Silver nanoparticle-induced cell damage via impaired mtROS-JNK/MnSOD signaling pathway

Abstract

This study investigated the mechanism of silver nanoparticle (AgNP) cytotoxicity from a mitochondrial perspective. The effect of AgNP on manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme, against oxidative stress has not been studied in detail. We demonstrated that AgNP decreased MnSOD mRNA level, protein expression, and activity in human Chang liver cells in a time-dependent manner. AgNP induced the production of mitochondrial reactive oxygen species (mtROS), particularly superoxide anion. AgNP was found to increase mitochondrial calcium level and disrupt mitochondrial function, leading to reduced ATP level, succinate dehydrogenase activity, and mitochondrial permeability. AgNP induced cytochrome c release from the mitochondria into the cytoplasm, attenuated the expression of the anti-apoptotic proteins phospho Bcl-2 and Mcl-1, and induced the expression of the pro-apoptotic proteins Bim and Bax. In addition, c-Jun N-terminal kinase (JNK) phosphorylation was significantly increased by AgNP. Treatment with elamipretide (a mitochondria-targeted antioxidant) and SP600125 (a JNK inhibitor) showed the involvement of MnSOD and JNK in these processes. These results indicated that AgNP damaged human Chang liver cells by destroying mitochondrial function through the accumulation of mtROS.

Keywords:

• Silver nanoparticle
• mitochondrial reactive oxygen species
• c-Jun N-terminal kinase
• mitochondrial superoxide dismutase
• mitochondrial dysfunction

Introduction

Since the discovery that silver nanoparticle (AgNP) products were harmful to human health, strong evidence supported the association between AgNP-mediated reactive oxygen species (ROS), subsequent oxidative stress, and cytotoxicity (Flores-López et al. 2019; Tortella et al. 2020; Salama et al. 2023). Many studies have demonstrated that the mechanism of AgNP-induced cytotoxicity was caused by oxidative stress, DNA damage, and apoptosis (Noga et al. 2023; Suthar et al. 2023).

Mitochondria are the main oxygen-consuming sites for oxidative phosphorylation to produce ATP, and ROS in mitochondria are produced as byproducts of oxygen (O₂) metabolism. Manganese superoxide dismutase (MnSOD) is a major mitochondrial antioxidant enzyme that scavenges ROS to protect mitochondria from oxidative stress and mitochondrial dysfunction (Latham et al. 2023). It also maintains the mitochondrial integrity of cells exposed to oxidative stress (Liu et al. 2020). MnSOD overexpression prevents oxidative stress-induced cell death and tissue damage (Tavleeva et al. 2022).

Mitochondrial ROS (mtROS) production is not only determined by ROS production and disposal rates but is also regulated by several factors, such as mitochondrial membrane potential (ΔΨm), mitochondrial metabolic state, and O₂ levels (Mailloux 2020). AgNP-induced ROS production is closely related to mitochondria (Zhang et al. 2022). Previously, we demonstrated that AgNP caused cytotoxicity by oxidative stress-induced apoptosis and damage to cellular components (Piao et al. 2011). In apoptosis, c-Jun N-terminal kinase (JNK) plays an important role in regulating mitochondrial pro- and anti-apoptotic proteins (Xu and Hu 2020). When JNK is phosphorylated, signaling is activated, and the activated JNK modulates cell survival or death via altering stress-related protein transcription (Liu et al. 2020). Previous studies have reported that MnSOD transcription is regulated via the mtROS-driven activation of JNK signaling (Liu et al. 2020; Trombetti et al. 2021). Although much research on cytotoxicity of AgNP have already been conducted, research on the mitochondria-related mechanism underlying AgNP-induced cell damage is still insufficient; hence, this study aimed to analyze this further.

Material and methods

Materials

AgNP was provided by Professor Jinhee Choi (Seoul University, Seoul, Republic of Korea) and characterized as previously described (Eom and Choi 2010). AgNP with a size smaller than 100 nm was evenly dispersed in the tetrahydrofuran (THF) solution by sonication for 3 h, stirred for 3 h to evaporate the THF, refilled with deionized water, and filtered through a cellulose membrane with a pore size of 100 nm to remove AgNP aggregates. Then, the remaining THF was detected using a nuclear magnetic resonance spectrometer until no free THF was detected. According to our previous study, an AgNP concentration of 4 μg/mL was set as the optimal concentration for this study (Piao et al. 2011). The other materials were listed in Table 1.

