Egyptian Journal of Basic and Clinical Pharmacology
Vol. 12 (2022), Article ID 101579, 9 pages
doi:10.32527/2022/101579
N-acetylcysteine Attenuates the Hepatotoxic Effect of Remdesivir in Rats
Abstract
Background: Remdesivir is approved for treatment of corona virus disease (COVID)-19. Transaminases elevations have been reported during remdesivir therapy. This study assessed the effects of remdesivir, N-acetylcysteine and their combination on the liver functions in rats. Methods: Rats were randomly assigned into: control, N-acetylcysteine 150 mg/kg, remdesivir 10mg/kg, remdesivir 20mg/kg and N-acetylcysteine150 mg/kg-remdesivir 20mg/kg groups. N-acetylcysteine was administered orally while remdesivir was injected i.p. once daily for ten days then alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total proteins and bilirubin levels were determined in serum. Glutathione (GSH), superoxide dismutase (SOD), malondialdhyde (MDA), tumor necrosis factor-α (TNF-α), caspase-3 and phosphorylated-Akt (p-Akt) levels were determined in the liver homogenate. Hematoxylin and eosin (H&E) sections from the liver were examined as well as immunohistochemical sections for detection of B-cell lymphoma 2 (Bcl-2) expression. Results: In dose dependent manner, remdesivir elevated ALT, AST and ALP while total proteins and bilirubin levels were not affected. GSH, SOD and p-Akt levels were decreased while MDA, TNF-α and caspase-3 levels were increased with hepatocyte degeneration and mononuclear inflammatory cells infiltration with decreased Bcl-2 expression. In N-acetylcysteine-remdesivir group; ALT, AST, ALP, MDA, TNF-α and caspase-3 levels were decreased while the levels of GSH, SOD and p-Akt were augmented with amelioration of histopathological changes and enhanced Bcl-2 expression. Conclusion: N-acetylcysteine prevented hepatotoxic effect of remdesivir through mitigation of oxidative stress, inflammation and apoptosis in rats.
1. Introduction
Coronavirus disease 2019 (COVID-19) results in pulmonary involvement however a direct liver injury has been reported. Consequently, it is better to monitor liver functions and hepatic safety of drugs utilized in the protocols of COVID-19 management to avoid drug-induced liver injuries (DILI) [1]. DILI ranges from asymptomatic laboratory abnormalities to acute liver cell failure and even death [2].
Remdesivir is a nucleoside RNA polymerase inhibitor. This broad-spectrum antiviral drug was FDA approved for treatment of COVID-19 in October 2020 [3]. According to ACTT-1, remdesivir-treated patients were recovered after a median of 10 days [4]. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) elevations have been reported during remdesivir therapy [5].
N-acetylcysteine, a glutathione (GSH) precursor, is a mucolytic drug as well as an effective antidote in paracetamol toxicity [6]. The drug restores the GSH levels and inhibits lipid peroxidation and scavenges reactive oxygen species (ROS) [7]. Cytokines and ROS induce acute inflammatory changes, hepatocyte apoptosis and necrosis [8]. Tumor necrosis factor-α (TNF-α) promotes damage of hepatocytes by proteases activation [9]. Caspase-3 is involved in apoptosis of liver cells [10]. On the other hand, the phosphatidylinositol-3-OH kinase (PI3K)–protein kinase B (Akt) pathway activation results in inhibition of oxidative injury and hepatocyte apoptosis [11]. N-acetylcysteine ameliorated liver ischemia/reperfusion injury [12]. The drug is approved in acetaminophen-induced hepatotoxicity and some guidelines recommend its use in non-acetaminophen-induced acute liver failure [13].
This study aimed to assess the effects remdesivir alone and N-acetylcysteine administration before remdesivir on the liver functions in rats as well as the mechanistics involved.
2. Materials and Methods
2.1. Drugs
Remdesivir vial (American Pharmaceutical Company Gilead). Remdesivir was injected, intraperitoneal (i.p.), once daily for 10 days. The administered doses of remdesivir were equivalent to anti-COVID-19 human doses; 20 mg/kg is equivalent to the loading dose (3.2 mg/kg) on day 1 of COVID-19 treatment protocol, while 10 mg/kg is equivalent to doses (1.6 mg/kg) on days 2-10 of the protocol [14] and converted to rat dose according to Nair and Jacob [15]. N-acetylcysteine powder, South Egypt Drug Industries Company, 6 October City, Egypt.
2.2. Animals
Male Wister rats weighing 200-230 gm were purchased from faculty of pharmacy animal house, Zagazig University, Egypt. Standard and hygienic environmental conditions were utilized in the animal care with free access to food and water ad libitum. The experimental protocols were reviewed and approved by the Faculty of Medicine local institutional animal care and use committee, Zagazig University, Egypt (Approval number: ZU-IACUC/3/F/86/21, date: 29-12-2021).
2.3. Experimental design
Rats were randomly divided into five equal experimental groups (six rats per each). Control (vehicle) group: Rats were administered distilled water 1mL/kg by i.p. injection. N-acetylcysteine group: Rats were administered N-acetylcysteine150 mg/kg (dissolved in distilled water) orally by gavage [16]. Remdesivir 10 group: Rats were administered remdesivir 10 mg/kg by i.p. injection. Remdesivir 20 group: Rats were administered remdesivir 20 mg/kg by i.p. injection. N-acetylcysteine-Remdesivir group: Rats were administered N-acetylcysteine150 mg/kg orally one hour before remdesivir 20 mg/kg injection. In all groups, the treatments were administered once daily for ten days for all groups.
In the 11th day, the animals were anesthetized by sevoflurane as 8% in 100% O2in an induction chamber. Blood samples were collected directly from the heart. Blood samples were kept for 30 min then centrifugation at 1,500×g for 15 min was done, then serum was separated for assessment of ALT, AST, ALP, total proteins and total bilirubin. The liver of each animal was excised and divided into two sets; one to be homogenized and the other for histopathological studies. For preparation of liver homogenate, the liver tissue was perfused with cold phosphate buffer saline, pH7.4 that contains 0.16 mg/ml heparin. The tissue was homogenated in 10 ml cold 50 mM potassium phosphate buffer, pH7.5 per gram tissue and centrifuged at 5000 r.b.m for 20 min. The supernatant was removed and stored at -80 C until assay.
3. Biochemical Studies
3.1. Assessment of ALT, AST, ALP, total bilirubin and total proteins
The ALT, AST and ALP enzymatic activities were determined by enzymatic kinetic method. The serum level of total bilirubin and total proteins were determined using colorimetric method. All the reagents were provided by Crescent diagnostic Company, King Saudi Arabia).
3.2. Determination of oxidant/antioxidant biomarkers in liver homogenate
The lipid peroxide in the form of malondialdhyde (Serial No. 1F4346D808) level and superoxide dismutase (Serial No. 745402C55B) enzymatic activity were assayed in liver tissue homogenate samples by ELISA kits (Cloud Clone Corp Company, Houston, TX, USA), according to the instructions of the manufacture, and the absorbance was measured at 450 nm using a microplate reader. The content of reduced glutathione (CAT. No is GR 2511, Biodiagnostic Company Egypt) in the liver tissue homogenate was determined by colorimetric method according to manufacture instructions [17].The method is based on the reduction of 5,5dithiobis (2-nitrobenzoic acid) with reduced glutathione to generate a yellow color compound that is proportional to the amount of reduced glutathione in the sample. Using a spectrophotometer, the absorbance was determined at 405 nm.
3.3. Determination of tumor necrosis factor-alpha (TNF-α) in liver homogenate
TNF-α (Serial No. 3898289A45) was measured by ELISA kits (Cloud Clone Corp Company, Houston, TX, USA) according to manufacture instructions and the absorbance was measured at 450 nm using a microplate reader [18].
3.4. Detection of caspase-3 concentrations in liver homogenate
Caspase-3 concentration was detected according to Wei et al. [19] by ELISA kits (serial No. MBS261814, My BioSource, San Deigo, USA). Caspase-3 antibody is coated onto a microplate then standards and samples are pipetted followed by washing then biotin-conjugated antibody specific for Casp-3 is added followed by washing. Horseradish peroxidase is added followed by washing then substrate solution is added and color developed is read using a microplate reader set to 450 nm.
3.5. Detection of phosphorylated Akt (p-Akt) concentrations in liver homogenate
The p-Akt concentration was assessed according to method of Boudewijn and Coffer [20] utilizing AKT [pS473] kit (DRG International, Inc. USA). AKT antibody was coated onto the wells of the microtiter strips then samples are pipetted. After washing, p-AKT is added then horseradish peroxidase-labeled anti-rabbit IgG (IgG HRP) is added. Washing is done then substrate solution is added and the produced color was read at 450 nm.
3.6. Histopathological study:
All liver tissue samples were fixed with 10% formalin. embedded in paraffin blocks, Consecutive 4-μm thick sections from formalin-fixed, paraffin-embedded tissue blocks were prepared and stained with hematoxylin and eosin (H&E) for histopathological evaluation [21].
3.7. Histopathological scoring of acute liver injury
Liver sections were graded numerically to assess histopathological parameters in acute hepatic injury [22], each parameter is scored from 0-3 as follow: portal inflammation, lobular necrosis or apoptosis, lobular inflammation, steatosis, ballooning and cholestasis. A combined score of all histopathological findings semi-quantitatively estimated given a maximum value of 18 defined as mild, moderate, and severe was applied to the hepatic injury.
0 indicating no abnormality,
1–6indicating mild injury,
7–12 indicating moderate injury
13–18 with severe liver injury.
Table 1: Effect of N-acetylcysteine, remdesivir, and N-acetylcysteine-remdesivir combination on the serum levels of AST, ALT, ALP, bilirubin, and total proteins.
Table 2: Effect of N-acetylcysteine, remdesivir, and N-acetylcysteine-remdesivir combination on hepatic GSH, SOD, TNF-α and MDA levels.
Table 3: Effect of N-acetylcysteine, remdesivir, and N-acetylcysteine-remdesivir administration on caspase-3 and p-Akt levels in liver homogenates in rats.
Table 4: Histopathological score of control, N-acetylcysteine, remdesivir, and N-acetylcysteine-remdesivir groups.
Table 5: Immunohistochemical (Bcl-2 positive cells) score of control, N-acetylcysteine, remdesivir, and N-acetylcysteine-remdesivir groups.
3.8. Immunohistochemical staining of Bcl-2:
For immunohistochemical detection of Bcl-2, (Thermo Fisher SCIENTIFIC Invitrogen: Bcl-2 Monoclonal Antibody was used by dilution 1:100. 4-m thick sections were deparaffinized in xylene and hydrated in a series of ethanols before being rinsed in tap water and phosphate buffered saline (PBS). Using biotin-free Bond Polymer Refine Detection, the main antibody binding to tissue slices was seen (Leica Microsystems). Slides were counterstained with hematoxylin after post-primary amplification (8 minutes; Leica Microsystems) and detection with the Novolink Polymer Detection System (15 minutes; Leica Microsystems) using 3, 3'-diaminobenzidine (Novocastra Laboratories; 1:50) (Leica Microsystems). All slides were immunostained blindly by the pathologist.
3.9. Evaluation of Bcl-2 expression in liver tissue:
The mean number of positive brown cytoplasmic stained cells in each section was counted in the hepatic tissue of ten high-power fields per slide (400x), then dividing that number by the total number of cells (stained and non-stained) inside the identical fields, multiplying by 100, and expressing the results as an average percentage number [23].
3.10. Statistical analysis
The SPSS 18.0 software suite for Windows was used to statistically analyze the results (SPSS Inc., Chicago, IL). The statistical analysis was done statistically using a one-way ANOVA followed by a post hoc Tukey test for multiple comparisons. The histopathological and immunohistochemical (non-parametric) data were analyzed using Kruskal-Wallis test. Statistical significance was set at p value <0.05.
4. Results
4.1. Effect of N-acetylcysteine, remdesivir and N-acetylcysteine-remdesivir combination on the serum levels of AST, ALT, ALP, total proteins and bilirubin.
