1 Center of Investigation in Chemistry, Department of Chemistry, Faculty of Sciences and 2 Department of Anatomy, Porto Medical School, University of Porto, Porto, Portugal
* Author to whom correspondence should be addressed at: Department of Anatomy, Porto Medical School, Al. Prof. Hernâni Monteiro, 4200319 Porto, Portugal. Tel.: +351 22 5096808; Fax: +351 22 5505640; E-mail: mmpb{at}med.up.pt
(Received 10 November 2003; In revised form 12 January 2004; accepted 26 March 2004)
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ABSTRACT |
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INTRODUCTION |
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This pigment is thought to be an innocuous end-product, but its excessive accumulation originates conglomerates occupying huge areas of cytoplasm that might mechanically interfere with the functioning of cell organelles (Constantinides et al., 1986; Sundelin and Nilsson, 2001
; Brunk and Terman, 2002a
). We have previously reported that long-term ethanol feeding results in precocious and progressive deposition of lipofuscin in cerebellar and hippocampal neurons, namely in Purkinje, and CA1 and CA3 pyramidal cells (Tavares and Paula-Barbosa, 1983
; Tavares et al., 1985
; Borges et al., 1986
). In line with previous observations (Constantinides et al., 1986
), we have also found that the use of antioxidants delays, or even prevents, the accumulation of lipofuscin (Paula-Barbosa et al., 1991
).
Flavanol compounds such as catechin monomers [(+)-catechin and ()-epicatechin)] and procyanidins (oligomers and polymers) are present in our diet both in foodstuffs and in beverages such as red wine, tea and fruit juices (Soleas et al., 1997; Santos-Buelga and Scalbert, 2000
; Scalbert and Williamson, 2000
). Flavanols from red wines, mainly catechin monomers and procyanidins dimers and trimers (de Freitas and Glories, 1999
; Santos-Buelga and Scalbert, 2000
; da Silva Porto et al., 2003
) display an important in-vitro antioxidant activity in several lipid systems and in particular against oxidation of LDL, higher than that evinced by the well known antioxidant
-tocopherol (Teissedre et al., 1996
; da Silva Porto et al., 2003
). The presence of flavanol molecules with powerful antioxidant activity in red wine (Frankel et al., 1993
; Roig et al., 1999
) was soon related to a variety of protective actions that wine can exert, despite the widely accepted alcohol-induced deleterious effects (Lukoyanov et al., 1999
; Cadete-Leite et al., 2003
). These beneficial effects were first recognized in the prevention of coronary heart diseases (Frankel et al., 1993
; Soleas et al., 1997
; German and Walzem, 2000
; van de Wiel et al., 2001
; Agarwal, 2002
). In the CNS, reports have also been made emphasizing the role of polyphenols in reducing the risk of strokes (Agarwal, 2002
) and neurodegenerative alterations (Sun et al., 1999
; Moosmann and Behl, 2002
; Sun et al., 2002
).
We thought it of interest to test the antioxidant efficacy of a grape seed extract comprised by catechin monomers and oligomeric procyanidins in the CNS by evaluating their effects on alcohol-induced neuronal lipofuscin formation. For this purpose, the total amount of neuronal lipofuscin was estimated using morphometric methods in hippocampal CA1 and CA3 pyramids and in cerebellar Purkinje cells of alcohol-fed rats, half of which were concomitantly ingesting a mixture of grape seed flavanols. Data were compared with those obtained from control rats, some of which were also ingesting the same polyphenols.
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MATERIALS AND METHODS |
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The composition of flavanol extract in catechin monomers and low-molecular-weight procyanidin oligomers was determined according to a procedure described before (de Freitas et al., 1998). The fraction containing these compounds was firstly purified by chromatography using a Toyoperl Gel (Tosoh) and the content of the following compounds was determined by HPLC (Merck-Hitachi L-7100) on a reverse-phase C18 column. Detection was conducted with a diode-array detector (Merck-Hitachi L-7450A) at 280 nm. The calibration curves were obtained by injecting standards.
These procedures allowed verification that the detailed composition of the flavanol extract used in this experiment was the following (weight per g dry weight): (+)-catechin, 0.056 g; ()-epicatechin, 0.050 g; ()-epicatechin-gallate, 0.016 g; dimers B1, 0.237 g; B2, 0.138 g; B3, 0.032 g; B4, 0.038 g; B5, 0.026 g; B8, 0.008 g; B2-3-O-gallate, 0.151 g (total weight: 0.769 g). The remaining components of the extract, weighing 0.321 g, were constituted by more complex procyanidins.
