2026 Volume 51 Issue 8 Pages 449-461
Benzene exposure is a major risk factor for hematologic malignancies, including acute myeloid leukemia (AML), through mechanisms involving genotoxicity, oxidative stress, and dysregulation of hematopoietic signaling pathways. This study investigated the protective effects of melatonin, vitamin C, and their combination against benzene-induced pre-leukemic alterations in rats, with emphasis on bone marrow genotoxicity, oxidative stress, and activin A/follistatin expression. Forty male Wistar rats were allocated into five groups (n=8): control, benzene, benzene + melatonin, benzene + vitamin C, and benzene + combined treatment. Genotoxicity was assessed using bone marrow micronucleus assays, while oxidative stress biomarkers, serum activin A/follistatin levels, and bone marrow gene expression were evaluated using biochemical assays, ELISA, and qPCR, respectively. Benzene exposure significantly increased micronucleus frequency, reduced %PCE and PCE/NCE ratio, elevated %NCE, and suppressed activin A and follistatin expression. Melatonin or vitamin C alone partially attenuated these abnormalities, whereas combined treatment demonstrated the strongest protective effects, including marked improvement of genotoxicity indices, restoration of catalase activity, and significant upregulation of activin A and follistatin gene expression. Combined melatonin and vitamin C supplementation attenuated benzene-associated pre-leukemic alterations, particularly genotoxic and oxidative stress-related abnormalities. Combined melatonin and vitamin C supplementation attenuated benzene-associated pre-leukemic alterations in rats, particularly genotoxic and oxidative stress-related abnormalities. These protective effects were accompanied by modulation of activin A/follistatin expression patterns, suggesting a potential association between restoration of this signaling axis and improved bone marrow homeostasis. However, further mechanistic studies are required to determine whether activin A/follistatin signaling directly contributes to the observed protective effects.
Pre-leukemia includes many bone marrow illnesses characterized by the uncontrolled proliferation of atypical white blood cells, frequently advancing to more severe forms such as acute myeloid leukemia (AML). AML, a highly common and fatal subtype in adults, originates from genetic abnormalities in hematopoietic stem cells, resulting in disrupted differentiation and a buildup of immature blasts in the bone marrow and peripheral circulation (Döhner et al., 2022). This leads to significant consequences like anemia, infections, and hemorrhage, with untreated instances associated with a dire prognosis and elevated fatality rates. Environmental pollutants, especially benzene, a recognized industrial solvent and carcinogen, are significantly associated with the development of AML by processes that include DNA damage, chromosomal abnormalities, and disruption of normal hematopoiesis. Animal models, particularly in rats, have demonstrated significant use in elucidating these pathways and discovering innovative therapies (Glass et al., 2003; Stenehjem et al., 2015).
The disruption of critical signaling pathways, notably the activin A-follistatin system, is fundamental to AML pathogenesis. Activin A, a constituent of the transforming growth factor-β (TGF-β) superfamily, exerts diverse effects on hematopoiesis by modulating cell proliferation, differentiation, and death (Blank and Karlsson, 2015). Its function is stringently controlled by follistatin, an endogenous antagonist that binds to activin A with high affinity, thus regulating downstream Smad signaling and preserving hematopoietic homeostasis. Disruptions in this axis—such as increased activin A or diminished follistatin—have been associated with leukemogenesis, facilitating unchecked blast growth and immune evasion in AML. Quantitative PCR (qPCR) tests, utilizing the 2^−ΔΔCt method, have demonstrated modified mRNA expression of these factors in leukemic cells, underscoring their potential as biomarkers and therapeutic targets (Aref et al., 2021; Synolaki et al., 2021).
Recent data identify melatonin as a versatile drug in cancer treatment, transcending its function in circadian rhythm regulation. This pineal hormone demonstrates significant antioxidant, anti-inflammatory, and pro-apoptotic properties, principally mediated by melatonin receptors MT1 and MT2 found on hematopoietic and immunological cells (Ahmad et al., 2023; Reiter et al., 2017). Activation of MT1 induces cell cycle arrest at the G2/M phase and reduces proliferation in leukemic blasts, whereas MT2 enhances apoptosis and strengthens immune surveillance. Research indicates that melatonin can alleviate oxidative stress in acute myeloid leukemia models by decreasing reactive oxygen species (ROS) and safeguarding against chemotherapy-related toxicities (Liu et al., 2016).
