Can disulfiram modulate liver injury in CCl4-induced fibrosis in rats?

Abstract

Aim:

This study aimed to evaluate the hepatoprotective and antifibrotic effects of DSF in a rat model of CCl₄-induced liver fibrosis.

Background:

Liver fibrosis is a chronic and potentially life-threatening condition characterized by fibroblast proliferation and excessive extracellular matrix deposition. Oxidative stress and inflammation are central mechanisms in its pathogenesis. Disulfiram (DSF), known for its antioxidant and potential antifibrotic properties, may attenuate liver injury.

Methods:

Thirty-five male Wistar rats were randomly divided into seven groups (n = 5 per group): Control, Oil, DSF 25 mg/kg, DSF 50 mg/kg, CCl₄, CCl₄ + DSF 25 mg/kg, and CCl₄ + DSF 50 mg/kg. After seven weeks of treatment, liver histopathology, serum liver enzymes (ALT, AST, ALP), and malondialdehyde (MDA) levels were assessed.

Results:

CCl₄ administration significantly increased serum ALT and AST levels compared with the Control group (p < 0.001). DSF at 25 mg/kg significantly reduced ALT (p = 0.0265), AST (p = 0.0334), and MDA (p < 0.001) compared with the CCl₄ group. Histopathological analysis revealed improved hepatocyte morphology in the low-dose DSF group (p = 0.0358). However, neither DSF dose produced a significant reduction in collagen deposition or vascular congestion, indicating no clear antifibrotic effect at these doses (>0.9999).

Conclusion:

Low-dose DSF demonstrated hepatoprotective effects through improvements in biochemical markers and hepatocyte morphology, but failed to significantly attenuate fibrosis. These findings suggest that lower DSF concentrations (< 25 mg/kg) may offer greater antifibrotic potential and should be further investigated in dose-response studies.

Introduction

Chronic liver diseases, such as hepatitis and fatty liver disease, lead to hepatocyte damage, resulting in the replacement of parenchymal tissue with fibrosis. The progression of fibrotic liver disease can culminate in liver cirrhosis and hepatocellular carcinoma. Furthermore, chronic portal hypertension, a consequence of liver fibrosis, primarily contributes to clinical complications such as bleeding and encephalopathy (1, 2). While the exact mechanisms underlying the development of liver fibrosis remain unclear, reactive oxygen species (ROS) undoubtedly play a significant role in the pathological alterations in the liver (3). Excessive free radicals can overwhelm the body’s antioxidant defenses and damage the biological molecules, including lipids in the liver cell membranes, which may reduce intracellular antioxidant enzymes (4).

Carbon tetrachloride (CCl₄) is a well-known hepatotoxin that induces liver necrosis, apoptosis, fibrosis, cirrhosis, and cancer. Cytochrome P450 enzymes primarily metabolize CCl₄ into toxic metabolites, such as the trichloromethyl radical (CCl₃), trichloromethylperoxy radical (CCl₃O₂), and phosgene (CCl₂O). Chronic administration of CCl₄ is one of the most prevalent methods employed to induce chronic liver damage (5). Disulfiram (DSF) has been utilized for over 60 years to treat alcoholism without severe side effects. It is currently under investigation in preclinical studies due to its anti-inflammatory properties and its potential role as a cancer-modulatory drug (6-8). DSF has demonstrated protective effects by inhibiting NLRP3 inflammasome activation, thereby modulating lysosomal degradation and ROS production (9). Besides that, DSF may mitigate fibrosis progression by inhibiting epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition, on the one hand, through the activation of the cyclooxygenase-2-prostaglandin E₂ axis, and on the other hand, by blocking gasdermin D cleavage and pyroptosis. It also inhibits macrophage migration and alters the expression of key profibrotic factors. Conversely, studies have shown that long-term administration of high doses of DSF can induce toxic effects on liver cells, manifested as increased levels of liver enzymes (ALT, AST), elevated blood bilirubin, tissue necrosis, and the induction of liver fibrosis (10). Although some recent studies have investigated the effects of DSF on liver fibrosis, their findings have been inconsistent, which may be related to differences in dosage, duration of administration, and experimental design. For example, Yang et al. demonstrated that low-dose DSF could attenuate CCl₄- and BDL-induced fibrosis in rats, primarily through inhibition of hepatic stellate cell activation (11). Other studies have reported DSF’s anti-inflammatory and antioxidant properties in various injury models (12, 13), while some clinical and experimental reports have described its potential hepatotoxicity at higher doses (10, 14). These conflicting results highlight the importance of carefully defining the therapeutic window of DSF. In the present study, we evaluated two different doses of DSF (25 and 50 mg/kg) in a CCl₄-induced liver fibrosis model, suggesting that dose selection may play a crucial role in modulating the hepatic effects of DSF.

