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. 2024 May 24;12(6):384.
doi: 10.3390/toxics12060384.

GC/MS-Based Metabolomic Analysis of A549 Cells Exposed to Emerging Organophosphate Flame Retardants

Mengyao Sun  1   2 , Xiao Chang  3 , Ying Gao  2 , Sisi Zou  2 , Shaomin Wang  2 , Hongmin Liu  4
Affiliations

GC/MS-Based Metabolomic Analysis of A549 Cells Exposed to Emerging Organophosphate Flame Retardants

Mengyao Sun et al. Toxics. .

Abstract

Emerging organophosphate flame retardants (eOPFRs) have attracted attention in recent times and are expected to gain extensive usage in the coming years. However, they may have adverse effects on organisms. Due to their novel nature, there are few relevant articles dealing with toxicological studies of the above eOPFRs, especially their information on the perturbation of cellular metabolism, which is, thus far, marginally understood. Our research initially assessed the cytotoxicity of eOPFRs, which include compounds like cresyl diphenyl phosphate (CDP), resorcinol bis(diphenyl phosphate) (RDP), triallyl phosphate (TAP), and pentaerythritol phosphate alcohol (PEPA). This evaluation was conducted using the methyl thiazolyl tetrazolium (MTT) assay. Subsequently, we utilized a gas chromatography/mass spectrometry (GC/MS)-based metabolomic approach to investigate the metabolic disruptions induced by these four eOPFRs in A549 cells. The MTT results showed that, at high concentrations of 1 mM, their cytotoxicity was ranked as CDP > TAP > RDP > PEPA. In addition, metabolic studies at low concentrations of 10 μM showed that the metabolic interference of CDP, TAP, and PEPA focuses on oxidative stress, amino acid metabolism, and energy metabolism, while RDP mainly affects energy metabolism-galactose metabolism and gluconeogenesis. Therefore, from the perspective of cytotoxicity and metabolic analysis, RDP may be a more promising alternative. Our experiments provide important insights into the possible metabolic effects of potential toxic substances and complement the evidence on the human health risks of eOPFRs.

Keywords: A549 cells; cytotoxicity; emerging organophosphate flame retardants; gas chromatography/mass spectrometry; metabolomic analysis.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
The dose-response curves revealed the cytotoxic effects of eOPFRs on A549 cells, which were fitted using a nonlinear model (inhibitor vs. normalized response—variable slope). The cell viability of A549 cells was evaluated using the MTT assay, with the results expressed as the mean ± SEM (n ≥ 3).
Figure 2
Figure 2
PCA of features derived from GC/MS metabolic profile in different eOPFR treatments: (A) solvent control group vs. CDP treatment group (R2X (cum) = 0.664, Q2 (cum) = 0.260); (B) solvent control group vs. RDP treatment group (R2X (cum) = 0.671, Q2 (cum) = 0.217); (C) solvent control group vs. TAP treatment group (R2X (cum) = 0.609, Q2 (cum) = 0.153); and (D) solvent control group vs. PEPA treatment group (R2X (cum) = 0.551, Q2 (cum) = 0.123). Note: All of the above PCA scores plots have 2 components.
Figure 3
Figure 3
OPLS-DA score plots of GC/MS data derived from the spectra of the cell sample in different eOPFR treatments and validation models for the supervised models generated from cell samples. (A) OPLS-DA scores plot for control and CDP-treated group (1 orthogonal and 1 predictive components, R2X (cum) = 0.660, R2Y (cum) = 0.954, Q2 (cum) = 0.831); (B) OPLS-DA scores plot for control and RDP-treated groups (1 orthogonal and 1 predictive components, R2X (cum) = 0.614, R2Y (cum) = 0.988, Q2 (cum) = 0.945); (C) OPLS-DA scores plot for control and TAP-treated groups (1 orthogonal and 1 predictive components, R2X (cum) = 0.583, R2Y (cum) = 0.995, Q2 (cum) = 0.934); and (D) OPLS-DA scores plot for control and PEPA-treated groups (1 orthogonal and 1 predictive components, R2X (cum) = 0.530, R2Y (cum) = 0.994, Q2 (cum) = 0.908).
Figure 4
Figure 4
Venn diagram of potential biomarkers of eOPFRs acting on A549 cells.
Figure 5
Figure 5
Summary plot of metabolic pathway analysis of eOPFRs treatments performed on A549 cells: (A) control group vs. CDP treatments; (B) control group vs. RDP treatments; (C) control group vs. TAP treatments; and (D) control group vs. PEPA treatments. (a) D-glutamine and D-glutamate metabolism; (b) glycine, serine, and threonine metabolism; (c) alanine, aspartate, and glutamate metabolism; (d) arginine and proline metabolism; (e) arginine biosynthesis; (f) glutathione metabolism; (g) glyoxylate and dicarboxylate metabolism; (h) inositol phosphate metabolism; (i) phenylalanine, tyrosine, and tryptophan biosynthesis; (j) phenylalanine metabolism; and (k) cysteine and methionine metabolism.
Figure 6
Figure 6
Summary plots of metabolite enrichment analysis of eOPFRs treatments performed on A549 cells (bar charts and network graphs): (A) control group vs. CDP treatments; (B) control group vs. RDP treatments; (C) control group vs. TAP treatments; and (D) control group vs. PEPA treatments.
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