Structure-activity analysis of diffusible lipid electrophiles associated with phospholipid peroxidation: 4-hydroxynonenal and 4-oxononenal analogues

Abstract

Electrophile-mediated disruption of cell signal-ing is involved in the pathogenesis of several diseases including atherosclerosis and cancer. Diffusible and membrane bound lipid electrophiles are known to modify DNA and protein substrates and modulate cellular pathways including ER stress, antioxidant response, DNA damage, heat shock, and apoptosis. Herein we report on a structure-activity relationship for several electrophilic analogues of 4-hydroxynonenal (HNE) and 4-oxononenal (ONE) with regard to toxicity and anti-inflammatory activity. The analogues studied were the oxidation products of HNE and ONE, HNEA/ONEA, the in vivo hydrolysis products of oxidized phosphatidylcholine, COOH-HNE/COOH-ONE, and their methyl esters, COOMe-HNE/ONE. The reactivity of each compound toward N-acetylcysteine was determined and compared to the toxicity toward a human colorectal carcinoma cell line (RKO) and a human monocytic leukemia cell line (THP-1). Further analysis was performed in differentiated THP-1 macrophages to assess changes in macrophage activation and pro-inflammatory signaling in response to each lipid electrophile. HNE/ONE analogues inhibited THP-1 macrophage production of the pro-inflammatory cytokines, IL-6, IL-1β, and TNFα, after lipopolysaccharide (LPS)/IFNγ activation. Inhibition of cytokine production was observed at submicromolar concentrations of several analogues with as little as 30 min of exposure. Phagocytosis of fluorescent beads was also inhibited by lipid electrophile treatment. Lipid electrophiles related to HNE/ONE are both toxic and anti-inflammatory, but the anti-inflammatory effects in human macrophages are observed at nontoxic concentrations. Neither toxicity nor anti-inflammatory activity are strongly correlated to the reactivity of the model nucleophile, N-acetylcysteine.

Scheme 1. Representative Electrophiles Derived from ω-6 PUFAs

Scheme 2. Synthesis of HNE, ONE, HNEA, and ONEA

Reagents: (a) Dess-Martin periodinane, CH₂Cl₂; (b) NaOH, H₂O/MeOH; (c) CrO₃, H₂O/H₂SO₄, acetone.

Scheme 3. Synthesis of COOH-HNE, COOH-ONE, and Corresponding Esters

Reagents: (a) 10-(Tetrahydro-2H-pyran-2-yloxy)decanal, piperidine, CH₃CN; (b) TBDMSCl, imidazole, DMF; (c) MgBr₂-Et₂O, ether; (d) bleach, Tempo, NaBr, Bu₄NBr, NaHCO₃, CH₂Cl₂/H₂O; (e) DIBAL-H, Et₃N, CH₂Cl₂; (f) Dess-Martin periodinane, CH₂Cl₂; (g) HF_aq, CH₃CN; (h) Diazald, KOH, H₂O/EtOH, ether.

Figure 1

THP-1 cell viability assay for select lipid electrophiles. THP-1 cells were treated with various concentrations of lipid electrophiles for 24 h, and cell viability was assayed using the fluorescent substrate calcein-AM. IC₅₀ values were determined from a plot of log of electrophile concentration versus percent of live cells as compared to a vehicle (DMSO) control.

Figure 2

Stability (open symbols) and reactivity (closed symbols) of HNE and ONE analogues. (A) HNE with (filled red triangle) and without (open red triangle) NAC, COOH-HNE with (filled blue circle) and without (open blue circle) NAC, COOMe-HNE with (filled black square) and without (open black square) NAC; (B) ONE with (filled red triangle) and without (open red triangle) NAC, COOH-ONE with (filled blue circle) and without (open blue circle) NAC, COOMe-ONE with (filled black square) and without (open black square) NAC; (C) HNEA with (filled blue circle) and without (open red triangle) NAC; (D) ONEA with (filled blue circle) and without (open red triangle) NAC. Reactions were carried out with 0.056 mM electrophile and 1 mM NAC in phosphate buffer (50 mM, pH 7.4) at 37 °C. The consumption of the electrophile was monitored by UV absorbance at 225 and 230 nm for HNE and ONE, respectively.

Figure 3

Effect of continuous vs transient HNE treatment on cytokine expression during LPS/IFNγ challenge. THP-1 cells were pretreated with various concentrations of HNE for 30 min, followed by either leaving in or washing out (w.o.) the electrophile, and 6 h of LPS challenge. HNE was assayed for its ability to inhibit IL-1β (A) and IL-6 (B) expression.

Figure 4

Inhibition of cytokine expression by HNE and ONE. IL-6 ELISA of conditioned media from THP-1 macrophages pretreated with HNE or ONE (30 min), washed, and challenged with LPS/IFNγ for 6 h (A). IL-1β (B) or TNFα (C) ELISA of media from THP-1 cells pretreated with HNE for 30 min, washed, and challenged with LPS for the times indicated (6 h LPS challenge for TNFα ELISA).

Figure 5

IL-6 Western blot of cell lysate from THP-1 macrophages pretreated with HNE, washed, and challenged with LPS for 6 h. The lysate was probed with either anti-IL-6 or antiactin antibodies.

Figure 6

Recovery of IL-6 synthesis by delaying the time between LPS challenge and HNE treatment. Western blot for IL-6 from THP-1 macrophage cell lysate. Cells were pretreated with 10 μM HNE or the vehicle control for 30 min followed by washing with PBS. Following washout of the electrophile, cells were challenged with LPS/IFN-γ after an incubation time of 0, 0.5, or 3 h. The cell lysate was probed with either anti-IL-6 or anti-actin antibodies.

Figure 7

Inhibition of cytokine expression following acute exposure to HNEA and ONEA. IL-1β ELISA (A), IL-6 ELISA (B), and TNFα ELISA (C) of conditioned media from THP-1 macrophages pretreated with HNEA or ONEA (30 min), washed, and challenged with LPS/IFNγ for 6 h.

Figure 8

Inhibition of cytokine expression following treatment with COOMe-HNE and COOMe-ONE. IL-1β ELISA (A), IL-6 ELISA (B), and TNFα ELISA (C) of conditioned media from THP-1 macrophages pretreated with COOMe-HNE or COOMe-ONE (30 min), washed, and challenged with LPS/IFNγ for 6 h.

Figure 9

Inhibition of phagocytosis by THP-1 macrophages following pretreatment with lipid electrophiles. Fluorescent microscopy images (GFP Filter) of THP-1 macrophage phagocytosis following 30 min of pretreatment with the DMSO vehicle, 10 μM HNE, or 0.5 μM ONE and washout with PBS. Phagocytosis of fluorescent polystyrene latex beads after 3 h (A). Quantification of fluorescent bead uptake by THP-1 macrophages following pretreatment with electrophiles, washout, and exposure to fluorescent beads for 3 or 6 h (B). * p-value <0.05; **p-value <0.01 as compared to the 3 h and 6 h vehicle (DMSO) control, respectively.