Evaluation of arsenic metabolism and tight junction injury after exposure to arsenite and monomethylarsonous acid using a rat in vitro blood–Brain barrier model

Hiroshi Yamauchi; Toshiaki Hitomi; Ayako Takata

doi:10.1371/journal.pone.0295154

. 2023 Nov 30;18(11):e0295154. doi: 10.1371/journal.pone.0295154

Evaluation of arsenic metabolism and tight junction injury after exposure to arsenite and monomethylarsonous acid using a rat in vitro blood–Brain barrier model

Hiroshi Yamauchi ^1,^*, Toshiaki Hitomi ¹, Ayako Takata ¹

Editor: Mária A Deli²

PMCID: PMC10688625 PMID: 38032905

Abstract

Experimental verification of impairment to cognitive abilities and cognitive dysfunction resulting from inorganic arsenic (iAs) exposure in children and adults is challenging. This study aimed to elucidate the effects of arsenite (iAs^III; 1, 10 and 20 μM) or monomethylarsonous acid (MMA^III; 0.1, 1 and 2 μM) exposure on arsenic metabolism and tight junction (TJ) function in the blood–brain barrier (BBB) using a rat in vitro-BBB model. The results showed that a small percentage (~15%) of iAs^III was oxidized or methylated within the BBB, suggesting the persistence of toxicity as iAs^III. Approximately 65% of MMA^III was converted to low-toxicity monomethylarsonic acid and dimethylarsenic acid via oxidation and methylation. Therefore, it is estimated that MMA^III causes TJ injury to the BBB at approximately 35% of the unconverted level. TJ injury of BBB after iAs^III or MMA^III exposure could be significantly assessed from decreased expression of claudin-5 and decreased transepithelial electrical resistance values. TJ injury in BBB was found to be significantly affected by MMA^III than iAs^III. Relatedly, the penetration rate in the BBB by 24 h of exposure was higher for MMA^III (53.1% ± 2.72%) than for iAs^III (43.3% ± 0.71%) (p < 0.01). Exposure to iAs^III or MMA^III induced an antioxidant stress response, with concentration-dependent increases in the expression of nuclear factor-erythroid 2-related factor 2 in astrocytes and heme oxygenase-1 in a group of vascular endothelial cells and pericytes, respectively. This study found that TJ injury at the BBB is closely related to the chemical form and species of arsenic; we believe that elucidation of methylation in the brain is essential to verify the impairment of cognitive abilities and cognitive dysfunction caused by iAs exposure.

Introduction

Chronic arsenic poisoning, caused by contamination of drinking water with inorganic arsenic (iAs), has occurred on a large scale in Asian and Latin American countries [1]. Epidemiological studies in Bangladesh [2,3], Taiwan [4], and Mexico [5] have identified the impairment of cognitive abilities of children as an emerging health issue in areas where chronic arsenic poisoning occurs. The WHO has also warned about the effects on cognitive development caused by iAs exposure during the fetal stage and infancy [6]. Furthermore, in non-iAs contaminated areas such as Spain [7] and the USA [8], the effects of dietary iAs on children’s cognitive abilities have also been studied. In this context, the European Food Safety Authority is concerned about the impact of even low concentrations of iAs in rice and processed rice products on the cognitive abilities of young children [9]. On the other hand, cognitive dysfunction in adults in areas known to be affected by chronic arsenic poisoning has also been reported [10–12]. Furthermore, a follow-up study of 23,000 subacute arsenic-poisoning patients who consumed iAs-contaminated powdered milk for three consecutive months while their blood–brain barrier (BBB) was immaturely revealed cognitive dysfunction that manifested when they were older [13]. These studies highlight that cognitive dysfunction from iAs exposure is a problem for children and adults.

Several technical problems exist when conducting experimental validation of findings from epidemiological studies. Animal studies of iAs exposure and central nervous system damage have confirmed that orally administered arsenite (iAs^III) can penetrate the BBB via the blood and migrate to the brain [14,15]. In addition, iAs^III has been reported to cause tight junction (TJ) injury [16] and, subsequently, neurons [17] as it penetrates the BBB. Detailed information on the permeability and TJ injury at the BBB is essential for elucidating how cognitive dysfunction is induced by iAs exposure. However, there are technical difficulties in measuring the quantitative permeability of iAs at the BBB in animal experiments and in assessing TJ injury. An in vitro-BBB model, developed from the triplicate culture of rat [18,19], pig [20,21], and mouse [22] primary brain cells (i.e., vascular endothelial cells, pericytes, and astrocytes), is a promising method for such validation studies. Recent studies have reported the application and effectiveness of the in vitro-BBB model, which enables the prediction of the permeability of different substances at the BBB [23,24]. Previous research results using rat in vitro-BBB models have included silver nanoparticles [25] and dioxin [26]. However, to date, no research on arsenic exposure and TJ injury of BBB have yet used this rat in vitro-BBB model.

It is hypothesized that the health consequences for patients with acute [27] and chronic arsenic poisoning depend on arsenic methylation capacity, which is the efficiency with which iAs are converted to dimethylarsenic acid (DMA^V) [28–30]. iAs are methylated by a methyltransferase (AS3MT) and glutathione to produce monomethylarsonous acid (MMA^III) as the first intermediate metabolite [31,32]. Importantly, MMA^III is considered more toxic than iAs^III [31–33]. On the other hand, MMA^III has been reported to be metabolized in human brain cells, resulting in its conversion to DMA^V [33].

We hypothesize that the impairment of cognitive abilities and cognitive dysfunction caused by iAs exposure occurs in response to TJ injury at the BBB. This is followed by penetration of iAs and its metabolites through the BBB, ultimately leading to glial cell and neuron injury. This study aimed to elucidate the mechanism of TJ injury at the BBB due to iAs^III or MMA^III treatment using a rat in vitro-BBB model.

Materials and methods

Chemicals

iAs^III (arsenite; arsenic trioxide, 99.995% purity, CAS No. 1327-53-3) was purchased from Sigma-Aldrich (St. Louis, MO, USA). MMA^III (CH₅AsO₂, ≥ 95% purity, CAS No. 25400-23-1) was purchased from Toronto Research Chemicals (Toronto, Canada). Arsenic 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 each experiment.

Rat in vitro-BBB model study

A rat in vitro-BBB model comprising three cell types (RBT-24H) was purchased from PharmaCo-Cell Company Ltd [18,19]. This rat in vitro-BBB model is produced by a triple co-culture of primary brain capillary endothelial cells, pericytes, and astrocytes from Wistar rats. In general, rats are not the best model for arsenic clearance studies because of the strong binding properties of rat erythrocytes to arsenic [34,35]. In the present study, we used primary cultured rat brain cells washed with intracerebrovascular blood to ensure that rat erythrocytes do not bind arsenic and affect clearance. The structure of the rat in vitro-BBB model 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 (Fig 1). BBB maturation status was then determined by measuring transendothelial electrical resistance (TEER) (Millicell ERS-2, Merck Millipore, Billerica, MA, USA). To do so, cells were first incubated at 37°C according to the manufacturer’s protocol until the TEER value was 150 Ω × cm² or higher. iAs^III (1, 10, and 20 μM) or MMA^III (0.1, 1, and 2 μM) were exposed in the luminal compartment (i.e., the blood side), and samples taken from the medium in the abluminal compartment (i.e., the brain side) at 6 h and 24 h time points. Samples for Western blot (WB) analysis were collected separately from vascular endothelial cells and pericyte group, or astrocytes. Cells and arsenic-containing solutions were then frozen in liquid nitrogen and stored at −80°C until further analysis.

Fig 1 — (A) Schematic representation of the rat *in vitro* BBB model (luminal compartment; blood side, abluminal compartment; brain side). (B) BBB formation was confirmed by measured transendothelial electrical resistance (TEER) values and by immunofluorescence staining the expression of TJ proteins [claudin-5 and zonula occludens-1 (ZO-1); green, Alexa Fluor 488].

Measurement of BBB intercellular transport

We measured the permeability coefficient of sodium fluorescein (Na-F), a measure of paracellular transport in the rat in vitro-BBB models at 24 h after iAs^III or MMA^III treatment. All measurements were performed according to the manufacturer’s protocol [18,19]. Na-F (10 μg/mL) solution was added to the luminal compartment of the insert, and this insert was then transferred to another 24-well plate containing DPBS-H [10 mM HEPES, 25 mM glucose in Dulbecco’s phosphate-buffered saline (DPBS)]. After incubation at 37°C for 30 min, the concentration of Na-F was measured using a microplate luminometer ARVOx4 2030 Multilabel Reader (Perkin Elmer, Waltham, MA, USA; excitation wavelength: 485 nm, emission wavelength: 535 nm). The permeability coefficient of Na-F was calculated using the following equation:

P_{a p p} (c m / s) = V_{A} / A x {[C]}_{L u m i n a l} x Δ {[C]}_{A b l u m i n a l} / Δ t

Here: P_app, permeability coefficient. V_A, volume of the abluminal chamber; A, membrane surface area; [C]_Luminal, initial luminal tracer concentration; [C]_Abluminal, abluminal tracer concentration; t, time of experiment.

