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Effect of nerolidol on cyclophosphamide-induced bone marrow and hematologic toxicity in Swiss albino mice

Open AccessPublished:January 24, 2020DOI:https://doi.org/10.1016/j.exphem.2020.01.007

      Highlights

      • Administration of cyclophosphamide (200 mg/kg, intraperitoneal) induces marked hematotoxicity, myelotoxicity, oxidative stress, inflammation, and histologic aberrations in femoral bone marrow of Swiss albino mice.
      • Nerolidol was evaluated for its myeloprotective and hematoprotective potential in cyclophosphamide-induced myelotoxic mice.
      • Administration of nerolidol ameliorated cyclophosphamide-induced hematotoxicity, myelotoxicity, oxidative stress, inflammation, and histologic aberrations in femoral bone marrow of Swiss albino mice.
      Cyclophosphamide (CP) is one of the commonly used anticancer drugs, but its use is limited by myelotoxicity. Nerolidol (NER) is a lipophilic, bioactive sesquiterpene reported to have neuroprotective, cardioprotective, gastroprotective, and renal protective potential, but its myeloprotective potential is underexplored. This study was aimed at evaluating the myeloid-protective potential of NER in CP-induced myelotoxic mice. NER 200 and 400 mg/kg was given orally from the first to the 14th day. CP 200 mg/kg was administered intravenously on the seventh day. At the end of the study, mice were humanly killed, and blood and bone marrow were collected and stored for hematologic, biochemical and histopathologic estimations. Bone marrow analysis revealed reduced bone marrow cellularity, α-esterase activity, colony-forming unit granulocyte–macrophage (CFU-GM) levels, colony-forming unit erythroid (CFU-E) levels, and burst-forming unit-erythroid (BFU-E) levels. Hematologic findings revealed reduced peripheral blood count and granulocyte-colony stimulating factor (G-CSF) levels, whereas biochemical analysis revealed increased malondialdehyde (MDA), tumor necrosis factor α (TNF-α), interleukin (IL)-6, and IL-1β levels and reduced superoxide dismutase (SOD), catalase (CAT), and IL-10 levels. Histopathologic study further strengthened our findings. Treatment with NER significantly reversed the hematotoxic and myelotoxic aberrations and retained the structural integrity of bone marrow. Findings of the current study suggest that NER is a potential therapeutic molecule that can mitigate CP-induced hematotoxic and myelotoxic manifestations. However, more detailed studies are needed to explicate the mechanism underlying its protective effect.
      Cancer is the third leading cause of death worldwide and the second common cause of death in developing countries [
      • Liu M
      • Tan H
      • Zhang X
      • et al.
      Hematopoietic effects and mechanisms of Fufang e׳jiao Jiang on radiotherapy and chemotherapy-induced myelosuppressed mice.
      ,
      • Thun MJ
      • DeLancey JO
      • Center MM
      • Jemal A
      • Ward EM
      The global burden of cancer: priorities for prevention.
      ]. To meet this alarming situation, many anticancer drugs have been developed. Cyclophosphamide (CP) is a drug that belongs to the class of alkylating agents [
      • Iqubal A
      • Iqubal MK
      • Sharma S
      • et al.
      Molecular mechanism involved in cyclophosphamide-induced cardiotoxicity: old drug with a new vision.
      ]. CP is a potent anticancer and immunosuppressant drug that shows promising therapeutic utility against hepatocarcinoma, lung carcinoma, brain tumor, acute myeloid leukemia, and autoimmune diseases [
      • Ashry NA
      • Gameil NM
      • Suddek GM
      Modulation of cyclophosphamide-induced early lung injury by allicin.
      ]. CP is a pro-drug with its two active metabolites, phosphoramide mustard (PM) and acrolein. PM is an anticancer metabolite, whereas acrolein is a toxic metabolite, responsible for hematological toxicity and myelotoxicity [
      • Brock N
      • Hohorst HJ
      Metabolism of cyclophosphamide.
      ]. CP or its metabolite reacts with glutathione (GSH), restricts its antioxidant activity, increases the production of reactive oxygen species, and causes lipid peroxidation that leads to oxidative stress [
      • Iqubal A
      • Iqubal MK
      • Sharma S
      • et al.
      Molecular mechanism involved in cyclophosphamide-induced cardiotoxicity: old drug with a new vision.
      ,
      • El-Naggar S
      • Ibrahim M
      • El-Tantawi H
      • Al-Sharkawi I
      Pretreatment with the micro-alga, Spirulina platensis ameliorates cyclophosphamide-induced hematological, liver and kidney toxicities in male mice.
      ,
      • Patra K
      • Bose S
      • Sarkar S
      • et al.
      Amelioration of cyclophosphamide induced myelosuppression and oxidative stress by cinnamic acid.
      ]. CP has also been reported to induce the production of inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β [
      • Alqahtani S
      • Mahmoud AM
      Gamma-glutamylcysteine ethyl ester protects against cyclophosphamide-induced liver injury and hematologic alterations via upregulation of PPARγ and attenuation of oxidative stress, inflammation, and apoptosis.
      ]. Additionally, it is well documented that balanced hematopoietic growth factors and hematopoietic inhibitory factors are crucial to an effective hematopoietic system as they regulate the growth, differentiation, and proliferation of hematopoietic stem cells (HSCs) to produce blood cells [
      • Wang H
      • Wang M
      • Chen J
      • et al.
      A polysaccharide from Strongylocentrotus nudus eggs protects against myelosuppression and immunosuppression in cyclophosphamide-treated mice.
      ].
      TNF-α, IL-6, and IL-1β are considered hematopoietic inhibitory factors, whereas IL-10 is considered a hematopoietic growth factor. CP causes alterations in the levels of these hematopoietic factors and results in myelotoxicity [
      • Liu M
      • Tan H
      • Zhang X
      • et al.
      Hematopoietic effects and mechanisms of Fufang e׳jiao Jiang on radiotherapy and chemotherapy-induced myelosuppressed mice.
      ]. Myelotoxicity manifests as direct damage to bone marrow cells and hematopoietic stem cells and reduced peripheral blood counts [
      • Raj S
      • Gothandam K
      Immunomodulatory activity of methanolic extract of Amorphophallus commutatus var. wayanadensis under normal and cyclophosphamide induced immunosuppressive conditions in mice models.
      ]. Consequences of myelotoxicity further result in reduced levels of red blood cells (RBCs), white blood cells (WBCs; neutropenia or granulocytopenia), and platelets (thrombocytopenia) and reduced hemoglobin (Hb) counts (anemia) [
      • Patra K
      • Bose S
      • Sarkar S
      • et al.
      Amelioration of cyclophosphamide induced myelosuppression and oxidative stress by cinnamic acid.
      ,
      • Alqahtani S
      • Mahmoud AM
      Gamma-glutamylcysteine ethyl ester protects against cyclophosphamide-induced liver injury and hematologic alterations via upregulation of PPARγ and attenuation of oxidative stress, inflammation, and apoptosis.
