Interaction of coexposure to inorganic arsenic and manganese: Tight junction injury of the blood–brain barrier and the relationship between oxidative stress and inflammatory cytokines in glial cells

Toshiaki Hitomi; Hiroko Okuda; Ayako Takata; Hiroshi Yamauchi

doi:10.1371/journal.pone.0330287

. 2025 Aug 25;20(8):e0330287. doi: 10.1371/journal.pone.0330287

Interaction of coexposure to inorganic arsenic and manganese: Tight junction injury of the blood–brain barrier and the relationship between oxidative stress and inflammatory cytokines in glial cells

Toshiaki Hitomi ^1,^#, Hiroko Okuda ^1,^#, Ayako Takata ¹, Hiroshi Yamauchi ^1,^*

Editor: Mária A Deli,²

PMCID: PMC12377611 PMID: 40853884

Abstract

Coexposure to inorganic arsenic (iAs) and manganese (Mn) may exacerbate cognitive dysfunction caused by iAs alone. In this study, we investigated the cytotoxicity of coexposure to iAs and Mn in glial cells and the expression and correlation between oxidative stress and inflammatory cytokines. Additionally, we assessed tight junction (TJ) injury using a rat in vitro blood-brain barrier (BBB) model. In glial cells, coexposure to iAs and Mn increased cytotoxicity compared to single exposure, suggesting a likely additive effect. iAs exposure significantly increased the expression of antioxidant stress markers, including nuclear factor erythroid 2–related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), relative to Mn exposure. Notably, HO-1 expression was further elevated under coexposure conditions, indicating a potential synergistic effect. Regarding inflammatory cytokines, expression of C-C motif chemokine ligand 2 (MCP-1) and interleukin-6 (IL-6) was slightly higher in the iAs exposure compared to Mn exposure. A synergistic effect was observed in the Mn concentration-dependent increase in IL-6 under coexposure. A significant positive correlation was found between Nrf2 or HO-1 and inflammatory cytokines (MCP-1 and IL-6) (p < 0.001), suggesting an interaction between oxidative stress and inflammatory cytokines. The BBB TJ injury was evaluated by measuring the transendothelial electrical resistance values and the Claudin-5 and zonula occludens-1. The results showed expression in iAs exposure but not in Mn exposure. Furthermore, Mn did not affect iAs-induced TJ injury. In conclusion, our findings demonstrate that coexposure to iAs and Mn exerts synergistic effects on oxidative stress and inflammatory cytokines in glial cells. These joint effects may increase the risk of neurotoxicity compared to single-iAs or Mn exposure.

Introduction

The global incidence of cognitive dysfunction is increasing, affecting both children and adults. Epidemiological studies increasingly highlight neurotoxic substances, such as inorganic arsenic (iAs) and manganese (Mn), as significant contributors. For instance, iAs exposure, primarily through contaminated groundwater, has been linked to cognitive dysfunction in children across Bangladesh [1–4], India [5,6], China [7,8], and Mexico [9,10]. Similarly, Mn exposure from groundwater and air contamination has been associated with cognitive dysfunction in children from diverse regions, including Bangladesh [11–14], China [15,16], Canada [17,18], Mexico [19,20], the United States [21,22], and Brazil [23,24]. Animal studies have demonstrated that exposure to iAs [25,26] or Mn [27–29] alone can induce cognitive dysfunction. Although the precise mechanisms underlying such cognitive dysfunction remain incompletely understood, accumulating evidence suggests that oxidative stress and proinflammatory cytokines may contribute to the common neurotoxic effects of iAs and Mn [25–29]. Notably, while iAs is strongly neurotoxic, Mn is an essential trace element [30] that becomes neurotoxic only at elevated concentrations. Furthermore, emerging evidence suggests that exposure to either iAs [25,31,33] or Mn [31–33] alone may contribute to the onset and progression of Alzheimer’s disease (AD). AD is a prominent neurodegenerative disorder primarily characterized by progressive cognitive decline, typically manifesting with aging in adulthood [34,35]. Despite these findings, a critical gap in our understanding persists regarding the long-term consequences of early-life exposure (i.e., fetal, infant, and childhood periods) to iAs and Mn on the subsequent development and progression of AD.

Previous studies [1–29] have primarily focused on cognitive dysfunction caused by individual exposure to neurotoxicants such as iAs and Mn. However, in real-world environments, coexposure to these toxicants is common, and such combined exposure may induce additive or synergistic neurotoxicity that cannot be predicted from single exposure studies alone. In particular, the mechanisms by which coexposure to iAs and Mn leads to cognitive impairment remain poorly understood, and comparative investigations with single-exposures are urgently needed. For example, in arsenic-contaminated regions of Bangladesh, groundwater is often polluted with both iAs [1–4] and Mn [11–14], and studies suggest that combined exposure may cause more severe cognitive deficits in children than single iAs exposures [36–38]. Although this is often viewed as a localized issue, iAs and Mn co-contamination is a global concern. Groundwater used for drinking and agriculture often contains both of these elements, raising concerns about potential health effects on populations numbering in the millions [39]. In the glacial aquifers of northern United States, high concentrations of iAs and Mn have also been detected, raising concerns about health risks through drinking water [40]. Furthermore, the European Food Safety Authority has expressed concern regarding the dietary intake and neurotoxicity of iAs [41] and Mn [42]. Despite the likelihood that exposure through groundwater and food may result from co-contamination, methodologies for assessing the health risks of coexposure remain underdeveloped. Addressing this gap requires innovative research strategies and analytical approaches. Cognitive dysfunction caused by coexposure to neurotoxicants should be recognized not merely as a regional issue, but as a broader public health risk affecting populations worldwide.

We are interested in the cognitive dysfunction caused by combined exposure to iAs and Mn and are attempting to clarify the mechanism of its occurrence. Our hypothesis for the mechanism of cognitive dysfunction induced by iAs exposure is as follows: first, oxidative stress induced by iAs triggers tight junction (TJ) injury in the blood–brain barrier (BBB), facilitating the entry of iAs into the brain. This, in turn, triggers oxidative stress and inflammatory cytokine responses in glial cells, leading to neuronal cell damage induced by inflammation and the development of various brain dysfunctions. We used a rat in vitro BBB model to confirm this hypothesis and analyze the relationship between iAs exposure and TJ injury in the BBB. We thus confirmed that iAs exposure increases TJ injury in a concentration-dependent manner [43].

Epidemiological studies [11–24] and animal [27–29] experiments have demonstrated that Mn is a neurotoxic substance. Neurodegenerative diseases caused by Mn exposure can be understood as a health effects problem on a larger scale than those caused by iAs exposure. Studies on animals [44,45] and rat in vitro BBB models [43] have confirmed that iAs enters the brain through the BBB. The routes by which Mn reaches the brain are the BBB [46], the blood–cerebrospinal fluid barrier (BCB) [47], and the olfactory nerve [48]. Although there are no studies directly comparing these three routes, it is believed that Mn is more likely to reach the brain via active transport through BCB than through the BBB. Both iAs and Mn have routes that the BBB can cross, indicating that the BBB is a potential target for these substances. Obtaining information on the joint effects of iAs and Mn on the BBB is important. If additive or synergistic effects occur between iAs and Mn, TJ injury to the BBB may increase, promoting the transfer of iAs and Mn to brain tissue and increasing their accumulation. Against this background, verifying the coexposure of iAs and Mn compared to their single exposure is important. To specifically address this issue, it is crucial to verify the damage to the BBB caused by coexposure to iAs and Mn using a rat in vitro BBB model [43]. Furthermore, it is essential to gather information on the joint effects of oxidative stress and inflammatory cytokines in glial cells and to analyze the results thoroughly. In this study, we evaluated the joint effects of iAs and Mn by distinguishing additive effects—where the combined outcome equals the sum of individual effects (E_iAs + E_Mn= E_{iAs + Mn})—from synergistic effects, where the combined outcome exceeds this sum (E_{iAs + Mn}> E_iAs + E_Mn). Here, E_iAs denotes the effect of exposure to iAs, E_Mn the effect of exposure to Mn, and E_{iAs + Mn} the effect of coexposure.

Therefore, this study was conducted with the following objectives: 1) Investigate the cytotoxicity in glial cells induced by single or coexposure to iAs and Mn. 2) Evaluate oxidative stress and inflammatory cytokine responses in glial cells following single or coexposure to iAs and Mn. 3) Examine the TJ injury induced by single or coexposure to iAs and Mn using a rat in vitro BBB model. By addressing these objectives, we anticipate clarifying the joint effects of iAs and Mn, particularly concerning their impact on BBB integrity and glial cell function, thereby contributing valuable insights into the mechanisms of coexposure-induced neurotoxicity.

Materials and methods

Chemicals

iAs (arsenite; arsenic trioxide, 99.995% purity, CAS No. 1327-53-3) and Mn (MnCl₂·4H₂O, ≥ 99% purity, CAS No. 13446-34-9) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions were prepared using cell culture-grade sterile water (Nacalai Tesque, Inc., Kyoto, Japan) and brain capillary endothelial cell culture medium (PharmaCo-Cell Company Ltd, Nagasaki, Japan). Diluted solutions were used for subsequent experiments.

Rat in vitro BBB model and sample collection

A rat in vitro BBB model comprising three cell types (RBT-24H) was purchased from PharmaCo-Cell Company Ltd. [49,50]. This model is based on a triple coculture of primary brain capillary endothelial cells, pericytes, and astrocytes isolated from Wistar rats. The rat in vitro BBB model structure consists of brain capillary endothelial cells on the top of a transwell filter in the insert portion, pericytes on the bottom, and astrocytes attached to the bottom of a 24-well plate. The maturation of the rat in vitro BBB model was determined by measuring the transendothelial electrical resistance (TEER) using the Millicell Electrical Resistance System (ERS)-2 Voltohmmeter (Merck Millipore, Billerica, MA, USA). Cells were incubated at 37°C according to the manufacturer’s protocol until the TEER value reached ≥150 Ω × cm², which indicates that the TJs are functioning properly [49,50].

The exposure concentrations were determined with reference to survey results from Bangladesh. In a survey on cognitive dysfunction in children, the concentrations of arsenic and Mn in groundwater in an area of chronic arsenic poisoning were reported as 117.8 ± 145.2 µg/L and 1,386 ± 927 µg/L, respectively [1]. For reference, 5 µM iAs corresponds to 378 µg/L, and 10 µM Mn corresponds to 549 µg/L. In this experiment, the lowest exposure concentrations (5 µM iAs and 10 µM Mn) were approximately 3-fold higher than the survey value for iAs and approximately 0.4-fold (i.e., 40%) of the survey value for Mn. Therefore, these concentrations were considered to reflect environmentally relevant exposure levels. Furthermore, in the Mn group, concentrations corresponding to 10-fold and 20-fold the measured value of 1,386 µg/L reported in the survey were also applied. As the experimental conditions, iAs alone (10 µM), Mn alone (10, 100, and 200 µM), and coexposure (iAs + Mn: 10 + 10, 10 + 100, and 10 + 200 µM) were applied in the luminal compartment (i.e., the blood side) for 24 h. The detailed procedures for BBB culture, treatment, and cell sample collection (primary brain capillary endothelial cells, pericytes, and astrocytes) were performed exactly as described in our previous study [43]. Next, we obtained the results of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), claudin-5, and zonula occludens-1 (ZO-1) in the western blot (WB) analysis related to the evaluation of oxidative stress and TJ injury in the BBB. Furthermore, the expression and localization of claudin-5 and ZO-1 were examined by immunocytochemistry to assess morphological TJ injury in the BBB.

Rat cerebral cortical mixed glial cell culture

Rat cerebral cortical mixed glial cells (i.e., glial cells) were purchased from PharmaCo-Cell Company Ltd. and used in the experiments. These glial cells, consisting of primary microglia and astrocytes derived from Wistar rats, originated from the same source as the rats used to construct the rat in vitro BBB model. These cells were cultured in brain capillary endothelial cell culture medium supplemented with 5 ng/mL recombinant rat granulocyte-macrophage colony-stimulating factor (GM-CSF; FUJIFILM Wako, Osaka, Japan) at 37°C in a humidified 5% CO₂ atmosphere. The culture medium was changed once every 3–4 days.

Cytotoxicity assay

The cytotoxicity of glial cells after chemical treatment was validated using the water-soluble tetrazolium salt-8 (WST-8) assay with the Cell Counting Kit-8 (CCK-8; Dojindo, Tokyo, Japan). On the first day, cells were seeded at a density of 20,000 cells/well in 100 µL of brain capillary endothelial cell culture medium in 96-well plates and incubated overnight at 37°C in a humidified 5% CO₂ atmosphere. The next day, the cells were treated with iAs alone (1 and 5 µM), Mn alone (10, 100, and 200 µM), or coexposure (iAs + Mn: 5 + 10, 5 + 100, and 5 + 200 µM). After 24 h of treatment, the CCK-8 solution was added to each well and incubated, after which the absorbance was measured at 450 nm using a microplate luminometer ARVOx4 2030 Multilabel Reader (Perkin Elmer, Waltham, MA, USA).

Real-time quantitative PCR

After the treatment of glial cells with iAs alone (5 µM), Mn alone (10, 100, and 200 µM), or coexposure (iAs + Mn: 5 + 10, 5 + 100, and 5 + 200 µM) for 24 h, total RNA was extracted using the FastGene^TM RNA Premium kit (Nippon Genetics, Tokyo, Japan) according to the manufacturer’s instructions. Next, cDNA was synthesized using 1 µg of total RNA using the PrimeScript RT Reagent Kit (Takara Bio, Shiga, Japan) according to the manufacturer’s protocol. Real-time quantitative PCR was conducted using the StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) with TB Green Premix Ex Taq II (Takara Bio). Primer sets from the Perfect Real-Time support system (Takara Bio, https://www.takara-bio.co.jp/research/prt/) were also used. Oxidative stress-related genes were analyzed using the primer sets targeting Nrf2 (nuclear factor erythroid 2 (NFE2) like basic leucine zipper (bZIP) transcription factor 2; Nfe2l2, mRNA; Primer Set ID: RA086678) and HO-1 (heme oxygenase 1; Hmox1, mRNA; RA086407). Inflammatory cytokines were analyzed using the primer sets targeting MCP-1 (C-C motif chemokine ligand 2; Ccl2, mRNA; RA011410), IL-1β (interleukin 1 beta; Il1b, mRNA; RA075332) and IL-6 (interleukin 6; Il6, mRNA; RA079019). The thermal cycling conditions were as follows: initial activation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s and annealing/extension at 60°C for 30 s. The expression levels of antioxidant stress-related and inflammatory cytokine genes were quantified. Gene expression in each sample was normalized to β-actin (actin, beta; Actb, mRNA; RA015375) and expressed relative to the control group.

