Manganese Exposure: Delayed Effects and Biomarkers in adult male and female Wistar rats

Abstract

Manganese (Mn) is an essential trace element required for various physiological processes, but excessive exposure can lead to neurotoxicity and motor impairments. This study investigated the immediate and delayed behavioral effects and potential tissue biomarkers, as well as the immediate oxidative and metabolic alterations, following subacute Mn intoxication in adult male and female Wistar rats. Animals received MnCl₂ (15 mg/kg, intraperitoneally, for 5 days per week over 4 weeks, totaling 30 days of exposure). Following this treatment, animals were allocated into two independent experimental endpoints. The first group was evaluated immediately after the end of the 30-day MnCl₂ exposure period. The second group underwent an additional 30-day Mn-free recovery period before evaluation, resulting in a total experimental duration of 60 days for this group. Corresponding control groups were run in parallel for both endpoints. After the exposure period, no significant motor coordination deficits were observed. However, motor impairments emerged after the recovery period, suggesting delayed neurotoxic effects. Oxidative stress markers revealed decreased non-protein thiol levels in the cortex and hippocampus, indicating Mn-induced antioxidant depletion, while mitochondrial complex activities varied by sex and brain regions. Although Mn concentrations in brain tissues returned to baseline after recovery, elevated Mn levels persisted in nails and teeth, suggesting that these tissues are promising non-invasive biomarkers of cumulative Mn exposure. These findings demonstrate that, within a predefined two-endpoint design, Mn neurotoxicity manifests in a time- and sex-dependent manner, highlighting distinct temporal patterns of functional and biochemical vulnerability. Thus, the identification of nails and teeth as reliable non-invasive biomarkers further underscores the complexity of Mn toxicity and highlights the importance of considering sex-specific responses in toxicological assessments.

Graphical Abstract

Summary of sex-dependent behavioral, biochemical, and bioaccumulation outcomes following subacute manganese (Mn) exposure in adult Wistar rats. Male and female rats were exposed to MnCl₂ (15 mg/kg/day, intraperitoneally) for 30 consecutive days and evaluated at two predefined experimental endpoints: immediately after the exposure period and after an additional 30-day Mn-free recovery interval. Immediate assessments revealed sex-dependent alterations in anxiety-like behavior and region-specific oxidative and mitochondrial changes. After the recovery period, delayed motor coordination impairments were evident, particularly in males, despite normalization of brain Mn levels. Mn bioaccumulation persisted in peripheral tissues, including hair, nails, and teeth, in both sexes. Overall, the results show that, within this two-endpoint design, Mn induces time-dependent and sex-specific neurotoxic effects, with nails and teeth emerging as reliable non-invasive biomarkers of cumulative Mn exposure.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12035-026-05812-0.

Introduction

Manganese (Mn) is an essential trace element required for multiple physiological processes, but when present in excess it becomes neurotoxic [1]. Toxic Mn accumulation may arise from genetic disorders, secondary to liver dysfunction, and/or environmental exposure exceeding the World Health Organization's recommended intake (0.7–10.9 mg/day), including contaminated drinking water or pesticide use [2–4].

The brain is particularly vulnerable to Mn excess, with preferential accumulation in basal ganglia structures, such as the striatum, substantia nigra, and globus pallidus. The clinical manifestations, collectively referred to as manganism, resemble Parkinsonian syndromes and include motor, neuropsychological, and cognitive deficits [4–9]. Importantly, adverse effects have also been reported in both humans [10, 11] and animal experimental models [12, 13], even at low-level exposure [6]. These effects can be modulated by factors such as age, sex, and ethnicity [14].

Although the mechanisms underlying Mn neurotoxicity are not fully defined, several lines of evidence implicate oxidative stress, mitochondrial dysfunction, neuroinflammation, and neurotransmitter disruption as central pathways contributing to neural injury [15–22].

Despite substantial evidence of Mn-induced neurotoxicity, few studies have systematically evaluated whether metabolic, biochemical, and long-term neurobehavioral alterations persist after exposure cessation or recover over time. Moreover, the potential utility of peripheral keratinized tissues, such as nails and teeth, as cumulative and non-invasive biomarkers of Mn exposure remains poorly understood. To address these gaps, we employed an in vivo model in adult male and female Wistar rats exposed to MnCl₂ for 30 days, using two predefined experimental endpoints: one cohort evaluated immediately at the end of exposure and a second cohort evaluated after an additional 30-day Mn-free recovery interval. This design enabled us to distinguish immediate from delayed effects on behavior, brain region-specific oxidative and mitochondrial alterations, and peripheral Mn bioaccumulation, allowing the characterization of sex-dependent vulnerabilities and the identification of reliable biomarkers of cumulative Mn exposure.

Material and Methods

Animals

Male and female adult Wistar rats were obtained from the Federal University of Rio Grande do Sul (UFRGS), Brazil, and acclimated to the housing conditions for 20 days upon arrival. Adult rats (8 weeks of age; body mass 200–300 g) were housed in the UNOESC animal facility under controlled conditions (22 ± 1 °C; 12-h light/dark cycle; 30–70% humidity). Animals had ad libitum access to water and standard chow containing 110 mg/kg of Mn. The experimental protocol was approved by the UNOESC Ethics Committee for Animal Research (protocol number 45/2016). All efforts were made to minimize the number of animals used and to reduce animal suffering.

