Comparative Study of the Protective Effects of Alpha-Lipoic Acid and Alpha-Tocopherol on Isoniazid-Induced Hepatitis in Mice

By Ikram Ullah Khan¹, Shabana Ali², Arooj Shahid¹, Muhammad Rafay Rasheed¹, Malik Sikandar Mehmood¹, Barkat Ullah Khan³

Affiliations

1. Department of Pharmacology, Army Medical College, Rawalpindi, Pakistan
2. Department of Pharmacology, NUST School of Health Sciences, Islamabad, Pakistan
3. Department of Physiology, Army Medical College, Rawalpindi, Pakistan

doi: 10.29271/jcpsp.2025.07.854

ABSTRACT
Objective: To assess the preventive role of alpha-tocopherol (vitamin E) and alpha-lipoic acid (ALA) in isoniazid-induced hepatitis.
Study Design: Experimental study.
Place and Duration of the Study: Department of Pharmacology, Army Medical College, National University of Medical Sciences, Rawalpindi, and National Institute of Health, Islamabad, Pakistan, from July 2020 to June 2022.
Methodology: A total of 150 mice were taken and divided into five groups, each containing 30 mice. Isoniazid was administered at a dose of 100 mg/kg orally for 28 days for the induction of hepatotoxicity. Alpha-lipoic acid and alpha-tocopherol were administered orally half an hour prior to the administration of isoniazid in a dose of 50 mg/kg and 100 mg/kg, respectively, alone and in combination for 28 days. Blood and liver samples were taken for biochemical and histopathological analysis, respectively. Statistical difference between serum levels of liver enzymes on day 0 and day 28 was analysed using paired t-test.
Results: Results showed protective effects of ALA and alpha tocopherol against isoniazid-induced hepatotoxicity by reducing the serum levels of bilirubin and liver enzymes. Histopathology also indicated preservation of liver architecture against isoniazid-induced hepatitis by ALA and alpha tocopherol which was indicated by reduced necrosis, steatosis, and portal inflammation.
Conclusion: The use of ALA and alpha-tocopherol may be helpful in preventing isoniazid-induced hepatotoxicity.

Key Words: Alpha lipoic acid, Alpha tocopherol, Isoniazid, Hepatitis.

INTRODUCTION

Tuberculosis (TB) is the leading cause of mortality from a single infectious agent in the world,¹ caused by mycobacterium tuberculosis, an acid-fast aerobic bacillus. According to WHO treatment guidelines 2022, the standard regimen for new cases of TB consists of 2-month intensive phase treatment with isoniazid (INH), rifampicin (RMP), pyrazinamide (PZA), and ethambutol (E), followed by 4-month continuation phase involving INH and RMP.² The continuation phase can be increased up to 6 months in countries having a higher incidence of TB relapse and prevalence of resistant organisms. The overall success of this regimen is more than 80% globally.³
 

Compliance, an important factor in determining the overall success of anti-tuberculosis therapy (ATT), is limited by the variety of adverse medicine reactions (ADRs) associated with ATT.⁴ Major adverse effects associated with first-line anti-TB agents include anorexia, vomiting, nausea, hyper-uricaemia (with PZA), peripheral neuropathy (with INH), and optic neuritis (with ethambutol).⁵

One of the major ADRs of ATT is agent-induced hepatotoxicity, having an incidence rate of 2-28%,⁶ up to 40%⁷ found in one study, with a mortality rate of 0.2%.⁷ The incidence of hepatotoxicity among ATT agents is highest with INH (56%), followed by RMP (34%) and PZA (10%).⁷ INH-induced hepatotoxicity is caused by the conversion of INH to toxic metabolites by hepatic enzymes N-acetyl transferase 2 (NAT2) and cytochrome P450 2E1 isoform (CYP2E1).⁸ These toxic metabolites cause hepatic damage via covalent bonding, oxidative stress, and metabolic dysfunction.^8,9

Drug-induced hepatotoxicity has many implications, which may sometimes lead to contraindications to isoniazid use. Overt hepatitis is an indication for switching to alternate treatment regimens which have potentially toxic effects as well as putting huge burden on healthcare expenditures.^10,11 There is a need to investigate potential remedy to the common and life-threatening adverse effects of these economical and relatively safer first-line agents such as INH.

