Hydroxychloroquine ameliorates dasatinib-induced liver injury via decrease in hepatic lymphocytes infiltration

Abstract

Dasatinib is an effective treatment for chronic myeloid leukemia. However, cases of idiosyncratic hepatotoxicity were reported. This study was conducted to investigate the chemopreventive effects of hydroxychloroquine against dasatinib-induced hepatotoxicity. Balb/c mice were randomly assigned into four groups; vehicle control (5% DMSO, i.p., n = 6), dasatinib (50 mg/kg; i.p., n = 6), hydroxychloroquine (10 mg/kg, i.p., n = 6), and hydroxychloroquine + dasatinib (10 mg/kg + 50 mg/kg; i.p., n = 6). Treatments were given once every 2 days for 14 days. Serum and histopathological assessments of liver architecture and fibrosis were performed using H&E, Masson’s trichrome, and reticulin staining. The infiltration of lymphocytes was assessed using immunohistochemistry. The gene expression of antioxidant enzymes (CAT, SOD-2, GPX-1) was assessed using real-time quantitative PCR. Dasatinib showed a significant increase in liver injury biomarkers (AST and ALT) with higher lymphocytes infiltration (as indicated by CD3⁺, CD4⁺, CD8⁺, and CD20⁺ immunohistochemistry). Hepatic tissue of Dasatinib group exhibited significant downregulation in the gene expression of antioxidant enzymes (CAT, SOD-2, and GPX-1) compared to the control group. However, the combination of hydroxychloroquine with dasatinib showed a slight increase in AST and ALT. Also, hydroxychloroquine + dasatinib treated mice showed a significant reduction in lymphocytes infiltration as compared to dasatinib. The results showed that dasatinib induces an immune response leading to an increase in lymphocytes infiltration which promotes hepatocyte destruction and persistent liver injury. The results also suggest that hydroxychloroquine ameliorates dasatinib-induced hepatotoxicity via reduction in hepatic infiltration of T and B immune cells.

Introduction

Dasatinib (DASA) is a chemotherapeutic agent that is prescribed for the treatment of chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphocytic leukemia by targeting and inhibiting breakpoint cluster region gene (BCR-ABL), platelet-derived growth factor receptors (PDGFR-α, PDGFR-β), and Scr family kinases. ¹ It belongs to the second generation of small tyrosine kinase inhibitors that are orally administered when leukemia patients are refractory to imatinib, the first-generation tyrosine kinase inhibitor. DASA is very effective in managing leukemia at either 100 mg or 140 mg once daily dose. ² However, administration of DASA has been reported to cause serious adverse reactions that affect multiple organs. Among these side effects are hepatotoxicity,^3,4 cardiotoxicity, ⁵ and nephrotoxicity. ⁶ In fact, DASA is primarily metabolized in the liver by the CYP 3A4 pathway as a major route of drug clearance. It is predicted that liver damage may be related to the production of toxic intermediates as a result of DASA metabolism. Also, DASA is subjected to drug-drug interactions as a result of being metabolized by CYP3A4. ⁷

Hepatotoxicity is a major concern associated with DASA in the clinic. Previous studies have reported that DASA induces elevation of serum aminotransferases.^3,8,9 In a phase II study, continuous administration of DASA is associated with an elevated level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in chronic myeloid leukemia patients. A similar result was also observed in another phase II study performed on 116 patients with chronic myeloid leukemia in myeloid blast crisis or lymphoid blast crisis. After 6 months of follow-up, 8% of patients treated with DASA experienced a grade 3 to 4 elevation of ALT, AST, and bilirubin activity. ¹⁰ Dose adjustments or temporary discontinuation of DASA is recommended if the serum levels of AST and ALT are 5 times the upper limit of normal. Therefore, hepatotoxicity associated with the use of this medication is now receiving considerable attention, and strategies that counteract DASA-induced liver injury may improve its clinical efficacy by ensuring the safe chronic administration. ¹¹

