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Cellular transport and secretion of the cyanobacterial neurotoxin BMAA into milk and egg: Implications for developmental neurotoxicity
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Environmental toxicology.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cyanobacterial amino acid β-N-methylamino-L-alanine (BMAA) is a neurotoxin implicated in the etiology of neurodegenerative diseases. Cyanobacteria are cosmopolitan organisms present in various environments. BMAA can cause long-term neurodegenerative alterations in rats exposed during the neonatal period, a period that corresponds to the last trimester and the first few years of life in humans. As BMAA has been reported to be bioaccumulated in the aquatic food chain and detected in mussels, crayfish and fish used for human consumption, the main aim of this thesis has been to investigate the final step in the mammalian food-chain, i.e. the transfer of BMAA into breast milk.

Autoradiographic imaging and mass spectrometry analysis showed an enantiomer-selective uptake of BMAA and that the neurotoxin was transferred from lactating mice and rat, via the milk, to the brain of the nursed pups. The results show that transport of BMAA may be disproportional to dose. In addition, BMAA was found present both as free amino acid and tightly associated to proteins in rat brains. Surprisingly, however, no association to milk proteins was found. In vitro studies of murine (HC11) and human (MCF7) mammary epithelial cells suggest that BMAA can pass the human mammary epithelium into milk. Additional transport studies on human intestinal, glioblastoma and neuroblastoma cells showed that L-BMAA was consistently favored over D-BMAA and that the transport was mediated by several amino acid transporters. We also demonstrated that egg-laying quail transfer BMAA to its offspring by deposition in the eggs, particularly in the yolk but also in the albumen. Furthermore, comparative analysis of carboxyl- and methyl-labeled [14C]-BMAA suggested that BMAA was not metabolized to a large degree.

Altogether, the results indicate that BMAA can be transferred from mothers, via the milk, to the brain of nursed human infants. Determinations of BMAA in mothers’ milk and cows’ milk are therefore warranted. We also propose that birds’ eggs could be an additional source of BMAA exposure in humans. It might therefore be of concern that mussels are increasingly used as feed in commercial egg production.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. , 72 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1316
Keyword [en]
BMAA, beta-N-methylamino-L-alanine, milk, secretion, amino acid transporter, autoradiography, metabolism
National Category
Developmental Biology Cell Biology
Research subject
Biology with specialization in Environmental Toxicology
Identifiers
URN: urn:nbn:se:uu:diva-265865ISBN: 978-91-554-9408-7 (print)OAI: oai:DiVA.org:uu-265865DiVA: diva2:866723
Public defence
2015-12-18, Friessalen, EBC, Norbyvägen 14, Uppsala, 09:30 (English)
Opponent
Supervisors
Available from: 2015-11-26 Created: 2015-11-03 Last updated: 2016-01-13
List of papers
1. Maternal Transfer of the Cyanobacterial Neurotoxin beta-N-Methylamino-L-Alanine (BMAA) via Milk to Suckling Offspring
Open this publication in new window or tab >>Maternal Transfer of the Cyanobacterial Neurotoxin beta-N-Methylamino-L-Alanine (BMAA) via Milk to Suckling Offspring
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 10, e78133- p.Article in journal (Refereed) Published
Abstract [en]

The cyanobacterial neurotoxin beta-N-methylamino-L-alanine (BMAA) has been implicated in the etiology of neurodegenerative disease and proposed to be biomagnified in terrestrial and aquatic food chains. We have previously shown that the neonatal period in rats, which in humans corresponds to the last trimester of pregnancy and the first few years of age, is a particularly sensitive period for exposure to BMAA. The present study aimed to examine the secretion of C-14-labeled L-and D-BMAA into milk in lactating mice and the subsequent transfer of BMAA into the developing brain. The results suggest that secretion into milk is an important elimination pathway of BMAA in lactating mothers and an efficient exposure route predominantly for L-BMAA but also for D-BMAA in suckling mice. Following secretion of [C-14] L-BMAA into milk, the levels of [C-14] L-BMAA in the brains of the suckling neonatal mice significantly exceeded the levels in the maternal brains. In vitro studies using the mouse mammary epithelial HC11 cell line confirmed a more efficient influx and efflux of L-BMAA than of D-BMAA in cells, suggesting enantiomer-selective transport. Competition experiments with other amino acids and a low sodium dependency of the influx suggests that the amino acid transporters LAT1 and LAT2 are involved in the transport of L-BMAA into milk. Given the persistent neurodevelopmental toxicity following injection of L-BMAA to neonatal rodent pups, the current results highlight the need to determine whether BMAA is enriched mother's and cow's milk.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-211448 (URN)10.1371/journal.pone.0078133 (DOI)000326037000089 ()
Available from: 2013-11-27 Created: 2013-11-25 Last updated: 2017-12-06Bibliographically approved
2. Transfer of developmental neurotoxin beta-N-methylamino-L-alanine (BMAA) via milk to nursed offspring: Studies by mass spectrometry and image analysis
Open this publication in new window or tab >>Transfer of developmental neurotoxin beta-N-methylamino-L-alanine (BMAA) via milk to nursed offspring: Studies by mass spectrometry and image analysis
2016 (English)In: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, Vol. 258, 108-114 p.Article in journal (Refereed) Published
Abstract [en]

