Ameliorative effect of alpha lipoic acid and royal jelly against the side effects of cyclophosphamide in liver of albino rats

Cyclophosphamide (CP) is a cytotoxic anticancer drug used for the treatment of neoplastic diseases. The present study aimed to examine biochemical, histological, and ultrastructural effects of CP on rat liver and determine the hepatoprotective effects of alpha-lipoic acid (LA) or royal jelly (RJ) against CP. The present study revealed that CP-induced significant increase in hepatic marker enzymes (ALT and AST), and elevation in malonaldehyde (MDA) was concomitant with a significant decrease of superoxide dismutase (SOD). It caused histopathological changes in the liver of rat including vacuolation, infiltration, degeneration, and necrosis. Ultrastructurally, the hepatocytes appeared degenerated with multiple small- and medium-sized lipid droplets in the cytoplasm. Kupffer cell showed a shrunken nucleus. Administration of LA and RJ resulted in an obvious improvement in the altered level of ALT, AST, MDA, and SOD activities when compared with the CP-treated group in addition to marked amelioration in histopathology and ultrastructure of the liver. LA is markedly effective than RJ in protecting rats against CP-induced biochemical, histopathological, and ultrastructural changes. This protection may be due to its antioxidant properties and scavenging abilities against active free radicals.


Background
Anticancer drugs are usually used for the treatment of cancer and have been detected in hospital effluents, wastewater treatment plant effluents, and river water samples in concentrations up to the μg L −1 range [1].
Cyclophosphamide (CP) is an alkylating agent with antineoplastic and immunosuppressive effects. One of its metabolites is acrolein which leads to toxic side effects such as cell death and oxidative stress [2]. Moreover, CP cause toxicity in healthy tissues in addition to cancerous tissues. CP in high doses leads to acute inflammation in the kidney, bladder damage, and liver damage as well as apoptosis [3,4].
CP is converted to acrolein and phosphoramide mustard, an agent that adds alkyl groups to oxygen and nitrogen atoms of guanine, one of the four nitrogen bases that form the DNA nucleotides, producing DNA crosslinks and introducing DNA breaks. Its cytotoxic and mutagenic effects principally occur in proliferating cells [5].
The liver is considered the center of drug metabolism and bioconversion, and the liver is rich in a collection of biotransformation enzymes. Drug bioconversion in the liver normally produces inactive metabolic end products which are excreted from the body but can also generate toxic metabolites [6]. Furthermore, the liver plays an essential role for the metabolism and detoxification of xenobiotics in the body [7] and enhanced production of free radicals, and oxidative stress can be induced during detoxification of xenobiotic [8].
CP leads to abnormal activities of serum enzymes that indicate cellular damage; this appears as an increase in serum transaminases ALT, AST, alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) [9]. Moreover, there were increasing levels of malonaldehyde (MDA) and GST and there was a decrease in glutathione (GSH) level and superoxide dismutase (SOD) activity [10].
CP induced histopathological hepatotoxicity in mice which is showed by inflammation in the central vein and pyknotic nuclei in hepatic cells [11], prominent intense cellular degeneration, cellular inflammation of hepatocytes with vascular congestion and sinusoidal dilatation, and the focal area of hepatic necrosis replaced by focal hepatic hemorrhage and leukocytic cells infiltration [10].
CP administration leads to ultrastructural changes such as plasma membrane disruption and widespread cytoplasmic degenerative changes. Also, some endothelial cells were exhausted of organelles, and their cytoplasm only displayed a few vesicular structures. Besides, damaged endothelial cells containing large electronlucent vacuoles were often seen. Many lysosomal bodies of irregular size and shape displayed breaks in their cytoplasmic membranes, and heterogeneous density appeared in numerous Kupffer cells [12].
Many antioxidants have recently been used to inhibit oxidative damage produced by high oxidative stress attributable to CP [13]. The administration of alpha-lipoic acid (LA) which is a common antioxidant is beneficial in numerous pathologies where ROS have been concerned [14,15]. Because LA is a strong antioxidant, it causes a common systemic improvement including liver health [16,17]. Besides, LA protects the liver from fat accumulation by suppressing appetite, increasing metabolism, and reducing body weight [18].
Lipoic acid (LA) was found in mitochondria as the coenzyme for mitochondrial dehydrogenase multienzyme complexes. Exogenous supplementation with LA has been reported to increase unbound lipoic acid levels, which reduce oxidative stress both in vitro and in vivo situations and act as a potent antioxidant [19].
Royal jelly (RJ) is a natural product produced by a bee and used in medicine. The chemical structure of RJ indicates the presence of many bioactive substances including 10-hydroxydecanoic acid and 24-methylenecholesterol and also several biological and pharmacological activities [20]. Furthermore, RJ administration provides sufficient protection against the rat brain tissue function and structure [21].
RJ is an effective antioxidant and has a free radical scavenging capacity [22,23]. Besides, it has been shown to possess numerous biological activities, such as antioxidative, antibacterial, immunomodulatory, anti-inflammatory, and tumoricidal activities, and also is commonly used in commercially available drugs and health foods, in addition to cosmetics, in many countries [24]. RJ contains the 57-kDa glycoprotein which stimulates the hepatocyte development and liver regeneration [25].
The current study carried out to study the histopathological, ultrastructure, and biochemical changes of CP effect on the liver and to estimate the possible protective role of LA and RJ in improving these changes.

