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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access  Full Text Article                                                                                                                                                                                            Research Article

Investigation of the Efficacy of Zonisamide in Alcohol Induced Hepatotoxicity

Nilüfer Dönmezdil *, Selçuk Tunik, Fırat Aşır , Senem Çetin Duran, Dilara Akinci, Enis Duran, Eda Yıldızhan, Nurullah Peker, Mehmet Obut 

Department of Histology and Embryology, Medical Faculty, Dicle University, Diyarbakır, Turkey

INTRODUCTION

Chronic alcohol use is globally recognized as a significant public health issue and is cited as a major contributor to numerous diseases and deaths ¹. According to the World Health Organization, alcohol consumption is responsible for millions of deaths and the emergence of hundreds of diseases annually ². Alcohol, similar to other addictive substances, has the potential to cause dependency across various age groups ³.

The impact of alcohol on the liver is particularly severe, with alcohol use disorder being a primary cause of diverse hepatic diseases ⁴. The prevalence of alcohol-related problems is closely linked to socio-cultural factors and must be addressed as a worldwide health concern ⁵.

The detrimental effects of chronic alcohol consumption on the liver are multifaceted. Alcohol directly acts as a hepatotoxin, damaging liver cells. Prolonged and excessive intake can lead to serious conditions such as fatty liver disease, alcoholic hepatitis, and cirrhosis, a terminal stage liver disease ⁵. Acetaldehyde, produced during alcohol metabolism, initiates cellular damage and subsequent inflammation, further exacerbating damage through increased oxidative stress and the suppression of antioxidant defenses in the liver ⁶.

Zonisamide, a sulfonamide-structured anticonvulsant used primarily for treating various neurological conditions, including epilepsy, modulates seizures by regulating abnormal brain electrical activity and reducing neuronal hyperexcitability ⁷. It is also noted for its anti-inflammatory and antioxidant properties, which may theoretically protect against alcohol-induced liver damage ⁸. This makes zonisamide a potential candidate for the prevention and treatment of liver injuries associated with alcohol use ⁹.

In recent years, there has been a surge in research into novel therapeutic agents aimed at treating chronic alcohol abuse and liver diseases ¹⁰. The potential hepatoprotective effects of zonisamide are drawing significant interest from the scientific and medical communities ¹¹. A comprehensive approach, encompassing everything from lifestyle modifications to pharmacological interventions, is required to prevent and treat alcohol-related liver injuries ^12,13. Understanding the role of zonisamide in this context could significantly aid in developing effective treatment strategies and provide new insights into managing alcohol-induced liver conditions ¹⁴.

MATERIAL AND METHOD

In this study, 40 male Wistar albino rats were used. The experimental animals were divided into 4 groups (n:10). The groups were formed as follows;

Group1: Sham group: The rats in this group were administered with an amount of saline equivalent to ethanol given orally 3 times a day for 4 days and zonisamide given orally 1 time a day. 

Group 2: Ethanol group: Rats in this group were given ethanol orally 3 times a day for 4 days; instead of zonisamide, saline equivalent to the dose of zonisamide was given once a day.   

Group3: Zonisamide group: Rats in this group were given zonisamide (100 mg/kg) orally once a day for 4 days; saline equivalent to the dose of ethanol was given 3 times a day.

Group4: Ethanol+Zonisamide group: Rats in this group were given ethanol orally 3 times a day for 4 days. At noon, 1 hour before the ethanol dose, 100 mg/kg zonisamide was administered once a day.

The experimental animals had unlimited access to food and water and were kept under control in 12 hours of daylight/12 hours of darkness, 8:00 am-8:00 pm, 23±2 oC. Zonisamide (Zalipin 100 mg) was obtained from Generica. Ethanol administration in the groups was based on Majchowicz's excessive alcohol protocol ¹⁵. Three hours after the last alcohol administration, the animals were sacrificed by intracardiac blood sampling under general anesthesia.

