3-Methylcholanthrene elicits DNA adduct formation in the CYP1A1 promoter region and attenuates reporter gene expression in rat H4IIE cells.
Cytochrome CYP1A (CYP1A) enzymes catalyze bioactivation of 3-methylcholanthrene (MC) to genotoxic metabolites. Here, we tested the hypothesis that CYP1A2 catalyzes formation of MC-DNA adducts that are preferentially formed in the promoter region of CYP1A1, resulting in modulation of CYP1A1 gene expression. MC bound covalently to plasmid DNA (50 micro g) containing human CYP1A1 promoter (pGL3-1A1), when incubated with wild-type (WT) liver microsomes (2 mg) and NAPPH 37 degrees C for 2h, giving rise to 9 adducts, as determined by (32)P-postlabeling. Eighty percent of adducts was located in the promoter region. Transient transfection of the adducted plasmids into rat hepatoma (H4IIE) cells for 16h, followed by MC (1 micro M) treatment for 24h inhibited reporter (luciferase) gene expression by 75%, compared to unadducted controls. Our results suggest that CYP1A2 plays a key role in sequence-specific MC-DNA adduct formation in the CYP1A1 promoter region, leading to attenuation of CYP1A1 gene expression.
Moorthy
Bhagavatula
B
Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA. bmoorthy@bcm.tmc.edu
Muthiah
Kathirvel
K
Fazili
Inayat S
IS
Kondraganti
Sudha R
SR
Wang
Lihua
L
Couroucli
Xanthi I
XI
Jiang
Weiwu
W
D000818:Animals; D045744:Cell Line, Tumor; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics*; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism*; D003580:Cytochromes; D018736:DNA Adducts / Q000378:metabolism*; D004790:Enzyme Induction; D005260:Female; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D008114:Liver Neoplasms, Experimental; D008297:Male; D008748:Methylcholanthrene / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D018345:Mice, Knockout; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D011401:Promoter Regions, Genetic / Q000187:drug effects; D051381:Rats; D014162:Transfection
D003580:Cytochromes; D018736:DNA Adducts; D008748:Methylcholanthrene; C017170:Cyp1a2 protein, rat; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2