Eur. J. Biochem. 271, 1671–1676 (2004) FEBS 2004
doi:10.1111/j.1432-1033.2004.04070.x
Effects of homocysteine on apoptosis-related proteins and
anti-oxidant systems in isolated human lymphocytes
Anna Mangiagalli1, Alberta Samuele1, Marie-Thérèse Armentero1, Eleonora Bazzini1, Giuseppe Nappi1,2
and Fabio Blandini1
1
Laboratory of Functional Neurochemistry, Neurological Institute ‘C. Mondino’, Pavia, Italy; 2Department of Neurology and
Otorhinolaryngology, University ‘La Sapienza’, Rome, Italy
Homocysteine (Hcy) is a nonprotein-forming sulphur amino
acid that plays an important role in remethylation and
trans-sulphuration processes. In recent years, it has been
suggested that increased levels of plasma Hcy may play a
role in the pathogenesis of various diseases, particularly
at the cardiovascular level. The pathogenic mechanism of
hyperhomocysteinemia, however, has not been clarified.
Because oxygen radicals can be generated by the autooxidation of this amino acid, it has been suggested that Hcy
may cause cellular damage through oxidative mechanisms,
ultimately leading to apoptotic cell death. In this study, we
sought to investigate the effects of Hcy on oxidative damage
and antioxidant agent levels, as well as on apoptosis-related
proteins and apoptosis occurrence in human cells. For this
purpose, we measured levels of Bcl-2, caspase-3 and caspase-9 activity, Cu/Zn superoxide dismutase, reduced
glutathione, lipid peroxidation [malondialdehyde and
4-hydroxy-2 (E)-nonenal concentrations], apoptotic singlestranded DNA and nuclear changes in human isolated
lymphocytes exposed to increasing concentrations of Hcy.
Incubation with Hcy did not induce significant changes in
any of these biomarkers. Therefore, our results do not
support the existence of a direct link between increased
levels of Hcy and the occurrence of a pro-apoptotic state
mediated by enhanced oxidative stress.
Increased levels of homocysteine (Hcy), a thiol-containing
amino acid involved in the methionine conservation cycle
and in trans-sulfuration pathways, have been associated
with increased risk for atherosclerotic and thrombotic
vascular diseases [1–3]. Increased Hcy levels have also been
reported in patients with neurodegenerative disorders, such
as Alzheimer’s disease [4,5] or Parkinson’s disease [6–9],
and the importance of a balanced Hcy metabolism for
the maintenance of neuronal homeostasis has been recently
pointed out [10].
Hcy concentrations may increase as a result of deficiency
in folate [11], vitamin B6 or vitamin B12 [12,13], or because of
genetic mutations, particularly within the genes that encode
the enzymes methylenetetrahydrofolate reductase [14] and
cystathionine b-synthase [15]. The cellular and molecular
mechanisms underlying the adverse effects of hyperhomocysteinemia have not been fully elucidated. Various authors
have suggested that the amino acid may act as a pro-oxidant
agent; indeed, the metabolism and metal-catalysed autooxidation of Hcy are paralleled by the formation of oxygenfree species, which may play a role in the endothelial damage
associated with hyperhomocysteinemia [16–19]. In addition,
in vitro studies have reported that Hcy, particularly at high
concentrations, induces apoptosis (programmed cell death)
in various cell lines [20,21]. This may be related, again, to the
putative pro-oxidant properties of the amino acid, as
oxidative stress is a major trigger of apoptosis [22].
Peripheral blood lymphocytes have been extensively used
to study the involvement of oxidative stress and/or apoptosis in the pathogenesis of numerous pathological conditions, including cardiovascular and neurodegenerative
disorders [23–26], as they provide a convenient and accessible model for in vitro studies. In addition, there is evidence
that the humoral and cellular immune systems play a role in
atherogenesis and that hyperhomocysteinemia may intervene in the phenomenon by inducing B lymphocyte
proliferation [27]. This effect would be mediated by the
production of intracellular reactive oxygen species, which
act as second messengers to regulate signal transduction
pathways controlling gene expression and post-translational
modification of proteins [27].
