The detailed molecular mechanisms of cytotoxicity and genotoxicity of cobalt as well as cobalt compounds are still unclear. Based on the previous reports, there are some major aspects which are described below: 1) Oxidative stress: CoNPs and cobalt ions can produce excessive ROS through Fenton reaction[27, 28], as well as enhancement of intracellular antioxidant enzyme (e.g. catalase, glutathione peroxidase and heme oxygenase-1 etc.) to degrade ROS. Harmful biological effects and irreversible damage are generally caused because of the oxidation of proteins, lipids, carbohydrates, and DNA, induced by excessive ROS. At the same time, the excessive ROS could promote the release of inflammatory factors, deplete glutathione, and reduce the activity of antioxidant stress-related proteins such as GPx4, superoxide dismutase, catalase etc. As a result, chromosomal damage of cells, apoptosis, and necrosis are caused through various pathways[28–31]. 2) Hypersensitivity: Cobalt, chromium, and nickel are the well known immunosensitizers. The adverse reactions of MoM hip prosthesis wear particles might be caused by the metal ion hypersensitivity[32, 33]. Earlier studies have demonstrated that patients with MoM hip prosthesis have higher risk of lymphopenia and metal hypersensitivity reactions[34]. In vivo studies have also confirmed that micron-scale rather than nano-scale chromium-nickel particles cause type IV hypersensitivity reactions[35]. 3) Autophagy and endoplasmic reticulum stress: Metal particles generated from the wears often induce stress in the endoplasmic reticulum, mediating the expression of a variety of inflammatory factors (TNF-α, IL-6 and IL-1β). It is found that the autophagy is involved in promoting the apoptosis of osteoblasts which are caused by wear particles and inhibits the plasma reticulum stress. Moreover, autophagy can reduce inflammatory factor secretion and osteoclast production and alleviate the effects of wear particles on osteoblasts[36–38]. 4) Destroy cell structure: CoNPs and cobalt ions could directly bind to proteins with multiple functions in the cells including redox system enzymes, metabolism, molecular transport, cell signaling, and organelles[39]. Additionally, they can induce oxidation and the loss of biological function, as well as destroy skeleton of cells directly, leading to chromosome condensation and aberrations. Cobalt could also bind to several proteins involved in cellular redox systems and active oxygen scavenging. Furthermore, cobalt has also been shown to interact with various receptors, ion channels, and biomolecules[40, 41]. 5) Replace essential metal ions: Earlier report demonstrated that cobalt can interact with various metal based proteins (e.g. zinc-finger protein, catalase etc.) and replace the corresponding metal ions (e.g. Mg2+, Ca2+, Zn2+, etc.). Therefore, the functions of these proteins or enzymes are lost or altered[39]. All these previous reports altogether suggest that CoNPs can induce cell death through oxidative stress.
Our study also confirmed that the administration of CoNPs toward Balb/3T3 cells led to enhancement in the intracellular ROS level as compared to the control group (Fig. 2). However, the exact molecular mechanism of how ROS causes the cell death remains unclear. Although CoNPs-treatment caused a significant increasement of apoptosis rate of Balb/3T3 cell as compared to the control egroup (9.78% VS. 4.75%) (Fig. 3a, b, e), it was still much lower than the cell death rate, as observed by the CCK-8 assay under the same conditions (9.78% vs. 45%) (Fig. 1f). These results suggest that non-apoptotic mechanisms are involved in CoNPs-mediated celluar death.
Ferroptosis is a process to regulate the cell death, discovered in recent years. It is different from other forms of cell death in terms of cellular morphology and molecular mechanism. It is characterized by iron-dependent accumulation of lipid hydroperoxide to lethal levels[42, 43]. The main features of ferroptosis are the cellular uptake of iron, formation of intracellular ROS through the Fenton reaction, excessive consumption of GSH in the cells, and the inactivation of GPx4. These process ultimately cause lipid peroxidation and production of lethal reactive oxygen species to promote the cell death[43]. Ferroptosis is majorly associated with iron ion imbalance, oxidative stress, and GSH homeostasis. Although it has been reported that Ca2+ influx and GSH depletion play an important role in ferroptosis[44] and oxidative death (Oxytosis), the specific mechanism by which iron ions induce cell death remains unclear[45].