Western blot analysis

For WB analysis, frozen transmembranes containing vascular endothelial cells and pericytes group, and astrocytes were lysed in CelLytic M (Sigma-Aldrich) containing a protease inhibitor cocktail (Nacalai Tesque, Inc.) to extract total protein. After obtaining total protein samples, their concentrations were determined using a CBB solution-based protein assay (Nacalai Tesque, Inc.). Each protein lysate (10 μg/lane) was loaded onto a NuPAGE Novex 3%–8% Tris-acetate gel (Life Technologies Corporation, Carlsbad, CA, USA) and electrophoresed. Protein bands were then transferred to a polyvinylidene fluoride (PVDF) membrane (Invitrogen, Carlsbad, CA, USA) and incubated in Bullet Blocking One (Nacalai Tesque, Inc.) for five minutes at room temperature to block nonspecific binding sites. Membranes were then incubated with the following primary antibodies: rabbit monoclonal anti-nuclear factor-erythroid 2-related factor 2 (Nrf2) (1:1,000, #33649; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit polyclonal anti-heme oxygenase-1 (HO-1) (1:1,000, 10701-1-AP; Proteintech Group, Inc., Chicago, IL, USA), mouse monoclonal anti- claudin-5 (1:1,000, 35–2500; Thermo Fisher Scientific, Rockford, IL, USA), rabbit polyclonal anti-ZO-1 (1:500, 61–7300; Thermo Fisher Scientific) and mouse monoclonal anti-β-actin (1:2,500, A5316; Sigma-Aldrich). All incubations were performed at 4°C and incubated with secondary antibodies (1:5,000, NA9311ML, or NA93401ML; GE Healthcare, Little Chalfont, UK) for 1 h at room temperature. Positive protein bands were enhanced using a chemiluminescence kit ECL Select Western Blotting Detection Reagent (GE Healthcare) or an ECL Prime Western Blotting Detection Reagent (GE Healthcare). Images were then acquired using an Image Quant LAS 4000 (GE Healthcare). Finally, band density quantification was performed using NIH ImageJ (National Institutes of Health, Bethesda, MD, USA).

Immunocytochemistry

We used immunocytochemistry to determine the presence of claudin-5 and ZO-1. For claudin-5, vascular endothelial cells on transwell inserts were first fixed with a 50% acetone/50% methanol (1:1, ice-cold) solution for one minute at room temperature, and for ZO-1, cells were fixed in 3% paraformaldehyde in DPBS (+/+), DPBS with calcium and magnesium, for 10 min at room temperature then treated with 0.1% Triton X-100 in DPBS (+/+) for 10 min at room temperature. Both cell samples were then washed in DPBS (+/+) and blocked overnight at 4°C with 3% BSA in DPBS (+/+). Primary antibodies [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)] were diluted in 0.1% BSA in DPBS then incubated at 37°C for 45 min. After washing, samples were incubated with a secondary antibody (1:200, A28175; Goat-anti-mouse IgG H&L, Alexa Fluor 488, Thermo Fisher Scientific) at 37°C for 45 min. DAPI nuclear staining was performed after washing. Finally, all specimens were imaged using a KEYENCE BZ-X710 fluorescence microscope (Keyence, Inc., Osaka, Japan). Immunofluorescence staining quantification was performed using NIH ImageJ.

Analysis of arsenic by HPLC-ICP-MS

All exposed arsenic samples were collected from the abluminal compartment (brain side) of the 24-well plate (Fig 1), transferred to a Falcon tube, and the volume was increased to 10 mL by adding Milli-Q water. Samples were filtered through 0.45 μm membrane filters (Hillex-HP, PES 33 mm, Non-sterile, Millipore, MA, USA) before analysis. Next, arsenic species in the filtered medium were separated by high-performance liquid chromatography (HPLC, Agilent 1260, Agilent Technologies, Inc., Tokyo, Japan). Separation by HPLC was carried out using a CAPCELL PAK C18 MG column (4.6 × 250 mm, OSAKA SODA, Osaka, Japan) and a mobile phase solvent (10 mmol/L sodium 1-butane sulfonate, 4 mmol/L tetramethylammonium hydroxide, 4 mmol/L malonic acid, 0.05% methanol, adjusted to pH 3.0). For HPLC, the column temperature was maintained at room temperature, the sample injection volume was fixed at 20 μL, and we used a flow rate of 0.75 mL/min. Arsenic was then detected via inductively coupled plasma mass spectrometry (ICP-MS, Agilent 8800, Agilent Technologies, Inc.).

Statistical analyses

Statistical analyses were performed using IBM SPSS Version 25.0 (IBM Corp, Armonk, NY, USA). All results are summarized as mean ± standard deviation (SD). Student’s t-tests were used to assess the statistical significance of differences in mean values of groups, and one-way ANOVAs with Tukey’s or Dunnett’s post hoc tests were used to compare two or more groups. Spearman’s rank correlation coefficient was used to calculate the association between pairs of variables. Finally, a p-value of <0.05 was considered statistically significant for the two-tailed test.

Results

Metabolism and penetration rate of arsenic

The blood side (i.e., the luminal compartment) of the rat in vitro-BBB model was incubated with 20 μM (3,960 ng/mL) iAs^III or 2 μM (248 ng/mL) MMA^III, and arsenic that crossed the BBB after 6 h or 24 h was then collected from the brain side (i.e., the abluminal compartment). Arsenic content was then quantified using HPLC-ICP-MS in different chemical forms. Penetration rate values at the BBB following iAs^III or MMA^III treatment are shown in Table 1. The total penetration rate of MMA^III (23.1% ± 7.76%) at 6 h was approximately twice that of iAs^III (12.9% ± 2.23%), but this difference was not statistically significant. In contrast, a trend toward increased total penetration rate was observed for MMA^III (53.1% ± 2.72%) compared to iAs^III (43.3% ± 0.71%) in the 24 h samples (p < 0.01). Next, we summarize the metabolism of iAs^III or MMA^III shown in Table 1. The final metabolite of iAs^III or MMA^III was DMA^V, but no dimethylarsinous acid was detected. The unconverted rate of iAs^III after 24 h was 84.6%, a characteristic of the low rate of metabolism observed. In contrast, for MMA^III the unconverted rates at 6 h and 24 h were 34.6% and 25.9%, respectively. These results for MMA^III suggested a feature that makes it more readily susceptible to methylation and oxidation reactions than iAs^III. Furthermore, these results suggest important information that MMA^III was not degraded to iAs in the BBB. In this study, iAs^III or MMA^III were oxidized and converted to arsenate (iAs^V) or monomethylarsonic acid (MMA^V), but possible oxidation reactions in the culture medium were also included.

Table 1. Penetration and percentage of arsenic metabolites after a single exposure to iAs^III or MMA^III in a rat in vitro-BBB model.

Arsenic	Time (h)	Penetration rate (%)
		TA	iAs^III	iAs^V	MMA^III	MMA^V	DMA^V
iAs ^III	0–6	12.9±2.23	11.2±1.81	1.11±0.10	n.d.	n.d.	0.90±0.73
			(86.9)	(8.72)			(4.35)
	0–24	43.3±0.71	36.6±1.07	5.75±0.04	n.d.	n.d.	1.42±0.46
			(84.6)	(13.3)			(2.18)
MMA ^III	0–6	23.1±7.76	n.d.	n.d.	7.63±0.79	8.27±1.55	4.03±0.52
					(34.6)	(36.9)	(28.5)
	0–24	53.1±2.72**	n.d.	n.d.	13.7±5.09	27.9±3.59	9.01±0.14
					(25.9)	(52.7)	(21.4)

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Abbreviations for the five arsenic species: arsenite (iAs^III); arsenate (iAs^V); monomethylarsonous acid (MMA^III); monomethylarsonic acid (MMA^V); dimethylarsenic acid (DMA^V). Total arsenic (TA) is the sum of the five arsenic species. The iAs^III or MMA^III doses added to the rat in vitro-BBB model (blood side) were 3,960 ng and 248 ng (as As). Values indicate mean ± SD (n = 3). Values in brackets indicate the percentage of arsenic in total penetration rate. Dimethylarsinous acid was not detected in all samples. Comparison of iAs^III and MMA^III; **p < 0.01. n.d., Not detected.

TJ injury at the BBB following exposure to iAs^III and MMA^III

Evaluation by TEER and Na-F

TJ injury at the BBB after a single exposure to iAs^III or MMA^III was evaluated by %TEER values. These values at 6 h and 24 h showed a statistically significant and concentration-dependent decrease in the iAs^III or MMA^III groups relative to the control group (p < 0.001) (Fig 2). Moreover, treatment with 1μM MMA^III reduced TEER significantly compared with 1μM iAs^III (p < 0.001). Furthermore, this trend was confirmed by treatment with higher concentrations of iAs^III (20 μM) or MMA^III (2 μM) (Fig 2). Our results show that MMA^III produced significantly stronger TJ injury to the BBB than iAs^III. Next, Na-F values (i.e., the permeability coefficient) were measured using the 24 h specimens that were also used for measuring TEER values. Na-F values after a single treatment with high concentrations of 20 μM iAs^III or 2 μM MMA^III were then elevated significantly compared to the control group by approximately threefold (p < 0.05) for 20 μM iAs^III and approximately 16-fold (p < 0.001) for 2 μM MMA^III, respectively (Fig 3). Furthermore, we observed a significant negative correlation between TEER and Na-F values following iAs^III or MMA^III treatment at 24 h (iAs^III, ρ = −0.818, p < 0.001; MMA^III, ρ = −0.847, p < 0.001).

Fig 2 — TEER was measured at 6 h and 24 h after exposure to iAs^III (1, 10, and 20 μM) or MMA^III (0.1, 1, and 2 μM). The %TEER was expressed as 100% of the mean of the control TEER value (100%). Results are presented as mean ± SD (n = 4). Comparisons between control and arsenic-treated groups were then performed using one-way ANOVAs with Tukey’s post hoc tests at a significance level of ***p < 0.001. Student’s t-tests were then used to compare TEER values after 6 h and 24 h treatment, and significance levels are indicated as follows: ^##p < 0.01 and ^###p < 0.001. Finally, a comparison of the iAs^III (1 μM) or MMA^III (1 μM) exposure groups was performed by a one-way ANOVA with Tukey’s post hoc test, with significance levels indicated as ^‡‡‡p < 0.001.

