Targeting of the respiratory chain by toxicants: beyond the toxicities to mitochondrial morphology

The mitochondrion is an important subcellular target of environmental toxicants.

Abstract

The mitochondrion is an important subcellular target of environmental toxicants. With environmental stress, a series of toxic effects on mitochondria are induced, which originate from the dynamic changes of mitochondrial fusion and fission, structure/membrane damage, and respiratory chain dysfunction. The toxic effects of various toxicants on mitochondrial morphology and intact membranes, and their determination of cell fate, have already been broadly studied and reported on. However, their effects on the integrity and function of the mitochondrial respiratory chain (RC) remain incompletely understood. Recently, Fan et al. and Yu et al. approached this topic by closely examining the mitochondrial toxicities, including the effect on the respiratory chain, induced by organic arsenical chemical 2-methoxy-4-(((4-(oxoarsanyl)phenyl)imino)methyl)phenol and thiourea gold(i) complexes (AuTuCl). Obviously, toxicant-induced dysfunction of the respiratory chain can hinder ATP production, and may elevate ROS generation. The increased ROS can further damage mtDNA, and consequently leads to inactivation of some RC protein-encoding mtDNA, generating a vicious circle of amplifying mitochondrial damage. We hope that these studies focused on RC structure and activity will broaden our view of mitochondrial toxicology and draw forth more profound mechanistic studies on the respiratory chain toxicity of environmental toxicants and their application in risk assessment.

Since the discovery of the mitochondrion over a century ago, numerous studies have revealed that mitochondria play a fundamental role in the maintenance of cell homeostasis. The mitochondrion is responsible for a series of metabolic functions either as an organelle of cells generating ATP or the main source of reactive oxygen species/reactive nitrogen species (ROS/RNS) in the cells. However, more evidence has indicated that the stress of various environmental toxicants, e.g. fine particulate matter (PM_2.5),1,2 cigarette smoke,3 bisphenol A,4 heavy metals,5–7 pesticides,8,9 ionizing radiation,10 and emerging toxicants such as nanoparticles11–14 can cause mitochondrial DNA (mtDNA) damage,3,10,14 changes to the structural integrity4,6,8 or dynamic morphology,1,2,11,13 dysregulation of ROS generation,1,5,13,14 and dysfunction of ATP production,3,8,10 which consequently result in toxicological effects and alteration of cell fates (Fig. 1). The dynamic changes of mitochondrial morphology have been primarily attributed to fusion and fission,15 which are the main processes determining mitochondrial shape, size and integrity in cells. In certain circumstances, mitochondrial fusion is a toxicant stress-induced adaptive response in cells, aiming to diminish cellular damage and increase survival through increasing metabolic efficiency and elevating ATP production, etc.15–17 Mitochondrial fission, in normal conditions, keeps in balance with fusion to create new mitochondria.15,16 Toxic stress can induce aberrant mitochondrial fission, through which fragmented mitochondria are generated.15,18 Mitophagy, selective autophagic degradation of damaged mitochondria, appears to be intimately linked to mitochondrial fission and fusion processes.15 Therefore, the fusion and fission processes are deemed to be a balanced mitochondrial dynamic: compensation of damage by fusion and elimination of damage by fission. Obviously, consistent exposure to toxicant stress may badly damage the structure and function of mitochondria, leading to decreased ATP yield and increased ROS production, and subsequently disrupt the intracellular homeostasis and eventually change the cell fate.

Fig. 1. Mitochondrial toxicity by environmental toxicants. With exposure to environmental toxicants, a series of toxic effects on mitochondria are induced, which originate from mitochondrial dynamic change in terms of fusion and fission, structure/membrane damage, and respiratory chain dysfunction. Under slight stress, fusion may exert an adaptive response to diminish cellular damage and increase survival, through increasing metabolic efficiency and enhancing ATP production. Mitophagy is induced when the separation of fragmented mitochondria is generated via fission. Dysfunction of the respiratory chain can hinder ATP production and elevate ROS generation, which can further damage mtDNA, and consequently leads to deficiency of mtDNA-coding respiratory chain complex proteins. In addition, mitochondrial membrane damage increases the permeabilization and formation of mitochondrial pores, allowing the release of cytochrome C into the cytosol to trigger apoptosis.

