Lineage-Selective Disturbance of Early Human Hematopoietic Progenitor Cell Differentiation by the Commonly Used Plasticizer Di-2-ethylhexyl Phthalate via Reactive Oxygen Species: Fatty Acid Oxidation Makes the Difference

Abstract

Exposure to ubiquitous endocrine-disrupting chemicals (EDCs) is a major public health concern. We analyzed the physiological impact of the EDC, di-2-ethylhexyl phthalate (DEHP), and found that its metabolite, mono-2-ethylhexyl phthalate (MEHP), had significant adverse effects on myeloid hematopoiesis at environmentally relevant concentrations. An analysis of the underlying mechanism revealed that MEHP promotes increases in reactive oxygen species (ROS) by reducing the activity of superoxide dismutase in all lineages, possibly via its actions at the aryl hydrocarbon receptor. This leads to a metabolic shift away from glycolysis toward the pentose phosphate pathway and ultimately results in the death of hematopoietic cells that rely on glycolysis for energy production. By contrast, cells that utilize fatty acid oxidation for energy production are not susceptible to this outcome due to their capacity to uncouple ATP production. These responses were also detected in non-hematopoietic cells exposed to alternate inducers of ROS.

Keywords: ROS quenching; di-2-ethylhexyl phthalate; endocrine-disrupting compounds; fatty acid oxidation; hematopoiesis; hematopoietic stem and progenitor cells; hematotoxicity; mono-2-ethylhexyl phthalate; reactive oxygen species.

Figure 1

Differential disruption of myeloid lineage differentiation by DEHP. (a,j,n) Expansion of erythrocyte (a), dendritic cell (i), and neutrophil (n) cultures in the presence of increasing concentrations of DEHP. (b) Cell pellet representing erythroid populations after 6 days of treatment with increasing concentrations of DEHP. (c) Impact of increasing concentrations of DEHP on the relative expression of HBB in differentiating erythrocytes after 6 days. (d–h) Expression of CD71 and CD235a in erythroid populations after 6 days of treatment with varying concentrations DEHP. From left to right: isotype control (d), vehicle control (e), 25.6 µM (f), 128.2 µM (g), and 256.41 µM DEHP (h). (i,m) Caspase activity in differentiated erythroid populations after 2 days (i) and in dendritic cell populations after 3 days (m) of treatment with varying concentrations of DEHP. Cells treated with staurosporine were included as positive controls. (k) Impact of DEHP on the relative expression of GPNMB in dendritic cell cultures. (l) Expression of CD14 on dendritic cells treated with 256.41 µM DEHP (red) compared to vehicle control (black); isotype control is as shown (grey-filled). (o) Expression of CD14 on neutrophils treated with 25.6 µM (red), 128.2 µM (green), and 256.41 µM DEHP (blue) compared to vehicle control (black); isotype control is as shown (grey). (p,q) Impact of DEHP on the relative expression of ELANE (p) and S100A8 (q) in neutrophils. Data are presented as mean ± standard error of the mean (SEM); individual replicates are visualized as black dots in bar charts in case of n = 3. Significance was determined by one-way ANOVA (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 2

DEHP alters the lipidome and suppresses glycolysis, glutaminolysis, and polyamine synthesis in erythrocytes and dendritic cells. (a–c) Erythrocyte metabolites altered (FDR-significant) after 2 days (a), 4 days (b), and 6 days (c) in culture with increasing concentrations of DEHP (d–f) Dendritic cell metabolites (FDR-significant) after 3 days (d), 8 days (e), and 11 days (f) in culture with increasing concentrations of DEHP. (g) Total ester glycerophospholipids (GPL) concentration in erythroid cultures treated with increasing concentrations of DEHP. (h) Total ether GPL concentration in erythroid cultures treated with increasing concentrations of DEHP. (i) Percent ether GPL content in erythroid cultures treated with increasing concentrations of DEHP. (j) Total ester GPL concentration in dendritic cell cultures treated with increasing concentrations of DEHP. (k) Total ether GPL concentration in dendritic cell cultures treated with increasing concentrations of DEHP. (l) Percent ether GPL content in dendritic cell cultures treated with increasing concentrations of DEHP. (m) Model depicting the interconnections between altered metabolites associated with glutaminolysis and polyamine synthesis. (n) Total ester GPL content in neutrophil cultures treated with increasing concentrations of DEHP. (o) Total ether GPL content in neutrophil cultures treated with increasing concentrations of DEHP. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 3