      ]. Decreases in the levels of hematopoietic cells further weaken the body's immune system, cause secondary infections, increase the risk of internal bleeding, and cause multi-organ damage [
      • Richardson PG
      • Riches ML
      • Kernan NA
      • et al.
      Phase 3 trial of defibrotide for the treatment of severe veno-occlusive disease and multi-organ failure.
      ]. Thus, management of these myelotoxic manifestations becomes an important task in maintaining the well-being of patients [
      • El-Sebaey AM
      • Abdelhamid FM
      • Abdalla OA
      Protective effects of garlic extract against hematological alterations, immunosuppression, hepatic oxidative stress, and renal damage induced by cyclophosphamide in rats.
      ]. Although today hematopoietic growth factors such as granulocyte–macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO), and thrombopoietin (TPO) are used to accelerate hematopoietic recovery during the course of chemotherapy [
      • Liu M
      • Tan H
      • Zhang X
      • et al.
      Hematopoietic effects and mechanisms of Fufang e׳jiao Jiang on radiotherapy and chemotherapy-induced myelosuppressed mice.
      ], certain limitations such as secondary myeloid leukemia, convulsion, secondary hypertension, arthralgia, allergic reactions, and high costs often limit their use [
      • Dygai A
      • Goldberg V
      • Artamonov A
      • et al.
      Effects and mechanisms of hemopoiesis-stimulating activity of immobilized oligonucleotides under conditions of cytostatic myelosuppression.
      ]. Thus, the need for a safe and cost-effective therapeutic molecule that can take care of these situations remains an attractive and hot target for researchers.
      In the recent scenario, a growing trend is exploration of the use of natural bioactive molecules with potent antioxidant and anti-inflammatory profiles to combat this myelotoxicity [
      • El-Naggar S
      • Ibrahim M
      • El-Tantawi H
      • Al-Sharkawi I
      Pretreatment with the micro-alga, Spirulina platensis ameliorates cyclophosphamide-induced hematological, liver and kidney toxicities in male mice.
      ,
      • Patra K
      • Bose S
      • Sarkar S
      • et al.
      Amelioration of cyclophosphamide induced myelosuppression and oxidative stress by cinnamic acid.
      ,
      • Alqahtani S
      • Mahmoud AM
      Gamma-glutamylcysteine ethyl ester protects against cyclophosphamide-induced liver injury and hematologic alterations via upregulation of PPARγ and attenuation of oxidative stress, inflammation, and apoptosis.
      ,
      • Wang H
      • Wang M
      • Chen J
      • et al.
      A polysaccharide from Strongylocentrotus nudus eggs protects against myelosuppression and immunosuppression in cyclophosphamide-treated mice.
      ,
      • Raj S
      • Gothandam K
      Immunomodulatory activity of methanolic extract of Amorphophallus commutatus var. wayanadensis under normal and cyclophosphamide induced immunosuppressive conditions in mice models.
      ,
      • Ayhanci A
      • Yaman S
      • Appak S
      • Gunes S
      Hematoprotective effect of seleno-L-methionine on cyclophosphamide toxicity in rats.
      ,
      • Anisimova N
      • Ustyuzhanina N
      • Bilan M
      • et al.
      Fucoidan and fucosylated chondroitin sulfate stimulate hematopoiesis in cyclophosphamide-induced mice.
      ,
      • Xu SF
      • Yu LM
      • Fan ZH
      • et al.
      Improvement of ginsenoside Rg1 on hematopoietic function in cyclophosphamide-induced myelosuppression mice.
      ]. Nerolidol is a naturally occurring acyclic sesquiterpene present in the essential oils of various plants [
      • Chan WK
      • Tan LTH
      • Chan KG
      • Lee LH
      • Goh BH
      Nerolidol: a sesquiterpene alcohol with multi-faceted pharmacological and biological activities.
      ]. Nerolidol is extensively used in cosmetics (perfumes and shampoos) and noncosmetic products (cleansers and detergents) [
      • Neto JDN
      • de Almeida AAC
      • da Silva Oliveira J
      • dos Santos PS
      • de Sousa DP
      • de Freitas RM
      Antioxidant effects of nerolidol in mice hippocampus after open field test.
      ,
      • De Carvalho RB
      • De Almeida AAC
      • Campelo NB
      • Lellis DROD
      • Nunes LCC
      Nerolidol and its pharmacological application in treating neurodegenerative diseases: a review.
      ]. The antioxidant and anti-inflammatory properties of nerolidol have been well documented [
      • Chan WK
      • Tan LTH
      • Chan KG
      • Lee LH
      • Goh BH
      Nerolidol: a sesquiterpene alcohol with multi-faceted pharmacological and biological activities.
      ]. Preclinical studies have reported the potent neuroprotective, cardioprotective, gonad-protective, renal-protective, antibacterial, antileishmanial, and anti-biofilm activities of nerolidol [
      • Neto JDN
      • de Almeida AAC
      • da Silva Oliveira J
      • dos Santos PS
      • de Sousa DP
      • de Freitas RM
      Antioxidant effects of nerolidol in mice hippocampus after open field test.
      ,
      • De Carvalho RB
      • De Almeida AAC
      • Campelo NB
      • Lellis DROD
      • Nunes LCC
      Nerolidol and its pharmacological application in treating neurodegenerative diseases: a review.
      ,
      • Melekoglu R
      • Ciftci O
      • Eraslan S
      • Cetin A
      • Basak N
      The beneficial effects of nerolidol and hesperidin on surgically induced endometriosis in a rat model.
      ,
      • Klopell FC
      • Lemos M
      • Sousa JPB
      • et al.
      Nerolidol, an antiulcer constituent from the essential oil of Baccharis dracunculifolia DC (Asteraceae).
      ,
      • Kaur D
      • Pahwa P
      • Goel RK
      Protective effect of nerolidol against pentylenetetrazol-induced kindling, oxidative stress and associated behavioral comorbidities in mice.
      ,
      • Javed H
      • Azimullah S
      • Khair SBA
      • Ojha S
      • Haque ME
      Neuroprotective effect of nerolidol against neuroinflammation and oxidative stress induced by rotenone.
      ,
      • Fonsêca DV
      • Salgado PR
      • de Carvalho FL
      • et al.
      Nerolidol exhibits antinociceptive and anti‐inflammatory activity: involvement of the GABA ergic system and proinflammatory cytokines.
      ,
      • Baldissera MD
      • Souza CF
      • Grando TH
      • et al.
      Nerolidol-loaded nanospheres prevent behavioral impairment via ameliorating Na+, K+-ATPase and AChE activities as well as reducing oxidative stress in the brain of Trypanosoma evansi-infected mice.
      ,
      • Arruda DC
      • D'Alexandri FL
      • Katzin AM
      • Uliana SR
      Antileishmanial activity of the terpene nerolidol.
      ]. However, to date, its effect on bone marrow and hematological toxicity remains unexplored. Therefore, we aimed to explore the myeloprotective and hematopoietic potency of nerolidol in a cyclophosphamide-induced myelotoxicity and hematotoxicity model in Swiss albino mice.

