Effects on and transfer across the blood-brain barrier in vitro—Comparison of organic and inorganic mercury species
© The Author(s). 2016
Received: 22 July 2016
Accepted: 19 November 2016
Published: 15 December 2016
Transport of methylmercury (MeHg) across the blood-brain barrier towards the brain side is well discussed in literature, while ethylmercury (EtHg) and inorganic mercury are not adequately characterized regarding their entry into the brain. Studies investigating a possible efflux out of the brain are not described to our knowledge.
This study compares, for the first time, effects of organic methylmercury chloride (MeHgCl), EtHg-containing thiomersal and inorganic Hg chloride (HgCl2) on as well as their transfer across a primary porcine in vitro model of the blood-brain barrier.
With respect to the barrier integrity, the barrier model exhibited a much higher sensitivity towards HgCl2 following basolateral incubation (brain-facing side) as compared to apical application (blood-facing side). These HgCl2 induced effects on the barrier integrity after brain side incubation are comparable to that of the organic species, although MeHgCl and thiomersal exerted much higher cytotoxic effects in the barrier building cells. Hg transfer rates following exposure to organic species in both directions argue for diffusion as transfer mechanism. Inorganic Hg application surprisingly resulted in a Hg transfer out of the brain-facing compartment.
In case of MeHgCl and thiomersal incubation, mercury crossed the barrier in both directions, with a slight accumulation in the basolateral, brain-facing compartment, after simultaneous incubation in both compartments. For HgCl2, our data provide first evidence that the blood-brain barrier transfers mercury out of the brain.
KeywordsOrganic mercury Inorganic mercury Methylmercury Thiomersal Mercuric mercury In vitro blood-brain barrier model
Mercury (Hg) is present in the environment due to both natural sources and anthropogenic activity. It exists as elemental Hg as well as inorganic and organic compounds, all incorporating different toxicological properties [1, 2]. Human exposure to organic species mainly results from the consumption of contaminated fish or seafood in the form of methylmercury (MeHg), the most frequently occurring organic species in the aquatic food chain . The use of the ethylmercury (EtHg) containing preservative thiomersal in medical preparations, including vaccines, represents a non-dietary route of human exposure towards organic Hg species . In contaminated terrestrial food, Hg is mainly present as inorganic Hg . In 2012, in accordance to the Joint FAO/WHO Expert Committee on Food Additives (JECFA)  the European Food Safety Authority (EFSA) Panel on Contaminants in the Food Chain established a tolerable weekly intake (TWI) of 4 μg/kg body weight (b.w.) for inorganic Hg . Based on new epidemiological data, the EFSA Panel reevaluated for MeHg the provisional tolerable weekly intake (PTWI) of 1.6 μg/kg b.w. (JECFA ) and established a TWI of 1.3 μg/kg b.w.. High fish consumers may exceed this TWI by up to six fold .
The central nervous system (CNS) represents the major target organ of organic Hg species exposure . Thiomersal shows a higher or at least similar toxicity compared to MeHg in brain associated cells in vitro (e.g. [8, 9]). However, the toxic potential of MeHg under in vivo conditions seems to be higher, which might be a consequence of different disposition kinetics .
The transport of MeHg across the blood-brain barrier to the brain side is well described in literature, while EtHg and inorganic Hg are not adequately characterized regarding their entry into the brain. Based on in vitro as well as in vivo studies an active transport mechanism of MeHg as a cysteine complex (MeHg-S-Cys) across the blood-brain barrier via the L-type neutral amino acid transport (LAT) system has been proposed [11–14]. It has to be noted that the transport of MeHg across the blood-brain barrier involves both, uptake into and efflux from brain endothelial cells. Heggland et al. postulated that LAT does not seem to be involved in the efflux of MeHg in vitro but that MeHg is transferred out of brain endothelial cells as a complex with glutathione (GSH), thereby using GSH-transporters . In 2013 Zimmermann et al. postulated a LAT system based uptake of MeHg-S-Cys as well as EtHg-S-Cys into C6 glioma cells, whereas MeHg and EtHg enter the cells by other mechanisms . Besides this active, energy dependent transfer of organic Hg species, transfer mechanisms can be passive depending on the Hg species . Following intramuscular injection of 20 μg thiomersal in mice, EtHg as well as inorganic Hg were identified in brain tissue indicating that EtHg can pass the blood-brain barrier . In vivo, a rapid metabolism of EtHg to inorganic Hg is described [10, 19]. Thus, because of a possible dealkylation of the organic compounds as well as an oxidation of elemental Hg, e.g. resulting from the outgassing of amalgam fillings, inorganic Hg has to be taken into account, when investigating mechanisms of Hg species dependent transfer into/out of the brain and Hg species induced neurotoxicity [20, 21]. Toimela et al. demonstrated transfer differences between mercury chloride (HgCl2) and methylmercury chloride (MeHgCl) within an in vitro blood-brain barrier model composed of rat brain endothelial cells accompanied with glia cells and neuronal SH-SY5Y cells as target cells . The authors concluded from cytotoxic effects on the neuronal cells in the brain-facing compartment that MeHgCl passed the barrier model because it exerted cytotoxic effects on the neuronal cells, while barrier-building cells were not affected. HgCl2 did not cause any toxicity to neuronal cells, as long the barrier cells were not affected .