Alpha lipoic acid (ALA), a di-thiol compound synthesised in mitochondria, is a naturally occurring antioxidant that scavenges a number of free radicals. ALA has shown a protective role in reversing the hepatic damage caused by several agents and toxins.^12-14 Alpha-tocopherols, a biologically active form of vitamin E, is a potent antioxidant which prevents lipid peroxidation and scavenges free radicals. It has been used to reverse hepatotoxicity caused by certain agents.^15,16

Both ALA and alpha-tocopherol have shown protective effects in isoniazid-induced hepatotoxicity in rats and rabbits, respectively.^13-16 Studies have shown that isoniazid-induced hepatotoxicity in mice closely resembles to that of a human’s hepatotoxicity model as compared to rats and rabbits.¹⁷ The contribution of INH and acetyl hydrazine (AcHz) covalent binding is determined by the rate of acetylation, and affinities of cytochromal enzymes for both the agents and their clearance. These steps are species-dependent and it has been found that INH-induced hepatitis in rats and rabbits does not resemble the human model as INH-induced hepatitis in humans is a delayed–onset and is characterised by centrilobular necrosis. This study was carried out to explore the potential hepatoprotective role of ALA and alpha-tocopherol in the INH-induced hepatitis model of mice.

METHODOLOGY

It was a laboratory-based experimental study conducted at the Department of Pharmacology and Therapeutics, Army Medical College, Rawalpindi, in collaboration with the National Institute of Health (NIH), Islamabad, and the Department of Chemical Pathology, Army Medical College, Rawalpindi, Pakistan, from July 2020 to June 2022. The procedures were performed after getting approval from the Ethics Committee of Army Medical College, Rawalpindi, Pakistan, known as the Centre for Research in Experimental and Applied Medicine (CREAM).

A total of 150 healthy male mice were selected by convenient, non-probability, random sampling technique and equally distributed into five groups, each containing 30 mice. Only male mice were included in the study, while mice having deranged liver enzymes were excluded from the study. Group A was labelled as the control group in which mice were fed on a normal diet ad libitum. Group B was taken as the disease control group in which INH was administered in a dose of 100 mg/kg orally for 28 days for the induction of hepatotoxicity. In Group C, ALA was administered orally via a gavage tube, half an hour prior to the administration of INH in a dose of 50 mg/kg for 28 days. The Group D was administered alpha-tocopherol (100 mg/kg) orally through a gavage tube initially for 1 week (to achieve steady state) and then half an hour prior to INH (100 mg/kg), once daily for four weeks. Mice in Group E were administered both ALA and Alpha-tocopherol before INH for four weeks.

On day zero and day 28, blood samples were taken from the tail vein of mice in clot activator tubes for biochemical analysis. Serum levels of bilirubin, alkaline phosphatase (ALP), alanine transaminase (ALT), and aspartate transferase (AST) were taken as biochemical parameters.

Animals were sacrificed on the 29^th day and liver samples were dissected out and preserved for histopathological analysis. Sections were examined under a high-power field for the presence or absence of necrosis, steatosis, portal triaditis, degeneration, fibrosis, and regeneration.

Analysis of data was done using SPSS version 21. Statistical difference between serum levels of liver enzymes on day 0 and day 28 was analysed using the paired t-test. Histopathological parameters were analysed for distribution of frequencies in each group.

RESULTS

Results of biochemical parameters are shown in Table I. Paired t-test showed that change in serum levels of liver enzymes and bilirubin in groups A, C, D, and E from day 0 to day 28 was insignificant, whereas in the Group B, bilirubin levels increased significantly from day 0 to day 28. Similarly, the levels of ALT, AST, and ALP also raised significantly in Group B but the levels remained clinically normal in groups C, D, and E.

The results of histological parameters are shown in Table II. Administration of INH for 28 days in Group B resulted in ballooning degeneration (80% of specimens, shown in Figure 1B), necrosis (50% of specimens), steatosis (80% of specimens, shown in Figure 1D) and portal triaditis (50% of specimens, shown in Figure 1C) on histological examination of mice liver.

Figure 1: Photomicrograph of the slide showing normal architecture (A), ballooning degeneration (B), portal triaditis (C), steatosis (D) (Stained with H & E. Examined with 10x power (A), 40x power (B-D).

Table I: Showing the comparison of liver enzymes in different groups using paired t-tests.
 

