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1INSERM U724, Vandoeuvre-lès-Nancy; 2Université Henri Poincaré, Faculté de Médecine, Vandoeuvre-lès-Nancy, France; and 3IRCCS, Oasi Maria SS, Institute for Research on Mental Retardation and Brain Aging, Troina, Italy
Submitted 24 April 2007 ; accepted in final form 27 August 2007
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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homocysteine; B vitamins; steatosis; oxidative stress
Homocysteine (HCY) is a metabolite of the essential amino acid methionine. Its levels may vary considerably among individuals according to genetic, dietary, and environmental factors, and elevated plasma concentrations have been identified as a risk factor for a wide range of pathological situations (30). In neonates, the risk of hyperhomocysteinemia (hHCY) is closely related to elevated maternal HCY concentrations during pregnancy that may be influenced by the dietary intake of B vitamins (42). A number of studies have reported an association between increased maternal plasma HCY and pregnancy complications, including recurrent miscarriages, preeclampsia, and placenta abruption (27). In the progeny, hHCY has been associated with fetal death, premature birth, intrauterine growth retardation, neural tube defects, and hepatic stress (2, 9). HCY production primarily occurs in the liver. The methionine cycle successively involves the synthesis of S-adenosylmethionine (SAM) from methionine by the enzyme methionine-adenosyltransferase (MAT) and the transmethylation reactions of a large number of substrates that use SAM as cosubstrate and produce S-adenosylhomocysteine (SAH). HCY originates from the hydrolysis of SAH, a reversible reaction catalysed by SAH hydrolase. HCY can be either remethylated to methionine by methionine synthase (MS), an enzyme that requires folate and vitamin B12, or catabolized via the transsulfuration pathway, which involves cystathionine β-synthase (CBS) to generate cysteine and glutathione (20). Additionally, HCY remethylation can be also catalyzed in the liver by betaine-homocysteine methyltransferase (BHMT).
It has previously been shown that hypoxia and/or ischemia may alter remethylation as well as adenosylation of HCY (3, 14, 15, 33). The resulting decreases in SAM concentration and MAT expression have been reported in the adult rat liver and in isolated rat hepatocytes exposed to moderate chronic hypoxia (3, 14, 15, 33). In the adult rat, a significant SAM deficit associated with DNA hypomethylation was recorded after 10 days of hypoxia (15). The authors suggested that hypoxia would result in a decreased methylation similar to that produced by a methyl-deficient diet. Nevertheless, several studies have shown that the liver's tolerance to hypoxia/ischemia can be augmented by prior exposure to a noninjurious preconditioning stimulus (34, 52). In addition, preconditioning strategy was shown to promote the release of hepatocyte growth factor and to improve regenerative capacity of the liver (6, 21). Considering that both hHCY and brief hypoxia may occur simultaneously in neonates, particularly in some populations at risk of malnutrition, and that hypoxia can affect HCY metabolism, we aimed to evaluate the potential effects of such a hypoxic preconditioning on hepatic stress in a rat model of nutritionally-induced hHCY during prenatal and suckling periods.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Animal treatments were conducted in accordance with internal guidelines for animal care and housing. Adult female Wistar rats (Charles River, l'Arbresle, France) were constantly maintained under standard laboratory conditions, with food and water available ad libitum. One month before pregnancy, they were exposed to a methyl-deficient diet, as previously described by Blaise et al. (7, 8). For this purpose, they were fed a diet deprived of vitamins B12 (0.0 µg/kg), B2 (0.34 mg/kg), folate (10.0 µg/kg), and choline (0.06 mg/kg) (n = 9) (Special Diet Service, Saint-Gratien, France). Control females were fed standard food (n = 9) (Maintenance Diet M20; Scientific Animal Food and Engineering, Villemoisson-sur-Orge, France) containing vitamins B12 (40 µg/kg), B2 (13 mg/kg), folate (900 µg/kg), methionine (4,800 mg/kg), and choline (2,100 mg/kg). According to the suppliers, protein sources were identical (and represent 26%) in both diets, whereas methionine and cystine levels were similar.