Cyclophosphamide (CP)
CP was dissolved in saline, orally treated in a dose of (5 mg/kg b.wt) and was purchased from Baxter Oncology Gmbh Kantstrasse2 D-33790 Halle, Germany.

Alpha-lipoic acid (LA)
LA was dissolved in 100 ml (25 ml di methyl sulpho oxide (DMSO) + 75 ml distilled water), and we used NaOH to neutralize the solution pH = 7 and it was given orally in a dose of 25 mg/kg b.wt three times a week for 4 weeks. LA was purchased from Sigma-Aldrich Chemical Company Inc., Product of China, stored at 2-8°C, MW 206.33, mp 60-62oC, assay ≥ 98.0% (HPLC).

Royal jelly (RJ)
RJ was dissolved in distilled water and was given orally in a dose of 1 g/kg b.wt three times a week for 4 weeks and was purchased from Safe for Pharmaceutical Products for Pharco Pharmaceuticals (stored at a temperature not exceeding 30°C).

Animals
Sixty-four white male albino rats (Rattusnorvegicus), weighing 140-170 g, were obtained from the Egyptian Organization for Biological Products and Vaccines. The animals were housed in plastic cages with wired covers and kept under normal laboratory conditions for the different periods used. The animals were supplied with water and classic rat chow ad libitum, and they were kept under observation for a week before the start of the experiment. In our study, animal care was carried out following the European Community Directive (86/609/ EEC) and national rules; this is in accordance with the NIH guidelines for care and use of laboratory animals, 8th edition. This was administrated by the committee of the Zoology Department, Beni-Seuf University, Egypt. At the end of the experiment, the rats were sacrificed by anesthesia inhalation under light diethyl ether (5%).
Then, the animals were divided into eight groups each of 8 rats.
Group 1. This group served as a control group and received saline orally. Group 2. This group received alkaline solution orally (to make the medium pH neutral).
Group 3. This group was given di methyl sulpho oxide (DMSO) orally.
Group 4. This group received an oral dose of lipoic acid (25 mg/kg b.wt) three times a week for 4 weeks [26].
Group 5. This group received an oral dose of royal jelly (1 g/kg b.wt) three times a week for 4 weeks [27].
Group 6. This group was orally treated with CP (5 mg/ kg b.wt) 3 days per week for 4 weeks [28].
Group 7. This group was orally treated with CP (5 mg/ kg b.wt) and then received an oral dose of lipoic acid (25 mg/kg b.wt) three times a week for 4 weeks.
Group 8. This group was orally treated with CP (5 mg/ kg b.wt) and then received an oral dose of royal jelly (1 g/kg b.wt) three times a week for 4 weeks.

