Hepatoprotective potential of N-acetyl cysteine in rats with phenytoin induced liver injury

Introduction

Phenytoin (PHT) is a medication used to treat neurological and psychiatric conditions, as well as epilepsy (antiepileptic).¹ PHT metabolites are associated with liver damage, including cholestatic hepatitis, cytotoxic hepatitis, or mixed reaction.^2,3 The exact mechanism of PHT-induced hepatotoxicity remains unclear.⁴ One of the proposed mechanisms is that PHT may cause significant liberation of reactive oxygen species in hepatic mitochondria, resulting in mitochondrial malfunction.^3,5 Another proposed mechanism is thought to be related to the activation of inflammatory pathways with the overproduction of pro-inflammatory cytokines.^6,7 N-acetyl cysteine (NAC) has been used in therapeutic contexts for numerous years. It has been employed in the management of several conditions, including paracetamol overdose, adriamycin-induced cardiotoxicity, ischemia-reperfusion injury, heavy metal poisoning, and idiopathic pulmonary fibrosis.^8,9 The presence of a principal function as an antioxidant, its free thiol group, enables it to interact with reactive oxygen and nitrogen species.^10,11 NAC possesses anti-inflammatory properties by inhibiting the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), a key player in the inflammatory cascade and immune response to oxidative stress. NAC inhibits the translocation of the NF-κB transcription factor and nuclear activation, which are essential for controlling the expression of genes that promote inflammation.^12,13 Research has demonstrated that NAC inhibits the secretion of inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, in macrophages that have been activated by lipopolysaccharide.¹⁴ That being said, a number of studies were carried out to investigate the potential hepatoprotective effects of NAC against various hepatotoxic substances, including carbamazepine,¹⁵ Adriamycin,¹⁶ anti-tuberculosis drugs,¹⁷ and others. This study aimed to evaluate the possible protective effects of NAC against PTH-induced hepatotoxicity.

Ethical approval

This study was conducted with the approval of the ethical committee of the College of Pharmacy, University of Kerbala on February 6, 2024 (Ref: 2024An.8).

Methods

Twenty male Wistar albino rats, weighing 120–170 g, were used in this study. The animals were kept in the animal house at the College of Pharmacy, University of Kerbala, where they had unlimited access to food and drink, 12-hour light/dark cycle. The animals were acclimated for seven days prior to the experiment, were in good health, and were free of specific pathogens. All animal procedures in this study were conducted in accordance with institutional and international guidelines for the ethical treatment of animals. They were divided randomly into four groups, each of which contained five animals, as follows:

The animals were maintained for 24 h following their last dose and then anesthetized with xylazine and ketamine (75 mg/kg). After the rats were anesthetized, a 5 cc syringe was used to draw blood from their left ventricle. After centrifuging the serum with an Eppendorf apparatus, it was chilled to -20°C for storage.¹⁸ For histological investigation, liver tissues were removed, washed with cold phosphate buffer (pH 7.4), weighed, and stored in 10% formalin for later analysis.¹⁹ Biochemical markers, including enzymes that measure total serum bilirubin TSB, ALT, AST, and alkaline phosphatase activity, were assessed via the colorimetric method, utilizing cell biolabs assay kits in compliance with the manufacturer’s protocols.

Throughout the study, no unanticipated or anticipated negative events were noted. All efforts were made to minimize animal suffering, including the use of appropriate anesthesia and analgesia, continuous health monitoring by trained personnel, and the implementation of humane endpoints when necessary. These measures were undertaken to ensure the ethical treatment of animals and the reliability of scientific outcome according to ARRIVE reporting guidelines.²⁰

Results

Effects of phenytoin and N-acetyl cysteine on the serum level of biochemical markers

The findings revealed a marked increase p < 0.05 in plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total serum bilirubin (TSB), and alkaline phosphatase (ALP) in the phenytoin-treated group when compared with the other groups. In addition, there was a significant decrease in the serum levels of ALT, AST, and TSB (except for ALP) in the phenytoin+ NAC group when compared with the phenytoin-treated group. Moreover, in the NAC only treated group, the levels of liver enzymes were low compared to the control group, but this reduction was significant only for ALP level, as shown in Table 1, Figure 1: (A-D).

Table 1. Effects of different treatment modalities on biochemical markers.

Parameters\Groups	ALT (U/L) Mean ± SD	AST (U/L) Mean ± SD	ALP (U/L) Mean ± SD	Total Bilirubin (mg/dl) Mean ± SD
Control	44.14 ± 2.79c,d	206.43 ± 15.64c	147.13 ± 7.59b,c,d	0.76 ± 0.15c
NAC	41.56 ± 4.44c,d	185.72 ± 8.84c	111.87 ± 11.93a,c,d	0.80 ± 0.11c
Phenytoin	64.16 ± 5.24a,b,d	269.64 ± 31.29a,b,d	172.16 ± 4.19a,b	1.30 ± 0.32a,b,d
Phenytoin+ NAC	51.74 ± 3.04a,b,c	207.04 ± 13.97c	170.82 ± 4.86a,b	0.72 ± 0.09c

a Significant from control,

b Significant from NAC,

c Significant from phenytoin, and

d Significant from Phenytoin+ NAC.

