New molecular and biochemical insights of doxorubicin-induced hepatotoxicity

Life Sciences 250 (2020) 117599

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Review article

New molecular and biochemical insights of doxorubicin-induced hepatotoxicity Pureti Lakshmi Prasanna1, Kaviyarasi Renu$, Abilash Valsala Gopalakrishnan

T

⁎

Department of Biomedical Sciences, School of Biosciences and Technology, VIT, Vellore, Tamil Nadu 632014, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Doxorubicin Hepatotoxicity Oxidative stress Molecular mechanisms and apoptosis

Chemotherapeutic antibiotic doxorubicin belongs to the anthracycline class, slaughters not only the cancer cells but also non-cancerous cells even in the non-targeted organs thereby resulting in the toxicity. The liver is primarily involved in the process of detoxification and this mini-review we focused mainly to investigate the molecular mechanisms heading hepatotoxicity caused due to doxorubicin administration. The alterations in the doxorubicin treated liver tissue include vacuolation of hepatocytes, degeneration of hepatocyte cords, bile duct hyperplasia and focal necrosis. About the literature conducted, hepatotoxicity caused by doxorubicin has been explained by estimating the levels of liver serum biomarkers, ROS production, antioxidant enzymes, lipid peroxidation, and mitochondrial dysfunction. The liver serum biomarkers such as ALT and AST, elated levels of free radicals inducing oxidative stress characterized by a surge in Nrf-2, FOXO-1 and HO-1 genes and diminution of anti-oxidant activity characterized by a decline in SOD, GPx, and CAT genes. The augmented levels of SGOT, SGPT, LDH, creatine kinase, direct and total bilirubin levels also reveal the toxicity in the hepatic tissue due to doxorubicin treatment. The molecular insight of hepatotoxicity is mainly due to the production of ROS, ameliorated oxidative stress and inflammation, deteriorated mitochondrial production and functioning, and enhanced apoptosis. Certain substances such as extracts from medicinal plants, natural products, and chemical substances have been shown to produce an alleviating effect against the doxorubicin-induced hepatotoxicity are also discussed.

1. Introduction Cancer is one of the deadliest diseases with an exponential mortality rate among the worldwide population [1]. Doxorubicin belongs to

anthracyclines class of compounds and is widely used as a chemotherapeutic drug to treat various types of cancers such as lymphomas, leukemias, and carcinomas of the breast, ovaries, thyroid, lungs [2–4]. A red-colored pigment extracted from the Streptococcus

Abbreviations: ACOX1, Peroxisomal acyl-coenzyme A oxidase 1; AHR, aryl hydrocarbon receptor; AIF, apoptosis-inducing factor; ALP, alanine phosphatase; ALT, alanine transaminase; AMP, adenosine monophosphate; AST, aspartate transaminase; ATGL, adipose triglyceride lipase; ATP, adenosine diphosphate; Bax, BCL2Associated X Protein; Brca2, breast cancer type 2; Cas 3, caspase; CAT, catalase; cdkn1a, Cyclin Dependent Kinase Inhibitor 1A; CTGF, connective tissue growth factor; Cu, Copper; Cyp1a1, Cytochrome P450 Family 1 Subfamily A Member 1; Cyp450, cytochrome P450; Cyt C, Cytochrome C; DDR, DNA damage response; DIABLO, (direct inhibitor of apoptosis protein (IAP)-binding protein with low PI; Fas, APO-1/APT/CD95; FAT, fatty acid translocase; Fe, Iron; Fe+2, ferrous ion; Fe+3, ferric ion; Fos and Jun, AP-1 Transcription Factor Subunit; FOXO1, Forkhead box protein O1; FOXO3, forkhead box protein O3; GGT, Gamma Glutamyl transpeptidase; GPx, glutathione peroxidase; GSH, glutathione; GST, Glutathione-S-transferase; H2O2, hydrogen peroxide; HDL, high density lipoprotein; Keap-1, Kelch-like ECH-associated protein 1; LDH, lactate dehydrogenase; LDL, low density lipoproteins; MDA, malondialdehyde; MDR-1, multi-drug resistance 1; MRP-1, multidrug resistance protein 1; NAFLD, Non-alcoholic fatty liver disease; NO, nitric oxide; NOS3, Nitric Oxide Synthase 3; NQO1, NAD(P)H Quinone Dehydrogenase 1; Nrf2, Nuclear factor erythroid 2-related factor 2; O2−, superoxide ion; PGC-1α, Peroxisome proliferator-activated receptor gamma coactivator 1 alpha; PPARγ, peroxisome proliferator-activated receptor γ; PXR, pregnane X receptor; RNS, reactive nitrogen species; ROS, reactive oxygen species; SGOT, Serum Glutamic Oxaloacetic Transaminase; SGPT, Serum Glutamic Pyruvic Transaminase; SIRT1, sirtuin 1; Smac, second mitochondria-derived activator of caspase; SOD1, superoxide dismutase 1; TBARS, Thiobarbituric acid reactive substances; TFAM, transcription factor A, mitochondrial; TG, triglycerides; TNFα, tumor necrosis factor alpha; Topo2a, topoisomerase 2 alpha; Topo2b, topoisomerase 2 beta; wee-1, Mitosis inhibitor protein kinase; XDH, Xanthine Dehydrogenase; γ-H2AX, H2A histone family member X ⁎ Corresponding author at: Department of Biomedical Sciences, School of Biosciences and Technology, VIT, Vellore, Tamil Nadu 632014, India E-mail address: [email protected] (A. Valsala Gopalakrishnan). $ Equal Contribution for first authors. https://doi.org/10.1016/j.lfs.2020.117599 Received 4 February 2020; Received in revised form 17 March 2020; Accepted 24 March 2020 Available online 29 March 2020 0024-3205/ © 2020 Elsevier Inc. All rights reserved.

