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Department of Metabolism and Pathological Biochemistry,
Department of Food and
*
Department of Ultrastructures, Istituto Superiore di Sanità, 00161 Rome, Italy
1To whom correspondence should be addressed at Metabolism and Pathological Biochemistry Department, Istituto Superiore di Sanità, Viale R. Elena, 299, 00161 Rome, Italy. E-mail: mbpsegr{at}net.iss.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • tyrosol • oxidized LDL • Caco-2 cells • antioxidant • polyphenols
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
Rice Evans et al. 1996
). In particular, phenolic
antioxidants, present in virgin olive oil, and responsible for the high
resistance of the oil to oxidation, can protect against LDL oxidation
(Wiseman et al. 1996
) similar to the phenolic compounds
present in red wine and green tea (Frankel et al. 1993
, Zhenhua et al. 1991
). These
compounds scavenge free radicals and break peroxidative chain
reactions.
Oxidative stress, defined as an overproduction of free radicals, or a
diminuition in antioxidant defense mechanisms, determines cellular
damage with functional alterations of the involved tissue. Free
radicals can attack any biochemical component of the cell, but lipids
are a major target. Lipid peroxidation of cell membranes and plasma
lipoproteins represents a primary event in the establishment of
oxidative stress. Susceptibility of LDL to oxidative modifications
depends on its fatty acid composition and cellular and extracellular
antioxidants, which serve to trap reactive oxygen species and to
inhibit the chain reaction of free radicals (Esterbauer et al. 1992
). Oxidized
LDL2
(ox-LDL) generate a mixture of compounds with potential cytotoxic
activity, including cholesteryl esters and phospholipid hydroperoxides,
end-products of lipid peroxidation such as malondialdehyde and
4-hydroxy-nonenal and a variety of cholesterol oxides
(Esterbauer et al. 1992
). Lipid peroxides and their
degradation products are involved in the inflammatory response as
chemoactractants and/or modulators of cytokines and enzymes
(Berliner et al. 1993
). If the rate of production
exceeds the capacity of endogenous antioxidant defenses, these toxic
oxidants may cause tissue injury and destruction with the consequent
onset of a chronic inflammatory pathology (Holvoet et al.
1994
).
Some epidemiological studies showed a high correlation between a diet
particularly rich in biophenols and a lower risk of cardiovascular
diseases (Hertog et al. 1993
). Phenolic compounds might
play an important role in preventing oxidative stress-linked
gastrointestinal diseases which are characterized by inflammatory
intestinal injury, such as Chron's disease and ulcerative colitis
(Grisham 1994
).
To determine the possible oxidative damage to intestinal mucosa and the
possible protective effect of phenolic dietary compounds, we evaluated
morphological and functional changes induced by ox-LDL in the human
colon adenocarcinoma cell line, Caco-2. This cell line spontaneously
undergoes full differentiation in vitro with enterocyte-like
features, both structurally (microvilli, tight junctions), and
functionally (brush border-associated enzymes, transport across the
surface membrane) (Pinto et al. 1983
). Since the
intestine is the primary site of exposure to substances present in
food, this cell line has been recognized as a suitable model for
evaluating the effect of nutrient components, for both normal dietary
constituents and additives, contaminants, toxicants and drugs,
(Artursson et al. 1994
, Dessì et al. 1997
, Koninks et al. 1992
, Leher and Lee 1993
).
Some progress has been made in identifying the role played by some
natural antioxidants in human nutrition; however, little information is
available on olive oil biophenols and their biological activity in cell
culture systems (Manna et al. 1997
). Here we examine the
protective effects exerted by the major phenolic compound present in
olive oil, tyrosol, (p-hydroxyphenylethanol) on Caco-2
cells. In spite of its relatively low antioxidant activity, tyrosol
showed a more stable protective effect against oxidation in our
preliminary studies, even in critical conditions, i.e., in the presence
of prexisting hydroperoxides, when other antioxidants, e.g., vitamin E
or other more active plant phenols, may actually become
pro-oxidants (Yamanaka et al. 1997
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LDL (1.019–1.063 kg/L) were prepared from freshly isolated
plasma pooled from different healthy human donors by density gradient
ultracentrifugation in the presence of 0.1 mmol/L of EDTA. The isolated
LDL were dialyzed against NaCl (0.15 mol/L) containing 10 mmol/L of
EDTA, and analyzed for protein by the Bradford method (Bradford 1976
), using bovine serum albumin as a standard. Aliquots of
LDL solution (2 g/L) were oxidized with 25 µmol/L of
CuSO4 for 18 h at 37°C. The same LDL preparations at
the same concentration, without exposure to copper, were used as native
LDL (n-LDL).
