JAIDS Journal of Acquired Immune Deficiency Syndromes:
Upregulatory Mechanisms Compensate for Mitochondrial DNA Depletion in Asymptomatic Individuals Receiving Stavudine Plus Didanosine
Miró, Òscar MD*; López, Sònia PhD*; Rodríguez de la Concepción, Marisa PhD†; Martínez, Esteban MD‡; Pedrol, Enric MD§; Garrabou, Glòria PhD*; Giralt, Marta PhD†; Cardellach, Francesc MD*; Gatell, Josep M. MD‡; Vilarroya, Francesc PhD†; Casademont, Jordi MD*
From the *Mitochondrial Research Laboratory, Muscle Research Unit, Department of Internal Medicine, Hospital Clinic, Institut d'Investigations Bioiuèdiques August Pi I Sunyer, Barcelona, Spain; †Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, Spain; ‡Department of Infectious Diseases, Hospital Clinic, Institut d'Investigations Bioiuèdiques August Pi I Sunyer, Barcelona, Spain; and §HIV Unit, Department of Internal Medicine, Fundació Hospital-Asil de Granollers, Granollers, Spain.
Received for publication April 16, 2004; accepted July 6, 2004.
Supported by the following grants: Fundación para la Investigación y la Prevención del Sida en España (FIPSE 3102-00 and 3161/00A), Fundació La Marató de TV3 (020210), Redes de Investigación en Mitocondrias (V2003-REDC06E-0) y Sida (Rg-173), and Suport a Grups de Recerca 2001/SGR/00379.
Reprints: Òscar Miró, Department of Internal Medicine, Hospital Clinic, Villarroel 170, 08036 Barcelona, Catalonia, Spain (e-mail: email@example.com).
Nucleoside analogue use is often related to mitochondrial DNA (mtDNA) depletion, but mitochondrial function is preserved in most asymptomatic patients. We determined whether homeostatic mechanisms are able to compensate for this mtDNA depletion in patients receiving stavudine plus didanosine (d4T + ddI), an antiretroviral combination with great in vitro and in vivo capacity to decrease mtDNA. We included 28 asymptomatic HIV-infected individuals: 17 subjects (cases) on a first-line antiretroviral regimen consisting of d4T + ddI as the nucleoside backbone plus nevirapine or nelfinavir for at least 6 months (mean: 16 ± 8 months) and 11 naive subjects (controls). We assessed the following in peripheral blood mononuclear cells: mitochondrial mass by citrate synthase activity, mtDNA content by real-time polymerase chain reaction, cytochrome c oxidase subunit II (COX-II) expression by Western blot analysis, and COX activity by spectrophotometry. The mitochondrial mass and mtDNA content of cases decreased when compared with controls, whether normalized per cell or per mitochondrion. Conversely, COX-II expression and COX activity were similar in cases and controls. COX-II expression was constant and independent of the mtDNA content, whereas it was closely related to COX activity. We concluded that treatment with dd4T + ddI is associated with decreased mitochondrial mass and mtDNA content but that COX-II expression and COX activity remain unaltered. These data suggest that upregulatory transcriptional or posttranscriptional mechanisms compensate for mtDNA depletion caused by d4T + ddI before profound mtDNA depletion occurs.
Mitochondrial DNA (mtDNA) depletion is a secondary effect inherent to most of the nucleoside reverse transcriptase inhibitors (NRTIs). The main reason for this fact is that NRTI triphosphates are also able to inhibit DNA γ-polymerase, the only enzyme that replicates mtDNA.1. Some reports have attributed a pathogenic role to mtDNA depletion in the long-term adverse effects associated with the use of NRTIs, especially hyperlactatemia2,3 and lipodystrophy.4-8 However, the cumulated lifetime doses of NRTIs at which these adverse effects develop have not been established. In fact, the reasons why some people do not develop these adverse effects, despite large doses of NRTIs, remain unknown, and few studies have evaluated the functional relevance of mtDNA depletion at the level of the mitochondrial respiratory chain (MRC). Thus, many questions arise as to the real role of mtDNA depletion in these adverse effects.9-11
One factor that may contribute to the inconsistent presentation of adverse effects, despite the constant presence of mtDNA depletion, may be the development of homeostatic mechanisms to compensate for this depletion. At least before profound mtDNA depletion occurs, these mechanisms may act at transcriptional or posttranscriptional levels to compensate for the mild to moderate decrease of the mtDNA content. The final objective of these hypothetic changes would be to maintain the MRC capacity unaltered, because all the mtDNA-encoded genes are devoted to the synthesis of proteins of MRC complexes. To extend this mechanism, we assessed the effects of the antiretroviral therapy beyond mtDNA content by determination of the expression of the human cytochrome c oxidase subunit II (COX-II, 1 of the 3 subunits of COX encoded by mtDNA) and COX activity. We chose HIV-infected individuals undergoing treatment with stavudine and didanosine (d4T + ddI) as the nucleoside backbone of a highly active antiretroviral therapy (HAART) regimen because of the great in vitro12 and in vivo13,14 capacity of this antiretroviral combination to decrease the mtDNA content.