Fig 3 — iAs^III (1, 10, and 20 μM) or MMA^III (0.1, 1, and 2 μM) permeability coefficients (Papp) of Na-F were measured after 24 h of treatment. Results are presented as mean ± SD (n = 4). Comparisons between control and arsenic-treated groups were performed using one-way ANOVAs with Dunnett’s post hoc tests at significance levels of *p < 0.05; ***p < 0.001.

Evaluation using claudin-5 and ZO-1

TJ injury at the BBB following a single treatment with iAs^III or MMA^III was evaluated by measuring the protein expression patterns of claudin-5 and ZO-1 by WB method. iAs^III or MMA^III exposure was found to induce a statistically significant concentration-dependent decrease in claudin-5 expression at both 6 h and 24 h compared to the control group (p < 0.05, 0.01, 0.001) (Fig 4A and 4B). However, the expression of claudin-5 in 1 μM iAs^III and MMA^III showed a decreasing pattern in MMA^III compared to iAs^III at both 6 h and 24 h, but there was no statistically significant difference between the two groups. On the other hand, ZO-1 expression after iAs^III or MMA^III treatment showed a sporadic decreasing trend (Fig 4A and 4C), but there was no statistically significant concentration-dependent decrease in ZO-1 expression at both 6 h and 24 h compared to the control group. In addition, ZO-1 expression after 20 μM iAs^III exposure decreased at 24 h compared to 6 h (p < 0.05). Next, the evaluation of the correlation between the expression patterns of claudin-5 and ZO-1 revealed a significant correlation between them in MMA^III-treated cells (6 h, ρ = 0.847, p < 0.001) but not in iAs^III-treated cells (Table 2).

Fig 4 — (A) WB analysis of claudin-5 and ZO-1 after 6 h and 24 h of iAs^III (1, 10, and 20 μM) or MMA^III (0.1, 1, and 2 μM) exposure. Expression levels of claudin-5 (B) and ZO-1 (C) were quantified by densitometry, standardized to β-actin, and compared with the control value of 1. Results are presented as the mean ± SD of three independent experiments (n = 3). Comparisons between control and arsenic-treated groups were performed using one-way ANOVAs with Tukey’s post hoc tests. The significance levels were: *p < 0.05; **p < 0.01; and ***p < 0.001. Finally, differences in the expression of claudin-5 or ZO-1 at 6 and 24 h were tested using Student’s t-test. The significance levels were: ^#p < 0.05.

Table 2. Spearman correlation coefficients (ρ) among TEER, claudin-5, and ZO-1.

Sample	Time (h)	Claudin-5		TEER
		iAs^III	MMA^III	iAs^III	MMA^III
Claudin-5	6			0.923^***	0.951^***
Claudin-5	24			0.797^**	0.977^***
ZO-1	6	0.371	0.874^***	0.406	0.860^***
ZO-1	24	0.448	0.657^*	0.790^**	0.564

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The measured values of TEER, claudin-5/β-actin, and ZO-1/β-actin were used to analyze rank correlation coefficients.

*p < 0.05

**p < 0.01

***p < 0.001.

Next, TJ injury at the BBB after a single exposure to high concentrations of iAs^III (20 μM) or MMA^III (2 μM) was evaluated by fluorescent immunostaining for the expression of the claudin-5 and ZO-1 proteins. Claudin-5 expression in the control group was observed as a zonal pattern around the plasma membrane. In the iAs^III- and MMA^III- treated groups, a decreased (i.e., sparse and weak) expression of claudin-5 was observed in the regions indicated by red arrows (Fig 5). Claudin-5 expression was decreased at 6 h and 24 h in both the iAs^III- and MMA^III-treated groups. In contrast, ZO-1 expression was mildly decreased following treatment with iAs^III (20 μM) or MMA^III (2 μM) relative to the control group (Fig 5A and 5B). For this phenomenon, quantitative analysis of immunofluorescence staining of claudin-5 and ZO-1 after exposure to iAs^III or MMA^III was performed (Fig 5C and 5D). Claudin-5 expression was found to be significantly reduced compared to the control (p < 0.05, 0.01, 0.001) (Fig 5C), supporting the results of fluorescent immunostaining (Fig 5A). In addition, the decrease in expression tended to be slightly stronger at 24 h than at 6 h (Fig 5C). In contrast, reduction of ZO-1 expression was observed after treatment with iAs^III or MMA^III, as well as claudin-5, compared to the control group (Fig 5B). Quantitative results of ZO-1 expression showed a significant decrease (p < 0.05, 0.001) compared to the control group (Fig 5D), supporting the results of fluorescent immunostaining (Fig 5B). In addition, there was a significant decrease in the expression of ZO-1 (Fig 5D), but it tended to be less specific than claudin-5 (Fig 5C).

Fig 5 — Representative immunofluorescence staining of (A) claudin-5 (green, Alexa Fluor 488) and (B) ZO-1 (green, Alexa Fluor 488), and cell nuclei (blue, DAPI) after 6 h and 24 h exposure to iAs^III (20 μM) or MMA^III (2 μM). Arrowheads indicate typical localization changes due to tight junction (TJ) damage. Scale bars: 20 μm. Quantitative analysis of immunofluorescence staining of claudin-5 (C) and ZO-1 (D) following iAs^III or MMA^III exposure. Four other photographs were obtained from the same membrane as those shown in (A) and (B). The % TJ protein expressed area was expressed as 100% of the mean of each control (100%). Results are presented as mean ± SD (n = 5). Comparisons between control and arsenic-treated groups were performed using one-way ANOVAs with Tukey’s post hoc tests. The significance levels were: *p < 0.05; **p < 0.01; and ***p < 0.001.

We understand that TEER value and claudin-5 reductions are significant indicators of TJ injury in the BBB. In this study, we found a significant correlation between TEER and claudin-5 expression (Table 2), with MMA^III-treated cells showing a significant relationship after 24 h of exposure (24 h, ρ = 0.977, p < 0.001). However, the correlation between TEER and ZO-1 expression was weaker than that between TEER and claudin-5 (Table 2).

Relationship between the Nrf2 and HO-1 expression and TJ injury

We evaluated the expression of Nrf2 in vascular endothelial cells, pericytes (i.e., the EP group), and astrocytes following treatment with iAs^III or MMA^III by the WB method. Exposure to low concentrations of iAs^III (1 μM) or MMA^III (0.1 μM) did not alter Nrf2 expression in the EP group or astrocytes compared with the control (Fig 6A and 6B). The 1μM MMA^III treatment showed a significant increase in Nrf2 expression in astrocytes at 6 h compared to the control (p < 0.01), and these values were significantly higher compared to those of the 1μM iAs^III treatment (p < 0.05). Moreover, exposure to high concentrations of iAs^III (10 and 20 μM) or MMA^III (2 μM) was found to increase Nrf2 expression significantly in both the EP group and astrocytes (p < 0.01, 0.001); this effect was particularly significant in astrocytes at the 6 h time point in both the iAs^III- and MMA^III-treated groups (Fig 6A and 6B). The expression of HO-1 in the EP group and astrocytes after treatment with iAs^III or MMA^III was then evaluated by the WB method. Exposure to iAs^III increased HO-1 expression significantly in both the EP group and astrocytes except treatment group with 1 μM concentration (Fig 6A and 6C). Exposure to MMA^III showed a significant concentration-dependent increase in HO-1 expression in the EP group at 6 h (p < 0.01, 0.001). However, we observed only a slight increase in HO-1 expression in the astrocytes after 6 h and 24 h (Fig 6A and 6C). Moreover, the increased expression of Nrf2 and HO-1 after iAs^III or MMA^III exposure is common, so we analyzed the correlation between the two groups (Table 3). Characteristically, the significant correlation between Nrf2 and HO-1 was stronger in the EP group than in astrocytes and more pronounced for iAs^III (6 h and 24 h AST, p < 0.01, 0.001) than for MMA^III (6 h and 24 h AST, p < 0.05) (Table 3).

Fig 6 — (A) WB analysis of Nrf2 and HO-1 images after 6 h and 24 h exposure to iAs^III (1, 10, and 20 μM) or MMA^III (0.1, 1, and 2 μM). EP: vascular endothelial cells and pericyte group; AST: astrocytes. The expression levels of Nrf2 (B) and HO-1 (C) were quantified by densitometry, standardized to β-actin, and compared to the control value of 1. Results are presented as the mean ± SD of three independent experiments (n = 3). Comparison of control and arsenic-treated groups was performed using one-way ANOVAs with Tukey’s post hoc tests. The significance levels were: *p < 0.05; **p < 0.01; ***p < 0.001. Moreover, Student’s t-tests were used to compare expression between EP and AST, and the significance levels were ^#p < 0.05 and ^##p < 0.01. Student’s t-tests were also used to compare 6 h and 24 h treatments, with significance levels of ^†p < 0.05; ^††p < 0.01. Finally, the comparison of iAs^III (1 μM) or MMA^III (1 μM) exposure groups was performed using one-way ANOVAs with Tukey’s post hoc tests, with a significance level of ^‡p < 0.05.

Table 3. Spearman correlation coefficients (ρ) among TEER, Nrf2, and HO-1.

Sample	Time (h) Cell group	Nrf2		TEER
Sample	Time (h) Cell group	iAs^III	MMA^III	iAs^III	MMA^III
Nrf2	6 h EP			-0.874^***	-0.657^*
	6 h AST			-0.874^***	-0.818^**
	24 h EP			-0.881^***	-0.961^***
	24 h AST			-0.811**	-0.835^***
HO-1	6 h EP	0.825^***	0.825^***	-0.951^***	-0.797^**
	6 h AST	0.874^***	0.678^*	-0.999^***	-0.510
	24 h EP	0.818^***	0.909^***	-0.958^***	-0.947^***
	24 h AST	0.762^**	0.580^*	-0.965^***	-0.821^**

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AST, astrocytes; EP, vascular endothelial cell and pericyte group.

