Protective Effects of Tamarillo (Solanum betaceum) Extract Against Apoptosis in Lead Acetate-Induced Mice Testicular Damage

Introduction

Tamarillo (Solanum betaceum Cav.) is a fruit species native to the Andean regions of South America, Southeast Asia, and the North Island of New Zealand.^1–3 Due to its high resemblance in its flesh texture to a tomato, it is also known as a “tree tomato”.² Ripened fruit broadly distinguished by the colors of yellow-orange, red, and purple exhibiting a slightly bitter, sour, and astringent taste with a characteristic aroma.⁴ These flavors originate from the fruit’s 70 volatile compounds and organic acids.^5–10 Among these compounds, phenolics, carotenoids, and anthocyanins are considered to be the main bioactive components for health-promoting benefits.¹¹ Furthermore, Solanum betaceum is also known for its high nutritional contents such as vitamins A, B6, and C, dietary fiber, and potassium, higher than lemon, banana, pomegranate, and blueberry.¹² The health benefits of Solanum betaceum consumption include antioxidative, antiproliferative, antinociceptive, anti-inflammatory, allergenicity, anti-obesity, and antimicrobial properties.¹³ Despite possessing numerous beneficial properties, Solanum betaceum has yet to be fully exploited due to its unique flavor and color, which remain unpopular.²

Flavonoid represents a remarkable group of plant secondary metabolites and has been widely studied for their potential to counteract the harmful effects of lead toxicity, which induces apoptosis and oxidative damage in various human tissues.¹⁴ In total, 800 variants of flavonoids have been explored since their first discovery in the 1930s.¹⁵ The pharmaceutical roles of flavonoids include cytotoxic, anticancer, anti-inflammatory, antiviral, antibacterial, cardioprotective, hepatoprotective, neuroprotective, antimalarial, antileishmanial, antitrypanosomal, and antiamoebic.^15–18 This is due to their free radical scavenging mechanism, metal chelation capabilities, and highly accurate protein-binding activity.¹⁹ Among these flavonoids, anthocyanin dominates the phenolic composition in Solanum betaceum.²⁰ Anthocyanins such as cyanidin, delphinidin, and pelargonidin rutinosides have been discovered in Solanum betaceum from Brazil, Colombia, Ecuador, and New Zealand.^4,21–23 Anthocyanins are water-soluble flavonoids generally utilized as a coloring pigment widely present in fruits and vegetables.²⁴ Anthocyanins extinguish reactive radical species by hydrogen atom transfer.^25–29

Concerning the anthocyanin levels in other natural sources, some studies have presented evidence of such compounds initiating protective mechanisms towards various diseases. One is anthocyanin extracted from purple yam potentially preventing lead-caused reproductive toxicity due to its antioxidant, anti-apoptotic properties, and JNK signaling pathway.³⁰ Furthermore, a study on anthocyanin (sourced from black beans) active role against varicocele-induced model presented the prevention of oxidative damage, which induces active spermatogenesis and production of high-quality sperm cells.³¹

This study aimed to analyze the histological profile of apoptosis, Caspase-3, and the superoxide dismutase (SOD) expression in the testis of mice exposed to lead acetate. Hence, deepening the understanding of the Solanum betaceum in testicular protective effect and exploring the possibility of its use in lowering infertility rates.

Materials and Methods

Materials and Equipments

The material used in this study were Balb/C male mice (Mus musculus), Solanum betaceum fruit, ethanol, filter paper, CMC powder, lead acetate, phosphate buffer saline (PBS), methanol, ether, aquabidest, xylol, paraffin, entellan^®, ketamine, xylazine, NaCl, Bouin’s fixative solution, hematoxylin counterstain, Terminal deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL; 12156792910, Roche), proteinase-K, hydrogen peroxide, SOD mouse monoclonal antibody (Sigma-Aldrich, SAB4200807) and caspase-3 mouse monoclonal antibody (Sigma-Aldrich, C5737), diaminobenzidine (DAB), Trekkie Avidin-HRP, and Trekkie Universal Link. All substances used were of analytical grade. Necessary instruments for gaining data were a refrigerator, rotary microtome, micro-scale, syringe, sonde, scalpel, pipette, micropipette, tweezers, object glass, pencil, light microscope, hand counter, timer, micrometer, aluminium foil, computer with statistical program.

