Efficiency of early intervention by N-acetyl cysteine on liver fibrosis and markers of hepatocellular carcinogenesis induced by diethylnitrosamine in mice

Efficiency of early intervention by N-acetyl cysteine on liver fibrosis and markers of hepatocellular carcinogenesis induced by diethylnitrosamine in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Efficiency of early intervention by N-acetyl cysteine on liver fibrosis and markers of hepatocellular carcinogenesis induced by diethylnitrosamine in mice Majid Jafari-Khorchani, Mohammad-Jalil Zare-Mehrjardi, Abdolamir Allameh This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7032706/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background N-acetylcysteine is a hepatoprotective agent with antioxidant and therapeutic potential. In this study, the effectiveness of early and late intervention with NAC in hepatocellular carcinogenesis (HCC) induced by diethyl nitrosamine (DEN) in mice has been evaluated. Methods Newborn mice (14-day-old) were divided into 4 groups (n = 4/group). Control, HCC group, early NAC intervention, and late NAC intervention group. NAC treatments were followed after HCC induction by DEN administration (50 mg/kg, i.p), followed by phenobarbitone (PB, 500 mg/L via drinking water). In the early intervention group, NAC (150 mg/kg) was given by gavage during 8–16 weeks after birth. In the late group intervention, NAC was given during 16–24 weeks of birth. After 7 months (28 weeks), mice were sacrificed; blood and liver tissues were collected. Liver damage markers, as well as serum levels of liver fibrosis biomarker, PIIINP (N-terminal propeptide type III collagen), and antioxidant capacity (TAC) were determined. Histology examination on liver biopsies, together with changes in tissue total oxidant factors and liver cells proliferation index (Ki67) were determined in liver tissues. Results Early intervention with NAC in mice during HCC induction resulted in a significant decrease in serum levels of liver damage markers, AST and ALT. This finding was corroborated with liver histology data, particularly tissue fibrosis. Intervention with NAC during HCC progression resulted in a significant decrease in serum PIIINP and hepatic total oxidative stress, GSH, and KI67 expression. NAC treatment also resulted in TAC overregulation. Conclusion Early treatment with NAC in HCC model of mice can improve liver fibrosis and cancer through antioxidant system. However, NAC intervention at later stages of HCC development encounters multiple molecular and cellular pathways with less therapeutic efficiency. Hence, treatment with NAC at the early stages of HCC, where oxidative stress is seriously disturbed, is beneficial in the prevention of tumor development. NAC intervention at early stage of HCC induction in mice greatly subsided HCC-related liver damage markers, along with liver fibrosis and cancer cell proliferation. This data suggests that NAC can efficiently delay and ameliorate tissue fibrosis and cancer promotion. HCC NOX4 PIIINP Oxidative stress Glutathione Diethylnitrosamine Liver fibrosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Antioxidant supplements are commonly used, with some studies suggesting potential anticancer properties. However, the efficacy and safety of antioxidant supplementation remain controversial, both in healthy individuals and in cancer patients [ 1 ]. Under normal conditions, cellular processes generate reactive oxygen species (ROS), which are neutralized by antioxidants to maintain genomic and protein integrity. In cancer cells, elevated metabolic activity leads to increased ROS production. To survive, cancer cells upregulate antioxidant defenses, enabling proliferation[ 2 ]. N-acetylcysteine is a hepatoprotective drug that functions as a precursor of cellular glutathione that subsides glutathione depletion during hepatotoxicity. NAC, by increasing cellular GSH, can protect liver against oxidative stress; Subsequently, NAC acts as a potent antioxidant factor. NAC is widely used as an alleviator of different diseases through the increase of glutathione. NAC is demonstrated to indicate antitumor properties in several cancers. However, NAC also promotes the growth of multiple cancers [ 3 – 6 ]. NAC is a well-known antidote to the toxicity of acetaminophen. It has been widely investigated for liver injuries; however, there are many conflicts regarding its beneficial effects and underlying mechanisms [ 7 – 9 ]. NAC is often prescribed for treatment purposes following drug toxicity. Different therapeutic effects have been assigned to NAC. NAC is an N-acetylated derivative of the natural amino acid L-cysteine. It is N-acetyl-L-amino acid and classified as an acetylcysteine derivative. It is a conjugate acid of N-acetyl-L-cysteinate. It has a role as an antidote to paracetamol poisoning [ 10 – 12 ], an anti-infective agent, an antioxidant, an antiviral drug [ 13 , 14 ], a mucolytic actions, a radical scavenger, a ferroptosis inhibitor and a geroprotector [ 15 – 17 ]. NAC is widely used to alleviate various diseases through glutathione elevation and has demonstrated antitumor properties in several cancers [ 3 – 6 , 18 ]. However, some evidences also show that NAC might promote the growth of certain malignancies. It has been reported that NAC exacerbated tumor growth in lung cancer and melanoma. These studies stated that low ROS levels might be an advantage for tumor cells. NAC reduces ROS, DNA damage, and p53, leading to exacerbating lung cancer cell growth and increasing invasion of human melanoma cells [ 19 – 21 ]. The mechanism of action of NAC is generally directly or by increasing intracellular GSH, especially in hepatic tissue. Regarding direct elimination capacity of ROS, NAC participates in the detoxification and reacts with highly oxidative radicals such as hydroxyl radicals (HO•). Moreover, NAC can act as a chelating agent in transition metals, including Cu²⁺ and Fe³⁺, and heavy metals such as Cd²⁺, Hg²⁺, and Pb²⁺ via thiol side chain, which forms complexes that are simply excreted. Although NAC is able to directly scavenge ROS, its reaction rates are lower than antioxidant enzymes such as Superoxide dismutase, catalase (CAT), and Glutathione peroxidase (GPx). Therefore, the direct capacity of ROS elimination by NAC is not as significant as its broader antioxidant activity [ 22 , 23 ]. Oxidative stress, which is considered a hallmark of liver cancer, appears at early stages of chemically induced hepatocellular carcinoma (HCC) in mice, but oxidative stress factors and related cellular damages remain active throughout the carcinogenesis process [ 24 , 25 ]. It was therefore assumed that intervention in HCC by NAC could ameliorate oxidative stress-related damage in liver and provide protective condition for subsequent damage. NAC, by improving antioxidant system, both enzymatic and non-enzymatic factors, can help suppress cancer cells and reduce drug resistance in tumor cells [ 4 , 26 , 27 ]. Regardless of the etiological factors in HCC, this malignancy is often associated with liver fibrosis, for which NAC is also effective in preventing tissue fibrosis [ 28 , 29 ], by inhibition of the expression of fibrogenic markers such as α-SMA and TGF-β, leading to decreased extracellular matrix accumulation and fibrosis [ 30 ]. The present study aimed to investigate the efficiency of NAC on chemically induced HCC in mice in the protection of liver damage related to tumor formation with emphasis on the antioxidant system. For this purpose, HCC was induced in a period of 7 months, which was observed by sequential changes through oxidative stress damage, liver fibrosis and HCC development. The efficiency of NAC intervention on HCC induction was compared at two-time schedules as early and late intervention. Materials and Methods Adult male and female C57BL/6 mice were purchased from Iran University of Medical Sciences, Tehran, Iran. Adult mice were mated and the newborns (Body weight 8 ± 0.5 g; 2-week-old) were used. Mice were kept at 22–25°C, four mice per cage, on a 12-hour light/dark cycle, with free access to water and standard chow diet. Experimental design In this study, HCC was induced in newborn C57BL/6 mice by DEN and PB treatments as described earlier [ 25 ]. Briefly, 14-day-old mice were randomly divided into four groups: 1) Control group (C), 2) HCC group received DEN and phenobarbital. Groups 3 and 4 were treated with NAC as described below. Each mouse (14 days old) was treated with a single i.p dose of 50 mg/kg of DEN (Sigma-Aldrich, USA), dissolved in 0.6% normal saline, into both sides of the peritoneal cavity. DEN injection was followed by PB administration that was dissolved in drinking water (500 mg/L) from 4 weeks after birth for a period of six months (Fig. 1 ). NAC intervention during DEN-induced HCC in mice Wherever indicated, each mouse received 150 mg/kg body weight of NAC (dissolved in Distilled Water) for 8 weeks via