|Ahead of print publication
Selenium abrogates tenofovir/lamivudine/efavirenz-induced hepatotoxicity in rats
Elias Adikwu1, Michael Ikechukwu Oraebosi2, Innocent Biradee3
1 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Niger Delta University, Bayelsa State, Nigeria
2 Department of Pharmacology and Therapeutics, Nile University of Nigeria, Abuja, Nigeria
3 Department of Biomedical Technology, School of Science Laboratory Technology, University of Port Harcourt, Rivers State, Nigeria
|Date of Submission||24-Jun-2020|
|Date of Decision||28-Jul-2020|
|Date of Acceptance||09-Aug-2020|
|Date of Web Publication||01-Apr-2021|
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Niger Delta University, Bayelsa State
Source of Support: None, Conflict of Interest: None
Background: The therapeutic benefit of tenofovir/lamivudine/efavirenz (TLE) in the treatment of human immunodeficiency virus can be truncated by the occurrence of hepatotoxicity. This study evaluated the protective effect of selenium (Se) against hepatotoxicity induced by TLE in albino rats. Materials and Methods: Adult male albino rats (n = 40) randomized into four groups (n = 10) were used. Group 1 (Control) orally received normal saline (0.2 mL) daily. Group 2 orally received Se (0.1 mg/kg) daily. Group 3 orally received TLE (8.6/8.6/17.1 mg/kg) daily. Group 4 orally received Se (0.1 mg/kg) and TLE (8.6/8.6/17.1 mg/kg) daily. All rats were treated for 90 days. After treatment, the rats were euthanized, and serum samples were centrifuged from blood samples and assessed for liver function markers. Liver samples were harvested and evaluated for morphological changes and biochemical parameters. Results: Impaired liver redox status in TLE-treated rats was characterized by remarkable (P < 0.001) decreases in glutathione peroxidase superoxide dismutase, catalase, and glutathione levels with remarkable (P < 0.001) increases in malondialdehyde levels when compared to control. The alterations in liver function markers were marked by remarkable (P < 0.001) increases in serum aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl transferase, conjugated bilirubin and total bilirubin levels when compared to control. Hepatocyte necrosis and fatty change were observed in TLE-treated rats. However, TLE-induced hepatotoxic changes were significantly (P < 0.01) reversed in Se supplemented rats when compared to TLE. Conclusion: Se may be clinically effective against hepatotoxicity caused by TLE.
Keywords: Antiretroviral, hepatotoxicity, mitigation, rats, selenium
| Introduction|| |
Tenofovir/lamivudine/efavirenze (TLE) is used for the treatment of human immunodeficiency virus (HIV). It is recommended by the European and American guidelines as a primary regimen for the treatment of HIV. The introduction of TLE has reduced the menace of HIV infection. It has shown an efficacy of 80% with less degree of virological failure and resistance. However, the life-long use of TLE may cause toxicities including central nervous system toxicity (depression, abnormal dreams, attention disorders, and dizziness) nephrotoxicity and hepatotoxicity. The hepatotoxic effect of TLE is of serious concern and has been attributed to efavirenz. Hepatotoxicity caused by TLE is often characterized by conspicuous alterations in serum liver function markers. Also, perturbations in liver histology characterized by necrosis, fatty and inflammatory cell infiltrations have been documented. The mechanisms by which TLE causes hepatotoxicity are not well understood, but mechanisms including direct cholestatic injury, endoplasmic reticulum stress, mitochondrial dysfunction, and oxidative stress have been speculated. Furthermore, immune-mediated mechanism due to the presence of inflammatory cell infiltrates has been reported.
Selenium (Se) has received considerable attention as an essential micronutrient. It influences the state of health of plants, animals, and humans. It is incorporated in selenoproteins during their translation as amino acid selenocysteine. The amino acid selenocysteines are involved in the syntheses of diverse selenoenzymes including glutathione peroxidase (GPx), iodothyronine deiodinases, and thioredoxin reductases. Se functions as a redox center which reduces hydrogen peroxide and phospholipid hydroperoxides to harmless products through the activity of Se dependent GPx. This action maintains membrane integrity,, prevents prostacyclin production and reduces the incapacitation of biomolecules such as proteins, lipids and DNA by oxidative stress., Se has lots of physiological functions which include cellular homeostasis, maintaining immune-endocrine function, and metabolic cycling. It has shown potential benefits in human disease conditions, especially chronic metabolic disorders such as hyperlipidemia and hyperglycemia. Therapeutic benefits in cancer, asthma, cardiovascular, and neurodegenerative disorders have also been reported in some studies. Se has shown potential protective effects against animal models of drug-induced toxicities including hepatotoxity, but with no study on its protective effect against TLE-induced hepatotoxicity. This study assessed the ability of Se to offer protection against a rat model of TLE-induced hepatotoxicity. Rat model is used experimentally to increase knowledge and to proffer possible remedies to biomedical problems that can be used clinically.
| Materials and Methods|| |
The directive (2010/63/EU) on the handling of animals for experimental purpose promulgated by the European Parliament and of the Council was used.
