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Oxidative stress and mitochondrial dysfunction in parafacial respiratory group induced by maternal cigarette smoke exposure in rat offspring
Author’s Accepted Manuscript Oxidative stress and mitochondrial dysfunction in parafacial respiratory group induced by maternal cigarette smoke exposure in rat offspring Fang Lei, Wen Wang, Yating Fu, Ji Wang, Yu Zheng www.elsevier.com
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S0891-5849(18)31071-2 https://doi.org/10.1016/j.freeradbiomed.2018.09.003 FRB13905
To appear in: Free Radical Biology and Medicine Received date: 16 June 2018 Revised date: 30 August 2018 Accepted date: 2 September 2018 Cite this article as: Fang Lei, Wen Wang, Yating Fu, Ji Wang and Yu Zheng, Oxidative stress and mitochondrial dysfunction in parafacial respiratory group induced by maternal cigarette smoke exposure in rat offspring, Free Radical Biology and Medicine, https://doi.org/10.1016/j.freeradbiomed.2018.09.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Oxidative stress and mitochondrial dysfunction in parafacial respiratory group induced by maternal cigarette smoke exposure in rat offspring
Fang Lei, Wen Wang, Yating Fu, Ji Wang, Yu Zheng* Department of physiology, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu, Sichuan 610041, P.R. China
* Corresponding author. Dr. Yu Zheng , Department of Physiology , West China School of Basic Medical Sciences and Forensic Medicine , Sichuan University , 3-17 Renmin South Road , Chengdu, Sichuan 610041. P.R. China
. Tel: 86 28 8550 2389; Fax: 86 28 8550 3204. E-mail:
[email protected]
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Abstract Cigarette smoke exposure (CSE) negatively affects neurodevelopment. We established a CSE rat model to determine how maternal CSE induces oxidative stress and mitochondrial dysfunction in parafacial respiratory group (pFRG) essential to central chemoreceptive regulation of normal breathing. Pregnant rats were exposed to cigarette smoke during gestational days 1-20, and the offspring were studied on postnatal day 2. Our data showed that maternal CSE resulted in elevated accumulation of ROS, which left a footprint on DNA and lipid with increases in 8-hydroxy-2’-deoxyguanosine and malondialdehyde contents. Furthermore, maternal CSE induced decreases in manganese superoxide dismutase, catalase and glutathione reductase activities as well as reduction in glutathione content in pFRG in the offspring. Moreover, maternal CSE led to mitochondrial ultrastructure changes, mitochondrial swelling, reduction in ATP generation, loss of mitochondrial membrane potential and increase in mitochondrial DNA copy number. These findings suggest that maternal exposure to cigarette smoke alters normal development of pFRG that is critical for normal respiratory control.
Graphical abstract
Key words: maternal cigarette smoke exposure; parafacial respiratory group; oxidative stress; mitochondrial dysfunction
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Introduction Parafacial respiratory group (pFRG) is one of the brainstem nuclei which are critical for normal respiratory control[1]. More notably, pFRG neurons are the most advocated central respiratory chemoreceptors playing a putative role in central respiratory chemoreception[2, 3]. Studies have demonstrated that impairment of pFRG development or function correlates with blunted responses to hypercapnia and acidification [4, 5]. Maternal cigarette smoking increases the risks of miscarriage, placental abruption, preterm birth, intrauterine growth retardation, respiratory disorders, and behavioral impairments[6-9]. Despite increased general education on these risks and government policies to ban smoking, maternal smoking during gestation is still common in both developed and developing countries[10]. Toxic agents in cigarette smoke can be transferred into the fetus via the placenta leading to underdevelopment of the fetus. Among these toxic substances, nicotine, as a vasoconstrictor, acts upon maternal cardiovascular system and reduce uterine blood flow, leading to deprivation of oxygen and nutrients in the fetus[11]. The consequent chronic hypoxia and malnutrition may alter the physiological development of organs and tissues, including the brain[12-14]. Human study has indicated that prenatal cigarette smoke exposure (CSE) negatively affects biological parameters of the developing brainstem, including ventrolateral respiratory nuclei in the medulla oblongata[15], which are closely associated with respiratory control[16]. It has been well known that most of the living organisms need oxygen to survive. Nevertheless, living organisms are constantly exposed to oxidants from endogenous metabolic processes, such as reactive oxygen species (ROS), a group of oxygen-derived byproducts released during mitochondrial oxidative phosphorylation to generate ATP[17]. In physiological condition, there is a balance between ROS production and their clearance in the system and no oxidative stress usually occurs. Oxidative stress develops when the intracellular antioxidants are unable to counteract the overproduction of ROS, leading to lipid peroxidation, DNA strand breaks and other forms of intracellular oxidative injuries, which are detrimental to cell structures and functions[18]. Mitochondria are the major intracellular source but also the most affected target of ROS[19].
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Mitochondria play a key role in development by supplying energy for the rapid fetal growth[20]. Thus, impaired mitochondrial homeostasis may fatally imperil energy metabolism and cell functions. Studies have demonstrated that oxidative stress with mitochondrial damage and dysfunction is implicated in numerous neurodegenerative diseases such as Alzheimer's disease and Parkinson disease[21-24]. Cigarette smoke contains a substantial amount of ROS[25], which may exceed intracellular antioxidant capacity leading to oxidative stress. Smoking has been considered to be strongly associated with elevated oxidative stress in smokers[26]. Maternal smoking during pregnancy can cause severe oxidative stress not only in the mother, but also in the offspring[27, 28], because of the diffusion of free radicals and harmful chemicals within cigarette smoke, such as nicotine, through the blood-placental barrier into the fetus[29]. Studies have indicated that maternal CSE decreases the antioxidant capacity in lungs[30] and kidneys[31, 32] in the offspring. However, it is unclear whether maternal CSE increases oxidative stress and mitochondrial perturbations in pFRG in the offspring. This study was designed to test the hypothesis that maternal CSE increases oxidative stress and mitochondrial dysfunction in pFRG, which is important for ventilatory control, in the offspring.
