Tài liệu Báo cáo khoa học: Antioxidant defences in cybrids harboring mtDNA mutations associated with Leber’s hereditary optic neuropathy docx

Antioxidant defences in cybrids harboring mtDNA
mutations associated with Leber’s hereditary optic
neuropathy
Maura Floreani
1
, Eleonora Napoli
1,2
, Andrea Martinuzzi
2
, Giorgia Pantano
2
, Valentina De Riva
2
,
Roberta Trevisan
1,2
, Elena Bisetto
1,2
, Lucia Valente
1
, Valerio Carelli
3
and Federica Dabbeni-Sala
1
1 Department of Pharmacology and Anesthesiology, Pharmacology Section, University of Padova, Italy
2 ‘E. Medea’ Scientific Institute, Conegliano Research Centre, Conegliano, Italy
3 Department of Neurological Sciences, University of Bologna, Italy
Leber’s hereditary optic neuropathy (LHON), the first
disease to be linked with a maternally inherited
mtDNA point mutation [1], is a genetic form of retinal
ganglion cell degeneration leading to loss of central

vision and optic nerve atrophy occurring predomin-
antly in young males [2]. Three pathogenic mutations
at nucleotides 11778, 3460 and 14484, affecting,
respectively, ND4, ND1 and ND6 subunit genes of
complex I of the respiratory chain, are most often
associated with the disease, even though other rare
pathogenic mutations have been reported [2]. Com-
plex I dysfunction is postulated to underlie LHON
pathogenesis. However, the details of complex I dys-
function in LHON and its consequences on cellular
Keywords
antioxidant enzymes; galactose medium;
GSH and GSSG; Leber’s hereditary optic
neuropathy (LHON) cybrids; oxidative stress
Correspondence
A. Martinuzzi, ‘E. Medea’ Scientific Institute,
Conegliano Research Centre, via Costa Alta
37, 31015 Conegliano (TV), Italy
E-mail:
(Received 2 October 2004, revised 13
December 2004, accepted 22 December
2004)
doi:10.1111/j.1742-4658.2004.04542.x
Oxidative stress and imbalance between free radical generation and detoxi-
fication may play a pivotal role in the pathogenesis of Leber’s hereditary
optic neuropathy (LHON). Mitochondria, carrying the homoplasmic
11778 ⁄ ND4, 3460 ⁄ ND1 and 14484 ⁄ ND6 mtDNA point mutations associ-
ated with LHON, were used to generate osteosarcoma-derived cybrids.
Enhanced mitochondrial production of reactive oxygen species has recently
been demonstrated in these cybrids [Beretta S, Mattavelli L, Sala G, Trem-

olizzo L, Schapira AHV, Martinuzzi A, Carelli V & Ferrarese C (2004)
Brain 127, 2183–2192]. The aim of this study was to characterize the anti-
oxidant defences of these LHON-affected cells. The activities of glutathione
peroxidase (GPx), glutathione reductase (GR), superoxide dismutases
(SOD) and catalase, and the amounts of glutathione (GSH) and oxidized
glutathione (GSSG) were measured in cybrids cultured both in glucose-rich
medium and galactose-rich medium. The latter is known to cause oxidative
stress and to trigger apoptotic death in these cells. In spite of reduced SOD
activities in all LHON cybrids, and of low GPx and GR activities in cells
with the most severe 3460 ⁄ ND1 and 11778 ⁄ ND4 mutations, GSH and
GSSG content were not significantly modified in LHON cybrids cultured
in glucose medium. In contrast, in galactose, GSSG concentrations
increased significantly in all cells, indicating severe oxidative stress, whereas
GR and MnSOD activities further decreased in all LHON cybrids. These
data suggest that, in cells carrying LHON mutations, there is a decrease in
antioxidant defences, which is especially evident in cells with mutations
associated with the most severe clinical phenotype. This is magnified by
stressful conditions such as exposure to galactose.
Abbreviations
CuZnSOD, cupper zinc superoxide dismutase; DMEM, Dulbecco’s modified Eagle’s medium; GSH, glutathione; GSSG, oxidized glutathione;
GPx, GSH peroxidase; GR, GSSG reductase; LHON, Leber’s hereditary optic neuropathy; MnSOD, manganese superoxide dismutase; ROS,
reactive oxygen species; SOD, superoxide dismutase.
1124 FEBS Journal 272 (2005) 1124–1135 ª 2005 FEBS
function, with particular reference to the specific ret-
inal ganglion cell degeneration, are still poorly under-
stood.
The biochemical phenotype of complex I dysfunc-
tion in LHON has been investigated in various
patient-derived tissues (lymphocytes, platelets, muscle)
and cell lines (fibroblasts and lymphoblasts) (reviewed

in [3]). Spectrophotometrically assessed complex I spe-
cific activity was essentially slightly affected by the
11778 ⁄ ND4 mutation and not at all by the
14484 ⁄ ND6 mutation, but the 3460 ⁄ ND1 mutation
consistently lowered the enzyme activity [4–6].
A useful experimental approach for the study of the
cellular phenotype associated with the LHON muta-
tions is provided by the transmitochondrial cytoplasmic
hybrid (cybrid) cellular model [6–9]. Cybrids are
obtained by fusing a rho° cell line (host) completely
devoid of mtDNA with cytoplasts produced by enuclea-
tion of cells (donor) derived from patients or controls
[10]. In this way, the mtDNA of a donor cell can be
studied in the context of a ‘neutral’ nuclear background.
The current knowledge suggests that the 3460 ⁄ ND1
and 11778 ⁄ ND4 mutations consistently decrease com-
plex I-driven respiration, whereas the 14484 ⁄ ND6
mutation induces a milder defect [6]. On the other
hand, complex I-dependent ATP synthesis is severely
affected in cybrids with all three common LHON
mutations (reviewed in [11]). However, the limited clin-
ical expression of LHON suggests that the total ATP
cellular content is compensated in most tissues. Con-
currently, there is a potential for stably increased
production of reactive oxygen species (ROS) [2,3,12].
Indeed an excess of ROS production, in particular
mitochondrial superoxide anion, has been observed in
neuronal (NT2) cybrid cells carrying the 11778 ⁄ ND4
and 3460 ⁄ ND1 LHON mutations, after retinoic acid-
induced differentiation [13] and, more recently, in

