Azoramide ameliorates cadmium-induced cytotoxicity by inhibiting endoplasmic reticulum stress and suppressing oxidative stress

Background Cadmium (Cd) is hazardous to human health because of its cytotoxicity and long biological half-life. Azoramide is a small molecular agent that targets the endoplasmic reticulum (ER) and moderates the unfolded protein response. However, its role in Cd-induced cytotoxicity remains unclear. This study was performed to investigate the protective effect of azoramide against Cd-induced cytotoxicity and elucidate its underlying mechanisms. Methods Inductively coupled plasma‒mass spectrometry was used to measure Cd concentrations in each tissue of ICR male mice. The human proximal tubule epithelial cell line HK-2 and the human retinal pigment epithelial cell line ARPE-19 were used in the in vitro study. Cell apoptosis was determined by DAPI staining, JC-1 staining, and annexin V/propidium iodide double staining. Intracellular oxidative stress was detected by MitoSOX red staining, western blot, and quantitative real-time PCR. Moreover, ER stress signaling, MAPK cascades, and autophagy signaling were analyzed by western blot. Results The present data showed that Cd accumulated in various organs of ICR mice, and the concentrations of Cd in the studied organs, from high to low, were as follows: liver > kidney > testis > lung > spleen > eye. Our study demonstrated that azoramide inhibited ER stress by promoting BiP expression and suppressing the PERK-eIF2α-CHOP pathway. Additionally, we also found that azoramide significantly decreased ER stress-associated radical oxidative species production, attenuated p38 MAPK and JNK signaling, and inhibited autophagy, thus suppressing apoptosis in HK-2 and ARPE-19 cells. Conclusion Our study investigated the effect of azoramide on Cd-induced cytotoxicity and revealed that azoramide may be a therapeutic drug for Cd poisoning.


In vivo experiments
Specific pathogen-free male (SPF) ICR mice weighing 22 to 25 g (aged 5 weeks) (n = 10) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.All mice were housed in individually ventilated cages under SPF conditions in a controlled environment (24 C, 55% humidity, and a 12-h day/night cycle) and given free access to drinking water and diet.All mice were kept in SPF facilities of Hangzhou Hibio Technology Company, and the experimental protocol was approved by the Animal Care and Use Committee of Hangzhou Hibio Technology Company (approval number HB2108002).The study was performed according to international, national and institutional rules for animal experiments.
The mice were allowed to acclimate for 1 week and were randomly divided into two groups.The concentrations of Cd were selected based on previous studies (Luo et al., 2016;Zhao et al., 2021).For cadmium exposure, ICR mice (n = 5) were intraperitoneally administered CdCl 2 (5 mg/kg) once a day for 7 days.The normal control (n = 5) received the same volume of normal saline.Twenty-four hours after the last injection, all mice were anesthetized with pentobarbital sodium (70 mg/kg), and blood samples were collected for subsequent analyses.The mice were then sacrificed by cervical dislocation, and organs (heart, liver, kidney, lung, spleen, testis, and eyes) were dissected, weighed, and stored at −80 C until analysis.No animal was excluded from analysis, but a mouse's spleen weight, a mouse's liver Cd concentration, and an animal's serum Cd concentration in the control group were not included in the analysis because of the failure of sample collection or preparation.The organ coefficient (relative organ weight) was calculated with the organto-body weight ratio (%).