Lipid extracts of native LDL and ox-LDL fractions, obtained as
described (Folch et al. 1957
), were analyzed for lipid
composition by gas–liquid chromatography. Oxidation resulted in a
reduction in arachidonic acid and polyunsaturated fatty acid levels by
about 51 and 35%, respectively. The extent of lipid peroxidation was
estimated as malondialdehyde and 4-hydroxyalkenals content by a
colorimetric kit (LPO 586; Bioxytech S.A., Bonneuil Sur Marne, France).
A mean value of 45.5 ± 8.2 nmol/mg LDL protein was measured in
oxidized samples vs. 4 ± 0.5 nmol/mg protein in native LDL.
Modification of LDL was tested by measuring the increase of
electrophoretic mobility on 0.5% of agarose gel and the fragmentation
of apolipoprotein (apo) B by SDS-PAGE (data not shown).
Caco-2 cell cultures. Human colon adenocarcinoma Caco-2 cells were obtained from the European Cell Culture collection (Salisbury, United Kingdom). Cells were grown in Dulbecco's modified Eagle medium (DMEM: Hyclone, Cramlington, United Kingdom) with 4.5 g/L of glucose supplemented with 10 mL/L nonessential aminoacids (Flow Laboratories, Irvine, Scotland), 0.2 mmol/L of L-glutamine (Flow), 5 x 104 IU/L of penicillin (Flow), 50 mg/L of streptomycin (Flow), and 100 mL/L of fetal calf serum (Flow), at 37°C in a humidified atmosphere of 5% CO2 in air. The cells were seeded at 3 x 105 in 25 cm2 tissue culture flasks (Falcon, Free Lake, NJ); routine cell passages were carried out twice a week by removing cells with a solution containing 2.5 g/L of trypsin and 0.2 g/L of EDTA, in calcium-free and magnesium-free PBS.
Experimental procedures and treatments
Low density lipoproteins, n-LDL or ox-LDL, were sterilized by filtration with a 0.4 µm Millipore membrane (Millipore Corporation, Bedford, MA) and added to the cell culture medium at a final concentration of 0.2 g/L. Tyrosol (Sigma, St. Louis, MO) was directly dissolved in the medium and sterilized in the same way.
On day 5 of culture, before cells reached confluency and began to differentiate, they were washed twice with serum-free medium. The medium was then replaced by DMEM containing 20 mL/L of Ultroser G (Flow), a lipoprotein-free serum substitute, and exposed to native or ox-LDL with and without phenolic compounds.
Preliminary experiments were performed to evaluate optimal cell treatment conditions regarding ox-LDL exposure time, phenol incubation time, and the most effective antioxidant concentrations (different exposure time of treatments and different concentrations of tyrosol were assessed). An ox-LDL exposure time of 24 h and/or 48 h, and 2 h of 0.5 mmol/L of phenol preincubation were chosen on the basis of these preliminary tests. All experiments included untreated cells (controls), cells treated with n-LDL, ox-LDL, tyrosol, and cells treated with LDL or ox-LDL in the presence of the phenol. Tyrosol was added 2 h before treatments with LDL and was present in the culture medium for the entire experimental period in all the experiments presented in this study.