Methods The experimental protocols were approved by the Animal Care and Use Committee of Sichuan University, and all studies were performed in accordance with the national institute of health guide for the care and use of laboratory animals (NIH publication No.8023) revised 1978. Animals Adult Sprague Dawley rats were obtained from Sichuan university experimental animal center. At the beginning of experiment, rats were kept in a temperature-controlled (~25C) room with a 12h light/dark cycle and had access to food and water ad libitum. Pregnancy was confirmed by the presence of spermatozoa on the vaginal smear and the following day was considered as gestational day 1. Pregnant rats were divided into 2 groups: Control and CSE. 2-day-old pups were used in the studies, and randomization was performed within each litter in both experimental groups. Maternal cigarette smoke exposure 4
Maternal CSE was designed to mimic active smoking during pregnancy as previously described[33]. Maternal CSE was initiated on day 1 of pregnancy and continued until gestational day 20. Pregnant rats were exposed in a CSE apparatus to tobacco smoke generated by lit cigarettes. The daily CSE was performed in two sessions, one in the morning starting at 9:00 and one in the afternoon starting at 16:00. For each session, the pregnant rats were exposed to a total of 10 cigarettes over a period of 60min: 2 lit cigarettes for 10 min followed by a 2-min interval per time, 5 times for one session. Pregnant rats in the Control group were placed into an identical apparatus, but received fresh air. Sample collection Animals at postnatal day 2 were sacrificed by decapitation after being anesthetized with ether inhalation. Brainstems were isolated, and then 900-μm-thick medullary slices containing pFRG were obtained by vibrating microtome (MA752, Campden Instrument LTD, UK), and pFRG was sampled from the slices and stored at −80C for further detection. Intracellular ROS assay Intracellular ROS was measured by a fluorescent dye dihydroethidium (DHE) with a DHE commercial kit (keyGEN BioTECH, Nanjing, China) according to the manufacturer’s instruction[34]. Briefly, the contents (5mg) of DHE were dissolved in 3.17ml dimethylsulfoxide to make a 5mM DHE reagent stock solution. Then the 5mM DHE reagent stock solution was diluted in PBS to make a 50μM DHE reagent working solution. The fresh frozen medulla was sectioned at 12μm, and the sections containing pFRG were collected and incubated with DHE reagent working solution for 30min at 37C in a humidified chamber protected from light, and then the sections were washed in PBS for 3 times. Fluorescent images were collected using a fluorescence microscope (Olympus BX51TR, Olympus Corp., Tokyo, Japan). All imaging parameters were kept constant during imaging. Fluorescent intensity was quantified with Image Pro-Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA). Data were expressed as mean fluorescence intensity. Immunohistological staining 8-hydroxy-2’-deoxyguanosine (8-OHdG) is known as an end product of oxidative damage to DNA and known to be a sensitive oxidative stress biomarker[35]. Therefore, we examined whether 5
maternal CSE induced an increase in 8-OHdG expression. One medulla oblongata was randomly selected from each litter and fixed in 4% paraformaldehyde at 4C overnight, and then was embedded in paraffin and sectioned. Sections (5μm) containing pFRG were collected. For immunohistological staining, sections were incubated with primary antibody against 8-OHdG (1:100, Santa Cruz Biotechnology, CA, USA) at 4C overnight, followed by anti-mouse IgG according to the manufacturer’s instructions. Nuclei were counterstained with hematoxylin. After dehydrating and mounting, light microscopy pictures were taken with a microscope (Olympus BX51TR, Olympus Corp., Tokyo, Japan). Immunoreactivity intensity was quantified with Image Pro-Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA). Data were expressed as mean optical densities. Measurement of lipid peroxide, antioxidant content and antioxidant enzyme activities A thiobarbituric acid reactive species assay based on the formation of malondialdehyde (MDA) was performed to quantify the amount of lipid peroxidation with a MDA assay kit (KeyGEN BioTECH, Nanjing, China). Briefly, 100μl of the sample was added to the reaction mixture, and the mixture was incubated for 40min in a water bath maintained at 95C. After cooling, the absorbance of the mixture was read at 532nm. 10nmol/ml MDA was used as a standard in the assay. The result was expressed as nmol/mgprot. Glutathione (GSH) concentration was determined with a GSH assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. In brief, the reaction reagents were added to the sample to observe the color development due to formation of thionitrobenzoate. The absorbance was measured at 412nm, and the data were expressed as μM/mgprot using GSH as a standard. The mitochondrial manganese superoxide dismutase (Mn-SOD) activity in pFRG was determined using a Mn-SOD assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. The assay was based on the ability of Mn-SOD, present in the samples, to inhibit the oxidation of hydroxylamine. In brief, 10μl of sample was added to the working solution, and incubated at 37C for 40min. The absorbance was measured at a wavelength of 550nm, and the Mn-SOD activity was calculated based on the ability of 1 unit of
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Mn-SOD to inhibit oxidation of hydroxylamine by 50%. The catalase (CAT) activity in pFRG was determined with an assay kit (KeyGEN BioTECH, Nanjing, China). 50μl of sample was added to the reaction buffer. The absorbance of the reaction mixture was monitored at 405nm. The enzyme activity was calculated based on the ability of 1 unit of CAT to decompose 1μmol H2O2. The glutathione reductase (GR) activity in pFRG was assayed with a GR assay kit (Beyotime, Shanghai, China). Briefly, 20μl of sample was added to the reaction mixture and the oxidation of NADPH was monitored at 412nm for 10min. The data were expressed as mU/mgprot. Isolation of mitochondria Mitochondria were isolated from fresh pFRG tissues by differential centrifugation using a tissue mitochondrial isolation kit (Beyotime, Shanghai, China). Briefly, a 10% pFRG homogenate was prepared in isolation buffer and centrifuged at 600×g for 5min at 4C to pellet the nuclear fraction. The resulting supernatant was centrifuged at 11,000×g for 10min at 4C to obtain the mitochondrial fraction. Assessment of mitochondrial swelling Mitochondrial swelling was assessed as previously described[36]. In brief, mitochondria (0.5mg/ml) were incubated in respiration buffer (containing 100mM KCl, 50mM sucrose, 10mM HEPES, and 5mM KH2PO4, pH 7.4 at 25C) with an addition of 10mM pyruvate/malate. Mitochondrial swelling was determined by measuring the absorbance of the suspension at 540nm, and the decrease of the absorbance of the suspension indicates mitochondrial swelling. Determination of mitochondrial membrane potential The mitochondrial membrane potential (MMP), an indicator of the inner and outer membrane integrity[37], was examined using the JC-1 staining kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. In mitochondria with high MMP, JC-1 forms aggregates eliciting characteristic red fluorescence; upon loss of MMP, these aggregates dismantle into green fluorescent monomers. Thus, the ratio of red to green fluorescence can represent the level of MMP[38]. 100μl of mitochondria containing 100μg of protein was incubated with JC-1 working 7
