Comparative hepatoprotective effects of Benzene Sulfonamide Derivatives in a rat model of ethylene glycol-induced liver injury

Abstract

Ethylene glycol (EG), an alcohol derivative, is metabolized into toxic compounds contributing to liver damage. This study investigated the therapeutic potential of two benzene sulfonamide derivatives—SBCL and SBF—against EG-induced hepatotoxicity in rats. Liver damage was induced using 0.12 ml of 5 % EG, followed by oral administration of the test compounds (10 mg/kg) every 48 h for 21 days.

Rats were divided into six groups (n = 5 per group): a negative control (NEG), a positive control (EG), and four treatment groups—SBCL in healthy rats (SBCL-NEG), SBCL in EG-treated rats (SBCL-EG), SBF in healthy rats (SBF-NEG), and SBF in EG-treated rats (SBF-EG). The two compounds influenced liver biomarkers, including ALT, ALP, serum oxalate, and citrate, as well as oxidative stress indicators such as nitric oxide (NO), lipid peroxidation (LPO), protein carbonyls (PC), and superoxide dismutase (SOD) activity. SBCL and SBF reduced ALT levels in healthy rats, indicating potential hepatoprotective effects under normal conditions. However, SBCL was associated with elevated oxidative stress markers and reduced antioxidant activity in EG-treated rats, suggesting possible pro-oxidant effects in degenerative liver tissue. In contrast, SBF showed a more stable protective profile in healthy and EG-treated rats. These findings recommend that benzene sulfonamide derivatives, especially SBF, may have therapeutic relevance in managing EG-induced liver injury. SBCL may exhibit context-dependent toxicity under pathological conditions. Overall, the study supports the potential of these compounds in modulating liver toxicity, but additional research is needed to clarify their safety in pathological conditions.

Graphical abstract

Highlights

• •Benzene sulfonamide derivatives demonstrated therapeutic effects in alleviating EG-induced liver damage in rats
• •SBCL and SBF have potential hepatoprotective effects.
• •SBCL and SBF treatment in the presence of liver damage reduced oxidative stress markers.
• •BCL showed a dual effect—beneficial under normal conditions but potentially harmful in the presence of hepatotoxicity.
• •SBF exhibited a more consistent protective profile.
• •SBF showing a more reliable protective effect compared to SBCL

1. Introduction

The liver plays a central role in metabolism, homeostasis, and detoxification, functioning in close physiological coordination with the kidneys through pathways such as the urea and ornithine cycles [1]. In this process, the liver converts toxins into bile and urea, facilitating digestion and renal excretion, respectively [2]. Beyond detoxification, both organs cooperate in regulating electrolyte balance, including sodium and calcium homeostasis [3]. Additionally, the liver, kidneys, and adrenal glands interact through hormonal pathways to maintain fluid and sodium balance, ensuring circulatory stability and overall homeostasis [4], [5].

Oxidative stress (OS) arises when there is an imbalance between the production of reactive oxygen species (ROS) and the body’s ability to neutralize them through antioxidant defenses. ROS are highly reactive molecules generated during normal metabolic processes, particularly in mitochondria, but their levels can spike under conditions of environmental stress, toxin exposure, or disease. The liver, a key organ in detoxification and metabolism, can suffer cellular damage when excessive OS surpasses its antioxidant defense capacity.

This damage manifests as lipid peroxidation, protein oxidation, and DNA damage, which impair liver function and contribute to inflammation, fibrosis, and apoptosis. Understanding the dynamics of OS in liver diseases has profound clinical implications. Measuring biomarkers of OS, such as malondialdehyde (MDA) or glutathione depletion, can provide prognostic insights and help guide management strategies. Moreover, enhancing the liver’s antioxidant defenses through dietary antioxidants, pharmacological interventions, or lifestyle modifications is a promising approach to mitigate OS and slow disease progression. This underscores the critical role of oxidative stress in liver health and disease management [6].

Ethylene glycol (EG) is often utilized as a flavoring agent in food and is a common ingredient in industrial fluids, antifreeze, and coolants for vehicle radiators [7]. In humans, EG is metabolized similarly to ethanol. Initially, it is converted to glycolaldehyde in the liver by alcohol dehydrogenase. Then, aldehyde dehydrogenase oxidizes glycolaldehyde to glycolate [8]. Then, glycolate is converted to glyoxalate by tartrate dehydrogenase, a key enzyme in EG metabolism, contributing to hepatic toxicity. Glyoxalate is further metabolized into glycolic acid, glycine, and oxalic acid. This leads to potential hepatic and renal toxic effects, such as increased hepatic fatty deposits, inflammation, glycogen quantity, and decreased carbohydrates. The formation of toxic metabolites results in severe metabolic acidosis and an increased concentration of intermediates that need detoxification, causing rapid depletion of glutathione (GSH) and glutathione-dependent detoxifying enzymes [9].

Glyoxalic acid may lead to the formation of crystalline oxalate in the renal tubules, which could be the primary cause of nephrotoxicity. This process lowers oxalate excretion and promotes the buildup of calcium oxalate in the renal tubules, leading to damage to the tubular epithelium and kidney tissue [10]. Additionally, oxalate crystals themselves are cytotoxic, directly harming the kidneys. The hepatotoxic effects of glyoxalic acid, with the formation of crystalline substances within hepatocytes, may contribute to hepatic steatosis. Although EG has been associated with fatty liver and cirrhosis in animal studies and human cases, no specific symptoms are definitively linked to its hepatotoxicity. EG-induced liver toxicity can manifest in two ways, depending on the exposure duration: acute toxic doses or chronic exposure from sources like seasoning, alcoholic beverages, and drug addiction [11].