      Methods

      Drugs and chemicals

      Cyclophosphamide (Endoxan, Batch No. AEU1040) was obtained from Baxter Oncology GmbH (Frankfurt, Germany). Nerolidol (CAS No. 7212-44-4, Lot STBG8020) was purchased from Sigma-Aldrich (St. Louis, MO). Enzyme-linked immunosorbent assay (ELISA) kits for interleukins and cytokines (IL-6, Catalog No. KB2068; IL-10, Catalog No KB2072; IL-1β, Catalog No. KB2063; TNF-α, Catalog No. KB2145) were procured from KRISHGEN Biosystems (Worli, Mumbai, India).

      Experimental animals

      Male Swiss albino mice (35–40 g) were obtained from the Central Animal House Facility of Jamia Hamdard. The experimental protocol was approved by the Institutional Animal Ethics Committee of Jamia Hamdard (IAEC/JH-1484). Experiments were conducted according to the National Institutes of Health's Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised in 1978), and all efforts were made to minimize animal suffering. Animals were allowed to acclimate for 1 week and were housed in standard polypropylene cages with access to a commercial standard pellet diet. Animals were maintained under controlled room temperature (23 ± 2°C) and relative humidity (60 ± 5%) on a 12-hour light/12-hour dark cycle in the Central Animal House Facility, Jamia Hamdard (New Delhi, India).

      Treatment protocol

      The doses of nerolidol (200 and 400 mg/kg, oral) and cyclophosphamide (200 mg/kg, intravenous), as well as the dosing schedules, were based on our previous report [
      • Iqubal A
      • Sharma S
      • Ansari MA
      • et al.
      Nerolidol attenuates cyclophosphamide-induced cardiac inflammation, apoptosis and fibrosis in Swiss Albino mice.
      ,
      • Iqubal A
      • Sharma S
      • Najmi AK
      • et al.
      Nerolidol ameliorates cyclophosphamide-induced oxidative stress, neuroinflammation and cognitive dysfunction: plausible role of Nrf2 and NF-κB.
      ]. Animals were divided into five groups (n = 6) treated as follows: (1) vehicle control: 0.1-mL Tween 80 oral for 14 days; (2) CP 200 (toxic): CP 200 mg/kg intravenously once on the seventh day; (3) NER 200 + CP 200: NER 200 mg/kg orally for 14 days and CP 200 mg/kg intravenously once on the seventh day; (4) NER 400 + CP 200: NER 400 mg/kg orally for 14 days and CP 200 mg/kg intravenously once on the seventh day; (5) NER alone: NER 400 mg/kg orally for 14 days.

      Isolation and preparation of bone marrow cells for antioxidant activities

      After the animals were humanly killed, bone marrow was collected from the femur, and homogenate was prepared according to the method proposed by El-Sayed et al. in 2010 [
      • El-Sayed E-SM
      • Abdel-Aziz A-AH
      • Helal GK
      • Saleh S
      • Saad AS
      Protective effect of N-acetylcysteine against carmustine-induced myelotoxicity in rats.
      ]. Briefly, a 23G needle was used to isolate bone marrow from the femoral bone, and the marrow was centrifuged at 10,000 rpm for 20 min at 4°C to obtain a clear supernatant. This clear supernatant was used to estimate thiobarbituric acid-reacting substances (TBARS), glutathione (GSH), and catalase (CAT) according to previously published methods [
      • Iqubal A
      • Sharma S
      • Ansari MA
      • et al.
      Nerolidol attenuates cyclophosphamide-induced cardiac inflammation, apoptosis and fibrosis in Swiss Albino mice.
      ,
      • Iqubal A
      • Sharma S
      • Najmi AK
      • et al.
      Nerolidol ameliorates cyclophosphamide-induced oxidative stress, neuroinflammation and cognitive dysfunction: plausible role of Nrf2 and NF-κB.
      ].

