Drug- induced hepatotoxicity is a common clinical problem caused by a large number of drugs with different mechanisms and variable outcomes. Deferasirox (DSX) was approved by the FDA in 2005 as an iron chelator for treatment of chronic iron overload in patients requiring multiple blood transfusions. However, the use of the drug was associated with post-market reports of hepatic injury with unknown mechanism of liver injury . In the present study, Deferasirox induced a significant increase in serum AST, ALT, ALP and total bilirubin. This is in agreement with pre-marketing studies that demonstrated marked elevation of liver transaminases in about one third of patients taking Deferasirox. These data indicate drug-induced hepatitis and liver failure. The rationale of the present findings is that elevated transaminases level is associated with the occurrence of hepatic injury while increased bilirubin level is an indication of the severity of the liver injury. This means that Deferasirox-induced liver injury was substantially enough to impair liver function.
Another study has found that Deferasirox caused death of a patient with sickle cell anemia due to hepatitis. The patient possesses polymorphism in the Abcc2 gene. This gene encodes MRP2 protein, thus genetic polymorphism in this gene is associated with increased susceptibility to Deferasirox- induced hepatotoxicity .
Also, it was found that Deferasirox (10–20 mg/kg) sickle cell anemia patients ≥40 years old caused severe changes in renal function or hepatotoxicity that resulted in discontinuation of the treatment. It was suggested that hepatotoxicity is related to the Haplotype MRP2 and the UGT1A1*6 genotype resulted in elevation of creatinine. Glucuronidation by UDP-glucuronosyltransferase 1A (UGT1A) subfamily which encodes UDP-glucuronosyltransferase. is the main metabolic pathway of Deferasirox. In bile, Deferasirox is then excreted through the multidrug resistance protein (MRP2) which is a transporter anion expressed in the hepatocytes membranes. MRP2 is encoded by the Abcc2 gene .
In contrast to the present findings, Miura et al., 2010 found that three cases of transfusion-mediated iron-overloaded patients with hematological diseases had elevated ALT levels. Treatment with DSX decreased and subsequently normalized elevated ALT levels. This occurred also with the abnormal AST level . Other parameters related to impaired liver function including ALP, γ-GTP, T-Bil, and Alb were not changed. However, we cannot rely on these findings that DSX may improve liver damage in those patients because of the small number of patients and lack of supportive histopathological findings of liver tissue examination.
Another study found that DFX at a dose of 100 mg/kg was the most effective in preventing elevation of ALT, AST enzymes and histopathologic changes induced by concanavalin A .
Several mechanisms may account for Deferasirox –induced hepatotoxicity such as production of reactive intermediates by cytochrome P450, stimulation of autoimmunity, apoptosis, and disruption of calcium homeostasis, canalicular injury and mitochondrial injury . This was supported by the findings that treatment of rats with Deferasirox induced a significant elevation of hepatic tissue MDA content with a significant reduction in hepatic tissue GSH content.
The most frequent mechanism for drug-induced liver injury is metabolism by cytochrome P450 that involves the formation of high energy reactive intermediates which then undergo conjugation process. Subsequent covalent bonding of these reactive intermediates to cellular components, such as proteins or nucleic acids produces cellular dysfunction . However, Deferasirox undergoes minimal metabolism by CYP-450 system which makes this mechanism unlikely for Deferasirox.
Immune- mediated liver toxicity may also account for Deferasirox- induced hepatotoxicity. The drug may bind to an enzyme and becomes immunologically active which then activates cellular and humoral immune responses leading to cellular death. This may account for the significant elevation of hepatic tissue IL-6 content in Deferasirox- treated group observed in the present study.
Cholestatic injury may be considered as another possible mechanism for Deferasirox-induced liver injury . The drug may block bile salt transport proteins in the canalicular membranes responsible for the generation of bile. This in turn leads to cholestasis and jaundice and accounts for the observation that total bilirubin is significantly elevated in Deferasirox group.
Another possible mechanism that may account for Deferasirox-induced liver injury is mitochondrial injury. The main characteristics of this injury are macrovesicular lesions, focal necrosis, fibrosis and cholestasis. Mitochondrial injury may result from inhibition of fatty acid β- oxidation.
