Metabolites of Organophosphate Flame Retardants and 2‑Ethylhexyl Tetrabromobenzoate in Urine from Paired Mothers and Toddlers

As a result of the polybrominated diphenyl ether (PBDE) ban in the mid-2000s, the chemical flame retardant market has moved toward alterative compounds including chlorinated alkyl and nonchlorinated aryl organophosphate flame retardants (OPFRs) as well as aromatic brominated compounds such as Firemaster 550 (FM550). Recent studies have shown that the OPFRs and Firemaster 550 components are frequently detected in polyurethane foams and in indoor dust. Some OPFRs are considered carcinogenic and/or neurodevelopmental toxicants, and children’s exposure to these compounds is a concern. OPFRs are readily metabolized and excreted in the urine as their dialkyl and diaryl compounds which function as biomarkers for OPFR exposure. Limited research has shown that adults are broadly exposed to OPFRs, but nothing is known about children’s exposure. Similarly, 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB), a FM550 component, is metabolized to tetrabromobenzoic acid (TBBA). The current study measured levels of bis­(1,3-dichloro-2-propyl) phosphate (BDCIPP), bis­(1-chloro-2-propyl) phosphate (BCIPP), diphenyl phosphate (DPHP), 2 alkylated DPHPs, and TBBA in urine collected in 2013 from 21 US mother-toddler pairs. BDCIPP, DPHP, and ip-DPHP were detected in 100%, 98%, and 96% of all individuals, whereas BCIPP and <i>tert</i>-butyl-DPHP (tb-DPHP) were only detected in 8% and 13%. Further, TBBA was detected in 27% of adults but 70% of children. Overall, children had higher urinary levels of BDCIPP, DPHP, ip-DPHP, and TBBA as compared to their mothers, suggesting higher exposure. For example, on average, BDCIPP levels in children were 4.9 times those of mothers. BDCIPP and DPHP levels in mother’s urine were also significantly correlated with levels in children’s urine, suggesting similar exposure routes, likely in the home environment. Various potential predictors of OPFR exposure were assessed using a questionnaire. In children some predictors of hand-mouth exposure were associated with elevated BDCIPP and DPHP levels (e.g., less frequent hand washing for BDCIPP). Overall, these trends are consistent with higher flame retardant levels in children as a result of increased hand-mouth behavior and elevated dust exposure

48 h PE fluorescence (CD38 surface expression) histograms for RA-resistant HL60 cells over 84 days.

<p>RA-resistant HL60 cells were labeled with PE-conjugated anti-CD38 and analyzed by flow cytometry. RA-resistant HL60 grown continuously in RA initially display elevated CD38 expression (histogram located in the gate P2). A growing subpopulation that does not express CD38, despite continual RA exposure, is indicated (by an arrow) as a low signal peak (top left graph). Over the course of 84 days, the number of cells that do not express CD38 continues to increase. The two distinct RA-resistant HL60 cells lines (bottom right graph), one with RA-inducible CD38 expression and one that has lost RA-inducible CD38 expression, were separated using Fluorescent Activated Cell Sorting (FACS) and the separated cell lines were termed R38+ and R38−, respectively.</p

Wright’s stain cytology for control and treated WT, R38+ and R38− HL60 cell lines.

<p>Control WT cells are round; RA-treated R38+ and R38− also retain a round, stem-like appearance. WT HL60 cells showed morphological changes consistent with differentiation toward granulocytes when treated with RA, PP2, or PP2+RA. Meanwhile the two RA-resistant HL60 cell lines, R38+ and R38−, both showed morphological changes consistent with differentiation only during PP2 and PP2+RA treatment, but not RA treatment alone.</p

Diagrams of the signaling proteins investigated in this study for each treatment case in R38+ and R38− compared to the RA-treated WT HL60 cells.

