The endocrine disruptor mono-(2-ethylhexyl) phthalate affects the differentiation of human liposarcoma cells (SW 872)

Abstract

Esters of phthalic acid (phthalates) are largely used in industrial plastics, medical devices, and pharmaceutical formulations. They are easily released from plastics into the environment and can be found in measurable levels in human fluids. Phthalates are agonists for peroxisome proliferator-activated receptors (PPARs), through which they regulate translocator protein (TSPO; 18 kDa) transcription in a tissue-specific manner. TSPO is a drug- and cholesterol-binding protein involved in mitochondrial respiration, steroid formation, and cell proliferation. TSPO has been shown to increase during differentiation and decrease during maturation in mouse adipocytes. The purpose of this study was to establish the effect of mono-(2-ethylhexyl) phthalate (MEHP) on the differentiation of human SW 872 preadipocyte cells, and examine the role of TSPO in the process. After 4 days of treatment with 10 µM MEHP, we observed changes in the transcription of acetyl-CoA carboxylase alpha, adenosine triphosphate citrate lyase, glucose transporters 1 and 4, and the S100 calcium binding protein B, all of which are markers of preadipocyte differentiation. These observed gene expression changes coincided with a decrease in cellular proliferation without affecting cellular triglyceride content. Taken together, these data suggest that MEHP exerts a differentiating effect on human preadipocytes. Interestingly, MEHP was able to temporarily increase TSPO mRNA levels through the PPAR-α and β/δ pathways. These results suggest that TSPO can be considered an important player in the differentiation process itself, or alternatively a factor whose presence is essential for adipocyte development.

Figure 1. Basal levels of (A) C/EBP-α, (B) GLUT4, (C) S100B, (D) PPAR-α, (E) PPAR-β/δ, (F) PPAR-γ, (G) TSPO, (H) PKCε mRNAs normalized to RPS18.

Cells were seeded and collected after the indicated time points; day 0 represents one day after the plating. Results are expressed in terms of mean and S.E.M., calculated from 3 independent experiments and presented as fold increase or decrease from the value measured on day 0. Significance (compared to day 0 values) was calculated using one-way ANOVA followed by Bonferroni's post hoc test; *p<0.05, **p<0.01, ***p<0.001.

Figure 2. Effect of MEHP and PMA on the gene transcription of TSPO, PKCε, PPAR-α, PPAr-β/δ, and PPAR-γ.

(A) Dose response of TSPO gene expression after 4, 8 and 12 days of treatment with 1, 10, and 50 µM MEHP. (B) Effect of 10 µM MEHP on PKCε gene expression after 4 days of treatment. (C) Effect of 10 µM MEHP on PPAR-α, PPAR-β/δ, and PPAR-γ gene expression after 4 days of treatment. (D) Effect of 10, 50, or 100 10 nM PMA on TSPO gene expression after 4 days of treatment. Cells were seeded for 24 h before treatment with MEHP (A–C) or PMA (D) and collected after the indicated time points (A) or after 4 days (B–D). qRT-PCR results are normalized to RPS18, expressed in terms of mean and S.E.M. calculated from 3 independent experiments, and presented as fold increase or decrease compared to control. One-way ANOVA followed by Bonferroni's post hoc test (A) or Student's t-test (B–D) was used to calculate statistical significance; *p<0.05, **p<0.01, ***p<0.001.

Figure 3. MEHP induces the differentiation of SW 872 cells.

(A) qRT-PCR products of ACACA, ACLY, GLUT1, GLUT4 and S100B mRNA normalized to RPS18 and presented as fold increase or decrease compared to control, 4 days after treatment with 10 µM MEHP. (B) Proliferation assay. Cells were seeded for 24 h before treatment with 10 µM MEHP and the assay was performed as described in section 2.4 (C) Cellular triglyceride content assay. Cells were seeded for 24 h before treatment with 10 µM MEHP and collected after 4 or 6 days to assay triglyceride content. D0, D4CTRL, and D4M10 represent untreated cells at day 0, untreated cells at day 4, and MEHP-treated cells at day 4, respectively. Results are expressed in terms of mean and S.E.M., calculated from three independent experiments. Student's t-test was used to calculate statistical significance compared to control; *p<0.05.

Figure 4. The effect of MEHP on TSPO gene expression is mediated by PPAR-α and PPAR-β/δ , and on PKCε gene expression by PPAR-α.

(A) PPAR-α, PPAR-β/δ, PPAR-γ and PKC mRNA levels are greatly reduced following treatment with gene-specific siRNAs compared to treatment with scrambled siRNA. (B) Effect of gene-specific siRNA treatment on TSPO transcription, with or without (+/−) MEHP, compared to similar treatment with scrambled siRNA (SCR). (C) Effect of gene-specific siRNA treatment on PKCε transcription, with or without (+/−) MEHP compared to similar treatment with scrambled siRNA (SCR). Cells were seeded for 24 h before treatment with MEHP, and then transfected with siRNA specific to PPAR-α, PPAR-β/δ, PPAR-γ, and PKCε knockdown. Cell lysates were collected after 4 days. qRT-PCR results are expressed in terms of mean and S.E.M., calculated from three independent experiments. Student's t-test was used to calculate statistical significance compared to scrambled siRNA control in the absence (*) or presence (#) of MEHP; *p<0.05, **p<0.01, ***p<0.001, ^#p<0.05.

Figure 5. MEHP treatment results in decreased TSPO protein level.

(A) Densitometric analysis of TSPO immunoblot. Cells were seeded for 24 h before treatment with 10 µM MEHP and collected at day 0 and after 4 days. D0, D4C, and D4M10 represent untreated cells at day 0, untreated cells at day 4, and MEHP-treated cells at day 4, respectively. (B) Saturation binding assay. Cells were seeded for 24 h before treatment with MEHP and collected after 4 days. D4C, and D4M10 represent untreated cells and MEHP-treated cells at day 4, respectively. Results are expressed in terms of mean and S.E.M., calculated from two independent experiments. Student's t-test was used to calculate statistical significance compared to control (*p<0.05), or to day 0 (^#p<0.05).

Figure 6. TSPO gene knockdown decreases PPAR-γ transcription and increases transcription of S100B, ACACA, and ACLY.

(A) TSPO mRNA levels following treatment with gene-specific siRNA, compared to similar treatment with scrambled siRNA. (B) Effect of gene-specific siRNA treatment on PPAR-γ, S100B, ACACA, and ACLY mRNA levels compared to treatment with scrambled siRNA. Cells were transfected with siRNA as described in section 2.5, and collected after 4 days. qRT-PCR results are expressed in terms of mean and S.E.M., calculated from three independent experiments. Student's t-test was used to calculate statistical significance compared to scrambled siRNA treatment; * p<0.05, **p<0.01, ***p<0.001.

Figure 7. Effects of MEHP on SW 872 human preadipocytes.

MEHP enhances differentiation, inhibits cellular proliferation, and decreases mitochondrial TSPO expression in SW 872 cells.