Dietary oxidized n-3 PUFA induce oxidative stress and inflammation: role of intestinal absorption of 4-HHE and reactivity in intestinal cells.

Dietary intake of long-chain n -3 PUFA is now widely advised for public health and in medical practice. However, PUFA are highly prone to oxidation, producing potentially deleterious 4-hydroxy-2-alkenals. Even so, the impact of consuming oxidized n -3 PUFA on metabolic oxidative stress and inflammation is poorly described. We therefore studied such effects and hypothesized the involvement of the intestinal absorption of 4-hydroxy-2-hexenal (4-HHE), an oxidized n -3 PUFA end-product. In vivo , 4 groups of mice were fed for 8 weeks high-fat-diets containing moderately oxidized or unoxidized n -3 PUFA. Other mice were orally administered 4-HHE and euthanized postprandially vs baseline mice. In vitro , human intestinal Caco-2/TC7 cells were incubated with 4-hydroxy-2-alkenals. Oxidized diet increased 4-HHE plasma levels in mice (up to 5-fold, P <0.01) compared with unoxidized diet. Oxidized diet enhanced plasma inflammatory markers and activation of NFkappaB in the small intestine together with decreasing Paneth cell number (up to 19% in the duodenum). Both in vivo and in vitro, intestinal absorption of 4-HHE occurred associated with 4-HHE-protein adducts and increased expression of glutathione peroxidase 2 (GPx2) and glucose-regulated protein 78 (GRP78). Consumption of oxidized n -3 PUFA results in 4-HHE accumulation in blood after its intestinal absorption and triggers oxidative stress and inflammation in the upper intestine.


INTRODUCTION
Chronic inflammation and oxidative stress are now recognized as major factors involved in the pathogenesis of several current diseases such as overweight, obesity and cardiovascular diseases (1)(2)(3). Elevated levels of pro-inflammatory cytokines and chemokines, such as interleukins (IL) and monocyte chemotactic protein-1 (MCP-1), are hallmarks of the metabolic syndrome (1,2). Several studies demonstrate the nutritional benefits of consuming long-chain (LC) n-3 PUFA from fish, in particular docosahexaenoic acid (DHA, 22:6 n-3) and eicosapentaenoic acid (EPA, 20:5 n-3) to protect against several pathologies (4)(5)(6)(7). Therefore, nutritional recommendations in Western societies have been established for n-3 PUFA intake, of 500 mg/day of EPA and DHA, to achieve nutrient adequacy and reduce incidence of chronic diseases, particularly cardiovascular diseases (8).
However, current studies reveal that the n-3 PUFA may not be devoid of risk. Possible harmful effects of high levels of n-3 PUFA on retinal membrane degeneration have been described by Tanito et al (9). Therefore, dietary LC n-3 PUFA are highly vulnerable to oxidation, which is one of the major problems in food chemistry and may decrease their nutritional value. Indeed, peroxidation causes loss of nutritional quality and further leads to the generation of genotoxic and cytotoxic compounds such as the 4-hydroxy-2-alkenals (10,11). 4-Hydroxy-2-hexenal (4-HHE) and 4-hydroxy-2-nonenal (4-HNE) are major endproducts derived from n-3 and n-6 PUFA peroxidation, respectively. In addition to being markers of lipid peroxidation in vivo, 4-HNE and 4-HHE induce noxious effects on biological systems. These lipid aldehydes are prone to react with thiols and amines moieties and make Schiff base and/or Michael adducts with biomolecules such as proteins, DNA and phospholipids (12,13). Numerous studies reported the genotoxicity and cytotoxicity of these 4-hydroxy-2-alkenals in pathophysiological contexts on tissues and cells (14)(15)(16), but nothing is known to date about the possible contribution of 4-hydroxy-2alkenals being present in food products, their fate after ingestion and metabolic effects.
In this context, the intestinal tract represents the first barrier of detoxification and of defense against oxidative stress. Thus, intestinal cells can be exposed to oxidized PUFA or to lipid peroxidation end-products. However, evidence is lacking to support unfavorable health effects of dietary oxidized n-3 PUFA and demonstrate their possible role in the generation of oxidative stress and inflammation. Furthermore, limited data is available to support the hidden assumption of intestinal absorption of dietary lipid oxidation byproducts.
Therefore, we hypothesized that long-term intake of limited amounts of oxidized n-3 PUFA in high-fat (HF) diets could exert harmful health effects, due to the absorption of their end-products such as 4-HHE by the small intestine; the end-products having been formed during the processing, storage and/or final handling of foods or after their ingestion. The aim of the present study was thus to investigate (i) the effects of oxidized n-3 PUFA diets compared with unoxidized diets on oxidative stress and inflammation in mice and (ii) the possible implication of the intestinal absorption of some PUFA oxidation end-products, namely 4-hydroxy-2-alkenals, through their effects on intestinal stress and inflammation, in vivo and in vitro. Because several types of LC n-3 PUFA sources are present in human food, we tested lipid mixtures containing EPA and DHA carried by either triacylglycerols (TG) or phospholipids (PL). Moreover, we analyzed different segments of the absorptive intestinal epithelium, especially the duodenum before the interaction with bile salts occurs and the jejunum that represents the major site of lipid absorption, but also the ileum to test whether some effects remain in this more distal segment.

