The zebrafish embryo as a model for chemically-induced steatosis: A case study with three pesticides.

There is an increasing incidence and prevalence of fatty liver disease in the western world, with steatosis as the most prevalent variant. Known causes of steatosis include exposure to food-borne chemicals, and overconsumption of alcohol, carbohydrates and fat, and it is a well-known side effect of certain pharmaceuticals such as tetracycline, amiodarone and tamoxifen (drug-induced hepatic steatosis). Mechanistic knowledge on chemical-induced steatosis has greatly evolved and has been organized into adverse outcome pathways (AOPs) describing the chain of events from first molecular interaction of a substance with a biological system to the adverse outcome, intrahepatic lipid accumulation. In this study, three known steatosis-inducing pesticides (imazalil, clothianidin, and thiacloprid) were tested for their ability to induce hepatic triglyceride accumulation in the zebrafish (Danio rerio) embryo (ZFE) at 5 days post fertilization, both as single compounds and equipotent binary mixtures. The results indicate that the ZFE is very well applicable as a higher tier testing model to confirm effects in downstream key events in AOPs, that is, chemically-induced triglyceride accumulation in the whole organism and production of visible steatosis. Moreover, dose addition could be concluded for binary mixtures of substances with similar and with dissimilar modes of action.

An expeditive and green chemo-enzymatic route to diester sinapoyl-l-malate analogues: sustainable bioinspired and biosourced UV filters and molecular heaters.

Sinapoyl malate, naturally present in plants, has proved to be an exceptional UV filter and molecular heater for plants. Although there are nowadays industrially relevant sustainable synthetic routes to sinapoyl malate, its incorporation into certain cosmetic formulations, as well as its adsorption on plant leaves, is limited by its hydrophilicity. To overcome these obstacles, it is important to find a way to effectively control the hydrophilic-lipophilic balance of sinapoyl malate to make it readily compatible with the cosmetic formulations and stick on the waxy cuticle of leaves. To this end, herein, we describe a highly regioselective chemo-enzymatic synthesis of sinapoyl malate analogues possessing fatty aliphatic chains of variable length, enabling the lipophilicity of the compounds to be modulated. The potential toxicity (i.e., mutagenicity, carcinogenicity, endocrine disruption, acute and repeated-dose toxicity), bioaccumulation, persistence and biodegradability potential of these new analogues were evaluated in silico, along with the study of their transient absorption spectroscopy, their photostability as well as their photodegradation products.

Adverse outcome pathway-based analysis of liver steatosis in vitro using human liver cell lines.

Here, we present an in vitro test battery to analyze chemicals for their potential to induce liver triglyceride accumulation, a hallmark of liver steatosis. We describe steps for using HepG2 and HepaRG human hepatoma cells in conjunction with a combination of several in vitro assays covering the different molecular initiating events and key events of the respective adverse outcome pathway. This protocol is suitable for assessing single substance effects as well as mixtures allowing their classification as steatotic or non-steatotic. For complete details on the use and execution of this protocol, please refer to Luckert et al. (2018),1 Lichtenstein et al. (2020),2 and Knebel et al. (2019).3.

Okadaic Acid Activates JAK/STAT Signaling to Affect Xenobiotic Metabolism in HepaRG Cells.

Okadaic acid (OA) is a marine biotoxin that is produced by algae and accumulates in filter-feeding shellfish, through which it enters the human food chain, leading to diarrheic shellfish poisoning (DSP) after ingestion. Furthermore, additional effects of OA have been observed, such as cytotoxicity. Additionally, a strong downregulation of the expression of xenobiotic-metabolizing enzymes in the liver can be observed. The underlying mechanisms of this, however, remain to be examined. In this study, we investigated a possible underlying mechanism of the downregulation of cytochrome P450 (CYP) enzymes and the nuclear receptors pregnane X receptor (PXR) and retinoid-X-receptor alpha (RXRα) by OA through NF-κB and subsequent JAK/STAT activation in human HepaRG hepatocarcinoma cells. Our data suggest an activation of NF-κB signaling and subsequent expression and release of interleukins, which then activate JAK-dependent signaling and thus STAT3. Moreover, using the NF-κB inhibitors JSH-23 and Methysticin and the JAK inhibitors Decernotinib and Tofacitinib, we were also able to demonstrate a connection between OA-induced NF-κB and JAK signaling and the downregulation of CYP enzymes. Overall, we provide clear evidence that the effect of OA on the expression of CYP enzymes in HepaRG cells is regulated through NF-κB and subsequent JAK signaling.

Use of transcriptomics in hazard identification and next generation risk assessment: A case study with clothianidin.

