Uridine supplementation antagonizes zalcitabine (DDC)-induced microvesicular ste

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Uridine supplementation antagonizes zalcitabine (DDC)-induced

microvesicular steatohepatitis in mice

Hepatology Jan 2007

Dirk Lebrecht 1, Yetlanezi A. Vargas-Infante 1, Bernhard Setzer 1,

Janbernd Kirschner 2, Ulrich A. 1 *

1Department of Rheumatology and Clinical Immunology, Medizinische

Universitätsklinik, Freiburg, Germany

2Department of Pediatrics, Medizinische Universitätsklinik,

Freiburg, Germany

“….Our study suggests that the dietary supplementation of uridine

attenuates the mitochondrial hepatotoxicity of zalcitabine in accord

with prior in vitro data. The mechanism of the beneficial effect of

uridine is not fully delineated…. HCV-infection (and HCV genotype I

in particular) has also been found to modestly decrease hepatic mtDNA.

[40][41]…. The effects of uridine on HCV related mitochondrial

dysfunction have however not been examined…. The clinical

experience of uridine supplementation for hepatotoxicity is very

limited but promising. In one HIV patient uridine supplementation was

described to reverse mitochondrial steatohepatitis despite continued

long-term antiretroviral treatment with stavudine.[42] Using a non-

invasive 13C-methionine breath test in HIV-positive individuals under

treatment with stavudine or zidovudine, a three day course of

Mitocnol was recently found to reproducibly enhance the function of

hepatic mitochondria over a period of 4 weeks.[43]….â€

Abstract

Zalcitabine is an antiretroviral nucleoside analogue that exhibits

long-term toxicity to hepatocytes by interfering with the replication

of mitochondrial DNA (mtDNA). Uridine antagonizes this effect in

vitro. In the present study we investigate the mechanisms of

zalcitabine-induced hepatotoxicity in mice and explore therapeutic

outcomes with oral uridine supplementation.

BalbC mice (7 weeks of age, 9 mice in each group) were fed 0.36

mg/kg/d of zalcitabine (corresponding to human dosing adapted for

body surface), or 13 mg/kg/d of zalcitabine. Both zalcitabine groups

were treated with or without Mitocnol (0.34 g/kg/d), a dietary

supplement with high bioavailability of uridine. Liver histology and

mitochondrial functions were assessed after 15 weeks. One mouse

exposed to high dose zalcitabine died at 19 weeks of age. Zalcitabine

induced a dose dependent microvesicular steatohepatitis with abundant

mitochondria. The organelles were enlarged and contained disrupted

cristae. Terminal transferase dUTP nick end labeling (TUNEL) assays

showed frequent hepatocyte apoptosis. mtDNA was depleted in liver

tissue, cytochrome c-oxidase but not succinate dehydrogenase

activities were decreased, superoxide and malondialdehyde were

elevated. The expression of COX I, an mtDNA-encoded respiratory chain

subunit was reduced, whereas COX IV, a nucleus-encoded subunit was

preserved.

Uridine supplementation normalized or attenuated all toxic

abnormalities in both zalcitabine groups, but had no effects when

given without zalcitabine. Uridine supplementation was without

apparent side effects.

“….Uridine had no intrinsic effect on mtDNA biogenesis, but when

administered with zalcitabine attenuated and in the case of low dose

zalcitabine fully abrogated mtDNA depletion…â€

Conclusion: Zalcitabine induces mtDNA-depletion in murine liver with

consequent respiratory chain dysfunction, up-regulated synthesis of

reactive oxygen species and microvesicular steatohepatitis. Uridine

supplementation attenuates this mitochondrial hepatotoxicity without

apparent intrinsic effects.

Discussion

Our investigations establish a new model of nucleoside analogue

mediated mitochondrial hepatotoxicity, provide insight into its

pathogenesis and suggest beneficial effects of uridine

supplementation.

In this model, zalcitabine induced a dose dependent mtDNA-depletion

and specifically impaired mtDNA-encoded respiratory chain functions,

in keeping with its inhibitory activity on polymerase-gamma.

Impairment of oxidative phosphorylation inhibits beta oxidation and

leads to an intracellular increase of triglycerides and non-

esterified fatty acids, the cause of microvesicular steatosis.[26] In

vitro investigations have suggested that residual wild-type mtDNA-

levels in the order of 20% protect from respiratory chain dysfunction.