The cyanobacterial non-proteinogenic amino acid beta-N-methylamino-L-alanine (BMAA) is proposed to be involved in the etiology of amyotrophic lateral sclerosis/parkinsonism dementia complex. When administered as single doses to neonatal rats, BMAA gives rise to cognitive and neurodegenerative impairments in the adult animal. Here, we employed mass spectrometry (LC-MS/MS) and autoradiographic imaging to examine the mother-to-pup transfer of BMAA in rats. The results show that unchanged BMAA was secreted into the milk and distributed to the suckling pups. The concentration of BMAA in pup stomach milk and the neonatal liver peaked after 8 h, while the concentration in the pup brain increased throughout the study period. About 1 and 6% of the BMAA recovered from adult liver and brain were released following hydrolysis, suggesting that this fraction was associated with protein. No association to milk protein was observed. Injection of rat pups with [methyl-C-14]-L-BMAA or [carboxyl-C-14]-L-BMAA resulted in highly similar distribution patterns, indicating no or low metabolic elimination of the methylamino- or carboxyl groups. In conclusion, BMAA is transported as a free amino acid to rat milk and suckling pups. The results strengthen the proposal that mothers' milk could be a source of exposure for BMAA in human infants. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Keyword
BMAA; Cyanobacterial neurotoxin, kinetics; Milk secretion; Developmental neurotoxicity; Mother-to-offspring transfer
National Category
Developmental Biology
Identifiers
urn:nbn:se:uu:diva-265847 (URN)10.1016/j.toxlet.2016.06.015 (DOI)000381648300012 ()27320960 (PubMedID)
Projects
Milk, secretion, BMAA, beta-N-methylamino-L-alanine, autoradiography, mass spectrometry
Funder
Swedish Research Council Formas
Available from: 2015-11-03 Created: 2015-11-03 Last updated: 2017-05-12Bibliographically approved
3. Potential transfer of neurotoxic amino acid beta-N-methylamino-L-alanine (BMAA) from mother to infant during breast-feeding: Predictions from human cell lines
Open this publication in new window or tab >>Potential transfer of neurotoxic amino acid beta-N-methylamino-L-alanine (BMAA) from mother to infant during breast-feeding: Predictions from human cell lines
2017 (English)In: Toxicology and Applied Pharmacology, ISSN 0041-008X, E-ISSN 1096-0333, Vol. 320, 40-50 p.Article in journal (Refereed) Published
Abstract [en]

β-N-methylamino-alanine (BMAA) is a non-protein amino acid produced by cyanobacteria, diatoms and dinoflagellates. BMAA has potential to biomagnify in a terrestrial food chain, and to bioaccumulate in fish and shellfish. We have reported that administration of [14C]l-BMAA to lactating mice and rats results in a mother to off-spring transfer via the milk. A preferential enantiomer-specific uptake of [14C]l-BMAA has also been demonstrated in differentiated murine mammary epithelium HC11 cells. These findings, together with neurotoxic effects of BMAA demonstrated both in vitro and in vivo, highlight the need to determine whether such transfer could also occur in humans. Here, we used four cell lines of human origin to examine and compare the transport of the two BMAA enantiomers in vitro. The uptake patterns of [14C]l- and [14C]d-BMAA in the human mammary MCF7 cell line were in agreement with the results in murine HC11 cells, suggesting a potential secretion of BMAA into human breast milk. The permeability coefficients for both [14C]l- and [14C]d-BMAA over monolayers of human intestinal Caco2 cells supported an efficient absorption from the human intestine. As a final step, transport experiments confirmed that [14C]l-and [14C]d-BMAA can be taken up by human SHSY5Y neuroblastoma cells and even more efficiently by human U343 glioblastoma cells. In competition experiments with various amino acids, the ASCT2 specific inhibitor benzylserine was the most effective inhibitor of [14C]l-BMAA uptake tested here. Altogether, our results suggest that BMAA can be transferred from an exposed mother, via the milk, to the brain of the nursed infant.