For this study, a total of 80 adult Wistar rats were used. Animals were prospectively allocated to two predefined experimental cohorts (immediate and recovery), each initially comprising n = 10 animals per sex per treatment group. Detailed allocation of animals across behavioral and biochemical endpoints is provided in Supplementary Table S1.

Chemicals

All reagents were of analytical grade or the highest purity available for pharmaceutical applications. Manganese chloride tetrahydrate (MnCl₂·4H₂O, MW 197.91) was obtained from Fisher Chemical (Fair Lawn, NJ). Nitric acid was purchased from VWR International (Radnor, PA), and perchloric acid was obtained from RICCA Chemical Company (Arlington, TX). Anhydrous disodium hydrogen phosphate (Na₂HPO₄), sodium phosphate monobasic (NaH₂PO₄), bovine serum albumin (BSA), ethylenediaminetetraacetic acid (EDTA), and sucrose were purchased from Sigma-Aldrich (St. Louis, MO). Manganese solutions were freshly prepared by dissolving MnCl₂·4H₂O in sterile 0.9% saline to achieve concentrations of 15 mg/kg. Solutions were prepared every two weeks and stored at 4 °C. Prior to intraperitoneal (i.p.) injection, the solutions were equilibrated to room temperature for 30 min.

Mn Treatment In Vivo

The subacute Mn experimental intoxication protocol was adapted from O’Neal et al. [23]. Animals were randomly assigned to two predefined experimental endpoints groups to distinguish immediate from delayed effects of Mn exposure. All treated animals received MnCl₂ (15 mg/kg, intraperitoneally (i.p)), once daily for five consecutive days per week over four weeks (30 days) to induce intoxication. Both cohorts underwent the same dosing regimen; however, the recovery cohort was maintained for an additional 30-day Mn-free period to assess delayed neurotoxic outcome (totaling 60 days). A matched control group for each endpoint was run in parallel and received i.p. injections of the vehicle (1 mL/kg of 0.9% sterile saline) (Scheme 1).

Scheme 1.

Experimental design and timeline of subacute manganese exposure and evaluation endpoints. Adult male and female Wistar rats received MnCl₂ (15 mg/kg, i.p.) once daily, five days per week, for four consecutive weeks. From the beginning of the study, animals were allocated into two predefined experimental endpoints. The immediate cohort was evaluated and euthanized at the end of the 30-day exposure period to assess early behavioral outcomes, brain region–specific oxidative and mitochondrial alterations, and Mn/Fe tissue accumulation. In parallel, the recovery cohort was maintained for an additional 30-day Mn-free recovery interval before evaluation (totaling 60 days), allowing the assessment of delayed behavioral effects, along with Mn and Fe levels in brain and peripheral tissues. Control groups were run in parallel for each endpoint and received vehicle injections. Behavioral tests, biochemical analyses, and metal quantification were performed according to the timeline indicated

Behavioral tests

Elevated Plus-maze

Anxiety-related behaviors were assessed separately in animals evaluated immediately after exposure and in those evaluated after a 30-day Mn-free recovery period using the elevated plus-maze (EPM). The EPM is a plus-shaped wooden apparatus consisting of two opposite open arms (50 cm × 10 cm) and two opposite enclosed arms (50 cm × 10 cm × 40 cm) extending from a central platform (10 cm × 10 cm). The maze is elevated 50 cm above the floor. Each animal was individually placed in the center of the maze, facing one of the enclosed arms, and allowed to explore freely for 5 min [24]. The following parameters were recorded: the frequency of open-arm entries, the frequency of enclosed-arm entries, and the time spent in open arms. An entry was defined when the animal placed all four paws into an arm. A decrease in open-arm entries and/or time spent in the open arms was interpreted as an anxiogenic effect. Final sample sizes for behavioral analyses are provided in Supplementary Table S2.

Rotarod

Motor coordination and balance were assessed separately in animals evaluated immediately after exposure and in those evaluated after a 30-day Mn-free recovery period using an accelerating rotarod apparatus (Insight Scientific Equipments, Ribeirão Preto, SP, Brazil). The rotarod consists of a grooved metal roller, 6 cm in diameter and 16 cm in height, divided into 9 cm wide compartments. During the adaptation phase, animals were placed on the non-rotating roller until they could maintain a stable position with all four paws for 30 s. To ensure the animals' aptitude for the task, a baseline test followed, where the rats were placed on the rotarod at a constant speed of 12 rpm for one min. Motor coordination was evaluated using an accelerated protocol in a single session, where the rotation speed incrementally increased from 0 to 40 rpm over a span of 300 s. The latency to fall, recorded in seconds, was measured [25]. Final sample sizes for behavioral analyses are provided in Supplementary Table S2.

Tissue Preparation for Analysis

At each experimental endpoint (immediate evaluation after 4 weeks of Mn exposure or recovery evaluation following 4 weeks Mn-free recovery period), the animals were anesthetized with ketamine (80 mg/kg) and xylazine (12 mg/kg) and euthanized by decapitation. Brain tissues and serum were rapidly collected and stored at − 80 °C for further analyses. Additionally, samples of hair, nails, and teeth were immediately collected and prepared for subsequent analysis.