The DEHP metabolite, MEHP, reduces SOD activity and increases ROS at environmentally relevant concentrations. (a–c) Relative fluorescence (DCF) after DEHP treatment of erythrocytes for 4 h (a), dendritic cells for 3 d (b), and neutrophils for 3 d (c). (d–i) Relative expression of aryl hydrocarbon receptor (AhR) protein in erythrocytes (d,e), dendritic cells (f,g), and neutrophils (h,i) in response to increasing concentrations of DEHP. (j–l) Concentrations of MEHP (j) and DEHP (k) detected in cell culture supernatants after treatment with 128.2 µM DEHP and other concentrations for 48 h (l). (m–o) Relative fluorescence (DCF) after MEHP or 2-EH treatment of erythrocytes for 4 h (m), dendritic cells for 3 d (n), and neutrophils for 3 d (o). (p–r) Relative SOD activity after DEHP or MEHP treatment of erythrocytes for 2 d (p), dendritic cells for 3 d (q), and neutrophils for 3 d (r). Data are presented as mean ± standard error of the mean (SEM); individual replicates are visualized as black dots in bar charts in case of n = 3. Significance was determined by one-way ANOVA (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 4

Increased oxidative stress leads to a shift from glycolysis to the pentose-phosphate pathway and ATP depletion in glycolytically active cells but not in cells capable of ATP production by fatty acid oxidation. (a–c) ATP levels after DEHP treatment of erythrocytes for 2d d (a), dendritic cells for 3 d (b), and neutrophils for 3 d (c). (d,e) NADPH levels after DEHP treatment of erythrocytes for 2 d (d) and dendritic cells for 3 d (e). (f,g,i) Relative fluorescence (DCF) in neutrophils (f), HepG2/C3A cells (g), and HUVECs (i) with or without preincubation with 5 µM etomoxir (ETO) and 250 µM N-acetylcysteine (NAC) or 10 µM butylated hydroxyanisole (BHA) for 2 h, followed by DEHP for 4 h (h,j). Relative fluorescence (DCF) in HepG2/C3A cells (h) and HUVECs (j) with or without preincubation with 5 µM ETO and 250 µM NAC or 10 µM BHA for 2 h, followed by tert-butyl hydroperoxide (TBHP) for 4 h. Data are presented as mean ± SEM; individual replicates are visualized as black dots in bar charts in case of n = 3. Statistical significance was determined by one-way ANOVA (* p < 0.05; ** p < 0.01; *** p < 0.001; n.s., not significant).

Figure 5

Active fatty acid oxidation increases the capacity for reactive oxygen species (ROS) quenching regardless of the nature of the stimulus. Model depicting the critical role of active fatty acid oxidation (FAO) in generating adenosine triphosphate (ATP) to support glucose (Glc) phosphorylation (to glucose-6-phosphate (G6P)), resulting in pentose phosphate pathway (PPP) activity. Administration of di-2-ethylhexyl phthalate (DEHP) results in the accumulation of ROS due to reduction of superoxide dismutase (SOD) activity mediated by its metabolite, mono-2-ethylhexyl phthalate (MEHP). The observed reduction in SOD activity may be the result of MEHP-dependent dysregulation of aryl hydrocarbon receptor (AhR) signaling. ROS can also be formed in response to unrelated mechanisms (e.g., in response to tert-butyl hydroperoxide (TBHP)). Increased concentrations of ROS redirect the glycolytic flux through PPP, leading to reduced levels of ATP (also via reduced tricarboxylic acid cycle (TCA) activity) in cells that are incapable of active FAO (left). Reduced levels of ATP result in reduced rates of glucose phosphorylation and NADPH generation, which are factors that ultimately lead to cell death. In cells capable of active FAO, ATP is generated by a mechanism that does not rely on glycolysis. These cells produce higher levels of NADPH and are thus able to quench toxic ROS (right).