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. 2012 Dec;120(12):1720-6.
doi: 10.1289/ehp.1205383. Epub 2012 Sep 22.

Triflumizole is an obesogen in mice that acts through peroxisome proliferator activated receptor gamma (PPARγ)

Xia Li  1 , Hang T Pham, Amanda S Janesick, Bruce Blumberg
Affiliations

Triflumizole is an obesogen in mice that acts through peroxisome proliferator activated receptor gamma (PPARγ)

Xia Li et al. Environ Health Perspect. 2012 Dec.

Abstract

Background: Triflumizole (TFZ) is an imidazole fungicide used on many food and ornamental crops. TFZ is not thought to be particularly toxic or carcinogenic, but little is known about its effect on development. TFZ is identified as a peroxisome proliferator activated receptor gamma (PPARγ) activator in ToxCast. Because PPARγ is a master regulator of adipogenesis, we hypothesized that TFZ would activate PPARγ, thereby inducing adipogenesis and weight gain in vivo.

Objectives: We sought to test the ability of TFZ to activate PPARγ and promote adipogenesis in vitro and in vivo.

Methods: We used transient transfection to test the ability of TFZ to activate PPARγ, and we used 3T3-L1 preadipocytes and human multipotent mesenchymal stromal stem cells (MSCs) to study the adipogenic capacity of TFZ in culture. We treated pregnant mice with three doses of TFZ and evaluated the effects on body weight, adipose depot weight, and MSC programming in the prenatally exposed offspring.

Discussion: TFZ induced adipogenesis in MSCs and in mouse 3T3-L1 preadipocytes. Prenatal exposure to levels of TFZ at approximately 400-fold below the reported no observed adverse effect level increased adipose depot weight. All doses of TFZ tested increased adipogenic gene expression in MSCs while inhibiting expression of osteogenic genes.

Conclusions: TFZ acts through a PPARγ-dependent mechanism to induce adipogenic differentiation in MSCs and preadipocytes at low nanomolar concentrations. Prenatal TFZ exposure increases adipose depot weight and diverts MSC fate toward the adipocyte lineage; therefore, we conclude that TFZ is an obesogen in vivo.

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Conflict of interest statement

B.B. is a named inventor on U.S. patents 5,861,274, 6,200,802, 6,815,168, and 7,250,273 related to PPARγ. The other authors declare they have no actual or potential competing financial interests.