Determination of Mn and/or Fe by Atomic Absorption Spectrometry

Mn and Fe concentrations were analyzed in animals from both endpoints. 100 mg of brain tissue, 50 mg each of hair and teeth, 20 mg of nails, and 300 μL of serum [26], were used. Tissues were digested in a 4:1 mixture of 65% nitric acid and perchloric acid for 24 h at room temperature. After digestion, samples were placed in an ultrasonic bath at 70 °C for 3 h, followed by the addition of 3.5 mL of 1% perchloric acid. The final solution was filtered through a 0.22 μm syringe filter. Metal concentrations were determined using a Perkin Elmer Atomic Absorption Spectrometer (Analyst 800®) calibrated with standard curves. Results were expressed as μg/g for solid tissues and μg/mL for serum.

Oxidative and Metabolic Parameters

Oxidative and metabolic parameters were assessed in brain tissues collected from animals in the immediate endpoint cohort and were homogenized in 10 volumes of 20 mM HEPES buffer (pH 7.4) and centrifuged at 5,000 × g for 10 min at 4 °C. The supernatant was used to determine thiobarbituric acid-reactive substances (TBARS) and non-protein thiol (NPSH) levels.

Measurement of Thiobarbituric Acid-reactive Substances (TBARS)

Lipid peroxidation was determined in the immediate endpoint cohort, using the TBARS method, as described by Esterbauer and Cheeseman [27]. Briefly, brain tissue homogenates were mixed with an equal volume of 10% trichloroacetic acid (TCA) and centrifuged at 3,000 × g for 10 min. An aliquot of the supernatant was incubated with an equal volume of 0.67% thiobarbituric acid (TBA) in 7.1% sodium sulfate and heated in a water bath for 45 min. The absorbance of the pink complex formed by the reaction of TBA with lipid peroxidation by-products was measured at 535 nm using a Synergy HTX microplate reader (BioTek®). A standard calibration curve was prepared with malondialdehyde (MDA) standards treated identically to the samples. TBARS levels were expressed as nmol MDA/mg of protein.

Non-protein Thiol Groups (NPSH) Measurement

NPSH groups, primarily glutathione (GSH), were determined in the immediate endpoint cohort, using Ellman’s reagent [28]. Previously prepared brain tissue supernatants were treated with an equal volume of 10% TCA and centrifuged. Aliquots of the supernatant were diluted in 80 mM sodium phosphate buffer (pH 7.4). To these diluted samples, 0.5 mM DTNB (5,5'-dithiobis-2-nitrobenzoic acid) was added. Absorbance of the colorimetric reaction between DTNB and thiol groups was measured at 412 nm using a Synergy HTX microplate reader (BioTek®) after a 10-min reaction. NPSH levels were expressed as nmol NPSH/mg of protein.

Mitochondrial Complex I and II Activities

Brain tissues from the immediate endpoint cohort were homogenized in 10 volumes of 4.4 mM potassium phosphate buffer (pH 7.4) containing 0.3 M sucrose, 5 mM MOPS, 1 mM EGTA, and 0.1% bovine serum albumin. Homogenates were centrifuged at 3,000 × g for 10 min at 4 °C. The supernatant was used to measure mitochondrial complex I and II activities, following the methods of Latini et al. [29] [29]. Complex I activity was determined by measuring NADH-dependent ferricyanide reduction at 420 nm [30]. Complex II activity (succinate–2,6-dichloroindophenol (DCIP) oxidoreductase) was measured as described by Fischer et al. [31]. Enzyme activities of the respiratory chain complexes were expressed as nmol/min/mg of protein, measured at 37 °C using a Synergy HTX microplate reader (BioTek®).

Protein Determination

Protein concentration was measured by Lowry method [32] using bovine serum albumin as the standard.

Statistical Analysis

Data are presented as mean ± standard error of mean (SEM). Body weight gain was analyzed using two-way repeated-measures ANOVA followed by Tukey’s post hoc test. Data from behavioral parameters and biochemical determinations were analyzed using one-way or two-way ANOVA, to verify the interaction between group and sex, both followed by Tukey’s post hoc test when p was significant. The “n” values represent the number of animals included in each test. Only significant p values were given in the text. Differences between groups and sexes were considered significant when p < 0.05. Statistical analyses were performed using the Statistica® version 9.0 software and the graphics were made using GraphPad Prism® software version 10 (GraphPad Software, San Diego, CA, USA).

Regarding sample size considerations, all groups initially comprised n = 10 animals per sex per treatment; however, subsets of animals were allocated to specific endpoints according to predefined methodological requirements, resulting in variation in final n across behavioral and biochemical analyses. Importantly, group sizes were balanced within each individual analysis. Two-way ANOVA models (including repeated-measures when applicable) were applied, and all relevant interaction terms were explicitly tested. Pearson’s correlation analyses were performed to evaluate associations between peripheral Mn levels and oxidative parameters. Detailed sample sizes per outcome are provided in Supplementary Table S1, significant interaction analyses are summarized in Supplementary Table S2, and correlation results are presented in Supplementary Table S3.