Tissue homogenate preparation
Blood was collected and centrifuged for obtaining the serum for ALT, AST analysis; then, liver homogenate was obtained by grinding a small piece (1 g) of freshly excised tissue in 10 volumes of 0.9% saline solution and was kept at − 20°C till use in the determination of superoxide dismutase (SOD), using the methods of [29], and lipid peroxidation (LPO) [30].

Histological preparations
After 4 weeks, the animals from each group were sacrificed under mild diethyl ether anesthesia. Small pieces of liver were fixed in 10% neutral buffered formalin solution for 24 h. Liver tissue specimens were embedded in paraffin using a conventional method and then cut. Paraffin sections of some liver were stained with hematoxylin and eosin [31].

Ultrastructural preparations
At the end of the experiment (4 weeks), specimens of the liver were cut into small pieces measuring about 1 mm 3 and directly fixed in fresh 3% glutaraldehydeformaldehyde at 4°C for 18-24 h. Then, the specimens were washed in phosphate buffer (pH 7.4) and then post-fixed in isotonic 1% osmium tetroxide for 1 h at 4°C [32]. Serial dehydration in alcohol was carried out. The specimens were then passed through propylene oxide solution and finally embedded in EPON epoxy resin. Semithin sections were cut from these blocks at 1.0 μm thickness by ultra-cut E Reichert-Jung ultramicrotome with the aid of glass knives, stained with toluidine blue stain, and examined by light microscope to detect the area of interest. Ultrathin sections (70-90 nm) were then prepared using the ultramicrotome glass knives, stained with uranyl acetate and lead citrate [33], and examined with a Joel JEM-2100 transmission electron microscope which worked at an accelerating voltage 80 kV.

Statistical analysis
Analysis of data was performed using student ANOVA test and comparing between means using LSD (least significant difference) as outlined by IBM Corp [34].. Results were expressed as mean ± standard error, and values of P > 0.05 were considered non-significantly different, while those P < 0.05 and P < 0.01 were significantly and highly significantly different, respectively.

Determination of liver function enzymes
3.1.1 Determination of serum alanine transaminase (ALT) and aspartate transaminase (AST) activities

Figures 1 and 2 indicate that ALT and AST activities
were markedly increased in the CP group in comparison to control, slightly alkaline solution, DMSO, LA, and RJ group. Besides, the administration of LA in concomitant with CP and also RJ in concomitant with CP showed observable amelioration in these activities nearly similar to the control group.

Determination of antioxidant parameters
The cyclophosphamide-treated group indicated a marked increase in lipid peroxidation in the form of malonaldehyde (MDA) and decrease in superoxide dismutase (SOD) in comparison with control, slightly alkaline solution, DMSO, CP+ LA, and CP+ RJ groups, although LA and RJ administration made a decrease in MDA and increase in SOD activity similar to control groups.
One-way ANOVA analysis of liver lipid peroxidation and superoxide dismutase of all groups are given in Figures 3 and 4. Nearly similar results in MDA activities were in the control, slightly alkaline solution, DMSO, CP+ LA, and CP+ RJ groups. CP administration resulted in a significant (P ˂ 0.001) increase in MDA level (44.89 ± 1.19 nmol/g tissue) when compared to the control group (9.55 ± 0.37 nmol/g tissue). And CP administration caused a significant decrease in SOD level (43.48 ± 1.39 U/g tissue) when compared to the control group (96.94 ± 1.12 U/g tissue). However, administration of Al and RJ to CPtreated rats revealed marked amelioration in the altered level of MDA and SOD activities when compared with the CP-treated group.