Several novel sulfonamide derivatives have been studied for their effects on oxidative stress and inflammation, which are directly implicated in liver damage. These compounds inhibit enzymes such as dihydrofolate reductase, disrupting the production of tetrahydrofolate (THF). By blocking THF synthesis, sulfonamides impair purine and thymine nucleotide biosynthesis, which hinders DNA synthesis and disrupts normal cellular function. Sulfamethoxazole, a widely used sulfonamide drug, inhibits dihydropteroate synthase (DHPS), a key enzyme in folate synthesis. This inhibition not only affects nucleic acid and protein synthesis but also promotes OS and inflammatory responses in hepatic cells [12]. The OS induced by sulfonamide derivatives can lead to the generation of ROS, exacerbating liver cell damage and contributing to conditions such as steatosis and hepatocyte apoptosis. These effects highlight the potential hepatotoxicity of sulfonamide-based compounds, emphasizing the need for careful evaluation of their impact on liver function in therapeutic contexts.

This study aimed to evaluate the potential therapeutic role of benzenesulfonamide derivatives on EG exposure's biochemical and physiological impacts on Sprague-Dawley rats, with a specific focus on hepatic toxicities. The research involved examining various hematological parameters, assessing liver enzymes, and analyzing oxidative stress markers, antioxidant levels, and inflammatory biomarkers. Additionally, histopathological and electron microscopy examinations were conducted to understand the cellular and tissue-level changes in the liver induced by EG.

2. Materials and methods

2.1. Chemicals

The compounds, exemplified by compound A [(N-(4-chlorophenyl)-4-isobutoxy-N-(1-methylpiperidin-4yl) benzene sulfonamide), SBCL] and compound B [(N-(4-fluorophenyl)-4-methoxy-N-(4-(4-methylpiperidin-4-yl) benzene sulfonamide), SBF], were chosen due to their distinct structures and pharmacological advantages [13]. Sigma Aldrich company was the main source of all the chemicals.

2.2. Animals

About 35 male Sprague Dawley Albino rats (National Research Centre (NRC), Cairo), weighing approximately 105 ± 10 g each, were kept in cages with stainless steel wire lids. They were given a week to acclimate to the laboratory environment, during which they had free access to food. Experiments were carried out under stable temperature and humidity conditions.

The rats were divided into two main groups: one group, consisting of 15 rats, underwent induction of liver toxicity, while the second group, comprising 15 rats, served as the control. This division enabled a comparative analysis between rats with induced liver stones and those without, to understand the effects of liver stone induction on various physiological parameters or biochemical markers. It is essential to highlight that all animal handling and experimentation strictly follow the regulations and guidelines set by the Research Ethics Committee of the Faculty of Veterinary Medicine at Suez Canal University in Ismailia, Egypt (Protocol number: 2019021).

The oral usage was delivered through gavage for 30 days, with doses administered every 48 h following the induction of liver damage using 0.12 ml of 5 % ethylene glycol, corresponding to approximately 6 g/kg body weight. This dosing regimen was selected to model chronic low-dose ethylene glycol exposure rather than acute intoxication, thereby mimicking environmentally or dietarily relevant exposure scenarios. Accordingly, this model induces sub-chronic liver injury, characterized by moderate biochemical and histopathological alterations, rather than severe acute toxicity

Subgroups were delineated as follows, with each subgroup consisting of a sample size of n = 5; NEG served as the negative control, and EG group was considered the positive control that received ethylene glycol for 21 consecutive days. Groups 3 (SBCL-NEG group) and 4 presented animals were treated with 10 mg/kg of SBCL, and rats of EG group were treated with SBCL, respectively. Group 5 was SBF-NEG animals without hepatocyte damage and received 10 mg/kg of SBF. However, group 6 was EG group treated with SBF.

This dose was chosen based on prior in vivo studies investigating benzene sulfonamide derivatives, where similar dosing demonstrated biological efficacy without inducing systemic toxicity. Additionally, using a single standardized dose allowed direct comparison between SBCL and SBF while minimizing confounding variables.

2.3. Sample collection

At the end of the experiment, all rat groups were anesthetized with 50 mg/kg Ketamine and 10 mg/kg Xylazine intraperitoneal. Blood samples were obtained using heparinized Hematocrit capillary tubes from the retro-orbital venous plexus and promptly transferred into tubes for serum separation. These samples were left to stand at a slanting angle for approximately 45 min at 4°C. Then, the serum was separated via centrifugation at 2500 rpm for 15 min. The obtained serum was preserved at a temperature of −20 °C and employed for evaluating specific biochemical parameters, levels of antioxidants, and OS; liver tissue samples that were inflammatory markers, and for histopathological alterations.

2.4. Liver enzymes assessment

All the parameters were determined using a Semi-Automated Chemistry Analyzer, BIOLAB ES-102 (Biomed Diagnostics). Reagents were provided by Diamond Diagnostics and Vitro Scientific Kit, Cairo, Egypt.

The kinetic method used to determine the serum alanine aminotransferase (ALT) [14]. Serum alkaline phosphatase (ALP) activity was evaluated according to the International Federation of Clinical Chemistry [15].

2.5. Citrate and oxalate assessment

The Colorimetric/Fluorometric citrate kit (ab83396) offers a straightforward and sensitive method for measuring citrate. During the assay, citrate is transformed into pyruvate through oxaloacetate. The pyruvate is then quantified by changing the colorless probe into a highly colored one that can be measured at 570 nm and fluoresces (Ex/Em: 535/587 nm) [16].

