NRTIs and mitochondrial toxicity

However, lipodystrophy-associated changes can occur in the absence of protease inhibitors, suggesting that other mechanisms are contributing to the development of the problems. At this stage, no one is sure if protease inhibitors contribute relatively little to the process, or if there are multiple potential pathways by which the syndrome can develop.

Dutch researchers led by Dr Kees Brinkman published a paper in The Lancet which argued that mitochondrial toxicity caused by the NRTIs is crucial in the development of fat and metabolic disorders associated with HAART. They drew parallels between HAART-related lipodystrophy and alcohol-related multiple symmetrical lipomatosis type 1 (MSL 1), both linked to mitochondrial dysfunction as well as abnormal fat accumulation and peripheral wasting. See also Lactic acidosis / acidaemia in A to Z of illnesses for more on other mitochondrial toxicities and the effects of elevated lactate levels.

Mitochondria are normal structures in human cells which are responsible for energy production. The mitochondria contain genetic material which may mutate to cause mitochondrial disease. Mutations in mitochondrial DNA cause disorders such as mitochondrial encephalopathy (ME), lactic acidosis, stroke-like episodes, a type of epilepsy and an eye disease called Leber's hereditary optic atrophy. Mitochondrial DNA is easily damaged, and cannot be repaired if polymerase gamma (a protein responsible for replication of mitochondria) is inhibited by nucleoside analogues. As mitochondrial DNA becomes more damaged, oxidative phosphorylation declines, leading to cell damage, toxicity and cell death. According to this theory, mitochondrial damage may lead to wasting of subcutaneous fat leading to fat loss in the legs, arms, buttocks and face. A different effect may occur if mitochondrial DNA in central fat cells is impaired; in this situation, lipids could accumulate within cells.

While at first sight this may appear contradictory, different NRTIs are likely to have different effects in different tissues, depending on the distribution and penetration of NRTIs into particular cells, the distribution of adipocytes and the type of fat in different locations, and the cellular capacity to break down NRTIs. ¹ It is already apparent, for example, that the NRTIs have different mitochondrial toxicities in other tissues, causing peripheral neuropathy, pancreatitis and myopathy in a drug-specific manner.

The Dutch team has also proposed that the extent to which various NRTIs inhibit mitochondrial polymerase gamma will affect the severity of mitochondrial toxicity associated with particular drugs.¹ Certainly a hierarchy of effects corresponds with the extent of polymerase gamma inhibition. However, a recent in vitro study found toxicity was not enhanced when d4T was added to ddI, although both drugs alone significantly impact on polymerase gamma. This suggests that inhibition of polymerase gamma may not be the key mechanism which produces mitochondrial toxicity. ²

An Australian group has closely examined fat tissue from people treated with PI and non-PI HAART. Electron microscopes revealed abnormal fat cell structure with prominent mitochondrial abnormalities, lipid accumulation in the cytoplasm and loss of fat cell volume. These changes were associated with fat cell loss and lipogranulomata formation. ³ The same group found an association between elevated lactate levels and the more rapid onset of lipoatrophy (lactate is elevated when mitochondrial DNA is damaged).

Researchers in Germany and Hawaii have found significantly reduced levels of mitochondrial DNA in the subcutaneous fat tissue of NRTI-treated individuals when compared with HIV-positive untreated controls, and there was also a significantly lower level in NRTI-treated patients who had experienced fat loss on treatment when compared with those on NRTIs who remained unaffected.^{4 5} Reduced levels of mitochondrial DNA are also associated with symptomatic high lactate in people taking NRTIs.⁶

The German group found that the extent of mitochondrial DNA reduction was significantly associated with the duration of NRTI therapy and Dr Ulrich Walker, who conducted the study, told the Second International Workshop on Lipodystrophy that if mitochondrial DNA continued to decline at the rate seen hitherto (if compared to the control group and assessed by average duration of therapy), mitochondrial DNA would be reduced by 50% within seven years. The reduction in mitochondrial DNA was significantly associated with d4T use, but it is important to note that this study only looked at 19 NRTI-treated patients, of whom 14 received d4T. There was also a trend towards reduced mitochondrial DNA in 3TC recipients. The study did not investigate the total duration of NRTI therapy and possible associations with NRTIs used prior to the current regimen, so these data should be treated with caution.

The German team has also studied the effect of the NRTIs on human fat cells in laboratory experiments. The NRTIs ddI, ddC and d4T were found to deplete mitochondrial DNA and affect cell growth, lipid levels, lactate production and the expression of a protein encoded by mitochondrial DNA known as cytochrome C oxidase subunit II (COX II). In contrast, AZT did not affect mitochondrial DNA or COX II but it did impair cell growth, and increase lactate and intracellular lipid levels. 3TC had no significant effects. ²

A study in mice found that while d4T did not cause mitochondrial DNA reduction in the adipose tissue of normal mice, obese mice experienced a 45% reduction in adipose mtDNA at peak plasma d4T concentrations three times lower than those expected in humans. ⁷ When the dose was increased fivefold, normal mice had lost nearly 50% of their mtDNA in liver and muscle tissue after six weeks of treatment.

