An in vitro bioassay for xenobiotics using the SXR-driven human CYP3A4/lacZ reporter gene.
The dose and time effect of nine xenobiotics, including 17beta-estradiol, corticosterone, dexamethasone, progesterone, nifedipine, bisphenol A, rifampicin, methamphetamine, and nicotine were investigated, in vitro, using human steroid and xenobiotics receptor (SXR)-binding sites on the human CYP3A4 promoter, which can enhance the linked lacZ reporter gene transcription. To test this, liver-specific SAP (human serum amyloid P component)-SXR (SAP/SXR) and human CYP3A4 promoter-regulated lacZ (hCYP3A4/lacZ) constructs were transiently transfected into HepG2 and NIH3T3 cells to compare the xenobiotic responsiveness between human and nonhuman cell lines. In the HepG2 cells, rifampicin, followed by corticosterone, nicotine, methamphetamine, and dexamethasone, exhibited enhanced levels of the lacZ transcript, whereas those of bisphenol A and nifedipine were found to be reduced. No significant responses were observed with 17beta-estradiol or progesterone. In addition, 17beta-estradiol and progesterone did not change the levels of the lacZ transcripts in the HepG2 cells, but did induce significant increases in the transcripts of the NIH3T3 cells. Treatment with corticosterone and dexamethasone, which were highly expressed in the HepG2 cells, did not affect the levels of the lacZ transcript in NIH3T3 cells. These results show that lacZ transcripts can be measured, rapidly and reproducibly, using reverse transcriptase-polymerase chain reaction (RT-PCR) based on the expression of the hCYP3A4/lacZ reporter gene, and was mediated by the SXR. Thus, this in vitro reporter gene bioassay is useful for measuring xenobiotic activities, and is a means to a better relevant bioassay, using human cells, human genes and human promoters, in order to get a closer look at actual human exposure.
Lee
Mi R
MR
Division of Laboratory Animal Resources, National Institute of Toxicological Research, Korea Food and Drug Administration, Seoul, Korea.
Kim
Yeon J
YJ
Hwang
Dae Y
DY
Kang
Tae S
TS
Hwang
Jin H
JH
Lim
Chae H
CH
Kang
Hyung K
HK
Goo
Jun S
JS
Lim
Hwa J
HJ
Ahn
Kwang S
KS
Cho
Jung S
JS
Chae
Kap R
KR
Kim
Yong K
YK
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D017930:Genes, Reporter / Q000187:drug effects*; D006801:Humans; D007763:Lac Operon; D051379:Mice; D041681:NIH 3T3 Cells; D000077297:Pregnane X Receptor; D011987:Receptors, Steroid / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D014162:Transfection; D015262:Xenobiotics / Q000633:toxicity*
D000077297:Pregnane X Receptor; D011987:Receptors, Steroid; D015262:Xenobiotics; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human