In the present study, a well-characterized primary porcine in vitro blood-brain barrier model was applied to compare the effects of MeHgCl, thiomersal and HgCl2 on the barrier as well as to characterize their transfer properties across this barrier model. In contrast to existing studies, we focused on both transfer into as well as transfer out of the brain.
Primary blood-brain barrier cell culture model
Isolation, cultivation and cryopreservation of primary porcine brain capillary endothelial cells (PBCECs) from brain tissue of freshly slaughtered pigs (from the slaughterhouse) was performed as previously described [23, 24]. On day in vitro (DIV) 2, PBCECs were thawed and seeded on rat tail collagen-coated Transwell® filter inserts with microporous polycarbonate membranes (1.12 cm2 growth area, 0.4 μM pore size; Corning, Wiesbaden, Germany) in plating medium (Medium 199 Earle supplemented with 10% newborn calf serum, 0.7 μM L-glutamine, 100 μg/mL gentamycin, 100 U/mL penicillin, 100 μg/mL streptomycin (all Biochrom, Berlin, Germany)) according to literature . After 2 days of proliferation (DIV 2–4) PBCECs reached confluence and differentiation was induced by replacing the plating medium with serum-free culture medium (Dulbecco’s modified Eagle’s medium/Ham’s F12 (1:1) with 4.1 mM L-glutamine, 100 μg/mL gentamycin, 100 U/mL penicillin, 100 μg/mL streptomycin (Biochrom) and 550 nM hydrocortisone (Sigma Aldrich, Deisenhofen, Germany)) . According to manufacturer’s information, this medium contains 15.75 mg/L (100 μM) L-cysteine*HCl and 17.24 mg/L (115 μM) L-methionine. All experiments were started after another 2 days of differentiation (DIV 6). The PBCECs built up a tight monolayer on the rat tail collagen-coated polycarbonate membranes comparable to the epithelium of a brain microvessel. The collagen coating on the filter corresponds to the basal membrane in vivo. Epithelial cells seeded on this surface in vitro develop tight junctions ensuring the polarity between apical and basolateral cell membranes. Thus, in this fully developed in vitro model of the blood-brain barrier the apical (upper) compartment refers to the lumen of the vessel in vivo and therefore mimics the blood side. Vice versa, the basolateral compartment represents the parenchymal side of the blood-brain barrier and mimics the brain side in vivo .
Preparation of Hg species stock solutions
Stock solutions of MeHgCl (>99.9% purity, Sigma Aldrich, Deisenhofen, Germany), thiomersal (>97% purity, Sigma Aldrich) and HgCl2 (>99.999% purity, Sigma Aldrich) were prepared in sterile distilled water shortly before each experiment. Thiomersal is well known to release ethylmercury (EtHg) in aqueous solutions .
For the evaluation of cytotoxic effects of the Hg species on PBCECs the neutral red uptake assay was performed to quantify the lysosomal integrity. This endpoint has been shown before to be both applicable to assess viability of compounds in PBCECs in general  and to be suitable to assess cytotoxicity of mercury species e.g. in human astrocytes . The cellular uptake of neutral red depends on the cell’s capacity to maintain pH gradients, which strongly depends on the cellular ATP level . Fonfria et al. demonstrated both, decreased intracellular ATP levels as well as decreased mitochondrial activity, in murine cerebellar granule cells following incubation of high MeHgCl and HgCl2 concentrations , providing additional evidence that lysosomal integrity is a suitable marker to assess Hg species induced cytotoxicity. Briefly, PBCECs were cultivated in rat tail collagen-coated 96 well culture plates under the same conditions as the cells seeded on Transwell® filter inserts. Cells were exposed to the respective Hg species on DIV 6. After 72 h the neutral red uptake assay was carried out according to literature .
