Glutathione Conjugation of Nitrogen Mustards: In vitro Study.

RATIONALE

This paper describes an in vitro study designed to identify metabolic biomarkers resulting from the conjugation of nitrogen mustards (NMs) with glutathione (GSH). The method developed is essential in providing evidence in the event of NM exposure in biomedical samples.

METHODS

The mass spectral characterization of the proposed NMs-GSH conjugates was performed with liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). The final reaction mixtures were analysed in the positive electrospray ionisation (ESI) at different incubation times.

RESULTS

This study identified three conjugates in addition to ethanolamines (EAs), the hydrolysis products of NMs. Monoglutathionyl, diglutathionyl and phosphorylated conjugates were produced for each of the NMs, bis(2-chloroethyl)ethylamine (HN1), bis(2-chloroethyl)methylamine (HN2), and tris(2-chloroethyl)amine (HN3). The monoglutathionyl conjugates consisted of HN1-GSH, HN2-GSH and HN3-GSH. The spontaneous and primary conjugates of diglutathionyl were HN1-GSH2, HN2-GSH2, and HN3-GSH2. These included phosphorylated conjugates, namely HN1-GSH-PO₄, HN2-GSH-PO₄, and HN3-GSH-PO₄ as they might have formed due to hydrolysis in phosphate buffer.

CONCLUSIONS

The mass spectral data of all conjugates formed in the presence of all NMs and GSH is reported in this study. These GSH metabolites can be used to confirm the NMs toxicity in biological samples such as urine.