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Assessment of mitochondrial toxicity by analysis of mitochondrial protein expression in mononuclear cells
Author(s) -
Lin ChenHan,
Sloan Derek D.,
Dang Chung H.,
Wagner Thor,
Cabrera Albert J. E.,
Tobin Nicole H.,
Frenkel Lisa M.,
Jerome Keith R.
Publication year - 2009
Publication title -
cytometry part b: clinical cytometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.646
H-Index - 61
eISSN - 1552-4957
pISSN - 1552-4949
DOI - 10.1002/cyto.b.20458
Subject(s) - mitochondrial toxicity , mitochondrial dna , microbiology and biotechnology , biology , mitochondrion , peripheral blood mononuclear cell , didanosine , ethidium bromide , stavudine , cytochrome c oxidase , dna , virology , biochemistry , in vitro , gene , viral load , virus , antiretroviral therapy
Background: Real‐time PCR has quantified decreased mitochondrial DNA levels in association with nucleoside reverse transcriptase inhibitor (NRTI) therapy of HIV‐infected populations. However, real‐time PCR is best suited to distinguish log differences in an analyte. In an effort to monitor individuals in more detail, we developed a flow cytometric assay to gauge mitochondrial function. Methods: Flow cytometric quantification of a mitochondrial DNA‐encoded mitochondrial protein (cytochrome c oxidase subunit I (COX‐I)) and a nuclear DNA‐encoded mitochondrial protein [ATP synthase subunit D (Sub‐D)] was optimized and validated. Results: Intra‐assay and interassay variability was low using peripheral blood mononuclear cells (PBMCs) (CV of 6.15% for COX‐I and 7.11% Sub‐D, and 9.38% and 9.83% for COX‐I and Sub‐D, respectively). Mitochondrial protein depletion was evident with in vitro treatment of cells with ethidium bromide (EtBr) and zalcitabine (ddC). Mitochondrial protein expression in 40 healthy adults clustered tightly. Depletion of mitochondrial protein, however, was neither detected in cryopreserved PBMC from NRTI‐treated children ( n = 9) nor in adults with a history of symptoms consistent with mitochondrial toxicity or ongoing treatment with didanosine (ddI) or stavudine (d4T) ( n = 51). Conclusions: A validated flow cytometric assay allows simultaneous detection of mitochondrial DNA and nuclear DNA encoded proteins at the single cell level, offering a method to monitor for mitochondrial function. Prospective studies are required to evaluate whether mitochondrial protein loss is observed in at‐risk patients prior to the onset of symptoms from mitochondrial dysfunction. © 2008 Clinical Cytometry Society