Histopathological observations of the liver 3.3.1 Liver of control rats
The histological examination of liver sections of control rats and alpha-lipoic acid-and royal jelly-treated rats showed their normal architecture in Fig. 5a-c, respectively. The normal liver consists of several poorly defined hepatic lobules. Each lobule is formed of cords of hepatocytes radiating from the central vein. The hepatic cords are separated by narrow blood sinusoids which are lined by Kupffer cells and endothelial cells. The hepatocytes are large polyhedral with acidophilic cytoplasm and darkly stained nuclei.

Liver of cyclophosphamide-treated rats
The portal vein was dilated and engorged with blood in addition to a marked proliferation of the bile duct (Fig. 6a).
In other areas, minute to large fat globules were seen intracellularly forming a signet ring appearance (Fig. 6b). The hepatocytes appeared enlarged with the presence of empty vacuoles within the cytoplasm surrounding the nuclei in multiple areas. These vacuoles were in the form of cloudy swelling and hydropic degeneration. Moreover, pyknosis also appeared (Fig. 6c). Necrosis with lysis of hepatocytes was also found in some areas (Fig. 6d). Multifocal leukocytic infiltrations were found in the hepatic parenchyma and vacuolar degeneration in hepatocytes (Fig. 6e).

Liver of rats treated with cyclophosphamide plus alpha-lipoic acid and royal jelly
In these cases, the liver of CP+ LA and CP+ RJ, respectively, showed improvement in comparison with CP group, central vein retained to normal structure, and both hepatocytes and blood sinusoids nearly appeared more or less similar to the control group (Fig.7a, b).

Ultrastructural observations of the liver 3.4.1 Liver of control rats
Ultrastructural examination of hepatocytes from animals of the control group revealed no destructive alterations with normal organelles that overcrowded the cytoplasm, particularly rough and smooth endoplasmic reticulum and mitochondria. The rough endoplasmic reticulum consists of closely packed parallel and flattened cristae. The nuclei appear spherical with one nucleolus and the normal pattern of chromatin distribution. Numerous electron-dense glycogen rosettes and bile duct with intact microvilli were detected (Fig. 8a, b). Sinusoid with normal Kupffer cell was seen (Fig. 8c).

Liver of cyclophosphamide-treated rats
Degenerated hepatocytes with multiple small-and medium-sized lipid droplets in the cytoplasm, also disorganization and decrease in glycogen, dilatation in ER, and electron-dense mitochondria, appeared in the cytoplasm (Fig. 9a). In other hepatocytes, there is marked variation in the size of mitochondria, where marked, swollen, and elongated mitochondria with illdefined cristae appeared (Fig. 9c). Moreover, dilated damaged bile duct was also seen (Fig. 9b). Vacuolations and marked collagen fibers were also observed (Fig. 9c). Kupffer cell showed a shrunken nucleus with an observed increase in the number of lysosomes in the cytoplasm (Fig. 9c, d).

Liver of rats treated with cyclophosphamide plus alpha-lipoic acid and royal jelly
The liver of CP-treated rats with alpha-lipoic acid showed marked recovery of the cytoplasm organelles and bile duct (Fig. 10a, b). Kupffer cells restored their normal structure (Fig. 10c). The liver of CP-treated rats with royal jelly showed marked recovery of the cytoplasm organelles except for a few lipid droplets (Fig. 10d). There was nearly normal appearance of the bile duct (Fig. 10e), but Kupffer cell showed a shrunken nucleus (Fig. 10f).