[27][28] We have however not found support for this mtDNA threshold

effect in vivo. The in vivo and in vitro discrepancy of the mtDNA

genotype phenotype correlation may be related to the possibility that

hepatocytes maintain ATP-synthesis more efficiently in vitro than in

vivo, because they can more efficiently compensate for their defect

in oxidative phosphorylation by increasing glycolysis due to the

ample supply of glucose in the culture medium.

Although mtDNA-depletion is probably sufficient to explain the onset

of liver pathology in humans,[29-31] additional and possibly

interconnected effects are likely to contribute to mitochondrial

injury.[32] We have observed an increase in hepatic ROS production.

It is likely that the increased ROS production originates at least in

part from the respiratory chain, because the decreased activity of

respiratory chain complexes partially blocks the flow of electrons,

allowing them to react with oxygen.[32] The enhanced mitochondrial

ROS formation can then directly attack mtDNA and respiratory chain

polypeptides, but also mitochondrial cardiolipin, the latter being

important for COX function and also releasing reactive lipid

peroxidation products.[32][33] Polymerase-gamma is also a target of

oxidative damage.[34] Through these effects, ROS may close several

vicious circles that contribute to the respiratory decline.

Our study suggests that the dietary supplementation of uridine

attenuates the mitochondrial hepatotoxicity of zalcitabine in accord

with prior in vitro data.[13] The mechanism of the beneficial effect

of uridine is not fully delineated but it is conceivable that uridine

itself or its metabolites disinhibit mtDNA replication by competing

with antiviral pyrimidine analogues at polymerase-gamma or other

steps of intracellular NRTI transport or phosphorylation.[13][14]

Uridine may alternatively correct an intracellular pyrimidine deficit

which in itself induces cell cycle arrest and apoptosis, as

demonstrated by experimental work with direct DHODH inhibitors.[16]

[17] The TUNEL results suggest that apoptosis can principally

accompany hepatic mtDNA-depletion, but further quantitative

conclusions from the observed extent of apoptosis should not be made.

The importance of the intracellular pyrimidine pools for the survival

of cells with a dysfunctional respiratory chain is also supported by

the fact that cells without a single molecule of mtDNA are rescued

from cell death and grow virtually normal, if the intracellular

pyrimidine pools are replenished by uridine.[27]

Extensive pharmacokinetic studies suggest that uridine concentrations

that are protective in vitro can be safely achieved with oral and

parenteral dosing.[14] Oral uridine supplementation (150 mg/kg/d) is

also recommended and safely used long-term in patients with

hereditary orotic aciduria, an inborn error of pyrimidine de novo

synthesis, in which uridine reverses megaloblastic anemia and other

symptoms.[35]

There is a theoretical concern, that high uridine levels are not only

able to compete with NRTIs at the level of polymerase-gamma but may

also do so at the level of HIV-reverse transcriptase. Phenotypic HIV-

resistance assays however did not find a negative effect of uridine

on viral suppression.[36][37] Clinical trials performed for other

indications did not detect a negative effect of uridine

supplementation on the antiretroviral efficacy of NRTI, although the

number of patients were few and follow-up was short.[38][39]

Our pilot study has several limitations. Serum pharmacokinetics of

zalcitabine was not performed because there is no reliable assay. The

optimal dose of uridine is also unknown because intramitochondrial

concentrations of NRTIs and of uridine metabolites cannot be

measured. It is also not possible to extend the observations from

mice to humans and from zalcitabine to the other more frequently used

NRTI. In vitro however, uridine does also antagonize the

mitochondrial toxicity of stavudine and zidovudine which like

zalcitabine are pyrimidine analogues, whereas the mitochondrial

toxicity of didanosine, a purine analogue was not abrogated.[13][24]

This observation underlines the hypothesis of a competitive effect of

uridine with pyrimidines.

HCV-infection (and HCV genotype I in particular) has also been found

to modestly decrease hepatic mtDNA.[40][41] An increased formation of

ROS is thought to represent an important contributor for the HCV-

mediated mtDNA decline. The effects of uridine on HCV related

mitochondrial dysfunction have however not been examined.

