Journal of The Arab Society for Medical Research

: 2019  |  Volume : 14  |  Issue : 1  |  Page : 14--24

Role of citicoline as a protective agent on toluene-induced toxicity in rats

Nermeen Shaffie, Marwa E Shabana 
 Department of Pathology, National Research Centre, Cairo, Egypt

Correspondence Address:
Marwa E Shabana
Department of Pathology, Medical Division, National Research Center, Cairo


Background/aim Toluene is used as an organic solvent; it has toxic effects on liver, renal, and neurological tissues. Citicoline has antioxidant effects. The aim of this study was to investigate the protective effect of citicoline against toluene-induced toxicity in rats. Material and methods A total of 24 rats were divided in four equal groups with six rats each. Rats in group 1 were the control. Rats in group 2 were exposed to toluene intraperitoneally at 900 mg/kg. Rats in group 3 were exposed to toluene 900 mg/kg in combination with citicoline at 150 mg/kg orally. The rats in group 4 were exposed to toluene 900 mg/kg in combination with citicoline 300 mg/kg orally. All treatments were used daily for 6 days. All rats were killed by decapitation. Tissue sections were stained with routine histological methods and examined under light microscope. In addition, sections were immunohistochemically staining with caspase-3 for liver and renal tissues and glial fibrillary acidic protein (GFAP) for brain tissue, and area percentage of positivity was measure by image analysis system. Results Histopathological and immunomorphometric studies of GFAP in brain and caspase-3 in liver and kidney were done. Toluene-treated groups showed vacuolar degeneration of hepatocytes and epithelial lining of renal tubules, neuronal damage, and neurodegeneration. There was an increase in caspase-3 in liver and kidney, which show marked increase in control positive rats that treated with toluene alone, but when large dose of citicoline 300 mg was added to toluene, the area percentage of caspase-3 was markedly decreased in liver and kidney. In addition, GFAP expression in brain tissue was decreased in toluene-treated control positive group, which then increased when treated with citicoline especially with a large dose. Conclusion The results of this study indicate that citicoline treatment can protect against toluene-induced toxicity in rats.

How to cite this article:
Shaffie N, Shabana ME. Role of citicoline as a protective agent on toluene-induced toxicity in rats.J Arab Soc Med Res 2019;14:14-24

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Shaffie N, Shabana ME. Role of citicoline as a protective agent on toluene-induced toxicity in rats. J Arab Soc Med Res [serial online] 2019 [cited 2020 Feb 18 ];14:14-24
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The abuse of volatile substances is an important problem especially in children in United States and many other countries. Exposure to inhalants occurs primarily via pulmonary absorption, and less absorption takes place through gastrointestinal tract or skin [1].

Substance abusers may have coexistent disorders such as acquired immune-deficiency syndrome, hepatic cirrhosis, traumatic brain injury, and nutritional deficiencies that affect neuropathological studies [2].

Organic solvents may produce cerebellar dysfunction, encephalopathy, cranial neuropathies, peripheral neuropathy, and parkinsonism. Of all inhalants, the greatest incidence of abuse of a single substance is found with toluene [3].

Toluene (methylbenzene) is an aromatic, clear, volatile colorless hydrocarbon. It is a component of many paints, glues, adhesives, inks, and cleaning fluids; it is also widely used as a cleaning and drying agent in the rubber and lumber industries, and in the dry cleaning and chemical industries [4].

After inhalation, toluene is rapidly absorbed through the lungs and then distributed throughout the body. It is estimated that 40–60% of inhaled toluene is rapidly absorbed. Oral absorption is complete, but its rate is slower than pulmonary absorption [5].

Following absorption, toluene has a high lipophilic activity. It is distributed quickly to highly perfused tissues such as brain and liver, with accumulation in tissues with high lipid content. Toluene has shown high concentrations in adrenals, adipose tissue, bone marrow, highly vascular tissues (e.g. kidney and liver), brain, and blood [6]. High levels of toluene were detected in the brain and liver tissue of people who died from sniffing glue [7].

Most of the inhaled toluene is metabolized in the liver by oxidation pathway and then excreted in urine as hippuric acid within 12 h after exposure. Metabolism of toluene produces free oxygen radicles and can mediate cellular damage. Approximately 7–20% of absorbed toluene is eliminated in the expired air unchanged [6].

Toluene, which is lipophilic, is thought to change lipid structure in the cell wall, increase membrane fluidity, and result in apoptosis [8]. Another study shows that toluene results in apoptosis by increasing reactive oxygen radicals, which are considered the initiating factor [9].