Results

Figure 1 illustrates the immediate effects of subacute Mn exposure, evaluated at the end of the 30-day treatment period, on body weight gain, motor coordination, and anxiety-like behavior. Compared to male controls, Mn exposure significantly reduced body weight gain in male rats during weeks 2, 3, and 4 of the treatment period. In contrast, no significant differences were observed in female, regardless of Mn treatment (Fig. 1a) (interaction between group and week: [F_(1,19) = 32.625; P < 0.001], interaction between group and sex: [F_(1,19) = 62.997; P < 0.001] and interaction between group, sex and week: [F_(1,19) = 8.66; P < 0.001]). Given that Mn intoxication causes Parkinson-like symptoms, motor coordination was evaluated using the Rotarod test. No differences in latency to fall were observed between Mn-treated and control animals of either sex in the immediate post-exposure assessment (Fig. 1b). Based on these observations, we next assessed whether Mn exposure alters anxiety-like and exploratory activity using the EPM test. No differences were observed in the number of entries into the closed arms of the EPM in the immediate post-exposure cohort (Fig. 1c). Mn-exposed males showed a significant lower percentage of open-arm entries compared to control males (Fig. 1d) [F_(3,19) = 5.049; P < 0.01], indicating increased anxiety-like behavior. This effect was not observed in females. Additionaly, Mn-treated females exhibited significantly higher percentage of open-arm entries compared to Mn-treated males (Fig. 1d) (interaction between group and sex: [F_(1,19) = 10.03; P < 0.01]). Similarly, Mn-treated females spent more time in the open arms compared to Mn-treated males after the 30-day treatment period (Fig. 1e) (interaction between group and sex: [F_(1,19) = 6,13; P < 0.05]).

Fig. 1.

Immediate effects of subacute Mn exposure (MnCl₂; 15 mg/kg i.p.; daily for 5 days/4 weeks) on body weight gain (a), motor coordenation (b) and anxiety-like behavior (c-e) in adult male and female Wistar rats. Data are expressed as mean ± standard error of the mean (10 animals/group for body weight gain and 6 animals/group for behavioral tests). Significant p-values are shown in each panel. For panel (a) ^∗p < 0.05 when compared to vehicle group between weeks of treatment using Two-way ANOVA for repeated measures followed by Tukey post-hoc test. For panel (b-e) one and Two-way ANOVA followed by Tukey post-hoc test were used for behavioral comparisons

Mn accumulation in the central nervous system (CNS) is associated with motor impairment. To determine whether Mn preferentially accumulated in specific brain structures during the immediate post-exposure period, Mn concentrations were quantified in the cortex, hippocampus, cerebellum and striatum (Fig. 2a-d). In females, Mn exposure significantly increased Mn concentration in the cortex (Fig. 2a) [F_(3,24) = 9.406; P < 0.001], hippocampus [F_(3,24) = 10.38; P < 0.001] (Fig. 2b), cerebellum [F_(3,25) = 29.81; P < 0.001] (Fig. 2c), and striatum [F_(3,25) = 33.1; P < 0.001]) (Fig. 2d). In males, Mn accumulation was detected primarily in the striatum (Fig. 2d) and, although not statistically significant, a trend toward increased Mn levels was observed in the cerebellum (p = 0.06) (Fig. 2c). Thus, two-way ANOVA confirmed sex-dependent Mn deposition patterns in the hippocampus (Fig. 2b) and cerebellum (Fig. 2c) of Mn treated groups (interaction between group and sex: hippocampus [F_(1,24) = 9.478; P < 0.01] and cerebellum [F_(1,24) = 4.911; P < 0.05]).

Fig. 2.

Immediate effect of subacute Mn exposure (MnCl₂; 15 mg/kg i.p.; daily for 5 days/4 weeks) on Mn concentrations in the cerebral cortex (a), hippocampus (b), cerebellum (c), striatum (d), hair (e), nail (f), tooth (g) and serum (h) in adult male and female Wistar rats. Data represent mean ± standard error of the mean (6 animals/group). Significant p-values and interaction effects are indicated in each panel. One and two-way ANOVA followed by Tukey post-hoc test was used for statistical comparisons

Given the well-established correlation between iron (Fe) and Mn homeostasis, Fe concentrations were assessed in the same brain regions at both predefined experimental endpoints, immediate after the exposure period and after the 30-day Mn-free recovery period (Suppl. Figure S1). Our analysis revealed that during the immediate post-exposure assessment cohort, Mn exposure let to a significantly lower Fe concentration in the striatum of females when comparing to their controls [F_(3,24) = 4.375; P < 0.05]) (Suppl. Fig. S1d). Sex-dependent patterns were also evident, with females showing reduced Fe levels in the hippocampus (Suppl. Fig. S1b) and increased Fe levels in the cerebellum (Suppl. Fig. S1c) relative to Mn-treated males (interaction between group and sex: hippocampus [F_(1,25) = 4.306; P < 0.05] and cerebellum [F_(1,25) = 12.53; P < 0.05]). In contrast, after the 30-day Mn-free recovery period, Fe concentration did not differ between Mn-treated and controls animals in any brain region (Suppl. Fig. S1e-h), suggesting normalization of Fe homeostasis over time despite the early sex-specific disruptions observed immediately after exposure.

To evaluate the usefulness of peripheral tissues as biomarkers of systemic Mn exposure, Mn concentrations were measured in hair, nails, teeth, and serum, during the immediate post-exposure assessment (Fig. 2e-h). Mn exposure led to a significant increase in Mn deposition in the hair (Fig. 2e) [F_(3,21) = 55.16; P < 0.001], nails (Fig. 2f) [F_(3,21) = 10.29; P < 0.001], and teeth (Fig. 2g) [F_(3,20) = 76.51; P < 0.01] in both male and females in comparison to their controls. Serum Mn did not differ between groups (Fig. 2h). Furthermore, Mn-treated females exhibited significantly higher Mn concentrations in hair compared to males (Fig. 2e). In contrast, males showed higher Mn levels in nails (Fig. 2f) and teeth (Fig. 2g) relative to females, indicating tissue-specific and sex-dependent patterns of Mn deposition (interaction between group and sex: hair [F_(1,21) = 5.594; P < 0.001], nails [F_(1,21) = 1.149; P < 0.05] and tooth [F_(1,20) = 12.748; P < 0.001]).