Vitamin C (ascorbic acid) has gained increasing attention as an adjunct in oncology because of its dual antioxidant and pro-oxidant properties at therapeutic concentrations. At high doses, particularly through intravenous administration, vitamin C may selectively generate hydrogen peroxide within malignant cells, exploiting their elevated oxidative vulnerability to induce apoptosis and inhibit tumor progression. In hematological malignancies, vitamin C has also been suggested to influence cellular differentiation and redox-sensitive signaling pathways, including mechanisms potentially related to epigenetic regulation and TET enzyme activity (Cimmino et al., 2017; Villagran et al., 2021). However, the precise contribution of these pathways to benzene-induced pre-leukemic alterations remains incompletely understood. When combined with other therapeutic agents, vitamin C has demonstrated synergistic potential in reducing leukemic burden and improving treatment responses in experimental AML models (Villagran et al., 2021).
Standard assays assess genotoxicity and oxidative damage in bone marrow by measuring polychromatic erythrocytes (PCEs), normochromatic erythrocytes (NCEs), micronucleated PCEs (MN-PCEs), and micronucleated NCEs (MN-NCEs). The PCE/NCE ratio functions as an indicator of erythropoietic activity, with variations indicating hematological toxicity. Biomarkers of oxidative stress, such as malondialdehyde (MDA) for lipid peroxidation, superoxide dismutase (SOD) and catalase for antioxidant defense, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) for DNA damage, elucidate disease processes and treatment responses. Increased blast percentages in peripheral blood are associated with the severity and prognosis of AML (Hayashi, 2016a; Valavanidis et al., 2009).
Notwithstanding these discoveries, the interaction between melatonin and vitamin C in regulating activin A and follistatin expression in benzene-induced pre-leukemia is still inadequately investigated. This work examines the joint impact on gene expression profiles and bone marrow characteristics in a rat model, with the objective of revealing synergistic protective mechanisms that may guide novel therapy options for AML.
All experimental procedures were conducted on male Wistar rats aged 8–10 weeks, weighing 180–220 g at the start of the study. The animals were obtained from a recognized breeding facility and acclimatized for one week before research. The subjects were accommodated in conventional polycarbonate cages (n = 4–5 per cage) under regulated environmental conditions: a 12-hr light/dark cycle, a temperature maintained at 22 ± 2°C, relative humidity of 50–60%, and unrestricted access to standard rodent chow and filtered water. To alleviate stress, cages were enhanced with bedding and nesting materials, and animals were handled with care during daily inspections. All protocols strictly complied with worldwide ethical standards for animal research, encompassing the ARRIVE guidelines and the concepts of the 3Rs (Replacement, Reduction, Refinement) (Kilkenny et al., 2010). The research received approval from the Institutional Animal Ethics Committee of Cihan University-Erbil (Approval Number: CUE-REC/2025/04), thereby adhering to national standards for laboratory animal welfare (Care and Animals, 1986).
ChemicalsBenzene (purity ≥99%, Chem Lab, Belgium) was diluted in a vehicle composed of 2-propanol and distilled water (1:5:5 v/v) for administration. Melatonin (Bioven Ingredients, India) was freshly made each day by dissolving the powder in distilled water to attain a concentration appropriate for a 10 mg/kg body weight dosage, thereafter vortex mixed to maintain uniformity. Ascorbic acid (vitamin C, YourHealthStore.co.uk, UK) was diluted in distilled water to provide a 200 mg/kg dosage. All solutions were preserved in light-resistant containers at 4°C and utilized within 24 hr of preparation to ensure stability.
Induction of pre-leukemia with benzenePre-leukemic alterations were elicited with intravenous injection of benzene via the tail vein. Each rat was administered 0.2 mL of the diluted benzene solution (1:5:5 ratio with 2-propanol and distilled water) bi-daily for a duration of 4 weeks. This dose regimen was chosen based on recognized models that simulate benzene-induced hematotoxicity and genotoxic precursors to leukemia, enabling the observation of changes in bone marrow function without immediate mortality (Khalade et al., 2010; Zhao et al., 2021). Animals were observed daily for indicators of distress, including as alterations in body weight, activity levels, and hematological parameters using tail vein blood sample. All adverse reactions were documented, and humane endpoints were established to guarantee animal welfare.