Preparation of unoxidized and oxidized lipid blends and mice diets
Four lipid blends were prepared at the labscale to obtain similar fatty acid composition and quantities in the four diets and similar amounts of glycerophospholipids and triacylglyerols. In these blends, DHA was supplied either in the form of triacylglycerols (TG diet) or phospholipids (PL diet), i.e., the name chosen for the diets reflects the type of molecules that carry long-chain n-3 PUFA in the diet. The oxidized lipid blends (TG-ox and PL-ox respectively) were prepared as described as follows: primary lipid mixtures for PL and TG groups were prepared with a small proportion of lard to maintain oxidability of PUFA. These preliminary oil mixtures were dispersed in 30% w/w relative to aqueous phase (mineral water; Evian) to prepare oil-in-water emulsions. The emulsions were then kept at 50°C in the dark with continuous shaking until oxidation level was considered as sufficient according to our previous experiments (estimated α-tocopherol contents of the blends decreased by 50%). Oxidized emulsions were then lyophilized and the resulting oxidized lipid mixtures completed with the necessary quantity of lard to reach the required final composition of lipid blends. The composition of the four diets is reported in Table 1.

Animals and diets
Male C57BL/6 mice (8 wk, 20g) were from Janvier SA (Le Genest Saint-Isle, France) and were housed in a temperature-controlled room (22°C) with a 12 h light/12 h dark cycles.

Caco-2/TC7 cell culture and treatment
Caco-2/TC7 cells, which are the widely used in vitro model of human origin to test intestinal absorption of lipids, were provided by Monique Rousset (Centre de Recherche des Cordeliers, Paris, France) and used between passage 35 and 45. Cells were seeded in 75-cm 2 flasks (Falcon, Becton Dickinson) until 80-90% confluence. They were grown in complete DMEM (Gibco) supplemented with 20% heat-inactivated FBS (Gibco), 1% nonessential amino acids (Gibco) and 1% antibiotics (penicillin/streptomycin, Gibco), and maintained under a 10% CO 2 atmosphere at 37°C. For experiments, cells were seeded at a density of 25×10 4 cells per filter on microporous (0.4 μm pore size) polyester filters (Transwell, Corning, USA) and grown to confluence in complete medium, which was routinely reached 7 days after seeding. The cells were used 21 days after seeding.
Monolayers were incubated with 4-HHE or 4-HNE in the apical compartment (1 to 100 µM brought in DMSO at 0.5% in the final medium) and the basolateral compartment receiving serum-free DMEM. After incubation, basolateral media and cells were collected.

Animal treatment with 4-HHE
After an overnight fast, three groups of 4 male C57/BL6 mice (8 weeks, 22 g) were given a single application of 4-HHE diluted in water via 0.5% DMSO by oral gavage at a dosage of 10 mg/kg b.w. and were sacrificed 1, 2, 4 h after gavage. A fourth group of mice was sacrificed immediately after gavage for the baseline control. For sacrifice, mice were anesthetized by IP injection of pentobarbital (35 mg/kg) and blood was collected by cardiac puncture with heparinized syringes. Plasma and small intestine mucosa (duodenum and jejunum) were collected.

Plasma triacylglycerols and NEFA measurements
Plasma triacyglycerols (TAG) were measured with the triglyceride PAP kit (BioMérieux France) as previously described (19). Plasma TAG concentration was calculated by subtracting the free glycerol in plasma measured with the glycerol UV-method (R-Biopharm/Boehringer, Mannheim, Germany). Plasma NEFA was measured using NEFA-C kit (Wako Chemicals, Neuss, Germany) (19).

