Elucidating the Role of the NRF2 pathway in Nitrogen Mustard Induced Toxicity in Mouse Epidermal Skin Cells

Abstract

Nitrogen mustard (NM), a structural analog of warfare agent sulfur mustard (SM), is a chemical vesicant that causes blistering of skin and damage to mucus membranes. NM is a strong alkylating and DNA damaging agent. Currently, we lack effective therapies to rescue skin injuries by NM and SM. The insight into the mechanisms of NM and SM‐induced skin injuries is crucial for the development of effective targeted therapies. Our lab’s previous studies suggest that skin toxicity from NM exposure can cause macromolecular damage and activation of signaling pathways, especially those related to inflammation, oxidative stress, and DNA damage. Therefore, targeting anti‐oxidative stress and anti‐inflammatory pathways such as the nuclear factor erythroid 2‐related (Nrf2) pathway can be essential to treat epidermal injuries from this vesicant exposure. Nrf2 is a key transcription factor that controls the basal and induced expression of an array of antioxidant response element–dependent genes to provide anti‐inflammatory and cytoprotective effects to cells. Thus, activating the Nrf2 pathway can be a targeted approach for the treatment of NM skin toxicity. To study the role of the Nrf2 pathway in NM‐induced toxicity, we used mouse skin epidermal JB6 cells. The JB6 cells were treated with 75µM of NM for 2, 6, 12 or 24 hours where approximately 50% of cell viability was achieved (MTT assay and trypan blue cell counting). Using qPCR analysis, we further studied the RNA expression of the Nrf2 pathway and its targeted genes including heme oxygenase‐1 (HO‐1), NADPH Quinone oxidoreductase enzyme (NQO‐1), Glutamate‐Cysteine Ligase Modifier Subunit (GCLM), Glutamate‐Cysteine Ligase Catalytic Subunit (GCLC) and Catalase. Our results showed an upregulation in RNA expression for HO‐1 at 2 and 6 hours post NM exposure, and a downregulation in Nrf2, NQO‐1, Catalase, GCLM, and GCLC. There was also an upregulation for HO‐1 and NQO‐1 at 12 hours post NM exposure. However, there was a downregulation in RNA expression for all these markers at 24 hours post NM exposure. To induce the activation of Nrf2 pathway and its targeted genes, we want to treat the cells with synthetic oleanane triterpenoid derivatives of 2‐cyano‐3,12‐dioxooleana‐1,9(11)‐dien‐28‐oic acid (CDDO), which are among the most potent Nrf2 activators. These results will help us further investigate in in vivo studies if triterpenoids via cytoprotective and antioxidant effects could be used for the treatment of acute as well as long‐term skin toxicity from NM exposure.