WB analysis

WB analysis was performed as described previously [43], including sample preparation, gel electrophoresis, membrane transfer, and antibody incubation. The following primary antibodies were used: rabbit monoclonal anti-Nrf2 (1:1000, #33649; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit polyclonal anti-HO-1 (1:1000, 10701–1-AP; Proteintech Group, Inc., Chicago, IL, USA), mouse monoclonal anti-claudin-5 (1:1000, 35–2500; Thermo Fisher Scientific), rabbit polyclonal anti-ZO-1 (1:500, 61–7300; Thermo Fisher Scientific), and mouse monoclonal anti-β-actin (1:2500, A5316; Sigma-Aldrich). Detection and quantification were performed as described previously [43].

Immunocytochemistry

The expression of claudin-5 and ZO-1 in vascular endothelial cells of the BBB was evaluated using immunocytochemistry as described previously [43]. Briefly, primary brain capillary endothelial cells on transwell inserts were fixed, permeabilized, and stained with specific primary and secondary antibodies. The following primary antibodies were used: mouse monoclonal anti-claudin-5 (1:100, 35–2500; Thermo Fisher Scientific) and mouse monoclonal anti-ZO-1 (1:100, 33–9100; Thermo Fisher Scientific). DAPI was used for nuclear staining, and fluorescence images were obtained using a BZ-X710 fluorescence microscope (Keyence, Osaka, Japan). The exposure time was standardized to 1/30 s as the imaging condition. After setting a threshold by eye, a line (70 µm) was arbitrarily set such that there were two or more adjacent cells as the region of interest in those images, and then the immunoreactivity of the crossed primary brain capillary endothelial cell membranes was quantitatively measured as a histogram using ImageJ (National Institutes of Health, MD, USA). Stained regions obtained as histogram peaks were averaged in each sample (n = 6, average 40–50 locations) and quantified. The counting method used in this analysis was a modification of the method described previously [51].

Statistical analyses

Statistical analyses were conducted using IBM SPSS Version 28.0 (IBM Corp, Armonk, NY, USA). All results are summarized as mean ± standard deviation (SD). One-way ANOVAs with Tukey’s post hoc tests were used for comparing two or more groups. Spearman’s rank correlation coefficient was used to calculate the association between pairs of variables. p < 0.05 was considered statistically significant for the two-tailed test.

Results

Cytotoxicity, oxidative stress, and inflammatory cytokines in glial cells

Cytotoxicity of iAs and Mn.

Cytotoxicity in glial cells was evaluated using the CCK-8 method at 24 h after exposure to iAs alone (1 and 5 µM) or Mn alone (10, 100, and 200 µM) or coexposure (iAs + Mn: 5 + 10, 100, and 200 µM) (Fig 1). Although no cytotoxicity was detected in the 1-µM iAs group, the 5-µM iAs group demonstrated significant cytotoxicity, resulting in an approximately 30% reduction in viability compared with that in the control group (p < 0.001; see S1 Table). Mn also exerted toxic effects (p < 0.001; see S1 Table) on the control group. Furthermore, there were no statistically significant differences in cytotoxicity between the 5-µM iAs and Mn groups. Coexposure to iAs and Mn significantly increased the cytotoxicity compared with that in the control group (p < 0.001; see S1 Table). There was an approximately 2.5-fold increase in toxicity compared with exposure to 5 µM iAs (p < 0.001; see S1 Table) or Mn (10, 100, and 200 µM) (p < 0.001; see S1 Table). This increase in toxicity due to coexposure to iAs and Mn suggested a likely additive effect.

Fig 1 — The cytotoxicity of iAs (1 and 5 µM) or Mn (10, 100, and 200 µM) alone and coexposure to (iAs + Mn: 5 + 10, 100, and 200 µM) was evaluated in glial cells using the CCK-8 method 24 h after exposure. Results are expressed as mean ± SD (n = 5). Comparisons of the control group, iAs and Mn alone groups, and iAs and Mn coexposure groups were performed using one-way ANOVA with Tukey’s post hoc tests. The levels of statistical significance were as follows: for control, ^***p < 0.001; for iAs, ^###p < 0.001. Moreover, the significance levels for each concentration of Mn and the corresponding coexposure were as follows: 10 µM, ^†††p < 0.001; 100 µM, ^‡‡‡p < 0.001; 200 µM, ^$$$p < 0.001. NS indicates no significant differences among the coexposure groups. Detailed statistical values are provided in S1 Table.

Oxidative stress induced by iAs and Mn.

In glial cells, real-time PCR analysis was performed to evaluate the mRNA expression levels of the antioxidant stress-related genes Nrf2 and HO-1 after exposure to iAs alone (5 µM) or Mn alone (10, 100, and 200 µM) or coexposure (iAs + Mn: 5 + 10, 100, and 200 µM). The Nrf2 mRNA expression was found to be approximately 1.8-fold higher in the iAs group than in the control group (p < 0.001; see S2 Table, panel A and Fig 2A). In contrast, there was no significant increase in the Mn group (Fig 2A). Coexposure to iAs and Mn resulted in a statistically significant increase in Nrf2 mRNA expression compared with that in the control group (p < 0.001; see S2 Table, panel A), and this pattern of Nrf2 mRNA expression was dependent on Mn concentration (Fig 2A). Similarly, HO-1 mRNA expression was approximately 3.5-fold higher in the iAs group than in the control group (p < 0.001; see S2 Table, panel B), whereas there was no increase in the Mn group (Fig 2B). Coexposure to iAs and Mn significantly increased HO-1 mRNA expression compared to the control group (p < 0.001; see S2 Table, panel B). Furthermore, a synergistic effect of 200 µM-Mn were suggested for the increasing trend of HO-1 mRNA expression (Fig 2B; see S2 Table, panel B). The correlation between Nrf2 and HO-1 mRNA expression among the groups is shown in Table 1. There was a significant correlation in the iAs group (ρ = 0.929, p < 0.001) and the iAs and Mn coexposure group (ρ = 0.809, p < 0.001). There was also a significant correlation in the Mn group (ρ = 0.635, p < 0.01); however, the correlation coefficient was lower than that in the iAs group, suggesting that the effect of Mn on oxidative stress is weaker than that of iAs.

Fig 2 — iAs alone (5 µM) or Mn alone (10, 100, and 200 µM) or coexposure (iAs + Mn: 5 + 10, 100, and 200 µM) treatment of glial cells for 24 h. After exposure, the expression of antioxidant stress-related genes *Nrf2* (A) and *HO-1* (B) was evaluated by real-time PCR and normalized to β-actin. Data are expressed as Nrf2/β-actin and HO-1/β-actin mRNA ratios. Results are expressed as mean ± SD (n = 4). Comparisons of the control group, iAs and Mn groups, and coexposure to iAs and Mn groups were performed using one-way ANOVA with Tukey’s post hoc tests. The levels of statistical significance were as follows: for control, ***p < 0.001; for iAs, ^##p < 0.01, ^###p < 0.001. Furthermore, the significance levels for each concentration of Mn and the corresponding coexposures were as follows: 10 µM, ^†††p < 0.001; 100 µM, ^‡‡‡p < 0.001; 200 µM, ^$$$p < 0.001. Detailed statistical values are provided in S2 Table, corresponding to Figs 2A and 2B.

Table 1. Spearman correlation coefficients (ρ) among Nrf2 mRNA, HO-1 mRNA, MCP-1 mRNA, IL-1β mRNA, and IL-6 mRNA expression levels.

	Group	HO-1	MCP-1	IL-1β	IL-6
Nrf2	iAs	0.929***	0.714*	0.905**	0.929***
	Mn	0.635**	0.479	0.662**	0.753***
	iAs + Mn	0.809***	0.717***	0.737***	0.809***
HO-1	iAs		0.690	0.833*	0.810*
	Mn		0.603*	0.497	0.459
	iAs + Mn		0.869***	0.871***	0.964***
MCP-1	iAs			0.762*	0.690
	Mn			0.309	0.747***
	iAs + Mn			0.877***	0.917***
IL-1β	iAs				0.786*
	Mn				0.747***
	iAs + Mn				0.902***

Open in a new tab

iAs group, n = 8; Mn group, n = 16; iAs + Mn group, n = 20.

*p < 0.05; **p < 0.01; ***p < 0.001.

Inflammatory cytokines induced by iAs and Mn.

The levels of inflammatory cytokines (MCP-1, IL-1β, and IL-6 mRNA) were evaluated by real-time PCR analysis after 24 h (Fig 3). MCP-1 mRNA expression was approximately two-fold higher in the iAs group than in the control group (p < 0.001; see S3 Table, panel A and Fig 3A). However, there was no increase in the Mn group (Fig 3A). Coexposure to iAs and Mn resulted in a significant increase in MCP-1 mRNA expression compared with that in the control group and iAs or Mn alone group (p < 0.001; see S3 Table, panel A and Fig 3A). The IL-1β mRNA expression remained unchanged in the iAs group compared with that in the control group (Fig 3B). The Mn group exhibited a significant difference only at high concentrations (200 µM, p < 0.001; see S3 Table, panel B and Fig 3B). The iAs and Mn coexposure group exhibited a clear trend of elevated IL-1β mRNA expression compared with the control group (p < 0.01, 0.001; see S3 Table, panel B and Fig 3B). The IL-6 mRNA expression was approximately two-fold higher in the iAs group than in the control group (p < 0.001; see S3 Table, panel C and Fig 3C). A slight increase in IL-6 mRNA expression was detected in the Mn group compared with that in the control group; however, a significant difference was observed in the high-concentration groups (100 µM, p < 0.01; 200 µM, p < 0.05; see S3 Table, panel C and Fig 3C). In contrast, the iAs and Mn coexposure group exhibited a significant increase in IL-6 mRNA expression compared with the control group and iAs or Mn alone group (p < 0.001; see S3 Table, panel C and Fig 3C), and this increase was dependent on Mn concentration. A trend toward increased IL-6 mRNA expression suggested an additive effect at 10 µM-Mn, while clear evidence of a synergistic effect was observed at 100 and 200 µM-Mn (Fig 3C; see S3 Table, panel C). Remarkably, regarding the expression of inflammatory cytokines in the iAs and Mn coexposure group, a significant correlation was detected between MCP-1 and IL-6 mRNA expression (ρ = 0.917, p < 0.001) and IL-1β and IL-6 mRNA expression (ρ = 0.902, p < 0.001) (Table 1). This suggests the importance of MCP-1 mRNA or IL-6 mRNA in investigating the levels of inflammatory cytokines.

Relationship between oxidative stress and inflammatory cytokines.

We analyzed the relationship between oxidative stress and inflammatory cytokines in the iAs or Mn alone groups and the iAs and Mn coexposure group. The Nrf2 mRNA and HO-1 mRNA expression levels were used as indicators of oxidative stress. As depicted in Fig 2 and S2 Table, iAs exposure induced oxidative stress, whereas Mn exposure did not. The expression of inflammatory cytokines was confirmed in the iAs alone group based on MCP-1 and IL-6 mRNA expression and in the Mn alone group based on IL-1β and IL-6 mRNA expression (Fig 3; see S3 Table). The relationship between oxidative stress and inflammatory cytokines was evaluated using the correlation presented in Table 1. A negative correlation between two indices suggests an inhibitory or antagonistic effect, whereas a positive correlation suggests a synergistic effect. We detected a positive correlation in all the experimental groups. The iAs and Mn coexposure group tended to exhibit a significant correlation compared with the iAs or Mn alone group. In particular, in the iAs and Mn coexposure group, there was a significant positive correlation between HO-1 and MCP-1 mRNA (ρ = 0.869, p < 0.001), HO-1 and IL-1β mRNA (ρ = 0.871, p < 0.001), and HO-1 and IL-6 mRNA (ρ = 0.964, p < 0.001) expression (Table 1).

Effect of single or combined exposure to iAs and Mn on BBB TJ injury

Expression of Nrf2 and HO-1 in BBB.

The rat in vitro BBB model was exposed to iAs (10 µM) or Mn (10, 100, and 200 µM) alone or iAs and Mn combination (iAs + Mn: 10 + 10, 100, and 200 µM), after which the protein expression levels of Nrf2 and HO-1 in the vascular endothelial cell and pericyte group at 24 h were evaluated by WB analysis (Fig 4A). Nrf2 protein expression was approximately two-fold higher in the iAs group than in the control group (p < 0.001; see S4 Table, panel A and Fig 4B). However, there was no increase in the Mn group (Fig 4B). The expression of Nrf2 protein in the iAs and Mn coexposure group showed a significant increased trend compared to the control group (p < 0.01, 0.001; see S4 Table, panel A and Fig 4B). However, the values in the coexposure group were at the same level as those in the iAs group, and no additive or synergistic effects were observed (Fig 4B). Furthermore, the expression of HO-1 protein in the iAs, Mn, and iAs+Mn groups showed a trend similar to that of Nrf2 protein expression (p < 0.05, 0.01, 0.001; see S4 Table, panel B and Fig 4C).

Fig 4 — **(A)**: Representative western blot images depicting Nrf2 and HO-1 protein expression after exposure to iAs alone (10 µM), Mn alone (10, 100, and 200 µM), and coexposure (iAs + Mn: 10 + 10, 100, and 200 µM) for 24 **h. (B and C)**: The expression levels of Nrf2 (B) and HO-1 (C) proteins were quantified by densitometry, standardized to β-actin, and compared with the control value of 1. Results are expressed as mean ± SD (n = 4). Comparison of the control group, iAs and Mn alone groups, and iAs and Mn coexposure group was performed using one-way ANOVA with Tukey’s post hoc tests. The levels of statistical significance were as follows: for control, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001; for iAs, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001. The significance levels for each concentration of Mn and the corresponding coexposures were as follows: 10 µM, ^†p < 0.05, ^††p < 0.01; 100 µM, ^‡‡‡p < 0.001; 200 µM, ^$$p < 0.01, ^$$$p < 0.001. NS indicates no significant differences among the coexposure groups. Detailed statistical values are provided in S4 Table, corresponding to Figs 4B and 4C.