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which constitute a part of membrane lipids, proteins, and DNA further resulting in higher lipid peroxidation [20,21]. The levels of lipid peroxidation are estimated by measuring the amount of malondialdehyde (MDA). It was reported that the doxorubicin-induced lipid peroxidation activity in the microsomes and mitochondria of hepatocytes was enhanced in the presence of Fe+2 ions. The ROS species generated by doxorubicin include the superoxide radicals, hydroxyl radicals, hydrogen peroxide, oxygen radicals, peroxynitrite, and dinitrogen trioxide. SOD detoxifies the superoxide radical by catalyzing the conversion of the O2– radical into H2O2 which is further detoxified into water molecule by the enzyme catalase [22].

peucetius strain produced an antibiotic against tumors in mice known as daunorubicin. This streptococcus species was further genetically manipulated to produce adriamycin, also called as doxorubicin. Doxorubicin possesses a broad spectrum of therapeutic index [3]. Doxorubicin is marketed under the brand name adriamycin which is available in both powdered and liquid form and typically administered through intravenous route in doses of 60 to 75 mg/m2 body surface area every 21 to 28 days. It is also available in liposomal form [5]. Apart from its chemotherapeutic activity, the traditional usage of doxorubicin has been limited due to its adverse effects on various organs which led to cardiotoxicity, hepatotoxicity, nephrotoxicity, fertility problems and induction of type-2-diabetes like condition, diabetic cardiomyopathy [6–8]. Due to its biphasic nature doxorubicin has been shown to induce hepatotoxicity at acute and sub-acute doses [9]. The molecular mechanisms involved in doxorubicin causing hepatotoxicity are mainly due to the production of reactive oxygen species (ROS) by the drug during its metabolism in the liver which results in imbalanced redox potential leading to oxidative stress, reduced levels of antioxidant enzymes, apoptosis, inflammation, and mitochondrial dysfunction. The liver is the major organ involved in the process of metabolism and detoxification of drugs. Doxorubicin can be administered directly or in the pegylated form called as doxorubicin hydrochloride (Caelyx) where the drug is encapsulated inside the liposomes whose surface is bounded with methoxy-polyethylene glycol groups thereby resulting in the targeted delivery to tumor sites to some extent, escaping the immunosurveillance, promoting the sustained release of drug for a longer time, and reduced drug content in the hepatic tissue [10,11]. The major advantage behind the liposomal formulation is it reduces the drug-induced toxicity to other healthy or non-targeted tissues whereas the liposomal form of the drug is restricted to undergo hepatic metabolism due to the size of the fenestrations present on the hepatic sinusoidal endothelium which acts as a barrier stands as a disadvantage thereby indicating reduced clearance and volume of distribution [11].

3. Structural abnormalities in doxorubicin treated liver Various reports were produced based on structural examination of liver tissue extracted from animal models treated with doxorubicin. The histopathological examination of hepatic tissue after doxorubicin treatment revealed marked bile duct hyperplasia, dilation of sinusoidal space, and central vein congestion [23], vacuolation of hepatocytes, dilatation of sinusoids, condensation of nuclei and degeneration of hepatocyte cords [24], cellular edema, focal necrosis, and de-arrangement of hepatic trabeculae [25] proliferation of biliary duct, parenchymal necrosis [26], dilation of intercellular spaces and vacuolization and swelling of mitochondria [27], lymphocyte infiltration [28]. 4. Molecular and biochemical alterations in the liver upon doxorubicin treatment Biochemical examination of doxorubicin treatment reported a significant increase in levels of Alanine Transaminase (ALT) and Aspartate Transaminase (AST) indicating damage to hepatic tissue is reported in different studies [29–31]. Doxorubicin possesses the ability to produce superoxide radicals and peroxynitrite radicals during its metabolism in the liver. Therefore, the ROS produced initiates lipid peroxidation which results in hepatic damage and leakage of the hepatic enzymes such as ALT and AST into the serum. Thus, the level of these enzymes serves as biomarkers for hepatotoxicity [32,33]. The increased ALT, AST and alkaline phosphatase (ALP) indicate hike in cellular permeability thereby leading to cytological disturbances. An increased amount of these enzymes indicates liver damage. The antioxidant enzymes provoke a defensive mechanism against the doxorubicin-induced oxidative stress through the production of ROS. Doxorubicin reduces the level of antioxidants such as glutathione (GSH), SOD, GPx and CAT due to the vigorous generation of ROS species thereby inhibiting the activity of defensive mechanism [24,33]. Doxorubicin significantly elevated the expression of intrinsic and extrinsic apoptotic molecules such as BCL2-Associated X Protein (Bax) and Fas respectively. The increased amount of these molecules induced the expression and formation of large amounts of caspase-3 that finally elicited apoptotic signaling either by intrinsic or extrinsic signaling pathways [30]. Doxorubicin not only plays a major role in inducing free radical production it also suppresses the detoxification capability of the hepatic tissue. According to a report, there is an increased level of SOD, GPx, and CAT increased during oxidative stress. There is a significant reduction in cytochrome P450 (CYP 450) and GSH levels in the rat liver tissue whereas glutathione level is increased in hepatocytes. The increased antioxidant enzyme activity indicates an increased detoxification process to eliminate the ROS produced by the doxorubicin thereby indicating hepatotoxicity [27]. The increased serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), lactate dehydrogenase (LDH), creatine kinase, direct and total bilirubin levels indicate hepatotoxicity induced due to doxorubicin administration [24,34,35]. The tumor cells possess an efficient antioxidant defense mechanism, where it inactivates the doxorubicin by undergoing conjugation reaction to survive oxidative stress induced by it [36]. The administration of doxorubicin along with taxanes increases

