Identifying sex-based disparities in porcine mitochondrial function

Mitochondria are the primary source of cellular energy. Mitochondrial DNA (mtDNA) copy number in peripheral blood leukocytes (PBLs) has high genetic heritability and inter-individual variation of mtDNA copy number in PBLs has been associated with the risk of several human cancers. In this study, we investigated into the link between mtDNA and clear-cell renal cell carcinoma (ccRCC) in a large population-based case control study. We measured relative mtDNA copy number in PBLs from 616 Caucasian ccRCC cases and 603 controls frequency-matched on age, gender, and ethnicity. In the analyses of controls only, there was a significant inverse correlation between age and mtDNA copy number (P=0.001); men had significantly lower mtDNA copy number than women (P<0.001); and both smoking and hypertension had a negative effect on mtDNA copy number (P=0.03 and 0.04, respectively). We found a significantly lower mtDNA copy number in cases compared to controls (mean±SD: 1.16±0.37 versus 1.21±0.39, P=0.04). Dichotomized at the median level of mtDNA copy number in controls, individuals with lower mtDNA copy number were at an increased risk of ccRCC (OR=1.20, 95% CI: 0.96-1.51, P=0.11) adjusting for age, gender, smoking status, BMI, and history of hypertension in a multivariate logistic regression analysis. In stratified analyses, the association between lower mtDNA copy number and increased ccRCC risks reached statistical significance in younger aged (age<60) individuals (OR=1.56, 95% CI: 1.12-2.15, P=0.01), women (OR=1.57, 95% CI: 1.06-2.35, P=0.03), and individuals without history of hypertension (OR=1.48, 95% CI: 1.08-2.04, P=0.02). Interestingly, there was a significant interaction between age and mtDNA copy number, and between a history of hypertension and mtDNA copy number in elevating ccRCC risk (P for interaction=0.02 and 0.03, respectively). In summary, our data showed that several established risk factors of ccRCC, including advanced age, male sex, smoking, and history of hypertension, negatively impacted mtDNA copy number in PBLs. Lower mtDNA copy number in PBLs was associated with significantly increased risk of ccRCC in younger aged individuals, women, never smokers, and those without a history of hypertension. There was a significant interaction between age and mtDNA, and a history of hypertension and mtDNA in elevating the risk of ccRCC. These findings indicate that the association between mtDNA copy number in PBLs and ccRCC risk may be modified by various host characteristics (age, gender, smoking, and history of hypertension), providing novel insight into the role of mtDNA in modulating RCC tumorigenesis. Citation Format: Xin Wang, Stephanie Melkonian, Jian Gu, David Chang, Nizar Tannir, Christopher Wood, Xifeng Wu. Mitochondrial DNA copy number in peripheral blood and the risk of clear-cell renal cell carcinoma: Effect of age, gender, smoking, and history of hypertension. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4320. doi:10.1158/1538-7445.AM2014-4320

Alterations in mitochondrial DNA (mtDNA) copy number are observed in human gliomas. However, whether variations in mtDNA copy number in whole blood play any role in glioma carcinogenesis is still largely unknown. In current study with 395 glioma patients and 425 healthy controls, we intended to investigate the association between mtDNA copy number in whole blood and glioma risk. Overall, we found that levels of mtDNA copy number were significantly higher in glioma cases than healthy controls (mean: 1.48 vs. 1.32, P < 0.01). In both cases and controls, levels of mtDNA copy number were inversely correlated with age (P < 0.01, respectively). And in cases, newly diagnosed, glioblastoma (GBM), and high grade glioma patients had significantly lower mtDNA copy number than their counterparts (P = 0.02, P < 0.01, and P = 0.04, respectively). In the multivariate analysis, elevated mtDNA copy number levels were associated with a 1.63-fold increased risk of glioma (adjusted odds ratio (OR) = 1.63, 95% confidence interval (CI) = 1.23-2.14). In further quartile analysis, study subjects who had highest levels of mtNDA copy number had 1.75-fold increased risk of gliomas (adjOR = 1.75, 95%CI = 1.18-2.61). In brief, our findings support the role of mtDNA copy number in the glioma carcinogenesis. © 2016 Wiley Periodicals, Inc.