gavage. Two groups of mice were subjected to NAC treatment. The early Intervention group received NAC from the age of 2 months (6 weeks after DEN administration), and the late Intervention group received NAC treatments at the age of 4 months (14 Weeks after DEN treatment). Development of liver fibrosis and HCC was monitored by histopathological examination carried out on few mice sacrificed at different time points. HCC induction was confirmed in mice treated for 7 months. Then, mice were anesthetized through i.p. injection of ketamine (100 mg/kg) and xylazine (20 mg/kg). Blood specimen was collected by cardiac puncture, serum was separated and stored at -80°C. Mice were then sacrificed by cervical dislocation, and liver tissue was removed, washed, and weighed. A portion of the liver tissue was fixed in 4% paraformaldehyde for histopathological analysis via hematoxylin and eosin (H&E) staining. The remaining liver tissue was immediately frozen at -80°C for molecular and biochemical analyses. Preparation of liver homogenate After measuring the weight of the liver tissue, 100 mg of each frozen sample was homogenized in 1 mL of normal saline to prepare a 10% (mg/mL) homogenate. The homogenate was centrifuged at 10,000 × g for 10 minutes at 4°C, and the clear supernatant was collected and stored for future analyses. Estimation of serum N-terminal propeptide type III collagen (PIIINP) Serum level of PIIINP, as the marker of tissue fibrosis was determined using a mouse-specific ELISA kit (Zellbio, Germany, Cat. # ZB-10691C-M9648). The assay was performed according to the manufacturer's instructions. In brief, the kit employs a sandwich ELISA technique in microplate wells pre-coated with PIIINP-specific antibodies. Serum samples were added to the wells, followed by a biotinylated antibody specific to PIIINP and an avidin-horseradish peroxidase conjugate. After incubation and washing steps, a chromogenic substrate was added and the enzymatic reaction was terminated with an acidic stop solution. The resulting color intensity was measured spectrophotometrically at 450 nm. PIIINP levels in the samples were quantified based on a standard curve prepared by different concentrations of standard solution provided in the assay kit. Estimation of biochemical markers in serum and liver homogenate Glutathione [ 31 ] levels was determined in liver homogenates using commercially available colorimetric assay kit (ZellBio GmbH, Cat. # ZB-GSH-69A) following the manufacturer's instructions. Briefly, liver tissue samples were homogenized and centrifuged. Samples and standards were added to a microplate, followed by the addition of assay reagents. After incubation, absorbance was measured at 405 nm. GSH concentrations were calculated using the standard curve and normalized by protein concentration of respective liver homogenate. Total oxidative stress levels were estimated in liver homogenates using a commercially available colorimetric assay kit (Navand Salamat, Cat. # NS-15017, Iran), following the manufacturer's protocol. Total antioxidant capacity (TAC) was estimated in serum samples by FRAP assay according to Benzie and Strain procedure [ 32 ]. This assay is based on the reduction of TPTZ-Fe3 + to TPTZ-Fe2 + by the antioxidant capacity of samples. The color change of the reaction was measured at a wavelength of 593 nm and the total antioxidant capacity was calculated based on a FeSO4 standard curve. Estimation of liver damage enzyme markers (ALT and AST) Alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were estimated in serum samples using colorimetric methods. The analysis was performed with commercially available assay kits. Expression of Ki-67 in liver tissue (immunohistochemistry) : Immunohistochemical (IHC) technique was performed on formalin-fixed, paraffin-embedded (FFPE) tissue sections. Tissue sections were mounted on poly-L-lysine-coated slides and deparaffinized. Endogenous peroxidase activity was quenched by immersing slides in 10% hydrogen peroxide solution in PBS for 10 minutes. Antigen retrieval was achieved by heating in sodium citrate buffer (10 mM, pH 6) at 95°C for 30 minutes. Following PBS washes, sections were incubated with Ki-67 antibody (MAD-000310QD-3, Master Diagnóstica, Spain 1:300) for 50 minutes at room temperature. After two PBS washes, slides were treated with secondary antibodies (MAD-000237Q, Master Diagnóstica, Spain dilution 1:1000) for 45 minutes at room temperature. Immunoreactivity was visualized using 3,3′-Diaminobenzidine as the chromogen, applied for 10 minutes at room temperature. Slides were then washed with tap water, counterstained with Mayer's hematoxylin, mounted, and examined under a light microscope for Ki-67 expression analysis. Statistical analysis All assays were performed in duplicate, and data are expressed as mean ± standard deviation (SD). Statistical analysis was conducted using GraphPad Prism software (version 8.0.1). Differences in parameters among the experimental groups were evaluated using one-way ANOVA followed by Tukey's post hoc test. Statistical significance was set at p < 0.05 for all analyses. Results Body weight and liver weight As shown in Fig. 2 A, body weight data have no significant differences between all groups. In the case of liver weight, there was a substantial increase in the HCC group compared to the control group (122%, p < 0.0001). Early NAC intervention reduced liver (25%) compared to the HCC group (p < 0.01). However, the liver weight in the late-treated group remained similar to the HCC group (Fig. 2 B). Histopathological data of liver tissues with H&E staining Microscopic examination of H&E-stained liver preparations showed normal morphology in the control group. In the HCC group, atypical nuclei, focal proliferation, and anisokaryosis were observed. Liver of early NAC treatment mice still showed slight atypical nuclei, but focal proliferation was not visible in this group. In the liver of late NAC treatment mice, numerous atypical nuclei and multinuclear cells were observable (Fig. 3 ). Effects of NAC treatment on expression of liver Ki-67 (IHC) The Ki67-positive cells in liver tissue of the HCC group were higher than in the control group (p < 0.001). Early treatment with NAC significantly reduced the proportion of Ki67-positive cells compared to the HCC and late treatment groups (82%, p < 0.001 and 69%, p < 0.01, respectively). Interestingly, the late NAC-treated group demonstrated a reduction in Ki67-positive cells compared to the HCC group (44%, p < 0.01, Fig. 4 ). Effects of NAC intervention on serum ALT and AST levels Serum ALT in the HCC group was significantly increased (~ 275%) as compared to the control group (P < 0.0001). Early NAC intervention significantly decreased the ALT by about 50% (p < 0.01). The decrease in the ALT in the early-treated group was also significant when compared to the late-treated group (P < 0.05) (Fig. 5 A). Serum AST was increased by 88% in the HCC group compared to the control group (p < 0.0001). Whereas, it was decreased by about 26% and 16% in the early NAC treatment group compared to the HCC and late NAC treatment groups (p < 0.001 and p < 0.01, respectively, Fig. 5 B). Effects of NAC intervention on serum PIIINP (N-terminal propeptide type III collagen) Serum PIIINP level was increased by 145% in the HCC group treated with DEN and PB compared to the control group (p < 0.001). NAC intervention during HCC development resulted in a significant decrease in PIIINP in mice treated with NAC compared to the HCC group. NAC treatment in the HCC group at the early stage resulted in 38% and 33% decrease of serum PIIINP when compared to that measured in the HCC group and late NAC intervention groups (p < 0.01 and p < 0.05, respectively) (Fig. 6 ). Effects of NAC treatment on total oxidative stress in liver homogenate During HCC development, there was a significant increase (about 50%) in hepatic total oxidative marker as compared to the control group (p < 0.001). Early and late administration of NAC during HCC development in mice resulted in a significant decrease in total oxidative status compared to the untreated HCC group (34% and 38%, respectively, p < 0.05) (Fig. 7 A). Changes in liver GSH content in NAC-treated mice. Hepatic GSH content was depleted by 38% in the HCC group (p < 0.01). NAC treatment at early and late stages of HCC development resulted in recovery of hepatic GSH by approximately 55% and 76%, respectively (p < 0.01 and p < 0.001, respectively) (Fig. 7 B). Effects of NAC treatment on serum Total Antioxidant Capacity (FRAP assay) Serum TAC was significantly declined (36% reduction) during the HCC development in mice (p < 0.0001). Treatment with NAC at the early stage of HCC development increased serum TAC by approximately 41% compared to that measured in the HCC group (p < 0.001). TAC in the late NAC-treated group was significantly increased vs the HCC group by about 43% (p < 0.001, Fig. 7 C). Discussion There are several reports on the role of NAC and its redox system in preventing and delaying liver cancer in animal models [ 4 , 26 , 33 ]. In the present study, attempts were made to examine the effects of NAC intervention during the chemically-induced hepatocellular carcinogenesis with emphasis on oxidative stress factors and liver fibrosis markers. NAC, as the precursor of glutathione, is also widely used to alleviate various diseases through glutathione elevation and has demonstrated antitumor properties in several cancers [ 18 , 34 ]. However, depending on the experimental protocols, particularly the time schedules of intervention, NAC may also promote the growth of certain malignancies [ 3 – 6 ]. It has been reported that NAC exacerbated tumor growth in lung cancer and melanoma[ 19 , 20 ]. Perhaps, in the case of early intervention in HCC development with NAC, the oxidative stress cascade is potentially important in the initiation process of tumorigenesis. In this stage, oxidative stress-related tissue damage probably imposes an accelerating influence on the exacerbation of HCC induction. Nevertheless, when NAC is given at later stages, the stage known as tumor progression, other pathways will be involved, such as multiple gene mutations and EMT which are complicated to be reversed by antioxidants. In this study, the effects of NAC intervention were compared at two different time schedules during HCC induction in mice. Microscopic examination of the liver in the Treatment of HCC group treated with NAC at the early stage of HCC showed some cellular damage. However, the nuclei had no signs of atypical features and were nearly uniform. At late NAC intervention stage, some atypical nuclei were noted, suggesting the initiation stage of HCC induction [ 25 ]. Histopathological data showed that focal cell proliferation in the HCC group was due to the HCC initiator feature of DEN and the promoter feature of PB. DEN, an electrophilic species, is subject to in vivo intricate chemical transformations and can interact with DNA bases to yield O6-ethylguanine [ 35 ]. DEN can stimulate the secretion of interleukin-6 (IL-6) from Kupffer cells (KCs). IL-6 is implicated in the pathogenesis of HCC through a mechanism reliant on signaling by STAT3 [ 36 ]. During this progression, the concentrations of lipids and glycogen and various enzymes associated with glycolytic processes, the oxidative pentose phosphate pathway, glycogen catabolism, and cell membrane function are impaired. All these alterations are linked mainly to the neoplastic transformation of hepatocytes. DEN is independently effective at inducing hepatocellular carcinoma in mouse models. Still, hepatotoxic substances like phenobarbital, which affect the expression of interleukin-22 (IL-22), hepatocyte growth factor (HGF), or macrophage inflammatory protein-1, increase the carcinogenicity of DEN [ 35 , 37 , 38 ]. Early NAC intervention prevented HCC induction more efficiently than NAC intervention after initiation stage. The result of liver damage enzymes (ALT and AST) also proved our histopathological observation. Early NAC intervention decreased the ALT and AST levels more significantly compared to the mice treated with NAC after 16 weeks of HCC onset. The same pattern was observed in the PIIINP results. PIIINP was significantly elevated in the serum of HCC-induced mice, which is a demonstrator of fibrogenesis and type III collagen synthesis during HCC induction [ 39 , 40 ]. PIIINP was more readily decreased in mice treated with NAC at early stage of HCC. Har-Zahav et al. showed that Type III collagen synthesis decreased with NAC intervention because of CD44 decline, which is crucial in various pathological processes such as immune response, wound healing, and cancer metastasis [ 3 ]. Early NAC intervention alleviated serum ALT and AST levels, indicating the direct effects of NAC on hepatocyte injury. As an antioxidant, NAC exerts hepatoprotective effects against injuries, chronic healing response, fibrogenesis, and HCC induction [ 7 ]. This also led to a decrease in serum PIIINP shown in 8-week-old mice treated with NAC. NAC did not significantly decrease PIIINP in late intervention, which suggests that after the initiation stage of HCC induction, antioxidants are less efficient compared to the condition where NAC is administered at earlier stages of HCC induction. Increased Ki67 expression, a cell proliferation marker, in the HCC group was modulated in mice treated with NAC at early stage. Although the Ki67 marker in late intervention was reduced, there was a remarkable difference between the effectiveness of the two intervention groups. Kim et al. showed that NADPH oxidase 4 (NOX4) knockout as a ROS producer accelerated HCC induction, whereas it attenuated fibrosis [ 41 ]. Aligning with this study, NAC treatment in early weeks decreased fibrosis-related markers, but controversially, it also inhibited HCC progression in line with the Hsiao et al. study that investigated the effect of GSH treatment [ 42 ]. In this regard, fibrosis, as a less complicated feature, is attenuated by antioxidant interventions, but HCC showed different responses by exposure to increased antioxidants. There are different mechanisms of action of NAC on tissue fibrosis and cancer development. Improvement of antioxidant system is probably the most relevant changes assigned to NAC properties. It has also been reported that NAC decreases P53 expression in the mouse liver as a tumor suppressor gene [ 3 , 43 ]. Late intervention with NAC was less efficient than early intervention, although it efficiently improved the redox balance. The results of oxidative stress-related indices showed that NAC increased antioxidant capacity. In the HCC group, total oxidative stress was increased, following the decrease in TAC and GSH, which were utterly compensated by NAC intervention in both groups treated with NAC. NAC is transported to hepatocytes and promotes GSH synthesis. Cysteine provided by NAC directly promotes the detoxification of ROS. NAC also upregulates nuclear factor erythroid 2-related factor 2 (Nrf2), as a transcription factor, and expression of antioxidative enzymes including quinone oxidoreductase (NQO1), γ-glutamatecysteine ligase (γ-GCS), and heme oxygenase-1 (HO-1), and facilitates glutathione synthesis [ 35 ]. According to Qiu et al, the effects of NAC showed that early NAC administration to the wild-type (WT) mice (starting at 1 month) effectively prevented HCC initiation, while late treatment (starting at 5 months) had no impact on tumor prevention [ 4 ]. Accumulating studies have shown that antioxidants even have tumor-promoting effects [ 1 , 44 ]. More molecular and biochemical studies are crucial to a better understanding of the impact of NAC on various cellular and molecular changes that occur during HCC development. Conclusion Overall, our data show that early treatment with NAC during HCC induction can improve liver fibrosis and cancer through antioxidant system. However, NAC intervention at later stages of HCC development encounters multiple molecular and cellular pathways with less therapeutic efficiency. Hence, treatment with NAC at early stages of HCC where oxidative stress is seriously disturbed is beneficial in prevention of tumor development. Abbreviations NAC N-acetylcysteine HCC Hepatocellular carcinoma DEN Diethylnitrosamine PB Phenobarbitone PIIINP N-terminal propeptide of type III collagen TAC Total Antioxidant Capacity GSH Glutathione AST Aspartate aminotransferase ALT Alanine aminotransferase ROS Reactive Oxygen Species HO• Hydroxyl radical CAT Catalase GPx Glutathione peroxidase SOD Superoxide dismutase α-SMA Alpha-smooth muscle actin TGF-β Transforming growth factor-beta FRAP Ferric reducing antioxidant power TPTZ 2,4,6-Tris(2-pyridyl)-s-triazine FFPE Formalin-fixed paraffin-embedded IHC Immunohistochemistry PBS Phosphate-buffered saline IL-6 Interleukin-6 STAT3 Signal Transducer and Activator of Transcription 3 EMT Epithelial-mesenchymal transition IL-22 Interleukin-22 HGF Hepatocyte growth factor Nrf2 Nuclear factor erythroid 2-related factor 2 NQO1 NAD(P)H quinone dehydrogenase 1 γ-GCS Gamma-glutamylcysteine synthetase HO-1 Heme oxygenase-1 WT Wild type Declarations Ethics approval All animal experiments and methods were carried out in accordance with the relevant guidelines of the Experimental Animal Care and Use Committee of Tarbiat Modares University (Code number: IR.MODARES.AEC.1402.025, date: 30/09/2023). Data Availability Statement The supporting data for the present study findings are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests Funding This study was funded by the Iran National Science Foundation (INSF) under grant number 4026761 and performed as Majid Jafari-Khorchani's PhD thesis in the Clinical Biochemistry Department at Tarbiat Modares University. Authors' contributions MJK and MJZM induced the HCC model, performed the experiments, and wrote the manuscript. AA conceived the concept, theorized the project, and interpreted the data. 