osteosarcoma-derived cybrid cell lines carrying the
three pathogenic mutations 11778 ⁄ ND4, 3460 ⁄ ND1
and 14484 ⁄ ND6 [14].
Under such conditions, oxidative stress may become
the prevalent pathological consequence of complex I
dysfunction and trigger apoptotic cell death [15]. In
accordance with this view, two recent studies using
different models showed the convergent result that
LHON pathogenic mutations predispose cells to apop-
tosis [16,17].
As oxidative stress and imbalance between free rad-
ical generation and detoxification may play a pivotal
role in LHON pathogenesis, the aim of this study was
to investigate the level and efficiency of antioxidant
defences in cells carrying the most common LHON
mutations. Therefore, mitochondria carrying the
homoplasmic 11778 ⁄ ND4, 3460 ⁄ ND1 and 14484 ⁄ ND6
mtDNA point mutations were used to generate osteo-
sarcoma-derived cybrids. In these cells, in which we
observed different extents of reduced oxygen consump-
tion, we measured the basal content of glutathione
(GSH), oxidized glutathione (GSSG) and the activities
of the antioxidant enzymes glutathione peroxidase
(GPx; EC 1.11.1.9), glutathione reductase (GR; EC
1.8.1.7), superoxide dismutase (SOD; EC 1.15.1.1) and
catalase (EC 1.11.1.6). For comparison, we also carried
out all determinations on cybrids repopulated with
control mitochondria. Furthermore, we measured the
same parameters in the same cybrids subjected to glu-
cose deprivation and galactose replacement in the cul-

ture medium, an experimental condition shown to
accelerate apoptotic death in cells bearing LHON
mutations [17]. As under these conditions the cells are
forced to rely on mitochondrial respiratory chain for
their ATP production, glucose replacement with galac-
tose represents an ideal system for studying a response
to metabolic ⁄ oxidative stress in cells showing impaired
mitochondrial function.
Results
Oxygen consumption in control and LHON
cybrids
The results reported in Fig. 1 allow a comparison of
oxygen consumption between multiple controls and
Fig. 1. Oxygen consumption in individual cybrid cell lines. Assay
conditions are described in Experimental procedures. Values are
expressed as fmol oxygen consumedÆmin
)1
per cell, and are means
from two to seven different clones each assessed in at least three
independent experiments. Cell lines are grouped into controls and
11778 ⁄ ND4 (11778), 3460 ⁄ ND1 (3460) and 14484 ⁄ ND6 (14484)
LHON cybrids. The dotted line represents the mean of each group.
***P < 0.001.
M. Floreani et al. Antioxidant defences in LHON cybrids
FEBS Journal 272 (2005) 1124–1135 ª 2005 FEBS 1125
LHON cybrids. Grouping all control cybrids, and
grouping LHON cybrids by pathogenic mutation, it is
evident that mitochondrial respiration is significantly
decreased in LHON cybrids carrying the most severe
11778 ⁄ ND4 and 3460 ⁄ ND1 point mutation, the mean

reduction in respiration compared with the controls
being 29.3% (P<0.001) and 33.5% (P<0.001),
respectively. In contrast, in 14484 ⁄ ND6 affected cybrids
the reduction was only 8.9% [P ¼ nonsignificant (NS)],
confirming the milder phenotype of the 14484 ⁄ ND6
mutation. As no detectable differences were observed
among the data obtained from different clones of the
same cell line, or among data obtained in single controls
and in single cell lines affected by the same mtDNA
mutation, we decided to carry out the following
experiments in a single clone representative of each
independent line (HPC7, control; HFF3, 11778 ⁄ ND4;
HMM12, 3460 ⁄ ND1; HL180, 14484 ⁄ ND6).
Antioxidant defences in cybrids incubated
in glucose medium
The pattern of antioxidant defences was evaluated in
cybrids maintained in basal culture conditions, i.e. in
the presence of high glucose concentration (25 mm;
glu-) in the medium, which was Dulbecco’s modified
Eagle’s medium (DMEM). The data reported in
Table 1 clearly indicate that both GSH and GSSG
concentrations were similar in all cybrids tested. GSH
concentration in cells bearing 3460 ⁄ ND1 and
11778 ⁄ ND4 mutations tended to be lower than in
other cybrids, but the observed differences were not
statistically significant. As expected, in basal condi-
tions, all cells maintained a very high ratio between
reduced and oxidized glutathione, the percentage of
GSSG with respect to total glutathione (GSH +
GSSG) being about 0.4 to 0.5% in all cell lines.

In Fig. 2 the activities of the glutathione-related
enzymes, GPx and GR, are reported. Total GPx activ-
ity measured in cells with a 3460 ⁄ ND1 mutation was
less than half of the activity present in controls. In
contrast, there were no significant differences from the
controls in the enzymatic activities of cells with
14484 ⁄ ND6 or 11778 ⁄ ND4 mutations. GR activity in
cells carrying the 3460 ⁄ ND1 or the 11778 ⁄ ND4 muta-
tion was significantly lower than in controls or cybrids
carrying the 14484 ⁄ ND6 mutation. Also in this case,
the activity present in cells bearing the mild
14484 ⁄ ND6 mutation was not different from that in
control cybrids.
When we assessed the activity of catalase, we did
not find any significant difference among the various
cybrid lines (data not shown).
To complete the analysis of antioxidant enzyme
profile in cybrids maintained in glu-DMEM, cytosolic
CuZnSOD and mitochondrial MnSOD were meas-
ured. CuZnSOD and MnSOD protein in control and
LHON-affected cybrids was quantified by Western
blot using a specific antiserum (Fig. 3). Densitometric
Table 1. GSH and GSSG content (nmol per mg protein) in control
and LHON-affected cybrid cells cultured in glu-DMEM. The results
are means ± SD from four independent experiments carried out in
duplicate on two dishes for each experiment.
Cell line GSH GSSG
% GSSG ⁄
(GSH + GSSG)
Controls 38.90 ± 4.10 0.16 ± 0.04 0.409

