Detection of heteroplasmic mitochondrial DNA in single mitochondria.

BACKGROUND: Mitochondrial DNA (mtDNA) genome mutations can lead to energy and respiratory-related disorders like myoclonic epilepsy with ragged red fiber disease (MERRF), mitochondrial myopathy, encephalopathy, lactic acidosis and stroke (MELAS) syndrome, and Leber's hereditary optic neuropathy (LHON). It is not well understood what effect the distribution of mutated mtDNA throughout the mitochondrial matrix has on the development of mitochondrial-based disorders. Insight into this complex sub-cellular heterogeneity may further our understanding of the development of mitochondria-related diseases. METHODOLOGY: This work describes a method for isolating individual mitochondria from single cells and performing molecular analysis on that single mitochondrion's DNA. An optical tweezer extracts a single mitochondrion from a lysed human HL-60 cell. Then a micron-sized femtopipette tip captures the mitochondrion for subsequent analysis. Multiple rounds of conventional DNA amplification and standard sequencing methods enable the detection of a heteroplasmic mixture in the mtDNA from a single mitochondrion. SIGNIFICANCE: Molecular analysis of mtDNA from the individually extracted mitochondrion demonstrates that a heteroplasmy is present in single mitochondria at various ratios consistent with the 50/50 heteroplasmy ratio found in single cells that contain multiple mitochondria.

Effects of menthol on tobacco smoke exposure, nicotine dependence, and NNAL glucuronidation.

Menthol is a controversial cigarette additive because its physiologic or pharmacologic effects may possibly increase the risk for cancer and its targeted market is the Black community. In a community-based cross-sectional study on 525 Black and White volunteers, we compared levels of urinary and plasma cotinine, plasma thiocyanate, urinary 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAL), and its detoxified form (NNAL-Gluc) between menthol and nonmenthol smokers. In regression models that adjusted for daily cigarette intake, no significant differences were observed in the concentration of these biomarkers by menthol status in both races. There was no significant association between high Fagerstrom nicotine dependence scores and the use of menthol cigarettes (odds ratio, 1.1; 95% confidence interval, 0.6-2.0), but an increased risk was observed with smoking a cigarette soon (

Association between haplotypes of manganese superoxide dismutase (SOD2), smoking, and lung cancer risk.

Tobacco smoke contains high concentrations of reactive oxygen species (ROS) that can damage DNA, proteins, and lipids. Manganese superoxide dismutase (SOD2) catalyzes the dismutation of superoxide radicals into hydrogen peroxide and protects against oxidative stress in lung tissues. Three tagSNPs were identified in one block of high linkage disequilibrium that spans the entire SOD2 gene and 5-kb promoter region. These tagSNPs, representing four haplotypes (TAA, TCA, TCG, CCG), were genotyped in 372 lung cancer cases and 605 controls. There was no association between the haplotype frequencies and the overall lung cancer risk. The TCG haplotype (6% in controls) was significantly associated with a lower risk of lung cancer in light smokers (