Elsevier

Toxicology Letters

Volume 206, Issue 3, 30 October 2011, Pages 289-299
Toxicology Letters

Mechanisms involved in lipid accumulation and apoptosis induced by 1-nitropyrene in Hepa1c1c7 cells

https://doi.org/10.1016/j.toxlet.2011.07.024Get rights and content

Abstract

1-Nitropyrene (1-NP) is a nitro-polycyclic aromatic hydrocarbon (nitro-PAH) present in diesel exhaust and bound to particular matter in urban air. We show that 1-NP and the referent PAH benzo(a)pyrene (BP) induce apoptosis and a lipid accumulation dependent on cytochrome P450 1A1-metabolites in mouse hepatoma cells, whereas 1-amino-pyrene had no effect. The caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk), inhibits 1-NP-induced apoptosis, but failed to alter 1-NP-triggered lipid accumulation determined by Nile red staining. We further show that cholesterol and fatty acid contents are modified after nitro-PAH exposure and that 1-NP-induced cholesterol level is partially involved in related apoptosis. In parallel, the activity of the stearoyl-CoA desaturase 1 (SCD1), determined by fatty acid analysis, and its expression are reduced by 1-NP. The role of SCD1 in 1-NP-induced apoptosis is demonstrated in cells down-expressing SCD1, in which an increased apoptosis is observed, whereas the SCD1 overexpression elicits the opposite effects. In contrast, changes in SCD1 gene expression have no effect on the induced lipid accumulation. Moreover, 1-NP increases the activity of the AMP-dependent protein kinase (AMPK) leading to a caspase-independent apoptosis. Overall, our study demonstrates that the 1-NP-induced apoptosis is caspase- and AMPK-dependent, and is associated to a decrease of SCD1 expression which results in an alteration of lipid homeostasis.

Highlights

► 1-Nitropyrene increases apoptosis and lipid accumulation in hepatoma cells. ► 1-NP modifies cholesterol and fatty acids content. ► 1-NP alters the expression and activities of two enzymes involved in lipid homeostatsis, the steaoyl-CoA desaturase 1 (SCD1) and the AMP-dependent protein kinase (AMPK). ► 1-NP-induced apoptosis is caspase- and AMPK-dependent, and is associated to a decrease of SCD1 expression.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs are a ubiquitous group of environmental pollutants. They are often produced by incomplete combustion of organic material including gasoline and diesel. In urban air, PAHs are not only often adsorbed on particular matter, but also found in soil and water. Humans are commonly exposed to such compounds not only via air, but also through diet. PAHs may exert a wide range of toxic effects, and some of them have been identified as carcinogenic to humans. Benzo[a]pyrene (BP) is an important carcinogenic PAH, whereas 1-nitropyrene (1-NP) is the most abundant nitro-PAH in diesel exhaust particles.

The deleterious effects of these environmental chemicals have partly been linked to the formation of reactive electrophilic metabolites. In the mouse hepatoma cell line Hepa1c1c7, these compounds have been found to increase cytochrome P-450 (CYP)1A1 expression (Landvik et al., 2007). The binding of BP, a well known ligand of the aryl hydrocarbon-receptor (AhR), to this receptor leads to its nuclear translocation and activation of AhR-dependent gene transcription such as CYP1A1. Nitro-PAHs require metabolic activation by nitroreduction or ring oxidation to react with DNA; but CYP enzymes are also reported to reduce nitro-PAHs (H.J. Kim et al., 2005).

Several reports demonstrate that BP causes apoptotic and necrotic cell death (Holme et al., 2007), while 1-NP was found to cause apoptosis as well as a non-apoptotic programmed cell death with “paraptotic” characteristics (Landvik et al., 2007, Asare et al., 2008). Recently, we also observed that 1-NP exposure resulted in a very marked increase of lipid droplets in Hepa1c1c7 cells (Asare et al., 2009); however the mechanisms involved are still unknown.