Proliferating activity
Each experiment was conducted by seeding 6 x 104 cells per well in 24 multiwell plates. On day 5 of culture, cells in 0.5 mL of DMEM containing 20 mL/L Ultroser G were exposed to n-LDL or ox-LDL without and with 0.25, 0.5, and 1 mmol/L of tyrosol for 44 h. After this incubation period, 100 µL of the same medium containing 1.85 kBq of [14C]thymidine (Amersham, Buckinghamshire, United Kingdom; sp. act.: 2.09 GBq/mmol) was added to each well. After a 4-h incubation, incorporation was stopped by 1 mL of 100 g/L trichloroacetic acid at 0°C. The wells were washed twice with 1 mL of PBS, and cells were fixed by 1 mL of methyl alcohol, for 10 min. The alcohol was then removed and the cell monolayer dissolved in 0.5 mL of 0.1 mol/L NaOH. Radioactivity was evaluated in 0.2 mL aliquots of NaOH extracts with a liquid scintillation spectrometer. [14C]Thymidine incorporation was expressed as a percentage of values observed in untreated controls.
Lactate dehydrogenase release
12 x 103 cells per well were seeded in 96-well microtiter plates. On day 5 of culture, cells were washed as described above, and the medium was replaced by 0.2 mL of medium containing 20 mL/L of Ultroser G. After a 2-h preincubation with three different concentrations of phenolic compound, cells were incubated with n-LDL or ox-LDL for 48 h. Plates were centrifuged at 250 x g for 4 min; 0.1 mL of supernatant aliquots was transferred to clean 96 multiwell plates and lactate dehydrogenase release was determined using a Sigma colorimetric kit. The absorbance was measured at a wavelength of 490 nm (reference wavelength: 690 nm) by a microplate reader (Novopath, Biorad, Hercules, CA). Lactate dehydrogenase (LDH) activity in the culture medium as a result of leakage from nonviable cells, was expressed as a percentage of total LDH activity, obtained by treating cells with 10 mL/L of Triton X-100 (Sigma), and corrected by the activity already present in the medium of untreated cells (spontaneous release).
Morphological analyses
For cytoskeletal analyses and scanning electron microscopy studies, control and treated cells were seeded on 13-mm diameter glass coverslips in separate wells (50 x 103 cells/well).
Light microscopy.
For fluorescence microscopy studies, cells were fixed with 37 g/L
formaldehyde in PBS (pH 7.4) for 10 min at room temperature. After
washing in the same buffer, the cells were permeabilized with 5 g/L of
Triton X-100 (Sigma) in PBS for 5 min at room temperature. For
cytoskeletal analyses, Caco-2 cells were stained with
fluorescein-phalloidin (Sigma) or with a mixture (1:1) of 
and
ß antitubulin antibodies (Sigma) at 37°C for 30 min. The first is a
toxin capable of binding directly to F-actin and is usually linked
with a fluorescent marker. The second is a mixture of and ß (1:1)
antitubulin antibodies capable of reacting with the cell microtubular
network. For the detection of tubulin, cells were subsequently
incubated with anti-mouse IgG-fluorescein linked whole antibody
(Sigma) at 37°C for 30 min. Finally, after washing, all the samples
were mounted with glycerol-PBS (2:1) and observed with a Nikon
Microphot (Nikon Corporation, Tokyo, Japan) fluorescence microscope.
For apoptosis evaluation, detached cells were first collected by
centrifugation (5 min) and resuspended in PBS. An aliquot of these
cells (40 mL) was seeded on polylysine-coated cover slips for 20
min, fixed with formaldehyde, permeabilized with Triton X-100 as
described above and stained with Hoechst 33258 fluorescent dye.
Quantitative evaluation of apoptotic cells, excluding isolated
apoptotic bodies, (Hoechst staining, Bursh et al. 1992
)
was performed by counting 300 cells at high magnification (500x) as
previously described (Malorni et al. 1993
).
Scanning electron microscopy. Control and treated cells were washed in PBS and fixed with 25 mL/L of glutaraldehyde in 0.1 mol/L of cacodylate buffer (pH 7.4) containing 30 g/L of sucrose at room temperature for 20 min. Following post-fixation in 10 mL/L of osmium tetroxide for 30 min, cells were dehydrated through graded ethanols, critical point dried in CO2 and gold coated by sputtering with a Balzer Union SCD 040 apparatus (Liechtenstein). The samples were then examined with a Cambridge 360 scanning electron microscope (Cambridge, United Kingdom).