14484 ⁄ ND4 mutants 40.88 ± 6.01 0.15 ± 0.04 0.365
3460 ⁄ ND1 mutants 34.16 ± 4.16 0.13 ± 0.01 0.379
11778 ⁄ ND6 mutants 35.29 ± 6.16 0.18 ± 0.12 0.513
Fig. 2. GPx and GR activities in control and LHON-affected cybrid
cells cultured in glucose-supplemented culture medium (glu-
DMEM). Culture and assay conditions are described in Experimen-
tal procedures. The results are means ± SD from four independent
experiments carried out in duplicate using two dishes for each
experiment. **P < 0.01; ***P < 0.001.
Antioxidant defences in LHON cybrids M. Floreani et al.
1126 FEBS Journal 272 (2005) 1124–1135 ª 2005 FEBS
analysis of the blots (Fig. 4A) shows a trend of
increase in CuZnSOD and MnSOD in LHON-affec-
ted cybrids with respect to controls, the difference
being significant (P<0.01) for CuZnSOD in cells
carrying the 11778 ⁄ ND4 mutation. However, the high
expression of SOD proteins in cybrids with LHON-
associated mutations did not correspond to higher
enzymatic activities. The results reported in Fig. 4B
in fact indicate that CuZnSOD activity (clear col-
umns) tended to be lower in all cybrid lines com-
pared with controls, whereas MnSOD activity (dotted
columns) was significantly (P<0.05) lower in cells
bearing the 14484 ⁄ ND6 mutation. When CuZnSOD
and MnSOD activities were normalized to the
respective protein amounts, assessed as densitometric
units, the activities of the enzymes were always lower
(P<0.05) in mutated cybrids than controls
(Fig. 4C).
Antioxidant defences in cybrids incubated

in galactose medium
GSSG concentrations were measured during a 24-h
time course experiment in cells cultured in gal-DMEM
(Fig. 5) and, for comparison, in glu-DMEM. In the
latter condition, GSSG did not change over time (data
not shown). In contrast, a marked time-dependent
increase in GSSG concentration was observed in cy-
brids cultured in galactose. Controls and 14484 ⁄ ND6
mutated cells showed similar behavior over time; after
10 to 16 h of incubation in gal-DMEM, GSSG con-
centrations had increased significantly (P<0.001)
Fig. 3. Western blotting analysis of content of CuZnSOD (16 kDa)
and MnSOD (25 kDa) proteins in control and LHON-affected cybrid
cells maintained in glucose-supplemented culture medium (glu-
DMEM). Tubulin (55 kDa) was used as a reference protein. The
blots depicted are representative of three separate experiments.
Fig. 4. CuZnSOD and MnSOD activities in control and LHON-affec-
ted cybrid cells cultured in glucose-supplemented culture medium
(glu-DMEM). (A) Densitometric quantification of CuZnSOD (unfilled
columns) and MnSOD (filled columns) in control and LHON-affected
cybrid cells. Results are means ± SD from three separate blots and
are expressed as arbitrary densitometric units normalized to tubulin.
(B) CuZnSOD (filled columns) and MnSOD (unfilled columns) activit-
ies were assayed as described by Oberley & Spitz [47], as des-
cribed in Experimental procedures. The activities are expressed in
UÆmg protein
)1
. The results are means ± SD from six independent
experiments carried out in duplicate using two dishes for each
experiment. (C) The activities of CuZnSOD (unfilled columns) and

MnSOD (filled columns) obtained in the cell lysates (40 lg protein)
were normalized to densitometric units calculated from the respect-
ive Western blot analysis carried out on the same amount of pro-
tein from the same cell lysate. The results are means ± SD from
three independent experiments. *P < 0.05; **P < 0.01.
M. Floreani et al. Antioxidant defences in LHON cybrids
FEBS Journal 272 (2005) 1124–1135 ª 2005 FEBS 1127
compared with the respective values observed in glu-
DMEM (data not shown). In contrast, in 3460 ⁄ ND1
mutated cells, GSSG had increased significantly
(P<0.05) after 6 h of treatment, and after 24 h the
GSSG concentration was about 30-fold higher than
that measured in glucose medium. Moreover, starting
at 6 h of incubation in galactose, these cells had signi-
ficantly (P<0.001) higher GSSG concentrations than
those measured at the same times in controls and
14484 ⁄ ND6 mutated cybrids. The increase in GSSG
concentration was even more marked in 11778 ⁄ ND4
affected cells; with respect to the concentrations found
in glucose-treated cells, the increase in GSSG began to
be significant (P<0.01) after 2 h of treatment and
peaked after 16 h, reaching a 45-fold increase. Between
2 and 16 h of the galactose challenge, GSSG concen-
trations in these cybrids were significantly (P<0.001)
higher than those measured at the same times in all
other cybrids. Compared with other mutant cybrids,
the GSSG concentration in cells with the 11778 ⁄ ND4
mutation tended to decrease after 16 h of treatment,
possibly indicating a severe cellular defect.
Cellular GSH did not decrease as a consequence of

