Inhibition of aryl hydrocarbon receptor transactivation and DNA adduct formation by CYP1 isoform-selective metabolic deactivation of benzo[a]pyrene

Vitamin D Receptor Activation Enhances Benzo[a]pyrene Metabolism via CYP1A1 Expression in Macrophages

“…For CYP1A1 expression, microsomes (S9 fraction) from cells were prepared as described previously (Uno et al, 2001;Endo et al, 2008). Western blot analysis was performed using a monoclonal anti-VDR antibody (Santa Cruz Biotechnology, Santa Cruz, CA), a polyclonal anti-AHR antibody (R&D Systems Inc., Minneapolis, MN), a polyclonal anti-CYP1A1 antibody (Daiichi Pure Chemicals, Tokyo, Japan), and a monoclonal anti-␤-actin antibody (SigmaAldrich), visualized with an enhanced chemiluminescence detection system or an alkaline phosphatase conjugate substrate system (Endo et al, 2008).…”

“…Extracts from microsomal fractions were subjected for enzyme activity assays. BaP hydroxylation was assayed as previously reported (Nebert and Gelboin, 1968;Endo et al, 2008). In brief, 200 g of microsomal protein was incubated in 100 mM potassium phosphate buffer (pH 7.4) containing 1 mg/ml bovine serum albumin, 80 M BaP, and 0.5 mM NADPH at 37°C for 20 min.…”

“…BaP-induced DNA adducts were determined by a 32 P-postlabeling method (Talaska et al, 1996;Uno et al, 2001;Endo et al, 2008). After DNA extraction from cells, hydrolysis to 3Ј-phosphodeoxynucleotides with micrococcal endonuclease and spleen phosphodiesterase, and removal of nonadducted 3Ј-phosphodeoxynuleotides with n-butanol extraction, the 3Ј-phosphodeoxynucleosides, were labeled at the 5Ј positions with […”

“…We examined BaP hydroxylation activity by detecting conversion of BaP to 3-hydroxy-BaP. CYP1A1, but not CYP1A2 or CYP1B1, mediate this cellular reaction (Endo et al, 2008). Cells were treated with BaP and/or 1,25(OH) 2 D 3 for 24 h, and microsomes were isolated for enzyme activity assays.…”

“…In contrast, BaP-induced DNA adducts are increased in the liver, and BaP clearance from the blood is slower in CYP1A1-null mice (Uno et al, 2001). Overexpression of CYP1A1 in hepatocytes suppresses BaP-induced DNA adduct formation and AHR transactivation (Endo et al, 2008). CYP1A1 may be involved in both metabolic activation and detoxification of BaP depending on conditions (Uno et al, 2006).…”