Plant Materials

Solanum betaceum fruit was collected from farmland in Wonosobo, Central Java, Indonesia (GPS coordinates: 7.3633°S, 109.9031°E). Identification was based on morphological characteristics using standard botanical references and confirmed by consensus among trained staff at the Research Centre for Biology of the Indonesian Institute of Science (LIPI). A voucher specimen (No. 724/IPH.1.01/If.07/III/2017) is deposited at the LIPI herbarium.

Preparation of Ethanolic Extract of S. betaceum

S. betaceum fruit was dried by a fresh air dryer. Dry powder was extracted by maceration using ethanol for three days at room temperature with solvent replacement every 24 hours. The liquid extract was filtered through filter paper to separate filtrate and residue. The liquid extract of S. betaceum was evaporated with a rotary vacuum evaporator to obtain a viscous extract. The ethanol extract of S. betaceum was added to the treatment diet as a suspension using 1% CMC with dose of 2 mL/200 g. According to previous protocols, S. betaceum extract was simultaneously administered to the treatment group three days before lead acetate exposure for 35 days according to previous protocols.³²

Preparation of Lead Acetate

Lead acetate was used at a dose of 75 mg/kg, dissolved in distilled water, and saved at room temperature.³²

Experimental Design

A post-test control group experimental study was conducted using 12-week-old male Balb/C mice (25–30 g) to evaluate the gonadoprotective effect of the extract of Solanum betaceum (tamarillo) crude extract (TCE). Mice showing signs of illness, mortality, or more than 10% body weight loss during the study were excluded. A total of 35 mice were randomly assigned into five groups (n = 7 per group): a negative control group (K-), a positive control (K+), and three treatment groups (P1, P2, P3). The K-group received distilled water orally throughout the study. In contrast, the K+ group received lead acetate at 75 mg/kg dissolved in distilled water, by oral gavage once daily for 32 consecutive days (starting day 4 to day 35). The treatment groups (P1, P2, P3) received TCE at doses of 100, 200, and 400 mg/kg, respectively, by oral gavage once daily for 35 days. In these groups, lead acetate (75 mg/kg) was administered one hour after the TCE dose, starting from day 4 through day 35. The pretreatment design was adapted from a previously published study.³³ Mice were acclimated for 7 days before the experiment, maintained under a 12-hour light/dark cycle at room temperature.³⁴ On day 36, mice were euthanized under intraperitoneal anesthesia using ketamine (70 mg/kg) and xylazine (20 mg/kg), and both testes were excised for further analysis.

Immunohistochemical Analysis

Median laparotomy was conducted, and the testes were collected, then washed in 0.9% physiological NaCl solution and placed in Bouin’s fixative solution. Testicular tissue was processed using the standard method of embedding with paraffin. Then, the tissue block was sliced using a rotary microtome with a thickness of 4–5 μm and stored in an incubator at 65°C for 3 minutes to complete tissue attachment.³⁵ Tissue sections were washed for 15 minutes with three changes of PBS between each step. After deparaffinization and rehydration, tissue sections were exposed to 3% H₂O₂ for 10 minutes and methanol for 3 minutes to inactivate endogenous peroxidase activity and then to 10% normal serum for 45–60 minutes to block non-specific proteins. Following rinsing with PBS, the tissue section was then incubated with a primary antibody of SOD or Caspase-3 for 48 hours at 4°C.³⁶ The result of the antigen-antibody reaction was visualized using DAB for 2 minutes, followed by washing and counterstaining with hematoxylin for 10 second and rinsing with aquabidest. The tissue sections were then dehydrated with a series of alcohol and cleared with xylol. The last step was mounting using entelan. As a control of staining, tissue sections were incubated with PBS instead of primary antibody. The control staining slide showed a negative reaction with minimal background staining. Positive SOD or Caspase-3 staining was observed as brown stains in the tissue under a light microscope. The qualitative observation of positive tissue reaction was based on the brown color intensity and distribution of the immunoreaction product in the testicular tissues. The quantitative observation was done by counting the number of cell nuclei that give different levels of brown color intensity in 10 random fields from each testicular tissue section with 400x magnification. A blue color in the cell nuclei indicates a negative reaction. ImageJ software was used to count various brown-stained cell nuclei. The percentage of SOD and caspase-3 expression were compared between groups.³⁷