Oxalate oxidase facilitates the conversion of oxalate into hydrogen peroxide and carbon dioxide. Hydrogen peroxide interacts with chromogenic compounds, resulting in colored products through the action of peroxidase (POD). These products exhibit a specific absorption peak at 550 nm, with the intensity of the color directly correlating to the oxalate content [17].

2.6. Oxidative/antioxidants biomarkers assessment

Quantification of serum lipid peroxidation (LPO) is determined by detecting malondialdehyde (MDA) [18]. The ELISA kit with the Sandwich method was used to measure serum nitric oxide (NO) levels. The NO concentrations were determined by measuring the optical density (OD) of the samples and comparing them to a standard curve [19].

Superoxide dismutase (SOD) inhibition activity in serum can be determined by a colorimetric method [20].

Serum glutathione (GSH) was evaluated using BioVision’s ApoGSHTM GSH colorimetric assay, which provides a convenient, colorimetric method for analyzing GSH using a microtiter plate reader. The GSH concentration was determined by measuring absorbance at 412 nm [21].

The sandwich-ELISA method was used for serum protein carbonyl (PC) determination [22].

2.7. Inflammatory biomarkers assessment

During this assay, the quantitative sandwich enzyme immunoassay technique was employed. Antibodies specific to interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor-alpha (TNF-α) were pre-coated onto a microplate. After stopping the color development, the intensity of the color was measured.

Rat serum alpha-fetoprotein (AFP) levels were measured using an ELISA kit (MyBioSource, USA, CAT. NO. TA MBS700622) following the method outlined by Maiolini and Masseyeff [23]. This ELISA analytical technique relies on AFP antibody-antigen interactions (immunosorbency) and an HRP colorimetric detection system to identify AFP antigen targets in samples, with a detection range of 15.6–1000 pg/ml.

2.8. Histopathological changes

For histopathological studies, liver tissues were immediately placid in 10 % neutral buffered formalin (Sigma Aldrich Chemical Company, USA). The tissues were processed using standard techniques and embedded in paraffin blocks. Sections were cut with a rotary microtome and stained with Ehrlich’s hematoxylin and eosin. The histological sections were examined under a light microscope to assess histopathological changes.

2.9. Electron microscope examination

Small pieces of liver were excised and then cut below a dissecting microscope in the presence of 2 % glutaraldehyde and processed for the electron microscope. The samples were examined with the transmission electron microscope (JEOL JEM-2100, Japan) within the Electron Microscope Unit in Mansoura Governorate, Egypt.

2.10. Statistical analysis

The data were analyzed using SPSS 11.0 for Windows. Tabulation and graphics of data were created using Microsoft Excel XP. All the data were expressed as mean ± standard error (SE). The statistical significance of differences was evaluated by using a One-Way Analysis of Variance (ANOVA) followed by Post-hoc Duncan to compare among groups. Differences were considered statistically significant at p < 0.05

3. Results

3.1. Biochemical markers

The effect of sulfonamide was examined using benzene derivatives, compound A (SBCL) and compound B (SBF), on induced liver damage in rats. Table 1 demonstrates how SBCL and SBF affect liver enzymes ALT and AST. Administering SBCL and SBF to rats without induced liver damage led to a statistically significant decrease in ALT levels. Specifically, ALT values dropped from 56.42 ± 3.69 IU/L in the control group (NEG) to 48.44 ± 1.68 IU/L in SBCL-NEG treatment group and 41.86 ± 0.63 IU/L in the SBF-NEG group. However, inducing liver damage resulted in a statistically meaningful decrease in ALT levels, with the EG group showing levels of 41.02 ± 1.74 IU/L.

Table 1.

Effect of compound A (SBCL) and compound B (SBF) treatment on liver functions of rats after induction of ethylene glycol.

Parameters	NEG	SBCL-NEG	SBF-NEG	EG-group	SBCL-EG	SBF-EG
ALT (IU/L)	56.42 ± 3.69	48.44 ± 1.68a	41.86 ± 0.63a	41.02 ± 1.74a	50.28 ± 1.73b,c	42.86 ± 287c
ALP (IU/L)	377.6 ± 12.60	421.2 ± 16.18a	343.6 ± 8.76a	326.2 ± 17.81a,b	361.6 ± 30.28	356 ± 22.31
Oxalates (mmol/l)	0.28 ± 0.18	0.10 ± 0.01a	0.10 ± 0.01a	0.38 ± 0.01b	0.20 ± 0.01b	0.25 ± 0.01
Citrate (μmol/ml)	2.15 ± 0.01	2.15 ± 0.01	2.15 ± 0.01	5.32 ± 0.01a,b	4.45 ± 0.01b	4.91 ± 0.01

Data were expressed as means ± SEM, n = 5. Data were statistically analyzed using One-way ANOVA, followed by Duncan multiple comparisons test, P ≤ 0.05. NEG, control negative; EG, control positive; SBCL-NEG, non-induced liver damage group was treated with compound A; SBF-NEG, non-induced liver damage group was treated with compound B; SBCL-EG, induced liver damage group was treated with compound A; SBF-EG, induced liver damage group was treated with compound B. ^a referred to the comparison of SBCL-NEG, SBF-NEG, or EG with NEG. ^b referred to the comparison of SBCL-EG, or SBF-EG, with EG. ^c referred to the comparison of SBCL-EG with SBF-EG as statistically significant. Alanine aminotransferase (ALT, IU/L), alkaline phosphatase (ALP, IU/L), oxalates (mmol/l), and citrate (μmol/ml).

After inducing liver damage, SBCL led to significant increase in ALT levels. Specifically, ALT levels increased from 41.02 ± 1.74 IU/L in the EG group to 50.28 ± 1.73 IU/L in the SBCL-EG-treated rat (Table 1). Moreover, rats treated with SBCL-EG (50.28 ± 1.73 IU/L) exhibited a marked increase in ALT levels compared to SBF-EG (42.86 ± 2.87 IU/L).

