2021 年 46 巻 8 号 p. 345-358
Pb exposure is a worldwide environmental contamination issue which has been of concern to more and more people. Exposure to environmental Pb and its compounds through food and respiratory routes causes toxic damage to the digestive, respiratory, cardiovascular and nervous systems, etc. Children and pregnant women are particularly vulnerable to Pb. Pb exposure significantly destroys children’s learning ability, intelligence and perception ability. Mitochondria are involved in various life processes of eukaryotes and are one of the most sensitive organelles to various injuries. There is no doubt that Pb-induced mitochondrial damage can widely affect various physiological processes and cause great harm. In this review, we summarized the toxic effects of Pb on mitochondria which led to various pathological processes. Pb induces mitochondrial dysfunction leading to the increased level of oxidative stress. In addition, Pb leads to cell apoptosis via mitochondrial permeability transition pore (MPTP) opening. Also, Pb can stimulate the development of mitochondria-mediated inflammatory responses. Furthermore, Pb triggers the germination of autophagy via the mitochondrial pathway and induces mitochondrial dysfunction, disturbing intracellular calcium homeostasis. In a word, we discussed the effects of Pb exposure on mitochondria, hoping to provide some references for further research and better therapeutic options for Pb exposure.
Pb is a natural toxic substance and people are often at high risks of Pb exposure. Pb deposits in our bones, liver, kidneys, brain and other tissues, causing great harm and leading to multiple lesions in various organs and systems, such as neurodegenerative diseases (Karri et al., 2018), cardiovascular diseases (Simões et al., 2017), reproductive system diseases (Hassan et al., 2019) and so on. Moreover, it has been demonstrated that Pb can affect the fetus through the placenta during pregnancy (Bocca et al., 2019) and infants through breast milk during lactation (Park et al., 2018), bringing incalculable damage to early-age development. Today, the problem of Pb exposure has received more and more public attention, making it a more urgent matter to study the underlying mechanism of Pb exposure and explore more possible therapeutic targets for Pb exposure.
Mitochondria seem to be sensitive targets of Pb exposure. Mitochondria are endosymbiotic organelles which support all complex life for various processes. At present, mitochondria remain highly reduced genomes in eukaryotic cells, which encode genes essential for cellular bioenergetics based on their evolution. Mitochondria are the powerhouses of cells. They are involved in metabolizing nutrients and ATP synthesis through the tricarboxylic acid cycle (TAC) and oxidative phosphorylation (Letts and Sazanov, 2017). Furthermore, mitochondria are one of three major Ca2+ storage sites (De Stefani et al., 2016) and play significant roles in the Ca2+ signaling pathway responsible for vital cellular processes such as cell proliferation, apoptosis and autophagy. In addition, mitochondria are significantly involved in metabolism. Mitochondrial dysfunction has been found to be associated with a considerable number of diseases, and strategies targeting mitochondria for therapy have progressed considerably (Bhatti et al., 2017). The existing evidence of Pb-induced mitochondrial damage suggests that anti-mitochondrial damage may be a potential therapeutic strategy for Pb exposure. In this review, we summarize the latest evidence that mitochondria are sensitive targets for Pb exposure and the possible biomarkers and molecular mechanisms of Pb-induced mitochondria damage.
More than 90% of O2 in the body is consumed in mitochondria. For the imperfect process of electron flow, a small amount of oxygen which was consumed by mitochondria is not completely reduced and results in the production of ROS such as hydrogen peroxide and superoxide anion. Among these, the most important is superoxide anion, mainly produced by protease complexes I and III in the respiratory chain of the inner mitochondrial membrane, which is regarded as “primary” ROS, and then they interact with many other compounds and produce “secondary” ROS (Bhatti et al., 2017). In a biological context, ROS are formed as natural by-products of the normal metabolism of oxygen, and play an important role in cell signaling and homeostasis (Zhang et al., 2016). Under physiological conditions, appropriate amounts of reactive oxygen species can promote immunity, repair, survival, and growth. However, ROS levels can increase dramatically during environmental stress such as UV, heat exposure and Pb exposure. Pb can induce increased levels of oxidative stress in mitochondria (Fig. 1). It has been proven that Pb can either directly or indirectly generate superfluous ROS and reactive nitrogen species (RNS) in organ pathophysiology in the mitochondria (Ahmad et al., 2018; Ma et al., 2017; Sousa and Soares, 2014). It can remarkably reduce the activities of mitochondrial respiratory chain enzyme complexes, especially complex II and complex III, while there is little impact on complex IV. This then leads to the decrease of ATP which suggests the failure in the capacity of oxidative phosphorylation to produce ATP (Ma et al., 2017).
Pb induces mitochondrial dysfunction leading to the increased level of oxidative stress. Pb remarkably reduced the activities of mitochondrial respiratory enzyme complex II and complex III. Pb induces the failure in the capacity of oxidative leading to the decrease synthetic of ATP. Pb can either directly or indirectly through the disturbance of eliminate ROS system to generate ROS and RNS in mitochondria. What’s more, Pb can decrease the fluidity of mitochondria membrane which is associated with the mitochondrial permeability transition (MPT). The increased level of oxidative stress negatively affects various components such as lipids, proteins, polysaccharide and deoxyribonucleic acid (DNA). Drugs such as ascorbic acid, cyanidin-3-O-glucoside, selenium, mangiferin and ferulic acid have been reported to have protective function in Pb induced oxidative stress in mitochondria.
