Research Article

The Role of Alpha Lipoic Acid and Tempol Against Liver Injury after Acetaminophen Subacute Exposure in Mice

Suhad Mohmmed Alrobyrai1 , Falah Muosa Al-Rekabi2*

1College of Veterinary Medicine, Iraq; 2University of Baghdad College of Veterinary Medicine Department of Physiology, Biochemistry and Pharmacology, Iraq.

Received | December 03, 2023; Accepted | June 06, 2024; Published | February 09, 2024

*Correspondence | Falah Muosa Al-Rekabi, University of Baghdad College of Veterinary Medicine Department of Physiology, Biochemistry and Pharmacology, Iraq; Email: [email protected]

Citation | Al-Rekabi FM, Alrobyrai SM (2024). The role of alpha lipoic acid and tempol against liver injury after acetaminophen subacute exposure in mice. Adv. Anim. Vet. Sci., 12(3):490-500.

DOI | https://dx.doi.org/10.17582/journal.aavs/2024/12.3.490.500

ISSN (Online) | 2307-8316

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Acetaminophen, also known as N-acetyl-para-aminophenol (APAP (was first used to treat both acute and chronic pain as an antipyretic and analgesic (Bandoli et al., 2020). It is also prescribed to patients for whom nonsteroidal anti-inflammatory drugs (NSAIDs) are contraindicated, such as those with gastric ulcers and bronchial asthma and has been used as a home analgesic for over three decades (Hegazy et al., 2021). In the early 1980s, in the United Kingdom, (APAP) paracetamol® supplanted aspirin as the most popular over-the-counter analgesic (Bertolini et al., 2006). The company’s medicines are offered in many dosage forms, such as syrup, injection, suppositories, regular pills, effervescent tablets, and others (Allen and Ansel, 2013). Adults commonly take 325–650 mg of APAP every 4–6 hours or 1 g of immediate-release oral formulations every 4–6 hours as required, not to exceed 4 g per day (McEvoy, 2007). In the case of viral diseases in cattle, such as three-day sickness, cowpox, and so on, paracetamol is occasionally used as an oral pain reliever in dogs. In severe pain dogs and small mammals, APAP is used as an analgesic or antipyretic. It may be especially useful for treating chronic pain conditions in dogs with renal dysfunction, APAP should be administered orally or rectally at a dose of 10-15 mg/kg every 8 hours (Hernández-Avalos et al., 2020). If used long-term, which is defined as being over five days, it might be prudent to reduce the frequency of administration to every 12 hours, particularly at the lower end of the dosing range. The recommended dose for mice, rats, gerbils, hamsters, guinea pigs, and chinchillas is 1-2 mg/mL in their drinking water (Plumb, 2018). The recommended dosage for horses is 0.04-0.1 mg/kg administered intramuscularly. The administration can be repeated every 6-12 hours, however, a longer interval of 36-48 hours is preferable (Duggan and Scott, 2009; Golf et al., 2011). For perioperative analgesia or treatment of laminitis, the dosage is 0.01-0.05 mg/kg or 0.04 mg/kg respectively, administered by intramuscular, subcutaneous, or intravenous route (Papich, 2020). Dogs suffering from chronic pain should receive analgesics containing Paracetamol at a dosage of 10 to 15 mg/kg on a regular basis (Otero, 2013). The exposure of subacute paracetamol has led to significant hepatotoxicity in young and old mice, as observed through the use of biochemistry (ALT) and histology (Kane et al., 2016). Administration of either a single or double dose of N-acetlcysteine (NAC) failed to provide protection against this toxicity from subacute paracetamol in either young or old mice (Wu et al., 2004). This discovery holds notable clinical implications for the treatment of paracetamol toxicity in patients of all ages, and suggests the pressing need for new treatments to combat subacute paracetamol toxicity (Vliegenthart et al., 2015). In another study investigation, it was observed that exposing mice to a dosage of 200 mg/kg of APAP increases their susceptibility to the hepatotoxic effects caused by APAP. This heightened vulnerability is thought to stem from the elevated formation of APAP-protein adducts in the liver, consequently intensifying mitochondrial dysfunction in hepatocytes (Duan et al., 2016). As delineated in the study conducted by Venkatesan et al. (2014) the sub-acute oral toxicity of acetaminophen in Sprague Dawley rats was evaluated using doses of 250, 500, and 1000 mg/kg body weight over a period of 28 days. The results of this investigation indicated that there were no significant changes in hematological parameters, urinalysis parameters, absolute and relative organ weights, and gross pathological changes observed in the treated groups compared to the control group. However, notable alterations were detected in specific biochemical parameters related to liver and kidney function, including AST, ALT, ALP, blood urea, creatinine, glucose, and proteins, particularly within the high dose group (1000 mg/kg). These changes suggested that acetaminophen induced a certain degree of hepatotoxicity and nephrotoxicity in the rats, though not to an extent leading to mortality or clinical manifestations. According to the authors observations, the “no observed adverse effect level” (NOAEL) for acetaminophen in Sprague Dawley rats was determined to be 500 mg/kg body weight (Venkatesan et al., 2014).

