Antioxidant capacity of N-acetylcysteine against the molecular and cytotoxic implications of cadmium chloride leading to hepatotoxicity and vital progression

Many studies have reported that cadmium (Cd) can induce liver cell injury; however, the toxicity mechanisms of Cd on the liver have not been fully explained. Thirty-two male albino rats were divided into four groups: the control group, the N-acetylcysteine (NAC) group orally as effervescent instant sachets with a concentration of 200 mg dissolved in distilled water and dosage was 200 mg/kg body weight freshly prepared, the cadmium chloride (CdCl2) group (treated with 3 mg/kg orally), and the N-acetylcysteine (NAC) + cadmium chloride group (treated with 200 mg/kg orally post to CdCl2) for 60 days. The NAC alone did not make notable changes in most of the parameters. The CdCl2 alone, compared to control, induced significant alterations in oxidative stress markers (increment in lipid peroxidation (LPO) and nitric oxide (NO)) and antioxidant defense system (decrement in superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (GPx)), which resulted in a downregulation of pro-apoptotic Bcl2-associated X protein (Bax) and caspase-3 and upregulation of anti-apoptotic B-cell leukemia/lymphoma 2 (Bcl2) protein as well as the survival fate of hepatic cells. Post-administration of NAC to CdCl2 resulted in a reduction in oxidative stress markers, shifting of cells from the G2/M phase to the G0/G1 inhibiting signal-regulated kinase activation, and impairment of the anti-apoptotic signaling pathway when compared to the CdCl2 group alone. Accordingly, the Bcl2/Bax ratio was reduced to 1.17-fold change, as an adaptive process to hepatic tissue injury. These findings demonstrated that NAC would attenuate the possibility of oxidative stress and cytotoxicity of hepatic tissue induced by CdCl2. Supplementary Information The online version contains supplementary material available at 10.1007/s11356-022-23823-x.

Cadmium toxicity causes severe damage in the gene expression process. The disruption results in creating reactive oxygen species, disrupting the oxidative stress, causing apoptosis, and halting the DNA repair process. These results contribute to carcinogenesis (Hartwig 2013;Waalkes 2003;Đukić-Ćosić et al. 2020;Albeltagy et al. 2021). One significant result is apoptosis, whereby cells are exposed to external stresses, causing programmed cell death (Gu et al. 2018;Cardinale et al. 2010). Another indication of apoptosis, showing damage in the pathways of the affected cells, would be the imbalance of the pro-apoptotic and anti-apoptotic (Zawlik et al. 2005). A measuring tool is used post an apoptotic stimulus, which is the ratio of Bax/Bcl-2, to determine the mortality fate of the cell (Mahdavi et al. 2018).
The oxidative stress and lipid peroxidation involved in the pathogenesis of various diseases and consequently the role Responsible Editor: Lotfi Aleya of free radical scavenging antioxidants in organ toxicity and carcinogenicity received much attention (Omata et al. 2010). On the other hand, a recent focus involves understanding the role of natural products with antioxidant activity to improve the hazardous effects of drugs and xenobiotics.
N-acetylcysteine (NAC) has been used as an antioxidant in several in vivo and in vitro studies and recorded antioxidant and radical scavenging activities (Ates et al. 2008). The molecular mechanism through which NAC exerts this activity has been discussed. In this regard, the mechanisms of NAC antioxidant activity may be directed toward oxidant species or indirect because of its ability to act as a precursor of l-cysteine, the building block in glutathione synthesis, and breaking disulfides to restore thiol pools (Mahmoud et al. 2019). NAC treatment offers protection against oxidative stress induced by different toxicants due to its dual role as a nucleophile and as a thiol donor (Suke et al. 2008). Additionally, NAC prevents cell death pathways and inflammation signaling in vital organs by elevating endogenous levels of glutathione and restraining mitochondrial membrane depolarization (Zafarullah et al. 2003;Abdel-Daim et al. 2019) with antifibrotic action (Salamon et al. 2019).
This study aimed to investigate the relevant mechanisms and pathways of Cd carcinogenicity by assessing the cell cycle progression, the Bax/Bcl-2 balance impairment, oxidative stress and histopathological changes of hepatic tissues, and the N-acetylcysteine direct antioxidant property and indirectly as a precursor in glutathione synthesis to mitigate the associated oxidative incidence.