Measurement of the concentration of Cd in mouse blood and tissues
The concentrations of Cd in ICR mouse blood and tissues, including the liver, spleen, lung, kidney, testis, and eye were analyzed by ICP−MS.The method for Cd detection using ICP −MS was established according to previously reported methods with minor modifications (Egger et al., 2019;Tai et al., 2022).Tissues (100 mg) were homogenized in RIPA lysis buffer (1,000 mL) (P0013C; Beyotime Biotechnology, Shanghai, China) with a tissue grinder.Tissue homogenate was centrifuged at 4 C, 5,000 rpm for 5 min.Then, 100 mL of supernatant or serum was digested with 68% HNO 3 in 3.9 mL and reacted in a 15 mL centrifuge tube for 5 min.Subsequently, the reaction solutions were diluted with deionized water and analyzed using a 7800 ICP−MS instrument (Agilent Instruments, Tokyo, Japan).The optimum instrument conditions were set as listed in Table 1.The protein concentration of the supernatant was determined using a BCA kit (P0010S; Beyotime, Shanghai, China).The Cd concentration in each organ or tissue was expressed in unit of mg/mg protein, while the serum Cd concentration was expressed in units of mg/L and mg/mg protein.

DAPI staining
Azoramide and Cd concentrations were selected according to previously reported works (Fu et al., 2015;Komoike, Inamura & Matsuoka, 2012;Zhang et al., 2019).HK-2 cells and ARPE-19 cells were incubated with azoramide (20 mM) for 5 h and then treated in the presence or absence of Cd (20 mM) for 24 h.After washing with phosphate-buffered saline (PBS) three times, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and stained with DAPI for 5 min in the dark.Cell nuclei were observed and photographed by fluorescence microscopy (IX53, Olympus, Tokyo, Japan).The healthy cell nucleus is uniformly stained and clear-edged, while the apoptotic nuclei show irregular edges around the nucleus, heavier staining, and nuclear pyknosis.According to the previously reported method (Jeon et al., 2023), the ratio of condensed nuclei to total nuclei was calculated and expressed as the apoptosis rate (% apoptosis) in each group, and at least three photos were included in the analysis.

Cell apoptosis analysis
Cell apoptosis was examined with flow cytometry (DxFLEX; Beckman Coulter, Suzhou, Jiangsu, China) following annexin V/propidium iodide (PI) double staining (CA1020; Solarbio, Beijing, China).After the indicated treatment, the cells were trypsinized and rinsed with PBS and then resuspended in 100 mL of binding buffer.Five microliters of annexin V and 5 mL of PI were sequentially added and incubated for 5 min in the dark at room temperature.Subsequently, cells were diluted with 400 mL binding buffer, and at least 10 4 cells were analyzed in each treatment.Annexin V-positive cells were regarded as apoptotic cells.Therefore, cells in quadrants 1 and 4 of the flow cytometry dot plots with quadrant markers were considered apoptotic.Apoptosis rate = (annexin V positive cell number/total cell number) × 100%.

Western blotting (WB)
Total proteins were extracted with RIPA lysis buffer (Beyotime, Shanghai, China), which contains 1 mM phenylmethanesulfonyl fluoride (ST506; Beyotime, Shanghai, China) and 1% protein phosphatase inhibitor cocktail (P1260; Solarbio, Beijing, China).The protein concentrations were determined with a BCA protein quantitation kit (P0010S; Beyotime, Shanghai, China).Next, 10 mg of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (ISEQ00010; Millipore, Burlington, MA, USA).After blocking with 5% nonfat milk at room temperature for 1 h, the membranes were incubated overnight at 4 C with the indicated primary antibodies.Following this, the membranes were incubated with their corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (115-035-044 and 111-035-003, respectively; dilution: 1:2,500 and 1:5,000, respectively; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature.Finally, the protein bands were visualized using the ChemiDox TM XRS+ system (Bio-Rad, Hercules, CA, USA) after exposure to Western ECL Substrate (1705061; Bio-Rad, Hercules, CA, USA) for 2 min.The optical density of each WB band was measured using Image Lab TM software (Bio-Rad, Hercules, CA, USA) and normalized to the densities of GAPDH for the same samples.The results are expressed as fold changes relative to the corresponding control, which was always considered as 1.

