PPARδ signaling mediates the cytotoxicity of DHA in H9c2 cells

Victor Samokhvalov, Igor Zlobine, Kristi L. Jamieson, Paul Jurasz, Christopher Chen, Kin Sing Stephen Lee, Bruce D. Hammock, John M. Seubert

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Docosahexaenoic acid (22:6n3, DHA) is an n-3 polyunsaturated fatty acid (PUFA) known to affect numerous biological functions. While DHA possesses many properties that impact cell survival such as suppressing cell growth and inducing apoptosis, the exact molecular and cellular mechanism(s) remain unknown. Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors that regulate many cell pathways including cell death. As DHA acts as a ligand to PPARs the aim of this study was to examine the involvement of PPARδ in DHA-mediated cytotoxicity toward H9c2 cells. Treatment with DHA (100. μM) resulted in a significant decline in cell viability, cellular metabolic activity and total antioxidant capacity coinciding with increased total proteasome activities and activity of released lactate dehydrogenase (LDH). No changes in reactive oxygen species (ROS) production or accumulation of lipid peroxidation products were observed but DHA promoted apoptotic cell death as detected by flow cytometry, increased caspase-3 activity and decreased phosphorylation of Akt. Importantly, DHA enhanced PPARδ DNA binding activity in H9c2 cells strongly signifying that the cytotoxic effect of DHA might be mediated via PPARδ signaling. Co-treatment with the selective PPARδ antagonist GSK 3787 (1. μM) abolished the cytotoxic effects of DHA in H9c2 cells. Cytotoxic effects of DHA were attenuated by co-treatment with myriocin, a selective inhibitor of serine palmitoyl transferase (SPT), preventing de novo ceramide biosynthesis. LC/MS analysis revealed that treatment with DHA resulted in the accumulation of ceramide, which was blocked by GSK 3787. Interestingly, inhibition of cytochrome P450 (CYP) oxidase with MS-PPOH (50. μM) abolished DHA-mediated cytotoxicity suggesting downstream metabolites as the active mediators. We further demonstrate that CYP oxidase metabolites of DHA, methyl epoxy docosapentaenoate (EDP methyl esters, 1. μM) (mix 1:1:1:1:1:1; 4,5-, 7,8-, 10,11-, 13,14-, 16,17- and 19,20-EDP methyl esters) and 19,20-EDP cause cytotoxicity via activation of PPARδ signaling leading to increased levels of intracellular ceramide. These results illustrate novel pathways for DHA-induced cytotoxicity that suggest an important role for CYP-derived metabolites, EDPs.

Original languageEnglish (US)
Pages (from-to)10-20
Number of pages11
JournalToxicology Letters
Volume232
Issue number1
DOIs
StatePublished - Jan 5 2015

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Peroxisome Proliferator-Activated Receptors
Cytotoxicity
Ceramides
Metabolites
Cytochrome P-450 Enzyme System
Electron Transport Complex IV
Cell death
Cell Survival
Oxidoreductases
Esters
Cell Death
Cells
Phosphorylation
Flow cytometry
Docosahexaenoic Acids
Biosynthesis
Omega-3 Fatty Acids
Cell growth
Proteasome Endopeptidase Complex
Cytoplasmic and Nuclear Receptors

Keywords

  • Apoptosis
  • Ceramide
  • Docosahexaenoic acid
  • Epoxydocosapentaenoic acids
  • H9c2 cells
  • PPARδ

ASJC Scopus subject areas

  • Toxicology

Cite this

Samokhvalov, V., Zlobine, I., Jamieson, K. L., Jurasz, P., Chen, C., Lee, K. S. S., ... Seubert, J. M. (2015). PPARδ signaling mediates the cytotoxicity of DHA in H9c2 cells. Toxicology Letters, 232(1), 10-20. https://doi.org/10.1016/j.toxlet.2014.09.029

PPARδ signaling mediates the cytotoxicity of DHA in H9c2 cells. / Samokhvalov, Victor; Zlobine, Igor; Jamieson, Kristi L.; Jurasz, Paul; Chen, Christopher; Lee, Kin Sing Stephen; Hammock, Bruce D.; Seubert, John M.

