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. 2011 Feb 15;251(1):41-9.
doi: 10.1016/j.taap.2010.11.013. Epub 2010 Dec 1.

Omega-3 fatty acid oxidation products prevent vascular endothelial cell activation by coplanar polychlorinated biphenyls

Zuzana Majkova  1 , Joseph Layne, Manjula Sunkara, Andrew J Morris, Michal Toborek, Bernhard Hennig
Affiliations

Omega-3 fatty acid oxidation products prevent vascular endothelial cell activation by coplanar polychlorinated biphenyls

Zuzana Majkova et al. Toxicol Appl Pharmacol. .

Abstract

Coplanar polychlorinated biphenyls (PCBs) may facilitate development of atherosclerosis by stimulating pro-inflammatory pathways in the vascular endothelium. Nutrition, including fish oil-derived long-chain omega-3 fatty acids, such as docosahexaenoic acid (DHA, 22:6ω-3), can reduce inflammation and thus the risk of atherosclerosis. We tested the hypothesis that cyclopentenone metabolites produced by oxidation of DHA can protect against PCB-induced endothelial cell dysfunction. Oxidized DHA (oxDHA) was prepared by incubation of the fatty acid with the free radical generator 2,2-azo-bis(2-amidinopropane) dihydrochloride (AAPH). Cellular pretreatment with oxDHA prevented production of superoxide induced by PCB77, and subsequent activation of nuclear factor-κB (NF-κB). A₄/J₄-neuroprostanes (NPs) were identified and quantitated using HPLC ESI tandem mass spectrometry. Levels of these NPs were markedly increased after DHA oxidation with AAPH. The protective actions of oxDHA were reversed by treatment with sodium borohydride (NaBH₄), which concurrently abrogated A₄/J₄-NP formation. Up-regulation of monocyte chemoattractant protein-1 (MCP-1) by PCB77 was markedly reduced by oxDHA, but not by un-oxidized DHA. These protective effects were proportional to the abundance of A₄/J₄ NPs in the oxidized DHA sample. Treatment of cells with oxidized eicosapentaenoic acid (EPA, 20:5ω-3) also reduced MCP-1 expression, but less than oxDHA. Treatment with DHA-derived cyclopentenones also increased DNA binding of NF-E2-related factor-2 (Nrf2) and downstream expression of NAD(P)H:quinone oxidoreductase (NQO1), similarly to the Nrf-2 activator sulforaphane. Furthermore, sulforaphane prevented PCB77-induced MCP-1 expression, suggesting that activation of Nrf-2 mediates the observed protection against PCB77 toxicity. Our data implicate A₄/J₄-NPs as mediators of omega-3 fatty acid-mediated protection against the endothelial toxicity of coplanar PCBs.

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Conflict of interest statement

Conflict of Interest Statement

The authors declare they have no actual or potential competing financial interests.

