N-Acetyl Cysteine Protects TMJ Chondrocytes from Oxidative Stress

Introduction

Inflammation of the temporomandibular joint (TMJ) is often treated with arthrocentesis, which is thought to have an anti-inflammatory effect (Nitzan and Price, 2001; Brennan and Ilankovan, 2006). TMJ arthrocentesis is generally applied to a TMJ closed-lock for pain-free manipulation (Nitzan et al., 1990). Arthrocentesis, by definition, refers to needle puncture of a joint space, and injection and lavage with a therapeutic substance, such as anesthetics, followed by joint manipulation (Frost and Kendell, 1999). At the end of lavage, steroids are often injected to alleviate intracapsular inflammation (Dimitroulis and Dolwick, 1996).

In this study, we focused on chondrocytes derived from cartilage formed on rat TMJ condylar bone. In response to inflammatory mediators or excessive mechanical stress, chondrocytes produce abnormal levels of reactive oxygen species (ROS), which are generally produced by immune cells during host defense (Tiku et al., 1990; Henrotin et al., 1993). The major ROS produced by chondrocytes is superoxide anion, which generates derivative radicals, including hydrogen peroxide (H₂O₂) (Hiran et al., 1997). In the presence of iron Fe²⁺ and H₂O₂, chondrocytes release hydroxyl radicals that cause oxidative stress in living systems (Kovacic and Jacintho, 2001; Valko et al., 2001). Cellular responses to ROS are dependent upon the cellular redox status. In some inflammatory conditions, when there is overproduction of ROS and a deficiency of enzymatic and non-enzymatic anti-oxidants, oxidative stress may cause DNA damage and apoptotic cell death (Schweikl et al., 2006; Hecquet and Malik, 2009). Furthermore, ROS modify proteins by oxidation of specific amino acids, leading to impaired biological activity, changes in protein structure, and accumulation of damaged proteins in tissue (Henrotin et al., 2003).

The glutathione redox cycle is regarded as the most important regulatory mechanism for controlling oxidative stress (Meister and Anderson, 1983). N-acetyl cysteine (NAC) is a cell-permeable glutathione derivative that promotes the glutathione redox cycle (Zafarullah et al., 2003). In addition to increasing cellular glutathione levels, NAC also works as a direct ROS scavenger.

To determine the therapeutic potential of NAC in TMJ disorders that are typically treated by arthrocentesis, we aimed, in this study, to demonstrate the protective potential of NAC in controlling oxidative stress in chondrocytes. We examined the effects of various concentrations of NAC on chondrocyte proliferation and differentiation under oxidative challenge with H₂O₂. Exposure of chondrocytes to H₂O₂ mimics the inflammatory state of the biological environment and is known to inhibit proteoglycan and DNA synthesis in chondrocytes (Baker et al., 1989; Vincent et al., 1989).

Materials & Methods

TMJ chondrocytes were isolated from condylar bone surfaces by previously described methods (Takigawa et al., 1984) with slight modifications. Treated cultures contained NAC at 2.5, 5, or 10 mM (in 1 mL culture medium); control cultures did not contain NAC. To simulate inflammatory conditions, we co-treated cultures with 100 µM H₂O₂. Cell metabolic activity, proliferation, gene expression, glycosaminoglycan (GAG) deposition, intracellular ROS level, and glutathione redox status were examined. Details of the cell culture and assay protocols can be found in the Appendix. The protocol for this study was approved by the University of California at Los Angeles Animal Research Committee.

Three independent cultures were prepared for the untreated control and experimental groups for each assay (n = 3). Cytomorphometry and ROS image-based analysis were performed with 5 cells (n = 5). We used one-way ANOVA, at a significance level of p < 0.05, to examine differences in the variables among the different experimental culture conditions and various NAC concentrations. When necessary, the Bonferroni multiple comparison test was used for post hoc evaluation.

Results

NAC Restored Cell Viability, Density, and Proliferation of TMJ Chondrocytes

Exposure to 100 µM H₂O₂ decreased cell density to half the level of that in the untreated control culture, and demonstrated a 30% lower rate of cell proliferative activity. However, the addition of 2.5 mM and 5 mM NAC restored cell density and proliferation to the equivalent levels of those of the untreated control (Fig. 1A). Flow cytometry revealed that the percentage of viable chondrocytes 24 hrs after seeding was 67.7% with 100 µM H₂O₂ exposure, 86.7% in cultures supplemented with 5 mM and 10 mM NAC, and 91.3% in untreated control cultures (Fig. 1B). In the 5 mM and 10 mM NAC-supplemented cultures, the numbers of late-apoptotic cells decreased considerably.

