Geert R. Verheyen

Automated analysis of contractility in the embryonic stem cell test, a novel approach to assess embryotoxicity

The embryonic stem cell test (EST) is an ECVAM-validated assay to detect embryotoxicity. The output of the assay is the effect of test compounds on the differentiation of murine-derived embryonic stem cells (D3 cells), recorded by visual analysis of contracting cardiomyocyte-like cells. Incorporation of a system to assess the contractility in an automated manner is proposed, to increase the throughput in the EST independent of observer bias. The automated system is based on image recording of each well, resulting in the area (pixels) and frequency of contractility (Hz). Four test compounds were assessed for their embryotoxic potency in the 96-well version of the EST, with both manual and automated analysis: 6-Aminonicotinamide, Valproic Acid, Boric Acid, and Penicillin G. There was no statistically significant difference in the outcome of both methods in the fraction of contractility (p<0.05), resulting in the same rank-order of Relative Embryotoxic Potency (REP) values: 6-aminonicotinamide (1)>valproic acid (0.007-0.013)>Boric Acid (0.002-0.005)>Penicillin G (0.00001). The automated image recording of contractile cardiomyocyte-like cells in the EST allows for an unbiased high throughput method to assess the embryotoxic potency of test compounds, resulting in an outcome comparable to manual analysis.

Screening for phospholipidosis induced by central nervous drugs: Comparing the predictivity of an in vitro assay to high throughput in silico assays

Drug-induced phospholipidosis is a side effect for which drug candidates can be screened in the drug discovery phase. The numerous in silico models that have been developed as a first line of screening are based on the characteristic physicochemical properties of phospholipidosis-inducing drugs, e.g. high logP and pK(b) values. However, applying these models on a predominantly high lipophilic, basic CNS chemistry results in a high false positive rate and consequently in a wrong classification of a large number of valuable drug candidates. Here, we tested 33 CNS-compounds (24 in vivo negative and 9 in vivo positive phospholipidosis-inducers) in our in house developed in vitro phospholipidosis screening assay (Mesens et al., 2009) and compared its predictivity with the outcome of three different, well established in silico prediction models. Our in vitro assay demonstrates an increased specificity of 79% over the in silico models (29%). Moreover, by considering the proposed plasma concentration at the efficacious dose we can show a clear correlation between the in vitro and in vivo occurrence of phospholipidosis, improving the specificity of prediction to 96%. Through its high predictive value, the in vitro low throughput assay is thus preferred above high throughput in silico assays, characterized by a high false positive rate.

MUTZ-3-derived dendritic cells as an in vitro alternative model to CD34+ progenitor-derived dendritic cells for testing of chemical sensitizers

The cytokine-dependent CD34(+) human acute myeloid leukaemia cell line MUTZ-3 was used to generate immature dendritic-like cells (MUTZ-3 DC) and their validity as an alternative to primary CD34(+) progenitor-derived DC (CD34-DC) for testing chemical-induced sensitization was assessed. Expression levels of the DC maturation markers HLA-DR, CD86, CD83 and CD11c were studied using flow cytometry after 24 and 48 h exposure to the model compound nickel sulphate (100 and 300 microM). No maturation of MUTZ-3 DC was observed, whereas significantly upregulated expression levels of CD83 and CD86 were noticed in CD34-DC after 24h treatment with 300 microM nickel sulphate compared to control cells. Differential expression of the cytokine genes IL1beta, IL6, IL8, CCL2, CCL3, CCL3L1, CCL4 was analyzed using real-time RT-PCR after 6, 10 and 24h of nickel sulphate exposure. In response to 100 microM nickel sulphate MUTZ-3 DC revealed slightly upregulated mRNA levels after 24h, whereas 300 microM induced transcription of CCL3, CCL3L1 and IL8 significantly after 6 or 10h. These cytokine data correspond to the previously observed effects of 100 microM nickel sulphate in CD34-DC. Our findings underline the stimulatory capacity of nickel sulphate in MUTZ-3 DC with regard to cytokine mRNA induction, but not surface marker expression. Compared to CD34-DC, however, the studied endpoint markers seemed to be less inducible, making the MUTZ-3 DC model in its presented form less suitable for in vitro testing of sensitization. Further assessment of MUTZ-3 DC using other differentiation protocols and an extended set of chemicals will be required to reveal whether this cell line may be a valid alternative model system to primary CD34-DC.

Phospholipidosis in Rats Treated with Amiodarone: Serum Biochemistry and Whole Genome Micro-Array Analysis Supporting the Lipid Traffic Jam Hypothesis and the Subsequent Rise of the Biomarker BMP

To provide mechanistic insight in the induction of phospholipidosis and the appearance of the proposed biomarker di-docosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate (BMP), rats were treated with 150 mg/kg amiodarone for 12 consecutive days and analyzed at three different time points (day 4, 9, and 12). Biochemical analysis of the serum revealed a significant increase in cholesterol and phospholipids at the three time points. Bio-analysis on the serum and urine detected a time-dependent increase in BMP, as high as 10-fold compared to vehicle-treated animals on day 12. Paralleling these increases, micro-array analysis on the liver of treated rats identified cholesterol biosynthesis and glycerophospholipid metabolism as highly modulated pathways. This modulation indicates that during phospholipidosis-induction interactions take place between the cationic amphiphilic drug and phospholipids at the level of BMP-rich internal membranes of endosomes, impeding cholesterol sorting and leading to an accumulation of internal membranes, converting into multilamellar bodies. This process shows analogy to Niemann-Pick disease type C (NPC). Whereas the NPC-induced lipid traffic jam is situated at the cholesterol sorting proteins NPC1 and NPC2, the amiodarone-induced traffic jam is thought to be located at the BMP level, demonstrating its role in the mechanism of phospholipidosis-induction and its significance for use as a biomarker.