Skip to main content
NCBI home page
Wiley Open Access Collection logoLink to Wiley Open Access Collection
. 2024 Dec 8;45(4):669–684. doi: 10.1002/jat.4739

Behavioral Alterations in Mice Exposed to Manganese via Drinking Water: Effects of Sex and a Lipopolysaccharide Challenge

Helaina D Ludwig 1,2, Jessica M Carpenter 1,3, Nikolay M Filipov 1,2,3,✉
PMCID: PMC11885083  PMID: 39647842

ABSTRACT

Manganese (Mn) is an essential and important metal; however, overexposures lead to adverse neurological outcomes. Nonoccupational Mn overexposure occurs primarily through consumption of Mn‐contaminated drinking water (DW). Sex differences in terms of nervous and immune systems' responsiveness to excessive Mn in the DW are understudied. Thus, this study investigated behavioral and sex differences in response to Mn DW treatment (0.4 g Mn/L for up to 8 weeks) and a lipopolysaccharide (LPS) challenge of adult C57BL/6 mice with GFP‐tagged monocytes/microglia. After 6 weeks, in motor function tests, Mn exposure resulted in decreased activity and gait deficits. In two different mood tests (open field test [OFT]/elevated zero maze), Mn‐exposed mice exhibited decreased fear/anxiety‐like behavior. Two weeks after behavioral assessment, when mice were challenged with LPS, circulating inflammatory cytokines, and acute phase proteins increased in both sexes. After 8 weeks of Mn exposure, liver and brain Mn levels were increased, but Mn alone did not affect circulating cytokines in either sex. Notably, Mn‐exposed/LPS‐challenged males had potentiated plasma cytokine output, whereas the reverse was seen in females. Males, but not females, continued to exhibit increased fearlessness (i.e., increased OFT center time), even when challenged with LPS. Overall, our results show that Mn DW exposure increases brain Mn levels and it leads to behavioral alterations in both sexes. However, males might be more susceptible to the effect of Mn on mood, and this effect is recalcitrant to an inflammagen challenge. Mn augmented post‐LPS cytokine production only in males, further indicating that important Mn effects are sex‐biased.

Keywords: acute phase proteins, behavioral deficits, cytokines, LPS, manganese, sex differences

Short abstract

Manganese (Mn) overexposure from contaminated drinking water is neurotoxic. Yet, sex differences in its effects are understudied. Male and female mice were exposed to Mn with drinking water (0.4 g/L for 8 weeks) and subsequently challenged with lipopolysaccharide (LPS). Mn exposure reduced motor activity and altered anxiety behaviors in both sexes. Notably, although brain and liver Mn were increased in both sexes, only males showed heightened cytokine responses post‐LPS and continued fearlessness‐like behavior. These findings suggest that Mn exposure impacts mood differently between sexes, indicating sex‐biased susceptibility.

1. Introduction

Manganese (Mn) is an essential metal, necessary for synthesis of amino acids, lipids, and carbohydrates (IOM 2001). It is also an important cofactor for multiple biological processes including free radical defense, bone formation, and metabolism (Chen, Bornhorst, and Aschner 2018; Erikson et al. 2007; IOM 2001). Mn can be transported across the blood–brain, as well as the blood–CSF barriers, by multiple transporters including, but not limited to, transferrin, SLC30A10, and divalent metal transporter‐1 (Aschner 2006; Aschner and Aschner 1990; Liu et al. 2021). Although an essential metal, overexposure to Mn is neurotoxic (Chen, Bornhorst, and Aschner 2018; Erikson et al. 2007). In the brain, Mn preferentially targets the basal ganglia and causes parkinsonism (Racette et al. 2017), which, at very high exposures, can result in manganese (Laohaudomchok et al. 2011). In manganism, cock‐walk gait is a specific clinical symptom (Chen, Bornhorst, and Aschner 2018; Laohaudomchok et al. 2011), whereas parkinsonism‐related symptoms associated with excess Mn exposure include rigidity, speech impairment, postural instability, and locomotor deficits, such as reduced response speed, tremors at rest, and gait alterations (Bowler et al. 2006; Bowler et al. 2007; Park et al. 2006; Sahni et al. 2007). Additionally, Mn overexposure causes mood alterations, for example, irritability, compulsive behaviors, anxiety, and/or depression in both humans (Bouchard et al. 2007; Bowler et al. 2003; Bowler et al. 2006; Laohaudomchok et al. 2011; Sahni et al. 2007) and laboratory animals (Dodd, Ward, and Klein 2005; Krishna et al. 2014; Liu et al. 2019).

Exposure level, route, and duration all influence the severity of Mn‐induced neurological dysfunction (Chen, Bornhorst, and Aschner 2018; Lucchini and Tieu 2023; Lucchini, Martin, and Doney 2009). In occupational settings, such as Mn mining operations or welding, inhaled Mn is the greatest concern (Elsner and Spangler 2005; Gonzalez‐Cuyar et al. 2014; Laohaudomchok et al. 2011; Utembe et al. 2015). Although occupational exposure to Mn remains a major concern, excess exposure for the general public via contaminated drinking water (DW) is increasingly associated with adverse neurological outcomes (Ljung and Vahter 2007). For instance, Bouchard et al. (2011) reported intellectual impairment in children exposed to Mn through DW, but not diet. Consumption of Mn‐contaminated well water correlated with memory deficits, lower intelligence scores, and increased infant mortality across multiple studies (Bouchard et al. 2011; Brna et al. 2011; Hafeman et al. 2007; Sahni et al. 2007; Wasserman et al. 2006; Woolf et al. 2002). Surface and groundwater Mn levels in the US range, respectively, 400–800 μg/L and 2900–5600 μg/L (ATSDR 2012). Excess levels of Mn DW (≥ 0.2 mg/L) are associated with adverse neurological outcomes in chronic exposure scenarios (ATSDR 2012) and at least 2.6 million Americans potentially drink water with elevated Mn (McMahon et al. 2019).

In rodents, peripheral lipopolysaccharide (LPS) administration increases proinflammatory cytokines in both circulation and the brain (Perry 2004). Additionally, it causes sickness behaviors characterized, among others, with decreased exploratory and rearing activity, as well as increased thigmotaxis and corner preference (Godbout et al. 2005; Krishna, Dodd, and Filipov 2016). Ongoing inflammation, or the presence of an inflammagen, such as LPS, can exacerbate the neurotoxic effects of Mn (Filipov, Seegal, and Lawrence 2005). That Mn treatment causes microglial overproduction of cytokines and other proinflammatory mediators is well established (Crittenden and Filipov 2008; Crittenden and Filipov 2011; Filipov and Dodd 2012; Filipov, Seegal, and Lawrence 2005; Zhao et al. 2009). However, the effects of Mn on peripheral cytokines or other inflammatory markers are not.

Behavioral studies centered on the consequences of Mn exposure via the increasingly relevant DW route (Avila et al. 2010; Krishna et al. 2014) are sparse. Mood and motor alterations are two behavioral domains affected throughout multiple studies. A common element that these behavior‐focused studies (Avila et al. 2010; Krishna et al. 2014; Sepúlveda et al. 2012) have is that they were male‐biased. At nonbehavioral level, Mn has been reported to have sex‐dependent toxicity (Chi et al. 2017; Dodd, Ward, and Klein 2005; Madison et al. 2011; Riojas‐Rodríguez et al. 2010; Zhang, Zhou, and Fu 2003). Females typically have higher blood Mn levels, likely due to higher absorption rates (Chen, Bornhorst, and Aschner 2018). Children exposed to excess Mn had lower intelligence scores than nonexposed children; girls were affected more than boys (Riojas‐Rodríguez et al. 2010). In school‐aged children, visuospatial learning also has sex‐specific vulnerability to neuroactive metals, predominantly Mn (Rechtman et al. 2020). Sex differences in Mn effects when exposure is in adulthood are understudied. Limited Mn exposure studies, in rodents, have been utilized to investigate behavioral sex differences and effects associated with Mn exposure (Liu et al. 2019; Moreno et al. 2009; Sepúlveda et al. 2012), with results ranging from no sex differences (Liu et al. 2019) to male‐specific hyperactivity (Moreno et al. 2009).

Immune differences between sexes, that is, cytokine production, could play a role in the responses to Mn and inflammatory stimuli. In this regard, adult females reportedly produce more anti‐inflammatory cytokines than males (Barrientos et al. 2019), but mount a more robust inflammatory response to the same inflammatory stimuli too (Bouman, Heineman, and Faas 2005). Middle‐aged female mice challenged with LPS exhibited a greater decrease in locomotor activity than males and had higher cytokine levels in response to LPS (Dockman et al. 2022). Non‐neurotoxicity studies reported that DW Mn exposure causes decreased fertility in males at 8000 mg/L, but not in females (Elbetieha et al. 2001), and results in sex‐specific gut microbiome alterations (20 mg/kg/day) (Chi et al. 2017). Neurologically, Mn led to greater changes in mouse striatal morphology in females compared with males injected with Mn subcutaneously (50 mg/kg) (Madison et al. 2011).

Whether Mn neurotoxicity is sex‐dependent in the context of DW exposure or inflammagen challenge is unknown. Hence, the main objectives of this study were to (i) evaluate the neurotoxic effects of Mn DW exposure on selected behavioral parameters, (ii) determine if the Mn effects are sex‐specific, and (iii) assess whether sex plays a role in the response of Mn‐exposed mice to an inflammagen.