N-acetylcysteine produced non-significant decrease in ALT (P=0.831), AST (P=0.998) and ALP (p=0.943) in relation to control group. Remdesivir 10 and 20 mg/kg significantly (p<0.001) increased ALT level in dose dependent manner from both control and N-acetylcysteine groups. Combination of N-acetylcysteine with remdesivir produced significant (p<0.001) decrease in ALT and AST levels from remdesivir groups while was insignificantly different from control (p=0.999, 0.999 and 0.978 respectively) and N-acetylcysteine (p=0.923, 981 and 0.724 respectively) groups. Total bilirubin and total proteins were not significantly changed in all treatment groups from control group. (Table 1)
4.2. Effect of N-acetylcysteine, remdesivir and N-acetylcysteine-remdesivir combination on hepatic GSH and SOD
N-acetylcysteine administration produced insignificant change in GSH content and SOD activities (p=0.058 and 0.725, respectively) in the liver tissue from control group. Remdesivir 10 mg/kg significantly (p<0.001) decreased GSH content and SOD activity from both control and N-acetylcysteine groups. Remdesivir 20 mg/kg showed significant decrease in GSH content SOD and catalase activities from N-acetylcysteine and control (p=0.00) groups and remdesivir 10 mg/kg group (p<0.05). In N-acetylcysteine-remdesivir 20mg/kg group, GSH content and SOD activities were significantly increased from remdesivir 10 and 20 mg/kg groups (p<0.001), while were insignificantly (p=0.998 and p=0.433) changed from control and N-acetylcysteine groups (Table 2)
4.3. Effect of N-acetylcysteine, remdesivir and N-acetylcysteine-remdesivir combination on hepatic TNF-α and MDA levels
TNF-α and MDA levels were insignificantly (p=0.969 and 0.644, respectively) changed in liver tissue in N-acetylcysteine group from control group. Administration of remdesivir 10 and 20mg/kg significantly (p<0.001) increased MDA and TNF-α levels from control and N-acetylcysteine groups. Administration of N-acetylcysteine-remdesivir 20 mg/kg combination demonstrated significant decrease (p<0.05) in TNF-α and MDA levels respectively from remdesivir 10 and 20 mg/kg groups, while were insignificant from control (0.966 and 0.922, respectively) and N-acetylcysteine (0.999 and 0.386, respectively) groups. (Table 2)
4.4. Effect of N-acetylcysteine, remdesivir and N-acetylcysteine-remdesivir combination on caspase-3 and p-Akt levels in the liver
N-acetylcysteine produced insignificant (p=0.977) change in caspase-3 level liver tissue homogenate from control group. Remdesivir 10/20 mg/kg produced a significant (p<0.001) increase in the apoptotic caspase-3 level from control and N-acetylcysteine groups. N-acetylcysteine administration with remdesivir 20mg/kg significantly (p<0.05) decreased caspase-3 level tissue from remdesivir10/20 groups but was insignificant from control (p= 1) and N-acetylcysteine (p=0.955) groups.