Within 24 h after delivery, the litter size was reduced to 10 pups for subsequent standardization of the study. One-half of the neonates were then placed for 5 min in a Plexiglas chamber thermoregulated at 36°C and flushed with 100% N2, whereas the remaining pups were exposed to 21% O2-79% N2 (normoxic conditions) for the same time (48). Pups were then allowed to recover for 20 min in normoxia before being returned to their dams. The hypoxic treatment did not result in any animal death and was previously demonstrated to be effective in preconditioning experiments (25). When used, the deficient diet was given to dams until weaning of their offspring, i.e., 21 days after birth. Pups were divided into four experimental groups (n = 6/group/age): C (control), rats not subjected to hypoxia and fed by a dam with standard diet; D (deficient), nonhypoxic rats fed by a dam with a deficient diet; H (hypoxia), rats exposed to hypoxia and fed by a dam receiving standard diet; and HD (hypoxia-deficient), rats exposed to hypoxia and fed by a dam with a deficient diet.
Sample Collection
For subsequent studies, pups were killed at 2, 5, or 21 days of age by exposure to excess halothane. Intracardiac blood samples were drawn for the measurement of vitamins and HCY plasma concentrations. The liver was rapidly collected, washed in Ringer-buffered solution (2 mM KCl, 125 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, 2 g/l glucose, pH 7.3), and frozen in liquid nitrogen. Liver cells were lysed at 4°C in 100 mM potassium phosphate buffer (pH 7.3) containing protease inhibitors (protease inhibitor cocktail; Sigma, St. Louis, MO). Protein samples were quantified according to Bradford (10).
Vitamin B12, Folate, Vitamin B2, and HCY Concentrations
Plasma concentrations of vitamin B12 and folate were determined by radiodilution isotope assay (simulTRAC-SNB; ICN Pharmaceuticals, Versailles, France) (7). Vitamin B2 status was assessed by measuring the glutathione reductase activity for determining the erythrocyte glutathione reductase activation coefficient (EGRAC) that corresponds to the ratio between enzyme activities determined with and without the addition of the cofactor FAD (4), and the liver glutathione reductase activation coefficient (LGRAC), according to Moat et al. (41). HCY plasma and liver concentrations were assessed by fluorescent polarization immunoassay (IMX System; Abbott, Fornebu, Norway), according to Amouzou et al. (1). For the measurement of HCY in the liver, crude homogenates were treated by dithiothreitol (DTT) to prevent HCY binding to proteins.
Enzyme Activities
All measurements described below were performed with tissue samples corresponding to 400 µg of total proteins.
CBS activity was measured by a method adapted from Taoka et al. (55). Proteins were incubated for 30 min at 37°C in a reactive solution containing 0.1 M Tris (pH 8.5), 1 mM cystathionine, 5 mM D,L-serine (Sigma), 1 mM pyridoxal 5'-phosphate, 0.25 µCi [14C]serine (Amersham Biosciences, Saclay, France), and 15 mM homocysteine (Sigma). After a washing with 20 mM maleic acid (pH 1.9) and 0.2 M LiCl, radiolabeled cystathionine was eluted with 50 mM acetic acid (pH 4.8) and 0.8 M LiCl into a Dowex 50WX2-200 gel (Sigma) and then quantified by means of a liquid scintillation analyzer (Packard Bioscience, Meriden, CT). MS activity was monitored by a method adapted from Chen et al. (16). Proteins were incubated in a buffered solution consisting of 100 mM KH2PO4 at pH 7.2, 25 mM DTT, 25 mM ascorbate, 0.5 mM SAM, 50 µM OH-cobalamin, 5 mM D,L-HCY (freshly prepared), and 250 µM [14CH3]methyl-tetrahydrofolate (
11,000 cpm/nmol). The reaction was stopped by adding cold water. After centrifugation, the supernatant was laid on a cationic gel (quaternary ammonium, AG1x8, Cl– form; Bio-Rad, Marne-la-Coquette, France). After elution, [14CH3]methionine was measured with PicoFluor-40 (Packard Bioscience).
Betaine-homocysteine methyltransferase (BHMT) activity was measured as described by Garrow (23). The activity of MAT was measured according to Chamberlin et al. (13), and methylene tetrahydrofolate reductase (MTHFR) activity was measured as reported by Kutzbach et al. (37).