Apoptosis is one of programmed cell death characterized by DNA fragmentation, membrane changes, cytoplasmic shrinkage, and cell death without lysis or damage to the adjacent cells [10]. Apoptotic pathways are categorized as being intrinsic (mitochondria mediated) or extrinsic (receptor mediated). Two main factors that elicit receptor-mediated apoptosis are Fas ligand and tumor necrosis factor. Mitochondria-mediated apoptosis, on the contrary, is triggered via the release of apoptotic factors from the mitochondrial intermembrane space into cytosol, such as cytochrome C [11].

Caspases take part in both the extrinsic and intrinsic apoptosis pathways. However, caspase-3, also known as the executioner caspase, is responsible for the morphological changes such as chromatin condensation, DNA breakdown laddering, and breakdown of membrane protein [12].

It is known that caspase-3 activity increases in liver damage caused by many organic solvents [13]. El-Nabi Kamel and Shehata [14] studied the effect of toluene on brain cortex, cerebellum, liver, kidney, and testis tissues. The investigators reported that the brain tissue was most affected [15]. There are several studies on substances that cause renal toxicity, and one of them is toluene [16].

It has been reported that toluene exposure leads to serious conditions such as metabolic acidosis, hematuria, hypokalemia, proteinuria, distal tubular renal acidosis, formation of renal stones, and pyuria because of nephrotoxicity [17]. It is obvious that toluene deteriorates the renal glomerular and tubular structures and causes renal emergence [18].

The most basic effect that may be observed by exposure to toluene is depression of the central nerve system. Additionally, toluene may cause permanent damage of the brain tissues. High concentrations of toluene may demonstrate certain effects such as loss of upright posture reflex, damage in psychomotor activities, and sedation [19].

Citicoline (cytidine diphosphocholine) is a mononucleotide composed of ribose, cytosine, pyrophosphate, and choline. As an endogenous compound, citicoline is an essential intermediate in the synthesis of cell membrane structural phospholipids [20].

Citicoline is composed of two essential molecules, choline and cytidine, the structural phospholipids of cell membranes. Phospholipids have a high turnover rate, which means that a cell requires continuous synthesis of these compounds to ensure adequate function of cell membranes. It appears that the ability of citicoline to improve phosphatidylcholine synthesis in the injured brain is the important component of citicoline neuroprotective capacity [21]. Citicoline is also an exogenous source for acetylcholine synthesis, a key neurotransmitter, and a member of the group of molecules that play important roles in cellular metabolism known as nucleotides. Moreover, several researchers reported the beneficial outcome of citicoline treatment in neurological disorders [22].

Citicoline has great benefit at the site of the lesion, which occurs because of neuronal cell damage, by decreasing the accumulation of free fatty acids. Citicoline shows neural restorative effects, presumably via action on the dopaminergic system of the central nervous system. Rats with substantia nigra lesions have been shown to regenerate nerve cells after treatment with citicoline, indicating its protective effect in this region. Further studies have proved that citicoline administration to rats increases striatal dopamine synthesis [23].

Furthermore, citicoline attenuates lipid peroxidation through attenuating the activation of phospholipase A2, thus reducing inflammation in neural tissues and other organs. Citicoline has been shown to have direct free-radical suppressive effects, as it has a suppressive effect on hydroxyl radical generation [24].

In the present study, we investigated the protective effects of citicoline on liver, renal, and neuronal damage in rats exposed to toluene.

 Materials and methods

Drug and chemicals

Cytidine diphosphocholine (citicoline, Somazina: 100 mg/ml oral solution; Ferrer International SA, Barcelona, Spain) was used in the study and dissolved in physiological saline to obtain the necessary doses. Toluene (Sigma, St Louis, Missouri, USA) was freshly prepared in paraffin oil to obtain the required doses.

Animal and ethical approval

Male Sprague-Dawley rats, weighing 180–200 g, were group-housed under controlled temperature and light conditions. Rats were allowed standard laboratory rodent chow and water ad libitum. The study followed the recommendations of the institutional Ethics Committee and that of the National Institutes of Health Guide for Care and Use of Laboratory animals, Medical Research Ethical Committee of National Research Centre, with approval no. 18186.

Study design

Rats were divided into four groups containing six animals in each group. Group 1 (negative control group) was treated with only 9% saline. Group 2 (control positive group) rats were treated with toluene (900 mg/kg) alone. Groups 3 and 4 rates were treated with toluene along with citicoline at 150 and 300 mg/kg, respectively. Drugs and vehicle were given orally on daily basis for 6 days. Rats were then euthanized by decapitation under ether anesthesia for tissue collection.