solution for 20min at 37C. After incubation, the sample fluorescence was measured and quantified by fluorescent microplate system. Examination of mitochondrial ultrastructure To assess the effect of maternal CSE on mitochondrial ultrastructure, transmission electron microscopy was applied. pFRG tissue was prefixed in 2.5% glutaraldehyde in phosphate buffer saline pH 7.4, and postfixed in 1% osmium tetroxide, and subsequently dehydrated in series acetone and embedded in Epon812. Ultrathin sections were stained with uranyl acetate and lead citrate, and then examined with a transmission electron microscope (H-600IV, Hitachi, Tokyo, Japan). Detection of ATP production ATP content of the samples was measured using an ATP commercial assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instruction. In brief, pFRG tissues were homogenized in boiling double distilled water (1:9, wt/vol). After centrifugation (3,500rpm, 10min), 15 μl supernatant was mixed with the ATP detection reagents. After incubation, the absorbance was measured at 636 nm, and the data were expressed as μg/gprot using 1mM ATP as a standard. Assessment of mitochondrial DNA copy number Mitochondrial DNA (mtDNA) copy number was measured using the real-time quantitative polymerase chain reaction (qPCR) as previously describled[39]. pFRG tissues were pooled from two animals. Genomic DNA was extracted by using the Universal Genomic DNA kit (CWBIO, Beijing, China). The purity and concentration of the extracted DNA was tested using Nanodrop™ 2000 (Thermo Fisher Scientific, Waltham, MA, USA). To quantify mtDNA copy number, two genes representing
either
mtDNA
or
nuclear
DNA
were
used,
ND1
(forward
primers
5’-TCCTCCTAATAAGCGGCTCCTTCTC-3’, reverse primers 5’-GGTCCTGCGGCGTATTCGAC-3’) and β-globin
(forward
primers
5’-CAGTACTTTAAGTTGGAAACG-3’,
reverse
primers
5’-
ATCAACATAATTGCAGAGC-3’). QPCR was performed on the platform of CFX96 TouchTM Real-Time PCR Detection system (BioRad Laboratories, CA, USA) with UltraSYBR Mixture Kit (CWBIO, Beijing, China). The reactions were performed in a total volume of 20μl including 10μl 2×UltraSYBR 8
Mixture, 1μl forward primer (4μM) and 1μl reverse primer (4μM), 1μl total genomic DNA (50ng/μl) and 7μl nuclease-free water. The amplification was preheated at 95C for 10min followed by a 40-cycle program of 15sec at 95C, 1min at 60C, 15sec at 95C, 1min at 60C, 15sec at 95C, 15sec at 60C. The threshold cycle (Ct) was collected using CFX ManagerTM software. The copy number of the mitochondrial gene ND1 was normalized to the single-copy nuclear gene, β-globin. Relative mtDNA copy number (RCN) was calculated by a comparative method, using the following equation: RCN=2∆Ct, where ∆Ct=Ctβ-globin- CtND1[39]. Statistical analysis Statistical analysis was performed using independent sample t test. All results were presented as mean ± SEM. Differences with a p value less than 0.05 were considered to be statistically significant.
Results Maternal CSE increases oxidative stress in pFRG in the offspring To detect whether maternal CSE causes oxidative stress in pFRG in the offspring, we examined intracellular ROS production in pFRG with a ROS fluorescent probe (Fig. 1A). Our result showed that intracellular ROS generation was significantly increased after maternal CSE compared with the Control group as indicated by increased mean fluorescence intensity (p<0.05, Fig 1B).
Fig. 1 Maternal CSE increased ROS generation in pFRG in the offspring. (A) Representative fluorescent images of ROS staining in pFRG in animals from the Control and CSE groups. (B) Comparison of mean fluorescence intensity of ROS between the Control and CSE groups. *p<0.05 v.s Control group. n=5. Scale bars represent 100μm.
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Maternal CSE increases DNA oxidation in pFRG in the offspring To examine the effect of elevated intracellular ROS production induced by maternal CSE on DNA damage, we next performed immunohistochemistry for 8-OHdG (Fig. 2A), which is known as an end product of oxidative damage to DNA and a sensitive oxidative stress biomarker. A remarkable increase of 8-OHdG was observed in pFRG from maternal cigarette smoke-exposed offspring compared with that from Control group (p<0.05, Fig. 2B).
Fig. 2 Maternal CSE augmented DNA oxidative damage in pFRG in the offspring. (A) Representative immunohistochemical staining of 8-OHdG in pFRG in animals from the Control and CSE groups. (B) Comparison of mean optical density (OD) values of 8-OHdG between the Control and CSE groups. *p<0.05 v.s Control group. n=5. Scale bars represent 40μm. Maternal CSE increases lipid peroxidation in pFRG in the offspring Then, to determine the detrimental effect of maternal CSE on lipid, the content of MDA, a marker of lipid peroxidation, was measured. As displayed in Fig. 3, the accumulation of MDA was markedly elevated in pFRG tissues from the animals subjected to maternal CSE in comparison with those from Control group (p<0.05).
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Fig. 3 Maternal CSE lipid peroxidation in pFRG in the offspring. Comparison of malondialdehyde (MDA) content between the Control and CSE groups. *p<0.05 v.s Control group. n=5 Maternal CSE reduces antioxidant capacity in pFRG in the offspring Oxidative stress is considered to be the disruption of redox signaling and regulation. Therefore, we examined the effect of maternal CSE on intracellular antioxidant capacity, subsequently. Our data showed that the activity of CAT, an antioxidant enzyme that catalyzes the decomposition of H2O2 with high efficiency, was decreased by maternal CSE (p<0.05, Fig. 4A). Likewise, the activity of Mn-SOD was significantly diminished in the animals subjected to maternal CSE (p<0.05, Fig. 4B). We also measured content of GSH, a dominant low-molecular-weight antioxidant in mammalian cells, and our result showed that maternal CSE significantly decreased GSH production (p<0.05, Fig. 4C). In addition, the activity of GR, an antioxidant enzyme that catalyzes the reduction of GSH, was detected as well. The data showed that maternal CSE markedly inhibited GR activity in the offspring (p<0.05, Fig. 4D).
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Fig. 4 Maternal CSE perturbed antioxidant defense system in pFRG in the offspring. Comparison of (A) catalase (CAT) activity, n=5, (B) mitochondrial manganese superoxide dismutase (Mn-SOD) activity, n=6, (C) glutathione (GSH) content, n=8, (D) glutathione reductase (GR) activity, n=6, in pFRG between the Control and CSE groups. *p<0.05 v.s Control group. Maternal CSE induces mitochondrial ultrastructure changes in pFRG in the offspring To test the effect of maternal CSE on mitochondrial morphology in pFRG, we examined mitochondrial ultrastructure using transmission electron microscope. Offspring from cigarette smoke-exposed mothers exhibited mitochondrial enlargement and swelling with a loss of matrix and cristae (Fig. 5).
