Change in mitochondrial endogenous superoxide production in smokers related to basal superoxide amount and mitochondrial DNA dynamics
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(2) TABLE OF CONTENTS ACKNOWLEGEMENTS . 3 . ABSTRACT (SPANISH) . 4 . Manuscript in progress: CHANGE IN MITOCHONDRIAL ENDOGENOUS SUPEROXIDE PRODUCTION IN SMOKERS RELATED TO BASAL SUPEROXIDE AMOUNT AND MITOCHONDRIAL DNA DYNAMICS 5 FINAL COMMENTS . 20 . SUPPLEMENTARY MATERIAL SUPPLEMENTARY MATERIAL 1: OCCUPATIONAL SURVEY SUPPLEMENTARY MATERIAL 2: INFORMED CONSENT SUPPLEMENTARY MATERIAL 3: ABSTRACT MONTREAL CONGRESS . 21 21 23 24 . . . . . 2 .
(3) ACKNOWLEGEMENTS First of all I want to thank Helena Groot and Diana Narváez for their unconditional help and support, for their teachings and advice. To Bennet Van Houten for his help and advice through out the process to standardize the QPCR technique. To Nicolás Giraldo for his contributions in the development and execution of the protocol to quantify mitochondrial superoxides. I would also like to acknowledge John Mario Gonzalez from the laboratory of basic medical sciences of the university for letting me use the flow cytometer and to Maria Mercedes Zambrano, Patricia del Portillo and Martha Cepeda from CorpoGen for letting me borrow the fluorescence microplate reader. Finally I would like to thank the volunteers of this study for their participation and to the members of my lab for their critical review of my work and making the lab a great place to be. . . . . 3 .
(4) ABSTRACT (SPANISH) El estrés oxidativo causa lesiones deletéreas en los componentes celulares que pueden llevar a un mal funcionamiento en la cadena de transporte de electrones en la mitocondria. Debido a que esta disfunción puede incrementar la producción de superóxidos mitocondriales y que éstos causan rupturas en el ADN, se ha propuesto el ADN de este organelo como un buen biomarcador del estrés oxidativo. El cigarrillo ha sido asociado a diferentes patologías a nivel sistémico, y en muchas de éstas se ha encontrado un incremento en el estrés oxidativo de las células. Para estudiar esta hipótesis, el objetivo del estudio era evaluar el efecto de fumar cigarrillo en la cantidad de superóxidos en la mitocondria y el contenido y daño del ADN mitocondrial. Se aislaron linfocitos de sangre periférica de 17 hombres fumadores y 32 no fumadores. Las lesiones y el contenido de ADN se evaluaron usando la técnica de PCR cuantitativa de fragmentos largos y la cuantificación de superóxidos mitocondriales se determinó por citometría de flujo con el fluorocromo MitoSOX. También se trataron los linfocitos con doxorubicina para inducir estrés oxidativo y se cuantificó el incremento en la producción de superóxidos. No se encontraron diferencias entre fumadores y no fumadores indicando que el ADN mitocondrial no es un biomarcador tan sensible del efecto de estrés oxidativo en linfocitos de sangre periférica. Sin embargo, encontramos que la cantidad de superóxidos basales, el contenido y lesiones en el ADN mitocondrial determinan el incremento en la producción de superóxidos en las células tratadas con doxorubicina. Esto evidencia que en los linfocitos humanos el ADN mitocondrial tiene diferentes mecanismos para controlar el incremento del estrés oxidativo. Por un lado, cuando la cantidad de superóxidos basales es más alta, se producen menos superóxidos después de tratar las células con doxorubicina, sugiriendo que estas células tienen los mecanismos de detoxificación o reparación aumentados. Por otro lado, mayor cantidad de DNA mitocondrial también se encontró asociada con una menor producción de superóxidos, probablemente porque evita la disfunción mitocondrial compensando el ADN defectuoso. Finalmente, nuestros resultados soportan la teoría del ciclo vicioso ya que los voluntarios que presentaban mayor cantidad de lesiones en el ADN generaban más superóxidos después del tratamiento. . . . 4 .