Therefore, future research should focus on developing novel approaches to address this issue (Kane et al., 2016). Non-acute APAP ingestions, also known as subacute or chronic APAP ingestions. Few researchs exist to guide treatment or define who must be screened, making it challenging to handle nonacute ingestions. The majority of chronic APAP ingestions that result in hepatotoxicity are nonacute. include people who are consuming supratherapeutic doses and are at risk of developing hepatitis (Daly et al., 2008; Jwied, 2009).Age, total dose, duration of use, presence of intercurrent febrile illness, starvation, co-administration of cytochrome P450-inducing drugs, underlying hepatic disease, and a person’s unique genetic makeup are all factors that have been proposed as potential risk factors for injury associated with chronic use ,hepatotoxicity risk associated with persistent consumption (Mohammad, 2013). On the basis of their medical history, clinical examination, and laboratory results, patients can be classified into three groups: those without hepatic injury and with no residual acetaminophen to be metabolized, those without injury but with some acetaminophen to be metabolized, and those with hepatotoxicity (Sztajnkrycer and Bond, 2001; Nafal et al., 2019). Alpha-lipoic acid exhibits strong antioxidant properties and demonstrates protective effects on the liver against injury (Hadi et al., 2020; Abd Hasan and Algareeb, 2022). Additionally employed to enhance the quality of semen after cryopreservation (Hamad, 2016). Most of the previous studies have focused on the acute toxicity of APAP and the role of antioxidants in preventing or reversing the liver damage caused by APAP overdose. However, there is a lack of research on the subacute toxicity of APAP, which is more relevant to the clinical scenario of chronic or repeated use of APAP for pain management. Moreover, the combination of ALA and Tempol as a potential hepato protective agent against APAP-induced subacute toxicity has not been explored before, to the best of our knowledge. Therefore, this research presents a novel and significant contribution to the field of pharmacology and toxicology, as it provides new insights into the mechanisms and therapeutic options for APAP-induced subacute liver injury.The objective of this research is to examine the potential hepatoprotective properties of Alpha Lipoic Acid (ALA) and tempol in murine model subjected to repeated exposure to acetaminophen(APAP). Furthermore, this investigation could elucidate the potential hazards associated with recurrent subacute APAP overdose, thereby contributing to the body of knowledge regarding its safe administration.

MATERIALS AND METHODS

Laboratory animals

Fifty male Bulb-C mice reached the age of 8 weeks and ranged in body weight from 20-30 grams . The mice used in this study were raised and housed under standard laboratory conditions. They were maintained on a 12-hour light/dark cycle, with ad libitum access to food and water. The temperature in the animal house was kept constant at 22 ± 2°C, with a relative humidity of 50 ± 10% (Huynh et al., 2005). All experimental procedures were conducted in accordance with the guidelines for the care and use of laboratory animals (Suckow et al., 2023). The mice were acclimatized to the laboratory conditions for a week prior to the commencement of the experiment. Every effort was made to minimize the number of laboratory animals used and their suffering. The health and behaviour of the mice were monitored daily by trained personnel. At the end of the experiment, the mice were humanely euthanized following the approved protocols (Allen-Worthington et al., 2015).

Experimental design

This experiment has been carried out with the participation of fifty male Bulb-C mice. They were divided into five equal groups. All experimental groups except (G1) were administered 700 mg/Kg.BW of APAP(SDI/Samara, Iraq) and treated orally via gastric gavage needle as follows for 30 days: (G1) administered distilled water only and considered negative control group (C-ve) while (G2) did not receive any treatment and considered positive control group (C+ve), G3(Par+Tempol) treated by 20 mg/Kg.BW of Tempol (Chem Cruz, USA) (Pınar et al., 2018), G4 (Par+ALA) treated by 20 mg/Kg.BW of Alpha lipoic acid (ALA) (Chemical point, Germany ) (Wang et al., 2018), while G5 (Par+tempol+ALA) treated by 10 mg/Kg.BW of each Tempol (Kwon et al., 2003) and ALA (Di Tucci et al., 2018). After that the laboratory animals have been rendered unconscious with diethyl ether (Thomas Baker, India).

Blood collection

Blood samples (1.5 mL) were collected via heart puncture (Rathkolb et al., 2013) from anesthetized mice by inhalation diethyl ether, using a 28-gauge, 4mm insulin syringe, taken after 30 days of treatment and one week post-treatment cessation. Samples were divided into anticoagulant-free gel tubes (gel tubes, Vaculab, China). and prothrombin time (PT) tubes (Al-Malik, Iraq). After a ١٠-minute incubation at room temperature, tubes were centrifuged at ٥٠٠٠ rpm for ١٥ minutes, separating serum and plasma, which were then stored at −١٨°C. These samples were utilized to assess biochemical markers (ALT, GGT, PT, INR) for liver function and clotting (Friedman et al., ٢٠١٤; Friedman, ٢٠٢٢).

Clinical chemistry

Half of each group was sacrificed after the 30-days treatment, and the remaining half after an additional week without treatment, for liver extraction and clinical chemistry analysis. A commercial kit for spectrophotometric methods was used to assess the Serum Alanine Aminotransferase (ALT) (Jourilabs, France), (Lala et al., 2021). Gamma Glutammyle Transferase (GGT) (Human Gesellschaft, Germany) (McCommis et al., 2015). Prothrombin time (PT) Biosam, UAE (Gosselin, 2023) and international normalized ratio(INR) levels (Yuan et al., 2023). ALT determination uses oxaloacetate hydrazone via 2,4-dinitrophenylhydrazine. ALT kit measures α-oxoglutarate + L-alanine reaction. GGT quantification/ GGT kit with a standardized kinetic colorimetric method. GGT catalyzes glutamyl group transfer from γ-glutamyl-3-carboxy-4-nitroanilide to form glutamylglycylglycine and 5-amino-2-nitrobenzoate. The Prothrombin Time (PT) Kit assesses blood coagulation using tissue thromboplastin and Ca++. PT measures activation time, aiding in hemorrhagic disorder estimation. Thromboplastin reagent addition to citrated plasma initiates clotting, and prolonged clot formation indicates extrinsic pathway factors deficiency.

Histopathology

The histopathological patterns of the liver of all experimental mice, were examined.The process begins with the euthanasia of the mouse by diethyl ether (Thomas Baker India). The euthanasia of the mice was conducted using diethyl ether within a close chamber, also known as a dissector (Nasution et al., 2023). This method involves the administration of diethyl ether vapor in a controlled environment, ensuring a rapid and humane method of euthanasia, until the mice loss of consciousness, followed by cessation of the heart and lung function (Meyer, 2015). Subsequently, a midline incision is made to expose the abdominal cavity, and enabling the careful extraction of the liver (Yokota et al., 2016). These organs are then immediately fixed in a 10% neutral buffered formalin) Alfa lab chemika China) for 48 h, following fixation, samples were sectioned to 0.5 cm thickness and placed in plastic cassettes (Werner et al., 2000). Dehydration and clearing of the tissues were automated using a histo- Line Laboratories ATP 1000 tissue processor (Italy). Subsequently, the dehydrated tissues were embedded in paraffin wax via a histo- line laboratory HESTION TEC 2900 embedding system, with temperature regulation managed by a TEC 2900 Thermal console) histo-line Laboratories, Italy (Jing et al., 2019). Tissue blocks were then sectioned at 4-5 µm thickness using a Hhistol line Laboratories, MRS3500 rotary microtome (Italy) (NODA and OGAWA, 1984). The sections were floated in a water bath and temperature -controlled hot plate, both regulated by the TEC 2900 Thermal console, before mounting on glass slides. staining was performed using Hematoxylin and Eosin (H and E, Dakocytomation, Denmark. The stained tissue sections were examined under a light microscope (Olympus Japan) at 40x magnifications for detailed histopathological assessment by a trained pathologist (Suvarna et al., 2018).