Next, we investigated the protein expression of Nrf2 and HO-1 in astrocytes under the same experimental conditions (Fig 5A). Following iAs exposure, Nrf2 protein expression increased to approximately 3-fold that of the control group (p < 0.05; see S5 Table, panel A and Fig 5B). In contrast, no increase in Nrf2 protein expression was observed in the Mn group compared to the control group (Fig 5B; see S5 Table, panel A). Coexposure to iAs and Mn resulted in Nrf2 protein expression compared to the control group (p < 0.001; see S5 Table, panel A) and the iAs group (p < 0.01, 0.001; see S5 Table, panel A). These findings suggest that there may be an additive effect of 10 and 100 µM-Mn on the increase in Nrf2 protein expression in the coexposure of iAs and Mn. Furthermore, a synergistic effect was suggested at 200 µM-Mn. On the other hand, following iAs exposure, the expression of HO-1 protein showed only a slight increase compared to the control group (p < 0.05; see S5 Table, panel B, and Fig 5C). In the Mn group, no significant increase in HO-1 protein expression was observed compared to the control group (see Fig 5C and S5 Table, panel B). Coexposure to iAs and Mn (10, 100, 200 µM-Mn) significantly increased HO-1 protein expression compared with the control and iAs groups (p < 0.001; see S5 Table, panel B). Furthermore, the increased expression of HO-1 protein with coexposure to iAs and Mn suggested a synergistic effect.

The expression of Nrf2 and HO-1 proteins in the BBB was more significant in astrocytes than in vascular endothelial cells and pericytes. In astrocytes exposed to a coexposure of iAs and Mn, the expression of Nrf2 and HO-1 increased, and it was clarified that iAs caused this effect. Additionally, the presence of synergistic effects was suggested (Figs 5B and 5C).

Evaluation of BBB TJ injury by TEER.

TJ injury in BBB can be evaluated by analyzing the changes in TEER values. We evaluated the TJ injury in BBB after exposure to iAs alone (10 µM), Mn alone (10, 100, and 200 µM), and coexposure (iAs + Mn: 10 + 10, 100, and 200 µM) for 24 h using %TEER values (Fig 6). The %TEER was expressed as 100% of the mean control TEER value. The %TEER values in the iAs group decreased by approximately 75% compared with that in the control group (p < 0.001; see S6 Table). In contrast, the Mn group showed no statistically significant decrease in %TEER values. Coexposure to iAs and Mn resulted in a reduction similar in %TEER to that observed after exposure to iAs alone (p < 0.001; see S6 Table). The %TEER values indicate that TJ injury can be attributed to iAs exposure, whereas Mn exposure does not appear to cause such injury. Therefore, it can be concluded that the TJ injury observed in the coexposure group is primarily due to the effect of iAs.

TEER was measured at 24 h after exposure to iAs alone (10 µM), Mn alone (10, 100, and 200 µM), and coexposure (iAs + Mn: 10 + 10, 100, and 200 µM). The %TEER values were calculated by normalizing each TEER measurement to the mean TEER value of the control group, which was defined as 100%. Results are expressed as mean ± SD (n = 4). Comparison of the control group, iAs and Mn alone groups, and iAs and Mn coexposure groups was performed using one-way ANOVA with Tukey’s post hoc tests. The levels of statistical significance were as follows: for control, ***p < 0.001; for iAs, ^###p < 0.001. The significance levels for each concentration of Mn and the corresponding coexposures were as follows: 10 µM, ^†††p < 0.001; 100 µM, ^‡‡‡p < 0.001; 200 µM, ^$$$p < 0.001. NS indicates no significant differences among the coexposure groups. Detailed statistical values are provided in S6 Table, corresponding to Fig 6.

Evaluation of the TJ proteins claudin-5 and ZO-1.

TJ injury in BBB in the vascular endothelial cell and pericyte groups after exposure to iAs alone (10 µM), Mn alone (10, 100, and 200 µM), and coexposure (iAs + Mn: 10 + 10, 100, and 200 µM) was evaluated by measuring the protein expression patterns of claudin-5 by WB analysis (Fig 7A). The iAs group showed a statistically significant decrease of approximately 60% in claudin-5 expression compared with that in the control group (p < 0.01; see S7 Table, panel A and Fig 7B). In contrast, the Mn groups showed no reduction in claudin-5 expression (Fig 7B; see S7 Table, panel A). The iAs and Mn coexposure group showed a statistically significant decrease in claudin-5 expression compared with that in the control group (p < 0.01; see S7 Table, panel A), a trend that was similar to that in the iAs group (Fig 7B; see S7 Table, panel A). TJ injury in BBB was evaluated by immunofluorescence staining for the expression of claudin-5 proteins (Fig 8). Claudin-5 expression in the control group (Fig 8Aa) was observed as a zonal pattern around the plasma membrane (yellow arrows). In the iAs group, a suppressed (i.e., sparse and weak) expression of claudin-5 was detected in the regions indicated by the red arrowhead (Fig 8Ab), which significantly reduced compared with that in the control group (p < 0.001; see S8 Table and Fig 8B). In contrast, in the Mn groups (Fig 8Ac-e), claudin-5 expression was observed as a banded pattern around the cell membrane, indicated by white arrows, which was not statistically significantly different compared with that in the control group (Fig 8B). In the iAs and Mn coexposure group (Fig 8Af-h), the expression of claudin-5 was observed in the regions indicated by the red arrowheads, which was suppressed compared with that in the control and Mn groups, similar to that in the iAs group (p < 0.001; see S8 Table and Fig 8B).

**(A)**: Representative immunofluorescence staining of claudin-5 (green, Alexa Fluor 488) after 24h exposure: a, control; b, iAs (10 µM); c-e, Mn groups (10, 100, and 200 µM); f-h, iAs and Mn coexposure groups (iAs + Mn: 10 + 10, 100, and 200 µM). The yellow arrow indicates typical TJ protein localization in the control group. Red arrowheads indicate altered TJ protein localization due to iAs-induced damage. White arrows indicate typical TJ protein localization after Mn exposure. Scale bars: 20 µm. **(B)**: Quantitative analysis of the immunofluorescence staining of claudin-5. Relative fluorescence intensity of claudin-5 is expressed as mean ± SD (n = 6). Comparison of the control group, iAs and Mn alone groups, and iAs and Mn coexposure groups was performed using one-way ANOVA with Tukey’s post hoc tests. The levels of statistical significance were as follows: for control, ***p < 0.001; for iAs, ^###p < 0.001. The significance levels for each concentration of Mn and the corresponding coexposures were as follows: 10 µM, ^†††p < 0.001; 100 µM, ^‡‡‡p < 0.001; 200 µM, ^$$$p < 0.001. NS indicates no significant differences among the coexposure groups. Detailed statistical values are provided in S8 Table, corresponding to Fig 8B.

We also investigated the expression of ZO-1 under the same experimental conditions (Fig 7A) and found a statistically significant decrease of approximately 60% in the iAs group compared with that in the control group (p < 0.001; see S7 Table, panel B and Fig 7C). However, the Mn groups (10 and 100 µM) showed no decrease in ZO-1 expression, but 200 µM-Mn was slightly decreased in the control group (p < 0.05; see S7 Table, panel B and Fig 7C). The iAs and Mn coexposure group showed a statistically significant decrease in ZO-1 expression compared with that in the control group (p < 0.001; see S7 Table, panel B), a trend that was similar to that in the iAs group (Fig 7C; see S7 Table, panel B). The results of fluorescence immunostaining for the expression of ZO-1 proteins are shown in Fig 9. The ZO-1 expression in the control group (Fig 9Aa) was observed as a zonal pattern around the plasma membrane (yellow arrows). In the iAs group, a suppressed (i.e., sparse and weak) expression of ZO-1 was observed in the regions indicated by the red arrowhead (Fig 9Ab), with no statistically significant difference in expression compared with that in the control group (Fig 9B; see S9 Table). In contrast, in the Mn groups (Fig 9Ac-e), the ZO-1 expression was observed as a banded pattern around the cell membrane, indicated by white arrows, and the expression was not statistically significantly different from that in the control group (Fig 9B; see S9 Table). In the iAs and Mn coexposure group (Fig 9Af-h), ZO-1 expression was observed in the regions indicated by red arrowheads and was suppressed compared with that in the control and Mn groups, similar to that in the iAs group (p < 0.001; see S9 Table, Fig 9B).

**(A)**: Representative immunofluorescence staining of ZO-1 (green, Alexa Fluor 488) after 24h exposure: a, control; b, iAs (10 µM); c-e, Mn groups (10, 100, and 200 µM); f-h, iAs and Mn coexposure groups (iAs + Mn: 10 + 10, 100, and 200 µM). The yellow arrow indicates typical TJ protein localization in the control group. Red arrowheads indicate altered TJ protein localization due to iAs-induced damage. White arrows indicate typical TJ protein localization after Mn exposure. Scale bars: 20 µm. **(B)**: Quantitative analysis of the immunofluorescence staining of ZO-1. Relative fluorescence intensity of ZO-1 is expressed as mean ± SD (n = 6). Comparison of the control group, iAs and Mn alone groups, and iAs and Mn coexposure groups was performed using one-way ANOVA with Tukey’s post hoc tests. The levels of statistical significance were as follows: for control, ***p < 0.001; for iAs, ^###p < 0.001. The significance levels for each concentration of Mn and the corresponding coexposures were as follows: 10 µM, ^†††p < 0.001; 100 µM, ^‡‡‡p < 0.001; 200 µM, ^$$$p < 0.001. NS indicates no significant differences among the coexposure groups. Detailed statistical values are provided in S9 Table, corresponding to Fig 9B.

The results of claudin-5 and ZO-1 expression clearly demonstrated that iAs exposure, and not Mn exposure, caused TJ injury. We speculated that the TJ injury observed in the iAs and Mn coexposure groups was caused by iAs. In other words, Mn did not affect TJ injury due to iAs exposure. The results of %TEER values (Fig 6; see S6 Table) also support this conclusion.

Discussion

Researchers widely recognize the importance of clarifying the biological effects of combined exposure to harmful substances. Epidemiological data suggest that combined exposure to iAs and Mn worsens cognitive dysfunction [36–38]. Research in this field is sparse, and regarding neurotoxicity, there have been studies on the effects of iAs and Mn [52], iAs and Pb [53], iAs, Mn, and Pb [54], and the effects of iAs, Mn, and Pb on motor activity [55], all of which are animal experiments using mice and rats. The joint effects of iAs and other elements suggest antagonistic, additive, or synergistic interactions; however, no definitive conclusion has been reached. Animal experiments are essential; however, there are difficulties involved in analyzing the results. Moreover, when examining the joint effects between toxic elements, the results become more complex when the three elements are targeted. Hence, we recognize the importance of obtaining highly reliable results for the two elements.

Cytotoxicity, oxidative stress, and inflammatory cytokines in glial cells coexposed to iAs and Mn

Cytotoxicity of coexposure to iAs and Mn.

We performed cytotoxicity assays using glial cells under exposure to iAs or Mn alone and coexposure to iAs and Mn. As depicted in Fig 1, the cytotoxicity of 5µM-iAs and 10µM-Mn was comparable, with no statistically significant difference. Notably, coexposure to iAs and Mn resulted in greater cytotoxicity than exposure to either iAs or Mn alone, suggesting an additive effect. Furthermore, the magnitude of this additive effect appeared to vary slightly depending on the Mn concentration. This study demonstrated an increase in toxicity of the combined exposure to iAs and Mn. These findings suggest that conventional methods and analysis are insufficient in epidemiological studies of chronic iAs poisoning and chronic Mn poisoning and that new methods need to be developed.

Oxidative stress in coexposure to iAs and Mn.

Studies have demonstrated that iAs exerts a potent oxidative stress effect, and the antioxidant stress factors Nrf2 and HO-1 are concomitantly expressed [56,57]. In contrast, studies assessing manganese-induced oxidative stress utilizing Nrf2 or HO-1 expression about manganese exposure and oxidative stress are generally less numerous than arsenic studies. Although oxidative stress may play a critical role, it may not be the initiating event [28]. Fig 2 shows the results of Nrf2 and HO-1 mRNA expression 24 h after exposure to iAs, Mn, or their combination in glial cells. The results of Nrf2 mRNA and HO-1 mRNA expression after iAs exposure revealed a substantial increase compared with that in the control group (p < 0.001). Conversely, Mn exposure did not alter the levels of Nrf2 mRNA and HO-1 mRNA expression compared with those in the control group. These results indicate that Mn-induced oxidative stress is significantly less pronounced than iAs-induced oxidative stress. Nevertheless, when Mn, which induces only mild oxidative stress, is coexposed with iAs, a notable enhancement in oxidative response is observed. Coexposure to iAs and Mn resulted in increased expression of HO-1 mRNA compared with iAs exposure alone (Fig 2B). This finding suggests that Mn may enhance iAs-induced oxidative stress through additive or synergistic effects. Although the underlying mechanism remains unclear, further investigation is warranted.

It is widely recognized that the Nrf2/HO-1 pathway plays a vital role in combating oxidative stress. Consequently, as shown in Table 1, we attempted to evaluate the intensity of oxidative stress based on the correlation coefficient between the expression levels of Nrf2 mRNA and HO-1 mRNA. The intensity of oxidative stress induced by Mn (ρ = 0.635, p < 0.01) was lower than that induced by iAs (ρ = 0.929, p < 0.001). However, the iAs and Mn coexposure group exhibited a correlation coefficient of 0.809 (p < 0.001). These results suggest that Mn itself has low oxidative stress effects, but that it enhances the oxidative stress caused by iAs.

Inflammatory cytokines in coexposure to iAs and Mn.

The amount of information on inflammatory cytokines induced by exposure to iAs or Mn and cognitive dysfunction is limited. In the case of iAs exposure, increased expression of IL-1β [58] and IL-6 [59] has been reported. With Mn exposure, increased expression of IL-1β has been observed [60]. In general, MCP-1 induces IL-1β and IL-6 [61]. As illustrated in Fig 3, exposure to iAs increased the MCP-1 mRNA expression, whereas this result was not observed in the Mn group. Subsequently, we confirmed the expression of IL-1β and IL-6 mRNA. Although there was no significant IL-1β mRNA expression in the iAs or Mn groups, there was an expression at the high concentration of 200 µM Mn. In contrast, there was a statistically significant expression of IL-6 mRNA in the iAs or Mn groups, similar to previous reports [59]. In the group exposed to the iAs and Mn combination, the expression of IL-6 mRNA and IL-1β mRNA (only at 5 µM iAs and 200 µM Mn) increased in conjunction with MCP-1 mRNA expression, indicating a characteristic dependent on Mn concentration (Fig 3). MCP-1 and IL-6 mRNA expression demonstrated a significant correlation (Table 1). The mechanism underlying these results remains unclear. In contrast, it has been confirmed that IL-6 levels increase in the brain tissue of mice exposed to a mixture of iAs, Mn, and Pb [55] compared with those observed with exposure to iAs or Mn alone. In this study, coexposure to iAs and Mn tended to increase IL-6 mRNA expression compared with exposure to iAs or Mn alone. Notably, a synergistic effect was clearly observed in the 100 and 200 µM Mn groups (Fig 3C). These results are consistent with findings from in vivo experiments in mice [55]. We believe that the role of inflammatory cytokines in brain tissue following coexposure to iAs and various other elements warrants further investigation.