PBCECs were cultivated in rat tail collagen-coated 24 well culture plates and cultured according to the cultivation in Transwell® filters. After 72 h incubation of the respective Hg species, PBCECs were washed twice with PBS (100 mM NaCl, 4.5 mM KCl, 7 mM Na2HPO4, 3 mM KH2PO4 (all Sigma Aldrich); pH 7.4) and incubated with 120 μL lysis buffer (RIPA-buffer; 0.01 M Tris, pH 7.6, 0.15 M NaCl, 0.001 M EDTA, 1% sodium desoxycholate, 0.1% (all Sigma Aldrich)) for 15 min on ice. After scrapping off and sonication, the suspension of lysed cells was centrifuged at 10 000 x g for 20 min at 4 °C. Total cellular Hg content was quantified by inductively coupled mass spectrometry (ICP-MS; Agilent 8800 ICP-QQQ, Agilent Technologies Deutschland GmbH, Boeblingen, Germany) in an aliquot of the supernatant. The Bradford assay was used to determine the cellular protein level.
Barrier integrity, capacitance and transfer measurements
The organic (0.01–3 μM) and the inorganic (0.01–100 μM) Hg species were applied on DIV 6 either to the apical (blood-facing) or to the basolateral (brain-facing) compartment of the in vitro blood-brain barrier model or to both compartments simultaneously by replacing 10% of the culture medium with fresh Hg species containing medium in the respective compartment. Barrier integrity was monitored during 72 h of incubation by measurement of the transendothelial electrical resistance (TEER) using the cellZscope (nanoAnalytics, Münster, Germany). Furthermore, the capacitance, which is related to the plasma membrane surface area, was recorded online (cellZscope; nanoAnalytics, Münster, Germany). Wells with TEER values of ≥ 600 Ω x cm2 and a capacitance of 0.45–0.6 μF/cm2 were used for the experiments providing a confluent PBCEC monolayer with tight barrier properties. Standard deviation of TEER values was < 18% and for capacitance values < 11% (not shown).
For transfer studies aliquots of both compartments were taken after 0, 3, 6, 24, 48 and 72 h in case of incubation on one side. In case of application on both sides simultaneously the first sample was collected after 1.5 h. The total Hg amount in these samples was quantified by ICP-MS. Hg transfer was calculated as % in relation to the total Hg content in both compartments.
For quantification of the total Hg content by ICP-MS, aliquots of the transfer studies and cellular bioavailability studies were diluted in a solution of 5% HNO3 + 5% HCl (v/v, both suprapur, Merck KGaA). An external calibration (1–150 ng/L; Hg standard for ICP, TraceCERT®, Fluka, Deisenhofen, Germany) was prepared in the same solution. Samples and calibration standards were incubated with rhodium (final concentration 10 ng/L; Merck KGaA, Darmstadt, Germany) as internal standard. A MicroMist nebulizer was used for sample introduction and gas flows were applied as follows: 15 L/min cool gas, 0.9 L/min auxiliary gas and 1 L/min nebulizer gas. The method exhibits a limit of detection of 0.8 ng/L and a limit of quantification of 2.9 ng/L calculated by the calibration method of the German Standard DIN standard 32645 .
Cytotoxicity and bioavailability in primary PBCECs
Cellular bioavailability of MeHgCl, thiomersal and HgCl2 in PCPECs after 72 h incubation
[μg Hg/mg protein]
0.010 ± 0.004
0.135 ± 0.051
0.807 ± 0.059
0.021 ± 0.002
0.084 ± 0.023
0.511 ± 0.107
0.004 ± 0.000
0.010 ± 0.002
0.047 ± 0.033
Evaluation of the barrier integrity and the capacitance
The applied well-characterized cell culture model of the blood-brain barrier is built up by fully differentiated PBCECs cultivated on Transwell® filter inserts between two fluid compartments filled with cell culture medium [23, 24]. The upper apical compartment refers to the blood side in vivo, whereas the lower basolateral compartment represents the brain side. The impact of the mercury species on the barrier integrity was assessed by online monitoring of the transendothelial electrical resistance (TEER) during the entire transfer experiment, since the electrical resistance correlates with the tightness of the barrier.