Methods

Animal and housing

This study obtained 35 male Wistar rats (220 ± 20 g) from the Animal Center of Babol University of Medical Sciences. The animals were housed in a controlled environment with a temperature maintained at 22°C, subjected to a 12-hour light-dark cycle, and provided unrestricted access to food and water. All experimental procedures were conducted in accordance with approval from the local ethics committee. (Code: IR.MUBABOL.REC.1399.231)

Dosing justification

Disulfiram was administered intraperitoneally at doses of 25 and 50 mg/kg. These doses were selected based on previous preclinical studies investigating DSF’s anti-inflammatory and antifibrotic effects across a broad dose range (4–100 mg/kg) in models of hepatic, pulmonary, and renal injury (11, 15-17).

Experimental design

The rats were randomly divided into seven groups, each consisting of five animals, as follows:

1. Control group: Received no intervention.

2. Oil group: Received intraperitoneal (IP) administration of olive oil (the vehicle for both CCl₄ and DSF) at a volume equivalent to the injection volume used in other groups.

3. DSF 25 group: Received IP administration of DSF (Sigma, Germany) at a dose of 25 mg/kg daily (in a volume of 200 μL olive oil) for seven weeks.

4. DSF 50 group: Received IP administration of DSF at a dose of 50 mg/kg daily (in a volume of 200 μL olive oil) for seven weeks.

5. CCl₄ group: Received IP administration of a 40% (v/v) solution of CCl₄ (Merck, Germany) in olive oil at an injection volume of 1 mL/kg (corresponding to a CCl₄ dose of ~636 mg/kg) twice a week for seven weeks.

6. CCl₄ + DSF 25 group: Received concurrent administration of CCl₄ (as described above) and DSF at a dose of 25 mg/kg (in a volume of 200 μL olive oil), daily for seven weeks.

CCl₄ + DSF 50 group: Received concurrent administration of CCl₄ (as described above) and DSF at a dose of 50 mg/kg (in a volume of 200 μL olive oil), daily for seven weeks.

Following the treatment period, all animals were anesthetized with a combination of ketamine and xylazine, and blood samples were collected from the heart of each animal. The entire liver was excised, with the middle lobe stored at -80°C for tissue extract preparation to measure MDA levels. The right lobe was preserved in 10% formalin for histopathological evaluation.

Liver histology evaluation

The tissue samples were placed in 10% formalin and replaced after 24 hours to ensure adequate fixation. Following proper fixation, tissue processing, and slide preparation, the samples were dehydrated in ascending concentrations of ethyl alcohol, cleared in xylene, and embedded in paraffin. Sections of 5 µm thickness were prepared for microscopic examination. Half of the slides were stained with hematoxylin and eosin (H&E), while the remaining were stained with Masson's trichrome stain.

Histopathological factors, including lobular structure deformation, cytoplasmic vacuolation, presence of necrotic cells near the central vein, vascular congestion, and foci of inflammatory cells, were graded based on the severity of liver tissue damage (0, absent; 1, mild intensity; 2, moderate intensity; 3, high intensity). Liver fibrosis was graded according to the Ishak fibrosis scoring system (0-8 grades) (18-21).