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Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7032706","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":524729896,"identity":"44eb37ee-ab79-4a8d-8ee3-f08c0d6f9eb5","order_by":0,"name":"Majid Jafari-Khorchani","email":"","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":false,"prefix":"","firstName":"Majid","middleName":"","lastName":"Jafari-Khorchani","suffix":""},{"id":524729897,"identity":"e3f4f7b3-e321-4034-9061-2bcb83792036","order_by":1,"name":"Mohammad-Jalil Zare-Mehrjardi","email":"","orcid":"","institution":"Tarbiat Modares 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13:50:35","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126533,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7032706/v1/7e75ef7f0ea5f319d620c95c.html"},{"id":92954554,"identity":"066a7d43-47ff-4ee4-bb77-c317291399fd","added_by":"auto","created_at":"2025-10-07 13:50:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":38599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe diagrammatic representation of the experimental procedure. \u003c/strong\u003eHCC was induced in mice at 14 days old following a single intraperitoneal i.p. injection of DEN with PB dissolved in drinking water. This study comprises 4 groups, including: 1) Control group, 2) HCC group received DEN (i.p. 50 mg/kg) and phenobarbitone (500 mg/ L drinking water), 3) The early NAC intervention group received DEN, PB, and NAC (150 mg/kg NAC via gavage from the 8\u003csup\u003eth\u003c/sup\u003e \u0026nbsp;to the 16\u003csup\u003eth\u003c/sup\u003e \u0026nbsp;week), and 4) late NAC intervention group received DEN, PB, and NAC (150 mg/kg NAC from the 16\u003csup\u003eth\u003c/sup\u003e \u0026nbsp;to the 24\u003csup\u003eth\u003c/sup\u003e \u0026nbsp;month). HCC: Hepatocellular carcinoma, DEN: Diethylnitrosamine, NAC: N-acetylcysteine.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7032706/v1/620c6bc983f7707b77d5671b.png"},{"id":92954555,"identity":"683100d9-7120-4836-997e-1f01ae51ce45","added_by":"auto","created_at":"2025-10-07 13:50:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":147324,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of body and liver weights in mice\u003c/strong\u003e \u003cstrong\u003etreated with NAC during hepatocellular carcinogenesis (HCC). \u003c/strong\u003eA) Mean body weight and B) Mean liver weight were recorded in different groups at 28\u003csup\u003eth\u003c/sup\u003e week. HCC was induced in mice at 14 days old following a single i.p. injection of DEN with PB dissolved in drinking water. The early NAC intervention group received 150 mg/kg NAC daily via gavage from the 8\u003csup\u003eth\u003c/sup\u003e to the 16\u003csup\u003eth\u003c/sup\u003e week, while the late NAC intervention group received the same dose of NAC from the 16\u003csup\u003eth\u003c/sup\u003e to the 24\u003csup\u003eth\u003c/sup\u003e week. Data are presented as mean ± SD. Statistical significance is indicated as follows: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7032706/v1/2d85e58129960b128eacfbf3.png"},{"id":92955486,"identity":"78a27ac7-04e4-4ef2-902c-d568c8f14b9e","added_by":"auto","created_at":"2025-10-07 13:58:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":469633,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of NAC intervention on liver tissue histopathology. \u003c/strong\u003eHepatocellular carcinoma (HCC) was induced in mice at 14 days old following a single i.p. injection of DEN with PB dissolved in drinking water. The early NAC intervention group received 150 mg/kg NAC daily via gavage from the 8\u003csup\u003eth\u003c/sup\u003e to the 16\u003csup\u003eth\u003c/sup\u003e week, while the late NAC intervention group received the same dose of NAC from the 16\u003csup\u003eth\u003c/sup\u003e to the 24\u003csup\u003eth\u003c/sup\u003e week. (A) The control group shows normal morphology in the liver cell and nucleus. (B) The HCC group depicts focal cellular proliferation (dotted line) accompanied by atypical nuclei(a) and anisokaryosis. (C) The early NAC intervention group indicates slight atypical nuclei (b). (D) The late NAC intervention group demonstrated numerous atypical nuclei (c) and multinuclear cells. Magnification: ×10, scale bar: 100 µm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7032706/v1/3a20878755826fe1ad2c46f2.png"},{"id":92954562,"identity":"c2664597-6510-41b5-b280-4bca887b08a0","added_by":"auto","created_at":"2025-10-07 13:50:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":360110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of NAC intervention on liver cell proliferation during hepatocellular carcinoma (HCC) development\u003c/strong\u003e. A) shows Ki-67 protein expression using Immunohistochemistry\u003cstrong\u003e (\u003c/strong\u003eIHC). HCC was induced in mice at 14 days old following a single i.p. injection of DEN with PB dissolved in drinking water. The early NAC intervention group received 150 mg/kg NAC daily via gavage from the 8\u003csup\u003eth\u003c/sup\u003e to the 16\u003csup\u003eth\u003c/sup\u003e week, while the late NAC intervention group received the same dose of NAC from the 16\u003csup\u003eth\u003c/sup\u003e to the 24\u003csup\u003eth\u003c/sup\u003e week. Arrowheads show Ki-67-positive cells. B) shows the Ki-67 protein expression (%) recorded by positive cells counting.\u0026nbsp; Data are presented as mean ± SD. Statistical significance is indicated as follows: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001. Magnification: ×10, Scale bars = 100 μm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7032706/v1/4274371387ee05bd3820c9cc.png"},{"id":92954558,"identity":"95f0160b-a1af-453e-b7ce-f56a5cb215c6","added_by":"auto","created_at":"2025-10-07 13:50:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":128875,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of NAC intervention on liver damage markers during hepatocellular carcinogenesis (HCC).\u003c/strong\u003e A) ALT and b) AST were measured in different groups at 28\u003csup\u003eth\u003c/sup\u003e week. HCC was induced in mice at 14 days old following a single i.p. injection of DEN with PB dissolved in drinking water. The early NAC intervention group received 150 mg/kg NAC daily via gavage from the 8\u003csup\u003eth\u003c/sup\u003e to the 16\u003csup\u003eth\u003c/sup\u003e week, while the late NAC intervention group received the same dose of NAC from the 16\u003csup\u003eth\u003c/sup\u003e to the 24\u003csup\u003eth\u003c/sup\u003e week.\u0026nbsp; Data are presented as mean ± SD. Statistical significance is indicated as follows: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7032706/v1/c482aec90edcb150e879ac44.png"},{"id":92955767,"identity":"d72971f7-4e5c-455f-94f1-4cd1a9028f99","added_by":"auto","created_at":"2025-10-07 14:06:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":61253,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of NAC intervention on serum levels of Procollagen III N-Terminal Peptide (PIIINP) during hepatocellular carcinogenesis (HCC)\u003c/strong\u003e. Serum PIIINP was measured via ELISA. HCC was induced in mice at 14 days old following a single i.p. injection of DEN with PB dissolved in drinking water. The early NAC intervention group received 150 mg/kg NAC daily via gavage from the 8\u003csup\u003eth\u003c/sup\u003e to the 16\u003csup\u003eth\u003c/sup\u003e week, while the late NAC intervention group received the same dose of NAC from the 16\u003csup\u003eth\u003c/sup\u003e to the 24\u003csup\u003eth\u003c/sup\u003e week. Data are presented as mean ± SD. Statistical significance is indicated as follows: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7032706/v1/09ceb9b7848136cc3dc2c0d0.png"},{"id":92954560,"identity":"5bbee45b-6295-4cc7-8dff-5cd5125e19fd","added_by":"auto","created_at":"2025-10-07 13:50:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":216373,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in oxidative stress-related factors in liver and serum of HCC mice following NAC intervention. \u003c/strong\u003eA) Liver total oxidant stress, B) Liver GSH content, and C) Serum total antioxidant capacity (TAC) were measured in experimental groups at 28\u003csup\u003eth\u003c/sup\u003e week. HCC was induced in mice at 14 days old following a single i.p. injection of DEN with PB dissolved in drinking water. The early NAC intervention group received 150 mg/kg NAC daily via gavage from the 8\u003csup\u003eth\u003c/sup\u003e to the 16\u003csup\u003eth\u003c/sup\u003e week, while the late NAC intervention group received the same dose of NAC from the 16\u003csup\u003eth\u003c/sup\u003e to the 24\u003csup\u003eth\u003c/sup\u003e week. Data are presented as mean ± SD. Statistical significance is indicated as follows: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7032706/v1/310e2b033d7a094e63b8589e.png"},{"id":93793069,"identity":"3ae68b0c-2f9e-4412-b44f-52289735bee6","added_by":"auto","created_at":"2025-10-17 