2. Doxorubicin - mechanism of action The doxorubicin intercalates between nitrogenous bases of DNA and inhibits the biosynthesis of macromolecules [12,13] which in turn leads to inhibition in the activity of topoisomerase II (Top II) enzyme due to which the replication process is disrupted. Thus, cancerous cells are ceased from cell division [1]. An enzymatic method, the doxorubicin undergoes a reversible oxidation process and produces a semiquinone form as an intermediate, catalyzed by the enzyme Nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) reductases which lead to the production of reactive oxygen species such as superoxide radicals. In the non-enzymatic method, the doxorubicin donates an electron to Ferric ion (Fe+3) and results in the formation of Ferrous (Fe+2)doxorubicin complex. The complex formed undergoes reduction with oxygen (O2) and produces ROS such as hydrogen peroxide (H2O2) and superoxide (O2−) which further results in oxidative stress [14,15]. The ROS produced causes membrane damage, DNA damage, lipid peroxidation and activates apoptotic pathways in both cancer and normal cells [16]. The pathway is regulated by the up-regulation of genes involved in the oxidation process and down-regulation of genes involved in the anti-oxidation process. The up-regulated genes involved in the oxidative process of doxorubicin to semiquinone form include NAD(P)H Quinone Dehydrogenase 1 (NQO1), Nitric Oxide Synthase 3 (NOS3), and Xanthine Dehydrogenase (XDH) whereas; the antioxidant genes superoxide dismutase 1 (SOD1), glutathione peroxidase (GPx), and catalase (CAT) are down-regulated thereby leading to an imbalance in the redox potential of the cell [2]. The nitric oxide (NO) exhibits both tumor progression as well as tumor suppression activities [17,18]. The NO produced can react with O2 or O2– and produce reactive nitrogen species (RNS) such as dinitrogen trioxide and peroxynitrite. The RNS generated further results in lipid peroxidation and DNA damage [19]. The ROS produced also reacts with the polyunsaturated fatty acids 2

Life Sciences 250 (2020) 117599

[9]

[9]

oxidative stress when compared with administration of doxorubicin alone where the increased levels are characterized by measuring superoxide dismutase and catalase levels [37]. An elevated level of triglycerides (TG), low-density lipoproteins (LDL), constitute bad cholesterol which builds up in arteries leading to conditions such as Nonalcoholic fatty liver disease (NAFLD), diabetes mellitus and renal diseases [24]. On the other hand, the decreased levels of High-density lipoproteins (HDL) can also be a cause for the above-mentioned disease conditions. The free radicals generated by the administration of doxorubicin leads to increased MDA levels which indicate lipid peroxidation and also serve as a biomarker for oxidative stress thereby resulting in liver damage [25]. The ROS species produced by the doxorubicin results in a chain of reactions producing free radical-induced damage to cell membrane which is an indicator of increased MDA levels [34]. Doxorubicin elevates the level of lipolysis marker such as aryl hydrocarbon receptor (AHR), decreased pregnane X receptor (PXR), peroxisome proliferator-activated receptor γ (PPARγ), fatty acid translocase (FAT), adipose triglyceride lipase (ATGL) and peroxisomal acyl-coenzyme A oxidase 1 (ACOX1) [7]. Doxorubicin decreases GSH, gammaglutamyl transpeptidase (GGT) levels and increases MDA, glutathioneS-transferase (GST) levels in both liver tissue and hepatocytes. On contrary to this, there is an augmented level of cysteine in the liver tissue but decreased in hepatocytes. Whereas, the elevated levels of ROS in hepatocytes but not altered in the tissue [36]. Doxorubicin elevates the level of apoptotic markers such as activated caspase 3 (Cas 3) and phosphorylated γ-H2AX (H2A histone family member X) levels in the rat liver. The elevated γ-H2AX serves as a biomarker for DNA damage response (DDR) [9,36]. Doxorubicin decreases the level of SOD levels, several number of kupffer cells, and the liver weight whereas there are an augmented level of thiols and thiobarbituric acid reactive substances (TBARS), which serves as a biomarker for lipid peroxidation. Further, the changes are established with coinciding histopathological findings [25]. Bulucu et al., in 2009 reported that doxorubicin reduces the level of trace elements such as iron (Fe) and copper (Cu) and attenuates the antioxidant levels and augments MDA levels [38]. Doxorubicin drastically increases the level of ALT, AST, and GGT which indicates hepatic damage [39]. Doxorubicin reduces the body weight and liver weight of the animals [40]. The doxorubicin augments the levels of biomarkers of hepatic damage such as LDH, AST, ALT, Cas-3, and apoptosis signaling molecules and attenuates antioxidant enzymes such as SOD and reduced GSH. The doxorubicin generated ROS activated the p53 signaling which led to the increased expression of Bax and Fas thereby resulting in apoptosis eventually leading to acute hepatic damage [40]. Doxorubicin upregulates the mRNA expression of connective tissue growth factor (CTGF) which is a profibrotic cytokine and tumor necrosis factoralpha (TNFα), proinflammatory cytokine. The genes such as heme oxygenase 1 (HO-1), GPx and multi-drug resistance 1 (MDR-1) and multidrug resistance protein 1 (MRP-1) are involved in the antioxidant defense system and drug transport respectively. The above genes are upregulated in acute and sub-acute doses; whereas, the quantification of mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) at an acute dose and Kelch-like ECH-associated protein 1 (Keap-1) at sub-acute dose remains unchanged but there is an increased expression of Nrf-2 at sub-acute and Keap-1 at acute dose. Further, there is an increased mRNA expression of cell cycle regulating genes such as cyclin-dependent kinase inhibitor 1A (cdkn1a), wee-1, apoptosis-inducing genes such as Fas, proto-oncogenes such as Fos (AP-1 Transcription Factor Subunit), Jun (AP-1 Transcription Factor Subunit), DNA damage repair genes such as breast cancer type 2 (Brca2), cytochrome P450 family 1 subfamily A member 1 (Cyp1a1), topological stress-relieving genes such as topoisomerase 2 alpha (Topo2a) and topoisomerase 2 beta (Topo2b). Apart from inducing DNA damage through oxidative stress, doxorubicin also increases the production of ceramides such as C16, C18 and C24:1, dihydro ceramides such as DHC18, and DHA24, sphingosine and dihydro-sphingosine, which indicates that there is a disturbance in sphingolipid metabolism [9]. Apart from the imbalances

