Yujuan Zhang,Jinhu Huang,Xiangxiu Li,Ci Fang,Liping Wang
Breast cancer resistance protein (BCRP), an ATP-binding cassette (ABC) half transporter encoded by theAbcg2gene, is reported to influence the pharmacokinetics of substrate drugs during clinical therapy. The aim of this study was to clarify the mechanisms that regulate the transcription of the chickenAbcg2gene through cloning and characterization of its promoter region. Results showed that theAbcg2gene is transcribed by a TATA-less promoter with several putative Sp1 sites upstream from two putative CpG islands. A luciferase reporter assay conducted both in chicken leghorn male hepatoma (LMH) cells and chicken primary hepatocytes mapped a basal promoter to nucleotides −110 to +30, which is responsible for the constitutive expression ofAbcg2. The 5′-region upstream of the basal promoter was characterized by both positive and negative regulatory domains. Further, using the cell-based reporter gene assay combined with RT-PCR and drug accumulation analysis, we found that four xenobiotics, daidzein, clotrimazole, ivermectin, and lipopolysaccharide (LPS), influence the expression and function of BCRP through significant regulation of theAbcg2gene promoter. Interaction sites with theAbcg2gene promoter of these four selected regulators were clarified by progressive deletions and mutation assays. This study shed some light on the regulatory mechanisms involved in chickenAbcg2gene expression and the results may have far-reaching significance regarding the usage and development of veterinary drugs.Keywords:chickenAbcg2;promoter;regulators;LMH;primary hepatocytes
Chlorpyrifos (CPF), an organophosphorus pesticide, is detected commonly in environments, where it is thought to be highly toxic to non-target organisms. However, the mechanism of CYP450s pathway mediated by nuclear receptors on CPF-induced apoptosis and necroptosis at the cellular level and the effect of CPF on the cytotoxicity of the chicken hepatocarcinoma cell line (LMH) has also not been reported in detail. Therefore, this experiment aims to explore whether CPF can improve apoptosis and necroptosis in LMH cells by activating the nuclear receptors/CYP450s axis. LMH cells, the subject of this study, were exposed to 5 μg/mL, 10 μg/mL, and 15 μg/mL doses of CPF. With the increase of CPF concentration, the increase of nuclear receptor level led to the up-regulation of CYP450s activity. With the massive production of ROS, the expression of apoptotic pathway genes (Bax, Caspase9, and Caspase3) enhanced, while Bcl-2 expression dropped sharply. The expression of programmed necroptosis genes (RIPK1, RIPK3, and MLKL) heightened, and Caspase8 reduced considerably. In short, our data suggests that excessive activation of nuclear receptors and CYP450s induced by CPF promotes ROS production, which directs apoptosis and programmed necroptosis in LMH cells. Graphical abstract
危险品化学品经营许可证（带存储）