Influence of Cinnamic Acid in Manganese Chloride-Induced Toxicity on some Heamatology and Reproductive Hormones in Female Rats

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1. Introduction

Manganese chloride is a potent environmental toxicant that disrupts the female reproductive system and hematological homeostasis in rats, primarily through oxidative stress and interference with the hypothalamic-pituitary-gonadal (HPG) axis (Zhu et al., 2016; Moreno et al., 2019) [11, 2]. Exposure to manganese chloride has been linked to reduced levels of reproductive hormones such as progesterone and estradiol, alongside hematological impairments like anemia and altered white blood cell counts (Olubodun et al., 2023) [3]. These effects are particularly pronounced in female rats due to sex-specific sensitivities to oxidative stress (Taylor et al., 2018) [6]. Cinnamic acid, a naturally occurring phenolic compound found in plants like cinnamon, exhibits antioxidant, anti-inflammatory, and metal-chelating properties, making it a promising candidate for mitigating heavy metal toxicity Yang et al., 2021) [8]. While cinnamic acid has shown protective effects against oxidative stress in reproductive and hematological systems, its specific role in counteracting manganese chloride–induced toxicity remains underexplored (Liu et al., 2023). This study investigates cinnamic acid’s efficacy in ameliorating manganese chloride–induced disruptions in progesterone, estradiol, and hematological parameters in female Wistar rats, aiming to provide insights into its therapeutic potential.

2. Materials and Methods

2.1. Preparation of Cinnamic Acid

Cinnamic acid (analytical grade) was dissolved daily in 5% dimethyl sulfoxide (DMSO) and diluted with distilled water to achieve concentrations of 50 mg/kg (low dose) and 100 mg/kg (high dose). Solutions were administered orally via gavage using a 5 ml syringe with an oral cannula, with doses adjusted weekly based on body weight (e.g., 10 mg for a 200 g rat at 50 mg/kg).

2.2. Preparation of Manganese Chloride

Manganese chloride (MnCl₂·4H₂O, analytical grade) was dissolved in distilled water to prepare a stock solution, stored in an amber-colored bottle to prevent light degradation. A daily oral dose of 10 mg/kg body weight was administered via gavage (e.g., 2 mg for a 200 g rat), adjusted weekly according to body weight.

2.3. Experimental Design

Thirty female Wistar rats (average weight 158–162 g) were acclimatized for two weeks and randomly assigned to six groups (n=5 per group) for a 90-day study: • Group A (Control): Fed grower mash and water ad libitum. • Group B (Manganese chloride only): Received 10 mg/kg manganese chloride daily. • Group C (Cinnamic acid low dose): Received 50 • Group D (Cinnamic acid high dose): Received 100 • Group E (Manganese chloride + cinnamic acid low dose): Received 10 mg/kg manganese chloride and 50 • Group F (Manganese chloride + cinnamic acid high dose): Received 10 mg/kg manganese chloride and 100 All procedures complied with Institutional Animal Care and Use Committee (IACUC) guidelines.

2.4. Blood Collection and Serum Tests

After 90 days, rats were fasted overnight, anesthetized with chloroform vapor, and sacrificed. Blood was collected via cardiac puncture using a 5 ml syringe with a 23G needle. Samples for hormonal assays were placed in plain tubes, clotted for 30 minutes, and centrifuged at 3000 rpm for 15 minutes to obtain serum, which was stored at −20°C. Hematological samples were collected in EDTA tubes and analyzed within 6 hours.

2.5. Biochemical Assays

Serum progesterone and estradiol levels were measured using ELISA kits with an ELISA microplate reader. Hematological parameters, including white blood cell count (WBC), lymphocyte percentage (LYM%), granulocyte count (GRAN#), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), and platelet count (PLT), were analyzed using an automated hematology analyzer.

2.6. Statistical Analysis

Data were analyzed using one-way ANOVA with Tukey’s post-hoc test. Results are expressed as mean ± standard deviation (SD), with significance set at p < 0.05.

3. Results and Discussion

3.1. Hormonal Parameters

Manganese chloride exposure (Group B) significantly reduced progesterone (2.45 ± 0.71 ng/mL) and estradiol (0.70 ± 0.22 pg/mL) compared to the control group (15.61 ± 2.19 ng/mL and 6.38 ± 2.77 pg/mL, respectively; p < 0.01) (Table 1). This finding aligns with Wang et al. (2019) [7], who reported manganese chloride–induced hormonal suppression resulting from oxidative stress and HPG axis dysregulation. Cinnamic acid alone (Groups C and D) maintained or elevated hormone levels, with the high-dose group (D) showing significant increases (67.74 ± 5.34 ng/mL for progesterone and 14.63 ± 0.20 pg/mL for estradiol; p < 0.01). Co-treatment with cinnamic acid (Groups E and F) partially restored hormone levels, with Group F showing better recovery (17.66 ± 0.46 ng/mL for progesterone and 2.19 ± 0.13 pg/mL for estradiol; p < 0.05), suggesting dose-dependent antioxidant effects (Yang et al., 2021) [8].