2. Materials and Methods

2.1. Reagents

All chemicals, unless stated otherwise, including manganese (MnCl2 .4H2O) and LPS ( Escherichia coli serotype 0111: B4), were purchased from Sigma Aldrich (St. Louis, MO).

2.2. Animals

The University of Georgia Institutional Animal Care and Use Committee (IACUC) approved all procedures that involved animal handling in advance, and they were in accordance with the latest National Institutes of Health and Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Homozygous male and female CX 3 CR1 GFP mice, on a C57BL/6 background (Jackson Labs, stock 005582), were used for all experiments. This strain of mice is behaviorally and immunologically indistinguishable from wild‐type C57BL/6 (Dockman et al. 2022). Same‐sex mice were group‐housed (2–5 per cage) with PicoLab Rodent Diet 20 (LabDiet, 5053) available ad libitum in an environmentally controlled room (22°C–24°C) with a relative humidity of 50%–70% and maintained on a 12 h light/dark cycle in an AAALAC accredited facility throughout the study. Mice were acclimated to using water bottles for 3 weeks. Then, they were randomly assigned to DW treatment groups and were 2 months old at treatment onset.

2.3. Animal Treatment and Tissue Collection

In total, 53 mice (males: 25 and females: 28) were exposed to vehicle control (NaCl; 0.4 g Na/L) or MnCl2 (0.4 g Mn/L) (n = 12–15/group) in deionized water for 8 weeks. Water bottles were changed weekly with freshly prepared control or Mn solutions. Body weight (BW), water intake, and estrus cycle stages (females only) were recorded weekly. The Mn DW concentration used in the current study was selected based on previous work that resulted in significant, human‐relevant Mn deposition in the brain (Avila et al. 2010; Krishna et al. 2014) and in behavioral alterations in C57BL/6 male mice (Avila et al. 2010; Krishna et al. 2014). Behavioral tests were carried out after 6 weeks of DW treatment (n = 12/group), while maintaining the mice on their respective DW treatments, and are described in detail below. At the end of the 8‐week exposure period, a subset within each sex and treatment group was randomly selected to receive an intraperitoneal (IP) injection of LPS ( E. coli serotype 0111: B4 1.5 × 1012 EU or 0.3 mg/kg BW) or saline vehicle (n = 6/group). Sickness behavior was assessed in an open field test (OFT) 4 h after LPS/vehicle treatment, and the mice were sacrificed 2 h later (6 h post‐LPS/vehicle treatment). Brains were extracted and processed similarly to Coban and Filipov (2007) and Carpenter et al. (2021). Briefly, brains were weighed, washed in ice‐cold HEPES‐buffered Hank's saline solution (pH 7.4), and separated longitudinally into two hemispheres; one half of the brain was fixed in 4% paraformaldehyde, cryoprotected, and stored at −80°C, whereas the other half was frozen on dry ice and stored at −80°C for future analyses. In addition, organs (liver, spleen, thymus, kidneys, and lymph nodes) and fat (subcutaneous, retroperitoneal, epididymal/ovarian, and brown adipose tissue) were collected, weighed, and stored at −80°C; plasma was collected from these mice and analyzed for tumor necrosis factor alpha (TNFα), interleukin 6 (IL‐6), interleukin 10 (IL‐10), C‐reactive protein (CRP), and serum amyloid A (SAA) using enzyme‐linked immunosorbent assays (ELISA) as described later.

2.4. Behavioral Tests

The tests used in this study, in the order presented below, evaluated locomotion and mood behavior in rodents that are known to be affected by Mn (Conrad et al. 2011; Krishna et al. 2014; Sepúlveda et al. 2012), and are both altered in Mn‐exposed humans (Bouchard et al. 2007). Male and female mice behavioral testing was carried out separately with all equipment receiving a thorough cleaning between testing the different sexes; all mice were subjected to tests in the same order. All animals were naïve to behavioral apparatuses prior to the start of testing. Tests were performed by a treatment‐blind experimenter in a room designated for behavioral testing, located in the same facility and near the room where the animals were housed.

2.4.1. OFT

An open field arena (25 cm × 25 cm × 40 cm, divided into a 16 square grid; Coulborn Instruments, Whitehall, PA) was used to conduct the OFT for a 30‐min period as in (Dockman et al. 2022; Krishna, Dodd, and Filipov 2016; Krishna et al. 2014). Limelight tracking software (Actimetrics, Wilmette, IL) was used to monitor and record videos of each test. ANY‐maze software (Stoelting Co., Wood Dale, IL) was used to assess and score mouse locomotor activity by an experimenter blinded to the treatments, per 5‐min interval and the total 30‐min test. Parameters of interest included distance traveled and the number of grid crossings for the first 5‐min total time (horizontal locomotor activity), time spent in the center versus the periphery (measures of anxiety‐like or fearlessness behavior), and the number of rearings (vertical activity). The same OFT, but ran for 15 min, was used to score and assess sickness behavior post‐LPS (detailed in Section 2.4.4).

2.4.2. Gait Test (GT)

Motor function was evaluated by utilizing the GT as described in Carpenter et al. (2021). Briefly, an 82 cm × 5.5 cm × 8 cm (l × w × h) runway was lined with a white paper strip; an empty cage with home cage bedding was placed at the end of the runway. Two trials, 5 min apart, were conducted—a training pre‐trial and a test trial that was used for subsequent statistical analyses. Before each trial, front and hind paws were painted with nontoxic red and black ink, respectively (Office Depot, item #839994 and #839967), and then, the mouse was allowed to traverse the runway. Between each trial, the runway was cleaned with 70% ethanol and lined with a new paper strip. Gait parameters (see Figure S1) that were measured included stride length, base width, interstep/intrastep distance, stride variability, total number of steps, and cadence (Carpenter et al. 2021; Mulherkar et al. 2013; Wang et al. 2017).

2.4.3. Elevated Zero Maze (EZM)

An EZM apparatus (50‐cm diameter; Stoelting Co., Wood Dale, IL) was used to assess anxiety‐like behaviors as in Carpenter et al. (2021), with locomotion also monitored. At the start of the test, mice were placed at the center of an open quadrant facing inward and allowed to explore the maze freely for 5 min. Parameters of interest included times spent in open and closed arms, latencies to enter or exit a closed area at the start of the test, and the number of head dips and stretch attend posture (SAP) attempts, as in Grewal et al. (1997). Mice were considered to be in a zone when 70% of the body was in an area. EZM parameters were tracked and scored using ANY‐Maze software (Stoelting) in a treatment blind manner.

2.4.4. Post‐LPS Challenge (Post‐LPS OFT)

At the end of the 8‐week exposure period, a subset of mice within each sex and treatment group were randomly selected to receive, via an IP injection, either saline vehicle or 0.3‐mg/kg BW LPS, which has been previously found to induce transient peripheral and central inflammatory response and accompanying behavioral changes in adult/middle‐aged mice on C57BL/6 background (Dockman et al. 2022; Krishna, Dodd, and Filipov 2016). Four hours post‐LPS/vehicle treatment, mice were subjected to a 15‐min OFT. Parameters of interest included locomotor activity, such as distance traveled and the number of grid crossings; rearing activity, that is, standing on hind legs to explore and assess the environment; and times spent in the center versus the periphery of the arena.

2.5. ELISA

Plasma concentrations of IL‐6, IL‐10, TNFα, CRP, and SAA were analyzed 6 h post‐LPS administration using mouse‐specific ELISA kits (Bio‐Techne, Minneapolis, MN) following the manufacturer's protocol. Samples and standards, namely, IL‐6 (1000–15.625 pg/mL), TNFα (2000–31.25 pg/mL), IL‐10 (2000–31.25 pg/mL), CRP (1500–23.4 pg/mL), and SAA (16,000–250 pg/mL), were run in duplicate. Absorbance (450‐nm analytical read; 570‐nm background correction read) was measured using an Epoch microplate spectrophotometer (BioTek Instruments, Winooski, VT), and the mean from the individual sample replicates was used for statistical analysis.

2.6. Liver and Brain Measurement of Mn and Fe

For analysis of tissue Mn and Fe, the liver median lobe and four to six random slices of the forebrain were weighed and digested in 1‐mL concentrated nitric acid for 2 h at 70°C and then brought to a total volume of 5 mL with ddH2O. An Inductively Coupled Argon Plasma‐Axially Viewed Optical Emission Spectophotometry (ICP‐AVOES) was used to determine Mn and Fe concentrations as in Dodd and Filipov (2011). Instrument values for Mn and Fe were reported as concentration in solution (ppm). The mean for each sample was expressed as μg Mn/g tissue and used for statistical analysis.

2.7. Statistical Analysis

All data were analyzed using Sigmaplot v12.5 (Systat Software, Inc., Chicago, IL); graphs were generated using GraphPad Prism v8.4.3 (San Diego, CA). Two‐way repeated measures (RM) or three‐way RM ANOVA (post‐LPS) were used to assess behavior parameters across 5‐min intervals for the OFTs. Body/organ weights, water intake, estrus staging, GT, EZM, and all ELISA data were analyzed by using two‐way ANOVA. For all ANOVAs, the F‐value is displayed as F (df, df) with degrees of freedom designated as (groups ‐1, total mice ‐groups). A Student–Newman–Keuls (SNK) post hoc comparison was run if a significant main effect or an interaction was detected, with statistical designation as (p‐value, q‐value). A p‐value of ≤ 0.05 was considered significant.

