Assessing the efficacy of antidote therapy against Amanita phalloides intoxication
Monieb Ahmed1, Inge A. M. Graaf1 , Bart G.J. Dekkers2, Daan J. Touw1,2, Sylvia Notenboom1

1 Division of Pharmacokinetics, Toxicology and Targeting, Groningen Research Institute for Pharmacy, University of Groningen, the Netherlands
2 Department of Clinical Pharmacy and Pharmacology, University of Groningen, University Medical Center Groningen, the Netherlands

Background: Amanita phalloides is one of the most toxic mushrooms in the world. Ingestion of α-amanitin, the main toxin of A. Phalloides, can result in acute liver and renal failure and even subsequent death in severe cases. Unfortunately, there is only weak clinical data for the effectiveness of the used antidotes benzylpenicillin and silibinin.

The aim of this study was to study the efficacy of these antidotes against α-amanitin toxicity in both kidney and liver, preferably using human tissue.

Methods: For this purpose, human, rat and porcine precision-cut kidney slices (PCKS) and human and rat precision-cut liver slices (PCLS) were exposed to 0.1;0.25;0.5, and 1µM α-amanitin for 24h and 48h. Besides, Silibin (100µM) or benzylpenicillin (0.5mМ) were given in combination with a toxic concentration of α-amanitin. The viability of PCLS and PCKS was evaluated by measurement of intracellular ATP and lactate dehydrogenase (LDH) leakage into the medium.

Results: PCKS remained viable after exposure to 0.1-1µM α-amanitin in all species. However, rat and human PCLS showed a decrease in intracellular ATP and an increase in LDH leakage after 24h and 48h exposure to either 0.5 or 1µM of α-amanitin, indicating toxicity. Preliminary results indicate that simultaneous exposure with silibinin prevents this decline in viability caused by α-amanitin better than Benzylpenicillin.

Conclusion: It appears that α-amanitin is not toxic to rat, porcine and human kidney. However, α-amanitin is toxic for rat and human liver. Silibinin seems to prevent the damage caused by α-amanitin in the liver, but further studies are needed.

Key words: α-amanitin, Benzylpenicillin, Silibinin, Amanita phalloides Intoxication, Precision-cut Liver slices, Precision-cut Kidney slices

CASE STUDY ON TOXICOKINETIC MODELLING OF ALKOXYETHANOLS IN ZEBRAFISH EMBRYOS
Renate Groot1,*, Ségolène Siméon2, Emiel Rorije1, Frédéric Bois2, Leo van der Ven1, Joop de Knecht1

1 – Rijksinstituut voor Volksgezondheid en Milieu (RIVM), Antonie van Leeuwenhoeklaan 9, Bilthoven, The Netherlands
2 – INERIS, METO unit, Parc ALATA BP2, 60550 Verneuil-en-Halatte, France
* - presenting author

Abstract
Until now, environmental and human risk assessments of chemicals have mostly been evaluated separately with their respective test models. However, a more integrated approach might be advantageous when adverse outcomes in the respective test systems have similar underlying pathways. At the intersection of both domains is the zebrafish embryo (ZFE): an ecotoxicological model with ever-increasing applications in human health assessment. In this internal RIVM project, alkoxyethanols and their metabolites were chosen to compare their developmental effects in ZFE and mammalian models. Database comparisons showed several discrepancies between these models, possibly caused by species sensitivity differences and/or toxicokinetic variations. The translation of embryo toxicity data for human health may be improved through computer-modelled toxicokinetics.
Specific factors complicate the use of ZFE for developmental toxicity: uncertainty about metabolism in the developing organism and unknown internal concentrations due to the possible barrier function of the chorion and unknown uptake mechanisms. These factors were explored through standardized ZFE toxicity assays combined with internal and external exposure and produced metabolites measurements (LCMS-MS).
The results show varying levels of metabolism during development, because overall the toxic potencies of parent compounds appear to increase with time. This matches with our hypothesis that, for the chemicals tested, the active metabolite exerts toxic effects in the embryo. Further research is focusing on quantifying parent compounds, possible intermediates and final metabolites in the medium and internally over time, and parameterizing a generic toxicokinetic model, which is based on physicochemical properties of the compounds and the specificities of the developing zebrafish.

Integrated in vitro assessment of lung toxicity and neurotoxicity of e-cigarette vapour
Yixuan Li1,2, Yvonne CM Staal2, Ilse Gosens2,, Flemming R Cassee2,, Harm J Heusinkveld1,2, Remco HS Westerink1,

1 Neurotoxicology Research Group, Toxicology and Pharmacology Division, Institute for Risk Assessment Sciences (IRAS), Utrecht University, Utrecht, The Netherlands.
2 National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands

The rapid increase of electronic cigarette (e-cigarette) use has raised concern of potential health risks of e-cigarette vapour containing nicotine and/or flavourings. Nicotine is highly addictive and known to modulate neuronal activity in the brain. Moreover, commonly used flavourings such as menthol and vanillin are also suggested to cause neuronal effects. Although oral exposure to flavourings is generally considered safe, toxicity upon inhalation exposure is largely unknown. Hence, an urgent need for an integrated assessment of lung- and neurotoxicity of e-cigarette vapour exists.