Metabolite Concentrations
The method for measuring SAM and SAH was adapted from Delabar et al. (19) and based on a reverse-phase liquid chromatography technique using a linear acetonitrile gradient. Proteins were precipitated with 0.2 N HClO4 and centrifuged, and the supernatant was filtered through 0.45 µm before injection on the column (Lichrospher, 100 RP-C18, 5 µm, 250 x 4 mm ID). The mobile phase was applied at a flow rate of 0.75 ml/min and consisted of 50 mM sodium phosphate (pH 3.2), 10 mM heptan sulfonate, and acetonitrile (10–20% from 0 to 20 min). Amounts of SAM and SAH were quantified using a UV detector (254 nm).
For total glutathione (GSH) determination, the reaction mixture contained 100 µl of homogenate, 2 µl of DTT (10 mM, Sigma) 15 µl of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB, 5 mM in PBS-EDTA 0.1 mM, Sigma). The mixture was vortexed briefly and incubated for 5 min at room temperature, and 5.5 µl of perchloric acid (60%, Sigma) was added. The mixture was vortexed again, incubated on ice for 10 min, and centrifuged at 20,000 g for 20 min at 4°C, and the supernatant was filtered through 0.45 µm. The separation is based on the Yasuhara method (61) with some modifications, and the HPLC system was provided by TSP (Thermo Electron, Cergy Pontoise, France). The sample (50 µl) was injected in a HPLC system that consisted of a high-pressure pump (TSP, P1000XR), an autosampler (TSP, AS100), and a C18 column (Resolve C18, 5 µm, 150 x 3.9 mm) maintained to 30°C at a flow rate of 0.8 ml/min (105 bar), and the signal was measured by UV absorbance (280 nm, TSP, UV2000). The amount of GSH was calculated using a range of standards and expressed as nanomoles per milligram of total proteins.
Markers of Oxidative Stress
First, global hepatic injury was estimated by serum transaminases, aspartate aminotransferase (ASAT), and alanine aminotransferase (ALAT), which were monitored according to Henry et al. (29) by using an Olympus AU 800 automated analyzer (Rungis, France).
Malondialdehyde (MDA), a marker of lipid peroxidation, was measured using a lipid peroxidation assay kit (Calbiochem, Meudon, France).
The liver superoxide dismutase (SOD) activities (Cu/Zn-SOD and Mn-SOD) were assessed with a Ransod kit (Randox, Oceanside, CA) according to the manufacturer's instructions. Glutathione peroxidase (GPX) activity was measured by means of a Kone Pro automate (Kone, Evry, France), and the assay was based on the method described by Paglia et al. (45). Cytochrome oxidase (COX) activity was monitored on liver tissue sections by the histochemical technique as described by Strazielle et al. (53). The COX staining intensity was quantified by densitometric analysis of the sections with a computer-assisted image analysis system (BIOCOM, Les Ulis, France) and standards were used to convert optical density into enzymatic activity reported in micromoles per minute per gram of tissue.
The liver glutathione reductase activity was measured according to Carlberg and Mannervik (11).
The activity of inducible-NO synthase (iNOS) was monitored by the conversion of [14C]arginine into [14C]citrulline, as reported by Kiedrowski et al. (35). The addition of EGTA, a Ca2+-chelating compound, was used to discriminate between the calcium-dependent (endothelial and neuronal NOS) and -independent (iNOS) isoforms (49). Radioactivity was measured by means of a Tri-Carb 1900CA scintillation counter (Packard Bioscience).
Histopathological Analyses
Histology. Cell density was measured after staining liver sections with the DNA fluorochrome 4,6-diamidino-2-phenylindole (DAPI; Sigma) (8). The number of cell nuclei was scored at 365 nm under fluorescence microscopy (Zeiss Axioscop, Strasbourg, France) by an observer without knowledge of the experimental conditions. Cells were counted at x40 magnification in three separate experiments, each investigating six distinct section areas delineated by an ocular grid of 1/400 mm2. Only cells with their nuclei present in the focal plane were counted. Numbers of cells was finally calculated per square millimeter.