Background: Graft-versus-host disease (GvHD) continues to be a major cause of transplant related mortality, morbidity and decreased quality of life post allogeneic hematopoietic cell transplant (HCT). Despite extensive use of histocompatibility antigen (HLA) matching between patients and donors and a variety of prophylactic regimens, the rates of GvHD have not declined substantially over the last decade. Recent studies have shown mitochondrial reactive oxygen species (ROS) plays an important role in T lymphocyte activation and initiation of the inflammatory response and represents a novel biological pathway that influences GvHD risk. We hypothesized that lower mitochondrial DNA (mtDNA) copy number that increases ROS related oxidative damage will be associated with acute and chronic GvHD. Methods: In a cohort of 201 patients who underwent allogeneic HCT for treatment of hematologic malignancies at the University of Minnesota between 2009 and 2012, we evaluated the association between donor post HCT mtDNA copy number at 7 time points (days 21, 28, 60, 100, 180, 365 and 730) and acute and chronic GvHD. Recipient blood was separated into CD3+ (T-lymphocytes) and CD15+ (neutrophil) fractions at each time point using magnetic bead separation. All recipients had 100% donor engraftment at the various time points for inclusion in the study. We used a quantitative real time PCR method, comparing the number of copies of the mitochondria- specific gene (ND1) to the number of copies of a nuclear gene (18s) to estimate mtDNA copy number. We used a linear mixed model adjusting for fixed effects of donor type (sibling, unrelated and cord blood donors), donor age, the interaction of GVHD status with time, and random effects for subject and assay run number. The outcome (CD3+/CD15+ mtDNA copy number) was log-transformed to improve model fit and normalize residual variance. Results: Overall CD3+ mtDNA copy number was lower among patients with grade II-IV acute GvHD (n = 90) as compared to those without acute GvHD (n = 79) (2.03 vs. 2.18, respectively; p = 0.02), while there were no significant differences in CD15+ mtDNA copy number between the two groups (p = 0.49). Similarly, CD3+ mtDNA copy number was lower among patients with chronic GvHD (n = 43) as compared to those without chronic GvHD (n = 79) (1.82 vs. 2.24, respectively; p = 0.01) while there were no significant differences in CD15+ mtDNA copy number between the two groups (p = 0.47). Conclusion: Our study shows that mtDNA is decreased in acute and chronic GVHD compared to transplant recipients without this complication, providing preliminary evidence that mtDNA copy number may serve as a biomarker for early detection and monitoring of GvHD. Future studies that evaluate the significance of altered mtDNA copy number on T lymphocyte function and ROS are needed to evaluate the utility of novel mt-based interventions for treatment of GvHD. Citation Format: Bharat Thyagarajan, Ryan Shanley, Pallavi Prakash, Helene Barcelo, Julie A. Ross, Michael R. Verneris, Mukta Arora. Lower T-lymphocyte mitochondrial DNA copy number is associated with graft versus host disease in patients undergoing hematopoietic cell transplantation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 600. doi:10.1158/1538-7445.AM2015-600

Previous epidemiological studies on mitochondrial DNA (mtDNA) copy number and risk of colorectal cancer have yielded mixed results. One study reported a positive association, another reported an inverse association, and a third, a U-shaped association of mtDNA copy number with colorectal cancer risk. Since oxidative stress is one of the proposed underlying mechanisms that may link mtDNA copy number variation to colorectal cancer risk, we conducted a matched case-control study of incident colorectal cancer nested within the Singapore Chinese Health Study. Pre-diagnostic blood and urine samples were obtained from 448 colorectal cancer cases and 869 age- and sex-matched controls. The mtDNA copy number in peripheral white cells was measured using real time PCR and urinary F2-isoprostanes were quantified using a liquid chromatography mass spectrometry based method. Conditional logistic regression was used to estimate associations of mtDNA copy number and urinary F2-isoprostanes with incident colorectal cancer. The odds ratios (OR) and 95% confidence intervals (95% CI) for those in the highest relative to those in the lowest quartiles of relative mtDNA copy numbers and urinary F2-isoprostanes were 1.02 (95% CI 0.72 - 1.43; p for trend = 0.56) and 1.11 (95% CI 0.78-1.59; p for trend = 0.35) respectively. Urinary F2-isoprostanes levels were not associated with the mtDNA copy number among the controls (r = 0.03; p = 0.33). The results of this prospective study suggest that white blood cell mtDNA copy number or urinary levels of F2-isoprostanes in biospecimens collected several years prior to the diagnosis of colorectal cancer may not be associated with risk of developing colorectal cancer. Citation Format: Bharat Thyagarajan, Renwei Wang, Woon-Puay Koh, Helene Barcelo, Jennifer Adams-Haduch, Weihua Guan, Roberd M. Bostick, Jian-Min Yuan, Myron D. Gross. Mitochondrial DNA copy number, urinary isoprostanes and colorectal cancer risk. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4281.