Balb/c mice 8.

9 mg/kg/week for 3 weeks (subacute model

Elevated levels of genes involved in drug transport, DNA damage repair, cell cycle progression and downregulation of antioxidants except for Nrf2

Oxidative stress enhanced by the downregulation of antioxidants

Upregulated genes - Mdr1, Mrp1, Cdkn1a, Wee1, Fas, Fos, Jun. Downregulated genes - H0–1, GPx-1, Upregulated genes - Mdr1, Mrp1, Wee1, Brca2, Cyp1a1,Topo2a1 and Topo2b Downregulated genes - H0–1, GPx-1 Oxidative stress enhanced by the downregulation of antioxidants Elevated levels of drug transport genes, cell cycle promoting genes and downregulation of antioxidants 10 mg/kg (acute model)

[44] – Increases oxidative stress Decreases cell viability 6.

7.

1 μM and 5 μM

HepG2 - human hepatocellular carcinoma Caco2 - Colorectal adenocarcinomas Balb/c mice

4

3

5

8 mg/kg

6 mg/kg/week for 3 weeks

15 mg/kg

Adult male Wistar rats (190–240 g) MaleSwiss albino mice weighing 21–25 g Female Sprague-Dawley rats 2

1 μM and 5 μM

[44] – Increases oxidative stress

[44]

[32]

Downregulation of Nrf2/HO-1 and upregulation of caspase-3 –

[73] –

Oxidative stress produced by the production of ROS Increases oxidative stress

[35] – ROS production by Doxorubicin through the enzymatic pathway Human 1.

50 mg/m2 and 75 mg/m2

Elevated levels of Direct and Total Bilirubin, serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) Elevated levels of liver serum biomarkers, MDA levels and decreased levels of antioxidant enzymes. Elevated levels of liver enzymes in serum (ALT, AST) and lipid peroxidation (MDA) Elevated levels of serum enzymes such an ALT, AST, LDH, α-HBDH, and α-HBDH/LDH Decreases cell viability

Oxidative stress

References Genes Molecular mechanism Characteristics Dosage of Doxorubicin administered Cell line/animal model S·No

Table 1 Hepatotoxic effects of doxorubicin at various doses on various experimental models.

P.L. Prasanna, et al.

3

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Fig. 1. Depiction of factors responsible for causing hepatotoxicity upon doxorubicin treatment.

in the levels of antioxidants and liver biomarkers upon doxorubicin treatment; physical exercise enhances the antioxidant defense mechanism and the levels of SOD, Catalase, GPx, GSH. Along with that, there is a decreased level of MDA and NO levels which indicate reduced lipid peroxidation [41]. The levels of urinary lipid metabolites such as MDA, formaldehyde, acetaldehyde, and acetone are elevated due to the increased production of free radicals after the administration of doxorubicin, which will eventually lead to lipid peroxidation. The ROS produced by the doxorubicin-induced lipid peroxidation in mitochondria and microsomes of hepatic tissue and also cause DNA single-strand breaks in the nucleus of hepatocytes [42]. The dosage of doxorubicin administered to various experimental models heading towards hepatotoxicity has been elucidated (represented in Table 1) and an overview of doxorubicin-induced hepatotoxicity has been represented in Fig. 1.