We designed a cross-sectional and observational study including HIV-infected patients on a first-line HAART regimen consisting of d4T + ddI as the nucleoside backbone (plus nelfinavir or nevirapine) for at least 6 months. A control group consisting of antiretroviral-naive HIV-infected patients was also studied. All patients were symptom-free regarding HIV disease and/or antiretroviral therapy at the time of inclusion in the study. None of the individuals receiving treatment had clinical data of lipodystrophy. Clinical and demographic data for each patient, including age, gender, CD4+ T-cell count, HIV-1 RNA copies, and duration of HAART, were recorded at the time of inclusion. Patients with a personal or familial history suggestive of mitochondrial disease or neuromuscular disorder were excluded. As reference values for all the mitochondrial experiments, we used data from 20 healthy individuals matched by age and gender who were not infected with HIV and had been previously assayed in our laboratory. The protocol was approved by the institutional ethics committee of each hospital, and all the patients provided written informed consent.
A total of 20 mL of venous blood was extracted from each patient, and peripheral blood mononuclear cells (PBMCs) were immediately isolated by Ficoll density gradient centrifugation (Histopaque-1077; Sigma Diagnostics, St. Louis, MO). The platelet count after the PBMC extraction procedure was confirmed to be less than 200 per PBMC in each case. Total DNA was obtained by a standard phenol-chloroform extraction procedure from an aliquot of PBMCs and was used for genetic studies. The remaining PBMCs were resuspended in 100 to 150 μL of phosphate-buffered saline (PBS) and used for Western blot studies and enzyme assays. Protein content was measured according to the protein-dye binding principle of Bradford.15
The quantity of mitochondria was estimated by means of spectrophotometric measurement at 412 nm (UVIKON 922; Kontron, Basel, Switzerland) of the citrate synthase (CS) activity (Enzyme Code (EC) 184.108.40.206), a mitochondrial matrix enzyme of the Krebs cycle, which remains highly constant in mitochondria and is considered to be a reliable marker of mitochondrial content.16-18 CS activity was expressed as nanomoles of reduced substrate per minute and per milligram of cell protein. The complete methodology has been described elsewhere.19
Mitochondrial DNA Quantification
For each DNA extract, the housekeeping r18S nuclear gene and the highly conserved mitochondrial ND2 gene were quantified separately by quantitative real-time polymerase chain reaction (PCR; LightCycler FastStart DNA Master SYBR Green I; Roche Molecular Biochemicals, Mannheim, Germany). Detailed conditions of the experiments have been reported extensively elsewhere.19 The results were expressed as the ratio of the mean mtDNA value of duplicate measurements to the mean nuclear DNA value (nDNA) of duplicate measurements (mtDNA/nDNA).20,21 The results of mtDNA content using the methodology described are related to cells. To normalize these results by the cellular mitochondrial content, we also calculated mtDNA content per mitochondria by dividing the mtDNA/nDNA ratio by CS activity.
Measurement of the Cytochrome C Oxidase Subunit II of Cytochrome C Oxidase
Crude protein extracts containing 20 μg of protein were mixed with a 1:5 volume of a solution containing 50% glycerol, 10% sodium dodecyl sulfate (SDS), 10% 2-mercaptoethanol, 0.5% bromophenol blue, and 0.5 M of Tris (pH 6.8), incubated at 90°C for 5 minutes and electrophoresed on 0.1% SDS/13% polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Blots were probed with a monoclonal antibody for the mtDNA-encoded human COX-II (A-6404; Molecular Probes, Eugene, OR) as well as with antibodies against voltage-dependent anion carrier (VDAC) or porin (Calbiochem Anti-Porin 31HL; Darmstadt, Germany) as a marker of mitochondrial protein loading and against β-actin (Sigma clone AC-15; St. Louis, MO) as a marker of overall cell protein loading. Immunoreactive material was detected by the enhanced chemiluminescence detection system and resulted in a 25-kd band for COX-II, a 31-kd band for VDAC, and a 47-kd band for β-actin as expected (Fig. 1). The intensity of signals was quantified by densitometric analysis (Phoretics 1D Software; Phoretics International LTD, Newcastle, England). The content of the COX-II subunit was normalized by the content of β-actin signal to establish the relative COX-II abundance per overall cell protein and by the nuclear-encoded mitochondrial protein VDAC to establish the relative COX-II abundance compared with overall mitochondria.
Cytochrome C Oxidase (Enzyme Code (EC) 220.127.116.11) Activity
The measurement of the specific enzyme activity was performed spectrophotometrically (UVIKON 922) at 37°C according to Rustin et al22 and was slightly modified for minute amounts of biologic samples.18,23 COX activity was expressed as nanomoles of oxidated substrate per minute and per milligram of cell protein. It was also calculated per mitochondrion by dividing absolute COX activity by CS activity.
Qualitative data were expressed as percentages and quantitative data as mean ± SD. Comparisons were carried out by using the χ2 test and unpaired Student t test for qualitative and quantitative variables, respectively. In the Student t test, normality of the distribution was ascertained using the Kolmogorov-Smirnov test before applying parametric tests. Linear regression analysis was performed to evaluate the relation between quantitative variables. In all cases, P values less than 0.05 were considered statistically significant.
We included 11 consecutive HIV-infected treatment-naive individuals (controls) and 17 consecutive HIV-infected individuals (cases) receiving a first-line HAART regimen containing d4T + ddI as the nucleoside backbone (9 of them receiving nevirapine and 8 of them taking nelfinavir as the third drug of the antiretroviral combination). The clinical characteristics are shown in Table 1. The 2 groups only differed in viral load, which was greater in untreated individuals.
CS activity was 126 ± 19 nmol/min/mg of protein for naive individuals and 92 ± 31 nmol/min/mg of protein for individuals receiving treatment (73% of control activity; P < 0.001), indicating decreased mitochondrial mass in patients on d4T + ddI. Individuals receiving d4T + ddI also showed a significant decrease in mtDNA content, which was expressed per cell (60% of control content; P < 0.01) or per mitochondrion (72% of control content; P < 0.05). Conversely, the expression of the COX-II subunit of COX (encoded by mtDNA) was similar in the 2 groups, regardless of expression per cell or per mitochondrion. Similarly, patients receiving d4T + ddI did not show a decrease in COX activity expressed per cell or per mitochondrion (Fig. 2). When we analyzed the cases according to the treatment that they were receiving, nevirapine or nelfinavir in combination with d4T + ddI, we did not find differences between the 2 subgroups for any of the studied mitochondrial parameters (data not shown). It is remarkable that although mtDNA content decreased in HIV-positive treatment-naive patients in comparison to uninfected individuals, the expression of COX-II and COX activity did not differ between the 2 groups.
Expression of the COX-II subunit was independent of the amount of mtDNA for the 2 groups of individuals, and this absence of a relation was confirmed per cell and per mitochondrion. In contrast, expression of the COX-II subunit and COX activity showed a close relation, being stronger in treatment-naive individuals than in subjects undergoing treatment with d4T + ddI (Fig. 3).
The time on antiretroviral treatment was associated with a significant decrease in mtDNA content, whereas the expression of COX-II was mild and not statistically significantly decreased and the activity of COX remained unaltered over time (Fig. 4). Interestingly, when we assessed the effects of HIV infection itself (by means of viral load) and immunologic status (by means of CD4+ T-cell count) on COX-II expression in the absence of antiretrovirals, we found a significantly greater decrease in the expression of COX-II in patients with greater viremia and poorer immunologic status (Fig. 5).