Drugs, chemicals and experimental protocol
Se capsules (Sodium selenite) (Bactolac Pharmaceuticals Inc., 7 Oser Avenue, Hauppauge, NY 11788, USA), TLE (Hetero Labs Limited Unit-111, 22-110, I.D.A, Jeedimetla, Hyderabad, India). The rats were sourced from the animal unit of the Department of Pharmacology and Toxicology, Faculty of Pharmacy, Niger Delta University, Nigeria. The rats were kept 5 per cage at 25°C ± 2°C and 12 h light/dark cycle with free access to chow and water. This study used Se (0.1 mg/kg) and TLE (8.6/8.6/17.1 mg/kg) suspended in 0.9% normal saline. Forty adult male albino rats (200–250 g) were randomized into four groups (n = 10). Group 1 (Control) orally received normal saline (0.2 mL) daily. Group 2 orally received Se (0.1 mg/kg) daily. Group 3 orally received TLE (8.6/8.6/17.1 mg/kg) daily. Group 4 orally received Se (0.1 mg/kg) and TLE (8.6/8.6/17.1 mg/kg) daily. All rats were treated for 90 days. The handling of animals to animal sacrifice lasted for 110 days.
After treatment, the rats were anesthetized, and blood samples were collected from the heart in sample containers. The blood samples were allowed to clot and serum samples were centrifuged from the clots and assessed for liver function markers. Liver samples were collected, rinsed in physiological solution and homogenized in 0.1 M Tris-HCl (pH 7.4). The homogenates were centrifuged at 2000 rpm for 20 min, and the supernatants were decanted and assessed for biochemical markers.
Total bilirubin (TB), gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST), conjugated bilirubin (CB), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and alanine aminotransferase (ALT) levels were evaluated using Konelab™ PRIME 60i automated biochemical analyzer (Thermo Scientific, Vantaa, Finland).
Liver oxidative stress marker assay
Malondialdehyde (MDA) was assayed according to Buege and Aust. Superoxide dismutase (SOD) was determined as described by Sun and Zigman. Catalase (CAT) was assayed using the method described by Aebi. Glutathione (GSH) was assayed as described by Sedlak and Lindsay. Glutathione peroxidase (GPx) was evaluated according to Rotruck et al.
Histology of the liver
Liver specimen were collected and fixed in 10% neutral-buffered formalin for 24 h. The fixed liver samples were dehydrated in ethanol solution of ascending concentrations. Liver specimens were sliced, paraffin-embedded, and sectioned (5 μm thick). The sections were stained with hematoxylin and eosin (Bio Lab Diagnostics Limited, Mumbai, India) for light microscopic evaluation.
GraphPad Prism 5.0 software (GraphPad Software Inc., La Jolla, CA, USA) was used for the data analysis. Data are expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was used to compare the experimental with control groups followed by Tukey's test. Significance was considered at P <0.01 and <0.001.
| Results|| |
Effect on serum biochemical parameters
Serum biochemical markers (ALT, GGT, AST, LDH, ALP, TB, and TB) were normal (P > 0.05) after the administration of Se in relation control [Table 1]. In contrast, the administration of TLE produced significant (P < 0.001) increases in serum biochemical parameters when compared to control [Table 1]. The percentage increases in serum biochemical markers caused by TLE represent (462.5%), GGT, (390.6%), AST (358.5%), LDH (393.3%), ALP (318.2%), TB (367.5%), and TB (324. 2%). In contrast to the observation in TLE administered rats, supplementation with Se led to significant decreases (P < 0.01) in serum biochemical markers when compared to TLE [Table 1].
|Table 1: Effect of selenium on liver function parameters of tenofovir/lamivudine/efavirenz-treated rats|
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Effect on liver tissue biochemical parameters
The administration of Se had no significant effects (P > 0.05) on liver tissue biochemical parameters (ALT, GGT, AST, LDH, and ALP) when compared to control [Table 2]. In contrast, TLE administration produced significant (P < 0.001) increases in the aforementioned biochemical parameters in relation to control [Table 2]. The percentage increases in liver tissue biochemical parameters produced by TLE were ALT (273.6%), GGT (270.2%), AST (313.7%), LDH (262.0%), and ALP (362.9%). However, Se supplementation produced significant (P < 0.01) decreases in liver tissue biochemical parameters when compared to TLE [Table 2].
|Table 2: Effect of selenium on liver tissue biochemical indices of tenofovir/lamivudine/efavirenz-treated rats|
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Effect on liver oxidative stress markers
Furthermore, in rats treated with Se, liver MDA and antioxidant levels (SOD, CAT, GSH, and GPx) were normal (P > 0.05) when compared to control [Table 3]. On the other hand, the administration of TLE significantly (P < 0.001) increased liver MDA and significantly (P < 0.001) decreased liver antioxidant levels in comparison to control [Table 3]. However, Se supplementation significantly (P < 0.001) decreased liver MDA level, and significantly (P < 0.01) increased liver antioxidant levels when compared to TLE [Table 3].
