Text

theses

Sulfur mustard and nitrogen mustard (mechlorethamine, HN2) are potent vesicants used in chemical warfare and cancer chemotherapy that primarily target skin, eye, and lung. These electrophilic, bifunctional alkylating agents cause oxidative stress and persistent tissue damage. Toxicity of related mustards, chlorambucil and melphalan, is limited by clearance from cells by multidrug resistance-associated protein 1 (MRP1/Mrp1), a transmembrane ATPase that stimulates efflux of glutathione-conjugated electrophiles. HN2 causes injury by covalently modifying biomolecules including glutathione. Monofunctional glutathione adducts contribute to cytotoxicity or are exported by MRP1/Mrp1. In A549 lung epithelial cells, which express MRP1 and MRP2, HN2 inhibits growth (IC5o = 0.18 µM), and inhibition of MRPs by MK-571 increases sensitivity to HN2 (IC50 = 0.045 µM). Similar effects are seen for other bifunctional mustards chlorambucil and melphalan. Using human embryonic kidney (HEK) 293 cells overexpressing MRP1 and MRP2, we determined that MRP1 provides resistance to HN2 while MRP2 does not protect the cells. HN2 inhibited MRP functional activity in both A549 cells and HEK cells overexpressing MRP1, and increased sensitivity to growth inhibition induced by MRP1/Mrp1 substrates etoposide, methotrexate, and vincristine in A549 cells. PAM212 cells and primary mouse keratinocytes express Mrp1 mRNA and protein. Activation of the transcription factor Nrf2 by sulforaphane increased Mrp1 mRNA, protein expression and activity in PAM212 cells and protected cells against HN2-induced growth inhibition (IC50 = 1 and 13 µM without and with sulforaphane, respectively). This protection was reversed by MK-571 (IC50 = 0.63 µM). Sulforaphane increased Mrp1 mRNA and protein expression and activity and decreased HN2 growth inhibition in primary keratinocytes (IC50 = 1.4 and 4.8 µM without and with sulforaphane, respectively). This attenuation was reversed by MK-571 (IC50 = 0.27 µM). MK-571 increases HN2-induced cytotoxicity in primary mouse keratinocytes (growth inhibition IC50 = 1.4 and 0.48 µM, without and with MK-571, respectively). Sulforaphane did not protect keratinocytes from Nrf2-/- mice (IC50 = 0.31 and 0.14 µM without and with sulforaphane, respectively). These data show MRP1/Mrp1-mediated efflux is important for regulating HN2 injury. Inhibiting MRP1/Mrp1 may increase mustard efficacy in cancer chemotherapy, while enhancing transport may represent a promising route to mitigate vesicant-induced cytotoxicity.

ETD_6566

Ph.D.

Includes bibliographical references

by Ronald G. Udasin

doi:10.7282/T38K7C30

ETD doctoral

The author owns the copyright to this work.