3. Results

3.1. BW, Water Intake, Organ Weight, and Estrus Cycle

BWs were unaffected by Mn (F(3,45) ≤ 1.579, p ≥ 0.1), except for an end‐of‐study, male‐specific, weight increase (F(1,23) = 5.498, p = 0.028; Figure 1A). Males in the Mn group gained about 20%, whereas control males' gain was 17% (Figure 1B). End female weight gain was about 20% in both groups (Figure 1B). Throughout the entire experimental period, water intake by both male and female Mn‐exposed mice was similar to their respective controls (Figure 2). There was a week‐dependent sex effect, such that during Week 4 (F(3,44) = 6.417, p = 0.028), males consumed more water than females. Mn consumption did not affect estrus cycle (F(1,22) ≤ 0.600, p ≥ 0.1; Figures 3 and 4); both Mn‐exposed and control female mice continued to cycle throughout the study. Eight weeks of Mn exposure also did not affect relative brain, kidney, liver, spleen, thymus, brown, or subcutaneous adipose tissue weights in either sex (F(3,44) ≤ 2.908, p ≥ 0.1; Table 1). Males, but not females, challenged with LPS had heavier livers (absolute; Table S1) and relative weights than vehicle controls (F(3,20) = 8.967, p = 0.007). In terms of relative organ weights, Mn‐exposed males, with and without LPS, had heavier livers than male controls (F(3,20) = 7.096, p = 0.015; Table 1) and only the Mn + LPS female livers were heavier than the rest (t = 3.586, p = 0.004). Interestingly, LPS‐challenged females had heavier spleens, both absolute (Table S1) and relative weight (Table 1) than saline controls (F(3,20) = 26.485, p < 0.001); this increased splenic weight was more pronounced in the females that were exposed to Mn (q = 4.562, p < 0.001; Table 1).

FIGURE 1.

FIGURE 1

Open in a new tab

Effect of Mn DW (0.4 g/L) exposure on body weight (BW) and on percent change in BW. (A) Average body weight; (B) percent change in BW. Data are presented as mean ± SEM. *Indicates a significant effect of Mn p < 0.05; n = 12/group.

FIGURE 2.

FIGURE 2

Open in a new tab

Effect of Mn DW (0.4 g/L) on water intake (mL/mouse/day) during the 8 weeks of treatment duration. Water intake is presented as mean ± SEM. # Indicates a significant sex difference p < 0.05, and ^ indicates a trending sex difference 0.05 < p < 0.10; n = 12/group.

FIGURE 3.

FIGURE 3

Open in a new tab

Average number of female mice in receptive (estrus and metestrus) or not receptive (diestrus and proestrus) stage of the estrus cycle.

FIGURE 4.

FIGURE 4

Open in a new tab

Percent of female mice in each stage of the estrus cycle throughout the 8‐week period (n = 12/group).

TABLE 1.

Relative tissue weight (mg/kg BW) for each treatment group after 8 weeks of Mn/saline DW exposure and 6 h post‐LPS challenge. Mean ± SEM. Bold indicates p < 0.05; n = 6/group.

Sex Organ/fat DW treatment—LPS challenge
Saline–saline Saline–LPS Mn–Saline Mn–LPS
Female Brain 21.6 ± 0.40 22.3 ± 0.72 21.0 ± 0.69 20.8 ± 0.64
Kidney 12.6 ± 0.12 13.5 ± 0.35 12.5 ± 0.20 13.3 ± 0.25
Liver 46.8 ± 0.94 46.5 ± 2.43 47.1 ± 1.28 51.4 ± 0.86
Spleen 3.4 ± 0.21 4.5 ± 0.21 3.1 ± 0.23 4.9 ± 0.34
Thymus 2.2 ± 0.09 2.2 ± 0.12 1.9 ± 0.24 1.9 ± 0.54
BAT 3.9 ± 0.26 3.9 ± 0.17 3.8 ± 0.31 4.7 ± 0.60
SQ 3.2 ± 0.21 3.7 ± 0.60 3.5 ± 0.71 3.1 ± 0.49
OT 9.3 ± 1.30 9.9 ± 0.98 8.0 ± 1.49 8.3 ± 0.71
RT 2.5 ± 0.41 3.2 ± 0.40 2.0 ± 0.35 2.9 ± 0.48
Male Brain 18.0 ± 0.33 17.5 ± 0.52 16.7 ± 0.47 17.0 ± 0.41
Kidney 13.6 ± 0.32 13.6 ± 0.44 13.0 ± 0.52 13.3 ± 0.31
Liver 41.3 ± 2.37 45.5 ± 0.72 44.0 ± 1.15 48.3 ± 0.86
Spleen 2.6 ± 0.11 2.6 ± 0.54 2.4 ± 0.18 3.1 ± 0.26
Thymus 1.1 ± 0.15 1.1 ± 0.16 1.2 ± 0.07 1.1 ± 0.05
BAT 3.5 ± 0.27 2.9 ± 0.13 2.3 ± 0.22 3.5 ± 0.27
SQ 2.5 ± 0.29 2.5 ± 0.29 3.2 ± 0.23 2.6 ± 0.39
ET 10 ± 0.55 10.5 ± 1.22 13.1 ± 2.23 13.1 ± 2.13
RT 3.3 ± 0.48 3.3 ± 0.36 4.9 ± 1.11 5.3 ± 1.29
Open in a new tab

Tissue abbreviations: BAT, brown adipose tissue; ET, epididymal adipose tissue; OT, ovarian adipose tissue; RT, retroperitoneal adipose tissue; SQ, subcutaneous adipose tissue.

3.2. Behavioral Analysis

3.2.1. OFT

After 6 weeks of Mn DW exposure, a two‐way ANOVA revealed an overall main effect of sex on locomotor activity with males being more active, with respect to total distance traveled (Figure 5A; F(3,44) = 11.039, p = 0.002), number of rearings (Figure 5B; F(3,44) = 14.334, p < 0.001), and grid crossings (F(3,44) = 9.037, p = 0.004; data not shown) regardless of DW treatment. Mn‐exposed mice exhibited a significant (F(3,44) = 5.189, p = 0.028) decrease in vertical activity (number of rearings) during the first 5‐min (Figure 5B) and also throughout the 30‐min OFT (F(3,44) = 4.251, p = 0.045; data not shown). Mn‐caused decrease of rearings was more pronounced in the females (q = 2.244, p = 0.03). Over the 30‐min testing duration, locomotor activity decreased (F(3,44) = 88.547, p < 0.001) irrespective of Mn and sex, as all mice habituated to the arena over time (Figure 6); there was also a strong trend for Mn‐exposed males to be more active early in the test (during the first 10 min; Figure 6; t = 1.989, p = 0.059).

FIGURE 5.

FIGURE 5

Open in a new tab

Open field test. Effect of Mn after 6 weeks of DW (0.4 g/L) exposure and/or sex differences on distance traveled (A), rearing activity (B), time spent in the periphery (C), or time spent in the center (D) of the open field arena (first 5 min). Data are presented as mean ± SEM. * Indicates a significant effect of Mn p < 0.05; # indicates a significant effect of sex p < 0.05. a Indicates p < 0.05 between saline and Mn within sex. n = 12/group.

FIGURE 6.

FIGURE 6

Open in a new tab

Open field test. Effect of Mn after 6 weeks of DW (0.4 g/L) exposure on total distance traveled per 5‐min interval in the open field arena. Data are presented as mean ± SEM. ♦ Indicates a significant effect of time; that is, habituation p < 0.05; # indicates a significant effect of sex p < 0.05; ^ indicates a trending Mn effect 0.05 < p < 0.10; n = 12/group.

Mn‐treatment was associated with decreased anxiety/increased fearlessness, that is, Mn‐exposed mice spent significantly more time in the center (Figure 5C; F(3,44) = 5.431, p = 0.024) and less time in the periphery (Figure 5D; F(3,44) = 4.129, p = 0.048) of the arena during the first 5‐min exploration phase of the OFT. This effect was more prominent in the Mn‐exposed males (q = 2.136, p = 0.038). There was also a main effect of sex, that is, female mice spent significantly more time in the center than male mice (F(3,44) = 12.958, p < 0.001).

3.2.2. GT

Mn‐exposed mice, regardless of sex, took more steps to complete the test (Figure 7A; F(3,44) = 4.120, p = 0.048) and exhibited a significantly shorter right‐to‐left hind paw interstep distance (Figure 7C; F(3,44) = 4.623, p = 0.037), the distance between consecutive right to left steps (Figure S1), and stride length (Figure 7B; F(3,44) = 7.779, p = 0.006), the distance between two consecutive pawprints of the same paw (Figure S1). Two‐way ANOVA revealed a main sex effect on fore/hind paw overlap, with females having a shorter overlap distance than males (Figure 7D; F(3,44) = 12.473, p < 0.001), the distance between a step's fore and hind paw (Figure S1).

FIGURE 7.

FIGURE 7

Open in a new tab

Gait test. Effect of Mn after 6 weeks of DW (0.4 g/L) exposure and/or sex differences on the total number of steps taken (A), average stride length (B), hind paw interstep distance (C), and average fore/hind paw overlap distance (D). Data are presented as mean ± SEM. * Indicates a significant effect of Mn p < 0.05; # indicates a significant effect of sex p < 0.05; n = 12/group.