In the present work, H&E-stained liver sections of Deferasirox group revealed disturbed hepatic lobular architecture, swollen hepatocytes with highly vacuolated cytoplasm and shrunken deeply stained pyknotic nuclei, extensive inflammatory cellular infiltration and proliferation of bile ducts. Similar results were obtained by other authors [39, 40] studying the damaging effect of zinc oxide nanoparticles and carbon tetrachloride on rat's liver, respectively.
Swelling of hepatocytes with vacuolation of their cytoplasm could be explained by ballooning degeneration associated with dilatation of endoplasmic reticulum. This dilatation results from mitochondrial dysfunction and ATP depletion, leading to loss of ion homeostasis and plasma membrane integrity resulting in fluid influx. The cytoplasm is partially rarefied along the cellular periphery and the cytoplasmic cytoskeleton clumps around the nucleus .
In cases of drug-induced liver injury, damaged hepatocytes release molecules called damage-associated molecular patterns. This results in activation of liver immune cells, including Kupffer cells, natural killer lymphocytes and dendritic cells thus which triggers an inflammatory cascade involving recruitment of neutrophils and monocyte from blood . This explanation coincides with the detection of extensive inflammatory cellular infiltration in the present study.
Activation of hepatic stem cells. These stem cells are activated in response to liver injury and differnetiated into progenitor cells for both hepatocytes and cholangiocytes. This ultimately leads to proliferation of bile ducts [43, 44].
In the current study, excessive deposition of collagen fibers in the portal area and around central veins was detected in Masson's trichrome-stained sections of Deferasirox group. This finding was confirmed by morphometric and statistical studies. There was a highly significant increase in the mean area percentage of collagen fibers in this group as compared to control group.
The process of liver fibrogenesis is initiated by damage of hepatocytes which results in recruitment of inflammatory cells and activation of Kupffer cells, with subsequent release of cytokines and growth factors [45, 46]. These factors stimulate differentiation of hepatic stellate cells into myofibroblasts-like cells that have the ability of synthesis and deposition of large amounts of collagen fibers resulting in liver fibrosis .
Intense caspase-3 immunoreaction in many hepatocytes of group II was confirmed statistically by a highly significant increase in the mean optical density of caspase-3 positive cells in this group as compared to control group.
Martin-Sanchez et al., 2017 studied the effect of Deferasirox on proximal renal tubules and proved that Deferasirox induced mitochondrial stress and caused loss of mitochondrial membrane potential. In addition, it downregulated the antiapoptotic protein BclxL while the levels of proapoptotic Bax were unchanged, leading to a decreased BclxL/Bax ratio which is known to predispose to apoptosis . Stressed mitochondria lead to release of cytochrome c into cytosol that causes activation of caspase-3 and apoptotic cell death .
The present data revealed that the group pretreated with curcumin exhibited significant reduction in the values of ALT, AST, ALP, total bilirubin, IL-6 and MDA levels with significant elevation in GSH compared to Deferasirox- treated group. In addition, histological, immunohistochemical and statistical findings exhibited partial improvement as compared to Deferasirox- treated group. These results support the evidence that curcumin possesses an antioxidant and hepatoprotective effects.
Curcumin is rich in curcuminoids which owing to their methoxy group1, 3 B-diketone moiety and phenolic hydroxyl can inhibit oxidation, and decrease the free radicals generation . In addition, Curcumin was found to inhibit NF-κB, which activates inflammatory cytokines and chemokines, leading to several inflammatory conditions . Also, curcumin is able to inhibit IL1, IL1B, IL6, IL8, tumor necrosis alpha, and cyclooxygenase pathways .
Kyung et al. used dimethylnitrosamine (DMN) for induction of liver cirrhosis in rats. The study found curcumin increased conductivity using a magnetic resonance-based electrical conductivity imaging method than cirrhotic tissues in the DMN-group. Curcumin also resulted in significant reduction of fibrosis and decreased inflammation compared with liver tissues treated with DMN . The hepatoprotective and anti-inflammatory effects of curcumin were associated with significant reduction in COX-2 expression in the DMN- group.