Animals and treatments
Studies were carried out in adult male Wistar rats (Charles River, Barcelona, Spain). Animals were individually housed and maintained throughout the experiment under room temperature of 2022°C and a daily photoperiod of 12 h light, between 07.00 and 19.00 hours. Prior to the experiment, rats had ad-libitum access to food and water. At 6 months of age, a total of 30 animals weighing between 550 and 665 g (average body weight, 601 ± 31 g) were randomly ascribed to the following groups, each consisting of 6 rats.
(1) Ethanol (Eth). These were rats with unrestricted access to a 20% (v/v) aqueous ethanol solution as the only available liquid source. Ethanol was introduced gradually, beginning with a 5% (v/v) ethanol solution on the first day and increasing 1% per day until the final concentration of 20% was attained. These rats had free access to standard laboratory pellet food (Letica, Barcelona, Spain). Food and fluid intake were measured every other day and the amounts consumed calculated.
(2) Ethanol/flavanol extract (Eth/FL). Ethanol was administered as in the previous group and the flavanol extract was added to the alcoholic drinking solution in a dosage of 200 mg/l. Due to the action of light upon the components of the extract the bottles of liquid were made opaque using aluminium foil and the solution was renewed every other day. Food and fluid intake were measured every other day and the amounts consumed calculated. No significant differences were found regarding the amount of food and fluid consumed when compared to the Eth group.
(3) Pair-fed (PFC). Rats from this group were given the same amount of food consumed by both ethanol-fed groups of rats. Sucrose was added to the drinking water to replace the isocalloric value of the ethanol. The percentage of sucrose in the water of the pair-fed controls was calculated taking into account the caloric values both of alcohol (1 g = 7 kcal) and sucrose (1 g = 4 kcal), as well as the volume of ethanol consumed by ethanol-fed rats (10 ml of ethanol = 7.9 g).
(4) Control (C). Animals with free access to standard laboratory chow and water throughout the experimental period.
(5) Control/flavanol extract (C/FL). Animals with free access to standard laboratory chow but with the flavanol extract added to the water in opaque bottles in the same dosage as in group 2. The rats had free access to the solution which was changed every other day.
Liquids consumed were supplemented with 300 mg/100 ml of vitamins (Vitamin Diet Fortification Mixture, ICN Biomedical, Cleveland, OH) and 500 mg/100 ml of minerals (Salt Mixture XIV, ICN Biomedical). During the first month of the experiment, animals were weighed every 3 days and thereafter every week. All procedures were carried out in compliance with the European Communities Council Directives of 24 November 1986 (86/609/EEC) and Portuguese Act no. 129/92. All efforts were made to minimize the number of animals used and their discomfort and suffering.
Blood ethanol concentrations were measured in the Eth and Eth/FL groups of rats using blood samples from the tail vein collected in the evening (20.00 hours) and morning (08.00 hours). Estimations were made weekly during the first month and thereafter monthly by using an enzymatic assay kit (Sigma, St Louis, MO). Blood samples obtained during the first month at 12.00 and 16.00 hours presented residual or undetectable ethanol, so no more collection of blood was done during the rest of the experimental period at these hours.
Tissue preparation
General procedures. After 26 weeks of treatment, at 12 months of age, the animals of all groups were anaesthetized with 3 ml/kg of a solution containing sodium pentobarbital (10 mg/ml) and chloral hydrate (40 mg/ml) given i.p. and killed by transcardiac perfusion of a fixative solution containing 1% paraformaldehyde and 1% glutaraldehyde in 0.12 mol/l phosphate buffer at pH 7.2. The brains were removed from the skulls, weighed and blocked by hand into right and left halves and the cerebellum isolated. After removal of the frontal and occipital poles, the blocks of tissue containing the right hippocampal formations and the cerebella were processed for electron microscopy.
