AJP - GI Information on EB 2010
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 293: G1252-G1261, 2007. First published October 4, 2007; doi:10.1152/ajpgi.00369.2007
0193-1857/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/6/G1252    most recent
00369.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Levy, E.
Right arrow Articles by Sinnett, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Levy, E.
Right arrow Articles by Sinnett, D.

MUCOSAL BIOLOGY

Biological role, protein expression, subcellular localization, and oxidative stress response of paraoxonase 2 in the intestine of humans and rats

Emile Levy,1 Karine Trudel,1 Moise Bendayan,2 Ernest Seidman,3 Edgard Delvin,4 Mounib Elchebly,4 Jean-Claude Lavoie,5 Louis-Philippe Precourt,1 Devendra Amre,5 and Daniel Sinnett5

Departments of 1Nutrition, 2Pathology and Cellular Biology, 4Biochemistry, and 5Pediatrics, Université de Montréal, Research Centre, CHU Sainte Justine, Montréal; and 3Research Institute, McGill University, Montréal, Québec, Canada

Submitted 13 August 2007 ; accepted in final form 29 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Oxidative stress is a cardinal manifestation of various intestinal disorders. However, very little knowledge is available on the intestine's inherent defense mechanisms against free radicals. This study was designed to determine the protein expression, subcellular localization and oxidative stress response of paraoxonase 2 (PON2), a member of a powerful antioxidant family in human and rat intestine. Biochemical and ultrastructural experiments all showed a substantial expression of PON2 in human and rat intestine. Western blot analysis disclosed higher levels of PON2 in the jejunum than in the duodenum, ileum, and colon. Cell fractionation revealed a predominant PON2 association with microsomes and lysosomes in the human jejunum, which differed from that in rats. PON2 was detected in the intestine as early as week 15 of gestation and was significantly increased by week 20. Iron ascorbate-mediated lipid peroxidation induced a marked decrease in PON2 expression in intestinal specimens coincidental to an abundant rise in malondialdehyde (MDA). On the other hand, preincubation with potent antioxidants, such as butylated hydroxytoluene, Trolox, and N-acetylcysteine, prevented iron-ascorbate-generating PON2 reduction in parallel with MDA suppression. Finally, the preincubation of permeabilized Caco-2 cells with purified PON2 led to a protection against iron-ascorbate-induced lipid peroxidation. These observations demonstrate that the human intestine is preferentially endowed with a marked PON2 expression compared with the rat intestine and this expression shows a developmental and intracellular pattern of distribution. Furthermore, our observations suggest PON2 protective effects against prooxidant stimuli in the small intestine.

subcellular organelles; antioxidants; enterocytes; Caco-2 cells

REDOX BALANCE, USUALLY OBSERVED in healthy subjects, is maintained by an equilibrium between prooxidants and antioxidants (21, 38). A decrease in antioxidant protection, an excess of reactive oxygen species (ROS), as well as a failure to repair oxidative damage lead to redox imbalance (21). The resulting accumulation of free radicals can have deleterious effects by reacting and oxidizing key organic substrates, such as polyunsaturated fatty acids, proteins, and DNA (6, 12, 50). This process, called oxidative stress, disturbs normal functioning and is involved in a wide spectrum of pathologies: cancer, atherosclerosis, cystic fibrosis, Alzheimer, and Parkinson (11, 26, 30, 39, 53, 55). Importantly, a variety of gastrointestinal diseases are also associated with ROS and oxidative stress (19, 41, 42). In fact, the gastrointestinal mucosa is repetitively exposed to luminal oxidants from ingested foods (13, 20, 41) and, despite the antioxidant properties of its mucus lining, there is a continuous generation of oxidative stress (20). Clearly, the ingestion and/or occurrence of peroxides may have implications for human health, particularly in the long term.

Antioxidants play a crucial role in preventing damage induced by oxidative stress through the neutralization of free radicals. Alimentary nonenzymatic oxidants, such as {alpha}-tocopherol, vitamin C, and retinoids, possess a chemical structure that allows the quenching of singlet oxygen and peroxides (25). In addition, humans are endowed with endogenous enzymatic antioxidants that include superoxide dismutase, which catalyzes the conversion of superoxides into oxygen and hydrogen peroxide and is less reactive toward organic molecules (36); catalase, a heme-containing enzyme, which allows the dismutation of hydrogen peroxide into water and oxygen (37); and glutathione peroxidase, which detoxifies activated oxygen through the catalysis of hydrogen peroxide reduction (1, 37). Recently, numerous investigators have focused on the paraoxonase (PON) protein family (2). Its distinct members (PON1, PON2, and PON3) are believed to be powerful attenuators of oxidative damage and highly atheroprotective (17, 32, 40). PON1 and PON3 circulate attached to high-density lipoprotein particles (15). They inhibit atherogenesis by hydrolyzing lipid hydroperoxides and by preventing low-density lipoprotein (LDL) oxidative modification (15). The distribution of PON2 in many tissues suggests the possibility of its playing an antioxidant role (15). PON2 is increased in cells harvested from 4-mo-old apolipoprotein (apo) E(–/–) animals exhibiting signs of oxidative stress (46). Additionally, the administration of PON2 to macrophages from apo E(–/–) mice reduced lipid peroxide content (46). Similarly, overexpression of PON2 in Hela cells prevents the formation of oxidized LDL (40, 45). Overall, these observations are indicative of the antioxidant properties of PON2.