Place, publisher, year, edition, pages
Elsevier, 2017
Keyword
BMAA, Cellular transport, Amino acid transporters, Breast milk, Neurodegeneration
National Category
Cell Biology Developmental Biology
Research subject
Biology with specialization in Environmental Toxicology
Identifiers
urn:nbn:se:uu:diva-265857 (URN)10.1016/j.taap.2017.02.004 (DOI)000396798200006 ()28174119 (PubMedID)
Funder
Swedish Research Council Formas
Available from: 2015-11-03 Created: 2015-11-03 Last updated: 2017-05-02Bibliographically approved
4. Protein association of the neurotoxin and non-protein amino acid BMAA (beta-N-methylamino-L-alanine) in the liver and brain following neonatal administration in rats
Open this publication in new window or tab >>Protein association of the neurotoxin and non-protein amino acid BMAA (beta-N-methylamino-L-alanine) in the liver and brain following neonatal administration in rats
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2014 (English)In: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, Vol. 226, no 1, 1-5 p.Article in journal (Refereed) Published
Abstract [en]

The environmental neurotoxin beta-N-methylamino-L-alanine (BMAA) is not an amino acid that is normally found in proteins. Our previous autoradiographic study of H-3-labeled BMAA in adult mice unexpectedly revealed a tissue distribution similar to that of protein amino acids. The aim of this study was to characterize the distribution of free and protein-bound BMAA in neonatal rat tissues following a short exposure using autoradiographic imaging and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The autoradiographic imaging of C-14-L-BMAA demonstrated a distinct uptake of radioactivity that was retained following acid extraction in tissues with a high rate of cell turnover and/or protein synthesis. The UHPLC-MS/MS analysis conclusively demonstrated a dose-dependent increase of protein-associated BMAA in neonatal rat tissues. The level of protein-associated BMAA in the liver was more than 10 times higher than that in brain regions not fully protected by the blood-brain barrier which may be due to the higher rate of protein synthesis in the liver. In conclusion, this study demonstrated that BMAA was associated with rat proteins suggesting that BMAA may be mis-incorporated into proteins. However, protein-associated BMAA seemed to be cleared over time, as none of the samples from adult rats had any detectable free or protein-associated BMAA.

Keyword
ALS/PDC, Cyanobacteria, Autoradiography, Mass spectrometry, Misincorporation, N-(2-aminoethyl) glycine
National Category
Medical and Health Sciences Natural Sciences
Identifiers
urn:nbn:se:uu:diva-222718 (URN)10.1016/j.toxlet.2014.01.027 (DOI)000332409000001 ()24472610 (PubMedID)
Funder
Swedish Research Council Formas
Available from: 2014-04-17 Created: 2014-04-14 Last updated: 2017-06-30Bibliographically approved
5. Deposition of cyanobacterial neurotoxin beta-N-methylamino-L-alanine (L-BMAA) in birds' egg: A potential source of BMAA exposure in humans
Open this publication in new window or tab >>Deposition of cyanobacterial neurotoxin beta-N-methylamino-L-alanine (L-BMAA) in birds' egg: A potential source of BMAA exposure in humans
(English)Manuscript (preprint) (Other academic)
Keyword
BMAA, beta-N-methylamino-L-alanine, quail, metabolism, autoradiography
National Category
Developmental Biology
Research subject
Biology with specialization in Environmental Toxicology
Identifiers
urn:nbn:se:uu:diva-265859 (URN)
Funder
Swedish Research Council Formas
Available from: 2015-11-03 Created: 2015-11-03 Last updated: 2016-01-13

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