Reduced glutathione measurements
To determine the intracellular reduced glutathione, a colorimetric assay for glutathione (Bioxytech GSH-400; Bonneuil Sur Marne, France) was used. After ox-LDL treatment, cells were detached from the substrate with EDTA (10 mmol/L) and Trypsin (2.5 g/L), centrifuged for 5 min at 1000 x g, washed twice with PBS, resuspended in the same buffer and homogenized by sonication for 30 s. Cell lysates were resuspended in 500 µL of ice-cold 50 mL/L metaphosphoric acid solution, centrifuged in a microfuge (3000 x g) at 4°C for 10 min. The supernatants were stored at 4°C until assayed. Reduced glutathione (GSH) was estimated at 400 nm by using a Beckman spectrophotometer (Beckman Instruments Inc., Fullrton, CA). Data, expressed as nmoles of GSH per µg of protein, were calculated on the basis of a GSH calibration curve determined according to protein content. This was obtained as follows: cells pellets, previously precipitated with ice-cold metaphosphoric acid solution, were resuspended in 500 µL of NaOH 1 mol/L. Proteins were measured according to manufacturer instructions (Protein assay; Biorad).
Statistical analysis
The data are presented as the arithmetic mean for each experimental point ± SEM. Statistical calculations were performed using a one-way ANOVA. Differences among groups were examined using the Bonferroni t-test, when the F value was significant. Differences with a P-value <0.05 or less were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).Moreover, treatment with ox-LDL and tyrosol resulted in extensive
protection of proliferative activity. The presence of tyrosol at three
different concentrations effectively reversed ox-LDL
antiproliferative activity (P < 0.01). LDH
release, (Fig. 2
)an indicator of cell membrane integrity, was significantly lower in
cell cultures preincubated with 0.25, 0.5 or 1 mmol/L tyrosol than in
culture monolayers treated only with ox-LDL (P < 0.01). Because tyrosol was so effective in counteracting
ox-LDL toxicity at 0.5 mmol/L, all further experiments focused on
the protective effect exerted at this concentration.
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A).After 24 h treatment with LDL, the cells showed an evident
flattening of the cell body on the substrate (Fig. 3B)
. In contrast,
after 24 h ox-LDL treatment, the cell monolayer appeared to be
altered, and cell-cell and cell-substrate contacts appeared to
be slightly modified with several retracted and rounded cells (Fig. 3C)
. After 48 h ox-LDL exposure, the cells detached from the
substrate and flowed freely in the culture medium (data not shown).
When the cells were treated with tyrosol 0.5 mmol/L in the absence
(Fig. 4
A)or in the presence (Fig. 4B)
of n-LDL, no changes were observed in
the cell layer ultrastructure. By contrast, morphological alterations
induced by ox-LDL were almost completely prevented when Caco-2
cells were pre-incubated for 2 h with tyrosol 0.5 mmol/L. The
cells appeared perfectly flat, with their typical cell-to-cell and
cell-to-substrate structure, with no sign of cell injury (Fig. 4C)
.
|
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A).This organization was enhanced by the appearance of numerous stress
fibers in cells treated with n-LDL (Fig. 5B)
. In contrast, after a
24-h treatment with ox-LDL, the cells showed marked alterations of
the actin network assembly represented by a rearrangement of filament
organization and distribution (Fig. 5C)
. Evaluation by fluorescence
analyses of the intracytoplasmic actin network in tyrosol exposed
cells, indicated that: (i) pre-exposure with this compound was
capable of "protecting" the actin skeleton from ox-LDL induced
injury (Fig. 6
C)and more importantly, (ii) tyrosol exposure was able per se to improve
actin stress fiber formation by promoting a well-organized network
assembly (Fig. 6A)
. Close structural relationships between increased
cytoskeletal organization induced by tyrosol (Fig. 6A)
and cell
flattening on the substrate are shown in Figure 4
.
|
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A).After treatment with tyrosol and/or n-LDL, the cells displayed a
well-organized network (Fig. 7B
, Fig. 8
A and
8B).In contrast, treatment with ox-LDL induced a rearrangement of
microtubules, which appear collapsed and partially depolymerized (Fig. 7C)
. Tyrosol was able to prevent ox-LDL effects, as shown by the
well-developed and organized microtubular apparatus (Fig. 8C)
.
Finally, apoptosis was evaluated in the different culture conditions.