<p>For a given protein, solid black arrows indicate increased expression while white arrows indicate increased phosphorylation. Dashed black lines indicate an interaction that has previously been demonstrated in this lab by immunoprecipitation and/or FRET, with the exception of Fgr:Slp76 and Fgr:c-Raf. These two interactions are implied by the known binding of Slp76 and c-Raf to Src-family kinase members. Dashed black lines with arrowheads indicate phosphorylation (kinase) events known in the literature. Solid-filled factors vs. white-filled factors serve to clarify the expression indicated by the black arrows. Gradient-filled factors indicate expression but to a lower level than the RA-treated WT case. Downstream effects (CD11b expression, p47^phox expression, etc) are written in black if they occur, grey if they do not occur, or in gradient if they occur but to a lesser extent than the RA-treated WT HL60 case. A: In RA-treated WT HL60, CD38 is upregulated, along with its intracellular binding partners Slp76, Vav1, c-Cbl, and Lyn. Fgr is also upregulated. MEK and ERK show increased phosphorylation, while c-Raf is upregulated and shows increased phosphorylation at S259, S621 and S289/296/301. Differentiation markers that occur include CD11b expression, cell cycle arrest, p47^phox expression and inducible ROS production. B: In RA-treated R38+ HL60, CD38 is upregulated, but not Vav1, c-Cbl, or Lyn. Fgr is not upregulated. MEK and ERK show increased phosphorylation; however c-Raf is not upregulated, nor shows increased phosphorylation. Increased CD11b expression, cell cycle arrest, p47^phox expression and inducible ROS production do not occur. C: In RA-treated R38− HL60, CD38 is not upregulated, nor Vav1, c-Cbl, or Lyn. Fgr is not upregulated. MEK and ERK show increased phosphorylation; however c-Raf is not upregulated, nor shows increased phosphorylation. Increased CD11b expression, cell cycle arrest, p47^phox expression and inducible ROS production do not occur. D: In PP2-treated R38+ HL60, CD38 is partially upregulated (indicated by the gradient CD38), and Slp76, Vav1, c-Cbl, and Lyn are upregulated. Fgr is not upregulated. MEK and ERK phosphorylation is decreased; however c-Raf is upregulated and shows increased phosphorylation. Increased cell cycle arrest occurs, but not increased CD11b expression, p47^phox expression or inducible ROS production. E: In PP2-treated R38− HL60, CD38 is not upregulated, but Slp76, Vav1, c-Cbl, and Lyn are upregulated. Fgr is not upregulated. MEK and ERK phosphorylation is decreased; however c-Raf is upregulated and shows increased phosphorylation. Increased cell cycle arrest occurs, but not increased CD11b expression, p47^phox expression or inducible ROS production. F: In PP2+RA-treated R38+ HL60, CD38 is upregulated, along with Slp76, Vav1, c-Cbl, and Lyn. Fgr is also upregulated. MEK and ERK phosphorylation is decreased; however c-Raf is upregulated and shows increased phosphorylation. Differentiation markers that occur include CD11b expression, cell cycle arrest, and p47^phox expression, but not inducible ROS production. G: In PP2+RA-treated R38− HL60, CD38 is partially upregulated, along with Slp76, Vav1, c-Cbl, and Lyn. Fgr is also upregulated. MEK and ERK phosphorylation is decreased; however c-Raf is upregulated and shows increased phosphorylation. Differentiation markers that occur include partial CD11b expression, cell cycle arrest, and p47^phox expression, but not inducible ROS production.</p

48 h Western blot data for control and RA-treated WT, R38+ and R38− HL60 cells.

<p>A representative blot is displayed above its respective bar graph, and each bar graph (error bars represent standard error) presents the fold change respective to each control. The fold change was calculated after performing densitometry across three or more repeated blots. Note that the scale of the y-axis for each bar graph differs. A: There was no change in total ERK or MEK levels for any cell line. RA induced MEK and ERK phosphorylation in all three cell lines. Only RA-treated WT HL60 cells showed upregulation of c-Raf expression. Also, only RA-treated WT HL60 cells exhibited increased c-Raf phosphorylation at S259, S621 and S289/296/301. Neither R38+ nor R38− displayed increased c-Raf expression or phosphorylation after RA treatment. B: RA-treated WT HL60 cells showed upregulation of Lyn, Fgr, Vav1, and c-Cbl expression. RA-inducible Slp76 expression was evident in R38+ and R38−. Immunoprecipitation of c-Cbl followed by blotting of CD38 reveals that there is little CD38 and c-Cbl interaction in RA-treated R38+ compared to RA-induced WT HL60. GAPDH (not shown) served as loading control; c-Cbl (not shown) served as control for c-Cbl immunoprecipitation.</p

Temporal Trends in Exposure to Organophosphate Flame Retardants in the United States

During the past decade, use of organophosphate compounds as flame retardants and plasticizers has increased. Numerous studies investigating biomarkers (i.e., urinary metabolites) demonstrate ubiquitous human exposure and suggest that human exposure may be increasing. To formally assess temporal trends, we combined data from 14 U.S. epidemiologic studies for which our laboratory group previously assessed exposure to two commonly used organophosphate compounds, tris­(1,3-dichloro-2-propyl) phosphate (TDCIPP) and triphenyl phosphate (TPHP). Using individual-level data and samples collected between 2002 and 2015, we assessed temporal and seasonal trends in urinary bis­(1,3-dichloro-2-propyl) phosphate (BDCIPP) and diphenyl phosphate (DPHP), the metabolites of TDCIPP and TPHP, respectively. Data suggest that BDCIPP concentrations have increased dramatically since 2002. Samples collected in 2014 and 2015 had BDCIPP concentrations that were more than 15 times higher than those collected in 2002 and 2003 (10^β = 16.5; 95% confidence interval from 9.64 to 28.3). Our results also demonstrate significant increases in DPHP levels; however, increases were much smaller than for BDCIPP. Additionally, results suggest that exposure varies seasonally, with significantly higher levels of exposure in summer for both TDCIPP and TPHP. Given these increases, more research is needed to determine whether the levels of exposure experienced by the general population are related to adverse health outcomes