In vitro cigarette smoke toxicity studies are traditionally performed with submerged cell models, not necessarily resembling a realistic human exposure scenario. The air-liquid interface (ALI) currently provides the most physiologically-relevant in vitro exposure method for inhaled substances. ALI-cultured human alveolar cells (A549) were used as lung model. After exposure to e-cigarette vapour, lung toxicity parameters such as cell cytotoxicity and production of pro-inflammatory cytokines, were assessed. To subsequently evaluate the neurotoxic potential of vapour components, the basolateral medium collected from the ALI system just after exposure was transferred to rat primary cortical cells cultured on micro-electrode array (MEA) to assess changes in neuronal network functionality. Using this innovative tandem approach it was demonstrated that exposure to e-cigarette vapour is able to induce changes in neuronal activity without overt lung toxicity.

This study demonstrates that it is possible to combine innovative in vitro techniques into an integrated test strategy for inhalation neurotoxicology.

This work was funded by the 3Rs Stimulus Fund and the Faculty of Veterinary Medicine (Utrecht University, The Netherlands).

Keywords: e-cigarettes, lung toxicity, neurotoxicity, micro-electrode array (MEA), air-liquid interface (ALI)

Can Precision Cut Tissue Slices be used as ex-vivo model for nanosafety studies?
A.Z. van der Meer, B. Abhimata, R. Bartucci, Y. Boersma, P. Olinga, A. Salvati

University of Groningen (RUG), Groningen Research Institute of Pharmacy

Keywords: nanoparticles, nanosafety, PCTS, viability, morphology

In the last decades, nanosafety has emerged as a novel field in toxicology and many in vivo and in vitro models have been used to assess effects of engineered nanomaterials (ENMs). Within this context, we explored the ex vivo model precision cut tissue slices (PCTS) as a potential model for nanosafety studies. PCTS are tissue slices with a diameter of 5 mm and a thickness of 200-300 µm and have been validated and used for toxicological studies. To test their potential use in nanosafety, we have used PCTS of mouse lungs, livers, and kidneys. PCTS were exposed to a panel of ENMs including carboxylated and amino-modified polystyrene (PS-COOH and PS-NH2), silica, and titania. Viability was determined by quantification of ATP in the tissue, normalized by total protein content. Relative gene expression of fibrosis and inflammation markers was determined using RT-qPCR, coupled with immuno-histochemical staining of Collagen type-1 in order to determine fibrotic response to ENMs at longer exposure times (72h exposure).
As expected, exposure to PS-COOH showed no significant effects, while exposure to silica resulted in a small decrease in viability and small increase in fibrotic marker expression. In accordance to literature, exposure to the PS-NH2 caused significant toxicity in all organs. In conclusion, PCTS can reproduce some results observed in in vitro tests for acute exposure to ENMs. In order to investigate eventual fibrotic responses after chronic exposure, further optimization to allow prolonged slice viability is required.

Effect of hyperthermia on neurotoxicity induced by illicit drugs
Michiel E Olijhoek1,*, Anne Zwartsen1,2, Laura Hondebrink2, Remco HS Westerink1

1 Neurotoxicology Research Group, Toxicology and Pharmacology Division, Institute for Risk Assessment Sciences (IRAS), Utrecht University, Utrecht, The Netherlands.
2 Dutch Poisons Information Center (DPIC), University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
* Presenting author

Cocaine and MDMA are amongst the most used illicit drugs, with a lifetime prevalence of up to 13% in young adults (aged 15-34). Both drugs are primarily used in the club scene, MDMA as a prosocial drug and cocaine as a stimulant. Their effects on various neuronal targets generate both desired effects and adverse side effects. One particular side effect of illicit drug use is hyperthermia. The drug-induced increase in body temperature may increase even more when users exert themselves during dance events in poorly ventilated areas. At emergency departments, temperatures up to 41˚C have been reported. However, most in vitro effects of illicit drugs have been measured at body temperature. It is unknown whether increased body temperature exacerbates neurotoxicity.

Recently, effects of illicit drugs on neuronal activity have been measured in primary cortical cultures grown on multi-well microelectrode arrays (mwMEA) at 37˚C. We therefore repeated these experiments and compared the effects of MDMA and cocaine at hyperthermia. Cells were exposed to MDMA or cocaine at 37˚C vs. 41˚C for 4h. Effects on neuronal activity were assessed at the start and end of the exposure. The exposure is followed by a wash-out period of 19h. Effects on neuronal activity and cell viability were measured again at 24h.

Our results indicate that hyperthermia increases the inhibitory effects of MDMA and cocaine on spontaneous neuronal activity at non-cytotoxic concentrations. In conclusion, the worsening of drug-induced neurotoxicity by hyperthermia should be taken into account in risk assessment of psychoactive substances.

Keywords: MDMA, cocaine, illicit drugs, hyperthermia, neurotoxicity.