ALP activity saw a notable rise in SBCL-NEG group but dropped in the SBF-NEG group. In animals treated with EG, there was a significant decrease in ALP activity compared to the NEG group. Treatment of induced liver damage rats with SBCL and SBF showed a no significant difference in ALP activities was observed. Comparing the effects of SBCL and SBF on ALP in damaged hepatocyte rats revealed a statistical increase in SBCL-EG and SBF groups (Table 1).

Moreover, administering SBCL and SBF to control rats caused a significant reduction of serum oxalate levels by approximately 2.5 times in SBCL compared to NEG. Similarly, treating rats with EG using SBCL or SBF caused decreased oxalate levels (Table 1). EG exposure caused a statistically significant increase in serum citrate levels (Table 1). However, treating rats with EG in combination with SBCL led to a statistically significant decrease in serum citrate levels.

The impacts of compound A (SBCL) and compound B (SBF) on GSH, SOD, NO, LPO, and PC levels were presented in Table 2. EG-treated rats resulted in a notable decrease in GSH levels by 39.8 % compared to the control group. Following SBCL and SBF, there was a significant increase in GSH concentration by approximately 1.5-fold, 1.3-fold, and 1.3-fold, respectively (Table 2).

Table 2.

Effect of compound A (SBCL) and compound B (SBF) treatment on serum antioxidants and oxidative stress markers of liver rats after induction of ethylene glycol.

Parameters	NEG	SBCL-NEG	SBF-NEG	EG-group	SBCL-EG	SBF-EG
GSH (ng/ml)	16.09 ± 0.10	15.97 ± 0.08	15.85 ± 0.07	9.68 ± 0.04a,b	12.53 ± 0.05b	12.96 ± 0.19b
SOD (U/ml)	3.95 ± 0.10	3.73 ± 0.03	3.66 ± 0.06	1.77 ± 0.03a,b	2.49 ± 0.02b	2.18 ± 0.04b
NO (pg/ml)	12.51 ± 0.06	12.56 ± 0.03	12.56 ± 0.04	32.71 ± 0.30a,b	18.97 ± 0.09b,c	16.05 ± 0.06b
LPO (nmol/ml)	0.67 ± 0.01	0.67 ± 0.01	0.66 ± 0.01	1.53 ± 0.01a,b	1.01 ± 0.01b	1.19 ± 0.01b
PC (ng/ml)	7.72 ± 0.03	7.62 ± 0.02	7.62 ± 0.02	19.70 ± 0.07a,b	13.94 ± 0.09b,c	10.01 ± 0.11b

Data were expressed as means ± SEM, n = 5. Data were statistically analyzed using One-way ANOVA, followed by Duncan multiple comparisons test P ≤ 0.05. NEG, control negative; EG, control positive; SBCL-NEG, non-induced liver damage group was treated with compound A; SBF-NEG, non-induced liver damage group was treated with compound B; SBCL-EG, induced liver damage group was treated with compound A; SBF-EG, induced liver damage group was treated with compound B. ^a referred to the comparison of SBCL-NEG, SBF-NEG, or EG with NEG. ^b referred to a comparison of SBCL-EG, or SBF-EG, with EG. ^c referred to the comparison of SBCL-EG with SBF-EG as statistically significant. Glutathione (GSH), superoxide dismutase (SOD), nitric oxide (NO), lipid peroxidation (LPO), and protein carbonyl (PC).

The SOD activities decreased significantly by 55 % in EG-rats compared to the control group. However, SOD activities increased by 1.4-fold and 1.2-fold with SBF and SBCL, respectively, compared to EG group, as illustrated in Table 2.

EG treatment led to a statistical increase in NO level by 2.6-fold, LPO by 2.3-fold, and PC by 2.6-fold compared to the negative controls (Table 2). SBCL and SBF resulted in a significant reduction of NO levels, with a decrease of 42 % for SBCL-EG and 51 % for SBF-EG. LPO levels decreased by 34 % for SBCL-EG and 22 % for SBF-EG compared to the positive control rats. Similarly, PC levels decreased by 29 % for SBCL-EG and 49 % for SBF-EG compared to the positive controls (Table 2).

The effect of SBCL and SBF on inflammatory markers IL-1β, IL-6, AFP, and TNF-α is shown in Table 3. The induction of EG in the rats led to a significant increase in IL-1β by nearly 3-fold than the negative control. However, the concentrations of IL-1β decreased when the treated rats with EG were treated with SBCL or SBF by 40.5 % and 27.7 %, respectively, as shown in Table 3.

Table 3.

Effect of compound A (SBCL) and compound B (SBF) treatment on serum inflammatory markers of the liver rats after induction of ethylene glycol.