Thus, when cell nuclei floating in the medium 24 and 48 h after
LDL treatment were analyzed for the presence of DNA fragmentation (or
chromatin clumping), which is indicative of programmed cell death or
apoptosis, control cell nuclei appeared as very few interphase nuclei,
floating freely in the supernatant (not shown). Ox-LDL treatment
(24 and 48 h) significantly induced (P < 0.01)
apoptosis compared controls (Fig. 9
A).Pre-exposure to tyrosol reduced apoptosis (P < 0.001) in cells treated with ox-LDL for 48 h. Cells exposed to
ox-LDL were characterized mainly by chromatin clumping and/or
condensation typical of apoptosis (Fig. 9B)
. In contrast, when tyrosol
was administered before ox-LDL exposure, only a small number of
cells with apoptotic morphology were visible (Fig. 9C)
. It is however
important to underline that the cell population undergoes loss of
cell-to-cell and cell-substrate contacts, during the 48 h
experimental period (48 h). In this study, detached cells were 1.6% ±
0.4 in controls, 19% ± 2 in ox-LDL treated cells and 5% ± 0.8
in cultures preincubated with tyrosol and then exposed to ox-LDL.
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). A marked depletion of this compound was found only in
ox-LDL-treated cells (0.03 nmol/µg protein). In fact,
tyrosol-protected cells had a high GSH content (0.28 nmol/µg
protein, three times that of control cells, 0.07 nmol/µg protein). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
Halliwell 1994
). Oxidation products of normal metabolism
can cause extensive damage to DNA, proteins and lipids; however many
repair processes are available to the cell, including enzyme and
structural defenses. The large group of natural antioxidants plays a
role in the protective mechanism (Block and Langseth 1994
, Rimm et al. 1993
). In this regard, high
consumption of fruit and vegetables is associated with a lowered risk
of degenerative diseases (Hertog et al. 1993
). At
present, however, there are little data to support the routine use of
exogenous natural antioxidants to prevent and/or treat these diseases.
The results shown here provide evidence that physiological
oxidative-stress inducers such as ox-LDL exert a cytotoxic
effect on an in vitro enterocyte-like cell system, and that
phenolic compounds with antioxidant properties, such as those contained
in virgin olive oil, may exert a protective effect. In fact, both
cytostatic and cytotoxic effects were observed: (i) the inhibition of
proliferation and (ii) the induction of apoptotic cell death. In
agreement with existing data (Agarwal et al. 1996
), we
hypothesize that ox-LDL-mediated cytotoxicity might be due to
intracellular pro-oxidant/antioxidant imbalance (Malorni et al. 1997
, Pirillo et al. 1997
, Viora et al. 1997
). In our experience, the pro-oxidant behavior shown by
ox-LDL dramatically affects plasma membrane and cytoskeletal
element integrity. These morphological changes, i.e., cell retraction
and rounding, are strictly related to functional alterations and lead
to the loss of cell-to-cell and cell-substrate contacts. These
modifications are considered to be typical features of an oxidative
stress-mediated injury (Bellomo et al. 1990
,
Hyslop et al. 1988
). It has been hypothesized that
cytoskeletal alterations (associated with cell retraction and
shrinking) might occur before cell detachment (Fiorentini et al. 1998
) and before the onset of any biochemically detectable sign
of DNA and nuclear fragmentation and plasma membrane leakage, and this
may be interpreted as one of the earliest changes in cell structure and
function caused by cholesterol oxides (Hughes 1994
). We
suggest that, as for other antioxidizing drugs such as
N-acetylcysteine (Malorni et al. 1995
and
Malorni et al.1996
), protection of these
subcellular structures from oxidative injury might be exerted by
pre-treatment of the cells with tyrosol. Moreover, we also observed
the detachment of Caco-2 cells from the substrate and, subsequently,
apoptosis. The temporal sequence of cell detachment and apoptotic cell
death observed here does not necessarily imply a causal relationship.
For instance, it was hypothesized that cells forced to extend
themselves over a large surface, i.e., spreading cells, survive better
than cells with a more rounded shape (Ruoslahti 1997
).
