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Summer Research Fellowship Programme of India's Science Academies

Investigating the role of N-Acetyl L-Cysteine and Hydrogen sulphide on DNA damage during senescence

Sukrit Mahajan

Department of Genetics, University of Delhi 110021

Prof Deepak Saini

Indian Institute of Science, Bengaluru 560012

Abstract

Aging can be defined as the time related deterioration of physiological functions essential for survival. At the cellular level when cells reach replicative exhaustion or get exposed to various stresses such as reactive oxygen species, telomere attrition among others, a state of irreversible cell cycle arrest is induced known as senescence. Progressive accumulation of senescent cells leads to organismal aging. DNA damage is one of the primary drivers for senescence and reactive oxygen species reinforce the senescent phenotype. In order to induce senescence, BrdU, a thymidine analog and/or ionizing radiation (IR) has been used. We investigated the effect of H2S and N-Acetyl L-Cysteine (NAC) on senescent cells in response to DNA damage. While the effects of these compounds are well studied in non-senescent cells, there is surprisingly little known about their role in DNA damage response during senescence. Therefore, we investigated the effect of H2S and NAC on DNA damage in the background of senescence by immunostaining for γ-H2AX foci, a classical marker of DNA damage response. Through our study, we show that H2S induces DNA damage in non-senescent cells, which is in line with previous studies. However, there was no change observed in the levels of DNA damage in senescent cells. In the lab, it has been shown that H2S levels are high in senescent cells in comparison to non-senescent cells. Interestingly, on reducing the cellular H2S levels by knocking down cystathionine γ-lyase (CSE), an enzyme required for H2S production, it was observed that the level of DNA damage increases in non-senescent cells in contrast to no change being observed in senescent cells. Similarly, on treatment with N-Acetyl L-Cysteine (NAC) we observed a significant decrease in γ-H2AX foci in senescent cells, while no difference was observed in non-senescent cells. In conclusion, our study helps in understanding the effect of H2S and NAC in senescent cells.

Keywords: senescence, DNA damage, H2S, N-Acetyl L-Cysteine, gamma H2AX 

Abbreviations

Abbreviations
BrdU 5-bromo-2'-deoxyuridine 
 CBSCysathionine β Synthase 
CSE Cystathionine γ-lyase 
γH2AX  gamma H2AX  
 H2SHydrogen Sulphide 
 MST3-mercaptopyruvate sulphur-transferase 
 NAC N-Acetyl L-Cysteine 

INTRODUCTION

Cellular Senescence

Aging is an inevitable part in the lifespan of an organism, which is associated with a progressive deterioration of physiological function and age-related disorders. A major driving force for aging is cellular senescence. Cellular senescence can be defined as a stress response towards various stimuli such as DNA damage, oncogene activation, dysfunctional mitochondria and metabolic imbalances, resulting in irreversible cell cycle arrest. Specifically, senescent cells accumulate high degree of DNA damage leading to upregulation of cell cycle proteins like p21 and p53. Though senescence is associated with genomic instability and is considered the primary cause of aging, it has been shown to be important during normal development and especially in tissue homeostasis (1). However, the chronic presence of senescent cells can result in induction of pathological inflammation of the tissues, leading to age-related disorders. This age related characteristic has been shown to extend to many tissues (2, 3). Several studies have shown that senescent epithelial cells and fibroblasts have been implicated in the progression of idiopathic pulmonary fibrosis (IPF), a cellular senescence associated disease (4). However, senescent lung epithelial cells which have been implicated in IPF remains a poorly understood phenomena.

Interestingly, in the course of finding an answer to overcome the phenomena of senescence, several studies have showcased the clearing of the senescent cells using compounds called senolytics, which resulted in alleviating IPF related dysfunction in mice (5). In the current study, we have utilized this idea in order to investigate the role of two compounds, namely N-Acetyl Cysteine (NAC) and hydrogen sulphide (H2S) during DNA damage in the background of senescence. Several studies report that these compounds have an anti-oxidant function and NAC specifically has been shown to have a protective role during oxidative stress (6, 7). Therefore, for our study we investigated whether, N-Acetyl L-Cysteine and H2S, two compounds implicated in various physiological phenomena, have any effect on DNA damage during senescence.