Quantification of hepatic fibrosis

Liver interstitial fibrosis was assessed in Masson’s trichrome–stained kidney sections. Images were acquired at 40× magnification and analyzed using Fiji (ImageJ, NIH, Bethesda, MD, USA). Collagen was isolated through color deconvolution (Masson’s Trichrome vector), and the collagen channel was subjected to fixed thresholding uniformly applied to all sections. For each animal, four randomly selected hepatic fields were analyzed, and the collagen-positive area fraction (%Area) was calculated (Figure 1).

Figure 1.

Quantitative assessment of hepatic fibrosis using Masson’s trichrome staining and Fiji/ImageJ analysis. Liver sections stained with Masson’s trichrome were examined at 40× magnification. (a) Original stained section; (b) collagen channel isolated using color deconvolution; (c) thresholded collagen-positive areas (highlighted in red) used for fibrosis quantification. Hepatic images were analyzed, and the collagen-positive area fraction (% area) was calculated for each specimen.

Measurement of liver enzymes (ALT, AST, and ALP)

At the end of the seven-week treatment period, all animals were anesthetized using ketamine and xylazine. Blood was drawn directly from the heart, and the samples were centrifuged at 3000 rpm/min to obtain serum, then stored at -80°C. The ALT, AST, and ALP levels were measured using a spectrophotometric method with a diagnostic kit, following the manufacturer’s instructions (Pars Azmoon Co., IR).

Measurement of malondialdehyde

The entire liver tissue was excised, and the middle lobe was stored at -80°C for tissue extract preparation to measure MDA levels. MDA levels were quantified using a spectrophotometric method with a diagnostic kit (Pars Azmoon Co., IR).

Data were analyzed using one-way ANOVA, Nested one-way ANOVA, and the nonparametric Kruskal-Wallis test. Statistical analysis was conducted using GraphPad Prism 9:00, with a p-value < 0.05 considered significant.

Results

Histopathology of the livers

The stained sections from each group were prepared using hematoxylin and eosin, as well as Masson's trichrome staining methods. Images were taken with a Microscope Eyepiece Camera and Zistlab software (ara-research.com). The Photos were examined to evaluate lobular structure, hepatocyte morphology, necrotic nuclei, inflammation, congestion, and fibrosis. Table 1 mentions the extent of injury and repair based on qualitative scoring (Table 1).

Table 1.

Qualitative histopathological parameters of the liver in the groups

Groups Histopathology	Control	Oil	DSF25	DSF50	CCL4	DSF25+CCL4	DSF50+CCL4
Disruption of liver lobule structure	-	-	-	+	+++	++	+++
Hepatocytes Vacuolation	-	-	-	+	+++	++	++
Necrotic cells in zone III	-	-	-	-	++	-	+
Inflammatory cell foci	-	-	-	-	-	-	+
Total vascular congestion*	-	+	-	-	+++	+++	+++
sinusoid collapse	-	-	-	-	+++	++	+++
Fibrosis grade	-	-	-	-	+++	+++	+++

C, Control group; Oil, administration of olive oil; DSF 25, receiving DSF 25 mg/kg; DSF 50, receiving DSF at a dose of 50 mg/kg; CCl₄, receiving 40% CCl₄ at a dose of 1 mg/kg; CCl₄ + DSF 25, receiving DSF at a dose of 25 mg/kg concurrently with CCl₄ injection; CCl₄ + DSF 50, receiving DSF at a dose of 50 mg/kg concurrently with CCl₄ injection.

*Total vascular congestion: the average congestion score of the central veins portal veins, and sinusoids

The liver exhibited a typical standard hepatic lobular structure. The nuclei of the hepatocytes showed euchromatin with normal-appearing cytoplasm and no evidence of vacuolization (dilated smooth endoplasmic reticulum) the portal and central veins and sinusoidal areas. In the hepatic zone III, around the central vein, there were no necrotic cells and no accumulation of inflammatory cells. There was no congestion in the portal and central veins and sinusoidal areas, and there was no evidence of sinusoidal collapse and hepatic fibrosis.