Four-micrometer sections of paraffin-embedded blocks were stained with hematoxylin-eosin and with special liver stains for analysis of collagen fibrosis (Sirius red stain), reticulin fibers (Gordon and Sweets stain) and iron (Perls stain). Histological analysis was performed using semiquantitative items focusing on necroinflammatory lesions, fibrosis, and steatosis, as usually done to score chronic hepatitis C activity in human pathology (32). The system devised for this study scored steatosis on the basis of the percentage of hepatocytes showing lipid accumulation as follows: 0 (none), 1 (<10%), 2 (10–30%), 3 (>30%).
Lipid staining with Oil red O was additionally used (38). For this purpose, sections of unfixed frozen liver tissue were incubated with 0.5% Oil red O for 3 min at room temperature. Tissue sections were then counterstained by dipping the slides in 0.01% DAPI. Quantification was performed by calculating the ratio between the number of lipid droplets and the number of nuclei stained by DAPI in three separate experiments, each investigating six distinct section areas.
Electron microscopy. The samples were dehydrated in a graded series of ethanol and then embedded in EPON resin. Ultrathin sections, stained with uranyl acetate and Reynold's lead citrate, were generated with Ultracut E (Reichert-Jung, Vienna, Austria) and finally observed with a transmission electronic microscope (Philips CM12, Eindhoven, Netherlands). Densities of lipid droplets and mitochondria were determined by three counts in seven areas of two sections per animal.
Immunohistochemistry. Immunohistochemical studies were performed on cryostat-cut 20-µm tissue sections mounted onto glass slides, as described by Daval. et al. (18). The presence of apoptosis was selectively analyzed in tissue sections by the Apostain method using monoclonal antibody to single-strand DNA (F7–26 generated from calf thymus single-strand DNA; AbCys, Paris, France) after DNA denaturation by heating in the presence of formamide, as previously described (8, 48). According to the manufacturer, this procedure allows the specific detection of apoptotic cells.
Bromodeoxyuridine (BrdU; Sigma) administration was used to investigate cell proliferation. BrdU was solubilized in 0.9% NaCl containing 0.007 N NaOH and administered intraperitoneally at 50 mg/kg. Animals were killed 24 h later by decapitation, and their livers were immediately frozen in methylbutane at –30°C. For BrdU immunostaining, DNA was first denatured by incubating liver sections in 2 N HCl for 45 min at room temperature followed by 10-min neutralization in 0.1 M sodium borate at pH 8.5. Tissue was rinsed in PBS for 10 min and then in PBS containing 10% bovine serum for 1 h and incubated overnight at 4°C with a mouse monoclonal antibody against BrdU (1:100; Oncogene Research Products, Boston, MA).
In all cases, labeled nuclei were scored in tissue sections counterstained by DAPI, and quantification was made by an independent investigator in four separate experiments with counts performed in four distinct areas of 100 cells (48).
Western Blotting of p53, p21, and p16
Samples corresponding to 40 µg of total proteins were electrophoresed on SDS-polyacrylamide gel (5% stacking, 12% running). In parallel, molecular weight standards were loaded into separate wells. After separation, proteins were transferred onto a polyvinylidene difluoride membrane (New England Nuclear, Boston, MA) using a Tris-glycine buffer (48 mM Tris base, 39 mM glycine, 0.037% SDS, and 20% methanol) by means of a wet electrotransferring unit (Bio-Rad) at 160 mA for 45 min. Subsequent experimental steps were performed using a commercially available kit with peroxidase-labeled secondary antibodies and the chemiluminescent substrate luminol (Boehringer, Mannheim, Germany). Nonspecific sites were blocked by incubating the membrane while shaking for 1 h in Tris-buffered saline (TBS) with 5% bovine serum albumin. Blots were then probed overnight at 4°C with p53, p21, or p16 monoclonal (except for p53) antibodies (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in TBS with 5% bovine serum albumin. After washing with TBS containing 0.1% Tween 20 (TBS-Tween), the membrane was incubated for 1 h with peroxidase-labeled secondary antibody (40 mU/ml, Boehringer) and washed four times in TBS-Tween. Chemiluminescent protein detection was then monitored according to the supplier's instructions by using Kodak X-Omat films (PerkinElmer, Courtaboeuf, France), and densitometric analyses were finally performed using a Biocom 200 imaging processor by using actin and glyceraldehyde-3-phosphate dehydrogenase as internal standards (Santa Cruz Biotechnology).