The present study demonstrates that although mtDNA depletion is clearly present in asymptomatic HIV-infected individuals treated with antiviral regimens containing d4T + ddI, this depletion is not associated with a decrease in expression of the COX-II subunit (encoded by mtDNA) or with a decrease in COX activity (complex IV of the MRC). Identical conclusions are achieved if the results are normalized per cell or per mitochondrion, which reasonably excludes any masking effect caused by changes in the whole mitochondrial content of the cell as a result of antiretroviral drugs. Thus, these data suggest that transcriptional (increased transcription of mRNA from mtDNA) or posttranscriptional (increased synthesis of protein from mRNA) mechanisms act to compensate for the loss in the number of mtDNA copies. Figure 2 is highly illustrative of the supposition that the amount of COX-II is maintained irrespective of the mtDNA content.
Our results are exclusively related to the conditions of the study (ie, to PBMCs of individuals receiving d4T + ddI for a mean of 16 months as a first-line therapy with no clinical evidence of drug-related long-term secondary effects). Therefore, our data do not mean that mtDNA depletion does not play a role in the adverse effects that may develop during long-term d4T + ddI treatment. PBMCs are not the target of such adverse effects; accordingly, they only represent a conservative estimate of what really occurs at target tissues. In addition, in patients receiving antiretrovirals for longer periods or those who have developed lipodystrophy or hyperlactatemia, the expression of mitochondrially encoded proteins and/or the activity of such proteins may be dramatically impaired. In fact, we found a tendency to a decline in COX-II expression in PBMCs in relation to the length of time on antiretroviral drugs. The results of the present study agree with the finding that in patients suffering from zidovudine myopathy, mtDNA depletion at the skeletal muscle level is accompanied by a clear decrease in COX-II expression in the sarcoplasm.24 In vitro studies have also shown a time- and dose-dependent mtDNA depletion caused by d4T and ddI on human hepatocellular carcinoma cell line (HepG), which preceded or coincided with a decline in COX-II expression.25 In any case, we believe that the absence of downstream effects caused by mtDNA depletion emphasizes the efficiency of mitochondria in compensating for antiretroviral toxicities, at least when mtDNA depletion is mild to moderate. This is in accordance with the hypothesis that only mtDNA defects involving more than 80% of the genetic material are able to induce MRC dysfunction.26
Compensatory mechanisms for mtDNA depletion have been proposed as an explanation for the lack of a close correlation between time on treatment and the probability of developing adverse effects. The intensity of such compensatory effects may vary from patient to patient according to risk factors such as the time on antiretroviral therapy, immunologic status, and/or degree of viremia. Interestingly, we found that although the expression of COX-II only showed a weak correlation with the first factor (time on antiretroviral therapy), the latter 2 factors (immunologic status and degree of viremia) were significantly associated with this expression. These findings suggest that toxic mitochondrial effects are not only limited to the inhibition of DNA γ-polymerase caused by antiretrovirals but to the combined effects of being immune compromised and having HIV infection and that receiving antiretrovirals could have a cumulative effect. Nowadays, it has been widely reported that HIV itself is also increasingly implicated in diverse and extensive mitochondrial disturbances,3,14,27-32 most of which are triggered by mitochondrially dependent apoptotic mechanisms. Consistent with those reports, the present study also postulates that HIV-infected patients naive to antiretrovirals have a decreased amount of mtDNA compared with uninfected people. Conversely, the expression of COX-II and COX activity seems to be less influenced than mtDNA content by the effects of HIV infection itself. Accordingly, adaptive mechanisms may be effective in withholding the decline in MRC function caused by mtDNA depletion as a result of administration of d4T + ddI in certain circumstances; however, in other cases, the collateral actions of antiretrovirals and/or HIV itself against mitochondria may be the final determinants leading to mitochondrial failure. In any case, the demonstration of up-regulatory mechanisms compensating for mtDNA depletion is an additional argument for the need to be cautious when using mtDNA quantification as the only tool to monitor the clinical relevance of the mitochondrial toxicity of antiretroviral drugs.
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