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Fig. 5 Maternal CSE induces mitochondrial ultrastructure change in pFRG in the offspring. Representative transmission electron microscopy (TEM) images from the (A) Control and (B) CSE groups. White arrows show normal mitochondria in the Control group, and black arrows show enlarged mitochondria and loss of matrix and cristae in the CSE offspring. Scale bars represent 0.5μm. Maternal CSE induces mitochondrial dysfunction in pFRG in the offspring To evaluate whether maternal CSE leads to mitochondrial dysfunction, we first examined mitochondrial swelling. By measuring the absorbance at 540nm, we found that maternal CSE resulted in decreased absorbance compared to the control group (p<0.05, Fig. 6A), suggesting that maternal CSE induced mitochondrial swelling, which indicated abnormal opening of mitochondrial permeability transition pore (mPTP)[36]. Meanwhile, we observed that the membrane potential was lost in the offspring from maternal CSE group as indicated by decrease in the ratio of red to green fluorescence (p<0.05, Fig. 6B). As disruption of mitochondrial homeostasis induced by oxidative stress can result in inhibition of mitochondrial bioenergetics, we subsequently examined the effect of maternal CSE on mitochondrial bioenergetics by measuring ATP generation in pFRG in the offspring. The data showed that maternal CSE detrimentally affect ATP production in pFRG in the offspring (p<0.05, Fig. 6C) It has been suggested that abnormal amount of mtDNA content, depletion or elevation, is associated with mitochondrial dysfunction. Thus, we investigated mtDNA copy number in pFRG 13
of neonatal rats. Our data showed that the relative mtDNA content was increased in pFRG in the offspring from CSE mothers (p<0.05, Fig. 6D).
Fig. 6 Maternal CSE induced mitochondrial dysfunction in pFRG in the offspring. (A) Comparison of mitochondrial swelling in pFRG between the Control and CSE groups, n=6. (B) Comparison of mitochondrial membrane potential (MMP) in pFRG between the Control and CSE groups, n=8. (C) Comparison of ATP content in pFRG between the Control and CSE groups, n=8. (D) Comparison of relative mitochondrial DNA (mtDNA) copy number in pFRG between the Control and CSE groups, n=6. *p<0.05 v.s Control group.
Discussion Maternal cigarette smoking during pregnancy has been shown to adversely affect brainstem development[15, 40] and has also been linked to an increased risk of sudden infant death syndrome[41], whose victims suffer a seriously decreased central chemosensitivity. In this study, with a rat model of maternal CSE, we demonstrated that maternal CSE can significantly increase 14
oxidative stress and impair mitochondrial function in pFRG in the offspring, which might be a possible reason for the impairment of central chemoreception induced by maternal CSE[42]. Cigarette smoke contains a large amount of ROS[25]. Previous study showed that maternal cigarette smoking during pregnancy increased brain oxidative damages in the offspring[43]. In this study, we found that maternal CSE enhanced ROS accumulation in pFRG, which provided direct evidence for increased oxidative stress in the offspring. Excessive free radicals directly interact with cellular biomolecules, such as DNA, causing oxidative DNA damage[18]. Previous study showed that mice from mothers exposed to cigarette smoke displayed increased DNA oxidation in kidneys[31]. In our present investigation in rat, we found that maternal CSE resulted in serious oxidative damage to DNA as evidenced by overexpression of 8-OHdG, the most frequently used biomarker of oxidative DNA damage, in pFRG in the offspring as compared with control ones. In addition, accumulating evidence show that excessive ROS leads to increase in MDA, an end-product of peroxidation reaction elicited by free radicals acting on lipid. The amount of MDA can be used to estimate the degree of lipid peroxidation in tissues. Herein, our result showed that maternal CSE increased MDA content indicating an overproduction of lipid peroxide in pFRG in the offspring. These results suggest that maternal CSE gives rise to oxidative damage to pFRG in the offspring. Oxidative stress occurs when excessive ROS accumulation overpowers intracellular antioxidant capacity. In this study, it was found that offspring subjected to maternal CSE showed reduced GSH production and activities of CAT, GR and Mn-SOD, vital antioxidants for intracellular antioxidant defense in pFRG. GSH is highly sensitive to the imbalance between the antioxidant system and ROS, and is important for cellular ROS scavenging[44]. The antioxidant effect of GSH involves two pathways: 1) non-enzymatically reacts with radicals in a direct manner; 2) acts as an electron donor in reducing peroxides catalyzed by glutathione peroxidases[45]. Within cells, GSH can be generated by reactions catalyzed by GR[46]. CAT is another important cellular antioxidant enzyme known to convert hydrogen peroxide into water and oxygen, resulting in ROS disposal[47]. Mn-SOD is an important enzyme for intracellular antioxidant defense, especially within the mitochondria that converts superoxide free radicals into hydrogen peroxide, which was transformed to water by catalases and other peroxidases[48]. It has also been elucidated that 15
Mn-SOD is a part of protein complex necessary for mitochondrial DNA repair[49]. Thus, Mn-SOD plays an essential role in various aspects of mitochondrial protection. Consistent with studies in kidneys[31, 32], we found that the activity of mitochondrial Mn-SOD was reduced in the offspring exposed to maternal cigarette smoking. The reduction in GSH generation, CAT, GR and Mn-SOD activities in pFRG in the current study indicates that maternal CSE decreased the antioxidant capacity in the offspring. Mitochondria are essential for energy production, however at the same time, ROS accumulates as byproducts of mitochondrial oxidative phosphorylation causing mitochondrial damage, which in turn results in an escalation of oxidative stress[18, 19]. It has been reported that excessive ROS contributes to abnormal mitochondrial membrane permeability and increased swelling[50]. Our data showed that increased swelling was detected in mitochondria from CSE offspring. It has been indicated that mitochondrial swelling is due to prolonged opening of mPTP[36]. Thus, increased swelling observed in the present study indicates an abnormal opening of mPTP, which is reported to cause an increase of permeability to solutes leading to disruption of solute homeostasis, drastic changes in mitochondrial ultrastructure and functional activity[51]. In this study, we in deed observed mitochondrial structure changes in animals subjected to maternal CSE with mitochondrial enlargement and a loss of matrix and cristae. We also observed that maternal CSE induced loss of MMP, likewise, maternal CSE resulted in an impediment to mitochondrial ATP production in the offspring. It has been shown that oxidative stress is associated with DNA damage, and the mitochondrial genome is more susceptible than the nuclear genome due to the lack of histone protection, reduced DNA repair capacity and close proximity to the ROS producing electron transport chain[31]. Previous study has indicated that abnormal mtDNA content, even without any detectable mtDNA mutations, can be indicative of mitochondrial dysfunction[18, 52]. Our finding, together with others[31, 53], supports that maternal CSE induces an increase in mtDNA level in response to oxidative stress in the offspring. The mtDNA amplification might be a compensatory mechanism in response to the decline of mitochondrial function[52, 54]. In conclusion, our study demonstrates that maternal CSE leads to oxidative stress and mitochondrial dysfunction in pFRG in rat offspring. Our results shine light on the consequence of 16
maternal smoking during pregnancy, and set the stage for further studies exploring the influence of maternal cigarette smoking on the development of brainstem in the offspring.
Acknowledgement We are grateful to Dr. G. Yang for his technical support in transmission electron microscopy. This work is supported by grants from the National Natural Science Foundation of China (No. 31471096).
Declarations of interest None.
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Highlights:
Maternal CS exposure increases oxidative damage in pFRG in rat offspring.
Maternal CS exposure decreases the antioxidant capacity in pFRG in rat offspring.