*p < 0.05

**p < 0.01

***p < 0.001.

Next, we analyzed the correlation between the oxidative stress response and BBB TJ injury after iAs^III or MMA^III exposure. We have already discussed that TEER values are adequate indicators of BBB TJ injury (Fig 2). In common with iAs^III and MMA^III exposure, significant negative correlations were identified between Nrf2 and TEER (iAs^III, p < 0.01, 0.001; MMA^III, p < 0.05, 0.01, 0.001) or HO-1 and TEER (iAs^III, p < 0.001; MMA^III, p < 0.01, 0.001) (Table 3). These results were commonly confirmed in the EP group and astrocytes. In other words, these significant negative correlation coefficients suggest a mechanism by which the anti-stress proteins Nrf2 and HO-1 are induced in a concentration-dependent manner against BBB TJ injury due to oxidative stress caused by iAs^III or MMA^III exposure. Until now, it has been technically challenging to verify TJ injury in BBB caused by iAs or their metabolites, but the rat in vitro BBB model has proven to be an effective and easy method for evaluation.

Discussion

We hypothesize that the damage to cognitive ability or cognitive dysfunction caused by exposure to iAs results from an initial TJ injury at the BBB, followed by stepwise injury to glial cells and neurons by iAs and their metabolites penetrating the BBB. In this study, a rat in vitro-BBB model was used to obtain the first information regarding penetration rate, TJ injury, and oxidative stress responses at the BBB following iAs^III and MMA^III exposure, as well as arsenic compounds the methylation and oxidative reactions within the BBB that are related to TJ injury.

Metabolism of iAs^III and MMA^III in the BBB

We understand that iAs are methylated in the liver and converted to MMA^V or DMA^V and that some of the arsenic is then transferred from the blood to brain tissue through the BBB [14,15]. In addition, iAs, MMA^V, and DMA^V detected in brain tissue are recognized as a portion of the blood penetrating the BBB. However, studies suggesting a methylation of iAs in brain tissue are limited. In one experiment, 0.1 μM sodium arsenite (iAs^III) was added to mouse brain slices, which were then observed for 24–72 h, and DMA^V was detected [36]. In another study, 2.5–10 mg/kg/day of sodium arsenite (iAs^III) was administered orally to mice for nine days, after which their brain tissue was evaluated, revealing the presence of DMA^V [37]. Moreover, the methylation of iAs^III and MMA^III in human astrocytes, microglia, and neurons has been demonstrated; however, to date no methylation has been observed in brain microvascular endothelial cells [33]. We predicted that this methylation capacity exists in primary cultured rat brain cells and used a rat in vitro-BBB model to examine the metabolism of iAs^III or MMA^III in the BBB. The structure of the rat in vitro-BBB model (Fig 1) involves a fundamental distinction between the luminal compartment (EP group) and the abluminal compartment (astrocytes region). Different concentrations of iAs^III or MMA^III were exposed to the luminal compartment and recovered from the abluminal compartment. HPLC-ICP-MS was then used to quantify the arsenic species recovered from the abluminal compartment. This experiment provided information on metabolism within the BBB, and penetration rate was also determined from total metabolite values and exposure.

Examination of samples recovered from the abluminal compartment of the rat in vitro-BBB model confirmed that iAs^III is methylated and converted to DMA^V (Table 1); this is consistent with previous analyses in mice. Furthermore, MMA^V was not detected in the abluminal compartment (astrocyte region) in our study. Interestingly, the conversion of iAs^III to MMA^V has not been confirmed in human astrocytes, microglia, or neurons [33]. Although the mechanism of iAs^III methylation in mouse brain tissue is speculative, it has been speculated that AS3MT is involved [36,37]. Although no relevant experiments have been performed in rats, AS3MT is also present in rat brain tissue [38]. Thus, in this study, methylation in the rat in vitro-BBB model is assumed to be an effect of AS3MT. Today, it is known that the metabolism of iAs is dependent on arsenic methylation capacity in patients with acute [27] or chronic arsenic poisoning [28–30]; however, the relationship between arsenic methylation capacity and iAs^III in human, mouse, or rat brain culture cells has not yet been assessed. Furthermore, validation of MMA^III metabolism in mouse brain tissue has yet to be conducted. Interestingly, the results of MMA^III methylation in human astrocytes, microglia, and neurons [33] are consistent with the results of the present study (Table 1). Therefore, methylation of iAs^III and MMA^III in brain tissue is assumed to be a likely mechanism of action in astrocytes. Furthermore, methylation in the rat in vitro-BBB model assumes a mechanism of action similar to the one-carbon metabolism [28–30]. We postulate that methylation of iAs^III and MMA^III in astrocytes may be a protective mechanism for glial cells and neurons. It may represent an independent methylation mechanism in brain tissue that protects against arsenic that has penetrated the BBB.

Next, we note that common oxidation reactions of iAs^III or MMA^III were observed within the BBB. In the present study, the oxidation reactions of iAs^III to iAs^V after exposure were low, with values of 8.72% and 13.3% at 6 h and 24 h, respectively. In contrast, the oxidation of MMA^III to MMA^V after exposure was higher, with values of 36.9% and 52.7% at 6 h and 24 h, respectively. In addition, the oxidation reaction of MMA^III to MMA^V was confirmed by the culture of human astrocytes, microglia, and neurons, but the conversion of iAs^III to iAs^V did not yield a clear result [33]. iAs^V has a lower acute toxicity than iAs^III. We believe that the oxidation reaction within the BBB is a detoxification mechanism similar to methylation. It should be noted that this experiment was not conducted under anaerobic conditions, and therefore other oxidation reactions in the culture medium may have affected the results and should therefore be considered.

In summation, approximately 15% of iAs^III was detoxified by oxidation and methylation, whereas the remaining 85% was highly toxic. Moreover, approximately 65% of MMA^III was detoxified by oxidation and methylation, with the remaining 35% retaining its potent toxicity. Taken together, we conclude from this information that the TJ injury at the BBB results from the toxic effect of unconverted iAs^III (85%) or MMA^III (35%).

TJ injury at the BBB following exposure to iAs^III and MMA^III

Although TEER values are generally used to measure TJ injury in cells, some studies have used TEER values to measure TJ injury in the BBB caused by iAs^III exposure. Recently, no change was reported in TEER values in response to exposure to 10 μM iAs^III in a porcine in vitro-BBB model manufactured from brain capillary endothelial cells [21]. Moreover, in a study using an in vitro blood–cerebrospinal fluid barrier model manufactured from porcine choroid plexus epithelial cells, a decrease in TEER values relative to the control was observed in response to 15 μM iAs^III exposure [39]. In this study, TEER values following treatment with 1 μM MMA^III showed a significant decrease compared with TEER values after 1 μM iAs^III treatment (p < 0.001). In addition, 2 μM MMA^III was found to cause a significant decrease in TEER values compared to 20 μM iAs^III (Fig 2) and a corresponding increase in Na-F values (Fig 3). Elevated Na-F levels suggest that the TJs at the BBB are destroyed and iAs^III or MMA^III penetrates the brain tissue. The rat in vitro-BBB model used in this study might have a broader range of sensitivity and utility than the previously reported in vitro-BBB models [21] and in vitro blood–cerebrospinal fluid barrier model [39].

Next, we investigated the effect of iAs^III or MMA^III exposure on BBB TJ proteins. In general, the membrane protein claudin-5 accumulates at the base of the gating function of BBB TJs [40], and ZO-1 exists as a backing protein [41]. Therefore, it has been hypothesized that a relationship exists between claudin-5 and ZO-1 expression. In the present study, claudin-5 expression was measured following treatment with 1–20 μM iAs^III or 0.1–2 μM MMA^III by the WB method. These data showed a concentration-dependent decrease in claudin-5 expression in both groups (Fig 4A and 4B). In addition, using a fluorescent immunostaining method, we observed claudin-5 expression after treatment with 20 μM iAs^III or 2 μM MMA^III. We found tears and defects in the string-like plasma membrane adhesion structure (Fig 5A). In a recent study, oral administration of iAs^III to mice 50 mg/L (397 μM) for one month decreased claudin-5 expression in the cerebral cortex [42]. Furthermore, in another study, iAs^III (0.15–15 mg/L) was administered orally to pregnant mice, and a decrease in occludin, claudin, ZO-1, and ZO-2 was observed in the brains of their offspring [16,43]. Taken together, these data show similarities between the results of the effect of TJ protein on mouse brain tissue and the results reported here.

This study evaluated the relationship between TJ injury and penetration rate in the BBB. Our TEER and Na-F results, along with the data characterizing claudin-5 and ZO-1 expression, may be evidence of TJ injury in the BBB following exposure to 20 μM iAs^III or 2 μM MMA^III (Figs 2–5). These results indicate that MMA^III treatment expresses TJ injury more intensely than iAs^III treatment. In addition, we observed a higher penetration rate (53.1% ± 2.72%) of MMA^III-treated cells than iAs^III-treated cells at the 24 h time point (43.3% ± 0.71%; p < 0.01; Table 1). To our knowledge, this is the first quantitative finding showing that the penetration rate fluctuates with the degree of TJ injury. However, since this approach is new, we understand the need for further study of rat in vitro-BBB models.