Histological assessment study

Liver, kidney, and brain samples of all animals were dissected immediately after death. The specimens were then fixed in 10% neutral-buffered formalin saline for at least 72 h. All the specimens were washed in tap water for half an hour and then dehydrated in ascending grades of alcohol, cleared in xylene, and embedded in paraffin. Serial sections of 6-µm thick were cut and stained with hematoxylin and eosin (H&E) for histopathological investigation.

Immunohistochemistry for glial fibrillary acidic protein and caspase-3

Paraffin-embedded sections were deparaffinized and hydrated. Immunohistochemistry for brain sections was performed with a mouse monoclonal glial fibrillary acidic protein (GFAP) antibody for detection of GFAP activity. For liver and kidney sections, immunohistochemistry investigation was performed with a mouse monoclonal caspase-3 antibody for detection of the caspase-3 activity. The paraffin sections were heated in a microwave oven (25 min at 720 W) for antigen retrieval and incubated with either anti-caspase (for liver and kidney samples) or anti-GFAP antibodies (for brain sections) (1 : 50 dilution) overnight at 4°C. After washing with PBS, followed by incubation with biotinylated goat-anti-rabbit-immunoglobulin G secondary antibodies (1 : 200 dilution; Dako Corp., Copenhagen, Denmark) and streptavidin/alkaline phosphatase complex (1 : 200 dilution; Dako Corp.) for 30 min at room temperature, the binding sites of antibody were visualized with DAB (Sigma). After washing with PBS, the samples were counterstained with H&E for 2–3 min and dehydrated by transferring them through increasing ethanol solutions (30, 50, 70, 80, 95, and 100% ethanol). Following dehydration, the slices were soaked twice in xylene at room temperature for 5 min, mounted, examined, and evaluated by high-power light microscope.

Histological and immunohistochemical studies

The studies were carried out on H&E-, GFAP-, and caspase-3-stained slides. Images were examined and photographed under a digital camera (Microscope Digital Camera DP70, Tokyo, and processed using Adobe Photoshop, version 8.0).

Immunomorphometric analysis

The morphometric analysis was performed at the Pathology Department, National Research Centre, using the Leica Qwin 500 Image Analyzer (Leica Imaging Systems Ltd, Cambridge, England,), which consists of Leica DM-LB microscope with JVC color video camera attached to a computer system Leica Q 500 IW.

Detection of glial fibrillary acidic protein and caspase-3 percentage area

Morphometric analysis was carried out on GFAP- and caspase-3-stained slides. The area to be measured is determined as an area per field in micrometer square, area fraction, and area percentage by using the interactive software of the system. The results appear automatically on the monitor in the form of a table with the total, mean, SD, SE, the minimum area, and the maximum area measured. The area is measured in 10 fields in each slide.

We started by detection of the marker color to be detected, and then the software forms a binary image for the area of stained by the marker. This area was measured using an objective lens with magnification of ×20.

Statistical analysis

Data were statistically described in terms of mean±SD, median and range, or frequencies (number of cases) and percentages when appropriate. Comparison of numerical variables between the study groups was done using the Mann–Whitney U test for independent samples for comparing two groups and Kruskal–Wallis test for comparing more than two groups. P values less than 0.05 was considered statistically significant. All statistical calculations were done using the computer program SPSS (2006, statistical package for the social sciences; SPSS Inc., Chicago, Illinois, USA) release 15 for Microsoft Windows.


Histopathological results

Treating animals with toluene affected all the examined tissues. For the cerebral cortex in brain tissue, toluene caused the nuclei of some neurons to be karyorrhectic and many neurons to be shrunken and dark ([Figure 1]b) when compared with the normal neurons ([Figure 1]a). Citicoline had an ameliorating effect for brain tissue in a dose-dependent manner. The low dose (150 mg/kg) slightly decreased the damaged neurons ([Figure 1]c), whereas the high dose (300 mg/kg) caused most of the affected neurons to appear normal ([Figure 1]d).{Figure 1}

In addition, examination of the hippocampal area clarified similar results. The normal structure of this area of brain tissue appears in [Figure 2]a. The damaging effect of toluene was in the form of decrease in thickness of this area. Many cells show darkly stained cytoplasm with small nuclei but with well-defined nucleoli ([Figure 2]b). Citicoline in its low dose caused slight decrease in damaged neurons, although disorganization of cell layers was observed ([Figure 2]c). The high dose of the drug nearly normalized the hippocampal area.{Figure 2}

