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. 2024 Mar 20:6:100164.
doi: 10.1016/j.crtox.2024.100164. eCollection 2024.

Investigation of in vitro biotransformation of tris (1-chloro-2-propyl) phosphate and confirmation in human urine

Fatima den Ouden  1 , Andrea Estévez-Danta  2 , Lidia Belova  1 , Celine Gys  1 , Anna Klimowska  1   3 , Maarten Roggeman  1 , Natan Van Wichelen  1 , José Benito Quintana  2 , Rosario Rodil  2 , Giulia Poma  1 , Adrian Covaci  1
Affiliations

Investigation of in vitro biotransformation of tris (1-chloro-2-propyl) phosphate and confirmation in human urine

Fatima den Ouden et al. Curr Res Toxicol. .

Abstract

Tris (1-chloro-2-propyl) phosphate (TCIPP) is one of the major organophosphate flame retardants present in the indoor and outdoor environment. Knowledge of biotransformation pathways is important to elucidate potential bioavailability and toxicity of TCIPP and to identify relevant biomarkers. This study aimed to identify TCIPP metabolites through in vitro human metabolism assays and finally to confirm these findings in urine samples from an occupationally exposed population to propose new biomarkers to accurately monitor exposure to TCIPP. TCIPP was incubated with human liver microsomes and human liver cytosol to identify Phase I and Phase II metabolites, by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Using a suspect-screening approach, the established biomarkers bis (1-chloro-2-propyl) hydrogen phosphate (BCIPP) and 1-hydroxy-2-propyl bis (1-chloro-2-propyl) phosphate (BCIPHIPP) were identified. In addition, carboxyethyl bis (1-chloro-2-propyl) phosphate (TCIPP-M1), bis (1-chloropropan-2-yl) (-oxopropan-2-yl) phosphate (TCIPP-M2) and 1-chloro-3-hydroxypropan-2-yl bis (1-chloropropan-2-yl) phosphate (TCIPP-M3) were identified. TCIPP-M2, an intermediate product, was not reported before in literature. In urine samples, apart from BCIPP and BCIPHIPP, TCIPP-M1 and TCIPP-M3 were identified for the first time. Interestingly, BCIPP showed the lowest detection frequency, likely due to the poor sensitivity for this compound. Therefore, TCIPP-M1 and TCIPP-M3 could serve as potential additional biomarkers to more efficiently monitor TCIPP exposure in humans.

Keywords: Human exposome; Human liver cytosol; Human liver microsomes; In vitro metabolism; In vivo metabolism; TCIPP.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

None
Graphical abstract
Fig. 1
Fig. 1
Overview of the experimental set-up of the in vitro metabolism assay used in this study. Created with BioRender.com.
Fig. 2
Fig. 2
Time trend of tris (1-chloro-2-propyl) phosphate (TCIPP) biotransformation. On the y-axis the response ratio (area metabolite/area internal standard) is displayed, the x-axis shows the incubation time. Time trends of five detected metabolites are shown namely 1-hydroxy-2-propyl bis (1-chloro-2-propyl) phosphate (BCIPHIPP), bis (1-chloro-2-propyl) hydrogen phosphate (BCIPP), carboxyethyl bis (1-chloro-2-propyl) phosphate (TCIPP-M1), bis (1-chloropropan-2-yl) (-oxopropan-2-yl) phosphate (TCIPP-M2) and 1-chloro-3-hydroxypropan-2-yl bis (1-chloropropan-2-yl) phosphate (TCIPP-M3). All time points consist of three replicates. Error bars are plotted but are not always visible due to the small size. Error bars represent the standard deviation of three replicates at each time point for each metabolite.
Fig. 3
Fig. 3
Chromatogram [M + H]+ and molecular structure (top) and MS/MS spectrum of carboxy ethyl bis (1-chloro-2-propyl) phosphate (TCIPP-M1) at 10 eV (bottom) in HLM samples. Found m/z values, mass errors and proposed structures are indicated.
Fig. 4
Fig. 4
Chromatogram [M + H]+ and molecular structure (top) and MS/MS spectrum at 10 eV (bottom) of product bis (1-chloropropan-2-yl) (-oxopropan-2-yl) phosphate (TCIPP-M2) in HLM samples. Found m/z values, mass errors and proposed structures are indicated.
Fig. 5
Fig. 5
Chromatogram [M + H]+ (top) and MS/MS spectrum at 10 eV (bottom) of 1-chloro-3-hydroxypropan-2-yl bis (1-chloropropan-2-yl) phosphate (TCIPP-M3) in an HLM sample. Found m/z values, mass errors and proposed structures are indicated.
Fig. 6
Fig. 6
Proposed biotransformation pathway of tris (1-chloro-2-propyl) phosphate (TCIPP) including the metabolites 1-hydroxy-2-propyl bis (1-chloro-2-propyl) phosphate (BCIPHIPP), bis (1-chloro-2-propyl) hydrogen phosphate (BCIPP), bis (1-chloro-2-propyl) phosphate (TCIPP-M1), bis (1-chloropropan-2-yl) (-oxopropan-2-yl) phosphate (TCIPP-M2) and 1-chloro-3-hydroxypropan-2-yl bis (1-chloropropan-2-yl) phosphate (TCIPP-M3).
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