Introduction

Doxorubicin (adriamycin), an antibiotic from the tetracyclic anthracycline class, was found in the cultures of Streptomyces peucetiues var. caesius (1, 2, and 3). Since the late 1960s, this has been one of the most efficient and popular chemotherapeutic medications (4 and 5). It is a cytotoxic compound that interacts with mammalian cells to inhibit cell growth and viability at low concentrations (6 and 7). It has a tetracyclic structure, which is linked to carbohydrates; removal of carbohydrates from it usually leads to loss of anti-tumor activity. In the treatment of cancers such as solid tumors, multiple myelomas, breast cancer, stomach cancer, ovarian cancer, testicular cancer, and bone cancer, doxorubicin is crucial (8,9, and10). Long-term use of this anticancer drug can lead to the accumulation of its metabolite, doxorubicinol, which can cause cardiomyopathy due to its cytotoxicity. This cardiotoxicity is dependent on the amount of doxorubicin and doxorubicinol accumulated in the body (11). Doxorubicinol has stronger cardiotoxic effects than doxorubicin (12). The mechanism of action of the drug is not entirely clear and may involve multiple actions (13). The anti-tumor activity of doxorubicin can intercalate between DNA bases, interfering with DNA replication and the transcription of RNA. During metabolism, doxorubicin (quinone) is reduced by a two-electron reaction to semiquinone and then to hydroquinone. Semiquinones react with molecular oxygen under aerobic conditions to form superoxide and hydrogen peroxide, which in turn form hydroxyl radicals in the presence of transition metals [iron, copper] (14, 15, and 16). Doxorubicin is classified as a Class III, so-called topoisomerase inhibitor. Double-stranded DNA can be either two loosely coiled molecules (negatively supercoiling) or two tightly coiled molecules (positively supercoiling) relative to its lowest energy state. (17). The enzyme topoisomerase plays a key role in temporarily cutting one strand. As a result, the other strand can pass the breakpoint, resulting in clipping the ends together, which is the backbone of replication. Doxorubicin inhibits the ability of the topoisomerase II enzyme to rejoin the severed ends, causing DNA strand breaks throughout dividing cells, a process that ultimately leads to cell death.

In our study, we examined enzymatic and non-enzymatic changes in the liver, kidney, and heart of the doxorubicin model compared to controls. Organelle changes under the influence of the anticancer drug doxorubicin were also examined using electron and light microscopy. The current investigation is fundamentally focused on understanding the toxic effects of the commonly used anticancer drug doxorubicin on antioxidant structures and detoxification systems in experimental rat models. This is to monitor general health and aims to demonstrate the efficacy of this drug in albino rats. Become a rat while receiving treatment. Several new approaches are being explored to improve activity and reduce the unwanted side effects of chemotherapy.

Materials and Methods

Animals

Experimental drugs

Doxorubicin is an anticancer drug (ADRIM) manufactured by Dahur India Ltd. (Pharmaceutical Sector, Solan-173205), India. Rats in the experimental group were given 18 intraperitoneal doses of 1 mg/kg three times a week. ¹⁸, group number II. The control group was Group No I., to which 0.5 ml/day of physiological saline was administered for a total of 52 days. (Me). Animal weights were monitored at weekly intervals.

Analytical Procedures

Extraction of tissue

The animals were kept in the same lab environment following treatment for two to three days. Before dissection, ether anesthesia was used to sedate the animals. The liver, kidney, and heart were taken after the rats had been killed, and any adhering fat on these tissues was cleaned off and washed with cooled normal saline (0.85%). These were then processed for structural and biochemical tests, including blotting, weighing, and processing.

A 10% homogenate was prepared by placing 0.5 g of tissue in 5 ml 0.1 M sodium phosphate buffer (pH 8.0). The homogenate was centrifuged at 9000 rpm for 20 minutes, and the resulting supernatant fraction was used for antioxidant enzyme measurements. Reductions in glutathione and lipid peroxidation were estimated using samples treated with 25% and 5% trichloroacetic acid (TCA). The enzymes cytochrome P450 and cytochrome b5 were evaluated from the microsomal fraction obtained after centrifugation at 105,000 g.

Measurement of malondialdehyde (MDA)

The thiobarbituric acid (TBA) assay was used to assess the concentration of MDA in the 10% homogenates of liver, kidney, and heart (made in 0.1 M sodium phosphate buffer pH 8.0). It is approximated using Esterbauer and Cheeseman’s methodology (19).