(5) Manuscript in progress: CHANGE IN MITOCHONDRIAL ENDOGENOUS SUPEROXIDE PRODUCTION IN SMOKERS RELATED TO BASAL SUPEROXIDE AMOUNT AND MITOCHONDRIAL DNA DYNAMICS ABSTRACT Oxidative stress causes deleterious lesions to cell components that can induce dysfunctions in the mitochondrial electron transport chain. This impairment can increase mitochondrial superoxide production and, due to its proximity, mitochondrial DNA has been proposed as a good biomarker of oxidative stress. Cigarette smoking is associated with different pathologies in a systemic level and many of these have been associated to an increase in oxidative stress. The aim of this study was to evaluate the effect of cigarette smoking in mitochondrial DNA content, DNA lesions and mitochondrial superoxide production. To achieve this, peripheral blood lymphocytes were studied from 17 healthy smokers and 32 non-‐smokers. Mitochondrial DNA lesions and content was evaluated with the long amplicon quantitative PCR and superoxide quantification was done by flow cytometry using MitoSOX fluoroprobe. In addition, isolated lymphocytes were treated with doxorubicin to induce oxidative stress and MitoSOX fluorescence was quantified. We found no differences between smokers and non-‐smokers indicating that mitochondrial DNA is not a very sensible biomarker of cigarette smoke effect in peripheral blood lymphocytes, but we report that the basal superoxide amount and mitochondrial DNA lesions and content determine the amount of superoxides produced by mitochondria after doxorubicin stimulation. This evidences that in human lymphocytes mitochondrial DNA has different mechanisms to control the superoxide production. First, when basal superoxide quantifications are higher, there is less superoxide production after the treatment, suggesting that these cells have better mechanisms to avoid mitochondrial dysfunction. Second, increased mitochondrial DNA content cause less superoxide production probably because it compensates the damage in the DNA and reduces the electron transport chain impairment. Finally, our results support the theory of the vicious cycle as people with more mitochondrial DNA lesions presented more superoxide production after treatment. . . 5 .
(6) INTRODUCTION Mitochondria are intracellular organelles with several functions, including the production of ATP through oxidative phosphorylation. They have their own circular, double stranded DNA (mtDNA); which in humans is composed of 16,569bp and codes for 22tRNAs, 2rRNAs and13 polypeptides, which are part of the Electron Transport Chain (ETC) enzymatic complexes [1]. Previous estimates based on isolated highly energized mitochondria suggested that as much as 2-‐4% of the oxygen consumed was converted into free oxygen radicals [2, 3]; however, more recent studies suggest that under normal physiological cellular conditions, superoxide (𝑂!! ) production is one or two orders of magnitude lower [4]. Mitochondrial dysfunction and oxidative stress have been associated with ageing, cancer, neurodegenerative and cardiovascular diseases, among others [4-‐8]. At the cellular level, oxidative stress causes the activation of redox-‐sensitive transcription factors that lead to the production of proinflamatory chemokines, activation of mitogenic protein kinases, opening of ion channels, lipid peroxidation and DNA oxidation [9]. Oxidative stress conditions can lead to a vicious cycle where it induces mitochondrial dysfunction, increasing the amount of 𝑂!! produced; which when converted to hydrogen peroxide by Manganese Superoxide Dismutates (MnSOD) can form highly reactive hydroxyl radicals by the Fenton reaction with reduced iron. [1]. For this reason mitochondria is one of the most important sources of endogenous Reactive Oxygen Species (ROS) and has an important role in the oxidative state of the cell [2]. Although mitochondria have base excision repair (BER) mechanisms that removes oxidative DNA lesions, under oxidative stress conditions they appear to be inactivated or are insufficient to repair the damage cascade [1, 10, 11]. mtDNA lesions block the RNA polymerase preventing transcription and therefore resulting in loss of the proteins of the electron transport chain [12]. Different studies have found an increase in mitochondrial mass and mtDNA content as a compensation mechanism [13-‐15], but recently it has been shown that defective mitochondria also induce a process of mtDNA depletion and mitophagy that appears to be mediated by ROS [16-‐18]. The long amplicon quantitative PCR (LA-‐QPCR or QPCR) technique described by Santos et. al. [19], allows the evaluation of mtDNA lesions specifically reducing possible sources of spurious damage to the DNA. Briefly, the technique is based on the decrease of the amplification of specific long DNA fragments due to lesions that block the polymerase progress. This technique has been used to show an accumulation of DNA damage in both the nucleus and mitochondria of patients with Friedreich’s ataxia [20] and an increase in mtDNA lesions in patients with macular degeneration [21], but it has not been used for biomonitoring genotoxic substances in human populations. Cigarette smoke contains more than 3,800 compounds that include ROS and chemicals that can form other reactive substances [22]. These compounds are responsible for the oxidative stress that is evidenced as an increase in lipid peroxidation of biological membranes and DNA damage [23]. Moreover, it has been demonstrated that cigarette smoke compounds affect mitochondrial function leading to an imbalance in the production of endogenous ROS, thereby increasing the oxidative stress [24]. Oxidative stress has been associated to most of the pathologies caused by cigarette smoking such as cardiovascular dysfunction, respiratory pathologies, immune imbalance and cancer [6, 25-‐29]. . . 6 .
