Introduction
Benzimidazole derivatives represent a significant class of heterocyclic compounds that have acquired extensive attention in pharmaceutical research due to their diverse biological activities, including antiparasitic, antifungal, antiviral, anti-inflammatory, and anticancer properties [1- 2]. The structural versatility of the benzimidazole scaffold makes it an attractive model for drug development, with modifications such as thiol, alkyl, or ether substituents often enhancing biological activity and selectivity [3- 4]. Among these modifications, bis-substituted benzimidazole compounds are particularly interesting due to their ability to interact with multiple cellular targets simultaneously.
Our laboratory has recently synthesized 1,5-bis-(mercaptobenzimidazolyl) diethylene glycol (BBO1), a novel bis-substituted benzimidazole derivative containing two thiol groups that may modulate cellular redox homeostasis. While the pharmacological potential of BBO1 requires investigation, comprehensive toxicological evaluation is essential to determine its safety profile prior to further investigations of its therapeutic applications.
Acute toxicity studies in laboratory animals, particularly Wistar rats, provide critical information regarding the median lethal dose (LD₅₀) and dose-response relationships following single-dose administration. These studies typically evaluate mortality, physiological parameters including body weight and food and water consumption, and behavioral parameters such as locomotor activity, anxiety, and depressive-like behaviors [5- 7]. Such comprehensive evaluation enables the detection of early toxicological manifestations and establishes the foundation for subsequent safety evaluations.
Oxidative stress (OS) is a key mechanism underlying chemical-induced toxicity, particularly in the central nervous system where neurons are highly susceptible to free radical damage due to their high metabolic activity and limited regenerative capacity [8- 9]. The brain regions most vulnerable to oxidative damage include the prefrontal cortex and hippocampus, which play crucial roles in cognitive and emotional functions. Toxicological assessment of oxidative stress typically involves measuring key biomarkers including nitric oxide (NO), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) which together provide insight into the balance between prooxidant generation and antioxidant defense mechanisms [10-13].
Therefore, the present study aims to evaluate the acute toxicity profile of BBO1 in male Wistar rats. This strain was selected due to its well-characterized physiology, high reproducibility, and frequent use in toxicological research [14]. The objective was to determine the lowest lethal dose (LLD), The median lethal dose (LD₅₀), of the absolute lethal dose (LD₁₀₀), and the no observed adverse effect level (NOAEL) , while monitoring physiological parameters, behavioral responses, and brain OS biomarkers.
Materials and Methods
1.Chemical characterization of BBO1
1,5-bis-(2-mercaptobenzimidazolyl)-3-oxapentane, is a novel symmetrical heterocyclic compound derived from benzimidazole (Figure 1). The molecule consists of two 2-mercaptobenzimidazole units linked by a diethylene glycol spacer containing one ether bridge (–O–) and two thioether linkages (–S–). Each benzimidazole ring bears a free thiol group (–SH), providing potential bidentate coordination sites for metal complexation.
BBO1 was synthesized through a nucleophilic substitution reaction between 2-mercaptobenzimidazole (Sigma-Aldrich - ref M3302) and diethylene glycol dichloride (Sigma-Aldrich - ref 35660), using potassium carbonate (K₂CO₃) as a base (Sigma-Aldrich- ref 60108). The reaction was carried out in dimethylformamide (DMF) (Sigma-Aldrich- ref 200-679-5). at room temperature for 24 hours. The crude product was isolated by filtration and purified through precipitation and recrystallization.
The compound was characterized using several analytical techniques, including: ¹H nuclear magnetic resonance (¹H NMR), mass spectrometry (MS), melting point determination, solubility and boiling point tests, and Fourier-transform infrared spectroscopy (FTIR). The molecular structure exhibits symmetry and flexibility, offering both chemical stability and promising coordination potential for applications in metal complexation or medicinal chemistry.
2.Experimental animals
This study was conducted using 30 male Wistar rats, aged 8 weeks and weighing 200 ± 20 g, obtained from the animal breeding facility of Ibn Tofail University, Morocco. Male rats were specifically selected to eliminate hormonal variables that could influence toxicological responses and to ensure consistency with established toxicological protocols for benzimidazole derivatives.