Statistical Analyses
Data were prospectively collected and analyzed with Statview 5 software for Windows (SAS Institute, Berkley, CA). Raw data were compared by using one-way analysis of variance with Fisher's test. In temporal studies, to allow comparisons between ages, Friedman's test was applied followed by Wilcoxon's test, according to Winer (60). Multiple regression, univariate (Z-test correlation), and multivariate correlation analyses were performed for correlation studies. A P value of <0.05 was considered to indicate statistical significance.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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In the conditions used, the hypoxic episode was found to transiently induce a significant hypoxemia (PO2 = 40.6 ± 5.3 vs. 62.5 ± 6.1 mmHg in controls, P < 0.01), along with mild hypercapnia (PCO2 = 49.2 ± 6.1 vs. 33.7 ± 5.8 mmHg, P < 0.05) and acidosis (pH = 7.23 ± 0.04 vs. 7.38 ± 0.02, P < 0.01). Several studies have reported that, depending on its characteristics, hypoxia can influence the cell cycle, promote cell proliferation, or induce apoptosis via the regulation of p53, p21, or p16 expression (36, 54, 58, 62). We therefore evaluated the consequences of brief hypoxia in the liver tissue. As in controls, hepatic cell density tended to decline with age (Fig. 1A). Whereas BrdU incorporation was progressively reduced during development, the number of apoptotic nuclei, as depicted by the Apostain F7–26-specific antibody, was augmented (Fig. 1A). However, no significant differences were found between control and hypoxia groups. In accord with the above observations, the expression profiles of p53, p21, and p16 proteins increased with age equally in both experimental groups (Fig. 1B).
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A physiological decline of the circulating levels of vitamin B12 and folate in control animals was observed during development (Table 1). Starting from postnatal day 2, reduction of vitamin B12 concentration was significantly more dramatic in pups exposed to the deficient diet than in controls, whereas folate concentration was significantly lowered at 5 and 21 days of age. Compared with controls, hypoxia by itself reduced concentrations of folates at 5 days (P = 0.0010) and 21 days (P = 0.0338), whereas no significant differences were measured in hypoxic pups for vitamin B12 concentrations. In parallel, vitamin B2 levels (reflected by EGRAC) remained unchanged.
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At 21 days, hepatic levels of both vitamins B12 and B2 (reflected by LGRAC) remained stable in the four experimental groups (Table 2). Tissue concentration of folate was decreased in the deficient group compared with controls, and hypoxia had no effect. Surprisingly, HCY concentration in the liver was not significantly affected by the deficient regimen. In the H + D group; however, HCY concentration was significantly higher than in the H group (P = 0.0117) and than in the D group (P = 0.0013).
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As illustrated in Fig. 2, developmental profiles of the various liver enzymes involved in HCY metabolism show a global decline in their activities, starting from postnatal day 2 for MS, MAT, and CBS and from postnatal day 5 for MTHFR and BHMT, the enzyme accounting for the hepatic alternative remethylation pathway. Brief neonatal hypoxia transiently reduced MAT and CBS activities in 2-day-old rat pups.
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The combination of transient hypoxia with exposure to the deficient diet had varying effects on enzymes. Compared with the D group, significantly enhanced activities of MTHFR, MS, and BHMT were recorded at various developmental stages, whereas MAT activity was reduced. Specifically in the H + D group, CBS activity was transiently stimulated at 2 and 5 days of age before falling at 21 days (Fig. 2).
Effects of Hypoxia on SAM, SAH, Glutathione Concentrations and iNOS Activity in Liver of Deficient Pups
In an attempt to clarify the influence of transient hypoxia in nutritionally deprived animals on enzyme activities, different substrates known to regulate HCY metabolic pathways, i.e., SAM, SAH, glutathione, and NO (as reflected by iNOS activity) were further analyzed in the liver of 21-day-old rats (Fig. 3).