After basolateral incubation (brain-facing side) the barrier integrity is irreversibly affected at 3 μM MeHgCl, 1 μM thiomersal and 1 μM HgCl2 (Fig. 2d – f). 1 and 2 μM MeHgCl strongly decreased TEER values to 15–35% but allowed a reconstitution of the barrier integrity to about 70%.
Transfer across the in vitro model of the blood-brain barrier
Transfer across the blood-brain barrier was assessed at 0.01, 0.1 and 1 μM of the Hg species. The barrier tightness was not affected by these concentrations, except for a basolateral incubation with 1 μM thiomersal or HgCl2.
The primary target organ of organic Hg species mediated toxicity is the CNS . In literature, the understanding of transfer mechanisms into the brain is limited to MeHg; first evidence exists for a comparable carrier mediated transfer mechanism for EtHg [11, 16]. To our knowledge, a possible efflux of organic and inorganic mercury species out of the brain side has yet not been evaluated. Although inorganic Hg does not seem to be able to cross the blood-brain barrier , it is present in the brain due to dealkylation of organic species or an oxidation of elemental Hg [18, 21]. Thus, the elucidation of an efflux of inorganic Hg besides the organic compounds is of special interest.
According to previous cytotoxicity studies in a human astrocytoma cell line , organic MeHgCl and thiomersal exerted in the present study stronger cytotoxic effects in the barrier building PBCECs as compared to inorganic HgCl2. The bioavailability studies, performed in subcytotoxic but transfer relevant concentrations, give evidence that this enhanced cytotoxicity is a result of a higher cellular Hg content following exposure to the organic compounds.
The TEER measurements clearly demonstrate differences in barrier integrity following basolateral incubation (brain side) as compared to apical (blood side) incubation. Barrier leakage caused by 3 μM of the organic and 100 μM of the inorganic species applied in the apical compartment correlated with the respective cytotoxic effects. In contrast, after basolateral application, the barrier was more sensitive towards all mercury species, but especially to inorganic HgCl2. This enhanced sensitivity of the barrier integrity towards basolateral HgCl2 application is not in line with the results of the cytotoxicity and bioavailability data, showing more than 10 fold higher cellular Hg concentrations after exposure to the organic species than to HgCl2.
Since in literature most studies investigating Hg mediated neurotoxicity focus on MeHg or EtHg, the effects of inorganic Hg species on brain endothelial cells are poorly understood. Oppedisano et al. demonstrated a more effective inhibition of the glutamine/amino acid transporter (ASCT2) , a carrier located at the abluminal site of the blood-brain barrier with brain to endothelium orientation, by HgCl2 as compared to MeHgCl . Fonfria et al. reported an alteration in neuronal glutamate transport associated with the excitatory amino acid transporter (EAAT3), a transporter with the same orientation as ASCT2 , induced by HgCl2 . It might be possible, at least because of steric advantage of the Hg2+ ion as compared to organic Hg species, that inorganic Hg ions generally exert stronger effects on transporters located to the abluminal side of the blood-brain barrier. Thus, a disruption of the barrier tightness following basolateral incubation might be a consequence of disturbed transport properties and homeostasis. This hypothesis needs to be proven in further studies.
