Absence of a p53 allele delays nitrogen mustard-induced early apoptosis and inflammation of murine skin

Abstract

Bifunctional alkylating agent sulfur mustard (SM) and its analog nitrogen mustard (NM) cause DNA damage leading to cell death, and potentially activating inflammation. Transcription factor p53 plays a critical role in DNA damage by regulating cell cycle progression and apoptosis. Earlier studies by our laboratory demonstrated phosphorylation of p53 at Ser15 and an increase in total p53 in epidermal cells both in vitro and in vivo following NM exposure. To elucidate the role of p53 in NM-induced skin toxicity, we employed SKH-1 hairless mice harboring wild type (WT) or heterozygous p53 (p53+/−). Exposure to NM (3.2 mg) caused a more profound increase in epidermal thickness and apoptotic cell death in WT relative to p53+/− mice at 24 h. However, by 72 h after exposure, there was a comparable increase in NM-induced epidermal cell death in both WT and p53+/− mice. Myeloperoxidase activity data showed that neutrophil infiltration was strongly enhanced in NM-exposed WT mice at 24 h persisting through 72 h of exposure. Conversely, robust NM-induced neutrophil infiltration (comparable to WT mice) was seen only at 72 h after exposure in p53+/− mice. Similarly, NM-exposure strongly induced macrophage and mast cell infiltration in WT, but not p53+/− mice. Together, these data indicate that early apoptosis and inflammation induced by NM in mouse skin are p53-dependent. Thus, targeting this pathway could be a novel strategy for developing countermeasures against vesicants-induced skin injury.

Myeloperoxidase deficiency attenuates nitrogen mustard-induced skin injuries

“…In the present study therefore, the role of MPO in these important events leading to NM-induced skin pathology was analyzed using MPO KO mice. Similar to earlier reports with SM and to our previous studies with CEES and NM, NM exposure in WT mice caused a robust NM-induced increase in neutrophil infiltration and MPO activity (Blank et al, 2000; Inturi et al, 2013; Jain et al, 2011b; Millard et al, 1997; Tewari-Singh et al, 2009; Vavra et al, 2004). However, this NM-induced increase in MPO activity was not observed in MPO KO mice confirming these mice were suitable for this study.…”

“…Our previous studies in mice have shown a profound increase in neutrophil infiltration and MPO activity in skin after dorsal exposure with CEES and NM at 12 h and 24 h time points (Inturi et al, 2013; Jain et al, 2011b; Tewari-Singh et al, 2009). To further investigate the role of the neutrophil-derived MPO enzyme in NM-induced skin injury, we employed a genetic approach using MPO KO mice where neutrophil infiltration and MPO activity were determined following NM exposure.…”

“…Previously, our studies have shown that CEES or NM exposure causes DNA damage leading to cell death of skin keratinocytes, and this could be a major contributor of skin injury (Inturi et al, 2013; Jain et al, 2011a; Pal et al, 2009; Tewari-Singh et al, 2010). Since MPO takes part in the production of free radicals which may cause DNA damage and apoptotic cell death, we examined the hypothesis that ablation of PMN and MPO activity would decrease the DNA damage and cell death following NM treatment.…”

“…We employed established NM-induced endpoints from our previous studies (Inturi et al, 2013 and unpublished data) in this study in C57BL/6J mice, since MPO KO mice were produced on a C57BL/6 background.…”

Nitrogen mustard exposure of murine skin induces DNA damage, oxidative stress and activation of MAPK/Akt-AP1 pathway leading to induction of inflammatory and proteolytic mediators

“…Their interaction with nucleic acids in DNA causes the formation of nonfunctional adducts and interstrand crosslinks resulting in DNA damage, which is the major lesion causing direct cytotoxicity or triggering downstream repair and/or cell death signaling pathways (Brimfield et al, 2009; Kehe and Szinicz, 2005). Studies with vesicating agents have shown that DNA damage and epidermal cell death involve the activation of p53-related signaling and DNA damage repair pathways, poly (ADP-ribose) polymerase (PARP), and calmodulin pathway (Inturi et al, 2013; Kehe et al, 2008; Korkmaz et al, 2006; Minsavage and Dillman, 2007; Rebholz et al, 2008; Ruff and Dillman, 2007; Tewari-Singh et al, 2010). Several reports indicate that SM exposure-induced skin toxic effects and DNA damage are associated with enhanced production of inflammatory cytokines including TNF α and chemokines, and increased oxidative stress as well nitric oxide production (Das et al, 2003; Kehe and Szinicz, 2005; Mukhopadhyay et al, 2006; Paromov et al, 2007), which could stimulate injury and escalate inflammatory responses (Kehe and Szinicz, 2005; Ruff and Dillman, 2007).…”