Introduction

Cancer incidence has increased due to population growth and aging as well as the increasing prevalence of risk factors such as smoking, obesity, and adoption of a Western lifestyle (1). Doxorubicin (DOX), an anthracycline antibiotic, is commonly used in the treatment of solid tumors in adult and pediatric patients, including breast, stomach, thyroid, liver, and ovarian cancers, as well as human hematological malignancies (2). The anticancer activity of DOX is mainly based on its intercalation into the DNA structure and the inhibition of topoisomerase II enzyme in rapidly proliferating cancer cells (3). However, most anticancer drugs, including DOX, act nonspecifically and damage both malignant and healthy tissues (4). The toxicity of DOX is attributed to mitochondrial dysfunction, reactive oxygen species (ROS) production, stimulation of apoptosis, lipid peroxidation, DNA damage, impaired calcium handling, and induction of p53 (5). Typical adverse effects of doxorubicin include myelosuppression, mouth ulcers, alopecia, fatigue, nausea, and vomiting. Additionally, DOX can cause disruption in lipid profile due to free radical-mediated processes, leading to dyslipidemia and an increased risk of cardiovascular disease (6). As the liver is the primary organ involved in detoxification and biotransformation of drugs, any damage to liver function is particularly significant (7). Such damage to the liver may occur due to oxidative stress and apoptosis (8).

Despite the development of new cancer drugs that have improved the life expectancy of cancer patients, the acute and long-term side effects of cancer chemotherapy remain an unsolved problem in the management of oncological diseases (9, 10). In this context, herbal medicine has gained attention as a potential complementary approach, owing to its perceived safety, efficacy, cost-effectiveness, and better compatibility with conventional treatments.

Jatropha tanjorensis, commonly called "hospital too far," is a member of the Euphorbiaceae family. It originated in Central America and has spread to many tropical and sub-tropical regions, including Africa, India, and North America (11). In several parts of Nigeria, the leaves of J. tanjorensis are locally consumed as a vegetable and added to daily meals, as well as used in treating diabetes mellitus due to their anti-hyperglycemic properties (12). Traditionally, a decoction of the leaves is used to treat anemia, diabetes, skin diseases, malaria, and cardiovascular diseases (13, 14). Antimicrobial studies of J. tanjorensis have showed that the aqueous extract of the leaves inhibits the gram-positive bacterium Staphylococcus aureus and the gram-negative bacterium Escherichia coli (15). J. tanjorensis possesses antioxidant properties and contains vitamins and ions, including phosphorus, selenium, and zinc (16, 17). Phytochemical analysis of J. tanjorensis leaves has shown that the plant is rich in cardiac glycosides, flavonoids, tannins, terpenoids, and saponins (16, 18).

Given the potential of J. tanjorensis as a natural therapeutic agent, this study aimed to evaluate the protective effects of J. tanjorensis against Doxorubicin-induced toxicity in the liver and spleen of rats.

Materials & Methods

Study Type and population:

This study employed an experimental design using 35 rats, which were randomly divided into 5 groups with 7 rats per group.

Chemicals and Reagents: Doxorubicin was obtained from Neon Laboratories Limited (India) as a solution for injection. All reagents and chemicals used were of analytical grade and highest purity.

Collection and Extraction of Jatropha tanjorensis Leaves: J. tanjorensis leaves were collected from a garden in Benin City, Edo State, Nigeria and botanically identified at the Department of Plant Biology and Biotechnology, Edo State University Uzairue, Nigeria. The collected leaves were thoroughly rinsed, air-dried at room temperature for four weeks, and pulverized into a fine powder using an electric blender. For aqueous extract preparation, 1 kg of the powdered plant material was soaked in 5 liters of double-distilled water for 48 hours at room temperature with daily stirring to ensure thorough extraction. After 48 hours, the mixture was filtered first through Whatman filter paper No. 42 (125 mm) followed by cotton wool. The resulting filtrate was concentrated to one-tenth of its original volume using a rotary evaporator at 40°C and then reduced to solid form using a water bath. The solid extract was weighed and dissolved in normal saline for daily administration to the rats.