Experimental designForty rats were randomly allocated to five groups (n = 8 per group) by stratified assignment to equalize body weights. A schematic overview of the experimental timeline, benzene exposure protocol, antioxidant treatment regimens, sample collection procedures, and downstream molecular and biochemical analyses is presented in Fig. 1.
Experimental design, dosing schedule, treatment groups, and analytical workflow in the benzene-induced pre-leukemic rat model. Rats were randomly allocated into five experimental groups (n = 8/group), including control, benzene-treated, melatonin-treated, vitamin C-treated, and combination-treated groups. The schematic illustrates the acclimatization period, benzene exposure protocol, antioxidant treatment regimens, sample collection procedures, and downstream analyses, including micronucleus assay, oxidative stress biomarker assessment, ELISA, and qRT-PCR gene expression analysis.
Group 1 (control) was administered oral distilled water daily. Group 2 (benzene-induced) received the benzene solution as previously detailed. Group 3 was administered benzene in conjunction with melatonin at a dosage of 10 mg/kg/day by oral route. Group 4 was administered benzene in conjunction with vitamin C at a dosage of 200 mg/kg/day orally. Group 5 was administered benzene along with a mixture of melatonin (10 mg/kg/day) and vitamin C (200 mg/kg/day) via oral route. Treatments were delivered through gavage for a duration of 4 weeks, commencing simultaneously with benzene induction. Dosing quantities were modified weekly according to body weight to ensure uniformity. This design facilitated the assessment of both individual and synergistic effects on benzene-induced changes.
Bone marrow smear preparationAt the end of the 4-week treatment period, rats were euthanized humanely by cervical dislocation under isoflurane anesthesia to minimize suffering. Bone marrow was harvested from the femurs by flushing with phosphate-buffered saline (PBS, pH 7.2) supplemented with 50% fetal bovine serum (FBS). The cell suspension was centrifuged at 3000 rpm for 10 min at 4°C, and the pellet was resuspended in a minimal volume of PBS. Smears were prepared by placing a drop of suspension on clean glass slides and spreading it evenly. Slides (at least five per animal) were air-dried for 10 min, fixed in absolute methanol for 5 min, and stained with a 1:1 mixture of May-Grünwald and Giemsa stains for 20 min. Bone marrow smears were examined under a light microscope using a 100× oil immersion objective lens, corresponding to a total magnification of 1,000× (Hayashi, 2016b).
Micronucleus assayGenotoxicity was evaluated with the bone marrow micronucleus assay, a conventional technique for identifying chromosomal damage (More et al., 2021). Subsequent to harvest, bone marrow cells were treated as previously described, and smears were produced and stained. Each rat was assessed for 1000 polychromatic erythrocytes (PCEs) and an equal number of normochromatic erythrocytes (NCEs) by two separate observers, who conducted the scoring blindly to reduce bias. The parameters included are: percentage of PCE (%PCE = PCEs / [PCEs + NCEs] × 100), percentage of NCE (%NCE = NCEs / [PCEs + NCEs] × 100), frequency of micronucleated PCE (MN-PCE = MN in PCEs / 1000 PCEs × 100), frequency of micronucleated NCE (MN-NCE = MN in NCEs / 1000 NCEs × 100), and the PCE/NCE ratio as a measure of erythropoietic activity. Micronuclei were recognized as diminutive, spherical entities exhibiting chromatin staining akin to that of the primary nucleus, yet distinct from it. Inter-observer variability was evaluated, and discrepancies were reconciled through consensus.
