Modification of Oxidative Stress Induced by Exposure to Arsenate in Rat Chondrocytes Treated With Chitosan–Glutathione Nanoparticles

ABSTRACT

Nanoparticles can modify the signaling pathways related to the toxic effects of xenobiotics and their toxicity by adding compounds that alter the oxidation–reduction state, such as glutathione (GSH). This work aimed to evaluate the antioxidant capacity of chitosan–glutathione nanoparticles (NPs Q‐GSH) to modify lipoperoxidation induced by sodium arsenate using a primary culture of rat chondrocytes as a model. NPs Q‐GSH were characterized in size (205.4 ± 33.35 nm), zeta potential (25.6 mV), and percentage of GSH (99.97%). To determine whether the toxicity of sodium arsenate was modified, chondrocytes were exposed to two concentrations of nanoparticles (0.08 and 0.64 μM GSH) for 2 h, followed by a 2‐h acute exposure to sodium arsenate at a concentration of 1 × 10⁻⁶ M, and the determinations associated with the redox state were carried out. ANOVA was performed, followed by multiple analyses of Fisher's means with a p < 0.05. Regarding the measurement of biomarkers, an increase in GSH levels was observed, which can be both synthesized by the cell and provided by the NPs Q‐GSH. A reduction in malondialdehyde levels was observed in the presence of an oxidizing agent, such as sodium arsenate, and subsequent exposure to NPs. Finally, the activity of the enzyme glutathione peroxidase increased significantly compared with untreated cells, which may be associated with GSH being its substrate and, therefore, favoring the activation of this enzyme. The activity of superoxide dismutase decreased, as did the levels of oxidized proteins. These systems could modify the signaling pathways associated with the redox state.

Short abstract

Some nanoparticulate systems have demonstrated significant biological activity in various experimental models. In this work, we demonstrate that in rat chondrocytes subjected to sodium arsenate‐induced redox imbalance, chitosan nanoparticles with glutathione can reduce the oxidative stress associated with xenobiotic exposure. The results showed significant differences in redox modulating enzymatic activity, decreasing the number of oxidized proteins without causing significant cytotoxicity. These results suggest that some modulatory signaling pathways may be involved.

1. Introduction

Nanoparticles are the most developed systems in nanotechnology. A nanoparticle is considered a discrete particle or entity with a length between 1–1000 nm (More et al. 2021) in at least one of its dimensions (length, width, or height).

GSH (l‐glutamyl‐l‐cysteinyl‐glycine) is a water‐soluble tripeptide formed by the amino acids glutamate, cysteine, and glycine. This molecule is an essential cellular antioxidant in all organs and tissues, especially in the liver, where the highest concentrations are found. GSH synthesis only occurs in the cytoplasm; once released, it cannot be reincorporated into the cell (Lu 2013).

GSH is found at an average concentration of 12 mM in mammalian cells. It has essential antioxidant functions because it participates in enzymatic inhibition, reduction of EROS, and inactivation of xenobiotics; it also controls membrane permeability and the transport of amino acids, it functions as a coenzyme, it intervenes in the process of apoptosis and synthesis of proteins, DNA, and RNA, in addition to regulating the formation and maintenance of the active form of certain enzymes. One of the most critical functions of GSH is to maintain the redox potential of the cell because it maintains the thiol groups of proteins in a reduced state and thus allows the generation of various intracellular signaling cascades (Biswas et al. 2006).

On the other hand, arsenic is a chemical compound that can significantly affect the inhibition of enzymatic activity. Its toxicity can be determined based on the form in which it is found. The mechanism by which arsenate (As(V)) damages the cell is believed to be related to its ability to compete with inorganic phosphate and form an unstable arsenate ester that is rapidly hydrolyzed (Tawfik and Viola 2011).

Arsenic is a metalloid that is biotransformed through oxidation–reduction and methylation reactions. These processes lead to bioactivation, generating more toxic methylated species and significant oxidative stress.