One of the most common causes of acute liver failure in clinic settings is the Drug Induced-Liver Injury (DILI). In the early phases of drug discovery and development, several leading and promising compounds were terminated, and their development was halted due to DILI. ¹² For example, Troglitazone was a promising antidiabetic medication that within a few years of clinical approval was withdrawn from the market due to its idiosyncratic hepatotoxicity. ¹³ The exact mechanism of DILI is still unknown. However, several observations suggest that administering a dose exceeding 50 mg on a daily basis of several medications such as valproic acid, ketoconazole, and isoniazid is associated with DILI. Other studies suggest that liver toxicity might be attributed to reactive metabolites that form during hepatic metabolism. ¹⁴ In addition, recent clinical studies have indicated the possibility of certain agents to induce immune-mediated mechanism of DILI. The immune-mediated liver injury is characterized by the presence of antibodies that are directed against hepatic proteins. The infiltration of several T-cells such as CD8⁺ to the hepatic tissues of DILI patients suggested the potential role of cell-mediated immunity as an underlying mechanism of hepatotoxicity. For instance, acetaminophen-induced liver toxicity is associated with the activation of Kupffer cells leading to increase in nitric oxide production. ¹⁵

Hydroxychloroquine (HCQ) has been traditionally used to treat malaria infection. It shows a safe and effective profile not only towards malaria infection but also against autoimmune diseases such as lupus erythematosus and rheumatoid arthritis.^16,17 Also, HCQ is shown to exert activity against different diseases such as cancer, type 2 diabetes, and autoimmune disease. ¹⁸ Recently, HCQ was primitively administered to manage patients with COVID-19 infection despite several studies showing no effects of HCQ against COVID-19 as compared to placebo. ¹⁹ In addition, HCQ has been shown to exert protective effects against heart and kidney hypertrophy in mouse models of systematic lupus erythematosus. ²⁰ It was suggested that these protective effects might be attributed to the ability of HCQ to reduce reactive oxygen species production ²⁰ as well as the infiltration of T-cells into kidney tissues. ²¹

Since HCQ is rarely associated with hepatotoxicity and it previously was shown to protect against renal damage, this study was designed to investigate the potential protective effects of HCQ against DASA-induced hepatotoxicity in a mouse model. The mechanisms by which DASA-induced hepatotoxicity was also explored including the recruitment of immune cells and the induction of oxidative stress.

Material and methods

Material and preparation

DASA was obtained from MCE^®(MedChemExpress LLC, New Jersey, USA). HCQ was obtained from Medical and Cosmetic Products Company Limited (Riyadh Pharma, Riyadh, Saudi Arabia). DASA was freshly prepared by dissolving it in 5% sterile DMSO whereas HCQ was 100% dissolved in normal saline.

Animal and experimental design

Twenty-four male BALB/C mice (approximately 12–14 weeks; 25–30 g) were obtained from the Animal Center, King Saud University, Riyadh, Saudi Arabia. All animals were kept in a controlled temperature and humidity in 12 h/12 h light/dark cycle rooms. The use of animals in this study was approved and performed in accordance with principles and guidelines set by King Saud University Animal Care and Use Committee (Approval# KSU-SE-20-47). Mice were randomly divided into four groups as followings: untreated control group (n = 6); DASA-injected group (50 mg/kg i.p., n = 6); HCQ-injected group (10 mg/kg, i.p., n = 6); and HCQ + DASA-injected group (HCQ 10 mg/kg, i.p., 2 h later followed by DASA 50 mg/kg, i.p., n = 6). The dose of HCQ was selected based on a previous study in which HCQ showed a protective effect in mouse kidney ²⁰ whereas DASA dose was selected based on our pilot study in which higher doses of DASA (>50 mg/kg) showed low tolerability, low survival rate, and high mortality incidences. All drugs were freshly prepared prior to the injection and each drug was given every other day for 14 days. At the end of the experiment, mice were euthanized by the administration of ketamine/xylazine cocktail (100 mg/kg + 10 mg/kg, i.p.) and euthanized.

Determination of liver biomarker enzymes

Blood samples were collected for the measurement of transaminases levels by cardiac puncture. The whole blood was transferred to serum-separating tubes to extract the serum for liver enzyme analysis. Then, blood was centrifuged at 2000× g at 4°C for 10 min to obtain serum. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using Reflotron® Plus Analyzer according to the manufacturer protocol (Catalog# 10745120-10745138 Roche Diagnostics, New York, USA).