Our results indicate a Hg transfer following organic MeHgCl and thiomersal exposure across the in vitro model of the blood-brain barrier towards the basolateral side of the endothelial cells. This is in contrast to inorganic HgCl2, which does not seem to be transferred towards the basolateral compartment in the case of an intact barrier. These results correspond to different in vivo studies, postulating a transfer of organic species into the brain [10, 18]. Since the respective culture media in the present study contains cysteine, a complex formation of organic species with cysteine is conceivable. These complexes have been shown before to enter the brain via a neutral amino acid carrier by mimicking the structure of methionine. Nevertheless, it has also been taken into account that the medium contains substantial levels of methionine, which has been discussed to disturb the transport of the respective organic mercury cysteine complexes [11, 16]. We can also not fully exclude that the applied lipophilic organic Hg species are not fully complexed to thiols and thus are capable to transfer across cell barriers by diffusion, whereas the hydrophilic inorganic HgCl2 is not expected to diffuse through cell membranes [11, 12]. Since total Hg blood concentrations of 0.6–30 μg/L (0.03–0.15 μM) are related to dietary Hg exposure in a population group with a wide range of seafood consumption and Hg is mainly present as MeHg in marine food , the application of 0.01 and 0.1 μM represent concentrations of physiological relevance. Burbacher et al. reported total blood Hg concentrations of 8–18 ng/mL (0.04–0.09 μM) 2 days after oral exposure of 20 μg/kg MeHg as MeHg hydroxide in infant monkeys. The intramuscular injection of one single thiomersal dose in humans (20 μg/kg) led to total blood Hg concentrations of 6–14 ng/L (0.03–0.07 μM) . The concentrations of HgCl2 used are of experimental importance to suggest that Hg2+ apparently tends to transfer more easily from the basolateral to the apical side of the blood-brain barrier model than from the apical to the basolateral side.
In a second approach, we investigated for the first time Hg transfer from the basolateral side to the apical side of the PBCECs (efflux). After application of the organic compounds in the basolateral compartment, a Hg transfer out of the brain-facing compartment was evident. Nevertheless, efflux rates were lower as compared to influx rates. Surprisingly, Hg transfer data after basolateral HgCl2 incubation indicate for an Hg efflux. These results are in accordance to the simultaneous application on both sides. Whereas the simultaneous application of organic species led to a slight accumulation on the basolateral side within the first 48 h, the inorganic species clearly accumulated in the apical compartment. Since a dealkylation of organic species in the brain, especially the in vivo observed rapid conversion of EtHg to inorganic Hg , as well as the oxidation of elemental Hg result in the presence of inorganic Hg in the brain, these findings might indicate a possible detoxification mechanism in Hg mediated neurotoxicity. Based on the efflux of inorganic Hg, the blood-brain barrier might protect the target organ brain from Hg induced neurotoxic damage. The results of the simultaneous incubation of the organic compounds argue for diffusion as transfer mechanism. According to uptake studies in brain endothelial cells  an active carrier mediated transport to the brain side as well as to the blood side can be excluded for MeHgCl and thiomersal.
Applying a well-characterized primary blood-brain barrier model, mercury influx and efflux were assessed, after exposure towards organic MeHgCl, thiomersal, and inorganic HgCl2. Our data indicate for the organic mercury species a transfer in both directions, with a slight accumulation on the basolateral side of the endothelial cells (brain side), thereby arguing for diffusion as transfer mechanism. HgCl2 was not able to cross the in vitro barrier towards the basolateral side, but partially transferred out of the basolateral compartment following basolateral incubation. Thus, the blood-brain barrier might play a role in preventing the target organ brain from Hg induced neurotoxic effects.
Central nervous system
Day in vitro
European Food Safety Authority
Joint FAO/WHO Expert Committee on Food Additives
MeHg as a cysteine complex
Primary porcine brain capillary endothelial cells
Provisional tolerable weekly intake
Transendothelial electrical resistance
Tolerable weekly intake
Availability of data and materials
All data generated or analysed during this study are included in this published article.
HL participitated in the design and coordination of the study, carried out the efflux studies and drafted the manuscript. JB participated in its design and coordination and prepared the PBCECs for the in vitro blood-brain barrier model. RF carried out the cytotoxicity and influx studies within her master thesis. GP carried out the ICP-MS analysis. HJG provided the theoretical and practical requirements for the preparation and cultivation of PBCECs within the in vitro blood-brain barrier model. TS designed and coordinated this study and helped to draft the manuscript. All authors read and approved the final manuscript.
The author(s) declare that they have no competing interests.