There is significant evidence of arsenic's ability to activate the Akt/Hif‐2α/NF‐κB pathways, leading to disruptions in articular cartilage homeostasis and altered chondrocyte pathways (Suminda et al. 2024).

In previous studies, we demonstrated the ability of Q‐GSH NPs to improve the cellular conditions of rat cartilage and the extracellular matrix after intra‐articular administration (López‐Barrera et al. 2019).

This work aims to evaluate the ability to reduce the toxic effects of Q‐GSH NPs exposed to arsenate and to modify the oxidant stress induced by exposure to arsenate, using the primary culture of rat chondrocytes as a model.

2. Materials and Methods

2.1. Chemicals

Pluronic F‐68 (Sigma‐Aldrich, CAS: 9003‐11‐6, St. Louis, MO, USA), reduced l‐glutathione (Sigma‐Aldrich, CAS: 70‐18‐8, purity: 99.5%, St. Louis, MO, USA), Chitosan, 75% deacetylated (Sigma‐Aldrich, CAS: 9012‐76‐4, St. Louis, MO, USA), sodium tripolyphosphate (STTP) (Sigma‐Aldrich, CAS: 7758‐29‐4, purity: 85%, St. Louis, MO, USA), 1% acetic acid pH 4.1 (Cedrosa, CAS: 64‐19‐7, purity: 99.93%, Naucalpan, Mexico, Mexico) were used. Chondrocyte culture was used in DMEM/F12 + Gluta MAX TM (1×) (Gibco, Thermo Fisher Scientific, Paisley, UK), Fetal Bovine Serum (Gibco, Thermo Fisher Scientific, Paisley, UK), Penicillin/Streptomycin (Gibco, Thermo Fisher Scientific, Paisley, UK), Collagenase type 1 (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA), Trypsin (10×) (Gibco, Thermo Fisher Scientific, Paisley, UK), for oxidative stress tests, 2‐Thiobarbituric acid (Sigma‐Aldrich, puruty: 98.5%, CAS: 504–17‐6, St. Louis, MO, USA), Hydrochloric acid (J.T. Baker, CAS: 7647‐01‐0, purity: 37.4%, Jaloxtoc, Mexico, Mexico) 37%, Malondialdehyde (dimethyl) (Sigma‐Aldrich, purity: 96%, CAS: 5578‐67‐6, St. Louis, MO, USA), 2,2′‐dinitro‐5‐5′‐dithiodibenzoicacid (Sigma‐Aldrich, purity: 99.3%, CAS: 69‐78‐3, St. Louis, MO, USA), 5‐sulfosalicylic acid dihydrate (Sigma‐Aldrich, purity: 99.2%, CAS: 5965‐83‐3, St. Louis, MO, USA) EDTA 0.5 mM (C10H14N2Na2O8.2 H2O) (J.T. Baker, CAS: 6381‐92‐6, purity: 37.4%, Jaloxtoc, Mexico, Mexico), Protein Assay dye Reagent concentrate (Bio‐rad, California, USA), Bovine serum albumin (Sigma‐Aldrich, St. Louis, MO, USA).

2.2. Preparation and Characterization of Nanoparticles

The chitosan nanoparticles with GSH (NP's Q–GSH) were prepared using the ionic gelation method, according to López‐Barrera et al. (2019). The determination of the particle size and Z potential of the NPs was carried out with the Nanosizer NANO–ZS90 equipment, Series MAL1109355, at a temperature of 25°C, through the Zetasiser software. The nanoparticles were ultracentrifuged at 27,000 rpm for 1 h to measure the percentage of GSH. Subsequently, the supernatant was separated and reacted with Ellman's reagent to indirectly measure the concentration of non‐encapsulated GSH.

2.3. Primary Chondrocyte Culture

Primary culture of chondrocytes was carried out from a cartilage sample obtained from a 3‐ to 4‐week‐old female Wistar rat from the animal facilities of the Faculty of Higher Studies Cuautitlan‐UNAM; the Institutional Committee approved the use of experimental animals for this trial for the Care and Use of Experimental Animals of the Faculty of Higher Studies Cuautitlan—UNAM CICUAE‐FESC C 23_30. The cartilage sample was treated as described by López‐Barrera et al. (2019); briefly, the tissue sample was mechanically homogenized and treated with 0.2% collagenase 1A/DMEM to degrade the extracellular matrix, cells obtained were centrifuged, and the pellet was suspended in DMEM/F12 medium and incubated at 37°C, 5% CO², and 90% relative humidity.