The aim of our study was to investigate, in vitro, whether
Hcy induces oxidative stress and/or apoptotic cell death in
human lymphocytes, by interfering with the regulatory
mechanisms of these pathological conditions. For this
purpose, we measured a number of oxidative stress-related
markers, including lipid peroxidation, antioxidant enzyme
Cu/Zn superoxide dismutase (Cu/Zn SOD) and reduced
glutathione (GSH), in isolated human lymphocytes exposed
Correspondence: F. Blandini, Laboratory of Functional
Neurochemistry, Neurological Institute C. Mondino,
Via Palestro 3, 27100 Pavia, Italy.
Fax: + 39 0382 380396, Tel.: + 39 0382 380333,
E-mail: [email protected]
Abbreviations: ANOVA, analysis of variance; Cu/Zn SOD, Cu/Zn
superoxide dismutase; GSH, reduced glutathione; ssDNA,
single-stranded DNA; Hcy, homocysteine; DTNB, 5,5¢-dithiobis
(2-nitrobenzoic acid).
(Received 8 October 2003, revised 9 February 2004,
accepted 27 February 2004)
Keywords: antioxidants; apoptosis; hyperhomocysteinemia;
oxidative damage.
FEBS 2004
1672 A. Mangiagalli et al. (Eur. J. Biochem. 271)
to increasing concentrations of Hcy. In the same samples,
we determined the effect of Hcy on a selected set of proteins
that participate in the apoptotic process with the opposite
role: Bcl-2, which regulates cell survival by counteracting
apoptosis [28]; caspase-9, which initiates cell death signalling
events [29]; and caspase-3, the major effector of the
apoptotic cascade [30,31]. We also investigated the actual
occurrence of apoptosis through the analysis of the typical
changes in DNA and membrane permeability associated
with the phenomenon.
Materials and methods
Chemicals
D,L-homocysteine,
)1
RPMI-1640 medium (modified with
20 mmolÆL Hepes and L-glutamine, without NaHCO3),
stabilized penicillin–streptomycin solution (10 000 UÆmL)1
penicillin and 10 mgÆmL)1 streptomycin), NaCl/Pi tablets,
Ellman’s reagent [5,5¢-dithiobis(2-nitrobenzoic acid);
(DTNB)], campthothecin, methanol and Trypan blue were
purchased from Sigma. Foetal bovine serum was from
BioWhittaker Inc., Yopro-1 and propidium iodide dyes
were provided by Molecular Probes Inc.
Sample preparation
Venous blood samples (30 mL) were obtained from 10
healthy volunteers ranging in age between 26 and 33 years
(mean ± SD, 29 ± 2.5 years). Experiments were carried
out with the understanding and written consent of each
subject. Blood was collected from the antecubital vein in
vacuum tubes containing EDTA (Terumo Europe, Belgium) and immediately centrifuged at low speed (250 g,
15 min, room temperature) to remove platelets. After
removal of the platelet-rich plasma layer, the original
volume was reconstituted with NaCl/Pi pH 7.4, and the
mixture layered over half of its volume of Lympholite-H.
The discontinuous gradient thus formed was centrifuged at
450 g for 20 min, at room temperature. After removal of the
upper layer, the lymphocyte band at the interface was
removed and washed twice with NaCl/Pi. Isolated lymphocytes were counted using an automated cell counter (MAXM, Beckman Coulter Inc.), and resuspended in modified
RPMI-1640 supplemented with 10% heat-inactivated foetal
bovine serum and 0.5% penicillin–streptomycin solution.
Different aliquots (each containing 2 · 106 lymphocytesÆmL)1) were prepared from each sample in 15 mL
sterile tubes. Increasing amounts of D,L-Hcy dissolved in
water were added, in duplicate, to the samples to obtain
final concentrations of 10, 100 and 1000 lM. Isolated
lymphocytes were then incubated for 24 h at 37 C in the
dark. In order to have positive controls for the apoptosis
study, subsets of lymphocytes were incubated with 10 lM
campthothecin, an apoptosis inducer, for 4 h at 37 C.
Further evaluation of the apoptotic process was carried out
on aliquots of lymphocytes incubated with D,L-Hcy at the
highest concentration (1000 lM).
After incubation, cell viability was verified using the
Trypan blue dye exclusion method and confirmed by
propidium iodide fluorescence microscopy (Axioskope-2,
Zeiss, Göttingen, Germany) [32].
Aliquots to be used for the assays of oxidative stress
markers and apoptosis regulatory proteins were pelleted
with a final centrifugation at 1000 g for 10 min and frozen,
after discarding the supernatant, at )80 C, until the assays
were carried out; the aliquots for the DNA analysis and
membrane permeability study underwent different treatments, according to the specific procedures (see below).