Animals were housed in the animal facility of the Faculty of Sciences, Ibn Tofail University (Kenitra, Morocco) under controlled environmental conditions: temperature 24 ± 2°C, humidity 55 ± 10%, and a 12 h light/12 h dark cycle. Rats had free access to standard chow (supplied by the company Alf Sahel) and water throughout the study period. A 7-day acclimatization period preceded all experimental procedures to minimize stress-related variables.
All experimental procedures were conducted in accordance with the Organization for Economic Cooperation and Development (OECD) guidelines and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Ethics Committee of Ibn Tofail University.
3.Acute toxicity evaluation
3.1.Experimental design
Acute toxicity assessment was conducted following the OECD guideline 423 for acute oral toxicity testing [15]. After the acclimatization period, rats were randomly assigned to six groups of five animals each: one control group receiving vehicle solution and five treatment groups receiving BBO1 at doses of 250, 500, 750, 1000 and, 1200 mg/kg body weight (Figure 2).
BBO1 was initially dissolved in 10% dimethyl sulfoxide (DMSO) was supplied by Sigma-Aldrich (ref. 472301, ≥99.9% purity, A.C.S. reagent grade). to prepare a stock solution at 120 mg/ml concentration. This stock solution was subsequently diluted with sterile saline to obtain the desired concentrations for each dose group. All injections were administered i.p. at a fixed volume of 1 ml per 100 g body weight to ensure consistent volume injection across groups. Control animals received an equivalent volume of vehicle solution (10% DMSO in saline) via the same route.
Animals were monitored for the first 4 hours post-injection, then twice daily for 14-days to assess mortality, behavioral changes, and clinical signs of toxicity. Parameters evaluated included signs such as imbalance, altered posture, scratching, hair loss, response to external stimuli, respiratory patterns, changes in body weight, food and water consumption, and signs of distress.
On day 14, rats in groups 1, 2, 3, 4 and 5 (Figure 2). underwent a series of neurobehavioral tests to evaluate the functional consequences of BBO1 exposure. The testing battery included the Elevated Plus Maze (EPM) for anxiety-like responses, the Forced Swimming Test (FST) for depressive-like behavior, and the Open Field Test (OFT) for locomotor activity evaluation. Following completion of behavioral testing, animals were subjected to an overnight fasting period (12-16 hours) to standardize metabolic conditions. Animals were anesthetized using chloral (sigma-aldrich, laborchemikalien Gmbh, Allemagne) administered i.p. at a dose of 0.5 ml per 100 g body weight. Animals were then euthanized by decapitation, and brain tissues were rapidly extracted. The prefrontal cortex (PFC) and hippocampus (HP) were carefully dissected and immediately processed for biochemical analysis of oxidative stress biomarkers, including MDA, CAT, SOD and NO.
3.2.clinical assessment and determination of toxicological parameters
acute toxicity assessment was conducted in accordance with the OECD guidelines for acute toxicity testing [16-17]. The experimental protocol involved administration of increasing doses of BBO1 to establish key toxicological thresholds and define dose-response relationships. All animals were monitored continuously until 100% mortality was observed within the 14-day observation period, enabling determination of the absolute lethal dose (LD₁₀₀).
The median lethal dose (LD₅₀) was calculated as the dose inducing mortality in 50% of the test animals within the observation period. LD₅₀ remains a critical indicator in toxicology for comparing the relative toxicity of compounds and was determined in accordance with the Globally Harmonized System (GHS) of Classification and Labelling of Chemicals, as outlined by the OECD [17].
The lowest lethal dose (LLD) is the smallest dose that results in the death of at least one animal, representing the threshold of observable lethality.
The no observed adverse effect level (NOAEL) is defined as the highest tested dose at which producing no statistically or biologically significant adverse effects compared to control animals [18-19]. This parameter is essential for risk assessment and for establishing safety margins for potential therapeutic applications.
Clinical assessments during the observation period included monitoring for signs of acute intoxication such as behavioral alterations, changes in food and water consumption, respiratory distress, and overall physical condition.
4.Behavioral assessment
4.1.Elevated Plus Maze (EPM)
The EPM was used to assess anxiety-like behavior in rodents following established protocols [20]. The apparatus consists of four arms (50 cm long x 10 cm wide) elevated 50 cm above the floor, with two open arms and two enclosed arms (40 cm high walls) arranged in a cross configuration. The maze was constructed of black wood and positioned in a dimly lit room to minimize external stimuli.