2.4. Experiment Design

To determine if the toxicity of sodium arsenate was modified, a dose–response curve was carried out using five different concentrations, of which two were chosen (0.08 and 0.64 μM of GSH). The following groups were obtained: negative control (cells without treatment), positive control (cells exposed to a concentration of 1 × 10⁻⁶ M of sodium arsenate for 2 h), empty nanoparticles, and nanoparticles loaded with GSH at concentrations of 0.08 and 0.64 μM of GSH for 2 h. Determinations of biomarkers associated with oxidative stress were conducted to evaluate the modification of the cellular redox state. Three independent experiments were performed in triplicate.

2.5. Biomarkers of Oxidative Stress

The DTNB (5,5′‐dithiobis‐(2‐nitrobenzoic acid)) technique was used to measure extracellular GSH levels; this reagent reacts with the thiol group of GSH to produce a yellow compound. The yellow compound produced is proportional to the amount of GSH in the sample and was read in the spectrophotometer at 450 nm. The TBARS test was carried out to evaluate lipoperoxidation, which measures lipid oxidation products at the membrane level, mainly malondialdehyde, which interacts with thiobarbituric acid, generating a pink adduct (J. Liu et al. 1997). It was read in the spectrophotometer at 540 nm. A reaction coupled with GRx was carried out using NADPH as an electron donor to measure the activity of glutathione peroxidase. An increase in GPx activity is observed with a decrease in absorbance at a wavelength of 340 nm. The activity of the glutathione reductase enzyme is quantified based on a coupled oxidation–reduction reaction. In this assay, GRx catalyzes the reduction of GSSH to GSH in a reaction dependent on NADPH, which is oxidized to NADP+. This oxidation is followed spectrophotometrically at a wavelength of 340 nm. The activity of the enzyme can be determined by the inhibition in the photoreduction of nitroblue tetrazolium (NBT) by superoxide radicals, which are generated by the autoxidation of hydroxylamine hydrochloride. Hydrochloride is an intermediate in the reduction of nitrate to ammonia and the oxidation of ammonia to nitrite, which is autoxidized by superoxide radicals. Accompanying the autoxidation of hydroxylamine at high pH, NBT is reduced in the presence of EDTA, which induces an increase in absorbance at 560 nm due to the accumulation of formazan (reduced NBT).

2.6. Statistical Analysis

Data were analyzed using a one‐way analysis of variance (ANOVA) followed by multiple comparisons of means according to the Fisher statistical test, considering a significant difference of p < 0.05 through the Origin lab 2024 program (Northampton, Massachusetts, USA).

3. Results

3.1. Characterization of Primary Culture and Cytotoxicity Assay

Figure 1 shows the results of the characterization of chondrocytes, where type II collagen and Sox9, characteristic proteins of these cells, are identified.

FIGURE 1.

Characterization of chondrocytes. (a) Collagen type II, (b) Sox 9.

In Figure 2, the cell viability observed at different nanoparticle concentrations is shown and determined using the crystal violet technique. Different letters indicate a significant difference between the means with a p < 0.05. The results show that none of the nanoparticle concentrations significantly decreased cell viability.

FIGURE 2.

Cell viability by the crystal violet technique (n = 9). (a) NPs Ch‐GSH; The numbers 1–5 (0.08‐, 0.16‐, 0.32‐, 0.64‐, and 1.28‐mM GSH), represent the different concentrations of GSH in the nanoparticles. (b) NPs Ch. The numbers 1–5, equivalent to the loaded NPs.

3.2. Characterization of Nanoparticles

In Figure 3, the graph shows the average size of the (a) nanoparticles and (b) the Z potential. Additionally, Table 1 presents the collected results.