glucose deprivation, but rather increased in some
cybrid lines (Fig. 5). In both controls and cells with the
14484 ⁄ ND6 mutation, GSH concentrations measured
after treatment with gal-DMEM were not significantly
different from those obtained in glu-DMEM (data not
shown), and no significant differences were observed at
any time in GSH concentrations between controls and
14484 ⁄ ND6 affected cybrids in gal-DMEM. In contrast,
GSH markedly increased in cells carrying the
3460 ⁄ ND1 and 11778 ⁄ ND4 mutations, which once
again showed similar behavior. A 12-h incubation in
galactose caused a significant (P<0.01 to 0.001)
increase in GSH concentration compared with the
values observed in glu-DMEM (data not shown).
The GSH concentration in both cell lines had doubled
after 16 to 24 h in gal-DMEM and was significantly
(P<0.01 to 0.001) higher than in controls
and 14484 ⁄ ND6 affected cells starting at 10 h of the
galactose challenge. However, in spite of this marked
increase in GSH concentration, the percentage of GSSG
with respect to total glutathione (GSSG + GSH)
in cybrids carrying the 3460 ⁄ ND1 and 11778 ⁄ ND4
mutations was significantly higher than that measured
in controls and cells with the 14484 ⁄ ND6 mutation
(Table 2), indicating a large imbalance in glutathione
homeostasis and conditions of extreme oxidative stress
in these cells.
Fig. 5. Time course of GSSG and GSH content in controls and
cybrids carrying the three primary LHON mutations incubated in
glucose-free ⁄ galactose-supplemented DMEM. Culture and assay

conditions are described in Experimental procedures. The results
are means ± SD from four independent experiments carried out in
duplicate using two dishes for each experiment. *P < 0.05;
**P < 0.01, ***P < 0.001.
Table 2. Percentage of GSSG vs. (GSH + GSSG) in control and
LHON-affected cybrid cells cultured in gal-DMEM for 6, 12 or 24 h.
The results, obtained from GSSG and GSH values reported in Fig. 5
are means ± SD from four independent experiments carried out in
duplicate on two dishes for each experiment.
Cell line
% GSSG ⁄ (GSH + GSSG)
6h 12h 24h
Controls 0.79 ± 0.04 1.96 ± 0.02 4.05 ± 0.02
14484 ⁄ ND4 mutants 0.63 ± 0.04 1.71 ± 0.02 4.15 ± 0.04
3460 ⁄ ND1 mutants 1.11 ± 0.01
a
2.17 ± 0.02
a
5.33 ± 0.05
a
11778 ⁄ ND6 mutants 6.08 ± 0.06
a
9.68 ± 0.10
a
5.82 ± 0.05
a
a
Significant difference from respective control value: P < 0.05.
Antioxidant defences in LHON cybrids M. Floreani et al.
1128 FEBS Journal 272 (2005) 1124–1135 ª 2005 FEBS

When we measured the activities of the antioxidant
enzymes in cybrids maintained for 24 h in galactose
medium, we found that GPx and catalase activities
were not different from those in basal conditions (data
not shown). In contrast, in some cybrid lines, GR and
SOD activities were significantly affected by glucose
deprivation. The results reported in Fig. 6 indicate that
the cells carrying the 3460 ⁄ ND1 or 11778 ⁄ ND4 muta-
tion had significantly lower GR activity than controls
and cybrids with the 14484 ⁄ ND6 mutation. Compar-
ison of these results with those reported in Fig. 2
shows that glucose deprivation caused a further
marked decrease in the already low GR activity pre-
sent in the 11778 ⁄ ND4 mutated cells, whereas the
trend of GR activity in the other cell lines was similar
to that observed when the cells were incubated in nor-
mal glucose medium.
Incubation in galactose caused a dramatic decrease
in SOD activity, particularly mitochondrial MnSOD,
in all LHON cybrids (Fig. 7). This decrease ranged
from 37% in the cells with the 14484 ⁄ ND6 mutation
to 68% in those with the 11778 ⁄ ND4 mutation, com-
pared with the controls. Comparison of the results
reported in Fig. 7 with the data in Fig. 4B clearly indi-
cates that MnSOD activity in control cells was not
affected by the incubation in gal-DMEM.
CuZn-SOD activity was less affected by glucose
deprivation. In gal-DMEM, only the 3460 ⁄ ND1
mutated cells had CuZn-SOD activity significantly dif-
ferent from that in controls, whereas in all other lines

it was similar to that observed in glu-DMEM (for
comparison, see Fig. 4B).
Discussion
A complex I-driven chronic increase in oxidative stress
has been suggested to be a relevant contributory factor
to retinal ganglion cell death and optic atrophy in
LHON [2,3,12]. The present results indicate that osteo-
sarcoma-derived cybrids carrying the three most com-
mon LHON pathogenic mutations in complex I subunit
genes show a partial respiratory defect, assessed as a
decrease in oxygen consumption, closely related to the
severity of the clinical spectrum of the disease [2,3]. In
fact, a 29 to 34% decrease in cell respiration is observed
in cells bearing the 11778 ⁄ ND4 and 3460 ⁄ ND1 muta-
tions, whereas a lower ( 9%) decrease in oxygen con-
sumption is present in cybrids with the 14484 ⁄ ND6
point mutation, compatible with the milder clinical phe-
notype [6]. In the same cybrids, a significant increase in
ROS production has recently been reported [14]; in par-
ticular, the highest ROS production was measured in
cybrids bearing the 3460 ⁄ ND1 mutation, followed by
11778 ⁄ ND4 and 14484 ⁄ ND6 mutations. In this study,
we also show, for the first time, that in these LHON-
affected cells there is low efficiency of the antioxidant
machinery, the 11778 ⁄ ND4 and 3460 ⁄ ND1 mutations
expressing clearly the most severe phenotype. The great-
est vulnerability of these cells to metabolic ⁄ oxidative
stress is magnified by glucose deprivation and galactose
replacement.
Antioxidant defences in cybrids cultured