Experimental Design/Procedure: Adult male Wistar albino rats (150-200g) were obtained and housed in cages under controlled temperature (22±2°C) with a 12-hour light-dark cycle. They were fed a standard laboratory diet and given free access to water. After a seven-day acclimatization period, the animals were randomly divided into five groups (7 rats per group):

·         Group 1 (Control): Received normal saline orally once daily for 12 days.

·         Group 2 (DOX): Received Doxorubicin injection (15 mg/kg) intraperitoneally on days 10, 11, and 12.

·         Group 3 (JT): Administered aqueous extract of J. tanjorensis (300 mg/kg) orally once daily for 12 days.

·         Group 4 (JT+DOX): Administered aqueous extract of J. tanjorensis (300 mg/kg) orally once daily for 12 days, followed by Doxorubicin (15 mg/kg) intraperitoneally on days 10, 11, and 12.

·         Group 5 (Vit C+DOX): Administered Vitamin C (100 mg/kg) orally once daily for 12 days, followed by Doxorubicin (15 mg/kg) intraperitoneally on days 10, 11, and 12.

The Doxorubicin dosage and administration protocol were based on the study by [Author et al., Year] (19). The J. tanjorensis dose (300 mg/kg) was selected based on the study by El-Moselhy et al. (2014) (20). Vitamin C was chosen as a positive control for its known hepatoprotective properties. All experimental animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Science and published by the National Institutes of Health (21).

Twenty-four hours after the final administration, the rats were euthanized and blood samples were collected in plain tubes. Samples were allowed to clot for 45 minutes before being centrifuged at 4000 rpm for 25 min to obtain sera for analysis. Serum samples were used to determine Aspartate Aminotransferase (AST), Alanine Transaminase (ALT), Acid Phosphatase (ACP), Albumin (ALB), Total Bilirubin (TB), and Total Protein (TP) levels.

Biochemical Analyses:

·         ACP activity was determined using a BIOSYSTEMS ACP assay kit following the manufacturer's protocol.

·         AST and ALT activities were determined using RANDOX kits according to the manufacturer's instructions based on the method described by Reitman and Frankel (1957) (22).

·         Albumin was determined using a RANDOX kit based on the Bromocresol green (BCG) method as described by Doumas et al. (1971) (23).

·         Total protein was determined using a RANDOX kit following the method described by Tietz (1995) (24).

·         Total bilirubin was determined using a RANDOX kit based on the method of Jendrassik and Grof (1938) (25).

Histopathological Analysis: Following euthanasia, liver and spleen tissues from all groups were excised, rinsed in normal saline, and fixed in 10% buffered formalin. Samples were then processed for histopathological examination at the Chemical Pathology Laboratory, University of Benin Teaching Hospital, Nigeria.

Statistical Analysis: Data were expressed as mean ± standard deviation. Differences among groups were determined by One-way ANOVA followed by [specific post-hoc test, e.g., Tukey's HSD] using Statistical Package for Social Sciences (SPSS) version 20. A probability level of less than 5% (P<0.05) was considered significant.

Results

The result of the Table 1 shown below established a significant increase in AST, ALT and ACP activities in Doxorubicin-administered rats compared to control, J. Tanjorensis and Vitamin C groups, an indication of hepatotoxicity. However, treatments with J. Tanjorensis and Vitamin C reduce AST, ALT and ACP activities compared to Doxorubicin-alone rats, an indication of hepatoprotection.

Table 1. Effects of aqueous leaf extract of J. Tanjorensis on liver function enzymes in Doxorubicin-induced toxicity Wistar rats

Values are expressed as Mean ± Standard Error of Mean, n=5. Values with different superscripts (a, b, c, d) down the column differ significantly at (P<0.05). AST-Aspartate Aminotransferase; ALT-Alanine Aminotransferase; ACP-Acid Phosphatase; Vit. C- Vitamin C; DOX- Doxorubicin.

The results shown in Table 2 revealed a significant decrease in the total protein and albumin of doxorubicin-treated group compared to the control and extract treated groups (p < 0.05). However, for groups administered J. Tanjorensis and Vitamin C, the values of total protein and albumin were significantly higher compared to the group that received only Doxorubicin. The result of Table 2 also showed that total bilirubin significantly increased in the doxorubicin-treated group compared to the control and extract group (p < 0.05). However, the group administered J. Tanjorensis and Vitamin C had total bilirubin with significantly lower values compared to the group that received only doxorubicin.

