Evaluation of the INS-1 832/13 cell line as a beta-cell based screening system to assess pollutant effects on beta-cell function.

Environmental pollutants have recently emerged as potential risk factors for metabolic diseases, urging systematic investigation of pollutant effects on metabolic disease processes. To enable risk assessment of these so-called metabolic disruptors the use of stable, robust and well-defined cell based screening systems has recently been encouraged. Since beta-cell (dys)functionality is central in diabetes pathophysiology, the need to develop beta-cell based pollutant screening systems is evident. In this context, the present research evaluated the strengths and weaknesses of the INS-1 832/13 pancreatic beta-cell line as diabetogenic pollutant screening system with a focus on beta-cell function. After optimization of exposure conditions, positive (exendin-4, glibenclamide) and negative (diazoxide) control compounds for acute insulin secretion responses were tested and those with the most profound effects were selected to allow potency estimations and ranking of pollutants. This was followed by a first explorative screening of acute bisphenol A and bis(2-ethylhexyl)phthalate effects. The same approach was applied for chronic exposures, focusing primarily on evaluation of acknowledged chronic stimulators (diazoxide, T0901317, exendin-4) or inhibitors (glibenclamide) of insulin secretion responses to select the most responsive ones for use as control compounds in a chronic pollutant testing framework. Our results showed that INS-1 832/13 cells responded conform previous observations regarding acute effects of control compounds on insulin secretion, while bisphenol A and bis(2-ethylhexyl)phthalate had limited acute effects. Furthermore, chronic exposure to known beta-cell reactive compounds resulted in deviating insulin secretion and insulin content profiles compared to previous reports. In conclusion, this INS-1 subclone appears to lack certain characteristics needed to respond appropriately to acute pollutant exposure or long term exposure to known beta-cell reactive compounds and thus seems to be, in our setting, inadequate as a diabetogenic pollutant screening system.

Unraveling the mode of action of an obesogen: mechanistic analysis of the model obesogen tributyltin in the 3T3-L1 cell line.

Obesogenic compounds are chemicals that have an influence on obesity development. This study was designed to unravel the molecular mechanisms of the model obesogen TBT, using microarray analysis in the 3T3-L1 in vitro system, and to evaluate the use of toxicogenomics for obesogen screening. The microarray results revealed enrichment of Gene Ontology terms involved in energy and fat metabolism after 10 days of TBT exposure. Pathway analysis unveiled PPAR signalling pathway as the sole pathway significantly enriched after 1 day and the most significantly enriched pathway after 10 days of exposure. To our knowledge, this is the first study delivering an in depth mechanistic outline of the mode of action of TBT as an obesogen, combining effects on both cell physiological and gene expression level. Furthermore, our results show that combining transcriptomics with 3T3-L1 cells is a promising tool for screening of potential obesogenic compounds.

Mechanistic evaluation of the insulin response in H4IIE hepatoma cells: new endpoints for toxicity testing?

This study was designed to evaluate if the rat H4IIE hepatoma cell line is a physiologically relevant model to study hepatic insulin responses to hint at its prospective application in pollutant-related insulin resistance research. DNA microarray analysis, real-time PCR and flow cytometric cell cycle analysis were used to assess the relevance of the insulin response in H4IIE cells. Insulin dose dependently stimulated H4IIE growth and time dependently altered the expression of the known insulin responsive genes: Fasn, Pck1 and Irs2. Microarray analysis performed on cells exposed to insulin (100nM) for 6h and 24h showed that genes related to carbohydrate and lipid metabolism were most profoundly afflicted, in accordance with in vivo hepatic insulin action. Since changes in carbohydrate and lipid metabolism are pivotal in the pathogenesis of insulin resistance, the presence of a physiological relevant insulin response in H4IIE cells pleads for further testing of its potential use in research on pollutant-driven insulin resistance.

Mechanistic profiling of the cAMP-dependent steroidogenic pathway in the H295R endocrine disrupter screening system: new endpoints for toxicity testing.

The need for implementation of effects on steroid synthesis and hormone processing in screening batteries of endocrine disruptive compounds is widely acknowledged. In this perspective, hormone profiling in the H295R adrenocortical cell system is extensively examined and recently OECD validated (TG 456) as a replacement of the minced testis assay. To further elucidate the complete mechanisms and endocrine responsiveness of this cell system, microarray-based gene expression profiling of the cAMP response pathway, one of the major pathways in steroidogenesis regulation, was examined in H295R cells. Next to the steroid synthesis pathway, a broader lipid metabolic pathway, including cholesterol uptake/biosynthesis, hormone metabolization and many hormone and nuclear receptors, are sensitive towards cAMP stimulation in this cell system. Moreover, these pathways were clearly dose and time responsive, indicating early regulation (10 h) of cholesterol uptake and mobilization genes and later expression (24-48 h) of cholesterol biosynthesis and steroid synthesis. Transcription network analysis suggested several important transcription factors that could be involved in regulation of the steroid hormone pathway, of which HNF4α, a broader lipid metabolism related transcription factor, might indicate some new transcription regulation patterns in this cell line. Overall we can conclude that the time dependent gene expression patterns of the strongly coordinated cholesterol supply and steroidogenesis pathways in the H295R cell system seem to reflect well the in vivo ACTH/cAMP signalling cascade in adrenal cells. Moreover, the completeness of the steroidogenic related pathways in terms of gene expression sensitivity, indicates the H295R cell line as a promising cell line in omics-based endocrine disruption screening.