Protective Role of Phoenix dactylifera Date Palm Pollen against Doxorubicin-Induced Toxicity in the Liver, Kidney, Testis and Biochemical Parameters in Male Rats

Date palm pollen (DPP) has a prophylactic role, known for its antioxidant effects. For this, the present study was designed to study the protective role of DPP against histological toxicity in hepatic, renal, and testicular tissues, as well as the level of (Total cholesterol, Triglyceride, Total proteins, Albumin, and testosterone hormone) in male white rats. The Iraqi date palm trees (Phoenix dactylifera L.) were selected from Salah Aldeen farms located at Salah Aldeen governorate. Pollen was collected from 20th March to 25th April 2019. This study used 20 male white rats that were distributed to four groups and weights close. All groups were given food and water continuously throughout the experiment, the second group (DPP) was given an orally administered suspension of date palm pollen (60 mg/kg) every day for four weeks, the third group intraperitoneal injections were given (10 mg/kg), and the fourth group (10 mg/kg) were given intraperitoneal injections one time per week and four weeks, in addition to that, orally-administered suspension of DPP (60 mg/kg) was given as well every day for four weeks. The results of the study showed a significant increase (P≤0.05) in the level of total cholesterol (TC), triglycerides (TG), and Original Research Article Hassan et al.; JPRI, 33(37B): 158-167, 2021; Article no.JPRI.70980 159 a significant decrease (P≤0.05) in the level of Total Protein, Albumin, and testosterone hormones, Current results suggest that the protective effect of (DPP) may by its antioxidant properties, and thus reduce the positive effects.


INTRODUCTION
Plant-derived crude medicines and nutraceuticals are commonly thought to have beneficial benefits in the treatment of liver and kidney disorders in herbal folk medicine. The fruits of the date palm Phoenix dactylifera L. (Arecaceae) are widely utilized in a variety of daily food across the world. Dates are high in dietary fiber, vitamins such vitamin C, B1, B2, and A, and minerals. To prove their usage in traditional medicine, several in vitro and in vivo investigations have been undertaken [1]. Antioxidant [2], anti-inflammatory [3], hepatoprotective, anticancer [4], nephroprotective [5], antimicrobial, antiallergic [6], immunostimulatory, antifungal [7], and gastrointestinal transition stimulatory activities have all been demonstrated in date extracts [8].
In an Alzheimer's disease transgenic mouse model, date supplementation proved to be beneficial [9]. The bioactive components of dates with strong antioxidant properties, such as tannins [10] and polyphenols (which comprise phenolic acids, hydroxybenzoic and hydroxycinnamic acids, flavonoid glycosides, and proanthocyanidins), are thought to be responsible for their pharmacological actions [11]. Date pit extract was additionally demonstrated to hinder DNA damage and mediate antigenotoxic effects [12]. Earlier research have revealed that date palm pollen (DPP) possesses allergic and antigenic components [13]. Despite this, mounting research suggests that DPP has a wide range of medicinally beneficial benefits. DPP has been demonstrated in clinical trials to decrease oral mucositis in people [14] and to have antiinflammatory and antiproliferative properties in rats [15]. Surprisingly, recent investigations in rats have found that DPP extracts reduce testicular dysfunction and thyroid dysfunction caused by cadmium poisoning [16]. DPP, on the other hand, has been found to affect reproductive characteristics in adult male rats [17].