Mitochondria-associated reactive oxygen species (ROS) measurements
After the designated treatment, mitochondrial ROS were assessed by MitoSOX TM Red (M36008; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) staining according to the manufacturer's instructions.Cells were collected and resuspended in 100 mL DMEM/ F12 medium and incubated with 2.5 mM MitoSOX TM Red in the dark at 37 C for 15 min.After washed with PBS three times, the cells were analyzed by flow cytometry (DxFLEX), and at least 10 4 cells were included in the analysis.CytExpert software (Beckman Coulter, Suzhou, Jiangsu, China) was used for measurement of the mean fluorescence intensity of each sample, and the results were expressed as fold changes relative to the control group.
Relative gene expression was calculated with the 2 −ΔΔCt method with GAPDH as a loading control.At least three independent samples were performed in each group, and each sample was measured in triplicate.

JC-1 staining
Disruption of mitochondrial inner transmembrane potential (DÉ m) triggers the activation of intrinsic apoptotic pathway.In this study, DÉ m was evaluated using JC-1, a dye that accumulates in mitochondria in a potential-dependent manner.When mitochondrial membrane integrity is compromised, the fluorescence emission of JC-1 shifts from red (aggregate) to green (monomer) (Li et al., 2016).The JC-1 staining kit (C2006) from Beyotime Company was used for this purpose.HK-2 cells and ARPE-19 cells were incubated with azoramide (20 mM) for 5 h and then treated with or without Cd (20 mM) for 24 h.JC-1 staining was performed according to the manufacturer's instructions.Briefly, cells were washed with PBS, incubated with JC-1 for 30 min at 37 C, and observed under an inverted fluorescence microscope (IX53, Olympus, Tokyo, Japan).

Inhibition of p38 MAPK and JNK
HK-2 cells and ARPE-19 cells were seeded in 6-well plates and incubated with Cd (20 mM) in the presence or absence of SB203580 (10 mM) or SP600125 (10 mM) for 24 h.Concentrations of the inhibitors were selected based on previous studies (Jin et al., 2023;Tang et al., 2023).Then, the cells were collected for WB analysis or stained with annexin V/ PI and measured by flow cytometry.

Statistical analysis
Data are expressed as the means ± standard deviations (SD).For statistical analysis, the unpaired Student's t test was used for data with only two groups; one-way analysis of variance followed by Tukey's multiple comparisons test was used for data containing more than two groups.All data were analyzed with GraphPad Prism software (version 9.0; San Diego, CA, USA), and p < 0.05 was considered statistically significant.
The heart coefficient, liver coefficient, and kidney coefficient were not significantly altered (Figs.1B-1D).However, the lung coefficient and spleen coefficient showed significant increases (Figs.1E and 1F).Notably, the testis coefficient showed a dramatic decrease following Cd treatment (Fig. 1G).
To assess Cd concentration in various organs of ICR mice, we employed ICP-MS.Our findings demonstrated that Cd concentration in the serum of control mice was 2.95 ± 5.46 mg/L (or 0.04 ± 0.08 mg/g protein) (Figs.1H and 1I).In the Cd exposure group, Cd concentration significantly increased to 44.22 ± 11.27 mg/L (or 0.72 ± 0.17 mg/g protein) in comparison to the control (Figs.1H and 1I).Notably, we found that Cd accumulated in the eye at a higher concentration (10.83 ± 9.14 mg/g protein vs 2.11 ± 0.62 mg/g protein in the control) (Fig. 1J).Among the organs tested, Cd concentration ranked highest in the liver (741.68 ± 81.65 mg/g protein), followed by the kidney (358.35 ± 28.24 mg/g protein), testis (153.52 ± 45.67 mg/g protein), lung (45.69 ± 19.65 mg/g protein), spleen (37.61 ± 13.11 mg/g protein), eye (10.83 ± 9.14 mg/g protein), and serum (0.72 ± 0.17 mg/g protein) (Fig. 1K).These data indicate that Cd tends to accumulate in multiple vital organs, with a preference for the liver and kidney.Intriguingly, the testis showed higher Cd concentration compared to the lung or spleen, confirming that Cd is reproductively toxic (Fig. 1K).