Also in agreements with our findings, a study using alcoholic-liver disease found that treatment with curcumin prevented both the pathological and biochemical changes induced by alcohol in an alcohol-induced liver damage (ALD) model as manifested by reduction of activation of NF-kB and the induction of cytokines, chemokines, COX-2, iNOS, and nitrotyrosine formation . It was suggested that curcumin prevented ALD by inhibiting lipid peroxidation, activation of NF-kB, and expression of proinflammatory mediators.
In consistent with the present findings, Ibrahim et al., found that the use of curcuminoids crude extract resulted in significant increases in antioxidant enzymes; super oxide dismutase, reduced glutathione, catalase and reduction in liver integrity enzyme biomarkers; aspartate transaminase, alanine transaminase and alkaline phosphatase in comparison to CCL4-induced liver damage . These data support our findings that curcumin preserves liver cell membranes integrity and prevents leakage and release of enzymes such as ALT, AST and ALP and was able to reverse the injurious effects of Deferasirox on the liver. In the present study, curcumin significantly reduced the liver tissue MDA and increased GSH content. This indicates that in addition to protecting the liver against toxicants, curcumin also enhance the antioxidant system of the liver, this will positively enhance the liver capability to fight toxicant.
In another study, it was found that curcuminoids significantly attenuated AFB1-cytotoxicity. The heaptoprotective mechanisms for curcumin against AFB1 toxicity are related to antioxidant response and anti-inflammatory potentials . In addition, curcumin has been reported to inhibit the expression of NF-kB dependent inflammatory cytokines, such as TNF-α, IL-6, IL-2, TGF-b, and MCP-1, and inflammation-promoting molecules such as COX-2 and iNOS in Kupffer cells .
The data of the present study showed that in comparison to Deferasirox group, rats pretreated with silymarin exhibited significant reduction in ALT, AST, ALP, total bilirubin, IL-6 and MDA contents with significant elevation of GSH content. These results were confirmed by the noticeable improvement detected in histological, immunohistochemical and statistical findings.
In accordance with our results, a previous study revealed that CCl4 administration resulted in significant elevation of intracellular enzymes, such as transaminases and serum alkaline phosphatase and an ethanolic plant extract of silymarin produced insignificance decreases in liver functions. The stabilization of these enzymes indicates the improvement of the functional status of the liver . Also, elevation of MDA and reduction of GSH by CCL4 have been reversed upon use of the extract which indicates an antioxidant potential of silymarin.
The protective effects of silymarin may be attributed to different mechanisms including; (1) scavenging of free radicals and chelating free Fe and Cu. (2) inhibition of ROS-producing enzymes and preventing the formation of free radical (3) activating antioxidant enzymes and non-enzymatic antioxidants, via transcription factors, such as Nrf2 and NF-κB (4) Inhibition of NF-κB pathways . It was found that silymarin protects the liver from various toxins such as arsenic, CCL-4, mycotoxins, thioacetamide, cisplatin, manganese, benzopyrene, doxorubicin and ethanol owing to its antioxidant and free radical scavenging properties .
Also, our data indicated that silymarin exhibited an anti-inflammatory potential which is evident by significant reduction of inflammatory cytokine IL-6. A large number of studies have demonstrated a regulatory effect of silymarin on the expression of NF-κB in various in vitro and in vivo models. The drug performs its anti-inflammatory activity by modulating the expression of pro-inflammatory genes such as cyclooxygenase, lipoxygenase, nitric oxide synthases and several cytokines .
Silymarin has been shown to have antioxidant, anti-inflammatory and antifibrotic activities in chronic liver disease, liver cirrhosis, non-alcoholic fatty liver and steatohepatitis. Silymarin exerts membrane-stabilizing and antioxidant activites. Furthermore, it reduces inflammatory reactions and inhibits liver fibrogenesis. Silymarin was found to significantly reduce tumor cell proliferation, angiogenesis as well as insulin resistance. In addition, it possesses an anti-atherosclerotic effect and suppresses TNF α-induced protein production and mRNA expression due to adhesion molecules .
In addition to its antioxidant and anti-inflammatory potentials, silymarin exhibits an anti-fibrotic property. This effect is associated with its ability to prevent the conversion of hepatic stellate cells into myofibroblasts. Silymarin down-regulates TGF-ß1 mRNA, inhibits NF-kB, and prevents the stimulation of hepatic stellate cells. These data indicate that Silymarin is able to slow down the progression of early fibrosis .