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Figure 1.

The number of chondrocytes, proliferative activity, and cell viability in the untreated control culture, 100 µM H₂O₂ treated culture, and cultures co-treated with 100 µM H₂O₂ + NAC at various concentrations of 2.5, 5.0, and 10 mM (H-N 2.5 mM, H-N 5 mM, and H-N 10 mM, respectively). (A) Cell density quantified with a hemocytometer, and proliferation rate measured by BrdU incorporation during mitosis. Data are shown as the mean ± SD (n = 3). *p < 0.05, indicating a statistically significant difference from the untreated control culture. (B) Cell viability and apoptosis analysis by AnnexinV-based flow cytometry in chondrocytes 24 hrs after seeding. Flow cytometric images are shown at the top. (C) Percentages of viable cells (quadrant 3) and late-apoptotic cells (quadrant 2) are shown at the bottom.

NAC Restored Initial Cell Behavior of TMJ Chondrocytes

Low-magnification confocal microscopic imaging after rhodamine phalloidin staining showed that the number of adherent cells in H₂O₂-treated cultures was substantially reduced compared with that in untreated control cultures after 24 hrs of incubation (Fig. 2A). However, more cells were found to be adherent in cultures co-treated with NAC. High-magnification images showed that the cells were clearly smaller in the H₂O₂-treated cultures than in the untreated cultures. Cells in the H₂O₂/NAC co-treated cultures were enlarged, with clearly stretched cell processes and developed cytoskeleton. Cytomorphometry showed that the cell area, perimeter, and Ferret’s diameter, which were reduced in H₂O₂ cultures, were significantly increased in NAC/H₂O₂ cultures, regardless of the NAC concentration. A majority of the parameters increased to the level comparable with that in control cultures (Fig. 2B).

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Figure 2.

Initial spread and cytoskeletal arrangement of chondrocytes 24 hrs after seeding in the untreated control culture, 100 µM H₂O₂-treated culture, and 100 µM H₂O₂ + NAC co-treated culture. (A) Top images are low-magnification confocal laser microscopic images (50x). Representative confocal microscopic images (600x) of cells stained with rhodamine phalloidin for actin filaments are shown below. (B) Cytomorphometric evaluations are presented. Data are shown as the mean ± SD (n = 5). *p < 0.05; **p < 0.01, indicating a statistically significant difference from the untreated control culture.

NAC Up-regulated Chondrogenetic Function, Even under Oxidative Stress

Cell metabolism, represented by WST1, was measured on culture day 2. The WST1 value, under exposure to H₂O₂, was decreased to less than half that of the untreated control. However, NAC supplementation in 100 µM H₂O₂ cultures increased cellular metabolism in an NAC dose-dependent manner, and in every case was significantly higher than in the untreated control cultures (Fig. 3A). When WST-1 values were standardized with the number of cells, NAC-mediated increase in cellular metabolic activity was more pronounced (Fig. 3A).

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Figure 3.

NAC-restored or enhanced function of chondrocytes. (A) Cell metabolic activity evaluated by WST1 staining on culture day 2. (B) Expression of collagen I, collagen II, and aggrecan genes analyzed by RT-PCR on culture day 10. The panels show representative electrophoresis images visualized with ethidium bromide staining. (C) Intensity of bands for each of the target genes, normalized with reference to GAPDH expression levels. Data are shown as the mean ± SD (n = 3). (D) Representative images of Alcian blue stain on culture days 7 and 14. (E) GAG colorimetry via detection of the stained Alcian blue dye. Data are shown as the mean ± SD for panel A, C, and E (n = 3). *p < 0.05; **p < 0.01, indicating a statistically significant difference from the untreated control culture.

Chondrogenic gene expression was reduced at culture day 10 with H₂O₂ (p < 0.05) (Figs. 3B, 3C). However, supplementation with NAC restored the expression levels of all genes tested to those of the untreated controls. The addition of 5 mM NAC was most effective, yielding more than 3-fold up-regulation of collagen II and aggrecan gene expression, compared with that in untreated controls (Fig. 3C).

