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Investigation of microcystin congener-dependent uptake into primary murine neurons.

Feurstein D, Kleinteich J, Heussner AH, Stemmer K, Dietrich DR - Environ. Health Perspect. (2010)

Bottom Line: MCs can cross neuronal cell membranes, with a subsequent decrease of PP activity.Of 15 mOatps, 12 were expressed at the mRNA level, but we found detectable protein levels for only two: mOatp1a5 (Slco1a5) and the known MC-LR transporter mOatp1b2 (Slco1b2).These data suggest mOatp-mediated uptake of MC congeners into neurons, thus corroborating earlier assumptions of the neurotoxic potential of MCs.

View Article: PubMed Central - PubMed

Affiliation: Human and Environmental Toxicology, University of Konstanz, Konstanz, Germany. daniel.dietrich@uni-konstanz.de

ABSTRACT

Background: Contamination of natural waters by toxic cyanobacteria is a growing problem worldwide, resulting in serious water pollution and human health hazards. Microcystins (MCs) represent a group of > 80 cyclic heptapeptides, mediating cytotoxicity via specific protein phosphatase (PP) inhibition at equimolar concentrations (comparable toxicodynamics). Because of the structure and size of MCs, active uptake into cells occurs via organic anion-transporting polypeptides (OATP/Oatp), as confirmed for liver-specific human OATP1B1 and OATP1B3, mouse Oatp1b2 (mOatp1b2), skate Oatp1d1, and the more widely distributed OATP1A2 expressed, for example, at the blood-brain barrier. Tissue-specific and cell-type-specific expression of OATP/Oatp transporters and specific transport of MC congeners (toxicokinetics) therefore appear prerequisite for the reported toxic effects in humans and other species upon MC exposure. Beyond hepatotoxicity induced by the MC-LR congener, the effects of other MC congeners, especially neuronal uptake and toxicity, are unknown.

Objectives: In this study we examined the expression of mOatps and the uptake of congeners MC-LR, MC-LW, and MC-LF in primary murine neurons.

Methods: Intracellular MC accumulation was indicated indirectly via uptake inhibition experiments and directly confirmed by Western blot analysis and a PP inhibition assay. Neuronal mOatp expression was verified at the mRNA and protein level.

Results: MCs can cross neuronal cell membranes, with a subsequent decrease of PP activity. Of 15 mOatps, 12 were expressed at the mRNA level, but we found detectable protein levels for only two: mOatp1a5 (Slco1a5) and the known MC-LR transporter mOatp1b2 (Slco1b2).

Conclusions: These data suggest mOatp-mediated uptake of MC congeners into neurons, thus corroborating earlier assumptions of the neurotoxic potential of MCs.

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Related in: MedlinePlus

WB analysis in neuronal membrane fractions (A–F) and immunodetection of mOatp1b2 in cultured neurons (G,H). M, protein marker. (A–F) WB analysis of mOatp1b2 (A), mOatp1a5 (B), mOatp2b1 (C), mOatp3a1 (D), mOatp1c1 (E), and mOatp4a1 (F). Liver and brain membrane fractions were used as positive controls, and β-actin was a loading control. (G,H) Immunodetection of mOatp1b2 in cultured neurons treated with (G) Hoechst (blue), rabbit anti-mOatp1b2, and fluorochrome-conjugated secondary antibody (red) or (H) Hoechst and fluorochrome-conjugated secondary antibody only (negative control).
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f4-ehp-118-1370: WB analysis in neuronal membrane fractions (A–F) and immunodetection of mOatp1b2 in cultured neurons (G,H). M, protein marker. (A–F) WB analysis of mOatp1b2 (A), mOatp1a5 (B), mOatp2b1 (C), mOatp3a1 (D), mOatp1c1 (E), and mOatp4a1 (F). Liver and brain membrane fractions were used as positive controls, and β-actin was a loading control. (G,H) Immunodetection of mOatp1b2 in cultured neurons treated with (G) Hoechst (blue), rabbit anti-mOatp1b2, and fluorochrome-conjugated secondary antibody (red) or (H) Hoechst and fluorochrome-conjugated secondary antibody only (negative control).

Mentions: We analyzed mOatp expression at the protein level by WB using crude membrane fractions of primary murine neurons, as well as mouse liver and brain homogenates. The expression of mOatp1b2 and mOatp1a5 was confirmed in neurons (Figure 4A,B) with immunopositive bands at approximately 80 and 60 kDa and 85 and 65 kDa, respectively. Expression of mOatp2b1 (Figure 4C), mOatp3a1 (Figure 4D), mOatp1c1 (Figure 4E), and mOatp4a1 (Figure 4F) was either absent or less than the limit of detection for the crude neuron membrane extract we used.