Liver plays a major role in lipid homeostasis, but hepatocytes are not a physiological site of lipid storage, and development of steatosis is usually associated with cellular dysfunction and apoptosis. Nevertheless, it is well known that several liver toxic xenobiotics may alter lipid metabolism by leading to accumulation of lipid droplets that can cause a fatty liver (Prince et al., 1993). An effect of the strong AhR ligand, TCDD, as well as some of the PAHs on lipid accumulation has previously been observed in human macrophages (Vogel et al., 2004, Podechard et al., 2009). Similarly, hepatic lipid accumulation was observed in mice exposed to TCDD (Shen et al., 1991, Matsumura et al., 1997). Interestingly, some recent findings have also suggested lipid droplet accumulation to be more directly involved in cell death processes (Schaffer, 2003). So, the link between lipid homeostasis and apoptosis induced by aromatic hydrocarbon (AH) pollutants remains to be characterized.

In liver cells, a prolonged activation of the AMP-dependent protein kinase (AMPK), an enzyme involved in lipid homeostasis, can trigger apoptosis and activation of caspase-3 (Meisse et al., 2002). AMPK can be activated by an inhibition of the stearoyl CoA desaturase (SCD1), a key enzyme of lipid metabolism catalyzing Δ9-desaturation of the saturated fatty acids (SFA) palmitate and stearate to the monounsaturated fatty acids (MUFA) palmitoleate and oleate, respectively (Scaglia et al., 2009). Furthermore, the ratio between SFA and MUFA seems to be involved in the regulation of cell growth, as loss of SCD1 has been reported to be associated with a reduced proliferation as well as an increased apoptosis (Scaglia and Igal, 2005, Scaglia and Igal, 2008, Minville-Walz et al., 2010).

In the present study, we have extended our previous observations showing that 1-NP induces lipid accumulation (Asare et al., 2009) and apoptosis (Solhaug et al., 2004, Landvik et al., 2007). We have sought to understand the relationship between lipid accumulation and apoptosis induced by PAHs. Therefore, we have compared the effects of 1-NP to those observed after exposure to its non-toxic amine metabolite 1-amino-pyrene (1-AP) and BP and, we have characterized the changes in lipid composition. Furthermore, the role of AMPK and SCD1 in the perturbation of lipid homeostasis and apoptosis induced by 1-NP was examined.

Section snippets

Chemicals

BP, 1-NP, 1-AP, Oil red O (ORO), N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and methyl-β-cyclodextrin D (MβCD) were provided by Sigma–Aldrich (St Louis, MO). TCDD was obtained from Cambridge Isotope Laboratories (Cambridge, MA). 5-Aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICAR), compound C (CpC) and cylic-pifithrin-α (PFT) were provided by Calbiochem (La Jolla, CA). Chemicals were

Effects of AHs on lipid content

We have recently reported that 1-NP induces accumulation of lipid droplets in Hepa1c1c7 cells (Asare et al., 2009). Thus, in order to elucidate possible triggering mechanism involved, we exposed the cells to some other AHs. The cells were examined for lipid droplets by light microscopy following ORO staining. BP and 1-NP which is known to be metabolized into reactive metabolites in these cells increased the number of ORO positive cells (Fig. 1A and B). Increased lipid accumulation was confirmed

Discussion

We previously reported that 1-NP is an important environmental pollutant that induced lipid accumulation as well as apoptosis in the Hepa1c1c7 cell line (Asare et al., 2009). In the present study, we show that this compound modifies the cellular lipid composition and reduces the expression and activity of SCD1. These changes result in an activation of AMPK, which seems to be involved in the apoptotic process. Thus, the results provide possible mechanistic explanations to how environmental

Conflict of interest statement

None declared.

Acknowledgements

For microscopy and flow cytometry analyses, we would like to thank respectively the Impac cells and the cytometry platforms of IFR140, University of Rennes 1, France.

This work was supported by the Ligue Nationale contre le Cancer (équipe labellisée). N. Podechard was a recipient of a fellowship from the “Région Bretagne”, and X. Tekpli a recipient of a fellowship from the French Ministry of Research and of the Association for Research on Cancer (ARC).

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