The administration of N-acetylcysteine produced insignificant (p=0.688) change in the anti-apoptotic p-Akt level from control group. The p-Akt level was significantly (p<0.05) decreased in the liver tissue homogenate by remdesivir 10 and 20 mg/kg administration from control and N-acetylcysteine groups. The p-Akt level was significantly (p<0.05) increased by administration of N-acetylcysteine along with remdesivir tissue from remdesivir 10/20 mg/kg groups but was insignificant from control (p=0.647) and N-acetylcysteine (p=1.00) groups. (Table 3)
5. Histopathological Findings
Light microscopic examination of the liver sections of control and N-acetylcysteine groups revealed normal lobular architecture showing central vein with radiating hepatic cords, consists of normal hepatocytes with eosinophilic cytoplasm and central open-faced nuclei (Figure 1 A,B). Administration of remdesivir produced hepatocyte degeneration in the form of cloudy swelling, fatty change, hepatic parenchymal and portal tract infiltration by mononuclear inflammatory cells and lobular necrosis. The median scores of remdesivir 10 and 20 mg/kg groups were 7 and 12 respectively that differ significantly from the control group (Figure 1 C-J). N-acetylcysteine-remdesivir group showed marked improvement in hepatic histoarchitecture, with minimal hepatocyte injury in the form of traces of mononuclear inflammatory cells infiltrates and mild cloudy swelling (Figure 1 K,L), with median score 2 that was significantly different from remdesivir groups. (Table 4)
5.1. The Bcl-2 expression in liver tissue
The median Bcl-2 positive cells score was significantly decreased in the remdesivir 10 and 20 groups to 7 and 3 respectively (Figure 2: C,D) compared to control and N-acetylcysteine groups scores, 39 and 38 respectively (Figure 2: A,B). In N-acetylcysteine-remdesivir group (Figure 2: E), the median Bcl-2 score was significantly increased to 36, compared to remdesivir groups. (Table 5)
6. Discussion
Drug induced liver injury (DILI) could be represented by acute hepatocellular hepatitis, cholestatic damage, or mixed liver injury [24]. ALT, AST, and ALP are located in the hepatocytes. These enzymes are released into the bloodstream in the case of hepatocellular damage [25]. The findings of this study demonstrated that remdesivir-induced AST, ALT, and ALP elevations, particularly at the higher dose. In addition, the histopathological results showed inflammation and hepatocyte necrosis with decreased Bcl-2 protein expression. The total proteins were not reduced by remdesivir treatment denoting that the drug did not impair the biosynthetic capacity of the liver. In the present findings, absence of bilirubin elevation after remdesivir treatment excluded the occurrence of cholestatic liver injury. In line with the current findings, Ghosh et al. [26] who reported that remdesivir-treated patients exhibited significantly higher transaminases and ALP levels than non-remdesivir-treated patients, while serum albumin levels were unaffected.
According to the current findings, remdesivir administration decreased GSH content, and SOD activities, especially with the higher dose, while increasing MDA and TNF-α levels. These findings indicated the occurrence of oxidative stress and lipid peroxidation in the hepatocyte membrane. In reality, conversion of superoxide anions to molecular oxygen and hydrogen peroxide consumes SOD [27]. TNF-α binds to its receptor forming TNFR1 complex, which activates the nuclear factor kappa B (NF-kB) pathway, causing apoptosis [28]. These data showed that remdesivir-induced liver damage overwhelmed the antioxidant capacity of the liver, resulting in hepatocyte necrosis and apoptosis.
The current study found that combining N-acetylcysteine with remdesivir 20 mg/kg prevented the increases in ALT, AST, ALP, MDA and TNF-α. In addition, this medication restored SOD activity as well as replenished GSH in the liver. Histopathological examination confirmed the protective activity of N-acetylcysteine on liver tissue by reducing inflammation and hepatocyte necrosis with enhanced Bcl-2 protein expression. To avoid liver cell damage during remdesivir treatment, the equilibrium between reactive oxygen species and antioxidant defense must be restored. N-acetylcysteine enhances endogenous antioxidant activity by increasing GSH production [29]. Also sulfhydryl groups of N-acetylcysteine block electrophilic free radicals. It has beneficial effects as a potent free radical scavenger [30].
The present findings are in harmony with the findings obtained by Skrzydlewska et al. [31] who reported the hepatoprotective effect of N-acetylcysteine in methanol-induced liver damage. They revealed that N-acetylcysteine reduced hepatic transaminases, ALP, and MDA levels while maintaining normal liver histology. Furthermore, N-acetylcysteine had a hepatoprotective activity on carbamazepine-induced hepatotoxicity via reducing lipid peroxidation [32]. Also the drug protected from trichloroethylene-induced hepatotoxicity in rats [33]. In clinical trials, early treatment of N-acetylcysteine after acetaminophen-induced liver damage resulted in glutathione replenishment in the liver [34]. The use of N-acetylcysteine in the treatment of non-acetaminophen-induced acute liver failure considerably reduced mortality [35]. Moreover, by blocking leukocyte chemotaxis, N-acetylcysteine had an anti-inflammatory effects [36].
According to the present findings remdesivir elevated apoptotic caspase-3 while lowered the prosurvival p-Akt levels as well as the anti-apoptotic Bcl-2 expression in the liver cells. The combination of N-acetylcysteine with remdesivir reduced caspase-3 while increasing p-Akt levels and Bcl-2 expression. These findings documented the involvement of apoptosis in remdesivir-induced liver damage, as well as the anti-apoptotic activity of N-acetylcysteine.