Genome instability in Maple Syrup Urine Disease correlates with impaired mitochondrial biogenesis

Background: Mitochondrial DNA (mtDNA) copy number variation is common in colorectal tumors and one case control study showed mtDNA copy number to be associated with increased colorectal cancer risk. Data on mtDNA copy number and colorectal cancer risk in prospective cohorts is lacking. Objectives: We evaluated the association between mtDNA copy number in peripheral blood and colorectal cancer risk among participants of the Singapore Chinese Health Study. Methods: We conducted a nested case-cohort study of 422 colorectal cancer cases (168 incident cases with pre-diagnostic blood and 254 prevalent cases with post-diagnostic blood) and 880 controls who were free of colorectal cancer at blood draw. The relative mtDNA copy number was measured using real time PCR. Analysis of covariance and logistic regression methods were employed to examine the association between mtDNA copy number and colorectal cancer risk. Results: mtDNA copy number was significantly higher among colorectal cancer cases than controls (geometric mean: 0.62 vs. 0.50, p&amp;lt;0.0001). There was a U shaped relationship between the mtDNA copy number and colorectal cancer risk. Individuals in the low and high mtDNA copy numbers were at increased risk of colorectal cancer; odds ratios (95% confidence interval) for developing colorectal cancer for the lowest and highest quartile of mtDNA copy numbers were 1.83 (1.16-2.90) and 3.54 (2.25-5.55), respectively, compared to those in the second quartile of mtDNA after adjustment for potential confounders (p for curvilinear trend &amp;lt;0.001). The lowest mtDNA copy number quartile was associated with colorectal cancer only in the pre-diagnostic blood samples (OR=3.22, 95% CI:1.51-6.85) but not in the post-diagnostic blood samples (OR=1.36, 95% CI:0.80-2.32), implying that cancer status impacts the mtDNA copy number-colorectal cancer risk association. Conclusions: This is the first study to show a prospective association between mtDNA copy number and colorectal cancer risk. Further studies are needed to confirm this association and understand the biological significance of these observations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4486. doi:1538-7445.AM2012-4486

Available evidence supports the role of reactive oxygen species in the pathogenesis of placental insufficiency, gestational diabetes mellitus (GDM), and other pregnancy complications. Abnormal placental mitochondrial function resulting from reactive oxygen species may also be an important precedent of adverse perinatal outcomes. We investigated the association of placental oxidative stress with placental mitochondrial DNA (mtDNA) copy number, an indicator of placental mitochondrial density and possible mitochondrial dysfunction, using samples collected from GDM cases and controls. 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, was measured in placentas of 19 GDM cases and 21 controls using a competitive immunoassay. Placental mtDNA copy number was determined using real-time quantitative PCR. Bivariate and multivariable linear regression procedures were used to evaluate associations of these two biomarkers. Placental DNA oxidation was positively associated with mtDNA copy number in both GDM and control placentas. After adjusting for maternal age, pre-pregnancy body mass index and gestational age at delivery, mtDNA copy number increased (beta = 67.0; 95% CI 27.8 - 106.2, p = 0.001) for every 0.1 ng/microg increase of placental 8-OHdG among GDM cases and controls. These cross sectional data suggest an association of placental mtDNA copy number with oxidative stress. The consequences of placental oxidative stress and mitochondrial dysfunction on the course and outcomes of pregnancy remain to be elucidated in larger prospective studies.