To further characterize Mn-induced metabolic alterations, immediate post-exposure activities of mitochondrial respiratory chain complexes I (CI) and II (CII) were measured across brain regions (Fig. 3). CI activity was selectively altered in a sex- and region- dependent manner, with a significant increased in the striatum of Mn-treated females compared to their Mn-treated counterparts [F_(3,27) = 9.849; P < 0.05] and to Mn-treated males (Fig. 3d) (interaction between group and sex: [F_(1,27) = 10.248; P < 0.001]). No differences were detected in the other brain regions. Regarding CII activity, distinct and sex-dependent immediate effects of Mn exposure were observed across brain regions (Fig. 3e-h). Mn-treated males exhibited a significant inhibition of cortical CII activity, whereas Mn-treated females showed a significant increased compared to their respective controls [F_(3,23) = 7.323; P < 0.01], resulting in a robust different sex-pattern (interaction between group and sex: [F_(1,23) = 21.829; P < 0.01]) (Fig. 3e). Furthermore, Mn exposure let to a significant reduction in CII activity in the male hippocampus [F_(3,23) = 4.534; P < 0.05] and female cerebellum [F_(3,24) = 11.09; P < 0.01] when compared to the controls (Fig. 3f and g). No significant alterations in CII activity were detected in the striatum (Fig. 3h).

Fig. 3.

Immediate effect of subacute Mn exposure (MnCl₂; 15 mg/kg i.p.; daily for 5 days/4 weeks) on mitochondrial respiratory chain complex I activity in the cerebral cortex (a), hippocampus (b), cerebellum (c), and striatum (d), and on complex II activity in the cerebral cortex (e), hippocampus (f), cerebellum (g), and striatum (h) in adult male and female Wistar rats. Data represent mean ± standard error of the mean (7 animals/group). Significant p-values are described in each panel. One and Two-way ANOVA followed by Tukey post-hoc test was used for statistical comparisons

Given the observed alterations in mitochondrial activity in the immediate post-exposure cohort, oxidative stress markers were assessed at the end of the 30-day Mn exposure period (Fig. 4). Lipid peroxidation analysis demonstrated no significant differences in TBARS levels between Mn-treated and control animals across any brain region (Fig. 4a-d). In contrast, the NPSH levels were significantly decreased in the cortex [F_(3,36) = 13.17; P < 0.001] and hippocampus [F_(3,36) = 13.33; P < 0.001] of Mn-treated females compared to controls (Fig. 4e and f). No NPSH content alterations were observed in the cerebellum (Fig. 4g) or striatum (Fig. 4h). These findings suggest selective oxidative vulnerability in specific brain regions following Mn exposure.

Fig. 4.

Immediate effects of subacute Mn exposure (MnCl₂; 15 mg/kg i.p.; daily for 5 days/4 weeks) on lipid peroxidation (TBARS) levels in the cerebral cortex (a), hippocampus (b), cerebellum (c), and striatum (d), and on non-protein thiol (NPSH) levels in the cerebral cortex (e), hippocampus (f), cerebellum (g), and striatum (h) in adult male and female Wistar rats. Data represent mean ± standard error of the mean (9 animals/group). Significant p-values are described in each panel. One and Two-way ANOVA followed by Tukey post-hoc test was used for statistical comparisons

Considering the importance of monitoring the progression or remission of toxic effects over time, and to distinguish immediate from delayed responses following exposure cessation, behavioral parameters were evaluated in a predefined recovery cohort after and additional 30-day Mn-free period. After this recovery interval, Mn-treated animals exhibited increased body weight gain when compared to their controls in all weeks evaluated. (Fig. 5a) (interaction between group and week: [F_(1,19) = 21.263; P < 0.001], interaction between group and sex: [F_(1,19) = 29.063; P < 0.001] and interaction between group, sex and week: [F_(1,19) = 4.606; P < 0.01]).

Fig. 5.

Delayed effects of subacute Mn exposure (MnCl₂; 15 mg/kg i.p.; daily for 5 days/4 weeks) on body weight gain (a), motor coordination (b) and anxiety-like behavior (c-e) after a 30-day Mn-free recovery period in adult male and female Wistar rats. Data represent mean ± standard error of the mean (10 animals/group for body weight gain and 5 animals/group for behavioral tests). Significant p-values are described in each panel. Two-way ANOVA for repeated measures followed by Tukey post-hoc test was used for body weight analysis (a). One and Two-way ANOVA followed by Tukey post-hoc test was used for behavioral outcomes (b-e)

Interestingly, after the 30-days Mn-free recovery period, Mn-treated males exhibited a shorter latency to fall compared to their controls, indicating a delayed impairment in motor coordination (Fig. 5b), whereas no difference was observed in females. Although no significant sex × group interaction was detected in the factorial two-way ANOVA model, one-way ANOVA across the four groups revealed a significant overall group effect [F_(3,16) = 14.26; P < 0.01], and post hoc comparisons confirmed that Mn-treated males performed worse than both their respective controls and Mn-treated females. In contrast, anxiety-like behaviors assessed at the delayed endpoint showed no significant differences between Mn-treated and control animals. The number of entries into the closed arms, open arms, and the time spent in the open arms of the elevated plus-maze did not differ between groups (Fig. 5c-e), indicating normalization of anxiety-related behavior following the recovery period.