Indeed, apoptosis is associated with alterations in cellular oxidant/antioxidant balance which is heavily influenced by glutathione [37]. Caspase-3 triggers the activation of proteases that break cytoskeleton structural proteins, leading to apoptosis [38]. Both intrinsic and extrinsic pathways of apoptosis are inhibited by p-Akt and Bcl-2, with cell survival signal transduction enhancement [39]. For instance, p-Akt suppresses the transcription factor forkheadboxO3 and glycogen synthase kinase-3 with subsequent block of apoptosis [40]. Furthermore, enhanced p-Akt and Bcl-2 expression prevented beta-amyloid induced neurotoxicity by mitigating neuronal apoptosis [41]. Moreover, N-acetylcysteine was reported to inhibit caspase-3 and enhance conjunctival wound healing with inhibition of apoptosis and mitigation of inflammation [42].
7. Conclusion
N-acetylcysteine administration before remdesivir mitigated the liver injury through replenishment of GSH with anti-inflammatory as well as anti-apoptotic effects through enhancement of prosurvival p-Akt and Bcl-2 with decreased TNF-α and caspase-3 in rats. Clinical studies are required to establish the hepatoprotective effect of N-acetylcysteine against remdesivir-induced liver injury.
Competing Interests
The authors declare no competing interests.
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Egyptian Journal of Basic and Clinical Pharmacology
Vol. 12 (2022), Article ID 101579, 9 pages
doi:10.32527/2022/101579
N-acetylcysteine Attenuates the Hepatotoxic Effect of Remdesivir in Rats
Abstract
Background: Remdesivir is approved for treatment of corona virus disease (COVID)-19. Transaminases elevations have been reported during remdesivir therapy. This study assessed the effects of remdesivir, N-acetylcysteine and their combination on the liver functions in rats. Methods: Rats were randomly assigned into: control, N-acetylcysteine 150 mg/kg, remdesivir 10mg/kg, remdesivir 20mg/kg and N-acetylcysteine150 mg/kg-remdesivir 20mg/kg groups. N-acetylcysteine was administered orally while remdesivir was injected i.p. once daily for ten days then alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total proteins and bilirubin levels were determined in serum. Glutathione (GSH), superoxide dismutase (SOD), malondialdhyde (MDA), tumor necrosis factor-α (TNF-α), caspase-3 and phosphorylated-Akt (p-Akt) levels were determined in the liver homogenate. Hematoxylin and eosin (H&E) sections from the liver were examined as well as immunohistochemical sections for detection of B-cell lymphoma 2 (Bcl-2) expression. Results: In dose dependent manner, remdesivir elevated ALT, AST and ALP while total proteins and bilirubin levels were not affected. GSH, SOD and p-Akt levels were decreased while MDA, TNF-α and caspase-3 levels were increased with hepatocyte degeneration and mononuclear inflammatory cells infiltration with decreased Bcl-2 expression. In N-acetylcysteine-remdesivir group; ALT, AST, ALP, MDA, TNF-α and caspase-3 levels were decreased while the levels of GSH, SOD and p-Akt were augmented with amelioration of histopathological changes and enhanced Bcl-2 expression. Conclusion: N-acetylcysteine prevented hepatotoxic effect of remdesivir through mitigation of oxidative stress, inflammation and apoptosis in rats.
Egyptian Journal of Basic and Clinical Pharmacology
Vol. 12 (2022), Article ID 101579, 9 pages
doi:10.32527/2022/101579
N-acetylcysteine Attenuates the Hepatotoxic Effect of Remdesivir in Rats
How to cite this article
Ahmed A. Abdelsameea, Mohamed Ali Alabiad, and Hussein M. Ali, "N-acetylcysteine Attenuates the Hepatotoxic Effect of Remdesivir in Rats," Egyptian Journal of Basic and Clinical Pharmacology, Vol. 12, Article ID 101579, 9 pages, 2022. doi:10.32527/2022/101579.