Experimental animals
Thirty-two male Wistar rats weighing 150-200 g (2-3 months) were purchased from the Faculty of Medicine, Medical Ain Shams Research Institute (MASRI), Cairo, Egypt. Animals were housed in polycarbonate boxes with steel wire tops, bedded with wood shavings. Ambient temperature was controlled at 22 ± 3 °C with relative humidity. Food and water were provided ad libitum. All animals were acclimatized for 1 week before the beginning of the experiment. Animal care and use followed the guidelines of Investigations & Ethics for Laboratory Animal Care at the Department of Zoology, Faculty of Science, Helwan University (approval no. HU2018/Z/FM1118-03).
Animals were divided into 4 groups (8 rats/each): the control group, received 10 ml/kg physiological saline (0.9% NaCl); the NAC group orally as effervescent instant sachets with a concentration of 200 mg dissolved in distilled water and dosage was 100 mg/kg body weight freshly prepared (Mahmoud et al. 2019); the CdCl 2 group, received 3 mg/kg body weight CdCl 2 according to Babaknejad et al. (2015); and the NAC + CdCl 2 group, received 100 mg/kg NAC (Mahmoud et al. 2019) 1 h post to CdCl 2 for 60 consecutive days.
At the end of the experiment, rats were sacrificed by cervical decapitation and dissected for obtaining the desired tissue. Blood samples were collected immediately from the inferior vena cava and left to clot in dry test tubes and then centrifuged at 3000 × g for 10 min to obtain serum. The obtained serum was stored at − 80 °C for further biochemical assays. The liver tissues were collected, a part was fixed in 10% buffered formalin for histopathological investigations, and another part was kept at − 80 °C for oxidative stress markers; ELISA technique and flow cytometry assay were also performed.

Quantitative real-time PCR analysis
RNA was isolated from freshly removed liver tissue using the TRIzol reagent (Qiagen, Germantown, MD, USA) following the instructions of the manufacturer. The RNA concentration was determined using NanoDrop, and then it was reverse transcribed into cDNA by using a kit of RevertAid™ H Minus Reverse Transcriptase supplied by Fermentas, Thermo Fisher Scientific Inc., Canada, according to the manufacturer's protocol. An SYBR Green PCR kit (Qiagen, Germany) was used to determine mRNA levels of Bcl2, Bax, and Casp3. Quantitative PCR was performed in triplicate on a ViiA™ 7 PCR system (Applied Biosystems, USA). The relative levels of mRNA were calculated by the 2 −ΔΔCt method, which was normalized to the mRNA level of the Gapdh housekeeping gene. Primer sequences are shown in Table 1.

Determination of cell cycle phases and apoptosis by flow cytometry
Liver cells (2 × 10 6 ) were resuspended in 1 ml ice-cold PBS and then permeated with 70% ice-cold ethanol at 4 °C for 24-48 h. Cells were washed twice by adding 2 ml cold PBS (1800 × g, 5 min). The cell pellets were resuspended in 300-500 µl PI/Triton X-100 staining solution (1000 µl of 0.1% Triton + 40 µl PI + 20 µl RNAse) and were incubated at 37 °C for 15 min. The cells were acquired using a BD FACS Canto II flow cytometry (BD Biosciences, USA), and the data were analyzed by BD FACS Diva software.

Determination of liver function
Transaminases (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) in serum samples were determined according to Reitman and Frankel (1957) while serum alkaline phosphatase (ALP) activity was assayed according to the method described by Belfield and Goldberg (1971).

Determination of oxidative stress markers
Grinding and homogenizing of the liver was performed by mixing with 10 mM phosphate buffer (pH 7.4). The supernatant was prepared from the liver by centrifugation of homogenate for 10 min (3000 × g) at 4 °C. The lipid peroxide (LPO) level was measured as described by Ohkawa et al. (1979). Nitric oxide (NO) level was detected according to the method of Green et al. (1982) that used the Griess reagent. The activity of reduced glutathione (GSH) was investigated using the method of Ellman (1959). The superoxide dismutase (SOD) level was evaluated following the standard technique described by Nishikimi et al. (1972). The catalase (CAT) activity was demonstrated following the method described by Aebi (1984). The activity of glutathione peroxidase (GPx) was determined according to the procedure described by Paglia and Valentine (1967).

