Fruit and vegetable consumption, cigarette smoke, and leukocyte mitochondrial DNA copy number

Abstract

Background: Mitochondrial dysfunction is an important component of the aging process and has been implicated in the development of many human diseases. Mitochondrial DNA copy number (mtDNAcn), an indirect biomarker of mitochondrial function, is sensitive to oxidative damage. Few population-based studies have investigated the impact of fruit and vegetable consumption and cigarette smoke (2 major sources of exogenous antioxidants and oxidants) on leukocyte mtDNAcn.

Objectives: We investigated the association between fruit and vegetable consumption, cigarette smoke, and leukocyte mtDNAcn based on data from the Nurses' Health Study (NHS).

Methods: Data from 2769 disease-free women in the NHS were used to examine the cross-sectional associations between dietary sources of antioxidants, cigarette smoke, and leukocyte mtDNAcn. In vitro cell-based experiments were conducted to support the findings from the population-based study.

Results: In the multivariable-adjusted model, both whole-fruit consumption and intake of flavanones (a group of antioxidants abundant in fruit) were positively associated with leukocyte mtDNAcn (P-trend = 0.005 and 0.02, respectively), whereas pack-years of smoking and smoking duration were inversely associated with leukocyte mtDNAcn (P-trend = 0.01 and 0.007, respectively). These findings are supported by in vitro cell-based experiments showing that the administration of naringin, a major flavanone in fruit, led to a substantial increase in mtDNAcn in human leukocytes, whereas exposure to nicotine-derived nitrosamine ketone, a key carcinogenic ingredient of cigarette smoke, resulted in a significant decrease in mtDNAcn of cells (all P < 0.05). Further in vitro studies showed that alterations in leukocyte mtDNAcn were functionally linked to the modulation of mitochondrial biogenesis and function.

Conclusions: Fruit consumption and intake of dietary flavanones were associated with increased leukocyte mtDNAcn, whereas cigarette smoking was associated with decreased leukocyte mtDNAcn, which is a promising biomarker for oxidative stress-related health outcomes.

FIGURE 1

Effect of naringin and NNK on mtDNAcn in HL-60 cells. Cells were treated with 100 μM naringin (A) or 200 μM NNK (B) for 24 h, and the relative mtDNAcn was analyzed by quantitative real-time PCR with a specific primer against mtDNA and β-actin, as described in Methods. The mtDNA genes ND1, ND2, COI, and COII were used to represent mtDNAcn. Values are means ± SDs from 3 independent experiments. *Different from control, P < 0.05 (Wilcoxon's rank-sum test). COI, cytochrome c oxidase subunit I; COII, cytochrome c oxidase subunit II; mtDNAcn, mitochondrial DNA copy number; ND1, nicotinamide adenine dinucleotide dehydrogenase subunit 1; ND2, nicotinamide adenine dinucleotide dehydrogenase subunit 2; NNK, nicotine-derived nitrosamine ketone; PCR, polymerase chain reaction.

FIGURE 2

Effect of naringin and NNK on intracellular ROS in the leukocytes. The ratio of GSSG compared with total GSH concentration, ratio of NAD(P)H compared with total NAD(P) concentration, TBARS assay for assessment of lipid peroxidation products, and intracellular peroxide production were measured in the HL-60 cells after the treatment with 100 μM naringin (A) or 200 μM NNK (B) for 24 h. Values represent the fold change over the levels observed in the control and are shown as means ± SDs of 3 independent experiments. *Different from control, P < 0.05 (Wilcoxon's rank-sum test). GSH, glutathione; GSHt, total glutathione; GSSG, oxidized glutathione; H₂O₂, intracellular peroxide; NADPt, total NAD(P); NNK, nicotine-derived nitrosamine ketone; ROS, reactive oxygen species; TBARS, thiobarbituric acid reactive substance.