There are antioxidant systems in mitochondria that eliminate reactive oxygen species, which are divided into the enzymatic systems and non-enzymatic systems. The enzymatic systems comprise superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx); the non-enzymatic systems are mainly glutathione (GSH) and vitamins C/E. When the two antagonistic forces of ROS and oxidants are imbalanced, and the damaging effect of ROS is more powerful than the compensatory effect of antioxidants, the cells can be damaged, which is called oxidative stress. Studies have indicated that Pb exposure could significantly inhibit the antioxidant system in mitochondria. The activities of GPX, SOD, CAT, GSH are obviously decreased while the activity of iNOS is opposite (Chi et al., 2017; Han et al., 2017; Pal et al., 2013). In addition, the oxygen is rapidly consumed due to the oxidation of the polyunsaturated fatty acid acyl chain via ROS. The addition of Pb increases oxygen consumption drastically and leads to the decrease of membrane fluidity, which is associated with the mitochondrial permeability transition (MPT). Lipid peroxidation and protein carbonylation are significant indicators of oxidative stress. Malondialdehyde (MDA), as a marker of the oxidative stress, can modify the amino acid residues and form stable adducts. In the meanwhile, it can also form covalent adducts with nucleic acids, and membrane lipids (Del Rio et al., 2005). Increases in the final product of lipid peroxidation and protein carbonylation (MDA) levels were observed under Pb exposure, indicating elevated oxidative stress (Ma et al., 2017; Pal et al., 2013).
Oxidative stress is a harmful process which can negatively affect various components such as lipids, proteins, polysaccharide and deoxyribonucleic acid (DNA) (Pizzino et al., 2017). Multiple studies support an interactional relationship between oxidative stress and inflammation (Biswas, 2016). Almost all human organs are vulnerable to damage by oxidative stress. The most common diseases induced by abnormal levels of oxidative stress are heart diseases (Peoples et al., 2019), cancer (Yang et al., 2016), diabetes (Jha et al., 2016), and neurodegenerative diseases (Islam, 2017) such as Alzheimer’s disease (Tönnies and Trushina, 2017) and Parkinson’s disease (Subramaniam and Chesselet, 2013) and so on. Also, there are many drugs have been proven to have a protective function in Pb-induced oxidative stress in mitochondria, such as ascorbic acid (Ahmad et al., 2018), cyanidin-3-O-glucoside (Wen et al., 2018), selenium (Han et al., 2017), mangiferin (Pal et al., 2013) and ferulic acid (Lalith Kumar and Muralidhara, 2014), etc. Altogether, a considerable number of drugs have been found to be able to alleviate the elevated levels of oxidative stress induced by Pb, and targeting mitochondrial damage to reduce intracellular ROS/RNS level must be a significant therapeutic approach for Pb exposure.
Mitochondrial permeability transition pore (MPTP), a protein complex present between inner and outer mitochondrial membranes, is a non-specific channel which plays an essential role in maintaining healthy mitochondria homeostasis (Halestrap, 2009). Abnormal MPTP opening can result in the destruction of mitochondria, ultimately leading to the pathological elimination of mitochondria and cells (Zorov et al., 2014). In addition, recent findings suggest that MPTP seems to be involved in aging (Panel et al., 2018). Existing evidence indicates that opening of the MPTP represents a potential therapeutic target in multiple human diseases such as ischaemia-reperfusion injury, neuromuscular diseases of childhood, and age-related neurodegenerative disease (Briston et al., 2019). The molecular composition of MPTP is not yet fully understood, but it is believed that MPTP is composed of voltage-dependent anion channel (VDAC), adenine nucleoside translocator protein (ANT), and cyclophilin-D (Cyp-D). In normal physiological conditions, MPTP allows substances with molecular weight less than 1.5 KD to pass freely and drive ATP synthase through oxidative phosphorylation to maintain mitochondrial membrane potential (MMP) and intracellular and extracellular ion balance (Halestrap, 2009). When the cell is under stress, such as ROS or Ca2+ overloading, MPTP is completely open, and consequently substances with molecular weight greater than 1.5 KD and soluble substances pass freely and non-selectively. The integrity of the inner membrane will be disrupted, causing the collapse of the MMP and mitochondrial swelling and finally leading to cell death (Bernardi et al., 2015). Proteins such as Cyt-c, apoptosis-initiating factor (AIF), and endonuclease are released into the cytoplasm and induce apoptosis by initiating caspase-dependent or -independent mechanisms.