Parameters

Group A

Group B

Group C

Group D

Group E

Day 0

Day 28

p-value

Day 0

Day 28

p-value

Day 0

Day 28

p-value

Day 0

Day 28

p-value

Day 0

Day 28

p-value

Serum bilirubin

1.7 ± 0.35

2.03 ± 0.93

0.06#

1.7 ± 0.35

2.1 ± 1.0

0.04*

1.7 ± 0.35

2.6 ± 0.67

<0.001*

1.6 ± 0.35

2.1 ± 0.46

<0.001*

1.7 ±   0.35

2.1 ±  0.21

<0.001*

Serum ALT

43.3 ± 13.6

43.8 ± 13.6

0.86#

42.9 ± 13.9

451.2 ± 44.9

<0.001*

42.9 ± 13.9

44.2 ± 7.8

0.68#

42.0 ± 12.1

54.6 ± 17.9

<0.14#

43.4 ±      12.4

59.5 ±   34.8

0.35*

Serum AST

60.5 ± 15.8

59.6 ± 16.3

0.81#

58.6 ± 16.5

98.2 ± 14.7

<0.001*

61.0 ± 16.1

80.9 ± 16.2

<0.001*

57.2 ± 15.9

72.0 ± 28.2

0.013*

57.8 ±    15.2

70.8 ±  22.7

<0.001*

Serum ALP

39.2 ± 2.8

39.4 ± 2.7

0.79#

39.4 ± 2.7

42.9 ± 2.5

<0.001*

39.2 ± 2.7

43.8 ± 1.9

<0.001*

39.4 ± 2.7

43.1 ± 2.7

<0.001*

39.2 ±   2.7

42.0 ±  3.1

<0.001*

p-values, as calculated by using paired t-test, *Statistically significant (p <0.05), # Statistically non-significant (p >0.05).

Table II: Distribution of histological parameters among groups.
 

Histological parameters	Group A (N = 30)	Group B (N = 30)	Group C (N = 30)	Group D (N = 30)	Group E (N = 30)
Ballooning degeneration	0	24 (80%)	16 (53%)	18 (60%)	3 (10%)
Necrosis	0	15 (50%)	3 (10%)	3 (10%)	1 (3%)
Steatosis	0	24 (80%)	18 (60%)	16 (53%)	9 (30%)
Portal triaditis	0	15 (50%)	3 (10%)	0 (0%)	0 (0%)
Fibrosis	0	0	0	0	0
Regeneration	0	0	0	0	0

Normal liver architecture is also shown in Figure 1A. Concurrent administration of ALA in Group C resulted in a preservation of liver histology characterised by a decreased frequency of ballooning degeneration (53%), necrosis (10%), steatosis (60%), and portal triaditis (10%). Similarly, adminis- tration of alpha-tocopherol also demonstrated hepato- protective role by decreasing ballooning degene-ration, steatosis, necrosis, and portal triaditis to 60, 10, 53%, and 0%, respectively. Combined administration of ALA and alpha- tocopherol to Group E also resulted in significant reduction of hepatotoxicity.

DISCUSSION

Drug-induced hepatotoxicity is one of the most significant adverse effects associated with anti-tuberculosis drugs. Search for an effective remedy for INH-induced hepatitis has been an active area of research for the last two decades. Studies have been conducted in various animal models using different agents to discover a possible solution to this problem. However, most of these studies were conducted on rat models and few on rabbit models. The contribution of INH and AcHz covalent binding is determined by the rate of acetylation, and affinities of cytochromal enzymes for both agents and their clearance. These steps are species-dependent, and it has been found that INH-induced hepatitis in rats and rabbits does not resemble the human model as INH-induced hepatitis in humans is a delayed–onset and is characterised by centrilobular necrosis.¹⁷ This study was conducted on the mice model which closely resembles to that of the human hepatitis model, as the relative concentrations of INH to (AcHz) are similar to that in humans.¹⁷

The dose of INH for induction of hepatitis was 100 mg/kg, which is similar to the dose used by Wessam et al. and administered orally.¹⁴ Hepatotoxicity was assessed by measuring serum levels of ALT, AST, ALP, and bilirubin. ALT levels three times the upper limit of normal were considered indicative of drug-induced liver injury. The degree of hepatic damage was also assessed by microscopic examination of liver samples.

This study evaluated the hepatoprotective role of ALA and alpha-tocopherol. Previous studies have shown the protective effects of alpha lipoic acid in rats^13,14 and ALA in rabbits.¹⁶ This study evaluated their effects in mice model and compared their effects with each other using serial biochemical.

The findings are in accordance with a previous study in which ALA reduced serum bilirubin levels in INH-induced hepatitis in rats.¹⁴ Tanaka et al. also showed a significant reduction in serum bilirubin levels by the administration of ALA in INH-induced hepatitis model of rats.¹³ Administration of alpha-tocopherol (Group D) also proved beneficial in preventing the hepatic injury induced by INH, indicated by the statistically non-significant change in serum levels of bilirubin on day 28. These findings are similar to a previous study conducted by Tayal et al. in albino rabbits, which also showed protective effects of alpha-tocopherol for INH- induced hepatitis, indicated by a significant reduction of serum bilirubin levels.¹⁶ Comparing the relative protection offered by ALA (Group C) with that of alpha-tocopherol (Group D), there was no significant difference between the two groups. Combined administration of ALA and alpha-tocopherol (Group E) proved no added advantage in hepato- protection compared to that provided by either agents alone.

