Most studies on the toxicity of aluminium, whether they have involved animal or cell experiments, have used AlCl3, Al2(SO4)3, and Al(NO3)3 (Foglio et al. 2012; Shrivastava 2012; Sood et al. 2012; Sun et al. 2009; Zhuang et al. 2016). APS has rarely been used. In China, only APS is allowed to be added to food as a leavening agent (Zhang et al. 2016). Therefore, to explore the mechanism of aluminium toxicity, APS was selected as the experimental reagent in this study.
The present study demonstrated that APS changed SF9 cell morphology, lowered cell viability, induced apoptosis, and altered gene expression. Treating SF9 cells with increasing APS concentrations caused the cells to become increasingly enlarged and clustered. As the incubation time increased, the cell membranes became more blurred, and the cellular contents flowed out. The IC50 of SF9 cells treated with APS for 72 hours was 0.334μg/ml (equivalent to 12.4μM) , which was similar to human lymphocytes 10-20μM (Aluminium chloride) (Paz et al. 2017). While the Chen et al. treated SH-SY5Y cells with Al maltolate for 24h, and the cell bodies, neuritis and viability decreased when the concentration of aluminium exceeds 400μM(Chen et al. 2011). After treating Rat gliatoma C6 cells with AlCl3 • H2O ( 1–8mM) for 24h, the rate of apoptotic and necrotic cells increased(Zhang et al. 2010). One of the reasons for the difference in these in vitro results is due to the different chemical properties of different Al-containing compounds. The bioavailability and solubility of aluminium are affected by its hydrolytic morphology(Crisponi et al. 2012). The main species at PH≤5 is the hexa-aquo [Al(H2O)6]3+. And new mononuclear species reported as [Al(OH)2]+ and [Al(OH)]2+ are formed by deprotonation of water molecules as the PH increases or the soluble neutral species Al(OH)3 neglects the coordinated water molecules. In addition, polynuclear species [Al2(OH)2]4+ and [Al3(OH)3]5+ are also formed depending on time and total aluminium concentration(Rubini et al. 2002). Therefore, to describe the solution chemical properties of aluminium in biological systems, it is necessary to consider the soluble and insoluble forms of hydroxy-oxygen complexes and the morphological equilibrium of complexes formed with other competing ligands (Crisponi et al. 2011). On the other hand, cell culture types may just differ in susceptibility to Al. For example, different degree of cell viability impairment was significantly related to the composition of cell membrane(Cecchi et al. 2005). The composition of membrane lipids is an important factor affecting the interaction between foreign substances and cells itself, and the resulting cell membrane disturbance is a key step in early cell death(Butterfield 2002). After crossing the cell membrane, the redox status and calcium ion levels of different cells are closely related to the susceptibility of cells to foreign poisons(Stefani and Dobson 2003). In this study, compared with the control group, the groups treated with increasing APS concentrations exhibited decreasing proportions of normal cells and gradually increasing proportions of early apoptotic cells, late apoptotic cells and dead cells. Consistent with previous findings on aluminium-induced splenic lymphocyte apoptosis (Zhuang et al. 2016), these changes indicate that aluminium induced SF9 cell apoptosis in a dose-dependent manner.
KEGG enrichment analysis of transcriptome results showed that the downregulated genes were mostly involved in the longevity regulation pathway, the PI3K-Akt signalling pathway, and protein processing in the endoplasmic reticulum. Among the upregulated genes, most genes were involved in pathways associated with the metabolism of CYP, the metabolism of glutathione, and the metabolism of xenobiotics by CYP.
Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase in the PI3K-related kinase (PIKK) family and plays important roles in cell growth, proliferation, survival and cell cycle regulation. mTOR forms the catalytic subunit of at least two functionally distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 consists of four parts: mTOR, regulatory-associated protein of mTOR (RAPTOR), GßL (mammalian lethal Sec13 protein 8, mLST8), and rapamycin-FKBP12 (Foster and Fingar 2010; Kim et al. 2003; Saxton and Sabatini 2017; Zhang et al. 2000). mTORC1 plays a key role in regulating cell growth, thus controlling the balance between anabolism and catabolism to enable cells to adapt to environmental conditions. It can promote efficient protein synthesis through the phosphorylation of p70S6 kinase 1 (S6K1) on its hydrophobic motif site and eIF4E Binding Protein (4EBP) at multiple sites (Gingras et al. 1999; Holz et al. 2005). The mTORC1 signal can regulate mRNA translation and most profoundly affects mRNA containing pyrimidines, including the mRNA of most of the genes involved in protein synthesis (Hsieh et al. 2012; Thoreen et al. 2012). RAPTOR, an mTOR binding protein in mTORC1 with a molecular weight of 150 kDa, contains multiple WD repeats. As a scaffold protein, it plays a pivotal role in the mTOR signalling pathway (Hara et al. 2002; Kim et al. 2002) and related mTOR pathways, such as the PI3K-Akt signalling pathway and the longevity regulating pathway. In our transcriptome results, RAPTOR gene expression was downregulated, which also influenced the function of mTORC1. The mechanistic target of mTOR coordinates cell growth and metabolism with nutrients and other environmental inputs. Aluminium ions have strong binding ability with amino acids and proteins (Dudev et al. 2018; Fan et al. 2010), which may inhibit the amino acid-sensitive mTOR pathway. The decreased level of RAPTOR weakens the phosphorylation of S6K1, further affects protein synthesis and inhibits cell growth(Kim et al. 2002). Moreover, muscle-specific RAPTOR or mTOR knockout mice show muscle atrophy and weight loss leading to early death(Risson et al. 2009). On the other hand, many studies have shown that inhibition of mTOR can promote autophagy and prolong life(Roczniak-Ferguson et al. 2012; Settembre et al. 2012). Here, autophagy could be a result of disrupted apoptotic machinery. However, for biological growth process, growth promotion should be the main line of regulation, and autophagy should be used as a compensation pathway to regulate abnormal growth and metabolism. RAPTOR/mTORC1 loss mice have normal white adipose tissue for the first several weeks of life, but lipodystrophy associated with hepatomegaly, hepatic steatosis and insulin intolerance soon developed(Lee et al. 2016). This indicates that the early inhibition of mTOR pathway may be helpful to cell health, but the continuous inhibition, coupled with the continuous deterioration of the nutritional environment, will eventually lead to cell damage and apoptosis. Excessive autophagy also shortened cell life, leading to death as the concentration of aluminium ions increased. Although autophagy and apoptosis are different forms of programmed cell death, many studies have specifically focused on the cross-talk that occurs between autophagy and apoptosis to understand how these two types of cell death are related to each other(Green et al. 2011; Noguchi et al. 2014). For example, Atg5, which is known to induce autophagy, converts autophagy to apoptosis through calpain-mediated cleavage(Rubinstein et al. 2011).
In addition, we found that the expression of PtdIns-dependent protein kinase-1 (PDPK1) and some small heat shock proteins (HSPs), including heat shock 70 kDa protein (HSPA1s or Hsp70s) and crystallin alpha B (CRYAB), was downregulated. PDPK1 is an important protein in the PI3K-Akt signalling pathway, which is related to cell proliferation and survival processes (Nicholson and Anderson 2002). The PI3K-Akt-mTOR pathway also affects protein synthesis (Ekizceli et al. 2017; Kenessey and Ojamaa 2006), which may be related to PDPK1 phosphorylation and activation of Akt and S6K1 (Avruch et al. 2001).Downregulation of PDPK1 expression could inhibit the phosphorylation of downstream signals, thus inhibiting cell proliferation and protein synthesis. Especially in the research of tumor inhibition, PDPK1 is often used as a potential therapeutic target to inhibit the proliferation of cancer cells by inhibiting its expression (Zhang and Yu 2019; Zhang and Zhong 2020). CRYAB is implicated in cell apoptosis by negatively regulating the activity of caspase-3 to promote or inhibit apoptosis. Downregulation of CRYAB protein expression showed enhanced apoptosis, accompanied by an increase in Caspase-3 (Kamradt and M. 2002; Stegh et al. 2008). On the other hand, the 70-kDa heat shock proteins (HSPA1s) are ubiquitous molecular chaperones that plays an active role in the folding and remodeling of various cellular proteins, and are also regulated by PI3K-Akt signalling pathway(Lulu et al. 2019). They act at almost all stages of a protein's life, from synthesis to degradation, and are essential for maintaining protein homeostasis(Balchin et al. 2016; Imamoglu et al. 2020; Rosenzweig et al. 2019).