The clinical experience of uridine supplementation for hepatotoxicity

is very limited but promising. In one HIV patient uridine

supplementation was described to reverse mitochondrial

steatohepatitis despite continued long-term antiretroviral treatment

with stavudine.[42] Using a non-invasive 13C-methionine breath test

in HIV-positive individuals under treatment with stavudine or

zidovudine, a three day course of Mitocnol was recently found to

reproducibly enhance the function of hepatic mitochondria over a

period of 4 weeks.[43]

We conclude that uridine has beneficial effects in this murine model

of pyrimidine analogue induced hepatotoxicity. Uridine

supplementation should be examined in a carefully monitored clinical

trial in HIV-infected patients who have pyrimidine analogue-induced

mitochondrial steatohepatitis and who cannot be switched to

antiretrovirals with a lower potential of hepatotoxicity.

Article Text

Mitochondrial hepatotoxicity is a complication in HIV patients taking

nucleoside analogue reverse transcriptase inhibitors (NRTI) as part

of their antiretroviral treatment. Individuals may experience

hepatomegaly, steatosis, steatohepatitis and even acute liver failure

with life-threatening lactic acidosis.[1-6] NRTI undergo

intracellular triphosphorylation and then inhibit polymerase-gamma,

the intramitochondrial enzyme which is responsible for the

replication of mitochondrial DNA (mtDNA).[7] Preclinical studies have

demonstrated that NRTI exposure to hepatocytes cause mtDNA depletion

and intracellular steatosis.[8][9] More recently, low hepatic mtDNA-

levels due to prolonged treatment with dideoxynucleoside NRTI

(didanosine, stavudine and zalcitabine) were confirmed in HIV/HCV

coinfected humans.[10] Acute catastrophic hepatotoxicity was also

observed in humans treated with fialuridine, an investigational

nucleoside analogue for chronic hepatitis B and potent inhibitor of

mtDNA synthesis.[11][12] Treatment options for these forms of

mitochondrial hepatotoxicity are limited. It is commonly recommended

to discontinue the responsible agent and to switch to less toxic

antiretrovirals. The recovery from mitochondrial toxicity however may

have a slow offset. Switching HIV drugs may also encounter compliance

problems and carry risks with regard to the potential development of

HIV-resistance or side effects.

We have recently discovered that uridine prevents and even reverses

mitochondrial toxicity in hepatocytes exposed to pyrimidine NRTI.[13]

The beneficial effects of uridine were dose-dependent and observed in

concentrations between 50-200 M.

The mechanism of the protective effects of uridine has not been fully

delineated.[14] Mitochondrial toxicity and related respiratory chain

dysfunction are thought to cause a deficiency of intracellular

pyrimidines, because a normal electron flux through the respiratory

chain is required for the activity of dihydroorotate dehydrogenase

(DHODH), an enzyme which is essential for pyrimidine de novo

synthesis.[15] There is evidence that low intracellular pyrimidine

levels can trigger cell cycle arrest and apoptosis.[16][17]

Intramitochondrial pyrimidine deficiency may also aggravate mtDNA-

depletion by allowing the triphosphorylated pyrimidine analogues to

compete more efficiently with their natural pyrimidine counterparts

at polymerase-gamma. Uridine can replenish pyrimidine pools by being

salvaged into pyrimidines distal from DHODH.[14]

The unknown in vivo effects of uridine on mitochondrial

hepatotoxicity were the aim of our study. We established a new mouse

model in which we used zalcitabine as the strongest polymerase-gamma

inhibitor among the pyrimidine NRTI.[7][8] As a source of uridine, we

used Mitocnol (NucleomaxX), a dietary supplement with a high

bioavailability of uridine.[18]

Abbreviations

COX, cytochrome c-oxidase; CS, citrate synthase; DHODH,

dihydroorotate dehydrogenase; GAPDH, glycerol aldehyde phosphate

dehydrogenase; MDA, malondialdehyde; MtDNA, mitochondrial DNA; nDNA,

nuclear DNA; NRTI, nucleoside analogue reverse transcriptase

inhibitor; ROS, reactive oxygen species; SDH, succinate dehydrogenase.

Materials and Methods

Animals.

