP2E1.展开更多
Cytochrome P450（CYP） 2El is a dual function monoxygenase with a crucial role in the metabolism of 6% of drugs on the market at present. The enzyme is of tremendous interest for its association with alcohol consumpti...Cytochrome P450（CYP） 2El is a dual function monoxygenase with a crucial role in the metabolism of 6% of drugs on the market at present. The enzyme is of tremendous interest for its association with alcohol consumption, diabetes, obesity and fasting. Despite the abundant experimental mutagenesis data, the molecular origin and the structural motifs for the enzymatic activity deficiencies have not been rationalized at the atomic level. In this regard, we have investigated the effects of mutation on the structural and energetic characteristics upon single point mutations in CYP2E1, N219D and $366C. The molecular dynamics（MD） simulation combined with quantum mechanics/molecular mechanics（QM/MM） and noncovalent interaction（NCI） analysis was carried out on CYP2EI and its two mutants. The results highlight the critical role of Phe207, which is responsible for both structural flexibility and energetic variation, shortening the gap between the theory and the experimentally observed results of enzymatic activity decrease, The underlying molecular mechanism of the enzymatic activity deficiencies for mutants may be attributed to the changes of spatial position of Phe207 in the two mutants. This work provides particular explanations to how mutations affect ligand-receptor interactions based on combined MD and QM/MM calculations. Furthermore, the mutational effects on the activity of CYP2E1 obtained in the present study are beneficial to both the experimental and the computational works of CYPs and may allow researchers to achieve desirable changes in enzymatic activity.展开更多
文摘Objective： To investigate whether CYP2E1 is responsible for the acrylamide metabolic activation in FIp-In CHO cell system. Methods： CYP2E1 cDNA was subcloned from the human liver full-length cDNA library and subsequently transfected into the FIp-In CHO cells to generate the stable transfectant of CYP2E1. The CYP2E1 mRNA expression was determined by RT-PCR. Acrylamide and its epoxide glycidamide induced cytotoxicity and cell cycle arrest in G2/M were conducted using MTS assay and flow cytometry, respectively. Results： In the CHO cell stably expressing CYP2E1 （CHO-2E1）, a -1.5 kb size of band was detected from the mRNA in the cells while no corresponding band in the CHO-vector cells, which indicated that CYP2E1 was successfully transfected in the CHO cells. Compared with the CHO-vector cells, acrylamide showed a concentration dependent loss of viability in the CHO-2E1 cells but no significant change of G2/M arrest was found. As expected, glycidamide induced similar profile of cytotoxicity in both of the cells, and G2/M arrest presented a concentration-dependent increased in the CHO-2E1 cells. Conclusion： The result suggested that CYP2E1 might be responsible for the acrylamide metabolism, and its metabolite glycidamide was a direct cytotoxic and genotoxic agent. It should be further considered whether acrylamide-induced toxicity is through its epoxide glycidamide in the presence of CYP2E1.
基金Supported by the National Natural Science Foundation of China（No.21273095）.
文摘Cytochrome P450（CYP） 2El is a dual function monoxygenase with a crucial role in the metabolism of 6% of drugs on the market at present. The enzyme is of tremendous interest for its association with alcohol consumption, diabetes, obesity and fasting. Despite the abundant experimental mutagenesis data, the molecular origin and the structural motifs for the enzymatic activity deficiencies have not been rationalized at the atomic level. In this regard, we have investigated the effects of mutation on the structural and energetic characteristics upon single point mutations in CYP2E1, N219D and $366C. The molecular dynamics（MD） simulation combined with quantum mechanics/molecular mechanics（QM/MM） and noncovalent interaction（NCI） analysis was carried out on CYP2EI and its two mutants. The results highlight the critical role of Phe207, which is responsible for both structural flexibility and energetic variation, shortening the gap between the theory and the experimentally observed results of enzymatic activity decrease, The underlying molecular mechanism of the enzymatic activity deficiencies for mutants may be attributed to the changes of spatial position of Phe207 in the two mutants. This work provides particular explanations to how mutations affect ligand-receptor interactions based on combined MD and QM/MM calculations. Furthermore, the mutational effects on the activity of CYP2E1 obtained in the present study are beneficial to both the experimental and the computational works of CYPs and may allow researchers to achieve desirable changes in enzymatic activity.