Epon embedding. After 2 h of post-fixation in the perfusion solution, the neocortex was dissected in order to free the right hippocampal formations that were sliced perpendicular to the septotemporal axis on a tissue chopper, at approximately 1 mm thick. The cerebella were parasagitally sliced on the tissue chopper, at the same thickness, to obtain blocks from the cerebellar vermal lobes IVVI (Larsell, 1952). All blocks were immediately processed for electron microscopy according to standard procedures (Palay and Chan-Palay, 1974
) and subsequently embedded in Epon according to the isector method in order to ensure isotropy (Madeira et al., 1995
). Each block was embedded in an isector of 10 mm diameter (Nyengaard and Gundersen, 1992
). Using a systematic random sampling procedure (Gundersen and Jensen, 1987
), six blocks containing the hippocampal formation and three blocks containing the cerebellar vermal lobules IVVI were selected per animal. From each of these blocks, eight serial sets of four 2-µm thick semithin IUR sections (Nyengaard and Gundersen, 1992
), containing the Purkinje cell layer and CA1 and CA3 pyramidal cell layers were obtained and stained with toluidine blue. Afterwards, hippocampal and cerebellar blocks containing the neuronal populations under study were trimmed in order to obtain ultrathin sections. From each animal three blocks were used. From each of them, 1012 serial ultrathin isotropic random sections containing the Purkinje cell layer, CA1 pyramidal cell layer and CA3 pyramidal layer were cut, collected on formvar-coated grids, and double stained with uranyl acetate and lead citrate.
Neuronal volumes
The mean somatic and mean nuclear volumes of Purkinje cells, CA1 and CA3 pyramidal neurons were estimated using IUR semithin sections (Nyengaard and Gundersen, 1992) and the nucleator as previously described in detail (Gundersen et al., 1988a
; Madeira et al., 1995
). Neurons were sampled using the disector (Madeira et al., 1995
; West and Gundersen, 1990
): all neurons that had nucleolus in the reference section but not in the look-up section were selected for measurements. On average 55 Purkinje cells, 70 CA1 pyramidal neurons and 65 CA3 pyramidal neurons were analysed in each animal. The coefficient of error (CE) of the individual estimates was calculated according to Gundersen et al. (1999)
. All the estimations were performed, at magnification of x2000, using the C.A.S.T.-Grid System software (Olympus DK, Denmark) and a Heidenhain MT-12 microcator (Heidenhain, Germany). The mean cytoplasmic volume was estimated subtracting the mean nuclear volume to the mean somatic volume.
Lipofuscin quantification
Using the ultrathin sections, 10 Purkinje cells presenting a nucleus from each animal were photographed using a systematic random sampling procedure at a primary magnification of x1800 and observed at a final magnification of x5400. Using the same method of selection, 12 CA1 and 12 CA3 pyramidal cells from each animal were photographed at a primary magnification of x2700 and observed at a final magnification of x8100. The volumetric density (Vv) of the lipofuscin granules was determined using point counting techniques (Weibel, 1980; Gundersen et al., 1988b
) utilizing an adequate plastic replica (Weibel, 1980
) in which the area associated with each point was 0.30 µm2. The fraction of cytoplasm occupied by the pigment was estimated in each micrograph from the total number of points that fell on the lipofuscin granules and from the total number of points that fell in the neuronal cytoplasm. The mean total volume of lipofuscin of the neuronal populations analysed from each animal was calculated multiplying the Vv of lipofuscin granules by the mean cytoplasmic volume.
All the estimations were performed by an observer who was blinded to experimental group.
Statistical analyses
To test for the effect of treatment, a one-way analysis of variance (ANOVA) was performed. Animals were used as replicates and the remainder mean square as the error term. Whenever appropriate, the NewmanKeuls post-hoc test was performed to examine differences between groups. Differences were considered to be significant if P < 0.05. Throughout the text, values are expressed as mean ± standard deviation (SD).
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RESULTS |
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Qualitative observations
The ultrastructural study of Purkinje cells and CA1 and CA3 pyramidal cells from all groups of rats showed two types of dense cytoplasmic membrane-bound lipofuscin granules (Fig. 3). Some, resembling primary lysosomes, were of small dimensions and presented a simple structural organization with uniformly dense matrix. Others were structurally more complex, with different sizes and irregular shapes. Their matrix was also fine and electron-dense. However, one or more lucent peripherally located vacuoles could often be recognized. The latter granules were preferentially localized in the apical poles of the neurons.