Statistical analysis
Data were presented as mean ± standard deviation (SD). Statistical analysis was conducted using one-way analysis of variance (ANOVA) using SPSS (Statistical Package for the Social Sciences), version 17, followed by Duncan multiple range test (DMRT) post hoc test for the least significant difference between group P < 0.05. Furthermore, Pearson's correlation coefficient (r) test was used for correlating data. Figure 1 represents the level of Casp3, Bax, and Bcl2 expressions in the liver tissue of rats in different groups. The data indicated a non-significant change in Casp3, Bax, and Bcl2 expressions in the NAC group (0.76-, 1.50-, and 1.25-fold changes), respectively, compared to the control group. Markedly, there was a significant upregulation in Bcl2 (1.54-fold change) and Bcl2/Bax (2.10-fold changes) and a non-significant change in Casp3 (1.22-fold change) and Bax (0.79-fold change) expression level after a daily treatment with CdCl 2 compared to their levels in the control group. Post-administration of NAC to CdCl 2 daily resulted in a significant upregulation in Casp3 level (3.18-fold change) and Bax (2.61-fold change) when compared to both control and CdCl 2 groups and a significant downregulation in Bcl2 (0.92-fold change) and Bcl2/Bax (1.17-fold change) when compared to the CdCl 2 group.

Results of apoptotic/anti-apoptotic markers by ELISA
The data indicated that NAC administration showed a nonsignificant change in apoptotic/anti-apoptotic marker. The CdCl 2 group showed a significant increase in Bcl2 protein level when compared to its level in the control group, whereas post-administration of NAC to CdCl 2 showed a significant decrease (P < 0.05) in Bcl2 protein level when compared to its level in both control and CdCl 2 groups. There was a significant decrease in the protein level of Bax and caspase-3 in the CdCl 2 group compared to the control group, whereas post-treatment of NAC to CdCl 2 showed a significant increase (P < 0.05) in both Bax and caspase-3 when compared to its level in the CdCl 2 group and the control group. The Bcl2/Bax ratio showed a significant increase in the CdCl 2 group when compared to the control group, whereas the NAC + CdCl 2 group showed a significant decrease (P < 0.05) in Bcl2/Bax ratio when compared to the control and CdCl 2 groups (Fig. 2). Figure 3 shows the flow analysis data of hepatocytes. NAC administration induced a significant change in both S and G 2 /M percentages when compared to the control values, while the number of cells in G 0 /G 1 phase fraction was statistically non-significant in the CdCl 2 group compared to the control group. However, the number of cells in S and G 2 /M phases increased significantly (P < 0.05) when compared to the control group. Interestingly, post-administration of NAC to CdCl 2 showed a significant decrease in cell numbers in S and G 2 /M phases when compared to the CdCl 2 group. Moreover, the number of cells at G 0 /G 1 was non-significant when compared to the CdCl 2 group.

Oxidative stress markers
Figures 4 and 5 show the levels of LPO, NO, enzymatic antioxidants, GSH, non-enzymatic antioxidants, and oxidative markers SOD, CAT, and GPx. NAC administration showed a non-significant change in LPO, NO, and GSH and a significant decrease in SOD, CAT, and GPx when compared with the control group. CdCl 2 induced oxidative stress, as evidenced by a significant (P < 0.05) elevation in LPO and NO levels. Data reported a significant decrease (P < 0.05) in both GSH content and activities of SOD, CAT, and GPx when compared with the control group. On the other hand, the NAC + CdCl 2 group indicated an antioxidant potential of NAC. The levels of LPO and NO decreased significantly (P < 0.05) when compared to the CdCl 2 group. Meanwhile, a significant increase (P < 0.05) was detected in the activities of SOD, CAT, GPx, and GSH.

Liver function enzymes
As shown in Fig. 6, NAC administration caused a non-significant change in ALT, AST, and ALP levels in the serum of the group compared to the control group; CdCl 2 administration caused an elevation in ALT, AST, and ALP levels significantly (P < 0.05) in the serum of the group compared Fig. 1 The mRNA expression levels for Casp3, Bax, and Bcl2 and Bcl2/Bax ratio. Data are expressed as mean ± SD. The relative expression is referenced to Gapdh (internal control) and represented as fold change (log2 scale). a: Significant change at P < 0.05 against the control group. b: Significant change at P < 0.05 against the CdCl 2 group to the control group. On the other hand, post-administration of NAC to CdCl 2 resulted in a significant decrease (P < 0.05) in AST and ALP significantly when compared to the CdCl 2 group. Pearson's correlation among different parameters had been tested during this experiment. Pro-apoptotic markers (Cas-3 and Bax) were found to be positively correlated with enzymatic and non-enzymatic molecules and negatively correlated with an anti-apoptotic marker (Bcl-2), oxidative stress markers (LPO and NO), and liver function parameters (ALT, AST, and ALP), while Bcl-2 and liver function parameters were found to be negatively correlated with enzymatic and non-enzymatic molecules and positively correlated with oxidative stress markers. Furthermore, enzymatic and non-enzymatic molecules (SOD, CAT, GPx, and GSH) were found to be positively correlated with pro-apoptotic markers and negatively correlated with Bcl-2, oxidative stress markers, and liver function parameters as indicated in Table 2. Figure 7A shows representative hematoxylin and eosin staining of normal rat liver tissue. Regarding the administration of NAC alone for 60 days, the hepatic lobules appeared more or less as the control one. The CdCl 2 group showed disturbance of the hepatic lobule, presence of hepatocyte focal necrosis, hydropic degeneration, and pyknotic nuclei (Fig. 7B). In case of post-administration of NAC to CdCl 2 , the hepatic lobules appeared more or less as control rats (Fig. 7C), although some sections showed dilated portal tract associated with congested blood vessels and necrotic hepatocytes. This can be attributed to the antioxidant and free radical quenching efficacy of NAC. The scoring summary of the liver histopathological alterations was presented as a supplementary data (Table S1). Fig. 2 The protein levels of caspase-3, Bax, Bcl2, and Bcl2/Bax ratio in liver tissue of adult male albino rats. Data are expressed as mean ± SD. a: Significant change at P < 0.05 against the control group. b: Significant change at P < 0.05 against the CdCl 2 group

