Lucia Vergauwen

Adverse Outcome Pathway (AOP) Development I: Strategies and Principles

An adverse outcome pathway (AOP) is a conceptual framework that organizes existing knowledge concerning biologically plausible, and empirically supported, links between molecular-level perturbation of a biological system and an adverse outcome at a level of biological organization of regulatory relevance. Systematic organization of information into AOP frameworks has potential to improve regulatory decision-making through greater integration and more meaningful use of mechanistic data. However, for the scientific community to collectively develop a useful AOP knowledgebase that encompasses toxicological contexts of concern to human health and ecological risk assessment, it is critical that AOPs be developed in accordance with a consistent set of core principles. Based on the experiences and scientific discourse among a group of AOP practitioners, we propose a set of five fundamental principles that guide AOP development: (1) AOPs are not chemical specific; (2) AOPs are modular and composed of reusable components-notably key events (KEs) and key event relationships (KERs); (3) an individual AOP, composed of a single sequence of KEs and KERs, is a pragmatic unit of AOP development and evaluation; (4) networks composed of multiple AOPs that share common KEs and KERs are likely to be the functional unit of prediction for most real-world scenarios; and (5) AOPs are living documents that will evolve over time as new knowledge is generated. The goal of the present article was to introduce some strategies for AOP development and detail the rationale behind these 5 key principles. Consideration of these principles addresses many of the current uncertainties regarding the AOP framework and its application and is intended to foster greater consistency in AOP development.

Structure-activity relationship assessment of four perfluorinated chemicals using a prolonged zebrafish early life stage test.

Perfluorinated compounds (PFCs) are a group of anthropogenic chemicals containing diverse functional groups and chain lengths. They are known to be persistent and bioaccumulative explaining their worldwide environmental presence. The toxicological information on these chemicals is still incomplete and insufficient to assess their environmental impact and structure-activity relationship. In the present study, the developmental effects of PFOS (perfluorooctane sulfonate, C8), PFOA (perfluorooctanoic acid, C8), PFBS (perfluorobutane sulfonate, C4) and PFBA (perfluorobutanoic acid, C4) were evaluated in zebrafish embryos (Danio rerio). The different chain lengths and functional groups of the selected chemicals made it possible to determine the structure-activity relationship of these compounds. PFCs with longer chain lengths (C8) tend to be more toxic than PFCs with shorter chain lengths (C4). Comparison based on the functional groups of compounds with the same chain length indicates that PFCs with a sulfonate group have a larger toxic potential than the ones with a carboxyl group. Furthermore, exposure to the different PFCs resulted in some general effects, such as deformations of the tail and an uninflated swim bladder, as well as in more specific effects which might be related to the structure of the tested chemicals. Oedemas and effects on length could only be detected in 8-carbon PFCs while malformations of the head were a more specific action of the sulfonated PFCs. Effects on hatching rate and success were found in PFOA exposed embryos and heart rates were affected after exposure to PFOS, PFOA and PFBS.

Long-term warm or cold acclimation elicits a specific transcriptional response and affects energy metabolism in zebrafish

Organisms are often forced to acclimate to changing environmental temperature. Temperature compensation mechanisms have been reported, which enable organisms to minimize some of the temperature related effects. To investigate this process, zebrafish (Danio rerio) were acclimated to a control (26 degrees C), an increased (34 degrees C) or a decreased (18 degrees C) temperature for 4, 14 and 28 days. In general, warm acclimation depleted energy stores and decreased the condition factor, while cold acclimation increased both. The energy parameters as well as the transcriptional responses (investigated using printed 15k microarrays and real time PCR) indicated that warm acclimation was particularly stressful. However, after 28 days of warm acclimation, energy stores had recovered from the initial depletion. This could have been facilitated by the observed downregulation of transcripts involved in catabolic processes. Transcriptional regulation seemed to be an important means of coordinating the temperature compensation process. We could distinguish an early response which was independent of the direction of the temperature change and a direction specific long-term response. The early response was characterized by the upregulation of defence mechanisms, tissue regeneration and hemopoiesis. In the long-term response there was a strong emphasis on compensating for the altered metabolic rate as well as cell structure and replacement.