Oxidative stress and inflammatory cytokines in interactions.

In both individual exposures to iAs or Mn, and particularly in their coexposure, activation of the antioxidant stress pathway Nrf2/HO-1 was significantly increased (Fig 2B; see S2 Table, panel B). Furthermore, a synergistic effect was suggested by the upregulation of IL-6 mRNA expression (Fig 3C; see S3 Table, panel C). While the precise interpretation of this phenomenon remains unclear, several notable findings were obtained. HO-1, which is induced by Nrf2, is known to play a suppressive role against oxidative stress and inflammatory cytokines [62]. In the coexposure group, significant positive correlations were observed between HO-1 expression and the expression of inflammatory cytokines (MCP-1, IL-1β, and IL-6), with correlation coefficients of ρ = 0.869, 0.871, and 0.964, respectively (all p < 0.001) (Table 1). These findings suggest that oxidative stress and inflammatory cytokines may interact synergistically in glial cells, potentially enhancing their toxic effects. Such coordinated activation in glial cells could exacerbate neuronal damage.

Our results provide experimental evidence supporting a critical issue raised by epidemiological studies in Bangladesh—namely, that coexposure to iAs and Mn may worsen cognitive dysfunction more than iAs exposure alone [36–38]. Although our findings support this possibility, further studies are needed to draw definitive conclusions.

TJ injury in BBB with coexposure to iAs and Mn.

iAs [41] and Mn [42] are ingested through the daily diet, and some of them accumulate in brain tissue. It has been confirmed that iAs can cross the BBB and be transferred to the brain tissue in experimental animals [44,45], although there is limited research. Studies using a rat in vitro BBB model have provided information on the permeability and metabolism of iAs across the BBB [43]. The known transfer routes of Mn to the brain tissue are the BBB [46], the BCB [47], and the olfactory nerve [48]. Based on research results regarding the transfer of iAs and Mn to brain tissue, it is possible to speculate that iAs and Mn share a common mechanism for passing through the BBB. We believe that information on TJ injury in the BBB is essential for research into cognitive dysfunction caused by iAs or Mn exposure. Therefore, our study focused on the joint effects between iAs and Mn in BBB TJ injury, a mechanism that has not yet been investigated.

In the rat in vitro BBB model exposed to iAs, the expression of the antioxidant stress factors Nrf2 and HO-1 was measured in the vascular endothelial cell and pericyte groups and astrocytes. Results demonstrated that the levels of Nrf2 and HO-1 expression in astrocytes were higher than those in the vascular endothelial cell and pericyte groups. This observation is consistent with findings from previous studies, further validating the reliability of the experimental approach [43]. To the best of our knowledge, there have been no reports on the behavior of oxidative stress caused by Mn in the vascular endothelial cell + pericyte group and astrocytes using Nrf2 and HO-1 as indicators. In this study, we measured Nrf2 and HO-1 expression levels in response to exposure to low, medium, and high concentrations of Mn (10, 100, and 200 µM). In both the vascular endothelial cell and pericyte group and the astrocyte group, there was no change in the concentrations of Nrf2 and HO-1 compared with those in the control group (Figs 4 and 5; see S4 and S5 Tables). This result suggests that Mn does not strongly induce oxidative stress within BBB. The mechanism underlying this phenomenon remains to be elucidated. Mn accumulates in brain tissue and has been reported to accumulate specifically in astrocytes [63]. Further research is necessary on the oxidative stress caused by the synergistic effect of iAs and Mn in astrocytes.

The TJ injury induced by iAs exposure was confirmed by the results of TEER measurements (Fig 6; see S6 Table), claudin-5 expression (Figs 7 and 8; see S7 and S8 Tables), and ZO-1 expression (Figs 7 and 9; see S7 and S9 Tables). These results were consistent with those of previous research [43]. Although it is known that Mn accumulates in the brain tissue [30], there is no information to confirm TJ injury in the BBB. In this study, Mn did not induce TJ injury in the BBB and did not exacerbate the TJ injury induced by iAs (Figs 7-9; see S7–S9 Tables). In other words, Mn did not exert any antagonistic or synergistic effects on TJ injury in the BBB caused by iAs. Nonetheless, it is crucial to acknowledge that Mn is an essential element and is consistently present in the brain tissue [30,42]. Hence, it is conceivable that situations arise where elements or substances exhibiting neurotoxicity come into contact with Mn. Therefore, when exploring the neurotoxicity of Mn, it is imperative to investigate the joint effects of Mn and other neurotoxic substances, particularly compared with iAs.

Conclusion

To date, limited information has been available regarding the effects of individual or combined exposure to iAs and Mn on BBB TJ injury. Our study demonstrated that iAs induces TJ disruption, whereas Mn alone did not exert such effects, nor did it significantly alter iAs-induced TJ injury. Furthermore, through cytotoxicity assays using glial cells and evaluation of antioxidant stress-related factors (Nrf2, HO-1) and pro-inflammatory cytokines, we obtained compelling evidence suggesting the presence of additive or synergistic effects under coexposure to iAs and Mn. Notably, coexposure resulted in greater toxicity than individual exposures. These findings indicate that coexposure to iAs and Mn may pose a higher risk of cognitive dysfunction than exposure to either iAs or Mn alone. Therefore, it is imperative to strengthen environmental risk assessment related to groundwater or dietary exposure, and to advance experimental research to further elucidate the mechanisms underlying these joint toxic effects.

Supporting information

S1 Table. Cytotoxicity of iAs or Mn alone and coexposure to iAs and Mn in glial cells.

*One-way ANOVA with Tukey’s post hoc tests. This table supports the statistical comparisons shown in Fig 1.

(XLSX)

pone.0330287.s001.xlsx^{(13.8KB, xlsx)}

S2 Table. Oxidative stress of iAs or Mn alone and coexposure to iAs and Mn in glial cells.

(A) Nrf2 mRNA expression. (B) HO-1 mRNA expression. *One-way ANOVA with Tukey’s post hoc tests.This table supports the statistical comparisons shown in Fig 2A and 2B.

(XLSX)

pone.0330287.s002.xlsx^{(14.6KB, xlsx)}

S3 Table. Inflammatory cytokines in iAs and Mn alone groups and iAs and Mn coexposure groups in glial cells.

(A) MCP-1 mRNA expression. (B) IL-1β mRNA expression. (C) IL-6 mRNA expression. *One-way ANOVA with Tukey’s post hoc tests. This table supports the statistical comparisons shown in Fig 3A–C.

(XLSX)

pone.0330287.s003.xlsx^{(15.7KB, xlsx)}

S4 Table. Expression of Nrf2 and HO-1 proteins in the vascular endothelial cell and pericyte groups after exposure to iAs or Mn alone and coexposure to iAs and Mn.

(A) Nrf2 protein expression. (B) HO-1 protein expression. *One-way ANOVA with Tukey’s post hoc tests. This table supports the statistical comparisons shown in Fig 4B and 4C.

(XLSX)

pone.0330287.s004.xlsx^{(14.7KB, xlsx)}

S5 Table. Expression of Nrf2 and HO-1 proteins in astrocytes after exposure to iAs or Mn alone and coexposure to iAs and Mn.

(A) Nrf2 protein expression. (B) HO-1 protein expression. *One-way ANOVA with Tukey’s post hoc tests. This table supports the statistical comparisons shown in Fig 5B and 5C.

(XLSX)

pone.0330287.s005.xlsx^{(14.7KB, xlsx)}

S6 Table. Changes in TEER after exposure to iAs or Mn alone, and coexposure to iAs and Mn.

*One-way ANOVA with Tukey’s post hoc tests. This table supports the statistical comparisons shown in Fig 6.

(XLSX)

pone.0330287.s006.xlsx^{(13.7KB, xlsx)}

S7 Table. Expression of the tight junction proteins claudin-5 and ZO-1 after exposure to iAs or Mn alone and coexposure to iAs and Mn.

(A) Claudin-5 protein expression. (B) ZO-1 protein expression. *One-way ANOVA with Tukey’s post hoc tests. This table supports the statistical comparisons shown in Fig 7B and 7C.

(XLSX)

pone.0330287.s007.xlsx^{(14.4KB, xlsx)}

S8 Table. Quantitative analysis of the immunofluorescence staining of claudin-5 after exposure to iAs or Mn alone and coexposure to iAs and Mn.

*One-way ANOVA with Tukey’s post hoc tests. This table supports the statistical comparisons shown in Fig 8B.

(XLSX)

pone.0330287.s008.xlsx^{(13.4KB, xlsx)}

S9 Table. Quantitative analysis of the immunofluorescence staining of ZO-1 after exposure to iAs or Mn alone and coexposure to iAs and Mn.

*One-way ANOVA with Tukey’s post hoc tests. This table supports the statistical comparisons shown in Fig 9B.

(XLSX)

pone.0330287.s009.xlsx^{(13.3KB, xlsx)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This study was financially supported by the Japan Society for the Promotion of Science (JSPS) (https://www.jsps.go.jp) in the form of KAKENHI grants received by AT (JP21H03185 and JP24K22214) and TH (JP21K10422). No additional external funding was received for this study.

