The G11778A LHON mutation does not enhance ethambutol cytotoxicity in a cybrid model

Abstract

Leber's hereditary optic neuropathy (LHON) is a maternally inherited mitochondrial disorder, leading to a selective loss of retinal ganglion cells (RGC) and degeneration of the optic nerve, which results in severe visual impairment or even blindness. The primary causes are point mutations of the mitochondrial DNA (mtDNA), associated with aminoacid exchanges in complex I of the electron transport chain (ETC), which are thought to disturb oxidative ATP generation in the mitochondria. The major side effect of the antibiotic ethambutol, commonly used in tuberculosis therapy, is a retinopathy, which may lead to selective RGC loss, if not detected in an early stage. Moreover, LHON was reported to be elicited by ethambutol in some mutation carriers. The present study intended to measure a possible synergism between mitochondrial dysfunction, caused by the most common LHON mutation (G11778A) and caused by ethambutol, which may lead to a higher cytotoxicity of the drug in LHON cells. An NT2/D1 teratoma-derived LHON cybrid line and the parental cells. Determination of ethambutol toxicity in both lines, using a microtiter tetrazolium assay, luminometric measurement of ATP/ADP ratios and determination of mtDNA copy numbers by Real-time PCR. Short-term ethambutol toxicity occurred only at micromolar concentrations, far beyond the estimated plasma peak concentrations of patients under antibiotic therapy. No significant difference occurred between both cell lines. The ATP/ADP ratios in the cybrids were surprisingly low, but showed no correlation with the mutational status of drug-treated cells. The mtDNA copy number of treated LHON and parental cells did not differ significantly. Ethambutol shows no synergism with the most common primary LHON mutation with respect to mitochondrial energy production or mtDNA replication in cybrid cells, although the issue of ATP decline should be further addressed in neuronally differentiated cybrids with complete OXPHOS dependency.