Oil Group: The liver tissue maintained its typical structure without cytoplasmic vacuolation. No necrotic cells were detected around the central vein. However, mild vascular congestion was noted in both the portal and central veins, with no evidence of fibrosis development and sinusoidal collapse.

DSF25 Group: Both the lobular structure of the liver tissue and cellular morphology appeared normal, without cytoplasmic vacuolation. No necrotic cells were observed around the central vein, and no inflammatory cell accumulation was found. Congestion was absent in the portal vein, central vein, and sinusoidal space. Fibrosis was also not discernible in the tissues.

DSF50 Group: Mild deformation was noted in the lobular structure and the vacuolation of hepatocytes (+). No necrotic cells were observed around the central vein, and no accumulation of inflammatory cells was found. While no congestion was observed in the portal vein, mild congestion was noted in the central vein (+) in some samples. The sinusoidal space appeared normal, with no signs of congestion. Tissue samples from this group revealed no evidence of fibrosis.

CCL4 Group: The lobular structure was deformed across all slides. Approximately 30% of hepatocytes (++) displayed pyknosis, mostly in zone III, with necrotic nuclei surrounding the central veins. Almost all hepatocytes (+++) showed vacuolation in their cytoplasm (swollen smooth endoplasmic reticulum). A few hepatocytes containing two nuclei were noted along the sinusoids. No accumulation of foci inflammation was observed. Vascular congestion was pronounced in both the portal and central veins (+++), and the sinusoids (++) were enhanced. The sinusoidal space had undergone severe collapse, accompanied by an increased density of type I collagen in the portal space, where grade III fibrosis, characterized by fibrous bridges between two portal triad spaces, was discernible. Furthermore, fibroses are characterized by fibrous bridges between the portal triad and the central vein. Fibrous bridges between the portal triad and central vein characterize a few grade IV fibroses.

CCL4+DFS25 Group: The lobular structure of the liver showed notable improvement compared to the CCL4 group, reaching a moderate score (++). Hepatocyte nuclei were euchromatic with 3-4 nucleoli. The average population of hepatocytes (++) exhibited vacuolar cytoplasm. A few areas of necrosis of hepatocytes around the central vein were noted, and a small proportion of hepatocyte cells contained two nuclei. A limited number of accumulations of inflammatory cells were present. The collapse of the sinusoidal space between hepatocyte cords was classified as severe (+++), accompanied by significant congestion in the portal triads and central veins (+++). In contrast, the sinusoids exhibited moderate congestion. Furthermore, fibrotic changes were observed in almost all cases of grade III fibrosis and a few cases of grade IV fibrosis.

CCL4+DSF50 Group: The lobular structure displayed deformation ranging from average to severe (+++). Mild (+) pyknosis of nuclei was observed in the liver tissues, while most hepatocytes exhibited euchromatic nuclei with more than two nucleoli. A moderate (++) population of hepatocytes showed dilated vacuolar cytoplasm. Both hepatocyte necrosis and mitotic divisions were mild. Inflammatory cell accumulation was also mild. Congestion in both the portal and central veins, as well as sinusoidal congestion, was severe. Notably, fibrotic changes consistent with grade III and one slide grade IV were evident in the samples.

Generally, we can summarize the mentioned alterations in hepatic histopathology parameters as follows: classic liver lobules, as shown in Figure 2; changes in hepatocyte morphology, as depicted in Figure 3; vascular congestion, as illustrated in Figure 4; and fibrosis, as presented in Figure 5, between the studied groups.

Figure 2.

Classic lobule structure in the studied groups. a, b, and c illustrate the classic lobule structure in liver tissue, delineated by the central vein at the core and the portal triad at its periphery, as indicated by the yellow lines and CL. This structure is distinctly visible in the control, D25, and D50 groups (a: control; b: D25; c: D50), while images d, e, and f depict the CCL4, CCL4+D25, and CCL4+D50 groups, respectively. All figures are magnified at 10X, with a scale bar of 50 µm.

Figure 3.