Cell adhesion and spreading are different and well-defined
processes, mainly dependent on the integrity and function of the
cytoskeleton, at least in terms of focal adhesion plaque assembly and
cell contractility (Burridge et al. 1997
; Nobes and Hall 1995
). The microfilament system plays a key role in
such processes including cell attachments. Tyrosol protects the cells
from detachment and thus, from apoptosis. This type of cell death has
recently been called anoikis (Frisch et al. 1996
),
referring to that homeless condition due to the loss of cell-cell
and cell-substrate contacts followed by cell detachment and finally
leading to cell lysis.
Taken together, these findings shed light on the mechanism by which ox-LDL exerts specific cytotoxic effects in enterocyte-like cells in culture. The interaction of LDL oxidant products with cells can prime the events characteristic of cell death by apoptosis.
The protection against cytotoxic damage and apoptosis exerted by tyrosol seems to be ascribable to a specific activity of the drug on intracellular antioxidant capability. In fact, on the basis of our results, this phenolic compound seems to be able to counteract intracellularly the ox-LDL-induced effects by modifying cell redox potential, while an extracellular activity seems to be excluded. Further experiments are in progress to evaluate the mechanisms leading to the alterations occurring in enzymes critical to GSH synthesis.
However, the powerful effect of tyrosol in this study is surprising because several studies have suggested that the antioxidant activity of this monophenol is weak. Also our data on the effect of tyrosol and other biophenols present in olive oil on the conjugated diene formation, monitored spectrophotometrically at 234 nm, in LDL oxidized by CuSO4, confirmed that finding. In fact, when oxidized in the presence of tyrosol, LDL showed an increase of lag-phase (130 ± 11 min vs. 85 ± 33 min in LDL alone), higher than that observed when vanillic acid was used (90 ± 11 min), and comparable to that obtained with o-coumaric acid (139 ± 23 min), but very low compared to those obtained when other biophenols, i.e., protocatecuic acid, caffeic acid, oleoeuropeine, were used (>500 min).
Moreover, studies on chemical structure/antioxidant activity
relationships showed that tyrosol can exert its effect only as
a hydroxyl radical scavenger or, at most, as an -tocopherol
regenerator (Rice Evans et al. 1996
). Other
polyphenols are able to quench lipid alkoxyl, peroxyl radicals and/or
to chelate metal ions, preventing metal-catalyzed formation of
initiating species. However, different methods of assessment, varying
substrate systems and different concentrations of antioxidants, have
all contributed to the complexity of this issue. When assessing
antioxidant activities against lipid oxidation, the relative
contribution of different factors should be considered: (i) direct
scavenging of the initiating species, (ii) the rate constant for
peroxyl radical scavenging and (iii) the partition coefficients of the
compounds that influence the accessibility to radicals in the
lipophilic phases. In a recent report, the order of efficacy of some
polyphenols as antioxidants in three different types of preparations
was studied, and a different hierarchy of antioxidant activities was
proposed (Saija et al. 1995
). The authors speculate that
the conflicting sequences of antioxidant potential in the different
systems depend on the capabilities of tested polyphenols to penetrate
and interact with lipid bilayers to different extents. Tyrosol is a
rather stable compound and, therefore, when compared with other
polyphenols, much less subject to autooxidation. With this in mind, we
demonstrated in a pilot study that the antioxidant capability of
tyrosol is also displayed under critical conditions. In fact, in the
presence of old LDL, when autoxidation phenomena had already started,
tyrosol maintained an unchanged antioxidant activity, while other more
active natural flavonoids show a drastic reduction of their antioxidant
effectiveness and sometimes even became pro-oxidants
(Yamanaka et al. 1997
).
In conclusion, the present results provide an important contribution to understanding the cytotoxic mechanisms underlying cellular damage induced by ox-LDL and related oxidative stress. Moreover, this study suggests the possible relevance of dietary intake of olive oil based on the capability of the major biophenol present, in lowering the risk of reactive oxygen metabolite-mediated diseases such as inflammatory bowel disease. However, to confirm this hypothesis, further studies are needed to better understand the molecular mechanisms involved in tyrosol action and its possible intracellular and/or extracellular scavenging activity, making use also of different experimental models.
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FOOTNOTES |
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Manuscript received December 1, 1998. Initial review completed December 22, 1998. Revision accepted March 29, 1999.
|
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