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80 nmol/mg protein, SAM concentration significantly decreased in the liver of D rats, an effect that was significantly potentiated by the additional exposure to neonatal hypoxia (P < 0.01 between groups D and HD). As a result, the SAM/SAH ratio, an index of global cell methylation, fell from 1.62 ± 0.30 in the control group to 0.61 ± 0.06 in the D group (P < 0.01), to finally reach 0.46 ± 0.06 in the HD group, with P < 0.01 between D and HD groups. Total glutathione was greatly affected in the liver of deficient pups, its concentration varying from 14.3 ± 3.2 in controls to 3.6 ± 0.9 nmol/mg protein (P = 0.0002). Similar concentrations were found in the D and HD groups, with no significant changes between H and control groups. Regarding the ratio of reduced (GSH) to oxidized (GSSG) glutathione, the reduced-form concentration tended to decrease in the HD group compared with the D group (0.92 ± 0.60 vs. 1.3 ± 0.5 nmol/mg protein, P = 0.0749); meanwhile, GSSG concentrations were higher in the HD group than in the D group (2.9 ± 0.2 vs. 2.3 ± 0.8 nmol/mg protein).
Concerning iNOS, its activity in the liver was transiently stimulated in the ensuing hours after exposure to hypoxia. By 2-h postexposure, enzyme activity was 13.29 ± 0.31 vs. 2.21 ± 0.64 nmol·h–1·mg–1 protein in controls (n = 4, P < 0.01). At 21 days of age, iNOS activity did not vary significantly among the three experimental groups C, D, and H, but was markedly and specifically increased in the livers of rats belonging to the HD group (Fig. 3).
Oxidative Stress in the Liver and Anatomopathological Consequences
HCY is known to exert cytotoxic effects through the induction of oxidative stress (50, 57). We therefore monitored some markers of oxidative stress and evaluated concomitant anatomopathological manifestations in the livers of rats from the various experimental groups at 21 days (Fig. 4).
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As a consequence of early exposure to the deficient diet, activities of most antioxidative enzymes were reduced: GPX (125.9 ± 41.8 vs. 382.2 ± 99.8 µmol·min–1·g protein–1, P = 0.0035), Mn-SOD (0.082 ± 0.018 vs. 0.112 ± 0.029 U/g protein, P = 0.0333), and GSH reductase (5.1 ± 1.6 vs. 7.8 ± 0.8, P = 0.0197; Fig. 4A). Cu/Zn-SOD activity was unaltered, and CO activity was slightly but significantly increased (P = 0.0183). Among the antioxidant enzymes studied, only GPX remained affected at 21 days following transient birth hypoxia, showing a significantly increased activity (601.3 ± 208.4 vs. 382.2 ± 99.8 µmol·min–1·g protein–1 protein in controls, P = 0.0098). Finally, hypoxia in D pups appeared to accentuate the deficiency-associated reduction of GSH reductase (P = 0.0322) and only tended to increase CO activity (P = 0.062).
Electron microscopic studies showed a physiological augmentation of the number of mitochondria in the hepatocytes during postnatal development. However, compared with control conditions, exposure to the deficient diet was associated with a noticeable increase in mitochondria density at postnatal day 21 (1.09 ± 0.09 vs. 0.58 ± 0.21 per µm2, P = 0.0047), a phenomenon that was found to be exacerbated by birth hypoxia (1.71 ± 0.08 per µm2) (Fig. 4, B and C). At this developmental stage, quantitative histological analyses did not reveal any changes in the density of hepatocytes or in the extent of apoptosis in the liver tissue among the different experimental groups (not shown). Nonetheless, a reduced incorporation of BrdU was noticed in the HD group (Fig. 4D), suggesting that cell proliferation is reduced.