      Isolation of blood serum for the estimation of inflammatory markers and G-CSF

      Animals were anesthetized terminally, and blood was collected by cardiac puncture and centrifuged at 15,000 rpm for 10 min to obtain serum. Serum was then transferred to the vials and used for the estimation of TNF-α, IL-6, IL-1β, IL-10, and granulocyte colony-stimulating factor (G-CSF) using commercially available ELISA kits according to the manufacturer's instructions [
      • Iqubal A
      • Sharma S
      • Ansari MA
      • et al.
      Nerolidol attenuates cyclophosphamide-induced cardiac inflammation, apoptosis and fibrosis in Swiss Albino mice.
      ].

      Estimation of bone marrow cellularity and α-esterase-positive cells

      After animals were humanly killed, bone marrow was collected from the femur into medium containing 2% fetal calf serum (FCS). Bone marrow cells were counted using a hemocytometer and expressed as total live cells. Then, on clean glass slides, the bone marrow smear was stained with para-rosaniline hydrochloride and counterstained with hematoxylin for determination of α-esterase-positive cells expressed as α-esterase-positive cells/4000 cells [
      • Alqahtani S
      • Mahmoud AM
      Gamma-glutamylcysteine ethyl ester protects against cyclophosphamide-induced liver injury and hematologic alterations via upregulation of PPARγ and attenuation of oxidative stress, inflammation, and apoptosis.
      ].

      Estimation of hematological parameters

      Hematological parameters were estimated by collecting blood from retro-orbital plexus. Blood was transferred into an EDTA vial and RBC, WBC, Hb, platelets, granulocytes, lymphocytes, and monocytes were estimated using an automated hematoanalyzer.

      Estimation of bone marrow hematopoietic progenitor cells for granulocytes/macrophages and hematopoietic progenitor cells for erythrocytes

      To estimate colony-forming units granulocytes–macrophages (CFU-GM), CFU erythrocytes (CFU-E), and burst-forming units erythrocytes (BFU-E), hematopoietic cell colony-forming unit assays were performed. Methylcellulose culture medium was incubated with 1 × 105 nucleated bone marrow cells at 37°C and 5% CO2 for 7 days. After 7 days, colonies of CFU-GM, CFU-E, and BFU-E were counted and expressed as number of colonies per 1 × 105 nucleated cells using a Motic inverted microscope [
      • Liu M
      • Tan H
      • Zhang X
      • et al.
      Hematopoietic effects and mechanisms of Fufang e׳jiao Jiang on radiotherapy and chemotherapy-induced myelosuppressed mice.
      ,
      • Yang Y
      • Xu S
      • Xu Q
      • et al.
      Protective effect of dammarane sapogenins against chemotherapy-induced myelosuppression in mice.
      ].

      Histopathologic analysis

      Femoral bone was removed, fixed in 4% formaldehyde, treated with formic acid–sodium citrate for decalcification, and embedded in paraffin wax. Five-micrometer–thick sections were cut transversely and stained with hematoxylin and eosin to detect histological alterations; further processing was according to the published method [
      • Sharma S
      • Khan V
      • Najmi AK
      • Alam O
      • Haque SE
      Prophylactic treatment with icariin prevents isoproterenol-induced myocardial oxidative stress via nuclear factor-like 2 activation.
      ]. Photomicrographs were taken with a computer-enabled Motic microscope.

      Statistical analysis

      Data are expressed as the mean ± SEM. A one-way analysis of variance (ANOVA) followed by a post hoc Tukey multiple comparison test was used to determine the significance of data. The toxic group, treated with CP 200 mg/kg intravenous (CP 200) was compared with the control group, whereas treatment groups administered nerolidol 200 and 400 mg/kg oral (NER 200 and NER 400) were compared with the CP 200 group. Values were considered statistically significant at p < 0.05. Statistical analyses were performed using Graph Pad Prism 4.0 software (Graph Pad Software San Diego, CA).

      Results

      Effect of nerolidol on lipid peroxidation and antioxidant enzymes

      Administration of CP 200 to the toxic group significantly increased the level of TBARS (p < 0.001) and reduced the levels of CAT and SOD (p < 0.001) as compared with the control group (Table 1). Administration of NER 400 mg/kg to the treatment group reduced the level of TBARS (p < 0.001) and increased the levels of CAT and SOD (p < 0.01) as compared with the CP 200–treated group. Administration of NER 200 mg/kg to the treatment group, however, was found ineffective in reversing these parameters to normal as compared with CP 200.
      Table 1Effect of nerolidol on cyclophosphamide-induced oxidative stress parameters
      Antioxidant parameterControlCP 200NER 200 + CP 200NER 400 + CP 200NER alone
      TBARS17.75 ± 1.3955.83 ± 1.83
      p < 0.001, significant vs. control.
      49.74 ± 3.2236.99 ± 1.94
      p < 0.001, significant vs. CP.
      17.31 ± 1.75
      SOD7.97 ± 0.452.96 ± 0.36
      p < 0.001, significant vs. control.
      4.31 ± 0.475.78 ± 0.56
      p < 0.01.
      7.48 ± 0.47
      CAT165.99 ± 6.0293.80 ± 2.67
      p < 0.001, significant vs. control.
      104.20 ± 4.31143.62 ± 3.38
      p < 0.001, significant vs. CP.
      170.94 ± 5.09
      Values are expressed as the mean ± SEM (n = 6). A one-way analysis of variance followed by Tukey multiple comparison test was used to determine the significance of data.
      a p < 0.001, significant vs. control.
      b p < 0.001, significant vs. CP.
      c p < 0.01.