Notably, compared to the well addressed and broadly reported studies of mitochondrial structure and morphology changes, the effects of environmental toxicants on the assembly and activity of mitochondrial RC supercomplexes, intact electron transport chain (ETC), and oxidative phosphorylation function remain incompletely understood and need to be intensively investigated. Recently, Fan et al.19 and Yu et al.,20 both from the State Key Laboratory of Virology, Wuhan University, China, approached this topic by closely examining the mitochondrial toxicities, including the effect on respiratory chain function, induced by organic arsenical chemical 2-methoxy-4-(((4-(oxoarsanyl)phenyl)imino)methyl)phenol (MOPIMP)19 and thiourea gold(i) complexes (AuTuCl).20

The generation of ATP in mitochondria requires a functional electron transport chain (ETC) and the oxidative phosphorylation system (OXPHOS). The proteins involved in this process are localized in the mitochondrial inner membrane and are organized into five multi-subunit complexes (I–V) referred to as the respiratory chain (RC). Complexes I to IV collectively form a functional electron transport chain, and complex V is the mitochondrial ATP synthase. Except for complex II, these supercomplexes have subunits encoded both in the mitochondrial genomic DNA and the nuclear genes. Complex I or NADH : ubiquinone reductase is the largest OXPHOS complex with 45 subunits, where electron transfer from NADH to coenzyme Q happens;21 complex II or succinate dehydrogenase (quinone) is shared between the tricarboxylic acid (TCA) cycle and the ETC and has no proton pumping activity;22 complex III or quinol-cytochrome c reductase executes electron transfer coupled to proton pumping;23 complex IV or cytochrome c oxidase (COX) catalyses the oxidation of cytochrome c and the reduction of oxygen to water, coupled to proton translocation;24 complex V is the ATP-synthesizing enzyme using the proton motive force generated by complexes I, III and IV.25 In addition to the generation of the majority of cellular ATP by oxidative phosphorylation, the respiratory chain also generates reactive oxygen species (ROS) as a byproduct. The mitochondrial RC-ETC is responsible for the majority of mtROS production. The primary sources of mtROS from the RC are complexes I and III.26,27 In vitro data showed that the superoxide generated by mitochondria was estimated to account for 0.2–2% of cellular oxygen consumption, resulting in organelle concentrations of superoxide of between 10 and 200 pM.28 Numerous methodologies and protocols to measure the structure and functions of mitochondria have been developed and systematically presented previously.29 Recently, a technique of modified native gel polyacrylamide gel electrophoresis (PAGE) in combination with immunoblotting, in-gel assays (IGA) and electroelution has been developed to investigate the functionality and composition of mitochondrial ETC supercomplexes.30 The RC function is tightly affected by RC components in terms of the structure, posttranslational modification and complex assembly. For example, as one of the main proteins, cytochrome c plays a crucial role in controlling redox signalling in mitochondrial oxidative phosphorylation. The mimetic phosphorylation modification of cytochrome c protein, by incorporating p-carboxymethyl-l-phenylalanine at the position of the amino acid residue tyrosine 97, was demonstrated to enhance the electron donation rate to cytochrome c oxidase within the respiratory chain. Simultaneously, this phosphomimetic modification decreased mitochondrial ROS production and lowered the caspase-3 activation activity.31

However, most studies regarding the association of mitochondria with human diseases and health have focused on mitochondrial genetics and pathways,32–34 and our understanding is still limited on the roles of the mitochondrial RC as a source of, and target for fighting diseases. Obviously, the mitochondrial RC is at the forefront of efforts to understand the physiology and physiopathology of mitochondria-related diseases. Accordingly, medicines that target the mitochondrial RC could influence mitochondrial functioning, including energy metabolism, ROS generation, diffusion of proteins, and other critical pathological processes. However, it should be mentioned that a potential unexpected contradiction could occur between in vivo and in vitro effects. For example, a profiling study of the in vivo effects of the compound GSK932121A, a suspected complex III electron transport chain (ETC) inhibitor, displayed an adapted response state of increased calcium retention capacity and basal oxygen consumption rate. This in vivo effect was suggested to have resulted from the initial insult in combination with compensatory changes made by the tissue in order to maintain energy production.35