TUNEL Analysis

Testicular cell apoptosis was evaluated using a TUNEL assay based on manufacture instructions. The TUNEL method detects fragmented DNA in the nucleus during apoptotic cell death in situ. Following the de-paraffinized and rehydrated of 5–6 μm thick tissue sections in xylene and graded ethanol series, the slides were treated for 30 min with 15 ug/mL proteinase-K and washed with PBS. Endogenous peroxidase activity was inhibited by 3% hydrogen peroxidase. Subsequently, the slides were incubated with 25 μL TUNEL solution (50 μL of enzyme solution and 450 μL label solution) for 60 minutes at 37°C. They were put into pre-warmed working strength wash buffer at room temperature for 10 minutes and incubated with blocking buffer for 30 minutes. Thorough washes in PBS separated each step. In one observation field, propidium iodide was added to compare apoptotic and non-apoptotic cells. Labelling was visualized using DAB chromogen, followed by hematoxylin counterstain, and sections were dehydrated, cleared, and mounted. Quantitative analysis of testicular apoptosis was estimated according to Hu et al³⁸ by direct observation through a fluorescence microscope (Olympus, Tokyo, Japan). TUNEL will only detect apoptotic cells and give green fluorescence, while propidium iodide will detect non-apoptotic cells and give red fluorescence.³⁸ Examination was carried out by counting positive cells at 400x magnification and counted in 10 random fields from each testicular tissue section.

Data Processing and Analysis

All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) 26.0. Data were initially tested for normality using the Shapiro–Wilk test. Variables with p>0.05 were considered normally distributed and analyzed using one-way ANOVA. For variables that did not meet the normal distribution (p<0.05), non-parametric tests were applied, specifically the Kruskal–Wallis, followed by Mann–Whitney U-tests. All data were presented as mean (±) standard deviation (SD), and statistical significance was accepted at p<0.05.

Results

Tamarillo Extract Decreased Apoptosis of Testicular Damage

Apoptotic cells in the testis of the control and treatment groups were identified by TUNEL assay, as shown in Figure 1. The highest increase in the percentage of apoptotic cells in relation to negative control was observed after lead acetate exposure of 8.257 ± 1.350. Only a few TUNEL-positive cells were observed in negative control animals of 1.286 ± 0.414. However, the number and signal density of TUNEL-positive germinal cells significantly decreased in TCE at the dose of 100 mg/kg and 400 mg/kg (Figure 2 and Table 1).

Table 1 Means Distribution of Apoptosis Expression Within Experimental Groups

Figure 1 Representative of photomicrograph of TUNEL staining in mice testis of negative control (a), positive control (b), TCE at the dose of 100 mg/kg (c); 200 mg/kg (d), and 400 mg/kg (e).

Figure 2 Means of testicular apoptosis expression by TUNEL assay. Quantitative analysis of apoptosis in the testis. The percentage of mouse testicular apoptosis was calculated as the ratio of apoptosis-positive seminiferous tubules to the total number of seminiferous tubules.

The effectiveness of TCE in protecting male reproductive organs was assessed by comparing the means of the control and treatment groups. Table 1 is grouped into similar means of testicular apoptosis-level distribution, A, B, and C, respectively. The treatment groups tended to have a lower apoptosis level than the positive control group suggesting an effective dose of TCE in this range.

The statistically significant differences among the means groups were tested with the Kruskal–Wallis test (Figure 3). The treatment group P3 was found to be the most similar to the negative control group, with no significant difference in means (p=0.115) (Figure 3). This suggests that TCE at the dose of 400 mg/kg reduces similar apoptosis levels as a negative control group.

Figure 3 Effect of tamarillo extract on apoptosis was measured by comparing differences between treatment groups with the negative control group. Level of significance of p<0.05 and p<0.01 is represented with * and ** respectively.

Tamarillo Extract Decreased Caspase-3 Expression in Testicular Damage

Various degrees of caspase-3 expression in the testes of all groups were detected by immunohistochemical staining (counterstained with hematoxylin). Caspase-3 facilitates the process of apoptosis in response to testicular damage causing infertility. Histological observation on the control group showed testicular cells were observable and normal testicular architecture. However, staining dramatically increased testicular damage in the mouse exposed to lead acetate. In the mouse, treated with tamarillo extract, the number and morphological integrity of testicular cells were preserved. Observations indicated that the testicular toxic effect of lead acetate was reduced by tamarillo extract (Figure 4).

