Alkylated epidermal creatine kinase as a biomarker for sulfur mustard exposure: comparison to adducts of albumin and DNA in an in vivo rat study

Abstract

Sulfur mustard (SM) is a chemical warfare agent which use is banned under international law and that has been used recently in Northern Iraq and Syria by the so-called Islamic State. SM induces the alkylation of endogenous proteins like albumin and hemoglobin thus forming covalent adducts that are targeted by bioanalytical methods for the verification of systemic poisoning. We herein report a novel biomarker, namely creatine kinase (CK) B-type, suitable as a local biomarker for SM exposure on the skin. Human and rat skin were proven to contain CK B-type by Western blot analysis. Following exposure to SM ex vivo, the CK-adduct was extracted from homogenates by immunomagnetic separation and proteolyzed afterwards. The cysteine residue Cys282 was found to be alkylated by the SM-specific hydroxyethylthioethyl (HETE)-moiety detected as the biomarker tetrapeptide TC(-HETE)PS. A selective and sensitive micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HRMS) method was developed to monitor local CK-adducts in an in vivo study with rats percutaneously exposed to SM. CK-adduct formation was compared to already established DNA- and systemic albumin biomarkers. CK- and DNA-adducts were successfully detected in biopsies of exposed rat skin as well as albumin-adducts in plasma. Relative biomarker concentrations make the CK-adduct highly appropriate as a local dermal biomarker. In summary, CK or rather Cys282 in CK B-type was identified as a new, additional dermal target of local SM exposures. To our knowledge, it is also the first time that HETE-albumin adducts, and HETE-DNA adducts were monitored simultaneously in an in vivo animal study.

Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro

“…Alignment of rmCK and hmCK using the Clustal Omega program (Sievers et al 2011) revealed an overall identity of the amino acid sequences of 96.6% with a complete matching of all Cys and Met positions. Cys 283 was recently identified as a target of alkylating agents including SM (Lüling et al 2021;Steinritz et al 2021). As Cys 283 in the active site of CK (Wang et al 2006) is supposed to bind creatine by electrostatic interactions between the free thiol-moiety of its side chain and the guanidine-group of creatine (Bong et al 2008), we assumed that a loss of CK activity is due to the alkylation of this residue after treatment with SM.…”

“…Our study confirms that Cys 283 of rmCK as well as diverse Met residues were alkylated by SM. The use of IM 70 (-HETE) TV and KSM 179 (-HETE)TE as biomarkers for the verification of SM exposure, in addition to the already reported use of TC 283 (-HETE)PS (Steinritz et al 2021), will be elaborated in future studies.…”

“…Cys 283 is located in the active site of CK and is susceptible by chemical modifications (Reddy et al 2000). After treatment of rmCK with SM, alkylated Cys 283 was detected present in the protonated tetrapeptide TC 283 (-HETE)PS ([M + H] + m/z 511.2) which is characterized by its product ions at m/z 105.037 and m/z 137.009 obtained by MS/HR MS (Lüling et al 2021;Steinritz et al 2021). Alkylation of Cys 74 , Cys 146 , and Cys 254 only occurred to a negligible extent (data not shown).…”

“…It was shown that alkylation of CK with iodoacetamide (IAA) or iodomethane, which both target free Cys residues, reduced the enzyme activity (Atherton et al 1970;Reddy and Watts 1978). Treatment of CK with the alkylating chemical warfare agent sulfur mustard (SM) resulted in the formation of the specific hydroxyethylthioethyl-(HETE)-moiety at Cys 283 (Lüling et al 2021;Steinritz et al 2021) but its effect on enzyme activity has not been addressed so far. This was investigated in the present study with particular focus on Cys 283 but also on reactive Met residues, that in principle might also be essential for the activity of certain enzymes (Rogers et al 1976) and were shown to be a potential target of alkylation by SM (Siegert et al 2019).…”

Alkylated glutamic acid and histidine derived from protein‐adducts indicate exposure to sulfur mustard in avian serum

“…Therefore, for legal as well as medical and forensic reasons, bioanalytical methods are mandatory to confirm exposure by the detection of biomarkers 6 . Those, derived from alkylation of endogenous proteins, for example, serum albumin (SA) by SM (adducts), are most valuable typically detected by micro liquid chromatography‐electrospray ionization tandem‐mass spectrometry (μLC‐ESI MS/MS) 2,7–12 . Diverse amino acids in proteins are known to be prone to alkylation especially including Cys, 9,10,12–14 Glu, 2 Met, 7,15 and His 16 .…”