in glu-DMEM
We first assessed the antioxidant defences of LHON
cybrids in glucose-supplemented medium (glu-DMEM),
Fig. 6. GR activity in control and LHON-affected cybrid cells cul-
tured for 24 h in glucose-free ⁄ galactose-supplemented culture
medium (gal-DMEM). Culture and assay conditions are described in
Experimental procedures. The results are means ± SD from four
independent experiments carried out in duplicate using two dishes
for each experiment. *P < 0.05; ***P<0.001.
a
P < 0.05, Signifi-
cant differences from values of cybrid cells bearing the 3460 or
11778 mutation.
Fig. 7. CuZnSOD (unfilled columns) and MnSOD activities (filled
columns) in control and LHON-affected cybrid cells cultured for
24 h in glucose-free ⁄ galactose-supplemented culture medium (gal-
DMEM).The activities are expressed in UÆmg protein
)1
,asdes-
cribed by Oberley & Spitz [47]. The results are means ± SD from
four independent experiments carried out in duplicate using two
dishes for each experiment. **P<0.01; ***P<0.001.
M. Floreani et al. Antioxidant defences in LHON cybrids
FEBS Journal 272 (2005) 1124–1135 ª 2005 FEBS 1129
a condition in which cells derive their energy mainly
from anaerobic glycolysis [18] and ATP production is
ensured even in the presence of mitochondrial dysfunc-
tion [19,20]. Under these culture conditions, LHON
cybrids grew normally, as previously reported [7,17].
However, some alterations in their antioxidant machin-

ery emerged, more clearly in cybrids carrying the
3460 ⁄ ND1 and 11778 ⁄ ND4 mutations in which GPx
and GR activities were significantly reduced. As GPx
and GR are proteins encoded by the nuclear genome of
the parental 143B.TK
–
cells, which is constant among
the cybrid cell lines compared in this study, the low
GPx and GR activities in 3460 ⁄ ND1 and 11778 ⁄ ND4
mutated cybrids may be ascribed to post-translational
events. It is well known that GR [21,22] and GPx [23]
activities are significantly decreased in the presence of
ROS or in a condition of drug-induced ROS genera-
tion. As increased generation of ROS has been
observed in the glu-DMEM cultured cybrids, partic-
ularly 3460 ⁄ ND1 and 11778 ⁄ ND4 mutated cells [14],
we can hypothesize that the decrease in GPx and GR
activities displayed by these mutants may reflect the
higher ROS concentrations. On the other hand, signifi-
cant increases in ROS production have also been
observed in NT2 neuronal-like cybrids carrying the
11778 ⁄ ND4 and 3460 ⁄ ND1 mutations [13] and in
human–ape xenomitochondrial cybrids partially defici-
ent in complex I [15]. A ‘chronic’ oxidative insult may
also explain the apparent contradiction of SOD results
in LHON cybrids. As shown by Western blot analysis,
the expression of CuZnSOD and MnSOD proteins
seems to be slightly increased in LHON cybrids
compared with controls, whereas the enzyme activities
are lower. Both MnSOD and CuZnSOD proteins are

possibly upregulated as a compensatory mechanism,
but may be partially inactivated by oxidative damage.
Complex I impairment in a variety of human diseases
has been previously reported in association with
MnSOD induction, although not all patients defective
in complex I displayed an increase in MnSOD activity
[24]. Furthermore, not all tissues have the same ability
to upregulate MnSOD, as suggested by the different
behavior of skeletal and cardiac muscle observed in the
ANT1-knockout animal model of oxidative stress [25].
Moreover, MnSOD and CuZnSOD expression increa-
ses several fold in Saccharomyces cerevisiae during
menadione-induced oxidative stress, without a parallel
increase in their activities [26]. CuZnSOD inactivation
has been ascribed to hydrogen peroxides [27], whereas
inactivation of MnSOD relates to peroxynitrite species,
generated by the interaction of superoxide anion
with nitric oxide [28], which can nitrate critical
tyrosine residues causing loss of enzyme activity [29].
Experiments are currently in progress in our laboratory
to evaluate protein nitrosylation in control and
mutated cybrids cultured in glu-DMEM.
Overall, our results indicate that, in spite of low effi-
ciency in some antioxidant enzymes, particularly evi-
dent in cells affected by the most severe LHON
pathogenic mutations, the cybrids maintained in glu-
cose-supplemented medium successfully manage the
increased generation of ROS. This is indicated by
GSH and GSSG concentrations, which are not signifi-
cantly modified in comparison with those of controls,

and by the normal cell growth [17].
Antioxidant defences in cybrids cultured
in gal-DMEM
When the cells were cultured in glucose-free ⁄ galactose-
supplemented medium, the situation dramatically
changed. The replacement of glucose by galactose in
the culture medium (gal-DMEM) forces the cells to
rely on oxidative phosphorylation for ATP production,
which in the case of LHON cybrids is severely
impaired when driven by complex I substrates [30].
Galactose medium induces a dramatic time-dependent
depletion of cellular ATP content [31] and a wave of
apoptotic cell death [17]. As peroxide scavenging via
pyruvate as well as via NADPH-dependent reactions is
decreased in these conditions, because the restricted
flow of galactose to glucose 6-phosphate decreases
NADPH availability [20,32], incubation in galactose is
expected to induce a metabolic ⁄ oxidative stress crisis,
which aggravates the pathogenicity of LHON muta-
tions. In fact, LHON cybrids incubated in gal-DMEM
show dramatic modifications in their antioxidant
defences. All LHON cybrids had GR activities that
were significantly lower than in control cybrids, with a
particularly large decrease in the 11778 ⁄ ND1 mutated
cells. Large modifications in glutathione homeostasis
were also evident in cybrids carrying 11778 ⁄ ND4 or
3460 ⁄ ND1, as reflected by their earlier and more
marked increases in GSSG concentration compared
with the control and 14484 ⁄ ND6 mutated cybrids.
This accumulation of GSSG is the likely result of