The number of studies suggesting the protective properties of PON2 is, however, limited, and there has been little insight into its role in animal and human physiology. In particular, only limited data are available on the intestine, even though this organ is exposed on a daily basis to high levels of oxidative stress and requires strong antioxidants, which may preserve important endocrine, metabolic, immunological, and absorptive functions. The major aims of the present investigation were to examine the protein expression, subcellular localization, ontogeny, role, and response to oxidative stress of PON2 in the human and rat intestine. Experiments were also carried out to explore whether powerful antioxidants may prevent the manifestation of oxidative stress and the depletion of PON2 following the exposure of microsomal fractions to iron-ascorbate.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Small intestine and large bowel tissues were obtained from fetuses ranging from 15 to 20 wk following legal or therapeutic abortion with informed patient consent. No tissues were collected from cases associated with known fetal abnormalities or fetal death. Intestinal samples were also obtained from patients (50–60 yr of age) who had undergone surgical resection of different areas of the digestive tract. Normal tissue found adjacent to the resected pathological tissues was used in all cases, as ascertained by routine hematoxylin-eosin staining. The human study protocol was approved by the Ethics Committee of Sainte-Justine Hospital and the Institutional Review Committee for the use of human material from the Centre Hospitalier Universitaire de Sherbrooke/Faculté de Médecine. All experimental animal procedures were authorized by the Institutional Animal Care Committee.

Preparation and specificity of PON2 antibody. The PON2 polyclonal antibody (Ab) was prepared by Invitrogen (Carlsbad, CA). It was generated against the PON2 region spanning amino acids 94–112. The specificity of the antibody was evaluated by various methods, including ELISA that recognized only PON2 among various proteins, Western blotting following the incubation of the Ab in the presence or absence of the 19-amino acid antigen, the omission of the primary PON2 Ab in Western blot, and the identification of the PON2 sequences following immunoprecipitation and SDS-PAGE. Noteworthy is the KEEKPRARELRISRGFDL epitope that allows cross-reaction between humans and rats. It is also important to note that pilot studies were initiated to demonstrate 1) the specificity of the PON2 polyclonal Ab in humans and rats and 2) the equivalent response to PON2 Ab from human and rat tissues, which were simultaneously tested by PAGE and Western blot.

Immunocytochemical labeling. Rat and human intestinal tissues were sampled and fixed by immersion in 0.1 mM phosphate-buffered 1% glutaraldehyde for 2 h. Tissues were then processed for embedding in Lowicryl at –30°C (4, 31). Tissue thin sections were cut, mounted on Parlodion-carbon coated grids, and processed for the protein A-gold immunolabeling according to previously described methods (4, 31). The anti-PON2 antibody was used at 1/10 dilution in combination with the 10-nm protein A-gold complex. Control of specificity was carried out by omitting the primary antibody step or by replacing the PON2 antibody with albumin.

Preparation of microsomes and subcellular fractions. Intestinal specimens were rinsed, homogenized, and centrifuged for 15 min at 12,000 g at 4°C to prepare microsome fractions, a technique described earlier (8, 9). The supernatant fraction was then centrifuged for 60 min at 100,000 g. The pellet was centrifuged for 60 min at 4°C. The washed microsomal pellets were quick frozen and stored at –80°C for later use. To isolate subcellular fractions, tissue homogenate was subjected to differential and discontinuous sucrose density gradient centrifugation.

Western blots. To assess the presence of PON2 and evaluate its mass, intestinal tissues were homogenized and adequately prepared for Western blotting as described previously (29). The Bradford assay (Bio-Rad), was used to estimate protein concentration. Proteins were denatured in sample buffer containing SDS and β-mercaptoethanol, separated on a 4–20% gradient SDS-PAGE, and electroblotted onto nitrocellulose membranes. Nonspecific binding sites of the membranes were blocked with defatted milk proteins followed by the addition of primary antibodies directed against PON2. The relative amount of primary antibody was detected with species-specific horseradish peroxidase-conjugated secondary antibody. Even if identical protein amounts of tissue homogenates were applied, the β-actin protein was used to confirm equal loading on SDS-PAGE (results not shown). Blots were developed and the mass of PON2 was quantitated using an HP Scanjet scanner equipped with a transparency adapter and software.

Effect of antioxidants. To determine whether lipid peroxidation was responsible for alterations in PON2 protein expression, the antioxidants butylated hydroxytoluene (BHT) (0.5 mM), N-acetylcysteine (NAC) (5 mM), and Trolox (0.5 mM) were added to microsomes or to the apical compartment of Caco-2 cells for 1 h before incubation with iron-ascorbate.

Estimation of lipid peroxidation. The amount of free malondialdehyde (MDA) formed during the reaction was determined by HPLC, as we previously described (6). Proteins were first precipitated with a 10% sodium tungstate (Na2WO4) solution (Aldrich, Milwaukee, WI). The protein-free supernatants were then reacted with an equivalent volume of 0.5% (wt/vol) thiobarbituric acid solution (TBA; Sigma, St. Louis, MO) at 90°C for 60 min. After cooling to room temperature, the pink chromogene [(TBA) 2-MDA] was extracted with 1-butanol and dried over a stream of nitrogen at 37°C. The dry extract was then resuspended in a potassium dihydrogen phosphate (KH2PO4)-methanol mobile phase (70:30, pH 7.0) before MDA determination by HPLC with fluorescence detection.

