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Mitochondrial Dysfunction and DNA Damage Accompany Enhanced Levels of Formaldehyde in Cultured Primary Human Fbroblasts Cristina A

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Mitochondrial Dysfunction and DNA Damage Accompany Enhanced Levels of Formaldehyde in Cultured Primary Human Fbroblasts Cristina A www.nature.com/scientificreports OPEN Mitochondrial dysfunction and DNA damage accompany enhanced levels of formaldehyde in cultured primary human fbroblasts Cristina A. Nadalutti1, Donna F. Stefanick1, Ming-Lang Zhao1, Julie K. Horton1, Rajendra Prasad1, Ashley M. Brooks2, Jack D. Grifth3 & Samuel H. Wilson1* Formaldehyde (FA) is a simple biological aldehyde that is produced inside cells by several processes such as demethylation of DNA and proteins, amino acid metabolism, lipid peroxidation and one carbon metabolism (1-C). Although accumulation of excess FA in cells is known to be cytotoxic, it is unknown if an increase in FA level might be associated with mitochondrial dysfunction. We choose to use primary human fbroblasts cells in culture (foreskin, FSK) as a physiological model to gain insight into whether an increase in the level of FA might afect cellular physiology, especially with regard to the mitochondrial compartment. FSK cells were exposed to increasing concentrations of FA, and diferent cellular parameters were studied. Elevation in intracellular FA level was achieved and was found to be cytotoxic by virtue of both apoptosis and necrosis and was accompanied by both G2/M arrest and reduction in the time spent in S phase. A gene expression assessment by microarray analysis revealed FA afected FSK cells by altering expression of many genes including genes involved in mitochondrial function and electron transport. We were surprised to observe increased DNA double-strand breaks (DSBs) in mitochondria after exposure to FA, as revealed by accumulation of γH2A.X and 53BP1 at mitochondrial DNA foci. This was associated with mitochondrial structural rearrangements, loss of mitochondrial membrane potential and activation of mitophagy. Collectively, these results indicate that an increase in the cellular level of FA can trigger mitochondrial DNA double-strand breaks and dysfunction. Genome stability is important for maintenance of normal cellular metabolism and is essential for cell survival. During evolution, cells have developed highly conserved mechanisms of DNA repair to prevent genetic altera- tions due to DNA damage caused by endogenous as well as exogenous sources1,2. Eukaryotic cells contain two genomes, nuclear and mitochondrial (mtDNA), and it is suggested that oxidative stress in mitochondria will also impose oxidative stress on the nucleus3–5. Terefore, oxidative stress may impact both genomes in a highly interconnected fashion. Mitochondria are double membrane organelles and generate most of the cell’s supply of adenosine triphos- phate (ATP), used as a source of chemical energy for cellular functions by establishing intricate genetic interac- tions with the nuclear compartment. However, among the approximately 1,200 proteins needed for mitochondrial metabolism, only a few are encoded by the mitochondrial genome6. Te mammalian mitochondrial DNA is a double-stranded, closed-circular molecule assembled into compact structures called nucleoids. Te mitochon- drial genome encodes 13 proteins that are key components of the oxidative phosphorylation system (OXPHOS), in addition to 2 rRNAs and 22 tRNAs, necessary for the mitochondrial translational machinery7. OXPHOS is characterized by multimeric complexes, that are responsible for the transfer of electrons to molecular oxygen, leading to protons being pumped into the inter-membrane space, and ultimately to complex V (also known as ATP synthase), for the generation of ATP. Many reactive molecules are produced during OXPHOS in the mito- chondrial matrix and the majority are formed when electrons leak from the electron transport chain and interact 1Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, 27709, USA. 2Center for Integrative Bioinformatics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, 27709, USA. 3Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. *email: [email protected] SCIENTIFIC REPORTS | (2020) 10:5575 | https://doi.org/10.1038/s41598-020-61477-2 1 www.nature.com/scientificreports/ www.nature.com/scientificreports with molecular oxygen creating reactive oxygen species (ROS). ROS oxidize membrane lipids to generate alde- hydes, which in turn chemically modify DNA bases that become abundant in aging and diseased human tissues. Tus, compared to the nuclear DNA, mtDNA is up to 100 times more vulnerable to damage8,9. Tis vulnerability is exacerbated by the absence of an efcient nucleotide excision repair system (NER), such that mitochondria appear to lack the ability to repair certain