The HO-1 is mainly responsible for the catalysis of heme and the production of antioxidants such as bilirubin and biliverdin [47]. In doxorubicin treatment, the genes responsible for antioxidant enzymes such as Nrf2 and HO-1 and apoptotic enzymes such as Cas-3 are downregulated and up-regulated respectively. The increased malondialdehyde levels result in the production of ROS, which further leads to a series of apoptotic events [32,48]. Doxorubicin is lipophilic and possesses DNA binding capacity which serves as the main reason for the accumulation of the drug in the hepatic nucleus thereby resulting in DNA damage [49].

5. Molecular mechanisms involved in doxorubicin-induced hepatotoxicity

The imbalanced redox potential created across the tissue leads to a series of events resulting in cell death. The ROS produced through doxorubicin treatment promotes apoptosis through the activation of p53, Cas 3 and Cytochrome C (Cyt C) in both cancerous and noncancerous cells [50]. The ROS induced activation of p53 and forkhead box protein O3 (FOXO3) triggers the activation of death promoting signals and suppresses the activity growth-promoting factors resulting in an imbalance in the expression of anti and pro-apoptotic signaling molecules thereby resulting in cell death and toxicity [51]. Doxorubicin is more resistant to the cell line HepG2 possessing wild type p53 and cell line Hep3B possessing deleted p53. The HepG2 initiated the apoptotic signaling through the p53 level; within 24 h after doxorubicin administration, whereas the Hep3B initiated the apoptotic signaling in a p53 independent manner. Whereas, in the case of the Huh-7 cell line possessing mutated p53, p53 levels were down-regulated [52]. In the case of the intrinsic signaling pathway, the Bax initiates apoptosis through mitochondrial signaling which triggers the activation of Cyt-C whereas the extrinsic signaling pathway is activated upon binding of Fas ligand to its receptor [53]. The mitochondrial signaling pathway is activated by the release of apoptosis promoting factors such as cytochrome c, apoptosis-inducing factor (AIF), and caspase activating factors such as Smac (second mitochondria-derived activator of caspase)/ DIABLO (direct inhibitor of apoptosis protein (IAP)-binding protein with low PI), Omi/HtrA2 or endonuclease G from the mitochondrial intermembrane space into the cytoplasm resulting in the formation of a complex, cytochrome c/Apaf-1/caspase-9 (Cyt C/Apaf-1/Cas 9) also termed as the apoptosome. The apoptosome complex formed triggers the activation of Cas 3 resulting in apoptosis [54–56]. The extrinsic pathway is initiated upon binding of Fas ligand to the Fas receptor which results in the trimerization and activation of caspase 8 (Cas 8). The activated Cas 8 undergoes series of cascade reactions and activates Cas 3 which eventually results in apoptosis [56,57].

5.2. Elated apoptosis during doxorubicin treatment triggered by p53 via extrinsic and intrinsic signaling pathways

The molecular mechanisms behind the doxorubicin hepatotoxicity and the dosage of the drugs administered are mentioned in Table 1. The elevated ALT, AST and GGT levels in the serum are a prime indicator for hepatic damage. Investigating into the molecular insights that doxorubicin-induced hepatic damage is initiated upon the activation of genes responsible for oxidative stress response, DNA damage, DNA repair, drug transport, a progression of the cell cycle, mitochondrial dysfunction and apoptosis [43]. It has been elucidated that an interconnection exists between oxidative stress, inflammation and lipid peroxidation induced by the ROS produced [44]. The toxic effects induced by doxorubicin on hepatic tissue involves an obstruction in the cell cycle of hepatocytes, imbalance redox potential induced by the ROS produced during the metabolism of the drug and disturbance in the respiratory chain that takes place in mitochondria [45]. The interaction existing among various genes concerning doxorubicin-induced hepatotoxicity has been represented in Fig. 2. 5.1. Augmentation of oxidative stress upon doxorubicin treatment in hepatic tissue via Nrf2/HO-1 pathway Oxidative stress is defined as an imbalance in the redox status of the cell which is caused due to the production of free radicals [46]. The response against oxidative stress created by the doxorubicin-induced free radicals has been initiated by the activation of the antioxidant mechanism through the Nrf2 gene which is responsible for regulating the activities of proteins of antioxidant system and enzymes involved in the process of detoxification. The genes present downstream are NAD (P)H, GST, and HO-1 which are involved in scavenging the cells against oxidative stress-induced by the doxorubicin, particularly in the liver. 4

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Fig. 2. The above figure represents the molecular mechanisms behind the doxorubicin-induced hepatotoxicity. The free radicals generated during the metabolism of doxorubicin results in the activation of oxidative stress, pro-inflammatory factors, and pro-apoptotic. The inflammatory signal is activated upon the NF-κB pathway through the inhibition of SIRT1 by the doxorubicin. The inflammatory signal and oxidative stress induced by the ROS results in the lipid peroxidation further leading to apoptosis. On the other hand, the ROS generated leads to elevated levels of liver enzyme biomarkers in the serum which serve as an indicator of liver damage. The SIRT1, NAD-dependent deacetylase is directly inhibited by the doxorubicin due to which p53 undergoes the process of acetylation and becomes activated resulting in activation of the apoptotic pathway. PGC1 α is deactivated upon inhibition of SIRT1 which drops the levels of mitochondrial biogenesis. Doxorubicin also reduces the phosphocreatine content which in turn reduces the ATP/ADP level resulting in mitochondrial dysfunction. SIRT1 also regulates the levels of antioxidants therefore, the antioxidant levels are dropped down due to the ROS production and also due to the inhibition of SIRT1. Eventually, elevated levels of ROS, pro-inflammatory and proapoptotic factors and suppressed levels of antioxidant enzymes, ATP/ADP, and mitochondrial biogenesis results in hepatotoxicity.