3.2.3. EZM

In the EZM, Mn‐exposed mice, specifically males, spent significantly more time in the open arms of the maze compared with controls (Figure 8A; F(3,44) = 4.946, p = 0.031), an indication of decreased anxiety/increased fearlessness. Latency to enter the closed arm was not significantly different between Mn treatments or sexes (F(3,44) = 0.0172, p = 0.896 and F(3,44) = 0.509, p = 0.479, respectively; data not shown). For SAP, one of the parameters used to assess risk assessment behavior, two‐way ANOVA revealed the overall main effects of Mn and sex. Mn‐exposed mice exhibited a decrease in the number of closed‐arm SAP attempts than saline controls (Figure 8C; F(3,44) = 6.326, p = 0.016). Female mice, regardless of Mn treatment, attempted more closed‐arm SAPs than males (F(3,44) = 30.620, p < 0.001). In terms of general EZM activity, Mn‐exposed mice displayed an increase in the number of closed and open arm entries, which was male driven (q = 2.145, p < 0.001; data not shown).

FIGURE 8.

FIGURE 8

Open in a new tab

Elevated zero maze. Effect of Mn after 6 weeks of DW (0.4 g/L) exposure and/or sex differences on time spent in the open arm(s) (A) and number of closed‐arm stretch attend posture (SAP) attempts (B). Data are presented as mean ± SEM. * Indicates a significant effect of Mn p < 0.05; # indicates a significant effect of sex p < 0.05; ^ indicates a trending effect of sex 0.05 < p < 0.10; n = 12/group.

3.2.4. Post‐LPS OFT

After 8 weeks of Mn DW exposure, a three‐way ANOVA revealed the main effects of sex and LPS on locomotor activity. Males were more active than females, regardless of Mn treatment or LPS challenge, with respect to the total distance traveled (Figure 9A; F(3,20) = 7.253, p = 0.01). This main effect of sex persisted from the 6‐week (pre‐LPS) time point (i.e., Figure 5A). Post‐LPS, the LPS‐challenged mice, regardless of sex or Mn treatment, traveled less distance (Figure 9A; F(3,20) = 5.352, p = 0.026) and had decreased vertical activity during the first 5‐min interval (Figure 9B; F(3,20) = 7.888, p = 0.008). Within sex, LPS‐challenged females, but not males, made fewer rearings than saline controls, an effect that approached significance (F(3,20) = 3.994, p = 0.053). Over the 15‐min testing duration, mice habituated and locomotor activity decreased irrespective of Mn, LPS, and sex (F(3,20) = 5.079, p = 0.03).

FIGURE 9.

FIGURE 9

Open in a new tab

Post‐LPS open field test. Effect of Mn after 8 weeks of DW (0.4 g/L) exposure, sex differences, and/or sickness behavior, four hours after a single dose of either saline or LPS (0.3 mg/kg BW), on distance traveled (first 5 min) (A), rearing activity (B), and times spent in the periphery (C) or in the center (D) of the open field arena. Data are presented as mean ± SEM. * Indicates a significant effect of Mn p < 0.05; # indicates a significant effect of sex p < 0.05; $ indicates a significant effect of LPS, p < 0.05; ^ a trend within female mice 0.05 < p < 0.10. a p < 0.05 for Mn effect within males challenged with saline or LPS, respectively; n = 6/group.

Interestingly, the Mn‐induced decreased time in the periphery and increased time in the center, which were observed during the OFT at 6 weeks (Figure 5C,D, respectively), were still present after 8 weeks of Mn DW exposure in the males, but not the females (Figure 9C,D; periphery and center times, respectively). There were overall main effects of Mn and sex on the times spent in the periphery and center of the arena. Mn‐exposed mice spent significantly more time in the center (Figure 9D; F(3,20) = 9.838, p = 0.003) and less time in the periphery (Figure 9C; F(3,20) = 11.181, p = 0.002) of the arena; this main effect was due to the Mn‐exposed males and independent of LPS (Figure 9D; q = 3.046, p = 0.004).

3.3. Plasma Cytokine and Acute Phase Protein ELISAs

In the absence of LPS, plasma IL‐6 and TNFα were undetectable, regardless of Mn treatment and sex (Figure 10A,B, respectively). Six hours after a single LPS administration, increased plasma IL‐6 and TNFα in both Mn and control mice were noted. A two‐way ANOVA revealed an overall main effect of sex, with females' plasma IL‐6 levels being greater than in males (Figure 10A; F(3,20) = 17.463, p < 0.001). Notably, within LPS, male mice that were treated with Mn had greater plasma IL‐6 and TNFα levels compared with saline + LPS males (Figure 10A; q = 6.133, p = 0.012 and Figure 10B; q = 2.190, p = 0.034, respectively); in the females, Mn diminished the LPS effect on both inflammatory cytokines (Figure 10A; q = 6.133, p < 0.001 and Figure 10B; q = 4.125, p < 0.001, respectively). Baseline IL‐10 (without LPS) was higher in the males than in the females (Figure 10C; F(3,20) = 17.816, p < 0.001), and it was unaffected by Mn (F(3,20) = 0.130, p = 0.72). After LPS, IL‐10 in the plasma decreased in the males (q = 4.811, p = 0.001), but it increased significantly in the females (Figure 10C; q = 9.237, p < 0.001). As was the case for TNFα and IL‐6, Mn‐treated/LPS‐challenged (Mn + LPS) female mice had lower IL‐10 (F(3,20) = 7.219, p = 0.01) than LPS‐treated control mice.

FIGURE 10.

FIGURE 10

Open in a new tab

Plasma cytokine and acute phase protein (APP) levels. Effect of Mn after 8 weeks of DW (0.4 g/L) exposure, sex differences, and/or effect of acute LPS administration (0.3 mg/kg BW) on levels of plasma cytokines and APPs 6 h after LPS administration, (A) IL‐6, (B) CRP, (C) TNFα, (D) SAA, and (E) IL‐10. Data are presented as mean ± SEM. # Indicates a significant effect of sex p < 0.05; * indicates a significant effect of Mn p < 0.05; $ indicates a significant effect of LPS p < 0.05; not detected (ND); n = 6/group.

In both sexes, plasma CRP significantly increased after LPS administration (Figure 10D; F(3,20) = 216.234, p < 0.001), and this increase was unaffected by Mn (F(3,20) = 0.291, p = 0.591). A three‐way ANOVA revealed an overall main effect of sex within the LPS‐administered groups, that is, male's plasma CRP levels were greater than in females (F(3,20) = 10.026, p = 0.002). In the absence of LPS, plasma SAA was undetectable, regardless of DW treatment and sex; LPS administration increased plasma SAA in both DW treatment groups and sexes. There was an overall main effect of sex, with plasma SAA being higher in males (Figure 10E; F(3,20) = 24.306, p < 0.001). Although DW Mn exposure did not increase plasma SAA levels post‐LPS compared with saline + LPS males, in females, similar to IL‐6, TNFα, and IL‐10, Mn diminished the LPS effects on SAA (Figure 10E; F(3,20) = 13.524, p < 0.001).

3.4. Liver and Brain Mn and Fe

After 8 weeks of Mn DW exposure, a three‐way ANOVA revealed the overall main effects of Mn and sex on liver Mn. Liver Mn levels were higher in Mn‐exposed mice, regardless of sex, in the absence of LPS (Figure 11A; F(3,20) = 106.033, p < 0.001).

FIGURE 11.

FIGURE 11

Open in a new tab

Liver and brain Mn and Fe levels. Effect of Mn after 8 weeks of DW (0.4 g/L) exposure, sex differences, and/or effect of acute LPS administration (0.3 mg/kg BW) on Mn and Fe levels in brain and liver tissues, 6 h after LPS administration, (A) liver Mn levels, (B) brain Mn levels, (C) liver Fe levels, and (D) brain Fe levels. Data are presented as mean ± SEM. * Indicates a significant effect of Mn p < 0.05; # indicates a significant effect of sex p < 0.05; $ indicates a significant effect of LPS p < 0.05; n = 6/group.

Saline‐challenged females had higher liver Mn regardless of DW treatment (Figure 11A; F(3,20) = 5.935, p = 0.019), however, this overall effect of sex was not seen among LPS‐challenged mice (F(3,20) = 0.482, p = 0.492). Three‐way ANOVA revealed an overall LPS main effect of LPS; LPS‐challenged mice had lower liver Mn levels than control mice (F(3,20) = 5.173, p = 0.028). This LPS effect was more pronounced in females, with Mn + LPS females having significantly lower Mn liver levels than Mn + saline females (Figure 11A; q = 5.822, p < 0.001). There were also overall main effects of sex and DW treatment on liver Fe levels; Fe was lower in the Mn‐exposed mice (Figure 11C; F(3,20) = 19.170, p < 0.001). This overall Mn main effect was driven by the Mn‐exposed females, which had significantly lower levels of liver Fe than controls (F(3,20) = 16.745(1), p < 0.001); in the males, there was only a trend in the same direction (F(3,20) = 3.313, p = 0.084). Female mice, regardless of LPS challenge or DW treatment, had higher liver Fe than males (Figure 11C; F(3,20) = 60.045, p < 0.001).

In the brain, Mn‐exposed mice, regardless of sex and LPS challenge, had higher levels of Mn than controls overall (Figure 11B; F(3,20) = 48.292, p < 0.001). Brain Fe levels were only significantly different within the female mice. Mn‐exposed females had significantly higher brain Fe levels than controls overall (Figure 11D; F(3,20) = 5.49, p = 0.03), but this effect was because of the greater levels in the absence of LPS (Figure 11D; q = 4.101, p = 0.009).

