Maternal transfer of the cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA) via milk to suckling offspring

Abstract

The cyanobacterial neurotoxin β-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 (14)C-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 [(14)C]L-BMAA into milk, the levels of [(14)C]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.

Figure 1. Tissue localization of [¹⁴C]-labeled L- and D-BMAA in nursing mouse dams.

Autoradiograms showing high levels of radioactivity in the mammary glands (MG) of nursing dams 24 h after injection of [¹⁴C]L-BMAA (A, B) or [¹⁴C]D-BMAA (C, D). Enlargements of the posterior mammary glands are shown in B and D. White areas correspond to high levels of radioactivity. BM  =  bone marrow, St  =  stomach, *  =  glandular part of stomach, In  =  lower gastrointestinal tract, Ki  =  kidney, Li  =  liver.

Figure 2. Tissue localization of [¹⁴C]-labeled L-BMAA in a lactating mouse dam with discontinued nursing.

Autoradiogram showing very high levels of radioactivity in the mammary glands (MG) and other tissues of a lactating dam that nursed for 2 h and was killed 24 h after injection of [¹⁴C]L-BMAA. Compare the level of radioactivity of the dam nursing for 24 h ( Figure 1A ). The high retention of labeled substance in the mammary glands and all other tissues in the dam with discontinued nursing is due to interruption of milk excretion. White areas correspond to high levels of radioactivity.

Figure 3. Tissue localization of [¹⁴C]-labeled L- and D-BMAA in suckling pups following a single administration to the nursing dams.

Autoradiograms showing very high levels of [¹⁴C]L- and [¹⁴C]D-BMAA enantiomers in coagulated milk filling the stomach (St) of pups nursed for 8 or 24 h. White areas correspond to high levels of radioactivity. Th  =  thymus, He  =  heart, St  =  stomach, In  =  lower gastrointestinal tract, Ki  =  kidney, Li  =  liver, Sp  =  spleen.

Figure 4. Levels of [¹⁴C]-labeled L- and D-BMAA in pup stomach milk and brain following administration to the nursing dams.

A Levels of radioactivity in coagulated stomach milk in suckling mouse pups nursed by dams given a single injection of [¹⁴C]L- and [¹⁴C]D-BMAA. The level of L-BMAA in the milk is significantly higher than D-BMAA suggesting a high secretion of the L-enantiomer into milk. B Levels of radioactivity in the brain of suckling mouse pups nursed by dams given a single injection of [¹⁴C]L- and [¹⁴C]D-BMAA. The level of L-BMAA in the brain of the pups is significantly higher compared with D-BMAA. At 24 h there were significantly higher levels of L-BMAA in the brains of suckling pups compared to the levels in the maternal brains. At the end of the experiment the levels of L-BMAA in the maternal brains were significantly higher than the levels of D-BMAA. Mean values (cpm/g coagulated milk or tissue) ± SD are plotted. *p<0.05 compared to D-BMAA within each time point, #p<0.05 compared to their pups, and φ φ p<0.01 compared to dams treated with L-BMAA (Mann-Whitney U-test). The data analysis is based on the dam as a statistical unit (2 pups/dam/time point).

Figure 5. Influx of [¹⁴C]-labeled L- and D-BMAA in mammary gland HC11 cells.

Levels of radioactivity in cultured undifferentiated and differentiated mammary gland cells exposed to [¹⁴C]-labeled BMAA (1 µM) during 15 min. [¹⁴C]L-BMAA is taken up at a significantly higher rate than [¹⁴C]D-BMAA in both undifferentiated and differentiated cells. Differentiation of the mammary gland cells to a secretory state significantly increases the uptake of [¹⁴C]L- but not [¹⁴C]D-BMAA. Mean values (cpm/mg protein) from seven experiments ± SD are plotted. ***p<0.001 compared to undifferentiated cells, and ^### p<0.001 compared to D-BMAA within same cell phenotype.

Figure 6. Transport of L- and D-BMAA in differentiated mammary gland HC11 cells.

A Influx of 1 µM [¹⁴C]L-BMAA or [¹⁴C]D-BMAA up to 60 min exposure in cultured mammary gland cells. The influx was highly reduced at incubation at 4°C, compared to incubation at 37°C. B Concentration dependent influx of [¹⁴C]L-BMAA in the presence of unlabeled L-BMAA (0.0003–5 mM) during 15 min exposure in cultured mammary gland cells. The influx of L-BMAA was saturable. C Influx of 1 µM [¹⁴C]L-BMAA or [¹⁴C]D-BMAA during 15 min exposure in cultured mammary gland cells. Replacement of sodium ions with 120 mM choline chloride did not have a major impact on the influx of [¹⁴C]L-BMAA. D Influx of 1 µM [¹⁴C]L-BMAA or [¹⁴C]D-BMAA during 15 min exposure in cultured mammary gland cells. The presence of competing amino acids reduced the influx of [¹⁴C]L-BMAA. E Efflux of 1 µM [¹⁴C]L-BMAA up to 80 min from cultured preloaded mammary gland cells. A time-dependent efflux is illustrated by decreasing concentrations in the cells and increasing concentrations in the cell medium. Representative experiments are shown in A-E. F Viability of mammary cells exposed to increasing concentrations of L-BMAA for 3 h, as determined by MTT assay. Data are presented as mean values (% of vehicle) ± SD (n = 3-4/data-point).