Azoramide protects cells against Cd-induced toxicity
To investigate the protective role of azoramide in Cd-induced cytotoxicity, two widely used cell types, the human proximal tubule epithelial cell line HK-2 and the human retinal pigment epithelial cell line ARPE-19, were employed in the study.We found that Cd exposure could significantly induce apoptotic nuclear morphological changes, and the proportion of nuclear shrinkage and the intensity of DAPI staining increased significantly (Figs.2A and 2B).Cleavage of PARP1 (C-PARP1), a widely used apoptotic marker protein, was elevated after Cd treatment in HK-2 cells (Fig. 2C).In contrast, azoramide coincubation significantly suppressed PARP1 cleavage (Fig. 2C), indicating that azoramide exerts a protective effect on Cd-exposed HK-2 cells.However, the expression of C-PARP1 was too low to be detectable in ARPE-19 cells in the present study.We further detected apoptosis by annexin V/PI double-staining.Cd treatment dramatically increased the cell apoptosis rate compared to the control group (Figs.2D and 2E).However, azoramide significantly alleviated Cd-induced cell apoptosis in HK-2 and ARPE-19 cells, and the apoptosis rate was reduced by nearly 20% (Figs.2D and 2E).Overall, these results suggest that azoramide can significantly protect against Cd-induced cytotoxicity.Azoramide inhibits Cd-induced ER stress Azoramide has been shown in previous studies to be a small molecule inhibitor of ER stress (Fu et al., 2015;Ke et al., 2020).In our investigation, we found that azoramide treatment significantly enhanced Cd-induced BiP expression (Figs. 3A and 3B).Additionally, our results showed that Cd increased the phosphorylation level of PERK, whereas azoramide incubation effectively attenuated this increase (Figs. 3C and 3D).Our data showed that Cd increased p-eIF2a expression in HK-2 and ARPE-19 cells (Figs. 3E and 3F).However, azoramide exacerbated the phosphorylation of eIF2a (Figs.3E and 3F), which may inhibit the translation process and alleviate the pressure of protein folding and processing within the ER.Moreover, our study revealed that Cd treatment elevated the mRNA expression of CHOP, a key mediator of programmed cell death (Figs.3G and 3H).Azoramide significantly inhibited the upregulation of CHOP (Figs. 3G and 3H).These results strongly suggest that the azoramide's protective effect against Cd toxicity is closely associated with its ability to inhibit ER stress.

Azoramide inhibits Cd-induced intracellular oxidative stress
It is well-known that ROS can be generated from oxidative phosphorylation in mitochondria (Li et al., 2015).As byproducts in mitochondria, excess ROS may disrupt the electron transport chain and result in mitochondrial dysfunction.We therefore measured mitochondrial ROS with MitoSOX red staining and found that Cd exposure significantly upregulated ROS production in mitochondria, while azoramide suppressed mitochondrial ROS elevation (Figs.4A and 4B).The Nrf2 pathway is one of the major cellular defense mechanisms against oxidative stress (Tonelli, Chio & Tuveson, 2018).To elucidate its role in azoramide-induced protection effects, we detected Nrf2 by WB.We found that Cd exposure promoted Nrf2 expression, whereas azoramide treatment diminished Cd-induced Nrf2 upregulation (Figs.4C and 4D).As a consequence, the expression of downstream targeted genes, such as HO-1, was inhibited in the presence of azoramide (Figs.4E and 4F).Furthermore, JC-1 staining revealed that normal cells exhibited an intact DÉ m, whereas Cd treatment induced markedly DÉ m loss, as indicated by a shift in JC-1  fluorescence from red to green (Figs.4G and 4H).Azoramide, however, dramatically attenuated Cd-induced DÉ m loss and promoted cell survival (Figs.4G and 4H).
Collectively, these data suggest that azoramide may suppress ROS production in mitochondria and inhibit intracellular oxidative stress, thus inhibiting Cd-induced mitochondrial injury.