4. Discussion

We explored the effects of DW Mn exposure on motor and mood‐related behaviors of both male and female mice, with the female estrus cycle monitored throughout the study. Additionally, we examined sex differences and the effects of Mn on response to an LPS challenge. Previous rodent studies investigating neurobehavioral consequences of Mn exposure vary in Mn doses/concentrations, types of behavioral tests, strain, and age of mice utilized, as well as duration in Mn treatment prior to behavior testing and routes of exposure, including IP injection (Fleming et al. 2018; Liu et al. 2019), oral gavage (Moreno et al. 2009), subcutaneous osmotic pump (Sepúlveda et al. 2012), and DW (Avila et al. 2010; Krishna et al. 2014), the latter employed here as well. Although study variations might factor in the differences between behavioral outcomes reported, common behavioral domains, that is, locomotor, are affected across multiple studies and exposure paradigms (Krishna et al. 2014; Liu et al. 2019; Moreno et al. 2009; Sepúlveda et al. 2012).

In the current study, Mn‐exposed mice exhibited decreased vertical locomotor activity, shown by fewer rearing attempts during the OFT. DW Mn‐exposed male mice here did not display as much early hyperactivity during the OFT, a DW Mn effect reported by Krishna et al. (2014), but there was a similar trend for the Mn‐exposed males to be hyperactive early in the OFT. Moreno et al. (2009) and Liu et al. (2019) did not find overall alterations in horizontal locomotor activity caused by Mn exposure. However, these studies either did not monitor (Liu et al. 2019) or observe (Moreno et al. 2009) changes in rearing activity. Although the current study used the same Mn concentration, exposure route, and similar timeline as Krishna et al. (2014), the present mice were 2–3 months younger; age has been found to be a factor in Mn‐induced neurological dysfunction (Moreno et al. 2009) and it might have contributed to the less robust hyperactivity effect observed here in the males.

Locomotor deficits induced by Mn were observed during gait testing. Mn‐exposed mice exhibited decreased stride and a left‐biased hind paw interstep distance. To compensate for these deficits, Mn‐exposed mice took more, but shorter, steps to complete the GT. Liu et al. (2019) reported a similar result, also demonstrating a left‐side decrease in stride. When Mn was delivered continuously via an osmotic pump (at 30 mg/kg BW/day) in Sepúlveda et al. (2012), Mn exposure resulted in increased stride and decreased fore/hind paw overlap 1 week after treatment. When Fleming et al. (2018) utilized a lower Mn concentration, as well as older mice, they did not report any gait alterations or sex differences. Liu et al. (2019) reported similar gait deficits, but sex differences during gait testing were not reported. In the current study, females had a smaller fore/hind paw overlap, likely due to them being smaller than males of this strain and age. During the EZM, conducted under minimally anxiogenic red lighting (Williams 1971), there was an Mn‐induced increase of general activity, driven more by males, evident by the increased number of closed and open arm entries. Under halogen light conditions, which are markedly more stressful (Williams 1971), among both sexes of Sprague–Dawley rats, such Mn effect was not observed (Amos‐Kroohs et al. 2016).

In terms of anxiety/fearlessness‐like behavior, our findings are consistent with previous OFT studies, where Mn exposure was via DW (Krishna et al. 2014) or oral gavage (Moreno et al. 2009). Namely, Mn‐exposed male mice spent more time in the center than in the periphery of the OFT. In line with the current study's OFT results, Mn‐exposed mice also spent more time in the open than closed arms of the EZM, a similar finding reported by Amos‐Kroohs et al. (2016) in Mn‐exposed Sprague–Dawley rats. In the current study, this effect was more pronounced among males than females. Interestingly, Mn exposure also reduced the number of SAP attempts, a type of risk assessment when the mouse elongates the body toward potential treatment sources (Grewal et al. 1997). The SAP reduction, together with the increased time in the center and decreased rearing attempts, another risk assessment behavior (Choleris et al. 2001), suggests that DW Mn causes increased fearlessness/risky behavior rather than decreased anxiety‐like behavior.

These results, taken together, indicate that consequences of subchronic Mn DW consumption include both locomotor and mood alterations in male and female mice, with some sex bias. It is worth noting that Mn exposure paradigms using an oral (DW) route, including ours, found that Mn‐exposed mice exhibited reduced anxiety‐like/increased fearlessness‐like behavior, fitting with reports of Mn‐exposed children having increased defiant/oppositional behaviors (Bouchard et al. 2007; Bowler et al. 2003; Laohaudomchok et al. 2011; Sahni et al. 2007).

In mice, LPS challenge results in decreased locomotion and behavioral alterations in the mood domain (Dockman et al. 2022; Krishna, Dodd, and Filipov 2016). In the present study, we confirmed that, in vivo, LPS exposure results in sickness behaviors in both sexes (i.e., less activity and increased anxiety‐like behavior). When we examined sex differences between LPS‐induced sickness behaviors, we found that males exposed to LPS were generally more active than females, regardless of Mn exposure. Although prior exposure to Mn did not influence post‐LPS locomotor activity among males, they did spend more time in the center, which is similar to reports of mood alterations/compulsive behavior exhibited by children exposed to Mn via DW (Bouchard et al. 2007; Bowler et al. 2003; Bowler et al. 2006; Laohaudomchok et al. 2011; Sahni et al. 2007). Indeed, both Mn + saline and Mn + LPS‐treated males spent more time in the open area of the OFT. Thus, Mn‐induced risky/oppositional behavior, in males specifically, is likely to persist despite other behavior‐modifying immune stimuli being present.

In order to investigate potential peripheral inflammatory alterations associated with the post‐LPS behaviors, we assessed selected plasma cytokine and APP levels. Mn alone did not influence plasma cytokine or APP levels. However, when in conjunction with LPS, Mn‐exposed males had a greater inflammatory response (increased IL‐6 and TNFα). By contrast, LPS, both with and without Mn, induced a significant increase in plasma CRP and SAA of both sexes. Interestingly, females displayed a more robust innate immune response to LPS than males, which relates to the observed greater display of sickness behavior among females. That said, females within the Mn + LPS group had significantly lower levels of IL‐6, TNFα, and IL‐10 than females in the saline + LPS group. It is interesting to note that plasma cytokine levels detected in male Mn + LPS and saline + LPS were opposite the cytokine levels produced in females of the respective treatment groups, with Mn potentiating the production of proinflammatory cytokines in the males while diminishing their levels among females. The increased cytokine and APP production in response to LPS, combined with the increased liver weight, suggests that DW Mn is potentiating the inflammatory response in male mice, in vivo, as it does in vitro (Filipov, Seegal, and Lawrence 2005).

Rodent studies investigating sex differences in inflammatory response to LPS have reported varied results including males with a stronger peripheral cytokine response (Kuo 2016), females responding with higher IL‐6 and TNFα and with decreased IL‐10 (Cai et al. 2016), and females with increased IL‐6 and IL‐10, but with decreased TNFα (Erickson et al. 2018). These differing reports may be due to mice strain (IL‐10+/+ and IL‐10−/−, CD‐1, or C57BL/6) and mice age (8–10 weeks and 12–14 months), as well as LPS source ( E. coli serotypes 0127:B8, O26:B6, or 0111:B4 and Salmonella typhimurium), LPS dose (0.2–5 mg/kg BW), or timing of post‐LPS challenge plasma collection (6 h, 1 week, or 28 h post‐LPS challenge) (Cai et al. 2016; Dockman et al. 2022; Erickson et al. 2018; Kuo 2016). It has been reported that females possess smaller infectious loads and stronger overall immunity compared to males and that females have had increased inflammation after ovariectomy (Iwasa et al. 2014; Mabley et al. 2005). However, females also tend to exhibit lower levels of inflammatory cytokines, which is influenced by the effects of estrogen (Bouman, Heineman, and Faas 2005; Darnall and Suarez 2009). Though saline‐administered mice did not have detectable levels of proinflammatory cytokines in either DW group, males with continual Mn exposure did produce a more robust innate immune response to LPS. This is in line with in vitro studies that have found Mn potentiates proinflammatory cytokine production in LPS‐treated microglia (Filipov, Seegal, and Lawrence 2005) and in vivo when Mn exposure is combined with the parkinsonian toxicant MPTP (Hammond et al. 2020). Although females, regardless of DW treatment, displayed typical sickness behaviors in response to LPS, Mn + LPS males continued to exhibit risky behavior suggesting the possibility that males may be more susceptible to and/or affected for a longer duration by Mn. Dockman et al. (2022) reported that females had a more robust response to LPS than males, with increases in IL‐6, TNFα, and IL‐10, similar to the current study, where female plasma cytokines after equivalent LPS dose and timing were greater than males, and in line with human data reporting a more robust female immune response to the same inflammatory stimuli compared with males (Bouman, Heineman, and Faas 2005). The anti‐inflammatory cytokine IL‐10 is important for rebalancing the immune response to a homeostatic state, and estrogen has been previously found to help accelerate this by inducing IL‐10 action (Norden et al. 2016). Oztan et al. (2019) and Kresovich et al. (2018) have previously reported that adult men who have been exposed to excess Mn via occupational/dietary routes have higher levels of proinflammatory cytokines in their blood than those in the control groups; our data, with the males, suggest that this is the case in nonoccupational DW exposures when Mn exposure is interfaced with another inflammatory challenge.