After the 30-day Mn-free recovery period, Mn levels in all brain region analyzed returned to levels comparable to control (Fig. 6a–d), indicating that the CNS no longer acted as a Mn depot at this delayed endpoint. A similar normalization pattern was observed for Fe concentrations across all brain regions (Supplementary Figure S1e-h). In contrast, at the same delayed time point, Mn concentration remained elevated in the female nails (Fig. 6f) [F_(3,16) = 11.29; P < 0.001] and in teeth of both sexes [F_(3,16) = 36.2; P < 0.001], indicating persistent retention in these keratinized tissues. Hair Mn showed a trend toward elevation after the recovery period (p = 0.06), although with greater inter-individual variability (Fig. 6e). Together, these findings indicate that nails and teeth provide robust markers of cumulative Mn retention, while hair may reflect exposure with greater biological variability.

Fig. 6.

Delayed effects of subacute Mn exposure (MnCl₂; 15 mg/kg i.p.; daily for 5 days/4 weeks) on Mn concentrations in the cerebral cortex (a), hippocampus (b), cerebellum (c), striatum (d), hair (e), nail (f), tooth (g) and serum (h) after a 30-day Mn-free recovery in adult male and female Wistar rats. Data represent mean ± standard error of the mean (5 animals/group). Significant p-values are described in each panel. One and Two-way ANOVA followed by Tukey post-hoc test was used for statistical comparisons

Among the peripheral biomarkers assessed at the delayed endpoint, hair Mn showed the highest number of significant correlations with oxidative stress markers across multiple brain regions (Suppl. Table S3). Despite moderate correlation strength (r ranging from ± 0.30 to 0.58), this pattern suggests that hair Mn may be particularly informative of functional neurotoxic burden. Its faster growth and incorporation of circulating metals may allow it to better mirror ongoing systemic dynamics, whereas teeth and nails may reflect longer-term accumulation.

Discussion

Mn is recognized for its neurotoxic potential, particularly affecting motor functions, mitochondrial activity, and redox homeostasis. In this study, we examined both the immediate effects observed at the end of the 30-day Mn exposure period and the delayed effects assessed after an additional 30-day Mn-free recovery interval in male and female rats. This predefined two-endpoint experimental design enabled a systematic evaluation of behavioral, biochemical, and bioaccumulation outcomes and facilitated the identification of sex-dependent vulnerabilities and reliable tissue biomarkers of Mn exposure.

Our findings demonstrated that Mn exposure produced delayed motor impairments that emerged only after a Mn-free recovery period, whereas no deficits were evident immediately after the exposure phase. This temporal dissociation suggests that compensatory neurobiological mechanisms may initially mitigate Mn neurotoxicity, but these mechanisms deteriorate over time, revealing latent dysfunction. Mn is known to accumulate preferentially in the basal ganglia structures such as the globus pallidus and substantia nigra pars reticulata, regions central to motor regulation [33], altering dopaminergic and GABAergic neurotransmission, two major pathways required for coordinated movement [8, 16, 19]. Therefore, even after Mn levels normalize in the CNS, structural and synaptic changes produced during exposure may persist and contribute to delayed motor deficits, consistent with previous evidence reporting latent Mn neurotoxicity [34]. It is important to contextualize these findings within the broader literature, which includes numerous reports describing motor deficits during or immediately after Mn exposure [12, 23, 34, 35], as well as studies showing partial recovery of motor function after exposure cessation in occupational and animal models [13, 33, 36, 37]. These studies suggest that the temporal profile of Mn-induced motor alterations varies with exposure paradigms and assessment timing, and that recovery trajectories are influenced by dose, duration, and the functional domain evaluated. In the present study, the absence of overt motor deficits immediately after exposure may reflect transient compensatory adaptations, whereas the delayed assessment captured a later window in which persistent synaptic or mitochondrial dysfunction became behaviorally evident. Thus, rather than contradicting previous work, our results align with a pattern of delayed and sex-dependent vulnerability under specific exposure and recovery conditions.

Sex-dependent biochemical changes further support differential vulnerability. Females exhibited decreased non-protein thiols in the cortex and hippocampus, indicating impaired antioxidant capacity, while mitochondrial complexes I and II displayed distinct patterns of activity modulation across sexes and brain regions. These findings align with established mechanisms of Mn toxicity involving oxidative stress and mitochondrial dysfunction [38, 39], altering antioxidant gene expression, including downregulation of SOD2, GPx, and HO-1 [38, 40], and interfering with Nrf2 signaling, a central regulator of redox homeostasis [41]. Mn can also disrupt epigenetic regulators involved in neuronal resilience and stress responses [40, 42] and impair mitochondrial biogenesis via the PGC-1α pathway, which has been implicated in Mn-associated neurodegeneration [12, 43]. Recent multi-omics studies demonstrate that Mn affects transcriptomic and proteomic networks involved in neuroinflammation, autophagy, mitophagy, and synaptic signaling [44–48], suggesting broad molecular remodeling that may persist beyond the exposure window. Moreover, Mn-induced neuroinflammation mediated by glial activation [15, 17], stimulation of TLR3/NFκB signalling [18], and NLRP3 inflammasome activation [49, 50] may contribute to long-lasting neurotoxic effects.

