Effect of Rifampicin and Isoniazid on Liver of Albino Rat: A Review

Liver is one of the largest and most vital organs in the human body located between the absorptive surface of the gastrointestinal tract responsible for the regulation of several crucial biochemical activities such as carbohydrates, proteins, amino acid and lipids metabolism. Being frequently exposed to toxic substances and therefore vulnerable to tissue damage. Any damage to liver is associated with various metabolic dysfunctions [1, 2, 3].  Tuberculosis (TB) is one of the leading global health threats caused by Mycobacterium tuberculosis infecting nearly about one third of the world’s population and is relatively resistant to many antibiotics [4, 5].

Rifampicin (RIF) and Isoniazid (ISO) drugs are the primary medications used for the management of TB, where liver injury is the major adverse effect [6]. RIF is a bacteriostatic semisynthetic antibiotic drug derived from Streptomyces mediterranei. RIF was introduced in 1967 as a main drug in addition to isoniazid, ethambutol, pyrazinamide and streptomycin for the treatment of tuberculosis and inactive meningitis [1]. RIF a brownish red crystalline powder, was first introduced as an effective agent for the treatment of TB in 1968 [2] and found to lower the growth of most positive and negative Gram bacteria [5]. RIF compounds include rifabutin, rifapentine, rifalazil and rifaximin [6].  RIF has a chemical formula C₄₃H₅₈ N₄O₁₂ and molecular weight of 822.9 g/mol [8] (Fig. 1).  Isoniazid is the most reliable and commonly used medication for tuberculosis. It is mycobactericidal in nature [8].  It remains inactive when administered in the body, later metabolized into a biologically active compound by a bacterial catalase peroxidase enzyme which pairs the isonicotinic acid with nicotinamide adenine dinucleotide (NADH) to form isonicotinic acyl-NADH complex [9]. ISO is a carbohydrazide obtained by formal condensation between pyridine-4-carboxylic acid and hydrazine. It derives from an isonicotinic acid. Isoniazid chemically known pyridine-4-carbohydrazide having chemical formula of C₆H₇N₃O with a molecular weight of 137.139g/mol. [8]  (Fig. 2).         

Fig .1 and 2 The Chemical structure of Rifampicin and Isoniazid [8].

METHODOLOGY

The present review is a thorough assessment of RIF and ISO toxicity and associated risks, with a particular emphasis on its effect on the liver. The literature includes both experimental and non-experimental data extracted by using databases and journals indexed in Google Scholar, Science Direct, PubMed, Web of science, covering studies published between 1994 and 2025. The extracted data was then analysed and compiled to assess rifampicin and isoniazid induced hepatotoxicity.

EFFECT OF RIF AND ISO

Liver damage in various gradations could be the cause of administration of both RIF and ISO [5]. ISO treatment causes skin eruption, jaundice, back pain, infection and optic nerve degeneration and destructive influence on the peripheral and the central nervous system.  Hepatotoxins transform into chemically reactive metabolites in the liver, that have the potential to interfere with other cellular components such as protein, lipids and nucleic acids leading to dysfunction [1]. RIF exposure may lead to a number of symptoms, such as thrombocytopenia, which is defined by skin and mucosal bleeding and metabolic acidosis. Oligouric disturbances in the kidney, spasms, jaundice like symptoms such as yellowing of skin and eyes due to bile component accumulation, edema around the eyes, skin rashes and facial inflammation are examples of RIF and ISO induced toxicity [10]. Hepatitis is one of the documented side effects of rifampicin, which also includes nausea, vomiting, lack of appetite, stomach cramps and diarrhoea. Other adverse effects, are vertigo, headache, exhaustion, disorientation and menstruation disruption [11].

Body and organ weight

The body weight of rats co-administered with isoniazid and rifampicin significantly (p < 0.001) decreased to 171.8 ± 3.12g compared to that of control to 218 ± 7.18 g. whereas liver weight significantly (p < 0.001) increased to 8.77 ± 0.11 g compared to that of control with 6.61 ± 0.21 g [12,13,2]. Increase of mean body weight and liver weight was very slow in RIF and ISO treated group as compared to control [14]. Decrease in mean final body weight, liver weight, length, width and thickness noted in treated group compared to control group [15]. Significant increase in liver/body weight index after RIF and ISO treatment was noted as compared to control [16].

Morphological and behavioural observation

Animals treated with ISO alone and in combination with RIF exhibited decreased food intake, quietness, less motor activity, rough hair coat, inactiveness and slow response to external stimuli [17] [18].