Parameters	NEG	SBCL-NEG	SBF-NEG	EG-group	SBCL-EG	SBF-EG
IL-1β (pg/ml)	140.28 ± 0.77	135.84 ± 0.44	137.7 ± 0.30	539.14 ± 5.52a,b	320.7 ± 2.39b,c	389.72 ± 2.61b,c
IL-6 (pg/ml)	3.69 ± 0.05	3.56 ± 0.03	3.60 ± 0.04	9.78 ± 0.04a,b	5.81 ± 0.04b	6.19 ± 0.06b
AFP (ng/ml)	5.24 ± 0.26	7.3 ± 0.14a	6.22 ± 0.12a	6.72 ± 0.26a,b	5.62 ± 0.39c	6.68 ± 0.30b
TNF-α (pg/ml)	9.85 ± 0.03	9.72 ± 0.02	9.69 ± 0.03	22.01 ± 0.27a,b	14.93 ± 0.04b,c	16.99 ± 0.06b,c

Data were expressed as means ± SEM, n = 5. Data were statistically analyzed using One-way ANOVA, followed by Duncan multiple comparisons test, P ≤ 0.05. NEG, control negative; EG, control positive; SBCL-NEG, non-induced liver damage group was treated with compound A; SBF-NEG, non-induced liver damage group was treated with compound B; SBCL-EG, induced liver damage group was treated with compound A; SBF-EG, induced liver damage group was treated with compound B. ^a referred to the comparison of SBCL-NEG, SBF-NEG, or EG with NEG. ^b referred to the comparison of SBCL-EG, or SBF-EG, with EG. ^c referred to the comparison of SBCL-EG with SBF-EG as statistically significant. IL-1β, Interleukin1β; IL-6, interleukin 6; AFP, Alpha fetoprotein; and TNF-α, tumor necrosis factor-alpha.

Moreover, IL-6 increased in treated rats with EG by 3-fold compared to normal animals. However, SBCL or SBF treatment decreased IL-6 by 40.6 % and 36.7 %, respectively (Table 3).

Treating rats without EG with SBCL, SBF, and EG resulted in statistically significant elevation of AFP levels by 1.4-fold, 1.2-fold, and 1.3-fold, respectively. However, after treating rats with induced liver stones with SBCL, AFP levels decreased significantly by 16 % in SBCL-EG groups (Table 3).

In rats, TNF-α levels rose by 2.2-fold following stone induction with EG. However, TNF-α levels decreased after usage with SBCL and SBF by 32.2 % and 22.8 %, respectively (Table 3).

3.2. Histological findings of the liver

Histological examination of the liver tissue of NEG, SBCL-NEG, and SBF-NEG revealed normal hepatic architecture with intact cords of hepatocytes, prominent nuclei, and no signs of inflammation or necrosis in the portal tracts (Fig. 1a, b, and 1c). In EG-treated rats, there were notable histopathological changes, vacuolation of the cytoplasm, congestion of the portal area, and proliferation of the bile duct (Fig. 1d). Histological examination of liver tissue of SBCL-EG showed vacuolation of the cytoplasm with mild congestion of the portal area, and proliferation of the bile duct (Fig. 1e). Histological examination of liver tissue of SBE-EG showed normal hepatic architecture with no signs of inflammation or necrosis surrounding the portal area (Fig. 1f).

Fig. 1.

Photomicrograph of liver tissue of (a) NEG: control negative; (b) SBCL-NEG, non-induced liver damage group treated with compound A; (c) SBF-NEG, non-induced liver damage group treated with compound B revealed normal hepatic architecture with intact cords of hepatocytes, prominent nuclei, and no signs of inflammation or necrosis in the portal area (PA). (d) EG, control positive showed hydrobic degeneration (HD) of the hepatocytes, congestion of the portal area (PA), accompanied by proliferation of the bile duct and infiltration of inflammatory cells. (e) SBCL-EG, induced liver damage group treated with compound A, showed mild dilation of the portal area (PA) with vacuolated cells (HD) and proliferated bile ducts. (f) SBF-EG, induced liver damage group treated with compound B showed normal hepatocytes surrounding the portal area (PA). (HE, 100X).

3.3. Ultrastructure changes

The ultrastructural examination of the liver sections of the NEG, SBCL-NEG, and SBF-NEG groups (Fig. 2a, b, and c) exhibited normal hepatocytes with round nucleus with evenly distributed chromatin, sometimes slightly condensed along the nuclear membrane in hepatocytes. The cytoplasm is easily distinguished by its mitochondria and dense glycogen-rich areas. The ultrastructural examination of the liver sections of EG-treated rats (Fig. 2d) showed cellular alteration, including shrunken, condensed necrotic nuclei were observed. The ultrastructural examination of the liver sections of SBCL-EG and SBF-EG showed the absence of ultrastructural alteration (Fig. 2e and f).

Fig. 2.

An electron micrograph of (a) NEG: control negative; (b) SBCL-NEG, non-induced liver damage group treated with compound A; (c) SBF-NEG, non-induced liver damage group treated with compound B revealed normal hepatocytes with round nucleus (N) with evenly distributed chromatin, slightly condensed along the nuclear membrane in hepatocytes; Rough endoplasmic reticulum appears as regions with ribosomes on the nuclear membranes. The cytoplasm is easily distinguished from mitochondria and some lipid droplets. (d) EG, control positive showed cellular alteration, including shrunken, condensed necrotic nuclei were observed (arrow). The cytoplasm contained lysosomes. (e) SBCL-EG, induced liver damage group treated with compound A, and (f) SBF-EG, induced liver damage group treated with compound B, showed hepatocytes with normally round nuclei (N) with evenly distributed chromatin, slightly condensed along the nuclear membrane in hepatocytes.

4. Discussion

This study is the first to evaluate the therapeutic potential of benzene sulfonamide derivatives SBCL and SBF in a rat model of ethylene glycol-induced liver toxicity. While EG metabolism is known to cause oxidative stress and liver damage, the dual behavior of SBCL, offering hepatoprotection in normal hepatic architecture but exacerbating injury in damaged ones, is a novel finding. Additionally, the consistent protective effect observed with SBF positions it as a promising candidate for further development. The study offers new insight into the compound-specific responses of liver tissue to sulfonamide derivatives under normal and toxic conditions.

The study examined the effects of sulfonamide compounds SBCL and SBF on various biochemical parameters and anti-inflammatory parameters in rats, with and without induced liver damage by EG.