Statistical analysis

The data was analyzed using SPSS version 26 by SPSS Inc. A two-way ANOVA was conducted to determine the group differences. The LSD test was employed to identify significant differences, which were declared at a p-value of less than 0.05. The results were presented as the mean ± standard error (SE) (IBM Corp,. Armonk, NY, USA).

Results and Discussion

Manifestations of subacute toxicity from APAP exposure

During the course of our research conducted on mice, we meticulously observed various clinical symptoms that were prominently evident, particularly in the Positive control Group as well as the G3 (Par+ Tempol) groups. These symptoms encompassed shallow breathing, a proclivity for lying and slow movement, sporadic convulsions, profound lethargy, that occurred within a week subsequent to the commencement of treatment. After that all toxicity signs have been relieved.

The effect of orally administration APAP, tempol, and lipoic acid for 30 days on body weight/g of male mice

The body weights of the positive control and (Par+Tempol) groups exhibited a significant (P < 0.05) decrease compared to other experimental groups at the initiation of treatment. After 30 days, the experimental groups, namely the positive control, (Par+Lipoic), and (Par+Lipoic+Tempol), demonstrated a significant (P < 0.05) decrease in body weight compared to the negative group and G5 (Par+Lipoic+Tempol). Intriguingly, animals in the treated group (Par+Lipoic+Tempol) showed a non-significant (P < 0.05) difference throughout this period compared to the negative control group. Moreover, only the animals in the (Par+Lipoic+Tempol) group displayed a non-significant (P < 0.05) change in body weight compared to the negative control group after one week of treatment withdrawal. Conversely, both mentioned groups (Par+Lipoic and Par+Lipoic+Tempol) demonstrated a significant (P < 0.05) increase in body weight compared to other experimental groups, the positive control, and Par+Tempol, over the same period. The body weights of all experimental groups showed non-significant (P < 0.05) changes when comparing their values after one week of treatment withdrawal with those at the initiation of treatment, except for the treated (Par+Tempol) group, which displayed a significant (P < 0.05) decrease across the periods after 30 days of treatment and one week of treatment withdrawal compared to their body weights at the initiation of treatment (Figure 1).

 

The effect of orally administered APAP, tempol, and lipoic acid for 30 days on alt(u/l) of experimental groups

The serum ALT activity in the treated groups, negative control (Par+Tempol), (Par+Lipoic), and (Par+Lipoic+Tempol), exhibited a significant (P < 0.05) decrease compared to the positive control group after 30 days of treatment. However, ALT serum activity demonstrated non-significant (P < 0.05) changes among all experimental groups after one week of treatment withdrawal. The positive control group showed a significant (P < 0.05) decrease in ALT serum activity after one week of treatment withdrawal compared to its activity after 30 days of treatment. In contrast, all other experimental groups displayed non-significant (P < 0.05) changes across the two periods of the experiment (Figure 2).

 

 

The effect of orally administered APAP, tempol, and lipoic acid for 30 days on GGT (U/L) in the experimental groups

The serum GGT activity after 30 days of treatment in the group treated with (Par+Lipoic+Tempol) exhibited non-significant differences (P < 0.05) compared to the positive control group but showed a significant increase (P < 0.05) compared to the negative control group. Conversely, the (Par+Tempol) group demonstrated a significant increase (P < 0.05) compared to other experimental groups. After one week of treatment withdrawal, the serum GGT activity displayed a significant decrease (P < 0.05) in the (Par+Tempol), (Par+Lipoic), and (Par+Lipoic+Tempol) groups compared to the positive control group. The treated groups (Par+Tempol) and (Par+Lipoic) showed a significant increase (P < 0.05) compared to the negative control group, while only the (Par+Lipoic+Tempol) group exhibited non-significant differences (P < 0.05) compared to the negative control group.Furthermore, the serum GGT activity demonstrated a significant increase (P < 0.05) after one week of treatment withdrawal in the positive control and (Par+Tempol) groups compared to their activity after 30 days of treatment. In contrast, the negative control, (Par+Lipoic), and (Par+Lipoic+Tempol) groups showed non-significant differences (P < 0.05) between the two periods of the experiment (Figure 3).

The effect of orally administered APAP, tempol, and lipoic acid for 30 days on PT/ sec of the experimental groups

The prothrombin time (PT) in all experimental groups exhibited a significant (P < 0.05) decrease, except for the (Par+Tempol) group, which displayed a non-significant (P < 0.05) difference compared to the positive control after 30 days of treatment. Notably, both the positive control and (Par+Tempol) groups showed a significant (P < 0.05) increase in PT compared to the negative control group. Conversely, the other treated groups, namely, (Par+Lipoic) and (Par+Lipoic+Tempol), demonstrated a significant (P < 0.05) decrease compared to the negative control group after the same treatment period. The PT of all experimental groups exhibited non-significant (P < 0.05) changes after one week of treatment withdrawal.When considering both periods of the experiment (30 days of treatment and one week of treatment withdrawal), only the positive control and (Par+Tempol) groups showed a significant (P < 0.05) decrease in PT, while the other experimental groups displayed non-significant (P < 0.05) changes (Figure 4).