Consent of publication
Ethics approval and consent to participate
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Rice KM, Walker Jr EM, Wu M, Gillette C, Blough ER. Environmental mercury and its toxic effects. J Prev Med Public Health. 2014;47:74–83.View ArticlePubMedPubMed CentralGoogle Scholar
- FAO/WHO. Safety evaluation of certain food additives and contaminants. Mercury. 2011;63:605–85. WHO Food Additives Series.Google Scholar
- Risher JF, Murray HE, Prince GR. Organic mercury compounds: human exposure and its relevance to public health. Toxicol Ind Health. 2002;18:109–60.View ArticlePubMedGoogle Scholar
- Geier DA, King PG, Hooker BS, Dorea JG, Kern JK, Sykes LK, Geier MR. Thimerosal: Clinical, epidemiologic and biochemical studies. Clin Chim Acta. 2015;444:212–20.View ArticlePubMedGoogle Scholar
- EFSA. Scientific Opinion on the risk for public health related to the presence of mercury and methylmercury in food. EFSA J. 2012;10:1–241.Google Scholar
- FAO/WHO. Safety evaluation of certain food additives and contaminants. Methylmercury. 2007;58:269–315. WHO Food Additives Series.Google Scholar
- Farina M, Avila DS, da Rocha JB, Aschner M. Metals, oxidative stress and neurodegeneration: a focus on iron, manganese and mercury. Neurochem Int. 2013;62:575–94.View ArticlePubMedGoogle Scholar
- Pieper I, Wehe CA, Bornhorst J, Ebert F, Leffers L, Holtkamp M, Hoseler P, Weber T, Mangerich A, Burkle A, Karst U, Schwerdtle T. Mechanisms of Hg species induced toxicity in cultured human astrocytes: genotoxicity and DNA-damage response. Metallomics. 2014;6:662–71.View ArticlePubMedGoogle Scholar
- Zimmer B, Lee G, Balmer NV, Meganathan K, Sachinidis A, Studer L, Leist M. Evaluation of developmental toxicants and signaling pathways in a functional test based on the migration of human neural crest cells. Environ Health Perspect. 2012;120:1116–22.View ArticlePubMedPubMed CentralGoogle Scholar
- Burbacher TM, Shen DD, Liberato N, Grant KS, Cernichiari E, Clarkson T. Comparison of blood and brain mercury levels in infant monkeys exposed to methylmercury or vaccines containing thimerosal. Environ Health Perspect. 2005;113:1015–21.View ArticlePubMedPubMed CentralGoogle Scholar
- Aschner M, Aschner JL. Mercury neurotoxicity: mechanisms of blood-brain barrier transport. Neurosci Biobehav Rev. 1990;14:169–76.View ArticlePubMedGoogle Scholar
- Zheng W, Aschner M, Ghersi-Egea JF. Brain barrier systems: a new frontier in metal neurotoxicological research. Toxicol Appl Pharmacol. 2003;192:1–11.View ArticlePubMedPubMed CentralGoogle Scholar
- Kanai Y, Endou H. Functional properties of multispecific amino acid transporters and their implications to transporter-mediated toxicity. J Toxicol Sci. 2003;28:1–17.View ArticlePubMedGoogle Scholar
- Yin Z, Jiang H, Syversen T, Rocha JB, Farina M, Aschner M. The methylmercury-L-cysteine conjugate is a substrate for the L-type large neutral amino acid transporter. J Neurochem. 2008;107:1083–90.PubMedPubMed CentralGoogle Scholar
- Heggland I, Kaur P, Syversen T. Uptake and efflux of methylmercury in vitro: comparison of transport mechanisms in C6, B35 and RBE4 cells. Toxicol In Vitro. 2009;23:1020–7.View ArticlePubMedGoogle Scholar
- Zimmermann LT, Santos DB, Naime AA, Leal RB, Dorea JG, Barbosa Jr F, Aschner M, Rocha JB, Farina M. Comparative study on methyl- and ethylmercury-induced toxicity in C6 glioma cells and the potential role of LAT-1 in mediating mercurial-thiol complexes uptake. Neurotoxicology. 2013;38:1–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Syversen T, Kaur P. The toxicology of mercury and its compounds. J Trace Elem Med Biol. 2012;26:215–26.View ArticlePubMedGoogle Scholar
- Carneiro MF, Oliveira Souza JM, Grotto D, Batista BL, de Oliveira Souza VC, Barbosa Jr F. A systematic study of the disposition and metabolism of mercury species in mice after exposure to low levels of thimerosal (ethylmercury). Environ Res. 2014;134C:218–27.View ArticleGoogle Scholar