Each rat was individually placed in the central platform (10 x 10 cm) facing an open arm and allowed to freely explore the arena for 5 minutes. Behavioral parameters recorded include the number of entries into open arms (EOA) and total time spent in open arms (TOA). An arm entry is defined as placement of all four paws into an arm. increased open-arm exploration (higher EOA and TOA values) is interpreted as reduced anxiety levels, while avoidance of open arms indicates elevated anxiety-like behavior.
4.2.Forced Swimming Test (FST)
The FST was conducted to assess depression-like behavior using established protocols [21]. The apparatus consists of a transparent cylindrical tank (50 cm height x 20 cm diameter) filled with warm water to a depth of 30 cm. water temperature was maintained at 25 ± 2°C throughout testing to ensure consistent conditions.
Each animal is placed in the FST cylinder and its behavior recorded for 5 minutes. The immobility time (IMT), defined as the total duration during which the animal remains motionless except for minimal movements necessary to maintain head above water surface. Increased IMT is interpreted as depression-like behavior indicative of behavioral despair.
4.3.Open Field Test (OFT)
The OFT was conducted to simultaneously assess locomotor activity and anxiety-related behavior [22]. The testing apparatus consists of a square wooden arena (80 x 80 x 50 cm) with black walls and floor, divided into 25 equal squares (9 central and 16 peripheral zones). The arena was illuminated with a 60W overhead lamp. Each rat was placed in the center of the arena and allowed to explore freely for 10 minutes. Behavioral was recorded using a ceiling-mounted camera connected to an automated tracking software system (ANY-maze). The following parameters were analyzed: number of central squares visited (NRC), total number of squares crossed (NTS), and time spent in the central area (TCA). Higher TCA and NRC values indicate lower anxiety levels, while NTS reflects general locomotor activity [23-24]. The arena was thoroughly cleaned with 70% ethanol between trials to eliminate olfactory cues.
5.Biochemical analysis
Neurochemical analyses were performed to evaluate oxidative stress status through the assessment of lipid peroxidation and antioxidant enzyme activities in the PFC and HP. Following behavioral testing and euthanasia, brain tissues were rapidly extracted and the target regions carefully dissected under sterile conditions on an ice-cold surface. Tissue samples were immediately rinsed with ice-cold phosphate-buffered saline (50 mM, pH 7.4) to remove blood contamination.
Brain tissue homogenates were centrifuged at 3000 rpm for 10 minutes at 4°C using a refrigerated centrifuge. The resulting supernatants were carefully collected and stored in aliquots at −20 °C until biochemical analysis.
Lipid peroxidation was evaluated by quantifying MDA levels using the thiobarbituric acid reactive substances (TBARS) method as described by Draper and Hadley [25]. Antioxidant enzyme activities were assessed using established spectrophotometric methods: SOD activity was measured according to Beauchamp and Fridovich [26], and CAT activity determined following the protocol of Aebi [27]. NO levels were evaluated using the Griess reagent method [28]. All analyses were performed in duplicates, and protein concentrations were determined using the Bradford assay to normalize enzyme activities and metabolite levels.
6.Statistical analysis
Statistical analyses were conducted using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA). Data normality was assessed using the Shapiro–Wilk test, and homogeneity of variances was verified using the Brown–Forsythe test prior to performing ANOVA. Between-group comparisons were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons.
For dose-response analysis, linear regression and probit analysis were employed to calculate LD₅₀ values with 95% confidence intervals. Behavioral and biochemical data are presented as mean ± standard error of the mean (SEM), while mortality data are expressed as percentages. Statistical significance was set at p < 0.05 for all analyses.
Results
1.Acute toxicity evaluation
During acute toxicity testing, administration of BBO1 at doses exceeding 750 mg/kg induced significant dose-dependent alterations in physical appearance and behavior, clearly indicating signs of systemic toxicity. Within the first hour following i.p. injection, rats exhibited progressive reduction in motor activity, with symptom severity directly correlating with administered dose. At the highest doses, epileptiform seizures were observed immediately preceding death demonstrating pronounced neurotoxic potential of BBO1 under acute exposure conditions.