Fan et al. reported that a short-term incubation of mitochondria in vitro with the organic arsenical MOPIMP promoted the inhibition of respiratory chain complexes I, II, III and IV, and with damage to the respiration process.19 The activity of complex IV, the terminal complex of the electron transport chain, was significantly destroyed even when treated with MOPIMP at a low concentration level. Yu et al. observed that AuTuCl altered the mitochondrial permeability transition as well as impairing the functioning of the mitochondrial respiratory chain.20 In particular, they explored the underlying mechanism of severe mitochondrial RC dysfunction by AuTuCl exposure through inhibiting the activities of complex II and complex IV, leading to a deficiency in ATP production and effusion of cytochrome c. These RC toxic effects possibly triggered mitochondria-dependent apoptosis in cells. Obviously, dysfunction of the respiratory chain directly resulted in the burst of ROS and hindered ATP production (Fig. 1).

Overall, the studies by Fan et al. and Yu et al. represent several significant avenues of research on the toxicology of environmental factors. First, they outline a series of methods, including the evaluation of respiratory chain complex activity, detection of mitochondrial membrane fluidity, measurement of the respiration rate, and microcalorimetry assay of mitochondrial metabolic thermogenesis, for identifying the mitotoxicity of environmental toxicants both in mitochondrial morphology dynamics and structure and the mitochondrial RC, providing new information on the effects of these compound on the RC. Second, they explored the underlying mechanism of arsenic compound or AuTuCl mitotoxicity, where the RC is a core target of toxicity. These results will help improve our understanding of RC function and clarify the molecular causes of clinically and genetically complex mitochondria-dependent diseases. Therefore, these studies have important implications for future studies of mitochondria-dependent diseases and support the concept of the RC as the cause of multiple detrimental effects of environmental factors.

We consider the articles by Fan et al.19 and Yu et al.20 as featured articles as they provided significant insights into the molecular function alterations of the RC by such toxicants, and have further enriched our knowledge on the molecular initiating events (MIE) and models of action (MOA) of environmental toxicants. Especially, the data from such studies are valuable in developing the Adverse Outcome Pathway (AOP) for mitochondrial toxicology. These studies also suggest that in hazard and risk assessments of the potential mitochondrial toxicants we should consider not only the damage and morphology changes to the mitochondrial structure but also the effects on intact RC complexes and activity. Additionally, from these studies some new questions have also been presented, such as the role of RC biogenesis in critical diseases associated with environmental pollution, whether there is a group of RC-specific toxic agents and how to identify these chemicals, how RC signalling mechanisms contribute to the development of therapeutics, the judgement of consistency between the in vivo and in vitro toxic effects to RC, the involvement and mechanism of ncRNA and epigenetic regulation in the RC toxic effects, how the RC controls mitochondrial biosynthesis and energy metabolism, and the signal pathways linking the RC alterations with the endpoints of cellular toxic responses, such as senescence, autophagy and growth inhibition. In addition, our knowledge is almost blank on the aspect of how toxicants affect the assembly and activity of RC complexes. We expect that, commensurate with advances in techniques, e.g. superresolution fluorescence microscopy in combination with protein immunoprecipitation, modified native-PAGE and proteomics for the study of RC complex assembly and RC protein–protein interactions, CRISPR-Cas9 and site-directed mutation technology for the verification of toxicant-targeted proteins in RC, RNA immunoprecipitation for identifying the RC activity-regulating ncRNAs as the toxic targets, mitochondrial multi-molecular omics, and additional researchers with a passion in this field, we will uncover the full view of RC toxicology and its applicability to risk assessment of environmental exposure in the near future.

Conflicts of interest

There are no conflicts to declare.