Table 1. Reagent list.

Reagent	Action	Product No.	Company	City/State/Country
MitoTracker™ red FM	Mitochondrial labeling and localization dye	M22425	Invitrogen™	Waltham/MA/USA
Dihydroethidium (DHE)	Superoxide anion indicator	D11347
Rhod-2 AM, cell-permeant	Calcium indicator	R1244
ATP determination kit	ATP detection	A22066
Mitochondrial ROS detection assay kit	Mitochondrial ROS detection	701600	Cayman Chemical	Ann Arbor/MI/USA
Mitochondrial PT pore assay kit	Mitochondrial PT pore assay	601430
Dihydrorhodamine 123 (DHR123)	Cell permeable ROS probe	D1054	Sigma-Aldrich	St. Louis/MO/USA
Actin	Total or cytoplasm protein loading control antibody	A2066
Elamipretide (MTP-131)	Cytochrome c peroxidase inhibitor	S9803	Selleck Chemicals	Houston/TX/USA
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)	Cell viability	0793-5G	Amresco LLC	Solon/OH/USA
Caspase-Glo®-3/7 assay kit	Caspase-3 activity	G8090	Promega	Madison/WI/USA
MnSOD	Antibody	ADI-SOD-111	Enzo Life Sciences	Plymouth Meeting/PA/USA
COX4 (20E8)	Mitochondria protein loading control antibody	sc-58348	Santa Cruz Biotechnology	Dallas/TA/USA
Bim (H-191)	Antibody	sc-11425
Bax (B-9)	Antibody	sc-7480
Cytochrome c	Antibody	#4272	Cell Signaling Technology	Beverly/MA/USA
Phospho Bcl-2 (Ser70)	Antibody	#2827
Mcl-1	Antibody	#4572
Phospho SAPK/JNK(Thr183/ Tyr185)	Antibody	#9251
SAPK/JNK	Antibody	#9252
Phospho c-Jun (Ser73)	Antibody	#3270
RedSafe™	Nucleic acid staining solution	21141	iNtRON Biotechnology	Seongnam/Gyeonggido/Republic of Korea
PRO-PREP^TM	Protein extraction solution	17081

Cell condition

Human Chang liver cells were purchased from the American Type Culture Collection (Rockville, MD, USA) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco Life Technologies, New York, NY, USA) and 1% antibiotic-antimycotic (Gibco). The experimental conditions were conducted according to Table 2.

Table 2. Cell experimental conditions.

Cell type	Cell seeding number		Application experiment		Processing time
Human Chang liver cell	60 ml dish	1.0×10^6 cells/dish	MnSOD (RT-PCR, western blot, activity)		0.5, 1, 3, 6, 12, 24 h
			Western blot (other), ATP (ATP determination kit), morphology		3, 6, 12, 24 h
	6 well plate	2.0×10^5 cells/well	Flow cytometry	Total ROS (H2DCFDA), mtPTP (TMRE)	1, 12 h
				Superoxide anion (DHE)	1, 3 h
				Mitochondrial Ca^2+ (Rhod-2 AM)	24 h
	24 well plate	0.6×10^5 cells/well	Succinate dehydrogenase activity (MTT), apoptotic body (Hoechst 33342)		24 h
	96 well plate	0.15×10^5 cells/well	Mitochondrial ROS detection (mitochondrial ROS detection assay kit)		1, 3, 6, 12 h
			Caspase-3 activity (Caspase Glo®-3/7 assay kit)		24 h
	4 well chamber slide	0.5×10^5 cells/well	Confocal microscopy	MnSOD, mitochondrial Ca^2+ (Rhod-2 AM), morphology (Mitotracker)	24 h
				mtROS (DHR123), superoxide anion (DHE)	3 h
				mtPTP (TMRE)	12 h
				Phospho JNK	1, 3, 12 h

MnSOD mRNA

MnSOD mRNA was detected using reverse transcription–polymerase chain reaction (RT-PCR). A total RNA extraction solution was added to the cells, the total RNA was isolated, and cDNA was synthesized. After PCR according to the primers and PCR conditions in Tables 3 and 4, the amplified products were separated on a 1% agarose gel and imaged using Amersham ImageQuant (GE HealthCare, Chicago, IL, USA).