After approval by the animal ethics board, female BALB/C mice were

purchased at River, Germany. The rodents received humane care

according to the NIH guidelines

(http://grants.nih.gov/grants/olaw/olaw.htm), were housed at a normal

night-day rhythm, and were fed a normal mouse chow ad libitum (SSniff

R/M-H, Spezialdiäten, Germany). At 7 weeks of age, the mice were

divided into six groups of nine animals each. Controls consisted of

mice without any treatment (group A). Group B mice received 340

mg/kg/d of Mitocnol (Pharma Nord, Vojens, Denmark) in the drinking

water. Groups C and D received zalcitabine (0.36 mg/kg/d) in the

drinking water. This daily dose corresponds to human dosage adjusted

to body area and was calculated on the basis of a daily liquid

consumption of 5 ml.[19] Further mice (groups E and F) received a

higher dose of zalcitabine (13 mg/kg/d). Groups D and F were co-

treated with 340 mg/kg/d of Mitocnol in the drinking water, whereas

groups C and E received no Mitocnol.

Observations for fluid consumption, clinical signs and mortality were

carried out daily; body weights were recorded weekly. All animals

were killed by cervical dislocation at 22 weeks of age, immediately

prior to organ collection and postmortem examination. Livers were

weighted, snap-frozen and cryopreserved in liquid nitrogen until

subsequent analysis. Aliquots were fixed in glutaraldehyde (3%) for

subsequent electron microscopy.

Histopathology, Mitochondrial Ultrastructure and Apoptosis.

The degree of liver fibrosis, steatosis and necroinflammatory

activity was scored with haematoxylin and eosin using the modified

histological activity index (HAI) and the nonalcoholic

steatohepatitis (NASH) scores.[20][21] The percentage of hepatocytes

displaying microvesicular steatosis was also assessed with oil red O.

The evaluating person was blinded to the group status. Two randomly

selected liver samples from each group were examined with electron

microscopy.[22]

For the detection of apoptosis, terminal transferase dUTP nick end

labeling (TUNEL) assays were applied on two randomly selected liver

sections (6 um) from each group. Apoptotic nuclei were identified

with the ChromaTidelexaFluor 488-5-dUTP (Molecular Probes, Eugene,

OR), and the Terminal Transferase Kit (Roche, Mannheim, Germany).

Slides were counterstained with Hoechst 33342 for 10 minutes (37°C)

and embedded with Vectashield (Vector Laboratories, Burlingame, CA)

mounting medium according to the manufacturer's instructions. Slides

were examined in a blinded fashion with a confocal microscope (Zeiss,

Oberkochen, Germany) at 488 nm and 350 nm.

Amount of Hepatic Lipids.

Lipids were freshly extracted from the livers using

methanol/chloroform/water according to the Bligh-Dyer method and

quantified spectrophotometrically using a sulfo-phospho-vanillin

reaction on lipid standards (Sigma, Taufkirchen, Germany) as

described.[23]

Enzyme Activity.

The enzymatic activity of cytochrome c-oxidase (COX), succinate

dehydrogenase (SDH) and citrate synthase (CS) were measured

spectrophotometrically in freshly prepared tissue extracts.[22] COX

is a multisubunit respiratory chain complex which is encoded by both

nuclear DNA (nDNA) and mtDNA, whereas SDH is a respiratory chain

enzyme which is encoded entirely by nDNA. CS is a nDNA-encoded

component of the Krebs-cycle and located in the mitochondrial matrix.

MtDNA-Encoded Respiratory Chain Protein.

The mtDNA encoded subunit I of cytochrome c-oxidase (COX I) was

quantified by immunoblot.[8] The COX I signal was normalized to the

expression of the subunit IV of cytochrome c-oxidase (COX IV), which

is encoded by nDNA. Blots were also probed with a third antibody

(Research Diagnostics Inc., Flanders, NJ) against glycerol aldehyde

phosphate dehydrogenase (GAPDH), an enzyme which is entirely encoded

in the nucleus.

MtDNA Copy Number.

Total DNA was extracted with the QIAamp DNA isolation kit (Qiagen,

Hilden, Germany). MtDNA and nDNA copy numbers were determined by

quantitative PCR.[24] Briefly, mtDNA was amplified between nucleotide

positions 2469 and 2542 and was quantified with a FAM-fluorophore

labeled probe. For the detection of nDNA we selected GAPDH between

nucleotide positions 494 and 671 and used a HEX-fluorophore labeled

probe.

Each 25 l reaction contained 20 ng of genomic DNA, 100 nM probe, 200

nM primers and Taq-man absolute Master Mix (Abgene, Hamburg,

Germany). Amplifications of mitochondrial and nuclear products were

performed as triplicates. Absolute mtDNA and nDNA copy numbers were

calculated using serial dilutions of plasmids with known copy numbers.