      Effect of nerolidol on Hb level and RBC, WBC, and platelet counts

      Administration of CP 200 to the toxic group resulted in a significant reduction in Hb level and RBC, WBC, and platelet counts as compared with the control groups (p < 0.001). Treatment with NER 400 significantly restored these parameters to normal as compared with the CP 200–treated groups (p < 0.01 for Hb, RBCs, and platelets and p < 0.001 for WBCs) as illustrated in Figure 1. Treatment with NER 200 had no protective effect against the reduced peripheral blood counts as compared with the CP 200–treated group.
      Figure 1
      Figure 1Effect of CP 200, NER 200, and NER 400 on hematologic parameters (Hb level and RBCs, WBCs, and platelet counts). Values are expressed as the mean ± SEM (n = 6). A one-way ANOVA followed by Tukey multiple comparison test was used to determine the significance of data. ###p < 0.001, significant vs. control; **p < 0.01, ***p < 0.001, significant vs. CP. ns=nonsignificant vs. CP.

      Effect of nerolidol on peripheral blood granulocyte, lymphocyte, and monocyte counts and G-CSF

      Administration of CP 200 to the toxic group significantly reduced levels of granulocytes, lymphocytes, monocytes, and G-CSF as compared with the control groups (p < 0.001). Treatment with NER 400 restored the levels of granulocytes such as eosinophils (p < 0.05), basophils (p < 0.01), neutrophils (p < 0.01), lymphocytes (p < 0.01), monocytes (p < 0.01), and G-CSF (p < 0.001), as illustrated in Figure 2. Treatment with NER 200 exhibited no protective effect against reduced levels of peripheral blood granulocytes lymphocytes, monocytes, and G-CSF as compared with the CP 200–treated groups.
      Figure 2
      Figure 2Effect of CP 200, NER 200, and NER 400 on peripheral blood granulocyte (eosinophils, basophils, neutrophils), lymphocyte, and monocyte counts. Values are expressed as the mean ± SEM (n = 6). A one-way ANOVA followed by Tukey multiple comparison test was used to determine the significance of data. ###p < 0.001, significant vs. control; *p < 0.05, **p < 0.01, ***p < 0.001, significant vs. CP. ns=nonsignificant vs. CP.

      Effect of nerolidol on bone marrow hematopoietic progenitor cells for granulocytes/macrophages and erythrocytes

      Administration of CP 200 to the toxic group significantly reduced CFU-GM, CFU-E and BFU-E (p < 0.001) as compared with the control group (Figure 3). Administration of NER 400 to the treatment group significantly increased CFU-GM (p < 0.05), CFU-E (p < 0.01), and BFU-E (p < 0.01) as compared with CP 200–treated group. Administration of NER 200 to the treatment group, however, was ineffective.
      Figure 3
      Figure 3Effect of CP 200, NER 200, and NER 400 on CFU-GM, CFU-E, and BFU-E. Values are expressed as the mean ± SEM (n = 6). A one-way ANOVA followed by Tukey multiple comparison test was used to determine the significance of data. ###p < 0.001, significant vs. control; *p < 0.05, **p < 0.01, significant vs. CP. ns=nonsignificant vs. CP.

      Effect of nerolidol on inflammatory cytokines

      Administration of CP 200 to the toxic group significantly increased levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) and decreased levels of the anti-inflammatory cytokine (IL-10) as compared with the control groups (p < 0.001, Figure 4). Treatment with NER 400 reduced the levels of pro-inflammatory cytokines (TNF-α, p < 0.001; IL-6 and IL-1β, p < 0.01) and increased levels of anti-inflammatory cytokines as compared with the CP 200–treated groups (IL-10, p < 0.001). Treatment with NER 200, however, did not prove effective against the increased levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) and the decreased level of the anti-inflammatory cytokine (IL-10) as compared with the CP 200–treated groups.
      Figure 4
      Figure 4Effect of CP 200, NER 200, and NER 400 on inflammatory parameters (TNF-α, IL-6, IL-1β, and IL-10). Values are expressed as the mean ± SEM (n = 6). A one-way ANOVA followed by Tukey multiple comparison test was used to determine the significance of data. ###p < 0.001 significant, vs. control; ⁎⁎p < 0.01, ⁎⁎⁎p < 0.001 significant vs. CP. ns=nonsignificant vs. CP.

      Effect of nerolidol on bone marrow cellularity and α-esterase activity

      Administration of CP 200 to the toxic group resulted in a significant reduction in bone marrow cellularity and α-esterase activity as compared with the control groups (p < 0.001). Treatment with NER 400 significantly restored the reduced bone marrow cellularity and α-esterase activity as compared with the CP 200–treated groups (p < 0.01), as illustrated in Figure 5. Treatment with NER 200, however, did not exhibit any protective activity as compared with the CP 200–treated groups.
      Figure 5
      Figure 5Top: Effect of CP 200, NER 200, and NER 400 on histologic aberrations in the bone marrow. Bottom: Effect on bone marrow cellularity and α-esterase activity. Values are expressed as the mean ± SEM (n = 6). A one-way ANOVA followed by Tukey multiple comparison test was used to determine the significance of data. ###p < 0.001, significant vs. control; **p < 0.01, ***p < 0.001, significant vs. CP. ns=nonsignificant vs. CP.