Table 2. Effects of aqueous extract of J. Tanjorensis on liver synthetic molecules in Doxorubicin-induced Wistar rats

Values are expressed as Mean ± Standard Error of Mean, n=3. Values with different superscripts (a, b, c, d) down the column differ significantly at (P<0.05). Vit. C- Vitamin C; DOX- Doxorubicin

The histopathological assessments of the liver were examined and showed the liver of the control and extract treated groups had visible centriole and hepatocytes with no necrosis, no congestion and no inflammation (Figure A and C). The histopathological evaluation of liver tissue in the Doxorubicin group revealed visible vascular congestion, hemorrhage, necrosis, vacuolar degeneration in hepatocytes, sinusoidal dilation, mononuclear cell infiltration in portal region and parenchyma, macro and microvascular steatosis, pyknotic and hyperchromatic nuclei in hepatocytes. However, the liver of rats given Doxorubicin and J. Tanjorensis or Vitamin C showed hepatocyte regeneration, reduced signs of liver damage, and mild degeneration of hepatocytes. Figures 1 and 2 represents Photomicrographs of five study groups in liver and spleen, respectively.

Fig. 1. A: Photomicrograph of the liver of control rats which received only normal saline showing normal hepatic architecture, with hepatocytes having a normal lobular appearance with central veins surrounded by radiating hepatic cords (X400 magnification). B: Photomicrograph of liver of rats which received doxorubicin (15 mg/kg) from day 10 for three consecutive days showing vascular congestion, hemorrhage, necrosis, vacuolar degeneration in hepatocytes, sinusoidal dilation, mononuclear cell infiltration in portal region and parenchyma, macro and microvesicular steatosis, pyknotic and hyperchromatic nuclei in hepatocytes (X400 magnification). C: Photomicrograph of liver of rats which received J. Tanjorensis (300 mg/kg) with normal hepatocytes, with visible nuclei. (X400 magnification). D: Photomicrograph of liver of rats which received J. Tanjorensis (300 mg/kg) and then doxorubicin (15mg/kg) showing reduced signs of liver damage; mild degeneration of hepatocytes (X400 magnification). E: Photomicrograph of liver of rats which received Vitamin C (100 mg/kg) and then doxorubicin (15mg/kg) (X400 magnification)

Histological analysis of the spleen of the control rats given saline alone showed a precise splenic organization with distinct identifiable regions of red and white pulp with prominent central artery as splenic cords, venous sinuses, and veins in the spleen (Figure 1). The group administered Doxorubicin alone at a dose of 15mg/kg from day 10 for three consecutive days showed white and red pulp with congestion and thickened dilatation, disintegrated peri-arteriolar lymphoid sheath, depletion of lymphoid follicle, and tangible macrophages that appears granulated, lymphoid depletion of the white pulp as well as severe necrosis of lymphoid follicles (Figure 2). The spleen of rats administered J. Tanjorensis alone at a dose of 300mg/kg showed normal splenic tissues with normal red and white pulp & peri-arteriolar lymphoid sheath (Figure 3), while the spleen of rats administered J. Tanjorensis at a dose of 300mg/kg or Vitamin C at a dose of 100mg/kg and thereafter given Doxorubicin from day 10 for three consecutive days showed recovering peri-arteriolar lymphoid sheath and restoration of splenic architecture (Figure 4 and 5).

Fig. 2. A: Photomicrograph of the spleen of control rats which received only normal saline showing white and red pulp with prominent central artery (X400 magnification)

B: Photomicrograph of spleen of rats which received doxorubicin (15 mg/kg) from day 10 for three consecutive days showing degeneration of white pulp, disintegrated peri-arteriolar lymphoid sheath, granuloma formation, severe necrosis of lymphoid follicles, thickened dilated and congested central arteries, development of vacuoles and progressing degeneration of splenic tissues (X400 magnification). C: Photomicrograph of spleen of rats which received J. Tanjorensis (300 mg/kg) showing normal Red Pulp, White Pulp and peri-arteriolar lymphoid sheath (X400 magnification). D: Photomicrograph of spleen of rats which received J. Tanjorensis (300 mg/kg) and then Doxorubicin (15mg/kg) showing recovering peri-arteriolar lymphoid sheath and restoration of splenic architecture (X400 magnification). E: Photomicrograph of spleen of rats which received Vitamin C (100 mg/kg) and then Doxorubicin showing restoration of splenic architecture (15mg/kg) (X400 magnification)

Discussion

The liver is a crucial metabolic organ that plays a key role in glycogen storage, detoxification, and protein synthesis (26). Doxorubicin (DOX) treatment is known to increase reactive oxygen species (ROS) production and oxidative damage, leading to hepatocellular injury (27). These factors contribute to the liver's susceptibility to drug-induced toxicity (28). Mounting evidence suggests that even low doses of DOX in rats can cause irreversible liver damage and elevate apoptotic processes in hepatic tissue (29).

