Overview on the Side Effects of Doxorubicin

1.1 Historical background

In the 1950s Italian based company; Farmitalia research laboratory began a research program in finding the anticancer compounds from soil-based microbes. In the process of research collected a soil sample from the castle named as castle Del monte, which was built in the 13th century. The collected soil sample contains new strains of bacterial species and isolated from it. The separated microbe recognised with the name Streptomyces peucetius which is typically produces a significant red pigment. The antibiotic produced from this bacterium discovered to be efficient in treating the tumours especially solid tumours while researching on mice. Since a group of French scientists found the same compound about at the same time, they agreed to call the antibiotic daunorubicin, referring to the two nations. In which, Dauni refers to the pre-Roman tribe who inhabited the position in Italy where the species of bacteria were isolated and ruby represents the colour in Italy. The clinical trials of daunorubicin were began in 1960s and confirmed as successful in treating the lymphoma and acute leukaemia [1, 2]. After a short note of the time, in 1967 daunorubicin was discovered to be cause fatal cardiotoxicity. Then, by using nitroso-N-methyl urethane, the Italian research company mutated the strains of Streptomyces peucetius and developed a new strain of bacterial species that produces 14-hydroxylated daunorubicin, also known as Adriamycin (named after the Adriatic Sea), then changed its name to doxorubicin, which has a strong therapeutic index, but cardiac toxicity remains [3].

1.2 Doxorubicin chemical structure and properties

Doxorubicin (DOX) is an anthracycline antibiotic structurally similar to Daunorubicin as natural anti-cancer antibiotic used in cancer treatment. Its anticancer effect produced intercalating with DNA and this will inhibit DNA transcription and replication; and by binding to the topoisomerase II enzyme and inhibit the resealing of the DNA fragments. The presence of sugar moiety attached to the anthracycline ring further enhances the binding to phosphate and sugar moieties in to DNA. This led to stops the proliferation of cancer cells in the host [4]. Besides, the presence of quinone moiety apart from contributing the cytolytic ability by generating the intermediate radicals, which further react with the oxygen and forms superoxide ions and these ions also shows a high tendency towards the damaging the cell membranes causes a dose-dependent the cardiac myopathy [5, 6].

The Doxorubicin is mainly used in case of patients suffering from Breast cancer, ovarian cancers, lung cancers, bladder cancers, leukaemia (acute lymphoblastic leukaemia, acute myeloid leukaemia) and AIDS-related Kaposi’s sarcoma and various solid tumours. DOX also used in combination with other agents in case of bone sarcomas, soft tissue sarcomas, uterus cancer, endoblastoma cancer, cervix cancer, pancreatic cancer, Ewing’s sarcoma, mesothelioma, multiple myeloma, Wilms tumour and in neuroblastoma [7, 8].

2.1 Hepatotoxicity

Liver is one of the essential organs of the body; it plays a major role in metabolism and detoxification of several drugs. This can explains why liver is the primary body organ affected by chemotherapy. Despite being cytostatic and cytotoxic effects on cancer cells DOX documented to accumulate in the various tissues include liver cells. In humans, it is estimated as 50% of DOX eliminated in un-exchanged form, the remainder dose metabolised through hydroxylation, semiquinone formation [9]. The major pathway for biotransformation of DOX is catalysed by the NADPH-dependent carbonyl reductase, Nitric oxide synthase, cytochrome P-450 reductase, aldo-keto reductase enzymes. The hydroxylation occurs at C-13 carbon in group commonly reaction referred as electron reduction forms the secondary alcohol metabolites [10, 11, 12, 13]. The metabolized intermediates in the presence of oxygen converted to carbonyl moieties resulting in generation of Superoxide anions and hydrogen peroxides causes peroxidation of lipids in membranes of cell, aggregation of proteins (Figure 1) [13, 14, 15].

The regenerative capacity of liver is more can cure the damage caused by various agents such as DOX, which causes damage and decreases the regeneration of liver cells by increasing the oxidative stress due to the radical generation by oxidation in hepatocytes. The generated radical causes decrease in GSH levels, damages in DNA and also act as secondary metabolites in in many metabolic pathways which includes in cell proliferation and cell death [16, 17, 18]. To overcome such situations liver employs the efflux mechanisms, the efflux of DOX is achieved through from liver by ATP dependent ABC proteins (P-glycoprotein) which increase the efflux of the intracellular DOX and maintain the homeostasis. The mechanism uses large quantity of energy but with the presence of the DOX in liver cells decreases the ATP production and increases the ADP and Pi within the cells [19, 20, 21]. Due to this effect sometimes liver cells can’t able to regenerate from DOX induced effects and causes hepatotoxicity.

2.2 Nephropathy

Besides maintaining the homeostasis by regulating the body fluids, kidneys work to reabsorb the low concentrations general constituents in the body and also remove the foreign substances like drugs or other kinds of agents. For this kind of reasons kidneys considered as metastatic organs of human beings [22]. The regenerative capacity of the kidneys is low when compared to the liver and highly susceptible to epithelial degeneration occurs at renal glomerulus where the filtration occurs may lead to the glomerulosclerosis [23].