Physiological, biochemical, and genome-wide transcriptional analysis reveals that elevated CO2 mitigates the impact of combined heat wave and drought stress in Arabidopsis thaliana at multiple organizational levels

Climate changes increasingly threaten plant growth and productivity. Such changes are complex and involve multiple environmental factors, including rising CO2 levels and climate extreme events. As the molecular and physiological mechanisms underlying plant responses to realistic future climate extreme conditions are still poorly understood, a multiple organizational level analysis (i.e. eco-physiological, biochemical, and transcriptional) was performed, using Arabidopsis exposed to incremental heat wave and water deficit under ambient and elevated CO2 . The climate extreme resulted in biomass reduction, photosynthesis inhibition, and considerable increases in stress parameters. Photosynthesis was a major target as demonstrated at the physiological and transcriptional levels. In contrast, the climate extreme treatment induced a protective effect on oxidative membrane damage, most likely as a result of strongly increased lipophilic antioxidants and membrane-protecting enzymes. Elevated CO2 significantly mitigated the negative impact of a combined heat and drought, as apparent in biomass reduction, photosynthesis inhibition, chlorophyll fluorescence decline, H2 O2 production, and protein oxidation. Analysis of enzymatic and molecular antioxidants revealed that the stress-mitigating CO2 effect operates through up-regulation of antioxidant defense metabolism, as well as by reduced photorespiration resulting in lowered oxidative pressure. Therefore, exposure to future climate extreme episodes will negatively impact plant growth and production, but elevated CO2 is likely to mitigate this effect.

Microarray-based transcriptomic analysis of differences between long-term gregarious and solitarious desert locusts

Desert locusts (Schistocerca gregaria) show an extreme form of phenotypic plasticity and can transform between a cryptic solitarious phase and a swarming gregarious phase. The two phases differ extensively in behavior, morphology and physiology but very little is known about the molecular basis of these differences. We used our recently generated Expressed Sequence Tag (EST) database derived from S. gregaria central nervous system (CNS) to design oligonucleotide microarrays and compare the expression of thousands of genes in the CNS of long-term gregarious and solitarious adult desert locusts. This identified 214 differentially expressed genes, of which 40% have been annotated to date. These include genes encoding proteins that are associated with CNS development and modeling, sensory perception, stress response and resistance, and fundamental cellular processes. Our microarray analysis has identified genes whose altered expression may enable locusts of either phase to deal with the different challenges they face. Genes for heat shock proteins and proteins which confer protection from infection were upregulated in gregarious locusts, which may allow them to respond to acute physiological challenges. By contrast the longer-lived solitarious locusts appear to be more strongly protected from the slowly accumulating effects of ageing by an upregulation of genes related to anti-oxidant systems, detoxification and anabolic renewal. Gregarious locusts also had a greater abundance of transcripts for proteins involved in sensory processing and in nervous system development and plasticity. Gregarious locusts live in a more complex sensory environment than solitarious locusts and may require a greater turnover of proteins involved in sensory transduction, and possibly greater neuronal plasticity.

Mechanistic toxicity study of perfluorooctanoic acid in zebrafish suggests mitochondrial dysfunction to play a key role in PFOA toxicity

The aquatic environment is an important site for perfluorooctanoic acid (PFOA) deposit. Nevertheless, the exact mode of action and its resulting toxicological effects in aquatic organisms remain largely unknown. To gain a better understanding of the mode of action of teleost PFOA toxicity, transcriptomics, proteomics, biochemical parameters and reproduction were integrated in this study. Male and female zebrafish were exposed to nominal concentrations of 0.1, 0.5 and 1 mg L(-1) PFOA for 4 and 28 d resulting in PFOA accumulation which was higher in males than in females. These gender-related differences were likely caused by different elimination rates due to distinct hormone levels and differences in transport activity by solute carriers. The general mode of action of PFOA was described as an increase of the mitochondrial membrane permeability followed by an impairment of aerobic ATP production. Depletion of liver glycogen stores together with altered expression levels of transcripts involved in carbohydrate metabolism, with emphasis on anaerobic metabolism, was probably a means of compensating for this decreased aerobic efficiency. The mitochondrial dysfunction further resulted in effects on oxidative stress and apoptosis at the gene transcript and protein level. As a consequence, evidence for the replacement of the affected cells and organelles to sustain tissue homeostasis was found at the transcript level, resulting in an even greater glycogen depletion. Despite this increase in metabolic expenditure, no effects on reproduction were found indicating that the fish seemed to cope with exposure to the tested concentrations of PFOA during the exposure period of 1 month.