Relationship between the oxidative stress response and TJ injury at the BBB

The activation of Nrf2 is known to inhibit oxidative stress that causes cognitive dysfunction [44], and several studies with anti-stress protein responses to brain neurons by iAs^III exposure have been reported. After oral administration of iAs^III to mice, activation of Nrf2 and HO-1 in the cerebral cortex has been observed [43,45], and a single intraperitoneal injection of iAs^III to mice has also shown activation responses of Nrf2 in the cortex [46]. This study evaluated the impact on Nrf2 and HO-1 expression following TJ injury in the BBB caused by iAs^III or MMA^III exposure (Fig 6). After treatment with iAs^III (10 and 20 μM) or MMA^III (2 μM), Nrf2 expression was increased significantly in astrocytes. Interestingly, HO-1, a downstream enzyme of Nrf2, was expressed in a concentration-dependent manner in the EP group, which is the site of TJ injury in the BBB. Demonstrating that the antioxidant stress response of Nrf2 and HO-1 plays a vital role in BBB TJ injury is an essential outcome of this study. Finally, we believe that the elimination of oxidative stress must also be thoroughly investigated in studies to elucidate cognitive dysfunction induced by iAs^III exposure.

Conclusion

The rat in vitro-BBB model can be a potential alternative to in vivo studies, especially in the study of metabolism and TJ injury in the BBB, as we have confirmed. Previously, iAs^III and MMA^III were found to methylation in human brain cells [33]. In the present rat in vitro-BBB model, the methylation and oxidative reaction mechanisms of iAs^III or MMA^III were observed, and their conversion to low toxicity MMA^V and DMA^V was confirmed, consistent with the results in human brain cells. Interestingly, the TJ injury of BBB was speculated to be the mechanism of unconverted iAs^III or MMA^III, which maintains substantial toxicity. Furthermore, MMA^III is more readily metabolized than iAs^III; however, TJ injury by unconverted MMA^III has been confirmed to be more potent than iAs^III. This study provided results that recognize the importance of further examining the toxic effects of MMA^III, a metabolite of iAs. Future research on iAs exposure-induced impairment of cognitive abilities and cognitive dysfunction should be conducted, with research focusing on metabolism functions at the BBB.

Supporting information

S1 Table. Changes in TEER following iAs^III or MMA^III exposure.

*One-way ANOVAs with Tukey’s post hoc tests between control and arsenic-treated groups. Comparison between iAs^III 1 μM and MMA^III 1 μM-treated group: p < 0.001 (both 6 h and 24 h treatment). ^#Student’s t-tests between 6 h and 24 h treatment.

(XLSX)

Click here for additional data file.^{(15.3KB, xlsx)}

S2 Table. Sodium fluorescein permeability following iAs^III or MMA^III exposure.

*One-way ANOVAs with Dunnett’s post hoc tests between control and arsenic-treated groups.

(XLSX)

Click here for additional data file.^{(13KB, xlsx)}

S3 Table. Expression of claudin-5 and ZO-1 following iAs^III or MMA^III exposure.

*One-way ANOVAs with Tukey’s post hoc tests between control and arsenic-treated groups. Comparison of expression of claudin-5 between iAs^III 1 μM and MMA^III 1 μM-treated group: p = 0.993 and 0.816 (6 h and 24 h treatment); Comparison of expression of ZO-1 between iAs^III 1 μM and MMA^III 1 μM-treated group: p = 0.976 and 0.072 (6 h and 24 h treatment). ^#Student’s t-tests between 6 h and 24 h treatment.

(XLSX)

Click here for additional data file.^{(16.2KB, xlsx)}

S4 Table. Quantitative analysis of immunofluorescence staining of claudin-5 and ZO-1 following iAs^III or MMA^III exposure.

*One-way ANOVAs with Tukey’s post hoc tests between control and arsenic-treated groups. ^#Student’s t-tests between 6 h and 24 h treatment.

(XLSX)

Click here for additional data file.^{(14.5KB, xlsx)}

S5 Table. Expression of Nrf2 and HO-1 following iAs^III or MMA^III exposure.

*One-way ANOVAs with Tukey’s post hoc tests between control and arsenic-treated groups. Comparison of expression of Nrf2 between iAs^III 1 μM and MMA^III 1 μM-treated group: p = 1.000 and 0.044 (EP group; 6 h and 24 h treatment); p = 0.023 and 0.997 (AST group; 6 h and 24 h treatment). Comparison of expression of HO-1 between iAs^III 1 μM and MMA^III 1 μM-treated group: p = 0.327 and 1.000 (EP group; 6 h and 24 h treatment); p = 0.952 and 0.910 (AST group; 6 h and 24 h treatment). ^#Student’s t-tests between 6 h and 24 h treatment. ^†Student’s t-tests between EP group and AST group.

(XLSX)

Click here for additional data file.^{(19KB, xlsx)}

Acknowledgments

The authors would like to thank Dr. Yang Cao for her technical assistance.

Data Availability

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

Funding Statement

This work was supported by JSPS KAKENHI Grant No. JP21H03185. Ayako Takata.” And we report Takata's role. Takata had the roles of Data Curation, Formal Analysis, Investigation, Supervision, Validation, Visualization, and Writing-Review & Editing.

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PLoS One. doi: 10.1371/journal.pone.0295154.r001

Decision Letter 0

Mária A Deli

27 Sep 2023

PONE-D-23-26879PLOS ONE

Evaluation of arsenic metabolism and tight junction injury after exposure to arsenite and monomethylarsonous acid using a rat in vitro blood–brain barrier modelPLOS ONE

Dear Dr. Yamauchi,

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Two experts have evaluated the manuscript. It has merit but needs to be improved. While no new experiment is needed, rewriting of the text, and image analysis of the junctional staining, among others are requested.

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

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Reviewer #1: The manuscript: “Evaluation of arsenic metabolism and tight junction injury after exposure to arsenite and monomethylarsonous acid using a rat in vitro blood–brain barrier model.” Describes the use of an in vitro blood brain barrier model and the impact of two arsenic compounds on the barrier integrity itself and their impact on defined tight junction proteins of the barrier. I really appreciate the model itself as it is a coculture of three highly important cell types of the barrier unit., namely endothelial cells, astrocytes and pericytes. There is on main issue from my side, as the rat is known to metabolise arsenic compounds more effectively, until the dimethylated and pentavalent metabolite DMA(V), that is known to be less toxic compared to for instance the trivalent and monomethylated metabolites. The rat as model to test for arsenic toxicity is not the best suitable one. Please add this into the conclusion. But the overall structure and information of the manuscript is very interesting, the introduction is written clear and leads into the topic, the material and methods section is clear as well. Please add here what constitution the DPBS has and what is the difference between DBPS(+/+) and DBPS. Regarding the sample preparation for the ICP measurements, did you check if you loose analyte after filtration due to binding of the arsenic compounds to the filter. As this is very likely, which filter material has been used? The link for better quality figures leads to wrong figures (e.g. the high quality figure for figure 1 is not available for me, and the one in the manuscript is too blurred). For all figure legends, you only show data +SD not +/-, please correct this. The abbreviation for sodium fluorescein (NaF) is miss-leading, as for chemists it would mean sodium fluoride, please change this. The unit for litre is a big letter (L). Regarding the TEER measurement, at 20 µM iAs(III) and 20 µM MMA(III) the TEER is so long that it can be concluded the barrier is broken, but why is the arsenic quantification in both compartments not the same as there is no barrier apparent anymore? To conclude, after some minor changes, this manuscript should be ready for publication, in my opinion.

Reviewer #2: This manuscript of Yamauchi et al. focuses on an important topic, investigation the tight junction (TJ) impairment of the blood-brain barrier (BBB) caused by arsenic exposure and also the possible metabolic mechanisms behind it. Authors chose proper model for their study, a rat in vitro triple co-culture model of the BBB. The experiments are well designed. However, some part of the manuscript is written poorly which results in confusion. Those parts should be rewritten.

Comments:

1.) The whole “Evaluation using Claudin-5 and ZO-1” paragraph should be rewritten or clarify. Line 296,297.: you claim that there was no difference in claudin-5 expression after treatment with 1 μM iAsIII or 1 μM MMAIII. Before that, it is said that: “iAsIII or MMAIII exposure was found to induce a statistically significant concentration-dependent decrease in Claudin-5 expression…”. If you mean difference between 1 μM iAsIII and MMAIII treatment groups 2-way ANOVA analysis should be used. Also line 299 is confusing. Do you mean treatment groups at 6h time point showed no significant decrease in contrast to claudin-5 expression (Fig. 4B)?

2.) The immunofluorescent staining of TJ proteins should be evaluated by quantification for intensity and morphology.

Minor:

1.) Line 80-81.: “In addition, iAsIII has been reported to HAVE tight junction (TJ) injury…”. Instead of “have” the word “cause” would be a better choice.

2.) Line 126, 129.: For the sake of simplicity, I would write the sample collection from the abluminal compartment for arsenic analysis in one sentence. Sentence in the line 129 is unnecessary.

3.) Figure 1. legend: there are a lot of information (TEER, arsenic exposure) which are not visualized on the figures, hence those are unnecessary. Missing the abbreviation of ZO-1.

4.) Line 148.: “…luminal compartment of the insert portion…” portion is unnecessary.

5.) Line 151.: the sentence is not correct. The concentration of NaF was measured, not the permeability coefficient. Furthermore, please elaborate what is ARVOx4, which wavelength was used.

6.) Table 1.: missing the abbreviations.

7.) Line 236.: dimethylarsinous acid was not mentioned before or explained why it is important.

8.) Figure 4. legend: not the difference in Claudin-5 and ZO-1 expression was tested with Student’s test but the difference in 6h and 24h if I understand correctly. Also “#p” is not indicated in the legend.

9.) In my opinion, using the “i.e.” regarding the concentrations in all the legends is needless.

10.) If claudin-5 is not the beginning of a sentence, it should be written in lowercase.

11.) Figure 6C.: meaning of different columns is missing from the panel. The increase that 1 μM iAsIII treatment caused was not significant (as you say it in line 354-355)?