Biochemical Analysis

Previous experimental studies on animals suggest that ISO administration in toxic doses distress hepatocellular membrane integrity. The effect of rifampicin at high dose of 100 mg/kg body weight for 6 days showed noteworthy rise in the level of γ-glutamyl transpeptidase, acid ribonuclease, acid phosphatase whereas the level of succinate dehydrogenase diminished. The serum level of RNA, total proteins and lipids, bilirubin, phospholipids, cholesterol, lipid peroxides increased while hepatic glycogen decreased and the lower dose of 50 mg/kg body weight showed less significant changes as compared to higher dose [19]. [20, 9] evaluated liver injury in rats induced by ISO and RIF and detected three times increase in serum aspartate aminotransaminase, alanine transaminase, alkaline phosphatase and bilirubin and altered the lipid levels in serum and liver compared to control group, while triglycerides were significantly elevated. In contrast, phospholipids were significantly decreased in liver. Serum bilirubin, peroxidation of lipids (P < 0.01) and low levels of hepatic non protein thiols (P < 0.01) in all the animals of RIF and ISO treated group significantly increased as compared to control [21,22]. According to [23] administration of RIF and ISO at a high and low dose of 250 and 50 mg/kg body weight respectively for 90 days daily resulted in abnormal rises or falls in the liver marker enzymes with mild hyperlipidemia, hypercholesterolemia and hyperuricemia, leading to membrane damage causing outflow of alanine transaminase, alkaline phosphatase and bilirubin, increased lipid peroxidation and glutathione homeostasis, and enhanced the CYP 2E1 mediated bioactivation mechanism [24]. [25] reported significant elevation in the level of oxidative stress and hepatocellular injury followed by RIF and ISO treatment. [26, 27] demonstrated significant increase in the thiobarbituric acid reactive substances, the oxidative stress markers along with liver marker enzymes in experimental rats. The RIF and ISO administered animals exhibited significantly (P<0.01) reduced hepatic glutathione levels at100 mg/kg body weight intra peritoneally.

[12] reported increased total bilirubin level, blood urea nitrogen, serum creatinine and serum uric acid along with glutathione pyruvate transaminase, oxaloacetate transaminase and alkaline phosphatase. [28] stated that RIF and ISO elevated the level of alanine aminotransferase activity, steatosis, apoptosis and oxidative stress markers in experimental animals, whereas decreased glutathione content provoked liver damage. Significant increase in the total cholesterol, triglycerides and low density lipoprotein cholesterol levels, whereas high density lipoprotein cholesterol level showed a significant decrease. The level of total proteins, albumin and alpha 1-globulin decreased significantly. But no noticeable changes were documented in the other elements of globulins such as alpha, beta and gamma globulin, as well as albumin to globulin ratio and creatinine level [29, 30]. [31] revealed significantly increased serum levels of liver marker enzymes. Whereas activity of superoxide dismutase and glutathione was reduced followed by ISO and RIF treatment that indicate the oxidative stress induced liver damage. The level of serum bilirubin, serum enzyme markers, total protein, albumin and total globulin increased as compared to control [1, 3, 16, 32, 18, 33, 15, 34, 35, 3]. Furthermore, mRNA levels of Fas, Acc and Scd-1genes, some significant genes responsible for fatty acid synthesis, class B scavenger receptor CD36 rise in RIF treated group. Inactivation of transcription factors SREBP-1c and LXR-α that regulate the genes for synthesis of hepatic fatty acid was reported in RIF treated group. Whereas hepatic PXR was activated quickly in RIF treated mice. Hepatic PPARγ, a downstream target of PXR, was transcriptionally upregulated. Thus, the augmented level of lipid synthesis in liver and absorption of fatty acids into liver from circulation collectively attributed to RIF induced hepatic lipid accumulation. This partially attributed to RIF induced up regulation of PPARγ and its target genes [36]. [37] analysed and enumerated various proteins and reported that a total of 29 and 40 proteins expressed significantly whereas proteins 27 and 118 were not expressed in response to 177 and 442.5 mg/kg RIF, respectively. Analysis by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enhancement showed up regulation of glutathione transferase activity and down regulation of proteins associated with arachidonic acid metabolism whereas signalling pathway of transcription factor activated by a ligand and regulator of adipogenesis and lipid metabolism, CYP enzymes, tripeptide glutathione, chemical carcinogenesis and relative proteins amplified in RIF treated liver.

