Nrf2 Regulates the Sensitivity of Mouse Keratinocytes to Nitrogen Mustard via Multidrug Resistance-Associated Protein 1 (Mrp1)

Toxicol Sci. 2016 Jan;149(1):202-12. doi: 10.1093/toxsci/kfv226. Epub 2015 Oct 9.


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.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Heme Oxygenase-1 / genetics
  • Isothiocyanates / pharmacology
  • Keratinocytes / drug effects*
  • Mechlorethamine / toxicity*
  • Membrane Proteins / genetics
  • Mice
  • Mice, Inbred C57BL
  • Multidrug Resistance-Associated Proteins / genetics
  • Multidrug Resistance-Associated Proteins / physiology*
  • NAD(P)H Dehydrogenase (Quinone) / genetics
  • NF-E2-Related Factor 2 / physiology*
  • Propionates / pharmacology
  • Quinolines / pharmacology
  • Sulfoxides


  • Isothiocyanates
  • Membrane Proteins
  • Multidrug Resistance-Associated Proteins
  • NF-E2-Related Factor 2
  • Nfe2l2 protein, mouse
  • Propionates
  • Quinolines
  • Sulfoxides
  • Mechlorethamine
  • verlukast
  • Heme Oxygenase-1
  • Hmox1 protein, mouse
  • NAD(P)H Dehydrogenase (Quinone)
  • Nqo1 protein, mouse
  • sulforaphane
  • multidrug resistance-associated protein 1