The hepatic function was observed by measuring the serum levels of ALT and ALP in the current study. The release of ALT and ALP into the bloodstream indicates damage to plasma and organelle membranes. The current study found a complex relationship between sulfonamide compounds (SBCL and SBF) and liver enzyme levels, particularly ALT, in rats with induced hepatotoxicity. In rats that did not have liver damage induced by EG, treatment with SBCL and SBF resulted in a notable reduction in ALT levels compared to the control group. This indicates a potential protective effect of these sulfonamide compounds on liver functions under normal conditions. However, when hepatotoxicity was induced, the effects of SBCL and SBF on ALT levels diverged. SBF showed no notable difference in ALT levels compared to the induced stone group (EG), while SBCL caused a significant increase in ALT levels. Therefore, SBCL action may exacerbate liver enzyme elevation under conditions of liver damage, potentially highlighting a risk of hepatotoxicity under such pathological conditions.

An increase in ALP levels is typically a characteristic indicator of obstructive hepatobiliary disease, such as cholestasis liver disease [24]. In our study, ALP activity showed different trends based on remedy and the presence of liver damage. In SBCL-NEG exposure group, ALP activity rose notably, while it decreased in the SBF-NEG group. In rats with induced EG, there was a significant decrease in ALP activity compared to the NEG group. Treatment with SBCL and SBF in stone-induced rats did not significantly alter ALP activities. The fall of ALT and ALP levels with the different regimens indicated the repair of hepatic tissue damage.

The pathophysiological mechanisms underlying the changes observed in the model of EG induction may be attributed to an elevation in urinary oxalate concentration. Specifically, EG is readily absorbed through the intestines and metabolized in the liver to oxalate, causing hyperoxaluria [25]. SBCL and SBF significantly reduced serum oxalate levels in rats without EG, with SBCL showing a reduction of approximately 2.5 times compared to NEG. This indicates the strong potential of these compounds in reducing oxalate, a critical factor in liver stone formation. Treatment the EG group with SBCL and SBF exhibited reduced oxalate levels.

Although the activities of ethylene glycol–metabolizing enzymes were not directly assessed in the present study, the marked reduction in serum oxalate and citrate levels following SBCL and SBF administration suggests a potential interference with EG metabolic pathways. These effects may be mediated through modulation of key enzymes involved in EG biotransformation, such as alcohol dehydrogenase (ADH) and/or lactate dehydrogenase (LDH), thereby limiting the conversion of EG into toxic intermediates including glyoxalate and oxalate. In addition, the observed enhancement of antioxidant defenses may indirectly suppress oxidative metabolism of EG intermediates, contributing to reduced metabolite accumulation. These proposed mechanisms are hypothesis-driven and warrant further investigation through targeted enzymatic and molecular studies.

The induction of liver damage led to a significant increase in serum citrate levels in EG animals by 2.47-fold compared to NEG rats. In rats receiving SBCL, serum citrate levels significantly declined. In EG-treated rats, SOD and GSH declined while LPO, NO, and PC elevated. Inducing liver damage significantly elevated NO, LPO, and PC levels by 2.6-fold, 2.3-fold, and 2.6-fold, respectively, compared to the control group. These increases reflect significant oxidative and nitrosative stress associated with the induction of EG. EG administration caused a decrease in GSH levels by 39.8 % compared to the control rats. Aggarwal, Gautam, Sharma and Singla [26] found that EG treated-group had an imbalance in the antioxidants. EG exposure resulted in a significant decrease in SOD activity by 55 % compared with the control group, indicating increased oxidative stress in these rats. SBCL and SBF administration significantly elevated GSH concentrations. Moreover, SOD activity, along with NO, LPO, and PC levels, were affected, highlighting the impact of SBCL and SBF on oxidative stress and antioxidant mechanisms.

SBF and SBCL administration increased SOD activity by 1.4-fold and 1.2-fold, respectively, compared to EG group. Despite these improvements, SOD levels with SBCL and SBF showed significant enhancement, underscoring the effectiveness of these compounds in boosting antioxidant defenses. SBCL and SBF led to significant reductions in NO levels, with decreases of 42 % and 51 %, respectively.

LPO levels were reduced by 34 % with SBCL and 22 % with SBF, while PC levels decreased by 29 % for SBCL and 49 % for SBF compared to the positive control group. These findings demonstrate that SBCL and SBF can partially ameliorate OS markers. Therefore, SBCL and SBF possess some antioxidative properties in mitigating OS in rats with liver damage.

This study explores the effects of sulfonamide compounds SBCL and SBF on inflammatory markers in rats with induced liver damage. The findings demonstrate notable differences in the modulation of inflammation by these compounds, contributing to our understanding of their therapeutic potential and highlighting the complexity of their actions.

Proinflammatory cytokines play a role in initiating and sustaining the immune response by activating inflammatory cells and inducing the release of other cytokines. TNF-α, IL-1, and IL-18 are key proinflammatory cytokines in immune response [27]. TNF-α, primarily produced by various cell types including monocytes/macrophages, Kupffer cells, skin keratinocytes, and T and B lymphocytes, interacts with high-affinity receptors in tissues such as the liver, muscle, intestines, and lungs. It acts as the most potent proinflammatory cytokine, stimulating the production of IL-1, IL-6, and chemokines [28]. IL-1ß, another pro-inflammatory cytokine, shares similar effects with TNF-α. Additionally, TNF-α enhances the release of IL-1α and IL-1ß, which are secreted by macrophages and endothelial cells. IL-1ß mediates the metabolic and physiological impacts of circulating TNF-α and contributes to local and systemic effects of acute and chronic inflammation.

