Determination of Phospholipidosis Potential Based on Gene Expression Analysis in HepG2 Cells

Abstract

Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and the concurrent development of concentric lamellar bodies. Recently, H. Sawada et al. (2005, Toxicol. Sci. 83, 282-292) identified 17 genes as potential biomarkers of PLD in HepG2 cells. The present study was undertaken to determine if this set of genes measured by quantitative PCR could be validated in the same cell line. The objective was also to investigate the dose-response relationship to further validate the assay and to select the concentrations to use for screening activities. In a first experiment (one concentration tested), out of the 17 genes, the best gene biomarkers of PLD (i.e., 11 genes) were selected for practical screening reasons. Based on these genes, 91.6% (i.e., 11 of 12) of the compounds known to induce PLD were identified as positive and all the negative compounds (i.e., five of five) were also confirmed. When the data obtained in the first experiment were compared to the data by Sawada et al., (2005) the coefficient of correlation calculated was slightly higher than 75%. In the second experiment (26 compounds [all 17 compounds from the first experiment plus 9 other compounds] tested at a minimum of three concentrations), 93.3% (14/15) of the compounds known to induce PLD were identified as such and all the negative controls (six compounds) were also confirmed. Three compounds likely to induce PLD were identified as positive in our assay. Finally, two compounds for which no data are available were also tested. When both experiments 1 and 2 were compared, the coefficient of correlation for 16 compounds tested at the same concentrations reached 87.7%. In conclusion, the present study further confirms the utility of gene expression in HepG2 cells to identify a potential to induce PLD. Finally, based on the data presented, researchers are encouraged to use a range of minimum three concentrations (e.g., 12.5, 25, and 50 microM) to screen for PLD in the human HepG2 cell line.

High-content screening imaging and real-time cellular impedance monitoring for the assessment of chemical’s bio-activation with regards hepatotoxicity

“…As concerns erythromycin, an antibiotic drug able to induce hepatitis, hepatic dysfunction, jaundice and necrosis of hepatocytes at high dose (Pessayre et al, 1985), our study underlined not only its pro-oxidant properties leading to cytotoxicity but also it leading to an increase of lysosomal activity, as already shown (Cox et al, 1988). Perturbation of lysosomal function has been correlated with phospholipidosis (Atienzar et al, 2007;Gerbaux et al, 1996) and then hepatic injury (Antherieu et al, 2012), making crucial an early detection of such an event for drug development.…”

Activins and follistatins: Emerging roles in liver physiology and cancer

“…We observed a diurnal variation of beta E mRNA depending on food consumption in the rat liver and a surge of beta E expression in response to bacterial lipopolysaccharide (LPS) stimulation was also described [1,89] . Additionally, beta E expression was found and confirmed to be elevated in HepG2 cells as a consequence of phospholipidosis, a lipid storage disorder [91,92] . Expression of activin beta E was also significantly increased in the lung following airway inflammation [93] and in brains of rats infected with Borna disease virus [94] .…”

Activins and activin antagonists in hepatocellular carcinoma

“…Increased βE expression has also been observed in hepatic fibrosis induced by CCl4 [75] . Recently, induction of βE expression has been described as a marker for phospholipidosis in HepG2 hepatoma cells [82] . Similar to βC, βE subunit knock-out mice and double knock-outs lacking both βC and βE expression developed normally and had no defects in liver function [76] .…”