Vanja Cejvanovic

Simvastatin and oxidative stress in humans: A randomized, double-blinded, placebo-controlled clinical trial

Simvastatin reduces the blood concentration of cholesterol by inhibiting hydroxymethylglutaryl-coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis, and thereby reduces the risk of cardiovascular disease. In addition, simvastatin treatment leads to a reduction in fluxes in mitochondrial respiratory complexes I and II and might thereby reduce the formation of reactive oxygen species, which have been implicated in the pathogenesis of arteriosclerosis. Therefore, we hypothesized that simvastatin may reduce oxidative stress in humans in vivo. We conducted a randomized, double-blinded, placebo-controlled study in which subjects were treated with either 40 mg of simvastatin or placebo for 14 days. The endpoints were six biomarkers for oxidative stress, which represent intracellular oxidative stress to nucleic acids, lipid peroxidation and plasma antioxidants, that were measured in urine and plasma samples. A total of 40 participants were included, of which 39 completed the trial. The observed differences between simvastatin and placebo groups in the primary outcomes, DNA and RNA oxidation, were small and nonsignificant (p=0.68), specifically, 3% in the simvastatin group compared to 7.1% in the placebo group for DNA oxidation and 7.3% in the simvastatin group compared to 3.4% in the placebo group. The differences in biomarkers related to plasma were not statistically significant between the treatments groups, with the exception of total vitamin E levels, which, as expected, were reduced in parallel with the reduction in plasma cholesterol. In healthy young male volunteers, short-term simvastatin treatment, which considerably reduces cholesterol, does not lead to a clinically relevant reduction in a panel of measures of oxidative stress. Whether simvastatin has effects on oxidative stress in diseased populations, such as diabetes or hemochromatosis, where oxidative stress is prominent, is unknown but seems unlikely.

Markers of oxidative stress in obese men with and without hypertension

Abstract Objectives: The aim of our study was to investigate if the 24-hour excretion of the urinary markers for oxidative stress to DNA and RNA, measured as 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydro-guanosine (8-oxoGuo), respectively, were increased in obese individuals with or without hypertension compared to lean controls. Methods: A total of 63 obese hypertensive men (obeseHT), 40 obese normotensive men (obeseNT) and 27 lean normotensive men (leanNT) were included in the study. Body mass index (BMI) was between 20.0 and 24.9 kg/m2 in leanNT participants and ≥30 kg/m2 in obese participants. Hypertension was defined as a mean 24-hour systolic ambulatory blood pressure (AMBP) ≥ 130 mmHg or a mean 24-hour diastolic AMBP ≥80 mmHg and normotension as mean 24-hour AMBP <130/80 mmHg. Twenty-four hour urinary 8-oxoGuo and 8-oxodG excretion (nmol/24 h) were measured by a validated liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS). Results: Urinary 8-oxoGuo excretion was (median and [interquartile range]) 30.8 [27.8–32.2] nmol/24 h in leanNT, 36.8 [31.3–40.2] nmol/24 h in obeseNT and 40.6 [31.7–48.5] nmol/24 h in obeseHT. The difference was statistically significant (p = .002) and post hoc tests showed a significant difference between leanNT and obeseHT (p = .001) as well as obeseNT (p = .002), whereas the two obese groups did not differ (p = .6). No statistically significant differences in 8-oxodG concentrations were observed between the three groups (p = .3). Conclusion: The measurement of urinary excretion of 8-oxoGuo suggests that obesity in men, but not hypertension, is associated with increased oxidative damage to RNA.