FIGURE 3.

Size of nanoparticles. The image shows the particle size: (a) NPs Ch‐GSH and (b) NPs Ch.

TABLE 1.

Characterization of nanoparticles. Particle size and Z potential of the NPs, as well as the average percentage of GSH encapsulation.

NPs	Size	Potential Z	% encapsulation
NPs Ch‐GSH	205.4 ± 33.35	25.6	99.97
NPs‐Ch	174.4 ± 94.07	20.8	—

3.3. Biomarkers of Oxidative Stress

Figure 4 shows the intracellular GSH levels, evidencing that nanoparticle exposure and the combined exposure of nanoparticles with arsenate increase intracellular GSH levels.

FIGURE 4.

Intracellular glutathione levels (n = 9). Effect of NPs Ch‐GSH and NPs Ch on intracellular glutathione levels in rat chondrocytes. Bars with different letters indicate significant differences between the means (Fisher p < 0.05). C− untreated cells C+ sodium arsenate (As), NPs Ch‐GSH[L]: Nanoparticles at low concentration (0.08 μM of GSH), NPs Ch‐GSH[H]: Nanoparticles at high concentration (0.64 μM of GSH), NPs Ch[L] and NPs Ch[H] NPs Ch equivalent to the loaded NPs at high and low GSH concentrations.

Figure 5 shows the levels of malondialdehyde. It is observed that combined exposure with nanoparticles significantly decreases the levels compared with both controls, particularly at the high concentration.

FIGURE 5.

Lipoperoxidation levels (n = 9). Effect of NPs Ch‐GSH and NPs Ch on lipoperoxidation levels in rat chondrocytes. Bars with different letters indicate significant differences between the means (Fisher p < 0.05), C− untreated cells C+ sodium arsenate (As), NPs Ch‐GSH[L]: Nanoparticles at low concentration (0.08 μM of GSH), NPs Ch‐GSH[H]: Nanoparticles at high concentration (0.64 μM of GSH), NPs Ch[L] and NPs Ch[H] NPs Ch equivalent to the loaded NPs at high and low GSH concentrations.

In Figure 6, GPx activity is reduced by the combined exposure of nanoparticles with arsenate. In contrast, exposure to a low concentration maintains levels like the positive control. Empty nanoparticles, in combination with arsenate, also significantly decreased the activity of this enzyme.

FIGURE 6.

Glutathione Peroxidase Activity (n = 9). Effect of NPs Ch‐GSH and NPs Ch on glutathione peroxidase activity in rat chondrocytes. Bars with different letters indicate significant differences between the means (Fisher p < 0.05). C− untreated cells C+ sodium arsenate (As), NPs Ch‐GSH[L]: Nanoparticles at low concentration (0.08 μM of GSH), NPs Ch‐GSH[H]: Nanoparticles at high concentration (0.64 μM of GSH), NPs Ch[L] and NPs Ch[H] NPs Ch equivalent to the loaded NPs at high and low GSH concentrations.

In Figure 7, it can be observed that nanoparticle exposure in both concentrations maintains activity like that of the control cells, and its combination with arsenic keeps the levels low, suggesting that the GSH present in the nanoparticles may be recognized by the cell, meaning it would not be necessary to regenerate more GSH through this enzyme.

FIGURE 7.

Glutathione reductase activity (n = 9). Effect of NPs Ch‐GSH and NPs Ch on glutathione reductase activity in rat chondrocytes. Bars with different letters indicate significant differences between the means (Fisher p < 0.05). C− untreated cells C+ sodium arsenate (As), NPs Ch‐GSH[L]: Nanoparticles at low concentration (0.08 μM of GSH), NPs Ch‐GSH[H]: Nanoparticles at high concentration (0.64 μM of GSH), NPs Ch[L] and NPs Ch[H] NPs Ch equivalent to the loaded NPs at high and low GSH concentrations.

In Figure 8, the results of superoxide dismutase (SOD) activity are shown, where it is observed that combined exposure to nanoparticles and arsenate significantly increases the activity of this enzyme.