Pb exposure can induce the typical morphological changes of mitochondria such as mitochondria swelling, the rupture of outer membrane as well as the disruption of mitochondrial cristae, which reflect that the severity of damage to mitochondria is progressively increased by Pb with dose- and time-dependent effects (Liu et al., 2016). Pb leads to the pathological opening of mitochondrial permeability transition pore and thereby to cell death (Fig. 2). The MMP is significantly decreased with different concentrations of Pb, which is the prerequisite leading to MPTP opening (Ma et al., 2017). In the meanwhile, the MPTP opening is accompanied by the changes of membrane fluidity, which reflect the changes of the mitochondrial membranes in dynamic properties as discussed earlier. In normal physiological conditions, Cyt-c is considered to be stored only in mitochondrial intermembrane cristae spaces. The opening of MPTP induced mitochondrial swelling and the release Cyt-c and the AIF to the cytoplasm, which activates the caspase family, leading to cell apoptosis. Studies show that the release of Cyt-c, AIF and endonuclease of DNA from mitochondria is increased by incubation with Pb (Liu et al., 2016; Ma et al., 2017; Ye et al., 2016). The addition of Pb decreased the fluidity of membrane at a high level (Ma et al., 2017). Furthermore, Cyp-D, VDAC and ANT simultaneously take part in MPTP regulation in the Pb exposure model. Pb-induced mitochondrial apoptosis relies on MPTP opening. The up-regulation of VDAC-1, ANT-1 and down-regulation of Cyp-D, VDAC-2 and ANT-2 at transcription, translation as well as post-translation levels are observed under Pb exposure, which contribute to their functional alteration and later lead to MPTP opening (Liu et al., 2016; Ye et al., 2016). Targeting Cyp-D, knocking out ppif (the coding gene of Cyp-D) inhibited MPTP opening and mitigated Pb-induced mitochondria dysfunction, which maintained ATP supply and alleviated oxidative stress as well as cell apoptosis (Ye et al., 2020). In addition, cell apoptosis can be significantly decreased by MPTP inhibitors, CsA, DIDS and BA, which respectively target Cyp-D, VDAC and ANT, the components of MPTP. They obviously ameliorate Pb-induced mitochondrial dysfunction, such as increased oxidative stress, MMP collapse and the release of Cyt c as well as AIF from mitochondria. Moreover, the descending ATP level and ascending ADP/ATP ratio induced by Pb can be markedly reversed by BA (Liu et al., 2016; Ye et al., 2016).
Pb induces pathological opening of mitochondrial permeability transition pore (MPTP). In normal physiological conditions, MPTP allows substances with molecular weight less than 1.5 KD pass freely, while those which are more than 1.5 KD pass freely and non-selectively under Pb exposure conditions. MMP, the prerequisite leading to MPTP opening, was significantly decreased with different concentrations of Pb. The opening of MPTP induces mitochondrial swelling and leads to the release Cyt-c the AIF and endonuclease of DNA from mitochondria to the cytoplasm, leading to cell apoptosis. The up-regulation of VDAC-1, ANT-1 and down-regulation of Cyp-D, VDAC-2 and ANT-2 at transcription, translation as well as post-translation levels are observed under Pb exposure, which contribute to their functional alteration and later lead to MPTP opening. Bcl-2 and Bcl-xl inhibit the formation of Bax-mediated nonspecific pores via competitive bonding to ANT. Pb significantly increases the expression of Bax, while Bcl-2 and Bcl-xl are markedly decreased.
In addition, mitochondria can control the stability of OMM and the formation of mitochondrial apoptotic channels through the balance between pro-apoptotic proteins such as Bax, Bad and Bak and anti-apoptotic proteins such as Bcl-2 and Bcl-xL (Nicotra and Parvez, 2002; Ying and Padanilam, 2016). The structure of VDAC can be changed physiologically by its interaction with the ANT (Belzacq et al., 2003; Vyssokikh and Brdiczka, 2003). Bcl-2 and Bcl-xl inhibit the formation of Bax-mediated nonspecific pores via competitive bonding to ANT (Chin et al., 2018; Shimizu et al., 2000). Studies have shown that Pb can significantly increase the expression of Bax, while Bcl-2 and Bcl-xl were markedly decreased (Deng et al., 2015; Mao et al., 2020; Rai et al., 2013). Moreover, due to the complex signaling networks which MPTP involves in, the pathological opening of MPTP is bound to be closely related to a series of biological processes such as oxidative stress (Zorov et al., 2014), autophagy (Zhou et al., 2019) and disturbance of intracellular calcium homeostasis (Agarwal et al., 2017), which provide a wide range of effects on MPTP for Pb, further expanding the damage of Pb poisoning to the body. Collectively, the evidence that Pb-induced MPTP opening leads to the increased levels of oxidative stress and apoptosis in cells, and that mitochondria-mediated damage to cells is ameliorated by MPTP inhibitors suggests a potential strategy for Pb exposure via targeting MPTP opening.
Inflammation is a common comprehensive pathological response to infection and tissue injuries. In the normal physiologic process, inflammation induces the increase of disease resistance function of the body, facilitating the removal of pro-inflammatory factors and repairing the damaged tissues, while in the pathologic context, the excessive inflammatory response leads to the degeneration and necrosis of tissue, even to chronic inflammatory diseases. Studies show that mitochondrial function or dysfunction plays a significant role in such an intricate operating system (Meyer et al., 2018; West, 2017). Based on the endosymbiosis theory, both mitochondria and the eukaryotic cells plausibly originate from prokaryotes by endosymbiosis four billion years ago, which is critical to the eukaryote complexity. Thus, mitochondrial functions are inseparably associated with the various fundamental cellular physiology processes, including inflammation (Dela Cruz and Kang, 2018; Meyer et al., 2018) and innate immunity (Rongvaux, 2018). Furthermore, recent progress has shown that inflammatory response and innate immunity share common features. Therefore, we inevitably talk about innate immunity when discussing the effects of Pb-induced mitochondrial damage on the development of inflammation, and there is no clear boundary between inflammation and innate immunity. The existing research results confirm the influence of Pb on the immune system (Weinberg et al., 2015). In the research of Gargioni et al. (2006), Pb induced DNA damage and cell death in macrophages. Increased memory T cell and other immune cells in Pb-exposed children suggest the effects of Pb on the development and differentiation of immune cells as well (Cao et al., 2018; Zheng et al., 2019).
Pb can induce mitochondrial damage that promotes inflammation via TLR4-stimulated signaling cascades (Fig. 3). Existing evidence has shown that mitochondria participate in TLR signaling to accelerate the development of inflammation (Banoth and Cassel, 2018), suggesting a possible mechanism of Pb-induced inflammation. Inflammation is typically triggered by pathogen-associated molecule patterns (PAMPs) during extraneous injection or damage-associated molecular patterns (DAMPs) during tissue injuries such as Pb-induced tissue injuries. Both of them are perceived by pattern recognition receptors (PRRs), a class of recognition molecules mainly expressed on the surface of innate immune cells. Pb-induced MPTP opening has been discussed ahead, leading to the effusion of Mitochondrial DAMPs (MTDs), including mtDNA, ATP, cardiolipin and formyl-peptides, which have been deeply researched as a mechanism by which DAMPs induce inflammation (Meyer et al., 2018).In addition, a total of 11 TLRs have been identified up to now, among which TLR4 is the most extensively studied in Pb exposure models. It is evidenced that MTDs can increase TLR4 expression (Schwacha et al., 2016). In the meanwhile, Pb-induced increased circulating MTDs have remarkable effects on TLR9 and TLR4 activation (Bomfim et al., 2012; Wu et al., 2017). In addition, released MTDs can also act on formyl peptide receptors (formyl peptide), NLRP3/NLRC4/AIM2 inflammasomes (mtDNA, ATP, cardiolipin and formyl-peptide) and cGAS/STING (mtDNA) respectively, triggering neutrophil activation, various chemokines expression which ultimately induce inflammatory outcomes (West, 2017). Except TLR3 employing TRIF, all TLRs use MyD88 while TLR4 can perform its functions though both MyD88 and TRIF, which involved in TIRAP and TRAM. PAMPs or DAMPs induced interaction of TLRTIR domains, recruiting another adaptor’s TIR domain such as MyD88TIR. About 30 residues linker between DD and TIR domains of MyD88 finally leads to the formation of helical ternary complexes with the Interleukin 1 Receptor Associated Kinases (IRAKs) (Ve et al., 2017). TLR4-MyD88-NF-κB axis has been reported to be a significant link of Pb-induced mitochondria- mediated inflammatory response (Chibowska et al., 2016). Upon the Pb exposure context, the increased expression of both TLR4 and MyD88 are detected (Liu et al., 2015), which leads to the activation of IRAK-4 and then the phosphorylation of IRAK-1. On one hand, active IRAK-1 binds to tumor necrosis factor receptor-associated factor (TRAF6) to activate the TAK1/TAB complex, which activates IκB kinase (IKK), eliminating the inhibition of IKK to NF-κB, which then lead to the higher expression level of multiple cytokines (Chen et al., 2017; Chibowska et al., 2016; Yang et al., 2019). On the other hand, activated TLR4 can take effects through TRAF6 translocating to the mitochondrion, leading to the ubiquitination of Evolutionarily Conserved Signaling Intermediate In Toll Pathway (ECSIT), which is highly required for NF-κB signaling (West et al., 2011). This may be one of the mechanisms by which Pb damages mitochondria, leading to the extensive pathological injuries. However, Lpsd mutation (a single-point mutation in TIR domain) may abolish the interaction of TLRs with the downstream MyD88 signaling via disrupting this recruitment (Xu et al., 2000). P38 MAPK cascade is also initiated via ASK1. In normal context, ASK1 stays inactive with thioredoxin-1 (Trx-1). However, increased ROS induced by Pb can facilitate the release of ASK1 from Trx-1 and consequently leading to the activation of JNK and p38 MAPK pathways (Zhao et al., 2019). Moreover, TNFR- mediated TRAF2 activation also result in the activation of ASK1, which forms a true feedback loop that promotes the development of inflammation (Liu et al., 2000). Then, Pb-induced NF-κB and P38 activation and translocation into the nucleus activate transcription factors and subsequently upregulate the expression of a variety of cytokines such as TNF-α, IL-1, IL-6, IL-8, IL-10 and IL-18, leading to the development of inflammation (Metryka et al., 2018). Furthermore, TLR4/TRIF/IRF3 and TLR9/IRF3 have been reported to be involved in inflammatory response. Pb-induced higher circulating level of MTDs stimulates STING binding to Type-I IRF3 and phosphorylated IRF3 and co-translocating into nucleus, which results in the increased expression of NOD-like receptor protein 3 (NLRP3), while TLR9 senses mtDNA and is activated as well (Li et al., 2019; West, 2017; Zhang et al., 2019).
Pb stimulates the development of mitochondria-mediated inflammatory responses. Pb can induce mitochondrial damage promotes inflammation via TLR4-stimulated signaling cascades. Upon the Pb exposure context, the increased expression of both TLR4 and MyD88 are detected which leads to the activation of IRAK-4 and then the phosphorylation of IRAK-1. In the one hand, active IRAK-1 binds to tumor necrosis factor receptor-associated factor (TRAF6) to active the TAK1/TAB complex, which activates IκB kinase (IKK), eliminating the inhibition of IKK to NF-κB. In the other hand, activated TLR4 can take effects through TRAF6 translocating to the mitochondrion, leading to the ubiquitination of Evolutionarily Conserved Signaling Intermediate In Toll Pathway (ECSIT) which is highly required for NF-κB signaling. What’s more, Pb induced increased ROS facilitate the release of ASK1 from Trx-1 and consequently leading to the activation of JNK and p38 MAPK pathways. Activated NF-κB and P38 translocate into the nucleus to activate transcription factors and upregulate the expression of a variety of cytokines such as TNF-α, IL-1, IL-6, IL-8, IL-10, IL-18 and iNOS, leading to the development of inflammation. Besides, TLR4 can perform its functions though both MyD88 and TRIF/IRF3. Pb induced release of mitochondrial components, including mtDNA, ATP, cardiolipin and formyl-peptide, may act as DAMPs to promote inflammation. What’s more, Pb exposure leads to the assembly of NLRP3 inflammasomes. Pb induced the increased expression of inflammasome associated components (NLRP3, IL-1βand IL-18). Pb exposure may activate NLRP3 inflammationsome by down-regulating VDAC1 and VDAC2. Mitochondria may act as a platform for inflammasome assembly. Colocalization of the inflammasomes in perinuclear regions of mitochondria has been found which may provide an evidence for the modulation mechanism of mitochondrial components to inflammasomes.
Pb exposure can also lead to the assembly of NLRP3 inflammasomes. Inflammasome is a large protein complex which senses a variety of PAMPs and DAMPS. The activation of inflammasome leads to the cleavage of pro-casepase1 and then to the maturation of IL-1β, IL-18 and Gasdermin D (GSDMD), which ultimately lead to the induction of pyroptosis (He et al., 2016), a new mode of programmed cell death. The initiation of NRLP3 inflammasome assembly is highly related to the activation of the TLR signaling pathway through Pb, which upregulates NLRP3 expression at transcription level and activates NLRP3 at post-transcription level by phosphorylation and deubiquitination (Song and Li, 2018). In addition, it has been observed that Pb induces the increased expression of inflammasome-associated components (NLRP3, IL-1β (Huo et al., 2019; Li et al., 2014) and IL-18 (Chibowska et al., 2016)), indicating the activation of NF-κB signaling, which is significantly associated with mitochondrial signaling. The following step is the oligomerization of NLRP3, leading to the assembly of apoptosis-associated speck-like protein (ASC) and procaspase-1. A study shows that relative mRNA expression of inflammation-related factors (NF-κB, TNF-α, COX-2, NLRP3, ASC, caspase-1, IL-1β, IL-6 and IL-18) after Pb treatment were significantly increased while mRNA level of IFN-γ was significantly decreased (Huang et al., 2020). A significant role for mitochondria in NLRP3 inflammasome activation has been revealed by other studies (Liu et al., 2018; Zhou et al., 2011). The study of Zhou et al. has shown the correlation between mitochondrial ROS and NLRP3 inflammasome activation, indicating a close contact between Pb-induced oxidative stress and inflammation. Furthermore, the elimination of VDAC1 and VDAC2 significantly reduced mitochondrial ROS and the secretion of IL-1β, while the down-regulation of VDAC3 seems to have nothing to do with the activity of NLRP3 inflammatory corpuscle. This is consistent with the down-regulation of VDAC1 and VDAC2 induced by Pb exposure. Pb exposure may activate NLRP3 inflammasome by down-regulating VDAC1 and VDAC2, while there is little effect of Pb on VDAC3 (Liu et al., 2016). However, the functional mitotic phagocytosis and autophagy system can inhibit the activation of NLRP3 inflammatory corpuscles via removing damaged mitochondria. This may be the mechanism of self-protection induced by Pb. Moreover, mitochondrial dysfunction may work upstream of NLRP3 activation by providing ROS for the initiation of NLRP3 oligomerization or relocate mitochondria to the proximity of NLRP3 by inducing α-tubulin acetylation. Mitochondria may act as a platform for inflammasome assembly, providing evidence for the modulation mechanism of mitochondrial components to inflammasomes (Yu and Lee, 2016; Zhou et al., 2011). Taken together, these findings suggest that Pb exposure can promote inflammatory response, and the revelation of the underlying mechanisms may provide possible treatment strategies for Pb exposure.
Autophagy is a self-sufficient mechanism of energy and macromolecular precursors providing in the cell by which some identified and marked “defectives” are transported to lysosomes for degradation. Since autophagy was proposed by Ashford and Porter in 1963, we have achieved something on the researching road of this mechanism. Autophagy can be categorized into three types: microautophagy, macroautophagy and chaperone-mediated-autophagy, playing an essential role in many physiological processes and disease, such as starvation adaptation, cellular quality control, oncogenesis, immunity, and prevention of neurodegeneration (Mizushima and Komatsu, 2011). Different typical stages of autophagy include i) initiation of autophages; ii) nucleation; iii) expansion and stretching of autophagic membranes, closure and fusion with lysosomes; iv) intracapsular degradation.
Mitochondria are proven to take part in various physiological and pathological processes, and show high plasticity under different conditions. Normally, the quantity, morphology, and function of mitochondria maintain a relatively stable state, which is called mitochondrial homeostasis. To ensure normal functioning, mitochondria are equipped with a luxurious defense system against damage, such as proteases, antioxidant systems and DNA repair enzymes, etc. When cells are under stress and conclusively lead to the broken mitochondrial homeostasis, a set of systems called mitochondrial quality control will selectively clear away the impaired mitochondria if the damage cannot be restored via the cellular repair mechanism (Yoo and Jung, 2018). Mitochondria are involved in the autophagic response, and mitophagy is one of the important pathways for mitochondrial quality control.
Multifarious mechanisms are employed by mitochondria to maintain their homeostasis, among which mitochondrial fusion and fission are of great significance to the repair of damaged mitochondria. The high dynamic and high plasticity of mitochondria confer the possibility of this key mechanism. Mitochondria exchange material between well-functioning and poorly-functioning mitochondria through a fusion process and segregate impaired components through a fission process. The fusion and fission of the mitochondria is mediated by the dynamic protein family GTPases. Two steps of mitochondrial fusion are the fusion of the outer membrane mediated by mitofusin proteins (MFN1 and MFN2) and the subsequent joining of the inner membrane mediated by mitochondrial dynamin like GTPase, OPA1 while mitochondrial fission is mediated by dynamin-related GTPase, DRP1. In the study of Han et al. (2017), Pb induces autophagy by influencing mitochondrial dynamics. The morphological damage of mitochondria and the formation of the autophagosomes are clear after Pb exposure. Pb treatment remarkably alters the expression of the mitochondrial dynamics-related genes, increasing the expression of mitochondrial fission factor (MFF) and Drp1 while the expression of MFN1, MFN2 and OPA1 is down-regulated. The whole process is powered by GTP hydrolysis. This process is related to the supply of cellular energy, while the disturbing effect of Pb on energy metabolic processes is evident (Ma et al., 2017), suggesting that mitochondrial fusion and fission may be one of the targets of Pb exposure. In the meanwhile, it has been reported that healthy and dynamic mitochondria contribute to the efficiency and renewal of mitochondria, which is the connected with mitochondrial fusion and fission, while Pb affects mitochondrial membrane fluidity (Ma et al., 2017). Furthermore, Pb significantly up-regulates the expression of Bcl-2, Caspase3, Atg5, Beclin-1, Dynein, and LC3-II while the expression of LC3-1 and mTOR are down-regulated, suggesting that autophagy is trigged by Pb. (Han et al., 2017; Liu et al., 2019). These proteins play important roles in autophagy-related signaling pathways (Mukhopadhyay et al., 2014; Parzych and Klionsky, 2014). Interestingly, although Pb increased the expression of autophagic markers in PC12, the genesis and activity of lysosomes are inhibited by Pb, leading to the autophagic injuries in neural cells (Gu et al., 2019). There are also studies that show the opposite result that Pb can damage cell viability, leading to the depletion of autophagy proteins LC3-II and Beclin 1 (Guan et al., 2020; Liu et al., 2019). Differences between these results may be due to species diversity, different lead exposure environments and experimental conditions. Thus, the effects of Pb on autophagy need further study.
Mitophagy, serving as a mechanism of mitochondria quality control, is implicated to govern various physiological processes of cells, including metabolic reprogramming, mitochondrial fission and fusion, cell cycle control as well as apoptosis. Studies in the last decade suggest that the mechanisms linking genetic or acquired defects in mitophagy to neurodegenerative diseases (Chu, 2019; Wang et al., 2019), cardiovascular diseases (Bravo-San Pedro et al., 2017), and cancer (Bernardini et al., 2017) are more complex than normal mitochondrial quality dysregulation. Thus, a key scientific question that urgently needs to be studied is which substances of mitophagy can induce mitophagy response under pathological conditions, and which molecules specifically mediate the activation of mitophagy pathway and play a role in the development of diseases.
PINK1/Parkin axis is one of the major signaling pathways proven to initiate autophagy. It has been demonstrated that Pb notably increases the proportion of LC3-II/LC3-I and decreases the expression level of COX IV (the marker of mitochondria) (Gu et al., 2018; Lv et al., 2015). Studies have revealed that Pb exposure accelerates mitophagy via PINK1/Parkin-related pathway (Fig. 4). PINK1 is a gene which is ubiquitously expressed in various tissues. However, it usually maintains an undetectable level through the way of post-transcription. Under normal conditions, PINK1 is constitutively translocated to the mitochondrial outer and inner membrane with assistance of the translocase of the outer membrane (TOM) and translocase of the inner membrane (TIM) complexes. Once PINK1 targets at inner membrane, N-terminal mitochondrial targeting sequence (MTS) of PINK1 will be cleaved by the mitochondrial processing peptidase (MPP), and then F104 sequence at the N-terminus is secondary cleaved by presenilin-associated rhomboid-like (PARL) protease (Jin et al., 2010). Finally, PINK1 is rapidly degraded by the N-end rule pathway via the ubiquitin–proteasome system. It has been proven that Pb can cause mitochondrial depolarization, which is a prerequisite of mitophagy (Ma et al., 2017). The accumulation of uncleaved PINK1 and increased recruitment of Parkin to mitochondrial outer membrane are obvious under Pb treatment (Gu et al., 2018). It should be especially pointed out that the mitochondrial membrane potential (MMP, ΔΨm) is a prerequisite of translocation of the N-terminal MTS to the inner membrane. Pb-induced distorted MMP leads to the stabilization of PINK1 located on the mitochondrial outer membrane by impairing the import of PINK1 to the inner membrane (Ma et al., 2017).
Pb triggers mitophagy via PINK1/Parkin pathway. PINK1 usually maintains an undetectable level through the way of post-transcription. Under normal conditions, PINK1 is constitutively translocated to the mitochondrial outer and inner membrane with assistance of the translocase of the outer membrane (TOM) and translocase of the inner membrane (TIM) complexes. Once PINK1 targets at inner membrane, it can be rapidly degraded by the N-end rule pathway via ubiquitin–proteasome system. Mitochondrial membrane potential (MMP, ΔΨm) is the prerequisites of translocation of the N-terminal MTS to the inner membrane. Pb induced distorted mitochondrial membrane potential (MMP) leads to the blockage of PINK1 import. Accumulated PINK1 can be auto-phosphorylated and activated, and phosphorylates Parkin on serine 65 (Ser65) in the UBL domain. Besides, Pb notably increased the proportion of LC3-II/LC3-I and the decreased the expression level of COX IV, the marker of mitochondria. In addition, ataxia telangiectasis mutated (ATM) may be a potential factor to influence the phosphorylation level of PINK1 and Parkin under Pb exposure. ATM knockdown can block Pb-induced mitophagy.
Parkin, a downstream target of PINK1, contains an auto-inhibitory ubiquitin-like (UBL) domain, and three really interesting new gene (RING) domains (Seirafi et al., 2015). Accumulated PINK1 induced by Pb can be auto-phosphorylated and activated, and phosphorylates Parkin on serine 65 (Ser65) in the UBL domain, which is of great importance but not sufficient yet for recruitment and activation of Parkin. PINK1 also indirectly regulates Parkin activation by ubiquitin (Koyano et al., 2014). Phosphorylated Parkin S65 bonding with phosphorylated ubiquitin S65 is an activated E3 ligase and ubiquitinates the various substrate transmitted by E2-conjugating enzyme (Seirafi et al., 2015), which is involved in mitophagy initiation and multiple other crucial signaling pathways. Research shows that ataxia telangiectasis mutated (ATM) may be a potential factor to influence the phosphorylation level of PINK1 and Parkin under Pb exposure. ATM knockdown can block Pb-induced mitophagy. (Gu et al., 2018). Adapter proteins (p62, OPTN, NDP52) recognize phosphorylated polyubiquitin chains on mitochondrial proteins and initiate autophage formation by binding to LC3. Furthermore, the PINK1-Parkin pathway regulates mitochondrial dynamics (Deng et al., 2008) and motility (Wang et al., 2011) by targeting MFN and Miro for proteasomal degradation. This may be another possibility that Pb induces autophagy by affecting mitochondrial kinetics as discussed earlier (Han et al., 2017). In addition, there are receptor-mediated mitophagy pathways. Moreover, BNIP3/NIX (Roperto et al., 2019) and FUNDC1 mitophagic receptors localize to the OMM and interact directly with LC3 to mediate mitochondrial clearance (Liu et al., 2014). In addition, it has been reported that Pb-induced ER stress may play a regulatory role in the upstream of mitophagy (Gao et al., 2020).
Taken together, these findings suggest that more studies are needed on how Pb induces mitophagy directly by affecting key proteins on this pathway or indirectly through other cross-pathways. Targeting mitophagy might provide a meaningful direction for the therapy of Pb exposure.