References

1.Wasserman GA, Liu X, Parvez F, Ahsan H, Factor-Litvak P, van Geen A, et al. Water arsenic exposure and children’s intellectual function in Araihazar, Bangladesh. Environ Health Perspect. 2004;112(13):1329–33. doi: 10.1289/ehp.6964 [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Wasserman GA, Liu X, Parvez F, Ahsan H, Factor-Litvak P, Kline J, et al. Water arsenic exposure and intellectual function in 6-year-old children in Araihazar, Bangladesh. Environ Health Perspect. 2007;115(2):285–9. doi: 10.1289/ehp.9501 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Hamadani JD, Tofail F, Nermell B, Gardner R, Shiraji S, Bottai M, et al. Critical windows of exposure for arsenic-associated impairment of cognitive function in pre-school girls and boys: a population-based cohort study. Int J Epidemiol. 2011;40(6):1593–604. doi: 10.1093/ije/dyr176 [DOI] [PubMed] [Google Scholar]
4.Wasserman GA, Liu X, Parvez F, Chen Y, Factor-Litvak P, LoIacono NJ, et al. A cross-sectional study of water arsenic exposure and intellectual function in adolescence in Araihazar, Bangladesh. Environment International. 2018;118:304–13. doi: 10.1016/j.envint.2018.05.037 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Manju R, Hegde AM, Parlees P, Keshan A. Environmental Arsenic Contamination and Its Effect on Intelligence Quotient of School Children in a Historic Gold Mining Area Hutti, North Karnataka, India: A Pilot Study. J Neurosci Rural Pract. 2017;8(3):364–7. doi: 10.4103/jnrp.jnrp_501_16 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Vaidya N, Holla B, Heron J, Sharma E, Zhang Y, Fernandes G, et al. Neurocognitive Analysis of Low-level Arsenic Exposure and Executive Function Mediated by Brain Anomalies Among Children, Adolescents, and Young Adults in India. JAMA Netw Open. 2023;6(5):e2312810. doi: 10.1001/jamanetworkopen.2023.12810 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Wang S-X, Wang Z-H, Cheng X-T, Li J, Sang Z-P, Zhang X-D, et al. Arsenic and fluoride exposure in drinking water: children’s IQ and growth in Shanyin county, Shanxi province, China. Environ Health Perspect. 2007;115(4):643–7. doi: 10.1289/ehp.9270 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Wang Y, Wang Y, Yan C. Gender differences in trace element exposures with cognitive abilities of school-aged children: a cohort study in Wujiang city, China. Environ Sci Pollut Res Int. 2022;29(43):64807–21. doi: 10.1007/s11356-022-20353-4 [DOI] [PubMed] [Google Scholar]
9.Rosado JL, Ronquillo D, Kordas K, Rojas O, Alatorre J, Lopez P, et al. Arsenic Exposure and Cognitive Performance in Mexican Schoolchildren. Environ Health Perspect. 2007;115(9):1371–5. doi: 10.1289/ehp.9961 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Limón-Pacheco JH, Jiménez-Córdova MI, Cárdenas-González M, Sánchez Retana IM, Gonsebatt ME, Del Razo LM. Potential Co-exposure to Arsenic and Fluoride and Biomonitoring Equivalents for Mexican Children. Ann Glob Health. 2018;84(2):257–73. doi: 10.29024/aogh.913 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Wasserman GA, Liu X, Parvez F, Ahsan H, Levy D, Factor-Litvak P, et al. Water manganese exposure and children’s intellectual function in Araihazar, Bangladesh. Environ Health Perspect. 2006;114(1):124–9. doi: 10.1289/ehp.8030 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Khan K, Factor-Litvak P, Wasserman GA, Liu X, Ahmed E, Parvez F, et al. Manganese Exposure from Drinking Water and Children’s Classroom Behavior in Bangladesh. Environ Health Perspect. 2011;119(10):1501–6. doi: 10.1289/ehp.1003397 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Khan K, Wasserman GA, Liu X, Ahmed E, Parvez F, Slavkovich V, et al. Manganese exposure from drinking water and children’s academic achievement. Neurotoxicology. 2012;33(1):91–7. doi: 10.1016/j.neuro.2011.12.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Rahman SM, Kippler M, Tofail F, Bölte S, Hamadani JD, Vahter M. Manganese in Drinking Water and Cognitive Abilities and Behavior at 10 Years of Age: A Prospective Cohort Study. Environ Health Perspect. 2017;125(5):057003. doi: 10.1289/EHP631 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Yu X-D, Zhang J, Yan C-H, Shen X-M. Prenatal exposure to manganese at environment relevant level and neonatal neurobehavioral development. Environ Res. 2014;133:232–8. doi: 10.1016/j.envres.2014.04.012 [DOI] [PubMed] [Google Scholar]
16.Yu X, Chen L, Wang C, Yang X, Gao Y, Tian Y. The role of cord blood BDNF in infant cognitive impairment induced by low-level prenatal manganese exposure: LW birth cohort, China. Chemosphere. 2016;163:446–51. doi: 10.1016/j.chemosphere.2016.07.095 [DOI] [PubMed] [Google Scholar]
17.Bouchard MF, Sauvé S, Barbeau B, Legrand M, Brodeur M-È, Bouffard T, et al. Intellectual impairment in school-age children exposed to manganese from drinking water. Environ Health Perspect. 2011;119(1):138–43. doi: 10.1289/ehp.1002321 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Oulhote Y, Mergler D, Barbeau B, Bellinger DC, Bouffard T, Brodeur M-È, et al. Neurobehavioral function in school-age children exposed to manganese in drinking water. Environ Health Perspect. 2014;122(12):1343–50. doi: 10.1289/ehp.1307918 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Lane JM, Curtin P, Chelonis JJ, Pantic I, Martinez-Medina S, Téllez-Rojo MM, et al. Prenatal manganese biomarkers and operant test battery performance in Mexican children: Effect modification by child sex. Environ Res. 2023;236(Pt 2):116880. doi: 10.1016/j.envres.2023.116880 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Lane JM, Liu SH, Pantic I, Martinez-Medina S, Téllez-Rojo MM, Amarasiriwardena C, et al. Sex-specific association between prenatal manganese exposure and working memory in school-aged children in Mexico city: An exploratory multi-media approach. Environmental Pollution. 2023;333:121965. doi: 10.1016/j.envpol.2023.121965 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Wasserman GA, Liu X, Loiacono NJ, Kline J, Factor-Litvak P, van Geen A, et al. A cross-sectional study of well water arsenic and child IQ in Maine schoolchildren. Environ Health. 2014;13(1):23. doi: 10.1186/1476-069X-13-23 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Haynes EN, Sucharew H, Kuhnell P, Alden J, Barnas M, Wright RO, et al. Manganese Exposure and Neurocognitive Outcomes in Rural School-Age Children: The Communities Actively Researching Exposure Study (Ohio, USA). Environ Health Perspect. 2015;123(10):1066–71. doi: 10.1289/ehp.1408993 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Nascimento S, Baierle M, Göethel G, Barth A, Brucker N, Charão M, et al. Associations among environmental exposure to manganese, neuropsychological performance, oxidative damage and kidney biomarkers in children. Environ Res. 2016;147:32–43. doi: 10.1016/j.envres.2016.01.035 [DOI] [PubMed] [Google Scholar]
24.Carvalho CFD, Oulhote Y, Martorelli M, Carvalho COD, Menezes-Filho JA, Argollo N, et al. Environmental manganese exposure and associations with memory, executive functions, and hyperactivity in Brazilian children. Neurotoxicology. 2018;69:253–9. doi: 10.1016/j.neuro.2018.02.002 [DOI] [PubMed] [Google Scholar]
25.Vázquez Cervantes GI, González Esquivel DF, Ramírez Ortega D, Blanco Ayala T, Ramos Chávez LA, López-López HE, et al. Mechanisms Associated with Cognitive and Behavioral Impairment Induced by Arsenic Exposure. Cells. 2023;12(21):2537. doi: 10.3390/cells12212537 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Thakur M, Rachamalla M, Niyogi S, Datusalia AK, Flora SJS. Molecular Mechanism of Arsenic-Induced Neurotoxicity including Neuronal Dysfunctions. IJMS. 2021;22(18):10077. doi: 10.3390/ijms221810077 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Smith DR, Strupp BJ. Animal Models of Childhood Exposure to Lead or Manganese: Evidence for Impaired Attention, Impulse Control, and Affect Regulation and Assessment of Potential Therapies. Neurotherapeutics. 2023;20(1):3–21. doi: 10.1007/s13311-023-01345-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Dorman DC. The Role of Oxidative Stress in Manganese Neurotoxicity: A Literature Review Focused on Contributions Made by Professor Michael Aschner. Biomolecules. 2023;13(8):1176. doi: 10.3390/biom13081176 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Balachandran RC, Mukhopadhyay S, McBride D, Veevers J, Harrison FE, Aschner M, et al. Brain manganese and the balance between essential roles and neurotoxicity. J Biol Chem. 2020;295(19):6312–29. doi: 10.1074/jbc.REV119.009453 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Baj J, Flieger W, Barbachowska A, Kowalska B, Flieger M, Forma A, et al. Consequences of Disturbing Manganese Homeostasis. Int J Mol Sci. 2023;24(19):14959. doi: 10.3390/ijms241914959 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Islam F, Shohag S, Akhter S, Islam MdR, Sultana S, Mitra S, et al. Exposure of metal toxicity in Alzheimer’s disease: An extensive review. Front Pharmacol. 2022;13. doi: 10.3389/fphar.2022.903099 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Kim H, Harrison FE, Aschner M, Bowman AB. Exposing the role of metals in neurological disorders: a focus on manganese. Trends Mol Med. 2022;28(7):555–68. doi: 10.1016/j.molmed.2022.04.011 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Ahmed G, Rahaman MdS, Perez E, Khan KM. Associations of Environmental Exposure to Arsenic, Manganese, Lead, and Cadmium with Alzheimer’s Disease: A Review of Recent Evidence from Mechanistic Studies. JoX. 2025;15(2):47. doi: 10.3390/jox15020047 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Selkoe DJ. Alzheimer’s disease is a synaptic failure. Science. 2002;298(5594):789–91. doi: 10.1126/science.1074069 [DOI] [PubMed] [Google Scholar]
35.Jack CR Jr, Bennett DA, Blennow K, Carrillo MC, Dunn B, Haeberlein SB, et al. NIA-AA Research Framework: Toward a biological definition of Alzheimer’s disease. Alzheimers Dement. 2018;14(4):535–62. doi: 10.1016/j.jalz.2018.02.018 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Wasserman GA, Liu X, Parvez F, Factor-Litvak P, Ahsan H, Levy D, et al. Arsenic and manganese exposure and children’s intellectual function. Neurotoxicology. 2011;32(4):450–7. doi: 10.1016/j.neuro.2011.03.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Wasserman GA, Liu X, Parvez F, Factor-Litvak P, Kline J, Siddique AB, et al. Child Intelligence and Reductions in Water Arsenic and Manganese: A Two-Year Follow-up Study in Bangladesh. Environ Health Perspect. 2016;124(7):1114–20. doi: 10.1289/ehp.1509974 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Saxena R, Gamble M, Wasserman GA, Liu X, Parvez F, Navas-Acien A, et al. Mixed metals exposure and cognitive function in Bangladeshi adolescents. Ecotoxicol Environ Saf. 2022;232:113229. doi: 10.1016/j.ecoenv.2022.113229 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Schaefer MV, Plaganas M, Abernathy MJ, Aiken ML, Garniwan A, Lee I, et al. Manganese, Arsenic, and Carbonate Interactions in Model Oxic Groundwater Systems. Environ Sci Technol. 2020;54(17):10621–9. doi: 10.1021/acs.est.0c02084 [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Erickson ML, Elliott SM, Brown CJ, Stackelberg PE, Ransom KM, Reddy JE, et al. Machine-Learning Predictions of High Arsenic and High Manganese at Drinking Water Depths of the Glacial Aquifer System, Northern Continental United States. Environ Sci Technol. 2021;55(9):5791–805. doi: 10.1021/acs.est.0c06740 [DOI] [PubMed] [Google Scholar]
41.EFSA Panel on Contaminants in the Food Chain (CONTAM), Schrenk D, Bignami M, Bodin L, Chipman JK, Del Mazo J, et al. Update of the risk assessment of inorganic arsenic in food. EFSA J. 2024;22(1):e8488. doi: 10.2903/j.efsa.2024.8488 [DOI] [PMC free article] [PubMed] [Google Scholar]
42.EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), Turck D, Bohn T, Castenmiller J, de Henauw S, Hirsch‐Ernst KI, Knutsen HK, et al. Scientific opinion on the tolerable upper intake level for manganese. EFS2. 2023;21(12):e8413. doi: 10.2903/j.efsa.2023.8413 [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Yamauchi H, Hitomi T, Takata A. Evaluation of arsenic metabolism and tight junction injury after exposure to arsenite and monomethylarsonous acid using a rat in vitro blood-Brain barrier model. PLoS One. 2023;18(11):e0295154. doi: 10.1371/journal.pone.0295154 [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Sánchez-Peña LC, Petrosyan P, Morales M, González NB, Gutiérrez-Ospina G, Del Razo LM, et al. Arsenic species, AS3MT amount, and AS3MT gene expression in different brain regions of mouse exposed to arsenite. Environ Res. 2010;110(5):428–34. doi: 10.1016/j.envres.2010.01.007 [DOI] [PubMed] [Google Scholar]
45.Ramos-ChÃ¡vez LA, RendÃ³n-LÃ³pez CRR, Zepeda A, Silva-Adaya D, Del Razo LM, Gonsebatt ME. Neurological effects of inorganic arsenic exposure: altered cysteine/glutamate transport, NMDA expression and spatial memory impairment. Front Cell Neurosci. 2015;9. doi: 10.3389/fncel.2015.00021 [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Nyarko-Danquah I, Pajarillo E, Digman A, Soliman KFA, Aschner M, Lee E. Manganese Accumulation in the Brain via Various Transporters and Its Neurotoxicity Mechanisms. Molecules. 2020;25(24):5880. doi: 10.3390/molecules25245880 [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Morgan SE, Schroten H, Ishikawa H, Zhao N. Localization of ZIP14 and ZIP8 in HIBCPP Cells. Brain Sci. 2020;10(8):534. doi: 10.3390/brainsci10080534 [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Thompson K, Molina RM, Donaghey T, Schwob JE, Brain JD, Wessling-Resnick M. Olfactory uptake of manganese requires DMT1 and is enhanced by anemia. FASEB J. 2007;21(1):223–30. doi: 10.1096/fj.06-6710com [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Nakagawa S, Deli MA, Nakao S, Honda M, Hayashi K, Nakaoke R, et al. Pericytes from brain microvessels strengthen the barrier integrity in primary cultures of rat brain endothelial cells. Cell Mol Neurobiol. 2007;27(6):687–94. doi: 10.1007/s10571-007-9195-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Nakagawa S, Deli MA, Kawaguchi H, Shimizudani T, Shimono T, Kittel A, et al. A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. Neurochem Int. 2009;54(3–4):253–63. doi: 10.1016/j.neuint.2008.12.002 [DOI] [PubMed] [Google Scholar]
51.Akin EJ, Higerd-Rusli GP, Mis MA, Tanaka BS, Adi T, Liu S, et al. Building sensory axons: Delivery and distribution of NaV1.7 channels and effects of inflammatory mediators. Sci Adv. 2019;5(10):eaax4755. doi: 10.1126/sciadv.aax4755 [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Biswas S, Anjum A, Banna HU, Rahman M, Siddique AE, Karim Y, et al. Manganese attenuates the effects of arsenic on neurobehavioral and biochemical changes in mice co-exposed to arsenic and manganese. Environ Sci Pollut Res Int. 2019;26(28):29257–66. doi: 10.1007/s11356-019-06112-y [DOI] [PubMed] [Google Scholar]
53.Aktar S, Jahan M, Alam S, Mohanto NC, Arefin A, Rahman A, et al. Individual and Combined Effects of Arsenic and Lead on Behavioral and Biochemical Changes in Mice. Biol Trace Elem Res. 2017;177(2):288–96. doi: 10.1007/s12011-016-0883-0 [DOI] [PubMed] [Google Scholar]
54.Kabir E, Shila TT, Islam J, Beauty SA, Islam F, Hossain S, et al. Concomitant Exposure to Lower Doses of Arsenic, Lead, and Manganese Induces Greater Synergistic Neurotoxicity Than Individual Metals in Mice. Biol Trace Elem Res. 2025;203(3):1571–81. doi: 10.1007/s12011-024-04260-y [DOI] [PubMed] [Google Scholar]
55.Andrade V, Mateus ML, Batoréu MC, Aschner M, Dos Santos AM. Toxic Mechanisms Underlying Motor Activity Changes Induced by a Mixture of Lead, Arsenic and Manganese. EC Pharmacol Toxicol. 2017;3(2):31–42. [PMC free article] [PubMed] [Google Scholar]
56.Zheng F, Gonçalves FM, Abiko Y, Li H, Kumagai Y, Aschner M. Redox toxicology of environmental chemicals causing oxidative stress. Redox Biol. 2020;34:101475. doi: 10.1016/j.redox.2020.101475 [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Buha A, Baralić K, Djukic-Cosic D, Bulat Z, Tinkov A, Panieri E, et al. The Role of Toxic Metals and Metalloids in Nrf2 Signaling. Antioxidants (Basel). 2021;10(5):630. doi: 10.3390/antiox10050630 [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Mao J, Yang J, Zhang Y, Li T, Wang C, Xu L, et al. Arsenic trioxide mediates HAPI microglia inflammatory response and subsequent neuron apoptosis through p38/JNK MAPK/STAT3 pathway. Toxicol Appl Pharmacol. 2016;303:79–89. doi: 10.1016/j.taap.2016.05.003 [DOI] [PubMed] [Google Scholar]
59.Chen G, Mao J, Zhao J, Zhang Y, Li T, Wang C, et al. Arsenic trioxide mediates HAPI microglia inflammatory response and the secretion of inflammatory cytokine IL-6 via Akt/NF-κB signaling pathway. Regul Toxicol Pharmacol. 2016;81:480–8. doi: 10.1016/j.yrtph.2016.09.027 [DOI] [PubMed] [Google Scholar]
60.Pajarillo E, Kim S, Digman A, Dutton M, Son D-S, Aschner M, et al. The role of microglial LRRK2 kinase in manganese-induced inflammatory neurotoxicity via NLRP3 inflammasome and RAB10-mediated autophagy dysfunction. J Biol Chem. 2023;299(7):104879. doi: 10.1016/j.jbc.2023.104879 [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Strecker J-K, Minnerup J, Gess B, Ringelstein EB, Schäbitz W-R, Schilling M. Monocyte chemoattractant protein-1-deficiency impairs the expression of IL-6, IL-1β and G-CSF after transient focal ischemia in mice. PLoS One. 2011;6(10):e25863. doi: 10.1371/journal.pone.0025863 [DOI] [PMC free article] [PubMed] [Google Scholar]
62.Saha S, Buttari B, Panieri E, Profumo E, Saso L. An Overview of Nrf2 Signaling Pathway and Its Role in Inflammation. Molecules. 2020;25(22):5474. doi: 10.3390/molecules25225474 [DOI] [PMC free article] [PubMed] [Google Scholar]
63.Soto-Verdugo J, Ortega A. Critical Involvement of Glial Cells in Manganese Neurotoxicity. BioMed Research International. 2021;2021(1). doi: 10.1155/2021/1596185 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0330287.r001

Decision Letter 0

Mária Deli

6 Jun 2025

Dear Dr. Yamauchi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses all the points raised during the review process.