FIGURE 8.

SOD activity (n = 9). Effect of NPs Ch‐GSH and NPs Ch on SOD activity in rat chondrocytes. Bars with different letters indicate significant differences between the means (Fisher p < 0.05). C− untreated cells C+ sodium arsenate (As), NPs Ch‐GSH[L]: Nanoparticles at low concentration (0.08 μM of GSH), NPs Ch‐GSH[H]: Nanoparticles at high concentration (0.64 μM of GSH), NPs Ch[L] and NPs Ch[H] NPs Ch equivalent to the loaded NPs at high and low GSH concentrations.

In Figure 9, exposure to nanoparticles at both doses substantially decreases oxidized protein levels, and their combination with arsenate keeps the levels below those of the positive control, suggesting that the GSH present may reduce cellular stress.

FIGURE 9.

Oxidized proteins (n = 9). Effect of NPs Ch‐GSH and NPs Ch on oxidized proteins in rat chondrocytes. Bars with different letters indicate significant differences between the means (Fisher p < 0.05). C− untreated cells C+ sodium arsenate (As), NPs Ch‐GSH[L]: Nanoparticles at low concentration (0.08 μM of GSH), NPs Ch‐GSH[H]: Nanoparticles at high concentration (0.64 μM of GSH), NPs Ch[L] and NPs Ch[H] NPs Ch equivalent to the loaded NPs at high and low GSH concentrations.

4. Discussion

4.1. Characterization of Nanoparticles

The ionic gelation method was used to prepare NPs Ch‐GSH and NPs Ch. The proposed mechanism for the formation of these nanoparticles states that the ionotropic gelation of chitosan occurs due to electrostatic interactions between the products of the dissociation of STPP in aqueous solution (P3O10‐5 and HP3O10‐4) with the groups (—NH₃ ⁺) of chitosan. Furthermore, the particle size of these systems is susceptible to pH and ionic strength (Jonassen et al. 2012). We worked at a pH of 4.1 because chitosan is soluble in solutions with a pH lower than 6.0 (H. Liu et al. 2012; Trapani et al. 2009).

For the characterization of the NPs, the Nanosizer NANO—ZS90 equipment, Series MAL1109355, was used through the Zetasiser software. This equipment uses dynamic light scattering to analyze particle size and charge (Z potential).

According to Goycoolea et al. (2016), the particle size in nanoparticulate systems with chitosan varies between 100 and 350 nm, and the zeta potential is between +25 and +35 mV. Zeta potential suggests the magnitude of repulsion or attraction of charge between particles. It is, therefore, a fundamental parameter determining the stability of a suspension of NPs (López‐Barrera et al. 2019), so the results indicated that both suspensions of NPs are stable.

Physiological barriers limit the effectiveness of nanoparticles, which is why particle size is essential to crossing these limits. Endocytosis is suggested as a possible mechanism to cross physiological barriers when NPs are smaller than 500 nm (Dıaz‐Torres et al. 2016). So, the nanoparticles under study could efficiently translocate (174–205 nm), release the negatively charged GSH, and release the encapsulated glutathione.

4.2. Challenge With Nanoparticles

The biological model of chondrocytes was characterized by identifying collagen and Sox9 proteins characteristic of these cells (Figure 1). As mentioned above, according to the physicochemical characteristics of NPs, such as their particle size and charge, endocytosis may be the process by which NPs favor their incorporation into cells. The endocytic pathway is a process of vesicle formation in the plasma membrane with extracellular content, which subsequently fuses with internal compartments, mainly endosomes (Buratta et al. 2020). As seen in Figure 4, there is a significant difference in the amount of intracellular GSH between the control systems and the rest of the systems containing NPs, where an increase in GSH inside the cell is favored.