Histopathological and immunohistochemical analysis of liver tissue

Immediately after euthanization, liver samples from each experimental group were immediately excised, washed with PBS, blotted, and weighed. Subsequently, liver tissues were fixed with 10% buffered formalin and sent to the Histopathology Department at the College of Medicine, King Saud University for processing and analysis. Briefly, samples were embedded in paraffin wax and subsequently were cut into 4 μm sections by a microtome. Then, sections were stained with hematoxylin and eosin (H&E), reticulin, and Masson’s Trichrome. Mounted slides were examined under a light microscope (Olympus BX63) and photographed using a DP80 digital camera. Images were analyzed using Cellsens Entry Imaging analysis Software. Immunohistochemistry was performed using an automated immunohistochemistry staining system (BenchMark XT slide Stainer, Ventana Medical System, Tucson, AZ, USA) following the manufacturer protocol. The primary antibodies were anti-CD3, anti-CD4, anti-CD8, anti-CD20 rabbit monoclonal antibodies (Ventana, Roche Diagnostics, New York, USA).

RNA isolation and cDNA synthesis

Samples of hepatic tissue were dissected, snap-frozen in liquid nitrogen, and stored at −80°C for subsequent gene expression analysis. Approximately, 50 mg of liver tissue was added to 1 mL of TRIzol reagent (Catalog# 15596018, Invitrogen, California, USA) and homogenized using a homogenizer according to the manufacture protocol. Briefly, samples were centrifuged for 5 min at 5,000×g at 4°C to remove cell debris. Then, the supernatant was transferred to a new tube and incubated with chloroform for 3 min. The samples were centrifuged for 15 min at 12,000×g at 4°C and the aqueous upper layer containing RNA was transferred into a new tube then mixed with isopropanol, and centrifuged for 15 min at 12,000×g at 4°C. A gel like pellet was formed and then mixed with 70% ethanol followed by centrifugation for 15 min at x g at 4°C. Then, the total RNA purity and integrity was estimated using Nanodrop™ 8000 spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA). An equal amount of RNA (500 ng) was used to obtain cDNA using MCE^® RT Master Mix for qPCR kit (Catalog# HY-K0510 A, MedChemExpress LLC, New Jersey, USA).

Determination of gene expression of antioxidant enzymes

Quantitative real-time PCR was performed to detect gene expression of catalase (CAT), superoxide dismutase-2 (SOD-2), and glutathione peroxidase (GPX-1) using Applied Biosystem™ 7500 Fast Real-time PCR system (Life Technologies, Waltham, MA, USA). Briefly, the total volume of each PCR reaction was 20 μL that included 10 μL SYBR Green qPCR Master Mix (Catalog # HY-K0501, MedChemExpress LLC, New Jersey, USA), 2 μL cDNA, 0.4 μL forward, and 0.4 μL reverse primer (Table1). The PCR running condition consisted of the initial denaturation stage of 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. GAPDH was used as an internal reference gene to normalize gene expression, and the fold change was calculated using 2^−ΔΔct method.

Table 1.

The sequence of primers used in PCR.

Gene	Symbol	Forward sequence 5′-3′	Reverse sequence 5′-3′
Superoxide dismutase 2	SOD2	GTA GAG CCT TGC CTG TCT TAT G	AAA CCC AGA GGC ACC ATT AC
Glutathione peroxidase	GPX1	CGA CAT TGC CTG GAA CTT TG	GGA CAG CAG GGT TTC TAT GT
Catalase	CAT	GAT GGT AAC TGG GAT CTT GTG G	GTG GGT TTC TCT TCT GGC TAT G
Glyceraldehyde 3- phosphate dehydrogenase	GAPDH	ACC ACA GTC CAT GCC ATC AC	TCC ACC ACC CTG TTG CTG TA

Statistical analysis

All data were presented as mean ± standard deviations (SD). All data and graphs were generated by GraphPad Prism version 9.2 (GraphPad Software, San Diego, CA, USA). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison post hoc test. p ≤ 0.05 was accepted as statistically significant.

Results

Effects of HCQ ± DASA on mice body weight, survival rate, and liver weight

Mice were weighed on the first day of the experiment and continued until the end of the treatment. There was no difference in body weight on day 1 of the treatment (p > 0.05). After 14 days of DASA treatment, however, mice showed a significant reduction in body weight in comparison to control (Figure 1(a)). HCQ alone or in the combination with DASA did not significantly change body weight compared to control. The survival rate was also monitored during the course of treatment (Figure 1(b)). In the course of the experiment, the survival rate in DASA groups was 50%, where 3 mice died on day 6, 11, 13. A single death was recorded in HCQ + DASA group on day 13. On the other hand, control and HCQ survived until the end of the experiment. Postmortem analysis of liver showed an increase in liver mass as well as the liver/body weight ratio of mice treated with DASA as compared to control (Figure 1(c) and (d)). Liver mass was reduced in mice treated with HCQ + DASA compared to DASA (Figure 1(c))

Figure 1.