Maternal CS exposure destroys mitochondrial ultrastructure in pFRG in rat offspring.
Maternal CS exposure induces mitochondrial dysfunction in pFRG in rat offspring.
23
PII: DOI: Reference:
S0891-5849(18)31071-2 https://doi.org/10.1016/j.freeradbiomed.2018.09.003 FRB13905
To appear in: Free Radical Biology and Medicine Received date: 16 June 2018 Revised date: 30 August 2018 Accepted date: 2 September 2018 Cite this article as: Fang Lei, Wen Wang, Yating Fu, Ji Wang and Yu Zheng, Oxidative stress and mitochondrial dysfunction in parafacial respiratory group induced by maternal cigarette smoke exposure in rat offspring, Free Radical Biology and Medicine, https://doi.org/10.1016/j.freeradbiomed.2018.09.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Oxidative stress and mitochondrial dysfunction in parafacial respiratory group induced by maternal cigarette smoke exposure in rat offspring
Fang Lei, Wen Wang, Yating Fu, Ji Wang, Yu Zheng* Department of physiology, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu, Sichuan 610041, P.R. China
* Corresponding author. Dr. Yu Zheng , Department of Physiology , West China School of Basic Medical Sciences and Forensic Medicine , Sichuan University , 3-17 Renmin South Road , Chengdu, Sichuan 610041. P.R. China
. Tel: 86 28 8550 2389; Fax: 86 28 8550 3204. E-mail:
[email protected]
1
Abstract Cigarette smoke exposure (CSE) negatively affects neurodevelopment. We established a CSE rat model to determine how maternal CSE induces oxidative stress and mitochondrial dysfunction in parafacial respiratory group (pFRG) essential to central chemoreceptive regulation of normal breathing. Pregnant rats were exposed to cigarette smoke during gestational days 1-20, and the offspring were studied on postnatal day 2. Our data showed that maternal CSE resulted in elevated accumulation of ROS, which left a footprint on DNA and lipid with increases in 8-hydroxy-2’-deoxyguanosine and malondialdehyde contents. Furthermore, maternal CSE induced decreases in manganese superoxide dismutase, catalase and glutathione reductase activities as well as reduction in glutathione content in pFRG in the offspring. Moreover, maternal CSE led to mitochondrial ultrastructure changes, mitochondrial swelling, reduction in ATP generation, loss of mitochondrial membrane potential and increase in mitochondrial DNA copy number. These findings suggest that maternal exposure to cigarette smoke alters normal development of pFRG that is critical for normal respiratory control.
Graphical abstract
Key words: maternal cigarette smoke exposure; parafacial respiratory group; oxidative stress; mitochondrial dysfunction
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Introduction Parafacial respiratory group (pFRG) is one of the brainstem nuclei which are critical for normal respiratory control[1]. More notably, pFRG neurons are the most advocated central respiratory chemoreceptors playing a putative role in central respiratory chemoreception[2, 3]. Studies have demonstrated that impairment of pFRG development or function correlates with blunted responses to hypercapnia and acidification [4, 5]. Maternal cigarette smoking increases the risks of miscarriage, placental abruption, preterm birth, intrauterine growth retardation, respiratory disorders, and behavioral impairments[6-9]. Despite increased general education on these risks and government policies to ban smoking, maternal smoking during gestation is still common in both developed and developing countries[10]. Toxic agents in cigarette smoke can be transferred into the fetus via the placenta leading to underdevelopment of the fetus. Among these toxic substances, nicotine, as a vasoconstrictor, acts upon maternal cardiovascular system and reduce uterine blood flow, leading to deprivation of oxygen and nutrients in the fetus[11]. The consequent chronic hypoxia and malnutrition may alter the physiological development of organs and tissues, including the brain[12-14]. Human study has indicated that prenatal cigarette smoke exposure (CSE) negatively affects biological parameters of the developing brainstem, including ventrolateral respiratory nuclei in the medulla oblongata[15], which are closely associated with respiratory control[16]. It has been well known that most of the living organisms need oxygen to survive. Nevertheless, living organisms are constantly exposed to oxidants from endogenous metabolic processes, such as reactive oxygen species (ROS), a group of oxygen-derived byproducts released during mitochondrial oxidative phosphorylation to generate ATP[17]. In physiological condition, there is a balance between ROS production and their clearance in the system and no oxidative stress usually occurs. Oxidative stress develops when the intracellular antioxidants are unable to counteract the overproduction of ROS, leading to lipid peroxidation, DNA strand breaks and other forms of intracellular oxidative injuries, which are detrimental to cell structures and functions[18]. Mitochondria are the major intracellular source but also the most affected target of ROS[19].
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Mitochondria play a key role in development by supplying energy for the rapid fetal growth[20]. Thus, impaired mitochondrial homeostasis may fatally imperil energy metabolism and cell functions. Studies have demonstrated that oxidative stress with mitochondrial damage and dysfunction is implicated in numerous neurodegenerative diseases such as Alzheimer's disease and Parkinson disease[21-24]. Cigarette smoke contains a substantial amount of ROS[25], which may exceed intracellular antioxidant capacity leading to oxidative stress. Smoking has been considered to be strongly associated with elevated oxidative stress in smokers[26]. Maternal smoking during pregnancy can cause severe oxidative stress not only in the mother, but also in the offspring[27, 28], because of the diffusion of free radicals and harmful chemicals within cigarette smoke, such as nicotine, through the blood-placental barrier into the fetus[29]. Studies have indicated that maternal CSE decreases the antioxidant capacity in lungs[30] and kidneys[31, 32] in the offspring. However, it is unclear whether maternal CSE increases oxidative stress and mitochondrial perturbations in pFRG in the offspring. This study was designed to test the hypothesis that maternal CSE increases oxidative stress and mitochondrial dysfunction in pFRG, which is important for ventilatory control, in the offspring.