This increase in GSH may be due to a response of the cells to an external stimulus such as nanoparticles and an increase in de novo synthesis in the face of the challenge. Naturally, the synthesis of GSH occurs in the cytosol from its precursor amino acids: glycine, cysteine, and glutamic acid; this synthesis occurs by the consecutive action of two enzymes: glutamate cysteine ligase (GCL) and glutathione synthetase (GS). Under normal physiological conditions, the rate of GSH synthesis is determined mainly by two factors: GCL activity and the availability of the substrate cysteine (Lu 2013).

Evidence suggests that NPs that only contain chitosan can have antioxidant activity due to the amino groups present in the molecule. They also have biocompatibility, low toxicity properties, and high bioavailability and affinity for metals and proteins. These could activate a cellular response observed with increased GSH levels (Figure 5). This was enhanced by the oxidizing agent (sodium arsenate) (Chowdhury et al. 2008).

In Figure 5, NPs at low concentrations increase the levels of MDA compared with untreated cells. However, they do not reach the levels induced by sodium arsenate. The mechanism of EROS production by arsenate could be a direct mechanism of induction of lipid peroxidation by arsenate or its metabolic intermediates; it could also be generated because of the inhibition of the activity of enzymes such as glutathione reductase, which is part of the cell's defense against oxidative damage (Chouchane and Snow 2001).

In high‐concentration NPs, MDA levels are maintained like those of untreated cells, which may suggest that lipoperoxidation damage is not being generated. When we combine NPs with the stressor agent, it can be observed that the levels decrease when compared with the positive control. Given that glutathione levels were increased, but MDA levels were decreased, it could be considered that part of that GSH is provided by the NPs and not from the cells and, therefore, is being used to reduce the damage induced by the arsenate. As mentioned, GSH is an endogenous antioxidant that can reduce the levels of lipoperoxidation and participates in the transformations of reactive oxygen species (ROS), catalyzing the reduction of peroxide or lipo peroxide, for which it uses GSH as a reducing agent (Vairetti et al. 2021).

GPx is an antioxidant enzyme that is activated by external and internal stimuli at the cellular level. This enzyme uses GSH to reduce hydrogen peroxides and lipid peroxides to generate water and lipid alcohols (Rahal et al. 2014). The activity of this enzyme was measured from a reaction coupled with glutathione reductase using NADPH as an intermediate.

Figure 7 shows that a high concentration favors an increase in the activity of this enzyme, which may be associated with the activation of the transcription factor Nrf2 because the mechanism by which GPx synthesis is induced is through the factor nuclear (derived from erythroid 2), this transcription factor regulates the expression of detoxifying and antioxidant enzyme genes by binding to DNA to a sequence known as ARE (antioxidant response element) that can be activated by external factors such as the presence of NPs or external agents such as arsenate. These effects promote cells to activate antioxidant events so NPs can activate the transcription factor (Nguyen et al. 2003). The activity remains like that of untreated cells with a low concentration, which could be associated with the fact that the low concentration of NPs may not activate other antioxidant mechanisms at the cellular level. This would corroborate the hypothesis that the observed increases in GSH are due to the NPs and not to a response induced by the cell. However, it would be necessary to measure the synthesis and expression of other molecules, such as the transcription factor Nrf2, to corroborate this.

Furthermore, GSH‐containing nanoparticles can deliver GSH directly to cells, reducing the dependence on glutathione reductase (GR) to regenerate GSH from its oxidized form (GSSG). This mechanism suggests that the cell, upon receiving exogenous antioxidants, could downregulate GR production to balance antioxidant processes, decreasing its expression and activity (Zhang et al. 2024). Antioxidant nanoparticles, particularly those containing GSH, have shown the ability to suppress ROS production, thereby reducing the burden on the cellular redox machinery (Martinelli et al. 2020).

Additionally, arsenate, known to generate an increase in ROS, can overload cellular antioxidant systems. This constant oxidative stress could lead to the inhibition of GR by depletion of its cofactors, such as NADPH, or by direct modification of the enzyme due to interactions with ROS or with arsenic itself. Arsenic salts can also interfere with regulating the expression of antioxidant genes, which could contribute to the decrease in GR in situations of chronic exposure (Salnikow and Zhitkovich 2008). These combined mechanisms suggest that both GSH nanoparticles and arsenic act together to reduce GR activity in cells.

