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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.

SUBMITTER: Pantazides BG

PROVIDER: S-EPMC8042785 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Chlorine is a widely available industrial chemical and involved in a substantial number of cases of poisoning. It has also been used as a chemical warfare agent in military conflicts. To enable forensic verification, the persistent biomarkers 3-chlorotyrosine and 3,5-dichlorotyrosine in biomedical samples could be detected. An important shortfall of these biomarkers, however, is the relatively high incidence of elevated levels of chlorinated tyrosine residues in individuals with inflammatory diseases who have not been exposed to chlorine. Therefore, more reliable biomarkers are necessary to distinguish between endogenous formation and exogeneous exposure. The present study aims to develop a novel diagnostic tool for identifying site-specific chlorinated peptides as a more unambiguous indicator of exogeneous chlorine exposure. Human blood plasma was exposed in vitro to various chlorine concentrations, and the plasma proteins were subsequently digested by pronase, trypsin, or pepsin. After sample preparation, the digests were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). In line with other studies, low levels of 3-chlorotyrosine and 3,5-dichlorotyrosine were found in blank plasma samples in this study. Therefore, 50 site-specific biomarkers were identified, which could be used as more unambiguous biomarkers for chlorine exposure. Chlorination of the peptides TY*ETTLEK, Y*KPGQTVK, Y*QQKPGQAPR, HY*EGSTVPEK, and Y*LY*EIAR could already be detected at moderate in vitro chlorine exposure levels. In addition, the latter two peptides were found to have dichlorinated fragments. Especially, Y*LY*EIAR, with a distinct chlorination pattern in the MS spectra, could potentially be used to differentiate exogeneous exposure from endogenous causes as other studies reported that this part of human serum albumin is nitrated rather than chlorinated under physiological conditions. In conclusion, trypsin digestion combined with high-resolution MS analysis of chlorinated peptides could constitute a valuable technique for the forensic verification of exposure to chlorine.

Project description:BackgroundPre-exposure prophylaxis (PrEP) trials using tenofovir-based regimens have demonstrated that high levels of adherence are required to evaluate efficacy; the incorporation of objective biomarkers of adherence in trial design has been essential to interpretation, given the inaccuracy of self-report. Antiretroviral measurements in scalp hair have been useful as a marker of long-term exposure in the HIV treatment setting, and hair samples are relatively easy and inexpensive to collect, transport, and store for analysis. To evaluate the relationship between dose and tenofovir concentrations in hair, we examined the dose proportionality of tenofovir in hair in healthy, HIV-uninfected adults.MethodsA phase I, crossover pharmacokinetic study was performed in 24 HIV-negative adults receiving directly-observed oral tenofovir tablets administered 2, 4, and 7 doses/week for 6 weeks, with a ≥3-week break between periods. Small samples of hair were collected after each six-week period and analyzed for tenofovir concentrations. Geometric-mean-ratios compared levels between each pair of dosing conditions. Intensive plasma pharmacokinetic studies were performed during the daily-dosing period to calculate areas-under-the-time-concentration curves (AUCs).ResultsOver 90% of doses were observed per protocol. Median tenofovir concentrations in hair increased monotonically with dose. A log-linear relationship was seen between dose and hair levels, with an estimated 76% (95% CI 60-93%) increase in hair level per 2-fold dose increase. Tenofovir plasma AUCs modestly predicted drug concentrations in hair.ConclusionsThis study found a strong linear relationship between frequency of dosing and tenofovir levels in scalp hair. The analysis of quantitative drug levels in hair has the potential to improve adherence measurement in the PrEP field and may be helpful in determining exposure thresholds for protection and explaining failures in PrEP trials. Hair measures for adherence monitoring may also facilitate adherence measurement in real-world settings and merit further investigation in upcoming PrEP implementation studies and programs.Trial registrationClinicalTrials.gov NCT00903084.

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

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