The Inflammatory Lipid, 2‐Chlorohexadecanal: Cell Toxicity and Potential Scavenger Protection

Hypochlorous acid is produced by either leukocyte myeloperoxidase activity or from chlorine gas reactions with water. 2‐Chlorofatty aldehydes (2‐ClFALD) are: 1) formed when hypochlorous acid attacks the plasma membrane phospholipid plasmalogen molecular subclass and 2) produced following leukocyte activation as well in the lung of mice exposed to chlorine gas. The biological role of 2‐ClFALD is largely unknown. Recently, our lab showed the electrophilic α‐chlorinated carbon of the 2‐ClFALD molecular species, 2‐chlorohexadecanal (2‐ClHDA) is targeted by nucleophilic attack of the cysteinyl‐sulfhydryl group of glutathione. Here, we synthesized an alkyne analog of 2‐ClHDA (2‐ClHDyA) to demonstrate 2‐ClHDyA protein modification in RAW 264.7 cells using click chemistry strategies. Furthermore, confocal microscopy showed that 2‐ClHDyA co‐localizes to the endoplasmic reticulum, Golgi, and mitochondria. Additionally, diffuse distribution of 2‐ClHDyA suggested cytoplasmic protein modification. The intensity of the fluorescence signal of 2‐ClHDyA increased in a dose‐dependent manner suggesting increased protein modification. Cell viability/metabolic activity assessed by 3‐(4,5‐Dimethyl‐2‐Thiazolyl)‐2,5‐Diphenyl‐2H‐Tetrazolium Bromide (MTT) test revealed that 2‐ClHDA reduced RAW 264.7 cell viability in a dose‐dependent manner. Cell viability was further reduced in RAW 264.7 cells treated with 2‐ClHDA in DMEM with Fetal Bovine Serum (FBS) supplementation. Click chemistry showed 2‐ClHDyA modified FBS proteins. These observations suggested 2‐ClFALD modified proteins might have accounted for cell death via cell signaling outside or inside the cell. Next, we investigated traditional aldehyde sequesterers including N‐acetyl cysteine (NAC), carnosine, and aminoguanidine to identify potential protectors that would prevent cellular 2‐ClFALD‐protein interaction resulting in reduced toxicity caused by 2‐ClFALD. Among tested sequesterers, NAC proved to be an effective sequesterer of 2‐ClHDA. NAC prevented 2‐ClHDyA interaction with FBS proteins and rescued cell death significantly. Next, we characterized the conjugate of 2‐ClHDA and NAC using direct infusion electrospray mass spectrometry. The conjugate appeared to be a result of a nucleophilic substitution reaction where nucleophilic sulfur of NAC attacked the alpha carbon of 2‐ClHDA leading to the release of chloride ion. These studies demonstrate that the interaction of 2‐ClFALD with cellular proteins with free sulfhydryl groups cause toxicity and the interaction of 2‐ClFALD with N‐acetyl cysteine may protect against toxicity caused by 2‐ClFALD. Support or Funding Information NIH grants: R01 GM115553, R01 GM129508, U01 ES026458, U01 ES027697 and U01 ES028182