Figures

Figure 1
Figure 1
Activation of PPARγ by TFZ in transient transfection assays. The ability of a graded dose series of TFZ to activate GAL4-mPPARγ was tested in transiently transfected COS7 cells. TFZ and ROSI were tested in 3-fold serial dilutions from 10–4 M through 10–11 M. Cytotoxicity, as measured by decreased β-galactosidase activity was observed at 3.3 × 10–5M for both ROSI and TFZ. Data are depicted as fold induction over vehicle (0.1% DMSO) controls (mean ± SE); data points represent triplicate transfections (three biological replicates), and results were verified in multiple experiments.
Figure 2
Figure 2
Effect of TFZ on adipogenesis in 3T3-L1 cells. The adipogenic effect of TFZ was tested in 3T3-L1 cells during MDI-induced adipocyte differentiation. Cells were treated with 0.1% DMSO (vehicle), 100 nM ROSI (positive control), or TFZ. Media were replaced every other day with freshly supplemented ligands. Seven days after differentiation was initiated, cells were fixed and stained with Oil Red O or processed for RNA extraction. (A) Lipid accumulation was assessed by measuring the percent of surface area in each well covered by Oil Red O–positive cells using Image J software. (B–F) Adipogenic gene expression determined by QPCR in cells collected on day 7 of differentiation. (B) FABP4, (C) ADIPOQ, (D) LEP, (E) LPL, and (F) FSP27. Data are presented as fold induction (mean ± SE) relative to DMSO vehicle for triplicate samples (three biological replicates), and results were verified in multiple experiments. One-way ANOVA was conducted for TFZ treatment groups and DMSO, followed by Dunnett’s post hoc test. Unpaired t-test was conducted for ROSI versus DMSO. *p < 0.05, **p < 0.01, and #p < 0.001 compared with DMSO vehicle.
Figure 3
Figure 3
Effect of TFZ on adipogenesis in human white adipose tissue–derived MSCs. Adipogenesis was induced in hMSCs by adipogenic cocktail for 14 days in the presence of 0.1% DMSO (vehicle), 0.5 µM ROSI (positive control), or TFZ at 0.1 µM or 1 µM. (A) Lipid accumulation is shown by Oil Red O staining in hMSCs after 14 days of differentiation and quantified by measuring the percentage of surface area with lipid-laden adipocytes. (B–F) Adipogenic gene expression was determined by QPCR. (B) FABP4, (C) ADIPOQ, (D) LEP, (E) LPL, and (F) FSP27. Data presented are fold induction (mean ± SE) relative to DMSO and represent triplicate samples (three biological replicates), and results were verified in multiple experiments. One-way ANOVA was conducted for TFZ treatment groups and DMSO, followed by Dunnett’s post hoc test. Unpaired t-test was conducted for ROSI versus DMSO. *p < 0.05, **p < 0.01, and #p < 0.001 compared with DMSO.
Figure 4
Figure 4
Effect of PPARγ antagonist T0070907 on TFZ-induced adipogenesis. (A–D) 3T3-L1 cells differentiated into mature adipocytes by the addition of MDI were treated with DMSO, 0.1 µM ROSI, or 1 µM or 10 µM TFZ, in the presence or absence of 1 µM T0070907. (A) Cells were stained with Oil Red O, and lipid accumulation was quantified by ImageJ software. Data are presented as area fraction (mean ± SE). (B–D) QPCR analysis of RNA extracted from 3T3-L1 cells was used to assess the expression of adipogenic genes FABP4 (B), LEP (C), and LPL (D). (E–H) hMSCs differentiated into mature adipocytes by MDII (IBMX, dexamethasone, insulin, and indomethacin) were treated with DMSO, 0.5 µM ROSI, 0.1 µM or 1 µM TFZ, in the presence or absence of 1 µM T0070907. (E) Lipid accumulation was quantified as described for A. (F–H) RNA was extracted from hMSCs and analyzed by QPCR. Data are presented as fold induction (mean ± SEM) compared with DMSO for triplicate samples (three biological replicates) and results were verified in multiple experiments. One-way ANOVA was conducted for TFZ treatment groups and DMSO, followed by Dunnett’s post hoc test. Unpaired t-test was conducted for the positive control (ROSI) versus DMSO, and one-way ANOVA was conducted for all groups, followed by Bonferroni post hoc test comparing +T0070907 versus –T0070907. *p < 0.05, **p < 0.01, and #p < 0.001 compared with DMSO. ##p < 0.5, †p < 0.01, and ††p < 0.001 for +T0070907 compared with –T0070907.
Figure 5
Figure 5
Effect of prenatal TFZ exposure on body weight, adiposity, and MSC programming in mice. Groups of three pregnant female CD1 mice were exposed to DMSO, ROSI, or TFZ via the drinking water; resulting male offspring were sacrificed at 8 weeks of age, and fat pads (epididymal, retroperitoneal, and subcutaneous) were collected and weighed. The numbers of exposed F1 offspring were as follows: DMSO, 17; ROSI, 14; 0.1 µM TFZ, 11; 1 µM TFZ, 15; and 10 µM TFZ, 14. (A) Body weight and (B) total fat depot weights were normalized to body weight and are expressed as the percentage of adiposity. (C–F) White adipose–derived MSCs were collected from mice; cells from mice derived from a single litter were pooled (three litters per treatment) and cultured until confluence. RNA was extracted and analyzed by QPCR; expression of PPARγ (C), ZPF423 (D), Pref‑1 (E), and FABP4 (F) was normalized to the housekeeping gene 36B4. Data are expressed as fold change (mean ± SE) relative to DMSO controls. One-way ANOVA was conducted for TFZ treatment groups and DMSO, followed by Dunnett’s post hoc test. Unpaired t-test was conducted for the positive control (ROSI) versus DMSO. *p < 0.5, and **p < 0.01 compared with DMSO.
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