Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases human hepatic stellate cell activation

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a halogenated aromatic hydrocarbon that elicits toxicity through the aryl hydrocarbon receptor (AhR). In the liver, gross markers of TCDD toxicity are attributed to AhR activation in parenchymal hepatocytes. However, less is known regarding the consequences of TCDD treatment on non-parenchymal cells in the liver. Hepatic stellate cells (HSCs) are non-parenchymal cells that store vitamin A when quiescent. Upon liver injury, activated HSCs lose this storage ability and instead function in the development and maintenance of inflammation and fibrosis through the production of pro-inflammatory mediators and collagen type I. Reports that TCDD exposure disrupts hepatic retinoid homeostasis and dysregulates extracellular matrix remodeling in the liver led us to speculate that TCDD treatment may disrupt HSC activity. The human HSC line LX-2 was used to test the hypothesis that TCDD treatment directly activates HSCs. Results indicate that exposure to 10nM TCDD almost completely inhibited lipid droplet storage in LX-2 cells cultured with retinol and palmitic acid. TCDD treatment also increased LX-2 cell proliferation, expression of α-smooth muscle actin, and production of monocyte chemoattractant protein-1 (MCP-1), all of which are characteristics of activated HSCs. However, TCDD treatment had no effect on Col1a1 mRNA levels in LX-2 cells stimulated with the potent profibrogenic mediator, transforming growth factor-β. The TCDD-mediated increase in LX-2 cell proliferation, but not MCP-1 production, was abolished when phosphoinositide 3-kinase was inhibited. These results indicate that HSCs are susceptible to direct modulation by TCDD and that TCDD likely increases HSC activation through a multi-faceted mechanism.

Keywords: Collagen; Hepatic stellate cell; Liver; MCP-1; TCDD.

Fig. 1. LX-2 cells express a functional AhR

LX-2 cells were treated with DMSO or 10 nM TCDD for 24 hr. Levels of AhR and Cyp1a1 mRNA were measured by RT-PCR. GAPDH was used as a loading control. Results are representative of four samples from each treatment group.

Fig. 2. TCDD treatment modulates lipid storage in LX-2 cells

(A) LX-2 cells were treated for 48 hours with 5 μM retinol (Ret) and 100 μM palmitic acid (PA) in the presence of DMSO or 10 nM TCDD. Cells were then stained with oil red O to visualize lipid droplets (arrows). (B) LX-2 cells were cultured with retinol and palmitic acid for 48 hr to induce lipid storage, and then TCDD (10 nM) or DMSO was added for an additional 48 hr. Magnification 200x. Results are representative of three samples from each treatment group.

Fig. 3. TCDD treatment increases LX-2 cell proliferation

Cells were treated with 10 nM TCDD or DMSO. Viable cells were enumerated based on trypan blue exclusion at 24, 48, 72 and 96 hr after treatment. Data represent the mean ± SEM (n=3). Results are representative of four different experiments. *p<0.05 when compared to DMSO-treated cells at same time point.

Fig. 4. TCDD treatment increases the production of MCP-1 by LX-2 cells

Cells were treated with 10 nM TCDD or DMSO for 24 hr. (A) MCP-1 protein levels in supernatants were measured by ELISA. Data represent the mean ± SEM (n=3). (B) MCP-1 mRNA levels were measured in cell lysates after 24-hr treatment by semi-quantitative RT-PCR. * p<0.05 when compared to DMSO-treated control (n=3).

Fig. 5. TCDD treatment increases αSMA expression in LX-2 cells

LX-2 cells were grown on glass-cover slips, treated with 10 nM TCDD or DMSO for 24 hr, and αSMA expression was detected by fluorescence microscopy (anti-αSMA, red; DAPI, blue). (A) Untreated LX-2 cells at 0 hr. (B) LX-2 cells treated for 24 hr with DMSO. (C) LX-2 cells treated for 24 hr with TCDD. Magnification 630x.

Fig. 6. TCDD treatment does not increase collagen type I production by LX-2 cells

LX-2 cells were cultured for 24 hr with 2.5 ng/ml TGF-β1 and either TCDD (10 nM) or DMSO. Col1a1 mRNA levels were measured by qRT-PCR. Samples were analyzed in duplicate from 5 biological replicates per treatment group. Relative quantification was estimated using the ??Ct method normalized to β-actin. Error bars represent 95% confidence intervals, *p<0.05.

Fig. 7. PI3K inhibition suppresses the effect of TCDD on LX-2 cell proliferation but not on MCP-1 production

Cells were pretreated for 30 min with 25 nM wortmannin (WM) to inhibit PI3K signaling prior to being cultured for 72 hr in TCDD (10 nM) or DMSO. (A) Average (mean ± SEM) number of viable cells in WM-treated cultures. (B) Average (mean ± SEM) levels of MCP-1 in the supernatant of WM-treated cells. Results are representative of three independent experiments, *p<0.05.