Changes in the body weight (a), survival rate (b), liver weight (c), and (d) liver mass/body weight ratio of experimental mice in various treatment groups. (a) Change in body weight was significant in mice treated with 50 mg/kg DASA compared to control (p < 0.05). (b) Kaplan-Meier estimates of the survival curve. In control group, no mice were lost due to death. However, 3 mice died in DASA-injected group on day 6, 11, and 13. One death was observed in mice pretreated with HCQ + DASA. Statistical significance was assessed using log-rank test. (c) Liver was excised and weighed at the end of the treatment. DASA group showed an increase in liver mass and liver mass/body weight ratio as compared to control. *p < 0.05, **p < 0.01, ***p < 0.001.

Effects of HCQ ± DASA on hepatic biomarkers of injury

The assessment of liver damage was measured by the elevation of ALT and AST levels. DASA administration showed a significant increase in the serum activity of liver transaminases (ALT and AST) as compared to the control group (Figure 2(a) and (b)). However, the combination of HCQ + DASA showed a significantly reduced levels of liver transaminases as compared to DASA-treated group (Figure 2(a) and (b)) suggesting attenuation of hepatotoxicity by HCQ. The treatment with HCQ per se did not affect ALT and AST levels in serum as compared to control group.

Figure 2.

Changes in the levels of (a) alanine aminotransferase (ALT), and (b) aspartate aminotransferase (AST) serum levels after 14 days of treatment. ALT and AST were significantly increased in DASA-treated mice. HCQ reversed DASA-induced increase in ALT and AST levels. *p < 0.05, **p < 0.01, ***p < 0.001.

Effects of HCQ ± DASA on hepatic structure & pathology

To assess the effect of different treatments on the architecture of liver tissue, the liver section was stained with H&E, Masson’ trichrome, and reticulin (Figure 3(a)–(c)). H&E staining of the hepatic section of the control group showed the normal appearance of hepatocytes with well-preserved liver architecture. On the contrary, DASA-treated mice revealed severe hepatitis with lymphocytic infiltration (Figure 3(a)). The combination of HCQ + DASA showed only mild chronic inflammation with minimal lymphocytic inflammation (Figure 3(a)). Furthermore, Masson’s trichrome staining, commonly used to stain type I collagen found in hepatic tissue, showed that DASA induced severe distortion of liver structure with an accumulation of collagen fibers, which is an indicative sign of liver fibrosis. The combination of HCQ with DASA revealed mild chronic hepatitis with a decrease in the deposition of collagen fibers which is a signal of reduced liver fibrosis (Figure 3(b)). Similar results were obtained with reticulin, a thin fiber composed of type III collagen, staining where control group showed normal reticulin framework. In DASA group, there was an increase in reticulin expression with the presence of perforated fibrous septa and porto-portal bridging fibrosis. The combination of HCQ + DASA showed chronic mild hepatitis with periportal fibrosis (Figure 3(c)). Overall, these data suggest that DASA causes liver inflammation and fibrosis, which are reversed by treatment with HCQ.

Figure 3.

Photomicrographs of liver histological structure using (a) H&E, (b) Masson’s trichrome, and (c) reticulin. Hepatic section of control group showed a normal liver structure with little collagen fiber deposition. The DASA group exhibited severe hepatitis with distorted liver architecture and abundant of collagen fiber deposition. The combination of HCQ + DASA showed mild hepatitis with a decrease in collagen fiber deposition. Dot indicates portal venule, arrowhead indicates arteriole, arrow indicates branch of bile duct, square indicates hepatocytes, and diamond indicates central vein (magnification ×200).

Effects of HCQ ± DASA on hepatic lymphocytes subpopulations

Next, immunostaining was performed to detect lymphocytic infiltrate in hepatic tissue in different treatment groups (Figure 4(a)–(d)). Immunohistochemistry of the hepatic section of DASA-injected group showed a significant increase in the recruitment of CD3⁺, CD4⁺, CD8⁺, and CD20⁺ lymphocytes than that in both the control and HCQ groups (Figure 4(a)–(d)). The population of these lymphocytes was significantly reduced in HCQ + DASA group as compared to DASA group (Figure 4(a)–(d)). These data suggest that infiltration of CD4⁺/CD8⁺ T cells and CD20⁺ B cells could be responsible for DASA-medicated hepatotoxicity, which can be attenuated upon HCQ administration.