Permeabilization of Caco-2 cells by digitonin. Caco-2 cells were obtained from the American Type Culture Collection (Manassas, VA). They were grown in minimum essential medium (MEM; GIBCO-BRL, Grand Island, NY) and supplemented with 10% fetal calf serum, penicillin (100 units/ml), streptomycin (100 µg/ml) and L-glutamine (4 mM). Medium was changed every 2–3 days. Cells (4.5 x 105 cells) were used after 15 days of confluence and permeabilized in Krebs-Ringer bicarbonate solution containing 10 mM magnesium dichloride (MgCl2), 0.1 potassium chloride (KCl), 1 mM Na-free EDTA, 1 mM ATP, 50 pg/ml digitonin, and a mixture of antiproteases (25 µg/ml phenylmethanesulfonyl fluoride, 1 µg/ml pepstatin, 10 µg/ml aprotinin, 10 µg/ml leupeptin S) for 15 min. Then, Caco-2 digitonized cells were extensively washed and preincubated for 30 min with 10 µg PON2 (purified from plasma by chromatography on DEAE-sepharose CL-6B) before they were cultured in MEM in the presence or absence of iron-ascorbate (100 µM/200 µM) for 24 h. Cells and media were collected and MDA was determined as described above.

Statistical analysis. All values were expressed as means ± SE. The data were evaluated by ANOVA, when appropriate, and the differences between the means were assessed by Student's two-tailed t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
First we evaluated PON2 protein mass in intestinal homogenates. As illustrated by the profiles reported in Fig. 1, PON2 protein mass was higher in humans than in rats for the same quantities of homogenate proteins loaded on SDS-PAGE. Additionally, its expression was revealed at varying degrees in the different regions of the small intestine. PON2 levels in the gastrointestinal tract predominated in the jejunum. In fact, the protein expression of PON2 in the different human regions of the intestine (duodenum, jejunum, ileum) was found to be 2.7-, 3.1-, and 2.7-fold greater than corresponding rat tissue (Fig. 1A). We have also assessed PON2 gene expression in human tissues to determine whether it parallels PON2 protein distribution. PCR evaluation showed that the profile of PON2 mRNA transcripts corroborated the pattern of the PON2 protein expression in the intestinal regions (Fig. 1B).


Figure 1
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 1. Pattern of paraoxonase 2 (PON2) distribution in different regions of human and rat intestine. The proteins of tissue homogenates (A) were fractionated by SDS-PAGE and electrotransferred onto nitrocellulose membranes. The blots were then incubated with the polyclonal antibody overnight at 4°C. Immunocomplexes were revealed by means horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin and an enhanced chemiluminescence kit. PON2 mass was quantitated by use of an HP Scanjet scanner equipped with a transparency adapter and software. The mRNA levels of human tissues (B) were also assessed to corroborate the PON2 protein mass findings. Representative PCR and Western blot illustrate the PON2 tissue distribution. D, duodenum; J, jejunum; I, ileum. Values are expressed as means ± SE for n = 4 in each group of experiments. *P < 0.02 vs. humans; **P < 0.05 vs. duodenum and ileum.

 
Subsequently, immunolabeling for PON2 was carried out on human intestine tissue using specific anti-PON2 antibody in combination with the protein A-gold complex. Labeling by gold particles was obtained over cellular membranes as well as intracellular compartments. In human intestinal tissues (Fig. 2), the labeling was associated with microvilli as well as along the basolateral membrane. Apical membrane-derived endosomal vesicles also displayed gold particles. Additionally, the rough endoplasmic reticulum-Golgi complex secretory pathway as well as lysosomes and mitochondria nuclei displayed some labeling, whereas lipid droplets were devoid of gold particles. Under the control condition in which the primary antibody was omitted, the labeling was totally abolished (results not shown). Overall, these observations provided support for the presence of PON2 protein in the intestine. Moreover, the high-resolution immunogold approach disclosed the intracellular distribution of PON2 in the enterocyte.


Figure 2
View larger version (112K):
[in this window]
[in a new window]

  		Fig. 2. Immunogold labeling of PON2 on human intestinal tissue. The labeling by gold particles is present over the microvilli (mv) and is particularly associated with their limiting membrane and the endosomal vesicles (end) (A). Intracellularly, the labeling is present over the rough endoplasmic reticulum (RER), the Golgi apparatus (G), and the lysosomes (Lys), as well as in mitochondria (M) and nuclei (N) (B, C, and D). Lipid droplets (L), however, are devoid of any labeling (D). Protein A-golds are also noted on the basolateral membrane (blm) (D).

 
We then examined the cellular localization of PON2 in the adult jejunum by conventional fractionation. As shown in Fig. 3, the pattern of PON2 distribution was different in rats and humans. In rats, PON2 was mostly associated with nuclei, mitochondria, lysosomes, and microsomes, whereas its expression in humans was predominant in lysosomes and microsomes. The question arose as to whether microsomal PON2 expression pattern paralleled the homogenate profile displayed in Fig. 1. Western blotting of microsomes exhibited similar PON2 observations (Fig. 4), and PON2 was also preeminent in the jejunum compared with the duodenum and ileum.


Figure 3
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 3. Subcellular localization of PON2 in jejunum from humans and rats. Subcellular fractions were prepared from homogenates by sucrose density gradient centrifugation. Samples of each fraction were subjected to SDS-PAGE and Western blotting. Values represent means ± SE for 4 separate experiments. *P < 0.005 vs. homogenates.

 

Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 4. Protein expression of PON2 in intestinal microsomes. Microsomes were obtained by subcellular fractionation and PON2 profile was examined by SDS-PAGE and Western blotting. Values represent means ± SE for n = 4 in each group of experiments. *P < 0.05 vs. humans; **P < 0.05 vs. duodenum and ileum.

