Exposure to toluene in the blood and tissues triggers oxidative stress due to free radical production ( Abouee-Mehrizi et al., 2023; Demir et al., 2017). Here, oxidative stress is mediated by benzyl alcohol, which induces cytochrome P450 (CYP) 2E1 ( Hanioka et al., 2010; Kim et al., 2015) to produce unpaired electrons that are reactive toward glycoproteins and lipoproteins ( Jomova et al., 2023). This leads to the formation of oxidized lipids, such as low-density lipoprotein (Ox-LDL) ( Guan et al., 2019; Stajković et al., 2009). Ox-LDL triggers monocyte entry into the intima, which is mediated by adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1). After entering the intima, monocytes differentiate into macrophages and scavenge Ox-LDL ( Toma et al., 2016; Yurdagul et al., 2016).

Oxidative stress from exposure to harmful chemicals, such as toluene, may lead to the development of CVD ( He et al., 2024; Kim et al., 2012; Miller, 2020). Endothelial dysfunction is an early phase of most CVDs, and therefore, can be used to predict CVDs ( Hayek et al., 2021; Smiljic, 2017). Endothelial oxidative stress is characterized by the formation of lipid peroxidation products, such as malondialdehyde (MDA), and a decrease in the activity of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) ( Abouee-Mehrizi et al., 2023; Gamez-Mendez et al., 2015; Mao et al., 2019). Moreover, endothelial dysfunction is characterized by a decreased bioavailability of nitric oxide (NO), which releases adhesion cells, including VCAM-1-capturing monocytes and T cells ( Ardiana et al., 2021; Gradinaru et al., 2015).

VCAM-1 is an adhesion molecule on the endothelial surface. VCAM-1 expression is induced during the inflammatory process by mediators such as reactive oxygen species (ROS) ( Cook-Mills et al., 2011; Mu et al., 2015). An increase in VCAM-1 indicates endothelial damage due to inflammatory dysregulation and is the beginning of atherosclerosis, leading to CVD ( Kaur et al., 2022; Smiljic, 2017). This study uses a fixed dose and time to analyze the effect of acute toluene toxicity in the air, namely Serum CYP2E1 and Ox-LDL. On the other hand, SOD, CAT, MDA, and VCAM-1from the coronary artery, were used as biomarkers. We analyzed the combined effects of all bio-markers with toluene exposure on coronary arteries. Our findings provide a basis for research on the adverse effects of toluene toxicity.

Toluene is one of the most produced and used industrial solvents worldwide. It is the most widely abused volatile substance that is toxic to several organs ( Camara-Lemarroy et al., 2015; Djurendic-Brenesel et al., 2016; Yasar et al., 2016). Toluene is extensively metabolized by CYP2E1, leading to the formation of toluene epoxides, which can generate free radicals ( Ji et al., 2017; Méausoone et al., 2021; Moro et al., 2012). At low levels of free radicals, intracellular antioxidants, such as SOD, CAT, and GSH-Px, can reduce oxidative stress-mediated cell damage ( Hamid et al., 2022; Jiménez-Garza et al., 2015). Oxidative stress due to toluene exposure is mediated by benzyl alcohol, which activates CYP2E1 to produce ROS in the form of free radicals, such as O ₂*, H ₂O ₂, and *OH that damage proteins, lipids, and DNA ( Jiménez-Garza et al., 2015; Kim et al., 2012; Kodavanti et al., 2015).

Increased ROS production due to toluene exposure can trigger oxidative stress, which is characterized by accumulation of non-enzymatic oxidative damage of cell function. Oxidative stress can be identified by increasing lipid peroxides, especially polyunsaturated fatty acids in biological membranes, which will form MDA ( Konuk et al., 2012; Yurdagul et al., 2016). Increased lipid peroxidase (MDA) can interfere with the cofactor endothelial NO synthase (eNOS), causing a decrease in eNOS production and decreased bioavailability of nitric oxide (NO). In the presence of oxygen (through the formation of NO ₂ and N ₂O ₃) or superoxide (through the formation of peroxynitrite), NO can cause oxidation of lipid components, resulting in the formation of Ox-LDL ( Ardiana et al., 2021; Shiroto et al., 2014; Stajković et al., 2009). The chemotactic activity of Ox-LDL stimulates the binding of monocytes to endothelial cells, thereby enhancing the adhesive properties of the endothelium, including over-expression of VCAM-1, representing a key step in endothelial dysfunction ( Gradinaru et al., 2015; Hamid et al., 2022; Ivanova et al., 2017).