Morphology of hepatocytes in the studied groups. a, b, and c: Images illustrate normal hepatocytes in the liver tissue, identified with yellow circles, unchanging cytoplasm, and euchromatic nuclei presented in the control, D25, and D50 groups, respectively. d: Hepatocyte image showing vacuolated cytoplasm (VH), from the CCL4 group. e and f: Images depict the significantly reduced level of vacuolation within the representative groups of CCL4+D25 and CCL4+D50, respectively. All figures magnify 40X with a scale bar of 50 µm.

Figure 4.

Vascular congestion in the studied groups. a: Empty blood vessels in liver tissue, evidenced by yellow stars, from the control, D25, and D50 groups. In image d, the CCL4 group exhibits noticeable vascular congestion in the central veins (cc) and the portal vein (PC). Conversely, images e and f, representing the CCL4+D25 and CCL4+D50 groups, respectively, show reduced vascular congestion. All figures are magnified at 10X, with a scale bar of 50 µm.

Figure 5.

Fibrosis in the studied groups. a, b, and c demonstrate that no fibrosis in liver tissue is present in the control, D25, and D50 groups. In Figure d, the CCL4 group exhibits grade 3 liver fibrosis, with fibrosis evident between the two portal triads (FP-P). Images e and f depict the CCL4+D25 and CCL4+D50 groups, respectively, where a fvisible. All figures are magnified at 10X, with a scale bar of 50 µm.

Analysis of histopathological parameters

The histopathological analysis of various parameters—including destruction of liver lobules, morphological changes of hepatocytes, degree of vascular congestion, and fibrosis—revealed a significant increase in the CCl4-treated group compared to the control, oil-treated groups, and the D25 and D50 groups (P < 0.0001) (Figure 6). When comparing the level of liver lobular structure destruction between the CCl4 group and the treatment groups (CCl4 + D25 and CCl4 + D50), a reduction in destruction was observed in both treatment groups; however, this reduction was not statistically significant (P 0.6539 for CCl4 + D25 and P > 0.9999 for CCl4 + D50 (Figure 2, Figure 6a).

Figure 6.

Charts related to liver histopathological parameters. a) Destruction of liver lobules, b) Changes in liver morphology, c) Vascular congestion, and d) Degree of fibrosis across different groups. (**** P<0.0001; * P<0.05; ns = not significant)

Cont, Control group; Oil, administration of olive oil; D25, receiving DSF 25 mg/kg; D50, receiving DSF at a dose of 50 mg/kg; CCl₄, receiving 40% CCl₄ at a dose of 1 mg/kg; CCl₄ + D25, receiving DSF at a dose of 25 mg/kg concurrently with CCl₄ injection; CCl₄ + D50, receiving DSF at a dose of 50 mg/kg concurrently with CCl₄ injection.

The liver morphology assessment, which included cytoplasmic vacuolation and necrotic nuclei, indicated improvements for the CCl4 + D25 and CCl4 + D50 groups compared to the CCl4 group. Of importance, the damage reduction for the CCl4 + D25 group was highly significant at P 0.0102, whereas that of the CCl4 + D50 group was not significant at P = 0.1315 (Figure 3, Figure 6b).

Furthermore, the average score of total vascular congestion in the CCl4 + D25 and CCl4 + D50 groups decreased compared to the CCl4 group; however, this reduction was not substantial in either treatment group (P > 0.9999) (Figures 4 and 6c). Lastly, the mean fibrosis score in the CCl4 + D25 and CCl4 + D50 groups did not show a significant alteration compared to the CCl4 P > 0.9999) (Figure 5, Figure 6d).

Quantitative analysis of hepatic fibrosis revealed a marked increase in the percentage of fibrotic area in the CCl₄ group compared to the control group (11.72 ± 0.9968% vs. 1.99 ± 0.1797%, P < 0.0001). Treatment with Disulfiram at 25 mg/kg (CCl₄+D25) resulted in a mean fibrosis percentage of 10.07 ± 0.3677%. In contrast, the 50 mg/kg dose group (CCl₄+D50) showed 11.57 ± 0.5188%, which was not significantly different compared to the CCl₄ group (P =0.2300, P > 0.9999) (Figure 7).