Drought Induces Distinct Growth Response, Protection and Recovery Mechanisms in the Maize Leaf Growth Zone

Drought inhibits cell division and expansion in the maize leaf growth zone by reducing antioxidant levels and increases photosynthetic capacity to allow for enhanced growth upon recovery. Drought is the most important crop yield-limiting factor, and detailed knowledge of its impact on plant growth regulation is crucial. The maize (Zea mays) leaf growth zone offers unique possibilities for studying the spatiotemporal regulation of developmental processes by transcriptional analyses and methods that require more material, such as metabolite and enzyme activity measurements. By means of a kinematic analysis, we show that drought inhibits maize leaf growth by inhibiting cell division in the meristem and cell expansion in the elongation zone. Through a microarray study, we observed the down-regulation of 32 of the 54 cell cycle genes, providing a basis for the inhibited cell division. We also found evidence for an up-regulation of the photosynthetic machinery and the antioxidant and redox systems. This was confirmed by increased chlorophyll content in mature cells and increased activity of antioxidant enzymes and metabolite levels across the growth zone, respectively. We demonstrate the functional significance of the identified transcriptional reprogramming by showing that increasing the antioxidant capacity in the proliferation zone, by overexpression of the Arabidopsis (Arabidopsis thaliana) iron-superoxide dismutase gene, increases leaf growth rate by stimulating cell division. We also show that the increased photosynthetic capacity leads to enhanced photosynthesis upon rewatering, facilitating the often-observed growth compensation.

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.

The potential of AOP networks for reproductive and developmental toxicity assay development.

Historically, the prediction of reproductive and developmental toxicity has largely relied on the use of animals. The adverse outcome pathway (AOP) framework forms a basis for the development of new non-animal test methods. It also provides biological context for mechanistic information from existing assays. However, a single AOP may not capture all events that contribute to any relevant toxic effect, even in single chemical exposure scenarios. AOP networks, defined as sets of AOPs sharing at least one common element, are capable of more realistically representing potential chemical effects. They provide information on interactions between AOPs and have the potential to reveal previously unknown links between biological pathways. Analysis of these AOP networks can aid the prioritization of assay development, whether the goal is to develop a single assay with predictive utility of multiple outcomes, or development of assays that are highly specific for a particular mode of action. This paper provides a brief overview of the AOPs related to reproductive and developmental toxicity currently available in the AOP Wiki (http://aopwiki.org), and gives an example of an AOP network based on five reproductive and developmental toxicity-related AOPs for fish to illustrate how AOP networks can be used for assay development and refinement.

Deiodinase Knockdown during Early Zebrafish Development Affects Growth, Development, Energy Metabolism, Motility and Phototransduction

Thyroid hormone (TH) balance is essential for vertebrate development. Deiodinase type 1 (D1) and type 2 (D2) increase and deiodinase type 3 (D3) decreases local intracellular levels of T3, the most important active TH. The role of deiodinase-mediated TH effects in early vertebrate development is only partially understood. Therefore, we investigated the role of deiodinases during early development of zebrafish until 96 hours post fertilization at the level of the transcriptome (microarray), biochemistry, morphology and physiology using morpholino (MO) knockdown. Knockdown of D1+D2 (D1D2MO) and knockdown of D3 (D3MO) both resulted in transcriptional regulation of energy metabolism and (muscle) development in abdomen and tail, together with reduced growth, impaired swim bladder inflation, reduced protein content and reduced motility. The reduced growth and impaired swim bladder inflation in D1D2MO could be due to lower levels of T3 which is known to drive growth and development. The pronounced upregulation of a large number of transcripts coding for key proteins in ATP-producing pathways in D1D2MO could reflect a compensatory response to a decreased metabolic rate, also typically linked to hypothyroidism. Compared to D1D2MO, the effects were more pronounced or more frequent in D3MO, in which hyperthyroidism is expected. More specifically, increased heart rate, delayed hatching and increased carbohydrate content were observed only in D3MO. An increase of the metabolic rate, a decrease of the metabolic efficiency and a stimulation of gluconeogenesis using amino acids as substrates may have been involved in the observed reduced protein content, growth and motility in D3MO larvae. Furthermore, expression of transcripts involved in purine metabolism coupled to vision was decreased in both knockdown conditions, suggesting that both may impair vision. This study provides new insights, not only into the role of deiodinases, but also into the importance of a correct TH balance during vertebrate embryonic development.