N-Acetyl Cysteine Restores the Diminished Activity of the Antioxidant Enzymatic System Caused by SARS-CoV-2 Infection: Preliminary Findings

SARS-CoV-2 infects type II pneumocytes and disrupts redox homeostasis by overproducing reactive oxygen species (ROS). N-acetyl cysteine (NAC) is a precursor of the synthesis of glutathione (GSH) and it restores the loss of redox homeostasis associated to viral infections. The aim of the study is to evaluate the effect of the treatment with NAC on the enzymatic antioxidant system in serum from patients infected by SARS-CoV-2. We evaluated the enzymatic activities of thioredoxin reductase (TrxR), glutathione peroxidase (GPx), -S-transferase (GST), and reductase (GR) by spectrophotometry and the concentrations of the glutathione (GSH), total antioxidant capacity (TAC), thiols, nitrites (NO2–), and lipid peroxidation (LPO) in serum. The activity of the extracellular super oxide dismutase (ecSOD) was determined by native polyacrylamide gels, and 3-nitrotyrosine (3-NT) was measured by ELISA. A decrease in the activities of the ecSOD, TrxR, GPx, GST GR, (p = 0 ≤ 0.1), and the GSH, TAC, thiols, and NO2– (p ≤ 0.001) concentrations and an increase in LPO and 3-NT (p = 0.001) concentrations were found in COVID-19 patients vs. healthy subjects. The treatment with NAC as an adjuvant therapy may contribute to a reduction in the OS associated to the infection by SARS-CoV-2 through the generation of GSH. GSH promotes the metabolic pathways that depend on it, thus contributing to an increase in TAC and to restore redox homeostasis.


Introduction
According to the WHO Coronavirus (COVID-19) Dashboard, there have been 761,402,282 confirmed cases of the disease worldwide until April 2023, and 6,887,000 deaths by the new type 2 coronavirus (SARS-CoV-2), that causes severe acute respiratory syndrome. Moreover, 13,317,121,247 vaccine doses have been applied worldwide. SARS-CoV-2 type 2 infects the pneumocytes in the alveoli of the lungs, by the attaching its S protein with the angiotensin-converting enzyme 2 (ACE2) receptor and the serine protease TMPRSS2, facilitating the fusion of the viral membrane with the pneumocyte cellular membrane [1]. The structural and non-structural (nsp) proteins of SARS-CoV-2 possess the genetic information needed to evade the immune response strategies of the host and to sequester the cellular machinery to favor replication [2]. After evading the immune response, the virus enters the cell by endocytosis and progressively triggers a cytokine storm that results in systemic hyper inflammation caused acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD). These conditions are associated with the SARS-CoV-2 infection. In this sense, NAC can exogenously increase H 2 S by a synergistic effect with GSH and protect from the damage in the lungs of the patients infected by SARS-CoV-2 [15]. Therefore, the aim of this study is to evaluate the effect of treatment with NAC before and after on some enzymes of the enzymatic antioxidant system in patients infected by SARS-CoV-2.

Demographic Characteristics of the Healthy Subjects
A total of 20 healthy subjects (HS) were included in this study, of which 10 (50%) were men and 10 (50%) were women. HS had a median age of 53 min 30 and max 63 ranges. The values of glucose, insulin, cholesterol, triglycerides, HDL, LDL, and creatinine are shown in Table 2. In these subjects, there was no suspicion of degenerative disorders, inflammatory disease, and/or autoimmune diseases. Renal failure, hypertension, cardiovascular disease, diabetes mellitus, and dyslipidemia were not present. Table 2 shows the biochemical characteristics of the HS. The results are shown median and min and max range.

Oxidative Stress Markers
TAC showed a significant decrease in the patients infected by SARS-CoV-2 before the NAC treatment in comparison with HS and after the treatment (p < 0.001). The GSH levels showed the same tendency as TAC with a significant decrease in the before the NAC treatment patients (p < 0.001) versus HS and after the NAC treatment. The total thiol groups was decreased in the patients infected by SARS-CoV-2 before the NAC treatment vs. HS (p < 0.001) but the treatment with NAC increased them, showing statistically significant changes (p ≤ 0.05). However, the median of LPO index showed a significant increase in the patients infected by SARS-CoV-2 before the NAC treatment (p = 0.01 and p ≤ 0.05) in comparison with HS and in the patients after the treatment. The NO 2 levels in the patients before the NAC treatment showed a significant decrease (p < 0.001) in comparison to HS and after the NAC treatment. Finally, the 3-NT concentration was significantly increased in the patients infected by SARS-CoV-2 before the NAC treatment with respect to HS (p < 0.001); however, the NAC treatment caused a significant decrease in these patients (p ≤ 0.05). The OS markers are shown in Table 3.    Figure 2C). However, the GR activity only showed a tendency to increase but without showing a significant change, Figure 2D. The activity of ALDH was increased in the patients infected by SARS-CoV-2 before the NAC treatment (p = 0.02) in comparison to HS, and the treatment with NAC did not modify it, despite a tendency to increase its activity, Figure 1B. before the NAC treatment (p = 0.02) in comparison to HS, and the treatment with NAC did not modify it, despite a tendency to increase its activity, Figure 1B.   before the NAC treatment (p = 0.02) in comparison to HS, and the treatment with NA did not modify it, despite a tendency to increase its activity, Figure 1B.

Discussion
NAC is a stable form of the non-essential amino acid cysteine, being a precursor of the GSH tripeptide-thiol. It increases the activities of redox enzymes that employ it including TrxR, GPx, GST, glutaredoxin, and GR. NAC also favors the activity of the enzyme GCL, which participates in the GSH synthesis [16]. It contains sulfur and acts as a stabilizer for the formation of protein structures through reduction of thiol bridges. It has mucolytic properties by breaking disulfide bonds in mucous secretions rendering them less viscous and it is currently being used in septic shock and EPOC [17]. NAC is also less expensive for the population; it has very low toxicity; and has been approved by the FDA. Diverse studies have shown that the administration of NAC by different routes (orally, inhaled or intravenously) can contribute to suppressing SARS-CoV-2 replication, decrease the infection, and strengthen the immune and antioxidant systems [18]. Potential therapeutic benefits of NAC include extracellular scavenging of free radicals, replenishing and increasing GSH, suppression of the cytokine storm, and protection of T cell, thus mitigating inflammation and tissue injury. The combination of NAC with administrations of antiviral agents could reduce the hospital admission rate, mechanical ventilation, and mortality [18].
In this sense, the aim of this study is to evaluate the effect of NAC treatment before and after on the activities of some enzymes of the enzymatic antioxidant system in patients infected by SARS-CoV-2.
Our results show that the activity of ecSOD is decreased in the patients infected by SARS-CoV-2. This isoform of the enzyme participates in the first line of detoxification against the O 2 in the blood reducing it to H 2 O 2 . It is expressed in the sub endothelial space, in the surface of the vascular smooth muscle cells in blood vessels, and may be secreted into the extracellular space, hence found in the blood. It has a direct action on ROS, thus limiting oxidative lung injury. Our results suggest that the activity of ecSOD may be compromised in the patients infected by SARS-CoV-2 and therefore this enzyme is incapable of dismutating two O 2 to H 2 O 2 , resulting in an increase in OS, as has been previously reported [19].
The patients infected by SARS-CoV-2 also show a deficiency in metalloids such as zinc, selenium, copper, and manganese among others. This deficiency can probably decrease the activity of some antioxidant metallo-enzymes including the SOD isoforms and selenoenzymes such as TrxR and GPx that contribute to an increased OS [15]. The ecSOD activity may also be decreased because of the excess of substrate, despite its great replacement capacity to dismutate O 2 - [20]. However, the NAC treatment increases the activity of ecSOD. This could be due to an indirect effect resulting from an increase in the GSH concentration, which lowers ROS, being an antioxidant molecule. In this same sense, the decrease in the activity of TrxR and GPx observed in the patients infected by SARS-CoV-2 can contribute to OS by abolishing detoxification of other ROS such as H 2 O 2. The levels of ROS are increased during hyper inflammation by SARS-CoV-2, due to the activity of the innate immune system to fight viral infection through the peroxidases family [21]. H 2 O 2 is the substrate for the isoforms of GPx and TrxR. However, the high concentration of this ROS may, in turn, lower the activities of GPx and TrxR. Therefore, our results show that a decrease in the activities of these enzymes could be due to an excessive increase in H 2 O 2 in the SARS-CoV-2 infection [21,22].
We also found that GSH was depleted in the patients infected by SARS-CoV-2 in this study, as has been previously reported [15]. In addition, the protease M pro , a cysteine protease of the SARS-CoV-2, which is encoded by the nsp5 virus, can also interact with GPx1 and with other seleno-enzymes such as TxrR1 and favor the abolition of their activities increasing OS. Other nsp proteins such as the 12 and 13 can interact with GCL and decrease the synthesis of GSH [22]. Furthermore, an increase in the TrxR activity could block the entrance of the virus via a reduction of the ACE2 receptor through decreased thioredoxin and other thiols groups [23]. In this sense, the ACE2 receptor of the host has three disulphide bonds through conserved cysteines that are required in an oxidative environment. The presence of these bonds renders thiols rusty and allows for the infiltration of the SARS-CoV-2 virus into the cells. However, disulfide bridges are mainly reduced by the thioredoxin system, and the decrease in the TrxR activity diminishes the thiol groups as shown in our results, thus favoring the entrance of the virus [23].
The antioxidant therapy with the NAC increases the activities of GPx, TrxR, and thiol groups and this could contribute to a decrease in OS and the severity of the disease by interfering with the entrance of the virus into the host cells. TrxR possesses a seleno-cysteine in its catalytic site and NAC may supply this amino acid. The virus may also decrease TrxR and the glutaredoxin systems, which could be a viral strategy to inhibit DNA synthesis. A reduction in the synthesis of DNA may increase the pool of ribonucleotides for RNA synthesis, thereby enhancing virion production, a process which is unsustainable without the participation of the TrxR and glutaredoxin systems [23,24].
On the other hand, few studies have paid attention to GR in SARS-CoV-2 infection. This enzyme is essential for the GSH regeneration. A study in 104 subjects showed that the activity of GR was decreased in the SARS-CoV-2 infection [25]. Our results show a decrease in the activity of this enzyme and, although there was a tendency of the activity of GR to increase due to treatment with NAC, it did not show a statistical difference. This suggests that the SARS-CoV-2 infection decreases the activity of this enzyme and may lead to an increase in GSSG and a decrease in GSH, which would contribute to the positive feedback process between the inflammatory process and OS. The reduction in the activity of GR could lead to neutrophil dysfunction during the infectious processes as has been previously reported in GR-deficient mice and humans [26]. The decreased activity of this enzyme in neutrophils mediates S-glutathionylation of the key protein NOX in vitro and in vivo [27]. The S-glutathionylation process regulates the GHS conjugation with a cysteine thiol group on target proteins and this may modulate their function. The GR activity is also essential to promote the innate immune response against microorganism [28]. Therefore, the treatment with NAC could increase the activity this enzyme and favor GSH regeneration, thus contributing to decrease OS.