Azoramide inhibits the activation of p38 MAPK and JNK signaling
Evidence suggests that the MAPK signaling pathway may play a role in Cd-induced cell damage (Kalariya et al., 2009).In our study, we observed that Cd treatment significantly increased the phosphorylation levels of p38 MAPK and JNK (Figs. 5A-5D).However, azoramide dramatically inhibited p-p38 MAPK and p-JNK expression, suggesting that azoramide may inhibit the activation of MAPK signaling (Figs.5A-5D).These findings  We found that Cd-induced PARP1 cleavage in HK-2 cells was diminished by SB203580, an inhibitor of p38 MAPK (Fig. 5E).Conversely, the JNK inhibitor SP600125 significantly promoted Cd-induced cleaved PARP1 expression (Fig. 5F).Consistently, apoptosis analysis showed that SB203580 decreased Cd-induced apoptosis rate in HK-2 and ARPE-19 cells (Figs. 5G and 5I), whereas SP600125 aggravated Cd-induced cell apoptosis (Figs.5H and 5J).These results suggest that p38 MAPK activation is toxic to HK-2 and ARPE-19 cells.In contrast, JNK activation is beneficial for cell survival.

Azoramide inhibits Cd-induced autophagy
Autophagy may also play a certain role in Cd-induced cell apoptosis (Zhang et al., 2019).