Assays
It has been reported that when thiol compounds, including
Hcy, are added to commonly used cell culture media, a
rapid, time-dependent loss of detectable –SH groups occurs,
thus reducing the biological effects of the compounds being
tested [33]. To assess the potential influence of such a
phenomenon, in a preliminary experiment we added 100 or
1000 lM Hcy to aliquots of the medium used in this study
(modified RPMI-1640, 10% heat-inactivated foetal bovine
serum, 0.5% penicillin–streptomycin solution). Aliquots
were incubated for increasing periods of time (up to 24 h)
and free reduced Hcy levels were determined spectrophotometrically using the traditional Ellman’s reagent (DTNB)
and measuring the absorbance at 412 nm.
For the determination of the oxidative stress markers and
apoptosis regulatory proteins, frozen lymphocyte pellets
were resuspended in 0.6 mL ice-cold NaCl/Pi and homogenized by ultrasound (Vibra Cell, Sonics & Materials, Inc.,
Danbury, CT, USA). Homogenates were then centrifuged
at 15 000 g, for 10 min at room temperature, and the
resulting supernatants were used for the biochemical assays.
Cu/Zn SOD and Bcl-2 were assayed using two commercially
available ELISA kits (Bender MedSystems Diagnostics
GmbH, Vienna, Austria) on a 550 Model Bio-Rad Microplate Reader (440 nm) (Bio-Rad Laboratories). Fluorometric microtitration was performed for the determination
of GSH levels (Chemicon International Inc.), caspase-3
(Molecular Probes Inc.) and caspase-9 (Oncogene) protease
activities, using a fluorimetric microplate reader SpectraMax
Gemini XS (Molecular Devices Co.). Lipid peroxidation
was evaluated using a colorimetric assay kit for the detection
of malondialdehyde and 4-hydroxyalkenals, which are
generated by the oxidation of polyunsaturated fatty acids
in cell-membrane phospholipids [34] (Calbiochem).
Apoptosis detection was carried out using the fluorescent
probe Yopro-1 iodide, which selectively binds to apoptotic
nuclei [32], and by measuring the formation of singlestranded (ss) DNA. For Yopro-1 labelling, the cell suspension was added directly with 5 lM of the fluorescent dye and
scanned by microtitration at 485/530 nm excitation/emission wavelengths [32]. For the ssDNA assay, lymphocytes
were fixed with methanol (80% methanol in NaCl/Pi) at
room temperature for 30 min and then transferred directly
to a microtiter plate, according to the ssDNA Apoptosis
ELISA Kit (Chemicon International Inc.); this procedure is
based on the selective, formamide-induced denaturation of
DNA, which identifies apoptotic cells [35,36] and subsequent staining of ssDNA, using a mixture of anti-ssDNA
mAb and peroxidase-conjugated antimouse IgM.
Plasma levels of total Hcy in the volunteers were measured
by HPLC with fluorometric detection, using a commercially
available kit (Bio-Rad). The HPLC system consisted of an
isocratic pump (Agilent 1100 series, Agilent Technologies,
FEBS 2004
Homocysteine and apoptosis in lymphocytes (Eur. J. Biochem. 271) 1673
Waldbronn, Germany) equipped with an analytical column
(70 · 3.2 mm i.d.) and a precolumn (microguardTM;
Bio-Rad). Excitation and emission wavelengths on the
fluorometric detector (Jasco Corp.) were 385 and 515 nm,
respectively. Data obtained from the detector were collected
and integrated by a dedicated personal computer, equipped
with a chromatography software package and an instrument
interface (CLINICAL DATA MANAGEMENT SYSTEM, Bio-Rad).
Table 3. Caspase-9 activity and Yopro-1 fluorescence, expressed as
arbitrary fluorescence units (AFU), in human isolated lymphocytes
incubated for 24 h in the presence of 1000 lM Hcy or 10 lM campthothecin (apoptosis inducer). ANOVA: Caspase-9 F ¼ 608.94,
P < 0.001; Yopro-1 F ¼ 11.62, P < 0.005. Fisher’s post hoc test vs.
untreated cells (mean ± SD): *P < 0.05 **P < 0.001.