production [63]. The doxorubicin accumulates in the mitochondria and reduces the levels of inorganic phosphate thereby reducing adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) levels [49]. The phosphocreatine which is referred to as an energy reservoir is required for the production of ATP whereas the doxorubicin rapidly degrades the phosphocreatine levels thereby resulting in decreased ATP production. Therefore, the decreased ATP/ADP ratio indicates hepatic injury. Apart from this, doxorubicin decreases the level of some adenine nucleotides, ATP, ADP, and AMP [64]. Apart from these, the mitochondrial membrane potential is reduced upon doxorubicin treatment which results in the activation of apoptosis through the mitochondrial signaling pathway with the release of cytochrome c [65]. Doxorubicin also down-regulated the expression of PGC-1α and transcription factor A, mitochondrial (TFAM) which is required for mitochondrial DNA replication. Therefore, the reduced expression levels of PGC-1α and TFAM resulted in reduced mitochondrial biogenesis [66].

5.3. Doxorubicin exacerbates inflammation in hepatic tissue via SIRT1/ FOXO1/NF-κB signaling pathway Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α), a key regulator of oxidative metabolism which upregulates the antioxidant defense mechanism through the activation of enzymes such as MnSOD and Catalase, is kept under the control of sirtuin 1 (SIRT1) through the process of histone deacetylation. Apart from maintaining the redox potential, the SIRT1 is also involved in the inflammatory process through the NF- κB signaling thereby resulting in inflammation [58]. The doxorubicin inhibits the SIRT1 activity, which leads to the activation of NF-κB signaling which leads to inflammation and also promotes the histone acetylation on p53 resulting in apoptosis. Therefore, the doxorubicin-induced hepatic oxidative stress, inflammation and apoptosis through the upregulation of Forkhead box protein O1 (FOXO1), Nrf2, HO-1, P53, and BAX and down-regulation of Keap1 and BCL-2 through SIRT1/FOXO1/NF-κB signaling pathway [59]. Therefore, it has been concluded that one of the reasons for doxorubicin-induced hepatic damage is through the activation of NF-κB by the ROS produced [27]. The high levels of ALT, AST in the serum indicate liver damage; whereas the decreased level of total protein and albumin are synthesized in the liver also indicate hepatic damage. The elevated liver index value indicates hepatomegaly which was confirmed by histopathological findings. The most widely accepted mechanism of doxorubicin-induced hepatotoxicity is through oxidative stress and apoptotic pathways.

6. Amelioration of doxorubicin-induced hepatotoxicity via natural products and chemicals Many reports have been published regarding the alleviating effect of certain substances against the hepatotoxicity induced by the anthracycline antibiotic drug doxorubicin. Various types of natural products and certain drugs have shown to produce a hepatoprotective effect eliminating the toxic effect of doxorubicin. Natural extracts from plants such as Acetyl-11-keto beta boswellic acid is a pentacyclic triterpenoid extracted from Boswellia serrata which possess antioxidant and antiinflammatory effect [67]. Therefore, the use of boswellic acids along with doxorubicin has shown to reduce the hepatotoxicity by inhibiting the inflammatory and apoptotic pathways in mice models. The antioxidant effect of boswellic acids has shown to reduce the ROS content by activating the Nrf2/H0–1 pathway thereby eliminating the oxidative stress-induced toxicity in the hepatic tissue [32]. Another potent natural plant extract is dioscin which is a triterpenoid saponin extracted from Dioscorea nipponica Makino which exhibits anti-apoptotic, antiinflammatory and hepatoprotective responses [68]. The doxorubicin administered male mice models have been shown to induce apoptosis and inflammation by inhibiting the SIRT1 activity. Whereby, the administration of dioscin along with doxorubicin-induced the down-regulation of inflammatory and apoptotic pathways by up-regulating the SIRT1 thereby inhibiting the apoptosis in off-targeted tissues. The experiment conducted in AML-12 cell line where the concentration of

5.4. Doxorubicin impairs mitochondrial function via PGC-1α and impairment in the energy metabolism of hepatic tissue For a drug to undergo metabolism in the liver, it requires a lot of energy to produce its metabolites [28]. Mitochondria also play a key role in the detoxification process therefore mitochondria are prone to develop mitochondriopathy due to oxidative stress [60]. Mitochondrial dysfunction and degeneration have been reported as a cause of doxorubicin administration [44]. Doxorubicin exhibits higher affinity with the inner mitochondrial membrane and enters the matrix region where it produces ROS and initiates apoptotic pathways disrupting mitochondrial function and energy metabolism pathways. The doxorubicin-induced mitochondrial fragmentation in the hepatic tissue was reported by [61]. The mitochondria are present in large numbers in hepatocytes and possess high respiratory activity [62]. The Doxorubicin reduces oxidative phosphorylation, and increase the level of ROS 5

6

Male Sprague-Dawley rats Male B6D2F1 mice

BALB/C mice

Male Wistar rats

Male Sprague-Dawley rats

10. 11.

12.

13.

14.

Male Sprague-Dawley rats Male Sprague-Dawley rats

5. 6.