Toxicological risk assessment is essential in the evaluation and authorization of different classes of chemical substances. Genotoxicity and mutagenicity testing are of highest priority and rely on established in vitro systems with bacterial and mammalian cells, sometimes followed by in vivo testing using rodent animal models. Transcriptomic approaches have recently also shown their value to determine transcript signatures specific for genotoxicity. Here, we studied how transcriptomic data, in combination with in vitro tests with human cells, can be used for the identification of genotoxic properties of test compounds. To this end, we used liver samples from a 28-day oral toxicity study in rats with the pesticidal active substances imazalil, thiacloprid, and clothianidin, a neonicotinoid-type insecticide with, amongst others, known hepatotoxic properties. Transcriptomic results were bioinformatically evaluated and pointed towards a genotoxic potential of clothianidin. In vitro Comet and γH2AX assays in human HepaRG hepatoma cells, complemented by in silico analyses of mutagenicity, were conducted as follow-up experiments to check if the genotoxicity alert from the transcriptomic study is in line with results from a battery of guideline genotoxicity studies. Our results illustrate the combined use of toxicogenomics, classic toxicological data and new approach methods in risk assessment. By means of a weight-of-evidence decision, we conclude that clothianidin does most likely not pose genotoxic risks to humans.

Investigating the in vitro steatotic mixture effects of similarly and dissimilarly acting test compounds using an adverse outcome pathway-based approach.

Within the EuroMix project, we have previously developed an adverse outcome pathway (AOP)-based in vitro assay toolbox to investigate the combined effects of liver steatosis-inducing compounds in human HepaRG hepatocarcinoma cells. In this study, we applied the toolbox to further investigate mixture effects of combinations, featuring either similarly acting or dissimilarly acting substances. The valproic acid structural analogs 2-propylheptanoic acid (PHP) and 2-propylhexanoic acid (PHX) were chosen for establishing mixtures of similarly acting substances, while a combination with the pesticidal active substance clothianidin (CTD) was chosen for establishing mixtures of dissimilarly acting compounds. We first determined relative potency factors (RPFs) for each compound based on triglyceride accumulation results. Thereafter, equipotent mixtures were tested for nuclear receptor activation in transfected HepG2 cells, while gene expression and triglyceride accumulation were investigated in HepaRG cells, following the proposed AOP for liver steatosis. Dose addition was observed for all combinations and endpoints tested, indicating the validity of the additivity assumption also in the case of the tested mixtures of dissimilarly acting substances. Gene expression results indicate that the existing steatosis AOP can still be refined with respect to the early key event (KE) of gene expression, in order to reflect the diversity of molecular mechanisms underlying the adverse outcome.

An eight-compound mixture but not corresponding concentrations of individual chemicals induces triglyceride accumulation in human liver cells.

In real life, organisms are exposed to complex mixtures of chemicals at low concentration levels, whereas research on toxicological effects is mostly focused on single compounds at comparably high doses. Mixture effects deviating from the assumption of additivity, especially synergistic effects, are of concern. In an adverse outcome pathway (AOP)-guided manner, we analyzed the accumulation of triglycerides in human HepaRG liver cells by a mixture of eight steatotic chemicals (amiodarone, benzoic acid, cyproconazole, flusilazole, imazalil, prochloraz, propiconazole and tebuconazole), each present below its individual effect concentration at 1-3 µM. Pronounced and significantly enhanced triglyceride accumulation was observed with the mixture, and similar effects were seen at the level of pregnane-X-receptor activation, a molecular initiating event leading to hepatic steatosis. Transcript pattern analysis indicated subtle pro-steatotic changes at low compound concentrations, which did not exert measurable effects on cellular triglycerides. Mathematical modeling of mixture effects indicated potentially more than additive behavior using a model for compounds with similar modes of action. The present data underline the usefulness of AOP-guided in vitro testing for the identification of mixture effects and highlight the need for further research on chemical mixtures and harmonization of data interpretation of mixture effects.

Transcriptomics analysis of hepatotoxicity induced by the pesticides imazalil, thiacloprid and clothianidin alone or in binary mixtures in a 28-day study in female Wistar rats.

Co-occurrence of pesticide residues in food commodities raises a potential safety issue as their mixture effects on human health are largely unknown. In a previous study, we reported the toxicological effects (pathology and histopathology) of imazalil (IMZ), thiacloprid (THI), and clothianidin (CTD) alone and in binary mixtures in a 28-day oral gavage study in female Wistar rats. Five dose levels (up to 350 mg/kg body weight/day) ranging from a typical toxicological reference value to a clear effect dose were applied. In the present study, we undertook a transcriptomics analysis of rat livers by means of total RNA sequencing (RNA-Seq). Bioinformatic data analysis involving Ingenuity Pathway Analysis (IPA) was used to gain mechanistic information on hepatotoxicity-related pathways affected after treatment with the pesticides, alone and in mixtures. Our data show that 2986 genes were differentially regulated by CTD while IMZ and THI had effects on 194 and 225 genes, respectively. All three individual compounds shared a common subset of genes whose network is associated with xenobiotic metabolism and nuclear receptor activation. Similar networks were retrieved for the mixtures. Alterations in the expression of individual genes were in line with the assumption of dose addition. Our results bring new insight into the hepatotoxicity mechanisms of IMZ, THI, and CTD and their mixtures.

