title: Mitochondrial toxicity of microcystin-LR on cultured cells: application to the analysis of contaminated water samples.
abstract: Microcystins (MC) are potent hepatic toxins delivered into the cells by organic anion transporting peptides (OATP) where they target protein phosphatases and mitochondria. We analyzed the effects of MC-LR on primary hepatocytes, HepG2, and Jurkat T cells, and isolated rat liver mitochondria by measuring changes in O(2) consumption by optical oxygen sensing technique. Respiration of fresh primary hepatocytes was inhibited by MC-LR with EC50 = 2.74 +/- 0.65 nM, whereas an uncoupling effect on mitochondrial state 2 and state 3 respiration was observed with glutamate/malate as a substrate. HepG2 and Jurkat T cells lacking OATP showed no sensitivity to MC-LR; however, facilitated delivery of MC-LR resulted in a marked enhancement of HepG2 O(2) consumption and inhibition of Jurkat O(2) consumption at >or=0.1 nM. The respiratory response did not coincide with changes in viability, total cellular ATP, extracellular acidification, ROS formation, or protein phosphorylation, which were detectable at higher MC-LR doses. Such prominent effect on cellular respiration was therefore used for the detection of MC-LR in environmental samples. A simple and sensitive screening assay for MC-LR toxicity was developed, which uses Jurkat cells, facilitated delivery of the toxin(s) and measurement on a fluorescent reader. The assay was applied to a panel of environmental samples suspected to contain MC and benchmarked against the ELISA test. It allowed identification of toxic samples and quantification of both nonspecific and MC-LR type of toxicity.
authors.affiliation: Biochemistry Department, University College Cork, College Road, Cork, Ireland.
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Jasionek
authors.forename: Alexander
authors.initials: A
authors.lastname: Zhdanov
authors.forename: John
authors.initials: J
authors.lastname: Davenport
authors.forename: Ludek
authors.initials: L
authors.lastname: Bláha
authors.forename: Dimitri B
authors.initials: DB
authors.lastname: Papkovsky
mesh_terms: D000143:Acids / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D004777:Environment; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D049408:Luminescent Agents / Q000378:metabolism; D008297:Male; D008387:Marine Toxins; D052998:Microcystins / Q000633:toxicity*; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism; D010101:Oxygen Consumption / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D014876:Water Pollution / Q000032:analysis*
chemical_list: D000143:Acids; D049408:Luminescent Agents; D008387:Marine Toxins; D052998:Microcystins; C057862:cyanoginosin LR