Recent studies suggest a link between insulin resistance and mitochondrial function in white fat cells. The aim of this study was to evaluate adipocyte mitochondrial DNA (mtDNA) copy number in relation to adipocyte and clinical variables that are related to insulin sensitivity. We studied a group of 148 healthy volunteers with a large inter-individual variation in BMI. Relative amounts of mtDNA and nuclear DNA were determined by quantitative RT-PCR. The mtDNA:nuclear DNA ratio reflects the tissue concentration of mtDNA per cell. The mtDNA copy number was enriched in adipocytes of adipose tissue and decreased slightly by ageing (p = 0.015) and increasing BMI (p = 0.004); however, it was not influenced by sex, energy-restricted diets or marked long-term weight reduction. Adipose mtDNA copy number was not independently related to resting energy expenditure, overall insulin sensitivity or adipocyte lipolysis. However, it showed a strong positive correlation with basal (p = 0.0012) and insulin-stimulated lipogenesis (p < 0.0001) in fat cells, independently of age and BMI, and a weak positive correlation with levels of mRNA from several genes involved in mitochondrial oxidative capacity (r = 0.2-0.3). The mtDNA copy number in human white fat cells is fairly stable within healthy individuals. It is not influenced by sex or weight loss and is not important for overall insulin sensitivity or energy expenditure at rest. However, it is strongly related to adipocyte lipogenesis and weakly to mitochondrial oxidative capacity, suggesting that adipocyte mitochondria are, above all, local regulators.

Growth hormone protects against ovarian granulosa cell apoptosis: Alleviation oxidative stress and enhancement mitochondrial function

Reactive oxygen and nitrogen species in patients with rheumatoid arthritis as potential biomarkers for disease activity and the role of antioxidants

BackgroundPolycystic ovary syndrome (PCOS) is an endocrine disorder diagnosed by anovulation hyperandro-genism. Hyperandrogenism increases apoptosis, which will eventually disturb follicular growth in PCOS patients.Since mitochondria regulate apoptosis, they might be affected by high incidence of follicular atresia. This may causeinfertility. Since vitamin D3 has been shown to improve the PCOS symptoms, the aim of study was to investigate theeffects vitamin D3 on mtDNA copy number, mitochondrial biogenesis, and membrane integrity of granulosa cells ina PCOS-induced mouse model.Materials and MethodsIn this experimental study, the PCOS mouse model was induced by dehydroepiandrosterone(DHEA). Granulosa cells after identification by follicle-stimulating hormone receptor (FSHR) were cultured in threegroups: 1. granulosa cells treated with vitamin D3 (100 nM for 24 hours), 2. granulosa cells without any treatments,3. Non-PCOS granulosa cells (control group). Mitochondrial biogenesis gene (TFAM) expression was comparedbetween different groups using real-time PCR. mtDNA copy number was also investigated by qPCR. The mitochon-drial structure was evaluated by transmission electron microscopy (TEM). Hormonal levels were measured by anenzymelinked immunosorbent assay (ELISA) kit.ResultsThe numbers of pre-antral and antral follicles increased in PCOS group in comparison with the non-PCOSgroup. Mitochondrial biogenesis genes were downregulated in granulosa cells of PCOS mice when compared to thenon-PCOS granulosa cells. However, treatment with vitamin D3 increased mtDNA expression levels of these genescompared to PCOS granulosa cells with no treatments. Most of the mitochondria in the PCOS group were sphericalwith almost no cristae. Our results showed that in the PCOS group treated with vitamin D3, the mtDNA copy numberincreased significantly in comparison to PCOS granulosa cells with no treatments.ConclusionAccording to this study, we can conclude, vitamin D3 improves mitochondrial biogenesis and membraneintegrity, mtDNA copy number in granulosa cells of PCOS mice which might improve follicular development andsubsequently oocyte quality.

Changes in the PGC-1α and mtDNA copy number may play a role in the development of pelvic organ prolapse in pre-menopausal patients

Objective: To investigate the effects of mitochondrial DNA (mtDNA) copy number in peripheral blood and related factors on the risk of hypertension in coal miners. Methods: A case-control study was conducted in 378 coal miners with hypertension and 325 healthy coal miners recruited from Datong Coal Mine Group. A standard questionnaire was used to collect their general information, such as demographic characteristics, habits and occupational history. Fluorescence quantitative PCR was performed to detect the copy number of mtDNA. Logistic regression model was applied for identifying the related risk factors of hypertension and analyzing the interaction between mtDNA copy number and risk factors. Results: The prevalence of hypertension of high mtDNA copy number was lower than mtDNA copy numberin 0-5.67 group, but the difference was not statistically significant (P=0.414). Alcohol drinking (OR=1.80, 95% CI: 1.26-2.56), family history of hypertension (OR=1.74, 95% CI: 1.20- 2.50), work shifts (OR=0.69, 95% CI: 0.48-0.99), education level (P=0.012) and family monthly income level (P=0.001) were related to the prevalence of hypertension. There were potential interactions between mtDNA copy number and alcohol drinking, family monthly income level, family history of hypertension, respectively. Alcohol drinking was a risk factor for hypertension [1.77 (1.25-2.50)]. Potential interactions between mtDNA copy number and alcohol drinking reduced the risk of hypertension (OR=1.20, 95% CI: 1.07-1.35). Family history of hypertension was a risk factor for hypertension [1.81(1.26-2.59)]. Potential interactions between mtDNA copy number and family history of hypertension reduced the risk of hypertension (OR=1.24, 95%CI: 1.09-1.41). Family monthly income level was a protect factor for hypertension [0.55(0.46-0.66)]. Potential interactions between mtDNA copy number and family monthly income level increased the protection role of hypertension (OR=0.90, 95% CI: 0.86-0.94). Conclusion: mtDNA copy number variation was not significantly associated with the prevalence of hypertension in coal miners, but mtDNA copy number showed multiplication interaction on the prevalence of hypertension with alcohol drinking, family monthly income level as well as family history of hypertension and made their influences weaken.