DOX interferes with the glandular podocytes of the kidney and cause nephropathy the most accepted mechanism behind the nephropathy is an accumulation of proteinuria in the kidney by the local passage of leaked proteins [23]. Increase in the structural changes in nephrons causes hypertension, steroid resistance, high incidents of renal failure and glomerular vacuolization, inflammation, tubular dilation, intestinal fibrosis, permeability differences in the glomerulus, and certain conditions like hypoalbuminemia, dyslipidemia, hypercoagulation, size differences in kidney most likely observed [24]. A study conducted on the DOX effect on the mitochondria by the Lebrecht suggested that DOX interfere the mitochondrial mtDNA in the kidney with ROS produced from it and accelerating the damaging of the nephron. Another study reports suggesting that DOX forming an iron-mediated anthracycline complex, which produces the ROS led to an increase in the oxidative lesions in the cells causing damage to the critical cellular components [25, 26].

The decreasing the levels of the GSH (Glutathione), vitamin E levels and other natural oxidant levels production from the liver cells enhances the nephropathic conditions which may initially affect the Bowman’s capsule thickness and the glomerular tuft of the nephron. The study conducted by Rook et al. [26] Reported as Angiotensin-converting enzyme is said to be one of the responsible factors for tissue damage triggered by the DOX therapy. The ACE is causing the pro-inflammatory, pro-fibrotic effects which make interference in the kidney and nephrons to maintain the glomerular pressure and filtration rate of blood [27, 28]. The cases of nephropathy and proteinuria are rare in humans susceptibility towards such condition based on the genetic makeup of the individual.

2.3 Neurotoxicity

The brain is the largest and most complex organ in the human body contains about 100 billion neurons with 1 trillion established connections throughout the body. DOX is not able to transfer through the blood–brain barrier (BBB), therefore DOX effects against the brain via indirect way [29, 30]. These effects include: depression, anxiety, decrease in motor functions, haemoglobin levels, perception skills affected, and menopausal status, visuospatial skills are affected through cancer chemotherapy. The recovery of the cognitive functions may take up to a year [31]. The DOX mediate increase TNF-α level (inflammatory cytokines produced by the macrophages/monocytes during the acute inflammation involved in many signalling pathways) in the brain at cortex and hippocampus of mice [32, 33, 34]. The mitochondrial activity, glutathione-S-transferase, GSH levels, and MnSOD levels in the brain are decreased and increase in levels of 4-hydroxynoneal (HNA), thiobarbituric acid reactive substances (TBARS), malondialdehyde (MDA) and increase in levels of protein carbonyl groups [35, 36, 37, 38], which causes increase the oxidative stress in the brain cells and further led to cause cell damage.

The MnSOD levels in the brain generally detoxify the oxygen free radicals, inactivated by the Nitric oxide (NO). The DOX indirectly increased the concentration of NO by overexpressing the Nitric oxide synthase enzyme [37]. A study conducted on the NOS dependent brain injury with DOC reinforcing the nitric oxide tissue damage [38]. The mitochondrial activity is very important in the brain because it is a powerhouse of cells (energy production) brain uses 20% of body glucose for energy production to conduct and maintain the regular activities [39]. The DOX induces generation of MDA, TBARS, and HNA which cause the decrease the mitochondrial activities. A study conducted on the DOX-induced toxicity on rats with 10 mg/kg dose, the rats died between 10 and 50 days with observed light microscopic studies reveals that specific changes in the ganglionic cells of the peripheral nervous system [40].

2.4 Cardiomyopathy

This side effect found to be a dose-dependent on DOX. The DOX-induced cardiotoxicity occurs acutely and chronically. The acute effects occur within one week period the patient may experience arrhythmia, hypotension, and super ventricular tachycardia. These abnormalities are generally reversible in a noticeable period [41]. The chronic effects are shown in only 1.7% of patients with a 50% mortality rate [42, 43]. The chronic effect of DOX such as congestive heart failure reported in a study, when the patients are treated with dose 500–550 mg/m² in more than 4% of patients when treated with the dose is 551–600 mg/m² 18% of patients cause the CHF, and almost 35% of patients observed with CHF when treated with >601 mg/m² [44, 45].

A study conducted by the Zordosky and EI-kadi on DOX-induced toxicity reported as the induction of Brain natriuretic peptides, atrial natriuretic peptides genes, monooxygenases, cytochrome P genes and hypertrophy markers responsible for the xenobiotics and certain endogenous substances [46]. The inductions of these genes are cause cardiac hypertrophy leading to heart failure and altered the arachidonic acid mechanisms. A study reported the DOX effects based on the concentrations, at low concentrations DOX dose (o.5–1 μM) causes the alterations in structural proteins (includes sarcomeric myosin, nuclear lamina), plasma membrane blebbing (causes change in cell shape), and mitochondrial depolarization and fragmentation. At high concentration causes (5–50 μM) causes the cytoplasm vacuolization, swelling of mitochondrial cells, promote the cellular alterations (Figure 2) at the cellular and nuclear membranes [47]. The DOX reportedly binds to the cardiolipin (a mitochondrial inner membrane component), which raises the accumulation of the DOX inside the mycoplasma when compared to the other body cells. The high concentration existence of the NADPH dehydrogenase inside the mitochondria initiates the redox reaction in the complex and promotes the production of the Reactive oxygen species. Myocytes are generally having low levels of anti-oxidants when compared to the other tissue cells, considerably DOX shows enhanced effects on the heart and cause toxicity [48, 49, 50].