- Barregard L, Rekic D, Horvat M, Elmberg L, Lundh T, Zachrisson O. Toxicokinetics of mercury after long-term repeated exposure to thimerosal-containing vaccine. Toxicol Sci. 2011;120:499–506.View ArticlePubMedGoogle Scholar
- Rodrigues JL, Serpeloni JM, Batista BL, Souza SS, Barbosa Jr F. Identification and distribution of mercury species in rat tissues following administration of thimerosal or methylmercury. Arch Toxicol. 2010;84:891–6.View ArticlePubMedGoogle Scholar
- Henriksson J, Tjalve H. Uptake of inorganic mercury in the olfactory bulbs via olfactory pathways in rats. Environ Res. 1998;77:130–40.View ArticlePubMedGoogle Scholar
- Toimela T, Maenpaa H, Mannerstrom M, Tahti H. Development of an in vitro blood-brain barrier model-cytotoxicity of mercury and aluminum. Toxicol Appl Pharmacol. 2004;195:73–82.View ArticlePubMedGoogle Scholar
- Franke H, Galla H, Beuckmann CT. Primary cultures of brain microvessel endothelial cells: a valid and flexible model to study drug transport through the blood-brain barrier in vitro. Brain Res Brain Res Protoc. 2000;5:248–56.View ArticlePubMedGoogle Scholar
- Kroll S, El-Gindi J, Thanabalasundaram G, Panpumthong P, Schrot S, Hartmann C, Galla HJ. Control of the blood-brain barrier by glucocorticoids and the cells of the neurovascular unit. Ann N Y Acad Sci. 2009;1165:228–39.View ArticlePubMedGoogle Scholar
- Bornhorst J, Wehe CA, Huwel S, Karst U, Galla HJ, Schwerdtle T. Impact of manganese on and transfer across blood-brain and blood-cerebrospinal fluid barrier in vitro. J Biol Chem. 2012;287:17140–51.View ArticlePubMedPubMed CentralGoogle Scholar
- Rempe R, Cramer S, Huwel S, Galla HJ. Transport of Poly(n-butylcyano-acrylate) nanoparticles across the blood-brain barrier in vitro and their influence on barrier integrity. Biochem Biophys Res Commun. 2011;406:64–9.View ArticlePubMedGoogle Scholar
- Trumpler S, Meermann B, Nowak S, Buscher W, Karst U, Sperling M. In vitro study of thimerosal reactions in human whole blood and plasma surrogate samples. J Trace Elem Med Biol. 2014;28:125–30.View ArticlePubMedGoogle Scholar
- Repetto G, del Peso A, Zurita JL. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc. 2008;3:1125–31.View ArticlePubMedGoogle Scholar
- Fonfria E, Vilaro MT, Babot Z, Rodriguez-Farre E, Sunol C. Mercury compounds disrupt neuronal glutamate transport in cultured mouse cerebellar granule cells. J Neurosci Res. 2005;79:545–53.View ArticlePubMedGoogle Scholar
- DIN32645. Chemical analysis: Decision limit, detection limit and determination limit under repeatability conditions—terms, methods, evaluation. Technical Report 1994.
- Clarkson TW, Magos L. The toxicology of mercury and its chemical compounds. Crit Rev Toxicol. 2006;36:609–62.View ArticlePubMedGoogle Scholar
- Bridges CC, Zalups RK. Transport of inorganic mercury and methylmercury in target tissues and organs. J Toxicol Environ Health B Crit Rev. 2010;13:385–410.View ArticlePubMedGoogle Scholar
- Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ. Structure and function of the blood-brain barrier. Neurobiol Dis. 2010;37:13–25.View ArticlePubMedGoogle Scholar
- Oppedisano F, Galluccio M, Indiveri C. Inactivation by Hg2+ and methylmercury of the glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Prediction of the involvement of a CXXC motif by homology modelling. Biochem Pharmacol. 2010;80:1266–73.View ArticlePubMedGoogle Scholar
- Jenssen MT, Brantsaeter AL, Haugen M, Meltzer HM, Larssen T, Kvalem HE, Birgisdottir BE, Thomassen Y, Ellingsen D, Alexander J, Knutsen HK. Dietary mercury exposure in a population with a wide range of fish consumption--self-capture of fish and regional differences are important determinants of mercury in blood. Sci Total Environ. 2012;439:220–9.View ArticlePubMedGoogle Scholar