Our results demonstrated that DOX administration (15 mg/kg) caused significant liver toxicity, as evidenced by elevated serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), confirming previous findings (30). However, co-administration of J. tanjorensis or Vitamin C with DOX improved liver function, as indicated by reduced AST and ALT levels compared to the DOX-only group. These findings are consistent with previous studies (31-33). The reduction in ALT and AST levels following treatment with J. tanjorensis leaf extract suggests hepatoprotection, possibly mediated by its constituent phytochemicals, particularly flavonoids, as previously reported (16).

Acid phosphatase (ACP), primarily located in cell lysosomes, serves as an indicator of cellular toxicity. Enhanced lysosomal membrane peroxidation can lead to membrane lysis and increased ACP release and activity (34). In our study, DOX administration (15 mg/kg for three consecutive days) significantly increased ACP activity compared to control and J. tanjorensis groups, indicating cell damage and lysosomal membrane instability. Notably, administration of J. tanjorensis or Vitamin C effectively decreased ACP activity compared to the DOX-only group, consistent with other studies (36, 37).

Liver synthetic function parameters, such as albumin and total protein, are reliable indicators of hepatic damage. Albumin, the most abundant serum protein, is primarily synthesized in the liver and serves various physiological functions, including osmotic pressure regulation, redox balance, and transport of fatty acids, bilirubin, drugs, hormones, and vitamins (38). Our results showed significantly reduced concentrations of total protein and albumin in DOX-administered rats compared to control and plant-treated groups, indicating impaired hepatic function. This reduction may be due to compromised hepatic synthesis, increased intestinal protein loss, or protein-losing nephropathy (39). These findings align with previous reports (31-33, 40). Importantly, treatment with J. tanjorensis or Vitamin C in DOX-administered rats resulted in significantly higher total protein and albumin levels compared to the DOX-only group, suggesting protective and antioxidant effects.

The elevated total bilirubin levels observed in the DOX-administered group compared to control and plant-treated groups further indicate liver dysfunction, specifically impaired bilirubin clearance. This finding is consistent with previous studies (41, 42). Treatment with J. tanjorensis or Vitamin C significantly decreased total bilirubin levels in DOX-administered rats, likely due to their protective effects against DOX-induced oxidative damage in the liver.

Our histological findings corroborated the biochemical results. The livers and spleens of DOX-administered rats exhibited histopathological features consistent with previous reports (32, 43). Notably, treatment with J. tanjorensis following DOX administration attenuated hepatic injury and degenerative changes in both the liver and spleen.

Conclusion

In conclusion, our study demonstrates that J. tanjorensis effectively mitigates DOX-induced biochemical and histological liver injury in rats. These findings suggest that J. tanjorensis has promising potential in ameliorating DOX-induced hepatotoxicity and spleen damage. To further validate these results and establish their clinical relevance, we recommend additional in vivo and in vitro studies with larger sample sizes in both animal models and human subjects. Moreover, investigations into the molecular mechanisms underlying the protective effects of J. tanjorensis against DOX-induced toxicity would provide valuable insights for potential therapeutic applications.

Acknowledgments

The authors humbly thank the staff in animal house unit of the Department of Biochemistry, Edo State University Uzairue where the in vivo study was carried out.

Financial support

None

Ethical Statement

This study was conducted in accordance with the ethical principles of the Declaration of Helsinki and according to the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Science and published by the National Institutes of Health. The study protocol was approved by the Department of Biochemistry, Faculty of Basic Medical Sciences, Edo State University Uzairue with Mat. No.: PGS/BCH/21003018.

Data availability

Data will be available as per request.

Conflict of interest

The authors have no conflict of interest in this study.