Cardiovascular and All-Cause Mortality Risk Associated With Urinary Excretion of 8-oxoGuo, a Biomarker for RNA Oxidation, in Patients With Type 2 Diabetes: A Prospective Cohort Study

OBJECTIVE Cardiovascular mortality risk remains high among patients with type 2 diabetes. Oxidative stress indicated by high urinary excretion of the biomarker for RNA oxidation, 8-oxo-7,8-dihydroguanosine (8-oxoGuo), is associated with an increased risk of death in newly diagnosed and treated patients. We assessed whether 8-oxoGuo is associated with specific cardiovascular and all-cause mortality risk. RESEARCH DESIGN AND METHODS Urinary biomarkers for nucleic acid oxidation were measured in a cohort of patients with type 2 diabetes aged ≥60 years (n = 1,863), along with biochemical measurements, questionnaire findings, and Central Person Registry information to estimate the hazard ratios (HRs) for log2-transformed RNA oxidation using Cox regression. RESULTS During the 5-year follow-up, 173 of 1,863 patients had died (9.3%), including 73 patients who died of cardiovascular disease (42.2%). Doubling of RNA oxidation was associated with an HR of all-cause mortality of 2.10 (95% CI 1.63–2.71; P < 0.001) and an HR of cardiovascular death of 1.82 (95% CI 1.20–2.77; P = 0.005) after multiple adjustments. The 5-year absolute risks (ARs) of all-cause mortality (AR 13.9 [95% CI 10.8–17.0] vs. AR 6.10 [95% CI 4.00–8.30]) and cardiovascular mortality (AR 5.49 [95% CI 3.44–7.55] vs. AR 3.16 [95% CI 1.59–4.73]) were approximately two times higher in the highest quartile of RNA oxidation than in the lowest quartile. CONCLUSIONS We conclude that high RNA oxidation is associated with all-cause and cardiovascular mortality risk in patients with type 2 diabetes. Targeting oxidative stress via interventions with long-term follow-up may reveal the predictive potential of the biomarker 8-oxoGuo.

Iron induced RNA-oxidation in the general population and in mouse tissue

ABSTRACT Iron promotes formation of hydroxyl radicals by the Fenton reaction, subsequently leading to potential oxidatively generated damage of nucleic acids. Oxidatively generated damage to RNA, measured as 8‐oxo‐7,8‐dihydroguanosine (8‐oxoGuo) in urine, is increased in patients with genetic iron overload, which have led us to test the hypothesis that high iron status, assessed by iron biomarkers and genetic disposition, increases urinary excretion of 8‐oxoGuo. In a general Danish population study we used a Mendelian randomization design with HFE genotypes as a proxy for iron status and supplemented with ex vivo experiments in mice muscle tissue exposed to iron(II) sulfate to attempt to clarify this hypothesis. The biomarkers ferritin, transferrin, and transferrin saturation (TS) were associated with 8‐oxoGuo (in linear univariable and multivariable regression analyses: P < 0.001). Mendelian randomization indicated a causal pathway between genetically elevated iron biomarkers (assessed by ferritin and TS) and high levels of 8‐oxoGuo. The ex vivo experiments showed a monotonically increase in 8‐oxoGuo with increased iron concentration (ANOVA: P = 0.0008) that was prevented with iron chelation (P = 0.01). Our results indicate a causal relationship between iron biomarkers and 8‐oxoGuo. Furthermore, the ex vivo experiment shows a mechanistic link between iron and 8‐oxoGuo formation. Both iron overload and the biomarker 8‐oxoGuo have been linked to e.g. diabetes, which merits future studies to investigate if iron induced 8‐oxoGuo is involved in disease development. Graphical abstract Figure. No caption available. HighlightsIron biomarkers are associated with urinary excretion of 8‐oxoGuo.Blood donor status and menopause status (in women) are associated with 8‐oxoGuo.There is an indication of causal relationship between iron biomarkers and 8‐oxoGuo population.Mouse muscle tissue exposed to iron results in increased 8‐oxoGuo.