Hepatotoxicity of the pesticides imazalil, thiacloprid and clothianidin - Individual and mixture effects in a 28-day study in female Wistar rats.

Humans are exposed to pesticide residues through various food products. As these residues can occur in mixtures, there is a need to investigate possible mixture effects on human health. Recent exposure studies revealed the preponderance of imazalil, thiacloprid, and clothianidin in food diets. In this study, we assessed their toxicity alone and in binary mixtures in a 28-day gavage study in female Wistar rats. Five dose levels (up to 350 mg/kg bw/day) ranging from a typical toxicological reference value to a clear effect dose were applied. Data show that the liver was a target organ of all pesticides and their mixtures. Increases in liver weight were observed and histopathological examination revealed centrilobular hepatocellular hypertrophy and cytoplasm degeneration for all treatment conditions. No accumulation of hepatic triglycerides was reported. Tissue residue analysis showed altered pesticide residues in the liver and the kidney when being in mixture as compared to the levels of pesticide residues for the single compound treatment, indicating possible toxicokinetic interactions. Overall, all mixtures appeared to follow the additivity concept, even though quantitative analysis was limited for some endpoints due to the semi-quantitative nature of the data, raising no specific concern for the risk assessment of the examined pesticides.

An adverse outcome pathway-based approach to assess steatotic mixture effects of hepatotoxic pesticides in vitro.

Exposure to complex chemical mixtures requires a tiered strategy for efficient mixture risk assessment. As a part of the EuroMix project we developed an adverse outcome pathway (AOP)-based assay toolbox to investigate the combined effects of the liver steatosis-inducing compounds imazalil, thiacloprid, and clothianidin in human HepaRG hepatocarcinoma cells. Compound-specific relative potency factors were determined using a benchmark dose approach. Equipotent mixtures were tested for nuclear receptor activation, gene and protein expression, and triglyceride accumulation, according to the molecular initiating events and key events proposed in the steatosis AOP. All three compounds affected the activity of nuclear receptors, but not key genes/proteins as proposed. Triglyceride accumulation was observed with three different methods. Mixture effects were in agreement with the assumption of dose additivity for all the combinations and endpoints tested. Compound-specific RPFs remained similar over the different endpoints studied downstream the AOP. Therefore, it might be possible to reduce testing to a smaller battery of key tests. The results demonstrate the suitability of our in vitro assay toolbox, integrated within an AOP framework and combined with the RPF approach, for the analysis of steatotic effects of chemical mixtures. However, mRNA results suggest that the steatosis AOP still needs improvement.

Metabolism of the Marine Phycotoxin PTX-2 and Its Effects on Hepatic Xenobiotic Metabolism: Activation of Nuclear Receptors and Modulation of the Phase I Cytochrome P450.

PTX-2 is a marine biotoxin frequently found in shellfish that can lead to food intoxication in humans. Information regarding PTX-2 metabolism is scarce, and little is known of its effect on xenobiotic-metabolizing enzymes (XME) or its molecular pathways. The aim of this study was consequently to examine PTX-2 Phase I metabolism using rat and human liver S9 fractions, and also to assess the capability of PTX-2: (i) to modulate the gene expression of a panel of Phase I (CYP450) and II (UGT, SULT, NAT, and GST) enzymes, as well as the Phase III or 0 (ABC and SLCO) transporters in the human hepatic HepaRG cell line using qPCR; (ii) to induce specific CYP450 in HepaRG cells measured by immunolabeling detection and the measurement of the cells' activities; and (iii) to activate nuclear receptors and induce CYP promoter activities in HEK-T and HepG2 transfected cell lines using transactivation and reporter gene assay, respectively. Our results indicate that PTX-2 hydroxylation occurred with both rat and human S9 fractions. Whereas PTX-2 mostly upregulated the gene expression of CYP1A1 and 1A2, no induction of these two CYP activities was observed. Lastly, PTX-2 did not act as an agonist of CAR or PXR. Due to its effects on some key XME, more attention should be paid to possible drug-drug interactions with phycotoxins, especially as shellfish can accumulate several phycotoxins as well as other kinds of contaminants.