Methods The experimental protocols were approved by the Animal Care and Use Committee of Sichuan University, and all studies were performed in accordance with the national institute of health guide for the care and use of laboratory animals (NIH publication No.8023) revised 1978. Animals Adult Sprague Dawley rats were obtained from Sichuan university experimental animal center. At the beginning of experiment, rats were kept in a temperature-controlled (~25C) room with a 12h light/dark cycle and had access to food and water ad libitum. Pregnancy was confirmed by the presence of spermatozoa on the vaginal smear and the following day was considered as gestational day 1. Pregnant rats were divided into 2 groups: Control and CSE. 2-day-old pups were used in the studies, and randomization was performed within each litter in both experimental groups. Maternal cigarette smoke exposure 4
Maternal CSE was designed to mimic active smoking during pregnancy as previously described[33]. Maternal CSE was initiated on day 1 of pregnancy and continued until gestational day 20. Pregnant rats were exposed in a CSE apparatus to tobacco smoke generated by lit cigarettes. The daily CSE was performed in two sessions, one in the morning starting at 9:00 and one in the afternoon starting at 16:00. For each session, the pregnant rats were exposed to a total of 10 cigarettes over a period of 60min: 2 lit cigarettes for 10 min followed by a 2-min interval per time, 5 times for one session. Pregnant rats in the Control group were placed into an identical apparatus, but received fresh air. Sample collection Animals at postnatal day 2 were sacrificed by decapitation after being anesthetized with ether inhalation. Brainstems were isolated, and then 900-μm-thick medullary slices containing pFRG were obtained by vibrating microtome (MA752, Campden Instrument LTD, UK), and pFRG was sampled from the slices and stored at −80C for further detection. Intracellular ROS assay Intracellular ROS was measured by a fluorescent dye dihydroethidium (DHE) with a DHE commercial kit (keyGEN BioTECH, Nanjing, China) according to the manufacturer’s instruction[34]. Briefly, the contents (5mg) of DHE were dissolved in 3.17ml dimethylsulfoxide to make a 5mM DHE reagent stock solution. Then the 5mM DHE reagent stock solution was diluted in PBS to make a 50μM DHE reagent working solution. The fresh frozen medulla was sectioned at 12μm, and the sections containing pFRG were collected and incubated with DHE reagent working solution for 30min at 37C in a humidified chamber protected from light, and then the sections were washed in PBS for 3 times. Fluorescent images were collected using a fluorescence microscope (Olympus BX51TR, Olympus Corp., Tokyo, Japan). All imaging parameters were kept constant during imaging. Fluorescent intensity was quantified with Image Pro-Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA). Data were expressed as mean fluorescence intensity. Immunohistological staining 8-hydroxy-2’-deoxyguanosine (8-OHdG) is known as an end product of oxidative damage to DNA and known to be a sensitive oxidative stress biomarker[35]. Therefore, we examined whether 5
maternal CSE induced an increase in 8-OHdG expression. One medulla oblongata was randomly selected from each litter and fixed in 4% paraformaldehyde at 4C overnight, and then was embedded in paraffin and sectioned. Sections (5μm) containing pFRG were collected. For immunohistological staining, sections were incubated with primary antibody against 8-OHdG (1:100, Santa Cruz Biotechnology, CA, USA) at 4C overnight, followed by anti-mouse IgG according to the manufacturer’s instructions. Nuclei were counterstained with hematoxylin. After dehydrating and mounting, light microscopy pictures were taken with a microscope (Olympus BX51TR, Olympus Corp., Tokyo, Japan). Immunoreactivity intensity was quantified with Image Pro-Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA). Data were expressed as mean optical densities. Measurement of lipid peroxide, antioxidant content and antioxidant enzyme activities A thiobarbituric acid reactive species assay based on the formation of malondialdehyde (MDA) was performed to quantify the amount of lipid peroxidation with a MDA assay kit (KeyGEN BioTECH, Nanjing, China). Briefly, 100μl of the sample was added to the reaction mixture, and the mixture was incubated for 40min in a water bath maintained at 95C. After cooling, the absorbance of the mixture was read at 532nm. 10nmol/ml MDA was used as a standard in the assay. The result was expressed as nmol/mgprot. Glutathione (GSH) concentration was determined with a GSH assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. In brief, the reaction reagents were added to the sample to observe the color development due to formation of thionitrobenzoate. The absorbance was measured at 412nm, and the data were expressed as μM/mgprot using GSH as a standard. The mitochondrial manganese superoxide dismutase (Mn-SOD) activity in pFRG was determined using a Mn-SOD assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. The assay was based on the ability of Mn-SOD, present in the samples, to inhibit the oxidation of hydroxylamine. In brief, 10μl of sample was added to the working solution, and incubated at 37C for 40min. The absorbance was measured at a wavelength of 550nm, and the Mn-SOD activity was calculated based on the ability of 1 unit of
6
Mn-SOD to inhibit oxidation of hydroxylamine by 50%. The catalase (CAT) activity in pFRG was determined with an assay kit (KeyGEN BioTECH, Nanjing, China). 50μl of sample was added to the reaction buffer. The absorbance of the reaction mixture was monitored at 405nm. The enzyme activity was calculated based on the ability of 1 unit of CAT to decompose 1μmol H2O2. The glutathione reductase (GR) activity in pFRG was assayed with a GR assay kit (Beyotime, Shanghai, China). Briefly, 20μl of sample was added to the reaction mixture and the oxidation of NADPH was monitored at 412nm for 10min. The data were expressed as mU/mgprot. Isolation of mitochondria Mitochondria were isolated from fresh pFRG tissues by differential centrifugation using a tissue mitochondrial isolation kit (Beyotime, Shanghai, China). Briefly, a 10% pFRG homogenate was prepared in isolation buffer and centrifuged at 600×g for 5min at 4C to pellet the nuclear fraction. The resulting supernatant was centrifuged at 11,000×g for 10min at 4C to obtain the mitochondrial fraction. Assessment of mitochondrial swelling Mitochondrial swelling was assessed as previously described[36]. In brief, mitochondria (0.5mg/ml) were incubated in respiration buffer (containing 100mM KCl, 50mM sucrose, 10mM HEPES, and 5mM KH2PO4, pH 7.4 at 25C) with an addition of 10mM pyruvate/malate. Mitochondrial swelling was determined by measuring the absorbance of the suspension at 540nm, and the decrease of the absorbance of the suspension indicates mitochondrial swelling. Determination of mitochondrial membrane potential The mitochondrial membrane potential (MMP), an indicator of the inner and outer membrane integrity[37], was examined using the JC-1 staining kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. In mitochondria with high MMP, JC-1 forms aggregates eliciting characteristic red fluorescence; upon loss of MMP, these aggregates dismantle into green fluorescent monomers. Thus, the ratio of red to green fluorescence can represent the level of MMP[38]. 100μl of mitochondria containing 100μg of protein was incubated with JC-1 working 7