In the same sense, the other enzymes that participate in the S-glutathionylation process showed a decrease in their activity in this study, including GST. GST is a superfamily of isoenzymes that includes the α, µ, π, ω, θ, δ, ς, σ, κ, and m isoforms. These enzymes are multifunctional and are found in plasma, cell membranes, cytoplasm, endoplasmic reticulum, mitochondria, and the nucleus [29]. They participate as protective enzymes for exogenous and endogenous cellular detoxification and neutralization of the xenobiotics, oxidized lipids, ROS, and they decrease the rate of LPO through GSH conjugation. This conjugation increases the solubility of the toxic products, facilitating their excretion from the cell. They also play an important role in idiopathic pulmonary fibrosis and lung fibro genesis [29]. Several studies have reported that SARS-CoV-2 decrease the activity of these enzymes [30]. A study in patients with COVID-19 ranging in age from 20 to 58 years showed a decrease in the GSTp1 and there was a correlation with a higher prevalence of deaths in males in comparison with female patients [31]. Another study showed that the frequency of GSTM1−/−, GSTT1−/−, and GSTM1−/−/GSTT1−/− was higher in severe COVID-19. In patients with both GSTM1 + /+ and GSTT1−/− genotypes there was a poor survival rate. Therefore, patients with COVID-19 having the GSTT1−/− genotype had a high mortality rate [32]. However, the treatment with NAC favored an increase in this enzyme in the present study. This effect may be attributed to the increase in the GSH concentration by the treatment because it is a substrate for this enzyme, resulting in a decreased LPO. Nevertheless, the decrease in the activity of this enzyme could favor the accumulation of the LPO products such as 4-hydroxy-2-transnonenal (4-HNE) or malondialdehyde. LPO is the result of the chain reaction of OHon the unsaturated fatty acid and it is increased in the presence of transition metals such as Fe. The COVID-19 patients develop ferroptosis due to hijacking of the function of mitochondria and the knockdown of the GPx 4 isoform by the virus [33]. Furthermore, the peroxidized lipids damage the cell membrane.
However, the GST isoforms are not the only enzymes involved in reducing the formation of reactive lipid aldehydes. Other enzymes such as the ALDH superfamily are also employed for detoxification [34]. These enzymes are expressed in most human tissues, including immune cells, and are crucial for the metabolism of endogenous aldehydes, such as formaldehyde and 4-HNE which are responsible for the elimination of non-reactive products. In this sense, our results show that the activity of ALDH was increased. The role of this enzyme in SARS-CoV-2 has not been reported and few works have addressed its participation in other viral infections. However, there is an increase in this enzyme in B and C hepatitis infection [35]. The ALDH increase in SARS-CoV-2 infection could be a compensatory mechanism of protection that tries to detoxify the excess of OS associated with 4-HNE. It could also be the result of the expression of interferon to fight infections [36]. The increase would also compensate for the GST decrease.
On the other hand, a decrease in the TAC reflects the alteration of the antioxidant enzymes, GSH, thiols and an increase in the LPO, NO 2 -, and 3-NT. However, the treatment with NAC may restore this alteration and, as a result, the increase in the TAC and redox homeostasis tend to balance themselves [37]. This could contribute to enhance the host's innate immune system.
Moreover, the loss of the redox homeostasis is associated with the expression of the inducible nitric oxide synthase (iNOS) and nitric oxide (NO) exacerbation that could lead to the formation of peroxynitrites (ONOO -). These ROS may affect enzymatic and protein functions by modifying tyrosine residues and/or thiol groups, leading to the formation of oxidized thiol groups and 3-NT. [38]. The increased formation of O 2 due to the loss of the activity of ecSOD may react with NO and decrease its concentration; however, it would increase ONOOwhich has a longer half-life than O 2 and is more toxic, resulting in NSS [39]. This contributes to the inflammatory process associated with the interleukin storm. In addition, the NO 2 is diminished, but the 3-NT is increased in COVID-19 [15]. Our results agree with what was previously described. In addition, decreased NO levels may induce elevated circulating pro-inflammatory cytokines, platelet aggregation, chemokine expression, proliferation of vascular smooth muscle cell, oxidation of LDL lipoprotein, expression of vascular cell adhesion molecule-1, stimulation of thrombolysis by monocyte chemotactic protein-1. These alterations contribute to damage the lungs. Although the treatment with NAC reverses these alterations, the exact mechanism by which this effect is produced is still unknown and further studies are required for elucidation.
Perspectives: When SARS-CoV-2 attaches to the ACE2 receptor, it leads to an increase in angiotensin 2 and a decrease in angiotensin 1-7 [40]. This change increases O 2 − and a positive feedback process between the OS and the inflammatory process that, in turn, causes endothelial dysfunction, favoring the increase in von Willebrand factor and causing thrombosis [41]. The NAC treatment that favors the GSH synthesis could reduce the von Willebrand factor that forms blood clots [42]. Blood clotting issues in patients infected by SARS-CoV-2 are currently being evaluated and the potential effectiveness of taking GSH alone orally and IV, rather than its precursor NAC is being tested. The alpha-lipoic acid may also represent another novel treatment approach to block NF-κB and treat the cytokine storm and respiratory distress in patients with pneumonia caused by infection with SARS-CoV-2. Therefore, NAC, GSH, and micro minerals can be an effective complement in the treatment against SARS-CoV-2 infection due to their fundamental role in reducing OS, reducing the inflammatory state, and restoring the homeostasis of blood coagulation [43,44].
This study also confirms that the application of antioxidant therapies such as NAC treatment could help in the prevention and mitigation of the loss of redox homeostasis in the patients infected by SARS-CoV-2, and thereby have a beneficial impact by decreasing the infection and improving patient survival. These relevant findings suggest the need of conducting multicentric studies or systematic studies providing therapies with antioxidants that may improve the loss of redox homeostasis. Figure 3 summarizes the NAC treatment in the patients infected by SARS-CoV-2.
conducting multicentric studies or systematic studies providing therapies with antioxidants that may improve the loss of redox homeostasis. Figure 3 summarizes the NAC treatment in the patients infected by SARS-CoV-2.

Study Limitations
One of the limitations of this study was the sample size. The time in which the antioxidant therapy with the NAC was given, could have also been longer, but due to the lack of budget, this was not possible. However, the results obtained in this study are promising. Therefore, we suggest that the treatment with NAC should be given for the double of the time used in this study. Another limitation is the absence of a control group matched by the presence of comorbidities such as obesity, diabetes, and hypertension, which may affect the results and the conclusion.

Description of the Population Studied
This was an open, prospective, analytical, and longitudinal study (before-after treatment) in 16 patients with COVID-19 who received NAC and the results were compared with those obtained in 20 healthy subjects that acted as a control group. Inclusion criteria: The patients with COVID-19 were 18 years old or more and were admitted to the ICU of the CITIBANAMEX Center and developed or not septic shock, secondary to moderate or severe pneumonia by COVID-19. The treatment was applied between August and September of 2020. Ethical approval was obtained by the local ethics committee on 19th August 2020 (Control-9867/2020, register REG. CONBIOETICA-09-CEI-011-20160627). The protocol was registered (TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT 04570254). Diagnostic criteria for septic shock were based in the Sepsis-3 consensus [45]. A written informed consent for enrollment or consent to use patient data was obtained

Study Limitations
One of the limitations of this study was the sample size. The time in which the antioxidant therapy with the NAC was given, could have also been longer, but due to the lack of budget, this was not possible. However, the results obtained in this study are promising. Therefore, we suggest that the treatment with NAC should be given for the double of the time used in this study. Another limitation is the absence of a control group matched by the presence of comorbidities such as obesity, diabetes, and hypertension, which may affect the results and the conclusion.

Description of the Population Studied
This was an open, prospective, analytical, and longitudinal study (before-after treatment) in 16 patients with COVID-19 who received NAC and the results were compared with those obtained in 20 healthy subjects that acted as a control group. Inclusion criteria: The patients with COVID-19 were 18 years old or more and were admitted to the ICU of the CITIBANAMEX Center and developed or not septic shock, secondary to moderate or severe pneumonia by COVID-19. The treatment was applied between August and September of 2020. Ethical approval was obtained by the local ethics committee on 19th August 2020 (Control-9867/2020, register REG. CONBIOETICA-09-CEI-011-20160627). The protocol was registered (TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT 04570254). Diagnostic criteria for septic shock were based in the Sepsis-3 consensus [45]. A written informed consent for enrollment or consent to use patient data was obtained from each patient or their legal surrogate, in accordance with the Helsinki declaration, as amended by the congress of Tokyo, Japan [46]. Patients considered to have septic shock had to have an acute increase of at least 2 points in the SOFA score [43], lactate levels ≥ 2 mmol/L, and they had to be dependent on a vasopressor for at least 2 h before the time of enrollment. To evaluate organ dysfunction, the SOFA score (neurologic, respiratory, hemodynamic, hepatic, and hematologic) was calculated at admission and during all of the days of treatment [47]. The hospitalized patients were classified as moderate or severe; this classification was decided according to their ventilatory status. Patients with the severe condition required invasive mechanical intubation according to the Berlin criteria for ARDS [48]. Exclusion criteria were: patients that were under chronic use (last 6 months) or recent use of steroids, statins, antioxidants, and NAC; patients younger than 18 years; patients that were not able to grant an informed consent, refused to be included; and also pregnant woman or breastfeeding. Figure 4 describes the study flow diagram.
from each patient or their legal surrogate, in accordance with the Helsinki declaration, as amended by the congress of Tokyo, Japan [46]. Patients considered to have septic shock had to have an acute increase of at least 2 points in the SOFA score [43], lactate levels ≥2 mmol/L, and they had to be dependent on a vasopressor for at least 2 h before the time of enrollment. To evaluate organ dysfunction, the SOFA score (neurologic, respiratory, hemodynamic, hepatic, and hematologic) was calculated at admission and during all of the days of treatment [47]. The hospitalized patients were classified as moderate or severe; this classification was decided according to their ventilatory status. Patients with the severe condition required invasive mechanical intubation according to the Berlin criteria for ARDS [48]. Exclusion criteria were: patients that were under chronic use (last 6 months) or recent use of steroids, statins, antioxidants, and NAC; patients younger than 18 years; patients that were not able to grant an informed consent, refused to be included; and also pregnant woman or breastfeeding. Figure 4 describes the study flow diagram. In all patients, the use of hydroxychloroquine or antivirals was not considered since they received individualized management according to a proposed algorithm [3]. At the time this study was carried out, the patients were not vaccinated against SARS-CoV-2 because a vaccine had not yet been developed. Some results related to this study were previously reported by Chavarría et al. during the before treatment and after treatment evaluation with NAC [49].

