Effects of Moringa oleifera, Curcumin, and Green Tea Extracts on Histopathological Changes in Mesenteric Lymph Nodes and Spleen of Albino Rats with Benzene-Induced Leukemia

Ahmed Alazzouni1, Ashraf Al-Brakati2,*, Sherif Rabie1, Mohamed Gabry1 and Basmaa Hassan1 1Department of Zoology and Entomology, Faculty of Science, Helwan University, Helwan, Cairo Governorate 11111, Egypt 2 Department of Human Anatomy, College of Medicine, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia Article Information Received 29 January 2021 Revised 23 March 2021 Accepted 18 April 2021 Available online 12 August 2021


INTRODUCTION
T he global prevalence of leuke mia is reported to be 5.8 and 4.3 per 100,000 person-years in men and women, respectively (Ahmadi et al., 2016). Leukemia refers to cancers that begin in blood-forming cells of the body; these abnormal cells grow and multiply in an uncontrolled manner (Roy et al., 2014). Leukemia is rooted in the Latin language and means "white blood"; it disrupts the body's natural immunity and erythropoiesis. Leukemia induces the accumulation of cells outside the bone marrow, forming masses in vital organs of the body, such as the brain, lymph node (LN), spleen, and liver (Varkesh et al., 2013). Chronic exposure to benzene is associated with a variety of hematological disorders, such as aplastic anemia, myeloproliferative disorders, and most detrimentally, leukemia, with acute myeloid leukemia (AML) the most common (Khalade et al., 2010). Cyclophosphamide (CP) is a synthetic alkylating agent chemically related to the nitrogen mustards (Netíková et al., 2018). It is effective against a wide spectrum of malignancies, such as leukemia, lymphoma, breast, lung, prostate, and ovarian cancers (Chen et al., 2018). The side effects of cytotoxic drugs are major obstacles for treatment success (Fares, 2015).
Many substances derived from dietary or medicinal plants are known to be effective and versatile chemopreventive and antitumor agents in a number of experimental models of carcinogenesis (Abdellatef et al., 2010). Cancer therapeutic agents block mechanisms involved in inducing cancer processes (Ke and Shen, 2017). Moringa oleifera (Family: Moringaceae) is a multipurpose tree that is used as a vegetable, spice, a source of cooking and cosmetic oil, and a medicinal plant (Abdellatef et al., 2010). Leaves of Moringa oleifera are rich in compounds with anticancer activity, including 4(4'-O-acetyl-Lrhamnopyranosyloxy) benzyl isothiocyanate, and 4-(-L-rhamnopyranosyloxy) benzyl glucosinolate (Abdellatef et al., 2010). The active constituents of turmeric are the flavonoid curcumin (diferuloylmethane) and various volatile oils (Verma et al., 2018). Curcumin is one of the most promising chemopreventive agents against a variety of human cancers, including colon, duodenal, stomach, prostate, leukemia, and breast cancers (Sinha et al., 2012). The most active constituents of green tea are polyphenols (catechins) (Musial et al., 2020). Green tea contains high quantities of several polyphenolic O n l i n e F i r s t A r t i c l e components, which have antioxidant and antimutagenic effects (Maiti et al., 2019). Epigallocatechin-3-gallate (EGCG) is the predominant polyphenol in catechin green tea extract (GTE) and plays a central role in the anticancer effects of green tea polyphenols. Recent studies have demonstrated that EGCG has anticancer effects in hematopoietic malignancy (Kumazoe et al., 2015). The present study was designed to demonstrate the anti-leukemic role of Moringa oleifera, curcumin and green tea in a benzene-induced leukemia model in albino rats through assessment of hematological parameters. Additionally, this study evaluated the ameliorative role of these herbal products in histopathological alterations in the spleen and LNs of leukemic rats.

Chemicals and extracts
Moringa oleifera leaf aqueous extract was obtained from the National Research Center (NRC), Dukki, Egypt. Curcumin was obtained as turmeric extract, standardized to contain 95% curcuminoids with high antioxidant power in 500 mg capsules, and manufactured by Puritan's pride, Inc., U.S.A. Green tea extract (GTE) tablets were obtained as super antioxidant green tea leaf, standardized to contain 500 mg GTE, yielding 175 mg EGCG, and manufactured by Source Naturals, Inc., U.S.A. Benzene Chromasolv was obtained as benzene with a purity > 99.9%, was used in chromatographic analysis, and purchased from Sigma-Aldrich Corp., St Louis, USA. CP (Endoxan) was obtained as 200 mg powder manufactured by Baxter Oncology (Germany, Alemania), and imported by Egydrug. Other chemicals and dyes were of analytical grade and were purchased from El-Gomhouria Co. (Cairo, Egypt).

Experimental design
A total of 70 female albino rats (Rattus norvegicus) weighing 200 ± 20 kg were purchased from the New Veterinary Office (Cairo, Egypt). Rats were housed in wired cages in a temperature-controlled environment with 12-h light/dark cycles. Food and water were freely available throughout the experiment. Rats were divided into the following groups (10 rats per group): Group 1 (control group) received only distilled water through the experimental period; Group 2 (benzene group, leukemia positive control) received 0.2 mL benzene solution (twice/ week) intravenously in the tail vein for 3 months according to Akanni et al. (2014); Group 3 (Moringa oleifera treated group) received 100 mg/kg/day Moringa oleifera extract by gastric tube for 1 month after leukemia induction, according to the dose used by Akanni et al. (2014); Group 4 (curcumin treated group) received 300 mg/kg/day orally for 1 month after leukemia induction, according to Tomita et al. (2006); Group 5 (GTE treated group) received 350 mg/kg/day GTE orally for 1 month after leukemia induction (Laurence and Bacharach, 2013); Group 6 (combined GTE + curcumin treated group) received 350 mg/kg/day GTE, and then given 300 mg/kg/day curcumin in a sequential fashion by gastric tube for 1 month according to Ghosh et al. (2009). Group 7 (CP treated leukemic group) received 7.5 mg/kg CP intraperitoneally every 48 h for 1 month after leukemia induction (Akanni et al., 2014).

Hematological assay
Leukemia induction was evaluated based on hematological parameters in the benzene-injected group compared to the control group. Blood was analyzed using a hematology analyzer for total white blood cells (WBCs), total red blood cells (RBCS), hemoglobin (Hb) concentration, packed cell volume (PCV), and Figurelet count.

Histological assay
Paraffin sections were observed for spleen and mesenteric lymph nodes (MLNs) and stained by routine hematoxylin and eosin staining (Culling and Albert, 2013). Mast cells within MLNs were stained by toluidine blue for mast cell count detection. Mast cells were counted from five different micrographs/group and the average mast cell value was used for each group. MLNs and spleen elastic fibers were stained with Orcein for morphometric analysis of elastic fibers area percent in the study groups. Five different micrographs/group were calculated using ImageJ version 1.50i, based on the masking of elastic fibers.

Statistical analysis
All numerical data were statistically analyzed and expressed as the mean ± standard deviation (SD). The level of statistical significance was set at p < 0.05, using a one-way analysis of variance test. Further comparisons among groups were conducted according to Tukey's post hoc test using the Statistical Package for the Social Sciences (SPSS) version 20.

Hematological parameters
In the present study, the complete blood count showed a significant increase in WBC counts (marked leukocytosis), and decrease in RBC counts, Hb, PCV, and Figurelet counts in the benzene-treated group when compared to the control group. These data suggested the successful induction of leukemia by intravenous injection of benzene Chromasolv (0.2 mL twice per week) in albino O n l i n e

F i r s t A r t i c l e
rats. In rats receiving 100 mg/kg/day Moringa oleifera, 300 mg/kg/day curcumin, 350 mg/kg GTE, combined curcumin and GTE, and 7.5 mg/kg/48 h CP, WBC counts decreased, while RBC counts, Hb, hematocrit, and Figurelet count increased (Table I). The data shows amelioration of blood parameters in all treatment groups, which suggests the potential role of these natural products as anti-leukemic agents, similar to anti-leukemic drugs (CP).