Dutch researchers have reviewed body composition and mtDNA levels in adipose tissue of 28 participants in a randomised comparison of d4T/3TC and AZT/3TC, in which patients added indinavir at week 12. The original diagnosis of lipoatrophy in this study was made by a physician who was blinded to the original study medication, to avoid bias. They found that despite a similar duration of drug exposure for d4T and AZT, d4T-treated patients had a significantly greater prevalence of lipoatrophy (82% vs 9%), significantly less peripheral fat as measured by DEXA and significantly lower levels of mtDNA in subcutaneous fat cells. Stavudine treated patients were 56 times more likely to have lipoatrophy (p = 0.0002), but protease inhibitor exposure did not affect the risk of lipoatrophy. The group also found a significant relationship between the amount of mtDNA in subcutaneous fat and the ratio of peripheral to total fat as measured by DEXA, leading the authors to conclude that mtDNA loss is associated with lipoatrophy, and that d4T is more strongly associated with lipoatrophy than AZT. ⁸

A limitation of this study is that it included only 62% of the patients originally randomised; ten withheld consent and seven were lost to follow-up. However, there was no significant difference between the characteristics of participants in this study and those of the entire trial group. Another possible limitation is that the relationship between mtDNA levels and the ratio of peripheral fat to total fat was only found to be significant in thigh fat samples; the relationship was not found to be significant in the fat sample taken from the lower back.

In the TARHEEL study, in which 118 individuals with fat wasting were switched from d4T to either AZT or abacavir, restoration of fat was accompanied by an increase in levels of mitochondrial DNA in PBMCs and muscle tissue. The study did not report on mtDNA levels in adipocytes however, and did not include a d4T-treated control group. ⁹

Australian researchers examined mitochondrial DNA levels in 23 HAART-treated patients with lipodystrophy and eleven treatment-naive HIV-positive controls matched for body mass index and found that mtDNA levels were significantly depleted only in d4T-treated patients (13% of control levels). Markers of adipocyte differentiation were also suppressed in d4T-treated patients, but upregulated in AZT-treated patients. The same group conducted a cross-sectional analysis of mitochondrial DNA levels in the adipose tissue of 60 patients, and longitudinal analysis of 8 patients to determine the relationship between severity of fat wasting and mtDNA levels. They found that patients treated with d4T were likely to suffer significantly greater mtDNA depletion than AZT-treated patients, but that NRTI-treated patients as a whole had significantly lower mtDNA levels than untreated patients, and that NRTI-treated patients experienced mtDNA reductions in adipocytes of between 14% and 81% within eight months of commencing therapy. ¹⁰

Updating these findings in 2004, David Nolan reported on what is now a substantial cohort of patients who have undergone fat biopsies. One hundred and three patients underwent 147 fat biopsies. Forty-one treatment-naive patients have been studied, together with 38 AZT-treated patients and 30 d4T-treated patients. The cohort has also gathered data from 24 patients who have started treatment with nucleoside analogue backbones that do not include AZT or d4T, reflecting a growing trend towards avoidance of thymidine analogues in first-line HIV treatment.

The mean mitochondrial DNA level at baseline in treatment-naive patients was 1427 copies per cell, with no significant difference according to race, CD4 cell percentage or gender.

Amongst patients treated with AZT or d4T, mitochondrial DNA levels in adipose tissue declined by at least 60% within six to twelve months and this change was statistically significant in comparison to treatment-naive patients.

In contrast, 17 patients who commenced treatment with an abacavir-containing regimen that excluded AZT showed no significant reduction in mitochondrial DNA levels compared to treatment-naive patients. Similarly, three patients who commenced tenofovir-containing regimens showed no evidence of mitochondrial DNA reduction.

A second longitudinal study, also carried out in Australia, also found that lipoatrophy was associated with mtDNA depletion, but found differential effects depending on the cells sampled. Fat biopsies were obtained from 122 patients.

Whilst current d4T, ddI or ddC treatment was associated with lowered limb fat mtDNA, only ddI and ddC were associated with lowered mtDNA in PBMCs. AZT had a greater effect on mtDNA in suprailiac limb fat than abacavir, 3TC or tenofovir, but a lesser effect than the dideoxynucleotides. No effect of age, duration of HIV infection, time on treatment, protease inhibitor treatment or viral load was seen on mtDNA levels.

Although most research has focussed on the effect of drugs on adipose tissue mitochondria, one study has shown that mitochondrial DNA levels in plasma PBMCs are predictive of later development of lipodystrophy.

The analysis was carried out by French researchers using stored blood samples from the ALBI study, which randomised treatment-naive patients to receive either AZT/3TC, AZT/3TC alternating with d4T/ddI, or d4T/ddI for 48 weeks. Stored samples containing peripheral blood mononuclear cells were available for 37 of 51 patients in the AZT/3TC arm and 40 of 51 patients in the d4T/ddI arm. Blood samples were drawn at baseline and when individuals had completed 48 weeks of treatment.