Figure 4.

Immunohistochemistry of CD3, CD4, CD8, and CD20 expression in liver tissue of different treatment groups. Normal groups exhibited low expression of CD3⁺, CD4⁺, and CD8⁺ T-cells and CD20⁺ B-cells. DASA treated group showed increased population of CD3⁺, CD4⁺, and CD8⁺ and CD20⁺ lymphocytes (dotted oval). HCQ + DASA revealed minimal recruitment of lymphocytes. Dot indicates portal venule, arrowhead indicates arteriole, arrow indicates branch of bile duct, square indicates hepatocytes and dotted oval indicated positive expression (magnification ×200).

Effects of HCQ ± DASA on hepatic antioxidant enzyme gene expression

To investigate whether DASA increases oxidative stress in hepatic tissues, we analyzed the gene expression of antioxidant markers including superoxide dismutase-2 (SOD-2), catalase (CAT), and glutathione peroxidase (GPX-1). DASA treated group showed a significant downregulation of CAT, SOD-2, and GPX-1 levels in the hepatic tissue as compared to control group (Figure 5(a)–(c)). HCQ group showed a slight increase in SOD-2 expression and a significant increase in both GPX-1 and CAT. Unexpectedly, adding HCQ to DASA showed no significant upregulation in the expression of these antioxidant markers as compared to DASA monotherapy (Figure 5(a)–(c)).

Figure 5.

Hepatic gene expression of (a) CAT, (b) GPX-1, and (c) SOD-2 in different treatment groups. DASA exhibited a significant reduction in CAT, GPX, SOD2 as compared to that in the control and HCQ groups. The combination of HCQ + DASA showed a slight non-significant increase in these genes (p > 0.05). *p < 0.05, **p < 0.01, ***p < 0.001.

Discussion

In the present study, we investigated the role of DASA in inducing hepatotoxicity and the potential protection of HCQ. The evidence that DASA induces liver tissue injury and damage was demonstrated by histopathological, biochemical, and immunohistochemical analysis. Mice receiving DASA treatment showed signs of hepatocellular damage as substantiated by the significant increase in liver mass weight and total serum transaminase levels as compared to the control. Our results showed that the continuous treatment of DASA was associated with an increase in serum levels of liver intracellular enzymes. The increased levels of AST and ALT were indicative of liver injury and damage. ²² Despite the fact that DASA belongs to targeted tyrosine kinase inhibitors which hypothetically have lower toxicity compared to the traditional chemotherapy, the results showed that DASA at 50 mg/kg caused induction of liver damage and destruction. Meanwhile, HCQ was shown to exert a prophylactic effect in attenuating the hepatocellular injury induced by DASA. Mice that were treated with HCQ and followed by DASA showed a normal liver weight as well as reduced transaminase levels similar to those of the control group.

DASA is shown to induce hepatotoxicity both in vitro and in vivo. It has been demonstrated that DASA reduced the cell viability of rat primary hepatocytes, induced the release of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), and triggered the ballooning degeneration of hepatocytes in Sprague-Dawley rats. ²³ Recent studies suggest that DASA exerts mitochondrial toxicity in rat hepatocytes by inducing lipid peroxidation and reducing the mitochondrial membrane potential. ²³ Another theory for DASA-induced hepatotoxicity is the formation of reactive metabolites that interferes with critical cell functions since DASA belongs to tyrosine kinase inhibitors which undergo intense hepatic metabolism by CYP3A4. ²⁴ It has been reported that oxidative stress and the generation of reactive oxygen species (ROS) have been implicated in the pathophysiology of DASA-induced toxicity. The resultant oxidative stress may then trigger the nuclear factor-kappa B (NF-κB) inflammatory pathway, which increases hepatic intracellular pro-inflammatory cytokines that exacerbate the toxic effect of DASA on hepatocytes. ²⁵ The role of NF-κB in inflammatory response by modulating many pro-inflammatory proteins has been involved. So, one of the unwanted responses during therapy with tyrosine kinase inhibitors is the overexpression of NF-κB ²⁶ Therefore, ROS scavengers and antioxidant molecules might have the capacity to protect against the deleterious effects induced by DASA. ²³