The liver is important in regulating body burdon, distribution, and elimination of Mn, and chronic exposure can result in excess Mn being distributed to other organs instead of removal (Chen, Bornhorst, and Aschner 2018; Huang et al. 2011). In the current study, Mn exposure, via DW, significantly increased levels of Mn in both the brain and liver of both sexes. Mn‐exposed mice had significantly increased liver and brain Mn levels, as well as decreased female‐biased liver iron levels; Mn exposure did not affect brain iron levels, in agreement with results by Alsulimani, Ye, and Kim (2015) and Huang et al. (2011), as well as with reports that Mn is an antagonist of iron (Garcia et al. 2006). Mn‐exposed males did have heavier liver weight; when challenged with LPS, there was an increase in liver Mn levels as well as increased levels of plasma cytokines, but no change in liver iron levels. Although Mn‐exposed females also had higher levels of Mn in the liver, when challenged with LPS, their liver Mn, but not brain Mn, decreased. Although Mn‐exposed females did have similar elevated levels of Mn in the brain as males, Mn‐exposed males displayed sustained behavioral dysfunction, possibly suggesting that the Mn‐induced behavioral effects of Mn within females may be more inconspicuous. Previous studies have indicated that human females have exhibited lower intelligence scores and slower visuospatial learning (Riojas‐Rodríguez et al. 2010; Rechtman et al. 2020), which may suggest parallels with the more subtle nature of the effects observed in Mn‐exposed females compared with males. Because the lasting male‐specific behavioral alterations do not appear to be due to greater Mn deposition in the brain or liver, in the future, we will extend this research to enclose the contribution of brain neurochemistry, as well as neuroinflammation, to investigate the sex differences of the effects of Mn observed in the current study. Because Mn is known to disrupt dopaminergic, glutamatergic, and GABAergic systems within the brain (Soares et al. 2020; Lin et al. 2020), we will focus on brain monoamine and amino acid neurotransmitters.

In conclusion, the current study characterized sex differences in behavioral deficits in Mn‐exposed mice and expanded the existing literature on potential risks of DW Mn overexposure. We have shown that Mn exposure through DW for a subchronic period induces behavioral alterations in mice. Although both sexes demonstrated neurobehavioral dysfunction, our results suggest that males may be more susceptible to continuous Mn exposure and exhibit persistent deficits. Males exposed to Mn via DW also displayed an augmented response to an inflammatory challenge, which suggests a male bias in this domain. Females, on the other hand, exhibited diminished cytokine response, which could impede the immune system's defense against pathogens. These sex differences in cytokine responses and certain behavioral deficits further suggest that Mn effects, at least in the context of DW exposure, are sex‐biased.

Author Contributions

N.M.F. conceived and designed the study. N.M.F., H.D.L., and J.M.C. assisted in the investigation and sample collection. H.D.L. performed sample and data analysis. N.M.F. and H.D.L. wrote the manuscript. All authors read/contributed editorially and approved the final manuscript version.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1 Gait Test paw placement designations on runway. Fore and hind paws were painted with a non‐toxic red and black ink, respectively. Parameter defintions: fore/hindpaw overlap is the distance between a step’s fore and hind paw; hind paw interstep distance (aka step length) is the distance between consecutive right to left steps; stride length is the distance between two consecutive pawprints of the same paw.

Table S1. Absolute tissue weight (g) for each treatment group after 8 weeks of Mn/saline DW treatment and 6 h post‐LPS challenge. Mean ± SEM. Bold indicates p < 0.05; n = 6/group.

JAT-45-669-s001.docx (580.7KB, docx)

Acknowledgments

This research was supported in part by NIH/HIEHS under project number R21ES026383 and with funds from the Lalita and Raghubir Sharma Distinguished Professorship in Toxicology (NMF). Partial assistantship support was provided by the Interdisciplinary Toxicology Program and Department of Physiology and Pharmacology. We also thank Jillian Allen and Dr. Ryan Mote for their technical assistance during this study. We also thank the UGA Trace Analysis lab for running the ICP‐AVOES tissue metal analysis.

Funding: This research was supported in part by NIH/HIEHS under project number R21ES026383 and with funds from the Lalita and Raghubir Sharma Distinguished Professorship in Toxicology (N.M.F.). Partial assistantship support was provided by the Interdisciplinary Toxicology Program and Department of Physiology and Pharmacology.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Agency for Toxic Substances and Disease Registry (ATSDR) . 2012. Toxicological Profile for Manganese. Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service. [PubMed] [Google Scholar]
  2. Alsulimani, H. H. , Ye Q., and Kim J.. 2015. “Effect of Hfe Deficiency on Memory Capacity and Motor Coordination After Manganese Exposure by Drinking Water in Mice.” Toxicology Research 31, no. 4: 347–354. 10.5487/tr.2015.31.4.347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Amos‐Kroohs, R. M. , Davenport L. L., Gutierrez A., Hufgard J. R., Vorhees C. V., and Williams M. T.. 2016. “Developmental Manganese Exposure in Combination With Developmental Stress and Iron Deficiency: Effects on Behavior and Monoamines.” Neurotoxicology and Teratology 56: 55–67. 10.1016/j.ntt.2016.06.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Aschner, M. 2006. “The Transport of Manganese Across the Blood–Brain Barrier.” Neurotoxicology 27, no. 3: 311–314. 10.1016/j.neuro.2005.09.002. [DOI] [PubMed] [Google Scholar]
  5. Aschner, M. , and Aschner J. L.. 1990. “Manganese Transport Across the Blood‐Brain Barrier: Relationship to Iron Homeostasis.” Brain Research Bulletin 24, no. 6: 857–860. 10.1016/0361-9230(90)90152-P. [DOI] [PubMed] [Google Scholar]
  6. Avila, D. S. , Colle D., Gubert P., et al. 2010. “A Possible Neuroprotective Action of a Vinylic Telluride Against Mn‐Induced Neurotoxicity.” Toxicological Sciences: An Official Journal of the Society of Toxicology 115, no. 1: 194–201. 10.1093/toxsci/kfq036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Barrientos, R. M. , Brunton P. J., Lenz K. M., Pyter L., and Spencer S. J.. 2019. “Neuroimmunology of the Female Brain Across the Lifespan: Plasticity to Psychopathology.” Brain, Behavior, and Immunity 79: 39–55. 10.1016/j.bbi.2019.03.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bouchard, M. , Laforest F., Vandelac L., Bellinger D., and Mergler D.. 2007. “Hair Manganese and Hyperactive Behaviors: Pilot Study of School‐Age Children Exposed Through Tap Water.” Environmental Health Perspectives 115, no. 1: 122–127. 10.1289/ehp.9504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bouchard, M. F. , Sauvé S., Barbeau B., et al. 2011. “Intellectual Impairment in School‐Age Children Exposed to Manganese From Drinking Water.” Environmental Health Perspectives 119, no. 1: 138–143. 10.1289/ehp.1002321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bouman, A. , Heineman M. J., and Faas M. M.. 2005. “Sex Hormones and the Immune Response in Humans.” Human Reproduction Update 11, no. 4: 411–423. 10.1093/humupd/dmi008. [DOI] [PubMed] [Google Scholar]
  11. Bowler, R. M. , Gysens S., Diamond E., Booty A., Hartney C., and Roels H. A.. 2003. “Neuropsychological Sequelae of Exposure to Welding Fumes in a Group of Occupationally Exposed Men.” International Journal of Hygiene and Environmental Health 206, no. 6: 517–529. 10.1078/1438-4639-00249. [DOI] [PubMed] [Google Scholar]
  12. Bowler, R. M. , Gysens S., Diamond E., Nakagawa S., Drezgic M., and Roels H. A.. 2006. “Manganese Exposure: Neuropsychological and Neurological Symptoms and Effects in Welders.” Neurotoxicology 27, no. 3: 315–326. 10.1016/j.neuro.2005.10.007. [DOI] [PubMed] [Google Scholar]
  13. Bowler, R. M. , Roels H. A., Nakagawa S., et al. 2007. “Dose‐Effect Relationships Between Manganese Exposure and Neurological, Neuropsychological and Pulmonary Function in Confined Space Bridge Welders.” Occupational and Environmental Medicine 64, no. 3: 167–177. 10.1136/oem.2006.028761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Brna, P. , Gordon K., Dooley J. M., and Price V.. 2011. “Manganese Toxicity in a Child With Iron Deficiency and Polycythemia.” Journal of Child Neurology 26, no. 7: 891–894. 10.1177/0883073810393962. [DOI] [PubMed] [Google Scholar]
  15. Cai, K. C. , van Mil S., Murray E., Mallet J.‐F., Matar C., and Ismail N.. 2016. “Age and sex Differences in Immune Response Following LPS Treatment in Mice.” Brain, Behavior, and Immunity 58: 327–337. 10.1016/j.bbi.2016.08.002. [DOI] [PubMed] [Google Scholar]
  16. Carpenter, J. M. , Brown K. A., Diaz A. N., et al. 2021. “Delayed Treatment With the Immunotherapeutic LNFPIII Ameliorates Multiple Neurological Deficits in a Pesticide‐Nerve Agent Prophylactic Mouse Model of Gulf War Illness.” Neurotoxicology and Teratology 87: 107012. 10.1016/j.ntt.2021.107012. [DOI] [PubMed] [Google Scholar]
  17. Chen, P. , Bornhorst J., and Aschner M.. 2018. “Manganese Metabolism in Humans.” Frontiers in Bioscience (Landmark Edition) 23: 1655–1679. [DOI] [PubMed] [Google Scholar]
  18. Chi, L. , Gao B., Bian X., Tu P., Ru H., and Lu K.. 2017. “Manganese‐Induced Sex‐Specific Gut Microbiome Perturbations in C57BL/6 Mice.” Toxicology and Applied Pharmacology 331: 142–153. 10.1016/j.taap.2017.06.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Choleris, E. , Thomas A. W., Kavaliers M., and Prato F. S.. 2001. “A Detailed Ethological Analysis of the Mouse Open Field Test: Effects of Diazepam, Chlordiazepoxide and an Extremely Low Frequency Pulsed Magnetic Field.” Neuroscience & Biobehavioral Reviews 25, no. 3: 235–260. 10.1016/S0149-7634(01)00011-2. [DOI] [PubMed] [Google Scholar]
  20. Coban, A. , and Filipov N. M.. 2007. “Dopaminergic Toxicity Associated With Oral Exposure to the Herbicide Atrazine in Juvenile Male C57BL/6 Mice.” Journal of Neurochemistry 100, no. 5: 1177–1187. Portico. 10.1111/j.1471-4159.2006.04294.x. [DOI] [PubMed] [Google Scholar]
  21. Conrad, K. L. , Louderback K. M., Gessner C. P., and Winder D. G.. 2011. “Stress‐Induced Alterations in Anxiety‐Like Behavior and Adaptations in Plasticity in the Bed Nucleus of the Stria Terminalis.” Physiology & Behavior 104, no. 2: 248–256. 10.1016/j.physbeh.2011.03.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Crittenden, P. L. , and Filipov N. M.. 2008. “Manganese‐Induced Potentiation of in Vitro Proinflammatory Cytokine Production by Activated Microglial Cells Is Associated With Persistent Activation of p38 MAPK.” Toxicology in Vitro: An International Journal Published in Association With BIBRA 22, no. 1: 18–27. 10.1016/j.tiv.2007.07.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Crittenden, P. L. , and Filipov N. M.. 2011. “Manganese Modulation of MAPK Pathways: Effects on Upstream Mitogen Activated Protein Kinase Kinases and Mitogen Activated Kinase Phosphatase‐1 in Microglial Cells.” Journal of Applied Toxicology: JAT 31, no. 1: 1–10. 10.1002/jat.1552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Darnall, B. D. , and Suarez E. C.. 2009. “Sex and Gender in Psychoneuroimmunology Research: Past, Present and Future.” Brain, Behavior, and Immunity 23, no. 5: 595–604. 10.1016/j.bbi.2009.02.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Dockman, R. L. , Carpenter J. M., Diaz A. N., Benbow R. A., and Filipov N. M.. 2022. “Sex Differences in Behavior, Response to LPS, and Glucose Homeostasis in Middle‐Aged Mice.” Behavioural Brain Research 418: 113628. 10.1016/j.bbr.2021.113628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Dodd, C. A. , and Filipov N. M.. 2011. “Manganese Potentiates LPS‐Induced Heme‐Oxygenase 1 in Microglia but Not Dopaminergic Cells: Role in Controlling Microglial Hydrogen Peroxide and Inflammatory Cytokine Output.” Neurotoxicology 32, no. 6: 683–692. 10.1016/j.neuro.2011.09.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Dodd, C. A. , Ward D. L., and Klein B. G.. 2005. “Basal Ganglia Accumulation and Motor Assessment Following Manganese Chloride Exposure in the C57BL/6 Mouse.” International Journal of Toxicology 24, no. 6: 389–397. 10.1080/10915810500366500. [DOI] [PubMed] [Google Scholar]
  28. Elbetieha, A. , Bataineh H., Darmani H., and Al‐Hamood M. H.. 2001. “Effects of Long‐Term Exposure to Manganese Chloride on Fertility of Male and Female Mice.” Toxicology Letters 119, no. 3: 193–201. 10.1016/S0378-4274(00)00312-X. [DOI] [PubMed] [Google Scholar]
  29. Elsner, R. J. F. , and Spangler J. G.. 2005. “Neurotoxicity of Inhaled Manganese: Public Health Danger in the Shower?” Medical Hypotheses 65, no. 3: 607–616. 10.1016/j.mehy.2005.01.043. [DOI] [PubMed] [Google Scholar]
  30. Erickson, M. A. , Liang W. S., Fernandez E. G., Bullock K. M., Thysell J. A., and Banks W. A.. 2018. “Genetics and sex Influence Peripheral and Central Innate Immune Responses and Blood‐Brain Barrier Integrity.” PLoS ONE 13, no. 10: e0205769. 10.1371/journal.pone.0205769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Erikson, K. M. , Thompson K., Aschner J., and Aschner M.. 2007. “Manganese Neurotoxicity: A Focus on the Neonate.” Pharmacology & Therapeutics 113, no. 2: 369–377. 10.1016/j.pharmthera.2006.09.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Filipov, N. M. , and Dodd C. A.. 2012. “Role of Glial Cells in Manganese Neurotoxicity.” Journal of Applied Toxicology 32, no. 5: 310–317. 10.1002/jat.1762. [DOI] [PubMed] [Google Scholar]
  33. Filipov, N. M. , Seegal R. F., and Lawrence D. A.. 2005. “Manganese Potentiates In Vitro Production of Proinflammatory Cytokines and Nitric Oxide by Microglia Through a Nuclear Factor Kappa B–Dependent Mechanism.” Toxicological Sciences 84, no. 1: 139–148. 10.1093/toxsci/kfi055. [DOI] [PubMed] [Google Scholar]
  34. Fleming, S. M. , Santiago N. A., Mullin E. J., et al. 2018. “The Effect of Manganese Exposure in Atp13a2‐Deficient Mice.” Neurotoxicology 64: 256–266. 10.1016/j.neuro.2017.06.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Garcia, S. J. , Gellein K., Syversen T., and Aschner M.. 2006. “A Manganese‐Enhanced Diet Alters Brain Metals and Transporters in the Developing Rat.” Toxicological Sciences: An Official Journal of the Society of Toxicology 92, no. 2: 516–525. 10.1093/toxsci/kfl017. [DOI] [PubMed] [Google Scholar]
  36. Godbout, J. P. , Chen J., Abraham J., et al. 2005. “Exaggerated Neuroinflammation and Sickness Behavior in Aged Mice After Activation of the Peripheral Innate Immune System.” FASEB Journal 19, no. 10: 1329–1331. 10.1096/fj.05-3776fje. [DOI] [PubMed] [Google Scholar]
  37. Gonzalez‐Cuyar, L. F. , Nelson G., Criswell S. R., et al. 2014. “Quantitative Neuropathology Associated With Chronic Manganese Exposure in South African Mine Workers.” Neurotoxicology 45: 260–266. 10.1016/j.neuro.2013.12.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Grewal, S. S. , Shepherd J. K., Bill D. J., Fletcher A., and Dourish C. T.. 1997. “Behavioural and Pharmacological Characterisation of the Canopy Stretched Attend Posture Test as a Model of Anxiety in Mice and Rats.” Psychopharmacology 133, no. 1: 29–38. 10.1007/s002130050367. [DOI] [PubMed] [Google Scholar]
