Fig 2: Overexpression of catalase reduces BaP reactive intermediates in endothelial microsomes.(A-B) MAECs obtained from wild-type (WT) mice and transgenic mice overexpressing human catalase (hCat) were incubated with 10 nM of TCDD for 4 h. Microsomes were isolated from TCDD-treated MAECs with serial centrifugations. The total and microsomal levels of CYP1A1, EH, GSTP and NQO1 were determined by western blot analysis, and expressed as a ratio of their immunoblot intensity relative to ß-actin. (C-D) Microsomes were isolated from TCDD-treated MAECs with serial centrifugations, and incubated with 1 µM of BaP for 1 h. BaP reactive metabolites, including 9-hydroxybenzo[a]pyrene (9-OH BaP), 3-hydroxy BaP(3-OH BaP), 9,10-dihydroxy BaP (9,10-diol), 7,8-dihydro BaP (7,8-diol), 4,5-dihydroxy BaP (4,5-diol), BaP-3,6-dione (3,6-dione) and BaP-6,12-dione (6,12-dione), in the microsomes were determined using a reverse phase HPLC system. Panel C shows a representative HPLC chromatogram of BaP metabolites, and panel D shows the level of BaP metabolites as expressed relative to the level of microsomal proteins. Values represent the mean ± SEM of five separate experiments in which MAECs were pooled from 4 mice. * P< 0.05 vs. the same genotype cells without TCDD treatment (control), and † P< 0.05 vs. WT cells treated with TCDD.

Fig 3: Localization of CYP1A1 and 1B1 in endoplasmic reticulum.MAECs obtained from wild-type (WT) mice and transgenic mice overexpressing human catalase (hCat) were incubated with 1 µM of BaP or culture medium alone as a control (ctrl) for 4 h. After the cells were fixed and permeabilized, they were immunostained with antibodies against CYP1A1 (1A1) or CYP1B1 (1B1), and then stained with endoplasmic reticulum (ER) marker Tio6(3). (A-B) Images were obtained using a Leica TCS SP2 confocal microscope. (C) The protein levels of CYP1A1 and 1B1 were determined by the fluorescence intensity (F.I.). (D) The ratio of CYP1A1 and 1B1 proteins in the ER versus those in the whole-cell was determined by the F.I. in the ER and the entire cell. * P< 0.05 vs. the same genotype cells without BaP treatment (ctrl); and † P< 0.05 vs. BaP-treated WT cells.

Fig 4: mRNA and protein expression of CYP enzymes, AOEs and cytokine IL-1β (A-C). (A) mRNA expression was analyzed in SVG cells (at 24, 48, and 72 h) treated with 1 μM concentration of BaP (daily treatment). (B) Representative western blots of IL-1β, AOEs (SOD1 and CAT), and CYP enzymes (CYP2A6, CYP1B1, CYP1A1) in SVG cells (Western blot at 24, 48, and 72 h) treated with 1 μM concentration of BaP (daily treatment). Untreated controls were considered as 100% in the densitometry analysis. (C) Densitometry analysis. Statistical analyses were carried out by using Student’s t-test. Results are expressed as means ± S.E.M of n = 4 experiments. **P ≤0.01, ***P ≤0.001 in comparison with untreated control.