Ferroptosis has widely been confirmed in different diseases such as tumors and neuropathy and is one of the current research hotspots. Due to the similarity of physical and chemical properties of cobalt and iron elements, the comparative study of the two elements is also meaningful. The mechanism of cobalt toxicity is highly similar to the characteristics of ferroptosis. The earlier study showed that cobalt treatment led to significant increasement of intracellular ROS, lower GSH level, as well as the activation of Nrf2 signaling pathway. However, different antioxidants (such as N-acetylcysteine, alpha-Tocopherol, etc.) could exhibit antagonistic effects on cobalt toxicity and iron death[43, 46–56]. Our study reveals that CoNPs could induce the production of excessive ROS in Balb/3T3 cells, overproduction of lipid peroxidation products (MDA), and enhancement of cellular ferrous ion concentration. Moreover, CoNPs-treatment led to the reduction of intracellular GSH level and GPx4 activity. All these results suggest that CoNPs can activate ferroptosis in Balb/3T3 cells by stimulating oxidative stress, or directly induce ferroptosis-like death. Similar views was also provided by Gupta et al. when studying Parkinson’s pathogenesis in neuronal cells[57].
According to different signaling pathways and molecular mechanisms, the current detoxification drugs for CoNPs toxicity in vitro or in vivo are following categories: 1) Antioxidants such as nitrogen-acetylcysteine (NAC)[50], L-ascorbic acid (LAA)[46], melatonin[58], alpha-tocopher[48], etc. 2) Antagonists, such as zinc[59, 60]; 3) Chelating agents, such as EDTA[20, 21] and dimercaptopropanol sulfonic acid sodium[22]; 4) Endoplasmic reticulum stress inhibitor, such as sodium 4-phenylbutyrate[36, 37]. Based on previous studies, anti-oxidative stress is the key direction of detoxification research. The ideal medicine could be one that can resist oxidative stress and chelate with the metal ions. Moreover, it should be soluble in both water and fat as well as safe in vivo[61]. With this speculation, we discovered that ALA could effectively attenuate the CoNPs induced ferroptosis-like cell death in Balb/3T3 cells.
Lipoid acid (LA) is unique among different natural antioxidants and is a highly effective therapeutic agent associated with oxidative damage[62]. ALA is a dithiol compound that usually binds to the lysine residue of mitochondrial α-keto acid dehydrogenase. It is well-known that cytoplasmic and mitochondrial dehydrogenase can quickly reduce LA into dihydrolipoic acid (DHLA)[63]. Previous reports have demonstrated that ALA could bind iron or any divalent metal. Therefore, its iron chelating capability decreases the amount of free iron in the body system, thereby reducing oxidative stress by scavenging free radicals. Moreover, ALA could alleviate cytotoxicity during iron overload through reduction of glutathione content, mitochondrial dysfunction, as well as gene expression of heme oxygenase-1b and superoxide dismutase[64].
In the latest generation of antioxidants, lipoic acid is able to scavenge a variety of free radicals including hydroxyl radicals, hypochlorous acid, oxygen singlet, and peroxyl radicals, due to its solubility in both water and fat[25, 65]. On the other hand, the conventional antioxidants such as vitamins A, E, C and selenium can only interact with one or two free radicals[66]. In theory, lipoic acid seems to be the most effective drug among all antioxidants[62]. Additionally, lipoic acid can also interact with GSH and recycle endogenous GSH, stabilizing the antioxidant capacity of cells [64,67–69,]. LA has changed arrangement on its fatty acid-like skeleton, which allows it to capture the copper and iron ions, thereby forming a stable complex with these metals to achieve chelation of iron, copper, cobalt, manganese, zinc, cadmium, lead, nickel, arsenic, mercury and other transition metal ions[26, 62, 69–71]. In the present study, we found that CoNPs significantly induced overproduction of intracellular ROS, enhancement of intracellular Fe2+ (Fig. 4), inhibition of GPx4 activity (Fig. 6), and reduction of reduced intracellular GSH (Fig. 5) as compared to the control group. Importantly, ALA can significantly antagonize the CoNPs induced above adverse results, thereby maintaining the intracellular GSH homeostasis, stabilizing the cell’s antioxidant capacity, and inhibiting the ferroptosis-like cell death induced by CoNPs.