Table 3. Primers.

Gene	Primer		Company	City/Country
MnSOD	Sense	5′-GACCTGCCTTACGACTATGG-3′	Bioneer	Daejeon/Republic of Korea
	Antisense	5′-GACCTTGCTCCTTATTGAAG-3′
GAPDH	Sense	5′-GTGGGCCGCCCTAGGCACCAGG-3′
	Antisense	5′-GGAGGAAGAGGATGCGGCAGTG-3′

Table 4. Polymerase chain reaction (PCR) reaction condition.

PCR process	Temperature	Time	Cycle
Initial denaturation	94 °C	5 min
Denaturation	94 °C	45 sec	35
Annealing	53 °C	45 sec
Extension	72 °C	60 sec
Extension	72 °C	7 min
	4 °C	∞

Protein expression

For western blot analysis, protein samples (30 µg) were subjected to electrophoresis, nitrocellulose membrane transfer, blocking with 3% bovine serum albumin, and then incubation with the primary antibodies MnSOD, COX4, cytochrome c, Bax, Bim, Mcl-1, phospho Bcl-2, JNK, phospho JNK, and actin. Secondary antibodies were used immunoglobulin-G–horseradish peroxidase secondary antibody conjugates (Pierce, Rockford, IL, USA). Protein bands were visualized with an enhanced chemiluminescence detection reagent (Amersham, Little Chalfont, Buckinghamshire, UK), which was then exposed to X-ray film in the dark. For immunocytochemistry, after fixing the cells with 100% ethanol, permeabilizing with 0.5% Triton X-100, and blocking with 5% fetal bovine serum, the cells were incubated with MnSOD or phospho JNK antibody, followed by incubation with a fluorescein isothiocyanate-coupled secondary antibody. MitoTracker™ Red FM staining was performed and slides were made via mounting on a medium containing 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired using a confocal microscope (FluoView FV1200; Olympus, Tokyo, Japan).

MnSOD activity

The cells were suspended in 10 mM phosphate buffer (pH 7.5) and sonicated twice for 15 sec on ice for lysis. Triton X-100 (1%) was added, and cells were incubated on ice for 10 min, followed by centrifugation at 5000 × g to determine the protein content in the supernatant. After adding potassium cyanide solution, the inhibition rate of epinephrine auto-oxidation was measured at 480 nm, and the quantity of enzyme required for 50% inhibition was defined as one unit of enzyme activity. MnSOD activity was expressed as units/mg protein.

Mitochondrial ROS and superoxide anion levels

Cells were treated as described in Table 2. First, mtROS were detected using a spectrofluorometer (Perkin Elmer LS-5B; Perkin Elmer, Waltham, MA, USA) according to the instructions of the mtROS detection kit (Cayman Chemical, Ann Arbor, MI, USA). Next, 20 μM dihydrorhodamine 123 (DHR123), for detection of mtROS, or dihydroethidium (DHE), for detection of superoxide anion, was added to 6-well plates and incubated for 30 min, the supernatant was aspirated, rinsed with phosphate-buffered saline (PBS), trypsinized, and harvested. The fluorescence intensity of mtROS was detected using a flow cytometer (Becton Dickinson, San Jose, CA, USA). Finally, 20 μM DHR123 or DHE was added to each slide well and incubated at 37 °C for another 30 min. After rinsing with PBS, the cells were mounted with a mounting medium (DAKO, Carpinteria, CA, USA). Images were acquired on a confocal microscope (FV1200; Olympus).

Mitochondrial Ca²⁺ level

Cells were treated as described in Table 2. Cells were incubated with the mitochondrial Ca²⁺ indicator Rhod-2 AM. Cell fluorescence was measured using a flow cytometer (Becton Dickinson) or confocal microscope (FV1200; Olympus) after mounting with a DAPI-containing mounting medium.

Intracellular ATP level

Cells were lysed for 30 min by adding 200 µL lysis buffer (25 mM Tris [pH 7.8], 270 mM sucrose, 1 mM EDTA) on ice and sonicated 3 times for 15 sec. The supernatant was collected by centrifugation at 16 000 × g for 10 min at 4 °C and measured with luciferase/luciferin ATP determination kit (Molecular Probes, Eugene, OR, USA).