Ca2+, as a versatile second messenger, plays an essential role in signaling networks, participating in the activation of various enzymes. Normally, intracellular calcium homeostasis is controlled by a combination of Ca2+ translocase and intracellular calcium pool systems. Intracellular Ca2+ level, buffering by proteins of wide variety, endoplasmic reticulum and mitochondria, is maintained around 10-7 M. When cells are damaged, the disturbance of the manipulation process endangers mitochondrial function and cytoskeletal structure, and ultimately activates irreversible catabolic processes of intracellular components. The abnormal activation of Ca2+ signaling system is an important mechanism which leads to cell death caused by poisons shuch as Pb.
Intracellular calcium signaling networks work in various physiological and pathological processes. However, both Ca2+ and Pb2+ are divalent metals with similar chemical properties and, thus, Pb2+ can compete with Ca2+ at the plasma membrane for transport systems, such as Ca2+ channels, and Ca2+ pumps, thereby affecting calcium entry and exit to perturb calcium homeostasis. How Pb2+ binds and regulates Ca2+ signaling proteins is a hot direction to study the mechanism of Pb exposure (Dudev et al., 2018). As early as the last century, it has been proven that alterations in IPRs binding by Pb could result in disturbance of intracellular calcium homeostasis (Chetty et al., 1996) and Pb-calcium interactions in cells indicated Pb toxicity (Simons, 1993). In the study of Wang et al. (Wang et al., 2015), Pb exposure can lead to the subcellular calcium redistribution in rat proximal tubular cells. The elevations of Ca2+ in cytoplasm and mitochondria were obvious while the level of Ca2+ in ER was decreased. Pb exposure increased the expression of IP3R-1 and IP3R-2, enhancing IP3-binding sensitivity which leads to Ca2+ release activity of ER and then mitochondrial Ca2+ concentration. Furthermore, the association between the increased level of mitochondrial Ca2+ and oxidative stress has been revealed (Starkov et al., 2004). On one hand, long-lasting Ca2+ signals in cytoplasm may result in accumulation of Ca2+ in the mitochondria. On the other hand, mitochondria close to the ER may sense the large and local cytoplasmic Ca2+ increases in proximity to the activated Ca2+ channels. However, the activation of mitochondrial Ca2+ uptake is transient (Hajnóczky et al., 2003). Mitochondrial calcium uniporter (MCU) complex is a selective inward rectifying channel which mediates Ca2+ influx into mitochondrial matrix and the fate of mitochondrial metabolism can be regulated by the inhibition or enhancement of MCU activation (Pathak and Trebak, 2018). Pb reduced the expression of MCU, leading to the increased level of oxidative stress in SH-SY5Y cells and the decreased mitochondrial Ca2+ uptake (Yang et al., 2014). In addition, MPTP is a Ca2+-activated channel. Prolonged openings of MPTP lead to depolarization, Ca2+ release and finally to cell death (Giorgio et al., 2018). However, the precise mechanism of how Pb regulates the expression of MCU still remains to be revealed by further studies. In addition, it has been confirmed that Bcl-2 family proteins can induce early redistribution of Ca2+ from ER to the mitochondria (Hajnóczky et al., 2003). Pb can significantly increase the expression of Bax, while Bcl-2 and Bcl-xl are markedly decreased (Deng et al., 2015). Moreover, mitochondrial calcium homeostasis inextricably correlates with metabolism. Ca2+ directly activates a-ketoglutarate dehydrogenase as well as isocitrate dehydrogenase, and indirectly activates pyruvate dehydrogenase via a Ca2+-dependent phosphatase, dephosphorylating the catalytic subunit of pyruvate dehydrogenase. Activated mitochondrial dehydrogenases raise the level of NADH results in the increased electron supply to the ETC and increasing the generation of ATP (Pathak and Trebak, 2018). This may be one way by which Pb affects the normal metabolism of cells. Pb may perform its toxic mechanism which induces mitochondrial dysfunction, disturbing intracellular calcium homeostasis, interfering with mitochondrial metabolism and then a variety of downstream signaling networks.
Pb does immense harm to our body’s health, especially the harm to our nervous system. Numerous studies on the mechanism by which Pb destroys the normal physiological activity have been carried out and we have taken quite a long journey on the “road”. Mitochondria are essential for eukaryotic life. They produce most of ATP, providing energy for the cell requirement. Mitochondria take part in pivotal metabolic pathways, fully integrating themselves into the signaling networks which control multiple cellular functions (Annesley and Fisher, 2019). Existing studies have demonstrated that Pb-induced physiological disorders and pathological progression are closely related to mitochondrial dysfunction. In this review, we only summarize the negative effects of Pb-induced mitochondria damage on oxidative stress, MPT, immunity, inflammation, autophagy and calcium homeostasis. In conclusion, Pb-induced mitochondria damage participates in many pathological processes, leading to the occurrence of various diseases. Focusing on how Pb induces mitochondrial damage and how to repair may provide more possibilities for the treatment of Pb exposure.
Conflict of interestThe authors declare that there is no conflict of interest.