Please submit your revised manuscript by Jul 21 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Mária A. Deli, M.D., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: No

**********

2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: GENERAL COMMENT

This manuscript addresses an important topic in environmental toxicology: the combined effects of co-exposure to arsenic (As) and manganese (Mn) on blood-brain barrier (BBB) integrity and oxidative stress. Investigating joint toxicity is highly relevant, as humans are often exposed to metal mixtures (for example, arsenic and manganese frequently co-contaminate drinking water sources. While the subject matter is noteworthy, the manuscript requires substantial improvement in justification, methodology improvement and interpretation of data. Provided the authors address the issues outlined below, the study’s publication could be justified.

MAJOR COMMENTS

INTRODUCTION

-The manuscript in its current form suffers from significant conceptual and presentation issues. The Introduction does not clearly justify the rationale for studying As–Mn joint toxicity: it omits key background information (e.g. both metals’ links to neurodegenerative disease) and fails to define the concept of joint toxic action.

-The introduction fails to compellingly justify why examining the joint toxicity of arsenic and manganese is necessary. The authors mention an example of arsenic poisoning in South America, which is not aligned with the study’s context or focus, and thus feels out of place. Instead, the introduction should highlight scenarios where As and Mn co-exposure is a realistic concern (for instance, co-occurrence of As and Mn in groundwater affecting millions) PMID: 32786605

-More importantly, the text does not mention that both arsenic and manganese are individually known neurotoxicants associated with cognitive impairment and even Alzheimer’s disease risk. The authors should include this to strengthen the rationale. As reviewed somewhere else, chronic exposure to either As or Mn can induce oxidative stress and other neuropathological processes implicated in neurodegeneration. PMID: 40278152

-The introduction is ambiguous about the knowledge gap. It also fails to state whether co-exposure to arsenic and manganese leads to additive, synergistic, or antagonistic effects on the BBB and brain, given that each metal alone can disrupt neurological function.

MATERIALS AND METHODS

-The Materials and Methods lack critical details such as the rationale for dose selection and clarity on experimental controls, raising concerns about reproducibility and the environmental relevance of the findings.

The manuscript does not explain how the chosen doses of arsenic and manganese were determined, which is a critical oversight. The authors must clarify whether the concentrations used in vitro (for both single and combined exposures) reflect environmentally relevant levels (e.g., comparable to concentrations found in blood or drinking water of exposed populations) or were chosen based on prior toxicological studies.

RESULTS

-The Results are described without sufficient critical analysis, for instance, manganese alone appears to have no significant effect on oxidative stress, yet the authors proceed to discuss “joint” effects without demonstrating a true interaction.

-The interpretation of the results, specifically regarding the interaction between arsenic and manganese as reported in several paragraphs of the results section, is a point of major concern. It is not scientifically justified to infer a meaningful interaction if one component is essentially inert (at the tested dose) for the measured endpoints. If Mn alone caused no significant change, then any effect observed in the As+Mn co-exposure group is likely driven solely by arsenic.

MINOR COMMENTS

-The reference to “South American arsenic poisoning” in the Introduction appears misaligned with the study’s focus. If the authors are aiming to set a global context, they should either generalize this point or provide a clearer connection. Otherwise, this example could be removed to tighten the narrative.

-As noted above, the authors should insert a brief definition of joint toxic actions (additivity, synergy, antagonism) for clarity

-It’s suggested to mention in the Introduction (or Discussion) that both As and Mn have been implicated as risk factors in neurodegenerative diseases like Alzheimer’s. The authors allude to cognitive effects but don’t specifically cite evidence. Including a sentence such as, “Notably, epidemiological and mechanistic studies have linked chronic arsenic and manganese exposures to............." would help clarify this aspect.

Reviewer #2: The investigation into the combined toxicity of arsenic and manganese presents an interesting and relevant approach. The study effectively demonstrates the cytotoxic effects of the combined exposure. However, several figures indicate minimal differences between the arsenic-only and the combined exposure groups. This suggests that the proposed pathways may not represent the primary mechanisms underlying the observed effects.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: Yes: Temitope Adebambo

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

PLoS One. 2025 Aug 25;20(8):e0330287. doi: 10.1371/journal.pone.0330287.r002

Author response to Decision Letter 1

15 Jul 2025

July 15, 2025

To Reviewer 1

Subject: Response to Reviewers - PONE-D-25-22817 - "Interaction of coexposure to inorganic arsenic and manganese: Tight junction injury of the blood–brain barrier and the relationship between oxidative stress and inflammatory cytokines in glial cells.

Dear Reviewer,

Thank you very much for your insightful and constructive comments, which have significantly contributed to improving the clarity, rigor, and overall quality of our manuscript. Below, we provide point-by-point responses and highlight the corresponding revisions in the manuscript.

GENERAL COMMENT

Thank you for recognizing the importance of our study. We agree that the manuscript required substantial improvements in justification, methodology, and data interpretation. We have carefully considered your suggestions and revised the manuscript accordingly.

MAJOR COMMENTS

INTRODUCTION

We appreciate these crucial comments. We acknowledge the shortcomings in our introduction and have revised it as follows:

Comment -1: The manuscript in its current form suffers from significant conceptual and presentation issues. The Introduction does not clearly justify the rationale for studying As–Mn joint toxicity: it omits key background information (e.g. both metals’ links to neurodegenerative disease) and fails to define the concept of joint toxic action.

Response:

Comment-1: Thank you very much for your valuable comment. We have revised the Introduction section to more clearly justify the rationale for studying the joint toxicity of arsenic (iAs) and manganese (Mn) (lines 52–56).

Specifically, we have added important background information indicating that both iAs and Mn are individually associated with cognitive dysfunction and are implicated in the development of neurodegenerative diseases such as Alzheimer’s disease (lines 58–67).

In accordance with the reviewers' comments and instructions, we have included the definition of “joint effects” in the introduction of this study.

“In this study, we evaluated the joint effects of iAs and Mn by distinguishing additive effects—where the combined outcome equals the sum of individual effects (EiAs + EMn = EiAs + Mn)—from synergistic effects, where the combined outcome exceeds this sum (EiAs + Mn > EiAs + EMn). Here, EiAs denotes the effect of exposure to iAs, EMn the effect of exposure to Mn, and EiAs + Mn the effect of coexposure.”

(lines 109-113).

Furthermore, we emphasized the real-world relevance of such coexposure by referencing environmental contamination cases (e.g., groundwater contamination in Bangladesh; Refs. 36–38), and noted that iAs and Mn can co-occur in both groundwater (Refs. 39, 40) and the diet (Refs. 41, 42), potentially affecting the general population. These revisions can be found in lines 68–85 of the revised manuscript.

Comment -2: The introduction fails to compellingly justify why examining the joint toxicity of arsenic and manganese is necessary. The authors mention an example of arsenic poisoning in South America, which is not aligned with the study’s context or focus, and thus feels out of place. Instead, the introduction should highlight scenarios where As and Mn co-exposure is a realistic concern (for instance, co-occurrence of As and Mn in groundwater affecting millions) PMID: 32786605

Response:

Comment-2:

We appreciate the reviewer’s insightful comment. As suggested, we removed the unrelated information regarding chronic arsenic poisoning, arsenic carcinogenicity, and lifestyle-related diseases, as these were not directly relevant to the scope of our study.

We also revised the introduction to more clearly justify the necessity of investigating the joint toxicity of iAs and Mn.

Specifically, we added information emphasizing realistic and high-impact scenarios where co-exposure to these elements is a serious concern. For instance, we cited a recent report (PMID: 32786605; Ref. 39) documenting the co-occurrence of arsenic and manganese in groundwater in Bangladesh, where millions of people are affected by this contamination.

(lines 75–80.)

This revision enhances the relevance and urgency of our research within the context of environmental health.

Comment -3: More importantly, the text does not mention that both arsenic and manganese are individually known neurotoxicants associated with cognitive impairment and even Alzheimer’s disease risk. The authors should include this to strengthen the rationale. As reviewed somewhere else, chronic exposure to either As or Mn can induce oxidative stress and other neuropathological processes implicated in neurodegeneration. PMID: 40278152

Response:

Comment-3:

We have addressed this comment in lines 62–67. We clearly stated that iAs and Mn are each known to be neurotoxic and are associated with cognitive dysfunction and an increased risk of Alzheimer’s disease. To support this, we cited a recent review (PMID: 40278152; Ref. 33) that discusses neuropathological mechanisms, such as oxidative stress, induced by chronic exposure to these metals. Furthermore, we supplemented this background with evidence from animal studies (lines 56–60) demonstrating cognitive dysfunction following exposure to iAs and Mn.

Comment -4: The introduction is ambiguous about the knowledge gap. It also fails to state whether co-exposure to arsenic and manganese leads to additive, synergistic, or antagonistic effects on the BBB and brain, given that each metal alone can disrupt neurological function.

Response:

Comment-4:

The corresponding revision is found in lines 97–107. We now clarify that while iAs is known to disrupt the BBB through tight junction (TJ) injury, there is limited information on the direct impact of Mn on BBB integrity. Given this knowledge gap, we emphasize the importance of exploring whether coexposure to iAs and Mn produces additive or synergistic effects on TJ injury.

MATERIALS AND METHODS

Comment -5:

The Materials and Methods lack critical details such as the rationale for dose selection and clarity on experimental controls, raising concerns about reproducibility and the environmental relevance of the findings. The manuscript does not explain how the chosen doses of arsenic and manganese were determined, which is a critical oversight. The authors must clarify whether the concentrations used in vitro (for both single and combined exposures) reflect environmentally relevant levels (e.g., comparable to concentrations found in blood or drinking water of exposed populations) or were chosen based on prior toxicological studies.

Response

Thank you for these important comments. To enhance the clarity and reproducibility of the Materials and Methods section, we have made the following changes:

Comment -5:

(lines 140–148.)

(Ref. 1) Wasserman GA, Liu X, Parvez F, Ahsan H, Factor-Litvak P, van Geen A, et al. Water arsenic exposure and children’s intellectual function in Araihazar, Bangladesh. Environ Health Perspect. 2004 Apr; 112(13):1329–1333. doi: 10.1289/ehp.6964.

RESULTS

We appreciate these crucial observations. To improve the interpretation and presentation of our results, we have made the following revisions:

Comment -6:

The Results are described without sufficient critical analysis, for instance, manganese alone appears to have no significant effect on oxidative stress, yet the authors proceed to discuss “joint” effects without demonstrating a true interaction.

The interpretation of the results, specifically regarding the interaction between arsenic and manganese as reported in several paragraphs of the results section, is a point of major concern. It is not scientifically justified to infer a meaningful interaction if one component is essentially inert (at the tested dose) for the measured endpoints.

Response

Comment -6:

There was a significant error in the description of the results. Specifically, for some of the iAs + Mn results, although “additive effect” was the appropriate term, it was ambiguously expressed as “synergistic effect.” We have thoroughly reviewed all results and clarified the distinction between additive and synergistic effects for iAs + Mn, revising and adding the relevant text in the results section.

The response is the same as that given in response to major comment-1. We set joint effects in this study as follows.

In accordance with the reviewers' comments and instructions, we have included the definition of “joint effects” in the introduction of this study.

(lines 109-113).

Additionally, we have revised and added text regarding “joint effects” throughout the results; the revised and newly added portions have been highlighted in yellow in the manuscript for clarity. Additionally, since we have changed the interpretation of additive and synergistic effects in the results, we have revised the relevant sections in the “Discussion,” highlighting the revised and added portions in yellow. Furthermore, we have revised the content of the ‘Abstract’ and ‘Conclusion’ sections to ensure accuracy.

Comment -7:

If Mn alone caused no significant change, then any effect observed in the As+Mn co-exposure group is likely driven solely by arsenic.

Response

Comment -7:

As pointed out by the reviewer, when no significant changes were observed following Mn exposure alone, it is reasonable to assume that specific effects in the coexposure group may primarily reflect the influence of iAs. In our study, this was indeed the case for specific endpoints, such as the expression of Nrf2 and HO-1 in glial cells and BBB-astrocytes, as well as the mRNA levels of MCP-1 and IL-6. However, our data also demonstrated that specific markers—particularly HO-1 and IL-6 in glial cells, and HO-1 in BBB-astrocytes—showed significantly greater responses in the coexposure group than in either of the single exposure groups. These findings suggest synergistic effects between iAs and Mn, despite Mn showing no significant impact when applied alone. Therefore, Mn may act as a potentiating factor under coexposure conditions, enhancing iAs-induced toxicity beyond the level expected from iAs alone. We have clarified this interpretation in the revised manuscript and emphasized that the identification of such interactions is a key contribution of this study to the understanding of joint iAs and Mn toxicity.

MINOR COMMENTS

Comment -8:

The reference to “South American arsenic poisoning” in the Introduction appears misaligned with the study’s focus. If the authors are aiming to set a global context, they should either generalize this point or provide a clearer connection. Otherwise, this example could be removed to tighten the narrative.

Response

Comment -8:

Thank you for your comment. By the reviewers' comments, descriptions of carcinogenicity and lifestyle-related diseases associated with chronic arsenic poisoning have been deleted. The occurrence of cognitive dysfunction due to exposure to iAs and Mn alone has been reorganized and described.

lines 52-56.

Comment -9:

As noted above, the authors should insert a brief definition of joint toxic actions (additivity, synergy, antagonism) for clarity.

Response

Comment -9:

Thank you for your comment. The response to this comment is described in major comment -1 and -6. We have addressed this comment in lines 109–113.

Comment -10:

It’s suggested to mention in the Introduction (or Discussion) that both As and Mn have been implicated as risk factors in neurodegenerative diseases like Alzheimer’s. The authors allude to cognitive effects but don’t specifically cite evidence. Including a sentence such as, “Notably, epidemiological and mechanistic studies have linked chronic arsenic and manganese exposures to...........” would help clarify this aspect.

Response

Comment -10:

Thank you for your comment. The response to this comment is described in major comment -3.

To Reviewer 2

Comments

The investigation into the combined toxicity of arsenic and manganese presents an interesting and relevant approach. The study effectively demonstrates the cytotoxic effects of the combined exposure. However, several figures indicate minimal differences between the arsenic-only and the combined exposure groups. This suggests that the proposed pathways may not represent the primary mechanisms underlying the observed effects.