Succinate dehydrogenase activity

Cells were treated as described in Table 2. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well and incubated for 4 h and centrifuged at 800 × g for 5 min; then, the supernatant was aspirated. Formazan crystals were dissolved by adding dimethyl sulfoxide to each well, and the absorbance was read at 540 nm using a microplate reader (Molecular Devices, San Jose, CA, USA).

Mitochondrial permeability pore

Cells were treated as described in Table 2. The mitochondrial permeability pore (mtPTP) was measured via flow cytometry or confocal microscopy using an mtPTP assay kit according to the manufacturer’s instructions.

Hoechst 33342 staining

Cells were treated as described in Table 2. Hoechst 33342 reagent (20 µM) was added for staining for 10 min, and images were acquired using a fluorescence microscope (Olympus).

Caspase-3 activity

Cells were treated as described in Table 2. Cells were tested using the Caspase-Glo®-3/7 assay kit according to the manufacturer’s instructions.

Mitochondria morphology

Cells were treated as described in Table 2. First, the cell shape was imaged at 100× magnification through a phase-contrast inverted microscope. Next, the cells were treated with 100 nM MitoTracker™ Red FM, mounted with DAPI-containing mounting medium, and imaged under a confocal microscope.

Statistical analysis

Test results were presented as mean ± standard error. SigmaStat version 3.5 (Systat Software Inc., San Jose, CA, USA) was used to perform analysis of variance and Tukey’s test. A p < 0.05 was considered significant.

Results

AgNP decreased MnSOD mRNA, protein expression, and activity

In human Chang liver cells, AgNP decreased MnSOD mRNA and protein expression in a time-dependent manner 3 h after treatment (Figure 1A and B). Confocal microscopy images showed that AgNP treatment reduced MnSOD expression after 24 h, but the mitochondrial targeting antioxidant elamipretide (MTP-131) restored it (Figure 1C). Similarly, MnSOD activity showed a significant downward trend beginning at 1 h (Figure 1D). This suggested that AgNP could disrupt the mitochondrial antioxidant system.

Figure 1. AgNP reduced MnSOD mRNA, protein expression, and activity. (A) MnSOD mRNA expression was analyzed using RT-PCR. GAPDH was used as a loading control. (B) MnSOD protein expression was analyzed using western blotting. COX4 was used as a mitochondrial loading control. (C) Confocal microscopic images using MitoTracker™ Red FM and DAPI staining showed the MnSOD expression (green), mitochondrial (red), nucleus locations (blue), and the merged image indicated the mitochondria localization of MnSOD. (D) MnSOD enzyme activity was expressed as units/mg protein. *^,**Significantly different from the control group (p < 0.05, p < 0.001).

AgNP induced mtROS production including superoxide anion

A decrease in the expression or activity of antioxidant enzymes, which are intracellular antioxidant defense systems, is also associated with the production of excessive ROS (Latham et al. 2023). AgNP induced significant mtROS production within 1–12 h, with the maximum amount generated at 3 h (Figure 2A). Antimycin A was used as a positive control for mtROS production. Additionally, when mtROS were detected using flow cytometer and confocal microscope after staining of the cell permeant reagent DHR123, AgNP induced mtROS production, which was significantly inhibited by the mitochondrial targeting antioxidant MTP-131 (Figure 2B and C). In addition, as a result of measuring using flow cytometer and confocal microscope using DHE, a superoxide anion indicator, AgNP induced high level of superoxide anion, but it was suppressed by SOD (Figure 2D and E). These results suggested that AgNP induced excessive ROS in mitochondria.

Figure 2. AgNP-induced mitochondrial ROS production, including superoxide anion. (A–C) mtROS generated were detected using (A) mtROS detection kit, (B) flow cytometry, or (C) confocal microscopy after treatment with DHR123. Antimycin a was used as a positive control for mtROS production. MTP-131 is a mitochondrial-targeting antioxidant. (D, E) Superoxide anion was detected using (D) flow cytometry and (E) confocal microscopy after staining with DHE. (B, D) FI indicated the fluorescence intensity. (A) *^,**^,***Significantly different from the control group (p < 0.05, p < 0.01, p < 0.001). (B) *^,**Significantly different from the control group (p < 0.001, p < 0.01), ^#,##significantly different from the AgNP group (p < 0.001, p < 0.01). (D) *^,**Significantly different from the control group (p < 0.01, p < 0.001), ^#,##significantly different from the AgNP group (p < 0.05, p < 0.01).