O n l i n e F i r s t A r t i c l e
Histological parameters Figure 1A shows the normal structure of the MLNs where cortex (C) (B cell zone), para cortex (PC) (T cell zone), and medulla (M). The cortex consists of lymphoid follicles (LF) and germinal centers (GC) with lymphatic follicles (LF) that contain germinal centers (GC) (Fig. 1B). Medulla consists of medullary cords (MC) and medullary sinuses (MS) (Fig. 1C). The histological architecture of the control spleen (Fig. 1D); consists of white pulp (WP) and red pulp (RP). WP mainly exists in LF with a central arteriole (CA) and a periarteriolar lymphatic sheath (PALS), GC, and marginal zone (MZ) (Fig. 1E). RP consists of splenic cords (SC) and venous sinuses (VS) in between (Fig. 1F). Figure 1G showed thin, delicate elastic fibers dispersed in MLNs and thin, delicate elastic fibers dispersed in the spleen (Fig. 1H). There are few normal mast cells in the pericapsular area surrounding MLN (Fig. 1I).

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A. Alazzouni et al.
restoration of the normal number and shape of mast cells (Figs. 3,4,5,6,7I). Table II shows significant increase in elastic fiber area in the LN (21.62 ± 2.15 %) and in red pulp spleen (18.77 ± 1.84 %) of the benzene-treated group when compared with their respective controls, and significant decrease in elastic fiber area in the groups treated with Moringa oleifera, curcumin, GTE, combined curcumin and GTE, and CP when compared to that in the leukemia positive control group. This shows great improvement in the mean area percent values in the treated groups. Table II also shows significant increase in mast cell counts (12.50 ± 3.44) in the MLN sections in the benzene-treated group compared to that of the control group (4.20 ± 1.03). There is also a significant decrease in the mast cell count in the groups treated with Moringa oleifera, curcumin, GTE, combined curcumin and GTE, and CP when compared to that in the leukemia-positive control group. Fig. 4. Photomicrographs of the curcumin treatment group showing: A, marked improvement in lymph node structure with cortex (C), paracortex (PC), and medulla (M) (×100); B, cortical lymphoid follicle (LF) with germinal center (GC) (×400); C, medulla with medullary cords (MC) and medullary sinuses (MS) (×400); D, marked improvement in spleen white pulp (WP) and red pulp (RP) structure (×100); E, WP with LF, central arteriole (CA), periarteriolar lymphatic sheath (PALS), GC, and marginal zone (MZ) (×400); F, RP with splenic cords (SC) and venous sinuses (VS) (hematoxylin and eosin, ×400); G, almost normal shape of thin, delicate elastic fibers in the lymph node (arrow) (Orcein, ×400); H, nearly normal shape of thin, elastic fibers (arrow) in the spleen (Orcein, ×400); I, almost normal mast cell size and numbers (arrows) in the pericapsular connective tissue of the lymph node (toluidine blue, ×400).

