Mitochondrial dysfunction and DNA damage accompany enhanced levels of formaldehyde in cultured primary human fibroblasts

Abstract

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 fibroblasts cells in culture (foreskin, FSK) as a physiological model to gain insight into whether an increase in the level of FA might affect cellular physiology, especially with regard to the mitochondrial compartment. FSK cells were exposed to increasing concentrations of FA, and different 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 affected 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 alterations due to DNA damage caused by endogenous as well as exogenous sources 1,2. 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 nucleus 3-5. Therefore, 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 triphosphate (ATP), used as a source of chemical energy for cellular functions by establishing intricate genetic interactions with the nuclear compartment. However, among the approximately 1,200 proteins needed for mitochondrial metabolism, only a few are encoded by the mitochondrial genome 6. The mammalian mitochondrial DNA is a double-stranded, closed-circular molecule assembled into compact structures called nucleoids. The mitochondrial 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 machinery 7. OXPHOS is characterized by multimeric complexes, that are responsible for the transfer of electrons to molecular oxygen, leading to protons being pumpe...

The Impact of Mitochondrial Deficiencies in Neuromuscular Diseases

“…In addition to triggering ER stress, if ROS damage overwhelms these repair mechanisms, MMP may decrease, thus potentially leading to the increase in the permeability of mitochondrial membranes. Higher permeability in mitochondrial membrane can activate the mitochondrial permeability transition pore (MPTP) and thus, the release of DAMPs to the cytosol (mtDNA), ceramides, formaldehyde) [ 137 , 138 ]. MPTP and ROS can also promote the activation of mitochondrial fission and mitophagy to eliminate damaged mitochondria ( Figure 2 ).…”

Using Human Primary Foreskin Fibroblasts to Study Cellular Damage and Mitochondrial Dysfunction

“…Mitochondrial ultrastructure is best revealed directly under the high resolution afforded by TEM; this has been successfully used to distinguish several mitochondrial features (Figs. 6 and 2; (Eustaquio et al., 2018; Nadalutti et al., 2020; Rybka et al., 2019). It is well established that, e.g., under oxidative stress conditions, mitochondrial outer and inner membranes reorganize; the mitochondrial membrane potential is lost, and mitochondria undergo fragmentation to allow the cellular clearance of damaged organelles through mitophagy (Luciani et al., 2020; Perrotta & Aquila, 2015).…”

“…We have successfully employed this approach to study cellular damage and mitochondrial dysfunction after deregulation of endogenous formaldehyde (FA) levels (Nadalutti et al., 2020) by FA exogenous addition. The cells isolated in Basic Protocol 1 have been sub‐cultured in T‐75 flasks, treated with different concentrations of FA for 24 hr, and prepared for TEM as described here.…”

“…These cells can proliferate and migrate to the site of injury, where they secrete collagen, glycoproteins, glycosaminoglycans, and fibers that support wound healing repair (Acharya et al., 2008). Consequently, fibroblasts represent an ideal in vivo cell model to study and investigate many cellular processes, for example, cell growth and differentiation, wound healing, fibrosis, and oxidative stress (Garrett, Baker Frost, & Feghali‐Bostwick, 2017; Nadalutti et al., 2020; Pizzino et al., 2017).…”

Human Cryptochrome Mediates Oxidative Stress Responses Modulated by Static and Electromagnetic Fields in Doxorubicin-Treated Cells

“…ROS are associated in in ammation-related disorders such as aging, asthma, cancer, diabetes, neurodegeneration, etc 41 . Oxidative stress leads to DNA damages in the both nuclear and mitochondrial genomes of cells 42 . Cells have defence mechanisms that are activated in response to ROS/oxidative stress signaling, including anti-oxidants scavengers containing glutathione (GSH), glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD), etc., and also DNA damage responses (DDR) 43,44 .…”