 
We also explored the ontogeny of PON2 in the human small and large intestine using equal protein amounts of tissue homogenates. An increase in PON2 was noted only in the proximal regions of the small intestine and colonic tissue as a function of fetal age (Fig. 5). The proximal small intestine and colon exhibited a significant progressive rise in PON2 protein content: 145 and 123%, respectively, at the end of week 20 compared with the value (100%) noted at the end of week 15. No marked ontogenic differences in PON2 protein expression were recorded in the other intestinal segments.


Figure 5
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 5. Developmental pattern of PON2 expression in the human intestine. Proximal and distal small intestine (A) and colon (B) were analyzed with similar protein amounts at the various periods of gestation. PON2 profile was examined by SDS-PAGE and Western blotting. Values represent means ± SE for n = 4 in each group of experiments. *P < 0.05 vs. proximal segment; **P < 0.005 vs. proximal region; ***P < 0.01 vs. 20 wk of gestation.

 
We next performed cell fractionation to examine the subcellular localization of PON2 in human fetal jejunum. This experimental approach revealed a PON2 distribution similar to that in rats with evidently less enrichment in lysosomes (Fig. 6).


Figure 6
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 6. Analyses of PON2 content in subcellular fractions. PON2 expression in cellular fractions obtained from fetal small intestine. The organelles were isolated from the proximal intestine (17 wk of gestation) using sucrose density gradient centrifugation. They were resolved by SDS-PAGE, electroblotted onto nitrocellulose membranes, and incubated with primary and secondary antibodies. Values represent means ± SE for n = 4 in each experimental group. *P < 0.05 vs. homogenates; **P < 0.005 vs. homogenates.

 
Given the growing importance of PON as a cardiovascular disease risk factor and its potential involvement in protection against free radicals, we expected the status of PON2 to be significantly decreased under oxidative stress. To test this hypothesis, we exposed rat jejunum microsomes to iron-ascorbate-mediated lipid peroxidation. Incubation with iron-ascorbate at the concentration of (100 µM/200 µM) resulted in a significant increase in MDA levels in rat (Fig. 7A). Preincubation with strong antioxidants, such as BHT, NAC, and Trolox, markedly suppressed the production of MDA, providing direct evidence for the ability of the iron-ascorbate system to provoke profound lipid peroxidation (Fig. 7B). Similar findings were noted in human jejunum microsomes (Fig. 8). It is important to note that the addition of iron-ascorbate to microsomes never decreased the protein expression of protein disulfide isomerase (results not shown), a microsomal marker, which suggests a selective response of PON2 to oxidative stress.


Figure 7
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 7. Effect of iron-ascorbate-induced lipid peroxidation on PON2 protein expression in rat jejunum microsomes. Microsomal fractions were incubated in the presence or absence of iron-ascorbate (Fe/Asc). A: oxidative stress was assessed by measuring malondialdehyde (MDA) as an index of lipid peroxidation. To determine whether lipid peroxidation was largely responsible for PON2 changes, various antioxidants [butylated hydroxytoluene (BHT), N-acetylcysteine (NAC), and Trolox] were added separately to the reaction mixture before incubation with iron-ascorbate. B: then PON2 mass was quantitated by SDS-PAGE and Western blotting. Values are expressed as means ± SE for n = 4 for each experimental group. *P < 0.05 vs. control; **P < 0.005 vs. control.

 

Figure 8
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 8. Effect of iron-ascorbate-induced lipid peroxidation on PON2 expression in human jejunum microsomes. Microsomal fractions were incubated in the presence or absence of iron-ascorbate. A: oxidative stress was assessed by measuring MDA as an index of lipid peroxidation. To determine whether lipid peroxidation was largely responsible for PON2 changes, various antioxidants (BHT, NAC, and Trolox) were added separately to the reaction mixture before incubation with iron-ascorbate. B: then PON2 mass was quantitated by SDS-PAGE and Western blotting. Values are expressed as means ± SE for n = 4 for each experimental group. *P < 0.05 vs. control; **P < 0.005 vs. control.

 
Similar studies were repeated with fetal proximal intestinal microsomes (Fig. 9) and integral Caco-2 cells (Fig. 10 ) and corroborated the capability of iron-ascorbate to reduce PON2 protein expression. Preincubation with BHT at a concentration of 0.5 mM prevented the PON2 protein decline observed with iron-ascorbate, which suggests the direct involvement of oxidative stress.


Figure 9
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 9. Effect of iron-ascorbate-induced lipid peroxidation on PON2 status in fetal proximal intestinal microsomes. Microsomal fractions were incubated in the presence or absence of iron-ascorbate. A: oxidative stress was assessed by measuring MDA as an index of lipid peroxidation. To determine whether lipid peroxidation was largely responsible for PON2 changes, various antioxidants (BHT, NAC, and Trolox) were added separately to the reaction mixture before incubation with iron-ascorbate. B: then PON2 mass was quantitated by SDS-PAGE and Western blotting. Values are expressed as means ± SE for n = 4 for each experimental group. *P < 0.05 vs. control; **P < 0.005 vs. control.

 

Figure 10
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 10. Effect of iron-ascorbate-induced lipid peroxidation on PON2 status in microsomes and Caco-2 cells. Iron ascorbate (100 µM/200 µM) was administered to Caco-2 cell microsomes (A) as well as to differentiated Caco-2 cells (B) for 30 min at 37°C. BHT was added in some experiments at the concentration of 0.5 mM to neutralize the oxidative stress. Cells were then harvested, washed, homogenized, and tested for PON2 mass by SDS-PAGE and Western blotting. Values are expressed as means ± SE for n = 4 for each experimental group. *P < 0.05 vs. control.

 
Finally, studies were undertaken to assess the direct role of PON2 in oxidative stress. To this end, plasma membranes of Caco-2 cells were permeabilized with digitonin to introduce purified PON2 before the addition of iron-ascorbate. As illustrated in Fig. 11, permeabilized Caco-2 cells had more PON2 protein content compared with normal cells not exposed to digitonin. Furthermore, Table 1 indicates that MDA production was markedly diminished in permeabilized cells and their media in the presence of iron-ascorbate. On the other hand, MDA levels were raised in permeabilized Caco-2 cells incubated with PON2 antibody (results not shown). Taken together, these findings reasonably suggest the protective action of PON2 against iron-ascorbate-induced lipid peroxidation.


Figure 11
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 11. Effect of digitonin on PON2 uptake in Caco-2 cells. Digitonin (50 pg/ml) was added to Caco-2 cells as described in MATERIALS AND METHODS. Following the incubation period, cells were washed, cultured in medium containing purified PON2 protein, homogenized, and tested for PON2 mass by SDS-PAGE and Western blotting. Control cells (Cont) were cultured in the same conditions, but without the addition of PON2. Values are expressed as means ± SE for n = 3 for each group. *P < 0.01 vs. control.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Lipid peroxidation in Caco-2 cells challenged with purified PON2

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The PON family consists of the three PON1, PON2, and PON3 members that share structural properties. Most of the information available on the PON family function derives from studies of PON1. We therefore initiated a comparative investigation of rats and humans with special respect to subcellular localization and ontogeny as well as the status of PON2 in response to oxidative stress. Our data clearly established that PON2 is 1) localized in different regions of the intestine, including the duodenum, jejunum, ileum, proximal colon, and distal colon; 2) present at higher concentrations in the human intestine than in rat counterparts; 3) distributed in various cellular organelles, such as nuclei, mitochondria, lysosomes, and microsomes; 4) more associated with mitochondria and lysosomes in rat jejunum and with lysosomes and microsomes in human jejunum; and 5) decreased in the presence of iron-ascorbate-induced lipid peroxidation and restored with antioxidants. In addition, our findings emphasize the protective antioxidant role of PON2 in permeabilized Caco-2 cells.

ROS are abundantly released by activated neutrophils that infiltrate the intestinal wall during the cascade of immunological events that produce intestinal inflammation. Moreover, their luminal concentrations can dramatically increase owing to environmental factors, such as dietary oxidant ingestion, intraluminal catalase-negative bacteria, and desquamated cell oxidases. Therefore, important cellular detoxification systems may adequately control the amplified generation of ROS, terminate radical injury, and repair damaged cellular elements. As documented in the present study, PON2 is abundantly expressed along the intestine. We speculate that PON2 may be a potentially important antioxidant that could prevent impairment or breakdown of membrane integrity, ensuring the maintenance of cellular homeostasis and functions. In line with this assumption, several workers have suggested that PON2 may possess a biochemical function similar to that of PON1 and PON3, given its genetic association with pathophysiological conditions, including variations in plasma lipoproteins (7, 22), glucose levels in fasting type 2 diabetics (35), neonatal birth weight (10), the risk of coronary heart disease (47), and decreased oxidative state in human endothelial cells (40).

To delineate the role of PON2, we decided to introduce purified PON2 protein in permeabilized Caco-2 epithelial cell monolayers to assess its protective action against iron-ascorbate-induced lipid peroxidation. Cell permeabilization has been extensively used to allow access of extracellular reagents to intracellular targets (18, 23, 33). Digitonin opens pores in the membrane that allow the diffusion of large molecules, such as antibodies or enzymes, into the cytoplasm (14, 27, 48, 51). The results shown in the present investigation reveal the potential of PON2 in reducing the production of MDA in digitonin-permeabilized cells, indicating its antioxidant role. Confirmation was obtained with PON2 antibodies that favored the induction of lipid peroxidation. These permeabilization experiments constitute a direct evidence for the antioxidant function of PON2 since most of the accessible studies have only been suggestive compared with the substantial information available on PON1. In agreement with our observations, a quite new article (published during the preparation of the present manuscript) has reported that PON2 represents an endogenous defense mechanism in vascular cells (24). In this work, overexpression of PON2 markedly reduced oxidative stress, whereas its knockdown increased ROS in human umbilical vein endothelial cell-derived EA.hy 926 cells.

Of considerable interest is the detection of PON2 in a number of intracellular compartments. Subcellular fractionation analyses revealed the distribution of PON2 in nuclei, mitochondria, lysosomes, and microsomes. If the role of PON2 truly lies in counteracting oxidative stress, its subcellular localization may suggest its active participation in the protection of organelle integrity and function during lipid peroxidation.

This work provides evidence that PON2 expression is higher in the jejunum than in the duodenum and ileum in adult humans and rats. Previous studies emphasized that the jejunum is the preferential site of fatty acid uptake, lipid resynthesis, and chylomicron assembly (28). Since the jejunal wall is continuously exposed to prooxidants present in the diet (13, 20, 41), PON2 likely plays a significant role in neutralizing the effects of prooxidant excess that trigger dramatic changes in intestinal epithelial cells, including the collapse of the cytoskeleton, disruption of tight junctions, and loss of mucosal barrier integrity (3, 34, 44). Very little information is available concerning the antioxidant status of the enterocyte following peroxidative attack directed toward the brush-border membrane. Using iron-ascorbate oxygen radical-generating system, we showed that oxidative stress did not induce changes in the Caco-2 cells antioxidant enzyme activity of superoxide dismutase, catalase, glutathione peroxidase, and glutathione transferase (6). In the present investigation, iron-ascorbate-mediated lipid peroxidation induced a diminished level of PON2 protein expression, pointing out the direct implication of this protein in diminishing lipid peroxidation. Other proteins may counteract oxidative stress capable of provoking the loss of enterocyte function. Tso's team (43, 52) has provided evidence of the powerful antioxidant capacity of apo A-IV, a protein produced by the small intestine. Shamir et al. (49) have shown that the gut could be the site of synthesis of PON family members. Collectively, all these antioxidant proteins may protect against oxidative stress that arises from dietary oxidized lipids, circulatory oxidized lipoproteins, luminal flora, or inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis.