Examination of liver tissue revealed the normal structure of this tissue, as shown in [Figure 3]a. Toluene had a toxic effect on liver tissue in the form of dilatation of blood vessels and sinusoids and strong acidophilic appearance of cytoplasm of some hepatocytes ([Figure 3]b). In rats treated with toluene and 150 mg citicoline, dilatation and degeneration of epithelial lining of tubules are still noticed ([Figure 3]c). The high dose of citicoline markedly decreased the vacuolar degenerated hepatocytes ([Figure 3]d).{Figure 3}

By examining the renal tissue, the normal structure of this tissue is observed in [Figure 4]a. The toxic effect of toluene on renal tissue was in the form of vacuolar degeneration of tubular epithelial lining with dilatation of some tubules ([Figure 4]b). Low dose of citicoline had a very mild ameliorating effect on renal tissue, as dilatation and degeneration of epithelial lining of tubules were still noticed ([Figure 4]c), whereas a large dose of citicoline showed no vacuolar degenerated tubular epithelial lining cells, although signs of edema and dilatation of tubules were observed ([Figure 4]d).{Figure 4}

Immunomorphometric results

Histopathological evaluation was done using quantitative morphometric analysis of the pathological changes using GFAP that was detected in cytoplasm of viable astrocytes marked by the blue color in image analyzer system to be measured as an area %. The data presented in [Table 1] reported that the maximum expression was in the normal control group (4.68±0.06) that received saline only. On the contrary, the lowest GFAP expression was in the second group (0.88±0.02) that was treated by toluene only, denoting its destructive effect of neuronal tissue. The area % of GFAP increased gradually by treatment with citicoline. Area % of GFAP has shown slight increase in the group that received toluene combined with citicoline (150 mg/kg) (1.23±0.54) when compared with the positive control group, whereas area % of GFAP has been increased in the group that received toluene combined with citicoline (300 mg/kg) (4.1±0.04) when compared with the positive control group, with increased percentage (73.4%) indicating the efficacy of citicoline in improving the neuronal tissue ([Figure 5], [Table 1]).{Table 1}{Figure 5}

Activated caspase-3 labeling was specific in delineating morphologically apoptotic cells, where its expression was localized in the cytoplasm of the apoptotic cells, which were marked by the blue color in image analyzer system to be measured as an area %. The data presented in [Table 2] reported that the least liver tissue caspase-3 expression was in the normal control group (11.9±0.01), whereas the maximum expression was in the second group (toluene only treated group) (46.5±0.06). On the contrary, liver tissue caspase-3 expression was lowered in the group treated by citicoline 150 mg/kg, which showed slight decrease in caspase-3 expression (30.8±0.07) compared with the group treated with citicoline 300 mg/kg (22.1±0.03) by a reduction percentage of 47.3%, which indicates liver tissue improvement ([Figure 6], [Table 2]).{Table 2}{Figure 6}

For kidney tissue, the caspase-3 expression in control negative group was 12.4±0.01, which increased in the control positive group (treated with toluene only) to 63.8±0.07. There were gradual decreases in caspase-3 expression according to the doses of citicoline; usage of citicoline 150 mg/kg with toluene showed slight decrease in caspase-3 expression (35.7±0.05). However, high dose of citicoline (300 mg/kg) markedly reduced this expression to 20.6±0.03, with reduction percentage of 67.7% ([Figure 7], [Table 2]). These results clarified that citicoline had a good ameliorating effect in its high dose for all tissues examined.{Figure 7}


In this study, we investigated the toxic effect of toluene on brain, liver, and renal tissues. In addition, we investigated the protective effect of citicoline against toluene toxicity. Toluene and its metabolites have toxic effect on many organs [25]. Most of toluene undergoes series of metabolism in liver, and then is finally excreted in urine [7]. Toluene and its metabolites are reported to cause cell damage via oxidative stress [26]. The main effect of toluene is on nervous system, but animals exposed to moderate or high levels of toluene may also show harmful effects in their liver, kidneys, and lungs [4].

Acute exposure to toluene can cause vertigo, headache, coma, or even death [16]. Kanter [9] and many experimental studies proved that inhalation of toluene can cause hemorrhage, gliosis, shrunken cytoplasm, dark pyknotic cells, and necrosis. These findings are in agreement with the result of our study, which demonstrated that toluene can cause hemorrhage, degeneration, gliosis, many pyknotic cells, and necrosis.

There are many studies on substances that cause renal toxicity owing to chronic exposure [16]. One of these substances is toluene, which can lead to metabolic acidosis, hematuria, hypokalemia, formation of renal stones, and pyuria as a result of nephrotoxicity [15]. Meydan et al. [27] proved that toluene exposure causes shrinkage in the glomerular tuft and increases in connective tissue at interstitial area.