15:24:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2525169,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7032706/v1/8f3f6214-bd63-4ea6-8e65-62f176e63fa7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Efficiency of early intervention by N-acetyl cysteine on liver fibrosis and markers of hepatocellular carcinogenesis induced by diethylnitrosamine in mice","fulltext":[{"header":"Background","content":"\u003cp\u003eAntioxidant supplements are commonly used, with some studies suggesting potential anticancer properties. However, the efficacy and safety of antioxidant supplementation remain controversial, both in healthy individuals and in cancer patients [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Under normal conditions, cellular processes generate reactive oxygen species (ROS), which are neutralized by antioxidants to maintain genomic and protein integrity. In cancer cells, elevated metabolic activity leads to increased ROS production. To survive, cancer cells upregulate antioxidant defenses, enabling proliferation[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eN-acetylcysteine is a hepatoprotective drug that functions as a precursor of cellular glutathione that subsides glutathione depletion during hepatotoxicity. NAC, by increasing cellular GSH, can protect liver against oxidative stress; Subsequently, NAC acts as a potent antioxidant factor. NAC is widely used as an alleviator of different diseases through the increase of glutathione. NAC is demonstrated to indicate antitumor properties in several cancers. However, NAC also promotes the growth of multiple cancers [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. NAC is a well-known antidote to the toxicity of acetaminophen. It has been widely investigated for liver injuries; however, there are many conflicts regarding its beneficial effects and underlying mechanisms [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNAC is often prescribed for treatment purposes following drug toxicity. Different therapeutic effects have been assigned to NAC. NAC is an N-acetylated derivative of the natural amino acid L-cysteine. It is N-acetyl-L-amino acid and classified as an acetylcysteine derivative. It is a conjugate acid of N-acetyl-L-cysteinate. It has a role as an antidote to paracetamol poisoning [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], an anti-infective agent, an antioxidant, an antiviral drug [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], a mucolytic actions, a radical scavenger, a ferroptosis inhibitor and a geroprotector [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNAC is widely used to alleviate various diseases through glutathione elevation and has demonstrated antitumor properties in several cancers [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, some evidences also show that NAC might promote the growth of certain malignancies. It has been reported that NAC exacerbated tumor growth in lung cancer and melanoma. These studies stated that low ROS levels might be an advantage for tumor cells. NAC reduces ROS, DNA damage, and p53, leading to exacerbating lung cancer cell growth and increasing invasion of human melanoma cells [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe mechanism of action of NAC is generally directly or by increasing intracellular GSH, especially in hepatic tissue. Regarding direct elimination capacity of ROS, NAC participates in the detoxification and reacts with highly oxidative radicals such as hydroxyl radicals (HO\u0026bull;). Moreover, NAC can act as a chelating agent in transition metals, including Cu\u0026sup2;⁺ and Fe\u0026sup3;⁺, and heavy metals such as Cd\u0026sup2;⁺, Hg\u0026sup2;⁺, and Pb\u0026sup2;⁺ via thiol side chain, which forms complexes that are simply excreted. Although NAC is able to directly scavenge ROS, its reaction rates are lower than antioxidant enzymes such as Superoxide dismutase, catalase (CAT), and Glutathione peroxidase (GPx). Therefore, the direct capacity of ROS elimination by NAC is not as significant as its broader antioxidant activity [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOxidative stress, which is considered a hallmark of liver cancer, appears at early stages of chemically induced hepatocellular carcinoma (HCC) in mice, but oxidative stress factors and related cellular damages remain active throughout the carcinogenesis process [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. It was therefore assumed that intervention in HCC by NAC could ameliorate oxidative stress-related damage in liver and provide protective condition for subsequent damage. NAC, by improving antioxidant system, both enzymatic and non-enzymatic factors, can help suppress cancer cells and reduce drug resistance in tumor cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Regardless of the etiological factors in HCC, this malignancy is often associated with liver fibrosis, for which NAC is also effective in preventing tissue fibrosis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], by inhibition of the expression of fibrogenic markers such as α-SMA and TGF-β, leading to decreased extracellular matrix accumulation and fibrosis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe present study aimed to investigate the efficiency of NAC on chemically induced HCC in mice in the protection of liver damage related to tumor formation with emphasis on the antioxidant system. For this purpose, HCC was induced in a period of 7 months, which was observed by sequential changes through oxidative stress damage, liver fibrosis and HCC development. The efficiency of NAC intervention on HCC induction was compared at two-time schedules as early and late intervention.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eAdult male and female C57BL/6 mice were purchased from Iran University of Medical Sciences, Tehran, Iran. Adult mice were mated and the newborns (Body weight 8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 g; 2-week-old) were used. Mice were kept at 22\u0026ndash;25\u0026deg;C, four mice per cage, on a 12-hour light/dark cycle, with free access to water and standard chow diet.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperimental design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn this study, HCC was induced in newborn C57BL/6 mice by DEN and PB treatments as described earlier [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Briefly, 14-day-old mice were randomly divided into four groups: 1) Control group (C), 2) HCC group received DEN and phenobarbital. Groups 3 and 4 were treated with NAC as described below. Each mouse (14 days old) was treated with a single i.p dose of 50 mg/kg of DEN (Sigma-Aldrich, USA), dissolved in 0.6% normal saline, into both sides of the peritoneal cavity. DEN injection was followed by PB administration that was dissolved in drinking water (500 mg/L) from 4 weeks after birth for a period of six months (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eNAC intervention during DEN-induced HCC in mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWherever indicated, each mouse received 150 mg/kg body weight of NAC (dissolved in Distilled Water) for 8 weeks via gavage. Two groups of mice were subjected to NAC treatment. The early Intervention group received NAC from the age of 2 months (6 weeks after DEN administration), and the late Intervention group received NAC treatments at the age of 4 months (14 Weeks after DEN treatment).\u003c/p\u003e\u003cp\u003eDevelopment of liver fibrosis and HCC was monitored by histopathological examination carried out on few mice sacrificed at different time points. HCC induction was confirmed in mice treated for 7 months. Then, mice were anesthetized through i.p. injection of ketamine (100 mg/kg) and xylazine (20 mg/kg). Blood specimen was collected by cardiac puncture, serum was separated and stored at -80\u0026deg;C. Mice were then sacrificed by cervical dislocation, and liver tissue was removed, washed, and weighed. A portion of the liver tissue was fixed in 4% paraformaldehyde for histopathological analysis via hematoxylin and eosin (H\u0026amp;E) staining. The remaining liver tissue was immediately frozen at -80\u0026deg;C for molecular and biochemical analyses.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of liver homogenate\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter measuring the weight of the liver tissue, 100 mg of each frozen sample was homogenized in 1 mL of normal saline to prepare a 10% (mg/mL) homogenate. The homogenate was centrifuged at 10,000 \u0026times; g for 10 minutes at 4\u0026deg;C, and the clear supernatant was collected and stored for future analyses.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEstimation of serum N-terminal propeptide type III collagen (PIIINP)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSerum level of PIIINP, as the marker of tissue fibrosis was determined using a mouse-specific ELISA kit (Zellbio, Germany, Cat. # ZB-10691C-M9648). The assay was performed according to the manufacturer's instructions. In brief, the kit employs a sandwich ELISA technique in microplate wells pre-coated with PIIINP-specific antibodies. Serum samples were added to the wells, followed by a biotinylated antibody specific to PIIINP and an avidin-horseradish peroxidase conjugate. After incubation and washing steps, a chromogenic substrate was added and the enzymatic reaction was terminated with an acidic stop solution. The resulting color intensity was measured spectrophotometrically at 450 nm. PIIINP levels in the samples were quantified based on a standard curve prepared by different concentrations of standard solution provided in the assay kit.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEstimation of biochemical markers in serum and liver homogenate\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGlutathione [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] levels was determined in liver homogenates using commercially available colorimetric assay kit (ZellBio GmbH, Cat. # ZB-GSH-69A) following the manufacturer's instructions. Briefly, liver tissue samples were homogenized and centrifuged. Samples and standards were added to a microplate, followed by the addition of assay reagents. After incubation, absorbance was measured at 405 nm. GSH concentrations were calculated using the standard curve and normalized by protein concentration of respective liver homogenate.\u003c/p\u003e\u003cp\u003eTotal oxidative stress levels were estimated in liver homogenates using a commercially available colorimetric assay kit (Navand Salamat, Cat. # NS-15017, Iran), following the manufacturer's protocol.\u003c/p\u003e\u003cp\u003eTotal antioxidant capacity (TAC) was estimated in serum samples by FRAP assay according to Benzie and Strain procedure [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This assay is based on the reduction of TPTZ-Fe3\u003csup\u003e+\u003c/sup\u003e to TPTZ-Fe2\u003csup\u003e+\u003c/sup\u003e by the antioxidant capacity of samples. The color change of the reaction was measured at a wavelength of 593 nm and the total antioxidant capacity was calculated based on a FeSO4 standard curve.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEstimation of liver damage enzyme markers (ALT and AST)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAlanine aminotransferase (ALT) and aspartate aminotransferase (AST), were estimated in serum samples using colorimetric methods. The analysis was performed with commercially available assay kits.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression of Ki-67 in liver tissue (immunohistochemistry)\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eImmunohistochemical (IHC) technique was performed on formalin-fixed, paraffin-embedded (FFPE) tissue sections. Tissue sections were mounted on poly-L-lysine-coated slides and deparaffinized. Endogenous peroxidase activity was quenched by immersing slides in 10% hydrogen peroxide solution in PBS for 10 minutes. Antigen retrieval was achieved by heating in sodium citrate buffer (10 mM, pH 6) at 95\u0026deg;C for 30 minutes. Following PBS washes, sections were incubated with Ki-67 antibody (MAD-000310QD-3, Master Diagn\u0026oacute;stica, Spain 1:300) for 50 minutes at room temperature. After two PBS washes, slides were treated with secondary antibodies (MAD-000237Q, Master Diagn\u0026oacute;stica, Spain dilution 1:1000) for 45 minutes at room temperature. Immunoreactivity was visualized using 3,3\u0026prime;-Diaminobenzidine as the chromogen, applied for 10 minutes at room temperature. Slides were then washed with tap water, counterstained with Mayer's hematoxylin, mounted, and examined under a light microscope for Ki-67 expression analysis.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll assays were performed in duplicate, and data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analysis was conducted using GraphPad Prism software (version 8.0.1). Differences in parameters among the experimental groups were evaluated using one-way ANOVA followed by Tukey's post hoc test. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all analyses.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eBody weight and liver weight\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, body weight data have no significant differences between all groups. In the case of liver weight, there was a substantial increase in the HCC group compared to the control group (122%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Early NAC intervention reduced liver (25%) compared to the HCC group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, the liver weight in the late-treated group remained similar to the HCC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHistopathological data of liver tissues with H\u0026amp;E staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMicroscopic examination of H\u0026amp;E-stained liver preparations showed normal morphology in the control group. In the HCC group, atypical nuclei, focal proliferation, and anisokaryosis were observed. Liver of early NAC treatment mice still showed slight atypical nuclei, but focal proliferation was not visible in this group. In the liver of late NAC treatment mice, numerous atypical nuclei and multinuclear cells were observable (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of NAC treatment on expression of liver Ki-67 (IHC)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe Ki67-positive cells in liver tissue of the HCC group were higher than in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Early treatment with NAC significantly reduced the proportion of Ki67-positive cells compared to the HCC and late treatment groups (82%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and 69%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively). Interestingly, the late NAC-treated group demonstrated a reduction in Ki67-positive cells compared to the HCC group (44%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of NAC intervention on serum ALT and AST levels\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSerum ALT in the HCC group was significantly increased (~\u0026thinsp;275%) as compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Early NAC intervention significantly decreased the ALT by about 50% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The decrease in the ALT in the early-treated group was also significant when compared to the late-treated group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSerum AST was increased by 88% in the HCC group compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Whereas, it was decreased by about 26% and 16% in the early NAC treatment group compared to the HCC and late NAC treatment groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of NAC intervention on serum PIIINP (N-terminal propeptide type III collagen)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSerum PIIINP level was increased by 145% in the HCC group treated with DEN and PB compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). NAC intervention during HCC development resulted in a significant decrease in PIIINP in mice treated with NAC compared to the HCC group. NAC treatment in the HCC group at the early stage resulted in 38% and 33% decrease of serum PIIINP when compared to that measured in the HCC group and late NAC intervention groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of NAC treatment on total oxidative stress in liver homogenate\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDuring HCC development, there was a significant increase (about 50%) in hepatic total oxidative marker as compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Early and late administration of NAC during HCC development in mice resulted in a significant decrease in total oxidative status compared to the untreated HCC group (34% and 38%, respectively, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eChanges in liver GSH content in NAC-treated mice.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHepatic GSH content was depleted by 38% in the HCC group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). NAC treatment at early and late stages of HCC development resulted in recovery of hepatic GSH by approximately 55% and 76%, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of NAC treatment on serum Total Antioxidant Capacity (FRAP assay)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSerum TAC was significantly declined (36% reduction) during the HCC development in mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003cp\u003eTreatment with NAC at the early stage of HCC development increased serum TAC by approximately 41% compared to that measured in the HCC group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). TAC in the late NAC-treated group was significantly increased vs the HCC group by about 43% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThere are several reports on the role of NAC and its redox system in preventing and delaying liver cancer in animal models [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In the present study, attempts were made to examine the effects of NAC intervention during the chemically-induced hepatocellular carcinogenesis with emphasis on oxidative stress factors and liver fibrosis markers.