Doxorubicin
(DOX), also known as hydroxydaunorubicin, is a potent anticancer drug that has gained widespread acclaim in recent years for its use in the treatment of a variety of hematological and solid malignancies. However, its harmful perspective, cardiotoxicity, hepatic damage, nephrotoxicity, and testis toxicities' have reticent in its clinical practice [18]. Doxorubicinol toxicity is linked to the creation of reactive oxygen species (ROS), which chelates with iron and affects bio-macromolecules as well as the production of free radicals [19]. Furthermore, ROS produces oxidative stress, lipid peroxidation, DNA breakage, and protein oxidation, all of which harm membranes and macromolecules [20]. It has also been linked to genotoxicity, as evidenced by enhanced cellular death and a strong affinity for chromosomal DNA and nucleosome eviction and replacement [21]. The toxicity of the DOX is represented by several factors that lead to liver damage, which are the disturbance of vital intracellular activities as a result of the metabolism of doxorubicin to the hepatotoxic doxorubicinol [22]. Dox-induced renal toxicity might be part of a multi-organ dysfunction assisted by the buildup of free radicals, which eventually leads to membrane lipid peroxidation [23]. DOX can cause nephrotoxicity by having a negative impact on renal tissue, since it accumulates mostly in the kidney; increasing the permeability of glomerular capillaries; and causing tubular degradation [24]. DOX has been shown to impair testicular tissue, causing testicular damage, apoptosis, and a reduction in DNA synthesis, according to research [25].
The present study was designed to examine the protective role of DPP against histological toxicity in hepatic, renal and testicular tissues, as well as the level of (Total cholesterol, Triglyceride, Total proteins, Albumin, and testosterone hormone) in male white rats.

Dosage Preparation of Date Palm Pollen (DPP)
Iraqi date palm trees (Phoenix dactylifera L.) were selected from Salah Aldeen farms located at Salah Aldeen governorate. Pollen was collected from 20th March to 25th April 2019. Any visible impurities, as well as spathe fragments, were eliminated. The pollens are removed from the kernels using a fine gauze filter and then kept chilled (4°C) in a well-closed container for 3 hours in an incubator at 35°C. [20] Sprague Dawley male white rats, Age (6 -8) months and weigh (1500 -1100) kilograms. Animals were placed in cages designed for this purpose and divided into [4] groups. These animals were subjected to laboratory conditions that included 12 hours of light and 12 hours of darkness. The degree of heat was established at (22 ± 2) ℃. Cages were taken into account, clean, and sterilized. The animals were left for two weeks to adapt to the new breeding conditions and to ensure that they were free of diseases, and given food and water continuously throughout the experiment.

Experiment Design
This study used [20] males from mature white rabbits distributed to [4] groups, each group included [5] animals with close weights: 1.The first group (control group): 2.The second group (The DPP group): This group was given an orally administered suspension of water and DPP date palm pollen (60 mg/kg) every day for four weeks.
3.The third group (The DOX group): This group was given intraperitoneal injections of doxorubicin were given (10 mg/kg) per week and for four weeks.

4.The fourth group (The DOX and DPP group):
This group was given intraperitoneal injections of doxorubicin were given (10 mg/kg) once a week and an orally administered suspension of DPP date palm pollen (60 mg/kg) every day for four weeks.
All groups were given food and water continuously throughout the experiment.

Collection of Blood Samples
Blood samples were taken four weeks after the trial began. After that, blood samples were taken from the heart, and the blood was collected in anticoagulant-free plastic tubes and kept at room temperature for 15 minutes to allow the blood to coagulate, after which the tubes were centrifuged at 3000 r / min for 15 minutes to get serum blood. The serum was frozen by freezing at a temperature of 20-°C until the required chemical analysis, then the Liver, Kidney, and Testes were removed for histological study.

Biochemical Tests
The Cholesterol and Triglycerides concentration was estimated on [26] using the analysis kit manufactured by the French company (Biolabs SA, France). The Total proteins and albumin was estimated by using the analysis kits manufactured by (Biolabs SA, France), and the estimation of the concentration of the testosterone based on the technique of Elisa sandwich using the analysis kits manufactured by (Monobind INC, U.S.A).

Histological Preparations
After the animals were dissected, the liver, kidney, and testes were removed and washed with a physiological solution. Samples were prepared using microscopic tissue sections [27]. Using hematoxylin and eosin. After completing the preparation of the microscopic tissue sections, they were examined by optical microscopy.

Statistical Analysis
Statistical analysis was conducted by ANOVA Analysis of Variance. The significant differences were determined according to Duncan's multiple ranges and at a significant level (P≤0.05) [28].