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Quantitative results
Neuronal volumes. Data from neuronal volumes from all groups of rats and neuronal populations are shown in Fig. 4. ANOVA did not detect any significant effect of treatment on the mean nuclear volume of Purkinje cells [F(4,25) = 1.69, P = 0.23], CA1 pyramidal cells [F(4,25) = 0.25, P = 0.90] and CA3 pyramidal cells [F(4,25) = 1.35, P = 0.32]. ANOVA also revealed that the estimates of the mean somatic volume of Purkinje cells [F(4,25) = 1.36, P = 0.31], CA1 pyramidal cells [F(4,25) = 1.81, P = 0.20] and CA3 pyramidal cells [F(4,25) = 0.26, P = 0.89] were not dependent on the effect of treatment. In addition, no treatment-related alterations were found in the estimates of the mean cytoplasmic volume of Purkinje cells [F(4,25) = 1.53, P = 0.27], CA1 pyramidal cells [F(4,25) = 2.84, P = 0.10] and CA3 pyramidal cells [F(4,25) = 0.25, P = 0.90].
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DISCUSSION |
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Lipid peroxidation is a self-perpetuating widespread process through which lipid components of the membranes from cell organelles are converted into lipid peroxides (Nordmann, 1994), an event which strikingly contributes towards the formation of lipofuscin pigment (Brunk and Terman, 2002a
,b
). The membrane debris derive from the cell membrane system, including organelles, while lysosomic ROS are the end-result of a complex oxidative chain of molecular events mainly initiated in mitochondria during energy production (Brunk et al., 1992
; Brunk and Terman, 2002a
). More specifically, Brunk et al. (1992
, 2002a
) proposed that H2O2 generated by mitochondria and, in smaller amounts by other organelles, pervade the lumen of lysosomes. In addition to a wide spectrum of hydrolytic enzymes, lysosomes are also endowed with iron and other metals arising from the digested membranous debris. Thus, Fenton-type reactions between H2O2 and Fe (II) take place leading to the production of the harmful hydroxyl radicals, which are capable of initiating processes of lipid peroxidation (Brunk et al., 1992
; Halliwell, 1994
, 2001
; Brunk and Terman, 2002a
).
This attractive hypothesis of lipofuscinogenesis allows a satisfactory explanation of the role of different organelles and molecular mechanisms in the production of the pigment: (1) the rate of H2O2 generation by mitochondria, (2) the efficiency of cytosol antioxidant activity, (3) the effect of chain-breaking antioxidants after the initiation of lysosomal lipid peroxidation and (4) the action of intralysosomal iron chelation. It is noticeable that our data obtained two decades ago in some way support the role of this recently described mitochondrial involvement in lipofuscin production. In fact, in a series of studies carried out in the hippocampal formation and in the cerebellum of animals alcohol-fed for 12 months (Tavares and Paula-Barbosa, 1983; Borges et al., 1986
) we have found that, in parallel with marked alterations in the lysosomic cell compartment (Paula-Barbosa et al., 1986
), there were numerous swollen mitochondria with few cristae and even profiles whose matrix was completely deteriorated and homogenized (Paula-Barbosa et al., 1986
). Interestingly, these changes are now thought to reflect impaired mitochondrial fission, probably as a consequence of the oxidative damage suffered by mitochondrial DNA (Brunk and Terman, 2002a
).
In view of the evidence yielded for the role of lipid peroxidation in the genesis of lipofuscin, it is conceivable that the increased amount of its deposits, that we observed in CA1 and CA3 hippocampal pyramids and in cerebellar Purkinje cells following chronic ethanol consumption, might be associated with the ethanol-induced increase in lipid peroxidation (Henderson et al., 1999). This might result from the enhanced formation of cell membrane debris as a consequence of the deleterious effects of ethanol upon the cell membrane compartment (Paula-Barbosa et al., 1991
) and/or of the alcohol-induced depletion of antioxidant protective mechanisms. Indeed, on the one hand, due to its marked lipophilic properties ethanol can easily penetrate into the cell membrane, which alters the structure of the bilipidic membrane layer and increases membrane fluidity (Paula-Barbosa et al., 1991
; Henderson et al., 1999
). On the other hand, it is well recognized that alcohol intake decreases the amount of cell antioxidants (Nordmann et al., 1992
), thereby reducing the strength of the cell protective antioxidative shield (Brunk and Terman, 2002a
).
