Polycyclic aromatic hydrocarbons and their effects on energy metabolism in liver cells

Kód výsledku v IS VaVaI

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Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Polycyclic aromatic hydrocarbons and their effects on energy metabolism in liver cells

Popis výsledku v původním jazyce

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants that exert diverse types of toxicities, including genotoxicity/carcinogenicity, tumor promotion, endocrine disruption, or hepatotoxicity. Recently, we have established a novel complex in vitro system for testing of acute toxicity responses induced by various xenobiotics. The system is based on RT-qPCR analysis of expression profiles of genes that are involved in cellular stress responses, including: early stress response; heat shock protein response; DNA damage response; unfolded protein response (related to endoplasmic reticulum stress); and immunotoxicity. The cellular stress markers represent integral parameters affected by various intracellular pathways and they are complementary to e.g. luciferase reporter gene (CALUX) assays detecting specific nuclear receptor activations. In order to detect novel modes of action of PAHs, we evaluated cellular stress markers after exposure of differentiated human HepaRG cells to selected environmental PAHs. Two potent AhR ligands, benzo[a]pyrene (BaP) and benzo[k]fluoranthene (BkF), were found to induce expression of early stress and genotoxicity markers: ATF3, EGR1, GDF15, CDKN1A/p21, and GADD45A mRNAs, while BaP alone increased levels of IL-6 mRNA. In the present work, we studied the impact of PAHs on nuclear receptors, such as PPARα (peroxisomal proliferator-activated receptor α), LXR (liver X receptor), and FXR (farnesoid X receptor), which are involved in the control of endogenous energy (glucose and lipid) metabolism. In this study, differentiated HepaRG cells were exposed to four individual PAHs, fluoranthene (Fla), pyrene (Pyr), BaP, and BkF, that were selected to represent both low-molecular-weight and high-molecular-weight PAHs, with distinct toxicological characteristics regarding the AhR activation and genotoxicity potential. The expression of major genes involved in various metabolic pathways (glucose transport, glycolysis, gluconeogenesis and glycogen metabolism; fatty acid synthesis, transport, activation, and oxidation; triglyceride synthesis, storage, and hydrolysis; ketogenesis and bile acid metabolism) were evaluated by RT-qPCR after 24 h or 72 h exposure. These results were compared to the effects of selective agonists of AhR (TCDD), PPARα (GW7647), LXR (GW3965), and FXR (GW4064), in order to reveal possible contribution of these receptors to the PAH-caused effects. The AhR ligands, including BaP, BkF and TCDD, dysregulated expression of genes involved in glucose and glycogen metabolism, and decreased mRNA levels of CYP4A11 and CYP7A11, two enzymes of fatty acid oxidation and bile acid synthesis. All of these effects persisted up to 72 h. Pyr, which is not genotoxic and does not activate AhR, increased mRNA levels of triglyceride metabolism genes. Finally, Fla did not cause any changes after 24 h, but it decreased CYP4A11, CYP7A1 and HMGCS2 after 72 h. None of the tested PAHs induced expression of PPARα-, LXR-, neither FXR-target genes. Nevertheless, BaP, BkF, and TCDD decreased both basal and induced expression of several PPARα-specific genes, which may suggest the existence of an inhibitory cross-talk between AhR and PPARα. This is supported by the observation that BkF, a potent AhR ligand, decreased expression of genes of fatty acid transport, β-oxidation and triglyceride metabolism after 72 h, in a similar manner as TCDD. Taken together, our study indicates that high-molecular-weight PAHs acting as efficient AhR ligands may deregulate human liver glucose and lipid metabolism genes and that determination of expression of cellular stress marker genes and the genes linked to energy metabolism is a novel approach suitable for in vitro toxicity profiling of xenobiotics such as environmental contaminants or their mixtures.

Název v anglickém jazyce

Polycyclic aromatic hydrocarbons and their effects on energy metabolism in liver cells

Popis výsledku anglicky

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants that exert diverse types of toxicities, including genotoxicity/carcinogenicity, tumor promotion, endocrine disruption, or hepatotoxicity. Recently, we have established a novel complex in vitro system for testing of acute toxicity responses induced by various xenobiotics. The system is based on RT-qPCR analysis of expression profiles of genes that are involved in cellular stress responses, including: early stress response; heat shock protein response; DNA damage response; unfolded protein response (related to endoplasmic reticulum stress); and immunotoxicity. The cellular stress markers represent integral parameters affected by various intracellular pathways and they are complementary to e.g. luciferase reporter gene (CALUX) assays detecting specific nuclear receptor activations. In order to detect novel modes of action of PAHs, we evaluated cellular stress markers after exposure of differentiated human HepaRG cells to selected environmental PAHs. Two potent AhR ligands, benzo[a]pyrene (BaP) and benzo[k]fluoranthene (BkF), were found to induce expression of early stress and genotoxicity markers: ATF3, EGR1, GDF15, CDKN1A/p21, and GADD45A mRNAs, while BaP alone increased levels of IL-6 mRNA. In the present work, we studied the impact of PAHs on nuclear receptors, such as PPARα (peroxisomal proliferator-activated receptor α), LXR (liver X receptor), and FXR (farnesoid X receptor), which are involved in the control of endogenous energy (glucose and lipid) metabolism. In this study, differentiated HepaRG cells were exposed to four individual PAHs, fluoranthene (Fla), pyrene (Pyr), BaP, and BkF, that were selected to represent both low-molecular-weight and high-molecular-weight PAHs, with distinct toxicological characteristics regarding the AhR activation and genotoxicity potential. The expression of major genes involved in various metabolic pathways (glucose transport, glycolysis, gluconeogenesis and glycogen metabolism; fatty acid synthesis, transport, activation, and oxidation; triglyceride synthesis, storage, and hydrolysis; ketogenesis and bile acid metabolism) were evaluated by RT-qPCR after 24 h or 72 h exposure. These results were compared to the effects of selective agonists of AhR (TCDD), PPARα (GW7647), LXR (GW3965), and FXR (GW4064), in order to reveal possible contribution of these receptors to the PAH-caused effects. The AhR ligands, including BaP, BkF and TCDD, dysregulated expression of genes involved in glucose and glycogen metabolism, and decreased mRNA levels of CYP4A11 and CYP7A11, two enzymes of fatty acid oxidation and bile acid synthesis. All of these effects persisted up to 72 h. Pyr, which is not genotoxic and does not activate AhR, increased mRNA levels of triglyceride metabolism genes. Finally, Fla did not cause any changes after 24 h, but it decreased CYP4A11, CYP7A1 and HMGCS2 after 72 h. None of the tested PAHs induced expression of PPARα-, LXR-, neither FXR-target genes. Nevertheless, BaP, BkF, and TCDD decreased both basal and induced expression of several PPARα-specific genes, which may suggest the existence of an inhibitory cross-talk between AhR and PPARα. This is supported by the observation that BkF, a potent AhR ligand, decreased expression of genes of fatty acid transport, β-oxidation and triglyceride metabolism after 72 h, in a similar manner as TCDD. Taken together, our study indicates that high-molecular-weight PAHs acting as efficient AhR ligands may deregulate human liver glucose and lipid metabolism genes and that determination of expression of cellular stress marker genes and the genes linked to energy metabolism is a novel approach suitable for in vitro toxicity profiling of xenobiotics such as environmental contaminants or their mixtures.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

30108 - Toxicology

Projekt

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Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2022

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů