HIV Articles  
Back 
 
 
HIV Itself Causes Mitochondrial Damage
 
 
  Download the PDF here
 
"HIV-1 infection itself has been associated with mitochondrial toxicity. Using an in-vitro assay to quantify the ratio of mitochondrial DNA to nuclear DNA, Cote and colleagues [12] demonstrated that asymptomatic antiretroviral-naive HIV-infected patients have evidence of more mitochondrial toxicity than uninfected controls."
 
12.Cote HC, Brumme ZL, Craib KJ, Alexander CS, Wynhoven B, Ting L, et al. Changes in mitochondrial DNA as a marker of nucleoside toxicity in HIV-infected patients. N Engl J Med 2002; 346:811-820.
 
Changes in Mitochondrial DNA as a Marker of Nucleoside Toxicity in HIV-Infected Patients - pdf attached
 
Helene C.F. Cote, Ph.D., Zabrina L. Brumme, B.S., Kevin J.P. Craib, M.Math., Christopher S. Alexander, Ph.D., Brian Wynhoven, B.S., Lillian Ting, B.S., Hubert Wong, Ph.D., Marianne Harris, M.D., P. Richard Harrigan, Ph.D., Michael V. O'Shaughnessy, Ph.D., and Julio S.G. Montaner, M.D.
 
"Mitochondrial DNA:nDNA ratios were significantly lower in HIV-infected, asymptomatic patients who had never received antiretroviral therapy than in non-HIV-infected controls (P<0.001), a difference that was not explained by the lower CD4 counts in the former group. This finding is consistent with the results of recent in vitro studies in which HIV-infected cells had signs of mitochondrial necrosis.34 Furthermore, a mitochondria-controlled mechanism of cell death has been postulated in HIV infection.35-38"
 
Longitudinal measurements of mtDNA:nDNA ratios in samples collected before, during, and after antiretroviral therapy are presented in Figure 4. Among the eight patients with symptomatic hyperlactatemia, the mean mtDNA:nDNA ratio during therapy (0.41±0.08) was significantly lower than that among both the non-HIV-infected subjects (P<0.001) and the HIV-infected, asymptomatic patients who had never received antiretroviral therapy (P<0.001). The mean mtDNA:nDNA ratio during therapy was also significantly lower than both the mean mtDNA:nDNA ratio when the patients were receiving no therapy (0.74±0.13, P=0.02)


Figure 3. Shows reduction in mtDNA for HIV+ compared to non-HIV-infected and to patients with mitochondrial toxicity on therapy and after stopping therapy


We describe a novel polymerase-chain-reaction (PCR) assay for mitochondrial DNA performed on samples of venous blood. We applied it to three groups of subjects: normal volunteers not infected with HIV, HIV-infected asymptomatic patients never treated with antiretroviral drugs, and HIV-infected patients with symptomatic, nucleoside-related hyperlactatemia. Finally, in the group of patients with hyperlactatemia, we quantified ratios of mitochondrial to nuclear DNA longitudinally before, during, and after the discontinuation of therapy.

ABSTRACT


Background- Nucleoside analogues can induce toxic effects on mitochondria by inhibiting the human DNA polymerase γ. The toxic effects can range from increased serum lactate levels to potentially fatal lactic acidosis. We studied changes in mitochondrial DNA relative to nuclear DNA in the peripheral-blood cells of patients with symptomatic, nucleoside-induced hyperlactatemia.

Methods- Total DNA was extracted from blood cells. A nuclear gene and a mitochondrial gene were quantified by real-time polymerase chain reaction. Three groups were studied: 24 controls not infected with the human immunodeficiency virus (HIV), 47 HIV-infected asymptomatic patients who had never been treated with antiretroviral drugs, and 8 HIV-infected patients who were receiving antiretroviral drugs and had symptomatic hyperlactatemia. The patients in the last group were studied longitudinally before, during, and after antiretroviral therapy.

Results- Symptomatic hyperlactatemia was associated with marked reductions in the ratios of mitochondrial to nuclear DNA, which, during therapy, averaged 68 percent lower than those of non-HIV-infected controls and 43 percent lower than those of HIV-infected asymptomatic patients never treated with antiretroviral drugs. After the discontinuation of antiretroviral therapy, there was a statistically significant increase in the ratio of mitochondrial to nuclear DNA (P=0.02). In the patients followed longitudinally, the decline in mitochondrial DNA preceded the increase in venous lactate levels.

Conclusions- Mitochondrial DNA levels are significantly decreased in patients with symptomatic, nucleoside-related hyperlactatemia, an effect that resolves on the discontinuation of therapy.

Nucleoside analogues that inhibit reverse transcriptase remain a cornerstone of antiretroviral therapy for human immunodeficiency virus (HIV) infection. These agents inhibit HIV replication but can also inhibit the human DNA polymerase γ1,2 and thereby replication of mitochondrial DNA, leading to depletion of mitochondrial DNA and drug toxicity.3-6 Mitochondrial toxicity is at least partially responsible for adverse effects such as lactic acidosis, hepatic steatosis, myopathy, cardiomyopathy, peripheral neuropathy,7 pancreatitis, and possibly the lipodystrophy syndrome.5,6,8-12 Treatment of other conditions, such as hepatitis or cancer, with nucleoside analogues can also result in mitochondrial toxicity.13,14

Early studies on zidovudine-induced myopathy found a decrease in total mitochondrial DNA in muscle-biopsy specimens.15-17 In vitro studies of various anti-HIV nucleoside analogues have also shown that they can cause a reduction in the mitochondrial content of human lymphoblastoid cells,18 CEM cells,19 and HepG2 cells.20 Mitochondrial toxicity leads to decreased oxidative phosphorylation, intracellular accumulation of lipids, and increases in lactic acid.3,21 Patients with hyperlactatemia due to mitochondrial toxicity may present with symptoms such as fatigue, shortness of breath, weight loss, lipid abnormalities, and hepatic steatosis. The syndrome of hyperlactatemia is now well known among HIV-infected patients receiving antiretroviral therapy, particularly in those receiving stavudine (d4T, or Zerit).22-25 Its presentation, severity, and frequency are distinct from those of lactic acidosis, which is a rare, acute, and potentially fatal adverse effect of nucleoside analogues.26-29 Whether chronic hyperlactatemia is a risk factor for progressive liver damage29 or lactic acidosis30 remains uncertain.

The gold standard for the diagnosis of nucleoside-related mitochondrial toxic effects is muscle or liver biopsy; however, biopsy is not practical for routine screening and monitoring. Random measurements of venous lactate have been used to monitor for mitochondrial damage,31,32 but the clinical usefulness of this method remains unclear. Lack of specificity is a problem, as is technical and physiologic variability.30

We describe a novel polymerase-chain-reaction (PCR) assay for mitochondrial DNA performed on samples of venous blood. We applied it to three groups of subjects: normal volunteers not infected with HIV, HIV-infected asymptomatic patients never treated with antiretroviral drugs, and HIV-infected patients with symptomatic, nucleoside-related hyperlactatemia. Finally, in the group of patients with hyperlactatemia, we quantified ratios of mitochondrial to nuclear DNA longitudinally before, during, and after the discontinuation of therapy.

Discussion

The assay we used to determine the mtDNA:nDNA ratio can be performed on peripheral blood collected fresh or previously frozen. The assay was most reliable in the low range of mtDNA:nDNA values (Figure 2C), which is the clinically relevant range. Mitochondrial DNA was significantly depleted in HIV-infected patients with symptomatic hyperlactatemia who were receiving antiretroviral therapy. The decrease in mitochondrial DNA preceded the rise in venous lactate levels, an observation that is consistent with the view that hyperlactatemia is a consequence of depletion of mitochondrial DNA, and it was reversible in all the patients we studied. Mitochondrial DNA:nDNA ratios were significantly lower in HIV-infected, asymptomatic patients who had never received antiretroviral therapy than in non-HIV-infected controls (P<0.001), a difference that was not explained by the lower CD4 counts in the former group. This finding is consistent with the results of recent in vitro studies in which HIV-infected cells had signs of mitochondrial necrosis.34 Furthermore, a mitochondria-controlled mechanism of cell death has been postulated in HIV infection.35-38

In inherited mitochondrial diseases, severe symptoms tend to occur when the levels of mitochondrial DNA reach approximately 20 percent of normal, which is similar to the level of depletion observed in HIV-infected patients with symptomatic hyperlactatemia who are receiving antiretroviral therapy. The in vivo doubling times of mitochondrial DNA estimated from this relatively small data set are similar to the time of approximately 35 days observed for cultured cells in which mitochondrial DNA had been depleted by ethidium bromide treatment.39

The fact that the mtDNA:nDNA ratios measured while the patients were not receiving antiretroviral therapy were similar to those observed once the patients resumed nucleoside-containing therapy without stavudine points toward a possible association between stavudine and mitochondrial toxicity.22-25,32 Lactate levels remained normal after the resumption of therapy without stavudine. In vitro, the triphosphated form of stavudine is incorporated into DNA more readily than other currently used nucleoside analogues and exerts the greatest inhibition on the human polymerase γ.2 This may explain the apparent association between mitochondrial toxicity and stavudine. Further studies are needed to establish the relative mitochondrial toxicity in vivo of various nucleoside analogues.

The frequency of lactic acidosis in patients treated with nucleoside analogues has been retrospectively estimated to lie between 1 and 2 cases per 1000 person-years.26 Another study with a broadened case definition of hyperlactatemia estimated the incidence at 20.9 cases per 1000 person-years of treatment.22 We found that approximately 20 percent of patients receiving antiretroviral therapy had elevated lactate levels in random venous samples.32 A validated quantitative mitochondrial DNA assay could be a useful tool to monitor and evaluate mitochondrial toxicity among HIV-infected patients receiving antiretroviral therapy, as well as among patients with other diseases, such as hepatitis and cancer, which are also treated with nucleoside analogues.

Results

Mitochondrial DNA Assay


The mitochondrial DNA assay made use of real-time PCR to quantify the mitochondrial DNA content of blood cells in relation to their nuclear DNA content. The standard curves for both the mitochondrial gene (CCOI) and a nuclear gene (ASPOLG) were generated by serial dilution of the male HIV-negative DNA pool and showed linearity over the range studied (Figure 1Figure 1Typical Real-Time Polymerase-Chain-Reaction Standard Curves Generated for the Nuclear Gene for the Human Polymerase γ Accessory Subunit and the Mitochondrial Gene Human Cytochrome-c Oxidase Subunit I with Use of Serial Dilutions of the Pooled DNA Extracts from HIV-Negative Male Volunteers.). The level of agreement of the duplicate measurements performed for this study is illustrated in Figure 2Figure 2Levels of Agreement between Duplicate Measurements of the Nuclear Gene for the Human Polymerase γ Accessory Subunit (Panel A), the Mitochondrial Gene Human Cytochrome-c Oxidase Subunit I (Panel B), and the Ratio of Mitochondrial to Nuclear DNA (Panel C) Derived from Them.. For the nuclear and the mitochondrial gene, respectively, 96 percent and 98 percent of the differences between duplicate measurements were less than 2 SD from the mean difference, with Pearson's correlation coefficients of 0.94 and 0.99 and coefficients of determination of 89 percent and 97 percent, respectively. For the mtDNA:nDNA ratio (Figure 2C), the level of agreement was highest at lower values of the ratio, and overall, 92 percent of duplicate measurements were less than 2 SD from the mean, with a Pearson's correlation coefficient of 0.72 and a coefficient of determination of 52 percent.

Population Comparison

The assay was applied to three groups of men: 24 non-HIV-infected controls, 47 HIV-infected, asymptomatic patients never treated with antiretroviral drugs, and 8 HIV-infected patients with symptomatic hyperlactatemia who were treated with antiretroviral drugs. Figure 3Figure 3Ratio of Mitochondrial to Nuclear DNA in the Three Groups of Subjects. presents the mtDNA:nDNA ratios obtained for the non-HIV-infected and HIV-infected control groups. The mean mtDNA:nDNA ratio among the non-HIV-infected controls was significantly higher than that among the HIV-infected asymptomatic patients never treated with antiretroviral drugs (P<0.001). Among the eight HIV-infected patients with symptomatic hyperlactatemia, the mean mtDNA:nDNA ratio of the last sample collected before antiretroviral therapy was discontinued was 0.28±0.06. This value was 22 percent of the value in the non-HIV-infected subjects (1.28±0.38) and 39 percent of the value in the HIV-infected asymptomatic patients never treated with antiretroviral drugs (0.72±0.19), and it was significantly lower than the value in either of these control groups (P<0.001).

Characteristics of the Patients

The eight patients receiving antiretroviral therapy (Table 1Table 1Characteristics of the Eight HIV-Infected Male Patients with Symptomatic Mitochondrial Toxic Effects and Their Antiretroviral Regimens.) had mitochondrial toxic effects characterized by varying degrees of progressive hyperlactatemia (Figure 4Figure 4Longitudinal Analysis of Venous Lactate Levels (Open Circles), mtDNA:nDNA Ratios (Solid Circles), and Antiretroviral-Drug Regimens (Bars) over Time in the Patients with Symptoms of Mitochondrial Toxicity.), fatigue, rapid weight loss, and a reduced anaerobic threshold during exercise testing.25 Their antiretroviral regimens varied from first-line to salvage therapy, and all were receiving stavudine. Antiretroviral therapy was interrupted in all eight patients as a result of the development of symptomatic hyperlactatemia. In all cases, lactate decreased and symptoms resolved on the discontinuation of antiretroviral therapy.

Longitudinal mtDNA:nDNA Ratios

Longitudinal measurements of mtDNA:nDNA ratios in samples collected before, during, and after antiretroviral therapy are presented in Figure 4. Among the eight patients with symptomatic hyperlactatemia, the mean mtDNA:nDNA ratio during therapy (0.41±0.08) was significantly lower than that among both the non-HIV-infected subjects (P<0.001) and the HIV-infected, asymptomatic patients who had never received antiretroviral therapy (P<0.001). The mean mtDNA:nDNA ratio during therapy was also significantly lower than both the mean mtDNA:nDNA ratio when the patients were receiving no therapy (0.74±0.13, P=0.02) and the mean mtDNA:nDNA ratio when the patients were not receiving stavudine (0.69±0.06, P=0.008). The mean mtDNA:nDNA ratio obtained for the HIV-infected, asymptomatic patients who had never received antiretroviral therapy was not significantly different from the mean mtDNA:nDNA ratio when the patients were receiving no therapy (P=0.43) or the mean mtDNA:nDNA ratio when the patients were not receiving stavudine (P=0.26).

The results of several laboratory tests were investigated to explore their possible relation with the mtDNA:nDNA ratio. Among the HIV-infected, asymptomatic patients who had never received antiretroviral therapy, CD4 cell counts were not significantly correlated with the mtDNA:nDNA ratio (Spearman's rho=0.08; P=0.59; mean CD4 cell count, 233±162 per cubic millimeter; range, 10 to 830). For the eight HIV-infected patients with symptomatic hyperlactatemia receiving antiretroviral therapy, there were also no significant correlations between the mtDNA:nDNA ratio and CD4 cell count (P=1.00; mean CD4 cell count, 191±146 per cubic millimeter; range, 10 to 620), platelet count (P=0.29), white-cell count (P=0.31), alanine aminotransferase level (P=0.25), aspartate aminotransferase level (P=0.08), albumin level (P=0.20), or the international normalized ratio (P=0.24).

Mitochondrial DNA and Lactate Levels

In Patients 1, 4, and 8 (Figure 4), the decrease in the mtDNA:nDNA ratio clearly preceded the development of hyperlactatemia (earlier data on lactate levels were unavailable for the other five patients). Similarly, in Patients 4, 6, and 8, the time required for the mtDNA:nDNA ratio to rebound was similar to or shorter than that needed for the hyperlactatemia to resolve (i.e., to reach the range of 0.5 to 2.1 mmol per liter). A weak correlation (Spearman's rho =-0.32, P=0.02) was observed between the lactate levels and the mtDNA:nDNA ratio.

On the basis of the limited mtDNA:nDNA data available from the eight HIV-infected patients with symptomatic hyperlactatemia who were receiving antiretroviral therapy, the maximal in vivo half-life of mitochondrial DNA was estimated to range from 4.5 weeks (Patient 3) to 8 weeks (Patient 4). The maximal in vivo doubling time of mitochondrial DNA was estimated to range from 4 weeks (Patient 5) to 16 weeks (Patient 1). Short lapses in therapy (as occurred in the cases of Patients 3, 4, and 7), as well as extremely low circulating drug levels (as in Patients 4, 6, and 7), were also associated with increasing levels of mitochondrial DNA. For these patients, the maximal time before lactate levels returned to the normal range after the discontinuation of antiretroviral treatment varied from 4 weeks (Patient 8) to 28 weeks (Patient 5).

 
 
 
 
  iconpaperstack view older Articles   Back to Top   www.natap.org