Figure 7.

Quantitative assessment of the hepatic fibrosis in experimental groups. Masson’s trichrome staining was employed to assess hepatic fibrosis, expressed as the percentage of collagen-positive area (% fibrosis). Data are presented as box-and-whisker plots (minimum to maximum) for five animals per group (Control, Oil, D25, D50, CCL4, CCL4+D25, and CCL4+D50). Statistical analysis was performed using nested one-way ANOVA followed by multiple comparisons. ****p < 0.0001 indicates significant differences between the Control and CCL4, CCL4+D25, and CCL4+D50 groups. No significant differences were observed between the CCL4 group and the treatment groups.

Analysis of congestion parameters among each group

The incidence of vascular congestion was highest in the group given CCL4, with a mean congestion in the portal vein of 3.000±0.000, and lowest in the sinusoids at 2.400±0.1309. A statistically significant difference was recorded between the sinusoids and the portal vein (P=0.0007) and between the sinusoids and the central vein (P=0.0429).

In the group receiving D50, the average vascular congestion score in the portal vein was 0.2282 ± 0.9333, statistically significant compared to the congestion scores in the central vein and sinusoids (P = 0.0254).

Furthermore, in the group receiving both CCL4 and D50, the average congestion score in the portal vein was 2.600±0.2390, which demonstrated a statistically significant difference compared to the sinusoid's congestion score (P=0.0002) (Table 2).

Table 2.

Comparison of vascular congestion among the central vein, the portal vein, and the sinusoids in each group

Groups	Central vein Congestion (Mean±SE)	Portal vein congestion (Mean±SE)	Sinusoid Congestion (Mean±SE)	P-value
Control	0.000	0.000	0.066±0.066	NS
Oil	0.33± 0.12	0.4± 0.13	0.13± 0.09	NS
D25	0.33±0.12	0.33±0.12	0.06±0.06	NS
D50	0.2±0.12	0.93±0.22	0.2±0.1	P =0.0254 (P&C) P =0.0254 (P&S)
CCL4	2.8±0.1	3±0.00	2.4±0.13	P=0.0429 (C&S) P=0.0007 (P&S)
CCL4+D25	2.26±0.11	2.6±0.13	2.2±1	NS
CCL4+D50	2±0.23	2.6±0.13	1.4±0.13	P=0.0002 (P&S)

The table above indicates significant differences in vascular congestion were observed among the portal vein, central vein, and sinusoids in the groups treated with D50, CCL4, and the combination of D50 and CCL4.

Oil, administration of olive oil; DSF 25, receiving DSF 25 mg/kg; DSF 50, receiving DSF at a dose of 50 mg/kg; CCl₄, receiving 40% CCl₄ at a dose of 1 mg/kg; CCl₄ + DSF 25, receiving DSF at a dose of 25 mg/kg concurrently with CCl₄ injection; CCl₄ + DSF 50, receiving DSF at a dose of 50 mg/kg concurrently with CCl₄ injection; NS, not significant

Analysis of serum liver enzymes and MDA

A significant reduction in ALT levels was observed in the CCL4+DSF25 group compared to the CCL4 group (P=0.0265). While the ALT levels in the CCL4+DSF50 group were lower than those in the CCL4 group, this difference did not reach statistical significance (P = 0.6614). Similarly, AST levels showed a notable decrease in the CCL4+DSF25 group compared to the CCL4 group (P = 0.0334). In contrast, the reduction in AST levels with DSF at 50 mg/kg was not statistically significant (P=0.9995 = 0.9995). In contrast, no significant difference in ALP levels was detected between the groups of CCL4+D25 (P > 0.9999) and CCL4+D50 (P = 0.9979). Additionally, MDA levels significantly decreased in the CCL4+DSF25 group compared to the CCL4 group (P < 0.001); in contrast, the CCL4+D50 group did not demonstrate a statistically significant decrease (P = 0.1306) (Figure 8).

Figure 8.