RESULTS
The result in Table 1 showed a significant increase (P≤0.05) in the level of (TC and TG) in the(DOX) animal group compared to the healthy control group. It is noted that the group of animals that gavage with (DOX+DPP), (DPP) showed a significant decrease (P≤0.05) in the level of (TC and TG) compared to the group of (DOX) animal. Table 1 also showed a significant decrease (P≤0.05) in the levels of (Total proteins and albumin) in the (DOX) animals group compared to the healthy control group. It is noted that the group of animals that gavage with (DOX+DPP), (DPP) showed a significant increase (P≤0.05) in total protein and albumin levels compared to the group of (DOX) animals. It also showed a significant decrease (P≤0.05) in total proteins and albumin levels in the (DOX) animals group compared to the healthy control group. It is noted that the group of animals that gavage with (DOX+DPP), (DPP) showed a significant increase (P≤0.05) in the level of (Total protein and albumin) compared to the group of (DOX) animals.

Histological Study of the Liver
Microscopic inspection of the healthy control group's liver tissue sections and (DPP) group showed the natural shapes of the Central vein (CV), Radial arrangements of hepatocytes (HC), and Sinusoids(S) as in (Fig. 1a and b). Whereas in the (DOX) group many histological changes included thickening wall (TW) of central vein, hepatocytes necrosis (N), degeneration(D), karyolysis (KL) the usual radial arrangements of the hepatocytes were segmentally lost, infiltration of lymphocytes between hepatocytes and hemolysis (HL), as shown in (Fig. 1c). As for the group (DOX+DPP), an improvement in the central vein greatly reduced chemotherapeuticinduced degeneration and necrosis, but the radial organization of the hepatocytes was mostly preserved with an increased number of individual kupffers cells, as demonstrated in (Fig. 1d).

Histological Study of Kidney
The normal forms of the glomerulus (G), Bowman's capsule (BC), proximal urinary tubules (PUT), and distal urinary tubules (DUT) were observed in the renal tissue sections of the healthy control and (DPP) groups ( Fig. 2a and b). The renal micrographs of the rats treated with (DOX) only exhibited severe tissue damage in the form of the most glomerulus (DG) and there bowman's capsules (DB), degeneration (D), and necrosis(N) numerous of proximal and distal urinary tubules, separate of fibrocystic cells (FB) as shown in (Fig. 2c). Animals revealed DOX +DPP almost showed nearly normal tubular and glomerular tissue architecture (Fig. 2d).

Histological Study of Testis
Normal seminiferous tubules (SNT) with normal spermatogenesis (SPG) and interstitial Leydig cells (LD) were seen in the testicular histological sections of the healthy control and (DPP) groups ( Fig. 3a and b). In the rats treated with (DOX) were affected by less spermatogenesis(SPG) and Leydig cells (LD) with necrotic materials (NM) as shown in (Fig. 3c). Except for the appearance of many intratubular vacuolations, seminiferous tubules in rats treated with (DOX+DPP) seemed virtually normal (Fig. 3d).

DISCUSSION
Chemotherapy for cancer has been linked to side effects in numerous organ systems, including the liver, kidneys, and testes. It was hypothesized that combining a chemotherapeutic medication with cell protecting agents might be an effective strategy to decrease the risk of side effects [29]. The current work investigates the previously unknown hepatic, renal, and testicular protective effects of date palm pollen (DPP) therapy in rats when they are exposed to doxorubicin toxicity. To this aim, DPP therapy in rats reduced DOXinduced changes in indicators of hepatic, renal, and testicular function. DPP therapy also reduced DOX-induced oxidative stress in hepatic, renal, and testicular tissue, resulting in less redox-sensitive tissue damage.