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DISCUSSION
In this study, complete blood count and differential leukocyte count showed a significant increase in WBC counts (marked leukocytosis), decreased RBC count (anemia), decreased Hb concentration (hemoglobinemia), decreased PCV, and decreased Figurelet count in the benzene-treated group when compared to that of the control group. This demonstrates the successful induction of leukemia in rats by benzene injection, which is in line with a previous leukemia model of Olufemiet al. (2014), who reported that leukemia was induced as significantly elevated WBC (leukocyte) counts and anemia in the control group.
The mechanism of leukemia induction is explained by Li et al. (2019), in which multiple key molecular and cellular events may be involved in myeloid leukemogenesis. Exposure to environmental factors, including chemicals and ionizing radiation, is a risk factor for AML and myelodysplastic syndrome. After exposure, benzene undergoes metabolism by cytochrome P450 2E1 and 2F1, mainly in the liver. Benzene exerts its toxicity through reactive metabolites, that is, catechol, hydroquinone, benzoquinone, and others. Fig. 6. Photomicrographs of the combined curcumin and green tea extract treatment group showing: A, obvious improvement in lymph node structure with cortex (C), paracortex (PC), and medulla (M) (×100); B, cortical lymphoid follicle (LF) with germinal center (GC) (×400); C, medulla with medullary cords (MC) and medullary sinuses (MS) (×400); D, obvious improvement in spleen white pulp (WP) and red pulp (RP) structure (×100); E, WP with LF, central arteriole (CA), periarteriolar lymphatic sheath (PALS), GC, and marginal zone (MZ) (×400); F, RP with splenic cords (SC) and venous sinuses (VS) (hematoxylin and eosin, ×400); G, almost normal shape of thin, delicate elastic fibers in the lymph node (arrow) (Orcein, ×400); H, nearly normal shape of thin, elastic fibers (arrow) in the spleen (Orcein, ×400); I, almost normal mast cell size and numbers (arrows) in the pericapsular connective tissue of the lymph node (toluidine blue, ×400).