increased ROS production, when this exceeds the
metabolic capabilities of GPx and GR. A burst of oxi-
dative stress resulting in enhanced formation of per-
oxynitrite [29] in LHON cybrids may also explain the
significant decrease in MnSOD activity observed after
a 24-h incubation in gal-DMEM.
In cybrids bearing the most severe 3460 ⁄ ND1 and
11778 ⁄ ND4 mutations, we found a time-dependent,
large increase in GSH. This is not surprising, as very
similar cellular behavior, i.e. concurrent increase in
Antioxidant defences in LHON cybrids M. Floreani et al.
1130 FEBS Journal 272 (2005) 1124–1135 ª 2005 FEBS
GSSG and GSH, has been observed in multidrug-resist-
ant human breast carcinoma cells treated with a glu-
cose-free medium [33]. This indicates that the cells in
galactose medium increase glutathione synthesis, in an
attempt to counteract the increased production of intra-
cellular pro-oxidants. As GR activity is greatly impaired
in 3460 ⁄ ND1 and 11778 ⁄ ND4 mutated cybrids, and
NADPH availability is decreased in gal-DMEM, the
GSH increase observed in these cells may be due to
de novo GSH synthesis, a two-step process catalyzed by
c-glutamylcysteine synthetase and GSH synthetase [34].
We did not directly measure c-glutamylcysteine syn-
thetase expression and ⁄ or activity in cybrids in our
experimental conditions. However, our hypothesis is
supported by a study reporting that rat lung epithelial
L2 cells respond to menadione-induced oxidative stress,
with a 2.5-fold increase in their GSH content obtained
through increased c-glutamylcysteine synthetase activity

[35] and increased transcription of the regulatory sub-
unit of c-glutamylcysteine synthetase itself [36]. In spite
of the increase in GSH, however, the cybrids harboring
the most severe 3460 ⁄ ND1 and 11778 ⁄ ND4 mtDNA
mutations failed to maintain the percentage of GSSG
vs. (GSH + GSSG) at values similar to those of the
controls or the cybrids with the 14484 ⁄ ND6 mutation,
indicating a situation of greater cellular distress. This
imbalance in glutathione homeostasis may be closely
connected with the apoptotic death of LHON cybrids
grown in galactose medium [17]. It is well known that
the redox state of thiols regulates the mitochondrial
permeability transition pore [37], its opening being
responsible for energy uncoupling, diminished intracel-
lular ATP concentrations, and release of cytochrome
c and other pro-apoptotic factors.
Conclusions
Our study shows that cybrids carrying the three most
common mtDNA point mutations associated with
LHON show evidence of low efficiency of some of the
antioxidant enzymes, probably because of post-transla-
tional events. The extent of this phenomenon seems to
be related to the severity of the biochemical defect
associated with the LHON mutation and possibly to
the amount of ROS generated by mitochondria, corre-
lating also with the clinical phenotype of the disease.
Thus, the 14484 ⁄ ND6 mutation, which is associated
with a benign visual prognosis and normal complex I
activity [5] and no significant decrease in oxygen con-
sumption (present data), has the lowest ROS produc-

tion [14] and the mildest impairment in antioxidant
activity (present results). In contrast, the 3460 ⁄ ND1
and the 11778 ⁄ ND4 mutations, which consistently
decrease complex I activity [3,6] and oxygen consump-
tion (present data) and are associated with severe
neuropathology [38], are associated with the highest
mitochondrial ROS production [14] and the least effi-
cient antioxidant defences (present results). However,
while in glucose medium, all LHON-affected cybrids
retain the ability to buffer their dysfunctions, as shown
by the lack of modification in glutathione homeostasis
and by their normal growth. However, the modifica-
tions described in the antioxidant machinery result in
greater vulnerability of the cells to metabolic ⁄ oxidative
stress, as seen in galactose medium. Under these condi-
tions, a further decrease in some antioxidant activities
(MnSOD and GR) and drastic impairment of glutathi-
one homeostasis occur, particularly in cybrids with
3460 ⁄ ND1 and 11778 ⁄ ND4 mutations. Taken together
these results provide further insight into the patho-
physiological consequences of LHON mutations, indi-
cating that the unstable balance in which LHON cells
live may be upset by any exogenous or endogenous
condition (e.g. exposure to drugs and ⁄ or toxins, or
changing hormonal status) that favors ROS genera-
tion. This may precipitate the cells into a metabolic
crisis which in vivo may lead to apoptotic death of ret-
inal ganglion cells and clinically expressed LHON.
Experimental procedures
Materials

Tissue culture reagents were purchased from Gibco-Invitro-
gen (Milan, Italy). Cumene hydroperoxide, NADPH, GSH,
GSSG, xanthine, xanthine oxidase (from buttermilk), gluta-
thione reductase (from baker’s yeast), nitroblue tetrazolium,
2-vinylpyridine and albumin were obtained from Sigma
Chemical Co. (St Louis, MO, USA). NaCl ⁄ P
i
from Oxoid
had the following composition: NaCl 8 gÆ L
)1
, KCl
0.2 gÆL
)1
,Na
2
HPO
4
1.15 gÆL
)1
and KH
2
PO
4
0.2 gÆL
)1
(pH 7.3). All other reagents were of analytical grade and
were used as received.
Construction and characterization of cybrid cell
lines
Cybrid cell lines were constructed from fibroblasts obtained,

after informed consent, from skin biopsies of six members
of five unrelated families with clinically and molecularly
defined LHON, previously reported [4,5], and from five
unrelated controls. Cells, grown in DMEM supplemented
with 10% fetal calf serum, were then enucleated and fused
with osteosarcoma-derived mtDNA-less cells (rho°206
derived from 143B.TK
–
, a gift from G. Attardi) as described
[10]. After selection in uridine-deprived bromodeoxyuridine-
M. Floreani et al. Antioxidant defences in LHON cybrids
FEBS Journal 272 (2005) 1124–1135 ª 2005 FEBS 1131
supplemented medium, several cybrid clones (a minimum of
two for each individual) repopulated with mtDNA from
each fibroblast cell line were isolated and expanded. More-
over, to avoid possible confounding effects due to different
functional profiles of different batches of 143B.Tk
–
, and to
indirectly check the unstable nuclear genome of the 143B
cells, we measured the respiratory capacity of 143B cells fro-
zen just after being brought from the donor laboratory
(1993) or after various periods of time in culture (frozen in
1996, 1999, 2002). The observed values were all within the
range of experimental variation (± 10%).
The cybrids have been regularly checked for the presence
of the LHON pathogenic mutations 3460 ⁄ ND1, 11778 ⁄ ND4
and 14484 ⁄ ND6 following the standard PCR ⁄ restriction
fragment length polymorphism method [16,17]. All LHON
cybrids used carried stably homoplasmic mtDNA mutations,