Response

Thank you for acknowledging the relevance of our study on the combined toxicity of arsenic and manganese and for appreciating that our study effectively demonstrates the cytotoxic effects of the combined exposure.

We revised the “Results” and “Discussion” sections of the manuscript to provide a more detailed and careful interpretation of the data. Specifically, we supplemented the explanation of the potential additive and synergistic effects of iAs and Mn in coexposure. Information in this field is limited, and we have discussed the possible mechanisms as thoroughly as possible; however, further research is still needed. The revised and newly added sections are highlighted in yellow for clarity in the “Revised Article with Changes Highlighted” file.

Thank you again for your time and effort in reviewing our manuscript. Your feedback has significantly helped us to improve this study.

Hiroshi Yamauchi, Ph. D.

Visiting Professor

Department of Preventive Medicine, St. Marianna University School of Medicine, Japan

Email: hyama@marianna-u.ac.jp

Telephone: +81-44-978-6392

Fax: +81-44-978-6393

Attachment

Submitted filename: Response to Reviewers.docx

pone.0330287.s010.docx^{(36.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0330287.r003

Decision Letter 1

Mária Deli

30 Jul 2025

Interaction of coexposure to inorganic arsenic and manganese: Tight junction injury of the blood–brain barrier and the relationship between oxidative stress and inflammatory cytokines in glial cells

PONE-D-25-22817R1

Dear Dr. Yamauchi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support .

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Mária A. Deli, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

**********

Reviewer #1: Authors have addressed the concerns I raised in the first round of reviews. The updated report now includes how Mn and Arsenic exposures are individually linked to neurodegenerative diseases.

The real-worlds relevance has also been highlighted and the ambiguity in the introduction has been addressed.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: Yes: Temitope H Adebambo

**********

PLoS One. doi: 10.1371/journal.pone.0330287.r004

Acceptance letter

Mária Deli

PONE-D-25-22817R1

PLOS ONE

Dear Dr. Yamauchi,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Mária A. Deli

Academic Editor

PLOS ONE

Associated Data

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

Supplementary Materials

S1 Table. Cytotoxicity of iAs or Mn alone and coexposure to iAs and Mn in glial cells.

*One-way ANOVA with Tukey’s post hoc tests. This table supports the statistical comparisons shown in Fig 1.

(XLSX)

pone.0330287.s001.xlsx^{(13.8KB, xlsx)}

S2 Table. Oxidative stress of iAs or Mn alone and coexposure to iAs and Mn in glial cells.

(A) Nrf2 mRNA expression. (B) HO-1 mRNA expression. *One-way ANOVA with Tukey’s post hoc tests.This table supports the statistical comparisons shown in Fig 2A and 2B.

(XLSX)

pone.0330287.s002.xlsx^{(14.6KB, xlsx)}

S3 Table. Inflammatory cytokines in iAs and Mn alone groups and iAs and Mn coexposure groups in glial cells.

(A) MCP-1 mRNA expression. (B) IL-1β mRNA expression. (C) IL-6 mRNA expression. *One-way ANOVA with Tukey’s post hoc tests. This table supports the statistical comparisons shown in Fig 3A–C.

(XLSX)

pone.0330287.s003.xlsx^{(15.7KB, xlsx)}

S4 Table. Expression of Nrf2 and HO-1 proteins in the vascular endothelial cell and pericyte groups after exposure to iAs or Mn alone and coexposure to iAs and Mn.

(A) Nrf2 protein expression. (B) HO-1 protein expression. *One-way ANOVA with Tukey’s post hoc tests. This table supports the statistical comparisons shown in Fig 4B and 4C.

(XLSX)

pone.0330287.s004.xlsx^{(14.7KB, xlsx)}

S5 Table. Expression of Nrf2 and HO-1 proteins in astrocytes after exposure to iAs or Mn alone and coexposure to iAs and Mn.

(A) Nrf2 protein expression. (B) HO-1 protein expression. *One-way ANOVA with Tukey’s post hoc tests. This table supports the statistical comparisons shown in Fig 5B and 5C.

(XLSX)

pone.0330287.s005.xlsx^{(14.7KB, xlsx)}

S6 Table. Changes in TEER after exposure to iAs or Mn alone, and coexposure to iAs and Mn.

*One-way ANOVA with Tukey’s post hoc tests. This table supports the statistical comparisons shown in Fig 6.

(XLSX)

pone.0330287.s006.xlsx^{(13.7KB, xlsx)}

S7 Table. Expression of the tight junction proteins claudin-5 and ZO-1 after exposure to iAs or Mn alone and coexposure to iAs and Mn.

(A) Claudin-5 protein expression. (B) ZO-1 protein expression. *One-way ANOVA with Tukey’s post hoc tests. This table supports the statistical comparisons shown in Fig 7B and 7C.

(XLSX)

pone.0330287.s007.xlsx^{(14.4KB, xlsx)}

S8 Table. Quantitative analysis of the immunofluorescence staining of claudin-5 after exposure to iAs or Mn alone and coexposure to iAs and Mn.

*One-way ANOVA with Tukey’s post hoc tests. This table supports the statistical comparisons shown in Fig 8B.

(XLSX)

pone.0330287.s008.xlsx^{(13.4KB, xlsx)}

S9 Table. Quantitative analysis of the immunofluorescence staining of ZO-1 after exposure to iAs or Mn alone and coexposure to iAs and Mn.

*One-way ANOVA with Tukey’s post hoc tests. This table supports the statistical comparisons shown in Fig 9B.

(XLSX)

pone.0330287.s009.xlsx^{(13.3KB, xlsx)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0330287.s010.docx^{(36.2KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[pone.0330287.ref001] 1.Wasserman GA, Liu X, Parvez F, Ahsan H, Factor-Litvak P, van Geen A, et al. Water arsenic exposure and children’s intellectual function in Araihazar, Bangladesh. Environ Health Perspect. 2004;112(13):1329–33. doi: 10.1289/ehp.6964 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref002] 2.Wasserman GA, Liu X, Parvez F, Ahsan H, Factor-Litvak P, Kline J, et al. Water arsenic exposure and intellectual function in 6-year-old children in Araihazar, Bangladesh. Environ Health Perspect. 2007;115(2):285–9. doi: 10.1289/ehp.9501 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref003] 3.Hamadani JD, Tofail F, Nermell B, Gardner R, Shiraji S, Bottai M, et al. Critical windows of exposure for arsenic-associated impairment of cognitive function in pre-school girls and boys: a population-based cohort study. Int J Epidemiol. 2011;40(6):1593–604. doi: 10.1093/ije/dyr176 [DOI] [PubMed] [Google Scholar]

[pone.0330287.ref004] 4.Wasserman GA, Liu X, Parvez F, Chen Y, Factor-Litvak P, LoIacono NJ, et al. A cross-sectional study of water arsenic exposure and intellectual function in adolescence in Araihazar, Bangladesh. Environment International. 2018;118:304–13. doi: 10.1016/j.envint.2018.05.037 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref005] 5.Manju R, Hegde AM, Parlees P, Keshan A. Environmental Arsenic Contamination and Its Effect on Intelligence Quotient of School Children in a Historic Gold Mining Area Hutti, North Karnataka, India: A Pilot Study. J Neurosci Rural Pract. 2017;8(3):364–7. doi: 10.4103/jnrp.jnrp_501_16 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref006] 6.Vaidya N, Holla B, Heron J, Sharma E, Zhang Y, Fernandes G, et al. Neurocognitive Analysis of Low-level Arsenic Exposure and Executive Function Mediated by Brain Anomalies Among Children, Adolescents, and Young Adults in India. JAMA Netw Open. 2023;6(5):e2312810. doi: 10.1001/jamanetworkopen.2023.12810 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref007] 7.Wang S-X, Wang Z-H, Cheng X-T, Li J, Sang Z-P, Zhang X-D, et al. Arsenic and fluoride exposure in drinking water: children’s IQ and growth in Shanyin county, Shanxi province, China. Environ Health Perspect. 2007;115(4):643–7. doi: 10.1289/ehp.9270 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref008] 8.Wang Y, Wang Y, Yan C. Gender differences in trace element exposures with cognitive abilities of school-aged children: a cohort study in Wujiang city, China. Environ Sci Pollut Res Int. 2022;29(43):64807–21. doi: 10.1007/s11356-022-20353-4 [DOI] [PubMed] [Google Scholar]

[pone.0330287.ref009] 9.Rosado JL, Ronquillo D, Kordas K, Rojas O, Alatorre J, Lopez P, et al. Arsenic Exposure and Cognitive Performance in Mexican Schoolchildren. Environ Health Perspect. 2007;115(9):1371–5. doi: 10.1289/ehp.9961 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref010] 10.Limón-Pacheco JH, Jiménez-Córdova MI, Cárdenas-González M, Sánchez Retana IM, Gonsebatt ME, Del Razo LM. Potential Co-exposure to Arsenic and Fluoride and Biomonitoring Equivalents for Mexican Children. Ann Glob Health. 2018;84(2):257–73. doi: 10.29024/aogh.913 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref011] 11.Wasserman GA, Liu X, Parvez F, Ahsan H, Levy D, Factor-Litvak P, et al. Water manganese exposure and children’s intellectual function in Araihazar, Bangladesh. Environ Health Perspect. 2006;114(1):124–9. doi: 10.1289/ehp.8030 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref012] 12.Khan K, Factor-Litvak P, Wasserman GA, Liu X, Ahmed E, Parvez F, et al. Manganese Exposure from Drinking Water and Children’s Classroom Behavior in Bangladesh. Environ Health Perspect. 2011;119(10):1501–6. doi: 10.1289/ehp.1003397 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref013] 13.Khan K, Wasserman GA, Liu X, Ahmed E, Parvez F, Slavkovich V, et al. Manganese exposure from drinking water and children’s academic achievement. Neurotoxicology. 2012;33(1):91–7. doi: 10.1016/j.neuro.2011.12.002 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref014] 14.Rahman SM, Kippler M, Tofail F, Bölte S, Hamadani JD, Vahter M. Manganese in Drinking Water and Cognitive Abilities and Behavior at 10 Years of Age: A Prospective Cohort Study. Environ Health Perspect. 2017;125(5):057003. doi: 10.1289/EHP631 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref015] 15.Yu X-D, Zhang J, Yan C-H, Shen X-M. Prenatal exposure to manganese at environment relevant level and neonatal neurobehavioral development. Environ Res. 2014;133:232–8. doi: 10.1016/j.envres.2014.04.012 [DOI] [PubMed] [Google Scholar]

[pone.0330287.ref016] 16.Yu X, Chen L, Wang C, Yang X, Gao Y, Tian Y. The role of cord blood BDNF in infant cognitive impairment induced by low-level prenatal manganese exposure: LW birth cohort, China. Chemosphere. 2016;163:446–51. doi: 10.1016/j.chemosphere.2016.07.095 [DOI] [PubMed] [Google Scholar]

[pone.0330287.ref017] 17.Bouchard MF, Sauvé S, Barbeau B, Legrand M, Brodeur M-È, Bouffard T, et al. Intellectual impairment in school-age children exposed to manganese from drinking water. Environ Health Perspect. 2011;119(1):138–43. doi: 10.1289/ehp.1002321 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref018] 18.Oulhote Y, Mergler D, Barbeau B, Bellinger DC, Bouffard T, Brodeur M-È, et al. Neurobehavioral function in school-age children exposed to manganese in drinking water. Environ Health Perspect. 2014;122(12):1343–50. doi: 10.1289/ehp.1307918 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref019] 19.Lane JM, Curtin P, Chelonis JJ, Pantic I, Martinez-Medina S, Téllez-Rojo MM, et al. Prenatal manganese biomarkers and operant test battery performance in Mexican children: Effect modification by child sex. Environ Res. 2023;236(Pt 2):116880. doi: 10.1016/j.envres.2023.116880 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref020] 20.Lane JM, Liu SH, Pantic I, Martinez-Medina S, Téllez-Rojo MM, Amarasiriwardena C, et al. Sex-specific association between prenatal manganese exposure and working memory in school-aged children in Mexico city: An exploratory multi-media approach. Environmental Pollution. 2023;333:121965. doi: 10.1016/j.envpol.2023.121965 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref021] 21.Wasserman GA, Liu X, Loiacono NJ, Kline J, Factor-Litvak P, van Geen A, et al. A cross-sectional study of well water arsenic and child IQ in Maine schoolchildren. Environ Health. 2014;13(1):23. doi: 10.1186/1476-069X-13-23 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref022] 22.Haynes EN, Sucharew H, Kuhnell P, Alden J, Barnas M, Wright RO, et al. Manganese Exposure and Neurocognitive Outcomes in Rural School-Age Children: The Communities Actively Researching Exposure Study (Ohio, USA). Environ Health Perspect. 2015;123(10):1066–71. doi: 10.1289/ehp.1408993 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref023] 23.Nascimento S, Baierle M, Göethel G, Barth A, Brucker N, Charão M, et al. Associations among environmental exposure to manganese, neuropsychological performance, oxidative damage and kidney biomarkers in children. Environ Res. 2016;147:32–43. doi: 10.1016/j.envres.2016.01.035 [DOI] [PubMed] [Google Scholar]

[pone.0330287.ref024] 24.Carvalho CFD, Oulhote Y, Martorelli M, Carvalho COD, Menezes-Filho JA, Argollo N, et al. Environmental manganese exposure and associations with memory, executive functions, and hyperactivity in Brazilian children. Neurotoxicology. 2018;69:253–9. doi: 10.1016/j.neuro.2018.02.002 [DOI] [PubMed] [Google Scholar]