AgNP increased mitochondrial Ca²⁺ level

ROS can induce Ca²⁺ release from the mitochondria, increasing the cytosolic Ca²⁺ level, which may lead to cell apoptosis (Ding et al. 2021). Flow cytometry results showed that the fluorescence intensity of mitochondrial Ca²⁺ measured using the Rhod-2 AM, a mitochondrial Ca²⁺ indicator, at 12 h after AgNP treatment was significantly higher than that of the control group (Figure 3A). In addition, confocal microscopy revealed that AgNP treatment significantly increased the red fluorescence intensity of Rhod-2 AM (Figure 3B). These data suggested that mitochondrial Ca²⁺ level increased due to mtROS generated by AgNP.

Figure 3. AgNP increased the mitochondrial Ca²⁺ level. (A, B) After Rhod-2 AM staining, the mitochondrial Ca²⁺ level was determined using (A) flow cytometry and (B) confocal microscopy. (A) FI indicated the fluorescence intensity of Rhod-2 AM. *Significantly different from the control group (p < 0.01). (B) Confocal microscopic images showed the mitochondrial Ca²⁺ level (red).

AgNP damaged the mitochondrial function

Mitochondrial dysfunction is characterized by ATP depletion, depolarization of mitochondrial action potential, and mitochondrial membrane permeability, and it can be caused by excessive mtROS and Ca²⁺ level (Matuz-Mares et al. 2022). AgNP reduced ATP level in a time-dependent manner (Figure 4A). These data suggested that AgNP impaired mitochondrial function by causing a loss of mitochondrial membrane integrity. Succinate dehydrogenase activity in the mitochondrial respiratory chain was assessed using an MTT assay. Succinate dehydrogenase activity was reduced by AgNP treatment in a time-dependent manner (from 3 to 24 h) compared with that in the control group, and succinate dehydrogenase activity was reduced to 44% at 24 h (Figure 4B). Flow cytometric analysis of mtPTP opening experiment showed that AgNP impaired mitochondrial permeability, whereas MTP-131 ameliorated AgNP-induced mitochondrial dysfunction (Figure 4C). Similar results were observed in images captured using confocal microscopy (Figure 4D). Overall, these results indicated that AgNP damaged mitochondrial function through loss of ATP level, succinate dehydrogenase activity, and permeabilization of mitochondrial membrane via excessive mtROS and elevation of Ca²⁺ level caused by AgNP.

Figure 4. AgNP damaged mitochondrial function. (A) The ATP content was determined using an ATP determination kit. *Significantly different from the control group (p < 0.001). (B) Succinate dehydrogenase activity was detected using an MTT reagent. *^,**Significantly different from the control group (p < 0.01, p < 0.001). (C, D) The mtPTP opening was measured using (C) flow cytometry or (D) confocal microscopy using the mitochondrial PT pore assay kit.

AgNP changed the expression of mitochondria-related proteins

Opening of the pore leads to ΔΨm loss, which induces the release of cytochrome c from the mitochondria (Zhao et al. 2020). AgNP-induced changes in mitochondrial apoptosis-related proteins were also measured. AgNP induced the release of cytochrome c from the mitochondria into the cytoplasm (Figure 5A). As shown in Figure 5B, AgNP caused a time-dependent decrease in Mcl-1 expression and an increase in Bim and Bax expression. However, phospho Bcl-2 was present until 12 h and then decreased sharply at 24 h. It could be seen that these changes in the expression of apoptotic proteins were closely related to mitochondrial damage.

Figure 5. AgNP changed the expression of mitochondria-related proteins. (A, B) Western blot analysis was performed for the detection of (A) cytochrome c and (B) phospho Bcl-2, Mcl-1, Bim, and Bax proteins. COX4 was used as mitochondrial loading control and actin was used as cytosol or total loading control.