Figures

Figure 1
Figure 1
Oxidized DHA prevents PCB77-induced superoxide production and activation of NF-κB. (A) Oxidized DHA (oxDHA) was generated via oxidation for 16 h in 2 mM AAPH. Cells were pre-treated with control or oxDHA (40 μM) for 4 h; followed by exposure to vehicle control (DMSO) or PCB77 (5 μM) for 8 h, or antimycin A (50 μM) for 6 h. Cells were then stained with DHE, and red fluorescence was assessed using microscope and quantified by ImageJ 1.42q. (B) Cells were pre-treated with control or oxDHA (40 μM) for 4 h, followed by exposure to control or PCB77 (5 μM) for 6 h. TNF-α (5 ng/mL) treatment for 2 h was used as a positive control. NF-κB DNA-binding was assessed by EMSA. 1, control; 2, PCB77; 3, oxDHA; 4, oxDHA + PCB77; 5, TNF-α; 6, p65 supershift. Data represent mean ± SEM of 3–4 independent experiments. Two-way ANOVA revealed a statistically significant interaction between oxDHA and PCB77. *Significantly different compared to vehicle control (p<0.05). #Significantly different compared to PCB77-treated control without oxDHA (p<0.05).
Figure 2
Figure 2
Analysis of A4/J4-NPs by tandem mass spectrometry.(A) Oxidized DHA sample was infused into the ion source of our instrument, a species of m/z 357 generated in negative mode ESI was subjected to CID, and daughter ions identified from m/z 50 to 400. (B) A 4/J4-NP levels were quantitated in samples containing 40 μM of controls, unoxidized DHA, AAPH-oxidized DHA (oxDHA), or sodium borohydride ( NaBH4)-reduced DHA (redDHA). Qunatification was performed by HPLC ESI MS/MS using d4 -isoprostane as a surrogate calibration standard.
Figure 3
Figure 3
Oxidized DHA prevents MCP-1 mRNA up-regulation by PCB77. (A) Cells were pre-treated with control or oxDHA (0–40 μM) for 4 h. After exposure to control or PCB77 (5 μM) for 24 hours, MCP-1 mRNA expression was assessed using RT-PCR. (B) DHA was oxidized for increasing periods of time, and levels of malondialdehyde (MDA) were assessed using TBARS assay (dashed line). Cells were pre-treated with control or oxDHA (40 μM) for 4 h, followed by PCB77 treatment and RT-PCR as described in (A). (C) DHA was oxidized by AAPH and/or reduced using NaBH4 (redDHA). Cells were pre -treated with respective controls, oxDHA, or redDHA for 4 h, followed by PCB77 treatment and RT-PCR as described in (A). Data represent the mean ± SEM of 4-5 independent experiments. Two-way ANOVA revealed a statistically significant interaction between oxDHA and PCB77. *Significantly different compared to vehicle control (p<0.05). #Significantly different compared to PCB77-treated control without oxDHA (p<0.05).
Figure 4
Figure 4
Inhibition of PCB77-induced MCP-1 up-regulation by oxDHA and oxEPA. (A) Cells were pre-exposed to control, oxDHA (40 μM), or oxEPA (40 μM) for 4 h, followed by exposure to control or PCB77 (5 μM) for 24 hours. MCP-1 mRNA expression was assessed using RT-PCR. (B) Cells were treated as in (A) and MCP-1 protein levels in cell culture media were assessed using ELISA. Data represent mean ± SEM of 4 independent experiments. Two-way ANOVA revealed a significant interaction between oxidized fatty acids and PCB77. *Significantly different compared to vehicle control (p<0.05). #Significantly different compared to PCB77-treated control without oxidized fatty acid (p<0.05).
Figure 5
Figure 5
Nrf2 activation in endothelial cells. (A) Cells were treated with control, oxDHA (40 μM), oxEPA (40 μM), or sulforaphane (SR, 10 μM), for 4 h; followed by an assessment of Nrf2 DNA-binding activity using EMSA. 1, control; 2, oxDHA; 3. oxEPA; 4, SR; 5, Nrf2 supershift. (B) Cells were treated for 24 h with control, oxDHA (40 μM), or SR (10 μM); and protein levels of NQO1 were measured by Western Blot. (C) Cells were pre-treated with SR (5 μM) for 4 h, followed by exposure to control or PCB77 (5 μM) for 24 hours. MCP-1 mRNA expression was assessed using RT-PCR. Data represent mean ± SEM of 3–5 independent experiments. One-way ANOVA revealed a statistically significant treatment effect (A and B), and two-way ANOVA revealed an interaction between SR and PCB77 (C). *Significantly different compared to vehicle control (p<0.05). #Significantly different compared to PCB77-treated control without SR (p<0.05).
Figure 6
Figure 6
Inhibiting Nrf2 negates protection by oxDHA against PCB77 -mediated induction of MCP-1. Cells were pre-treated with the Nrf2 inhibitor all-trans retinoic acid (ATRA, 0.5 μM) (Wang et al., 2007)prior to exposure to oxDHA (40 μM) and PCB77 (5 μM). Data represent mean ± SEM of 3 independent experiments.*Significantly different compared to respective vehicle controls(p<0.05). #Significantly different compared to PCB77 -treated control (p<0.05).
Figure 7
Figure 7
Scheme illustrating the inhibition of PCB77-induced endothelial inflammatory response by A4/J4-NPs. A4/J4-NPs bind Keap1 in the cytoplasm thus increasing Nrf2 nuclear translocation and DNA-binding activity. This leads to induction of antioxidant defense genes, including NQO1. Antioxidant enzymes (NQO1) inhibit ROS production by PCB77, leading to inhibition ofPCB77 -induced NF-κB activation and expression of pro-inflammatory genes, such as MCP-1.
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References

    1. Ahn YH, Hwang Y, Liu H, Wang XJ, Zhang Y, Stephenson KK, Boronina TN, Cole RN, Dinkova-Kostova AT, Talalay P, Cole PA. Electrophilic tuning of the chemoprotective natural product sulforaphane. Proc Natl Acad Sci U S A. 2010;107:9590–9595. - PMC - PubMed
    1. Aiello RJ, Bourassa PA, Lindsey S, Weng W, Natoli E, Rollins BJ, Milos PM. Monocyte chemoattractant protein-1 accelerates atherosclerosis in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 1999;19:1518–1525. - PubMed
    1. Arsenescu V, Arsenescu RI, King V, Swanson H, Cassis LA. Polychlorinated biphenyl-77 induces adipocyte differentiation and proinflammatory adipokines and promotes obesity and atherosclerosis. Environ Health Perspect. 2008;116:761–768. - PMC - PubMed
    1. Chaudhary A, Mishra A, Sethi S. Oxidized omega-3 fatty acids inhibit pro-inflammatory responses in glomerular endothelial cells. Nephron Exp Nephrol. 2004;97:e136–145. - PubMed
    1. Daidoji T, Gozu K, Iwano H, Inoue H, Yokota H. UDP-glucuronosyltransferase isoforms catalyzing glucuronidation of hydroxy-polychlorinated biphenyls in rat. Drug Metab Dispos. 2005;33:1466–1476. - PubMed

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