title: Nrf2 Regulates the Sensitivity of Mouse Keratinocytes to Nitrogen Mustard via Multidrug Resistance-Associated Protein 1 (Mrp1).
abstract: Sulfur mustard and nitrogen mustard (mechlorethamine, HN2) are potent vesicants developed as chemical warfare agents. These electrophilic, bifunctional alkylating agents cause skin injury, including inflammation, edema, and blistering. HN2 covalently modifies macromolecules such as DNA, RNA, and proteins or is scavenged by glutathione, forming adducts that can contribute to toxicity. Multidrug resistance-associated protein 1 (Mrp1/MRP1) is a transmembrane ATPase known to efflux glutathione-conjugated electrophiles. In the present studies, we examined the effects of modulating Mrp1-mediated transport activity on the sensitivity of primary and PAM212 mouse keratinocytes to HN2. Primary keratinocytes, and to a lesser extent, PAM212 cells, express Mrp1 mRNA and protein and possess Mrp1 functional activity, as measured by calcein efflux. Sulforaphane, an activator of Nrf2, increased Mrp1 mRNA, protein, and functional activity in primary keratinocytes and PAM212 cells and decreased their sensitivity to HN2-induced growth inhibition (IC(50) = 1.4 and 4.8 µM in primary keratinocytes and 1 and 13 µM in PAM212 cells, in the absence and presence of sulforaphane, respectively). The Mrp1 inhibitor, MK-571, reversed the effects of sulforaphane on HN2-induced growth inhibition in both primary keratinocytes and PAM212 cells. In primary keratinocytes from Nrf2(-/-) mice, sulforaphane had no impact on Mrp1 expression or activity, or on sensitivity to HN2, demonstrating that its effects depend on Nrf2. These data suggest that Mrp1-mediated efflux is important in regulating HN2-induced keratinocyte growth inhibition. Enhancing HN2 efflux from keratinocytes may represent a novel strategy for mitigating vesicant-induced cytotoxicity.
authors.affiliation: *Department of Pharmacology and Toxicology, Rutgers University, Piscataway, New Jersey;
authors.forename: Ronald G
authors.initials: RG
authors.lastname: Udasin
authors.affiliation: *Department of Pharmacology and Toxicology, Rutgers University, Piscataway, New Jersey;
authors.forename: Xia
authors.initials: X
authors.lastname: Wen
authors.affiliation: *Department of Pharmacology and Toxicology, Rutgers University, Piscataway, New Jersey;
authors.forename: Kristin M
authors.initials: KM
authors.lastname: Bircsak
authors.affiliation: *Department of Pharmacology and Toxicology, Rutgers University, Piscataway, New Jersey;
authors.forename: Lauren M
authors.initials: LM
authors.lastname: Aleksunes
authors.affiliation: Department of Environmental Health Science, New York Medical College, Valhalla, New York;
authors.forename: Michael P
authors.initials: MP
authors.lastname: Shakarjian
authors.affiliation: Department of Pharmaceutics, Rutgers University, Piscataway, New Jersey; and.
authors.forename: Ah-Ng Tony
authors.initials: AN
authors.lastname: Kong
authors.affiliation: Department of Environmental Health Science, New York Medical College, Valhalla, New York;
authors.forename: Diane E
authors.initials: DE
authors.lastname: Heck
authors.affiliation: *Department of Pharmacology and Toxicology, Rutgers University, Piscataway, New Jersey;
authors.forename: Debra L
authors.initials: DL
authors.lastname: Laskin
authors.affiliation: §Department of Environmental and Occupational Medicine, Rutgers University Robert Wood Johnson Medical School, Piscataway, New Jersey. jlaskin@eohsi.rutgers.edu.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Laskin
mesh_terms: D000818:Animals; D051547:Heme Oxygenase-1 / Q000235:genetics; D017879:Isothiocyanates / Q000494:pharmacology; D015603:Keratinocytes / Q000187:drug effects*; D008466:Mechlorethamine / Q000633:toxicity*; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000502:physiology*; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics; D051267:NF-E2-Related Factor 2 / Q000502:physiology*; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D013454:Sulfoxides
chemical_list: D051547:Heme Oxygenase-1; D017879:Isothiocyanates; D008466:Mechlorethamine; D008565:Membrane Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D016660:NAD(P)H Dehydrogenase (Quinone); D051267:NF-E2-Related Factor 2; D011422:Propionates; D011804:Quinolines; D013454:Sulfoxides; C495636:Nfe2l2 protein, mouse; C059141:verlukast; C496542:Hmox1 protein, mouse; C491732:Nqo1 protein, mouse; C016766:sulforaphane; C421109:multidrug resistance-associated protein 1
keywords: alkylating agents < agents; chemical & biological weapons < agents; cutaneous or skin toxicity < systems toxicology; glutathione < biotransformation and toxicokinetics; membrane biology < systems toxicology; xenobiotic transporters < biotransformation and toxicokinetics