A human relevant mixture of persistent organic pollutants (POPs) and perfluorooctane sulfonic acid (PFOS) differentially affect glutamate induced excitotoxic responses in chicken cerebellum granule neurons (CGNs) in vitro.

Primary cultures of cerebellar granule neurons (CGNs) derived from chicken embryos were used to explore the effects on developmental neurotoxicity by a complex defined mixture of persistent organic pollutants (POPs). Its chemical composition and concentrations were based on blood levels in the Norwegian/Scandinavian population. Perfluorooctane sulfonic acid (PFOS) alone, its most abundant compound was also evaluated. Different stages of CGNs maturation, between day in vitro (DIV) 1, 3, and 5 were exposed to the POP mixture, or PFOS alone. Their combination with glutamate, an excitatory endogenous neurotransmitter important in neurodevelopment, also known to cause excitotoxicity was evaluated. Outcomes with the mixture at 500x blood levels were compared to PFOS at its corresponding concentration of 20 µM. The POP mixture reduced tetrazolium salt (MTT) conversion at earlier stages of maturation, compared to PFOS alone. Glutamate-induced excitotoxicity was enhanced above the level of that induced by glutamate alone, especially in mature CGNs at DIV5. Glutathione (GSH) concentrations seemed to set the level of sensitivity for the toxic insults from exposures to the pollutants. The role of N-methyl-D-aspartate receptor (NMDA-R) mediated calcium influx in pollutant exposures was investigated using the non-competitive and competitive receptor antagonists MK-801 and CGP 39551. Observations indicate a calcium-independent, but still NMDA-R dependent mechanism in the absence of glutamate, and a calcium- and NMDA-R dependent one in the presence of glutamate. The outcomes for the POP mixture cannot be explained by PFOS alone, indicating that other chemicals in the mixture contribute its overall effect.

Effects of defined mixtures of POPs and endocrine disruptors on the steroid metabolome of the human H295R adrenocortical cell line.

The presence of environmental pollutants in our ecosystem may impose harmful health effects to wildlife and humans. Several of these toxic chemicals have a potential to interfere with the endocrine system. The adrenal cortex has been identified as the main target organ affected by endocrine disrupting chemicals. The aim of this work was to assess exposure effects of defined and environmentally relevant mixtures of chlorinated, brominated and perfluorinated chemicals on steroidogenesis, using the H295R adrenocortical cell line model in combination with a newly developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method. By using this approach, we could simultaneously analyze 19 of the steroids in the steroid biosynthesis pathway, revealing a deeper insight into possible disruption of steroidogenesis. Our results showed a noticeable down-regulation in steroid production when cells were exposed to the highest concentration of a mixture of brominated and fluorinated compounds (10,000-times human blood values). In contrast, up-regulation was observed with estrone under the same experimental condition, as well as with some other steroids when cells were exposed to a perfluorinated mixture (1000-times human blood values), and the mixture of chlorinated and fluorinated compounds. Interestingly, the low concentration of the perfluorinated mixture alone produced a significant, albeit small, down-regulation of pregnenolone, and the total mixture a similar effect on 17-hydroxypregnenolone. Other mixtures resulted in only slight deviations from the control. Indication of synergistic effects were noted when we used a statistical model to improve data interpretation. A potential for adverse outcomes of human exposures is indicated, pointing to the need for further investigation into these mixtures.

Effects of defined mixtures of persistent organic pollutants (POPs) on multiple cellular responses in the human hepatocarcinoma cell line, HepG2, using high content analysis screening.

Persistent organic pollutants (POPs) are toxic substances, highly resistant to environmental degradation, which can bio-accumulate and have long-range atmospheric transport potential. Most studies focus on single compound effects, however as humans are exposed to several POPs simultaneously, investigating exposure effects of real life POP mixtures on human health is necessary. A defined mixture of POPs was used, where the compound concentration reflected its contribution to the levels seen in Scandinavian human serum (total mix). Several sub mixtures representing different classes of POPs were also constructed. The perfluorinated (PFC) mixture contained six perfluorinated compounds, brominated (Br) mixture contained seven brominated compounds, chlorinated (Cl) mixture contained polychlorinated biphenyls and also p,p'-dichlorodiphenyldichloroethylene, hexachlorobenzene, three chlordanes, three hexachlorocyclohexanes and dieldrin. Human hepatocarcinoma (HepG2) cells were used for 2h and 48h exposures to the seven mixtures and analysis on a CellInsight™ NXT High Content Screening platform. Multiple cytotoxic endpoints were investigated: cell number, nuclear intensity and area, mitochondrial mass and membrane potential (MMP) and reactive oxygen species (ROS). Both the Br and Cl mixtures induced ROS production but did not lead to apoptosis. The PFC mixture induced ROS production and likely induced cell apoptosis accompanied by the dissipation of MMP. Synergistic effects were evident for ROS induction when cells were exposed to the PFC+Br mixture in comparison to the effects of the individual mixtures. No significant effects were detected in the Br+Cl, PFC+Cl or total mixtures, which contain the same concentrations of chlorinated compounds as the Cl mixture plus additional compounds; highlighting the need for further exploration of POP mixtures in risk assessment.