Serum ALT levels were also raised significantly in Group B compared to Group A on day 28. Levels of ALT in groups C, D, and E were less than that of Group B on day 28. Serum ALT levels in Group C on day 28 were statistically not significant compared to day 0, indicating the meliorating action of ALA on INH-induced hepatitis. The probable reason for these effects is the attenuation of free radical generation and scavenging of reactive oxygen species inside the hepatocytes, ultimately preventing the liver injury associated with INH. These findings are in accordance with previous studies in which administration of ALA in rats reduced serum ALT levels in INH-induced hepatitis.^13,14 Administration of ALA also showed a similar hepatoprotective role indicated by a statistically significant reduction in serum ALT levels in the concanavalin A-induced hepatitis model in mice.¹² Administration of alpha-tocopherol also proved beneficial in preventing the hepatic injury induced by INH, indicated by the statistically non-significant change in serum levels of serum ALT levels on day-28. These findings are similar to a previous study conducted on a rabbit’s model which showed similar protective effects of alpha-tocopherol for INH-induced hepatitis.¹⁶ Comparing the relative protection offered by ALA with that of alpha-tocopherol, there was no significant difference between the two groups. Similarly, combined administration of ALA and alpha-tocopherol proved no significant advantage in hepato-protection against INH compared to that provided by either agents alone.

Serum levels of AST and ALP did not rise to clinically significant levels (which is indicated by more than three times the normal value) in this study after administration of INH to Group B. This is in contrast to previous studies which showed a clinically significant increase in AST levels in the drug- administered group in the INH-induced hepatitis model in rats¹⁴ and rabbits.¹⁶ The probable reason for this might be because INH was administered for 28 days in this study, causing acute liver injury, which is often associated with raised ALT. Raised serum AST and ALP is rather more associated with chronic liver injury compared to acute liver injury.¹⁸

Liver injury induced by INH was judged by ballooning degeneration, steatosis, portal triaditis, necrosis, and fibrosis, observed under high and low-power microscopy. INH treatment was associated with marked histological changes in Group B indicated by ballooning degeneration, steatosis, portal triaditis, and necrosis. Administration of ALA and alpha-tocopherol led to marked preservation of liver histology in groups C, D, and E, indicated by a reduced number of specimens showing ballooning degeneration, steatosis, portal triaditis, and necrosis. These results are similar to that of a previous study conducted by Wessam et al. in rat model of INH-induced hepatitis, in which administration of ALA resulted in marked preservation of liver histology, indicated by reduced inflammatory infiltrates in the portal triad area and necrosis.¹⁴ Another study also showed similar hepatoprotection in the INH-induced hepatitis model in rabbits, indicated by decreased necrosis and steatosis.¹⁶ However, fibrosis was not seen in any of the liver specimens in the current study. The probable explanation may be the reason that fibrosis is more marked in chronic inflammation, while INH-induced liver injury is predominantly of acute nature. This finding is in accordance with that of previous studies in which fibrosis was not seen in any of the liver speci-mens.^14,19,20 Similarly, the specimen did not reveal any signs of regeneration. A study conducted previously on a rat model of INH-rifampicin induced hepatitis also showed similar findings in which regeneration was not seen in any of the specimens treated with silymarin.¹⁹ This may be because of the shorter duration of treatment with the antioxidants. When the groups were compared with each other, it was found that, unlike the biochemical parameters, the effects of the combination of ALA and alpha- tocopherol on histological parameters were more pronounced as compared to either agents alone. On the other hand, the effects of ALA on the preservation of liver histology were similar to that of alpha- tocopherol.

These findings support the potential role of ALA and alpha- tocopherol as antidote for INH-induced hepatitis. However, the study is limited by certain factors. The exact molecular mechanism of the toxicity and its reversal by these agents could not be explored due to the limitation of time and resources. Similarly, the oxidative stress markers could also be helpful in comparing the antioxidant potential of the two agents and further justifying their use in drug-induced hepatitis.

CONCLUSION

Both ALA and alpha-tocopherol have protective effects in INH- induced hepatitis. However, further large-scale animal and human studies need to be conducted to further strengthen this conclusion.

ETHICAL  APPROVAL:
The procedures were performed after getting approval from the Ethics Committee of the Army Medical College, Rawalpindi, Pakistan, known as the Centre for Research in Experimental and Applied Medicine.

PATIENTS’ CONSENT:
The study is animal-based and carried out in NIH under the international guidelines of animal studies. Patient consent is not applicable in the subject case.

COMPETING  INTEREST:
The authors declared no conflict of interest.

AUTHORS’ CONTRIBUTION:
IUK: Conception of idea, manuscript writing, and principal investigator.
SA: Substantial contribution to the design of the work.
AS: Data analysis and interpretation.
MRR: Acquisition and analysis of the data.
MSM: Substantial contribution to the manuscript writing.
BUK: Conception and design of the work.
All authors approved the final version of the manuscript to be published.

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