      Effect of nerolidol on histological aberrations in the bone marrow

      The histopathologic study revealed the normal and uniform architecture of periosteum, cavities, and trabeculae. Hematoxylin and eosin (H&E) stain also revealed abundant bone marrow cells in the control and NER alone groups. Administration of CP 200 resulted in significant structural damage to bone marrow and reduced the number of bone marrow cells as compared with the control groups. Treatment with NER, in a dose-dependent manner, maintained the structural organization of bone marrow and significantly increased the number of bone marrow cells as compared with the CP 200–treated groups (Figure 5).

      Discussion

      The numbers of patients with various types of cancer are increasing daily, leading to the development of great quantities of anticancer drugs [
      • Thun MJ
      • DeLancey JO
      • Center MM
      • Jemal A
      • Ward EM
      The global burden of cancer: priorities for prevention.
      ]. The clinical outcomes associated with these drugs, however, are limited by their unwanted side effects [
      • Patra K
      • Bose S
      • Sarkar S
      • et al.
      Amelioration of cyclophosphamide induced myelosuppression and oxidative stress by cinnamic acid.
      ]. Therefore, specific adjuvant therapies are needed that can reduce the side effects of these drugs without interfering with their anticancer properties. Today more attention is being given to naturally occurring bioactive molecules as they possess significant antioxidant, anti-inflammatory, and anti-apoptotic potential [
      • Wang Z
      • Qi F
      • Cui Y
      • et al.
      An update on Chinese herbal medicines as adjuvant treatment of anticancer therapeutics.
      ]. CP is one of the most myelotoxic anticancer drugs, causes severe oxidative stress and inflammation, and alters the hematopoietic activity of bone marrow by producing reactive oxygen species and inflammatory cytokines [
      • Iqubal A
      • Haque SE
      • Sharma S
      • Ansari MA
      • Khan V
      • Iqubal MK
      Clinical updates on drug-induced cardiotoxicity.
      ]. Thus, the current study was designed to explore the myeloprotective and hematopoietic potency of nerolidol in CP-induced myelotoxic Swiss albino mice. The current study revealed significant myeloprotection and amelioration of hematological toxicity as assessed by estimation of bone marrow cellularity, α-esterase activity, CFU-GM, CFU-E, BFU-E, total blood count, G-CSF, oxidative stress, and inflammatory cytokines. Histopathologic analysis of femoral bone marrow further substantiated these findings.
      Bone marrow is one of the essential components of the body, responsible for maintenance of immunity and other homeostatic functions through production of RBCs, Hb, WBCs (granulocytes and agranulocytes), and platelets [
      • Raj S
      • Gothandam K
      Immunomodulatory activity of methanolic extract of Amorphophallus commutatus var. wayanadensis under normal and cyclophosphamide induced immunosuppressive conditions in mice models.
      ]. Myelosuppression or myelotoxicity more often predisposes individuals to life-threatening conditions, such as neutropenia, thrombocytopenia, septicemia, and multi-organ failure [
      • Richardson PG
      • Riches ML
      • Kernan NA
      • et al.
      Phase 3 trial of defibrotide for the treatment of severe veno-occlusive disease and multi-organ failure.
      ]. One of the reported mechanisms of CP-induced myelosuppression and hematological toxicity is the effect of CP or its metabolites on the DNA of bone marrow cells. CP inhibits DNA replication, which suppresses the ability of these bone marrow cells to produce peripheral blood cells. It is important to understand that the development and maturation of peripheral blood cells depend on the function of hematopoietic stem cells and hematopoietic progenitor cells in the bone marrow [
      • Liu M
      • Tan H
      • Zhang X
      • et al.
      Hematopoietic effects and mechanisms of Fufang e׳jiao Jiang on radiotherapy and chemotherapy-induced myelosuppressed mice.
      ]. It is further well established that circulating peripheral blood cells have fixed and limited life spans, and therefore, their continuous replacement is required for effective homeostasis of the body system. Thus, the absolute count of these peripheral blood cells directly reflects the function of bone marrow [
      • Carey PJ
      Drug-induced myelosuppression.
      ]. CP-induced bone marrow toxicity is much harder to treat; strict control of infection and use of intravenous antibiotics, anti-fungal agents, and GM-CSF are some of the preventive approaches [
      • Alqahtani S
      • Mahmoud AM
      Gamma-glutamylcysteine ethyl ester protects against cyclophosphamide-induced liver injury and hematologic alterations via upregulation of PPARγ and attenuation of oxidative stress, inflammation, and apoptosis.
      ]. However, the use of GM-CSF and adjuvant therapy is often limited by high costs and serious side effects [
      • Dygai A
      • Goldberg V
      • Artamonov A
      • et al.
      Effects and mechanisms of hemopoiesis-stimulating activity of immobilized oligonucleotides under conditions of cytostatic myelosuppression.
      ].
      There is growing evidence that hematotoxicity and bone marrow toxicity occur on exposure to CP, and the underlying mechanism is attributed to the oxidative stress [
      • Patra K
      • Bose S
      • Sarkar S
      • et al.
      Amelioration of cyclophosphamide induced myelosuppression and oxidative stress by cinnamic acid.
      ,
      • Ayhanci A
      • Yaman S
      • Appak S
      • Gunes S
      Hematoprotective effect of seleno-L-methionine on cyclophosphamide toxicity in rats.
      ]. The antioxidant enzyme system remains a first-line defense for cellular myeloid tissues on exposure to a toxic stimulus such as CP or its metabolites [
      • Patra K
      • Bose S
      • Sarkar S
      • et al.