12.) In Figure 6 related paragraph, please correct the references for the proper figure letter (e.g. line 345.: Fig 6AB; line 354.: Fig 6AC…).

13.) Instead of the word transmittance, “penetration” is more proper.

14.) Below Tables text should be written continuously, not in separate lines.

15.) Line 251.: Instead of (), I would write “Values in brackets indicate…”.

16.) The signs indicating significance (*, #, …) should be positioned above the columns in Figure 2, 3.

17.) Line 275.: The %TEER was expressed as 100% of the mean of control TEER value (100%).

18.) Paragraph from 319-327.: instead of “suppressed” other words should be used, for example decreased.

19.) Line 338.: instead of “owing”, please use “due”.

20.) Line 353, 354.: “…increased HO-1 expression significantly in both the EP group and astrocytes (p < 0.05, 0.01, 0.001)…” I would add to the sentence: except in treatment group with 1 μM concentration.

21.) There is no need to add (p < 0.05, 0.01, 0.001) in the text, only in the legends.

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PLoS One. 2023 Nov 30;18(11):e0295154. doi: 10.1371/journal.pone.0295154.r002

Author response to Decision Letter 0

7 Nov 2023

Confirmed.

We will obey the academic editors and PLOS ONE in all matters.

November 7, 2023

Response to Reviewers

Dear Reviewers,

Thank you for the thoughtful and constructive feedback you provided regarding our manuscript. The following is a point-by-point discussion of our responses to the issues that were raised in the reviews.

Response to Reviewer #1：

We understood Reviewer #1's comment to be 8 points. We have also described our response to each of the comments below.

Comment 1: The rat as model to test for arsenic toxicity is not the best suitable one. Please add this into the conclusion.

Reviewer 1 comments are important knowledge for arsenic researchers to understand, and we do understand them.

We have extensive experience in arsenic metabolism in patients with acute and chronic arsenic poisoning. On the other hand, we also have extensive experience in studying the metabolism, accumulation, and excretion of inorganic and methylated arsenic compounds in animals (hamsters, mice, and rats). We understand from experience that rats are a special kind of animal, with strong binding of arsenic to erythrocytes, making them unsuitable for urinary excretion studies. However, it is widely understood that rats, like hamsters and mice, have no problems studying arsenic metabolism. Furthermore, our study used primary cultured rat brain cells, but the blood was thoroughly washed away.

Next, we added two papers relevant to the reviewers' comments: Vahter and Marafante et al. report the following observations. In rats, urinary excretion is slower than in mammals such as humans, mice, and hamsters because the metabolically produced DMAV is retained in the erythrocytes, and arsenic is stored in the body for a longer time.

34. Vahter M. Biotransformation of trivalent and pentavalent inorganic arsenic in mice and rats. Environ Res. 1981;25(2): 286-93. https://doi.org/10.1016/0013-9351(81)90030-X

PMID: 7274192.

35. Marafante E, Bertolero F, Edel J, Pietra R, Sabbioni E. Intracellular interaction and biotransformation of arsenite in rats and rabbits. Sci Total Environ. 1982; 24(1): 27-39. https://doi.org/10.1016/0048-9697(82)90055-9 PMID: 7112092.

The “Revised Manuscript with Track Changes” describes the revised article.

The following sentence was added.

Corrected sentence. Line 119 to 122.: In general, rats are not the best model for arsenic clearance studies because of the strong binding properties of rat erythrocytes to arsenic [34, 35]. In the present study, we used primary cultured rat brain cells washed with intracerebrovascular blood to ensure that rat erythrocytes do not bind arsenic and affect clearance.

Comment 2: Please add here what constitution the DPBS has and what is the difference between DBPS(+/+) and DBPS

Here is the response to the comment and the revised sentence. DPBS (+/+) is DPBS with additional calcium and magnesium.

Original sentence. Line 186 to 190.: For claudin-5, vascular endothelial cells on transwell inserts were first fixed with a 50% acetone/50% methanol (1:1, ice-cold) solution for one minute at room temperature, and for ZO-1, cells were fixed in 3% paraformaldehyde in DPBS (+/+), DPBS with calcium and magnesium, for 10 min at room temperature then treated with 0.1% Triton X-100 in DPBS (+/+) for 10 min at room temperature.

Corrected sentence. Line 190 to 194.: For claudin-5, vascular endothelial cells on transwell inserts were first fixed with a 50% acetone/50% methanol (1:1, ice-cold) solution for one minute at room temperature, and for ZO-1, cells were fixed in 3% paraformaldehyde in DPBS (+/+), DPBS with calcium and magnesium, for 10 min at room temperature then treated with 0.1% Triton X-100 in DPBS (+/+) for 10 min at room temperature.

Comment 3: Regarding the sample preparation for the ICP measurements, did you check if you loose analyte after filtration due to binding of the arsenic compounds to the filter. As this is very likely, which filter material has been used?

We have experience with this method of arsenic measurement in a wide variety of samples, including human urine, cell culture medium, and natural water. However, we have not applied this method to samples containing high concentrations of protein. We only use samples that can be diluted with Milli-Q water. The measurement of arsenic in human urine using 0.45 µm membrane filters is commonly found among researchers using the HPLC-ICP- MS method.

The 0.45 µm membrane filters are made from the device material, polypropylene; the material, hydrophilic polyethersulfone, can be found in the Millipore Filters product catalog.

In this experiment, the adsorption rate of arsenic on 0.45 µm membrane filters has not been verified in detail using five arsenic mixtures and samples collected from the rat in vitro-BBB model. However, we have previously demonstrated the recovery rate using the standard addition method with human urine and confirmed that it is >90%.

　 Original sentence. Line 203 to 204.: Samples were filtered through 0.45 µm membrane filters (Millipore, MA, USA) before analysis.

Corrected sentence. Line 208 to 209.: Samples were filtered through 0.45 µm membrane filters (Hillex-HP, PES 33 mm, Non-sterile, Millipore, MA, USA) before analysis.

Comment 4: The link for better quality figures leads to wrong figures (e.g. the high quality figure for figure 1 is not available for me, and the one in the manuscript is too blurred).

Figures 1 to 6 have changed to high-quality TIFF files.

Comment 5: For all figure legends, you only show data +SD not +/-, please correct this.

In Figures 2 to 6, ±SD was added to all graph legends.

Comment 6: The abbreviation for sodium fluorescein (NaF) is miss-leading, as for chemists it would mean sodium fluoride, please change this.

NaF is mistaken for sodium fluoride, so we used sodium fluorescein (Na-F). In the manuscript, we changed to Na-F in 13 places.

Relatedly, we have deleted “sodium” in Line 264.

Original sentence. Line 264 to 266.: Next, sodium NaF values (i.e., the permeability coefficient) were measured using the 24 h specimens that were also used for measuring TEER values.

Corrected sentence. Line 269 to 271.: Next, sodium Na-F values (i.e., the permeability coefficient) were measured using the 24 h specimens that were also used for measuring TEER values.

Comment 7: The unit for litre is a big letter (L).

The unit of liter was changed to a large letter (L).

Comment 8: Regarding the TEER measurement, at 20 µM iAs(III) and 20 µM MMA(III) the TEER is so long that it can be concluded the barrier is broken, but why is the arsenic quantification in both compartments not the same as there is no barrier apparent anymore?

It was difficult to understand this reviewer's comment, and we hope we can respond accurately. First of all, the reviewer's comment “20 µM MMA(III)” is mistaken, the correct value is “2 µM MMA(III)”. So, the comment, “but why is the arsenic quantification in both compartments not the same as there is no barrier apparent anymore?” may not be a valid premise. That is exposure of the rat in vitro-BBB model to 20 µM iAs (III) or 2 µM MMA(III) results in different arsenic determination values in both compartments. We hope you will understand our answer.

Response to Reviewer #2：

Reviewer #2. Comments:

In response to Reviewer 2's comment 1), all relevant text was revised as instructed. The revised contents are shown below.

Original sentence. Line 292 to 302.: TJ injury at the BBB following a single treatment with iAsIII or MMAIII was evaluated by measuring the protein expression patterns of Claudin-5 and ZO-1 by WB method. iAsIII or MMAIII exposure was found to induce a statistically significant concentration-dependent decrease in Claudin-5 expression at both 6 h and 24 h compared to the control group (p < 0.05, 0.01, 0.001) (Fig 4A, B). Interestingly, we found no significant difference in Claudin-5 expression following 1 µM iAsIII or 1 µM MMAIII treatment. On the other hand, ZO-1 expression after iAsIII or MMAIII treatment showed a decreasing trend (Fig 4A, C). However, it was not significant compared to Claudin-5 expression. Next, the evaluation of the correlation between the expression patterns of Claudin-5 and ZO-1 revealed a significant correlation between them in MMAIII-treated cells (6 h, ρ = 0.847, p < 0.001) but not in iAsIII-treated cells (Table 2).

Corrected sentence. Line 297 to 310.: TJ injury at the BBB following a single treatment with iAsIII or MMAIII was evaluated by measuring the protein expression patterns of claudin-5 and ZO-1 by WB method. iAsIII or MMAIII exposure was found to induce a statistically significant concentration-dependent decrease in claudin-5 expression at both 6 h and 24 h compared to the control group (p < 0.05, 0.01, 0.001) (Fig 4A, B). However, the expression of claudin-5 in 1 µM iAsIII and MMAIII showed a decreasing pattern in MMAIII compared to iAsIII at both 6 h and 24 h, but there was no statistically significant difference between the two groups. On the other hand, ZO-1 expression after iAsIII or MMAIII treatment showed a sporadic decreasing trend (Fig 4A, C), but there was no statistically significant concentration-dependent decrease in ZO-1 expression at both 6 h and 24 h compared to the control group. In addition, ZO-1 expression after 20 µM iAsIII exposure decreased at 24 h compared to 6 h (p < 0.05). Next, the evaluation of the correlation between the expression patterns of claudin-5 and ZO-1 revealed a significant correlation between them in MMAIII-treated cells (6 h, ρ = 0.847, p < 0.001) but not in iAsIII-treated cells (Table 2).

