Exposure to chlorine gas (Cl2) presents a significant threat to public health. Chlorine is a leading chemical produced by volume in the US. Exposure to Cl2 has occurred as a result of train derailments, accidental misuse by swimming pool maintenance workers, accidents at water treatment facilities, and chemical warfare. Intentional exposure is a major concern since Cl2 is both a chemical warfare agent and potential chemical terror agent. Diagnostic biomarkers are needed to determine the extent of Cl2 exposure to humans and to predict outcomes, which ultimately could lead to improved therapeutic support and countermeasures. The Ford lab discovered that both Cl2 and HOCl target host plasmalogen lipids, resulting in 2-chlorofatty aldehyde (2-CLFALD) and 2-chlorofatty acid (2-CLFA) production. These chlorinated lipids accumulate to robust levels in lung and plasma of mice exposed to sub-lethal amounts of chlorine gas. In recent unpublished studies we have shown plasma 2-CLFA levels are elevated 40-fold in a five-subject cohort of humans exposed to chlorine at a water treatment facility near Birmingham, Alabama. Taken together, we have shown chlorinated lipids derived from plasmalogen oxidation are currently the best biomarkers of Cl2 exposure to humans and have the potential to be used to predict future outcomes that could lead to improved care following exposure.!Plasma chlorinated lipids are quantified using LC/MS detection employing a triple quadrupole instrument. The dependence on LC/MS detection is a limitation in implementing field analyses of Cl2 exposure using these biomarkers. The proposed studies will address this problem by identifying chlorolipid-modified proteins produced following Cl2 exposure. Future studies producing antibodies to chlorolipid-modified proteins could then be used to develop diagnostic kits. It is proposed that chlorinated lipids covalently modify proteins. The identification of specific proteins and amino acid residues modified by chlorinated lipids will be used in future studies to develop antibodies that can be used for rapid detection assays. Additionally, it is envisioned that identifying chlorinated lipid-modified proteins as a result of Cl2 exposure may provide new targets for future investigations to develop countermeasures to Cl2 exposure. There are three specific aims. Specific Aim 1 will identify amino acid motifs and proteins modified by 2-CLFALD. Specific Aim 2 will identify amino acid motifs and proteins modified by 2-CLFA. Specific Aim 3 will identify chlorinated lipid-modified proteins in lung and plasma from mice exposed to Cl2. These studies will identify chlorinated lipid-modified proteins, and show that they are present in plasma of Cl2 exposed mice. Furthermore, these proposed R21 studies have the potential to lead to new investigations in the future to develop improved countermeasures for, and improve outcomes of, Cl2 exposure.

The proposed studies will discover proteins modified by chlorinated lipids that are biomarkers of chlorine gas exposure. Ultimately, chlorinated lipid-modified proteins can be used to create antibodies to be used in rapid detection assays as well as to probe as new countermeasure therapeutic targets.