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Changes in Fat Mitochondrial DNA and Function in Subjects Randomized to Abacavir-Lamivudine or Tenofovir DF-Emtricitabine With Atazanavir-Ritonavir or Efavirenz: AIDS Clinical Trials Group Study A5224s, Substudy of A5202
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The Journal of Infectious Diseases, Volume 207, Issue 4, 15 February 2013
 The effect of nonthymidine nucleoside reverse-transcriptase inhibitors (NRTIs) on fat mitochondrial DNA (mtDNA) content and function is unclear.
Methods. A5202 randomized antiretroviral therapy-naive human immunodeficiency virus-infected subjects to abacavir-lamivudine (ABC/3TC) versus tenofovir DF-emtricitabine (TDF/FTC) with efavirenz (EFV) or atazanavir-ritonavir (ATV/r). A5224s, substudy of A5202, enrolled 269 subjects with fat measurements by dual-energy x-ray absorptiometry and computed tomography. A subset of subjects underwent fat biopsies at baseline and week 96 for mtDNA content (real-time polymerase chain reaction) and oxidative phosphorylation nicotinamide adenine dinucleotide (reduced) dehydrogenase (complex I) and cytochrome c oxidase (complex IV) activity levels (immunoassays). Intent-to-treat analyses were performed using analysis of variance and paired t tests.
Results. Fifty-six subjects (87% male; median age, 39 years) were included; their median body mass index, CD4 cell count, and fat mtDNA level were 26 kg/m2, 227 cells/μL, and 1197 copies/cell, respectively. Fat mtDNA content decreased within the ABC/3TC and TDF/FTC groups (combining EFV and ATV/r arms; median change, -341 [interquartile range, -848 to 190; P = .03] and -400 [-661 to -221; P < .001] copies/cell, respectively), but these changes did not differ significantly between the 2 groups (P = .57). Complex I and IV activity decreased significantly in the TDF/FTC group (median change, -12.45 [interquartile range, -24.70 to 2.90; P = .003] and -8.25 [-13.90 to -1.30; P < .001], optical density × 103/µg, respectively) but not the ABC/3TC group. Differences between the ABC/3TC and TDF/FTC groups were significant for complex I (P = .03).
Conclusions. ABC/3TC and TDF/FTC significantly and similarly decreased fat mtDNA content, but only TDF/FTC decreased complex I and complex IV activity levels.

This report, for the first time, details changes in mtDNA and mitochondrial function among subjects randomized to 1 of 4 commonly used nonthymidine NRTI-containing initial ART regimens. We found that ART initiation with both ABC/3TC- and TDF/FTC-containing regimens results in a significant decrease in fat mtDNA levels. In addition, in subjects treated with TDF/FTC (but not ABC/3TC), there was evidence of mitochondrial respiratory chain dysfunction, as assessed by decreases in both complex I and complex IV activity levels. In this same group (TDF/FTC treated), mitochondrial alterations were correlated with gains in subcutaneous and visceral abdominal fat. We also found in EFV- and ATV/r-containing arms significant but similar declines in fat mtDNA and in oxidative phosphorylation complex IV activity levels.
To date, no data are available on longitudinal fat mitochondrial changes in ART-naive subjects. This current study did not enroll an ART-naive control group who underwent serial biopsies without starting ART. Therefore, we cannot firmly conclude that the observed declines in mitochondrial indices are due to the effect of ART (and not HIV itself). However, we believe that these were unlikely to be due to HIV-1 infection alone. Indeed untreated HIV-1 infection has been linked in most studies but not all [9], to declines in mtDNA levels and in oxidative phosphorylation enzymes in peripheral blood mononuclear cells [10-12] or in adipose tissue [13]. Miura et al have also reported that mtDNA levels in HIV-1-infected individuals were inversely correlated with HIV-1 RNA levels [14]. Therefore, treating HIV-1 infection and suppressing the virus is expected to improve mitochondrial indices and not worsen them, as seen in our study.
The lack of correlation between the decline in mitochondrial indices and changes in fat content in the overall group should not necessarily lessen concerns about potential mitochondrial toxicity of nonthymidine NRTIs. Our study was relatively short, and it is conceivable that nonthymidine NRTI-induced mitochondrial toxicity is mild compared with that seen with thymidine NRTIs, resulting in slower development of clinical phenotypes such as lipoatrophy. It is also possible that the mitochondrial toxicity seen in our study could become problematic in subjects receiving other medications known to affect mitochondrial function, such as metformin [15], ethambutol [16], or linezolid [17], or in those exposed to excessive alcohol, a known mitochondrial toxin.
Moreover, in the TDF/FTC-treated group (regardless of the ATV/r vs EFV randomization), we found consistent inverse correlations between the activity levels of oxidative phosphorylation complex I and complex IV and several measures of fat content, at both visceral and subcutaneous levels, suggesting that lower mitochondrial function is associated with generalized fat gain in this TDF/FTC-treated group. Similar associations between complex I and complex IV activity levels and limb and trunk fat were recently reported from another study of TDF-based regimens [18]. Although the pathogenesis of lipohypertrophy is poorly understood, the role of mitochondrial toxicity has not been ruled out. Indeed, we and others have found significant mitochondrial abnormalities in dorsocervical adipose tissue, even from HIV-infected persons with lipoatrophy [19-20]. In this mitochondrial substudy of A5224s, the body fat composition results are overall consistent with the main A5224s results [2]. Limb fat and visceral fat increased significantly in the ATV/r arms but not in the EFV arms. In ACTG A5142, a randomized study in which patients received NRTIs of choice, including thymidine NRTIs in many, EFV was associated with higher rates of lipoatrophy than the PI lopinavir-ritonavir [21]. This finding suggested that EFV may have been associated with greater mitochondrial toxicity than lopinavir-ritonavir. This concept was further corroborated by a study showing that EFV inhibited mitochondrial function, including complex I activity, in human hepatic cells in vitro [22]. However, in our study, we found similar decreases in all mitochondrial indices with EFV- and ATV/r-containing arms.
Our study has some limitations, including small sample size and a significant number of premature study discontinuations before week 96. We recorded mitochondrial indices only at 2 time points and not beyond 96 weeks. Consequently, we could not exclude early improvements followed by worsening of these measures over time. All of these limitations are frequently encountered in studies requiring invasive tissue sampling, and it is not realistic to obtain a sufficient quantity of fat tissue at closely spaced time intervals in a substantial number of subjects.
In conclusion, we have shown significant perturbation in mitochondrial indices after 96 weeks of nonthymidine NRTI-containing regimens which were assigned randomly. In the TDF/FTC group, changes in oxidative phosphorylation complex I and complex IV activity levels consistently were inversely correlated with changes in several objective measures of body fat, including in both subcutaneous and visceral compartments. Ongoing investigations will clarify the impact of these regimens on fat apoptosis and oxidative stress. Given the anticipated use of life-long ART and the central role of NRTIs in current regimens, larger and longer studies are needed to better characterize the mitochondrial and body composition effects of nonthymidine NRTI-containing regimens. Finally, studies investigating other potential consequences of mitochondrial abnormalities, such as frailty and premature aging, are much needed.

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