A liver steatosis was present in all animals exposed to the deficient diet, with a Knodell score of 1.6 ± 0.8 (vs. 0.4 ± 0.2 in C, P < 0.0001). Steatosis was identified as microvesicular, without fibrosis, portal or septal inflammation, intralobular necrosis, or dysplasia (Fig. 5). Lipid accumulation in hepatocytes was confirmed by Oil red O staining and by electron microscopy. Neonatal enhanced the severity of steatosis in D pups (Fig. 5). Also, a significant increase of lipid droplets reported by total cells was recorded in the HD group following Oil red O staining compared with the D group (269.3 ± 48.6 vs. 132.6 ± 20.0, P = 0.0048).
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Correlation Studies
Correlation studies were performed at 21 days between enzyme activities and various factors susceptible to regulate these enzymes (i.e, SAM, SAH, SAM/SAH, total GSH, oxidized/reduced GSH, iNOS, folate, vitamin B12). Regarding the first step of the transsulfuration pathway, CBS activity was positively correlated with total GSH (CI 95%: 0.332 to 0.902, P = 0.0016) and SAM/SAH ratio (CI 95%: 0.275 to 0.831, P = 0.0016), and was negatively correlated with iNOS activity (CI 95%: –0.841 to –0.394, P = 0.0004). MTHFR activity was significantly correlated with folate (CI 95%: 0.042 to 0.670, P = 0.0297) and vitamin B12 (CI 95%: 0.445 to 0.870, P < 0.0001) concentrations. Regarding the remethylation pathway, MS activity was correlated with vitamin B12 (CI 95%: 0.139 to 0.720, P = 0.0076) and with glutathione (CI 95%: 0.310 to 0.878, P = 0.0016), whereas BHMT activity was independent of all the factors tested. MAT activity was positively correlated with total GSH (CI 95%: 0.090 to 0.801, P = 0.0209) and negatively correlated with iNOS (CI 95%: –0.867 to –0.377, P = 0.0003). Accordingly, plasma concentration of HCY was significantly correlated with concentrations of folate, vitamin B12, SAM, and iNOS activity in univariate analysis, whereas multivariate analysis showed that vitamin B12 availability is the main determinant of HCY levels (not shown).
Regarding hepatic stress at 21 days of age, activities of GPX, GSH reductase, and Mn-SOD, but not Cu/Zn-SOD, were significantly associated with HCY plasma concentration. Significant correlations were also found between HCY levels and the severity of hepatic steatosis (P < 0.0001), whereas elevated HCY concentrations were correlated with increased densities of mitochondria in the liver (P = 0.0002).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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By itself, early exposure to the deficient diet amplified the naturally occurring decline of HCY remethylation and transsulfuration pathways during development by influencing the corresponding enzyme activities (28, 44). By using the same model, deficient diet-associated disturbances in the one-carbon metabolism have been previously documented (7). This finally led to an elevation of HCY plasma concentration equivalent to that recorded in vitamin B-deficient humans (30). Exposure to the deficient diet was associated with oxidative stress in the liver, in line with reduced antioxidant capacities (as reflected by lower GSH levels, and decreased GPX, GSH reductase, and Mn-SOD activities) and increased lipid peroxidation (as shown by MDA). In parallel, histopathological analyses showed structural alterations in the liver tissue with a noticeable steatosis. An elevation of ASAT activity was recorded in the serum of deficient rats. In fact, ASAT elevation may reflect the liver steatosis, as this parameter has been previously reported as a good marker of non-alcoholic hepatosteatosis in humans (39). It is known that impaired HCY metabolism is associated with chronic liver disease, and the relationships among hyperhomocysteinemia, oxidative stress, lipid peroxidation, and liver disease have been largely documented (31, 50, 59). Nonetheless, the presence of steatosis was usually reported in the case of very high concentrations of HCY, such as those observed in CBS-deficient individuals, and our data suggest that chronic defects may also occur with early moderate hyperhomocysteinemia. It is noteworthy, however, that, if elevated HCY can certainly account for the deleterious effects observed in our model, other factors could participate to excess fat accumulation in the liver. Indeed, HCY accumulation was not recorded in the livers of deficient pups, and the effects of methyl-deficient diet may be related to decreased SAM/SAH ratio and subsequent inhibition of transmethylation reactions. Also, a decrease in phosphatidylcholine concentration may contribute to inhibit fat export.
