title: Aggregate culture: A more accurate predictor of microcystin toxicity for risk assessment.
abstract: Aggregate or spheroid culture has emerged as a more biologically relevant method for screening pharmaceutical compounds and understanding exact mechanism of action. Here in, the aggregate approach applied to the freshwater toxins, microcystins, further unearths exact mechanism(s) of toxicity and provides a markedly improved in vitro predictor of toxicity. Microcystins result in acute intoxication by binding covalently to protein phosphatase 1/2A, resulting in hepatocellular necrosis, hemorrhaging and death. Hepatocellular uptake by organic anion transporting polypeptides (OATPs), in addition to other intracellular sequelae, is considered essential for toxicity. In aggregate HepG2, expression of OAT1B1 and OATP1B3 significantly increased relative to monolayer culture. Uptake of two fluorescently labeled substrates significantly increased in aggregates compared with monolayer, confirmed by inhibition of uptake with known competitive substrates. Increased reaction oxygen species (ROS) production occurred following a three-hour exposure of microcystin LR at concentrations from 100 nM to 100 μM, with reversal by ROS scavengers, in contrast with no response in monolayers. These results suggest monolayer culture inadequately predict intracellular effects of microcystins and support evidence that aggregate culture more closely approximates in vivo form and function. The approach results in more reliable prediction of microcystin toxicity in vitro.
authors.affiliation: Department of Molecular Biosciences, School of Veterinary Medicine, 1089 Veterinary Medicine Drive, University of California, Davis, CA 95616, USA.
authors.forename: Amber F
authors.initials: AF
authors.lastname: Roegner
authors.affiliation: Department of Molecular Biosciences, School of Veterinary Medicine, 1089 Veterinary Medicine Drive, University of California, Davis, CA 95616, USA. Electronic address: bpuschner@ucdavis.edu.
authors.forename: Birgit
authors.initials: B
authors.lastname: Puschner
mesh_terms: D018929:Cell Culture Techniques; D002470:Cell Survival; D056945:Hep G2 Cells; D006801:Humans; D027381:Liver-Specific Organic Anion Transporter 1; D052998:Microcystins / Q000032:analysis / Q000633:toxicity*; D055616:Microtechnology; D027361:Organic Anion Transporters / Q000378:metabolism; D029362:Organic Anion Transporters, Sodium-Independent / Q000378:metabolism; D017382:Reactive Oxygen Species; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D027381:Liver-Specific Organic Anion Transporter 1; D052998:Microcystins; D027361:Organic Anion Transporters; D029362:Organic Anion Transporters, Sodium-Independent; D017382:Reactive Oxygen Species; C503999:SLCO1B1 protein, human; C421209:SLCO1B3 protein, human; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3
keywords: Aggregate; Cell culture model; Cyanotoxins; Microcystins; OATP1B1; OATP1B3