reconfirmed every 3 to 5 months.
Moreover, a functional check of the cybrids was carried
out by determination of oxygen consumption, as described
previously [39]. Briefly, the rate of oxygen consumption was
measured in intact cells with a Gilson 5 ⁄ 6 oxygraph on
samples of (4–5) · 10
6
cells in 1.85 mL DMEM lacking glu-
cose supplemented with 5% dialyzed fetal calf serum at
37 °C. By this method, we tested five control lines, three
lines with the 11778 ⁄ ND4 mutation, two lines with
3460 ⁄ ND1 and two with 14484 ⁄ ND6 mutations. Each line
is the result of a cybridization from a different individual,
and each bar shown in the graph of Fig. 1 is the result of
averaging data obtained from two to seven different clones
each assessed in at least three independent experiments.
A representative clone of each independent line (HPC7:
control; HFF3: 11778 ⁄ ND4; HMM12: 3460 ⁄ ND1; HL180:
14484 ⁄ ND6) was used for the extensive study detailed in
the following sections.
Culture conditions
Cybrid cell lines were grown in DMEM supplemented with
10% fetal calf serum, 2 mml-glutamine, 100 UÆmL
)1
penicil-
lin, 100 lgÆmL
)1
streptomycin and 0.1 mgÆmL
)1
bromode-

oxyuridine. For the experiments, the cells ( 1.8 · 10
6
) were
plated on 10-cm Petri dishes in 8 mL of the above reported
DMEM and maintained at 37 °C in an incubator under a
humidified 5% CO
2
atmosphere. After 24 h, the culture
medium was replaced with fresh complete DMEM supple-
mented with 25 mm glucose (glu-DMEM) or with glucose-
free DMEM supplemented with 5 mm galactose, 5 mm
sodium pyruvate and 5% fetal calf serum (gal-DMEM), as
described by Ghelli et al. [17]. The cells were incubated at
37 °C for 24 more hours.
Measurement of GSH and GSSG
Cellular GSH and GSSG concentrations were measured
enzymatically [40], as described previously [41]. Briefly, the
assay is based on the determination of a chromophoric
product, 2-nitro-5-thiobenzoic acid, resulting from the reac-
tion of 5,5¢-dithiobis-(2-nitrobenzoic acid) with GSH. In
this reaction, GSH is oxidized to GSSG, which is then
reconverted into GSH in the presence of glutathione reduc-
tase and NADPH. The rate of 2-nitro-5-thiobenzoic acid
formation is measured spectrophotometrically at 412 nm.
The cells [ (5–6) · 10
6
for each determination] were
washed once with NaCl ⁄ P
i
and then treated with 6% meta-

phosphoric acid (1 mL per dish) at room temperature.
After 10 min, the acid extract was collected, centrifuged for
5 min at 18000 g at 4 °C, and processed. The cellular debris
remaining on the plate was solubilized with 0.5 m KOH
and assayed for protein content as described by Lowry
et al. [42]. For total glutathione determination, the above
acid extract was diluted (1 : 6) in 6% metaphosphoric acid;
thereafter to 0.1 mL supernatant were added 0.75 mL 0.1 m
potassium phosphate ⁄ 5mm EDTA buffer (pH 7.4),
0.05 mL 10 mm 5,5¢-dithiobis-(2-nitrobenzoic acid) (pre-
pared in 0.1 m phosphate buffer) and 0.08 mL 5 mm
NADPH. After a 3-min equilibration period at 25 °C, the
reaction was started by the addition of 2 U glutathione
reductase (type III; Sigma; from bakers yeast; diluted in
0.1 m phosphate ⁄ EDTA buffer). Product formation was
recorded continuously at 412 nm (for 3 min at 25 °C) with
a Shimadzu UV-160 spectrophotometer. The total amount
of GSH in the samples was determined from a standard
curve obtained by plotting known amounts (0.05 to
0.4 lgÆ mL
)1
) of GSH against the rate of change in A
412
.
GSH standards were prepared daily in 6% metaphosphoric
acid and diluted in phosphate ⁄ EDTA buffer (pH 7.4).
For GSSG measurement, soon after preparation, the
supernatant of acid extract was treated for derivatization
with 2-vinylpyridine at room temperature for 60 min. In a
typical experiment, 0.15 mL supernatant was treated with

3 lL undiluted 2-vinylpyridine. Then 9 lL triethanolamine
was added. The mixture was vigorously mixed, and the pH
was checked; it was generally between 6 and 7. After
60 min, 0.1 mL aliquots of the samples were assayed by the
procedure described above for total GSH measurement.
The amount of GSSG was quantified from a standard
curve obtained by plotting known amounts of GSSG (0.05
to 0.20 lgÆmL
)1
) against the rate of change in absorbance.
GSH present in the samples was calculated as the differ-
ence between total glutathione and GSSG concentrations.
Assay of antioxidant enzyme activities
For measurement of GPx, GR and catalase activities,
monolayer cells [ (2–3) · 10
6
] were washed three times
with NaCl ⁄ P
i
before treatment directly on the dish with a
solution consisting of 0.25 m sucrose, 10 mm Tris ⁄ HCl
(pH 7.5), 1 mm EDTA, 0.5 mm phenylmethanesulfonyl
fluoride, 0.5 mm 1,4-dithio-dl-threitol and 0.1% (v ⁄ v) Non-
idet (solution A), to obtain complete lysis of intracellular
Antioxidant defences in LHON cybrids M. Floreani et al.
1132 FEBS Journal 272 (2005) 1124–1135 ª 2005 FEBS
organelles. Cells were then scraped from the plate, and the
samples were centrifuged for 30 min at 105 000 g. Protein
measurements [42] and enzyme assays were carried out on
the clear supernatant fractions.

