Chlorine is a toxic industrial chemical with a history of use as a chemical weapon. Chlorine is also produced, stored, and transported in bulk making it a high-priority pulmonary threat in the USA. Due to the high reactivity of chlorine, few biomarkers exist to identify exposure in clinical and environmental samples. Our laboratory evaluates acute chlorine exposure in clinical samples by measuring 3-chlorotyrosine (Cl-Tyr) and 3,5-dichlorotyrosine (Cl2-Tyr) using liquid chromatography tandem mass spectrometry (LC-MS/MS). Individuals can have elevated biomarker levels due to their environment and chronic health conditions, but levels are significantly lower in individuals exposed to chlorine. Historically these biomarkers have been evaluated in serum, plasma, blood, and bronchoalveolar lavage (BAL) fluid. We report the expansion into hair and lung tissue samples using our newly developed tissue homogenization protocol which fits seamlessly with our current chlorinated tyrosine quantitative assay. Furthermore, we have updated the chlorinated tyrosine assay to improve throughput and ruggedness and reduce sample volume requirements. The improved assay was used to measure chlorinated tyrosine levels in 198 mice exposed to either chlorine gas or air. From this animal study, we compared Cl-Tyr and Cl2-Tyr levels among three matrices (i.e., lung, hair, and blood) and found that hair had the most abundant chlorine exposure biomarkers. Furthermore, we captured the first timeline of each analyte in the lung, hair, and blood samples. In mice exposed to chlorine gas, both Cl-Tyr and Cl2-Tyr were present in blood and lung samples up to 24 h and up to 30 days in hair samples.
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- 3,5 Cl2-Tyr:
liquid chromatography tandem mass spectrometry
Chemical Weapons Convention
Organisation for the Prohibition of Chemical Weapons
high-performance liquid chromatography
U.S. Food and Drug Administration
Centers for Disease Control and Prevention
Biomedical Advanced Research and Development Authority
American Veterinary Medical Association
Institutional Animal Care and Use Committee
mobile phase A
mobile phase B
- R 2 :
coefficient of determination
limit of detection
lower limit of quantitation
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We want to thank Tonya Tuberville and Stephanie Boles for their assistance in testing different homogenization techniques. Furthermore, we want to thank Samantha Isenberg for reviewing all the chromatography as a quality control officer and for providing insightful feedback.
This work was supported by the Centers for Disease Control and Prevention and the Center for Preparedness and Response (PID 14782). Development of a Murine Model of Pulmonary Chlorine Exposure Injury was funded by the Task Order contract HHSO100201500004I to Battelle Memorial Institute by Biomedical Advanced Research and Development Authority (BARDA).
Conflict of interest
The authors declare that they have no conflicts of interest.
All animals were maintained under the Battelle animal care and use program accredited by the Association for Assessment and Accreditation for Laboratory Animal Care International. This care and use program was in accordance with guidelines set forth in the “Guide for the Care and Use of Laboratory Animals,” National Research Council, and/or the regulations and standards promulgated by the Agricultural Research Service, United States Department of Agriculture (USDA), pursuant to the Laboratory Animal Welfare Act of August 24, 1966, as amended.
Source of biological materials
All biological materials were either purchased from vendors or gifted to the CDC as part of a collaborative effort between CDC, Battelle Memorial Institute, and BARDA aimed at the development of a murine model of pulmonary chlorine exposure injury.
The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention or the Biomedical Advanced Research and Development Authority. Use of trade names is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, or the US Department of Health and Human Services.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Data from the analysis of all animal samples is attached in the supplemental data table.
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Pantazides, B.G., Crow, B.S., Quiñones-González, J. et al. Development of a clinical assay to measure chlorinated tyrosine in hair and tissue samples using a mouse chlorine inhalation exposure model. Anal Bioanal Chem 413, 1765–1776 (2021). https://doi.org/10.1007/s00216-020-03146-x
- Chlorine gas
- In vivo mouse study
- Hair and lung tissue