Characterization of chemical-induced sterile inflammation in vitro: application of the model compound ketoconazole in a human hepatic co-culture system.

Liver injury as a result of a sterile inflammation is closely linked to the activation of immune cells, including macrophages, by damaged hepatocytes. This interaction between immune cells and hepatocytes is as yet not considered in any of the in vitro test systems applied during the generation of new drugs. Here, we established and characterized a novel in vitro co-culture model with two human cell lines, HepG2 and differentiated THP-1. Ketoconazole, an antifungal drug known for its hepatotoxicity, was used as a model compound in the testing of the co-culture. Single cultures of HepG2 and THP-1 cells were studied as controls. Different metabolism patterns of ketoconazole were observed for the single and co-culture incubations as well as for the different cell types. The main metabolite N-deacetyl ketoconazole was found in cell pellets, but not in supernatants of cell cultures. Global proteome analysis showed that the NRF2-mediated stress response and the CXCL8 (IL-8) pathway were induced by ketoconazole treatment under co-culture conditions. The upregulation and ketoconazole-induced secretion of several pro-inflammatory cytokines, including CXCL8, TNF-α and CCL3, was observed in the co-culture system only, but not in single cell cultures. Taking together, we provide evidence that the co-culture model applied might be suitable to serve as tool for the prediction of chemical-induced sterile inflammation in liver tissue in vivo.

Accumulation of distinct persistent organic pollutants is associated with adipose tissue inflammation.

Hydrophobic environmental chemicals bio-accumulate in adipose tissue (AT) in animals and humans, but their impact on diseases related to adipose tissue dysfunction remains unclear. Moreover, visceral rather than subcutaneous (SC) fat deposition is more closely associated with cardio-metabolic diseases such as type 2 diabetes, fatty liver and cardiovascular diseases. We therefore tested the hypotheses that environmental chemicals bio-accumulate in a fat depot specific pattern and that these patterns are related AT inflammation and obesity comorbidities. First, we developed an extraction method for detecting and quantifying a set of 9 persistent organic pollutants (POPs) in human AT. The quantified chemicals exhibit KOW coefficients from 4 to 7. Paired abdominal omental and SC AT samples were obtained from 54 individuals (30 women, 24 men) with a wide range of body mass index (BMI, 16-70 kg/m2) during laparoscopic abdominal surgeries. Among the POPs are classical halogenated substances like Dichlorodiphenyldichloroethylene (DDE) and polychlorinated biphenyls (PCBs), but also fragrance substances. We find that AT concentrations of these chemicals are neither significantly different between visceral and SC fat depots nor between women and men. However, AT bio-accumulation of distinct POPs significantly correlates with AT macrophage infiltration, adipocyte size and parameters of glucose metabolism. In both fat depots, the strongest correlations of POPs (Ethyl- tetradecanoate, 4,4'-Diisopropylbiphenyl, 2-Phenyltetralin, 2,2',4,4',5,5'-Hexachlorobiphenyl, Hexachlorobenzene) and AT macrophage infiltration were detected in lean individuals. In men with obesity, abundance of POPs correlated with the duration of obesity. Additional significant associations between AT POPs and parameters of glycemia, insulin sensitivity, and inflammation suggest that specific environmental chemicals may contribute to AT dysfunction, adipocyte hypertrophy, impaired glucose metabolism, systemic inflammation and variation in fat distribution, but not to obesity.

DEHP deregulates adipokine levels and impairs fatty acid storage in human SGBS-adipocytes.

DEHP is a plasticizer which has been used in plastic products of everyday use for decades. Studies in mice and murine cell culture models identified DEHP as an endocrine disruptor that may also act as an obesogen. As this is of high concern in respect of the worldwide obesity epidemic, our aim is the translation of these findings into a human model system. On the basis of DOHaD, we investigated the influence of an environmentally relevant dose of DEHP [50 µg/ml] on adipogenesis in the human cell culture model SGBS. Pre-adipocytes were exposed to DEHP and differentiated into mature adipocytes. At different stages of differentiation, markers of adipogenesis like GLUT4, FABP4, LPL and PPARs, and of signaling pathways like AMPK/ACC2, JAK/STAT and MAPK were analyzed. Functional markers like adipokine secretion and triglyceride content as well as ROS production were measured in mature adipocytes. We found significantly lower expression levels of adipogenic markers, a reduction in lipid accumulation, higher leptin- and reduced adiponectin levels in the supernatant of treated adipocytes. Moreover, ROS production was significantly elevated after DEHP-exposure. In conclusion, DEHP led to lower grade of adipogenic differentiation in human SGBS-adipocytes under the chosen conditions.