Inducing liver damage significantly increases IL-1β levels, nearly 3.0-fold higher than the negative control. This is consistent with other studies indicating that IL-1β is a key pro-inflammatory cytokine elevated in liver diseases and other renal pathologies [29], [30]. SBCL and SBF reduced IL-1β levels by 40.5 % and 27.7 %, respectively. The greater reduction observed with SBCL highlights its stronger anti-inflammatory properties compared to SBF.

IL-6 levels also increased threefold in EG-treated animals, supported by literature describing IL-6 as a critical mediator in inflammatory responses and liver injury [30]. SBCL and SBF significantly decreased IL-6 levels by 40.6 % and 36.7 %, respectively. This decrease indicated that SBCL is more effective than SBF, which might have additional mechanisms contributing to its anti-inflammatory effects. The superior efficacy of SBCL in reducing inflammatory markers compared to SBF can likely be attributed to several factors. SBCL may have an enhanced ability to modulate oxidative stress and nitric oxide production more effectively. Additionally, its unique structural characteristics could facilitate better cellular penetration, stronger antioxidant activity, and more effective modulation of key inflammatory pathways [31]. Furthermore, potential differences in pharmacokinetics may allow SBCL to maintain therapeutic levels in tissues for longer, providing sustained anti-inflammatory effects [32].

Interestingly, AFP levels increased in rats treated with SBCL, SBF, and EG, suggesting that these may cause some degree of stress or damage, prompting an AFP response. AFP is often a marker of hepatic regeneration or damage. After inducing liver damage, SBCL decreased AFP levels by 16 %. TNF-α levels rose by 2.2-fold following EG induction, reflecting its role as a major pro-inflammatory cytokine in renal pathology. SBCL and SBF decreased TNF-α levels by 32.2 % and 22.8 %, respectively, indicating their potent anti-inflammatory action.

The observed ability of SBCL and SBF to reduce inflammatory markers such as IL-1β, IL-6, and TNF-α supports existing research on the anti-inflammatory potential of sulfonamide derivatives.

However, the study also highlights the differences between these compounds. While all applications were effective, SBCL consistently outperformed SBF in reducing inflammatory markers. These findings could inform future therapeutic strategies for managing liver damage and associated inflammation.

The histological and ultrastructural findings provide essential insight into the underlying liver damage, which can be closely correlated with the observed changes in liver enzymes and oxidative stress markers. Histological alterations in hepatocytes, such as necrosis, cytoplasmic vacuolization, and inflammatory cell infiltration, are indicative of hepatocellular injury and can explain the significant elevations in liver enzymes like ALT and ALP. The degenerative changes and alterations observed in the present study would indicate the local action of EG on the liver, as it is considered the main site of detoxification of toxic materials of administered substances [33]. The increased levels of ALT, a marker of hepatocellular damage, are reflective of the destruction of hepatocytes [34], as seen in histological sections.

The release of ALT into the bloodstream occurs when hepatocytes are injured, and the extent of damage observed histologically supports this biochemical finding. In parallel, ALP levels, which are typically elevated in cases of biliary damage [35], can be explained by histological evidence of bile duct proliferation or cholestasis. The presence of biliary proliferation and disrupted bile flow, as observed in histological sections, correlates with the increased ALP levels, signifying a disruption in the biliary structures that exacerbates liver injury.

Oxidative stress, which plays a central role in liver damage, is reflected in the biochemical markers and the histological observations. Elevated levels of LPO, NO, and PC are indicative of oxidative damage to hepatocytes, leading to cellular necrosis [36]. These biochemical markers of oxidative stress are mirrored in the histological and ultrastructural findings of hepatocellular swelling, mitochondrial damage, and lipid accumulation, which are common consequences of ROS-causing injury. The correlation between oxidative stress markers and histological alterations in liver tissue supports the notion that ROS contributes significantly to liver damage in EG-induced model, resulting in biochemical and histological evidence of injury. Moreover, the histological signs of inflammation, such as mononuclear cell infiltration and portal area inflammation, align with the elevated levels of pro-inflammatory cytokines and oxidative stress markers, indicating that inflammation plays a crucial role in the progression of liver injury.

The correlation between histological changes and biochemical data provides a robust framework for understanding the extent of liver damage and the therapeutic potential of various compounds. SBF and SBCL reduce histological signs of hepatocyte damage, such as lessened necrosis or reduced inflammation; these improvements would be expected to correlate with a decrease in ALT, ALP, and oxidative stress markers. This alignment between histological improvements and biochemical markers would support the hepatotherapeutic effects of SBF and SBCL, offering visual evidence of their effectiveness in mitigating liver injury.

This study highlights the differential effects of SBCL and SBF on liver enzymes, serum oxalate, citrate levels, and OS markers in rats with and without EG induction. Administering SBCL and SBF to rats without EG led to a decrease in ALT levels, indicating a potential hepatoprotective effect of these compounds under normal conditions. Inducing liver damage by EG significantly elevates OS as evidenced by the marked increases in NO, LPO, and PC levels and the significant decrease in SOD activity. SBCL appears to have a dual role, showing beneficial effects under normal conditions but potentially harmful effects in the presence of EG. Our findings indicate that while SBCL reduced ALT levels and improved antioxidant status in normal rats, it exacerbated ALT elevation and oxidative stress markers (NO, LPO, PC) in EG-damaged liver tissue. This dual behavior may be explained by three different mechanisms: a) altered hepatic redox balance in injured liver, where SBCL may shift from an antioxidant to a pro-oxidant role due to excessive ROS generation, b) impaired detoxification capacity in EG-injured hepatocytes, leading to accumulation of reactive metabolites, and c) structure-dependent redox cycling, where the chlorophenyl moiety in SBCL may enhance oxidative reactions under pathological conditions. Accordingly, we now explicitly describe SBCL as exhibiting context-dependent toxicity, and we emphasize the importance of cautious evaluation before therapeutic translation.

SBF demonstrates a more consistent protective profile. SBCL and SBF show the most robust antioxidative effects, significantly enhancing SOD activity and reducing NO, LPO, and PC levels. The data indicated that Benzene Sulfonamide Derivatives had therapeutic effects in rats with experimental EG-induced liver damage. Further studies are needed to explore the mechanisms through which these compounds exert their effects.

The current study parallel with our previous study that found both benzene sulfonamide derivatives improved kidney function, reduced stone formation, and preserved renal histological architecture, with SBCl showing superior antioxidant and anti-inflammatory effects. These results highlight their potential as effective and safer alternatives for managing nephrolithiasis and preventing related renal damage [37].

4.1. Study limitation

This study is the first to evaluate the effects of the benzene sulfonamide derivatives SBCL and SBF on ethylene glycol (EG)-induced liver toxicity in rats. While it provides valuable insights into their biochemical, anti-inflammatory, and antioxidative impacts, several limitations should be acknowledged:

• 1.Lack of Dose-Response Assessment: Only single doses of SBCL and SBF were tested. Without a dose-response analysis, it is unclear whether the observed effects are dose-dependent or if lower or higher doses might yield different toxicity or therapeutic profiles.
• 2.No Comparison to Standard Treatments: The study does not compare SBCL or SBF to known hepatoprotective or anti-inflammatory agents. Including a positive control could have helped benchmark the therapeutic value of these compounds.
• 3.Dual Nature of SBCL Not Fully Explored: The contrasting behavior of SBCL—hepatoprotective in normal liver and hepatotoxic in damaged liver—raises important safety concerns. Further investigation is needed to clarify the conditions under which SBCL transitions from therapeutic to harmful.
• 4.Pharmacokinetic and Bioavailability Data Missing: There was no evaluation of the pharmacokinetics or tissue distribution of SBCL and SBF. Understanding absorption, metabolism, and clearance is essential for drug development. This point will be cover in the future work.

These limitations highlight the need for expanded studies involving multiple dosing regimens, and in-depth mechanistic analyses before clinical translation can be considered.

5. Conclusion

The mechanism of action of the two benzene sulfonamide derivatives—SBCL (compound A) and SBF (compound B)—in ethylene glycol (EG)-induced liver damage appears to involve antioxidant and anti-inflammatory pathways. In control rats, compounds, particularly SBF, reduced ALT levels, indicating a baseline hepatoprotective effect. In EG liver injury, SBF maintained stable liver enzyme profiles and effectively reduced oxidative stress markers such as NO, LPO, and PC, while enhancing antioxidant defenses including GSH and SOD. SBCL, however, showed dual behavior: while it improved antioxidant markers to some extent, it also increased ALT levels and ALP activity in EG-treated rats, suggesting potential pro-oxidant effects under stress conditions. The two compounds significantly reduced inflammatory cytokines (IL-1β, IL-6, TNF-α) and minimized histological and ultrastructural liver damage, with SBF showing more consistent protective effects. Therefore, SBF suppresses oxidative stress and inflammation, whereas SBCL’s effect may be dose- or context-dependent, potentially switching from protective to harmful in liver tissue.

List of Abbreviations

AFP: alpha-fetoprotein, ALP: alkaline phosphates; ALT: alanine aminotransferase; compound A: (N-(4-chlorophenyl)-4-isobutoxy-N-(1-methylpiperidin-4yl) benzene sulfonamide); compound B (N-(4-fluorophenyl)-4-methoxy-N-(4-(4-methylpiperidin-4-yl) benzene sulfonamide); CV: central vein; DHPS: dihydropteroate synthase; EG: Ethylene glycol; K: Kupffer cells; IL-1β: interleukin 1β; IL-6: interleukin 6; LPO: lipid peroxidation; GSH; glutathione; N: necrotic cells; NEG: control negative; NO; nitric oxide; OD: optical density; OS: oxidative stress, PA portal area; PC: protein carbonyl; POD: peroxidase; SBCL-NEG: non-induced liver damage group treated with compound A; SBF-NEG: non-induced liver damage group treated with compound B; SOD: Superoxide dismutase; THF: tetrahydrofolate; TNF-α: tumor necrosis factor-alpha.

Ethics approval and consent to participate

All experimental procedures involving animals were conducted by the ethical standards of institutional and national guidelines. The study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Faculty of Veterinary Medicine at Suez Canal University in Ismailia, Egypt, under the protocol number [Protocol ID: 2019021], and all procedures complied with the ethical principles stated in the Declaration of Helsinki for the humane treatment of animals used in biomedical research.

Authors' contributions

All authors have made significant contributions to the conception and design of the study, acquisition of data, or analysis and interpretation of data. All authors were involved in drafting or revising the article critically for important intellectual content and approved the final version to be published. Each author agrees to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Funding

This study did not receive any dedicated funding from public, commercial, or non-profit organizations.

CRediT authorship contribution statement

Heba N. Gad El-Hak: Writing – review & editing, Writing – original draft, Supervision, Methodology, Investigation, Data curation. Zohour I. Nabil: Validation, Supervision, Investigation, Conceptualization. Rasha A. Al-Eisa: Software, Resources, Funding acquisition, Conceptualization. Ahmed M. Elgendy: Methodology, Investigation, Formal analysis, Data curation. El-Shenawy nahla: Writing – review & editing, Writing – original draft, Validation, Supervision, Investigation.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Handling Editor: Prof. L.H. Lash

Data availability

Data will be made available on request.