Detection of SARS-CoV-2 by qRT-PCR Technique
Paired nasopharyngeal and saliva swab samples were collected from 16 patients infected by SARS-CoV-2. Samples were classified as positive for SARS-CoV-2 when both the N1 and N2 protein primer-probe sets were detected. For the presence of the SARS-CoV-2 virus, specific probes for the detection of the virus in conjunction with the real-time reverse transcriptase polymerase chain reaction technique (qRT-PCR) were utilized.  In all patients, the use of hydroxychloroquine or antivirals was not considered since they received individualized management according to a proposed algorithm [3]. At the time this study was carried out, the patients were not vaccinated against SARS-CoV-2 because a vaccine had not yet been developed. Some results related to this study were previously reported by Chavarría et al. during the before treatment and after treatment evaluation with NAC [49].

Detection of SARS-CoV-2 by qRT-PCR Technique
Paired nasopharyngeal and saliva swab samples were collected from 16 patients infected by SARS-CoV-2. Samples were classified as positive for SARS-CoV-2 when both the N1 and N2 protein primer-probe sets were detected. For the presence of the SARS-CoV-2 virus, specific probes for the detection of the virus in conjunction with the real-time reverse transcriptase polymerase chain reaction technique (qRT-PCR) were utilized.

Healthy Subject (Control Group)
Twenty healthy subjects (HS) were matched by age and gender. HS were negative for SARS-CoV-2. The collection of peripheral blood samples was carried out by venopunction. In these subjects, there was no suspicion of inflammatory disease or presence of degenerative disorders such as thyroid and autoimmune diseases, diabetes mellitus, dyslipidemia, and arterial hypertension. The intake of some medications that could interfere with the results of the study as antioxidants drugs and NSAIDs was considered, and the drugs were suspended 48 h before the obtainment of the sample. Control subjects reported not having any disease. Biochemical variables were also determined in these subjects such as glucose, insulin, cholesterol, triglycerides, HDL, LDL, and creatinine levels, to confirm that they really did not have alterations that could modify the results.