(7) The genotoxic effect of cigarette smoke has been evidenced with techniques like sister chromatid exchanges and the Comet assay that evaluate nuclear DNA (nDNA) damage [30]; although nDNA is not an ideal biomarker to evaluate exposure and smoke effects in DNA, as smokers present higher rates of DNA repair [31]. For these reasons, some authors have hypothesized that mtDNA damage could be a good biomarker of tobacco smoke and that studies with mtDNA damage could contribute to the knowledge of molecular epidemiology of cancer. Most of the studies have evaluated mutations, mtDNA content and presence of oxidized bases, but oxidative damage to mtDNA apparently presents more single and double strand breaks. Therefore, the aim of this study was to evaluate the effect of cigarette smoking in mtDNA content, mtDNA lesions and mitochondrial superoxide production. . . 7 .
(8) MATERIALS AND METHODS Participants and sample collection 49 male volunteers, between the age range of 18-‐35, were recruited via advertisement at Universidad de los Andes, Bogotá, Colombia, for a study of smoking behavior and effects on the DNA. They were divided in two groups: smokers (n= 17), were daily smokers with a stable smoking pattern of at least 1 year, must not have had a prior history of cancer, concurrent infection, do intense physical activity (more than 12 hours per week) nor be consuming drugs that might affect the immune system response. On the other hand, the group of non-‐smokers (n= 32), defined as not having smoked more than 100 cigarettes in their lifetime, and must not live with an active smoker. All the volunteers signed an informed consent previously approved by the University’s ethics committee and a survey about their smoking status and lifestyle. Blood samples were collected by standard phlebotomy procedures. Mitochondrial DNA quantitative PCR assay Total genomic DNA from whole blood was isolated using the PAXgene blood DNA isolation kit (PreAnalytiX/QIAGEN) as previously described [20]. Briefly an 8.5mL sample of whole blood was collected in BD collection tubes with EDTA (BD Bioscience) and was immediately transferred to a processing tube containing a lysing solution. Lysed red and white blood cells were centrifuged, and the resulting pellet of nuclei and mitochondria was washed and resuspended. After digestion with protease, DNA was precipitated with the addition of isopropanol, washed with ethanol and resuspended in the elution buffer. DNA was diluted to a final concentration of 3 ± 0.3 ng/μL. Blood DNA was successfully extracted and its purity and quality was verified by absorbance at 260/280nm in nanodrop spectrophotometer and agarose gel electrophoresis, respectively. DNA lesion frequencies were calculated as previously described [19, 20, 32]. An 8.9kb fragment of mtDNA was amplified, quantified using the picogreen reagent (Invitrogen) and verified by agarose gel electrophoresis. The amplification of smokers (As) was compared to the amplification of non-‐smokers (Ans) obtaining a relative amplification ratio. Assuming a random distribution of lesions and using the Poisson equation [where λ is the average lesion frequency for the non – damaged template (i.e., the zero class; x =0)], the average lesion per DNA strand was determined using the equation λ = -‐ln(As)/(Ans). Amplification of mitochondrial fragment was normalized to mitochondrial copy number by the amplification of a short mitochondrial fragment (221bp), which due to its small size is probably free of damage. Each PCR run included a positive control with the same DNA for all the runs of the study in order to guarantee the efficiency of the PCR, a 50% control with half of the DNA concentration of the positive control to guarantee that the PCR is in the quantitative range and a negative control. For each volunteer, triplicate amplifications of the long mitochondrial fragment were conducted in each PCR run and two independent runs were conducted. The small fragment was amplified by triplicate. Only one person conducted the DNA extractions and PCRs. Superoxide quantification using MitoSOX Mononuclear cells were isolated from whole blood using ficoll gradient to 10 smokers and 23 non-‐ smokers. Briefly, 3mL sample of whole blood was collected using BD collection tubes with sodium heparin, diluted in 5mL of Roswell Park Memorial Institute (RPMI, Genaxxon Bioscience) cell culture media, supplemented with 2% of Fetal Bovine Serum (FBS), and added to the Histopaque 1077 (Sigma . . 8 .