Discussion
Cyclophosphamide (CP) has been used in the clinical treatment of various types of cancer and autoimmune diseases and also used as an immunosuppressant after organ transplantations (e.g., bone marrow transplantations) [35]. The side effect of this drug is fatal hepatic damages in humans and experimental animals [36]. CP cause toxicity in healthy tissues in addition to in cancerous tissues [4]. The liver plays an essential role in the metabolism and detoxification of xenobiotic in the body [7]. Metabolism of CP occurs in the liver and undergoes metabolic activation by cytochrome p450 isoenzyme [37]. Enzymatic activities of serum ALT, AST, LDH, and ALP were considered sensitive indicators of hepatic injury [38] and the injury to the hepatocytes alters their transport function and membrane permeability, leading to leakage of enzymes from the cells [39]. Moreover, these enzymes under normal conditions are present in high concentrations in the liver and are released into the circulation during hepatocyte necrosis or membrane damage, as indicated by elevated serum enzyme levels [40]. So an increase in the serum ALT, AST, LDH, and ALP activities in CP-treated rats indicated hepatic cell damage. This is clear in our study as CP administration caused a significant increase in the activities of serum ALT and AST; this indicates liver damage induced by the drug and these observations are consistent also with Tripathi et al. [41].
Oxidative stress is the result of an imbalance between ROS generation and the intracellular capacity for removing ROS, subsequently leading to excessive damage in the cell [42].
Lipid peroxidation is one of the main indexes of oxidative damage started by ROS, and it has been related to the altered membrane structure and enzyme inactivation. It is initiated by the abstraction of a hydrogen atom from the side chain of polyunsaturated fatty acids in the membrane [43].
The present data exposed that CP administration produced a marked oxidative impact, as evidenced by the significant increase in LPO as well as a decrease in SOD levels as showed in the CP-treated group. These results concords with Caglayan et al. [44] and Ghosh et al. [45] who illustrated that CP-induced lipid peroxidation. The reduction in the activities of the SOD and increase in LPO could reflect the adverse effects of CP, which imbalanced the antioxidant system in the liver tissue. CP administration has been demonstrated to be an excellent model to produce syndromes of both oxidative stress and hepatic damage [46]. The increase in lipid peroxides might result from increased production of free radicals and a decrease in antioxidant status. The oxidative stress observed in our study is in line with other reports [47], where it has been implicated in CP-induced hepatotoxicity. The generation of free radicals and other ROS as well as lipid peroxidation have been reported to be the major mechanisms in CP toxicity [13,48]. Such oxidative stress generates biochemical and physiological disturbances [49].
In the present study, liver revealed that CP induced a severe damaging effect on liver tissues. Abnormal histological findings were found in liver tissues such as infiltration, congestion, fatty changes, edema, vascular degeneration, and necrosis. These findings coincide well with the results obtained by Yagmurca et al. [13,49,50], Sakr et al. [50], Ince et al. [13], and Cengiz et al. [51] who illustrated that CP induction causes liver damage showed by dark staining, shrinkage, irregularity in the nucleus boundaries and increase in eosinophilia in the cytoplasm, as well as increases in congestion and erythrocytes accumulation in the vascular structures.
Ultrastructurally, the hepatocytes of the CP-treated group appeared degenerated with multiple small-and medium-sized lipid droplets; other hepatocytes showed swollen mitochondria with ill-defined cristae, marked collagen fibers, and damaged bile duct also appeared. Kupffer cell showed a shrunken nucleus and an increase in the number of lysosomes in the cytoplasm. These findings were confirmed by Zimnoch et al. [52] and Sulkowska et al. [53] who illustrated that CP treatment leads to a marked polymorphism of the mitochondria and condensation of their matrix, segmentary blurring of the structure of the surrounding membranes, the presence of osmophilic intramitochondrial bodies and paracrystalline structures usually arranged along the organelles. The rough endoplasmic reticulum was focally degranulated, while the smooth endoplasmic reticulum looks as if proliferated and hepatocytes showed regression of changes and gradual normalization. Alpha-lipoic acid (LA) is a universal antioxidant. Its administration is beneficial in various pathologies in which ROS have been implicated [14,15]. LA through its powerful antioxidant activity causes a general systemic improvement including liver health [16,54].
