Assessment of mitochondrial toxicity by analysis of mitochondrial protein expression in mononuclear cells.
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.
Lin
Chen-Han
CH
Vaccine and Infectious Disease Institute, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA.
Sloan
Derek D
DD
Dang
Chung H
CH
Wagner
Thor
T
Cabrera
Albert J E
AJ
Tobin
Nicole H
NH
Frenkel
Lisa M
LM
Jerome
Keith R
KR
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000328:Adult; D002648:Child; D002675:Child, Preschool; D004272:DNA, Mitochondrial / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism; D004996:Ethidium / Q000633:toxicity*; D005434:Flow Cytometry; D006801:Humans; D007223:Infant; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D008875:Middle Aged; D008928:Mitochondria / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D055815:Young Adult; D016047:Zalcitabine / Q000633:toxicity*
D004272:DNA, Mitochondrial; D024101:Mitochondrial Proteins; D016047:Zalcitabine; D003576:Electron Transport Complex IV; D000251:Adenosine Triphosphatases; D004996:Ethidium