Therapeutic Management
The treatment applied during hospitalization was chosen according to standard maneuvers considering the requirements of each individual patient, the hemodynamic and electrolyte demands, and the ventilator demands. Treatment was initiated during the first hour after admission and before the recognition of the presence or absence of septic shock. Cultures were performed on admission before starting the administration of a broad-spectrum intravenous antibiotic.
Management with crystalloid solutions and/or albumin was considered depending on the hemodynamic status, by means of dynamic indicators. Use of vasopressors to maintain a media arterial pressure (MAP) ≥65 mmHg was given if necessary. Norepinephrine (NE) was the first option and/or vasopressin was used when there was a need to increase the MAP or reduce the NE dose. Inotropic drugs were administered in cases of myocardial dysfunction (dobutamine). Transfusion of blood packs were applied in case of a decrease in hemoglobin (<7.0 g/dL) in the absence of myocardial ischemia, severe hypoxemia, or severe bleeding. Mechanical ventilation with tidal volumes of 6 mL/kg was used in ARDS patients [50]. Plateau pressure was maintained at ≤30 cm H 2 O, and alveolar conduction pressure of ≤13 cm H 2 O. Positive end expiration pressure titration was managed by the use of the FiO 2 /PEEP (fraction inspired of oxygen/positive end expiration pressure). The treatment with anticoagulants was based on the Thachil guidelines [51]. The management with the prone position was necessary in patients with PaO 2 /FiO 2 of ≤150 [52]. In all patients, the standard therapeutic management with dexamethasone 8 mg i.v., every 24 h for 7 days was applied between day 1 and 21 of the onset of symptoms when not contraindicated, and pentoxifylline tablets of 400 mg every 12 h by oral route or nasal-enteral tube for 5 days [53]. It was counter indicated when there was a requirement of O 2 > 3 L, progressive requirement of =2, PAO 2 /FiO 2 ≤250 mmHg, O 2 use plus bilateral infiltrates in the radiography, O 2 use plus DHL ≤250 U/L or ferritin ≥300 or DD ≥1000 ng/mL, CPK ≥2 times the upper normal value. The following conditions were not considered as counter indications or relative counter indications: glucose >250 mg/dL with hypoglycemic, hypokalemia <3.3 meq, blood pressure >155/95 mmHg with antihypertensive treatment, glaucoma, triglycerides >500 mg/dL (start treatment), history of known peptic ulcer or bleeding from recent gastrointestinal tract, untreated or decompensated dementia or psychiatric illness, use of non-potassium sparing diuretics or use of inhaled B2 agonists. The next conditions were monitored at follow-up: pre-prandial capillary glucometer (7-13-19 hrs.) for 10 days, even in fasting patients, MAP per shift and basal potassium every 72 h.

Doses of the NAC Therapy
Effervescent tablets of NAC 600 mg were used every 12 h by the oral route or nasalenteral tube for 5 days. To administer the NAC therapy, a medical management algorithm was used considering the presence of comorbidities in each patient [3]. The NAC was adjusted to each comorbid condition or to the presence of potential allergies or heart rhythm disorders due to each individual history. All data entry was monitored at the coordinating center, with site visits for source data verification.

Blood Sample Obtainment and Storage
Blood samples were obtained from each patient that entered the hospital. The blood samples of both COVID-19 patients and HS were centrifuged for 20 min at 936 g and 4 • C. The serum of the samples was placed in aliquots and stored at −30 • C. Laboratory tests were carried out for the COVID-19 patients to determine the acute-phase reactants, urea nitrogen, creatinine, glucose, hemoglobin, leukocytes, lymphocytes, platelets, albumin, Ddimer, fibrinogen, ferritin, C-reactive protein, procalcitonin, and interleukin-6 (IL-6). Data from the patient's medical history including demographic, prior illnesses to SARS-CoV-2 infection, COVID-19 test result, whether mechanical ventilation was used, and treatment type given were used for the results analysis. Additionally, other biochemical variables were determined in serum from the patients infected by SARS-CoV-2 and HS such as OS markers and antioxidant enzymatic system.

Oxidative Stress Markers
Evaluation of Total Antioxidant Capacity (TAC) In total, 100 µL of serum was suspended in 1.5 mL of a reaction mixture prepared as follows: 300 mM acetate buffer pH 3.6, 20 mM FeCl 3 6 H 2 O, and 10 mM of 2,4,6-tris-2pyridyl-s-triazine were dissolved in 40 mM HCl. These reactants were added in a ratio of 10:1:1 v/v, respectively. After mixing, the samples were incubated at 37 • C for 15 min in the dark. The absorbance was measured at 593 nm [15].

Glutathione Levels (GSH)
This test determines the oxidation of GSH by means of Ellman's reagent to form a yellow chromophore called 5 -thio-2-nitrobenzoic (TNB) and is quantified by a spectrophotometer. The rate of formation of TNB is proportional to the concentration of GSH in the sample. About 100 µL of serum was used according to a previously described method by Ellman. The calibration curve was obtained with solution GSH 1 mg/1 mL and the absorbance was measured at 412 nm [15].

Determination of Total Thiol Groups
This test consists of the Ellman's reagent reaction with a thiol group, commonly a thiolate, producing thoil-nitrobenzoate through potassium hydride. The technique used was previously described by Erel and Neselioglu [53], with some modifications carried out in our laboratory as previously reported [15]. About 50 µL of serum was used for the determination and the absorbance was measured at 415 nm. The calibration curve was obtained with solution GSSG 1 mg/1 mL and the absorbance was measured at 415 nm.