Although our aim was not to dissect these pathways in detail, the sex-dependent biochemical alterations observed here likely reflect differential engagement of Mn-responsive mechanisms previously described in the literature. Recent mechanistic studies support the interpretation that delayed neurochemical and behavioral alterations may arise from persistent mitochondrial and regulatory dysfunction induced by Mn. Harischandra et al. [46] demonstrated that Mn disrupts PINK1/Parkin-dependent mitophagy, promoting the accumulation of damaged mitochondria and long-term bioenergetic instability, a pattern consistent with the time-dependent modulation of mitochondrial complex I and II activities observed in our study. Complementarily, Liu et al. [47] identified Mn-induced S-nitrosylation of PINK1 as a critical upstream event impairing mitochondrial quality control, reinforcing the concept that mitochondrial dysfunction may persist even after brain Mn levels normalize. Moreover, epigenetic repression of antioxidant and mitochondrial regulatory genes, as reported by Lindner et al. [42], provides a plausible explanation for the sustained reduction in NPSH and the sex-specific metabolic vulnerability identified here. Together, these findings offer a coherent mechanistic framework linking our delayed functional outcomes to established molecular pathways of Mn neurotoxicity.

Mn-induced neurotoxicity is closely linked to disturbances in metal homeostasis, particularly through the dysregulation of Mn transporters. Among them, SLC30A10 functions as a Mn efflux transporter, limiting intracellular Mn accumulation, whereas SLC39A14 and SLC39A8 mediate Mn uptake into cells [51]. More recently, SLC39A11 has been identified as a sex-specific transporter regulating Mn metabolism and longevity, suggesting an additional layer of sex-dependent Mn regulation of homeostasis [52]. Consistent with these roles, biallelic mutations in SLC30A10 cause systemic Mn overload leading to early-onset parkinsonism, dystonia, polycythemia, and liver disease [53–55], findings further corroborated in experimental work demonstrating its essential role in biliary and intestinal Mn excretion [56]. Conversely, mutations in SLC39A14 result in severe Mn accumulation in the brain and progressive motor dysfunction, a phenotype confirmed in ZIP14-deficient mice exhibiting Mn overload and motor impairments [57], indicating that ZIP14 is necessary for regulating Mn absorption and systemic balance [58]. Taken together, these findings support the view that Mn clearance involves coordinated transporter activity, with SLC30A10-mediated efflux acting in concert with ZIP14-dependent tissue uptake [51, 57, 59].

Importantly, several Mn transporters display sex-dependent expression and regulation, which may help explain the differential susceptibility observed between male and female subjects. For instance, lower expression of SLC30A10 in females has been proposed to reduce Mn efflux capacity, favoring retention and oxidative vulnerability in regions such as the hippocampus and cortex. Moreover, polymorphisms in SLC39A8 have been linked to altered Mn transport and increased risk for neurodevelopmental and inflammatory disorders, with evidence suggesting modulation by sex hormones, where women exhibit greater Mn absorption than men [40, 59–61]. These findings align with our observation of higher Mn accumulation in females.

Although estrogen is often considered neuroprotective in Mn toxicity, our results demonstrate a more nuanced scenario. Estrogen levels fluctuate across the estrous (in rodents) or menstrual (in humans) cycle, dynamically influencing Mn uptake, distribution, and clearance. Estrogen can increase DMT1 and ZIP8 expression, enhancing Mn uptake into cells, particularly within basal ganglia [62, 63]. In humans, women show a higher prevalence of elevated Mn levels, likely due to greater Mn absorption secondary to lower iron stores, a physiological relationship also explaining the marked increase in Mn levels during pregnancy [23, 64, 65].

Furthermore, although estrogen can promote antioxidant responses, its actions are region-specific, dose-dependent, and receptor-dependent [66–68]. If receptor expression is regionally low or Mn disrupts downstream signaling pathways, estrogen´s protective effects may be attenuated or lost [36], consistent with the blunted antioxidant response observed here.

Sex-specific responses to Mn toxicity are further supported by previous work from our group. Richter Schmitz et al. (2019) demonstrated that males and females exhibit distinct oxidative and mitochondrial profiles following Mn exposure. Male rats exhibited more pronounced oxidative stress in the liver and kidney, whereas females showed increased mitochondrial complex I activity in the kidney, suggesting divergent mitochondrial adaptations or vulnerabilities across sexes [69]. Taken together, these findings indicate that Mn neurotoxicity does not confer a uniform sex bias, but rather reveals sex-dependent vulnerability across distinct biological domains, with females showing greater biochemical and metabolic sensitivity, while males exhibit more pronounced delayed functional impairment.