The antioxidant level differences were observed in rats under DOX treatment based on the age differences, younger Fischer rats contain more levels of antioxidants when compared to old Fischer rats. A recent study stated the involvement of the Toll-like receptor TLR-4 (a specific receptor in the immune system generally recognise the multiple bacterial antigens and plays a major role in the maturation of the phagosomes) [51]. The increase in TLR-4 expression in the DOX-induced Cardiomyocytes, when studied the cardiomyopathic cells in humans and animals. The deficiency of TLR shows decreased in lipid peroxidation and nitrotyrosine levels in cardiomyopathic cells. The other study on the glutathione peroxidase 1 (GPx) enzyme is present in both cytosol and mitochondria play a major role in the detoxification. The study conducted with the insertion of DOX on non-GPx and wild type mice, the results showed based on the study on myocytes of the non-GPx mice having the high concentration of the DOX deposits in cells, when compared to wild type mice [52, 53, 54].

The oxidative stress is a major cause for the exhibiting the cardiotoxicity, involved the generation of higher amounts of ROS cause the cellar alterations and damage are referred to as oxidative stress. The ROS is countered by the anti-oxidant system in the body, in cancer patients under the DOX chemotherapy observed the decreased the levels of GSH, TRAP levels in the body. The ROS is generation is catalysed by NADPH oxidase enzyme [55, 56].

In mitochondrial cells, the same reaction is mediated by NADH –ubiquinone oxidoreductase enzyme.

The generated oxygen radical undergo dimutation with hydrohen molecules and forms hydrogen peroxide reaction is mediated by the SOD enzyme [55].

The generated less active hydrogen peroxide is removed by the enzymes like catalase, glutathione peroxidase.

The generated oxygen radicals combine with the H2O2 and form the highly active hydroxyl radicals. The H2O2 also reacted with the ferrous ions resulting in the formation of ferric ions and reactive hydroxyl radicals [55].

Under stress conditions, oxygen radical facilitates the ferrous iron from the ferric ion. The iron, under normal conditions sequestered within the ferritin (a globular protein and forms the nanocage with the metal-protein complexes) but with regards to DOX when converted to its semi-quinone form complexes with iron-free radical and converted to DOX forms while generating the oxygen free radical. The generated complexes block the iron-free regulating proteins (IRP), and then these IRPs bound to the iron-responsive elements in mRNA ferritin. The tremendous amounts of free iron releases and gain complexes with the DOX. This specific condition magnifies the production of ROS in cells [57, 58, 59].

The ROS acts as secondary signalling molecules shows direct effects on the lipids, proteins, DNA, and RNA in various pathways involved in cell proliferation, cell death and maintain the homeostasis. It is domineering to maintain the levels of ROS in the body, in case of the heart the effect is maximum by ROS due to lack of efficient levels of anti-oxidants in myocytes. The conditions such as cellular hypertrophy, alterations in the gene expressions, ventricular remodelling, the extracellular matrix of the mitochondria transformation, calcium transient perturbation and cell death activation such kinds of pathological changes may be observed in myocytes lead the death of cells.

2.5 Mechanisms involved at different levels of cardio toxicity

2.6 Diagnosis

The DOX-induced cardiomyopathy consists of a complete examination of the cardiovascular system for detecting the symptoms, such as S3 gallop and elevated jugular vapour pressure, T wave impairments; low voltage QRS complexes are measured.

• Electrocardiography combined with Doopler studies used to study early diagnostic symptoms of the cardiac myopathy through the measure of latero-ventricular dysfunction.

• Radionuclide ventriculography used to access the latero-ventricular systolic and diastolic function. Observes the cardiac adrenergic denervation occurs in case of doxorubicin induced cardiomyopathy.

• Metaiodobenzylguanidine based nuclear imaging can be employed to assess cardiac adrenergic denervation occurred trough the DOX based cardiomyopathy.

• The DOX treated patients are sensitive to the indium labelled monoclonal antimyosin antibodies (myosin an ATP dependent superfamily of motor proteins major role in muscle contraction and motility) used to detect the cardiac myopathy, myocarditis, chagas heart disease ischemic myocardium, and kawasaki heart disease [85].

• The measurement of the cardiac enzymes and neurohormones are used for detecting the heart failure but not a characteristic feature of the DOX-induced cardiomyopathy [86].

• The presence of endomyocardial biopsy is the best route for detecting the DOX-induced diseases, according to the grade of biopsy severity of the disease is measured [87, 88]. It is invasive and requires experience for recognising the results become a disadvantage for this technique.