title: Epoxyeicosatrienoic acids (EETs) form adducts with DNA in vitro.
abstract: Epoxyeicosatrienoic acids (EETs) are potent lipid mediators formed by cytochrome P450 epoxygenases from arachidonic acid. They consist of four regioisomers of cis-epoxyeicosatrienoic acids: 5,6-, 8,9-, 11,12- and 14,15-EET. Here we investigated whether these triene epoxides are electrophilic enough to form covalent adducts with DNA in vitro. Using the thin-layer chromatography (TLC) (32)P-postlabelling method for adduct detection we studied the reaction of individual deoxynucleoside 3'-monophosphates and calf thymus DNA with the four racemic EETs. Under physiological conditions (pH 7.4) only ±11,12-EET11,12-EET formed adducts with DNA in a dose dependent manner detectable by the (32)P-postlabelling method. However, when pre-incubated at pH 4 all four racemic EETs were capable to bind to DNA forming several adducts. Under these conditions highest DNA adduct levels were found with ±11,12-EET followed by ±5,6-EET, ±8,9-EET, and ±14,15-EET, all of them two orders of magnitude higher (between 3 and 1 adducts per 10(5) normal nucleotides) than those obtained with ±11,12-EET at pH 7.4. Similar DNA adduct patterns consisting of up to seven spots were observed with all four racemic EETs the most abundant adducts being derived from the reaction with deoxyguanosine and deoxyadenosine. In summary, when analysed by the (32)P-postlabelling method all four racemic EETs formed multiple DNA adducts after activation by acidic pH, only ±11,12-EET produced DNA adducts in aqueous solution at neutral pH. Therefore, we conclude from our in vitro studies that EETs might be endogenous genotoxic compounds.
authors.affiliation: Division of Preventive Oncology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120 Heidelberg, Germany.
authors.forename: Dorothee J
authors.initials: DJ
authors.lastname: Funk
authors.affiliation: Goethe-University, Institute of Pharmaceutical Chemistry/Center for Drug Research, Development, and Safety (ZAFES), Frankfurt/Main, Germany.
authors.forename: Bernd L
authors.initials: BL
authors.lastname: Sorg
authors.affiliation: Division of Radiopharmaceutical Chemistry, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Kopka
authors.affiliation: Division of Radiopharmaceutical Chemistry, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Electronic address: h.schmeiser@dkfz.de.
authors.forename: Heinz H
authors.initials: HH
authors.lastname: Schmeiser
mesh_terms: D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D002417:Cattle; D004247:DNA / Q000737:chemistry*; D018736:DNA Adducts / Q000138:chemical synthesis*; D003838:Deoxyadenine Nucleotides / Q000737:chemistry; D003848:Deoxyguanine Nucleotides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010761:Phosphorus Radioisotopes / Q000737:chemistry; D012996:Solutions; D013237:Stereoisomerism
chemical_list: D018736:DNA Adducts; D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D010761:Phosphorus Radioisotopes; D012996:Solutions; C046783:11,12-epoxy-5,8,14-eicosatrienoic acid; C012873:2'-deoxy-5'-adenosine monophosphate; C040776:5,6-epoxy-8,11,14-eicosatrienoic acid; C050715:8,9-epoxyeicosatrienoic acid; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid; D004247:DNA; C427203:calf thymus DNA; D015126:8,11,14-Eicosatrienoic Acid
keywords: (32)P-postlabelling; Arachidonic acid; Cytochrome P450; DNA damage; EETs; Lipid mediator