On the other hand, exposure to nanoparticles can generate a dual effect. Some nanoparticles can generate additional ROS, exacerbating oxidative stress; therefore, stimulating more significant SOD activity (Figure 8). However, nanoparticles with antioxidant properties or loaded with molecules such as GSH could indirectly enhance the antioxidant response, including SOD, by reducing the ROS load. In this way, SOD activity increases as a compensatory mechanism to avoid the oxidative damage produced by arsenate and the presence of ROS induced by nanoparticles.

The decrease in levels of oxidized proteins (Figure 9) in chondrocytes exposed to arsenate and then treated with GSH nanoparticles can be explained by several mechanisms related to the neutralization of oxidative stress. Arsenate produces ROS, which damage cellular proteins and other biomolecules. GSH, a key antioxidant in cells, directly neutralizes ROS, preventing oxidative damage (Kennedy et al. 2020). Nanoparticles loaded with GSH can improve their intracellular bioavailability, enhancing their ability to reduce oxidative stress. Likewise, GSH is essential for functioning antioxidant enzymes such as glutathione peroxidase, which eliminates peroxides and protects proteins from oxidation (Vašková et al. 2023).

Furthermore, the GSH contained in the nanoparticles under study could activate the Nrf2 signaling pathway, which regulates the expression of antioxidant and detoxification genes, promoting a protective response against oxidative stress (Thiruvengadam et al. 2023). This process reduces protein oxidation by limiting ROS formation and restoring redox balance. It is also possible that GSH acts as a chelator, removing toxic metals such as arsenic and preventing the formation of additional free radicals. Together, these mechanisms contribute to the decrease in oxidized proteins in chondrocytes by reversing the oxidative effects induced by arsenate and restoring cellular homeostasis (López‐Barrera et al. 2019). Some studies show arsenic's abrasive capacity and its relationship with cartilage aging (Chung et al. 2020). Considering the physicochemical nature of the nanoparticles under study and their antioxidant capacity, we believe that the results of this work allow us to lay the foundations for the study of the redox modulation pathways related to the significant cellular effects that occur in arsenic exposure, given that the toxicological impact and mechanism of arsenic exposure on articular chondrocytes remain unclear. Furthermore, we consider the nanoparticles under study as a model for the disposition of an active ingredient that may contribute to the well‐being of chondrocytes in individuals with osteoarthritis.

5. Conclusions

Nanoparticles prepared by ionotropic gelation and characterized by dynamic light scattering have an optimal particle size and zeta potential to be a stable suspension. They can encapsulate GSH without generating cytotoxic damage inside the cell, and it was proven that they can do so without generating cytotoxic damage. Therefore, the viability of the chondrocytes was not modified when the nanoparticles were administered; they could also cross the cell membrane to release the encapsulated GSH.

Sodium arsenate was shown to be a stressor agent that increases enzymatic activity and can generate oxidative stress. However, it was demonstrated that Q‐GSH NPs at a concentration of 0.64 μM have a greater antioxidant capacity by reducing lipoperoxidation levels and increasing glutathione peroxidase activity.

It was identified that Q NPs also have antioxidant properties. They are generated by chitosan, a biopolymer that could activate a cellular response observed with increased GSH levels.

In this work, the antioxidant capacity of GSH nanoparticles was evaluated in rat chondrocytes, assessing the activity of the GPx enzyme and the levels of lipoperoxidation. Although they proved to be stable systems, more tests could be carried out, such as the activity of catalase, glutathione reductase, ROS, and the enzyme SOD, to have greater specificity in the data.

Conflicts of Interest

The authors declare no conflicts of interest.

Barrera, L. , García V., Rosas J., Soria‐Castro E., Torres R., and Noguera P.. 2025. “Modification of Oxidative Stress Induced by Exposure to Arsenate in Rat Chondrocytes Treated With Chitosan–Glutathione Nanoparticles.” Journal of Applied Toxicology 45, no. 11: 2391–2399. 10.1002/jat.4847.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.