The delayed onset of motor impairments observed in our study underscores the importance of long-term monitoring for individuals at risk of Mn intoxication, such as workers in welding, mining, and battery manufacturing industries [70–72]. The identification of easy-to-collect, reliable, and precise biomarkers is essential for early detection and prevention of Mn-induced neurotoxicity [73–76]. In our study, hair, nails, and teeth showed elevated Mn levels after a 30-day recovery period. These tissues capture cumulative exposure and provide historical evidence of Mn accumulation that is not reflected in blood, which represents only ongoing or recent exposure [52, 75, 76]. Importantly, systematic reviews identify hair, nails, and teeth as reliable biomarkers for differentiating exposure levels in occupational and environmental settings [77]. Additionally, studies in exposed workers validate Mn in nails and hair as stable indicators of chronic Mn exposure [78]. Together, these findings emphasize the potential of nails and teeth as non-invasive, persistent biomarkers suitable for longitudinal monitoring and evaluation of interventions aimed at reducing Mn body burden. Although hair Mn did not show the same degree of statistical robustness in retention after recovery, it exhibited the strongest and most consistent correlations with oxidative stress markers. This suggests that hair may better reflect ongoing functional neurotoxic burden, whereas nails and teeth may serve as more stable indicators of cumulative exposure.

In peripheral keratinized tissues such as nails and teeth, Mn becomes incorporated during tissue formation. For nails, this process reflects a time-averaged systemic Mn exposure over weeks to months, depending on nail growth rate (79). In teeth, Mn deposition occurs during dentin and enamel mineralization, particularly during early developmental stages, where Mn may substitute for calcium or phosphate within hydroxyapatite (80). Because these tissues are metabolically inert after formation, Mn deposition is preserved and reflects historical systemic exposure. Although Mn incorporation is largely passive and concentration-dependent, systemic Mn-binding proteins and local transporter expression may influence accumulation (81). Notably, SLC30A10 expression in epithelial and exocrine tissues suggests a potential mechanism for Mn sequestration into keratinized structures [75, 77]. Although we did not identify published evidence describing sex differences in Mn incorporation into keratinized tissues, our findings suggest this possibility, given that females exhibited higher systemic Mn accumulation in several brain regions and greater overall Mn burden after exposure. These patterns raise the hypothesis that sex-dependent toxicokinetics may also influence Mn deposition in nails and teeth, which should be explored in future studies.

A key methodological consideration is the choice of intraperitoneal (i.p.) injection, which ensures precise dosing and uniform systemic bioavailability across animals. While this approach enhances experimental control, it bypasses physiologically relevant barriers associated with human exposures, such as gastrointestinal absorption and pulmonary deposition [33, 78].

Occupational exposures typically occur through inhalation, allowing Mn to reach the brain via olfactory transport and partially bypass the blood–brain barrier. In contrast, ingested Mn undergoes regulated intestinal uptake and first-pass hepatic metabolism [33, 40]. Additionally, species-specific differences also limit extrapolation to humans, as rodents absorb Mn more efficiently from the gut and differ in hepatic-biliary Mn handling compared to humans [36, 78].

Although our findings provide valuable insights into delayed Mn-induced motor deficits, potential biomarkers, and sex-specific susceptibility, some limitations must be acknowledged. The molecular pathways underlying these impairments are not fully defined and warrant further exploration, particularly regarding the reversibility of Mn-induced neurotoxicity and the identification of targeted therapeutic strategies. Additionally, biochemical parameters were not assessed after the 30-day recovery period because the study was primarily designed to evaluate behavioral outcomes and identify peripheral biomarkers of cumulative Mn exposure. Moreover, the amount of available biological material after tissue allocation for the planned analyses was insufficient to reliable perform the additional biochemical assays across all brain regions. Despite these considerations, our study demonstrates clear sex-dependent effects of Mn on oxidative stress, mitochondrial function, and behavior. Furthermore, the identification of nails and teeth as promising biomarkers provides new opportunities for non-invasive monitoring of cumulative Mn exposure, with particular relevance for at-risk occupational populations such as welders, miners, and battery workers.

Conclusions

This study demonstrates that Mn exposure induces delayed motor impairments associated with oxidative stress and mitochondrial dysfunction, exhibiting distinct sex-dependent effects. Female rats were more susceptible to oxidative and mitochondrial dysfunction, whereas males showed greater impairments in motor coordination after the recovery period. In addition, peripheral keratinized tissues, particularly nails and teeth, emerged as promising non-invasive biomarkers for monitoring cumulative Mn exposure, while hair Mn showed stronger associations with biochemical alterations. Overall, our findings support a pattern of Mn neurotoxicity characterized by sex-dependent vulnerability rather than a uniform susceptibility, with females displaying greater biochemical sensitivity and males exhibiting more pronounced delayed functional impairment.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

All authors contributed to the study conception and design. T.E designed, planned, and performed the animal experimental model; carried out biochemical measurements and data analyses; prepared the initial figures; and revised the manuscript. D.P. performed the animal experimental model; carried out the behavioral tests and revised the manuscript. V.Z. performed the animal experimental model; carried out the behavioral tests and revised the manuscript. K.G.V. performed the animal experimental model and carried out biochemical measurements and data analyses. A.R.B. performed statistics and revised the manuscript. A.M. performed the Mn and Zn concentration analysis. D.C. performed the animal experimental model; carried out the behavioral tests; performed the statistical analyses; wrote and revised the manuscript. A.P.R. coordinated all experimental procedures and data analyses, prepared the final version of the figures, wrote the manuscript, was responsible for financial support acquisition, and provided the required infrastructure.

Funding

The Article Processing Charge (APC) for the publication of this research was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) (ROR identifier: 00x0ma614). This work was supported by grants from FAPESC (Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina) by FAPESC nº 15/2023 (Multilab).

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval

All procedures on animals were approved by UNOESC animal ethics committee under n° 45/2016 protocol.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing Interests

The authors declare no competing interests.