The atherogen 3-methylcholanthrene induces multiple DNA adducts in mouse aortic smooth muscle cells: role of cytochrome P4501B1.
OBJECTIVE
3-Methylcholanthrene (MC), a polycylic aromatic hydrocarbon, induces atherogenesis in mice fed an atherogenic diet. In this study, we tested the hypothesis that MC would induce DNA adducts in mouse aortic smooth muscle cells (SMCs) and that cytochrome P4501B1 (CYP1B1) plays an important role in the activation of MC to genotoxic intermediates.


METHODS
Cultured SMCs were treated with MC or the vehicle dimethyl sulfoxide (DMSO), and DNA was isolated after 24 h. In some experiments, the cells were pre-treated with the CYP1B1 inhibitor 1-ethynylpyrene (EP) prior to exposure to MC. DNA adducts were determined by the 32P-postlabeling assay. Aryl hydrocarbon hydroxylase assay was measured by fluorimetry.


RESULTS
MC induced formation of 12 DNA adducts that were not observed in DMSO-treated cells. DNA adduct formation was dose-dependent, with maximum response observed at 3 microM. Pre-treatment of cells with EP dramatically suppressed DNA adduct formation by MC. MC treatment caused induction of CYP1B1, but not CYP1A1.


CONCLUSIONS
The induction of high levels of multiple DNA adducts in SMCs by MC suggests that SMCs have a functional enzymatic machinery capable of metabolically activating MC to genotoxic metabolites. The significant inhibition by EP of MC-induced DNA adduct formation indicated that CYP1B1 was the primary CYP enzyme responsible for formation of genotoxic metabolites that may play a role in the induction of atherosclerosis by MC.
Moorthy
Bhagavatula
B
Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. bmoorthy@bcm.tmc.edu
Miller
Kimberly P
KP
Jiang
Weiwu
W
Ramos
Kenneth S
KS
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects*; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D001579:Benzopyrene Hydroxylase / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D065633:Cytochrome P-450 CYP1B1; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D018736:DNA Adducts / Q000096:biosynthesis*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D008748:Methylcholanthrene / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D011721:Pyrenes / Q000494:pharmacology
D065607:Cytochrome P-450 Enzyme Inhibitors; D018736:DNA Adducts; D004791:Enzyme Inhibitors; D011721:Pyrenes; C042881:1-ethynylpyrene; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D001579:Benzopyrene Hydroxylase; D001189:Aryl Hydrocarbon Hydroxylases; C585498:Cyp1b1 protein, mouse; D065633:Cytochrome P-450 CYP1B1