DISCUSSION
Humans are exposed to Cd by consuming contaminated food and drink.As a nonbiodegradable heavy metal, Cd exposure may lead to fatal implications for life (Genchi  , 2020).In this study, we measured Cd concentrations in different organs by using ICP-MS.Our findings demonstrate that the liver and kidney may be two preferable organs for Cd accumulation in the body, which is consistent with previous studies conducted in mice or humans (Egger et al., 2019;Tai et al., 2022;Winiarska-Mieczan & Kwiecien, 2016).Additionally, we observed that the Cd concentration in the testis was higher than in the lung and spleen.Accumulated evidence suggests high sensitivity of the testes to Cd (Ali et al., 2022).Furthermore, our study suggests that the relatively high accumulation of Cd in the testis may also contribute to testicular atrophy (weight loss).We also confirmed that Cd accumulation in the eye, supporting the notion that Cd may be involved in the development of certain ocular diseases.Prior studies have shown that Cd accumulation in the retina may be closely related to the pathogenesis of age-related macular degeneration (Kalariya et al., 2009;Zhang et al., 2019).Accumulation of Cd in various organs leads to tissue injury and organ dysfunction.Treatment strategies for Cd poisoning include promoting Cd excretion with chelating agents and attenuating its toxic effects using small molecules like melatonin and morin (Annie et al., 2023;Xie et al., 2022).Cd has been shown to disrupt cell metabolic activities and induce excessive accumulation of ROS, resulting in cell apoptosis (Zhang et al., 2019(Zhang et al., , 2020;;Zhao et al., 2021).Emerging evidence indicates that the ER may be the target organelle for Cd toxicity (Zhang et al., 2019;Zhao et al., 2021), and inhibition of ER stress has been identified as an effective strategy against Cd-induced cytotoxicity.Previous studies have reported that certain ER stress inhibitors, such as tauroursodeoxycholic acid and salubrinal, exert protective effects against Cd-induced cell injury (Chen et al., 2019;Komoike, Inamura & Matsuoka, 2012).However, these small molecules may lack mechanistic specificity and have limited success in clinical applications (Hetz, Chevet & Harding, 2013).By contrast, azoramide, an ER-targeted small-molecule compound, has been found to activate ER chaperone capacity and protect cells against ER stress (Fu et al., 2015).Our study demonstrates that azoramide has the potential to serve as an effective therapeutic agent against Cd-induced toxicity.
Azoramide significantly mitigated Cd-induced cell apoptosis in both HK-2 and ARPE-19 cells, suggesting that its protective effect is independent of cell type.Our results also indicate that azoramide treatment attenuated Cd-induced Nrf2 activation, suggesting that it may not exert its protective mechanism through Nrf2 signaling.Considering that Nrf2 expression is regulated by intracellular ROS levels (Tonelli, Chio & Tuveson, 2018), our findings suggest that azoramide may suppress Nrf2 activation by inhibiting ROS generation.
When unfolded proteins accumulate in the lumen during ER stress, PERK dissociates from the ER-resident molecular chaperone BiP and is autophosphorylated (Li et al., 2015).PERK phosphorylates eIF2a and hampers the global protein synthesis, thus relieving ER stress.In addition, p-eIF2a selectively enhances the translation of activating transcription Factor 4 (ATF4), which activates the expression of genes involved in antioxidant responses, amino acid biosynthesis, and transport to reestablish cell hemostasis and promote cell survival (Zhang et al., 2022).Our data showed that azoramide enhanced Cd-induced BiP expression, which boosts ER protein folding acutely and binds with PERK and further inhibits Cd-induced PERK phosphorylation.It is important to note that azoramide does not fully increase chaperone capacity without PERK activity, despite attenuating stress-induced PERK expression (Fu et al., 2015;Miao et al., 2022;Okatan et al., 2019).We observed that azoramide treatment dramatically promotes Cd-induced eIF2a phosphorylation and induces cell protective effects, which aligns with previously reported studies (Fu et al., 2015).It should be emphasized that multiple eIF2a kinases, including PERK, GCN2 (general control non-derepressible-2), PKR (double-stranded RNA activated protein kinase), and HRI (heme-regulated inhibitor), can affect the phosphorylation of eIF2a in response to different stresses (Halliday, Hughes & Mallucci, 2017).Therefore, the azoramide-associated eIF2a phosphorylation observed in our study may be induced by the activation of other eIF2a kinases, excluding PERK.
Acute and severe ER stress can induce cell apoptosis through the upregulation of CHOP, resulting in the downregulation of the antiapoptotic protein B-cell lymphoma-2 (Bcl-2) and increased production of the proapoptotic protein Bcl-2 interacting mediator of cell death (Bim) (Fu et al., 2015;Li et al., 2015).A study has confirmed that azoramide can protect cell from CHOP induction (Fu et al., 2015).Consistent with these findings, our study demonstrates that azoramide dramatically mitigates ER stress and significantly suppresses Cd-induced CHOP expression.Additionally, we observed that azoramide, as an ER stress inhibitor, mitigated Cd-induced oxidative stress.This suggests that Cd-activated ER stress may trigger ROS generation in mitochondria.Moreover, CHOP can upregulate endoplasmic oxidoreductin-1 (ERO1), further enhancing intracellular oxidative stress (Ong & Logue, 2023).ERO1 catalyzes the reduction of oxygen into hydrogen peroxide (H 2 O 2 ) during oxidative protein folding.The increase in ERO1 and H 2 O 2 contributes to the upregulation of ROS-sensitive Ca 2+ channels, such as the inositol-1,4,5-trisphosphate receptor (IP3R), resulting in increased Ca 2+ leakage from the ER to mitochondria.This process promotes metabolism and aggravates oxidative stress (Cao & Kaufman, 2014;Decuypere et al., 2011;Marciniak et al., 2004).Furthermore, the diffusion of ROS from mitochondria can attack the ER, further exacerbating ER stress (Decuypere et al., 2011).Thus, a vicious cycle between the ER and mitochondria is formed, triggering cell apoptosis.Consequently, our study demonstrates that azoramide treatment alleviates oxidative stress by restoring ER hemostasis, ultimately inhibiting mitochondrial dysfunction and cell apoptosis.
JNK and p38 MAPK are preferentially activated in response to environmental stress, particularly oxidative stress (Cao et al., 2014).Previous studies have suggested that Cd induces activation of JNK and p38 MAPK, but their roles in cell survival remain unclear (Kalariya et al., 2009).This study clarifies that p38 MAPK signaling promotes Cd-induced cell apoptosis, while the JNK signaling pathway attenuates Cd-induced cytotoxicity.These findings indicate that inhibiting p38 MAPK and activating JNK may be a promising approach to counter Cd-induced cytotoxicity.Furthermore, our study demonstrates that azoramide significantly reduces JNK and p38 MAPK phosphorylation.Since oxidative stress is an upstream event that triggers JNK and p38 MAPK signaling, and azoramide can decrease ROS generation, we conclude that the inhibitory effects of azoramide on JNK and p38 MAPK may be attributed to its suppression of ER stress-associated oxidative stress.