D,L-Hcy
(lM)
0
1000
Campthothecin (10 lM)
Statistical analysis
One-way analysis of variance (ANOVA) and Fisher’s
post hoc test were used to evaluate the effect of increasing
concentrations of Hcy on the lymphocyte levels of the
variables considered in this study. The minimum level of
statistical significance was set at P < 0.05.
Results and Discussion
In this study, we assessed the direct influence of increasing
concentrations of Hcy on various mechanisms that subserve
cellular and apoptotic processes in isolated human lymphocytes. The mean (± SD) basal level of total plasma Hcy in
the subjects participating in the study was 6.8 ± 1.3 lM
(range, 5.6–8.9 lM). A preliminary experiment, conducted
to verify the time-dependent changes of free reduced Hcy
levels in the medium (RPMI), showed a progressive
decrease, peaking at 24 h ()90%), when the medium was
spiked with 100 lM Hcy; however, when 1000 lM Hcy was
added to the medium, only a moderate ()40%), late
reduction was found after incubation (Table 1). This
partially confirmed previous observations [33], but also
Table 1. Determination of free reduced Hcy levels (lM) in RPMI
aliquots spiked with 100 or 1000 lM Hcy and incubated for increasing
periods of time (up to 24 h).
Time
100 lM Hcy
1000 lM Hcy
0
1h
2h
4h
8h
12 h
24 h
100
53
49
30
24
15
10
1000
906
884
840
786
734
603
Caspase-9 (AFU)
Yopro-1 (AFU)
22462 ± 256.2
18972 ± 556.8
38670 ± 907.2**
114.4 ± 5.3
109.8 ± 17.6
141.4 ± 7.2*
excluded an early, significant loss of Hcy from the
medium, particularly when using the highest concentration
(1000 lM).
As shown in Table 2, the 24-h incubation of isolated
lymphocytes with increasing concentrations of Hcy (10,
100 and 1000 lM) did not induce statistically significant
changes in any of the variables considered. In fact, we
found that Hcy does not modify the lymphocyte levels of
GSH, while inducing modest, not significant increases in
the levels of Cu/Zn SOD or lipid peroxidation when
lymphocytes were incubated with 100 lM Hcy. Similarly,
a slight increase was observed in the activity of caspase-3,
a major effector of apoptosis, for Hcy concentrations of
10 lM, while intracellular levels of antiapoptotic protein
Bcl-2 were unchanged. We also failed to demonstrate any
significant Hcy-induced increase in the lymphocyte levels
of ssDNA, a specific biochemical marker of ongoing
apoptosis [36–38].
Analogously, incubation of lymphocytes with 1000 lM
Hcy did not cause significant modifications of Yopro-1
fluorescence or increases in caspase-9 activity (Table 3).
Our findings seem, therefore, to contradict the assumption that hyperhomocysteinemia exerts its cytotoxic effects
by interacting with the apoptotic regulatory mechanisms
and/or increasing cellular levels of oxidative stress, as
previously suggested by in vitro and in vivo studies
[16–21,39–42]. The reasons for this discrepancy are unclear.
One possible explanation is that most of the diseases
associated with hyperhomocysteinemia involve the cardiovascular system. In fact, it has recently been reported that
the endothelial and smooth muscle cells of the cardiovascuar system may have limited ability to process Hcy, due to
the low expression of cystathionine b-synthase [42], the key
Table 2. Mean (± SD) levels of Bcl-2, caspase-3 activity, Cu/Zn SOD, GSH, MDA + 4-HNE (lipid peroxidation) and ssDNA in human isolated
lymphocytes, incubated for 24 h in the presence of increasing concentrations of homocysteine. The last row shows changes in apoptosis-related markers
(mean ± SD) following incubation with 10 lM campthothecin (apoptosis inducer). ly, lymphocytes; AMC, 7-amino-4-methylcoumarin. ANOVA:
Caspase-3: F ¼ 23.9, P < 0.001; ssDNA: F ¼ 3.0, P < 0.05. Fisher’s post hoc test vs. untreated cells (mean ± SD): *P < 0.05 **P < 0.001.