\u003c/p\u003e\u003cp\u003eNAC, as the precursor of glutathione, is also widely used to alleviate various diseases through glutathione elevation and has demonstrated antitumor properties in several cancers [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, depending on the experimental protocols, particularly the time schedules of intervention, NAC may also promote the growth of certain malignancies [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It has been reported that NAC exacerbated tumor growth in lung cancer and melanoma[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Perhaps, in the case of early intervention in HCC development with NAC, the oxidative stress cascade is potentially important in the initiation process of tumorigenesis. In this stage, oxidative stress-related tissue damage probably imposes an accelerating influence on the exacerbation of HCC induction. Nevertheless, when NAC is given at later stages, the stage known as tumor progression, other pathways will be involved, such as multiple gene mutations and EMT which are complicated to be reversed by antioxidants.\u003c/p\u003e\u003cp\u003eIn this study, the effects of NAC intervention were compared at two different time schedules during HCC induction in mice. Microscopic examination of the liver in the Treatment of HCC group treated with NAC at the early stage of HCC showed some cellular damage. However, the nuclei had no signs of atypical features and were nearly uniform. At late NAC intervention stage, some atypical nuclei were noted, suggesting the initiation stage of HCC induction [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHistopathological data showed that focal cell proliferation in the HCC group was due to the HCC initiator feature of DEN and the promoter feature of PB. DEN, an electrophilic species, is subject to in vivo intricate chemical transformations and can interact with DNA bases to yield O6-ethylguanine [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. DEN can stimulate the secretion of interleukin-6 (IL-6) from Kupffer cells (KCs). IL-6 is implicated in the pathogenesis of HCC through a mechanism reliant on signaling by STAT3 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. During this progression, the concentrations of lipids and glycogen and various enzymes associated with glycolytic processes, the oxidative pentose phosphate pathway, glycogen catabolism, and cell membrane function are impaired. All these alterations are linked mainly to the neoplastic transformation of hepatocytes. DEN is independently effective at inducing hepatocellular carcinoma in mouse models. Still, hepatotoxic substances like phenobarbital, which affect the expression of interleukin-22 (IL-22), hepatocyte growth factor (HGF), or macrophage inflammatory protein-1, increase the carcinogenicity of DEN [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEarly NAC intervention prevented HCC induction more efficiently than NAC intervention after initiation stage. The result of liver damage enzymes (ALT and AST) also proved our histopathological observation.\u003c/p\u003e\u003cp\u003eEarly NAC intervention decreased the ALT and AST levels more significantly compared to the mice treated with NAC after 16 weeks of HCC onset. The same pattern was observed in the PIIINP results. PIIINP was significantly elevated in the serum of HCC-induced mice, which is a demonstrator of fibrogenesis and type III collagen synthesis during HCC induction [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. PIIINP was more readily decreased in mice treated with NAC at early stage of HCC. Har-Zahav et al. showed that Type III collagen synthesis decreased with NAC intervention because of CD44 decline, which is crucial in various pathological processes such as immune response, wound healing, and cancer metastasis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEarly NAC intervention alleviated serum ALT and AST levels, indicating the direct effects of NAC on hepatocyte injury. As an antioxidant, NAC exerts hepatoprotective effects against injuries, chronic healing response, fibrogenesis, and HCC induction [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This also led to a decrease in serum PIIINP shown in 8-week-old mice treated with NAC. NAC did not significantly decrease PIIINP in late intervention, which suggests that after the initiation stage of HCC induction, antioxidants are less efficient compared to the condition where NAC is administered at earlier stages of HCC induction.\u003c/p\u003e\u003cp\u003eIncreased Ki67 expression, a cell proliferation marker, in the HCC group was modulated in mice treated with NAC at early stage. Although the Ki67 marker in late intervention was reduced, there was a remarkable difference between the effectiveness of the two intervention groups.\u003c/p\u003e\u003cp\u003eKim et al. showed that NADPH oxidase 4 (NOX4) knockout as a ROS producer accelerated HCC induction, whereas it attenuated fibrosis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Aligning with this study, NAC treatment in early weeks decreased fibrosis-related markers, but controversially, it also inhibited HCC progression in line with the Hsiao et al. study that investigated the effect of GSH treatment [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In this regard, fibrosis, as a less complicated feature, is attenuated by antioxidant interventions, but HCC showed different responses by exposure to increased antioxidants. There are different mechanisms of action of NAC on tissue fibrosis and cancer development. Improvement of antioxidant system is probably the most relevant changes assigned to NAC properties. It has also been reported that NAC decreases P53 expression in the mouse liver as a tumor suppressor gene [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLate intervention with NAC was less efficient than early intervention, although it efficiently improved the redox balance. The results of oxidative stress-related indices showed that NAC increased antioxidant capacity. In the HCC group, total oxidative stress was increased, following the decrease in TAC and GSH, which were utterly compensated by NAC intervention in both groups treated with NAC. NAC is transported to hepatocytes and promotes GSH synthesis. Cysteine provided by NAC directly promotes the detoxification of ROS. NAC also upregulates nuclear factor erythroid 2-related factor 2 (Nrf2), as a transcription factor, and expression of antioxidative enzymes including quinone oxidoreductase (NQO1), γ-glutamatecysteine ligase (γ-GCS), and heme oxygenase-1 (HO-1), and facilitates glutathione synthesis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAccording to Qiu et al, the effects of NAC showed that early NAC administration to the wild-type (WT) mice (starting at 1 month) effectively prevented HCC initiation, while late treatment (starting at 5 months) had no impact on tumor prevention [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Accumulating studies have shown that antioxidants even have tumor-promoting effects [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. More molecular and biochemical studies are crucial to a better understanding of the impact of NAC on various cellular and molecular changes that occur during HCC development.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOverall, our data show that early treatment with NAC during HCC induction can improve liver fibrosis and cancer through antioxidant system. However, NAC intervention at later stages of HCC development encounters multiple molecular and cellular pathways with less therapeutic efficiency. Hence, treatment with NAC at early stages of HCC where oxidative stress is seriously disturbed is beneficial in prevention of tumor development.