Discussion
Oxidative stress plays a major role in Cd-induced organ toxicity and carcinogenicity (Rafati Rahimzadeh et al. 2017;Cuypers et al. 2010). Cadmium incidentally reasons ROS generation and subsequently oxidative damage of lipids, proteins, and DNA (Matovic et al. 2015;Yang and Shu 2015). These deleterious effects occurred via its inhibitory effect on the antioxidant defense system (enzymatic and non-enzymatic antioxidants) (Thevenod and Lee 2013;Matovic et al. 2011).
The present study firmly resolved many aspects, such as DNA content, cell cycle progression phases, ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax, biochemical changes, and correlated histological changes to evaluate Fig. 4 The effect of NAC and/or CdCl 2 administered on LPO, NO, and GSH levels in liver tissue of adult male albino rats. Data are expressed as mean ± SD. a: Significant change at P < 0.05 against the control group. b: Significant change at P < 0.05 against the CdCl 2 group Fig. 5 The effect of NAC and/or CdCl 2 administered on SOD, CAT, and GPx activities in liver tissue of adult male albino rats. Data are expressed as mean ± SD. a: Significant change at P < 0.05 against the control group. b: Significant change at P < 0.05 against the CdCl 2 group Fig. 6 The effect of NAC and/or CdCl 2 administered on ALT, AST, and ALP activities in the serum of adult male albino rats. Data are expressed as mean ± SD. a: Significant change at P < 0.05 against the control group. b: Significant change at P < 0.05 against the CdCl 2 group the therapeutic potential of NAC against Cd-induced toxicity. CdCl 2 could shift the cell cycle from G 0 /G 1 to S and G 2 /M phases and trigger the S and G 2 /M phase cell cycle arrest. Post-administration of NAC to CdCl 2 showed a significant decrease in cell accumulation in S and G 2 /M phases compared to the CdCl 2 group. The present results are in concordance with Odewumi et al. (2011) and Kim et al. (2005). No significant changes were noted in pro-apoptotic Bax and Casp3 in the CdCl 2 group. However, a significant increase in Bcl2 and Bcl2/Bax ratio in the CdCl 2 -treated group suggests  hepatocytes, infiltration of inflammatory cells (red star), hydropic degeneration (white arrows), and pyknotic nuclei (yellow arrows). D A photomicrograph of a section of the liver of a rat administrated with cadmium and N-acetylcysteine showing improvement in the structure of the hepatic lobule. However, there are some apoptotic hepatocytes (white arrows), pyknotic nuclei (yellow arrows), infiltration of inflammatory cells (red star), and fat deposition (red arrows) (H&E stain, magnification = 400 ×) the tendency of hepatocytes toward resisting apoptosis. These results are in agreement with the previous studies on Cd-induced genotoxicity and carcinogenicity (Bjorklund et al. 2019;Buha et al. 2018). Post-administration of NAC to CdCl 2 triggered the apoptotic pathway through a slight elevation in the levels of Bax and Cas-3 and a significant decrease in the Bcl2/Bax ratio when compared to the CdCl 2 group. Two signs were reported to indicate Cd-induced hepatotoxicity: the first one is the boosted activity of liver enzyme biomarkers, while the second is the historical tracking of liver structure changes. The results of the present study were found to be corresponding with a previous study carried out by Ramaiah (2007). The study shows that the increased circulating liver marker enzymes are an accurate sign to determine the extent of hepatic damage (Ramaiah 2007). When detailing the results in comparison to the control group, a noticeable change was found in liver function enzymes which would be indicated by increased levels in ALT and AST and a decrease in ALP. In the conducted study, NAC significantly improved the circulating levels of hepatic enzymes and markedly improved the histological structure of the liver, suggesting membrane stabilizing and potent hepatoprotective potential of NAC.
Several key results of CdCl 2 treatment matched several previously published data, among which is the marked increase in NO, reacting with superoxide, forming hydroxyl radical and peroxynitrite which is a potent oxidant. The second notable result is the cellular GSH levels diminishing, while the last key result concluded a substantial increase in LPO (L'Hoste et al. 2009;Nair et al. 2015). Hence, it is safe to conclude that oxidative stress and lipid peroxidation are commonly acknowledged in the pathogenesis of various diseases. Another key conclusion is that the role of free radical scavenging antioxidants managed to gain attention and increase in reputation.