 

The effect of orally administered APAP, tempol, and lipoic acid for 30 days on INR of the experimental groups

After 30 days of treatment, the International Normalized Ratio (INR) results revealed a significant (P < 0.05) decrease in the treated groups, negative control, (Par+Lipoic), and (Par+Lipoic+Tempol), compared to the positive control group. Conversely, the treated group (Par+Tempol) displayed a non-significant (P < 0.05) change when compared to the positive control. Following one week of treatment withdrawal, the experimental groups, namely, negative control, (Par+Lipoic), (Par+Tempol), and (Par+Lipoic+Tempol), exhibited a significant (P < 0.05) decrease compared to the positive control group.Within-group analysis demonstrated that the INR values of the positive control, (Par+Lipoic), and (Par+Lipoic+Tempol) groups experienced a significant (P < 0.05) decrease after one week of treatment withdrawal compared to their values after 30 days of treatment. In contrast, the treated group (Par+Lipoic) showed a non-significant (P < 0.05) change across the two periods of the experiment (Figure 5).

 

Histopathology 

In the positive control group exposed orally to 700 mg/kg BW APAP, after 30 days of treatment liver histopathology shows dilated central vein with moderate mononuclear cell infiltration (lymphocytes and macrophages) with marked Centre lobular necrosis (Figure 6). After one week of withdrawal, the presence of a dilated central vein accompanied by lymphocytic cuffing and degenerated and necrotic. In the (Par+Tempol) group, liver histopathological examination after 30 days of treatment shows severe mononuclear cell infiltration )lymphocytes and macrophages( with marked necrotic hepatocytes (coagulative N) (Figure ٧). For the (Par+ALA) group, the liver histopathological examination after 30 days shows central vein (C.V) with moderate mononuclear cell infiltration and mild centrelobular necrosis (coagulative N) (Figure 8). In the (Par+Tempol+Lipoic Acid) group, shows mild mononuclear cell infiltration (lymphocytes and macrophages) with moderate vacuolar degeneration (Figure 9).

 

 

The APAP group (positive control group) had a substantial increase in ALT and GGT levels, indicating liver injury and cell necrosis, which could be attributed to the development of an APAP toxic metabolite, NAPQI (Azarmehr et al., 2019; Yang et al., 2021; Hameed and Hassan, 2022). The introduction of acetaminophen demonstrated effectiveness in inducing oxidative stress and

 

 