Since we have recently reported obvious HCY-associated apoptosis in the brain of 21-day-old rats exposed to the methyl-deficient diet during gestational and suckling periods (8), we aimed to investigate whether the same treatment could also induce liver apoptosis. According to our findings, early exposure to the deficient diet did not trigger apoptotic death in hepatocytes. Similarly, Robert et al. (50) did not observe apoptosis in livers of CBS–/– mice, although the proapoptotic ratio Bax/Bcl-2 was substantially increased. The authors suggested that the activation of some protective pathways might counteract apoptotic death. Interestingly, we showed that the density of mitochondria was notably increased in rat hepatocytes. Since oxidative stress is classically associated with mitochondrial dysfunction, it can be hypothesized that injured hepatic cells may react by promoting mitochondrial biogenesis.
When applied within 24 h after birth, brief hypoxia did not alter global development of the rats, as previously reported (48). In the liver of non-deficient animals, no patent damage could be observed following exposure to hypoxia. Similarly to normal conditions, neither physiological apoptosis nor cell proliferation were affected during the postnatal period during which the expression of the regulators of the cell cycle and apoptosis p53, p21, and p16 progressively increased, in line with liver maturation and cell recycling (5, 47). Except for MAT and CBS activities, which were transiently reduced at 2 days, HCY metabolism was comparable within hypoxia and control groups, and preconditioning hypoxia did not affect homocysteinemia. In good agreement with our own observations, expression of the MAT gene has been found to decrease in response to chronic hypoxia in isolated rat hepatocytes (3, 15), and it has been shown that this effect of hypoxia is under the influence of NO production (22).
When hypoxia was combined with the deficient diet, ASAT elevation was paralleled by an unexpected lower ALAT activity. Also, in these conditions, hypoxia appeared to modulate HCY metabolism temporally. First, at 2 days of age, it was associated with enhanced activities of enzymes implicated in HCY degradation, i.e., MS and CBS, and decreased MAT activity. In this respect, various transcription factors are known to be sensitive to regulation by hypoxia (26). It is possible that preconditioning-activated signaling pathways converge to act on transcription factors that drive the genomic response and/or to induce posttranslational modifications of existing proteins, in addition to the combined effects of the deficient diet.
At postnatal day 21, despite there being no apparent changes in enzyme activities in response to hypoxia alone, the combination of hypoxia with the deficient diet was associated with a significant augmentation of HCY liver concentration. We postulate that iNOS-associated production of NO may play a critical role in this observation. It has been shown that NO participates in the protective effects of ischemia preconditioning in the liver, and various mechanisms have been described (21, 46). Nevertheless, the functions of NO remain equivocal, since this compound interacts with the superoxide anion radical to form peroxynitrite (ONOO–), a reactive nitrogen species that causes extensive oxidative damage (12). As previously documented in other experimental models (21), hypoxia transiently stimulated iNOS activity in the rat liver. At 21 days of age, hypoxia or the methyl-deficient diet, when applied separately, had no obvious influence on iNOS activity, but their conjunction markedly augmented the enzyme activity, a process associated with exacerbated effects on liver histopathology and mitochondria density. In this respect, cellular insult and DNA damage following anoxia/reoxygenation were very recently localized and correlated with iNOS expression levels in the rat heart (17), and NO production has been shown to trigger mitochondrial biogenesis in various tissues through the activation of guanylate cyclase and generation of cGMP (43). The role of NO is further supported by the demonstration that iNOS is required for the development of alcohol-dependent hepatotoxicity in mice (40, 56). Taken together, these findings warrant further investigations to elucidate the participation of NO in the potentiation by postnatal hypoxia of liver injury related to an early exposure to methyl-deficient diet.
In conclusion, our results show that a short episode of hypoxia around birth may aggravate the long-term adverse effects of impaired HCY metabolism in the liver resulting from an inadequate vitamin status during pregnancy and would finally influence the severity of steatosis in the developing subject. This emphasizes the impact of early environmental factors on the risk of developing chronic diseases later in life, an emerging field of investigation for future prevention.
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FOOTNOTES |
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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.
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T mutation of the gene encoding methylenetetrahydrofolate reductase in coastal West Africa. Am J Clin Nutr 79: 619–624, 2004.
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