Assessment of the time-dependent dermatotoxicity of mechlorethamine using the mouse ear vesicant model

Abstract

Mechlorethamine (HN2) is an alkylating agent and sulfur mustard gas mimetic which is also used in anticancer therapy. HN2 is associated with skin inflammation and blistering which can lead to secondary infections. The purpose of the present study was to investigate the time-dependent dermatotoxicity of HN2 using the mouse ear vesicant model (MEVM). To this end, our operational definition of dermatotoxicity included tissue responses to HN2 consistent with an increase in the wet weights of mouse ear punch biopsies, an increase in the morphometric thickness of H&E stained ear sections and histopathologic observations including tissue edema, inflammatory cell infiltration and vesication. The ears of male Swiss Webster mice were topically exposed to a single dose of HN2 (0.5 μmol/ear) or DMSO vehicle (5 μl/ear) or left untreated (naive). Mice were then euthanized at 15 min, 1, 2, 4, 8 or 24 hr following HN2 exposure. Compared to control ears, mouse ears exposed to HN2 at all time points showed an increase in wet weights, morphometric thickness, edema, inflammatory cell infiltration and signs of vesication. The incidence in tissue vesication sharply increased between 4 and 8 hr after exposure, revealing that tissue vesication is well established by 8 hr and remains elevated at 24 hr after exposure. It is noteworthy that, compared to control ears, mouse ears treated with DMSO vehicle alone also exhibited an increase in wet weights and morphometric thickness at 15 min, 1, 2 and 4 hr following treatment; however, these vehicle effects begin to subside after 4 hr. The results obtained here using the MEVM provide a more holistic understanding of the kinetics of vesication, and indicate that time points earlier than 24 hr may prove useful not only for investigating the complex mechanisms involved in vesication but also for assessing the effects of vesicant countermeasures.

Combination of ebselen and hydrocortisone substantially reduces nitrogen mustard-induced cutaneous injury

“…At least one of the cutaneous responses to HN2 observed in mouse skin involves increased tissue expression of MMP-9, a matrix metalloproteinase that directly degrades extracellular matrix (ECM) proteins and activates cytokines and chemokines to regulate tissue remodeling in response to chemical injury. Our lab ( Cuffari et al, 2018 , Tumu et al, 2020 ) and others ( Kumar et al, 2015 , Wormser et al, 2002 ) have found that HN2 increases the expression of MMP-9 in mouse skin. In addition, we ( Tumu et al, 2020 ) and others ( Biyashev et al, 2020 , Composto et al, 2016 , Kumar et al, 2015 ) have observed that HN2 increases the tissue expression of inducible nitric oxide synthase (iNOS) in mouse skin, which, in turn, is important for generating cytotoxic levels of nitric oxide in the skin ( Composto et al, 2016 , Nguyen and Soulika, 2019 ).…”

“…In the present study, we used the MEVM to investigate cutaneous responses to HN2. We observed and confirmed that 24 hr after a single topical application of HN2, mouse ear skin exhibited massive immune cell infiltration, necrosis, increased TUNEL activity (an indicator of DNA injury), an upregulation of the tissue injury-associated matrix metalloproteinase 9 (MMP-9) ( Cuffari et al, 2018 , Kumar et al, 2015 , Tumu et al, 2020 , Wormser et al, 2002 ) and an increased tissue presence of the proinflammatory enzyme inducible nitric oxide synthase (iNOS) ( Biyashev et al, 2020 , Composto et al, 2016 , Kumar et al, 2015 , Tumu et al, 2020 ). MMP-9 is a proteolytic enzyme capable of degrading extracellular matrix proteins such as denatured collagen (gelatin) and collagen types IV, V, VII, IX and X ( van der Jagt et al, 2006 ).…”

“…When mouse ear skin is exposed to HN2, robust cutaneous responses including hyperplasia, neutrophil infiltration, DEJ disruption (vesication) and epidermal necrosis are observed within a 24 hr period ( Casillas et al, 1997 , Casillas et al, 2000 , Cuffari et al, 2018 , Dachir et al, 2002 , Tumu et al, 2020 ). In our studies, DMSO was used as the vehicle for both HN2 (vesicant) and the test compounds (EB-1 and HC).…”

“…When 5 µl of this solution is applied to the inner surface of a mouse ear, it is equal to a HN2 dose of 0.5 μmol/ear. This topical dose was used here because it has previously been found by our laboratory to robustly induce the formation of microblisters in Swiss Webster mice ( Cuffari et al, 2018 , Lulla et al, 2014 , Tumu et al, 2020 ) and C57BL mice ( Sparavalo, 2013 ). Ebselen: Five microliter (5 μl) volumes of 0.025 mg/μl, 0.037 mg/μl, 0.050 mg/μl, 0.075 mg/μl and 0.100 mg/μl EB-1 test solutions were applied to the ear (inner, ventral surface) to obtain the test doses of 0.125 mg/ear, 0.187 mg/ear, 0.250 mg/ear, 0.375 mg/ear and 0.500 mg/ear, respectively.…”

“…When 5 µl of this solution is applied to the inner surface of a mouse ear, it is equal to a HN2 dose of 0.5 μmol/ear. This topical dose was used here because it has previously been found by our laboratory to robustly induce the formation of microblisters in Swiss Webster mice ( Cuffari et al, 2018 , Lulla et al, 2014 , Tumu et al, 2020 ) and C57BL mice ( Sparavalo, 2013 ).…”