|Table 3: Effect of selenium on liver oxidative stress markers of tenofovir/lamivudine/efavirenz -treated rats|
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Effect on liver histology
Normal liver histology was observed in control rats [Figure 1]a. Hepatocyte necrosis [Figure 1]b and fatty change were observed in the liver of TLE-treated rats [Figure 1]c. However, decreased hepatocyte necrosis and the absence of fatty change were observed in the liver of Se supplemented rats [Figure 1]d.
|Figure 1: (a) Liver of rat in the control group showing normal histology. (b) Liver of rat treated with tenofovir/lamivudine/efavirenz showing hepatocyte necrosis. (c) Liver of rat treated with tenofovir/lamivudine/efavirenz showing hepatocyte necrosis and fatty change. (d) Liver of rat treated selenium and tenofovir/lamivudine/efavirenz showing hepatocyte necrosis (H and E, ×400)|
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| Discussion|| |
The occurrence of toxicities is a challenge with the use of highly active antiretroviral therapy (HAART) in the treatment of HIV. TLE is an essential HAART regimen that has reduced HIV associated death, but causes hepatotoxicity, which is a major health concern. A number of studies have attributed hepatotoxicity caused by TLE to its efavirenz component which belongs to the NNRTIs family. The speculated mechanisms by which NNRTIs cause hepatotoxicity include mitochondrial damage, endoplasmic reticulum stress, and inflammation. A number of studies reported possible benefits that can be derived from the antioxidant and anti-inflammatory activities of Se. This study examined the potential of Se to prevent hepatotoxicity caused by TLE in rats. Alterations in serum biochemical markers (ALT, LDH, AST, GGT, ALP, CB, and TB) and liver histology are primarily used as yardsticks for drug-induced hepatotoxicity. Experimentally, studies have shown that the impact of oxidative stress on drug-induced hepatotoxicity can be ascertained by the assessments of LPO index (MDA) and endogenous antioxidants. In this study, all evaluated parameters were normal in rats administered with Se. In contrast, the administration of TLE caused notable hepatotoxicity. This was characterized by elevations in serum and liver tissue biochemical parameters (ALT, LDH, AST, GGT, ALP, CB, and TB). Hepatic damage caused by TLE was accompanied by perturbation in liver histology characterized by hepatocyte necrosis and fatty change. Also, elevated MDA and decreased antioxidant (SOD, GPx, CAT, and GSH) levels occurred in the liver of TLE administered rats. The aforementioned observations are common features of hepatotoxicity caused by TLE.,
The elevations in serum and liver tissue biochemical parameters in TLE administered rats are evidence of the destruction of hepatocyte membrane causing the leakage and the release of these parameters into the blood. Studies have associated MDA, an index of LPO with ROS-induced tissue injury. Hence, the elevated level of liver MDA attest to the involvement of LPO in TLE-induced hepatotoxicity through the oxidation of hepatic polyunsaturated fatty acid by ROS. Antioxidants are present in biological systems to incapacitate the excess activities of ROS that may disrupt biomolecular function. The uncontrollable activities of ROS may compromise antioxidant function via incapacitation thereby increasing the vulnerability of biomolecules to damage by ROS-induced oxidative stress. Therefore, the reductions in liver endogenous antioxidants in TLE-treated rats clearly established that oxidative stress is one of the mechanisms by which TLE causes hepatotoxicity. TLE-induced hepatocyte necrosis might have resulted from the overwhelming activities of ROS which might have caused damage to hepatic biomolecules (lipids, proteins, and DNA). However, TLE-induced hepatotoxicity was mitigated by Se pretreatment. This was accompanied by decreased serum and liver tissue biochemical parameters and liver MDA with increased antioxidant activity. Furthermore, reduction in hepatocyte necrosis and the absence of fatty change were observed in Se-supplemented rats. These observations attest to the protective potential of Se against TLE-induced hepatotoxicity. This finding correlates with the reported protective effect of Se against silver nanoparticle-induced hepatotoxicity in rats. The observation in this study is also in unison with the reported protective effect of Se against a rat model of tebuconazole-induced hepatotoxicity.
In this study, the hepatoprotective effect of Se might have occurred through its inhibitory activity on hepatic oxidative stress induced by TLE. It might have scavenged and neutralized hepatic ROS generated by TLE. Se is an element that is involved in many biochemical processes such as the syntheses of coenzyme Q, GPx, and thioredoxin reductase., GPx is an antioxidant enzyme that renders hydrogen peroxide and organic hydroperoxides harmless. It protects surrounding tissues from ROS produced by the respiratory burst of macrophages and neutrophils during phagocytosis. Furthermore, Se, as a component of thioredoxin reductase has an important role in protecting cells from toxicants, especially oxidants and electrophiles. In addition, Se can inhibit platelet aggregation, oxidative modification of lipids, inflammation and endoplasmic reticulum stress.
| Conclusion|| |
Se may be effective against TLE associated hepatotoxicity.
The authors are grateful to Dr. Yibala Obuma of the Department of Medical Laboratory Sciences, Niger Delta University, Nigeria for tissue handling.
Financial support and sponsorship
Conflicts of interest
The authors declare no conflicts of interest.
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[Table 1], [Table 2], [Table 3]