Lipid Peroxidation and Superoxide Production.

Malondialdehyde (MDA) is one of the end products of lipid

peroxidation and an indicator of free radical production and

oxidative stress. MDA was spectrophotometrically quantified in

tissues with an assay for thiobarbituric acid reactive material.[25]

Superoxide production was measured in situ on transverse tissue

sections with the oxidative fluorescent dye dihydroethidium (Sigma,

Taufkirchen, Germany).[22] The intensity of the fluorescence was

quantified using Scion Image (Scion Corp).

Statistics.

Group means were compared ANOVA and Kruskal-Wallis ANOVA on the

ranks, followed by unpaired t tests or Wilcoxon Mann-Whitney tests,

as appropriate. Correlations were computed by nonlinear exponential

regression analysis. Graphics and calculations were performed using

the Sigma Plot 2000 version 8.0 (SPSS Inc.) and the Sigma Stat

version 3.1 (Jandel Inc.) packages.

Results

Macroscopic and Microscopic Pathology.

The daily fluid consumption of the mice was unaffected by any

treatment. One of the nine mice receiving high dose zalcitabine

without uridine (group E) died in week 21. This animal was excluded

from the analysis because we were unable to control for postmortem

time. The maximal medium body weight of group E was reached at week

19 and at that time point was lower compared to all other groups.

From week 19 until week 22 group E mice lost about 2% of body weight,

whereas there was a 11% gain in the controls (P = 0.004) and a 3%

gain in group F (P = 0.04). The liver weight of group E animals was

elevated by 19% compared to controls (P < 0.001). The liver weight of

all other groups did not differ from controls (Table 1).

The degree of hepatotoxicity as histologically assessed with the

necroinflammatory score was substantially elevated in all groups

treated with zalcitabine, dose dependent and most prominent in group

E (Fig. 1A). Coadministration of uridine reduced the

necroinflammatory score in both the high dose (P < 0.001) and the low

dose (P = 0.02) zalcitabine groups.

Zalcitabine also caused a dose dependent liver steatosis as evidenced

by elevated steatosis scores (Fig. 1B). On oil red staining, the

steatosis was microvesicular and panacinar (Fig. 2B). In group E,

there was also mild mononuclear infiltration with predominantly

perisinusoidal and just in a few cases periportal involvement. The

cytoplasm contained structures consistent with megamitochondria.

Lipogranulomas were not observed. Uridine alone had no intrinsic

effect on liver steatosis, but when given with zalcitabine, reduced

steatosis score in both dose groups (group C vs. D, P = 0.02; group E

vs. group F, P < 0.001, Fig. 1B and Fig. 2C). Among all animals, the

steatosis score correlated positively with the necroinflammatory

score (r = 0.86, P < 0.001).

On electron microscopy the hepatocytes of the zalcitabine high dose

group contained increased numbers of mitochondria (Fig. 2E). The

organelles were enlarged and had disrupted cristae. There were small

intracytoplasmic vacuoles, consistent with fat storage. Uridine

supplementation abrogated this ultrastructural damage; only a slight

increase in mitochondrial number was observed (Fig. 2F). The

mitochondria of the low dose zalcitabine group appeared to also have

a slight increase in number; the organelles in all other groups did

not show ultrastructural abnormalities (not shown).

Apoptotic nuclei were abundant in the zalcitabine high dose group

(Fig. 2K) and to a lesser amount in the low dose group (not shown).

Uridine supplementation prevented the zalcitabine induced apoptosis

(Fig. 2L) but when given without zalcitabine had no effects on

apoptosis (not shown).

Hepatic Lipids.

As expected, the steatosis score was positively correlated with the

amount of intrahepatic lipids (r = 0.73, P < 0.001) among all mice.

Zalcitabine increased the amount of hepatic lipids in a dose

dependent fashion (Fig. 1B). Uridine alone had no effect on hepatic

lipids but when co administered with zalcitabine reduced the elevated

hepatic lipid content in the low and the high dose zalcitabine

groups. Hepatic lipids in the low dose zalcitabine plus uridine group

did not statistically differ from controls (P = 0.07). Hepatic lipids

correlated positively with the necroinflammatory score among all

animals (r = 0.76; P < 0.001; Fig. 3A).

Enzyme Activities.

