Identification of the proteins related to cytochrome P450 induced by fenvalerate in a Trichoplusia ni cell line.
In order to reveal the metabolic reaction to the presence of fenvalerate mediated by P450 in insects, we used the trypan blue exclusion technique and 3-(4,5-dimethylthiazol)-2,5-diphenyltrazolium bromide (MTT) reduction assay to assess the vitality of Trichoplusia ni (Tn) cells treated with fenvalerate, and observed dose- and time-dependent changes in total cellular P450s. In addition, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to identify the proteins involved in the fenvalerate reaction process. Finally, the cDNA of P450 fragments was cloned and real-time RT-PCR was performed. Our data showed that at the 0-15 mumol/L challenge concentration of fenvalerate, at which the vitality of Tn cells was not affected (p > 0.05), there was a tendency toward a dose- and time-response of total cellular P450s, which peaked at the 9 h (p < 0.05) and 12 h (p < 0.01) time points following 12.5 mumol/L stimulation with fenvalerate. The 2-DE assay detected more than 1300 protein spots in each two-dimensional gel, of which 33 spots displayed significant differences. Among the changed spots, three isoforms of P450 were identified. One of the three P450 cDNA fragments (CYP4L4) was cloned and sequenced, and its expression in treated Tn cells increased significantly (p < 0.01). It was found that fenvalerate induced the expression of P450s in insect cells. This suggests that fenvalerate could be metabolized by CYP4L4 through a hydroxylation reaction in insect cells.
Fang
Xin-Kui
XK
School of Agriculture and Biology, Shanghai Jiaotong University, 800 Dongchuan Road, 200240 Shanghai, PR China.
Huang
Dan-Feng
DF
Wang
Zhao-Xia
ZX
Wan
Chun-Ling
CL
Sun
Tao
T
Xu
Wang-Jie
WJ
Liu
Chun-Yan
CY
Zhou
Pei
P
Qiao
Zhong-Dong
ZD
D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004790:Enzyme Induction / Q000187:drug effects; D019476:Insect Proteins / Q000032:analysis* / Q000737:chemistry; D009036:Moths / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D009570:Nitriles / Q000494:pharmacology*; D011722:Pyrethrins / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D019476:Insect Proteins; D009570:Nitriles; D011722:Pyrethrins; C460222:cytochrome P-450 CYP4L4 (cabbage armyworm); D003577:Cytochrome P-450 Enzyme System; C017690:fenvalerate