helix-distorting lesions induced in mtDNA by environmental genotox- ins and endogenous metabolites10–13. Terefore, the mtDNA lesions produced, if not repaired and/or eliminated by mitophagy, can afect mtDNA replication and transcription, leading to mitochondrial dysfunction14–17. During the last decades, increasing research has focused on formaldehyde (FA), a naturally occurring organic compound, and on its efects on human health. Te National Toxicology Program listed FA as a potential human carcinogen in 1981, based on evidence from studies in experimental animal models. Since then, several additional cancer studies have been published, and FA was classifed as a human carcinogen in 200618,19. FA covalently binds to DNA and forms DNA monoadducts, DNA-DNA interactions, DNA-protein and DNA-glutathione crosslinks20 that can lead to genotoxic stress if not removed2,21,22. In humans, the enzymes alcohol dehydrogenases (ADH2/ ADH5), the glutathione system, and the Fanconi anemia group D2 protein (FANCD2), a DNA crosslink repair protein, provide defense mechanisms against potential genotoxic efects caused by FA21,23,24. Until recently, it was believed that small amounts of FA in bodily fuids of healthy people were derived mainly from consumption of alcoholic beverages. However, recent data have shown that FA naturally occurs as a normal byproduct of metabolism25,26, and altered levels of FA may correlate with adverse conditions, such as cancer and neurodegenerative disorders27–30. Te full range of physiological roles of FA is still unclear, however, as are the consequences of FA accumulation within biological systems. Tere have been somewhat limited investigations connecting intracellular FA metabolism with physiol- ogy24,27,29,31–35. In the present study, we aimed to investigate how primary foreskin (FSK) fbroblast cells responded afer exposure to increasing concentrations of FA, especially with regard to the mitochondrial compartment. Te results provide insight into the biological processes that modulate FA efects, in vivo. We utilized human FSK cells in order to provide a physiological model to study the endogenous deregulation of FA and to investigate the attendant metabolic adaptations. Material and Methods Cell culture. Primary cultures of human fbroblasts (FSK) were cultured in Dulbecco’s modifed Eagle’s medium (DMEM) (Gibco, Carlsbad, California), supplemented with 10% FBS (Hyclone, Fisher Scientific, Canada, USA), 100 nM non-essential amino acids (Gibco) and 50 μg/ml of penicillin/streptomycin in a humid- ifed incubator at 37 °C in an atmosphere of 5% CO2. Mycoplasma testing was performed using a MycoAlert Mycoplasma detection kit (Lonza, Rockland, ME). Te fbroblast cells were placed in culture at the Lineberger® Comprehensive Cancer Center of the University of North Carolina at Chapel Hill (UNC) by members of the labo- ratory of Dr. J. Grifth. All of the experimental protocols used in the production of the FSK cells were approved by the UNC Institutional Review Board (01-0705). Te FSK cells used in the current study are lefover samples from the previously IRB-approved work, and the FSK cells were stored frozen. Since the cells were primary cultures, they were used only until passage 6. Te methods for the use of FSK cells were under guidelines and regulations of the National Institute of Environmental Health Sciences, National Institutes of Health. Human participants were not involved in the present study, and human tissue was not used. Determination of cell viability. Cell viability of FSK cells was determined by a crystal violet (CV) assay, as previously described32, with minor modifcations. Briefy, cells were plated on 6-well plates at the density of 1 × 105 cells per well. At 80% confuence, the cells were exposed to 250 μM or 500 μM FA, for 24 h, 48 h and 72 h and then stained with a solution of CV in 50% methanol (Met-OH) for 1 h at 25 oC. Cells were washed three times with water and CV dye retained by viable cells solubilized in 100% Met-OH. Subsequently, 100 μl of CV solubi- lized cells were transferred into a 96-well plate and the absorbance was read with a microplate reader at 570 nm (Biotek Synergy Multi-mode microplate reader, Winooski, VT, USA). Te fuorescence intensity was taken as proportional to the number of viable cells. Te results were expressed as percentage of control cells without any treatment. Average values were calculated from n = 3 wells in each study group. All experiments were performed with diferent cell batches and repeated at least three times. Evaluation of endogenous levels of FA. Te endogenous levels of FA in FSK cells under diferent exper- imental conditions were determined with a fuorometric FA assay (Sigma) according to the manufacturer’s instructions. Briefy, 25 × 106 cells for each study group were washed three times with phosphate-bufered saline
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