The total amount of protein

Using the Lowry et al. method, the total protein content in doxorubicin-treated samples was examined and contrasted with controls (20).

Measurement of antioxidants: reduced glutathione (GSH)

GSH plays a role in maintaining cellular redox potential in living systems. GSH is according to Moron et al. The developed method was highly regarded (21).

Antioxidant Enzyme Activity: Glutathione Peroxidase (GPx)

 GPx (EC 1:11:9) plays an instrumental role in maintaining the redox state during acute oxidative stress. The protective role of GPx is associated with antioxidant enzymes (22).

The supernatant was further used to determine the GPx enzyme by the method of Rotruck et al. (23).

Antioxidant Enzyme Activity: Glutathione Reductase (GR)

The activity of GR (EC 1.6.4.2) was estimated by the method of Racker (24).

Antioxidant Enzyme Activity: Catalase (CAT)

CAT (EC 1.11.1.6) activity was measured in accordance with Aebi’s instructions (25).

Drug Metabolizing Enzymes’ Activities

Antioxidant Enzyme Determination: Glutathione-S-Transferase (G-S-T)

The supernatant was further used for GST (EC 2.5.18) according to Habig’s method (26).

Antioxidant Enzymes Determination: Cytochrome P₄₅₀ (CYTp450) and Cytochrome b₅ (CYTb5)

CYTp450 (EC 14.14.1) and CYTb5 (EC 1.6.2.2) were tested in accordance with Omura and Sato’s techniques (27).

STRUCTURAL STUDIES

Light microscopy

Light microscopy of hepatic and renal tissues was observed and studied by the paraffin method. The liver and kidney were cut into small pieces and fixed in Bouin’s fixative (28) for 24-28 hours.

To remove excess Bouin’s fixative, the tissues were removed from the fixative and thoroughly washed with distilled water for several hours. After fixation, samples were washed with 70% alcohol to remove excess picric acid from the tissue and dehydrated through a graded series of ethanols. Tissues were decarburized using a series of graded xylene-alcohol mixtures, and the dealcoholized clear tissues were stored overnight at 58–60 °C in filtered molten paraffin. Finally, the tissue was embedded in filtered molten paraffin. Serial 3.0 μm thick sections of all tissues were obtained using a rotary microtome (Microm, model #HM 310), and sections were stained with hematoxylin followed by a secondary stain, eosin. Slides were mounted using DPX mounting medium as glue, and sections were viewed under a microscope and photographed.

Electron microscopy

The techniques for histological processing of biological material for electron microscopy are basically similar to those used for light microscopy. Compared to light microscopy, the use of electron microscopy gives higher resolution and magnifications.

Transmission electron microscopy 

Liver and kidney tissues were removed from rats, cut into 1 mm pieces with a drop of 3% glutaraldehyde, and immersed in fresh, ice-cold fixative for 2 hours and then in 0.1 M cacodylate buffer for 4 hours. Tissues were then briefly rinsed in buffer and post-osmicated with 1% osmic acid for 1–2 hours. The tissues were then dehydrated in a series of increasing alcohols, followed by dehydration in propylene oxide, and finally embedded in polymerized resin at 60 °C. After that, Araldite blocks were prepared, and 1 m sections were cut with a glass knife on the LKB-2000S, an ultramicrotome mounted on glass slides and stained with buffered toluidine blue. Appropriate areas were selected using a light microscope. Finally, ultra-thin sections of selected blocks were cut with a diamond knife, recorded on copper grids, and stained with uranium acetate and lead citrate for final viewing. Ultrathin sections were scanned and photographed with a JEM-JEOL 100S electron microscope.

Statistics

The data are presented as (mean ± SEM). Statistical analysis was performed using a t-test. Statistically significant at (p<0.05).

Results

Table 1: Doxorubicin-induced body weight and organ weight changes in male rats (mean ± SEM)

 Table 2: Effect of doxorubicin on hepatic antioxidant, antioxidant enzymes, and protein profile of male rats (mean ± SEM).

 Table 3: Effect of doxorubicin on renal antioxidant, antioxidant enzymes, and protein profile of male rats (mean ± SEM).

 Table 4: Effect of doxorubicin on cardiac antioxidant, antioxidant enzymes, and protein profile of male rats (mean ± SEM).

 Doxorubicin-treated rat liver under a light microscope.