The researchers found that:

• The mean mitochondrial DNA content fell from 5130 copies/cell at baseline to 2450 copies/cell at week 48.
• Although mitochondrial DNA levels were not significantly different at weeks 0 and 24, by week 48 they were significantly lower in the d4T/ddI group (1950 vs. 3020 copies/cell).
• Thirty-nine per cent of patients had at least one symptom of lipodystrophy 30 months after starting treatment. Forty-four per cent of these patients had mitochondrial DNA levels below 1410 copies/cell, compared to 7% of those without lipodystrophy. The odds ratio (OR) of lipodystrophy at month 30 in patients with mitochondrial DNA below 1410 copies/cell was 9.8. Thirty-five per cent of those originally randomised to AZT were still taking the drug at month 30, compared to 63% of the d4T group.
• Although d4T/ddI treatment was significantly associated with lipodystrophy (OR 2.3), after adjustment for mitochondrial DNA at week 48, there was no significant difference in the risk of lipodystrophy in d4T/ddI-treated patients when compared to AZT and 3TC-treated patients.

Evaluating mitochondrial DNA levels in this population as a predictor of lipodystrophy excludes any complicating effect of a third drug that might also influence the way in which lipodystrophy manifests itself, but does not reflect clinical practice today, nor the effect of protease inhibitors on mitochondrial function. ¹¹

While the theory of mitochondrial toxicity has considerable currency, not all research supports this theory. For example, a comparative study found no evidence that antiretroviral treatment was causing mitochondrial DNA mutations in people with HIV. ¹² This provided support for earlier findings that linked mitochondrial abnormalities to HIV infection itself. ^{13 14} Other studies have shown that some people with lipoatrophy have no reduction in mitochondrial DNA compared with a control group, ¹⁵ whereas some HIV-negative people do have lower than average levels, suggesting wide variability within the population (and/or the possibility that mtDNA may be distributed at random levels in fat tissue). In addition, a recent study has shown that measuring levels of mitochondrial DNA does not predict the development of lipoatrophy or other drug toxicities associated with nucleoside analogues. ¹⁶ In this study, the extent of mitochondrial DNA depletion was not associated with the presence of lipoatrophy, and one third of the nucleoside analogue-treated patients in the study showed an increase in mitochondrial DNA levels.

Although mitochondrial DNA may be depleted, several groups have shown that mitochondria are actually working harder (as measured by fat oxidation) in patients receiving protease inhibitors plus NRTIs, possibly in response to hypermetabolism. ^{17 18} This, rather than NRTI toxicity, might also explain why mitochondria show altered shape (morphology) in patients with lipodystrophy.

Experience in the field of inherited mitochondrial disorder has shown that the brain is commonly affected (either in the form of encephalitis or dementia), but the only nervous system disorder seen with NRTI-associated mitochondrial toxicity is peripheral neuropathy. Indeed, nucleoside analogues have a protective effect against the development of HIV-associated dementia.

Mitochondrial DNA mutations associated with fat accumulation in Madelung's syndrome are not seen in HIV lipodystrophy.

Elevated lactate levels in patients taking NRTIs, often cited as a marker of NRTI toxicity to mitochondrial DNA, may be a consequence of reduced clearance of lactate in the liver as a consequence of elevated triglyceride levels and hepatic steatosis (fat accumulation in the liver). ¹⁹ Finally, adipocytes are poor at phosphorylating the thymidine analogues AZT and d4T ^{20 21} in comparison to activated T-lymphocytes.

One group of researchers has developed a variation on the mitochondrial toxicity theory. The theory is that low levels of high density lipoprotein (HDL) plus mitochondrial toxicity lead to fat redistribution. HDL plays an important role in clearing lipids to the liver and is often low during HIV infection. The theory is that mitochondrial toxicity promotes the loss of white fat and the expansion of brown fat (which contains more mitochondria and may be less susceptible to mitochondrial toxicity). An inverse relationship was found between HDL and visceral fat confirming the relationship between low HDL and lipodystrophy. ²²

Mitochondrial activity is also impaired by HIV infection itself. In a Spanish study samples of peripheral blood mononuclear cells were obtained from 25 HIV-positive patients who had never taken antiretroviral drugs and 25 age and sex matched HIV-negative individuals. The HIV-positive patients had been diagnosed with HIV infection for a mean of 44 months, had a median CD4 cell count of 317 cells/mm³ and an HIV viral load of 100,000 copies/ml.

Investigators assessed mitochondrial DNA content, activity in the mitochondrial respiratory chain, the activity of a key enzyme involved in mitochondrial function, and lipid peroxidation, a marker of oxidative damage.

Mitochondrial DNA content was 23% lower in the HIV-positive patients (p < 0.05) than the HIV-negative controls. Activity in the mitochondrial respiratory chain (MRC) was also significantly lower in the HIV-infected individuals, MRC complex III being decreased by 38% (p < 0.001) and MRC complex IV by 19% (p = 0.001). ²³

The investigators suggest that the effect of HIV on mitochondria may increase their vulnerability to the effects of nucleoside analogues, although no association was found between CD4 cell count and mitochondrial DNA content.