Tissue Tracking 

The liver tissues were first placed in 10% neutral formalin solution and routine paraffin tissue follow-up was started. After fixation (24 hours), the tissues were kept under running tap water for 6 hours. The tissues were passed through 50%, 70%, 80%, 90%, 96% and absolute ethyl alcohol series and kept in xylene 3 times for 45 minutes for transparency. Finally, the tissues were placed in paraffin oil at 58°C for infiltration. The tissues were then embedded in paraffin blocks and sections of 4-6 μm thickness were taken for Hematoxylin-Eosin staining using a microtome (catalog number: Leica RM2265, Wetzlar, Germany). 

Hematoxylin-Eosin Staining 

Liver tissue sections from paraffin blocks were placed in a bain-marie set at 37°C. The sections were kept in an oven at 58-62°C for 6 hours to melt the excess paraffin on the slide. The sections were deparaffinized in xylene for 3x15 minutes. The sections were passed through decreasing alcohol series (100%, 96%, 90%, 70%, 50% ethyl alcohol) for 10 minutes each and then brought to distilled water and kept for 5 minutes. Harris Hematoxylin stain was applied for 8 minutes and then the sections were washed under running water for 5 minutes. The sections were rinsed and kept in alcoholic eosin stain for 6 minutes. The sections were rapidly immersed in increasing alcohol series (80%, 90%, 96% ethyl alcohol series) and kept in absolute alcohol for 2 minutes ¹⁶. Finally, the sections were soaked in xylene for 3x15 minutes and Entellan was dripped onto the tissue and covered with a coverslip.

Findings

Hematoxylin Eosin staining of the liver sections of the groups is shown in Figure 1. In the sections of the control group, the vena centralis was centrally located in the middle of the liver lobule, and the sinuses were normal and not dilated. Hepatocytes were generally binucleated and hexagonal in appearance (Figure 1a). In the zonisamide group, findings similar to the control group were observed. In this group, liver histology and vascular structures were normal. Hepatocytes were arranged radially (Figure 1b). In the ethanol group, vascular dilatation and congestion and impaired vascular integrity in the vena centralis, dilatation and congestion in the hepatic sinuses, leukocyte infiltration, pyknosis and degeneration in hepatocytes were observed due to alcohol toxicity. Disorders in the radial arrangement organization of hepatocytes and dense vacuolar structures in the cytoplasm were noted (Figure 1c). In the group treated with zonisamide after ethanol toxicity, the pathologies healed and restoration of liver tissue was observed. Reduction in vascular dilatation and congestion, regeneration in hepatocytes and normal appearance of the vena centralis were observed (Figure 1d).

Figure 1. Sections of liver tissue. A) Control, B) Zonisamide, C) Ethanol, D)

Ethanol+Zonisamide group. Zonisamide decreased alcohol toxicity. s: sinusoids, v: vena centralis, arrow: leukocyte infiltration, arrowhead: hepatocytes

DISCUSSION

In the findings of this study, normal liver tissue and vascular structures were observed in the control and zonisamide groups, while histopathologic findings in the liver tissue due to alcohol use were recorded in the ethanol group. It was observed that zonisamide treatment histologically improved the pathologies caused by alcohol toxicity and provided tissue regeneration.  

It can be concluded that zonisamide reduces alcohol-induced cellular damage by protecting the structural integrity of hepatocytes and by supporting cytoprotective effects and cell survival.

The findings of this study provide important evidence for the protective effects of zonisamide against alcohol-induced liver injury. These results show several similarities and differences when compared with previous studies in the literature. For example, a study by He et al. focused on the antioxidant properties of zonisamide and showed that this drug protects nerve cells against oxidative stress ¹⁷. This finding suggests that the antioxidant properties of zonisamide are not limited to the nervous system and may also protect against oxidative damage in other organs such as the liver. This is consistent with the findings obtained in our study, where zonisamide was observed to reduce alcohol-induced oxidative stress and protect the structural integrity of liver tissue.