      Amelioration of cyclophosphamide induced myelosuppression and oxidative stress by cinnamic acid.
      ]. Lipid peroxidation and increased malondialdehyde (MDA) levels are well-established markers of oxidative stress [
      • Cengiz M
      Boric acid protects against cyclophosphamide-induced oxidative stress and renal damage in rats.
      ]. An increased MDA level not only reduces levels of members of the endogenous antioxidant defense system such as SOD, CAT, and GSH, but also increases the production of inflammatory cytokines by promoting the nuclear translocation of NF-kB and induces apoptosis by releasing cytochrome c and activating caspases [
      • Ansari MA
      • Iqubal A
      • Ekbbal R
      • Haque SE
      Effects of nimodipine, vinpocetine and their combination on isoproterenol-induced myocardial infarction in rats.
      ,
      • Iqubal A
      • Sharma S
      • Sharma K
      • et al.
      Intranasally administered pitavastatin ameliorates pentylenetetrazol-induced neuroinflammation, oxidative stress and cognitive dysfunction.
      ,
      • Iqubal A
      • Iqubal MK
      • Haquea SE
      Various rodent models for inducing hepatotoxicity and evaluating hepatoprotective drugs.
      ,
      • Iqubal A
      • Iqubal M
      • Haque S
      Experimental hepatotoxicity inducing agents: a review.
      ]. SOD normally converts superoxide radicals into H2O2, which dissociate into H2O and O2 in the presence of CAT. GSH also reduces the level of H2O2 and is itself oxidized to GSSG in presence of glutathione peroxidase [
      • Khan V
      • Sharma S
      • Bhandari U
      • Ali SM
      • Haque SE
      Raspberry ketone protects against isoproterenol-induced myocardial infarction in rats.
      ,
      • Khan V
      • Sharma S
      • Bhandari U
      • Sharma N
      • Rishi V
      • Haque SE
      Suppression of isoproterenol-induced cardiotoxicity in rats by raspberry ketone via activation of peroxisome proliferator activated receptor-α.
      ]. Oxidized GSSG is then converted back into GSH by utilizing NADPH in the presence of glutathione reductase. Thus, the optimum levels of these antioxidant enzymes are required to combat oxidative stress and reduce damage to the bone marrow. In the present study, when we administered CP 200, we observed a significant elevation in MDA levels and reduction in SOD and CAT levels, as outlined in Table 1. Thus, on one hand, increased lipid peroxidation and a reduced antioxidant defense system increase the level of inflammatory cytokines and induce apoptosis in bone marrow cells, and on the other hand, increased free radicals interfere with the DNA replication of bone marrow cells, arrest their cell division, and inhibit the formation of new blood cells from the hematopoietic stem cells. Our findings agree with an earlier article that reported a similar effect of CP on oxidative stress and related bone marrow toxicity [
      • Patra K
      • Bose S
      • Sarkar S
      • et al.
      Amelioration of cyclophosphamide induced myelosuppression and oxidative stress by cinnamic acid.
      ,
      • Alqahtani S
      • Mahmoud AM
      Gamma-glutamylcysteine ethyl ester protects against cyclophosphamide-induced liver injury and hematologic alterations via upregulation of PPARγ and attenuation of oxidative stress, inflammation, and apoptosis.
      ,
      • El-Sebaey AM
      • Abdelhamid FM
      • Abdalla OA
      Protective effects of garlic extract against hematological alterations, immunosuppression, hepatic oxidative stress, and renal damage induced by cyclophosphamide in rats.
      ]. When we administered CP 200, we also found significant reductions in RBC, WBC, granulocyte, lymphocyte, monocyte, and platelet counts and Hb levels, in agreement with earlier findings [
      • Liu M
      • Tan H
      • Zhang X
      • et al.
      Hematopoietic effects and mechanisms of Fufang e׳jiao Jiang on radiotherapy and chemotherapy-induced myelosuppressed mice.
      ,
      • El-Naggar S
      • Ibrahim M
      • El-Tantawi H
      • Al-Sharkawi I
      Pretreatment with the micro-alga, Spirulina platensis ameliorates cyclophosphamide-induced hematological, liver and kidney toxicities in male mice.
      ,
      • Patra K
      • Bose S
      • Sarkar S
      • et al.
      Amelioration of cyclophosphamide induced myelosuppression and oxidative stress by cinnamic acid.
      ,
      • Alqahtani S
      • Mahmoud AM
      Gamma-glutamylcysteine ethyl ester protects against cyclophosphamide-induced liver injury and hematologic alterations via upregulation of PPARγ and attenuation of oxidative stress, inflammation, and apoptosis.
      ,
      • Wang H
      • Wang M
      • Chen J
      • et al.
      A polysaccharide from Strongylocentrotus nudus eggs protects against myelosuppression and immunosuppression in cyclophosphamide-treated mice.
      ,
      • Raj S
      • Gothandam K
      Immunomodulatory activity of methanolic extract of Amorphophallus commutatus var. wayanadensis under normal and cyclophosphamide induced immunosuppressive conditions in mice models.
      ,
      • Ayhanci A
      • Yaman S
      • Appak S
      • Gunes S
      Hematoprotective effect of seleno-L-methionine on cyclophosphamide toxicity in rats.
      ,
      • Anisimova N
      • Ustyuzhanina N
      • Bilan M
      • et al.
      Fucoidan and fucosylated chondroitin sulfate stimulate hematopoiesis in cyclophosphamide-induced mice.
      ,
      • Xu SF
      • Yu LM
      • Fan ZH
      • et al.
      Improvement of ginsenoside Rg1 on hematopoietic function in cyclophosphamide-induced myelosuppression mice.
      ,
      • Cengiz M
      Hematoprotective effect of boron on cyclophosphamide toxicity in rats.
      ]. Treatment with NER 400 reduced the level of MDA, increased the levels of CAT, SOD, granulocytes, lymphocytes, and monocytes, and reversed myelotoxic and hematological manifestations. NER 200, however, was found ineffective as outlined in Table 1 and illustrated in Figures 1 and 2.