Erbium oxide nanoparticles (Er2O3-NPs) have attracted significant attention for their unique physicochemical properties, including high surface area, biocompatibility, and stability. However, the impact of Er2O3-NPs on lymphoma cells (LCs) has not been explored, making this an innovative avenue for exploration. Therefore, the current study aimed to explore the influence of Er2O3-NPs on cell viability, genomic and mitochondrial DNA integrity, reactive oxygen species (ROS) generation and apoptosis induction in human U937 LCs. Er2O3-NPs were characterized using X-ray diffraction (XRD) and transmission electron microscopy (TEM). The effect of Er2O3-NPs on cell viability and genomic DNA integrity was estimated after 48 h using the WST-1 cytotoxicity and alkaline Comet assays, respectively. The generation level of reactive oxygen species (ROS) and mitochondrial membrane potential were also analyzed. Flow Cytometry was used to assess apoptosis induction and quantitative RT-PCR was conducted to measure the apoptotic (p53), anti-apoptotic (Bcl2), and mitochondrial (ND3) gene expression. Our results demonstrated the purity and well distribution of Er2O3-NPs and revealed that Er2O3-NPs induce strong cytotoxicity on U937 cells, as evidenced by a concentration-dependent reduction in cell viability with an IC50 value of 3.20 µg/ml. Exposure of U937 LCs to the IC50 concentration (3.20 µg/ml) of Er2O3-NPs promoted excessive ROS generation, leading to dramatic damage to genomic DNA and mitochondrial membrane potential, as well as marked dysregulation of apoptotic (p53), anti-apoptotic (Bcl2) and mitochondrial ND3 gene expression. This cascade of events triggered both apoptosis and necrosis in Er2O3-NPs-treated U937 LCs. In conclusion, these findings highlight the strong in vitro cytotoxic potential of Er2O3-NPs against highly aggressive U937 LCs, mediated by excessive ROS production, which leads to severe genomic DNA and mitochondrial membrane damage, as well as profound alterations in apoptotic, anti-apoptotic and mitochondrial gene expression. Future research is needed to further investigate the potential use of Er2O3-NPs in treating lymphoma and to optimize their therapeutic efficacy.

Pancreatic beta cell function has been shown to decline with age in man. Depletion of mitochondrial DNA (mtDNA) copy number is associated with impaired insulin secretion in pancreatic beta cell lines, and decreased mtDNA copy number has been observed with age in skeletal muscle in man. We investigated whether mtDNA copy number decreases with age in human pancreatic beta cells, which might in turn contribute to the age-related decline in insulin secretory capacity. We quantified mtDNA copy number in isolated human islet preparations from 15 pancreas donors aged between 17 and 75 years. Islets (n = 20) were individually hand-picked and pooled from each donor isolate for the quantification of mtDNA copy number and deleted mtDNA (%), which were determined using real-time PCR methods. There was a significant negative correlation between mtDNA copy number and islet donor age (r = -0.53, p = 0.044). mtDNA copy number was significantly decreased in islet preparations from donors aged > or =50 years (n = 8) compared with those aged <50 years (n = 7) (median [interquartile range]: 418 [236-503] vs 596 [554-729] mtDNA copy number/diploid genome; p = 0.032). None of the islet preparations harboured high levels of deleted mtDNA affecting the major arc. Given the correlation between mtDNA content and respiratory chain activity, the age-related decrease in mtDNA copy number that we observed in human pancreatic islet preparations may contribute to the age-dependent decline in pancreatic beta cell insulin secretory capacity.