  39. Hafeman, D. , Factor‐Litvak P., Cheng Z., van Geen A., and Ahsan H.. 2007. “Association Between Manganese Exposure Through Drinking Water and Infant Mortality in Bangladesh.” Environmental Health Perspectives 115, no. 7: 1107–1112. 10.1289/ehp.10051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Hammond, S. L. , Bantle C. M., Popichak K. A., et al. 2020. “NF‐κB Signaling in Astrocytes Modulates Brain Inflammation and Neuronal Injury Following Sequential Exposure to Manganese and MPTP During Development and Aging.” Toxicological Sciences 177, no. 2: 506–520. 10.1093/toxsci/kfaa115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Huang, P. , Chen C., Wang H., et al. 2011. “Manganese Effects in the Liver Following Subacute or Subchronic Manganese Chloride Exposure in Rats.” Ecotoxicology and Environmental Safety 74, no. 4: 615–622. 10.1016/j.ecoenv.2010.08.011. [DOI] [PubMed] [Google Scholar]
  42. IOM . 2001. Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington, DC: The National Academies Press. 10.17226/10026. [DOI] [PubMed] [Google Scholar]
  43. Iwasa, T. , Matsuzaki T., Kinouchi R., et al. 2014. “Changes in Central and Peripheral Inflammatory Responses to Lipopolysaccharide in Ovariectomized Female Rats.” Cytokine 65, no. 1: 65–73. 10.1016/j.cyto.2013.10.004. [DOI] [PubMed] [Google Scholar]
  44. Kresovich, J. K. , Bulka C. M., Joyce B. T., et al. 2018. “The Inflammatory Potential of Dietary Manganese in a Cohort of Elderly Men.” Biological Trace Element Research 183, no. 1: 49–57. 10.1007/s12011-017-1127-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Krishna, S. , Dodd C. A., and Filipov N. M.. 2016. “Behavioral and Monoamine Perturbations in Adult Male Mice With Chronic Inflammation Induced by Repeated Peripheral Lipopolysaccharide Administration.” Behavioural Brain Research 302: 279–290. 10.1016/j.bbr.2016.01.038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Krishna, S. , Dodd C. A., Hekmatyar S. K., and Filipov N. M.. 2014. “Brain Deposition and Neurotoxicity of Manganese in Adult Mice Exposed via the Drinking Water.” Archives of Toxicology 88, no. 1: 47–64. 10.1007/s00204-013-1088-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Kuo, S.‐M. 2016. “Gender Difference in Bacteria Endotoxin‐Induced Inflammatory and Anorexic Responses.” PLoS ONE 11, no. 9: e0162971. 10.1371/journal.pone.0162971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Laohaudomchok, W. , Lin X., Herrick R. F., et al. 2011. “Neuropsychological Effects of Low‐Level Manganese Exposure in Welders.” Neurotoxicology 32, no. 2: 171–179. 10.1016/j.neuro.2010.12.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Lin, M. , Colon‐Perez L. M., Sambo D. O., et al. 2020. “Mechanism of Manganese Dysregulation of Dopamine Neuronal Activity.” The Journal of Neuroscience 40, no. 30: 5871–5891. 10.1523/jneurosci.2830-19.2020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Liu, C. , Jursa T., Aschner M., Smith D. R., and Mukhopadhyay S.. 2021. “Up‐Regulation of the Manganese Transporter SLC30A10 by Hypoxia‐Inducible Factors Defines a Homeostatic Response to Manganese Toxicity.” Proceedings of the National Academy of Sciences of the United States of America 118, no. 35: e2107673118. 10.1073/pnas.2107673118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Liu, K. , Jing M.‐J., Liu C., et al. 2019. “Effect of Trehalose on Manganese‐Induced Mitochondrial Dysfunction and Neuronal Cell Damage in Mice.” Basic & Clinical Pharmacology & Toxicology 125, no. 6: 536–547. 10.1111/bcpt.13316. [DOI] [PubMed] [Google Scholar]
  52. Ljung, K. , and Vahter M.. 2007. “Time to Re‐Evaluate the Guideline Value for Manganese in Drinking Water?” Environmental Health Perspectives 115, no. 11: 1533–1538. 10.1289/ehp.10316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Lucchini, R. , and Tieu K.. 2023. “Manganese‐Induced Parkinsonism: Evidence From Epidemiological and Experimental Studies.” Biomolecules 13, no. 8: 1190. 10.3390/biom13081190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Lucchini, R. G. , Martin C. J., and Doney B. C.. 2009. “From Manganism to Manganese‐Induced Parkinsonism: A Conceptual Model Based on the Evolution of Exposure.” Neuromolecular Medicine 11, no. 4: 311–321. 10.1007/s12017-009-8108-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Mabley, J. G. , Horváth E. M., Murthy K. G. K., et al. 2005. “Gender Differences in the Endotoxin‐Induced Inflammatory and Vascular Responses: Potential Role of Poly (ADP‐Ribose) Polymerase Activation.” Journal of Pharmacology and Experimental Therapeutics 315, no. 2: 812–820. 10.1124/jpet.105.090480. [DOI] [PubMed] [Google Scholar]
  56. Madison, J. L. , Wegrzynowicz M., Aschner M., and Bowman A. B.. 2011. “Gender and Manganese Exposure Interactions on Mouse Striatal Neuron Morphology.” Neurotoxicology 32, no. 6: 896–906. 10.1016/j.neuro.2011.05.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. McMahon, P. B. , Belitz K., Reddy J. E., and Johnson T. D.. 2019. “Elevated Manganese Concentrations in United States Groundwater, Role of Land Surface–Soil–Aquifer Connections.” Environmental Science & Technology 53, no. 1: 29–38. 10.1021/acs.est.8b04055. [DOI] [PubMed] [Google Scholar]
  58. Moreno, J. A. , Yeomans E. C., Streifel K. M., Brattin B. L., Taylor R. J., and Tjalkens R. B.. 2009. “Age‐Dependent Susceptibility to Manganese‐Induced Neurological Dysfunction.” Toxicological Sciences 112, no. 2: 394–404. 10.1093/toxsci/kfp220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Mulherkar, S. , Liu F., Chen Q., et al. 2013. “The Small GTPase RhoA Is Required for Proper Locomotor Circuit Assembly.” PLoS ONE 8, no. 6: e67015. 10.1371/journal.pone.0067015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Norden, D. M. , Trojanowski P. J., Villanueva E., Navarro E., and Godbout J. P.. 2016. “Sequential Activation of Microglia and Astrocyte Cytokine Expression Precedes Increased iba‐1 or GFAP Immunoreactivity Following Systemic Immune Challenge.” Glia 64, no. 2: 300–316. 10.1002/glia.22930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Oztan, O. , Turksoy V. A., Daltaban I. S., et al. 2019. “Pro‐Inflammatory Cytokine and Vascular Adhesion Molecule Levels in Manganese and Lead‐Exposed Workers.” International Journal of Immunotherapy and Cancer Research 5, no. 1: 1–7. 10.17352/2455-8591.000020. [DOI] [Google Scholar]
  62. Park, R. M. , Bowler R. M., Eggerth D. E., et al. 2006. “Issues in Neurological Risk Assessment for Occupational Exposures: The Bay Bridge Welders.” Neurotoxicology 27, no. 3: 373–384. 10.1016/j.neuro.2005.10.010. [DOI] [PubMed] [Google Scholar]
  63. Perry, V. H. 2004. “The Influence of Systemic Inflammation on Inflammation in the Brain: Implications for Chronic Neurodegenerative Disease.” Brain, Behavior, and Immunity 18, no. 5: 407–413. 10.1016/j.bbi.2004.01.004. [DOI] [PubMed] [Google Scholar]
  64. Racette, B. A. , Searles Nielsen S., Criswell S. R., et al. 2017. “Dose‐Dependent Progression of Parkinsonism in Manganese‐Exposed Welders.” Neurology 88, no. 4: 344–351. 10.1212/WNL.0000000000003533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Rechtman, E. , Curtin P., Papazaharias D. M., et al. 2020. “Sex‐Specific Associations Between Co‐Exposure to Multiple Metals and Visuospatial Learning in Early Adolescence.” Translational Psychiatry 10, no. 1: 358. 10.1038/s41398-020-01041-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Riojas‐Rodríguez, H. , Solís‐Vivanco R., Schilmann A., et al. 2010. “Intellectual Function in Mexican Children Living in a Mining Area and Environmentally Exposed to Manganese.” Environmental Health Perspectives 118, no. 10: 1465–1470. 10.1289/ehp.0901229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Sahni, V. , Léger Y., Panaro L., et al. 2007. “Case Report: A Metabolic Disorder Presenting as Pediatric Manganism.” Environmental Health Perspectives 115, no. 12: 1776–1779. 10.1289/ehp.10421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Sepúlveda, M. R. , Dresselaers T., Vangheluwe P., et al. 2012. “Evaluation of Manganese Uptake and Toxicity in Mouse Brain During Continuous MnCl2 Administration Using Osmotic Pumps.” Contrast Media & Molecular Imaging 7, no. 4: 426–434. 10.1002/cmmi.1469. [DOI] [PubMed] [Google Scholar]
  69. Soares, A. T. G. , Silva A. de C., Tinkov A. A., et al. 2020. “The Impact of Manganese on Neurotransmitter Systems.” Journal of Trace Elements in Medicine and Biology 61: 126554. 10.1016/j.jtemb.2020.126554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Utembe, W. , Faustman E. M., Matatiele P., and Gulumian M.. 2015. “Hazards Identified and the Need for Health Risk Assessment in the South African Mining Industry.” Human & Experimental Toxicology 34, no. 12: 1212–1221. 10.1177/0960327115600370. [DOI] [PubMed] [Google Scholar]
  71. Wang, X. , Wang Q. M., Meng Z., Yin Z., Luo X., and Yu D.. 2017. “Gait Disorder as a Predictor of Spatial Learning and Memory Impairment in Aged Mice.” PeerJ 5: e2854. 10.7717/peerj.2854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Wasserman, G. A. , Liu X., Parvez F., et al. 2006. “Water Manganese Exposure and Children's Intellectual Function in Araihazar, Bangladesh.” Environmental Health Perspectives 114, no. 1: 124–129. 10.1289/ehp.8030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Williams, D. I. 1971. “Maze Exploration in the Rat Under Different Levels of Illumination.” Animal Behaviour 19, no. 2: 365–367. 10.1016/S0003-3472(71)80018-0. [DOI] [PubMed] [Google Scholar]
  74. Woolf, A. , Wright R., Amarasiriwardena C., and Bellinger D.. 2002. “A Child With Chronic Manganese Exposure From Drinking Water.” Environmental Health Perspectives 110, no. 6: 613–616. 10.1289/ehp.02110613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Zhang, S. , Zhou Z., and Fu J.. 2003. “Effect of Manganese Chloride Exposure on Liver and Brain Mitochondria Function in Rats.” Environmental Research 93, no. 2: 149–157. 10.1016/S0013-9351(03)00109-9. [DOI] [PubMed] [Google Scholar]
  76. Zhao, F. , Cai T., Liu M., Zheng G., Luo W., and Chen J.. 2009. “Manganese Induces Dopaminergic Neurodegeneration via Microglial Activation in a Rat Model of Manganism.” Toxicological Sciences 107, no. 1: 156–164. 10.1093/toxsci/kfn213. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 Gait Test paw placement designations on runway. Fore and hind paws were painted with a non‐toxic red and black ink, respectively. Parameter defintions: fore/hindpaw overlap is the distance between a step’s fore and hind paw; hind paw interstep distance (aka step length) is the distance between consecutive right to left steps; stride length is the distance between two consecutive pawprints of the same paw.

Table S1. Absolute tissue weight (g) for each treatment group after 8 weeks of Mn/saline DW treatment and 6 h post‐LPS challenge. Mean ± SEM. Bold indicates p < 0.05; n = 6/group.

JAT-45-669-s001.docx (580.7KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from Journal of Applied Toxicology are provided here courtesy of Wiley

RESOURCES