      It is well known that HPCs are the primary stem cells responsible for hematopoiesis in the red bone marrow. Under normal physiological conditions, HPCs give rise to myeloid cells (eosinophils, basophils, neutrophils, monocytes, erythrocytes, and platelets) and lymphoid cells (T cells, B cells, and natural killer cells). CFUs constitute a subtype of HPCs and differentiate into CFU-GM to produce granulocytes and macrophages. HPCs also differentiate into BFU-E and then into CFU-E. HPCs also produce erythrocytes. During myelosuppression, the levels of HPCs and different CFUs are reduced. Granulocyte colony-stimulating factor (G-CSF or CSF3) is chemically a glycoprotein and functionally a cytokine, produced by the immune cells. G-CSF is considered one of the potent inducers of HPCs such as CFU-GM and participates in the proliferation, differentiation, and release of granulocytes into the systemic circulation. Studies have reported significant reductions in the levels of different CFUs and G-CSF during chemotherapy that have been found to be responsible for neutropenia and high mortality rates. Supplementation with a recombinant G-CSF or G-CSF analogue such as filgrastim is an approved therapeutic approach to counteract these myelotoxic manifestations. In the current study, we explored the myelotoxic effect of cyclophosphamide and myeloprotective effect of nerolidol by performing hematopoietic progenitor CFU assays for CFU-GM, CFU-E, and BFU-E and an enzyme-linked immunosorbent assay for G-CSF. Our findings indicated significant declines in the numbers of CFU-GM, CFU-E, and BFU-E in bone marrow cells and a reduced level of G-CSF in the serum on treatment with CP 200. Administration of NER 400 significantly increased the numbers of CFU-GM, CFU-E, and BFU-E whereas NER 200 was found to be ineffective, as illustrated in Figures 2 and 3.
      Further, to determine the mechanism of myelotoxicity, we studied the effect of pro-inflammatory (TNFα, IL-1β, and IL-6) and anti-inflammatory (IL-10) cytokines, as these cytokines are very well documented to alter myeloid activities [
      • Alqahtani S
      • Mahmoud AM
      Gamma-glutamylcysteine ethyl ester protects against cyclophosphamide-induced liver injury and hematologic alterations via upregulation of PPARγ and attenuation of oxidative stress, inflammation, and apoptosis.
      ]. Alterations of the activity of these cytokines lead to myeloid inflammation and myeloid apoptosis, as they are positively related to the activation of NF-kB and caspases [
      • Iqubal A
      • Iqubal MK
      • Sharma S
      • et al.
      Molecular mechanism involved in cyclophosphamide-induced cardiotoxicity: old drug with a new vision.
      ]. NF-kB and caspases inhibit the activity of bone marrow cells, reduce peripheral blood counts, and thus are myelotoxic [
      • Alqahtani S
      • Mahmoud AM
      Gamma-glutamylcysteine ethyl ester protects against cyclophosphamide-induced liver injury and hematologic alterations via upregulation of PPARγ and attenuation of oxidative stress, inflammation, and apoptosis.
      ]. In the present study, when we administered CP 200, we observed significant elevations in the levels of TNF-α, IL-1β, and IL-6 and reduction in the level of IL-10, which was in agreement with earlier reports [
      • Wang H
      • Wang M
      • Chen J
      • et al.
      A polysaccharide from Strongylocentrotus nudus eggs protects against myelosuppression and immunosuppression in cyclophosphamide-treated mice.
      ,
      • Raj S
      • Gothandam K
      Immunomodulatory activity of methanolic extract of Amorphophallus commutatus var. wayanadensis under normal and cyclophosphamide induced immunosuppressive conditions in mice models.
      ,
      • Richardson PG
      • Riches ML
      • Kernan NA
      • et al.
      Phase 3 trial of defibrotide for the treatment of severe veno-occlusive disease and multi-organ failure.
      ,
      • El-Sayed E-SM
      • Abdel-Aziz A-AH
      • Helal GK
      • Saleh S
      • Saad AS
      Protective effect of N-acetylcysteine against carmustine-induced myelotoxicity in rats.
      ]. Treatment with NER 400 very well reversed the level of cytokines toward normal, whereas NER 200 was found ineffective, as illustrated in Figure 4.
      To further validate the myelotoxic effect of CP and myeloprotective effect of nerolidol, we performed H&E staining of femoral bone marrow. H&E staining clearly revealed significant damage to bone marrow cells and trabecular bone. Histological findings also revealed that CP administration caused a reduction in bone marrow count, which was in accordance with the previous findings [
      • Liu M
      • Tan H
      • Zhang X
      • et al.
      Hematopoietic effects and mechanisms of Fufang e׳jiao Jiang on radiotherapy and chemotherapy-induced myelosuppressed mice.
      ,
      • Xu SF
      • Yu LM
      • Fan ZH
      • et al.
      Improvement of ginsenoside Rg1 on hematopoietic function in cyclophosphamide-induced myelosuppression mice.
      ]. Treatment with NER 400 significantly reversed these histological aberrations of bone marrow toward normal and thus exhibited myeloprotective activity, as illustrated in Figure 5.

      Conclusions

      The findings of the present study revealed the myeloprotective potency of nerolidol against CP-induced bone marrow toxicity. The study was based on the analysis of biochemical agents (MDA, SOD, CAT, TNF-α, IL-1β, IL-6, IL-10), hematological agents (RBCs, WBCs, platelets, granulocytes, lymphocytes, Hb level), hematopoietic growth factor (G-CSF), bone marrow cell types (CFU-GM, CFU-E, and BFU-E), and histopathologic findings of femoral bone marrow. Thus, this study opens a gateway to further exploration and consideration of this drug as an adjuvant in CP-induced bone marrow toxicity.

      Acknowledgments

      We thank Jamia Hamdard for providing the necessary facilities to perform the experiment.

      Conflict of interest disclosure

      The authors declare no conflicts of interest.

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