modifying specific biological markers associated with paracetamol-induced liver injury in rats, achieved through a single injection of paracetamol (500 mg/kg) (Jahan et al., 2023). This is consistent with the findings of the present study on the hepatoprotective effect of nicorandil against acetaminophen-induced oxidative stress (Abd Al-Zahra et al., 2009; Al-Okialy, 2018) and hepatotoxicity in mice, where an elevation in levels of liver enzymes, specifically Alanine Aminotransferase (ALT) and Gamma-Glutamyl Transferase (GGT), was observed. These findings align with the results obtained from the comprehensive investigation and serve as confirmation there of (Govindaraju et al., 2021). A study suggested that mice exposed to APAP exhibited clear signs of liver damage, as indicated by hepatic necrosis and an increase in ALT levels in wild-type C57BL/6 mice (Liao et al., 2023). This is confirmed in the present study, concerning ALT and GGT serum activity, as well as various deleterious histopathological findings in mice dosed with Paracetamol only. In contrast to the decrease in the serum activities of these enzymes in all treated groups (Par+Tempol), (Par + Lipoic), and (Par + ALA + Tempol) along the period of the current study, histopathological findings like necrosis have been observed in mice given a hazardous dose of APAP, 700mg/Kg.BW (positive control group). The ameliorating effect of different agents used in the current study, referring to histopathological patterns of different treated groups (Par+Tempol), (Par + Lipoic), and (Par + ALA + Tempol), shows important improvement in liver histology when necrotic lesions disappeared. ALA has the capability to influence the expression and activities of cytochrome P450 enzymes (P450s), which play a crucial role in the metabolism of APAP and other pharmaceuticals. A research study conducted by (Ewees et al., 2019) has shed light on the impact of APAP-induced liver injury on the expression and activities of various P450s, including CYP2E1, 3A11, 1A2, and 2C29, in the liver of mice in a manner that is dependent on the age of the subjects. The study has put forth the hypothesis that these alterations could potentially affect the effectiveness of drugs that undergo metabolism by P450 enzymes. Conversely, it has been demonstrated that ALA has the ability to inhibit the activity of CYP2E1 and stimulate the expression of CYP1A2 in the liver of mice (Jahan et al., 2023). Bao et al. (2020) Consequently, it can be speculated that ALA might possess advantageous properties in terms of safeguarding against and treating APAP-induced liver injury by exerting control over the levels of P450 enzymes. On the contrary, the treated group (Par+ Tempol) showed an increase in the serum activity of GGT, in accordance with the findings of a separate investigation focusing on the impact of Tempol in relation to the renal toxicity induced by cisplatin. It can be concluded that the results obtained from the current study are in alignment with the finding of (Ge et al., 2019) who observed. The observations made during the study on the treated groups, which consisted of (Par + ALA + Tempol), revealed a clear and noticeable decrease in the levels of ALT. This outcome is in accordance with the findings of an ongoing research, which demonstrated that Tempol has the ability to diminish the levels of ALT in CCl4-intoxicated Rats (Jahan et al., 2023). The TD50 of orally administered APAP for mice is estimated at 736 mg/kg BW, derived from the logarithmic equation. However, due to the lack of specific studies on APAP’s TD50 value, establishing correlations or comparative analyses with existing literature is currently unfeasible. It’s noteworthy that the LD50 of acetaminophen in mice, administered orally, is 1120.28 mg/kg BW, determined with a 24-hour observational period (Aseel, 2021). Alpha-lipoic acid, renowned for its remarkable therapeutic potential, has emerged as a compelling avenue in the field of hepatotoxicity treatment. Hepatotoxicity encompasses a wide range of liver impairments caused by diverse factors, including the hazardous effects of toxic substances such as Acetaminophen. In the conducted study, rats were administered a daily oral dose of Acetaminophen at 100mg/kg (Mohammed et al., ٢٠٢١). Alpha-lipoic acid (ALA) exhibited antioxidant activity in rats experimentally induced with oxidative stress by H2O2 (Eidan, 2017). In a separate study, it was observed that the administration of Tempol resulted in the increase of various antioxidant proteins, such as superoxide dismutase, catalase, and glutathione. Additionally, the phosphorylation of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt), as well as the protein expression of nuclear factor erythroid 2-related factor (Nrf 2) and heme oxygenase-1 (HO-1), were all significantly elevated due to the treatment with Tempol (Ge et al., 2019). These findings strongly indicate that Tempol plays a protective role against APAP-induced hepatotoxicity through the activation of the PI3K/Akt/Nrf2 pathway. Furthermore, it was observed that Tempol treatment led to a decrease in the expression of pro-apoptotic proteins (cleaved caspase-3 and Bax) and an increase in the expression of the anti-apoptotic protein Bcl-2 in the liver. Moreover, the number of apoptotic cells, as determined by TUNEL staining, was significantly reduced following Tempol treatment, suggesting its potential to prevent apoptosis (Zhao et al., 2022). Overall, the evidence suggests that Tempol has the ability to restore normal liver function in mice with APAP-induced acute hepatotoxicity by activating the PI3K/Akt/Nrf2 pathway, thereby enhancing the antioxidant response and inhibiting hepatic apoptosis. Studies have provided evidence that lipoic acid exhibits a potential therapeutic benefit in treating the liver structural and chemical aberrations instigated by an overdose of the analgesic drug APAP in male albino mice (Tiiba et al., 2022). Besides, studies have revealed that lipoic acid possesses the ability to prevent APAP-induced liver damage by reducing necrosis, inflammation, and oxidative stress in the liver and promoting liver regeneration. This underscores the potential of lipoic acid to be employed as an effective therapeutic agent for treating APAP overdose-induced liver damage. Also, a certain study that is in agreement with histopathological findings in the current study, has found that results of the study indicate that it can mitigate the harm to the liver morphology and ultrastructure induced by hepatotoxicity caused by fluoride (Zhao et al., 2022). In a scientific investigation involving rodents that had sustained liver damage due to lipopolysaccharide injection, it was discovered that lipoic acid served to regulate the equilibrium of the oxidant/antioxidant status and avert the occurrence of oxidative stress (Cimen et al., 2019; Zalzala and Kareem, 2021; Al-Azzawi and Baraaj, 2016). These results are supported with those of the present study, which found that APAP administration elevated liver biomarker enzymes while lipoic acid and Tempol therapy reduced all of the changed values. The outcomes are reported (Abdel-Zaher et al., 2008; Jahan et al., 2023). The findings of the study revealed that lipoic acid and Tempol have the potential to mitigate the formation of NO as well as oxidative damage in the liver and kidney. This is made possible through the reduction of lipid peroxidation and glutathione peroxidase (GSH-Px) activity, along with an increase in intracellular GSH depletion. The results of the present study are in alignment with the study providing further evidence that serum ALT activity experiences an elevation when specific medications, such as APAP, are administered. The aforementioned study, which investigated the impact of various drugs on liver function, observed a consistent rise in ALT levels in response to the usage of certain pharmaceuticals, including APAP. Therefore, reinforcing the notion that drug-induced liver injury can manifest through an increase in serum ALT activity (Mehrpour et al., 2023). At therapeutic doses, APAP is generally regarded as safe, with no significant adverse effects. However, it is crucial to note that at higher doses such as the dosage rate 700 mg/Kg.BW that was administered to mice subacutely, APAP may lead to centrilobular liver necrosis, a severe condition characterized by the death of liver cells in the centrilobular region (Etemadi et al., 2023). It has been proposed that NAPQI, a metabolite that is highly reactive, is responsible for the depletion of glutathione through a reaction known as conjugation, in which it forms a covalent bond with proteins. This suggests that NAPQI plays a crucial role in the regulation of glutathione levels within the body. Additionally, this reactive metabolite has been found to bind to proteins, further illustrating its impact on cellular processes. The depletion of glutathione caused by NAPQI can lead to an increased oxidative stress response, as the detoxification of reactive oxygen and nitrogen species is hindered. This imbalance in the body’s antioxidant defenses can result in an elevated level of oxidative stress, which has been implicated in a variety of pathological conditions (Fatemeh, 2023). Furthermore, the perturbation of calcium metabolism has been suggested to be associated with the increased oxidative stress caused by glutathione deficiency. Altogether, these findings highlight the intricate relationship between NAPQI, glutathione, oxidative stress, and calcium metabolism, providing insights into the mechanisms underlying various diseases and potentially paving the way for the development of novel therapeutic strategies (Karthivashan et al., 2015).Fulminant hepatic failure (FHF), a severe and life-threatening condition, is frequently characterized by elevated levels of PT and INR, which are indicators of impaired blood clotting. In this particular study, a present a case involving a 36-year-old female patient who experienced FHF as a consequence of an acetaminophen overdose (Yoon et al., 2023). When compared to the PT recorded in the positive control group (APAP) and (Par+Tempol) treated group mice had a considerably longer prothrombin time in contrast to (Par+ ALA) treated mice, Lipoic treated animals in the current study, that showed a decrease in PT. We believe that LA has a more positive effect than Tempol. Moreover, the treatment group, (Par+ ALA +Tempol), had a very close value to the (Par+Lipoic) with a slight difference.

Conclusions and Recommendations

The findings of this study underscore the potential of alpha-lipoic acid and tempol in mitigating the subacute toxicity associated with Acetaminophen (APAP) exposure. The observed ameliorative effects suggest a promising confluence of these compounds in counteracting APAP-induced toxicity. However, the precise mechanisms of these effects remain to be elucidated. It is imperative to note that these findings are preliminary and should be interpreted with caution until validated by further research. The potential clinical application of these findings could revolutionize the management of APAP-induced toxicity, underscoring the need for continued research in this area. However, it is important to note that the study was conducted on mice, and further research is needed to determine wether these findings can be applied to humans.

ACKNOWLEDGMENTS

The authors extend their deepest appreciation to Prof. Dr. Bushra. I. Al-Kaisi, a distinguished consultant pathologist, for her indispensable contribution in the interpretation of the histopathology slides.

Novelty Statement

This study investigates the hepatoprotective effects of Alpha Lipoic Acid (ALA) and Tempol against subacutely repeated acetaminophen (APAP) exposure in a murine model. The findings reveal a significant increase in body weight and decreased levels of biochemical markers after withdrawal, suggesting the potential of ALA and Tempol in mitigating APAP-induced subacute toxicity. These results contribute to the understanding of novel therapeutic approaches for managing APAP-induced liver injury and emphasize the need for further research in this area.

aUthor’s contribution

FMKA-R: Conceptualized and designed the study, analyzed the data, supervised the project, and contributed to the final version of the manuscript. SMHA-R: Performed the experiments and wrote the initial draft of the manuscript. All authors reviewed and approved the final manuscript.

