Concurrent Inflammation Augments Antimalarial Drugs-Induced Liver Injury in Rats

2016 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2016, 6(4), 617-625 doi: 10.15171/apb.2016.076 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Introduction
Chloroquine and amodiaquine are widely administered against malaria. 1 Chloroquine is also used in the management of lupus erythematosus and rheumatoid arthritis. 2,3Chloroquine is one of the most effective disease-modifying antirheumatic drugs (DMARDs). 5modiaquine is used in the prophylaxis and treatment of malaria, especially against chloroquine-resistant isolates of Plasmodium falciparum. 4,5][8][9][10] Several cases of antimalarial drugsinduced liver injury are reported, but there is no clear mechanism for antimalarial drugs hepatotoxicity.2][13][14] Bioactivation of amodiaquine to a quinone imine metabolite, 15 and oxidative stress have also been suggested to be involved in the development of amodiaquine-induced hepatotoxicity. 16,17rug-inflammation interaction is an intriguing model for investigating the mechanisms of drug-induced liver injury. 18,19It has been reported that a modest and noninjurious inflammation interacts with a small dose of drugs and induces hepatotoxicity. 20,21Bacterial lipopolysaccharide (LPS, Endotoxin) is widely applied in this model to induce a modest inflammation. 206][27] This might interact with malaria drug therapy and finally, leads to drug-induced liver injury.The current investigation was designed to evaluate if a modest inflammation interacts with nonhepatotoxic doses of antimalarial drugs to induce liver injury in rats.

Animals
Male Sprague-Dawley rats (200-250 g weight, n=85), were obtained from laboratory animals breeding center, Shiraz University of Medical Sciences (Shiraz, Iran).Animals were housed in cages on wood bedding at an ambient temperature of 23±1 °C and had access to food and water ad libitum.Animals received humane care and use and were handled according to the animal handling protocol at Shiraz University of Medical Sciences, approved by a local ethics committee (#93-7076).

Serum biochemistry and liver histopathology
Animals were anesthetized (pentobarbital, 50 mg/kg, i.p) and blood samples were collected from the abdominal vena cava.The liver was removed and washed in cooledsodium chloride 0.9% (w/v).Serum samples were prepared by centrifugation (3000 g, 15 min, 4°C) and stored at -70°C for further analysis of ALT, LDH, AST, bilirubin, and TNF-α.For tissue histopathological evaluation, samples of liver were fixed in neutralbuffered formalin solution (0.4% sodium phosphate monobasic, NaH 2 PO 4 , 0.64% sodium phosphate dibasic, Na 2 HPO 4 , and 10% formaldehyde in distilled water). 31araffin-embedded sections of liver were prepared and stained with hematoxylin and eosin (H&E) before light microscope viewing.

Lipid peroxidation
Thiobarbituric acid reactive substances (TBARS) were measured as an index of lipid peroxidation in liver tissue. 34Briefly, the reaction mixture was consisted of 0.5 mL of 10% liver homogenate, 3 mL of metaphosphoric acid (1%, w/v) and 1 mL of thiobarbituric acid (1% , w/v).The mixture was heated (100°C, 45 minutes).Then, 4 mL of n-butanol was added to the reaction mixture and vigorously mixed.After centrifugation in 765g for 5 minutes, the absorbance of developed color in n-butanol phase was measured at 532 nm using an Ultrospec 2000 ® UV spectrophotometer. 35

Protein carbonylation in liver tissue
The oxidative damage of proteins was assessed by dinitrophenylhydrazine (DNPH) method. 36Briefly, a 10% liver homogenate (w/v) was prepared in phosphate buffer solution (pH 7.5) containing 0.1% Triton X-100.The homogenate was centrifuged (700 g, 10 min, 4°C).Then, 500 µl aliquots of the resulting supernatant were added to 300 µl of 2, 4-dinitrophenylhydrazine (DNPH) (10 mM, dissolved in 2 M HCl).The samples were incubated in dark with vortexing every 10 minutes (1 hour, 25°C).Then, 100 µL of trichloroacetic acid (20% w/v) was added.Tubes were centrifuged at 11,000 rpm for 3 minutes, and the supernatant discarded.The pellet was washed 3 times with 1 ml ethanol: ethyl acetate (1:1 v: v).Samples were left 10 minutes before centrifugation and the supernatant was discarded each time.The precipitate was redissolved in 600 µl of guanidine solution (8 M in 20 mM potassium phosphate, pH=2.3) and incubated for 15 minutes at 37°C.Finally, the solution was centrifuged (11,000 rpm for 3 min) and its absorbance was determined at 370 nm using an Ultrospec 2000 ® UV spectrophotometer (Uppsala, Sweden). 37,38eloperoxidase enzyme (MPO) activity in liver tissue MPO activity was assessed as an index of tissue inflammatory cells accumulation.To measure liver tissue MPO activity, 500 mg of tissue was homogenized in a solution containing 0.5% hexadecyl trimethyl ammonium bromide (HTAB) dissolved in potassium phosphate buffer (50 mM, pH=6) and then centrifuged (3000 g, 20 min, 4°C).Afterward, 100 µl of the supernatant was added to 2.9 mL of potassium phosphate buffer solution (50 mM, pH=6) containing 0.167 mg/ml of O-dianisidine hydrochloride and 0.0005% H 2 O 2 .After 5 min of incubation, the reaction was stopped with 0.1 ml of 1.2 M hydrochloric acid.The rate of change in absorbance was measured by a spectrophotometer (Ultrospec 2000 ® UV) at 400 nm.Myeloperoxidase activity was expressed in units per 100 mg weight of wet liver tissue. 39