The treatment of H₂O₂ reduced the total glycosaminoglycan (GAG) production by 70% from the level of control cultures at day 7, and this GAG level did not increase, even by day 14 of culture (Figs. 3D, 3E). Co-treatment with NAC increased the GAG level significantly, and the GAG level was increased during the duration of the culture; particularly, 5 mM NAC fully restored these levels to those of the untreated controls.

NAC-mediated Increase of Anti-oxidant Capacity

To understand the effects of cellular direct scavenging by NAC, we measured H₂O₂ concentrations in culture medium containing H₂O₂ and 2.5, 5, or 10 mM NAC (Fig. 4A). In the medium to which 100 µM H₂O₂ was added, approximately 94 µM H₂O₂ was detected. Adding NAC at 2.5, 5, and 10 mM decreased the H₂O₂ content to 84, 79, and 67 µM, respectively, after incubation for 30 min (Fig. 4A).

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Figure 4.

Anti-oxidant capacity of NAC and intracellular ROS level. (A) We measured H₂O₂ in culture medium without cells to determine the degree of direct H₂O₂ scavenging by NAC. Bar indicates H₂O₂ concentration. (B) Total intracellular glutathione concentration in chondrocytes 24 hrs after seeding. (C) Intracellular ROS level evaluated by DCF-DA fluorophotometry in chondrocytes 24 hrs after seeding. Data shown are the means ± SD (n = 3). (D) Representative dual-stained confocal laser microscopic images (600x) obtained with DCF-DA (top images) and rhodamine for actin filaments (bottom images). (E) Intracellular ROS level per cell area evaluated by densitometry with the confocal microscopic images. Data shown are the means ± SD (n = 3 for panels A-C, and 5 for panel E). *p < 0.05; **p < 0.01, indicating a statistically significant difference from the untreated control culture.

The total amount of glutathione 24 hrs after seeding was reduced by treatment with 100 µM H₂O₂ to less than 30% of the untreated control culture. Adding NAC increased the glutathione level in a dose-dependent manner (Fig. 4B). Intracellular ROS was increased 1.5 times in the culture with H₂O₂. NAC addition reduced the intracellular ROS to a level equal to that of the untreated control culture (Fig. 4C). Confocal microscopy revealed that the ROS signal was stronger in H₂O₂-treated cells than in untreated cells; NAC addition reduced the ROS signal clearly (Fig. 4D). The ROS level relative to the cell area was substantially increased by the treatment with H₂O₂ and reduced in a NAC concentration-dependent manner (Fig. 4E).

Discussion

Basic and clinical research on the control of oxidative stress in dental treatment has rarely been reported. This study demonstrated that oxidative stress from H₂O₂ induced apoptosis and suppressed proliferation and chondrogenic function of TMJ chondrocytes. In inflammatory conditions, inflammatory cytokines, such as IL-1 and TNF-α, activate signaling cascades in the cytoplasm (Okamoto et al., 2008). Directly, or via induction of the Ras pathway, the GTPase Rac is activated and associated with NADPH-oxidase, which generates superoxide (Hiran et al., 1997; Henrotin et al., 2003). Superoxide, or the dismutation product of H₂O₂, regulates mitogen-regulated kinase (MAPK) pathways, including extracellular signal-regulated protein kinase (pERK) and p38 MAPK cascades (Fialkow et al., 1994). These signaling events can naturally be inhibited by anti-oxidants; however, when there is an overproduction of ROS, both the activity of transcription factors and subsequent gene expression are modulated (Henrotin et al., 2003). Here, we reported that type II collagen and aggrecan gene expression are down-regulated by exposure to ROS in chondrocytes. Further, exposure of chondrocytes to H₂O₂ inhibited proteoglycan and DNA synthesis. In pathological conditions, such as osteoarthritis or rheumatoid arthritis, inhibition of hyaluronic acid synthesis and activation of proteinases that degrade extracellular matrix components of articulate cartilage are both caused by overproduction of ROS (Baker et al., 1989). We used 100 µM H₂O₂ because this concentration significantly decreased proliferation of TMJ chondrocytes in our preliminary study. The H₂O₂-induced decrease in cell proliferation and GAG deposition to less than half of the untreated control revealed a critical H₂O₂-induced inflammatory effect.