D,L-Hcy
(lM)
0
10
100
1000
Campthothecin (10 lM)
Bcl-2
(UÆ106 ly)1)
Caspase-3
(pmol AMCÆ106 ly)1)
Cu/Zn SOD
(ngÆ106 ly)1)
GSH
(nmolÆ106 ly)1)
MDA + 4HNE
(pmolÆ106 ly)1)
ssDNA
(ngÆ106 ly)1)
14.6
15.3
15.3
15.7
879.2
974.4
893.3
814.4
1744.1
67.7
63.4
81.8
70.3
3.6
3.8
3.6
3.7
11.3
10.1
12.9
5.7
872.4
864.3
942.4
878.5
961.9
±
±
±
±
2.0
1.1
0.6
2.0
±
±
±
±
±
97.5
170.9
186.8
179.1
224.6**
±
±
±
±
11.4
8.0
32.5
12.9
±
±
±
±
0.4
0.5
0.6
0.6
±
±
±
±
7.0
1.3
4.8
1.5
±
±
±
±
±
94.1
37.0
51.1
58.7
18.7*
FEBS 2004
1674 A. Mangiagalli et al. (Eur. J. Biochem. 271)
enzyme in the catabolic transformation of Hcy into cysteine
[43]. The impairment of such an important Hcy removal
system may account for an increased susceptibility of
endothelial and smooth muscle cells, compared to lymphocytes. Zhang et al. have recently reported that, in isolated
murine splenic T lymphocytes, exposure to increasing Hcy
concentrations enhances the production of reactive oxygen
species induced by Concanavalin A, but, on the other hand,
reduces Concanavalin A-induced apoptosis [44]. In addition, Fenech [45] has reported that the micronucleus
frequency, an index of genetic damage, in human lymphocytes is positively correlated with plasma Hcy and inversely
correlated with plasma vitamin B12. Therefore, it may be
hypothesized that combination of increased Hcy levels with
other concurrent agents (reduced vitamin B12 and/or folate
levels, for example) is required for the intracellular damage
to occur, at least in lymphocytes.
It must also be noted that about 99% of circulating Hcy
exists in various oxidized forms, indistinguishable by most
Hcy assays. Therefore, the cytotoxic potential of Hcy may
be due to alternative forms of Hcy. However, although free
reduced Hcy represents the minor circulating component,
it may reliably be considered the most active form for the
vascular endothelial function in vivo [46]. In addition,
aminothiol species, including Hcy, comprise a dynamic
system referred to as redox thiol status, which is involved in
the extracellular antioxidant defence system. Hyperhomocysteinemia may cause changes in the redox thiol status and
imbalance between pro-oxidant and antioxidant process
[47,48]. On the other hand, the pro-oxidant potential of
homocysteine itself has been recently questioned. Zappacosta et al. [49], for example, have shown that the oxidative
catabolism of Hcy does not produce significant amounts of
H2O2, as previously reported by other authors [50–52] and
Sengupta et al. have further questioned the role of circulating Hcy as a substantial source of reactive oxygen species
[53,54]; moreover, Hcy proved able to counteract the effects
of powerful oxidizing species, such as hypochlorite, peroxynitrite and ferrylmyoglobin, in addition to counteracting, as
mentioned above, Concanavalin A-induced apoptosis [44].
This is not entirely surprising if one considers that thiolic
compounds, such as Hcy itself, are generally considered to
be powerful antioxidants [55]. Another recent study, which
confirmed the association between hyperhomocysteinemia
and coronary artery disease, showed also enhanced plasma
levels of malondialdehyde, an index of lipid peroxidation, in
these patients. The authors, however, failed to demonstrate
any direct correlation between Hcy and malondialdehyde
levels [56].
All of these considerations have led to the hypothesis that
hyperhomocysteinemia, rather than playing a causative
role, may simply be a marker for tissue damage or repair,
particularly at the cardiovascular level [57]. The Hcy
increase associated, for example, with coronary artery
disease may be therefore an epiphenomenon of the disease
itself [58,59].
In conclusion, the incubation of human, isolated
lymphocytes with increasing concentrations of Hcy did
not induce significant changes in the intracellular levels of
caspase-3 and 9, Bcl-2, Cu/Zn SOD, GSH or lipid
peroxides, or in the occurrence of apoptosis. These data
seem therefore to question the hypothesized influence of
Hcy, at least as an isolated agent, on the regulatory
mechanisms of oxidative stress and apoptotic cell death,
which have been suggested to underlie hyperhomocysteinemia-related citotoxicity.
Acknowledgements
This study was supported by grant ICS030.9 (Italian Ministry of
Health). The authors thank R. Fancellu for his assistance in data
analysis.
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