Determination of Lipid Peroxidation (LPO) Levels, Aldehyde Deshydrogenase Activity (ALDH) and 3-Nitrotyrosine (3-NT)
This test is based on the malondialdehyde reaction, a secondary product of the oxidation of fatty acids with three or more bonds, with thiobarbituric acid in an acid medium and at high temperature, generating a pink-colored product. A total of 100 µL of serum was used for this determination and the absorbance was measured at 532 nm [52]. The activity of ALDH was determined through the colorimetric assay kit provided by Sigma-Aldrich (MAK082-1 KT), and 50 µL of serum was used for the determination. The 3-nitrotyrosine (3-NT) determination was carried out with a kit provided by LifeSpan BioSciencies, Seattle, WA, USA kit No. LS-40120). This assay is based on the competitive ELISA principle and is measured at a wavelength of 450 nm, using a visible light micro plate reader (Stat Fax 3200 Awareness Technology, Palm City, FL, USA), and 25 µL of serum was used for this determination.

Nitrites (NO 2 -)
The NO 2 levels in serum were determined by the Griess reaction and a total of 100 µL of serum was used. NO 2 − reacts in acid media with sulfanilic acid to form a diazonium cation which is then coupled to the aromatic amine 1-naphthylamine to produce a red-violet coloration that can be read on the spectrophotometer at 540 nm.

Determinations of TrxR, GPx, GST and GR Activities
The TrxR activity was determined using 50 µL of serum according to the method described by Pérez Torres et al. [54]. The sample was incubated and monitored at 412 nm for 6 min at 37 • C. To evaluate the GPx, GST, and GR activities, 50 µL of serum was used as was previously described [54]. The samples were incubated and monitored at 340 nm for 6 min at 37 • C.
The activity of extracellular super oxide dismutase (EcSOD) was determined in serum through non-denaturing gel electrophoresis according to the method previously report by Rodríguez-Fierros et al. [55]. A total of 25 µL of serum was applied directly in nondenaturing 10% polyacrylamide gels. The electrophoresis was carried out at 120 volts for 4 h. Subsequently, the gel was incubated with 2.45 mM nitro blue tetrazolium (NBT) solution for 20 min, then incubated with 28 mM EDTA solution, containing 36 mM potassium phosphate (pH 7.8) and 0.028 mM riboflavin, and exposed 10 min by UV light. Purified SOD from bovine erythrocytes with a specific activity of 112 U/mg of protein (Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control for calculating the activity of the enzyme.

Interleukin-6 Concentration
IL-6 levels were measured in plasma samples by an enzyme-linked immunosorbent assay (ELISA) using a commercial kit according to the manufacturer's instructions (BioLegend, San Diego, CA, USA), and 50 µL of serum was used for this determination.

Statistical Analysis
Continuous variables were expressed as median, first quartile, second quartile, interquartile range with minimum and maximum range. Categorical variables were expressed as frequencies and percentages. SigmaPlot ® version 14.5. (Systat Software Inc., San Jose, CA 95131, USA, EE.UU, North First Street, Suite 360, Jandel Corporation, San Jose, CA, USA) was used to generate the analysis and graphs. Continuous variables were compared with the Mann-Whitney U rank sum test followed by the normality test (Shapiro-Wilk) between HS vs. before the NAC treatment SARS-CoV-2 infection and by Kruskall-Wallis test before the NAC treatment SARS-CoV-2 infection vs. after the NAC treatment SARS-CoV-2 infection. Differences were considered statistically significant when p ≤ 0.05.

Conclusions
The treatment with NAC as an adjuvant therapy may contribute to reducing the OS associated with SARS-CoV-2 infection by restoring the loss of the redox homeostasis through the generation of GSH. Moreover, GSH promotes the metabolic pathways that depend on it, contributing to an increase in the TAC and to restore the redox homeostasis.

Institutional Review Board Statement:
The studies involving human participants were reviewed and approved by ethical approval from the local ethics committee on 19 August 2020 (Control-9867/2020, register REG. CONBIOETICA-09 CEI-011-20160627). A written informed consent for enrollment or consent to use patient data was obtained from each patient or their legal surrogate. The protocol was registered (TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT 04570254).

Informed Consent Statement:
The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

Data Availability Statement:
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.