3.2 Hematological Parameters

Cinnamic acid alone (Groups C and D) maintained most parameters close to control levels, with Group D showing a significant increase in PLT (1141.33 ± 466.88 ×10⁹/L vs. 609.00 ± 91.55 ×10⁹/L; p < 0.05), suggesting enhanced platelet production, as noted by Sadeghi et al. (2021). Co-treatment with cinnamic acid (Groups E and F) mitigated the effects of manganese chloride, with Group F (high-dose cinnamic acid) showing significant improvements in RBC (7.59 ± 0.46 ×10¹²/L), HGB (13.50 ± 0.74 g/dL), HCT (43.93 ± 8.24%), GRAN% (3.67 ± 0.06%), GRAN# (0.20 ± 0.10 ×10⁹/L), and P-LCR (9.07 ± 4.70%) (p < 0.05 compared to Group B). LYM% in Group F (84.37 ± 0.63%) also improved significantly (p < 0.05), indicating reduced inflammation. Other parameters, including WBC, MID%, LYM#, MID#, MCV, MCH, MCHC, RDW-SD, RDW-CV, MPV, PDW, and PCT, showed no significant differences (p > 0.05), suggesting that cinnamic acid’s protective effects are parameter-specific. These findings support cinnamic acid’s role in counteracting manganese chloride–induced oxidative stress and inflammation, likely through its antioxidant and metal-chelating mechanisms (Zhang et al., 2022) [9].

Table 2: Influence of Cinnamic Acid in Manganese Chloride Induced Toxicity on Hematological Parameters and in Female Rats Hematology Parameter A (Control) B (MnCl) only C (CA low dose) D (CA high dose) E (MnCl + CA Low dose)

3.3. Discussion

The hormonal suppression observed in Group B reflects manganese chloride’s disruption of steroidogenic enzymes and hypothalamic-pituitary-gonadal (HPG) axis function, as reported by Zhao et al. (2021) [10]. The restoration of hormone levels by cinnamic acid, particularly at high doses, suggests that its antioxidant properties effectively mitigate oxidative stress in ovarian tissues (Chen et al., 2022) [1]. Hematological changes in Group B, including reduced RBC, HGB, and HCT, alongside increased GRAN%, GRAN#, RDW-SD, RDW-CV, and P-LCR, indicate anemia, inflammation, and altered red cell morphology—findings consistent with Olubodun et al. (2023) [3] and Pereira et al. (2020) [4]. The significant recovery observed in Group F (RBC, HGB, HCT, GRAN%, GRAN#, LYM%, and P-LCR) supports cinnamic acid’s role in reducing erythrocyte damage and inflammation, likely through its antioxidant and metal-chelating mechanisms (Zhang et al., 2022) [9]. Non-significant changes in WBC, MID%, LYM#, MID#, MCV, MCH, MCHC, MPV, PDW, and PCT suggest selective toxicity and protection, possibly due to manganese chloride’s targeted impact on erythropoiesis and granulocyte activation (Silva et al., 2021) [5]. The elevated PLT observed in Group D aligns with findings by Sadeghi et al. (2021), though variability in other parameters indicates potential individual response differences. The 90-day study duration and single manganese chloride dose may limit insight into chronic toxicity, while missing data for some parameters (e.g., PDW, PCT in Group D) suggest areas requiring further investigation (Rocha et al., 2021).

4. Conclusion

This study demonstrates that manganese chloride induces significant reproductive and hematological toxicity in female Wistar rats, characterized by reductions in progesterone, estradiol, RBC, HGB, and HCT, alongside increases in GRAN%, GRAN#, RDW-SD, RDW-CV, and P-LCR. Cinnamic acid, particularly at 100 mg/kg, effectively mitigates these effects, restoring hormonal balance and improving hematological parameters through its antioxidant and metal-chelating properties.

These findings highlight cinnamic acid’s therapeutic potential against manganese chloride–induced toxicity. However, further research is needed to optimize dosing, elucidate underlying mechanisms, and assess long-term safety and efficacy in chronic exposure models.