Figure 1. The serum parameters of all groups of rats as follows: A: alanine aminotransferase (ALT), B: aspartate aminotransferase (AST), C: alkaline phosphatase (ALP), D: total bilirubin.

The results indicate the mean value ± standard deviation of the mean. a: significant from control, b: significant from NAC, c: significant from phenytoin, d: significant from Phenytoin+ NAC.

Results of histopathology

In this experimental setting, the liver sections obtained from the control group displayed normal histological characteristics that were distinguished by a central vein encircled by hepatocytes arranged in a radial fashion. In contrast, liver samples from the phenytoin-treated group showed prominent features, such as substantial congestion, hepatocyte degeneration, biliary stasis, vacuolated cytoplasm, localized necrosis, and inflammatory cell infiltration (neutrophils, lymphocytes, and eosinophils). The phenytoin and N-acetylcysteine groups showed considerable reductions in inflammatory cells, necrosis, and degeneration in the liver. Moreover, normal hepatocyte plates and lobular architecture remained intact. Liver slices from the N-acetyl cysteine group showed no significant abnormal features ( Figure 2: a-d).

Figure 2. Cross sections (liver)

for (a): control groups sections clarify central vein (CV), and normal hepatocyte (H). (b): Phenytoin group sections reveal necrosis (N) with biliary stasis (B.S), and focal degenerations (F.D), alongside inflammatory cells infilteration. (c): The (Phenytoin + N-acetylcysteine) group exhibits a reduction in necrosis, degeneration, and decrease in number of inflammatory cells. (d): N-acetylcysteine group show no remarkable pathology. H & E 40X.

Discussion

Phenytoin is one of the most widely used antiepileptic drugs in the management of various types of epilepsy. It is frequently utilized in outpatient settings and almost all emergency services worldwide. It is increasingly being utilized in therapeutic treatment of neuropathic pain,²² hiccups, migraines, and wound healing;²³ however, its dosage should be adjusted to reduce the risk of unwanted effects, which could lead to its withdrawal even if it is beneficial.²⁴ Phenytoin is commonly included in the list of ten major causes of drug-induced acute liver failure. More than 10% of cases of acute phenytoin hepatitis accompanied by jaundice lead to fatality. If signs of jaundice or liver illness occur early during treatment, it is advisable to halt the use of phenytoin.^23–25 Liver maintenance was evaluated by measuring ALT, AST, and ALP levels, which are enzymes that are mainly expressed at higher levels in the cytoplasm. During liver injury, these enzymes are released into the bloodstream in accordance with the severity of the liver damage.²⁶ Serum bilirubin level is an additional conventional marker of liver damage. The findings of our investigation indicate that phenytoin therapy resulted in varying degrees of biochemical changes in the liver enzymes of rats relative to other groups. Additionally, Phenytoin caused hepatic necrosis with focal degeneration and biliary stasis in rat livers, as shown by histopathological analysis. The obtained results were consistent with the findings of the investigated biochemical parameters. This study confirmed the hepatotoxic effects of phenytoin treatment. N-acetylcysteine (NAC) is a synthetic form of cysteine. It is widely recognized as an anti-inflammatory and antioxidant agent that provides hepatoprotection against liver injury caused by paracetamol.⁹ Different mechanisms have been suggested to be involved in the pathogenesis of phenytoin-induced hepatotoxicity, including oxidative stress and depletion of antioxidants.³ This provided a rationale for investigating the potential preventive benefits of NAC against PHT-induced hepatic damage caused by phenytoin. NAC mainly enhances the formation of glutathione and removes reactive oxygen species (ROS) generated during oxidative stress.^9,12,16 Moreover, various studies have examined the probable mechanisms that may explain the positive benefits of NAC in cases of nonparacetamol overdose. Previous studies have demonstrated that NAC stimulates guanylate cyclase activation, probably due to its anti-inflammatory, antioxidant, inotropic, and vasodilatory properties that enhance hepatic blood circulation and oxygen supply to essential organs.²⁷ NAC can mitigate endoplasmic reticulum tension and enhance mitochondrial function, both of which aid in liver protection against damage.^28,29 The same results were observed in cell cultures treated with tuberculosis medicines,³⁰ which further enhanced the potential positive effects of this agent in protecting the liver. The current investigation found that concomitant administration of NAC and phenytoin for 45 days led to a considerable decrease in phenytoin-induced hepatotoxicity, as indicated by the reduction in TSB, ALT, and AST levels. Moreover, NAC restored normal liver histopathology. These results are in agreement with those of previous studies that have demonstrated the hepatoprotective effects of NAC against several hepatotoxic conditions. Eftikhari et al.,³¹ found that NAC effectively reduced the increased levels of ALT and AST in an animal model of liver injury associated with risperidone. Additionally, NAC has demonstrated a defense mechanism against liver damage caused by other hepatotoxic medicines and chemicals, including adriamycin,¹⁶ azathioprine,³² and dimethyl nitrosamine.³³ In summary, NAC at a daily dose of (200) revealed a valuable effect against phenytoin-induced toxicity, probably because NAC acts as a scavenger of free radicals and mediates the oxidative stress pathway.

Conclusion

This study concluded that concomitant administration of NAC and phenytoin lowers the risk of phenytoin-induced hepatotoxicity. Moreover, this study confirmed that NAC is relatively safe when administered for a relatively prolonged period.