Intriguingly, cellular senescence, though an integral part of a cell, cannot be characterized by a unique marker. Thus, several assays and markers have been designated to distinguish senescence from other cellular phenomena, namely senescence associated β-galactosidase activity, high expression of cell cycle proteins like p53 and p21, DNA damage response through the formation of γH2AX foci and increase in the cell size. For the current study, we have used γH2AX as a marker of cellular senescence in order to understand whether NAC and H2S, have any role to play in DNA damage response during senescence.

N-Acetyl L-Cysteine: Scavenger of Reactive Oxygen Species

N-Acetyl L-Cysteine (NAC) has been used as a dietary supplement for many years (8). It is known to be an effective free radical scavenger and a major contributor to the maintenance of the glutathione status in cells. Being a precursor of glutathione, it is also involved in cellular detoxification. Recent reports have shown that NAC treatment leads to increase in the levels of many other antioxidants, with a concomitant reduction in pro-oxidant levels in old rats (9). An important part of the same study provides evidence that NAC slows down the rate of respiratory failure development during inspiratory resistive loading while another report shows that NAC has a positive effect in balancing the overall redox status of the cell (7). In addition, it was also revealed that NAC showcases an antioxidant potential in CD4+ T cells derived from healthy young (25–30 year olds) and older aged (55–60 year olds) donors (10). The group also observed a significantly higher intracellular GSH: GSSG ratio, reduced levels of oxidative DNA damage and increased proliferative capacity on treatment with N-Acetyl L-Cysteine. All these studies taken together provide a strong platform for studying the effect of NAC on DNA damage during senescence and investigate whether it possesses protective effects. If effective, it would open doors to providing better and more promising treatment regimens in age related disorders.

H2S: A Ubiquitous Gaso Transmitter

It is well known that H2S belongs to a family of gaso-transmitters, similar to nitric oxide and carbon monoxide. H2S is produced by the action of three different enzymes via independent reactions: Cystathionine γ Lyase (CSE), Cysathionine β Synthase (CBS) and 3-mercaptopyruvate sulphur-transferase (3-MST). While CBS is predominantly expressed in the nervous system, CSE is known to be produced ubiquitously in most tissues (11). Many reports have provided evidence of it being differentially expressed in various tissues such as neuronal, cardiovascular, respiratory, renal and gastrointestinal and proposed its cyto-protective roles as deregulation of H2S production has been implicated in pathophysiology of cardiovascular diseases, shock and burn injuries among others (11). Interestingly, contrasting reports show how H2S production by the bacteria in the gut flora results in damage to the colonic epithelium (12). These confounding effects of the gaso-transmitter, are quite intriguing. Moreover, while it has been well established that H2S induces DNA damage in non-senescent cells and is considered pro-apoptotic, it has also been shown that H2S donors reduce inflammation and alleviate oxidative stress in neurons by scavenging ROS and stimulating glutathione synthesis (13). Therefore, we were interested in studying the role of H2S in the mammalian system using human lung cells as a model.

DNA Damage and H2AX

DNA damage is one of the main hallmarks of aging and senescence and it becomes extremely important to investigate whether these compounds have any effect on DNA damage. Several reports have shown the association of DNA damage with senescence through the upregulation of an important tumour suppressor, p53. p53 levels rise during senescence when compared to non-senescent cells. However, a more direct read out of DNA damage is the phosphorylation of the histone H2A.X. H2A.X gets phosphorylated at Ser139 in mammalian cells and is referred to as γH2AX. Thus for our study we have used γH2AX foci formation as a read out to study the effect of NAC and H2S on DNA damage during senescence in mammalian cells through immunostaining and whether induction of H2S or treatment with NAC has any effect on the phosphorylation status of this protein.

MATERIALS AND METHODS

Cell Lines

A549 and HeLa cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal bovine serum (FBS), 100 μg/ml streptomycin, and 100 U/mL penicillin (Duchefa).

Inhibtion of CSE Expression

We used HEK293T cells for virus generation and synthesis of shRNA against cystathione synthase γ (CSE). HEK293 cells were cultured till they were 80% confluent and lentiviral transfer vectors and packaging vectors, PAX and PMD containing the gene of interest which codes for the shRNA against CSE, were transfected into HEK293T cells. The cells were incubated for 48 hours with the vectors, after which the cells were pelleted and the supernatant collected and stored at -80 ºC. Alternatively, the supernatant could be added to the medium culturing A549 cells. 10cm dishes coated with polybrene (10ug/ml) for 30 minutes were used for culturing A549 cells in 2% DMEM. The cells were infected with the virus for 72 hours. The virus medium was replenished with DMEM + 10% FBS. Stable cell lines were selected using puromycin (1-10 ug/ml). The cells were cultured with the viral gene coding for shRNA against CSE, incorporated successfully into the cells.

Immunofluorescence

Cells were fixed using 4% paraformaldehyde (PFA) for 15 mins at room temperature, followed by permeabilization using 1% Triton X-100 for 15 min at room temperature. Blocking was performed using 10% Fetal Bovine Serum (FBS) for 1 hour at room temperature. Primary antibodies were diluted in antibody dilution buffer (1% BSA, 0.3% Triton, 1X PBS) and incubated at room temperature for 1 hour. Primary antibody used was γH2AX (Cell Signalling Technology). Secondary antibody (TritC) was used and incubated for an hour at room temperature before counterstaining with DAPI for 15 minutes. The coverslips were mounted using mounting medium containing an antifade reagent DABCO. Images were acquired using IX83 Olympus Microscope. Images were analysed using Fiji (ImageJ). Foci counting was performed using the cell counter Plug-In.

Statistical Analysis

Samples were analysed for test of significance using Mann Whitney U Test. Error bars were plotted using standard error of mean. The difference between two samples was considered statistically significant if P < 0.05 (*), if P < 0.01 (**), if P < 0.001 (***) and if P<0.0001 (****).

RESULTS

NAC Treatment Leads to Reduction of γ-H2AX Foci in Senescent Cells

NAC has been shown to be a scavenger of reactive oxygen species. We wanted to understand its role during senescence by using γ-H2AX foci as a marker. We induced senescence in HeLa cells by treating them with BrdU for 48 and 96 hours respectively. Following the treatment, the cells were treated with 10mM NAC for 24 hours and then fixed for immunofluorescence. On treatment with BrdU, significant increase in the number of γH2AX foci was observed, compared to non-senescent cells. In figure 1B, on comparing lane 1 with 2 and 3, it is evident that a significant amount of increase in γH2AX foci was due to the onset and maintenance of senescence. However, there was no significant difference in the number of foci per cell between 48 and 96 hours treated BrdU samples. Moreover, on treating the non-senescent cells with NAC, no difference was observed in the γ-H2AX foci (Fig 1B, Compare Lanes 1 and 4). However, a drastic decrease in the foci was evidenced in 48 hours and 96 hour senescent cells, on being treated with NAC. (Figure 1A-B; Compare lanes 2 and 3 with lanes 5 and 6 respectively).

Similarly, the study was performed on A549 lung carcinoma cells, which became our primary model to study DNA damage during senescence. Since it is already known that senescence may lead to lung fibrosis, a major concern in old age diseases, investigating the effect of H2S in lung epithelial cells would be a step towards understanding the role of H2S in lung fibrosis. We induced senescence in these cells by irradiating them with 7 Greys of ionizing radiation. Following which, the cells were incubated for 96 hours under cell culture conditions and then treated with 10mM of NAC for 4 hours. We observed an increase in the γ-H2AX foci in senescent cells (Figure 1C), on comparing them with non-senescent cells (Fig 1D; Lanes 1 and 3). On treating the cells with 10mM NAC, a small degree of damage was observed in non-senescent cells with a concomitant decrease in γ-H2AX foci in the senescent cells (Figure 1D; Compare lane 1 with 2 and lane 3 with 4). Interestingly, no reports have suggested an increase in γ-H2AX foci on treatment with NAC in non-senescent cells. Following these results, it became important to understand the effect of H2S, which has been shown to be a major by-product of NAC metabolism and shown to increase DNA damage in non-senescent cells, similar to NAC in the present system of study.

    A: NAC treatment leads to reduction in γ-H2AX foci in senescent cells. Senescence was induced in HeLa cells using 100μMBrdU for 48 and 96 hours followed by 24 hours of 10mM N-acetyl cysteine treatment.  Representative images of non-senescent HeLa cells and senescent cells (treated with BrdU) with nucleus stained with DAPI and γ-H2AX stained with anti TRITC antibody
      Figure 1B: Quantitative analysis showing reduction in γ-H2AX foci in senescent HeLa cells on treatment with NAC. Each point on the graph represents a single cell and the number of foci per cell have been averaged out in each sample.
        Figure 1C: NAC treatment leads to reduction of γ-H2AX foci in senescent A549 cells
        Representative images showing the effect of N-acetyl cysteine on non-senescent and senescent
        cells. The nucleus has been stained with DAPI and γ-H2AX protein with anti-TRITC antibody.
          Figure 1D: Quantitative analysis for treatment of non-senescent and senescent A549 cells with N-acetyl cysteine. Error bars were plotted using standard error of mean. The difference between two samples was considered statistically significant if P < 0.05 (*), if P < 0.01 (**), if P < 0.001 (***) and if P<0.0001 (****). (n>100, N=3)

          Treatment with Na2S Leads to DNA Damage in Non-Senescent A549 Cells

          Following our results, which showed an increase in DNA damage in non-senescent cells on treatment with NAC, we wanted to study the effect of a by-product of NAC metabolism, as previously described. It is well known that NAC follows a pathway which leads to synthesis of H2S. Therefore, we used a donor of H2S, namely Na2S to understand the effect of the gaso-transmitter on senescence and whether it has a similar effect on senescent cells, like N-acetyl cysteine. Senescence was induced in A549 cells as previously described (Section 3.1) and cells were treated with 200μm of Na2S for 4 hours after 96 hours of incubation post irradiation. On performing the analysis, we observed a significant increase in γ-H2AX foci in senescent cells, on comparing them to non-senescent cells (Figure 2B, lane 1 and 2) as has been already shown in Figure 1. Interestingly, on treatment with Na2S, we observed an increase in the γ-H2AX foci in non-senescent cells, while there was no change observed in the senescent cells. This result was in contrast to what had been observed with NAC, thus suggesting that H2S had been acting on the DNA through a mechanism different from NAC. While the current result does show that H2S is leading to increase in DNA damage in non-senescent cells, there is no significant effect of the gaso-transmitter on senescent cells. The former result of H2S affecting non-senescent cells is on the same lines as reported by previous studies investigating the effect of H2S on non-senescent cells (14, 15).

          External_Image_3.png
            Figure 2A: Na2S treatment causes DNA damage in non-senescent cells. Representative images for cells stained with DAPI and TRITC (γ-H2AX
            foci) to compare the levels of DNA damage between non-senescent and senescent cells on being treated with Na2S.  
               Figure 2B: Quantitative analysis for treatment of non-senescent (NS) and senescent cells(S) treated with Na2S. The difference between two samples was considered statistically significant if P < 0.05 (*), if P < 0.01 (**), if P < 0.001 (***) and if P<0.0001 (****). (n>100, N=3)