[pone.0330287.ref025] 25.Vázquez Cervantes GI, González Esquivel DF, Ramírez Ortega D, Blanco Ayala T, Ramos Chávez LA, López-López HE, et al. Mechanisms Associated with Cognitive and Behavioral Impairment Induced by Arsenic Exposure. Cells. 2023;12(21):2537. doi: 10.3390/cells12212537 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref026] 26.Thakur M, Rachamalla M, Niyogi S, Datusalia AK, Flora SJS. Molecular Mechanism of Arsenic-Induced Neurotoxicity including Neuronal Dysfunctions. IJMS. 2021;22(18):10077. doi: 10.3390/ijms221810077 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref027] 27.Smith DR, Strupp BJ. Animal Models of Childhood Exposure to Lead or Manganese: Evidence for Impaired Attention, Impulse Control, and Affect Regulation and Assessment of Potential Therapies. Neurotherapeutics. 2023;20(1):3–21. doi: 10.1007/s13311-023-01345-9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref028] 28.Dorman DC. The Role of Oxidative Stress in Manganese Neurotoxicity: A Literature Review Focused on Contributions Made by Professor Michael Aschner. Biomolecules. 2023;13(8):1176. doi: 10.3390/biom13081176 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref029] 29.Balachandran RC, Mukhopadhyay S, McBride D, Veevers J, Harrison FE, Aschner M, et al. Brain manganese and the balance between essential roles and neurotoxicity. J Biol Chem. 2020;295(19):6312–29. doi: 10.1074/jbc.REV119.009453 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref030] 30.Baj J, Flieger W, Barbachowska A, Kowalska B, Flieger M, Forma A, et al. Consequences of Disturbing Manganese Homeostasis. Int J Mol Sci. 2023;24(19):14959. doi: 10.3390/ijms241914959 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref031] 31.Islam F, Shohag S, Akhter S, Islam MdR, Sultana S, Mitra S, et al. Exposure of metal toxicity in Alzheimer’s disease: An extensive review. Front Pharmacol. 2022;13. doi: 10.3389/fphar.2022.903099 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref032] 32.Kim H, Harrison FE, Aschner M, Bowman AB. Exposing the role of metals in neurological disorders: a focus on manganese. Trends Mol Med. 2022;28(7):555–68. doi: 10.1016/j.molmed.2022.04.011 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref033] 33.Ahmed G, Rahaman MdS, Perez E, Khan KM. Associations of Environmental Exposure to Arsenic, Manganese, Lead, and Cadmium with Alzheimer’s Disease: A Review of Recent Evidence from Mechanistic Studies. JoX. 2025;15(2):47. doi: 10.3390/jox15020047 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref034] 34.Selkoe DJ. Alzheimer’s disease is a synaptic failure. Science. 2002;298(5594):789–91. doi: 10.1126/science.1074069 [DOI] [PubMed] [Google Scholar]

[pone.0330287.ref035] 35.Jack CR Jr, Bennett DA, Blennow K, Carrillo MC, Dunn B, Haeberlein SB, et al. NIA-AA Research Framework: Toward a biological definition of Alzheimer’s disease. Alzheimers Dement. 2018;14(4):535–62. doi: 10.1016/j.jalz.2018.02.018 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref036] 36.Wasserman GA, Liu X, Parvez F, Factor-Litvak P, Ahsan H, Levy D, et al. Arsenic and manganese exposure and children’s intellectual function. Neurotoxicology. 2011;32(4):450–7. doi: 10.1016/j.neuro.2011.03.009 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref037] 37.Wasserman GA, Liu X, Parvez F, Factor-Litvak P, Kline J, Siddique AB, et al. Child Intelligence and Reductions in Water Arsenic and Manganese: A Two-Year Follow-up Study in Bangladesh. Environ Health Perspect. 2016;124(7):1114–20. doi: 10.1289/ehp.1509974 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref038] 38.Saxena R, Gamble M, Wasserman GA, Liu X, Parvez F, Navas-Acien A, et al. Mixed metals exposure and cognitive function in Bangladeshi adolescents. Ecotoxicol Environ Saf. 2022;232:113229. doi: 10.1016/j.ecoenv.2022.113229 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref039] 39.Schaefer MV, Plaganas M, Abernathy MJ, Aiken ML, Garniwan A, Lee I, et al. Manganese, Arsenic, and Carbonate Interactions in Model Oxic Groundwater Systems. Environ Sci Technol. 2020;54(17):10621–9. doi: 10.1021/acs.est.0c02084 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref040] 40.Erickson ML, Elliott SM, Brown CJ, Stackelberg PE, Ransom KM, Reddy JE, et al. Machine-Learning Predictions of High Arsenic and High Manganese at Drinking Water Depths of the Glacial Aquifer System, Northern Continental United States. Environ Sci Technol. 2021;55(9):5791–805. doi: 10.1021/acs.est.0c06740 [DOI] [PubMed] [Google Scholar]

[pone.0330287.ref041] 41.EFSA Panel on Contaminants in the Food Chain (CONTAM), Schrenk D, Bignami M, Bodin L, Chipman JK, Del Mazo J, et al. Update of the risk assessment of inorganic arsenic in food. EFSA J. 2024;22(1):e8488. doi: 10.2903/j.efsa.2024.8488 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref042] 42.EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), Turck D, Bohn T, Castenmiller J, de Henauw S, Hirsch‐Ernst KI, Knutsen HK, et al. Scientific opinion on the tolerable upper intake level for manganese. EFS2. 2023;21(12):e8413. doi: 10.2903/j.efsa.2023.8413 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref043] 43.Yamauchi H, Hitomi T, Takata A. Evaluation of arsenic metabolism and tight junction injury after exposure to arsenite and monomethylarsonous acid using a rat in vitro blood-Brain barrier model. PLoS One. 2023;18(11):e0295154. doi: 10.1371/journal.pone.0295154 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref044] 44.Sánchez-Peña LC, Petrosyan P, Morales M, González NB, Gutiérrez-Ospina G, Del Razo LM, et al. Arsenic species, AS3MT amount, and AS3MT gene expression in different brain regions of mouse exposed to arsenite. Environ Res. 2010;110(5):428–34. doi: 10.1016/j.envres.2010.01.007 [DOI] [PubMed] [Google Scholar]

[pone.0330287.ref045] 45.Ramos-ChÃ¡vez LA, RendÃ³n-LÃ³pez CRR, Zepeda A, Silva-Adaya D, Del Razo LM, Gonsebatt ME. Neurological effects of inorganic arsenic exposure: altered cysteine/glutamate transport, NMDA expression and spatial memory impairment. Front Cell Neurosci. 2015;9. doi: 10.3389/fncel.2015.00021 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref046] 46.Nyarko-Danquah I, Pajarillo E, Digman A, Soliman KFA, Aschner M, Lee E. Manganese Accumulation in the Brain via Various Transporters and Its Neurotoxicity Mechanisms. Molecules. 2020;25(24):5880. doi: 10.3390/molecules25245880 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref047] 47.Morgan SE, Schroten H, Ishikawa H, Zhao N. Localization of ZIP14 and ZIP8 in HIBCPP Cells. Brain Sci. 2020;10(8):534. doi: 10.3390/brainsci10080534 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref048] 48.Thompson K, Molina RM, Donaghey T, Schwob JE, Brain JD, Wessling-Resnick M. Olfactory uptake of manganese requires DMT1 and is enhanced by anemia. FASEB J. 2007;21(1):223–30. doi: 10.1096/fj.06-6710com [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref049] 49.Nakagawa S, Deli MA, Nakao S, Honda M, Hayashi K, Nakaoke R, et al. Pericytes from brain microvessels strengthen the barrier integrity in primary cultures of rat brain endothelial cells. Cell Mol Neurobiol. 2007;27(6):687–94. doi: 10.1007/s10571-007-9195-4 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref050] 50.Nakagawa S, Deli MA, Kawaguchi H, Shimizudani T, Shimono T, Kittel A, et al. A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. Neurochem Int. 2009;54(3–4):253–63. doi: 10.1016/j.neuint.2008.12.002 [DOI] [PubMed] [Google Scholar]

[pone.0330287.ref051] 51.Akin EJ, Higerd-Rusli GP, Mis MA, Tanaka BS, Adi T, Liu S, et al. Building sensory axons: Delivery and distribution of NaV1.7 channels and effects of inflammatory mediators. Sci Adv. 2019;5(10):eaax4755. doi: 10.1126/sciadv.aax4755 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref052] 52.Biswas S, Anjum A, Banna HU, Rahman M, Siddique AE, Karim Y, et al. Manganese attenuates the effects of arsenic on neurobehavioral and biochemical changes in mice co-exposed to arsenic and manganese. Environ Sci Pollut Res Int. 2019;26(28):29257–66. doi: 10.1007/s11356-019-06112-y [DOI] [PubMed] [Google Scholar]

[pone.0330287.ref053] 53.Aktar S, Jahan M, Alam S, Mohanto NC, Arefin A, Rahman A, et al. Individual and Combined Effects of Arsenic and Lead on Behavioral and Biochemical Changes in Mice. Biol Trace Elem Res. 2017;177(2):288–96. doi: 10.1007/s12011-016-0883-0 [DOI] [PubMed] [Google Scholar]

[pone.0330287.ref054] 54.Kabir E, Shila TT, Islam J, Beauty SA, Islam F, Hossain S, et al. Concomitant Exposure to Lower Doses of Arsenic, Lead, and Manganese Induces Greater Synergistic Neurotoxicity Than Individual Metals in Mice. Biol Trace Elem Res. 2025;203(3):1571–81. doi: 10.1007/s12011-024-04260-y [DOI] [PubMed] [Google Scholar]

[pone.0330287.ref055] 55.Andrade V, Mateus ML, Batoréu MC, Aschner M, Dos Santos AM. Toxic Mechanisms Underlying Motor Activity Changes Induced by a Mixture of Lead, Arsenic and Manganese. EC Pharmacol Toxicol. 2017;3(2):31–42. [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref056] 56.Zheng F, Gonçalves FM, Abiko Y, Li H, Kumagai Y, Aschner M. Redox toxicology of environmental chemicals causing oxidative stress. Redox Biol. 2020;34:101475. doi: 10.1016/j.redox.2020.101475 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref057] 57.Buha A, Baralić K, Djukic-Cosic D, Bulat Z, Tinkov A, Panieri E, et al. The Role of Toxic Metals and Metalloids in Nrf2 Signaling. Antioxidants (Basel). 2021;10(5):630. doi: 10.3390/antiox10050630 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref058] 58.Mao J, Yang J, Zhang Y, Li T, Wang C, Xu L, et al. Arsenic trioxide mediates HAPI microglia inflammatory response and subsequent neuron apoptosis through p38/JNK MAPK/STAT3 pathway. Toxicol Appl Pharmacol. 2016;303:79–89. doi: 10.1016/j.taap.2016.05.003 [DOI] [PubMed] [Google Scholar]

[pone.0330287.ref059] 59.Chen G, Mao J, Zhao J, Zhang Y, Li T, Wang C, et al. Arsenic trioxide mediates HAPI microglia inflammatory response and the secretion of inflammatory cytokine IL-6 via Akt/NF-κB signaling pathway. Regul Toxicol Pharmacol. 2016;81:480–8. doi: 10.1016/j.yrtph.2016.09.027 [DOI] [PubMed] [Google Scholar]

[pone.0330287.ref060] 60.Pajarillo E, Kim S, Digman A, Dutton M, Son D-S, Aschner M, et al. The role of microglial LRRK2 kinase in manganese-induced inflammatory neurotoxicity via NLRP3 inflammasome and RAB10-mediated autophagy dysfunction. J Biol Chem. 2023;299(7):104879. doi: 10.1016/j.jbc.2023.104879 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref061] 61.Strecker J-K, Minnerup J, Gess B, Ringelstein EB, Schäbitz W-R, Schilling M. Monocyte chemoattractant protein-1-deficiency impairs the expression of IL-6, IL-1β and G-CSF after transient focal ischemia in mice. PLoS One. 2011;6(10):e25863. doi: 10.1371/journal.pone.0025863 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref062] 62.Saha S, Buttari B, Panieri E, Profumo E, Saso L. An Overview of Nrf2 Signaling Pathway and Its Role in Inflammation. Molecules. 2020;25(22):5474. doi: 10.3390/molecules25225474 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0330287.ref063] 63.Soto-Verdugo J, Ortega A. Critical Involvement of Glial Cells in Manganese Neurotoxicity. BioMed Research International. 2021;2021(1). doi: 10.1155/2021/1596185 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Interaction of coexposure to inorganic arsenic and manganese: Tight junction injury of the blood–brain barrier and the relationship between oxidative stress and inflammatory cytokines in glial cells

Toshiaki Hitomi

Hiroko Okuda

Ayako Takata

Hiroshi Yamauchi

Roles

Abstract

Introduction

Materials and methods

Chemicals

Rat in vitro BBB model and sample collection

Rat cerebral cortical mixed glial cell culture

Cytotoxicity assay

Real-time quantitative PCR

WB analysis

Immunocytochemistry

Statistical analyses

Results

Cytotoxicity, oxidative stress, and inflammatory cytokines in glial cells

Cytotoxicity of iAs and Mn.

Fig 1. Cytotoxicity of iAs or Mn alone and coexposure to iAs and Mn in glial cells.

Oxidative stress induced by iAs and Mn.

Fig 2. Oxidative stress of iAs or Mn alone and coexposure to iAs and Mn in glial cells.

Table 1. Spearman correlation coefficients (ρ) among Nrf2 mRNA, HO-1 mRNA, MCP-1 mRNA, IL-1β mRNA, and IL-6 mRNA expression levels.

Inflammatory cytokines induced by iAs and Mn.

Fig 3. Inflammatory cytokines in iAs and Mn alone groups and iAs and Mn coexposure groups in glial cells.

Relationship between oxidative stress and inflammatory cytokines.

Effect of single or combined exposure to iAs and Mn on BBB TJ injury

Expression of Nrf2 and HO-1 in BBB.

Fig 4. Expression of Nrf2 and HO-1 proteins in the vascular endothelial cell and pericyte groups after exposure to iAs or Mn alone and coexposure to iAs and Mn.

Fig 5. Expression of Nrf2 and HO-1 proteins in astrocytes after exposure to iAs or Mn alone and coexposure to iAs and Mn.

Evaluation of BBB TJ injury by TEER.

Fig 6. Changes in TEER after exposure to iAs or Mn alone, and coexposure to iAs and Mn.

Evaluation of the TJ proteins claudin-5 and ZO-1.

Fig 7. Expression of the tight junction proteins claudin-5 and ZO-1 after exposure to iAs or Mn alone and coexposure to iAs and Mn.

Fig 8. Altered localization and expression of the tight junction protein claudin-5 by iAs or Mn alone and coexposure to iAs and Mn.

Fig 9. Altered localization and expression of the tight junction protein ZO-1 by iAs or Mn alone and coexposure iAs and Mn.

Discussion

Cytotoxicity, oxidative stress, and inflammatory cytokines in glial cells coexposed to iAs and Mn

Cytotoxicity of coexposure to iAs and Mn.

Oxidative stress in coexposure to iAs and Mn.

Inflammatory cytokines in coexposure to iAs and Mn.

Oxidative stress and inflammatory cytokines in interactions.

TJ injury in BBB with coexposure to iAs and Mn.

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Mária Deli

Roles

Author response to Decision Letter 1

Decision Letter 1

Mária Deli

Roles

Acceptance letter

Mária Deli

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases