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Departments of
*
Nutrition,
Biochemistry and
**
Pediatrics, University of Montreal and Centre de Recherche, Hôpital Sainte-Justine, Montréal, QC, Canada
2To whom correspondence should be addressed. E-mail: levye{at}justine.umontreal.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • BHT • malondialdehyde • lipoproteins • apolipoproteins • Caco-2 cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Their ability to move electrons constitutes the basis for the initiation and propagation of many toxic free-radical reactions. The most abundant transition metal in biologic tissues is iron, a potent catalyst of oxidative stress that can rapidly generate free radical–mediated injury (2
). The deleterious effects of chronic iron excess are well established in genetic disorders, and the pathophysiologic importance of iron-mediated oxygen free-radicals has been implicated in many human conditions (3
).
The gastrointestinal mucosa is subject to prolonged oxidative stress from reactive oxygen species generated during aerobic metabolism (4
). The influx of neutrophils and monocytes associated with inflammation can generate further reactive oxygen species via respiratory burst enzymes as well as those involved in prostaglandin and leukotriene metabolism (5
). In addition, the intestine is constantly exposed to various luminal oxidants originating from ingested nutrients (6
,7
). Key among these is the simultaneous consumption of iron salts and ascorbic acid, which can cause oxidative damage to biomolecules (8
). We therefore hypothesized that peroxidative attack directed against enterocyte brush border membranes could lead to perturbations in intestinal transport. In previous studies, we demonstrated that iron or iron + ascorbate impairs lipoprotein metabolism (9
,10
) and intestinal fat absorption (11
). However, we could not conclude that the observed changes were provoked by iron-mediated lipid peroxidation because antioxidants were not employed in these experiments to quench lipid peroxidation. The aim of the present experiments was to elaborate on these observations 1) by comparing the potential of selected antioxidants to scavenge lipid peroxidation caused by iron + ascorbate; and 2) by evaluating the role of antioxidant buffering capacity in normalizing the intracellular phase of lipid transport (lipid synthesis, apolipoprotein biogenesis as well as lipoprotein assembly and secretion).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Caco-2 cells (American Type Culture Collection, Rockville, MD) were grown at 37°C in minimum essential medium (MEM)3
as described previously (12
). For individual experiments, confluent cells were plated at 1 x 106 cells/well on 24.5-mm polycarbonate Transwell filter inserts with 0.4-µm pores in MEM supplemented with 5% fetal bovine serum. Cultures were maintained for 20 d, an ideal period for lipid synthesis studies (12
).
Lipid peroxidation and antioxidants.
Caco-2 cells were cultured in the presence or absence of Fe2+ + ascorbate added to the medium in the apical compartment. Incubation periods were terminated with 0.2% BHT (Sigma Chemical, St. Louis, MO) to measure malondialdehyde (MDA) as an index of lipid peroxidation by HPLC as described previously (9
). To determine whether lipid peroxidation was responsible for the alterations caused by iron + ascorbate, various antioxidants were added to the apical compartment separately for 1 h before incubation with iron + ascorbate. The antioxidants tested were catalase (2–10 g/L), mannitol (20–100 µmol/L), tocopherol (200–1000 µmol/L) and BHT (50–250 µmol/L).
Lipid and lipoprotein production.
Caco-2 cell lipid synthesis and secretion were assayed with radiolabeled [14C]-oleic acid as described previously (12
–14
). The final oleic acid concentration was 0.7 mmol/L (16.6 KBq)/well, added to the upper compartment in the presence of Fe2+ + ascorbate. For cholesterol biogenesis, [14C]-acetate (1994 GBq) was employed as a precursor (14
). At the end of a 24-h incubation period, cells were treated for lipid extraction by standard methods (12
–14
), and the various lipid classes from homogenates and media were then separated by TLC and counted as previously described (12
,14
). Cell protein was quantified by the method of Lowry et al. (15
). To determine the effects of Fe2+ + ascorbate and BHT on lipoprotein production, Caco-2 cells were incubated with the radioactive lipid substrate as above and de novo synthesized lipoproteins were then isolated by sequential ultracentrifugation using a TL-100 ultracentrifuge as described previously (13
).
De novo apolipoprotein synthesis.
The effect of Fe2+ + ascorbate and BHT on newly synthesized and secreted apolipoproteins was assessed with [35S]-methionine (100 mCi/L) (16
). After a 3-h incubation, immunoprecipitation from the medium and the cell lysate was performed in the presence of excess polyclonal antibodies to human apolipoproteins (Boehringer Mannheim, Mannheim, Germany) at 4°C overnight. Samples were analyzed by a linear 4–15% polyacrylamide gradient preceded by a 3% stacking gel, and gels were sectioned into 2-mm slices and counted as previously described (16
).
Enzyme and transfer activity evaluation.
The activities of ß-hydroxy-ß-methyl glutarate (HMG)-CoA reductase, acyl CoA:cholesterol acyltransferase (ACAT) and microsomal triglyceride transfer protein (MTP) were determined in isolated microsomes, whereas the measurement of liver (L)- and intestine (I)-fatty acid binding protein (FABP) was carried out in homogenates as previously described (11
,14
,17
,18
).
Statistical analysis.
All values were expressed as the mean ± SEM. Differences between treatment groups were analyzed by one-way ANOVA. When significant tests were found (P < 0.05), group differences were further evaluated by the two-tailed Student’s t test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Iron + ascorbate induced lipid peroxidation fourfold (P < 0.0001) compared with control cells cultured without the peroxidant (Fig. 1
). The efficiency of various antioxidants in preventing or reducing lipid peroxidation induced by iron + ascorbate was then evaluated in a dose-response study (Fig. 2
). BHT suppressed cellular peroxidation at 100 µmol/L and greater concentrations, whereas catalase, mannitol and tocopherol did not cause such large decreases at the concentrations tested. Because BHT maintains its cellular antioxidant activity near baseline values and even at higher concentrations, it was selected for the subsequent experiments.
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The incubation of Caco-2 cells with iron + ascorbate consistently decreased their phospholipid and triglyceride levels (Fig. 3A
). The addition of BHT to the apical compartment prevented the iron + ascorbate–mediated reduction in [14C]-oleic acid incorporation into these lipid classes. However, BHT did not normalize the abnormal basolateral lipid secretion caused by iron + ascorbate (Fig. 3
B).
|
). Iron + ascorbate enhanced synthesis (Fig. 4
A) and secretion (Fig. 4
B) of free and esterified cholesterol. The addition of BHT normalized cholesterol synthesis and export.
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Given the alterations noted in the incorporation of [14C]-acetate into cholesterol, we assessed the key enzyme activity in regulating cholesterol synthesis and esterification. Iron + ascorbate increased HMG-CoA reductase activity 100% (P < 0.05). Normal activities resulted from the addition of BHT to the culture medium (data not shown). ACAT activity was insensitive to iron + ascorbate supplementation (results not shown). Because FABP and MTP play a crucial role in lipoprotein assembly, we examined their modulation by iron + ascorbate to delineate their involvement in the abnormalities of lipid transport noted above. No differences were observed in cellular I- and L-FABP and MTP protein mass when Caco-2 cells were incubated with iron + ascorbate, with or without BHT (results not shown).
Apolipoprotein production.
We found an abnormal profile of apolipoprotein biogenesis in response to iron + ascorbate (Fig. 5
). Iron + ascorbate decreased the amounts of cellular apolipoproteins (apo) B-48, A-IV and A-I compared with control wells (Fig. 5
A). BHT normalized the cellular content of newly synthesized apolipoproteins. Radiolabeled apo B-100, apo B-48 and apo A-IV in the basolateral media of Caco-2 cell cultured with iron + ascorbate were lower than control values (Fig. 5
B). The addition of BHT restored the level of apo A-IV to normal and partially corrected the apo B-48 level, but did not alter the decline in apo B-100 due to iron + ascorbate.
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As anticipated from the lipid changes mentioned above, lipoprotein exocytosis was altered by iron + ascorbate (Fig. 6
). The secretion of chylomicrons, VLDL, LDL and HDL fractions all decreased significantly. Transport of chylomicrons and VLDL was partially normalized and that of HDL normalized by the presence of BHT (Fig. 6)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The addition of iron + ascorbate to the apical compartment impairs lipid, apolipoprotein and lipoprotein synthesis as reported previously (11
). Treatment with the antioxidant BHT did not completely prevent or abolish all of the adverse effects of iron + ascorbate.
In the current experiments, we employed iron + ascorbate, a well-established model for the induction of lipid peroxidation (10
,11
). It initiates peroxidation, as demonstrated by the increased values of MDA, probably by producing highly reactive hydroxyl radicals from hydrogen peroxide via Fenton-type reactions. Ascorbic acid can amplify the oxidative potential of iron by promoting metal ion–induced lipid peroxidation (19
). In the experiments described herein, Fe2+ + ascorbate was very effective in inducing lipid peroxidation, as demonstrated by high MDA levels, a well-established measure of lipid peroxidation. Supporting evidence was provided by the scavenger activity of BHT as well as catalase, mannitol and 
-tocopherol. BHT was selected as an antioxidant for the balance of the experiments because it was observed to be a powerful agent inhibiting iron-mediated oxidative stress. BHT has long been widely used as an antioxidant to preserve and stabilize the freshness, nutritive value, flavor and color of foods (20
).
One of the main objectives of this study was to ascertain whether the impaired lipid transport was due to lipid peroxidation, rather than the direct result of iron. In addition to iron-catalyzed peroxidation, iron + ascorbate impaired lipid esterification, cholesterol synthesis and apolipoprotein production. Potential mechanisms involved in these changes include the direct effects of free radicals on the physical properties of the endoplasmic reticulum membrane. This is where HMG-CoA reductase (the key regulatory enzyme in cholesterol synthesis), ACAT (cholesterol esterifying enzyme) and glycerol-3-phosphate acyltransferase (the rate-limiting enzyme in triglyceride esterification) are located and where apolipoprotein production takes place. Our recent studies demonstrated that the peroxidative reactions resulted in an alteration in the unsaturation/saturation ratio of hepatic membrane phospholipid fatty acids and microsomal membrane fluidity, which in turn influenced membrane enzyme activities (9
,10
). We therefore contend that iron + ascorbate–mediated peroxidative changes in the polyunsaturated fatty acids of membrane phospholipids affected endoplasmic reticulum integrity, thereby resulting in abnormal lipid and apolipoprotein synthesis.
Cholesterol is essential for cell integrity and function. The experiments performed in this study were aimed at defining whether lipid peroxidation could modify cholesterol synthesis. De novo cholesterogenesis, assessed by the incorporation of [14C]-acetate, was significantly increased. Confirmation was obtained by directly measuring microsomal HMG-CoA reductase activity. As mentioned before, lipid peroxidation may alter the immediate environment of HMG-CoA reductase, thereby affecting its function. On the other hand, ACAT activity was resistant to iron + ascorbate, which reasonably implies that these two enzymes react to lipid peroxidation differently.
The antioxidant BHT did not fully restore the intracellular processes disturbed by Fe2+ + ascorbate. It is thus conceivable that alternative mechanisms participated in the enterocyte pathway derangements. A plausible mechanism may involve direct interaction with iron. It has already been reported that various proteins are modified by the presence of iron (21
).
In conclusion, our data demonstrate that iron + ascorbate alters Caco-2 cell function. Intracellular lipid transport was affected by lipid peroxidation because, in association with the high levels of MDA and BHT, there was a modification of lipid esterification, cholesterol synthesis, apolipoprotein biogenesis and lipoprotein production. However, additional mechanisms related to iron per se may be involved, given the inability of BHT to fully correct iron-mediated intestinal fat transport disturbances.
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FOOTNOTES |
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3 Abbreviations used: ACAT, acyl CoA:cholesterol acyltransferase; FABP, fatty acid binding protein; HMG, ß-hydroxy-ß-methyl glutarate; MDA, malondialdehyde; MEM, minimum essential medium; MTP, microsomal triglyceride transfer protein.
Manuscript received 17 October 2001. Initial review completed 20 November 2001. Revision accepted 23 January 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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