(9) Aldrich) solution. After centrifugation, mononuclear cells were isolated and washed twice with RPMI + FBS 2% followed by centrifugation and resuspended in 1mL of RPMI + FBS 10%. Cell viability was evaluated using Trypan Blue dye assay and cells were diluted to a concentration of 107 cells/mL. Basal 𝑂!! , reported here as basal MitoSOX fluorescence, was evaluated as previously described [33, 34]. 1.5mL of cells were washed twice with Hanks Balanced Salt Solution (HBSS) + FBS 2% and resuspended in 500μL of the same solution. MitoSOX (Molecular Probes, Invitrogen) 10μM was prepared following manufacturer’s instructions and 500μL were added to the cells and incubated during 30 minutes at 37°C and 5% CO2. After MitoSOX loading, cells were washed with HBSS + FBS 2%, resuspended in 500μL of HBSS + FBS 2% to analyze by flow cytometry. To induce 𝑂!! production as positive control, 1.5mL of cells were incubated with 500μL of doxorubicin (20μM – Ebewe Pharma) for 2 hours at 37°C and 5% CO2 atmosphere. Then MitoSOX was loaded as previously described. All the procedures were conducted in the dark to avoid photobleaching. The change in MitoSOX fluorescence after the incubation with doxorubicin was used as a measure of the increase in 𝑂!! production after oxidative stress stimulation. Samples were acquired in a FACS CANTO II flow cytometer (BD Bioscience) equipped with a 488nm argon laser. Data were analyzed with FACSDiva software (BD Bioscience). At least 5x104 cells were acquired in the lymphocyte population gate according to their forward scatter (FSC) versus side scatter (SCC) features. Dead cells were excluded by light scatter (FSC-‐H versus FSH-‐A). MitoSOX fluorescence was read in the FL2 channel and mean fluorescence intensity was measured. Statistical Analysis To determine the homogeneity of the study populations, a randomized t-‐test and chi-‐square using Monte Carlo simulation was done. The effect of the different variables measured in the survey on mt DNA content, mtDNA lesions, basal 𝑂!! and the response to oxidative stress was evaluated using a randomized ANOVA model. Multiple linear regression was done and p-‐values for each variable was calculated based on the randomized variables. Correlations were done using the spearman correlation test. Most of the statistical analyses were based in randomization to reduce the effect of the differences between sample sizes and non – normal distribution of the variables evaluated. All the statistical analyses were performed using the R 2.12.2 statistical software. . . 9 .
(10) RESULTS Base line characteristics of the smoker and non-‐smoker groups are shown in Table I. Groups are similar regarding age, psychoactive drug consumption, exercise habits and fruit and vegetable consumption. The only difference between them was the amount of alcohol intake during the weekend (weekend index) where smokers tend to drink more. Relative mitochondrial DNA lesions were calculated using the QPCR technique [19, 20]. A short mitochondria fragment (221pb) was amplified as a measure of mitochondrial copy number. There were no differences between relative mtDNA lesions and content by smoking status, exercise, fruit and vegetable consumption (Table II). However, there were differences in mtDNA content between people that consume psychoactive substances and people that do not (Table II). Also, there was a small positive correlation between relative mtDNA lesions and age (rho=0.305, p value=0.0393) (Figure 1a) and an interaction effect of exercise and fruit and vegetable consumption in mtDNA lesions (p-‐ value=0.0453) (Figure 1b), although time of exercise had no effect. Mitochondrial 𝑂!! were evaluated staining the cells with MitoSOX fluoroprobe, which is directed to the mitochondria and produces red fluorescence when oxidized by 𝑂!! and excited at 510nm. To induce oxidative stress and as a positive control, cells were incubated for 2 hours with doxorubicin (20μM), a chemotherapy drug that induces mitochondrial 𝑂!! production. The net increase in 𝑂!! production was evaluated subtracting basal MitoSOX fluorescence to MitoSOX fluorescence after incubation with doxorubicin. This was used as a measure of the response to induced oxidative stress. There were no differences between smoking status, exercise, and alcohol intake, psychoactive drugs or fruits and vegetables (Table II). Nonetheless, there was a strong effect of basal MitoSOX fluorescence (p value <0.0001), relative mtDNA content (p value=0.0042) and relative mtDNA lesions/10Kb (p value=0.0188) in the increase in MitoSOX fluorescence (adjusted R2 of the model= 0.8845) (Figure 2), where the basal MitoSOX fluorescence and relative mtDNA content have a negative slope (Figure 2 a and c) and the relative mtDNA lesions/10Kb has a positive slope (Figure 2b). There was no correlation between the predictor variables. In addition, when discriminated by the relative mtDNA lesions/10Kb there are 3 clusters (Figure 2b) where people with less relative mtDNA lesions, more mtDNA content and basal MitoSOX fluorescence presented less 𝑂!! production when incubated with doxorubicin; people with more relative mtDNA lesions and less mtDNA content and basal MitoSOX fluorescence presented more 𝑂!! production when incubated with doxorubicin and there is a group in the middle that had no apparent pattern. We found no correlation between the lifestyle characteristics evaluated in the survey and each of the clusters found. . . 10 .