Serum levels of hepatic enzymes (ALT, AST, ALP) and MDA in the given treatment groups. a) Representatives of ALT levels, which are statistically significant in CCL4+D25 against the CCL4 group. b) Represents AST levels in which notable differences occurred in CCL4+D25 against the CCL4 groups. c) Represents ALP values with no significant difference among the groups. d) Shows MDA level, the significant difference in CCL4+D25 compared to the group that received CCL4 (**** P<0.0001; * P<0.05; ns = not significant).

Cont, Control group; Oil, administration of olive oil; D25, receiving DSF 25 mg/kg; D50, receiving DSF at a dose of 50 mg/kg; CCl₄, receiving 40% CCl₄ at a dose of 1 mg/kg; CCl₄ + D25, receiving DSF at a dose of 25 mg/kg concurrently with CCl₄ injection; CCl₄ + D50, receiving DSF at a dose of 50 mg/kg concurrently with CCl₄ injection.

Discussion

The present study investigated the therapeutic potential of disulfiram at doses of 25 and 50 mg/kg administered daily over seven weeks in a CCl₄-induced model of hepatic fibrosis in rats, using comprehensive histopathological and biochemical evaluations. CCl₄, a well-established hepatotoxin, induces fibrosis through its hepatic biotransformation into reactive trichloromethyl and peroxyl radicals, generating oxidative stress and triggering fibrogenic cascades (5). Consistent with this mechanism, our model successfully reproduced the characteristic features of hepatic injury and fibrosis progression.

Disulfiram, a classic ALDH inhibitor commonly used in the management of alcohol dependence, exhibits a broad pharmacological profile encompassing anti-inflammatory, antineoplastic, and antimicrobial properties (17). However, its role in fibrotic disorders remains controversial. While clinical evidence suggests that DSF-induced hepatotoxicity occurs in predisposed individuals (22), preclinical studies have reported beneficial effects through the modulation of oxidative stress and inflammatory signaling in multiple organ systems (15, 23-25). This apparent contradiction highlights the importance of dose-dependent and tissue-specific evaluations of DSF’s biological activity.

Our study utilized disulfiram at doses of 25 and 50 mg/kg, a range consistent with previously reported anti-inflammatory and tissue-protective effects in models of hepatic, pulmonary, and renal injury (16, 17, 26). While Yang et al. demonstrated antifibrotic activity at a lower dose of 4 mg/kg (11), most studies in pulmonary and hepatic fibrosis have employed intraperitoneal doses between 10-50 mg/kg/day (17, 26), with oral administration requiring higher doses of 150-200 mg/kg/day due to pharmacokinetic considerations (23).

The investigation revealed a distinct dose-response relationship, with DSF at 25 mg/kg demonstrating superior hepatoprotective efficacy compared to the 50 mg/kg dose. Treatment with the lower dose significantly ameliorated histopathological alterations, as evidenced by restored hepatocyte morphology, reduced cytoplasmic vacuolation, and nuclear necrosis (P = 0.0358). This histological improvement correlated with a substantial attenuation of hepatic injury biomarkers, as indicated by reduced serum ALT (P = 0.0265) and AST (P = 0.0334) levels. The hepatoprotective mechanism appears to involve substantial mitigation of oxidative stress, demonstrated by a marked decrease in MDA levels in the CCl₄+DSF25 group compared to the CCl₄ control (P<0.01). This reduction in lipid peroxidation suggests that DSF has the capacity to counteract oxidative damage in hepatic tissue.

Notably, our model demonstrated significant hepatoprotection without coregression, contrasting with Yang et al.'s report of direct anti-fibrotic effects (11). Our findings suggest that DSF primarily exerts hepatoprotective and anti-inflammatory effects during active injury phases, rather than directly reversing collagen deposition or matrix accumulation. Therefore, its therapeutic efficacy may depend on disease stage, tissue type, and local redox status. Quantitative collagen analysis (Figure 7) further confirmed the absence of significant fibrosis regression across DSF-treated groups, supporting the notion that hepatocellular protection precedes any measurable anti-fibrotic response.