Complete mortality (100%) was observed in animals receiving 1200 mg/kg within 30 to 90 minutes following injection, establishing this dose as the LD100. Mortality data across all dose groups are summarized in Table 1 and Figure 3.
No clinical signs of toxicity were observed in animals treated with 250 or 500 mg/kg BBO1 throughout the 14-day observation period. The 750 mg/kg dose represents the threshold for initial physiological and behavioral alterations without mortality, thus establishing this dose as the maximum tolerated dose (MTD). Based on the absence of adverse effects, NOAEL was established at 500 mg/kg. Consequently, comprehensive behavioral and biochemical evaluations were conducted in animals receiving doses up to 750 mg/kg.
2.Effect of BBO1 on body weight
Body weight was monitored once a week (Day 0, 1, 7, and 14) in all experimental groups throughout the 14-day period following BBO1 administration (Figure 4). Baseline body weights (Day 0) were comparable across all groups, ranging from 200 to 210g. Animals receiving i.p. injections of BBO1 at 250 and 500 mg/kg maintained normal weight gain patterns similar to control group throughout the study period, with no significant differences observed. In contrast, rats treated with higher doses (750 and 1000 mg/kg) exhibited significant body weight reduction compared to control group (p < 0.001). This dose-dependent weight loss became apparent on day 7 and was maintained at day 14, indicating sustained metabolic disruption at these dosage levels.
3.Effects of BBO1 administration on food and water consumption
Food and water consumption were monitored throughout the observation period to assess potential effects on basic physiological functions. Animals treated with 250 and 500 mg/kg BBO1 showed no significant change in daily food (g/day/rat) or water (mL/day/rat) intake compared to control group, indicating minimal impact on feeding behavior and fluid homeostasis at these doses.
Conversely, animals receiving higher doses (750 and 1000 mg/kg) showed marked and persistent reductions in both food and water consumption (p < 0.001) throughout the observation period (Figure 5). This dose-dependent decrease correlated with the observed body weight loss and likely contributed to the overall deterioration in physiological status at these dosage levels.
4.Effect of BBO1 on anxiety-like behavior and locomotor activity in the OFT
Our findings revealed dose-dependent alterations in both anxiety and locomotor activity following BBO1 administration (Figure 6). Animals treated with high doses (750 and 1000 mg/kg) exhibited significantly reduced exploration of the central area, as evidenced by decreased NRC compared to control animals (p < 0.001) (Figure 6A). In contrast, no significant differences were observed between the control group and the treated groups receiving lower doses (250 and 500 mg/kg).
Similarly, TCA analysis revealed a significant reduction of the time spent in the central area in groups treated with 750 and 1000 mg/kg of BBO1 compared to the controls (p < 0.001) (Figure 6B). animals treated with 250 or 500 mg/kg exhibited no statistically significant differences in TCA relative to the control group. These behavioral patterns indicate increased anxiety-like behaviors at doses ≥ 750 mg/kg.
Furthermore, general locomotor activity, assessed by NTS parameter, revealed significant hypoactivity in animals receiving higher doses (750 and 1000 mg/kg BBO1) compared to controls (p < 0.001) (Figure 6C). In contrast, no significant differences were observed in 250 and 500 mg/kg groups. These findings demonstrate a clear dose-dependent reduction in spontaneous motor activity, with the threshold for locomotor impairment at 750 mg/kg of BBO1.
5.Effects of BBO1 on depressive-like behavior in FST
Depressive-like behavior assessment using the FST revealed dose-dependent alterations of behavioral despair responses following BBO1 administration. Animals treated with 750 and 1000 mg/kg showed a significant increase in IMT compared to control animals (p < 0.001), indicating enhanced depressive-like behavior at these doses (Figure 7). This behavioral pattern suggests that BBO1 exposure at higher concentrations may induce neurochemical alterations associated with depressive-like states.
In contrast, rats receiving lower doses (250 and 500 mg/kg) showed no significant differences in IMT compared to the control group, indicating that depressive-like behaviors are not expressed at these doses levels. These findings establish a clear threshold effect, with depression-like manifestations emerging at doses ≥ 750 mg/kg.
