Development of a clinical assay to measure chlorinated tyrosine in hair and tissue samples using a mouse chlorine inhalation exposure model

Abstract

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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Abbreviations

Cl-Tyr:

3-chlorotyrosine

3,5 Cl2-Tyr:

3,5-dichlorotyrosine

LC-MS/MS:

liquid chromatography tandem mass spectrometry

BAL:

bronchoalveolar lavage

CWC:

Chemical Weapons Convention

OPCW:

Organisation for the Prohibition of Chemical Weapons

HPLC:

high-performance liquid chromatography

FDA:

U.S. Food and Drug Administration

ISTD:

internal standard

QC:

quality control

QCL:

QC low

QCM:

QC mid

QCH:

QC high

MB:

matrix blank

CDC:

Centers for Disease Control and Prevention

BARDA:

Biomedical Advanced Research and Development Authority

UV:

ultraviolet

AVMA:

American Veterinary Medical Association

IACUC:

Institutional Animal Care and Use Committee

MPA:

mobile phase A

MPB:

mobile phase B

R 2 :

coefficient of determination

LOD:

limit of detection

LLOQ:

lower limit of quantitation

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Acknowledgments

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.

Funding

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).

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Corresponding author

Correspondence to Brian S. Crow.

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Conflict of interest

The authors declare that they have no conflicts of interest.

Ethics approval

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.

Disclaimer

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.

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Supplementary information

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

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Keywords

  • Chlorine gas
  • 3-Chlorotyrosine
  • 3,5-Dichlorotyrosine
  • In vivo mouse study
  • LC-MS/MS
  • Hair and lung tissue