In animal cells, glutathione (GSH, γ-glutamyl-cysteinyl-glycine) is the most abundant low-molecular weight thiol, most of which is present in the cytosol. GSH/GSH disulfide (GSSG) is a major redox couple (Jones 2002) that plays crucial roles in nutrient metabolism, antioxidant defence, and other essential pathways. GSH metabolism is an important process in ageing and many other diseases (including Parkinson’s disease, Alzheimer’s disease, cancer, and liver disease) (Guoyao et al. 2004). In our study, GSH metabolism was identified as one of the pathways associated with many differentially expressed genes, mainly GSH transferase genes, including GSH S-transferase (GST), prostaglandin-H2 D-isomerase (HPGDS) and pyrimidodiazepine synthase (SE). The GST superfamily is a superfamily of multifunctional proteins that play fundamental roles in the cellular detoxification of exogenous and endogenous compounds. The main method for detoxification involves the catalysis of the binding of GSH with compounds that have an electrophilic centre to form nontoxic derivatives (Coleman et al. 1997; Frova 2006). After entering the cell, aluminium ions will form complexes with biomolecules, and proteins are the main targets. In the blood, for example, transferrin is the primary binding agent (Wróbel et al. 1995). Aluminium ions can induce the formation of backbone ring structures in a variety of peptide structures through simultaneous bonds with oxygen and nitrogen atoms. These ring structures lead to irreversible denaturation of proteins and destabilization of the proteins (Song et al. 2014). Meanwhile, aluminium can induce the formation of oxygen free radicals, which leads to oxidative damage of mitochondria through the production of reactive oxygen species (ROS) and the accumulation of iron, thus leading to the internal apoptosis of cells (Willhite et al. 2014; Wu et al. 2012; Yu et al. 2019). Aconitase is a key enzyme in mitochondria, and aluminium can also induce mitochondrial dysfunction by decreasing the activity of aconitase through increased oxidative stress or Fe-S cluster perturbation(Kumar et al. 2009; Middaugh et al. 2005). In our study, aluminium ions cause overexpression of GSTS, HPGDS and SE. These changes affect the biological functions of GSH, thus improving the sensitivity of TNF-induced apoptosis and decreasing the GSH/GSSG ratio, which is associated with inhibition of nuclear factor-kappaB (NF-κb) transactivation(Matsumaru et al. 2003) . The mode of cell death includes increased cathepsin activity and the release of cytochrome C (CYC) in mitochondria, and GSH regulation is one of the necessary conditions for CYC to promote apoptosis (Brown and Borutaite 2008). In this study, many upregulated DEGs are associated with CYP-related pathways. The main CYP system is the microsomal P450 system. CYP is a superfamily of monooxygenases and predominantly localized in endoplasmic reticulum membranes, where they metabolize all kinds of xenobiotic and endogenous compounds. CYPs are also present in subcellular compartments such as plasma membrane and mitochondria(Ahn and Yun 2010). Many xenobiotic inducers can induce the uncoupling of CYP and catalyze the cyclic production of ROS, leading to multiple toxic effects(Zangar et al. 2004). Here, aluminium, as an exogenous substance, may be involved in the above pathway, leading to cell apoptosis. Several studies have also shown that exposure to aluminium inhibits the CYP system(Cao et al. 2020; Zhu et al. 2013).