Antioxidants and free radicals biomarkersBlood samples were collected via cardiac puncture at euthanasia, allowed to clot, and centrifuged at 3000 rpm for 10 min to obtain serum. Oxidative stress biomarkers were quantified using commercial ELISA kits (Sunlong Biotech, China) following the manufacturer’s protocols. Superoxide dismutase (SOD), catalase (CAT), and 8-hydroxy-2′-deoxyguanosine (8-OHdG) were measured in duplicate with intra-assay CV <5%. Malondialdehyde (MDA) was assessed using the thiobarbituric acid (TBA) method: 150 µL serum was mixed with 10% trichloroacetic acid and 0.67% TBA, heated at 95°C for 45 min, cooled, and centrifuged. Supernatant absorbance was measured at 532 nm, and MDA concentration was calculated using the molar extinction coefficient of 1.56 × 10^5 M^-1 cm^-1 according to the method described by Ore and Akinloye (Ore and Akinloye, 2019). Serum activin A and follistatin levels were quantified by sandwich ELISA (Sunlong Biotech), with samples diluted 1:5, incubated on capture antibody-coated plates, and developed using TMB substrate. Absorbance at 450 nm was interpolated against a four-parameter logistic standard curve.
Gene expression analysis of activin A and follistatinTotal RNA was isolated from bone marrow cells utilizing the QIAamp RNA Blood Mini Kit (Qiagen), incorporating on-column DNase treatment to eradicate genomic contamination. The integrity of RNA was confirmed using agarose gel electrophoresis and NanoDrop spectrophotometry, with an A260/A280 ratio exceeding 1.8. Complementary DNA (cDNA) was generated from 1 µg of RNA utilizing the UltraScript Reverse Transcriptase Kit (PCR Biosystems) in a 20 µL reaction volume. Quantitative PCR (qPCR) was conducted on a StepOnePlus instrument (Applied Biosystems) utilizing SYBR Green master mix and gene-specific primers for Activin A (Inhba), follistatin (Fst), and β-actin (Actb) as the reference gene. Cycling parameters: 95°C for 10 min, succeeded by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Relative mRNA expression levels were quantified using the 2−ΔΔCt method with β-actin as the reference gene and the control group serving as the calibrator (El-Far et al.). Melt curve analysis validated amplicon specificity, and no-template controls were incorporated.
Gene expression analysis of activin A and follistatinTotal RNA was isolated from bone marrow cells utilizing the QIAamp RNA Blood Mini Kit (Qiagen), incorporating on-column DNase treatment to eradicate genomic contamination. RNA integrity was confirmed through agarose gel electrophoresis and NanoDrop spectrophotometry (A260/A280 ratio >1.8) (Svingen et al., 2015). Complementary DNA (cDNA) was generated from 1 µg of RNA utilizing the UltraScript Reverse Transcriptase Kit (PCR Biosystems) in a 20 µL reaction volume. Quantitative PCR (qPCR) was done on a StepOnePlus machine (Applied Biosystems) using SYBR Green master mix and gene-specific primers for Activin A (Inhba), follistatin (Fst), and β-actin (Actb) as the housekeeping gene. Cycling conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Relative expression was estimated using the 2^{-ΔΔCt} technique, normalized to β-actin and the control group (Livak and Schmittgen, 2001). Melt curve analysis showed amplicon specificity, and no-template controls were added.
Statistical analysisData were analyzed using GraphPad Prism version 9.0.0. Normality was assessed with the Shapiro-Wilk test, and homogeneity of variances with Levene’s test. One-way ANOVA followed by Tukey’s post-hoc test was used for multiple comparisons. Results are presented as mean ± SEM, with p < 0.05 considered statistically significant. Power analysis confirmed the sample size provided >80% power to detect moderate effect sizes.
Benzene exposure led to a pronounced disruption in bone marrow erythrocyte profiles. Compared to the control group, the benzene-treated rats exhibited a significant reduction in the percentage of polychromatic erythrocytes (%PCE; p < 0.0001) and a corresponding elevation in the percentage of normochromatic erythrocytes (%NCE; p < 0.0001), as illustrated in Fig. 1. Individual treatments with melatonin (10 mg/kg) or vitamin C (200 mg/kg) partially reversed these shifts, each increasing %PCE and decreasing %NCE relative to the benzene group (p < 0.0001 for both), though no significant difference was observed between these two monotherapies. Notably, the combined melatonin and vitamin C regimen yielded the most substantial improvements, with %PCE values approaching control levels and %NCE markedly reduced compared to all other groups (p < 0.0001).