Ethical statemen

This study was approved by the decision of the ethics University of Baghdad, College of Veterinary Medicine Animal Care and Use Committee, with numbered P.G.1247.

Conflict of interest

The authors have declared no conflict of interest.

REFERENCES

Abd-Al-Zahra JI, Ismael DK, Al-Shawi NN (2009). Preventive effects of different doses of pentoxyfilline against ccl4-induced liver toxicity in rats. Iraqi J. Pharm. Sci., 39.

Abd-Hasan RR, Algareeb AI (2022). Hepatoprotective effects of alpha-lipoic acid, Vitamin C alone, or in combination on methotrexate-induced liver injury. Mustansiriya Med. J., 21(1): 41–47. https://doi.org/10.4103/mj.mj_23_21

Al-Azzawi AFS, Baraaj AH (2016). Histological and biochemical study of nigella sativa seeds effects on liver of male albino rats treated with rifampicin. Iraqi J. Sci., 57(4C): 2829–2839.

Allen L, Ansel HC (2013). Ansel’s pharmaceutical dosage forms and drug delivery systems. Lippincott Williams and Wilkins.

Allen-Worthington KH, Brice AK, Marx JO, Hankenson FC (2015). Intraperitoneal injection of ethanol for the euthanasia of laboratory mice (Mus musculus) and rats (Rattus norvegicus). J. Am. Assoc. Lab. Anim. Sci., 54(6): 769–778.

Al-Okialy BN (2018). Role of alpha lipoic acid in oxidant/antioxidant status and gene expression of glutathione reductase in hydrogen peroxide exposed rats: (Part-2): Mohammad Saleh Alwan and Baraa Najim Al-Okialy. Iraqi J. Vet. Med., 42(2): 50–57. https://doi.org/10.30539/iraqijvm.v42i2.287

Azarmehr N, Afshar P, Moradi M, Sadeghi H, Sadeghi H, Alipoor B, Khalvati B, Barmoudeh Z, Abbaszadeh-Goudarzi K, Doustimotlagh AH (2019). Hepatoprotective and antioxidant activity of watercress extract on acetaminophen-induced hepatotoxicity in rats. Heliyon, 5(7). https://doi.org/10.1016/j.heliyon.2019.e02072

Aseel HM (2021). Therapeutic Trial of Liver Injury Parallel Acetaminophen Administration in Mice. University of Baghdad.

Bandoli G, Palmsten K, Chambers C (2020). Acetaminophen use in pregnancy: Examining prevalence, timing, and indication of use in a prospective birth cohort. Paediat. Perinat. Epidemiol., 34(3): 237–246. https://doi.org/10.1111/ppe.12595

Bertolini A, Ferrari A, Ottani A, Guerzoni S, Tacchi R, Leone S (2006). Paracetamol: New vistas of an old drug. CNS Drug Rev., 12(3-4): 250–275. https://doi.org/10.1111/j.1527-3458.2006.00250.x

Cimen B, Cimen L, Cetin I, Cetin A (2019). Alpha-lipoic acid alleviates lipopolysaccharide-induced liver damage in rats via antioxidant effect. Dicle Tıp Dergisi, 46(1): 125–132. https://doi.org/10.5798/dicletip.534849

Daly FFS, Fountain JS, Murray L, Graudins A, Buckley NA (2008). Guidelines for the management of paracetamol poisoning in Australia and New Zealand-explanation and elaboration. Med. J. Australia, 188(5): 296. https://doi.org/10.5694/j.1326-5377.2008.tb01625.x

Di Tucci C, Di Feliciantonio M, Vena F, Capone C, Schiavi MC, Pietrangeli D, Muzii L, Benedetti PP (2018). Alpha lipoic acid in obstetrics and gynecology. Gynecol. Endocrinol., 34(9): 729–733. https://doi.org/10.1080/09513590.2018.1462320

Duan L, Davis JS, Woolbright BL, Du K, Cahkraborty M, Weemhoff J, Jaeschke H, Bourdi M (2016). Differential susceptibility to acetaminophen-induced liver injury in sub-strains of C57BL/6 mice: 6N versus 6J. Food Chem. Toxicol., 98: 107–118. https://doi.org/10.1016/j.fct.2016.10.021

Duggan ST, Scott LJ (2009). Intravenous paracetamol (acetaminophen). Drugs, 69: 101–113. https://doi.org/10.2165/00003495-200969010-00007

Eidan SM (2017). Effect of adding Manganese chloride and Co-enzymes (α-lipoic acid and Q10) on post-cryopreservation semen quality characteristics of Holstein bulls: Eidan, SM 1; Sultan, OAA 1; Ibrahim, FF 2; Hurish, KF 2 and Naseer, HH 2. Iraqi J. Vet. Med., 41(2): 86–93. https://doi.org/10.30539/iraqijvm.v41i2.53

Etemadi Y, Akakpo JY, Ramachandran A, Jaeschke H (2023). Nrf2 as a therapeutic target in acetaminophen hepatotoxicity: A case study with sulforaphane. J. Biochem. Mol. Toxicol., e23505. https://doi.org/10.1002/jbt.23505

Ewees MG, Messiha BAS, Abdel-Bakky MS, Bayoumi AMA, Abo-Saif AA (2019). Tempol, a superoxide dismutase mimetic agent, reduces cisplatin-induced nephrotoxicity in rats. Drug Chem. Toxicol., 42(6): 657–664. https://doi.org/10.1080/01480545.2018.1485688

Fatemeh K (2023). Studies on glutathione conjugation reactions of some cyclic chalcone analogs to seek reactivity-biological activity relationships.

Friedman LS (2022). Tests of the liver’s biosynthetic capacity (eg, albumin, coagulation factors, prothrombin time). Literat. Rev. Curr. Through.

Friedman LS, Chopra S, Travis AC (2014). Approach to the patient with abnormal liver biochemical and function tests. Up to Date, 4.

