verlukast and sulforaphane

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.

Topics: Animals; Heme Oxygenase-1; Isothiocyanates; Keratinocytes; Mechlorethamine; Membrane Proteins; Mice; Mice, Inbred C57BL; Multidrug Resistance-Associated Proteins; NAD(P)H Dehydrogenase (Quinone); NF-E2-Related Factor 2; Propionates; Quinolines; Sulfoxides

The kidney secretes various xenobiotics through a well-established transport system. The transcription factor NF-E2-related factor 2 (NRF2) up-regulates a subset of genes encoding antioxidant and detoxification proteins. Kelch-like ECH-associated protein 1 (KEAP1) down-regulates NRF2 by facilitating continuous degradation of NRF2 protein. Here, we investigated the role of NRF2 in the expression of renal drug transporters by using a stable KEAP1 knockdown renal tubular HK-2 cell line (shKEAP1). KEAP1 knockdown resulted in a significant increase in the expression of four renal transporters, namely, multidrug resistance protein 1 (MDR1; ABCB1), breast cancer resistance protein (BCRP; ABCG2), multidrug resistance-associated protein 2 (MRP2; ABCC2), and MRP3 (ABCC3). In western blot and immunocytochemical analyses, protein levels of these transporters were also significantly higher in the knockdown group. Consequently, shKEAP1 cells released more Hoechst 33342 fluorescent dye and doxorubicin, and they were more resistant to doxorubicin than the control cells. In addition, cisplatin resistance of shKEAP1 decreased upon co-incubation with a transporter inhibitor. Whereas, a short term incubation (24h) with sulforaphane did not show noticeable changes in the expression of transporter. Collectively, these results indicate that NRF2 regulates the expression of MDR1, BCRP, MRP2, and MRP3 in human tubular epithelial cells. Altered expression of these transporters affects drug secretion in these cells, which may result in the renal cellular damage upon exposure to nephrotoxic xenobiotics.

Topics: ATP-Binding Cassette Transporters; Benzimidazoles; Cell Line; Cisplatin; Doxorubicin; Gene Knockdown Techniques; Humans; Intracellular Signaling Peptides and Proteins; Isothiocyanates; Kelch-Like ECH-Associated Protein 1; Multidrug Resistance-Associated Protein 2; NF-E2-Related Factor 2; Propionates; Quinolines; Sulfoxides