In: Toxicology Letters, Vol. 232, No. 1, 05.01.2015, p. 10-20.

Research output: Contribution to journalArticle

Samokhvalov, V, Zlobine, I, Jamieson, KL, Jurasz, P, Chen, C, Lee, KSS, Hammock, BD & Seubert, JM 2015, 'PPARδ signaling mediates the cytotoxicity of DHA in H9c2 cells', Toxicology Letters, vol. 232, no. 1, pp. 10-20. https://doi.org/10.1016/j.toxlet.2014.09.029
Samokhvalov V, Zlobine I, Jamieson KL, Jurasz P, Chen C, Lee KSS et al. PPARδ signaling mediates the cytotoxicity of DHA in H9c2 cells. Toxicology Letters. 2015 Jan 5;232(1):10-20. https://doi.org/10.1016/j.toxlet.2014.09.029
Samokhvalov, Victor ; Zlobine, Igor ; Jamieson, Kristi L. ; Jurasz, Paul ; Chen, Christopher ; Lee, Kin Sing Stephen ; Hammock, Bruce D. ; Seubert, John M. / PPARδ signaling mediates the cytotoxicity of DHA in H9c2 cells. In: Toxicology Letters. 2015 ; Vol. 232, No. 1. pp. 10-20.
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abstract = "Docosahexaenoic acid (22:6n3, DHA) is an n-3 polyunsaturated fatty acid (PUFA) known to affect numerous biological functions. While DHA possesses many properties that impact cell survival such as suppressing cell growth and inducing apoptosis, the exact molecular and cellular mechanism(s) remain unknown. Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors that regulate many cell pathways including cell death. As DHA acts as a ligand to PPARs the aim of this study was to examine the involvement of PPARδ in DHA-mediated cytotoxicity toward H9c2 cells. Treatment with DHA (100. μM) resulted in a significant decline in cell viability, cellular metabolic activity and total antioxidant capacity coinciding with increased total proteasome activities and activity of released lactate dehydrogenase (LDH). No changes in reactive oxygen species (ROS) production or accumulation of lipid peroxidation products were observed but DHA promoted apoptotic cell death as detected by flow cytometry, increased caspase-3 activity and decreased phosphorylation of Akt. Importantly, DHA enhanced PPARδ DNA binding activity in H9c2 cells strongly signifying that the cytotoxic effect of DHA might be mediated via PPARδ signaling. Co-treatment with the selective PPARδ antagonist GSK 3787 (1. μM) abolished the cytotoxic effects of DHA in H9c2 cells. Cytotoxic effects of DHA were attenuated by co-treatment with myriocin, a selective inhibitor of serine palmitoyl transferase (SPT), preventing de novo ceramide biosynthesis. LC/MS analysis revealed that treatment with DHA resulted in the accumulation of ceramide, which was blocked by GSK 3787. Interestingly, inhibition of cytochrome P450 (CYP) oxidase with MS-PPOH (50. μM) abolished DHA-mediated cytotoxicity suggesting downstream metabolites as the active mediators. We further demonstrate that CYP oxidase metabolites of DHA, methyl epoxy docosapentaenoate (EDP methyl esters, 1. μM) (mix 1:1:1:1:1:1; 4,5-, 7,8-, 10,11-, 13,14-, 16,17- and 19,20-EDP methyl esters) and 19,20-EDP cause cytotoxicity via activation of PPARδ signaling leading to increased levels of intracellular ceramide. These results illustrate novel pathways for DHA-induced cytotoxicity that suggest an important role for CYP-derived metabolites, EDPs.",
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AU - Chen, Christopher

AU - Lee, Kin Sing Stephen

AU - Hammock, Bruce D.

AU - Seubert, John M.

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