Effect of the Endogenous AHR ligand KA on CY2B and CYP1A Gene Expression Sample Clauses

Effect of the Endogenous AHR ligand KA on CY2B and CYP1A Gene Expression. Tryptophan metabolites and derivatives have been shown to activate the AHR (Xxxxx-Xxxxxxxx, et al., 1998) (Xxxxxx & Xxxxxxxxx, 2008). A recent study by XxXxxxxx and colleagues reported kynurenic acid (KA), a product of tryptophan oxidation, as a potent endogenous ligand for human AHR (2010). Kynurenic acid was shown to increase CYP1A mRNA levels in HepG2 hepatoma cells as well as being capable of binding AHR in competition binding assays (XxXxxxxx B. , et al., 2010). In an attempt to see if an endogenous ligand of AHR would have the same repressive effects on CYP2B1 expression, as in the case with 3MC and β-NF, hepatocytes were co-treated with varying concentration of KA with or without 1 mM PB. Kynurenic acid did not significantly increase CYP1A2 expression until the 100 µM concentration (Figure 3.3g). Increasing the concentration of KA above 1 mM was not done due to concern with cell viability. In a subsequent experiment with varying concentrations of KA (10 nM-1mM) and 1 mM PB, KA attenuated CYP2B1 mRNA expression at the 1 mM concentration. (Figure 3.3h). After 48 hours of exposure to 1 mM PB and 100 µM KA, CYP2B protein levels were probed. Canonically, PB alone dramatically increased CYP2B expression while with co-treatment with PB and KA there was not a significant decrease in CYP2B protein expression. KA alone did not induce CYP2B nor did it decrease CYP2B when compared to control (Figure 3.3i). * Relative Level to Control 9 CYP1A2 mRNA Expression 7 6 5 4 3 2 1 0 Concentration of KA log (nM) Figure 3.3g: Dose-dependent response of KA on CYP1A2 mRNA expression. Primary rat hepatocytes were treated with increasing concentrations of KA (10 nM -100 µM) for 24 hours after which CYP1A2 levels were observed via real-time RT-PCR. Symbol (*) indicates significance from control (p<0.05). Percent Maximal Induction 120 100 80 60 40 20 0 CYP2B1 mRNA Expression * Concentration of Kynurenic Acid log(nM) Figure 3.3h: Effect of concomitant treatment with 1 mm PB and increasing concentrations of KA on CYP2B1 mRNA expression. Cells were co-treated for 24 hours after which CYP2B1 levels were determined via real time RT-PCR. Symbol (*) indicates significance to 1 mM PB alone. CYP2B GAPDH CON 1 mM PB 100 µM KA 100 µM KA/1mM XX XXX0X0 Protein Expression 3000 *2500 2000 1500 1000 00 0 0 0 # 0 0 XXX XX XX XX/XX Treatments Relative Protein Levels (to Control) Figure 3.3i: Effect of PB and KA co-treatment on CYP2B protein expression. Primary rat hepatocytes were co-treated w...