Interestingly, the flavanols used in this study prevented the formation of lipofuscin in animals submitted to alcohol consumption, but not in water drinking animals, despite the larger amount of flavanols ingested. This finding suggests that the antioxidant effects of the flavanols are only effective under conditions of enhanced oxidative stress (i.e. only when additional protection is required to maintain the normal cellular pro-oxidative and antioxidative balance) (Sun et al., 1999, 2002
). This assumption is supported by our data obtained during aging (unpublished results). In this regard and for the sake of consistency, we cannot discard the possibility that some flavanol compounds tested might be absorbed at the intestinal level only after its mucosa was lesioned by ethanol exposure. On the other hand, the higher solubility of these compounds in hydroalcoholic solution may help their absorption. Nevertheless, the bioavailability of flavanols is a controversial subject because many authors believe that flavanols might not be absorbed before being metabolized by the intestinal microflora (Scalbert and Williamson, 2000
). The resulting low-molecular-weight polyphenolic metabolites might be absorbed through the intestinal barrier and be metabolized once more in the liver, yielding compounds that could act in situ as biological antioxidants (Scalbert and Williamson, 2000
).
The flavanols tested in this study display powerful antioxidant properties because they are able to form stable antioxidant-derived radicals (da Silva Porto et al., 2003). However, it is worth noting that most of existing data concerning the biological properties of these compounds was obtained on the grounds of in-vitro studies and pains are required in extrapolating to in-vivo experimental paradigms. Some of us (da Silva Porto et al., 2003
) have recently performed in-vitro studies to assess the antioxidant activity of some flavanols in the process of lipid peroxidation of human low density lipoprotein (LDL). Briefly, it was found that the flavanols used in this study act against LDL oxidation at three different levels: (1) by scavenging peroxyl radicals and forming stable phenoxyl radicals, which interrupt the propagation of the lipid chain, (2) by regenerating some endogenous antioxidants, such as
-tocopherol, which represent the first LDL line defence against oxidative stress, and (3) by chelating some catalytic metal ions such Fe (III), Cu (II) and Al (III), present in LDL intima or plasma, thus inhibiting the initiation of lipid peroxidation.
To shed light on the in-vivo antioxidant mechanisms of flavanols and/or its metabolites, we must bear in mind how antioxidant defences in the liver and brain can counteract enhanced pro-oxidant processes. This might occur by (1) inhibiting free radical production due to chelating iron derivatives, (2) trapping free radicals themselves, (3) interrupting the peroxidation process, and (4) reinforcing the natural antioxidant defences (Nordmann et al., 1992). The role of iron chelation by flavanols deserves to be emphasized as it has been demonstrated that ethanol increases the iron content in liver and cerebellum (Rouach et al., 1990
; Sergent et al., 1995
). Should this be the case, then iron can exacerbate oxidative stress by catalysing the conversion of superoxide and hydrogen peroxide to more potent oxidants such as hydroxyl radicals (Rouach et al., 1990
; Sergent et al., 1995
).
If, in addition, we assume that flavanols are bioavailable in the brain, then the likelihood that the mechanisms of action of these compounds might be the same in vivo and in vitro is high. Supporting this view, recent data showed that flavanols and some of their metabolites are capable of crossing the bloodbrain barrier (Youdim et al., 2003) and metabolites of epicatechin were found in rat brain after oral ingestion (Abd El Mohsen et al., 2002
). Should that be the case it is tantamount saying why flavanols exert the reported powerful antioxidant effect against the increased formation of lipofuscin following chronic alcohol consumption: all steps of lipid peroxidation can be counteracted by this antioxidant.
The yielded results unequivocally demonstrate that these compounds possess, directly or indirectly by their metabolites, striking in-vivo antioxidant effects in the brain, which fits data from other in-vivo (Roig et al., 1999; Sun et al., 1999
, 2002
; Simonyi et al., 2002
) and in-vitro studies (Frankel et al., 1993
; da Silva Porto et al., 2003
). It has likewise been unequivocally shown that antioxidant food compounds may have major significance in the prevention of a number of pathologies, including cardiovascular diseases and CNS disorders (for details see Nordmann et al., 1992
; Mossmann and Behl, 2002
). In this regard, it has recently been demonstrated that polyphenols contained in red wine stimulate genes which are known to slow the pace of aging and to increase lifespan by negatively regulating the p53 tumour suppressor activity (Howitz et al., 2003
). Be this as it may, our data hold true with the view that moderate drinking of wine, despite the unequivocal alcohol toxicity to almost every tissue in the body (Badawy, 2001
), paradoxically decreases the total mortality in middle-aged and elderly people (Soleas et al., 1997
; German and Walzem, 2000
).
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ACKNOWLEDGEMENTS |
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