Fatty acid analysis
Total lipids were extracted from 35 µL of plasma as described previously (19). The organic phase was evaporated under N 2 and total fatty acids were transesterified using boron trifluoride in methanol (BF3/methanol) (19). The FA methyl esters were then analyzed by GC using a DELSI instrument model DI 200 equipped with a fused silica capillary SP-2380 column (60 m x 0.22 mm). Heptadecanoic acid (C17:0, Sigma, France) was used as an internal standard.

Quantification of free malondialdehyde (MDA) and hydroperoxides in oil mixtures
Thiobarbituric acid-MDA adducts were separated using a method adapted from different authors (20,21) by HPLC and measured by fluorimetry using an external calibration curve (excitation 535 nm, emission 555 nm). Hydroperoxides were quantified in lipid blends according to a method adapted from Nourooz-Zahed et al (22).

Quantitative PCR Analysis
Total RNA was extracted from Caco-2/TC7, duodenum, jejunum and ileum of mice using the NucleoSpin ® RNA/Protein kit (Macherey Nagel, Duren, Germany). cDNAs were synthesized from 1 µg of total RNA in the presence of 100 units of Superscript II (invitrogen) with a mixture of random hexamers and oligo (dt) primers (Promega, Charbonnières, France). The amount of target mRNAs was measured by RT, followed by real-time PCR, using a Rotor-Gene Q (Qiagen, France). The amount of target mRNAs was measured by RT, followed by real-time PCR, using a Rotor-Gene Q (Qiagen, France).

Western blot analysis
Total proteins from Caco-2/TC7 and jejunum of mice were extracted with the NucleoSpin ® The sections were then counterstained and mounted.

Quantitative analysis for Paneth cells
The Paneth cell lineage was analyzed by assessing the percentage of crypt cross sections with Paneth cells (per 80 crypts). For this purpose, 4-6 sections were analyzed per mouse.
A crypt was considered when it was cut along or nearly along the length of the crypt lumen (at least two-thirds of the length of the crypt). All slides were analyzed by a single investigator who was blinded to the treatment groups. was performed by densitometric analysis of specific spots on immunoblots using Quantity One software.

Statistical analysis
All data are presented as means ± SEM and were analysed using Statview 5.0 software (Abacus Concept, Berkeley). One-way ANOVA followed by Fisher PLSD was used (i) For the dietary study to compare PL, PL-ox, TG and TG-ox groups, (ii) for the gavage study, to compare plasma alkenal concentrations as a function of time and (iii) for the Caco-2 cell studies, to compare treatment effects. Two-way ANOVA followed by Fisher PLSD was used to compare oxidized vs. unoxidized groups globally in the dietary study (mice of oxidized groups vs. mice of unoxidized groups). Differences were considered significant at the P < 0.05 level. Table 2 shows that we succeeded in producing lipid mixtures containing different amounts of oxidation products, especially 4-HHE which was significantly higher in oxidized vs unoxidized lipid blends. MDA and hydroperoxides in oxidized oils were in a reasonable range considered as acceptable for human consumption. Noticeably, oxidation did not impact the fatty acid profile ( Table 2); n-3 PUFA content and n-6/n-3 ratio were consistent with dietary recommendations. Mice in oxidized and unoxidized groups did not differ in final body weight gain, liver weight, white adipose tissue, plasma TAG or plasma NEFA (Table 3).

Aldehyde stress and inflammatory markers in plasma of mice fed oxidized n-3 diets
Because consuming oxidized n-3 diets could contribute to circulating biomarkers of lipid peroxidation such as 4-HHE and 4-HNE, we measured these 4-hydroxy-2-alkenals in plasma. Figure 1A  Regarding metabolic inflammation, we show higher concentrations in plasma of the proinflammatory cytokine IL-6 ( Fig. 1C) and of the chemokine MCP-1 (Fig. 1D) in oxidized groups.
Altogether, high-fat diet containing realistically oxidized n-3 PUFA induced the highest amounts of 4-HHE and of inflammatory markers in plasma. We therefore questioned whether oxidized oils would affect the small intestine, which is the primary defense line of the host regarding ingested products.