AgNP activated JNK signaling

We have previously verified that AgNP induced apoptosis via a caspase-dependent pathway involving the mitochondria (Piao et al. 2011). AgNP induced JNK phosphorylation in a time-dependent manner (from 3 to 24 h) (Figure 6A and B). Treatment with the mitochondrial targeting antioxidant MTP-131 and JNK inhibitor SP600125 confirmed the involvement of AgNP in mitochondrial damage and the JNK signaling in cell damage (Figure 6C–E). As shown in Figure 6C, cell viability decreased after AgNP treatment and was significantly recovered by treatment with MTP-131 or SP600125. Measurement of apoptotic body formation through Hoechst 33342 was also found to be protected by MTP-131 or SP600125 (Figure 6D). Caspase-3 activity was inhibited by MTP-131 and SP600125 (Figure 6E). These data confirmed that AgNP necessarily underwent JNK signaling.

Figure 6. AgNP activated JNK signaling. (A) Western blot analysis was performed to detect phospho JNK protein. JNK was used as loading control. (B) Phospho JNK was assessed using immunocytochemistry using a phospho JNK antibody and a fluorescein isothiocyanate-conjugated secondary antibody. Confocal microscopic image showed the phospho JNK expression (green), nucleus location (blue), and the merged image indicated the localization of phospho JNK. (C) Cell viability was detected using the MTT reagent. (D) Apoptotic bodies were detected by Hoechst 33342. The arrows indicated apoptotic bodies. (E) Caspase-3 activity was detected using the Caspase-Glo®-3/7 assay kit. (C, E) *Significantly different from the control group (p < 0.001), ^#significantly different from the AgNP group (p < 0.001).

AgNP changed mitochondria morphology

We observed time-dependent changes in cells after AgNP treatment using phase-contrast microscope. Morphological changes in damaged cells that did not completely match those of the control cells began to appear at 3 h, with clear changes observed after 6 h (Figure 7A). To demonstrate the effect of AgNP on mitochondrial damage, we further assessed the AgNP-induced changes in the mitochondrial morphological distribution via confocal microscopy using MitoTracker™ Red FM and DAPI dyes. In control cells, the mitochondria primarily contained spherical structures concentrated around the nucleus rather than being distributed throughout the cytoplasm; however, in AgNP-treated cells, this shape appeared distorted (Figure 7B).

Figure 7. AgNP changed the mitochondrial morphology. (A) AgNP-induced changes in cells were observed over time using a phase-contrast microscope (magnification 100×). (B) Changes in mitochondria morphology were observed using MitoTracker™ Red FM and DAPI staining. Confocal microscopic image showed the mitochondrial (red), nucleus locations (blue), and the merged image indicated the mitochondria localization.

Discussion

In this study, we investigated that AgNP-induced human Chang liver cytotoxicity was related to reduced MnSOD expression and mitochondrial dysfunction caused by mtROS accumulation. Results showed that mtROS were significantly produced after 1 h of AgNP treatment, which continued until 12 h.

SODs are enzymes that cause the dismutation (or partitioning) of superoxide anion (O₂⁻) into ordinary O₂ molecules or hydrogen peroxide; three subtypes of SOD have been identified, including copper-zinc SOD, MnSOD, and extracellular SOD. Among these, MnSOD initially scavenges superoxide anion from the mitochondrial matrix, thereby protecting mitochondria and other cellular components from oxidative stress (Palma et al. 2020; Liu et al. 2022). The physiological importance of MnSOD is highlighted in the finding that, in contrast to other SOD isoforms, MnSOD deficiency causes early neonatal death in gene-knockout mice (Liu et al. 2022). AgNP reduced MnSOD level in a time-dependent manner, which proved the downregulation of MnSOD via the accumulation of mtROS.

Mitochondria are ATP synthesis sites and the core of cellular energy metabolism. Ca²⁺ acts at several levels within organelles, is a key regulator of mitochondrial function, and stimulates ATP synthesis. Increased ROS levels owing to environmental stress increase mitochondrial Ca²⁺ level, causing mitochondrial dysfunction (Matuz-Mares et al. 2022). Our results showed that AgNP caused a significant increase in Ca²⁺ concentration at 12 h. However, studies have reported that Ca²⁺ accumulation in the mitochondrial matrix can increase ROS generation, triggering the permeability transition pore and cytochrome c release, thereby inducing apoptosis (Matuz-Mares et al. 2022). Therefore, mtROS production and Ca²⁺ accumulation have a mutually promoting relationship that maintains homeostasis via regulating intracellular antioxidants, such as MnSOD. The loss of ΔΨm causes the release of cytochrome c and activates the apoptotic pathway (Popov 2023). In previous study, we also demonstrated that AgNP destroyed ΔΨm (Piao et al. 2011).