[2,18,14] reported that the level of antioxidants like superoxide dismutase and catalase in RIF and ISO treated groups were significantly decreased whereas liver marker enzymes increased significantly (p < 0.001). The serum level of protein and albumin decreased significantly (p < 0.001). 50% decline in the ratio of albumin to globulin to that of its original value observed in RIF treated group as compared to control group. The serum cholesterol, bilirubin and malondialdehyde level significantly increased (P <0.001), in RIF treated group compared to control.

 [38] reported that activation in the hepatic stellate cell markers increased can be correlated with RIF and ISO treatment with the increased liver collagen and significant periportal fibrosis. These changes are concomitant with hepatocytic apoptosis, increased activity of hepatic cytochrome P450 2E1, NADPH oxidase and oxidative stress simultaneously that leads to the development of liver fibrosis.

[6] co?administered RIF and ISO and observed significantly (P<0.01) upregulated NF?κB DNA binding activity and expression of tumor necrosis factor alpha mRNA whereas bile salt export pump was significantly up and downregulated respectively. The protein expression of bile salt export pump was found to be significantly decreased. Liver injury induced by antituberculosis drugs, RIF and ISO may be ascribed to dysfunction of mitochondria, drug metabolic enzymes, oxidative stress, accumulation of protoporphyrin IX, stress in endoplasmic reticulum, disparity in bile transport and immune response, cholestasis and accumulation of lipid in the liver. ISO induced liver toxicity can be enhanced by RIF by regulating the expression of drug metabolizing enzymes and transporters. Whereas, ISO can provoke RIF induced liver damage by declining the serum bilirubin level [39]. Significant increase in cytokines interleukin- 6 and 1beta, tumor necrosis factor alpha, and caspases 3 that execute the apoptosis detected after RIF and ISO treatment [34].

Histopathological evidences

Histopathological studies of liver showed coagulative necrosis and inflammation of the hepatic artery, portal vein and bile duct, with piecemeal necrosis, infiltration of lymphocytes and neutrophils, congestion of the blood vessels, the central vein, hepatic sinusoids and interlobular septums with severe infiltration of inflammatory cells in RIF and ISO treated group [20, 21, 15]. [26] reported cell swelling, congestion and fatty degeneration of the liver cells and portal triaditis observed in RIF and ISO treated group. Neutrophil infiltration in the centrilobular region with portal triaditis, vacuolated cytoplasm and swollen sinusoids with some lymphatic accumulations were reported in RIF and ISO treated group [27, 24, 12, 29]. [30, 37] demonstrated that RIF and ISO treatment resulted in fatty degeneration, inflammation, distended sinusoids, necrosis and hypertrophy in hepatocytes. Hepatotoxicity induced by ISO alone causes necrosis and inflammatory cell infiltration with steatotis, cholestatic, thick wall of blood vessels showing congestion and mononuclear cells aggregation with large granulomatous lesion in liver parenchyma and lipid accumulation, which was evaluated by lysochrome diazo dye in RIF and ISO treated rats [40, 31]. [35, 36] noticed significant hepatocyte hypertrophy with prominent increase in cell size along with hepatocytes with enlarged and binucleated hepatocytes. [16] examined severe damage in the hepatic architecture with prominent polymorphonuclear inflammatory infiltration, cytoplasmic vacuolation, interlobular hemorrhage, dilated hepatic sinusoids with degenerated hepatocytes and moderately dilatation and congestion in central vein, disorganized cord of hepatocytes. Focal necrosis in the hepatic parenchyma with odema, some pyknotic hepatocytes, few bundles of fibrous tissue, hepatocytes merged with each other forming eosinophilic syncytial masses. Ballooning degeneration of hepatocytes with vacuolated cytoplasm and indistinct cell boundaries observed in RIF and ISO treated animals [31]. Histopathological alterations induced by RIF and ISO showed inflammation in the periportal region of hepatocytes [2]. Histopathology of liver in rifampicin treated group exhibited lipid accumulation, cell necrosis, enlarged central vein, increased sinusoidal spaces and disrupted portal vein that indicates damaged histoarchitechture. Rat treated with ISO indivdually or in combination with RIF induce liver alterations showing necrosis, immune cells infiltration, portal expansion and dilation, sinusoidal congestion, deranged structure, such as portal inflammation, fatty degeneration, necrosis, dilated sinusoidal capillaries, disrupted central vein and Kupffer cells inflammation and thus concluded that co-administration of RIF and ISO cause hepatotoxic effect which causes injury to the liver parenchyma. [4, 32, 39, 15, 17].

Table 1: Hepatotoxicity of rifampicin and isoniazid