2.) The immunofluorescent staining of TJ proteins should be evaluated by quantification for intensity and morphology.　

In response to reviewer 2 comment 2), we followed the instructions and added new quantitative immunofluorescence staining results for claudin-5 and ZO-1 after iAsIII or MMAIII exposure (Fig. 5C, D). The following sentence was added.

Corrected sentence. Line 335 to 345.: For this phenomenon, quantitative analysis of immunofluorescence staining of claudin-5 and ZO-1 after exposure to iAsIII or MMAIII was performed (Fig 5C, D). Claudin-5 expression was found to be significantly reduced compared to the control (p < 0.05, 0.01, 0.001) (Fig 5C), supporting the results of fluorescent immunostaining (Fig 5A). In addition, the decrease in expression tended to be slightly stronger at 24 h than at 6 h (Fig 5C). In contrast, reduction of ZO-1 expression was observed after treatment with iAsIII or MMAIII, as well as claudin-5, compared to the control group (Fig 5B). Quantitative results of ZO-1 expression showed a significant decrease (p < 0.05, 0.001) compared to the control group (Fig 5D), supporting the results of fluorescent immunostaining (Fig 5B). In addition, there was a significant decrease in the expression of ZO-1 (Fig 5D), but it tended to be less specific than claudin-5 (Fig 5C).

Relatedly, we have added explanations of Figures 5C and 5D to the legend of Figure 5. The following sentence was added.

Corrected sentence. Line 357 to 363.: Quantitative analysis of immunofluorescence staining of claudin-5 (C) and ZO-1 (D) following iAsIII or MMAIII exposure. Four other photographs were obtained from the same membrane as those shown in (A) and (B). The % TJ protein expressed area was expressed as 100% of the mean of each control (100%). Results are presented as mean ± SD (n = 5). Comparisons between control and arsenic-treated groups were performed using one-way ANOVAs with Tukey’s post hoc tests. The significance levels were: *p < 0.05; **p < 0.01; and ***p < 0.001.

We have also added relevant supplementary text in the Immunocytochemistry section of the Methods. The following sentence was added.

Corrected sentence. Line 202 to 203.: Immunofluorescence staining quantification was performed using NIH ImageJ.　

Minor:

In response to the reviewers' minor comments, we have made deletions, corrections, and additions in 1) through 21).

1.) Line 80-81.: “In addition, iAsIII has been reported to HAVE tight junction (TJ) injury…”. Instead of “have” the word “cause” would be a better choice.

Corrected sentence. Line 81.: Changed to “cause”.

2.) Line 126, 129.: For the sake of simplicity, I would write the sample collection from the abluminal compartment for arsenic analysis in one sentence. Sentence in the line 129 is unnecessary.

The original sentence is shown below, with the exception of “Samples for arsenic analysis were collected from the abluminal compartment of the 24-well plates”. The following changes have been made.

　 Original sentence. Line 126 to 130.: and samples taken from the medium in the abluminal compartment (i.e., the brain side) at 6 h and 24 h time points. Samples for Western blot (WB) analysis were collected separately from vascular endothelial cells and pericyte group, or astrocytes. Samples for arsenic analysis were collected from the abluminal compartment of the 24-well plates.

Corrected sentence. Line 129 to 133.: and samples taken from the medium in the abluminal compartment (i.e., the brain side) at 6 h and 24 h time points. Samples for Western blot (WB) analysis were collected separately from vascular endothelial cells and pericyte group, or astrocytes. Samples for arsenic analysis were collected from the abluminal compartment of the 24-well plates.

3.) Figure 1. legend: there are a lot of information (TEER, arsenic exposure) which are not visualized on the figures, hence those are unnecessary. Missing the abbreviation of ZO-1.

Duplicate descriptions of TEER and arsenic concentrations were deleted.

　 Original sentence. Line 135 to 142.: (B) BBB formation was confirmed by measured transendothelial electrical resistance (TEER) values and by immunofluorescence staining the expression of TJ proteins (Claudin-5 and ZO-1; green, Alexa Fluor 488). Mature BBB kits with TEER values greater than 150 Ω × cm2 were used in the experiments. Before arsenic exposure, Claudin-5 and ZO-1 exhibited continuous linear localization (green, Alexa Fluor 488) at the cell membrane (red arrow). Cell nuclei (blue, DAPI) were similarly identified via immunofluorescence staining. iAsIII (i.e., 1, 10, and 20 µM) or MMAIII (i.e., 0.1, 1, and 2 µM) was exposed in the luminal compartment (blood side).

Corrected sentence. Line 138 to 145.: (B) BBB formation was confirmed by measured transendothelial electrical resistance (TEER) values and by immunofluorescence staining the expression of TJ proteins [claudin-5 and zonula occludens-1 (ZO-1); green, Alexa Fluor 488]. Mature BBB kits with TEER values greater than 150 Ω × cm2 were used in the experiments. Before arsenic exposure, Claudin-5 and ZO-1 exhibited continuous linear localization (green, Alexa Fluor 488) at the cell membrane (red arrow). Cell nuclei (blue, DAPI) were similarly identified via immunofluorescence staining. iAsIII (i.e., 1, 10, and 20 µM) or MMAIII (i.e., 0.1, 1, and 2 µM) was exposed in the luminal compartment (blood side).

And the abbreviation of zonula occludens-1 (ZO-1) was corrected.

Corrected sentence. Line 140.: zonula occludens-1 (ZO-1).

4.) Line 148.: “…luminal compartment of the insert portion…” portion is unnecessary.

Corrected sentence. Line 151.: Deleted “portion”.

5.) Line 151.: the sentence is not correct. The concentration of NaF was measured, not the permeability coefficient. Furthermore, please elaborate what is ARVOx4, which wavelength was used.

Following the instructions, the sentences were changed as follows.

　 Original sentence. Line 150 to 152: After incubation at 37°C for 30 min, the permeability coefficient of NaF was measured using ARVOx4 (Perkin Elmer, Waltham, MA, USA).

Corrected sentence. Line 153 to 156.: After incubation at 37°C for 30 min, the concentration of Na-F was measured using a microplate luminometer ARVOx4 2030 Multilabel Reader (Perkin Elmer, Waltham, MA, USA; excitation wavelength: 485 nm, emission wavelength: 535 nm).

6.) Table 1.: missing the abbreviations.

Abbreviations for the five arsenic species are appended in Table 1. The meaning of “Total” is also added.

Corrected sentence. Line 253 to 255.: Abbreviations for the five arsenic species: arsenite (iAsIII); arsenate (iAsV); monomethylarsonous acid (MMAIII); monomethylarsonic acid (MMAV); dimethylarsenic acid (DMAV). Total is the sum of the five arsenic species.

7.) Line 236.: dimethylarsinous acid was not mentioned before or explained why it is important.

In response to this point, we have the following opinion: dimethylarsenic acid (DMAV) is known to be the final metabolite in patients with acute and chronic arsenic poisoning caused by inorganic arsenic, and we have provided information on these in the Introduction (lines 93-95). And from metabolism experiments using human brain cells, the final metabolite of monomethylarsonous acid (MMAIII) is also DMAV. This information is given in the Introduction (lines 98-99). Against this background, we did not consider it necessary to include information on DMAV in the Results section (line 241).

The reviewer 2 comments are correct, and we made a mistake in the notation of the legend in Fig 4. We have corrected it as follows.

Original sentence. Line 311 to 312.: Finally, the difference in Claudin-5, and ZO-1 expression at 6 h and 24 h was tested using Student’s t-tests.

Corrected sentence. Line 319 to 321.: Finally, differences in the expression of claudin-5 or ZO-1 at 6 and 24 h were tested using Student's t-test. The significance levels were: #p < 0.05.

9.) In my opinion, using the “i.e.” regarding the concentrations in all the legends is needless.

We have removed “i.e.” as noted. Relatedly, we removed “i.e.” regarding the concentrations in the full text.

10.) If claudin-5 is not the beginning of a sentence, it should be written in lowercase.

We changed the text to “claudin-5” as indicated.

11.) Figure 6C.: meaning of different columns is missing from the panel. The increase that 1 μM iAsIII treatment caused was not significant (as you say it in line 354-355)?

The reviewer 2 comment is correct, we did not append a legend to Fig 6C. Fig 6C is newly appended with legends for 6h EP, 6h AST, 24h EP, and 24h AST.

Next, we have corrected it as follows.

　 Original sentence. Line 353 to 354.: Exposure to iAsIII increased HO-1 expression significantly in both the EP group and astrocytes (p < 0.05, 0.01, 0.001) (Fig 6).

Corrected sentence. Line 378 to 380.: Exposure to iAsIII increased HO-1 expression

significantly in both the EP group and astrocytes except treatment group with 1 µM

concentration (Fig 6A, C).

Relatedly, the legend in Fig. 6 was also changed.

　 Original sentence. Line 353 to 354.: The levels statistical significance were as follows:

*p < 0.05; **p < 0.01; ***p < 0.001. Moreover, Student’s t-tests were used to compare expression between EP and AST, and here significance levels were #p < 0.05 and ##p < 0.01.

Corrected sentence. Line 408 to 410.: The significance levels were: *p < 0.05; **p <

0.01; ***p < 0.001. Moreover, Student’s t-tests were used to compare expression

between EP and AST, and the significance levels were #p < 0.05 and ##p < 0.01.