Crohn's disease and ulcerative colitis are two severe disorders characterized by chronic intestinal inflammation with increased risks of developing colorectal carcinoma (16). Intestinal damage in inflammatory bowel diseases has been related to increased free radical production resulting from impaired antioxidant defense, respiratory burst of infiltrating phagocytic cells, and pathogenic flora (19, 30, 54). Thus scavenging ROS is considered to be critical for regulating intestinal inflammation. Additional work is needed to highlight the status and elucidate the role of PON2 in these chronic inflammatory diseases.

Rats have largely been used to exploit their physiological uniqueness in addressing biomedical and nutritional research issues. Very often, findings in lipid metabolism and lipid peroxidation in rats are inadequately extrapolated to humans. Since many aspects relative to these fields in rats are not identical to humans (5), we have addressed the pattern of distribution in the intestine, the intracellular localization, and the response to oxidative stress of PON2 in rats and humans. Our findings could document various quantitative and qualitative changes in the two species; thus the rat may not represent an appropriate model for the prediction of PON2 localization and antioxidative effects in humans.

In conclusion, our data demonstrated the presence of PON2 in all the regions of the intestine and in different subcellular compartments. Microsomal PON2 protein expression was reduced by iron-ascorbate-induced lipid peroxidation whereas pretreatment with powerful antioxidants abolished PON2 decline. According to our experimental data, the cell-associated PON2 probably acts to decrease ROS that could otherwise cause deleterious effects on various intracellular organelles.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Crohn's and Colitis Foundation of Canada.