Further analysis of genotoxic markers revealed that benzene significantly lowered the PCE/NCE ratio while elevating the frequencies of micronucleated PCE (%MN-PCE) and micronucleated NCE (%MN-NCE; all p < 0.0001 versus control), indicative of chromosomal instability and impaired erythropoiesis (Fig. 2).
Combined effects of melatonin and vitamin C on percentages of polychromatic erythrocytes (%PCE) and normochromatic erythrocytes (%NCE) in benzene-exposed rats. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the benzene-treated group.
All interventions ameliorated these effects to varying degrees, but the combination therapy demonstrated superior efficacy in restoring the PCE/NCE ratio and minimizing micronucleus formation. Representative photomicrographs confirmed the morphological distinctions among PCE, NCE, MN-PCE, and MN-NCE, supporting the quantitative scoring criteria used in the assay (Fig. 3). These findings highlight the potential of combined antioxidant supplementation to mitigate benzene-induced hematotoxicity more effectively than single agents.
Combined effects of melatonin and vitamin C on micronucleated polychromatic erythrocytes (%MN-PCE), micronucleated normochromatic erythrocytes (%MN-NCE), and PCE/NCE ratio in benzene-exposed rats. Data are expressed as mean ± SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the benzene-treated group.
Serum levels of Activin A were significantly diminished in the benzene-exposed group compared to controls (p < 0.01), with no notable differences among the melatonin, vitamin C, or combination treatment groups for this parameter. In contrast, follistatin levels remained largely unchanged across most groups but were markedly elevated in the benzene + melatonin + vitamin C group relative to all others (p < 0.0001), suggesting a synergistic upregulation (Fig. 4).
Representative microscopic identification of polychromatic erythrocytes (PCE), normochromatic erythrocytes (NCE), micronucleated polychromatic erythrocytes (MN-PCE), and micronucleated normochromatic erythrocytes (MN-NCE) in bone marrow smears of benzene-exposed rats. Benzene exposure induced increased micronucleus formation and altered erythrocyte morphology, whereas treatment with melatonin (MEL), vitamin C (Vit C), and their combination partially restored normal cellular morphology. Representative images were obtained from stained bone marrow smears and analyzed under light microscopy.
Regarding oxidative stress indicators, 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels increased in the benzene + melatonin (p < 0.001) and benzene + vitamin C (p < 0.01) groups compared to benzene alone, while the combination group showed only a modest, non-significant elevation. Catalase activity was reduced by benzene exposure (p < 0.05 versus control) but substantially restored in the melatonin-treated group (p < 0.01). Superoxide dismutase (SOD) levels did not vary significantly among groups. Unexpectedly, malondialdehyde (MDA) concentrations were lower in all benzene-exposed groups than in controls (p < 0.01), with a slight rebound in the combination group (p < 0.05 versus individual treatments), though still below baseline (Fig. 5 and Fig. 6). These patterns underscore differential impacts on oxidative pathways, with combined therapy offering the most balanced restoration.
Effects of melatonin and vitamin C on serum follistatin and activin A levels in benzene-exposed rats. (A) Serum activin A concentration and (B) serum follistatin concentration were determined using ELISA assays. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the benzene-treated group.
Effects of melatonin and vitamin C on oxidative stress biomarkers in benzene-exposed rats. (A) 8-hydroxy-2′-deoxyguanosine (8-OHdG), (B) superoxide dismutase (SOD), (C) catalase activity, and (D) malondialdehyde (MDA). Data are expressed as mean ± SD. Statistical significance was analyzed using one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the benzene-treated group.
Relative gene expression analysis using the 2^(-ΔΔCt) method demonstrated significant benzene-induced suppression of both Activin A and follistatin expression in bone marrow cells compared with controls (Fig. 7). Activin A expression decreased to approximately 0.43-fold of control levels in the benzene group, whereas follistatin expression declined more markedly to approximately 0.26-fold. Melatonin treatment alone did not substantially restore gene expression and was associated with persistently low Activin A and follistatin levels. In contrast, vitamin C treatment significantly enhanced expression of both genes relative to benzene exposure alone. The combined melatonin and vitamin C treatment produced the strongest transcriptional response, with Activin A and follistatin expression markedly elevated above benzene-treated levels and approaching or exceeding control values. These findings suggest that combined antioxidant treatment partially restores benzene-associated suppression of hematopoietic regulatory gene expression, although the precise mechanistic contribution of this pathway requires further investigation.