6.Effects of BBO1 on anxiety-like behavior in the EPM
Elevated plus maze test confirms the anxiogenic effects of BBO1 observed in the OFT. Animals exposed to higher doses (750 and 1000 mg/kg) showed significant decrease in open arm entries (OAE), indicating heightened anxiety levels compared to the control group (p < 0.001). In contrast, animals treated with lower doses (250 and 500 mg/kg) did not show any significant change in OAE relative to controls.
Similarly, the analysis of time spent in the open arms (TOA), revealed marked reduction in TOA in animals receiving 750 and 1000 mg/kg BBO1 (p < 0.001). This avoidance behavior further confirms increased anxiety-like behavior at higher doses. No significant differences in TOA were observed between the control group and those treated with the lower doses (250 and 500 mg/kg), reinforcing the dose-dependent nature of BBO1-induced anxiogenic effects.
7.Impact of BBO1 on oxidative stress parameters in PFC and HP
7.1 Lipid peroxidation assessment (MDA levels)
Malondialdehyde (MDA) levels were significantly increased in both the PFC and HP following administration of 750 and 1000 mg/kg BBO1 compared to controls (p<0.001) (Figure 9). This dose-dependent elevation in MDA concentrations indicates increased lipid peroxidation in these brain regions. No significant changes in MDA levels were observed in animals treated with 250 and 500 mg/kg compared to controls. These results demonstrate that lipid peroxidation occurs at doses ≥ 750 mg/kg, which corresponds to the threshold for behavioral and physiological toxicity.
7.2 Nitric oxide concentrations
Nitric oxide (NO) levels were significantly increased in both the PFC and HP following treatment with 750 and 1000 mg/kg BBO1 compared to controls (p < 0.001), with the highest concentrations observed at 1000 mg/kg (Figure 10). Animals treated with 250 and 500 mg/kg showed no significant differences in NO levels compared to controls in either brain region.
7.3 Antioxidant enzyme activities
Superoxide dismutase (SOD) and catalase (CAT) activities were significantly increased in both the PFC and HP following treatment with 750 and 1000 mg/kg BBO1 compared to controls (p < 0.001) (Figure 11). Animals treated with lower doses (250 and 500 mg/kg) showed no significant changes in antioxidant enzyme activities in either brain region compared to controls.
Discussion
The present study provides comprehensive characterization of the acute toxicity profile of BBO1, a novel bis-substituted benzimidazole derivative, in male Wistar rats. Integrated behavioral, physiological, and biochemical analyses demonstrate dose-dependent neurotoxicity and systemic toxicity at doses above 750 mg/kg. These findings establish safety thresholds and provide preliminary data for future therapeutic development of this benzimidazole analog.
Mortality analysis revealed dose-dependent lethality following BBO1 administration. Total mortality occurred at 1200 mg/kg within 30-90 minutes post-injection, establishing this dose as the LD100. No deaths were observed at doses ≤ 750 mg/kg, while one animal died at 1000 mg/kg (20%) allowed for the estimation of the LLD at 1000 mg/kg and the LD50 at 1,071.4 mg/kg. These findings align with OECD and GHS classification guidelines for acute toxicity classification and are comparable to reported LD50 values for structurally related benzimidazole derivatives, which typically range from 800-1500 mg/kg in rodent models [29-30].
Clinical observations revealed progressive neurological deterioration in animals receiving doses exceeding 750 mg/kg. Reduced locomotor activity emerged within the first hour post-administration, demonstrating a dose-dependent temporal relationship that progressed to generalized seizure activity preceding death in the 1200 mg/kg group. These rapid-onset neurological manifestations indicate acute central nervous system (CNS) toxicity, likely mediated through perturbation of neuronal membrane excitability or disruption of cellular redox homeostasis. The progression from behavioral depression to seizure activity suggests the involvement of multiple pathophysiological mechanisms, including potential interference with neurotransmitter systems or direct cytotoxic effects on neural tissue. In contrast, no signs of toxicity were observed in animals receiving 250 or 500 mg/kg. These animals remained asymptomatic throughout the observation period, establishing a clear toxicological threshold.
Toxicological threshold determination revealed that 750 mg/kg represents the MTD, characterized by initial but non-lethal manifestations of toxicity encompassing both behavioral and physiological parameters. Conversely, 500 mg/kg was established as the NOAEL, as no significant alterations were observed in this group. These thresholds are essential for defining safety margins in future investigations.