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eNAC\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eN-acetylcysteine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eHCC\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHepatocellular carcinoma\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eDEN\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDiethylnitrosamine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003ePB\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePhenobarbitone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003ePIIINP\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eN-terminal propeptide of type III collagen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eTAC\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTotal Antioxidant Capacity\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eGSH\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGlutathione\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eAST\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAspartate aminotransferase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eALT\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAlanine aminotransferase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eROS\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eReactive Oxygen Species\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eHO\u0026bull;\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHydroxyl radical\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eCAT\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCatalase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eGPx\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGlutathione peroxidase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eSOD\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSuperoxide dismutase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eα-SMA\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAlpha-smooth muscle actin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eTGF-β\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTransforming growth factor-beta\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eFRAP\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFerric reducing antioxidant power\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eTPTZ\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e2,4,6-Tris(2-pyridyl)-s-triazine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eFFPE\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFormalin-fixed paraffin-embedded\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eIHC\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eImmunohistochemistry\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003ePBS\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePhosphate-buffered saline\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eIL-6\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInterleukin-6\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eSTAT3\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSignal Transducer and Activator of Transcription 3\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eEMT\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEpithelial-mesenchymal transition\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eIL-22\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInterleukin-22\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eHGF\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHepatocyte growth factor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eNrf2\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNuclear factor erythroid 2-related factor 2\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eNQO1\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNAD(P)H quinone dehydrogenase 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eγ-GCS\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGamma-glutamylcysteine synthetase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eHO-1\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHeme oxygenase-1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eWT\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eWild type\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments and methods were carried out in accordance with the relevant guidelines of the Experimental Animal Care and Use Committee of Tarbiat Modares University (Code number: IR.MODARES.AEC.1402.025, date: 30/09/2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe supporting data for the present study findings are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Iran National Science Foundation (INSF) under grant number 4026761 and performed as Majid Jafari-Khorchani's PhD thesis in the Clinical Biochemistry Department at Tarbiat Modares University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMJK and MJZM\u0026nbsp;induced the HCC model, performed the experiments, and wrote the manuscript. AA conceived the concept, theorized the project, and interpreted the data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully appreciate the Iran National Science Foundation (INSF), Tehran, Iran, for supporting this study (Grant number 4026761). s\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang VX, Sze KM-F, Chan L-K, Ho DW-H, Tsui Y-M, Chiu Y-T, Lee E, Husain A, Huang H, Tian L: Antioxidant supplements promote tumor formation and growth and confer drug resistance in hepatocellular carcinoma by reducing intracellular ROS and induction of TMBIM1. \u003cem\u003eCell \u0026amp; bioscience \u003c/em\u003e2021, 11:1-20.\u003c/li\u003e\n\u003cli\u003eKaiser J: Antioxidants could spur tumors by acting on cancer gene. In \u003cem\u003eBook Antioxidants could spur tumors by acting on cancer gene\u003c/em\u003e (Editor ed.^eds.). 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In \u003cem\u003eInternational Liver Cancer Association (ILCA) Annual Conference 2021\u003c/em\u003e. International Liver Cancer Association (ILCA). 2021\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HCC, NOX4, PIIINP, Oxidative stress, Glutathione, Diethylnitrosamine, Liver fibrosis","lastPublishedDoi":"10.21203/rs.3.rs-7032706/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7032706/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eN-acetylcysteine is a hepatoprotective agent with antioxidant and therapeutic potential. In this study, the effectiveness of early and late intervention with NAC in hepatocellular carcinogenesis (HCC) induced by diethyl nitrosamine (DEN) in mice has been evaluated.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eNewborn mice (14-day-old) were divided into 4 groups (n\u0026thinsp;=\u0026thinsp;4/group). Control, HCC group, early NAC intervention, and late NAC intervention group. NAC treatments were followed after HCC induction by DEN administration (50 mg/kg, i.p), followed by phenobarbitone (PB, 500 mg/L via drinking water). In the early intervention group, NAC (150 mg/kg) was given by gavage during 8\u0026ndash;16 weeks after birth. In the late group intervention, NAC was given during 16\u0026ndash;24 weeks of birth. After 7 months (28 weeks), mice were sacrificed; blood and liver tissues were collected. Liver damage markers, as well as serum levels of liver fibrosis biomarker, PIIINP (N-terminal propeptide type III collagen), and antioxidant capacity (TAC) were determined. Histology examination on liver biopsies, together with changes in tissue total oxidant factors and liver cells proliferation index (Ki67) were determined in liver tissues.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eEarly intervention with NAC in mice during HCC induction resulted in a significant decrease in serum levels of liver damage markers, AST and ALT. This finding was corroborated with liver histology data, particularly tissue fibrosis. Intervention with NAC during HCC progression resulted in a significant decrease in serum PIIINP and hepatic total oxidative stress, GSH, and KI67 expression. NAC treatment also resulted in TAC overregulation.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eEarly treatment with NAC in HCC model of mice can improve liver fibrosis and cancer through antioxidant system. However, NAC intervention at later stages of HCC development encounters multiple molecular and cellular pathways with less therapeutic efficiency. Hence, treatment with NAC at the early stages of HCC, where oxidative stress is seriously disturbed, is beneficial in the prevention of tumor development. NAC intervention at early stage of HCC induction in mice greatly subsided HCC-related liver damage markers, along with liver fibrosis and cancer cell proliferation. This data suggests that NAC can efficiently delay and ameliorate tissue fibrosis and cancer promotion.\u003c/p\u003e","manuscriptTitle":"Efficiency of early intervention by N-acetyl cysteine on liver fibrosis and markers of hepatocellular carcinogenesis induced by diethylnitrosamine in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-07 13:50:30","doi":"10.21203/rs.3.rs-7032706/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cb3d35ea-85e0-4e47-87dd-0f06679d479a","owner":[],"postedDate":"October 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-17T15:23:47+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-07 13:50:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7032706","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7032706","identity":"rs-7032706","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}