solution for 20min at 37C. After incubation, the sample fluorescence was measured and quantified by fluorescent microplate system. Examination of mitochondrial ultrastructure To assess the effect of maternal CSE on mitochondrial ultrastructure, transmission electron microscopy was applied. pFRG tissue was prefixed in 2.5% glutaraldehyde in phosphate buffer saline pH 7.4, and postfixed in 1% osmium tetroxide, and subsequently dehydrated in series acetone and embedded in Epon812. Ultrathin sections were stained with uranyl acetate and lead citrate, and then examined with a transmission electron microscope (H-600IV, Hitachi, Tokyo, Japan). Detection of ATP production ATP content of the samples was measured using an ATP commercial assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instruction. In brief, pFRG tissues were homogenized in boiling double distilled water (1:9, wt/vol). After centrifugation (3,500rpm, 10min), 15 μl supernatant was mixed with the ATP detection reagents. After incubation, the absorbance was measured at 636 nm, and the data were expressed as μg/gprot using 1mM ATP as a standard. Assessment of mitochondrial DNA copy number Mitochondrial DNA (mtDNA) copy number was measured using the real-time quantitative polymerase chain reaction (qPCR) as previously describled[39]. pFRG tissues were pooled from two animals. Genomic DNA was extracted by using the Universal Genomic DNA kit (CWBIO, Beijing, China). The purity and concentration of the extracted DNA was tested using Nanodrop™ 2000 (Thermo Fisher Scientific, Waltham, MA, USA). To quantify mtDNA copy number, two genes representing
either
mtDNA
or
nuclear
DNA
were
used,
ND1
(forward
primers
5’-TCCTCCTAATAAGCGGCTCCTTCTC-3’, reverse primers 5’-GGTCCTGCGGCGTATTCGAC-3’) and β-globin
(forward
primers
5’-CAGTACTTTAAGTTGGAAACG-3’,
reverse
primers
5’-
ATCAACATAATTGCAGAGC-3’). QPCR was performed on the platform of CFX96 TouchTM Real-Time PCR Detection system (BioRad Laboratories, CA, USA) with UltraSYBR Mixture Kit (CWBIO, Beijing, China). The reactions were performed in a total volume of 20μl including 10μl 2×UltraSYBR 8
Mixture, 1μl forward primer (4μM) and 1μl reverse primer (4μM), 1μl total genomic DNA (50ng/μl) and 7μl nuclease-free water. The amplification was preheated at 95C for 10min followed by a 40-cycle program of 15sec at 95C, 1min at 60C, 15sec at 95C, 1min at 60C, 15sec at 95C, 15sec at 60C. The threshold cycle (Ct) was collected using CFX ManagerTM software. The copy number of the mitochondrial gene ND1 was normalized to the single-copy nuclear gene, β-globin. Relative mtDNA copy number (RCN) was calculated by a comparative method, using the following equation: RCN=2∆Ct, where ∆Ct=Ctβ-globin- CtND1[39]. Statistical analysis Statistical analysis was performed using independent sample t test. All results were presented as mean ± SEM. Differences with a p value less than 0.05 were considered to be statistically significant.
Results Maternal CSE increases oxidative stress in pFRG in the offspring To detect whether maternal CSE causes oxidative stress in pFRG in the offspring, we examined intracellular ROS production in pFRG with a ROS fluorescent probe (Fig. 1A). Our result showed that intracellular ROS generation was significantly increased after maternal CSE compared with the Control group as indicated by increased mean fluorescence intensity (p<0.05, Fig 1B).
Fig. 1 Maternal CSE increased ROS generation in pFRG in the offspring. (A) Representative fluorescent images of ROS staining in pFRG in animals from the Control and CSE groups. (B) Comparison of mean fluorescence intensity of ROS between the Control and CSE groups. *p<0.05 v.s Control group. n=5. Scale bars represent 100μm.
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Maternal CSE increases DNA oxidation in pFRG in the offspring To examine the effect of elevated intracellular ROS production induced by maternal CSE on DNA damage, we next performed immunohistochemistry for 8-OHdG (Fig. 2A), which is known as an end product of oxidative damage to DNA and a sensitive oxidative stress biomarker. A remarkable increase of 8-OHdG was observed in pFRG from maternal cigarette smoke-exposed offspring compared with that from Control group (p<0.05, Fig. 2B).
Fig. 2 Maternal CSE augmented DNA oxidative damage in pFRG in the offspring. (A) Representative immunohistochemical staining of 8-OHdG in pFRG in animals from the Control and CSE groups. (B) Comparison of mean optical density (OD) values of 8-OHdG between the Control and CSE groups. *p<0.05 v.s Control group. n=5. Scale bars represent 40μm. Maternal CSE increases lipid peroxidation in pFRG in the offspring Then, to determine the detrimental effect of maternal CSE on lipid, the content of MDA, a marker of lipid peroxidation, was measured. As displayed in Fig. 3, the accumulation of MDA was markedly elevated in pFRG tissues from the animals subjected to maternal CSE in comparison with those from Control group (p<0.05).
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Fig. 3 Maternal CSE lipid peroxidation in pFRG in the offspring. Comparison of malondialdehyde (MDA) content between the Control and CSE groups. *p<0.05 v.s Control group. n=5 Maternal CSE reduces antioxidant capacity in pFRG in the offspring Oxidative stress is considered to be the disruption of redox signaling and regulation. Therefore, we examined the effect of maternal CSE on intracellular antioxidant capacity, subsequently. Our data showed that the activity of CAT, an antioxidant enzyme that catalyzes the decomposition of H2O2 with high efficiency, was decreased by maternal CSE (p<0.05, Fig. 4A). Likewise, the activity of Mn-SOD was significantly diminished in the animals subjected to maternal CSE (p<0.05, Fig. 4B). We also measured content of GSH, a dominant low-molecular-weight antioxidant in mammalian cells, and our result showed that maternal CSE significantly decreased GSH production (p<0.05, Fig. 4C). In addition, the activity of GR, an antioxidant enzyme that catalyzes the reduction of GSH, was detected as well. The data showed that maternal CSE markedly inhibited GR activity in the offspring (p<0.05, Fig. 4D).
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Fig. 4 Maternal CSE perturbed antioxidant defense system in pFRG in the offspring. Comparison of (A) catalase (CAT) activity, n=5, (B) mitochondrial manganese superoxide dismutase (Mn-SOD) activity, n=6, (C) glutathione (GSH) content, n=8, (D) glutathione reductase (GR) activity, n=6, in pFRG between the Control and CSE groups. *p<0.05 v.s Control group. Maternal CSE induces mitochondrial ultrastructure changes in pFRG in the offspring To test the effect of maternal CSE on mitochondrial morphology in pFRG, we examined mitochondrial ultrastructure using transmission electron microscope. Offspring from cigarette smoke-exposed mothers exhibited mitochondrial enlargement and swelling with a loss of matrix and cristae (Fig. 5).
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Fig. 5 Maternal CSE induces mitochondrial ultrastructure change in pFRG in the offspring. Representative transmission electron microscopy (TEM) images from the (A) Control and (B) CSE groups. White arrows show normal mitochondria in the Control group, and black arrows show enlarged mitochondria and loss of matrix and cristae in the CSE offspring. Scale bars represent 0.5μm. Maternal CSE induces mitochondrial dysfunction in pFRG in the offspring To evaluate whether maternal CSE leads to mitochondrial dysfunction, we first examined mitochondrial swelling. By measuring the absorbance at 540nm, we found that maternal CSE resulted in decreased absorbance compared to the control group (p<0.05, Fig. 6A), suggesting that maternal CSE induced mitochondrial swelling, which indicated abnormal opening of mitochondrial permeability transition pore (mPTP)[36]. Meanwhile, we observed that the membrane potential was lost in the offspring from maternal CSE group as indicated by decrease in the ratio of red to green fluorescence (p<0.05, Fig. 6B). As disruption of mitochondrial homeostasis induced by oxidative stress can result in inhibition of mitochondrial bioenergetics, we subsequently examined the effect of maternal CSE on mitochondrial bioenergetics by measuring ATP generation in pFRG in the offspring. The data showed that maternal CSE detrimentally affect ATP production in pFRG in the offspring (p<0.05, Fig. 6C) It has been suggested that abnormal amount of mtDNA content, depletion or elevation, is associated with mitochondrial dysfunction. Thus, we investigated mtDNA copy number in pFRG 13
of neonatal rats. Our data showed that the relative mtDNA content was increased in pFRG in the offspring from CSE mothers (p<0.05, Fig. 6D).