Total GPx activity was measured by the coupled enzyme
procedure with glutathione reductase, as described by Proh-
aska & Ganther [43], using cumene hydroperoxide as sub-
strate. Enzyme activity was monitored by following the
disappearance of NADPH at 340 nm for 3 min at 25 °C.
The incubation medium (final volume 1 mL) had the follow-
ing composition: 50 mm KH
2
PO
4
(pH 7.0), 3 mm EDTA,
1mm KCN, 1 mm GSH, 0.1 mm NADPH, 2 U glutathione
reductase and  300 lg protein. After a 3-min equilibration
period at 25 °C, the reaction was started by the addition of
0.1 mm cumene hydroperoxide dissolved in ethanol. The
specific activity was calculated by using a molar absorption
coefficient obtained from a standard curve of NADPH (0.02
to 0.1 l mol Æ mL
)1
), and GPx activity was expressed in nmol
NADPH consumed per mg proteinÆmin
)1
.
GR activity was measured by the method of Carlberg &
Mannervik [44], by following the rate of oxidation of
NADPH by GSSG at 340 nm for 3 min at 25 °C. The reac-
tion mixture (final volume 1 mL) contained: 0.1 m KH
2
PO
4

(pH 7.6), 0.5 mm EDTA, 1 mm GSSG, 0.1 mm NADPH,
and  300 lg protein. The specific activity was calculated
by using a molar absorption coefficient obtained from a
standard curve of NADPH (0.02 to 0.1 lmolÆmL
)1
), and
GR activity was expressed in nmol NADPH consumed per
mg proteinÆmin
)1
.
Total catalase activity was assayed by the method of
Aebi [45]. Activity was measured by monitoring, for 30 s at
25 °C, the decomposition of 10 mm H
2
O
2
at 240 nm in a
medium (final volume 1 mL) consisting of 50 mm phos-
phate buffer (pH 7.0) and  100 lg protein. Catalase activ-
ity was expressed as U per mg protein, assuming that 1 U
catalase decomposes 1 lmol H
2
O
2
Æmin
)1
.
For MnSOD and CuZnSOD assays, after treatment of
2 · 10
6

cells with solution A, an aliquot (0.6 mL) of cell
lysate was sonicated, on ice (2 · 30 s bursts) with a Labsonic
U2000 sonicator (B. Braun Biotech International, Melsun-
gen, Germany) and then centrifuged for 30 min at 105 000 g
as described by Siemankowski et al. [46]. The supernatant
was collected and dialyzed overnight in cold double-distilled
water to remove small interfering substances. Enzyme assays
were carried out by the method of Oberley & Spitz [47], with
minor modifications. Briefly, in 1 mL medium consisting of
50 mm KH
2
PO
4
(pH 7.8) and 0.1 mm EDTA, a superoxide-
generating system (0.15 mm xanthine plus 0.02 U xanthine
oxidase) was used together with 50 lm nitroblue tetrazolium
to monitor superoxide formation by following the changes in
colorimetric absorbance at 560 nm for 5 min at 25 °C. The
catalytic activities of the samples were evaluated as their abil-
ity to inhibit the rate of nitroblue tetrazolium reduction;
increasing amounts of protein (5 to 150 lg) were added
to each sample until maximum inhibition was obtained.
SOD activity was expressed as U per mg protein, 1 U SOD
activity being defined as the amount of protein causing
half-maximal inhibition of the rate of nitroblue tetrazolium
reduction. To assess MnSOD activity, cell fractions were
preincubated for 60 min at 0 °C in the presence of 5 m m
KCN, which produces total inhibition of Cu ⁄ ZnSOD. The
latter activity was calculated as the difference between activit-
ies in the absence and presence of KCN.

Western blot assay of CuZnSOD and MnSOD
On the same cybrid cell lysates, obtained as reported above
and used for SOD activity assays, Western blotting was car-
ried as described by Dieterich et al. [48]. Equal amounts of
protein (40 lg per lane) denatured in electrophoresis buffer
containing 100 mm Tris ⁄ HCl (pH 6.8), 8 mm dithiothreitol,
2% SDS, 2% glycerol, and 0.05% bromophenol blue at
95 °C were electrophoresed on SDS ⁄ polyacrylamide gel
(10% gel) and electrotransferred to a poly(vinylidene difluo-
ride) membrane (Bio-Rad). The membrane was blocked
with 5% nonfat dry milk in 0.02 m Tris ⁄ HCl buffer, pH 7.5,
containing 0.137 m NaCl and 0.1% (v ⁄ v) Tween 20 (TBST)
for 1 h at room temperature and then incubated with sheep
anti-(human CuZnSOD) IgG (Calbiochem, Darmstadt, Ger-
many) diluted 1 : 1000, anti-(human MnSOD) IgG diluted
1 : 10 000 (gift from John Guy, University of Florida), or
anti-(human tubulin) IgG (Sigma) diluted 1 : 1000. After
being washed with NaCl ⁄ P
i
⁄ 0.1%Tween, the blots were
incubated with horseradish peroxidase-conjugated anti-
sheep IgG (Calbiochem) diluted 1 : 10 000 at room tem-
perature for 1 h. After a final wash of the membrane with
TBST, the immunoreactivity was detected with enhanced
chemiluminescence detection solution (Sigma). The immuno-
reactivity was detected in the same way as described above.
Quantitative analysis of the blots was carried out using the
image j software (http//rsb.info.nih.gov ⁄ ij ⁄ ).
Statistical analysis
Results are expressed as arithmetic means ± SD. Compari-

sons were made by one-way analysis of variance. A P value
of less than 0.05 was considered significant.
Acknowledgements
This work was supported, in part, by grants from
MIUR (Italy). The financial support of Telethon Italy
(project No. GGP02323 to VC and AM) is gratefully
acknowledged.
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