Research on alcohol-induced liver disease requires ongoing evaluation and comparison of treatment approaches. For example, a study by Pandey et al. examined the potential benefits of antioxidant-based therapies such as silymarin, vitamin E and polyphenols in alleviating alcohol-induced liver injury ¹⁸. This study suggests that antioxidants may play important roles in the prevention and treatment of liver injury by neutralizing free radicals and reducing inflammation. However, another study by Huang et al. emphasized that zonisamide may have protective effects on alcohol-induced liver injury ¹⁰. The study by Huang et al. investigates whether zonisamide (ZNS) therapy reduces metabolic outcomes and nonalcoholic fatty liver disease in epilepsy patients. ZNS is a chemical with antiepileptic and antioxidant activities. This study evaluates the efficacy of ZNS therapy in reducing obesity and lowering vascular disease risks. The results of the study are characterized by ≥5% body weight reduction observed in patients after 12 and 24 weeks of ZNS therapy. ZNS treatment showed a significant reduction in serum levels of body weight, body mass index (BMI), HbA1c, triglycerides, hs-CRP and hepatic steatosis index (HSI) when adjusted for age, gender, time and baseline values. These results suggest that ZNS may be beneficial in patients with obesity and metabolic syndrome with high vascular risk. The results of the study support that agents that specifically modulate alcohol metabolism and oxidative stress may exert positive effects on the liver. However, a direct comparison of the results of these studies emphasizes the complexity of therapeutic strategies used in the management of alcohol-induced liver disease and that the effectiveness of these approaches may vary according to individual disease states. The findings of Huang and colleagues are a sign that the different findings in the existing literature indicate that more research is needed to support the protective effects of specific agents such as ZNS against alcohol-induced liver injury.

Our findings demonstrate a potential hepatoprotective effect of zonisamide against alcohol-induced liver injury and contrast with previous concerns regarding its hepatotoxicity. Although zonisamide, a sulfonamide anticonvulsant, is generally recognized for its neuroprotective roles, concerns regarding its hepatotoxic risks have persisted due to limited and inconsistent findings in the literature. For example, a review study by Kamitaki et al. highlights this uncertainty by noting that although zonisamide has been associated with potential liver injury, conclusive evidence is sparse and hepatotoxic risk has not been adequately characterized ¹⁹. Our data contribute to this dialog by suggesting that, contrary to these hesitations, zonisamide may in fact play a protective role in the context of alcohol-induced liver injury. This protective effect may be attributed to its anti-inflammatory and antioxidant properties, which may reduce oxidative stress and cellular damage typically exacerbated by alcohol consumption. These observations warrant a reassessment of the hepatotoxic risk profile of zonisamide and emphasize the need for further studies to clarify its safety and efficacy in liver protection.

In the literature, there are many studies on the efficacy of various ingredients with antioxidant properties for the management of alcohol-induced liver injury. For example, a study by Granado et al. demonstrated the potential benefits of certain antioxidants in reducing alcohol-induced oxidative stress and improving liver function ²⁰. This study is consistent with our findings supporting that the protective effects of zonisamide against alcohol-induced liver injury may be mediated through antioxidant mechanisms.

CONCLUSION

This study, which evaluated the effects of zonisamide on alcohol-induced liver injury, presents results that are both consistent with the existing literature and different in several aspects. These differences call for a more in-depth investigation of the effects of zonisamide on the liver and the discovery of new approaches for the treatment of alcohol-induced liver injury. In this context, further research is needed to obtain more comprehensive information on the potential therapeutic use of zonisamide.

Acknowledgement

This study was part of doctoral thesis of Ph.D. candidate Nilüfer Dönmezdil. The ethical approval was taken from Local Ethics Committee of Animal experimantation (DUSAM), Dicle University (approval number: 75, date: 28/02/2023).

Conflıcts of Interest:

The authors do not have any conflicts of interests.

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