In this study, we examined the expression of proteins involved in the mitochondria-related apoptotic pathways. The Bcl-2 family proteins, such as Bcl-2, Mcl-1, Bim, and Bax, perform a crucial role in regulating mitochondrial apoptosis by either inhibiting or promoting cell death. The interaction and balancing between Bcl-2 family members modulate the permeability of the outer layer of the mitochondrial membrane, triggering the release of activators of caspase cascade throughout the cytosol and subsequently triggering cell death (Czabotar and Garcia-Saez 2023). In general, Bcl-2 phosphorylation is known to effectively inhibit its activity and reduce its ability to prevent apoptosis, but phosphorylation at the serine 70 site increases the activity of Bcl-2 and decreases the apoptosis effect (Green 2022). JNK activation has been shown to lead to Bcl-2 phosphorylation and degradation, which activates caspase 9 and follows apoptosis (Xu and Hu 2020). After AgNP treatment, JNK began to be phosphorylated as early as 3 h, expression of anti-apoptotic proteins phospho Bcl-2 (serine 70) and Mcl-1 decreased at 24 h, and the expression of pro-apoptotic proteins Bim and Bax increased. AgNP induced the release of cytochrome c from the mitochondria into the cytosol. Mitochondrial injury is followed by depletion of intracellular ATP level. Succinate dehydrogenase, which is present in the mitochondrial respiratory chain of the mitochondrial membrane, is an indicator of mitochondrial membrane integrity (Cao et al. 2023). AgNP decreased ATP activity and succinate dehydrogenase levels in a time-dependent manner. Treatment with MTP-131, a mitochondria-targeting antioxidant, or SP600125, a JNK inhibitor, restored mitochondrial permeability, apoptotic bodies, and caspase-3 activity increased by AgNP, showing that the cell damage process of AgNP occurs through the mitochondria antioxidant system and JNK signaling. According to research, low levels of ROS intermediates lead to transient activation of JNK, but high ROS levels can activate the JNK pathway and prolong the activation time of JNK (Zhang et al. 2016). Our results showed that since the activation of JNK and the expression of MnSOD were significantly regulated from 3 h of AgNP treatment, it could be inferred that the accumulation of mtROS induced by AgNP activated JNK and reduced MnSOD, leading to cell damage. Regarding the study of the signaling pathway between JNK and MnSOD, mtROS can stimulate the transcription of MnSOD by activating JNK signaling (Liu et al. 2020), or MnSOD can effectively inhibit the mtROS-JNK-FOXO1 pathway, ultimately reducing cell death (Zhang et al. 2023). However, in our system, the phosphorylation of JNK and the inactivation of MnSOD by excess mtROS occurred almost simultaneously. We believe that either scenario is possible and depends on the degree of antioxidant system imbalance between mtROS and MnSOD. According to our observations, in vitro condition may not reflect the actual behavior or function in the liver and AgNP may have interactions with other tissues in the human body. In vitro assessment may not strongly represent the hazardous condition of AgNP or reflect the toxic effects due to the long-term exposure. Therefore, it is necessary to conduct in vivo assessment and clinical evaluation representing long-term exposure and dose-dependent exposure in further studies.

Conclusion

This study showed that AgNPs induced mitochondrial dysfunction through mtROS-JNK/MnSOD signaling, resulting in human Chang liver cell damage. Therefore, strengthening the antioxidant ability of MnSOD may prevent cell damage caused by AgNPs.

Abbreviations
AgNP	=	silver nanoparticle
ΔΨm	=	mitochondrial membrane potential
DAPI	=	4′,6-diamidino-2-phenylindole
DHE	=	dihydroethidium
DHR123	=	dihydrorhodamine 123
JNK	=	c-Jun N-terminal kinase
MnSOD	=	manganese superoxide dismutase
mtPTP	=	mitochondrial permeability pore
MTP-131	=	elamipretide
mtROS	=	mitochondrial reactive oxygen species
RT-PCR	=	reverse transcription–polymerase chain reaction
THF	=	tetrahydrofuran.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF), funded by the Ministry of Education [NRF-2021R1I1A1A01047212, RS-2023-00270936].