Figure 4 Representative of photomicrograph of Caspase-3 immunohistochemical staining in mice testis of negative control (a), positive control (b), TCE at the dose of 100 mg/kg (c); 200 mg/kg (d), and 400 mg/kg (e).

Almost no specific Caspase-3 immunoreactivity was found in the TCE-treated at the dose of 200 mg/kg. However, heavily labeled Caspase-3-positive cells were seen in mice exposed to lead acetate compared to other experimental groups (Figure 5).

Figure 5 Mean of mouse testicular caspase-3 expression by immunohistochemical staining Quantitative analysis of apoptosis in the testis. The percentage of mouse testicular caspase-3 was calculated as the ratio of caspase-3-positive nuclei to the total number of cell nuclei.

The mean intensity of caspase-3 staining was compared among experimental groups to analyze the amelioration of testicular damage of the tamarillo extract-treated group upon lead acetate exposure and the insignificant means distribution was grouped into Table 2. Lead acetate enhances the highest level of Caspase-3 expression of 5.886b ± 1.472. However, the treatment groups expressed lower testicular Caspase-3 than negative control, suggesting the protective effect of tamarillo extract in male mice exposed to testicular damage.

Table 2 Means Distribution of Caspase-3 Expression Within Experimental Groups

The statistically significant differences among the means groups were tested with the Kruskal–Wallis test. This study showed that the TCE-treated group at the dose of 200 mg/kg significantly lower Caspase-3 expression than the negative control group (p=0.05) (Figure 6).

Figure 6 Effect of tamarillo extract towards caspase-3 expression measured by comparing differences between treatment groups with the negative control group. Level of significance of p<0.05 and p<0.01 is represented with * and ** respectively.

Tamarillo Extract Increased SOD Expression in Testicular Damage

Tissue levels of antioxidant enzyme, SOD in each group are observed histologically in Figure 7. Morphological analysis of the mice seminiferous tubules in experimental groups showed mainly in all germ cells, especially in the adluminal compartment.

Figure 7 Representative of photomicrograph of SOD immunohistochemical staining in mice testis of negative control (a), positive control (b), TCE at the dose of 100 mg/kg (c); 200 mg/kg (d), and 400 mg/kg (e).

The highest increase in the percentage of SOD expression was in the negative control group at 8.571 ± 1.694, while the lowest increase was in the TCE-treated group at the dose of 100 mg/kg at 2.514 ± 1.014 (Figure 8, Table 3).

Table 3 Means Distribution of SOD Expression Within Experimental Groups

Figure 8 Means of mouse testicular SOD expression by immunohistochemical staining Quantitative analysis of apoptosis in the testis. The percentage of mouse testicular SOD was calculated as the ratio of SOD-positive nuclei to the total number of cell nuclei.

Table 3 is grouped based on the similar means of mouse testicular SOD intensity to show the identical antioxidant activity with the negative control group. The higher dose of TCE treatment showed increasing SOD expression in response to the same damage of testicular tissue induced by lead acetate.

TCE treatment at the dose of 400 mg/kg and negative control demonstrated comparable SOD results (p=0.262) (Figure 9), suggesting the effective dose of TCE to exert antioxidant activity to counter oxidative damage induced by lead acetate.

Figure 9 Effect of tamarillo extract towards SOD expression measured by comparing differences between treatment groups with the negative control group. Level of significance of p<0.05 and p<0.01 is represented with * and ** respectively.