Figure 1
Figure 1 Accumulation of Cd in different tissues of ICR mice.ICR mice were intraperitoneally administered CdCl 2 (5 mg/kg) once a day for 7 days.(A) Mouse body weights were recorded before (on Day 0) and after (on Day 7) Cd treatment (n = 5).(B-G) The ratios (%) of organ weights (heart, liver, kidney, lung, spleen, and testis) to body weights were calculated as organ coefficients and measured after Cd treatment for 7 days (n = 4 or 5).(H and I) Cd concentration in the serum was measured with ICP-MS and expressed as mg/L or mg/g protein (n = 4 or 5).(J) Cd concentration in the eyes was measured with ICP-MS (n = 5).(K) The Cd level in various tissue samples was detected by ICP-MS (n = 4 or 5).Ã p < 0.05, ÃÃ p < 0.01, ÃÃÃ p < 0.001 for the indicated comparisons.Full-size  DOI: 10.7717/peerj.16844/fig-1

Figure 2
Figure 2 Azoramide protects cells against Cd-induced toxicity.(A and B) HK-2 and ARPE-19 cells were stained with DAPI, and representative images were captured under an inverted fluorescence microscope.The ratio of apoptotic nuclei to total nuclei was calculated and expressed as the apoptosis rate; each value is presented as the mean ± SD of at least three fields of view under the microscope.(C) HK-2 cells were pretreated with 20 mM azoramide for 5 h and coincubated with Cd (20 mM) for 24 h.PARP1 and C-PARP1 were detected by WB, and corresponding densitometric analysis was conducted (n = 3).(D and E) HK-2 and ARPE-19 cells were preincubated with azoramide (20 mM) for 5 h and subsequently treated with or without Cd (20 mM) for 24 h.Cell apoptosis was assessed with flow cytometry after annexin V/PI staining (n = 3 or 6).ÃÃ p < 0.01, ÃÃÃ p < 0.001 for the indicated comparisons.Full-size  DOI: 10.7717/peerj.16844/fig-2

Figure 4 21 Figure 5
Figure 4 Azoramide inhibits Cd-induced intracellular oxidative stress.HK-2 and ARPE-19 cells were preincubated with azoramide (20 mM) for 5 h and subsequently treated with or without Cd (20 mM) for 24 h.The cells were then stained with MitoSOX and analyzed by flow cytometry (n = 3 or 5) (A and B); the expression of Nrf2 was measured by WB (n = 3) (C and D).HK-2 and ARPE-19 cells were preincubated with azoramide (20 mM) for 5 h and then treated with or without Cd (15 mM) for 24 h.The expression of HO-1 was detected by qPCR (n = 3 or 6) (E and F).(G and H) After treatment with Cd (20 mM) and azoramide (20 mM), mitochondrial function in HK-2 and ARPE-19 cells was determined by staining with the ΔΨm sensitive dye JC-1.Representative images were captured under an inverted fluoresce microscope.ÃÃ p < 0.01, ÃÃÃ p < 0.001 for the indicated comparisons.Full-size  DOI: 10.7717/peerj.16844/fig-4

Table 1
Operating parameters for the 7800 ICP-MS.

Table 2
Body and organ weights of the mice.