Statistical analysis
The results are expressed as a Mean±SEM.A one-way analysis of variance (ANOVA) with Tukey's multiple comparisons as a post hoc test was used to assess the significance of differences between mean values.A p< 0.05 was considered to be statistically significant.

Results
The current investigation was designed to examine the effects of bacterial endotoxin (LPS) on amodiaquine (AQ) and/or chloroquine (CQ)-induced liver injury.It was found that AQ (25, 50, and 100 mg/kg, oral) caused no significant changes in serum level of liver injury biomarkers when it was administered alone (Figure 1).CQ administration (25, 50, and 100 mg/kg, oral) also caused no significant elevation in ALT, LDH, AST, and serum total bilirubin as compared to control animals (Figure 2).Rats were pre-treated with a non-injurious dose of bacterial endotoxin (100 µg/kg of LPS, i.p).Then, animals received AQ (25, 50, and 100 mg/kg, oral) and/or CQ (25, 50, and 100 mg/kg, oral) (Figure 1).Interestingly, it was found that AQ and CQ caused a significant and severe elevation in serum ALT, LDH, and AST level when these drugs were administered to LPS-treated animals (Figure 1 and 2).No significant changes in serum bilirubin levels were identified between different groups (Figure 3).A significant elevation in serum TNF-α was detected in LPS and drugtreated animals (Figure 4).It was found that the sole administration of AQ (25, 50, and 100 mg/kg, oral) and/or CQ (25, 50, and 100 mg/kg, oral) caused no significant changes in liver glutathione content, lipid peroxidation, and protein carbonylation (Table 1).When animals were pre-treated with LPS (100 µg/kg, i.p), significant increase in lipid peroxidation and protein carbonylation, along with liver tissue glutathione depletion were detected after AQ (25, 50, and 100 mg/kg, oral) and/or CQ (25, 50, and 100 mg/kg, oral) administration (Table 1).Histopathological evaluation of liver tissue in AQ (25, 50 and 100 mg/kg) and CQ (25, 50 and 100 mg/kg)-treated animals revealed no significant changes in comparison with control animals (Figure 5).On the other hand, significant inflammatory cell infiltration (hepatitis and endotheliitis), and sinusoidal congestion were detected when LPS-treated animals received AQ (100 mg/kg) and/or CQ (100 mg/kg) (Figure 5).Assessment of MPO activity in liver tissue revealed a high amount of the enzyme in the liver of LPS-treated rats (Table 1).No significant differences were detected between different LPS and/or drug-treated groups in liver tissue MPO activity (Table 1).The effect of macrophage (Kupffer cells) inhibition was also investigated in antimalarial drugs and LPS-treated animals.We found that methyl palmitate (2 g/kg, i.v, 44 hours before LPS administration) prevented AQ and CQinduced elevation in serum level of liver injury biomarkers (Figure 1 and 2), but it had no significant effect on the serum level of TNF-α (Figure 4).Furthermore, lipid peroxidation, protein carbonylation, and liver glutathione depletion were mitigated in methyl palmitate-treated animals (Table 1).It might be noteworthy that the hepatotoxic effect of antimalarial drugs was not dose-dependent in any of the assessed parameters in drug-inflammation interaction model.The comparison between two investigated antimalarial drugs was made based on serum biochemical parameters.No significant differences were found between the severity of liver injury induced by chloroquine and amodiaquine in drug-inflammation interaction model as judged by serum ALT (P > 0.05), LDH (P > 0.05), and AST (P > 0.05).Furthermore, there was also no significant difference in serum TNF-α level when amodiaquine or chloroquine was compared in the LPS-treated animals (P > 0.05) (Figure 4).