Ge Z, Wang C, Zhang J, Li X, Hu J (2019). Tempol protects against acetaminophen induced acute hepatotoxicity by inhibiting oxidative stress and apoptosis. Front. Physiol., 10: 660. https://doi.org/10.3389/fphys.2019.00660

Golf M, Daniels SE, Onel E (2011). A phase 3, randomized, placebo-controlled trial of DepoFoam® bupivacaine (extended-release bupivacaine local analgesic) in bunionectomy. Adv. Ther., 28: 776–788. https://doi.org/10.1007/s12325-011-0052-y

Govindaraju P, Karan SC, Govindaraj N (2021). Effect of hydroalcoholic root extract of aerva lanata on acetaminophen induced hepatotoxicity in wistar rats. J. Clin. Diagn. Res., 15(9). https://doi.org/10.7860/JCDR/2021/49837.15351

Hadi RA, Mohammed MM, Salman IN (2020). Studying the effect of adding alpha lipoic acid to gabapentin to improve nerve conduction velocity and glycemic control of patients with diabetic neuropathy (sample of Iraqi population). Al-Mustansiriyah J. Pharma. Sci., 20(1). https://doi.org/10.32947/ajps.v20i1.685

Hamad ZM (2016). Protective role of Nigella sativa oil on renal damage induced by acetaminophen in male rats. Iraqi J. Vet. Med., 40(2): 77–81. https://doi.org/10.30539/iraqijvm.v40i2.116

Hameed HA, Hassan AF (2022). The prophylactic anti-inflammatory effect of omega-7 against paracetamol-induced liver injury in rats. Iraqi J. Vet. Med., 46(2): 43–47. https://doi.org/10.30539/ijvm.v46i2.1412

Hegazy A, Abd Al-Hameed EA, El-Wafaey D, Khorshed O (2021). Effect of paracetamol administration on the rat kidney structure: A morphological study. Zagazig Univ. Med. J., 27(4): 567–576.

Hernández-Avalos I, Valverde A, Ibancovichi-Camarillo JA, Sánchez-Aparicio P, Recillas-Morales S, Osorio-Avalos J, Rodríguez-Velázquez D, Miranda-Cortés AE (2020). Clinical evaluation of postoperative analgesia, cardiorespiratory parameters and changes in liver and renal function tests of paracetamol compared to meloxicam and carprofen in dogs undergoing ovariohysterectomy. PLoS One, 15(2): e0223697. https://doi.org/10.1371/journal.pone.0223697

Huynh TTT, Aarnink AJA, Verstegen MWA, Gerrits WJJ, Heetkamp MJW, Kemp B, Canh TT (2005). Effects of increasing temperatures on physiological changes in pigs at different relative humidities. J. Anim. Sci., 83(6): 1385–1396. https://doi.org/10.2527/2005.8361385x

Jahan I, Islam MD, Sarif S, Amena IJ, Shuvo AUH, Akter N, Chowdhury FI, Akter R, Ahmed I, Khan F (2023). Tempol alters antioxidant enzyme function, modulates multiple genes expression, and ameliorates hepatic and renal impairment in carbon tetrachloride (CCl4)-intoxicated rats. Livers, 3(1): 105–120. https://doi.org/10.3390/livers3010010

Jing D, Yi Y, Luo W, Zhang S, Yuan Q, Wang J, Lachika E, Zhao Z, Zhao H (2019). Tissue clearing and its application to bone and dental tissues. J. Dental Res., 98(6): 621–631. https://doi.org/10.1177/0022034519844510

Jwied AH (2009). Hepatoprotective effect of the aqueous extract of camellia sinensis against methotrexate-induced liver damage in rats. Iraqi J. Pharm. Sci., 18(2): 73–79. https://doi.org/10.31351/vol18iss2pp73-79

Kane AE, Huizer-Pajkos A, Mach J, McKenzie C, Mitchell SJ, de Cabo R, Jones B, Cogger V, Le Couteur DG, Hilmer SN (2016). N-acetyl cysteine does not prevent liver toxicity from chronic low-dose plus subacute high-dose paracetamol exposure in young or old mice. Fund. Clin. Pharmacol., 30(3): 263–275. https://doi.org/10.1111/fcp.12184

Karthivashan G, Arulselvan P, Fakurazi S (2015). Pathways involved in acetaminophen hepatotoxicity with specific targets for inhibition/downregulation. RSC Adv., 5(76): 62040–62051. https://doi.org/10.1039/C5RA07838E

Kwon TH, Chao DL, Malloy K, Sun D, Alessandri B, Bullock MR (2003). Tempol, a novel stable nitroxide, reduces brain damage and free radical production, after acute subdural hematoma in the rat. J. Neurotrauma, 20(4): 337–345. https://doi.org/10.1089/089771503765172291

Liao J, Lu Q, Li Z, Li J, Zhao Q, Li J (2023). Acetaminophen-induced liver injury: Molecular mechanism and treatments from natural products. Front. Pharmacol., 14: 1122632. https://doi.org/10.3389/fphar.2023.1122632

McCommis KS, Chen Z, Fu X, McDonald WG, Colca JR, Kletzien RF, Burgess SC, Finck BN (2015). Loss of mitochondrial pyruvate carrier 2 in the liver leads to defects in gluconeogenesis and compensation via pyruvate-alanine cycling. Cell Metab., 22(4): 682–694. https://doi.org/10.1016/j.cmet.2015.07.028

McEvoy GK (2007). American hospital formulary service. AHFS Drug Information. American society of health-system pharmacists, Bethesda, MD, 2184.

Mehrpour O, Hoyte C, Goss F, Shirazi FM, Nakhaee S (2023). Decision tree algorithm can determine the outcome of repeated supratherapeutic ingestion (RSTI) exposure to acetaminophen: Review of 4500 national poison data system cases. Drug Chem. Toxicol., 46(4): 692–698. https://doi.org/10.1080/01480545.2022.2083149

Meyer RE (2015). Euthanasia and humane killing. Veterinary anesthesia and analgesia: The fifth edition of lumb and jones, pp. 130–143. https://doi.org/10.1002/9781119421375.ch6

Mohammed S.O (2021). Therapeutic effects of N-acetylcysteine and alpha-lipoic acid on hepatotoxicity in rats after subacute exposure to acetaminophen. University of Baghdad.