This discrepancy may stem from methodological variations in fibrosis induction protocols or treatment dose and duration. However, our findings show partial concordance with Okabe et al., who found that DSF suppressed macrophage-mediated inflammation in pulmonary fibrosis (23), suggesting conserved anti-inflammatory mechanisms across tissue types.

The observed dissociation between hepatoprotection and fibrosis regression suggests that DSF may primarily target hepatocyte viability and inflammatory pathways during the established injury phase, rather than directly affecting collagen deposition. This mechanistic distinction underscores the context-dependent nature of DSF's therapeutic effects and emphasizes the need for precise dosing strategies tailored to specific pathological stages.

Our results delineate a narrow therapeutic window for DSF, wherein the 50 mg/kg dose not only exhibited diminished hepatoprotection but also promoted the formation of inflammatory foci, indicating potential toxicity at elevated concentrations (17).

The dose-response relationship observed herein may be explained through distinct molecular mechanisms. At the optimal 25 mg/kg dose, DSF maintains redox homeostasis through balanced antioxidant activity. However, at 50 mg/kg, the therapeutic balance shifts toward toxicity, potentially mediated through copper-DDC complex formation that exacerbates oxidative damage and initiates inflammatory cascades (27). This transition is further characterized by overwhelmed cellular antioxidant defenses, mitochondrial dysfunction, and activation of pro-apoptotic pathways (27).

The mechanistic shift at higher doses is corroborated by enhanced neutrophil infiltration and upregulation of pro-inflammatory cytokines, which collectively counteract the therapeutic potential of DSF. This behavior elucidates apparent contradictions in the literature, where clinical reports describe hepatotoxicity at elevated doses (22). While controlled studies demonstrate protection within appropriate dosing ranges (15).

Regarding antifibrotic efficacy, our findings differ from several reports in other organ systems. While Okabe et al. demonstrated DSF-mediated suppression of macrophage infiltration and fibrosis in pulmonary models (23), the anti-fibrotic efficacy of DSF in lung tissue has been further established by Hamidi et al., Karimzadeh et al., who identified modulation of RASSF1A and ZEB-1 as key mechanistic (15, 26), our hepatic model showed significant hepatoprotection without comparable fibrosis regression. Similarly, studies in renal (24) and orbital (25) fibrosis models reported positive outcomes, suggesting tissue-specific variations in DSF's antifibrotic potential.

The observed dissociation between hepatoprotection and fibrosis regression, coupled with the dose-dependent effects on sinusoidal congestion and architecture, underscores the complex pharmacodynamics of DSF in hepatic tissue. These findings collectively suggest that DSF's therapeutic profile is highly context-dependent, influenced by tissue type, disease stage, and dosing parameters.

Conclusion

In summary, administration of 25 mg/kg DSF improved hepatocyte morphology and serum liver enzyme levels, indicating a reduction in liver injury. Although no reduction in fibrosis was observed, the histopathological improvements suggest that DSF may exert beneficial effects at appropriate doses. Considering the dose-dependent nature of DSF responses, future studies should focus on identifying optimal dosing strategies to enhance its potential impact on liver injury.

Limitations of the study

This study has several limitations. First, oxidative stress assessment was limited to MDA measurement, which, although widely accepted, provides only a partial representation of redox balance. Second, no molecular analyses were conducted to explore pathways potentially involved in DSF’s effects, such as NLRP3 inflammasome activation, COX-2/PGE₂ modulation, or EMT regulation. Third, the relatively small sample size (n = 5 per group) may have limited the statistical power to detect subtle fibrotic differences.

Despite these limitations, consistent partial histopathological and biochemical improvements support DSF’s hepatoprotective activity. Future investigations integrating molecular profiling, larger cohorts, and comprehensive oxidative/antioxidant panels are strongly recommended to clarify the mechanistic role and therapeutic potential of DSF in hepatic fibrogenesis.

Funding

This research was supported by the Research and Technology Empowerment Committee of Babol University of Medical Sciences under grant number 9910223.

Conflict of interests

There is no conflict of interest for authors of this article.