Serum CYP2E1 concentration in the experimental groups was higher than in the control group, although this difference was not statistically significant. CYP2E1 is a key enzyme involved in toluene toxicity ( Harjumäki et al., 2021; Hartman et al., 2017), because toluene regulates its metabolism by inducing CYP2E1 activity. This was because of altering protein stability, causing toxicity or cell damage through the production of toxic metabolites. In addition, free radicals can cause oxidative stress. According to Guan et al., (2019) and Lu et al., (2012), CYP2E1 bioactivation could trigger apoptosis, which induces tissue and cell damage. During toluene biotransformation, SOD activity decreases, resulting in the accumulation of metabolic intermediates and byproducts of oxygen-derived free radicals: O ₂*and OH*or their derivatives, such as H ₂O ₂ ( Ji et al., 2017; Kim et al., 2012; Stajković et al., 2009). When toluene concentration is higher, more H ₂O ₂ is produced, which cannot be rapidly degraded, and eventually, inactivates SOD.

Excessive free radicals inactivate antioxidant enzymes CAT, GSH-Px, etc., because accumulated H ₂O ₂ poisons CAT, resulting in a significant decrease in CAT expression ( Hamid et al., 2022; Stajković et al., 2009). Consistently, in this study, significantly lower CAT expression in the experimental group (compared with the control group) correlated with decreased SOD. A decrease in CAT activity is associated with an increase in VCAM-1 expression because high doses of toluene lead to increased H ₂O ₂ formation ( Kaur et al., 2022). This stimulates an increase in O ₂* to induce oxidative stress and endothelial dysfunction ( Coyle & Kader, 2007; Habas & Shang, 2018).

Malondialdehyde (MDA), one of the byproducts of lipid peroxidation, is used as a marker of oxidative stress. In this study, the positive correlation between the expression of MDA metabolites and toluene levels may be due to an increase in lipid peroxidase resulting from increased toluene levels ( Konuk et al., 2012). Under conditions of high oxidative stress, the expression of MDA metabolites increases, which induces an increase in vascular endothelial growth factor and stimulates caveolin-1. This inhibits eNOS, decreases eNOS production, and reduces the bioavailability of NOS ( Moraes et al., 2014; Shiroto et al., 2014; Ye et al., 2016). Imbalance in NOS production results in a proinflammatory response, characterized by increased VCAM-1.

Several studies have found that toluene exposure can interfere with lipid function, such as increased plasma LDL ( Konuk et al., 2012; Stajković et al., 2009) and triglycerides ( Kim et al., 2012; Won et al., 2011). High triglyceride levels, reflecting increased concentrations of lipoproteins, including LDL, can exacerbate endothelial inflammation and promote monocyte infiltration into the arterial wall ( Wang et al., 2013). Our results show that mean serum Ox-LDL levels were higher in the experimental group, although this difference was not statistically significant. Given that not all LDL molecules are of the same size. Some, such as small-dense LDL, may be more susceptible to chemical modifications that increase their atherogenicity ( Ivanova et al., 2017).