Fig. 6 Maternal CSE induced mitochondrial dysfunction in pFRG in the offspring. (A) Comparison of mitochondrial swelling in pFRG between the Control and CSE groups, n=6. (B) Comparison of mitochondrial membrane potential (MMP) in pFRG between the Control and CSE groups, n=8. (C) Comparison of ATP content in pFRG between the Control and CSE groups, n=8. (D) Comparison of relative mitochondrial DNA (mtDNA) copy number in pFRG between the Control and CSE groups, n=6. *p<0.05 v.s Control group.
Discussion Maternal cigarette smoking during pregnancy has been shown to adversely affect brainstem development[15, 40] and has also been linked to an increased risk of sudden infant death syndrome[41], whose victims suffer a seriously decreased central chemosensitivity. In this study, with a rat model of maternal CSE, we demonstrated that maternal CSE can significantly increase 14
oxidative stress and impair mitochondrial function in pFRG in the offspring, which might be a possible reason for the impairment of central chemoreception induced by maternal CSE[42]. Cigarette smoke contains a large amount of ROS[25]. Previous study showed that maternal cigarette smoking during pregnancy increased brain oxidative damages in the offspring[43]. In this study, we found that maternal CSE enhanced ROS accumulation in pFRG, which provided direct evidence for increased oxidative stress in the offspring. Excessive free radicals directly interact with cellular biomolecules, such as DNA, causing oxidative DNA damage[18]. Previous study showed that mice from mothers exposed to cigarette smoke displayed increased DNA oxidation in kidneys[31]. In our present investigation in rat, we found that maternal CSE resulted in serious oxidative damage to DNA as evidenced by overexpression of 8-OHdG, the most frequently used biomarker of oxidative DNA damage, in pFRG in the offspring as compared with control ones. In addition, accumulating evidence show that excessive ROS leads to increase in MDA, an end-product of peroxidation reaction elicited by free radicals acting on lipid. The amount of MDA can be used to estimate the degree of lipid peroxidation in tissues. Herein, our result showed that maternal CSE increased MDA content indicating an overproduction of lipid peroxide in pFRG in the offspring. These results suggest that maternal CSE gives rise to oxidative damage to pFRG in the offspring. Oxidative stress occurs when excessive ROS accumulation overpowers intracellular antioxidant capacity. In this study, it was found that offspring subjected to maternal CSE showed reduced GSH production and activities of CAT, GR and Mn-SOD, vital antioxidants for intracellular antioxidant defense in pFRG. GSH is highly sensitive to the imbalance between the antioxidant system and ROS, and is important for cellular ROS scavenging[44]. The antioxidant effect of GSH involves two pathways: 1) non-enzymatically reacts with radicals in a direct manner; 2) acts as an electron donor in reducing peroxides catalyzed by glutathione peroxidases[45]. Within cells, GSH can be generated by reactions catalyzed by GR[46]. CAT is another important cellular antioxidant enzyme known to convert hydrogen peroxide into water and oxygen, resulting in ROS disposal[47]. Mn-SOD is an important enzyme for intracellular antioxidant defense, especially within the mitochondria that converts superoxide free radicals into hydrogen peroxide, which was transformed to water by catalases and other peroxidases[48]. It has also been elucidated that 15
Mn-SOD is a part of protein complex necessary for mitochondrial DNA repair[49]. Thus, Mn-SOD plays an essential role in various aspects of mitochondrial protection. Consistent with studies in kidneys[31, 32], we found that the activity of mitochondrial Mn-SOD was reduced in the offspring exposed to maternal cigarette smoking. The reduction in GSH generation, CAT, GR and Mn-SOD activities in pFRG in the current study indicates that maternal CSE decreased the antioxidant capacity in the offspring. Mitochondria are essential for energy production, however at the same time, ROS accumulates as byproducts of mitochondrial oxidative phosphorylation causing mitochondrial damage, which in turn results in an escalation of oxidative stress[18, 19]. It has been reported that excessive ROS contributes to abnormal mitochondrial membrane permeability and increased swelling[50]. Our data showed that increased swelling was detected in mitochondria from CSE offspring. It has been indicated that mitochondrial swelling is due to prolonged opening of mPTP[36]. Thus, increased swelling observed in the present study indicates an abnormal opening of mPTP, which is reported to cause an increase of permeability to solutes leading to disruption of solute homeostasis, drastic changes in mitochondrial ultrastructure and functional activity[51]. In this study, we in deed observed mitochondrial structure changes in animals subjected to maternal CSE with mitochondrial enlargement and a loss of matrix and cristae. We also observed that maternal CSE induced loss of MMP, likewise, maternal CSE resulted in an impediment to mitochondrial ATP production in the offspring. It has been shown that oxidative stress is associated with DNA damage, and the mitochondrial genome is more susceptible than the nuclear genome due to the lack of histone protection, reduced DNA repair capacity and close proximity to the ROS producing electron transport chain[31]. Previous study has indicated that abnormal mtDNA content, even without any detectable mtDNA mutations, can be indicative of mitochondrial dysfunction[18, 52]. Our finding, together with others[31, 53], supports that maternal CSE induces an increase in mtDNA level in response to oxidative stress in the offspring. The mtDNA amplification might be a compensatory mechanism in response to the decline of mitochondrial function[52, 54]. In conclusion, our study demonstrates that maternal CSE leads to oxidative stress and mitochondrial dysfunction in pFRG in rat offspring. Our results shine light on the consequence of 16
maternal smoking during pregnancy, and set the stage for further studies exploring the influence of maternal cigarette smoking on the development of brainstem in the offspring.
Acknowledgement We are grateful to Dr. G. Yang for his technical support in transmission electron microscopy. This work is supported by grants from the National Natural Science Foundation of China (No. 31471096).
Declarations of interest None.
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Highlights:
Maternal CS exposure increases oxidative damage in pFRG in rat offspring.
Maternal CS exposure decreases the antioxidant capacity in pFRG in rat offspring.
Maternal CS exposure destroys mitochondrial ultrastructure in pFRG in rat offspring.
Maternal CS exposure induces mitochondrial dysfunction in pFRG in rat offspring.
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