12.) In Figure 6 related paragraph, please correct the references for the proper figure letter (e.g. line 345.: Fig 6AB; line 354.: Fig 6AC…).

Corrected sentence. Line 367 to 383.: The reviewer's comment is correct, we made a mistake in the notation of Fig 6, correcting it to Fig 6A, B or Fig 6A, C, respectively.

13.) Instead of the word transmittance, “penetration” is more proper.

Corrected sentence Lines 50, 230, 235, 236, 238, 251, 252, 257, 424, 448, 500, 518, 522, 525.: Following the instructions, we changed “transmittance” to “penetration or penetration rate” and applied it to the text and Table 1.

Relatedly, we have changed “migrates into” to “penetrates”.

Original sentence. Line 473 to 474.: Elevated NaF levels suggest that the TJs at the

BBB are destroyed and iAsIII or MMAIII migrates into the brain tissue.

Corrected sentence. Line 499 to 500.: Elevated Na-F levels suggest that the TJs at the BBB are destroyed and iAsIII or MMAIII penetrates the brain tissue.

14.) Below Tables text should be written continuously, not in separate lines.

Following the instructions, the text below the table was written continuously.

15.) Line 251.: Instead of (), I would write “Values in brackets indicate…”.

The legend in Table 1 has been changed according to the instructions, and the following sentences have also been adjusted.

Original sentence. Line 251 to 254.:

() values indicate the percentage of arsenic in total transmittance.

n.d., Not detected.

dimethylarsinous acid was not detected in all samples.

Comparison of iAsIII and MMAIII; **, p < 0.01.　

Corrected sentence. Line 257 to 259.: Values in brackets indicate the percentage of arsenic in total penetration rate. Dimethylarsinous acid was not detected in all samples. Comparison of iAsIII and MMAIII; **p < 0.01. n.d., Not detected.　

16.) The signs indicating significance (*, #, …) should be positioned above the columns in Figure 2, 3.

We understood the reviewer 2 comments positively. Because all the significance level signs in Figures 2 and 3 are above the columns. Relatedly, in Figures 4B, C, 5C, D, and 6B, C, the significance level signs are located above the columns as in Figures 2 and 3.

17.) Line 275.: The %TEER was expressed as 100% of the mean of control TEER value (100%).

Followed instructions and corrected.

Corrected sentence. Line 280.: The %TEER was expressed as 100% of the mean of the control TEER value (100%).

18.) Paragraph from 319-327.: instead of “suppressed” other words should be used, for example decreased.　

Changed “suppressed” to “decreased” in three places in paragraphs 319-327 as instructed.

　 Original sentence. Line 319 to 327.: S Next, TJ injury at the BBB after a single exposure to high concentrations of iAsIII (20 µM) or MMAIII (2 µM) was evaluated by fluorescent immunostaining for the expression of the Claudin-5 and ZO-1 proteins. Claudin-5 expression in the control group was observed as a zonal pattern around the plasma membrane. In the iAsIII- and MMAIII- treated groups, a suppressed (i.e., sparse and weak) expression of Claudin-5 was observed in the regions indicated by red arrows (Fig 5). Claudin-5 expression was suppressed at 6 h and 24 h in both the iAsIII- and MMAIII-treated groups. In contrast, ZO-1 expression was mildly suppressed following treatment with iAsIII (20 µM) or MMAIII (2 µM) relative to the control group (Fig 5).

Corrected sentence. Line 327 to 335.: Next, TJ injury at the BBB after a single exposure to high concentrations of iAsIII (20 µM) or MMAIII (2 µM) was evaluated by fluorescent immunostaining for the expression of the claudin-5 and ZO-1 proteins. Claudin-5 expression in the control group was observed as a zonal pattern around the plasma membrane. In the iAsIII- and MMAIII- treated groups, a decreased (i.e., sparse and weak) expression of claudin-5 was observed in the regions indicated by red arrows (Fig 5). Claudin-5 expression was decreased at 6 h and 24 h in both the iAsIII- and MMAIII-treated groups. In contrast, ZO-1 expression was mildly decreased following treatment with iAsIII (20 µM) or MMAIII (2 µM) relative to the control group (Fig 5A, B).

19.) Line 338.: instead of “owing”, please use “due”.

Corrected sentence. Line 356.: Followed the instructions and changed “owing” to “due”.

21.) There is no need to add (p < 0.05, 0.01, 0.001) in the text, only in the legends.

Follow the instructions and answer 20 and 21) together.

Original sentence. Line 353 to 354.: Exposure to iAsIII increased HO-1 expression significantly in both the EP group and astrocytes (p < 0.05, 0.01, 0.001) (Fig 6).

Corrected sentence. Line 378 to 380.: Exposure to iAsIII increased HO-1 expression significantly in both the EP group and astrocytes except treatment group with 1 µM concentration (Fig 6A, C).

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(45.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0295154.r003

Decision Letter 1

Mária A Deli

16 Nov 2023

PONE-D-23-26879; Revised Manuscript, Research article

Evaluation of arsenic metabolism and tight junction injury after exposure to arsenite and monomethylarsonous acid using a rat in vitro blood–brain barrier model

PONE-D-23-26879R1

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PLoS One. doi: 10.1371/journal.pone.0295154.r004

Acceptance letter

Mária A Deli

20 Nov 2023

PONE-D-23-26879R1

Evaluation of arsenic metabolism and tight junction injury after exposure to arsenite and monomethylarsonous acid using a rat in vitro blood–brain barrier model

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 with our production department.

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Associated Data

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

Supplementary Materials

S1 Table. Changes in TEER following iAs^III or MMA^III exposure.

(XLSX)

Click here for additional data file.^{(15.3KB, xlsx)}

S2 Table. Sodium fluorescein permeability following iAs^III or MMA^III exposure.

*One-way ANOVAs with Dunnett’s post hoc tests between control and arsenic-treated groups.

(XLSX)

Click here for additional data file.^{(13KB, xlsx)}

S3 Table. Expression of claudin-5 and ZO-1 following iAs^III or MMA^III exposure.

(XLSX)

Click here for additional data file.^{(16.2KB, xlsx)}

S4 Table. Quantitative analysis of immunofluorescence staining of claudin-5 and ZO-1 following iAs^III or MMA^III exposure.

*One-way ANOVAs with Tukey’s post hoc tests between control and arsenic-treated groups. ^#Student’s t-tests between 6 h and 24 h treatment.

(XLSX)

Click here for additional data file.^{(14.5KB, xlsx)}

S5 Table. Expression of Nrf2 and HO-1 following iAs^III or MMA^III exposure.

(XLSX)

Click here for additional data file.^{(19KB, xlsx)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(45.7KB, docx)}

Data Availability Statement

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

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PERMALINK

Evaluation of arsenic metabolism and tight junction injury after exposure to arsenite and monomethylarsonous acid using a rat in vitro blood–Brain barrier model

Hiroshi Yamauchi

Toshiaki Hitomi

Ayako Takata

Roles

Abstract

Introduction

Materials and methods

Chemicals

Rat in vitro-BBB model study

Fig 1. Structure of the rat in vitro BBB model and experimental setup.

Measurement of BBB intercellular transport

Western blot analysis

Immunocytochemistry

Analysis of arsenic by HPLC-ICP-MS

Statistical analyses

Results

Metabolism and penetration rate of arsenic

Table 1. Penetration and percentage of arsenic metabolites after a single exposure to iAsIII or MMAIII in a rat in vitro-BBB model.

TJ injury at the BBB following exposure to iAsIII and MMAIII

Evaluation by TEER and Na-F

Fig 2. Changes in TEER following iAsIII or MMAIII exposure.

Fig 3. Evaluation of sodium fluorescein (Na-F) permeability caused by iAsIII or MMAIII exposure.

Evaluation using claudin-5 and ZO-1

Fig 4. Expression of the tight junction proteins claudin-5 and ZO-1 following iAsIII or MMAIII exposure.

Table 2. Spearman correlation coefficients (ρ) among TEER, claudin-5, and ZO-1.

Fig 5. Altered localization and expression of the tight junction proteins claudin-5 and ZO-1 followed by iAsIII or MMAIII exposure.

Relationship between the Nrf2 and HO-1 expression and TJ injury

Fig 6. Expression of Nrf2 and HO-1 in the vascular endothelial cell and pericyte group and astrocytes following iAsIII or MMAIII exposure.

Table 3. Spearman correlation coefficients (ρ) among TEER, Nrf2, and HO-1.

Discussion

Metabolism of iAsIII and MMAIII in the BBB

TJ injury at the BBB following exposure to iAsIII and MMAIII

Relationship between the oxidative stress response and TJ injury at the BBB

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Mária A Deli

Roles

Author response to Decision Letter 0

Decision Letter 1

Mária A Deli

Roles

Acceptance letter

Mária A Deli

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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RESOURCES

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Table 1. Penetration and percentage of arsenic metabolites after a single exposure to iAs^III or MMA^III in a rat in vitro-BBB model.

TJ injury at the BBB following exposure to iAs^III and MMA^III

Fig 2. Changes in TEER following iAs^III or MMA^III exposure.

Fig 3. Evaluation of sodium fluorescein (Na-F) permeability caused by iAs^III or MMA^III exposure.

Fig 4. Expression of the tight junction proteins claudin-5 and ZO-1 following iAs^III or MMA^III exposure.

Fig 5. Altered localization and expression of the tight junction proteins claudin-5 and ZO-1 followed by iAs^III or MMA^III exposure.

Fig 6. Expression of Nrf2 and HO-1 in the vascular endothelial cell and pericyte group and astrocytes following iAs^III or MMA^III exposure.

Metabolism of iAs^III and MMA^III in the BBB

TJ injury at the BBB following exposure to iAs^III and MMA^III