VCAM-1 is expressed in the endothelium, indicating endothelial damage ( Ardiana et al., 2021; Kaur et al., 2022). Our results showed that toluene exposure increased VCAM-1 expression starting from experimental group X1 (low dose). This increase in VCAM-1 expression is associated with a decrease in CAT activity ( Coyle & Kader, 2007; Habas & Shang, 2018) and increase in MDA metabolites ( Moraes et al., 2014; Ye et al., 2016). Toluene is lipophilic increasing and, therefore, can change the structure of cell membrane lipids, increasing oxygen free radicals and H ₂O ₂ ( Hamid et al., 2022; Konuk et al., 2012). The host antioxidant system cannot neutralize increased production of ROS. Furthermore, H ₂O ₂ reacts with Fe ⁺² and Cu+2 to form hydroxyl free radicals through the Fenton and Haber–Weiss reactions. OH* is a very reactive species and initiates a chain reaction with one hydrogen atom from the cell membrane forlipid peroxidation. Products of lipid peroxidation include MDA ( Habas & Shang, 2018; Stajković et al., 2009).

The overall aim of this study was to analyze the impact of toluene exposure on coronary artery response. We found that oxidative stress due to free radicals originating from toluene exposure in the air adversely affects the coronary artery.

This study shows that exposure to airborne toluene adversely affects the coronary arteries. Moreover, it also may increase the risk of endothelial dysfunction. Toluene exposure has toxic effects on the cardiovascular system, closely related to increased oxidative stress in the coronary artery. Low SOD and CAT expression with high MDA and VCAM-1 expression after toluene exposure may increase the risk of developing endothelial dysfunction, which reflects the onset of atherosclerosis as a cause of CVD. This adds to the evidence that toluene exposure causes CVD through the effects of oxidative stress on coronary arteries.

This study followed the principle of ethics proven with the ethical approval test. The ethical approval certificate number was 801/HRECC.FODM/X/2022. The experimental protocol was approved by The Health Research Ethical Clearance Committee (HRECC), Faculty of Dentistry, Airlangga, on October 26, 2022.

Enago has also checked this paper as an institution that provided a proofreading service on September 27, 2023. The certificate of proofreading has been uploaded to the Zenodo repository. The link to the certificate can be accessed on https://doi.org/10.5281/zenodo.13727263.

Animal ethics: 1.

ANIMAL TREATMENT Blood Collection

After euthanasia, rats will be collected in special plastic bags and wooden boxes, to then be burned in waste disposal at the Faculty of Veterinary Medicine, Universitas Airlangga.

The animal ethics certificate includes a link to the dataset accessible below: https://zenodo.org/records/11438933

The Animal Care and Use Committee (ACUC) of the Faculty of Dentistry, Universitas Airlangga approved the protocol of all procedures described in this study on October 26, 2022. All experimental procedures were performed in accordance with the ARRIVE guidelines ( Maryiantari, 2022). Tissue dissection and collection were performed by experienced laboratory technicians from the Department of Biochemistry, Faculty of Medicine, Universitas Airlangga. The rats were fasted for 3 h and weighed using a precision electronic balance. Additionally, they were anesthetized by intramuscular injection of 100 mg/kg body weight of ketamine (Ket-A-100 ^®, AgroVet, Peru) and 20 mg/kg body weight of xylazine (Interchemie werken “De Adelaar” B.V. The Netherlands. The protocol has been previously used and described by ( Bhatia et al., 2022; Olson et al., 2013).

After the reflex disappeared, the mice were placed in a supine position on the operating table, and blood was collected by cardiac puncture using a disposable terumo syringe with a 5 ml 23G needle, which was then placed in a tube without anticoagulant. After blood collection, the heart was cut, cleaned with isotonic sodium chloride solution, and placed in a tube containing 10% neutral buffered formalin (NBF-10%), then sent to the pathological anatomy laboratory of Universitas Airlangga. The blood in the tube was allowed to clot naturally (about 30 to 60 minutes) and then centrifuged at 3,500 rpm for 10 minutes to separate the serum. The blood was then poured into Eppendorf tubes and stored at -20°C until analysis of CYP2E1 enzyme levels and Ox-LDL serum was performed at the Tropical Blood Disease Research Center/TDRC Universitas Airlangga. Finally, the remains of the animals were collected in special plastic bags and wooden crates, then cremated at the landfill of the Faculty of Veterinary Medicine, Universitas Airlangga to avoid environmental pollution.