Relative fold changes in Activin A and follistatin gene expression (2−ΔΔCt) across experimental groups in the benzene-induced pre-leukemic rat model. Relative mRNA expression levels of (A) Activin A and (B) follistatin were quantified by qPCR, normalized to β-actin, and calculated using the 2−ΔΔCt method with the control group serving as the calibrator. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the benzene-treated group.
The present study demonstrated the significant mitigation of benzene-induced pre-leukemic changes in rats by combined supplementation of melatonin and vitamin C, mainly bone marrow genotoxicity, erythropoietic disruption, oxidative imbalance, and activin A/follistatin axis dysregulation. The combination regimen induced the most significant increase of the micronucleus frequency, restoration of the PCE/NCE ratio and recovery of the activin A and follistatin gene expression among all treatment groups suggesting a synergistic protective interaction between the two antioxidants.
Benzene exposure markedly changed the homeostasis of bone marrow as evidenced by the pronounced decrease in %PCE and increase in %NCE, the increased frequencies of MN-PCE and MN-NCE and the suppressed ratio of PCE/NCE. These results are consistent with established models of benzene-induced hematotoxicity and leukemogenic transformation, in which reactive benzene metabolites including hydroquinone and benzoquinone induce DNA strand breaks, chromosomal instability and impaired erythropoiesis (Bao et al., 2025). Sustained genotoxic stress in hematopoietic stem and progenitor cells is believed to be a key initiating event in the pathogenesis of myelodysplastic syndromes and acute myeloid leukemia (Mohammed et al., 2025; Muhialdin et al., 2023).
Partial improvement of these hematological abnormalities was observed following treatment with melatonin or vitamin C alone, but combined treatment demonstrated significantly greater protective efficacy. The reduction in micronucleus formation in the combination group suggests an increase in protection against chromosomal fragmentation and mitotic dysfunction. This may be a synergistic effect due to the complementary antioxidant mechanisms of melatonin and vitamin C. Melatonin is a potent mitochondrial targeted free radical scavenger and upregulates endogenous antioxidant enzyme activity, while vitamin C scavenges extracellular reactive oxygen species and enhances redox recycling. This combination of mechanisms might thus offer broader protection against benzene-induced oxidative and genotoxic damage in the bone marrow tissue (Zhao et al., 2021; Zhou et al., 2025).
The oxidative stress results are also in accordance with this interpretation. Benzene exposure significantly decreased catalase activity which is a sign of impaired enzymatic antioxidant defense. Melatonin treatment groups showed significant recovery of catalase levels, consistent with prior research demonstrating melatonin-mediated up-regulation of antioxidant enzymes and preservation of mitochondrial integrity in leukemia and toxicology models (Cao et al., 2025; Wang et al., 2022). In contrast, SOD levels were relatively stable among groups, suggesting that oxidative imbalance induced by benzene in this model may preferentially affect hydrogen peroxide detoxification pathways rather than superoxide metabolism (Darwiche et al., 2020; Ge et al., 2026; Zhao et al., 2025).
Interestingly, the 8-OHdG and MDA responses were less uniform than expected. Benzene exposure caused oxidative disruption, but the anticipated parallel increase of all oxidative biomarkers was not consistently seen. Similar inconsistencies have been reported in complex hematotoxic and pre-neoplastic conditions where adaptive cellular responses, altered lipid metabolism, compensatory antioxidant activation and selective oxidative targeting may influence behavior of biomarkers independently (Zhao et al., 2021; Zhou et al., 2025). The relatively modest increase of 8-OHdG in the combination group may therefore indicate a balanced redox state rather than persistent oxidative injury. Similarly, the decrease in the MDA levels in benzene groups might also be attributed to the change in the utilization of membrane lipids or adaptive metabolic responses in chronic benzene exposure (Zhou et al., 2025).