NAC exerts its anti-oxidant effect by 2 mechanisms: One is direct scavenging; and the other is by providing cysteine to cells, to modulate intracellular glutathione redox status. We have shown that 2.5 mM and 5 mM NAC directly scavenged about 20% of the H₂O₂ in culture in 30 min. This meant that at least 80% of the H₂O₂ remained available and permeated the cell membranes. This concentration of H₂O₂ may consume a certain amount of glutathione; however, the addition of 5 mM and 10 mM NAC not only restored glutathione levels but also elevated the amount of glutathione synthesis above that of the untreated controls, enabling the number of late-apoptotic cells to be restored. A previous study reported that NAC inhibited nitric-oxide-induced apoptosis of chondrocytes, which was mediated by glutathione (Nakagawa et al., 2010). Depletion of glutathione has been shown to substantially reduce the viability of bovine chondrocytes after challenge with H₂O₂ plus nitric oxide, while facilitating apoptosis of human chondrocytes that were exposed to peroxynitrite (Clancy et al., 1997; Del Carlo and Loeser, 2002). The findings in the present study are consistent with these previous results, strongly suggesting that retention of intracellular glutathione levels may be crucial for the maintenance of cellular homeostasis under the conditions of oxidative stress. The present study measured only total glutathione, although redox status (e.g., GSH/GSSG ratio) can be deduced from the results of a prior study (Sato et al., 2009). The proportion of oxidized glutathione, GSSG, increased with H₂O₂ exposure; the addition of 2.5 mM or 5 mM NAC decreased the amount of GSSG to almost zero (Sato et al., 2009). The GSH redox status has been reported to play an important role in the differentiation and phenotype expression of some cell types (Therond et al., 2000; Kim et al., 2004). NAC may inhibit activation of redox-sensitive transcription factors, such as activator protein-1 (AP-1), c-Jun, and nuclear factor kappa B (NF-κB), by altering intracellular redox status. These transcriptional factors are thought to play a key role in mediating cell differentiation (Kim et al., 2001; Zafarullah et al., 2003; Schweikl et al., 2006). Supplementation with 10 mM NAC increased the direct scavenging effects and glutathione synthesis to the highest level among the tested conditions. It did not produce the best effect for proliferative activity compared with supplementation with 2.5 mM or 5 mM NAC. Little information is available to explain the degrading effects of high concentrations of NAC on chondrocyte proliferation, so we cannot conclude that 10 mM NAC represented an overdose. However, this result suggests a range over which NAC concentrations will be optimal, a subject to be explored in future studies. Because we confirmed that the amount of intracellular ROS and glutathione responded to the addition of NAC dose-dependently, it should be very plausible that NAC mediated rescue of chondrocyte viability as well as the restoration of their function, caused at least partially by the role of NAC as an anti-oxidant agent. However, the expression of chondrocyte-related genes was increased with NAC to a level even higher than the baseline level without H₂O₂. A possible explanation would be that physically manipulating cells during the ordinary protocol of cell culture may cause oxidative stress, and the basic culture media per se may contain the source of oxidants, which may have been reduced or eliminated by the addition of NAC. Another possibility would be an independent pathway of NAC to influence the differentiation and other functions of chondrocytes. Further studies should address the effect of NAC treatment alone in chondrocytes, with a particular focus on profiling the differentiation marker genes and their interaction with the transcription factors mentioned above.

ROS-induced oxidative stress has been implicated in the pathogenesis of temporomandibular diseases (TMDs). In synovial fluid from patients with degenerative TMD, the reduction of synovial fluid viscosity may be due to oxidative structural alteration of hyaluronic acid (Saari et al., 1990). The generation of ROS in a TMD patient’s synovial fluid is typically detected by electron spin resonance analysis (Lee et al., 2004). A recent experimental study, testing in vivo administration of NAC on chondrocytes in knee joints in a rat osteoarthritis model, indicated that NAC significantly prevented cartilage destruction and chondrocyte apoptosis (Nakagawa et al., 2010), suggesting the therapeutic potential of NAC for clinical use. Combined with earlier results, the present in vitro study may have provided the data that justify the future in vivo testing of NAC. Delivery methods of NAC as well as optimal concentration of NAC need to be explored. The effect of NAC not only on chondrocytes but also on other cell types and tissues in TMJ also needs to be determined.