The mean ratio of hepatic COX and SDH-activity is an index of mtDNA

encoded respiratory chain function and was reduced in all mice

treated with zalcitabine (Table 1). In the low dose zalcitabine group

the mean COX/SDH-ratio was 45% of control values and 21% in the high

dose zalcitabine group. Uridine supplementation per se did not alter

the COX/SDH-ratio, but when given in conjunction with zalcitabine

attenuated although not fully normalized respiratory dysfunction. The

enzymatic activity of CS did not differ among all groups. Among all

mice, the COX/SDH-ratio correlated negatively with the steatosis

score (r = -0.79, P < 0.001), the necroinflammatory score (r = -0.86;

P < 0.001) and the amount of hepatic lipids (r = -0.78, P < 0.001,

Fig 3B).

MtDNA-Encoded Respiratory Chain Protein.

The expression of the mtDNA-encoded cytochrome c-oxidase subunit was

normalized for the expression of the nDNA-encoded subunit by

calculating the COX I/COX IV-ratio. The mean COX I/COX IV-ratio was

reduced in the high dose zalcitabine group (Table 1) compared to

controls (P = 0.007). Co-treatment with uridine significantly

improved and normalized the COX I/COX IV-ratio. In the low dose

zalcitabine group, the COX I/COX IV was 14% lower but not

significantly different from controls. The COX I/COX IV ratio was

inversely correlated with the steatosis score (r = -0.34, P = 0.04),

the necroinflammatory score (r = -0.35; P = 0.01) and the lipid

content (r = -0.4; P = 0.01). The COX IV/GAPDH ratio did not

statistically differ between all groups (Table 1). Taken together,

these results indicate that the respiratory chain defect is

restricted to the mtDNA-encoded respiratory chain subunit, whereas

the nDNA-encoded subunit is preserved.

MtDNA Content.

The mean wild mtDNA copy number was reduced in mice treated with low

and high dose zalcitabine (79% ± 16%, P = 0.02 and 63% ± 7%, P <

0.001 of control values, respectively). Uridine had no intrinsic

effect on mtDNA biogenesis, but when administered with zalcitabine

attenuated and in the case of low dose zalcitabine fully abrogated

mtDNA depletion (Table 1). Among all groups, liver mtDNA copy numbers

were inversely correlated with the steatosis score (r = -0.69, P <

0.001), the necroinflammatory score (r = -0.63; P < 0.001) and the

amount of hepatic lipids (r = -0.54, P < 0.001, Fig 3C), and

positively correlated with the COX/SDH-ratio (r = 0.6, P < 0.001).

Intrahepatic Reactive Oxygen Species.

Mean MDA levels are an indirect indicator of the formation of

reactive oxygen species (ROS) and were elevated in the livers of the

low and high dose zalcitabine group (161% ± 55%, P = 0.01 and 215%

± 57%, P < 0.001 of control values, respectively). Mitocnol alone

did not induce an elevation in mean MDA levels (Table 1) but

normalized the elevated MDA content in mice exposed to both

zalcitabine doses. MDA levels were positively correlated with the

steatosis score (r = 0.63, P < 0.001) and the amount of intrahepatic

lipids (r = 0.69, P < 0.001). MDA levels were negatively correlated

with the COX/SDH ratio (r = -0.67, P < 0.001), the COX I/COX IV ratio

(r = -0.67, P < 0.001) and mtDNA copy numbers (r = -0.62, P < 0.001).

Among the livers of all mice, superoxide content was highly

correlated with MDA levels (r = 0.9, P < 0.001). Similar to MDA

levels, superoxide was increased by a factor of 2.1 and 4.2 in the

low and high dose zalcitabine groups when compared to controls. In

the zalcitabine low dose group; superoxide content was normalized by

uridine coadministration although uridine did not appear to affect

ROS production when given without zalcitabine. In the zalcitabine

high dose group, uridine reduced superoxide content. Superoxide

levels correlated positively with the steatosis score (r = 0.77, P <

0.001) and the amount of intrahepatic lipids (r = 0.69, P < 0.001,

Fig. 3D), whereas inverse correlations were identified between

superoxide content and the COX/SDH-activity ratio (r = -0.73, P <

0.001), the COXI/COXIV ratio (r = -0.48, P = 0.002), and hepatic

mtDNA copies (r = -0.59, P < 0.001).