The effect of empagliflozin on oxidative nucleic acid modifications in patients with type 2 diabetes: protocol for a randomised, double-blinded, placebo-controlled trial

Introduction Cardiovascular disease is the leading cause of morbidity and mortality in patients with type 2 diabetes (T2D). Although glycaemic control reduces microvascular complications, the effect of intensive treatment strategies or individual drugs on macrovascular diseases is still debated. RNA oxidation is associated with increased mortality in patients with T2D. Inspired by animal studies showing effect of a sodium-glucose cotransporter-2 (SGLT-2) inhibitor (empagliflozin) on oxidative stress and a recent trial evaluating empagliflozin that demonstrated improved cardiovascular outcomes in patients with T2D at high risk of cardiovascular events, we hypothesise that empagliflozin lowers oxidative stress. Methods and analysis In this randomised, double-blinded and placebo-controlled study, 34 adult males with T2D will be randomised (1:1) to empagliflozin or placebo once daily for 14 days as add-on to ongoing therapy. The primary endpoints will be changes in 24-hour urinary excretion of 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) determined before and after intervention (by ultra-performance liquid chromatography tandem mass-spectrometry). Additionally, fasting levels of malondialdehyde (MDA) will be determined in plasma before and after intervention (by high-performance liquid chromatography). Further, the plasma levels of iron, transferrin, transferrin-saturation, and ferritin are determined to correlate the iron metabolism to the markers of oxidative modifications. Ethics and dissemination The study protocol has been approved by the Regional Committee on Biomedical Research Ethics (approval number H-16017433), the Danish Medicines Agency, and the Danish Data Protection Agency, and will be carried out under the surveillance and guidance of the GCP unit at Bispebjerg Frederiksberg Hospital, University of Copenhagen in compliance with the ICH-GCP guidelines and in accordance with the Declaration of Helsinki. The results of this study will be presented at national and international conferences, and submitted to a peer-reviewed international journal with authorship in accordance with Internation Committee of Medical Journal Editors (ICMJE) Recommendations state. Trial registration Study name: EMPOX; Pre-results: clinicaltrials.gov (NCT02890745). Protocol version 5.1 - August, 2016.

Increased oxidation of RNA despite reduced mitochondrial respiration after chronic electroconvulsive stimulation of rat brain tissue

Major depressive disorder (MDD) affects 350 million people worldwide and is a serious socio-economic burden. The most efficient treatment of MDD is electroconvulsive therapy (ECT), which has been shown to influence the oxidative status believed to be part of the pathophysiology of MDD. We investigated the effects of chronic electroconvulsive stimulation (ECS) on mitochondrial respiration and mitochondrial hydrogen peroxide production, RNA oxidation, and the content of mitochondria in the piriform cortex of the rat. We found reductions of mitochondrial respiration in respiratory states 2 and 3 by 33% and 32%, respectively, and a 23% reduction in electron transfer capacity. RNA oxidation, as measured by 8-oxo-7,8-dihydroguanosine, was increased by 58%, while mitochondrial production of H2O2 was unaffected. The increased oxidative stress may thus be ascribed to extra-mitochondrial sources.

RNA oxidation and iron levels in patients with diabetes

Aim: The urinary biomarker for oxidative stress to RNA, 8‐oxo‐7,8‐dihydro‐guanosine (8‐oxoGuo) is associated with mortality in patients with type 2 diabetes. Iron has also been linked to diabetes. In individuals with untreated hereditary iron overload it has been observed that 8‐oxoGuo was higher compared to controls. In the current study, we hypothesized that 8‐oxoGuo was associated with diagnosis of diabetes, and that iron confounded this association. Methods: Participants from a general Danish population were included in the study (n = 3567). UPLC‐MS/MS method was used for 8‐oxoGuo (nmol/mmol creatinine) measurement in spot urine. Iron biomarkers included total plasma iron, ferritin, transferrin saturation (TS) and transferrin. Results: 8‐oxoGuo was 17% higher in diabetes patients (n = 208) compared to non‐diabetes controls. Unadjusted logistic regression model showed an odds ratio of diabetes of 1.38 (95%CI:1.21–1.57, P < 0.0001) per unit increase of 8‐oxoGuo. When the model was adjusted for possible confounders the odds ratio was 1.09 (95%CI:0.94–1.26, P = 0.24). When additional adjustment was performed including ferritin, TS, or transferrin, respectively, the OR were 1.14 (95%CI:0.97–1.33, P = 0.09), 1.10 (95%CI: 0.95–1.28, P = 0.18), and 1.17 (95%CI:1.01–1.38, P = 0.04). Conclusions: Our study indicates that 8‐oxoGuo is higher in diabetes patients. The lack of association between 8‐oxoGuo and diabetes in the adjusted model may be due to the cross‐sectional design including post‐treatment bias. Our data did not show consistent effect of all iron biomarkers in relation to diabetes. Most likely, the iron biomarkers were affected by inflammation thus not reflecting true iron levels. Graphical abstract Figure. No caption available. HighlightsUrinary 8‐oxoGuo was 17% higher in diabetes patients compared to controls.High 8‐oxoGuo was associated with higher odds for having diabetes.Irons effects on the association between 8‐oxoGuo and diabetes was not clear.

