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

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

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-

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.

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

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

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

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

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

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

[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-

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,

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-

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

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

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.

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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