Reduced glutathione (GSH) is involved in maintaining the balance of cellular redox processes (Lushchak 2012) facilitating the production of ROS which lead to oxidative stress and increased lipid peroxidation (Clement et al. 2018). After NAC post-treatment, there were a significant decline in LPO and NO expression level ) and a significant increase in GSH (Correa et al. 2011) when compared to the CdCl 2 group. GSH levels, which resulted in less oxidative stress and attenuated cell necrosis, are in concordance with published data (Mahmoud et al. 2019;Wang et al. 2020). The antioxidant enzyme activities (SOD, CAT, GPx) were decreased significantly in the CdCl 2 group when compared with the control group and these data agree with previous studies (Mumtaz et al. 2020;Almeer et al. 2018;Ezedom et al. 2020). Meanwhile, adding NAC to CdCl 2 resulted in a significant increase in the activity of SOD, CAT, and GPx enzymes when compared to their level in the CdCl 2 group. These are consistent with Samuni et al. (2013) and Atagana and Asagba (2014). These enzymes are the primary antioxidant enzymes of the endogenous defense systems that protect cells from oxidative damage and exposure to cadmium causes inhibition of them. Cd can generate free radicals through decreasing the activities of antioxidant enzymes such as CAT, GPx, and SOD, or intracellular levels of antioxidants such as glutathione (Chen and Shaikh 2009).
The increased levels of these antioxidant enzymes with post-treatment of NAC could play a role to scavenge the oxygen radicals produced in the liver, which are in agreement with previous studies (El-Tarras Ael et al. 2016;Rani et al. 2014;Singh et al. 2012). Adding NAC to CdCl 2 significantly increased tissue SOD, CAT, and GPx activities which are consistent with Esrefoglu et al. (2006) and Samuni et al. (2013).
CdCl 2 induced liver injury involving oxidative and nitrosative stresses potentiating the damage to the liver tissues as validated by our histological results. The liver tissue in the CdCl 2 group showed a disturbance of the hepatic lobule, presence of focal necrosis of the hepatocytes, hydropic degeneration, and pyknotic nuclei. Meanwhile, the posttreatment with NAC attenuates the functional deterioration of the cells and reduces cell necrosis and our results are supported by previously published data (Skvarc et al. 2017;Cusumano et al. 2015;Sangsefidi et al. 2020;Esrefoglu et al. 2006;Abdel-Daim et al. 2019). In the current study, the histopathological changes are correlated with the biochemical changes.

Study limitations
Limitations of the present study were the dose-dependent effect that is not evaluated and the short duration of the study which might limit the outcomes precisely. Despite these limitations, this experimental study illustrated the carcinogenicity of cadmium, which is linked to the initiation of rodent cancer in the liver by defecting the oxidant/antioxidant balance, cell cycle, and programmed cell death pathway. However, the findings of this study are likely to translate to other species or systems, including human cancers.

Conclusion
In conclusion, the present results clearly validate the effectiveness of NAC in protecting rat liver against oxidative stress resulting from CdCl 2 intoxication. This is followed by a rising in GSH content as well as the SOD activity, while decreasing the LPO and NO levels. Decreased LPO suggests membrane firmness, which correlates with the increased activity of SOD. The mechanism by which NAC applies its antioxidant activity may be explained throughout stimulating the expression of a range of phase II antioxidant defense genes including the two subunits that control GSH synthesis. In addition, NAC opposes proliferation activity, lowering Bcl2/Bax ratio and upregulating Casp3, which leads to apoptotic fate. Data availability All relevant data are within the paper.

Declarations
Ethics approval All studies were performed following the rules of the Helwan University Institutional Animal Care and Use Committee (IACUC) (approval no. HU2018/Z/FM1118-03).

Competing interests The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.