Figure 1: Light micrographs of rat livers treated with doxorubicin for 8 weeks show the presence of vacuolated hepatocytes (*).  Click here to view figure

Figure 2: A light micrograph of a rat liver treated with doxorubicin for 8 weeks shows a narrow portal space (PS).  Click here to view figure

Doxorubicin-treated rat kidneys under a light microscope.

Figure 3: Light micrographs of rat kidneys treated with doxorubicin for 8 weeks show subtle changes in the tubules (T). Glomeruli (G) were observed. Lumen (L) and vacuolization (*) are observed. Photographed at magnification (x 40). Click here to view figure

Figure 4: Light micrographs of rat kidneys treated with doxorubicin for 8 weeks show dilated proximal tubules (PCT). Nuclei (N) and prominent nuclei (NU) can be seen. Note the lipid droplets in some tubules. Photographed at magnification (x 40). Click here to view figure

Doxorubicin-treated rat liver photographed under an electron microscope.            

Figure 5: Electron micrographs of rat livers treated with doxorubicin for 8 weeks show that hepatocytes contain many organelles. Many intact mitochondria were observed.  Click here to view figure

Figure 6: Electron micrographs of livers from rats treated with doxorubicin for 8 weeks clearly show the nucleus (N) near the center of the figure, and the cytoplasm consists of smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), as well as mitochondria (M). Lysosomes (Lys) are seen.  Click here to view figure

Figure 7: Electron micrographs of rat livers treated with doxorubicin for 8 weeks show that mitochondria (M) are present throughout the cytoplasm, with a smooth endoplasmic reticulum (SER). Lipid droplets (LD) were also found in the cytoplasm.  Click here to view figure

Figure 8: Electron micrographs of rat livers treated with doxorubicin for 8 weeks show cytoplasmic accumulation of large mitochondria (M). Lysosomes were also recognized.  Click here to view figure

Doxorubicin-treated rat kidney in electron micrograph form.

Figure 9: Electron micrographs of rat kidneys treated with doxorubicin for 8 weeks. Electron micrographs of several lysosomes (Ly) were visible and the lumen (L) was also evident.  Click here to view figure

Figure 10: Electron micrographs of rat kidneys treated with doxorubicin for 8 weeks. Electron micrographs of epithelial microvilli (Mv) are clearly visible.  Click here to view figure

Figure 11: Electron micrographs of rat kidneys treated with doxorubicin for 8 weeks. Electron micrographs of nuclei (N) are present, microvilli (Mv) are evident, and lysosomes (Ly) are visible. Image magnification (x3,000). Click here to view figure

Figure 12: Electron micrographs of rat kidneys treated with doxorubicin for 8 weeks. Electron micrographs of less elaborately shaped cells and mitochondria (M) have a more circular profile than subsequent complex sections. Image magnification (x3,000). Click here to view figure

Figure 13: Electron micrographs of rat kidneys treated with doxorubicin for 8 weeks. Note the presence of epithelial microvilli (MV). The proximal tubule (PCT) is a prominent core of tubular lining that shows dense chromatin. Photographed at magnification (x 2,500). Click here to view figure

General observations 

The body weights of doxorubicin rats were significantly lower than those of the corresponding controls. Body organs such as liver weights showed non-substantial increases in doxorubicin-treated rats with respect to controls. Kidney weights showed a non-significant decrease in the doxorubicin-treated group in comparison to the control group. After doxorubicin induction, the heart weight had a non-significant decrease compared to controls. Relevant data are collected in (Table 1).

Antioxidant and Antioxidant Enzyme Activity

Eight weeks after doxorubicin injection, GSH activity showed a non-significant increase in liver tissue, but a non-significant decrease in kidney tissue. On the other hand, heart tissue increased significantly. The data are shown in (Tables 2, 3, and 4).

Concentrations of MDA as an index of lipid peroxidation were measured in three organs. Total MDA in liver, kidney, and heart tissue formed in the doxorubicin-treated groups showed a significant decrease compared to the control group (Tables 2, 3, and 4). 

The activity of G-S-T was assessed and found to be significantly increased in liver, kidney, and heart tissues treated with the anticancer drug doxorubicin compared to control rats (Tables 2, 3, and 4).

Eight weeks after doxorubicin injection, the activity of GPx showed a significant decrease in liver, kidney, and heart tissues compared to their control counterparts (Tables 2, 3, and 4).

CAT activity in liver tissue was significantly increased in doxorubicin-treated samples, whereas CAT activity in kidney and heart tissue of doxorubicin-treated rats was significantly lower than that of control rats (Tables 2, 3, and 4).