Mohammad AAW (2013). Effect of different doses of paracetamol on liver enzyme activities in white Albino male mice. Eng. Technol. J., 31:(7 Part (B) Scientific). https://doi.org/10.30684/etj.31.7B.6

Nafal RH, Al-Warid HS, Al-Sultan HJ (2019). Seroprevalence of toxoplasmosis in patients with chronic liver disease in Baghdad. Iraqi J. Sci., pp. 1667–1672. https://doi.org/10.24996/ijs.2019.60.8.2

Nasution MHF, Kalanjati VP, Abdurachman A, Aditya D, Pamungkas DBB, Syamhadi MR (2023). The effects of methotrexate, Moringa oleifera leaf extract, and andrographis paniculata leaf extract on the testes of hyperglycemic wistar rats. Trop. J. Natl. Prod. Res., 7(6). https://doi.org/10.26538/tjnpr/v7i6.12

Noda T, Ogawa K (1984). Golgi apparatus is one continuous organelle in pancreatic exocrine cell of mouse. Acta Histochem. Cytoch., 17(4): 435–451. https://doi.org/10.1267/ahc.17.435

Otero PE (2013). The latest in pain management. In World Small Animal Veterinary Association World Congress Proceedings.

Papich MG (2020). Papich handbook of veterinary drugs-e-book. Elsevier Health Sciences.

Pınar N, Kaplan M, Özgür T, Özcan O (2018). Ameliorating effects of tempol on methotrexate-induced liver injury in rats. Biomed. Pharmacoth., 102: 758–764. https://doi.org/10.1016/j.biopha.2018.03.147

Plumb DC (2018). Plumb’s veterinary drug handbook: Desk. John Wiley and Sons.

Rathkolb B, Fuchs H, Gailus-Durner V, Aigner B, Wolf E, Hrabě de Angelis M (2013). Blood collection from mice and hematological analyses on mouse blood. Curr. Prot. Mouse Biol., 3(2): 101–119. https://doi.org/10.1002/9780470942390.mo130054

Suckow MA, Hashway S, Pritchett-Corning KR (2023). The laboratory mouse. CRC press. https://doi.org/10.1201/9780429353086

Suvarna KS, Layton C, Bancroft JD (2018). Bancroft’s theory and practice of histological techniques. Elsevier health sciences.

Sztajnkrycer MJ, Bond GR (2001). Chronic acetaminophen overdosing in children: risk assessment and management. Curr. Opin. Pediat., 13(2): 177–182. https://doi.org/10.1097/00008480-200104000-00016

Tiiba JDI, Sule MB, Ahmadu PU, Naamawu A, Fuseini M, Raymond A, Osei-Amoah E, Bobrtaa PC, Bacheyie PP, Abdulai M (2022). The potential of alpha lipoic acid to maintain liver function tests in xenobiotic-induced liver toxicity: A narrative review. Trop. J. Natl. Prod. Res., 6(7). https://doi.org/10.26538/tjnpr/v6i7.1

Venkatesan PS, Deecaraman M, Vijayalakshmi M, Sakthivelan SM (2014). Sub-acute toxicity studies of acetaminophen in Sprague Dawley rats. Biol. Pharma. Bull., 37(7): 1184–1190. https://doi.org/10.1248/bpb.b14-00066

Vliegenthart ADB, Shaffer JM, Clarke JI, Peeters LEJ, Caporali A, Bateman DN, Wood DM, Dargan PI, Craig DG, Moore JK (2015). Comprehensive microRNA profiling in acetaminophen toxicity identifies novel circulating biomarkers for human liver and kidney injury. Sci. Rep., 5(1): 15501. https://doi.org/10.1038/srep15501

Wang Q, Lv C, Sun Y, Han X, Wang S, Mao Z, Xin Y, Zhang B (2018). The role of alpha-lipoic acid in the pathomechanism of acute ischemic stroke. Cell. Physiol. Biochem., 48(1): 42–53. https://doi.org/10.1159/000491661

Werner M, Chott A, Fabiano A, Battifora H (2000). Effect of formalin tissue fixation and processing on immunohistochemistry. Am. J. Surg. Pathol., 24(7): 1016–1019. https://doi.org/10.1097/00000478-200007000-00014

Wu Z, MacPhee MIA, Oliveira GDB (2004). Reactive oxygen species in the initiation of IL-4 driven autoimmunity as a potential therapeutic target. Curr. Pharma. Design, 10(8): 899–913. https://doi.org/10.2174/1381612043452875

Yang Z, Wu W, Ou P, Wu M, Zeng F, Zhou B, Wu S (2021). MiR-122-5p knockdown protects against APAP-mediated liver injury through up-regulating NDRG3. Mol. Cell. Biochem., 476: 1257–1267. https://doi.org/10.1007/s11010-020-03988-0

Yokota S, Ueki S, Ono Y, Kasahara N, Pérez-Gutiérrez A, Kimura S, Yoshida O, Murase N, Yasuda Y, Geller DA (2016). Orthotopic mouse liver transplantation to study liver biology and allograft tolerance. Nat. Protoc., 11(7): 1163–1174. https://doi.org/10.1038/nprot.2016.073

Yoon U, Lai M, Nguyen T, Elia E (2023). A 36-year-old woman with acute liver failure following acetaminophen overdose, raised INR of 8.7, and normal blood viscosity measured by rotational thromboelastometry (ROTEM). Am. J. Case Rep., 24: e938500-1. https://doi.org/10.12659/AJCR.938500

Zalzala MH, Kareem VS (2021). The safranal effect against cyclophosphamide-induced liver injury. Iraqi J. Pharma. Sci., 30(2): 208–213. https://doi.org/10.31351/vol30iss2pp208-213

Zhao Y, Liu X, Liang C, Pei T, Guo M, Wang J, Zhang J (2022). α-lipoic acid alleviated fluoride-induced hepatocyte injury via inhibiting ferroptosis. J. Agric. Food Chem., 70(50): 15962–15971. https://doi.org/10.1021/acs.jafc.2c07484