The effect of smoking on the urinary excretion of 8-oxodG and 8-oxoGuo in patients with type 2 diabetes

Abstract Over the past decades, attention has been paid to understanding the impact of oxidative stress and related modifications of DNA and RNA on various human health risks. A recent meta-analysis comprising 1915 smokers and 3462 non-smokers found a significantly higher level of DNA oxidation measured as urinary 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG) excretion in smokers compared with non-smokers in a healthy population. We aimed to investigate if an increased urinary excretion of 8-oxodG in smokers versus never smokers and former smokers could be verified in a population with type 2 diabetes. Additionally, we measured RNA oxidation levels through urinary excretion of 8-oxo-7, 8-dihydroguanosine (8-oxoGuo). Our study included urinary samples from 2721 type 2 diabetic patients, analyzed using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Logistic regression was used to examine the relationship between daily smokers (n = 462) versus former (n = 1341) and never smokers (n = 918) regarding the RNA and DNA oxidation, respectively. We did not find any significant effect of smoking on urinary excretion of 8-oxodG or 8-oxoGuo in our study. Due to a sparse study area, it is still too early to draw any conclusions on smoking and RNA-oxidation. Regarding DNA oxidation, our study suggests that the effect of smoking seen in healthy populations might be attenuated in patients with type 2 diabetes.

Survival Biomarker 8-OxoGuo Is Not Associated with Insulin or Metformin Treatment in Type 2 Diabetes Patients

Objective In type 2 diabetes (T2D) we have previously shown that urinary markers of RNA oxidation (8-oxo-7,8-dihydroguanosine [8-oxoGuo]), but not DNA oxidation (8-oxo-7,8-dihydro-2’-deoxyguanosine [8-oxodG]) is associated with increased mortality in patients with newly diagnosed and well-established T2D (Diabetes Care. 2013 Mar;36(3):669–76). Since it is unknown which pharmacological therapy affects 8-oxoGuo and 8-oxodG, we investigated whether insulin or metformin treatment is associated with increased or decreased levels of urinary markers of nucleic oxidation in T2D patients. Research design and methods: Urine samples were analyzed for 8-oxoGuo and 8-oxodG by UPLC-MS/MS in a cohort of 2727 patients with known T2D. Medical information was derived from health examination questionnaires. Associations were tested with T-test and Cox regression models. Results 701 of the patients received insulin. There was no difference in mean value of 8-oxoGuo between insulin treated patients and controls, but insulin treated patients had lower mean value of 8-oxodG (p 0.05). Metformin treated patients had 0.58 (0.45-0.75) hazard ratio for overall death in the Cox model (p Conclusion 8-oxoGuo was not associated with neither insulin nor metformin treatment. The increased risk of death shown in T2D patients with high levels of 8-oxoGuo could not be explained by commonly used antidiabetic drugs. Further detailed studies of antidiabetic medications and their effects on oxidative stress are warranted.