At the molecular level, benzene exposure was associated with suppression of activin A and follistatin gene expression in bone marrow cells, while combining melatonin and vitamin C treatment markedly increased the expression of both genes (He et al., 2020; Portale et al., 2019). However, the present findings should be interpreted cautiously. Although the combination therapy simultaneously improved genotoxicity indices and restored activin A/follistatin expression, the current experimental design does not allow determination of a direct causal relationship between these molecular alterations and the observed phenotypic protection. Moreover, an important consideration raised by the present findings is that the observed alterations in activin A/follistatin expression did not demonstrate a strict linear relationship with all oxidative stress markers or genotoxicity indices. In particular, serum protein concentrations and bone marrow mRNA expression patterns were not fully concordant across treatment groups. Therefore, the current data do not establish a direct causal role for activin A/follistatin signaling in mediating the anti-genotoxic effects of melatonin and vitamin C.
Instead, our data suggests that the reinstatement of the activin A/follistatin axis may be part of a broader protective response coupled with improved bone marrow homeostasis. The disconnect between circulating protein levels and local gene expression may be an expression of the complex biology of the activin/follistatin system such as post-transcriptional regulation, differential secretion kinetics, protein turnover, tissue compartmentalization and systemic inflammatory feedback mechanisms (Guo et al., 2024; Travaglini et al., 2022). Moreover, the decrease of oxidative stress might not necessarily correlate linearly with hematopoietic signaling responses in multifactorial pre-leukemic states.
Importantly, although causality cannot be inferred, the combination treatment consistently led to (i) the greatest decrease in micronucleus formation; (ii) the strongest recovery of erythropoietic indices; and (iii) the highest upregulation of activin A and follistatin expression. Taken together, these observations suggest a potential biological association between modulation of this signaling axis and amelioration of benzene-induced marrow injury.
An especially notable finding of the present study was the marked restoration of follistatin expression following combined melatonin and vitamin C treatment, which was substantially greater than that observed with either intervention alone. Given the established role of follistatin as a high-affinity endogenous antagonist of activin signaling, this pronounced upregulation may reflect an adaptive protective response aimed at restoring hematopoietic homeostasis under benzene-induced marrow stress. Previous studies have suggested that dysregulation of the activin A/follistatin axis contributes to abnormal hematopoietic differentiation, inflammatory signaling, and leukemogenic progression in myeloid malignancies (Zhao et al., 2021; Zhou et al., 2025). Therefore, the observed recovery of follistatin expression in the combination group may be biologically relevant to the attenuation of marrow injury and erythropoietic disruption observed in the present model.
Nevertheless, the current findings should be interpreted cautiously. Although follistatin upregulation closely paralleled improvements in micronucleus frequency and erythropoietic indices, the study design does not permit determination of whether follistatin acts as a direct mechanistic mediator or rather represents part of a broader coordinated protective response involving oxidative stress reduction, inflammatory modulation, and restoration of bone marrow homeostasis. Additional mechanistic investigations involving pathway inhibition, receptor blockade, or gene-silencing approaches will be required to determine the precise contribution of follistatin signaling to the protective effects of melatonin and vitamin C.
The enhanced protective efficacy of the combined treatment may involve mechanisms beyond direct antioxidant activity alone. Previous studies have suggested that melatonin and vitamin C can modulate multiple signaling pathways associated with leukemic progression and hematopoietic regulation (El-Far et al., 2023; Talib et al., 2025). Melatonin has been reported to influence inflammatory transcription factors, mitochondrial function, and receptor-mediated signaling through MT1/MT2 pathways, whereas vitamin C has been proposed to participate in redox-sensitive and epigenetic regulatory processes, including pathways associated with TET enzyme activity and hematopoietic differentiation (Guo et al., 2024; Travaglini et al., 2022). However, it is important to emphasize that the present study did not directly evaluate TET activity, DNA methylation, hydroxymethylation, or other epigenetic endpoints. Therefore, any involvement of epigenetic regulation in the observed protective effects should currently be considered speculative and hypothesis-generating rather than experimentally demonstrated.
