title: Patulin-induced cellular toxicity: a vital fluorescence study.
abstract: The mechanisms of patulin-induced cellular toxicity in an immortalized rat granulosa cell line were examined using several vital fluorescence bioassays. Monochlorobimane and 5-chloromethylfluorescein diacetate were used to monitor cellular glutathione (GSH) levels and revealed dose- and time-dependent depletion of GSH by patulin. A significant reduction in the fluorescence of the monochlorobimane-GSH conjugate by 0.1 microM patulin was observed between 1 and 2 hr. Similar GSH depletion by the mycotoxin was also observed in parallel studies on a liver (Clone 9) and a renal (LLC-PK1) cell line, although reduction of fluorescence occurred within 1 hr at the same dosage. Analysis of the electrical potential-dependent partitioning of rhodamine 123 into mitochondria also revealed significant effects of patulin within 1 hr at 0.1 microM. An initial dose-dependent reduction in mitochondrial fluorescence was followed by loss of selective partitioning of the fluorophore into mitochondria at higher doses and/or a longer exposure of cells to patulin. The reduction in mitochondrial fluorescence was paralleled by a dose-dependent decrease in intracellular pH detected with 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Analysis of [Ca2+]i with indo-1 and fluo-3 revealed a significant dose-dependent influx of Ca2+ at 10 microM and an alteration of the pattern of ionomycin-induced Ca2+ influx at 1.0 microM following patulin treatment. A carboxyfluorescein fluorescence photobleaching assay was used to examine the effects of patulin on gap junction-mediated intercellular communication. Dose-dependent reduction in intercellular communication was observed within 2 hr with 1.0 microM patulin. These observations indicate that the fluorescence assays used in this study provide a sensitive index of toxicity caused by exposure to patulin. Further, the toxic effects of patulin may involve direct effects on cellular glutathione levels and mitochondrial function in addition to direct effects on the plasma membrane.
authors.affiliation: Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station 77843-4458.
authors.forename: R C
authors.initials: RC
authors.lastname: Burghardt
authors.forename: R
authors.initials: R
authors.lastname: Barhoumi
authors.forename: E H
authors.initials: EH
authors.lastname: Lewis
authors.forename: R H
authors.initials: RH
authors.lastname: Bailey
authors.forename: K A
authors.initials: KA
authors.lastname: Pyle
authors.forename: B A
authors.initials: BA
authors.lastname: Clement
authors.forename: T D
authors.initials: TD
authors.lastname: Phillips
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002450:Cell Communication / Q000187:drug effects; D005260:Female; D005453:Fluorescence; D005978:Glutathione / Q000378:metabolism; D006106:Granulosa Cell Tumor / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D006706:Homeostasis / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007365:Intercellular Junctions / Q000187:drug effects; D007425:Intracellular Membranes / Q000187:drug effects; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D010051:Ovarian Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D010365:Patulin / Q000633:toxicity*; D051381:Rats; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D010365:Patulin; D005978:Glutathione; D002118:Calcium