title: Reduced formation of depurinating estrogen-DNA adducts by sulforaphane or KEAP1 disruption in human mammary epithelial MCF-10A cells.
abstract: Sulforaphane (SFN) is a potent inducer of detoxication enzymes such as NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione-S-transferase (GST) via the Kelch-like erythroid-derived protein with CNC homology-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) signaling pathway. NQO1 reduces the carcinogenic estrogen metabolite, catechol estrogen-3,4-quinone, whereas GSTs detoxify it through conjugation with glutathione. These 3,4-quinones can react with DNA to form depurinating DNA adducts. Thus, SFN may alter estrogen metabolism and thus protect against estrogen-mediated DNA damage and carcinogenesis. Human breast epithelial MCF-10A cells were treated with either vehicle or SFN and either estradiol (E2) or its metabolite 4-hydroxyestradiol (4-OHE2). 4-Hydroxy-derived estrogen metabolites and depurinating DNA adducts formed from E2 and its interconvertable metabolite estrone (E1) were analyzed by mass spectrometry. Levels of the depurinated adducts, 4-OHE1/2-1-N3Adenine and 4-OHE1/2-1-N7Guanine, were reduced by 60% in SFN-treated cells, whereas levels of 4-OCH3E1/2 and 4-OHE1/2-glutathione conjugates increased. To constitutively enhance the expression of Nrf2-regulated genes, cells were treated with either scrambled or siKEAP1 RNA. Following E2 or 4-OHE2 treatments, levels of the adenine and guanine adducts dropped 60-70% in siKEAP1-treated cells, whereas 4-OHE1/2-glutathione conjugates increased. However, 4-OCH3E1/2 decreased 50% after siKEAP1 treatment. Thus, treatment with SFN or siKEAP1 has similar effects on reduction of depurinating estrogen-DNA adduct levels following estrogen challenge. However, these pharmacologic and genetic approaches have different effects on estrogen metabolism to O-methyl and glutathione conjugates. Activation of the Nrf2 pathway, especially elevated NQO1, may account for some but not all of the protective effects of SFN against estrogen-mediated DNA damage.
authors.affiliation: Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.forename: Muhammad
authors.initials: M
authors.lastname: Zahid
authors.forename: Yong
authors.initials: Y
authors.lastname: Liao
authors.forename: Eleanor G
authors.initials: EG
authors.lastname: Rogan
authors.forename: Ercole L
authors.initials: EL
authors.lastname: Cavalieri
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Davidson
authors.forename: James D
authors.initials: JD
authors.lastname: Yager
authors.forename: Kala
authors.initials: K
authors.lastname: Visvanathan
authors.forename: John D
authors.initials: JD
authors.lastname: Groopman
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Kensler
mesh_terms: D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D017209:Apoptosis; D015153:Blotting, Western; D001940:Breast / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D018736:DNA Adducts / Q000187:drug effects*; D004967:Estrogens / Q000378:metabolism*; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017879:Isothiocyanates / Q000494:pharmacology*; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013454:Sulfoxides; D053719:Tandem Mass Spectrometry
chemical_list: D016588:Anticarcinogenic Agents; D018736:DNA Adducts; D004967:Estrogens; D047908:Intracellular Signaling Peptides and Proteins; D017879:Isothiocyanates; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013454:Sulfoxides; C016766:sulforaphane