Effects of oxidized diets on markers of stress and inflammation in the small intestine
We analyzed the small intestine mucosa regarding the expression of genes that are known to be specifically involved in cell defense against oxidative stress and detoxification. The expression of mGPx2, a gastrointestinal glutathione peroxidase (GI-GPx) mainly expressed in the intestine, was increased specifically in the jejunum of oxidized groups (Fig. 2D,   P<0.05). Moreover, we examined the gene expression of two markers that are linked to endoplasmic reticulum (ER) stress. Mice fed oxidized diets exhibited a significantly higher expression of pro-survival factor glucose-regulated protein 78 (GRP78) both in the duodenum and in the jejunum ( Fig. 2B; E, P<0.05). The pro-apoptotic C/EBP homologous protein (CHOP) was significantly higher in the jejunum of oxidized groups (Fig. 2F).
However, on the whole down in the ileum, no effects of n-3 PUDA oxidation was observed. Regarding inflammation, results show that the jejunum of mice fed oxidized diet exhibited an increased phosphorylation of NF-κB P65 (Fig. 3A, P<0.0001), a major transcription factor involved in inflammation, associated with a significant increase of phosphorylated IkappaB alpha (IκBα), an inhibitor NF-κB protein (Fig. 3B, P<0.0001).
The activation of NF-κB required the phosphorylation and degradation of IκB protein that prevents the nuclear translocation of active NF-κB. We also analyzed Paneth cells in the duodenum, which are involved in innate immunity by sensing bacteria and by discharging antimicrobial peptides including -defensins. Paneth cell number was significantly decreased after oxidized vs unoxidized diets (Fig. 3C). Mocreover, the gene expression of

Kinetics of intestinal absorption of 4-HHE in mice and protein modifications in the small intestine
To test the hypothesis that the increased plasma level of 4-HHE following consumption of oxidized diet could be partly explained by the absorption of 4-HHE in the small intestine we measured the levels of 4-HHE in the plasma of fasted mice after gavage with 4-HHE.

We quantified protein carbonylation and the formation of aldehyde-protein adducts. Cell incubated for 24 h with increasing concentrations of 4-HHE or 4-HNE exhibited an
increased level of reactive carbonyl group in proteins in a dose-dependent manner from 50 µM (P<0.0001) vs untreated cells (Fig. 6 A,B). Fig. 6C also reveals a significant increase of HNE-protein adducts after incubating cells for 2 h with 4-HNE from 10 µM in a dose-dependent manner vs untreated cells. Similar treatment using 4-HHE resulted in increased amounts of HHE-protein adducts from 50 µM (P<0.05) (Fig. 6D).

GPx2 and ER stress-linked gene expression in Caco-2/TC7 cells
We then sought to determine whether some antioxidant systems were changed due to 4hydroxy-2-alkenals. Fig. 7 (A, B) shows that mGPx2 expression tended to increase after 24 This is slightly lower than in Surh et al. study (25), nevertheless our experimental conditions were relevant considering realistic food intakes.
Most of the biological effects of 4-hydroxy-2-alkenals are due to their capacity to react with the nucleophilic sites of proteins to form Michael or Schiff base protein adducts (13,26), which can be implicated in several diseases such as diabetes (27), atherosclerosis (28) and neurodegenerative diseases (14,16). Here we found that when 4-HHE was orally GRP78 is widely considered as a common regulator/sensor of ER stress. Overexpression of GRP78 is induced by a variety of environmental stress conditions leading to impairment of essential ER functions in order to maintain cell viability against oxidative stress and apoptosis (38). In addition, ER stress induces genes encoding non ER-proteins such as CHOP associated with growth arrest and induction to apoptosis (39). ER stress may also result from protein denaturation due to carbonylation and/or covalent adducts formation We cannot rule out that observed effects can be due to other bioactive species than 4-hydroxy-alkenals. For example, oxidized phospholipids by themselves have been reported to be proinflammatory (42,43); advanced oxidation protein products could also contribute to observed effects (44,45).
In conclusion, our study demonstrates in mice that long-term ingestion of oxidized n-3 PUFA induces an accumulation of 4-HHE in plasma, which can be partly due to 4  Bars represents means ± SEM of n=5-7 mice. * P<0.05. ANOVA followed by Fisher test.    ANOVA followed by Fisher test. ANOVA followed by Fisher test.   Data are mean ± SEM for n=7-9 per group. Abbreviations: WAT, white adipose tissue.