Discussion

This study was intended to investigate the effectiveness of tamarillo extract in protecting male fertility following lead acetate exposure. Findings indicate that oral administration of lead acetate significantly increased biomarkers of apoptosis in mice testicular tissue compared to the untreated group. Lead is a major human health hazard present in the environments and biological systems which can affect the gonadal structure and functions and can cause fertility alteration.^39,40 Our results agreed that mice exposed to the lead acetate showed irregularity in spermatogenesis, reduced plasma testosterone concentration, and degeneration of seminiferous tubules.^41,42

Recent biological agents have been investigated for gonadoprotective effects on testicular function such as dragon fruit,⁴² moringa,⁴³ chrysin,⁴⁴ and melatonin.⁴⁵ In the present study, the addition of tamarillo improved testicular histopathological changes induced by lead. Peroxidative injury and cellular macromolecules cause abnormalities in testicular tissue and the male reproductive system.⁴⁶ The mechanism of lead-induced testicular toxicity is due to the imbalance between the production of reactive oxygen species (ROS) and the scavenging capacity of antioxidants in the testes, therefore testicles are dependent on antioxidant agents to fight oxidative stress-induced damage.^39,46

The results of the present study clearly showed that the levels of apoptosis and Caspase-3 are remarkably increased in mice treated with lead acetate. Lead promotes free radicals and lowers the effects of antioxidants within the cells.⁴⁶ The imbalance of free radical species produced by lead exposure beyond cellular protective capacity leads to oxidative stress, ultimately resulting in cell apoptosis.⁴⁷ Lipid membranes are targeted for oxidative damage produced by xenobiotics including heavy metals.⁴⁸ Furthermore, lead exposure causes several antioxidant enzyme alterations by inhibiting functional SH groups, such as d-aminolevulinic dehydrogenase, superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT).^46,49 Pretreatment of TCE administration in P1, P2, and P3 provides a strategy to mitigate the antioxidant capacity of TCE to ameliorate lead-induced free radicals.

Apoptosis is a physiological process of programmed cell deletion, which contributes to maintaining the cell number in testicular tissue and removing damaged cells. However, excessive apoptosis could cause male reproductive dysfunction.⁵⁰ A study reported that a distinct increase of Caspase-3 in time- and dose-dependent manner induced apoptosis of germ cells, suggesting the degree of differences was correlated with the time of lead loading and exposure time to induce damage.⁵¹ Our results showed TCE 400 mg/kg is not only lower Caspase-3 to normal levels but also improve the degraded DNA in testicular cells caused by lead acetate.

Several studies used antioxidant evaluation as a marker of high levels of free radicals.^52–56 Administration of antioxidants results in increasing the activity of spermatogenesis and steroidogenesis in the testis, thereby preserving fertility. Antioxidants from tamarillo extract provide protective effects in maintaining testicular structure and function.

Free radical damage due to lead exposure depletes antioxidant reserves.⁵⁷ Cytosolic enzyme, catalase, and glutathione peroxidase enzymes break down H₂O₂, H₂O, and O₂. Meanwhile, the superoxide dismutase (SOD) enzyme catalyzes the dismutase reaction and ultimately converts anion radicals into H₂O₂.⁵⁸ In the present study, a combination of administration of TCE 400 mg/kg and lead acetate, the level of SOD was increased compared to its level in rats treated only with lead. Decreased SOD expression in testicular tissue may reflect cellular oxidative stress or compensatory mechanism in the response to cellular apoptosis. The activities of testicular SOD were significantly reduced in the lead-exposed mice, while the addition of Artemisia annua extract to lead acetate significantly improved the level of SOD.⁴⁴ Exogenous antioxidants such as vitamins C and E were also reported to have capabilities in preventing damage to germ cells and testicular tissue caused by excessive free radicals after extended lead acetate exposure.⁵⁸ A study reported the activity of antioxidants collected from tamarillo extracts with a dosage of IC50 was 1162,608 ppm, which can decelerate lipid peroxidation reaction which is caused by the increase of ROS induced by lead acetate exposure.⁵⁹ The capability of tamarillo extract to maintain testicular SOD expression seems to be dependent on the dosage. The highest dosage of tamarillo extract 400 mg/kg was comparable to the untreated group, suggesting the critical dose for TCE exerting antioxidant properties against oxidative stress caused by lead acetate. This effect could be due to the presence of anthocyanin and flavonoids, which can fend off various free radicals.⁶⁰

Conclusion

This study showed that the administration of Solanum betaceum had a significant effect on maintaining testicular structure. Tamarillo exhibited a protective effect on the reproductive system by mitigating lead acetate-induced oxidative stress and excessive cell apoptosis. This might be due to the improved superoxide dismutase level and decreased caspase-3 expression in the testis of male mice.