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 micro-tissues 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 24 h exposure to as little as 1 nM benzo(a)pyrene, and after a 24 h 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 24 h. 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 1 μM or 5 μM 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.

A novel human 3D lung microtissue model for nanoparticle-induced cell-matrix alterations

“…Samples were lysed with TRI Reagent (MRC, Cincinnati, OH) and stored at − 80 °C. Total RNA was then isolated as described previously [46]. For analysis of gene expression using Human Fibrosis RT 2 Profiler PCR Arrays (PAHS-120Z), DNase treated RNAs isolated from 3D microtissues were cleaned through RNeasy® MinElute™ columns, then converted to cDNAs and used for analysis according to vender specifications (Qiagen, Germantown, MD).…”

Impact of emerging, high-production-volume graphene-based materials on the bioavailability of benzo(a)pyrene to brine shrimp and fish liver cells

“…PLHC-1 cells (ATCC #CRL-2406) in monolayer were maintained in EMEM (ATCC) + 5% fetal calf serum + penicillin and streptomycin (Gibco, 50U/mL each) at 30°C with CO 2 . 36 For all exposures to benzo(a)pyrene, cells were plated in glass vials with or without glass coverslips. Plated cells were directly exposed to treatment media for 24 or 72 hr.…”

“…For in situ detection of Cyp1a protein expression, immunofluorescence was performed as described previously. 36 Samples were counterstained with Hoechst 33342 and imaged using confocal fluorescence and brightfield differential interference contrast (DIC). To pseudocolor nanomaterials in the images, DIC micrographs were inverted and adjusted to emphasize black regions of the original image using ImageJ software.…”

“…For quantification of cyp1a and abcc2 gene expression by qRT-PCR, cells were collected for RNA isolation and processed as described previously, with the addition of the abcc2 primer set (Table S2). 36…”

“…35 The fish liver cell line PLHC-1 allows us to detect the cellular response to benzo(a)pyrene with high sensitivity using protein and gene expression of the xenobiotic metabolism enzyme primarily responsible for this metabolism, Cyp1a. 36 We also evaluated expression of the transporter Abcc2, which is responsible for the cellular excretion of compounds like benzo(a)pyrene metabolites and has been reported to be upregulated after PAH exposure. 37, 38 Researchers have previously demonstrated graphene-mediated inhibition of related cellular transporters, potentially increasing co-contaminant toxicity by interfering with cellular excretion of damaging metabolites.…”