LA scavenges the singlet oxygen, hydrogen peroxide, hydroxyl radicals, and also chelates the ferrous ion involved in the production of hydroxyl radicals [19,55] and treatment of free radical-related diseases. It is a fact that thiols protect cells against CP-induced LPO [46]. The above reports corroborate well with our findings in experimental hepatotoxicity as treatment with LA showed improvement in levels of LPO and SOD near control groups and elevating level of liver enzymes ALT and AST. From these observations, it is possible to conclude that LA may reduce oxidative stress in CPinduced hepatotoxicity by alleviating lipid peroxidation through scavenging of free radicals or by enhancing the activities of antioxidant enzymes which then detoxify free radicals [19]. So, LA is considered a cytoprotectant in CP-induced hepatic oxidative injury [9].
In our study, rats treated with LA showed an improvement in the histopathology and the ultrastructure of liver sections comparable with CP-treated rats as LA resulted in a marked recovery of the cytoplasm organelles and Kupffer cells restored their normal structure. This protection maybe because of the protective effects of LA against various hepatic injuries [56]. Royal jelly (RJ) is a secretion of the hypopharyngeal glands of honeybee workers [57]. It is considered a traditional product commonly used to supplement the medical treatment of various diseases [58]. RJ also has antioxidant [22] and hepatoprotective effects [59].
In the present study, RJ administration, together with CP, leads to elevation of liver enzymes ALT and AST as well as improvement in the level of MDA and SOD in comparison with the CP-treated group. This improvement may be because RJ significantly improved the indices of hepatotoxicity and lipid peroxidation [60] and also because of the hepatoprotective effect of RJ against chemicals known to cause liver damage [61,62].
RJ produced a significant protective effect on the liver by decreasing the level of lipid peroxidation (MDA) [63]. In the groups that were administered RJ in association with CP, an improvement was observed in some oxidative stress parameters and certain other biochemical parameters [63].
In our study, liver sections of the RJ-treated group showed an improvement in the hepatic lesions as central vein appeared to have normal structure and both hepatocytes and blood sinusoids nearly appeared similar to control. RJ is a hepatoprotective agent to exhibit a marked protective effect on liver tissue [64]. RJ has been demonstrated to possess numerous pharmacological activities in vitro, in vivo in experimental animals, and through clinical studies, including vasodilative and hypotensive activities, an increase of growth rate, and antitumor, antimicrobial, anti-inflammatory, antihypercholesterolemic activities. Besides, recently, immunomodulatory activities and estrogen-like effects have been published [61].
Treated rats with RJ showed an improvement in the ultrastructure of liver sections comparable with CP- Fig. 9 a-e Electron micrographs of ultrathin section of the liver of CP-treated rats. a Degenerated hepatocytes having multiple small-and medium-sized lipid droplets (LD) and disorganization and decrease in glycogen electron-dense mitochondria (M). b Dilated damaged bile canaliculus (arrow), some lipid droplets (LD), and swelling and elongated mitochondria with ill-defined cristae (M). c, d Shrinked nucleus of Kupffer cell, collagen fibers (C), and vacuolation (arrow head) (scale bar = 2 μm, 500 nm, 2 μm, 2 μm, respectively) Abdul-Hamid et al. Beni-Suef University Journal of Basic and Applied Sciences (2020) 9:28 treated rats, whereas there was a noticeable regaining of the cytoplasm organelles except for a few lipid droplets and nearly normal appearance of bile duct, but Kupffer cell showed a shrunken nucleus. This improvement was because RJ contain the 57-kDa glycoprotein which stimulates hepatocyte development and liver regeneration [65] and also was a result of its component vitamins, antioxidant vitamins A, E, and C; vitamin D; and vitamin B complex [25]. These vitamins themselves had an anticancer effect [66][67][68]. Little studies founded about the side effects of CP on the ultrastructure of the liver and the protective role of LA and RJ on the ultrastructure of the liver. Our study recommended that alpha-lipoic acid and royal jelly protect against CP-induced liver damage by their antioxidant effects. Additionally, this study suggested that alpha-lipoic acid and royal jelly may be used parallel with CP to improve CP-induced injuries in biochemical, histopathology, and ultrastructure of the liver and oxidative stress parameters. Lipoic acid-treated animals showed minimal histologic abnormalities than royal jelly.