The doxorubicin-treated group showed a significant decrease in CYTp450 levels in liver and kidney tissues, and a significant increase in CYTp450 levels in heart tissue compared to control rats (Tables 2, 3, and 4).

Upon treatment with doxorubicin, a significant increase in CYTb5 was observed in liver and heart tissue, whereas levels of CYTb5 in kidney tissue were significantly decreased compared to their control counterparts (Tables 2, 3, and 4).

For doxorubicin, total protein in liver and kidney tissues showed a significant increase, whereas total protein content in the heart fraction did not show a significant decrease compared to the control group (Tables 2, 3, and 4).

Histological effects of doxorubicin on hepatic tissue

In the present study, it was evident that the liver cells displayed few distinct changes as a result of treatment with doxorubicin, detected in the tissues by light microscopy (Figures 1 and 2).

Electron microscopy data obtained from this study showed that after doxorubicin induction, rats exhibited slight structural changes, numerous mitochondria, continuity between rough and smooth endoplasmic reticulum, the presence of lipid droplets of different densities, and very strong evidence of microvilli. A long sine wave marked the room. Most organelles are visible (Figures 5, 6, 7, and 8).

Histological effects of doxorubicin on renal tissue

Eight weeks after doxorubicin treatment, kidney sections were examined under a light microscope and evaluated blindly. The renal cortex was nearly normal in appearance, and the glomeruli and glomerular quadrants showed non-significant changes in renal-treated rats. This implies that the production of toxic oxygen is one of the mechanisms leading to kidney tissue damage, which is not seen in this case (Figures 3 and 4).

Electron micrographs show little change in the histological aspect of the lumen, with fewer recognizable microvilli, fewer lysosomes, and recognizable mitochondria. The core appears very clearly and in large quantities. Also of note is the proximal convoluted tube. This is seen in the doxorubicin model (Figures 9, 10, 11, 12, and 13).

Discussion

GSH is involved in the protection of cellular redox potential against oxidative stress in biological systems. It is also known to be involved in maintaining normal kidney structure and function (Anand et al., (35).

According to Zaman et al. (36), the effectiveness of medications like doxorubicin and cisplatin in cancer cell lines is decreased by the reduction in intracellular GSH levels caused by its oxidation by ROS. Previous studies have shown that doxorubicin sensitivity increases and free radical damage to cells increases when cellular reduced glutathione levels are low (37).

According to reports, reactive oxygen intermediates (ROI) are crucial to doxorubicin toxicity. Increased free radical generation, reduced ROI breakdown by antioxidant enzymes (AOE), or a combination of the two can all contribute to increased ROI concentration under pathological conditions. An increase in production has the power to start lipid peroxidation (38).

Lipid peroxidation is an influential cardiotoxicity index of doxorubicin, especially under conditions of acute exposure. Doxorubicin was found to be more selective in inducing this oxidative damage in the heart compared to other organs such as the liver in mice (39). Cisplatin and transplatin decreased lipid peroxidation in the liver, kidney, and platelets (40 and 41). Our study showed that the concentration of MDA as an indicator of lipid peroxidation in liver, kidney, and heart tissues showed a significant decrease compared with the control group. Possibly due to the low levels of free radicals produced by this drug or in these tissues mentioned above, cell membranes are less susceptible to oxidative damage by free radicals.

Pre-irradiation treatment with P. headroom improved liver G-S-T, according to Mittal et al. (43). According to Awasthi et al., (44), GSH creates a complex molecule with doxorubicin in the presence of a G-S-T catalyst to create a drug conjugate that is less hazardous than the parent chemical. The data from our investigations are consistent with those from two prior studies.

A key role of G-S-T is the detoxification of endogenous products of lipid peroxidation, such as 4-hydroxy-2-nonenal. Human G-S-TA 4-4 has very high activity towards this substrate and is critical in physiology for protecting against oxidative stress induced by endogenous lipid peroxidation. It is possible that will play a role (45).

Shepherd et al. (46) noted that glutathione reductase activity increased in liver tissue, which is consistent with our report of a group of liver, kidney, and heart tissues that were treated with doxorubicin. Increased levels of free radicals or a metabolite of an anticancer medication may cause enzyme inactivation.

 Antioxidant enzyme (GPx) parameters serve as one of the indicators of oxidative stress on tissues after doxorubicin treatment in rats. The GPx reduction of the anticancer drug doxorubicin plays a protective role, consistent with previous reports by Staherin et al. (22). I agree.