              Na2S Leads to Reduction of γ-H2AX Foci in CSE Knockdown System

              In the previous section, we observed a significant increase in γ-H2AX foci in non-senescent cells on treatment with H2S although there was no effect of the gaso-transmitter in the senescent cells. Since, increasing the levels of H2S did not cause any effect on the senescent cells, we wanted to investigate whether decreasing the levels had any observable phenotype. Thus, we downregulated one of the enzymes (the cell line with the downregulated gene was already available in the lab), namely, cystathionine synthase gamma (CSE), which is predominantly involved in the synthesis of H2S. On downregulating the expression of the enzyme (Section 2.2) we saw an increase in the γH2AX foci in non-senescent cells (Figure 3B, Lane 1 and 5), which was concurrently observed in cells treated with the H2S donor. However, on downregulating the enzyme in senescent cells, there was no observable change in the level of DNA damage (Figure 3B, Lane 2 and 6). Interestingly, on addition of H2S in non-senescent and senescent cells in which CSE was knocked down, we observed a significant decrease in the γH2AX foci in both the cell samples. (Figure 3B, Compare Lane 5 to lane 7 and lane 6 to lane 8). The result has been further discussed under Section 4.

                Figure 3A. Na2S leads to reduction of γ-H2AX foci in CSE knockdown system.  Representative images of non-senescent and senescent cells treated with Na2S in the background of CSE knockdown. The nucleus is stained with DAPI and γ-H2AX protein stained with anti-TRITC antibody.
                  Figure 3B. Quantitative analysis of non-senescent and senescent cells treated with Na2S in the background of CSE knockdown. NS (Non-senescent), S (Senescent), CSE (Cystathione γ lyase siRNA).  Lane 1: NS, Lane 2: NS+Na2S, Lane 3: NS+200μM Na2S, Lane 4: S+200μM Na2S, Lane 5: NS+CSE, Lane 6: S+CSE, Lane 7: NS+CSE+200μM Na2S, Lane 8: S+CSE+200μM Na2S. The difference between two samples was considered statistically significant if P < 0.05 (*), if P < 0.01 (**), if P < 0.001 (***) and if P<0.0001 (****). (n>100, N=3)

                  Na2S Treatment Does Not Cause Any Significant Effect on Senescent Cells

                  On observing an increase in γ-H2AX foci, on both reduction and addition of H2S in the biological system, it became important to understand what was the optimum level of H2S required in the cells. We performed a dose related study for the same by treating the cells with 50, 100, 200 and 400μM of Na2S. On treating the cells with 50μM of Na2S, a significant increase in DNA damage was observed while there was no difference in the levels of in the γH2AX foci, between 50 and 100μM Na2S treated samples. However, on doubling the dosage from 100 to 200 and further to 400μM we observed a substantial increase in the γ-H2AX foci on comparing it to the untreated non-senescent cells (Figure 4B, Compare Lanes 1 to 2,3,4 and 5). Interestingly, in senescent cells, 50μM of Na2S did not have any effect on the γ-H2AX foci while increasing the levels two fold, led to a slight decrease in DNA damage in comparison to 50μM Na2S treated samples. As previously observed in Figure 2 and 3, there was no difference when senescent cells were treated with 200μM of Na2S in comparison to untreated senescent cells. The damage did seem to increase slightly when the dose was increased from 100μM to 400μM however there was no significant difference observed in the levels of senescent and Na2S treated senescent cells. These results suggest that there is an intricate mechanism involved in regulating the levels of H2S inside the cell and it might be one of the major reasons for the fluctuations observed in DNA damage during senescence.

                    Figure 4. Quantitative analysis for treatment of non-senescent and senescent cells with Na2S. NS (Non-senescent), S (Senescent). Lane 1: NS, Lane 2-5NS+Na2S (50,100,200,400μM respectively). Lane 6: S, Lane 7-10: S+ Na2S (50,100,200,400μM respectively). The difference between two samples was considered statistically significant if P < 0.05 (*), if P < 0.01 (**), if P < 0.001 (***) and if P<0.0001 (****).(n>100, N=3)

                    DISCUSSION

                    The current study was undertaken to investigate the effect of N-acetyl cysteine and H2S on DNA damage during senescence. The cells were treated with BrdU or Ionizing radiation to induce senescence in mammalian cells and the effect of the compounds on these cells was studied using γH2AX foci as a marker of senescence. Our results provide evidence that NAC reduces the levels of DNA damage in the senescent cells. However, varying degrees of damage is also observed in non-senescent cells in the A549 cell line (Figure 1C-D). This result was in contrast to what was observed in the HeLa cell line and thus we went ahead to understand whether H2S has a similar effect on senescent cells in A549 cells.