(11) DISCUSSION During the electron transport chain some electrons leak and produce 𝑂!! , which can be converted into more reactive species. When the oxidative balance is lost, and mitochondrial detoxification mechanisms are not efficient, ROS can oxidize DNA and proteins. This leads to a deficiency in transcription and ultimately to deregulations of the ETC that causes more endogenous ROS production [1, 2]. Oxidative damage induces a broad spectrum of lesions such as single and double strand breaks, abasic sites and base oxidative damage (e.g. thymine glycol or 8-‐oxodG). Non-‐oxidative lesions such as bulky adducts can also induce endogenous ROS production as they can induce mutations or uncouple transcription. There are three possible responses of mtDNA to avoid producing an excess of endogenous ROS under stress conditions. First it tries to detoxify 𝑂!! and repair the oxidative and non-‐oxidative DNA damage [12, 35]. As DNA lesions cause transcription to arrest, mtDNA increases its DNA content to compensate the transcription deficiencies and keep the ETC working correctly [12, 13, 36]. Finally, mitochondria that cannot compensate the damage can degrade its DNA, cause autophagy (i.e. mitophagy) and ultimately, apoptosis [37]. The QPCR technique has been successfully used to identify nDNA and mtDNA lesions in humans [20, 21] and in different organisms as a biomonitoring assay (reviewed in Meyer et. al [38] and Hunter et. al. [39]). Nonetheless, this is the first time the assay has been used for biomonitoring genotoxic substances in human populations. This technique only detects lesions that can stop the thermostable polymerase and many base oxidative damage are too small to interrupt replication; nevertheless, most of mtDNA lesions due to oxidative damage have been reported to be strand breaks [37]. MitoSOX is the mitochondrial targeted analog of hydroethidine (HE), which is specifically oxidized by 𝑂!! in the mitochondria and forms the red fluorescent product 2-‐hydroethidine [40]. HE has been proposed as the gold standard to evaluate intracellular 𝑂!! in cardiovascular studies [41] and MitoSOX has been used to monitor their production in different cell lines and tissues [42, 43]. In this study we adapted the flow cytometry quantitation of MitoSOX fluorescence to evaluate mitochondrial 𝑂!! production in peripheral blood lymphocytes. To ensure the assay was working correctly and evaluate mitochondrial response to oxidative stress we treated lymphocytes of each volunteer with doxorubicin, which induces mitochondrial dysfunction and 𝑂!! production. As cigarette smoking has been associated with an increase in oxidative stress, and it appears to have a central role in tobacco pathogenesis, different authors have hypothesized that mtDNA could be a good biomarker of tobacco’s exposure and effect. At the mitochondrial level, it has been shown that specific components of tobacco smoke such as N-‐nitrosamines and acrolein induce mitochondrial dysfunction and contribute to the increase of oxidative stress through endogenous ROS production [44, 45]. Nonetheless, non-‐oxidative lesions can also be induced by components as Polycyclic Aromatic Hydrocarbons (PAHs) that bind directly to DNA and forming bulky adducts and has a greater affinity for mtDNA than nDNA. Previous studies reported an increase in mtDNA mutations, content and lesions lesions in bronchoalveolar cells of smokers compared to non – smokers [14, 22, 46]. Since cigarette smoke effects are systemic (i.e. not just in respiratory system) we hypothesized that its components must travel by blood flow and therefore its effects could be evaluated in peripheral blood lymphocytes. This was assessed in mouse models where cell’s 𝑂!! was increased in lymphocytes [47]. Our results show no . . 11 .