Mature male albino rats Rats

AML-12 Male Sprague-dawley rats

3. 4.

8. 9.

Male mice

2.

Male albino rats

Mice

1.

7.

Experimental model

S·No

10 mg/kg

15 mg/kg

10 mg/kg

2.5 mg/kg 20 mg/kg

2 mg/kg/week for 6 weeks 2.5 mg/kg

7.5 mg/kg/week for 6 weeks

5 mg/kg/week 20 mg/kg

5.0 uM 5 mg/kg/week

15 mg/kg

6 mg/kg/week (i.p)

Dosage of Doxorubicin administered Boswellic acids (extracted from Boswellia serrata) – 125, 250 and 500 mg/kg/day p.o Dioscin (extracted from Dioscoreanipponica Makino) – 15, 30 and 60 mg/kg/14 days. Dioscin – 50, 100, and 200 ng/ml Vitamin-E 200 IU/kg/week Catechin 200 IU/kg/week Erdosteine 10 mg/kg Turmeric extract 200 mg/kg 10 days before doxorubicin administration 1 ml of ginger extract (24 mg/ml) 3times/week for 6 weeks Berberine (extracted from Coptis chinensis) – 60 mg/kg 1 h after doxorubicin administration Fermented Cordyceps(Cordyceps Sinensis)– 0.47, 1.2, 3 g/kg/day Theanine 2.5–10 mg/kg/day for 4 days Lovastatin 10 mg/kg Atorvastatin 10 mg/kg Pravastatin 20 mg/kg

Type and dosage Alleviating agent administered

Table 2 Alleviating effect of various substances on doxorubicin-induced hepatotoxicity.

Reduced nitrosative stress, downregulation of apoptotic and upregulation of antioxidant activities Upregulation of antioxidant pathway and free radical scavenging effect.

Reduced pro-fibrotic and pro-inflammatory effects.

Upregulation of hepatic energy metabolism Downregulation of the apoptotic pathway

Upregulation of anti-inflammatory and anti-oxidant levels Reduce ROS levels

Increased antioxidant levels. Inhibition of inflammatory response reduced NO production by inhibition of iNOS resulting in decreased RNS. Upregulation of anti-inflammatory and anti-oxidant levels

Increased cell viability Increased antioxidant levels.

Reduced inflammation and apoptosis through the upregulation of SIRT1.

Reduced ROS, upregulated Nrf2/HO-1 pathway

Characteristics

[24]

[73]

[9]

[28] [30]

[72] [33]

[71]

[27] [22]

[59] [27]

[58]

[32]

References

P.L. Prasanna, et al.

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doxorubicin was 5.0 μM and the concentration of dioscin was 50, 100 and 200 ng/ml has been shown to increase the cell viability thereby indicating the same results that were observed in the animal models [59]. Vitamin E is an essential component possessing antioxidant activity and is supplemented through the diet [58] and catechin is a phenolic compound possessing antioxidant activity and iron-chelating effect supplemented through the diet and fruits are rich sources of catechin [69]. When male Sprague-Dawley rats were treated in a combination of doxorubicin (5 mg/kg/week) with vitamin E (200 IU/kg/ week) and doxorubicin (5 mg/kg/week) with catechin (200 IU/kg/ week), it was found that the toxicity caused due to the administration of doxorubicin on hepatic tissue was neutralized due to the antioxidant effect of vitamin E and catechin which were confirmed by estimating the levels of antioxidant enzymes and histopathological changes in the hepatic tissue [27]. Erdosteine is a mucolytic thiol derivative consisting of two thiol groups that are involved in the scavenging of free radicals thus conferring antioxidant properties [70]. Male Sprague-Dawley rats treated with doxorubicin (20 mg/kg) and erdosteine (10 mg/kg) has been found to reduce the inflammatory response by scavenging the free radicals before the activation of the immune system and also inhibited the synthesis of NO by iNOS which otherwise reacts with superoxide radicals and results in the production of peroxynitrite thereby reducing the tissue injury. The plants Curcuma longa and Zingiber officinale belongs to the family Zingiberaceae whose roots are termed as rhizomes possess anti-inflammatory and antioxidant properties are shown to reduce the toxicity induced by doxorubicin on the liver. Male albino rats were given 200 mg/kg of turmeric extract for 10 days before doxorubicin (7.5 mg/kg for 30 days) administration [71]. Mature male albino rats were administered with doxorubicin (2 mg/kg/week for 6 weeks) followed by 1 ml of ginger extract (24 mg/ml) 3 times/week for 6 weeks [72]. A traditional Chinese herb Coptis Chinensis extract from berberine is an isoquinoline alkaloid that has been shown to have antiinflammatory, antitumor activity by combating ROS formation. The liver damage in experimental rats due to doxorubicin (2.5 mg/kg) administration was attenuated with the administration of Berberine (60 mg/kg 1 h after doxorubicin administration) which further confirms by measuring the liver enzyme biomarkers such as ALT and AST [33]. Another Chinese traditional herb Cordyceps Sinensis (0.75 g/kg/d, 1.2 g/kg/d, 3.00 g/kg/d) was administered along with doxorubicin (2.5 mg/kg) to male Sprague-Dawley rats to attenuate the decline in hepatic energy metabolism and restore to its normal function [28]. It was reported by Nagai et al. 2015 that when male B6D2F1 mice aged 5 weeks doxorubicin plus theanine was administered at a dose of 20 mg/kg doxorubicin on the 1st day followed by theanine (2.5–10 mg/ kg/day for 4 days, i.p.) was administered which eventually showed reduced apoptotic activity thereby eliminating hepatic injury [30]. Statins are the lipid or cholesterol-lowering drugs which when given along with doxorubicin has shown to reduce the toxicity induced in the hepatic tissue by exhibiting inhibitory activity on pro-inflammatory, nitrosative and pro-fibrotic pathways and up-regulating the antioxidant pathways. This was proved by administering statins such as lovastatin (BALB/c mice - 10 mg/kg), pravastatin (male Sprague - Dawley rats 20 mg/kg), and atorvastatin (male Wistar rats- 10 mg/kg/day) along with doxorubicin (10–15 mg/kg) [9,24,73]. Apart from the abovementioned products recently published, the article mentioned that physical exercise has been shown to decline the toxicity induced by doxorubicin. For this male Sprague - Dawley rats were trained in physical activity and then given administered with doxorubicin (10 mg/kg and 20 mg/kg) has shown to have better mitochondrial activity, decrease in oxidative stress, production of antioxidants and improved energy production through the increased consumption of oxygen thus conferring resistance to liver against doxorubicin treatment [41,66]. The hepatoprotective effects of various natural and chemical substances have been elucidated in Table 2.

