The role of organic anion transporting polypeptides (OATPs/SLCOs) in the toxicity of different microcystin congeners in vitro: a comparison of primary human hepatocytes and OATP-transfected HEK293 cells.

Cellular uptake of microcystins (MCs), a family of cyclic cyanobacterial heptapeptide toxins, occurs via specific organic anion transporting polypeptides (OATPs), where MCs inhibit serine/threonine-specific protein phosphatase (PP). Despite comparable PP-inhibitory capacity, MCs differ greatly in their acute toxicity, thus raising the question whether this discrepancy results from MC-specific toxikokinetic rather than toxicodynamic differences. OATP-mediated uptake of MC congeners MCLR, -RR, -LW and -LF was compared in primary human hepatocytes and HEK293 cells stably expressing recombinant human OATP1B1/SLCO1B1 and OATP1B3/SLCO1B3 in the presence/absence of OATP substrates taurocholate (TC) and bromosulfophthalein (BSP) and measuring PP-inhibition and cytotoxicity. Control vector expressing HEK293 were resistant to MC cytotoxicity, while TC and BSP competition experiments reduced MC cytotoxicity in HEK293-OATP transfectants, thus confirming the requirement of OATPs for trans-membrane transport. Despite comparable PP-inhibiting capabilities, MCLW and -LF elicited cytotoxic effects at lower equimolar concentrations than MCLR and MCRR, hence suggesting congener selective transport into HEK293-OATP transfectants and primary human hepatocytes. Primary human hepatocytes appeared one order of magnitude more sensitive to MC congeners than the corresponding HEK293 -OATP transfectants. Although the latter maybe due to a much lower level of PPs in primary human hepatocytes, the presence of OATPs other than 1B1 or 1B3 may have added to an increased uptake of MCs. In view of the high sensitivity of human hepatocytes and currently MCLR-only based risk calculations, the actual risk of human MC-intoxication and ensuing liver damage could be underestimated in freshwater cyanobacterial blooms where MCLW and-LF predominate.

Toxin mixture in cyanobacterial blooms--a critical comparison of reality with current procedures employed in human health risk assessment.

Cyanobacteria are the oldest life forms on earth known to produce a broad spectrum of secondary metabolites. The functions/advantages of most of these secondary metabolites (peptides and alkaloids) are unknown, however, some of them have adverse effects in humans and wildlife, especially when ingested, inhaled or upon dermal exposure. Surprisingly, some of these cyanobacteria are ingested voluntarily. Indeed, for centuries mankind has used cyanobacteria as a protein source, primarily Spirulina species. However, recently also Aphanizomenon flos-aquae are used for the production of so called blue green algae supplements (BGAS), supposedly efficacious for treatment of various diseases and afflictions. Unfortunately, traces of neurotoxins and protein phosphatases (inhibiting compounds) have been detected in BGAS, making these health supplements a good example for human exposure to a mixture of cyanobacterial toxins in a complex matrix. The discussion of this and other possible exposure scenarios, e.g. drinking water, contact during recreational activity, or consumption of contaminated food, can provide insight into the question of whether or not our current risk assessment schemes for cyanobacterial blooms and the toxins contained therein suffice for protection of human health.

Analytical and functional characterization of microcystins [Asp3]MC-RR and [Asp3,Dhb7]MC-RR: consequences for risk assessment?

The microcystin (MC) producing P. rubescens occurs in pre-alpine lakes and may impact fishery success, bathing, and raw water quality. P. rubescens extracts, characterized via LC-MS, contained the two MC-RR variants [Asp3]MC-RR and [Asp3,Dhb7]MC-RR. The protein-phosphatase-inhibition assay (cPPIA with phosphatases 1 and 2A) in its capability to quantify [Asp3]MC-RR, [Asp3,Dhb7]MC-RR, and MC-RR was compared to HPLC-DAD and anti-Adda-ELISA. The IC50 values (PP1 and PP2A) determined for MC-LR, MC-RR, and [Asp3]MC-RR were in the same range (1.9-3.8 and 0.45-0.75 nM). A 50-fold higher concentration of [Asp3,Dhb7]MC-RR (29.8 nM) was necessary to inhibit the PP2A by 50%. The PP1-IC50 of [Asp3,Dhb7]MC-RR was 22-fold higher (56.4 nM) than those of the other MCs, suggesting that specific structural characteristics are responsible for its weaker PPI capacity. Western blots demonstrated that [Asp3,Dhb7]MC-RR does not covalently bind to PP1. [Asp3,Dhb7]MC-RR has comparable in vivo LD50 values to MC-RR, despite a far lower PP-inhibiting capacity, suggesting that toxicodynamic and toxicokinetic characteristics of [Asp3,Dhb7]MC-RR are responsible for its high in vivo toxicity. The data demonstrate that cPPIA analysis of [Asp3,Dhb7]MC-RR-containing samples prevent reliable MC determination and lead to underestimation of potential toxicity.