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  3rd International Workshop on Adverse Drug Reactions and Lipodystrophy in HIV
Athens, Greece - October 2001
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Mitochondrial Toxicity and Lipodystrophy
  It appears as though there are multiple contributing factors for lipodystrophy. A number of studies have suggested that NRTIs contribute to lipodystrophy by causing mitochondrial damage to cells. Examples of mitochondrial damage are neuropathy and pancreatitis. In the first study described below the results suggest long-term use of NRTIs may result in increasing accumulation of mitochondrial damage to cells. In the second study discussed below data from Cecilia Shikuma's small study suggests that mitochondrial toxicity is associated with lipodystrophy. This suggests a mechanism for NRTIs contributing to lipodystrophy.
Ulrich Walker reported on "Increased long-term mitochondrial toxicity in pyrimidine nucleoside combinations", in vitro (test tube experiment). For background Walker reported that some NRTIs cause depletion of mitochondrial (mt) DNA in liver and other tissues by inhibiting polymerase-y. This may provoke clinically relevant lactic acidosis, steatohepatitis (fat in liver), and infrequently liver failure. His objective here was to evaluate the long-term mitochondrial toxicity of NRTI-combinations.
Walker reported that mtDNA depletion was most rapid and pronounced with ddC and declined in order of ddI>d4T>3TC=AZT. AZT and AZT/3TC in combination behaved peculiarly in increasing lactate and cell death independent of a decline of mtDNA and without dramatically augmenting intracellular lipids. The toxicity of all the other NRTIs and their combinations was largely related to mtDNA depletion, which preceded or coincided with a decline in COX II-expression, a decrease in cell growth, increased lactate production and increased intracellular lipids. The effects of the 3TC/AZT, 3TC/d4T, and ddC/d4T combinations were more pronounced than those of either component alone. The combination of ddI and d4T was not more toxic than ddI alone. In some tests the steady-state levels of mitochondrial damage were not reached at day 30. At some concentrations of ddI, of d4T and virtually all tested NRTI-combinations lactate, cell growth, and mtDNA depletion continued to worsen. No effects were observed with efavirenz. In sum, walker said the in vitro data indicate possible additive or synergistic long-term mitochondrial toxicity of the pyrimidine nuke combination. Mitochondrial damage due to some NRTI-concentration/combinations can worsen beyond one month of incubation. The implication is that mitochondrial damage accumulates over time. The longer patients are taking NRTIs it's possible mitochondrial damage may increase. It has also been speculated that 50% or more mitochondrial damage is needed to be seen before real damage can occur. Healthy persons also experience mitochindrial damage. It can be passed on genetically. Presumably, mt varies by individual, some persons may experience higher rates than others. Mitochondrial damage can occur in a variety of cells in the liver, muscles, etc. Ribavirin is a NRTI and there is controversy over whether or not it can lead to mitocondrial toxicity.

Quantitative Testing of Mitochondrial Toxicity
Cecilia Shikuma reported on "Competitive PCR-analysis of subcutaneous adipose tissue mitochondrial DNA from individuals with HAART associated lipidystrophy".
For background, Shikuma said semi-quantitative analysis for mitochondrial toxicity to DNA has previously been reported from individuals with HAART associated lipodystrophy. Because the cause of HIV-associated lipodystrophy is unknown and studies have shown that mitochondrial toxicity may come from HAART, a more quantitative mtDNA assessment of affected tissue may help in determining the cause of fat redistribution.
Shikuma used double competitive PCR to determine the number of copies of mtDNA per cell from biopsy specimens in cells in stomach tissue from HIV-infected individuals with lipodystrophy. A total of 29 patients were recruited, 10 subjects with lipodystrophy:
    HIV   ART- Naive   Non-Lipo   Lipo
N   7   5   7   10
MtDNA Copies/Cell Mean   1057   280   219   59
Log10 mtDNA Mean   2.89   2.42   1.74   1.45
% Mean Change
Compared to HIV
  ---   -16.0%   -39.6%   -49.8%
  Shikuma summarized, re-analysis of abdominal wall subcutaneous adipose tissue (in the belly) mt DNA content by the competitive PCR method confirm our previous report of significant mtDNA decrease in lipodystrophic subjects with fat wasting compared to HIV- subjects and antiretroviral naïve subjects. The decrease in comparison to non-lipodystrophic individuals did not reach significance (perhaps due to the small number of patients in the study). Since the difference between the non-lipo and lipo groups was not significant increased mt depletion due to lipodystrophy is suggestive. I suppose you could say that both groups (non-lipo and lipo) are ART experienced and the increased mt depletion could be due to HIV therapy, and not necessarily due to lipodystrophy.
A mean decrease of 49% in subcutaneous adipose (belly) tissue mtDNA copies/cell was found by this method in individuals with lipodystrophy compared to HIV negative persons.