O n l i n e F i r s t A r t i c l e
Effects of Moringa oleifera, Curcumin and Green Tea on Albino Rats 9 Fig. 7. Photomicrographs of the cyclophosphamide treatment group showing: A, slight improvement in the whole structure of the lymph node (×100); B, cortical changes with some lymphocytic depletion in germinal centers and some necrotic degenerated cells (ghost cell) (arrows) (×400); C, medulla with medullary cords (MC) and medullary sinuses (MS) (×400); D, slight improvement in spleen white pulp and red pulp structure (×100); E, white pulp with dilated central arteriole (CA) in non-active lymphoid follicle (LF) (×400); F, red pulp with dilated and congested venous sinuses (VS) and splenic cords (SC) (hematoxylin and eosin, ×400); G, almost normal shape of thin, delicate elastic fibers in the lymph node (arrow) (Orcein, ×400); H, nearly normal shape of thin, elastic fibers (arrow) in the spleen (Orcein, ×400); I, almost normal mast cell size and numbers (arrows) in the pericapsular connective tissue of the lymph node (toluidine blue, ×400).
Benzene and its metabolites redistribute and accumulate in bone marrow tissue, where they exert their selective toxicity to hematopoietic stem cells or progenitor cells (Khalade et al., 2010). The resultant molecular and cellular effects include oxidative stress, changes in gene expression and function, and a disrupted balance in proliferation, differentiation, apoptosis, and cytogenetic abnormalities, such as chromosomal aberrations (Maiti et al., 2019;Musial et al., 2020). Benzene metabolites interact with hematopoietic cells at different differentiation stages in bone marrow, resulting in genetic, chromosomal, or epigenetic abnormalities, genomic instability, and altered proliferation and differentiation of hematopoietic stem cells, leading to the formation of mutated hematopoietic cells and subsequent clonal evolution to leukemia (Roy et al., 2014). The amelioration of blood parameters measured by treatment with Moringa oleifera, curcumin, and GTE revealed the anti-leukemic action of these herbs. The anti-leukemic action of Moringa oleifera is supported by Akanni et al. (2014) leukemic conditions in the affected rats owing to its bioactive constituents in benzene-induced leukemia rats. Additionally, the authors reported that the extract might be an active, natural, and non-toxic anticancer drug lead. Hashim et al. (2012) has showed that curcumin has cytotoxic activity against leukemia cell lines and possesses anticancer activity by inhibiting cell growth activity, which show characteristic features of apoptosis. A population-based study by Kuo et al. (2009) has revealed that drinking large amounts of green tea containing more than 550 units of catechins decreases the risk of leukemia. EGCG also induced necrosis-like cell death in chronic myeloid leukemia cells, overcoming apoptosis resistance (Yao et al., 2017).
In the present study, Benzene Chromasolv caused a histological damages of the LNs and spleen, which also increased the percentage of non-organized thick elastic fibers in the LN and spleen, and elicited a strong inflammatory response represented by elevated mast cell counts in LN sections. These findings are in accordance with Akanni et al. (2014), who found that groups exposed to benzene carcinogen show varying lesions of the heart (mild to marked), coronary congestion, severe vacuolar degeneration and necrosis of hepatocytes with cellular infiltration by mononuclear cells, and diffuse tubular degeneration and necrosis with renal interstitial hemorrhage. Additionally, Hetal (2016) has found that liver tissues exhibit severe damage, such as sinusoidal dilation and necrosis due to benzene exposure. The various lesions in the LNs and spleen in this study are attributed to the DNA damaging ability of the agent (Nakajima et al., 2019).
Increased mast cells in the benzene group are explained by Sureshkumar et al. (2005), who concluded that the general pathological response to a toxicant triggers acute inflammatory immune processes. The authors reported that these changes were most probably due to the irritative effect of benzene, recruitment of inflammatory cells, and release of many inflammatory mediators, reactive oxygen species (ROS), or free radicals. A possible explanation for the ameliorative role of Moringa oleifera, curcumin, GTE, and combined curcumin and GTE treatment on the toxic effects of Benzene Chromasolv on LN and spleen structure could be the involvement of their antioxidant and scavenging properties. Antioxidants provide protection or remediation by scavenging ROS that damage DNA and initiate diseases, such as cancer (Aziz et al., 2019).
In the present study, CP treatment caused toxic changes in the LN and spleen structure, which was suggestive of the deleterious effects of CP on normal cells after prolonged usage. These findings are supported by Lee et al. (2010), who concluded that CP has severe and life-threatening adverse effects, including AML, bladder cancer, and permanent infertility, especially at higher doses. Thus, the aggravated damage to the organs of rats co-treated with CP and Benzene Chromasolv was suggestive of the deleterious effects of CP (anticancer drug) on normal cells after prolonged usage. The increased elastic fiber area percentage (elastosis) in the benzenetreated group is explained by Jimenez et al. (2006), who discussed the causes of solar elastosis as "thickening, degraded elastic fibers, and tangled shapes of elastic fibers lead to increased amounts of degraded elastic fibers" due to premature proteolytic degradation and impaired remodeling by extracellular matrix components.
The observed loss of physiologically relevant elastic fibers is also affected by the fact that fully differentiated (adult) dermal fibroblasts lose their ability to synthesize new elastin and thus cannot replace damaged elastic fibers (Roy et al., 2014). As elastic fibers are solely responsible for elasticity/resilience, there is an obvious need for the development of methods that might protect existing elastic fibers from premature degradation by elastolytic proteinases and facilitate new elastogenesis. Another possible mechanism is proposed by Philips et al. (2004), who reported that benzene and its derivatives increase the expression of elastin, synthesized primarily by fibroblasts. In the present study, Moringa oleifera, curcumin, and green tea treatment restored the normal elastic fiber formation/degradation machinery in both LNs and spleens, as represented by a decrease in the elevated elastic fiber area percent. This may be explained by the fact that polyphenols synergistically enhance elastogenesis induced by selected elastogenic compounds in dermal fibroblast cultures (Jimenez et al., 2006).

CONCLUSION
From the present study, it appears that Moringa oleifera, curcumin, GTE, and combined curcumin and GTE treatment could play an ameliorative and curative function against benzene-induced histological changes in the LN and spleen sections of leukemic rats.

ACKNOWLEDGEMENT
This work was supported by grants from the Taif University Researchers Supporting Program (project number: TURSP-2020/151), Taif University, Saudi Arabia.

Statement of conflict of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

O n l i n e F i r s t A r t i c l e
Effects of Moringa oleifera, Curcumin and Green Tea on Albino Rats