The present findings provide strong evidence that BBO1 administration at doses exceeding 750 mg/kg induces multisystem toxicity affecting both peripheral physiology and central nervous system function. The observed reduction in body weight gain at 750 and 1000 mg/kg reflects metabolic disruption and systemic stress responses characteristic of toxicity.
Similar findings have been reported for structurally related benzimidazole derivatives, which cause anorexia, reduced food intake, and energy imbalance in acute toxicity studies [31-32]. The concomitant reductions in food and water consumption during the first week post-exposure likely result from gastrointestinal irritation or centrally mediated appetite suppression [33]
neurobehavioral assessment showed dose-dependent impairments of many behaviors. BBO1 administration induced diminished locomotor activity, reduced exploratory behavior in novel environments, and enhanced behavioral despair responses, collectively indicating both anxiogenic and depressogenic properties.
These behavior alterations align with CNS toxicity patterns documented for structurally similar benzimidazole compounds [34-35]. Specifically, the marked reduction in open-arm entries and exploration observed in EPM and OFT provide convergent evidence for increased anxiety-like behavior at toxic dose levels, consistent with established behavioral pharmacology principles [36].
The observed neurobehavioral deficits were further corroborated by comprehensive oxidative stress biomarker analysis, revealing significant neurochemical perturbations in both the PFC and HP. BBO1 administration resulted in marked elevation of NO concentrations in both the PFC and HP, indicating activation of nitrosative stress pathways [37]. Excessive NO production can contribute to neuronal dysfunction through peroxynitrite-mediated oxidative damage and mitochondrial respiratory chain impairment [38]. The concurrent upregulation of SOD and CAT activities likely represents an adaptive antioxidant response attempting to mitigate reactive oxygen species (ROS) overproduction and accumulation. However, the significant elevation in MDA levels within these brain regions (the PFC and HP) confirms the occurrence of lipid peroxidation and subsequent cellular membrane integrity compromise [39-40], suggesting that endogenous antioxidant defenses were overwhelmed at toxic dose levels.
Taken together, these findings establish a comprehensive toxicological profile for BBO1 characterized by rapid-onset of CNS toxicity at doses ≥750 mg/kg, dose-dependent mortality at ≥1000 mg/kg, and acute biochemical alterations consistent with oxidative stress-mediated cellular damage. The convergence of behavioral, physiological, and neurochemical evidence supports a multifactorial toxicity mechanism involving disruption of cellular redox homeostasis and subsequent neuronal dysfunction. These results emphasize the critical importance of rigorous dose optimization in future pharmacological applications of BBO1 and highlight the necessity for comprehensive chronic toxicity studies, mechanistic elucidation, and safety assessment protocols prior to any potential therapeutic applications of BBO1 or related benzimidazole derivatives.
Conclusions
This study characterized the acute toxicity profile of BBO1, a novel bis-substituted benzimidazole derivative, in male Wistar rats. BB01 demonstrated dose-dependent toxicity with an LD50 of 1071.4 mg/kg, a MTD of 750 mg/kg, and a NOAEL of 500 mg/kg. toxic manifestations included weight loss, reduced food and water consumption, anxiety- and depressive-like behaviors, and significant oxidative stress in critical brain regions, characterized by elevated NO, MDA, SOD, and CAT levels in the PFC and HP.
The identification of clear toxicological thresholds provides essential safety parameters for future preclinical development of BBO1 and related benzimidazole derivatives. These findings emphasize the importance of assessing neurotoxicity and oxidative stress in early-stage drug development, particularly for compounds targeting CNS applications like benzimidazole derivatives.
This study has several limitations that should be considered. The use of male rats only limits the generalizability of our findings, as sex differences in drug metabolism and toxicity are common. Additionally, our acute exposure model does not address potential effects of repeated dosing or long-term exposure. Future studies should include chronic toxicity evaluation, pharmacokinetic analysis, and investigation of the molecular mechanisms underlying BBO1-induced oxidative stress. Evaluation of therapeutic potential at safe doses, assessment of sex-specific effects, and exploration of protective strategies against neurotoxicity are valuable research priorities for the development of this benzimidazole derivative.
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