Fig. 1. All sections of liver stained with (H&E) 400x, the control group (A) and the group treated with DPP (B) show the normal stretcher central vein (CV), Hepatocytes (HC), Sinusoids (S), Kupffer cells (KC). Figure(C): section of the liver of the group treated with DOX only shows Thickens of central vein wall (TW), Degeneration (D), Karyolysis (KL), Fibrosis (F), Congestion (COV). Figure (D): section of the liver of the group treated with DOX and DPP showing the normal stretcher of central vein (CV), Kupffer cells (KC), Hepatocytes (HC), and sinusoids (S)
Doxorubicin-based chemotherapy is used to treat a wide spectrum of solid tumors and hematological cancers. It's also linked to organ toxicity, including the heart, liver, kidneys, and testicles [30]. It has also been linked to genotoxicity, as evidenced by enhanced cellular death and a strong affinity for chromosomal DNA and nucleosome eviction and replacement. DOX is converted in the liver to doxorubicinol, which is poisonous [31]. DOX targets CYP 450-mediated hepatic glutathione depletion; downregulation of hepatic glutathione causes mitochondrial dysfunction, reactive oxygen species production, and tissue necrosis [32]. In this study, the cholesterol and triglyceride levels in the doxorubicin-treated group (G3) were significantly higher than in the healthy control group. As a result of these alterations, the doxorubicin therapy may cause an increase in oxidative stress and lipid peroxidation [33], It might also be related to the harmful impact of DOX on the liver [34]. DOX-induced acute nephrotoxicity is mostly regulated by proximal tubule cell damage, which is still being studied [35]. Chemotherapy-induced severe renal tubular deficiencies result in acute renal failure [36]. The underlying reasons of DOX-induced renal damage might be inflammation, which increases the formation of reactive oxygen species (ROS), oxidative stress, apoptosis, and a reduction in antioxidant enzymes in the kidney [37]. Total protein and albumin levels in the afflicted animal group (G3) are significantly lower than in the healthy control group. The reason for this is due to doxorubicininduced nephrotoxicity, which resulted in renal failure. The iron-anthracyclin complex causes an increase in the formation of free radicals, which causes severe damage to vital molecules, a b c d resulting in a defect in nephron function and protein loss in the urine [38]. DOX's anticancer and toxic actions are caused by the production of intracellular ROS and free radicals, which intercalated with DNA and inhibited topoisomerase [39]. Due to the high polyunsaturated fatty acid content of mammalian spermatozoa and the intrinsic lack of the superoxide dismutase (SOD) family in the testis, spermatozoa may be more susceptible to free radical damage [40]. ROS and their metabolites can damage DNA, proteins, and lipids, as well as disrupt enzymatic systems, induce cell death, and eventually lead to a drop in sperm parameters, which is associated with male infertility [41]. In the afflicted animal group (G3), testosterone levels are much lower than in the healthy control group. Furthermore, El-Maddawy et al [42] treatment with DOX caused a decrease in the testosterone concentration. DPP generated a substantial decrease in high lipid profiles and a significant rise in total protein, albumin, growth, and testosterone hormone levels, according to our findings. DPP therapy, on the other hand, has a strong antioxidant impact on DOX-induced changes in hepatic, renal, and testicular malondialdehyde, as well as non-protein sulfhydryl concentrations, according to our findings. The redox sensitivity of DPP's protective properties may be mediated by one or more of its bioactive components. DPP was shown to be nutrient-dense, including amino acids, vitamins, and minerals. Surprisingly, DPP was discovered to contain antioxidant phytochemicals like as rutin, flavonoids, and phenolic compounds [43]. In animal models, these phytochemicals have also been demonstrated to have hepato-and nephroprotective properties. DPP therapy, it turns out, can reduce the negative consequences of cadmium-induced testicular damage by preventing increased oxidative stress [44]. DPP's protective effects on the liver, kidneys, and testes have not been documented to our knowledge. Additional putative mechanisms that may contribute to the protective effects of DPP treatment include its previously reported. DOX poisoning causes an inflammatory reaction in the hepatic tissue, therefore it has anti-inflammatory properties [45]. In animal models, compelling evidence shows that DPP bioactive components have anti-inflammatory properties. As a result, it's tempting to hypothesize about DPP's and its components' anti-inflammatory properties [46].

CONCLUSIONS
The administration with Date Palm Pollen prevented oxidative damage in the hepatic, renal, and testicular tissue of the experimental rabbits. DPP's protective effect can be attributed to its a b c d membrane stabilizing, antioxidant, and antihyperlipidemic properties, which are mediated by biochemical marker regulation. The powerful and beneficial components in DPP, including as flavonoids, vitamins, amino acids, polyphenols, carotenoids, minerals, fatty acids, and organic acids, can be attributed to a wide range of pharmacological effects. Furthermore, the findings imply that DPP might be useful in identifying treatment potential for DOX-induced hepatic, renal, and testicular damage.

CONSENT
It is not applicable.

ETHICAL APPROVAL
All animals used in this study was authorized by the ethical clearance from the local committee of Samarra University.