In Vitro Metabolism of Isopropylated and tert-Butylated Triarylphosphate Esters Using Human Liver Subcellular Fractions

Abstract

Isopropylated and tert-butylated triarylphosphate esters (ITPs and TBPPs, respectively) are plasticizers and flame retardants that are ubiquitous in indoor environments; however, no studies to date have characterized their metabolism. Using human liver subcellular S9 fractions, phase I and II in vitro metabolism of triphenyl phosphate (TPHP), 4-tert-butylphenyl diphenyl phosphate (4tBPDPP), 2-isopropylphenyl diphenyl phosphate (2IPPDPP), and 4-isopropylphenyl diphenyl phosphate (4IPPDPP) was investigated at 1 and 10 μM doses. Parent depletion and the formation of known or suspected metabolites (e.g., likely hydrolysis or hydroxylated products), including diphenyl phosphate (DPHP), hydroxyl-triphenyl phosphate (OH-TPHP), isopropylphenyl phenyl phosphate (ip-PPP), and tert-butylphenyl phenyl phosphate (tb-PPP), were monitored and quantified via GC/MS or LC-MS/MS. tb-PPP and its conjugates were identified as the major in vitro metabolites of 4tBPDPP and accounted for 71% and 49%, respectively, of the parent molecule that was metabolized during the incubation. While the mass balance between parents and metabolites was conserved for TPHP and 4tBPDPP, approximately 20% of the initial parent mass was unaccounted for after quantifying suspected metabolites of 2IPPDPP and 4IPPDPP that had authentic standards available. Two novel ITP metabolites, mono-isopropenylphenyl diphenyl phosphate and hydroxy-isopropylphenyl diphenyl phosphate, were tentatively identified by high-resolution mass spectrometry and screened for in recently collected human urine where mono-isopropenylphenyl diphenyl phosphate was detected in one of nine samples analyzed. This study provides insight into the biological fate of ITP and TBPP isomers in human tissues and is useful in identifying appropriate biomarkers of exposure to monitor, particularly in support of epidemiological studies.

Figure 1.

Depletion rates are calculated in kinetic experiments for each compound. A: Assuming zero-order kinetics; B: Assuming first-order kinetics. Means (n=2) are plotted for each analyte and bars depict standard error. Slope lines of best fit are plotted and were calculated using linear regression; 95% confidence intervals for the best-fit lines are also shown by dashed lines. Numerical depletion rates are found in Table 2.

Figure 2.

In Vitro Biotransformation of TPHP. A: 1 μM; B: 10 μM. Means (n=3) are plotted for each analyte and error bars depict standard deviation of the total mass balance for each treatment. A dashed line separates inactive and active treatments. Total mass was significantly different from the unprocessed spike for the +s9 treatment (ANOVA; p <0.0001).

Figure 3.

In Vitro Biotransformation of 4tBPDPP. A: 1 μM; B: 10 μM. Means (n=3) are plotted for each analyte and error bars depict standard deviation of the total mass balance for each treatment. A dashed line separates inactive and active treatments. Total mass was not significantly different for any of the treatment groups compared to the unprocessed spike.

Figure 4.

In Vitro Biotransformation of 2IPPDPP. A: 1 μM; B: 10 μM. Means (n=3) are plotted for each analyte and error bars depict standard deviation of the total mass balance for each treatment. A dashed line separates inactive and active treatments. Asterisks denote a significant difference in total mass compared to the unprocessed spike (ANOVA; p <0.05).

Figure 5.

In Vitro Biotransformation of 4IPPDPP. A: 1 μM; B: 10 μM. Means (n=3) are plotted for each analyte and error bars depict standard deviation of the total mass balance for each treatment. A dashed line separates inactive and active treatments. Asterisks denote a significant difference in total mass compared to the unprocessed spike (ANOVA; p <0.05).

Figure 6.

Q Exactive GC Orbitrap GC-MS/MS chromatograms and spectra used to identify novel ITP metabolites with ThermoFisher Excalibur software. A-E are from 2IPPDPP in vitro metabolism samples and F-J are from 4IPPDPP in vitro metabolism samples. A/F: total ion chromatogram (TIC); B/G: extracted ion chromatogram (EIC; m/z = 365.5–366.5); C/H: observed mass spectra (RT = 14.21 or 16.32 min); D/I: simulated mass spectra for C₂₁H₁₉O₄P (exact mass = 366.10155); E: structure of mono-(o-isopropenylphenyl)-diphenyl phosphate and identification information (RT = 14.21 min, exact mass = 366.1015, δppm = −0.29); J: structure of mono-(p-isopropenylphenyl)-diphenyl phosphate and identification information (RT = 16.31 min, exact mass = 366.1015, δppm = −0.44).

Figure 7.

Orbitrap Fusion LC-MS chromatograms and spectra used to identify novel ITP metabolites with ThermoFisher Excalibur software. A-E are from 2IPPDPP in vitro metabolism samples and F-J are from 4IPPDPP in vitro metabolism samples. A/F: total ion chromatogram (TIC); B/G: extracted ion chromatogram (EIC; m/z = 385.0–385.5); C/H: observed mass spectra (RT = 7.05 or 7.11 min); D/I: simulated mass spectra for C₂₁H₂₂O₅P (exact mass = 385.11994); E: structure of hydroxy-o-isopropylphenyl diphenyl phosphate and identification information (RT = 7.05 min, exact mass = 385.1201, δppm = 0.31); J: structure of hydroxy-p-isoproplylphenyl diphenyl phosphate and identification information (RT = 7.11 min, exact mass = 385.1200, δppm = 0.23).While hydroxylation is shown in each of the structures to occur at the alkylated phenyl ring, it could also occur at an unsubstituted ring or at the isopropyl group.