Discussion
The current investigation was designed to evaluate the effect of concurrent inflammation on the antimalarial drugs-induced liver injury.Several investigations indicated that the threshold for hepatotoxicity from many xenobiotics was lowered by co-exposure to LPS. 22,23,[40][41][42][43] The ability of LPS to stimulate an inflammatory response may account for its pathogenicity in the liver.LPS is a potent activator of liver tissue macrophages (Kupffer cells) through toll-like receptors (TLRs) (Figure 6). 446][47] Although many parameters of the inflammatory response contribute to liver injury, 48 one well-studied pathway is the production of TNF-α.In many models of hepatotoxicity, the elevated TNF-α level is correlated with liver injury. 49We found that serum TNF-α level was significantly elevated in LPS-treated animals (Figure 4).Other cytokines rather than TNF-α (e.g IL-1 β and INF-γ) produced by other inflammatory cells (e.g.natural killers, T-helpers), might also be involved in the liver injury in drug-inflammation interaction model. 23,49,50n the current study, we found that Kupffer cells inhibition by methyl palmitate alleviated antimalarial drugs-induced liver injury in LPS-treated animals.These findings indicate that Kupffer cells might be involved in antimalarial drugs hepatotoxicity.However, methyl palmitate administration caused no significant changes in serum TNF-α level (Figure 4).This might indicate the role of other inflammatory cells such as neutrophils in the situation.It was found that serum TNF-α level was higher with drugs than LPS (Figure 4).Although these changes were not statistically significant (Figure 4), but it might mention the role of increased inflammatory cell aggregation in liver tissue when LPS-treated animals received antimalarial drugs.Hence, we might be able to propose that drug-induced tissue injury might recruit more inflammatory cells to the liver, which leads to more inflammation and cytokine production.We found that methyl palmitate significantly decreased the level of ALT, LDH, and AST levels.This might indicate the role of inflammatory cells such as macrophages, which are inhibited by methyl palmitate, in antimalarial drugs-induced liver injury.On the other hand, the pathogenic role of other inflammatory cells such as neutrophils in the drug-inflammation interaction model is also mentioned in previous investigations. 23,50ence, neutrophils and other immune cells might also be responsible for the liver injury induced by AQ and CQ.Inhibition of these cells (e.g.macrophage inhibition by methyl palmitate) might prevent drug bioactivation, at least in part, and consequently prevent liver injury (Figure 6).Applying broader acting inflammatory cell inhibitors and antibodies might shed light on the role of the immune system in antimalarial drugs hepatotoxicity.Furthermore, using inbreed animals are better choices to judge the role of immune system-mediated mechanisms of liver injury.On the other side, we might be able to suggest that immunosuppressant drugs might have a role in the treatment of antimalarial drugs-induced hepatotoxicity.Serum bilirubin is usually used as a biomarker of biliary tree injury. 35,51As bilirubin level didn't change significantly in the current investigation (Figure 3), we might be able to declare that the nature of antimalarial drugs-induced hepatotoxicity in the drug-inflammation interaction model is predominantly of the hepatocellular type rather than cholestatic. 35,51he role of peroxidase enzymes in adverse drug reactions has been reviewed previously. 52MPO is a peroxidase enzyme present in neutrophils, monocytes, and tissue macrophages (Figure 6). 52The MPO activity assessment in the current investigation indicated accumulation of inflammatory cells in the liver tissue (Table 1, Figure 5).4][55] Interestingly, it has been found that amodiaquine-induced cytotoxicity was hastened in an in vitro model of isolated rat hepatocytes in the presence of peroxidase enzyme. 17Hence, reactive metabolites produced in the inflamed liver might implicate in antimalarial drugs-induced liver injury.The role of inflammatory cells and myeloperoxidase enzyme in amodiaquine and chloroquine metabolism in liver and its relevance to hepatotoxicity in inflammation interaction model need further experiments to be precisely cleared (Figure 6).6][27] Tissue inflammation is an inseparable part of malaria disease. 56On the other hand, inflammatory stress might sensitize the liver to xenobiotics-induced injury. 57Hence, we might be able to suggest that inflammatory stress occurring during antimalarial drug therapy might participate in antimalarial drugs-induced liver injury in patients and enhance the sensitivity to antimalarial drugs.Investigating the effect of inflammation on antimalarial drugs hepatotoxicity in a broader time frame will enhance our understanding of the mechanism of toxicity and the pathological pattern of liver injury induced by Induced Liver InjuryAdvanced Pharmaceutical Bulletin, 2016, 6(4), 617-625

Figure 3 .
Figure 3. Serum level of total bilirubin.LPS: lipopolysaccharide; CQ: Chloroquine; AQ: Amodiaquine; Meth.Palm: Methyl palmitate.Data are given as Mean±SEM for five animals in each group.No significant changes were detected between serum total bilirubin levels of different experimental groups.

Figure 5 .
Figure 5. Photomicrographs of liver histopathological changes in LPS and antimalarial drugs-treated animals.A: Control rat liver; showed no significant histopathological lesions.B: LPS-treated rats; inflammatory cells infiltration (yellow arrow) was detected in this group.C: LPS+ Amodiaquine (100 mg/kg); massive inflammatory cells aggregation and hepatitis and sinusoidal congestion (red arrow) were found in this group (Yellow arrows).D: LPS+ Chloroquine (100 mg/kg); inflammatory cells infiltration and hepatitis (Yellow arrows), in addition to sinusoidal congestion (Red arrow) are also evident in this group.

Table 1 .
Hepatic lipid peroxidation and glutathione content in different experimental groups.