(12) differences between smokers and non-‐smokers in mtDNA lesions, mtDNA content or the amount of basal 𝑂!! , indicating that probably the effects of cigarette smoking are not increasing the endogenous ROS production in lymphocytes and correspondingly, the oxidative lesions and mtDNA content are not increased. The fact that volunteers were young people with a mild smoking pattern (mean pack years were 3.97) and that blood is not a tissue directly exposed to cigarette smoke, might account for the differences found in our study. Probably, due to continuous exposure to cigarette smoke components at a low dose, antioxidant systems and DNA repair mechanisms are elevated, as it has been observed in nDNA [31]. Also, lifespan of the cells, mtDNA rechange and mitochondrial biogenesis/degradation equilibrium might be different in bronchoalveolar cells and peripheral blood lymphocytes. This results indicate that mtDNA from peripheral blood lymphocytes is not a sensible method to evaluate the early effect of cigarette smoking. In agreement with the mitochondrial and free radical theory of ageing we found an increase of mtDNA lesions with age [48], but it was not correlated with an increase in the amount of basal 𝑂!! or changes in mtDNA content. Studies on the effect of exercise on mtDNA content show mixed results [49, 50], although there are no studies on the effect on the integrity of mtDNA. On the other hand, fruits and vegetables are known to contain antioxidants and induce mitochondrial biogenesis but how they might interact with exercise to increase or reduce mtDNA lesions needs further studies. We could not determine which lifestyle habits correlated with the amount basal 𝑂!! production in the mitochondria. Nonetheless, we found that the amount of basal 𝑂!! , mtDNA content and mtDNA lesions had a strong effect in the increase of 𝑂!! production. Our results show that people with high basal 𝑂!! , produce less 𝑂!! when stimulated with doxorubicin (Figure 2a). This might suggest that these cells are adapted to high oxidative stress conditions and therefore respond faster, this goes in agreement with Grishco et. al [51] that found that cells adapted to increased ROS levels, had higher concentrations of AP endonuclease, one of the enzymes of BER mechanism. Since we are observing less 𝑂!! production in response to oxidative stress induction, probably the activity or expression of detoxification enzymes are increased. We also found that induced 𝑂!! production increases with mtDNA lesions, which supports the hypothesis of the vicious cycle, where damaged mtDNA induces more endogenous ROS production (Figure 2b). Finally, higher amounts of mtDNA content can reduce the 𝑂!! production cascade, probably by avoiding the ETC dysfunction (Figure 2c). In Figure 2b it is evident the interaction of the three variables. On the lower left corner, there is a cluster people that presented higher amounts of 𝑂!! and mtDNA content, had less lesions and therefore produced less ROS in response to doxorubicin. On the higher left corner, the opposite occurs. As far as we know, this is the first study to report the interaction between mtDNA dynamics and basal 𝑂!! production in the response to oxidative stress in human cells. Although further studies should be conducted to determine which factors could affect these parameters, this may predict each person’s response to oxidative stress, which is frequent in different pathologies such as cancer or cardiovascular disorders, and direct a more accurate therapy. Further studies should be conducted to evaluate the change in mtDNA dynamics (i.e. mtDNA content and integrity) after the treatment with doxorubicin as here we only evaluated the baseline conditions. This could help to elucidate how the three possible responses of mtDNA to oxidative stress are acting. In summary, our results show that cigarette smoke does not have a determinant effect in the endogenous ROS production of peripheral blood lymphocytes, mtDNA lesions and content; therefore, it mtDNA of peripheral blood lymphocytes is not a sensible biomarker of the effect of cigarette smoke. . . 12 .
(13) More interestingly, we found that basal endogenous ROS production, mtDNA content and integrity are determinant in the response to induced oxidative stress. Further studies with a bigger sample size should be conducted to understand which lifestyle or genetic factor are determinant in the three baseline conditions. . . 13 .
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