In this study, the combination of HCQ with DASA showed minimal liver histological abnormalities with a slight increase in serum levels of AST and ALT. These results suggest a protective role of HCQ in countering the liver injury inflicted by DASA treatment. A possible mechanism for HCQ protective effects on the liver is that HCQ inhibits cellular infiltration and scavenges reactive oxygen species produced by inflammatory cells. ²⁷ In fact, the failure of the endogenous antioxidant defense mechanisms encourages the formation of excessive free radicals leading to tissue damage. Additionally, the destructive roles of lipid peroxidation and oxidative stress could be responsible for tissue inflammation and damage. Many studies have reported that HCQ can reduce tissue damage and inflammation resulting from all these deleterious functions due to its antioxidant property. This feature contributes to minimizing tissue damage by decreasing levels of myeloperoxidase (MOA) and malondialdehyde (MDA), and elevating of the level of antioxidant enzymes such as glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT). ²⁸ HCQ can be considered as an immunosuppressant agent utilized in many autoimmune disorders like systemic lupus erythematosus and rheumatoid arthritis. This immunosuppressant activity represents its capacity to interfere with the processing and manifestation of antigens in addition to reducing immune responses by inhibiting the formation of peptide-MHC protein complexes. Also, HCQ suppresses the upregulation of pro-inflammatory cytokines like (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). All these events take place after the administration of HCQ to diminish the improvement of autoimmune disease activity. ²⁹

The impact of HCQ on the liver as a hepatoprotective agent seems to be attributed to its anti-inflammatory properties as well as its ability to reduce the percentage of infiltrating immune cells. It has been shown that HCQ triggers apoptosis in all T-cell subpopulations of peripheral blood lymphocytes. ³⁰ HCQ is shown to protect the liver from ischemia/reperfusion injury by inhibition of inflammatory response via inhibiting the toll-like receptor (TLR-9) and NF-κB-mediated inflammatory pathways ³¹ On the other hand, inhibition of autophagy by HCQ promotes apoptosis via the activation of pro-apoptotic pathways. So, chloroquine is well known as an autophagy and inflammation inhibitor with dual roles to perform hepatoprotective effect.^32,33

There are two types of drug-induced liver injury. Type A is intrinsic toxicity that occurs as a direct result of administering a high dose of the drug whereas type B is an idiosyncratic reaction that occurs unpredictably to the therapeutic medication.^12,34 In this regard, our results suggested that DASA induced immune-mediated hepatitis. This conclusion was supported by the observation that immune-mediated liver injury has a shorter latency (1–6 weeks) compared to non-immune mediated liver injury. ³⁵ In addition, lymphocytes infiltration was noticeably higher in the liver of mice that were treated with DASA. Our results suggested that DASA either directly or through its metabolites initiated an immune response, which exacerbates liver injury. It has previously been shown that the recruitment of lymphocytes increases in response to inflammation. ³⁶ Innate immune cells such as Kupffer cells, dendritic cells, and natural killer cells are activated by the damaged liver cells which subsequently promote the production of IL-13, IL-10, TGF-β, and TNF-γ. ³⁷ In addition, the adaptive immune cells can also be activated by certain drugs. The mechanism of such activation is poorly understood. It is believed that certain drugs will induce liver injury causing the release of hepatocyte content such as DNA, RNA, and heat shock proteins which act as substrates for Toll-like receptors triggering cytokines and chemokines formation. Such cytokines and chemokines can activate other blood lymphocytes and recruit them to liver vasculature which can be extravasated into hepatocytes. As a result of lymphocyte extravasation, a potent generation of reactive oxygen species triggering hepatocytes cytotoxicity leading to aggravated liver injury. ³⁸ In addition, DASA treatment thought activation of B lymphocyte might have worsened fibrosis. In this regard, Novobrantseva et al. showed that treatment with CCL-4, a known liver cytotoxic agent, is associated with greater fibrosis in the presence of B cells in wild-type mice compared to B cell-deficient mice. ³⁹

Conclusion

To our knowledge, our study is the first to demonstrate that the hepatotoxicity associated with DASA is an immune-mediated drug-induced liver injury. Adding HCQ plays a curial role in protecting liver tissue against the infiltration of inflammatory and immune cells. We concluded that there was a significant sign of hepatotoxicity in DASA-treated groups; whereas the group that was pretreated with HCQ showed a protective effect. Our results indicated that Dasatinib can induce hepatic inflammation that progresses to fibrosis and cirrhosis. Further studies are warranted to explore the exact mechanisms by which hydroxychloroquine reduces the infiltration of immune cells into liver tissue.