   ACKNOWLEDGMENTS
 
The authors are grateful to Schohraya Spahis for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Levy, GI-Nutrition Unit, CHU Sainte-Justine, 3175 Côte Ste-Catherine, Montreal, Quebec, Canada, H3T 1C5 (e-mail: emile.levy{at}recherche-ste-justine.qc.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Arthur JR. The glutathione peroxidases. Cell Mol Life Sci 57: 1825–1835, 2000.[CrossRef][Web of Science][Medline]
  2. Aviram M, Rosenblat M. Paraoxonases and cardiovascular diseases: pharmacological and nutritional influences. Curr Opin Lipidol 16: 393–399, 2005.[Web of Science][Medline]
  3. Banan A, Fields JZ, Zhang Y, Keshavarzian A. iNOS upregulation mediates oxidant-induced disruption of F-actin and barrier of intestinal monolayers. Am J Physiol Gastrointest Liver Physiol 280: G1234–G1246, 2001.[Abstract/Free Full Text]
  4. Bendayan M. Colloidal gold post-embedding immunocytochemistry. Prog Histochem Cytochem 29: 1–159, 1995.[Web of Science][Medline]
  5. Bergen WG, Mersmann HJ. Comparative aspects of lipid metabolism: impact on contemporary research and use of animal models. J Nutr 135: 2499–2502, 2005.[Abstract/Free Full Text]
  6. Bernotti S, Seidman E, Sinnett D, Brunet S, Dionne S, Delvin E, Levy E. Inflammatory reaction without endogenous antioxidant response in Caco-2 cells exposed to iron/ascorbate-mediated lipid peroxidation. Am J Physiol Gastrointest Liver Physiol 285: G898–G906, 2003.[Abstract/Free Full Text]
  7. Boright AP, Connelly PW, Brunt JH, Scherer SW, Tsui LC, Hegele RA. Genetic variation in paraoxonase-1 and paraoxonase-2 is associated with variation in plasma lipoproteins in Alberta Hutterites. Atherosclerosis 139: 131–136, 1998.[CrossRef][Web of Science][Medline]
  8. Brunet S, Thibault L, Delvin E, Yotov W, Bendayan M, Levy E. Dietary iron overload and induced lipid peroxidation are associated with impaired plasma lipid transport and hepatic sterol metabolism in rats. Hepatology 29: 1809–1817, 1999.[CrossRef][Web of Science][Medline]
  9. Brunet S, Thibault L, Lepage G, Seidman EG, Dube N, Levy E. Modulation of endoplasmic reticulum-bound cholesterol regulatory enzymes by iron/ascorbate-mediated lipid peroxidation. Free Radic Biol Med 28: 46–54, 2000.[CrossRef][Web of Science][Medline]
  10. Busch CP, Ramdath DD, Ramsewak S, Hegele RA. Association of PON2 variation with birth weight in Trinidadian neonates of South Asian ancestry. Pharmacogenetics 9: 351–356, 1999.[Web of Science][Medline]
  11. Choi J, Rees HD, Weintraub ST, Levey AI, Chin LS, Li L. Oxidative modifications and aggregation of Cu,Zn-superoxide dismutase associated with Alzheimer and Parkinson diseases. J Biol Chem 280: 11648–11655, 2005.[Abstract/Free Full Text]
  12. Cochrane CG. Cellular injury by oxidants. Am J Med 91: 23S–30S, 1991.[Medline]
  13. Cross CE, Halliwell B, Allen A. Antioxidant protection: a function of tracheobronchial and gastrointestinal mucus. Lancet 1: 1328–1330, 1984.[CrossRef][Web of Science][Medline]
  14. Csala M, Banhegyi G, Braun L, Szirmai R, Burchell A, Burchell B, Benedetti A, Mandl J. Beta-glucuronidase latency in isolated murine hepatocytes. Biochem Pharmacol 59: 801–805, 2000.[CrossRef][Web of Science][Medline]
  15. Draganov DI, Stetson PL, Watson CE, Billecke SS, La Du BN. Rabbit serum paraoxonase 3 (PON3) is a high density lipoprotein-associated lactonase and protects low density lipoprotein against oxidation. J Biol Chem 275: 33435–33442, 2000.[Abstract/Free Full Text]
  16. Eaden J. Review article: colorectal carcinoma and inflammatory bowel disease. Aliment Pharmacol Ther 20, Suppl 4: 24–30, 2004.
  17. Getz GS, Reardon CA. Paraoxonase, a cardioprotective enzyme: continuing issues. Curr Opin Lipidol 15: 261–267, 2004.[CrossRef][Web of Science][Medline]
  18. Goncalves CA, Gottfried C, Dunkley PR. The use of permeabilized cells to assay protein phosphorylation and catecholamine release. Neurochem Res 25: 885–894, 2000.[CrossRef][Web of Science][Medline]
  19. Grisham MB. Oxidants and free radicals in inflammatory bowel disease. Lancet 344: 859–861, 1994.[CrossRef][Web of Science][Medline]
  20. Grisham MB, Von Ritter C, Smith BF, Lamont JT, Granger DN. Interaction between oxygen radicals and gastric mucin. Am J Physiol Gastrointest Liver Physiol 253: G93–G96, 1987.[Abstract/Free Full Text]
  21. Halliwell B. Free radicals and antioxidants: a personal view. Nutr Rev 52: 253–265, 1994.[Web of Science][Medline]
  22. Hegele RA, Harris SB, Connelly PW, Hanley AJ, Tsui LC, Zinman B, Scherer SW. Genetic variation in paraoxonase-2 is associated with variation in plasma lipoproteins in Canadian Oji-Cree. Clin Genet 54: 394–399, 1998.[Web of Science][Medline]
  23. Hersey SJ, Perez A. Permeable cell models in stimulus-secretion coupling. Annu Rev Physiol 52: 345–361, 1990.[CrossRef][Web of Science][Medline]
  24. Horke S, Witte I, Wilgenbus P, Kruger M, Strand D, Forstermann U. Paraoxonase-2 reduces oxidative stress in vascular cells and decreases endoplasmic reticulum stress-induced caspase activation. Circulation 115: 2055–2064, 2007.[Abstract/Free Full Text]
  25. Hosaka S, Obuki M, Nakajima J, Suzuki M. Comparative study of antioxidants as quenchers or scavengers of reactive oxygen species based on quenching of MCLA-dependent chemiluminescence. Luminescence 20: 419–427, 2005.[CrossRef][Web of Science][Medline]
  26. Kehrer JP. Free radicals as mediators of tissue injury and disease. Crit Rev Toxicol 23: 21–48, 1993.[Web of Science][Medline]
  27. Lepers A, Cacan R, Verbert A. Permeabilized cells as a way of gaining access to intracellular organelles: an approach to glycosylation reactions. Biochimie 72: 1–5, 1990.[Medline]
  28. Levy E. The 1991 Borden Award Lecture. Selected aspects of intraluminal and intracellular phases of intestinal fat absorption. Can J Physiol Pharmacol 70: 413–419, 1992.[Web of Science][Medline]
  29. Levy E, Menard D, Suc I, Delvin E, Marcil V, Brissette L, Thibault L, Bendayan M. Ontogeny, immunolocalisation, distribution and function of SR-BI in the human intestine. J Cell Sci 117: 327–337, 2004.[Abstract/Free Full Text]
