title: A 3D fish liver model for aquatic toxicology: Morphological changes and Cyp1a induction in PLHC-1 microtissues after repeated benzo(a)pyrene exposures.
abstract: To identify the potential environmental impacts of aquatic pollutants, rapid and sensitive screening tools are needed to assess adaptive and toxic responses. This study characterizes a novel fish liver microtissue model, produced with the cell line PLHC-1, as an in vitro aquatic toxicity testing platform. These 3D microtissues remain viable and stable throughout the 8-day testing period and relative to 2D monolayers, show increased basal expression of the xenobiotic metabolizing enzyme cytochrome P450 1A (Cyp1a). To evaluate pulsed, low-dose exposures at environmentally relevant concentrations, microtissue responsiveness to the model toxicant benzo(a)pyrene was assessed after single and repeated exposures for determination of both immediate and persistent effects. Significant induction of Cyp1a gene and protein expression was detected after a single 24h exposure to as little as 1nM benzo(a)pyrene, and after a 24h recovery period, Cyp1a expression declined in a dose-dependent manner. However, cell death continued to increase during the recovery period and alterations in microtissue architecture occurred at higher concentrations. To evaluate a pulsed or repeated exposure scenario, microtissues were exposed to benzo(a)pyrene, allowed to recover, then exposed a second time for 24h. Following pre-exposure to benzo(a)pyrene, cyp1a expression remained equally inducible and the pattern and level of Cyp1a protein response to a second exposure were comparable. However, pre-exposure to 1μM or 5μM of benzo(a)pyrene resulted in increased cell death, greater disruption of microtissue architecture, and alterations in cell morphology. Together, this study demonstrates the capabilities of this PLHC-1 microtissue model for sensitive assessment of liver toxicants over time and following single and repeated exposures.
authors.affiliation: Department of Pathology & Laboratory Medicine, Brown University, Providence, RI 02912, USA. Electronic address: april_rodd@brown.edu.
authors.forename: April L
authors.initials: AL
authors.lastname: Rodd
authors.affiliation: Department of Pathology & Laboratory Medicine, Brown University, Providence, RI 02912, USA.
authors.forename: Norma J
authors.initials: NJ
authors.lastname: Messier
authors.affiliation: Department of Pathology & Laboratory Medicine, Brown University, Providence, RI 02912, USA.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Vaslet
authors.affiliation: Department of Pathology & Laboratory Medicine, Brown University, Providence, RI 02912, USA.
authors.forename: Agnes B
authors.initials: AB
authors.lastname: Kane
mesh_terms: D000818:Animals; D001564:Benzo(a)pyrene / Q000633:toxicity*; D002460:Cell Line; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis* / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004781:Environmental Exposure / Q000032:analysis*; D004790:Enzyme Induction / Q000187:drug effects; D005399:Fishes / Q000033:anatomy & histology*; D021621:Imaging, Three-Dimensional; D020287:In Situ Nick-End Labeling; D008099:Liver / Q000033:anatomy & histology* / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D009929:Organ Size / Q000187:drug effects; D014874:Water Pollutants, Chemical / Q000633:toxicity*
chemical_list: D014874:Water Pollutants, Chemical; D001564:Benzo(a)pyrene; D003577:Cytochrome P-450 Enzyme System; D019363:Cytochrome P-450 CYP1A1
keywords: Animal alternatives; Cytochrome P450 1A (Cyp1a); Fish; Hepatocytes; Morphology; Polycyclic aromatic hydrocarbons (PAHs)