7. Conclusion Eventually, we conclude that anthracycline antibiotic doxorubicin confers toxicity to hepatic tissue which was proved by many scientific reports. The main reason underlying doxorubicin-induced hepatotoxicity is the production of free radicals in the off-target tissues. The incentives regarding the toxicity are oxidative stress, inflammation, and apoptosis due to ROS production, mitochondrial dysfunction causing imbalanced energy status in the cell eventually leading to apoptosis. The oxidative stress caused due to the production of free radicals is mediated via Nrf-2/HO-1 pathway, inflammation caused due to the ROS generation, established by the elevated levels of malondialdehyde was explained via SIRT1/FOXO1/NF-κB signaling pathway, elevated apoptosis characterized by DNA fragmentation via the activation of extrinsic and intrinsic signaling pathways through the activation of p53, mitochondrial dysfunction via deregulation of electron transport chain diminished ATP production and slashed mitochondrial biogenesis via downregulation of PGC-1α and TFAM which is required for mitochondrial DNA replication. This issue should be overwhelmed by upgrading the conveyance of the medication to the focused-on hand with the goal that the medication can have its viability to the fullest at the focused tumor. The use of medicinal plant extracts, herbs along with a chemotherapeutic drug to alleviate the toxic effect of the drug is considered as an appreciable way to treat drug side effects. Further research is needed to elucidate the molecular mechanisms more clearly to improve the drug efficacy on targeted sites and eradicate the toxicity at the off-target sites. Funding The authors thank VIT for providing “VIT SEED GRANT” for carrying out this work. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The author Kaviyarasi Renu is grateful to ICMR for providing financial assistance during this tenure. The authors thank VIT, Vellore, Tamilnadu, India, for supporting this work. References [1] S. Rivankar, An overview of doxorubicin formulations in cancer therapy, J. Cancer Res. Ther. 10 (2014) 853–858. [2] C.F. Thorn, C. Oshiro, S. Marsh, T. Hernandez-Boussard, H. McLeod, T.E. Klein, R.B. Altman, Doxorubicin pathways: pharmacodynamics and adverse effects, Pharmacogenet. Genomics 21 (2011) 440–446. [3] F. Arcamone, G. Cassinelli, G. Fantini, A. Grein, P. Orezzi, C. Pol, C. Spalla, Adriamycin, 14-Hydroxydaunomycin, a new antitumor antibiotic from S. peucetius var. caesius, Biotechnol. Bioeng. 67 (2000) 704–713. [4] H. Cortes-Funes, C. Coronado, Role of anthracyclines in the era of targeted therapy, Cardiovasc. Toxicol. 7 (2007) 56–60. [5] N.I.o. Health, Livertox: Clinical And Research Information on Drug-induced Liver Injury, Nih.gov, 2017, https://livertox.nih.gov. [6] K. Renu, A.V. Gopalakrishnan, Deciphering the molecular mechanism during doxorubicin-mediated oxidative stress, apoptosis through Nrf2 and PGC-1α in a rat testicular milieu, Reprod. Biol. 19 (2019) 22–37. [7] K. Renu, K. Sruthy, S. Parthiban, S. Sugunapriyadharshini, A. George, T.P. PB, S. Suman, V. Abilash, S. Arunachalam, Elevated lipolysis in adipose tissue by doxorubicin via PPARα activation associated with hepatic steatosis and insulin resistance, Eur. J. Pharmacol. 843 (2019) 162–176. [8] K. Renu, V. Abilash, T.P. PB, S. Arunachalam, Molecular mechanism of doxorubicininduced cardiomyopathy–an update, Eur. J. Pharmacol. 818 (2018) 241–253. [9] C. Henninger, J. Huelsenbeck, S. Huelsenbeck, S. Grösch, A. Schad, K.J. Lackner, B. Kaina, G. Fritz, The lipid lowering drug lovastatin protects against doxorubicininduced hepatotoxicity, Toxicol. Appl. Pharmacol. 261 (2012) 66–73.

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