Investigation of microcystin congener-dependent uptake into primary murine neurons.

Feurstein D, Kleinteich J, Heussner AH, Stemmer K, Dietrich DR - Environ. Health Perspect. (2010)

WB analysis in neuronal membrane fractions (A–F) and immunodetection of mOatp1b2 in cultured neurons (G,H). M, protein marker. (A–F) WB analysis of mOatp1b2 (A), mOatp1a5 (B), mOatp2b1 (C), mOatp3a1 (D), mOatp1c1 (E), and mOatp4a1 (F). Liver and brain membrane fractions were used as positive controls, and β-actin was a loading control. (G,H) Immunodetection of mOatp1b2 in cultured neurons treated with (G) Hoechst (blue), rabbit anti-mOatp1b2, and fluorochrome-conjugated secondary antibody (red) or (H) Hoechst and fluorochrome-conjugated secondary antibody only (negative control).
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2957914&req=5

f4-ehp-118-1370: WB analysis in neuronal membrane fractions (A–F) and immunodetection of mOatp1b2 in cultured neurons (G,H). M, protein marker. (A–F) WB analysis of mOatp1b2 (A), mOatp1a5 (B), mOatp2b1 (C), mOatp3a1 (D), mOatp1c1 (E), and mOatp4a1 (F). Liver and brain membrane fractions were used as positive controls, and β-actin was a loading control. (G,H) Immunodetection of mOatp1b2 in cultured neurons treated with (G) Hoechst (blue), rabbit anti-mOatp1b2, and fluorochrome-conjugated secondary antibody (red) or (H) Hoechst and fluorochrome-conjugated secondary antibody only (negative control).
Mentions: We analyzed mOatp expression at the protein level by WB using crude membrane fractions of primary murine neurons, as well as mouse liver and brain homogenates. The expression of mOatp1b2 and mOatp1a5 was confirmed in neurons (Figure 4A,B) with immunopositive bands at approximately 80 and 60 kDa and 85 and 65 kDa, respectively. Expression of mOatp2b1 (Figure 4C), mOatp3a1 (Figure 4D), mOatp1c1 (Figure 4E), and mOatp4a1 (Figure 4F) was either absent or less than the limit of detection for the crude neuron membrane extract we used.

Bottom Line: MCs can cross neuronal cell membranes, with a subsequent decrease of PP activity.Of 15 mOatps, 12 were expressed at the mRNA level, but we found detectable protein levels for only two: mOatp1a5 (Slco1a5) and the known MC-LR transporter mOatp1b2 (Slco1b2).These data suggest mOatp-mediated uptake of MC congeners into neurons, thus corroborating earlier assumptions of the neurotoxic potential of MCs.

View Article: PubMed Central - PubMed

Affiliation: Human and Environmental Toxicology, University of Konstanz, Konstanz, Germany. daniel.dietrich@uni-konstanz.de

ABSTRACT

Background: Contamination of natural waters by toxic cyanobacteria is a growing problem worldwide, resulting in serious water pollution and human health hazards. Microcystins (MCs) represent a group of > 80 cyclic heptapeptides, mediating cytotoxicity via specific protein phosphatase (PP) inhibition at equimolar concentrations (comparable toxicodynamics). Because of the structure and size of MCs, active uptake into cells occurs via organic anion-transporting polypeptides (OATP/Oatp), as confirmed for liver-specific human OATP1B1 and OATP1B3, mouse Oatp1b2 (mOatp1b2), skate Oatp1d1, and the more widely distributed OATP1A2 expressed, for example, at the blood-brain barrier. Tissue-specific and cell-type-specific expression of OATP/Oatp transporters and specific transport of MC congeners (toxicokinetics) therefore appear prerequisite for the reported toxic effects in humans and other species upon MC exposure. Beyond hepatotoxicity induced by the MC-LR congener, the effects of other MC congeners, especially neuronal uptake and toxicity, are unknown.

Objectives: In this study we examined the expression of mOatps and the uptake of congeners MC-LR, MC-LW, and MC-LF in primary murine neurons.

Methods: Intracellular MC accumulation was indicated indirectly via uptake inhibition experiments and directly confirmed by Western blot analysis and a PP inhibition assay. Neuronal mOatp expression was verified at the mRNA and protein level.

Results: MCs can cross neuronal cell membranes, with a subsequent decrease of PP activity. Of 15 mOatps, 12 were expressed at the mRNA level, but we found detectable protein levels for only two: mOatp1a5 (Slco1a5) and the known MC-LR transporter mOatp1b2 (Slco1b2).

Conclusions: These data suggest mOatp-mediated uptake of MC congeners into neurons, thus corroborating earlier assumptions of the neurotoxic potential of MCs.

Show MeSH
Related in: MedlinePlus