Cross-linking of thioredoxin reductase by the sulfur mustard analogue mechlorethamine (methylbis(2-chloroethyl)amine) in human lung epithelial cells and rat lung: selective inhibition of disulfide reduction but not redox cycling

Chem Res Toxicol. 2014 Jan 21;27(1):61-75. doi: 10.1021/tx400329a. Epub 2013 Dec 9.


Oxidative stress plays a key role in mechlorethamine (methylbis(2-chloroethyl)amine, HN2) toxicity. The thioredoxin system, consisting of thioredoxin reductase (TrxR), thioredoxin, and NADPH, is important in redox regulation and protection against oxidative stress. HN2 contains two electrophilic side chains that can react with nucleophilic sites in proteins, leading to changes in their structure and function. We report that HN2 inhibits the cytosolic (TrxR1) and mitochondrial (TrxR2) forms of TrxR in A549 lung epithelial cells. TrxR exists as homodimers under native conditions; monomers can be detected by denaturing and reducing SDS-PAGE followed by western blotting. HN2 treatment caused marked decreases in TrxR1 and TrxR2 monomers along with increases in dimers and oligomers under reducing conditions, indicating that HN2 cross-links TrxR. Cross-links were also observed in rat lung after HN2 treatment. Using purified TrxR1, NADPH reduced, but not oxidized, enzyme was inhibited and cross-linked by HN2. LC-MS/MS analysis of TrxR1 demonstrated that HN2 adducted cysteine- and selenocysteine-containing redox centers forming monoadducts, intramolecule and intermolecule cross-links, resulting in enzyme inhibition. HN2 cross-links two dimeric subunits through intermolecular binding to cysteine 59 in one subunit of the dimer and selenocysteine 498 in the other subunit, confirming the close proximity of the N- and C-terminal redox centers of adjacent subunits. Despite cross-linking and inhibition of TrxR activity by HN2, TrxR continued to mediate menadione redox cycling and generated reactive oxygen species. These data suggest that disruption of the thioredoxin system contributes to oxidative stress and tissue injury induced by HN2.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cells, Cultured
  • Cross-Linking Reagents / chemistry*
  • Cross-Linking Reagents / metabolism
  • Cross-Linking Reagents / pharmacology*
  • Disulfides / antagonists & inhibitors*
  • Disulfides / chemistry
  • Disulfides / metabolism
  • Epithelial Cells / drug effects*
  • Epithelial Cells / enzymology
  • Epithelial Cells / metabolism
  • Humans
  • Lung / cytology*
  • Lung / drug effects*
  • Lung / enzymology
  • Lung / metabolism
  • Male
  • Mechlorethamine / chemistry*
  • Mechlorethamine / metabolism
  • Mechlorethamine / pharmacology*
  • Models, Molecular
  • Oxidation-Reduction / drug effects
  • Rats
  • Rats, Wistar
  • Thioredoxin-Disulfide Reductase / antagonists & inhibitors*
  • Thioredoxin-Disulfide Reductase / chemistry*
  • Thioredoxin-Disulfide Reductase / metabolism


  • Cross-Linking Reagents
  • Disulfides
  • Mechlorethamine
  • Thioredoxin-Disulfide Reductase