It is important to note that benzene-induced genotoxicity is a highly multifactorial process that goes beyond the dysregulation of the activin A/follistatin axis alone. Several interrelated mechanisms have been implicated in benzene-associated hematotoxicity and leukemogenesis, including ROS overproduction, mitochondrial dysfunction, lipid peroxidation, DNA strand breakage, GPX4 inhibition-mediated ferroptosis induction, inflammatory signaling activation, and epigenetic dysregulation of hematopoietic stem cell (HSC) homeostasis (El-Far et al., 2023; Talib et al., 2025). Thus, the protective effects obtained by the combined treatment of melatonin and vitamin C are likely mediated by several complementary pathways and not by a single molecular mechanism.
In particular, melatonin has been shown to affect mitochondrial integrity, Nrf2-dependent antioxidant signaling, inflammatory cytokine production, and apoptosis-related pathways, while vitamin C can regulate TET-mediated DNA hydroxymethylation, redox cycling, and selective oxidative targeting of abnormal hematopoietic cells (Zhao et al., 2021; Zhou et al., 2025). The concomitant modulation of these processes may act in concert to reduce chromosome instability and promote better erythropoietic recovery in the present model (El-Far et al., 2023). In addition, the study did not assess other biomarkers of genetic damage and leukemogenic progression such as γ-H2AX, p53 activation, comet assay parameters, caspase signaling, Bax/Bcl-2 balance, and ferroptosis-related markers including GPX4 and lipid ROS, which should be examined in future studies to elucidate the mechanisms underlying the protective effects observed here.
Several limitations should be considered in the present findings. First, this experimental model is a pre-leukemic benzene-induced state and not fully established AML and thus may not fully reproduce the biological heterogeneity of human leukemia. Second, the follow-up time for the study was relatively short and long-term leukemic transformation or survival outcomes were not evaluated. Third, the measurements of serum activin A and follistatin were not accompanied by functional analyses of the pathway. Another limitation of the present study is the absence of direct epigenetic analyses. Although TET-associated pathways and epigenetic regulation were discussed based on previously published literature, no assessment of TET enzyme activity, DNA methylation/hydroxymethylation, histone modification, or chromatin remodeling was performed. Consequently, the study cannot determine whether epigenetic modulation contributed to the observed protective effects of melatonin and vitamin C.
Most importantly, no mechanistic inhibition experiments, receptor blockade studies, or gene-silencing approaches were performed to directly determine whether activin A/follistatin signaling functionally mediates the observed protective effects. Thus, the activin A/follistatin findings should be currently interpreted as associative, rather than causative. Future investigations with pathway-specific interventions and protein-level bone marrow analyses will be necessary to elucidate the precise mechanistic contribution of this signaling axis in benzene-induced leukemogenesis.
In conclusion, the combined administration of melatonin and vitamin C supplementation significantly reduced the pre-leukemic changes induced by benzene in rats, especially the genotoxic and oxidative alterations in the bone marrow tissue. These protective effects were associated with restoration of activin A and follistatin expression pattern, suggesting a potential link between modulation of this signaling axis and improved hematopoietic homeostasis. The results support the potential value of combined antioxidant therapy as a low-toxicity adjunctive strategy against benzene-associated hematotoxicity and early leukemogenic changes.
The authors gratefully acknowledge the support provided by the Department of Biology, Faculty of Science and Health, Koya University, and the Department of Biology, College of Science, Salahaddin University-Erbil, for providing the necessary laboratory facilities and resources. We also thank the Institutional Animal Ethics Committee of Cihan University-Erbil for approving the study protocol.
FundingThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
Conflict of interestThe 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.
Data availabilityThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
Author contributionsMohammed M. Hussein M. Raouf: Conceptualization, Methodology, Investigation, Formal analysis, Writing – original draft, Writing – review & editing, Visualization, Supervision, Project administration.
Ismail M. Maulood: Methodology, Validation, Resources, Writing – review & editing.
Zrar Saleem Marzani: Methodology, Investigation, Data curation, Writing – review & editing.
All authors read and approved of the final manuscript.
Ethical approval and consent to participateAll procedures involving animals were approved by the Institutional Animal Ethics Committee of Cihan University-Erbil (Approval Number: CUE-REC/2025/04) and conducted in accordance with international ethical guidelines for animal research, including the ARRIVE guidelines and the 3Rs principles.
Patient consent for publicationNot applicable (no human participants involved).