                    On treating cells with Na2S, an increase in the γH2AX foci was observed in non-senescent cells, which was in line with previous studies. Interestingly though, Na2S did not cause any observable effect on the senescent cells. One of the possible explanations could be that senescent cells already possess a high level of intracellular H2S (as found in the lab) in comparison to non-senescent cells and therefore on treating with H2S no effect is observed in senescent cells. However, H2S treatment leads to increase in γH2AX foci although the levels of H2S are low in non-senescent cells. To better understand this result, the gene for cystathione gamma lyase was knocked down (obtained from lab) which is one of the enzymes responsible for H2S synthesis in the cell. By knocking down the gene in non-senescent cells, we observed an increase in the levels of DNA damage which was comparable to that observed when cells were treated with H2S. Thus, it means that the levels of intracellular H2S are tightly regulated in the system and any perturbation in the levels of the gaso-transmitter would lead to induction of DNA damage. However, what was quite interesting to note was that on downregulation of the enzyme in senescent cells, there was no effect observed in the γH2AX foci, which was in coherence with the result that showcased no effect of H2S on senescent cells. It is possible that other enzymes, namely Cystathione β synthase or 3-mercaptopyruvate sulfurtransferase might be involved in maintaining the levels of H2S in the absence of CSE. It is also possible that the levels of H2S are already in the saturating range in the cells that downregulation of the enzyme might not be affecting the cell. Interestingly, when these knocked down cells were treated with H2S, a drastic decrease in the γH2AX foci was observed in both non-senescent and senescent cells. These results require further investigation as it is important to study the status of CBS and MST in these cells. It is possible that these enzymes might be maintaining the levels of H2S in these cells when CSE is knocked down or when H2S is provided in the system. Another plausible explanation is that H2S production might be tightly regulated through a feedback loop which needs to be uncovered.

                    Since we observed a wide range of effects of H2S in the cells, in the presence and absence of CSE, we wanted to understand whether H2S has a threshold value wherein it can cause an observable effect on the cells at the level of the DNA damage response. Therefore, we treated the cells with a range of Na2S, from 50μM to 400μM. Interestingly, while a gradual increase was observed in the γH2AX foci with increasing amounts of Na2S in non-senescent cells, there was no significant effect observed in the senescent cells. These results provide a strong evidence that while the levels of H2S are strictly regulated in the cells, any dysregulation in the non-senescent cells will cause a direct effect on the levels of γH2AX foci.

                    In conclusion, we believe that while NAC is a potential compound for decreasing the level of DNA damage in senescent cells, further analysis needs to be done using H2S since more than one pathway is involved in regulating the levels of H2S in the system. Therefore, it is important that the action of H2S be studied with respect to senescent cells and further investigations be made at the molecular level with respect to regulation of the production of H2S intracellularly.

                    ACKNOWLEDGEMENTS

                    I would firstly like to thank the Indian Academy of Sciences for giving me the opportunity to work in one of the best institutes of the country. It was truly a learning experience which I shall carry forward in my career. I express my gratitude to the man who made all this possible: Mr C S Ravikumar. His sincerity towards the students made for another valuable lesson.

                    It was a pleasure working with Kavya Gupta who not only pushed the boundaries further in order to make me realize how much I was truly capable of but also instilled in me the confidence to get things done. I would also like to thank Suramya Asthana. She has been instrumental in making me understand the project at hand and guiding me during the period of my stay. I wish her the best in all her future endeavours. This note would be incomplete without me acknowledging the other members of the lab (Srivatsa, Abraham and Rahul) who not only treated me as an equal but also provided me with an environment where I could enjoy the science and the discussions that happened along with it.

                    Keeping the best for the last, I was truly privileged to have been given the opportunity to work in Saini Lab. Prof Deepak Saini has had a lasting impression on me because of his positive attitude towards all his students. To have known him and worked under his supervision at the very start of my research career makes me truly inspired to have a career in academia. It would surely be a privilege to work with him in the future.

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