  30. Levy E, Rizwan Y, Thibault L, Lepage G, Brunet S, Bouthillier L, Seidman E. Altered lipid profile, lipoprotein composition, and oxidant and antioxidant status in pediatric Crohn disease. Am J Clin Nutr 71: 807–815, 2000.[Abstract/Free Full Text]
  31. Levy E, Stan S, Delvin E, Menard D, Shoulders C, Garofalo C, Slight I, Seidman E, Mayer G, Bendayan M. Localization of microsomal triglyceride transfer protein in the Golgi: possible role in the assembly of chylomicrons. J Biol Chem 277: 16470–16477, 2002.[Abstract/Free Full Text]
  32. Li HL, Liu DP, Liang CC. Paraoxonase gene polymorphisms, oxidative stress, and diseases. J Mol Med 81: 766–779, 2003.[CrossRef][Web of Science][Medline]
  33. Liu J, Xiao N, DeFranco DB. Use of digitonin-permeabilized cells in studies of steroid receptor subnuclear trafficking. Methods 19: 403–409, 1999.[CrossRef][Web of Science][Medline]
  34. Lum H, Roebuck KA. Oxidant stress and endothelial cell dysfunction. Am J Physiol Cell Physiol 280: C719–C741, 2001.[Abstract/Free Full Text]
  35. Mackness B, Durrington PN, Abuashia B, Boulton AJ, Mackness MI. Low paraoxonase activity in type II diabetes mellitus complicated by retinopathy. Clin Sci (Lond) 98: 355–363, 2000.[Medline]
  36. Maier CM, Chan PH. Role of superoxide dismutases in oxidative damage and neurodegenerative disorders. Neuroscientist 8: 323–334, 2002.[Abstract/Free Full Text]
  37. Mates JM, Perez-Gomez C, Nunez de Castro I. Antioxidant enzymes and human diseases. Clin Biochem 32: 595–603, 1999.[CrossRef][Web of Science][Medline]
  38. McCall MR, Frei B. Can antioxidant vitamins materially reduce oxidative damage in humans? Free Radic Biol Med 26: 1034–1053, 1999.[CrossRef][Web of Science][Medline]
  39. Moreira PI, Smith MA, Zhu X, Honda K, Lee HG, Aliev G, Perry G. Oxidative damage and Alzheimer's disease: are antioxidant therapies useful? Drug News Perspect 18: 13–19, 2005.[CrossRef][Web of Science][Medline]
  40. Ng CJ, Wadleigh DJ, Gangopadhyay A, Hama S, Grijalva VR, Navab M, Fogelman AM, Reddy ST. Paraoxonase-2 is a ubiquitously expressed protein with antioxidant properties and is capable of preventing cell-mediated oxidative modification of low density lipoprotein. J Biol Chem 276: 44444–44449, 2001.[Abstract/Free Full Text]
  41. Parks DA. Oxygen radicals: mediators of gastrointestinal pathophysiology. Gut 30: 293–298, 1989.[Free Full Text]
  42. Pavlick KP, Laroux FS, Fuseler J, Wolf RE, Gray L, Hoffman J, Grisham MB. Role of reactive metabolites of oxygen and nitrogen in inflammatory bowel disease. Free Radic Biol Med 33: 311–322, 2002.[Web of Science][Medline]
  43. Qin X, Swertfeger DK, Zheng S, Hui DY, Tso P. Apolipoprotein AIV: a potent endogenous inhibitor of lipid oxidation. Am J Physiol Heart Circ Physiol 274: H1836–H1840, 1998.[Abstract/Free Full Text]
  44. Rao RK, Baker RD, Baker SS, Gupta A, Holycross M. Oxidant-induced disruption of intestinal epithelial barrier function: role of protein tyrosine phosphorylation. Am J Physiol Gastrointest Liver Physiol 273: G812–G823, 1997.[Abstract/Free Full Text]
  45. Reddy ST, Wadleigh DJ, Grijalva V, Ng C, Hama S, Gangopadhyay A, Shih DM, Lusis AJ, Navab M, Fogelman AM. Human paraoxonase-3 is an HDL-associated enzyme with biological activity similar to paraoxonase-1 protein but is not regulated by oxidized lipids. Arterioscler Thromb Vasc Biol 21: 542–547, 2001.[Abstract/Free Full Text]
  46. Rosenblat M, Draganov D, Watson CE, Bisgaier CL, La Du BN, Aviram M. Mouse macrophage paraoxonase 2 activity is increased whereas cellular paraoxonase 3 activity is decreased under oxidative stress. Arterioscler Thromb Vasc Biol 23: 468–474, 2003.[Abstract/Free Full Text]
  47. Sanghera DK, Aston CE, Saha N, Kamboh MI. DNA polymorphisms in two paraoxonase genes (PON1 and PON2) are associated with the risk of coronary heart disease. Am J Hum Genet 62: 36–44, 1998.[CrossRef][Web of Science][Medline]
  48. Schulz I. Permeabilizing cells: some methods and applications for the study of intracellular processes. Methods Enzymol 192: 280–300, 1990.[Medline]
  49. Shamir R, Hartman C, Karry R, Pavlotzky E, Eliakim R, Lachter J, Suissa A, Aviram M. Paraoxonases (PONs) 1, 2, and 3 are expressed in human and mouse gastrointestinal tract and in Caco-2 cell line: selective secretion of PON1 and PON2. Free Radic Biol Med 39: 336–344, 2005.[CrossRef][Web of Science][Medline]
  50. Stadtman ER. Protein oxidation in aging and age-related diseases. Ann NY Acad Sci 928: 22–38, 2001.[Web of Science][Medline]
  51. Tang BL, Tan AE, Lim LK, Lee SS, Low DY, Hong W. Syntaxin 12, a member of the syntaxin family localized to the endosome. J Biol Chem 273: 6944–6950, 1998.[Abstract/Free Full Text]
  52. Tso P, Liu M. Apolipoprotein A-IV, food intake, and obesity. Physiol Behav 83: 631–643, 2004.[CrossRef][Medline]
  53. Valko M, Morris H, Cronin MT. Metals, toxicity and oxidative stress. Curr Med Chem 12: 1161–1208, 2005.[CrossRef][Web of Science][Medline]
  54. Wendland BE, Aghdassi E, Tam C, Carrrier J, Steinhart AH, Wolman SL, Baron D, Allard JP. Lipid peroxidation and plasma antioxidant micronutrients in Crohn disease. Am J Clin Nutr 74: 259–264, 2001.[Abstract/Free Full Text]
  55. Wood LG, Fitzgerald DA, Gibson PG, Cooper DM, Collins CE, Garg ML. Oxidative stress in cystic fibrosis: dietary and metabolic factors. J Am Coll Nutr 20: 157–165, 2001.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
G. Fortunato, M. D. Di Taranto, U. M. Bracale, L. Del Guercio, F. Carbone, C. Mazzaccara, A. Morgante, F. P. D'Armiento, M. D'Armiento, M. Porcellini, et al.
Decreased Paraoxonase-2 Expression in Human Carotids During the Progression of Atherosclerosis
Arterioscler Thromb Vasc Biol, March 1, 2008; 28(3): 594 - 600.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/6/G1252    most recent
00369.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Levy, E.
Right arrow Articles by Sinnett, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Levy, E.
Right arrow Articles by Sinnett, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.