In our study, we studied the pathological effect of toluene on renal tissue and found vacuolar degeneration of tubular epithelial lining and tubular dilatation and widening of urinary space denoting edema, which cause pressure on the glomerular tuft, leading to decrease its size. This goes with many studies such as Afravy et al. [28].

Ayan et al. [15] detected that high level of toluene causes hepatocyte degeneration, mononuclear cell infiltration, and apoptosis. This coincides with our results. Álvarez et al. [24] mentioned that toluene causes central and mid zonal hepatic necrosis, balloon degeneration, fatty degeneration, and periportal fibrosis, but in our result, we had moderate change in the form of apoptosis and dilatation of sinusoids and blood vessels.

Citicoline, a nucleoside derivative, is intermediate in membrane phospholipid biosynthesis. Citicoline is a cholinergic drug widely studied owing to its properties against brain and cardiovascular reperfusion [29].

Citicoline is composed of two essential molecules, cytidine and choline, the structural phospholipids of cell membranes. The protective effect of citicoline might be explained by its role in membrane biosynthesis as it can protect or regenerate the damaged organelles’ membranes (lysosomes) inside cells, preventing cell degeneration. Phospholipids have a high turnover rate, which means that a cell requires continuous synthesis of these compounds to ensure adequate function of cell membranes [30].

It has been proved from many studies that citicoline improves deficits that result from various conditions in human and experimental animals [30].

The ability of citicoline to protect against toluene toxicity was demonstrated on histopathological examination and by measurement of caspase-3 and GFAP immunoreactivity. Citicoline reduced pathological changes in a dose-dependent manner in each organ we studied. Our results demonstrated that citicoline-treated group showed improvement in pathological changes caused by toluene such as decreased neuronal damage, deceased apoptosis, and degenerated hepatocytes even returning to normal especially with high doses. Similar results were proved by Li and Yuan [31].

Caspases are aspartate-specific cysteine proteases and members of the interleukin-1 (IL-1) β-converting enzyme family. These enzymes are produced in inactive forms and are activated in a protease cascade. Caspases are often classified into initiator (caspase-1, −2, −4, −5, −8, −9, −10, −11, −12) and effector caspases (caspase-3, −6, −7). The latter ones, with caspase-3 being considered the most important member, are responsible for the execution of apoptosis or programmed cell death [31]. Apoptosis is a form of programmed cell death characterized by DNA fragmentation, cytoplasmic shrinkage, membrane changes, and cell death without lysis or damage to the neighboring cells [10]. Apoptosis is important during development, for normal tissue homeostasis, and as a protective mechanism to get rid of damaged cells owing to diseases or toxins. On the contrary, inappropriate apoptosis has been implicated in the development of cancer, neurodegenerative disorders, and autoimmune diseases [32]. Caspases take part in both the intrinsic and extrinsic apoptosis pathways. However, caspase-3, also known as the executioner caspase, is responsible for the morphological changes such as chromatin condensation, DNA breakdown and laddering, and breakdown of membrane proteins [12].

In this study, administration of toluene was found to induce strong caspase-3 immunostaining, suggesting the involvement of caspase-3 activation. Our findings also indicated the ability of citicoline to inhibit the activation of caspase-3 in the liver and kidney of toluene-treated rats. In this context, citicoline was shown to decrease the expression of caspase-3.

GFAP is considered as an astrocytic marker. This protein is a major protein constituent of glial filaments, which are important in astrocyte cytoskeleton. Increased GFAP expression is indicative of astrogliosis, a process in which astrocytes become activated in response to a variety of central nervous system insults [31]. Reactive astrocytes produce various cytokines such as IL-1, IL-6, IL-10, TNF-α and −β, interferon-α and −β, and neurotrophic factors (e.g. fibroblast growth factor, platelet-derived growth factor, and nerve growth factor) [32]. Reactive astrocytes form the glial scar and with cytokines and growth factors released from these cells play an important role in limiting brain damage, but they might also contribute to neuropathology [33],[34],[35]. We found that toluene exposure decreased immunostaining for GFAP in star-shaped glial cells and their processes. These data suggested inhibition of GFAP in glial cells by toluene. Rats treated with citicoline showed GFAP expression in glial cells and their processes, similar to saline-treated rats. GFAP immunostaining increased in rats treated with toluene along with citicoline (though not to the normal value). It is likely that the increase in GFAP expression by citicoline is a consequence of neuroprotection by the dye, and several researchers reported the beneficial outcome of citicoline treatment in neurological disorders [22].


In conclusion, findings in this current study indicate a protective action of citicoline against toxic effect of toluene. It must be taken by workers who deal with toluene to protect them from adverse effect.

Conflicts of interest

There are no conflicts of interest.


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