Catalase levels in liver tissue were increased in models of the anticancer drug doxorubicin, suggesting quenching of hydrogen peroxide. The findings of our study are consistent with those of the authors Yin et al. (47). Presumably, this attack by free radicals and reactive oxygen species on hepatic doxorubicin was potent enough to enhance the activity of catalase. Mean CAT concentrations are already significantly reduced in kidney and heart tissue. Branden et al. (38) concluded that after 10 weeks of doxorubicin treatment in rats, CAT levels were significantly reduced, supporting our results.

According to Bachur et al. (48), the enzyme CYTp450 is in charge of doxorubicin’s one-electron reduction into a semi-quinone free radical.

It’s possible that reduced CYTp450 levels have slowed down the metabolism of the medication doxorubicin. In the instance of rats treated with hepatic and renal tissues, our result agrees with Nebert and Russell’s (49) findings the elevated levels of cytochrome p450 in the cardiac tissue of the doxorubicin-treated groups may be the result of an increase in both the substrate and their metabolism.

Nebert and Russell (49) reported that CYTp450 and b5 play critical roles in the process of xenobiotic biotransformation and bioactivation. There is a possibility that cytochrome b5 depletion indicates the involvement of ROS in renal doxorubicin-induced enzyme inactivation, while cytochrome b5 may be involved in liver and heart cases. This is due to the strong interactions between this enzyme and different CYT-p450 species.

The cell’s overall protein patterns vary in response to medication therapy. These changes in protein levels may be caused by drug-induced denaturation and degradation of existing proteins or by the inducible synthesis of newly synthesized or existing proteins.

In the current study, it is evident that doxorubicin treatment causes small, visible alterations in the liver cells that can be seen when the tissues are microscopically examined. There are several freely available experimental models. Zicca et al. (50) showed that cisplatin-induced rat parenchyma exhibits structural alterations, which leads to hepatic injury, which contrasts with our findings. After administering CCl4 to rats, Bridges et al. (51) observed that altered liver function occurred. According to recent studies, doxorubicin therapy causes minor liver damage. After receiving doxorubicin treatment, the renal cortex displayed an almost normal appearance. In contrast, the glomeruli and glomeruli quadrants showed little change in the kidney. In a case of 10 weeks of doxorubicin treatment, Branden et al.’s (38) findings matched those of our doxorubicin model.

At the level of light microscopy, long-term doxorubicin therapy does not result in any appreciable modifications, while at the level of electron microscopy, histological points of view show little change in histological, ultrastructural, and biochemical characteristics.

In summary, our data demonstrate that in the doxorubicin model, anticancer drug-induced organelle changes are observed by light and electron microscopy. Enzymatic and non-enzymatic changes were examined in the liver, kidney, and heart using doxorubicin, compared to controls, and highlighted.

The current study aims primarily to understand the antioxidant architecture of commonly used anticancer drugs and their toxic effects on the detoxification system in experimental rat models. Finally, we examined the effects of anticancer agents such as doxorubicin on albino rats.

Conclusion

According to the findings of the current study, the anticancer drug Doxorubicin (Adriamycin) has a significant influence on rats used as an animal model, and these findings are crucial for understanding the mechanism of toxicity on many organs like the liver, kidney, and heart.

Free radicals cause tissue oxidative stress, which can be prevented by altering enzyme and antioxidant levels. This research illustrates the effectiveness of a key chemotherapeutic drug in the management of mammalian cancer.

A combination of doxorubicin and etoposide along with cisplatin, 4-hydroxyperoxycyclophamide, and vindesine has been reported to successfully kill tumors with synergistic antitumor activity in vitro (52). According to our results, it can be assumed that the combination of these drugs, doxorubicin and etoposide, or other drugs such as cisplatin, can improve activity and minimize side effects. 

Acknowledgment

I would like to thank Princess Tharwat University College, affiliated with Balqa Applied University, Muhawish Yusuf, Amman, Jordan, for providing help, assistance, and support throughout my research. We thank the Dean and Vice Dean of Princess Tharwat University College, affiliated with Balqa Applied University, Muhawish Yusuf, Amman, Jordan, for making available research, and development facilities to undertake the present work. And technical support with regard to the computation of the data is gratefully acknowledged.

Conflict of Interest

The author declares that there are no conflicts of interest relevant to this article.

Funding Source

There is no fund was received.

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