Farmaceutische, Biomedische en Dier­geneeskundige Wetenschappen

Voorbije doctoraten

Departement Diergeneeskundige Wetenschappen

An adverse outcome pathway-based tiered testing strategy for the assessment of thyroid hormone disruption in fish - Evelyn Stinckens (22/09/2020)

​Evelyn Stinckens

  • 22 september 2020
  • Promotoren: Prof. Dries Knapen, Dr. Hilda Witters en Dr. Lucia Vergauwen

Abstract

Thyroid hormones (THs) play a crucial role in the regulation of vertebrate development and homeostatic processes. A growing number of environmental pollutants are known to adversely affect the TH system. Disruption of this system is increasingly being recognized as an important endocrine disrupting mode of action that can cause a wide variety of adverse effects.

Major gaps have been identified in the tools available for the hazard and risk assessment of TH disrupting substances. The scientific community is currently challenged with developing new or improved testing approaches to evaluate TH disruption in fish. Therefore, the overall aim of this thesis was the development of a tiered testing strategy for the assessment of TH disruption in fish. We used the Adverse Outcome Pathway (AOP) framework for guiding our work.

First, we constructed AOPs leading from thyroperoxidase (TPO, essential for TH synthesis) and deiodinase (DIO, critical for TH activation) inhibition to impaired swim bladder inflation of zebrafish and fathead minnow through decreased thyroid hormone concentrations, ultimately affecting survival probability and population growth.

Next, we provided additional biological and toxicological data supporting the mechanisms and processes captured in the AOPs. We performed in chemico assays targeting the specific molecular initiating events, DIO and TPO inhibition, and we evaluated the use of the resulting in chemico data for predicting higher level in vivo endpoints. We demonstrated that the combined information of the enzyme inhibition assays can be used as a tool to reliably predict the biological effects on swim bladder inflation with only few outliers. This work thus provides an example of how the AOP framework and associated data generation can address current TH disruption testing challenges in the context of fish early-life stage assays, and fish tests in general.

Finally, we demonstrate how different assays covering essential biological processes along the continuum of the AOP network can be implemented in a tiered screening and testing approach for TH disruption in fish. The addition of the associated assays to existing test guidelines allows to increase their diagnostic value for the assessment of TH disruption. Further expanding the applicability domain of our AOP network would be of great value for screening and testing of TH disrupting compounds.

Drug disposition in the zebrafish embryo and larva: focus on cytochrome P450 activity - Evy Verbueken (16/12/2019)

​Evy Verbueken

  • 16 december 2019
  • Promotoren: Prof. Steven Van Cruchten en Prof. Chris Van Ginneken

Abstract

The zebrafish embryo is increasingly used as an alternative model to screen new drug candidates and environmental pollutants for developmental toxicity (i.e. teratogenicity). Since the zebrafish is not considered to be a test animal until it reaches the stage of independent feeding, the zebrafish embryo developmental toxicity assay (ZEDTA) fits within the 3Rs (i.e. Replacement, Reduction and Refinement) concept as described within laboratory animal sciences.

The externally developing zebrafish embryo cannot rely on maternal metabolism unlike the mammalian embryo. Hence, the zebrafish embryo gets directly exposed to the parent compound and depends on its own drug–metabolizing capacity for the detoxification or bioactivation of xenobiotics. In this respect, knowledge of the intrinsic biotransformation capacity during zebrafish organogenesis, which coincides with the exposure window of the ZEDTA, is key in order to correctly interpret the outcome of the ZEDTA. However, the overall results of studies described in literature regarding the xenobiotic–metabolizing capacity of zebrafish embryos are contradictory.

Hence, the main goal of this doctoral project was to characterize drug disposition in zebrafish during organogenesis with a main focus on cytochrome P450 (CYP)–mediated metabolism since the latter enzymes are responsible for the oxidation of the majority of marketed drugs. To this end, the thesis investigates the ontogeny of CYP enzymes on mRNA as well as on activity level, and to a lesser extent also of the expression levels of two major phase II enzymes and a drug transporter, i.e. abcb4, at different time–points during zebrafish organogenesis and beyond.

This project mainly showed that CYP–mediated biotransformation of xenobiotics appears to be immature during a major part of the ZEDTA exposure window (i.e. 4–120 h post–fertilization (hpf)). Moreover, the mRNA expression levels of the phase II enzymes and abcb4 reached maximum expression levels by the end of zebrafish organogenesis. These findings can have a profound impact on the predictivity of the ZEDTA for human safety assessment in the drug development process, especially in case of proteratogenic compounds that require bioactivation to exert their teratogenic potential.

A solution to overcome the immature biotransformation capacity of zebrafish embryos is to co–incubate the ZEDTA with a human–derived external metabolic activating system (MAS), such as human liver microsomes, during the entire exposure window of the ZEDTA. However, the co–incubation method with the external MAS needs to be further optimized and validated before it can be used in regulatory developmental toxicity testing.

Advancing the zebrafish embryo test for estrogen disruptor screening - Ellen Michiels (06/12/2019)

​Ellen Michiels

  • 6 december 2019
  • Promotoren: Prof. Dries Knapen en Prof. Steven Van Cruchten

Abstract

The presence of endocrine disrupting compounds (EDCs) in the environment is a major concern. EDCs directly interact with the endocrine system, leading to important adverse effects in humans and wildlife (e.g, related to reproduction, energy metabolism and growth, behaviour, etc.). To identify these compounds, several regulatory strategies and frameworks have been developed in many parts of the world. Various assays are available to assess the endocrine disrupting potential of compounds, and several methods are used to administer the compounds. Within the context of non-mammalian assays, fish are often used as model organisms.

Almost all current fish assays use adult animals, but there is increasing public pressure to reduce or even avoid the use of animal testing. Therefore, significant efforts are being made to develop new, alternative testing methods as well as to optimize existing methods for the assessment of endocrine disrupting compounds. The development of a fish embryo test for the detection of EDCs may offer a particular advantage in this context, since fish embryos are not considered test animals until the stage of free feeding while they do offer the biological relevance gained from using a model organism with an intact endocrine system.

Assays using fish and fish embryos are mostly performed via aquatic exposure. However, many EDCs are hydrophobic compounds. Aquatic exposure may therefore not always be the most biologically relevant exposure method. In addition, the use of hydrophobic compounds in an aquatic exposure set-up brings several practical challenges as well. In the present work, we used an existing fish embryo test guideline as a basis for the development of an alternative test system for the assessment of  EDCs.

Specifically, the Fish Embryo Acute Toxicity (FET) Test (OECD Test Guideline 236) was supplemented with a number of specific endpoints relevant to the screening and testing of estrogen disrupting compounds. In addition, we suggest a combination of aquatic exposure (i.e, as currently required in the FET test) and administration of the compound under consideration via micro-injection. Micro-injection into the yolk of the embryo can possibly mimic maternal transfer, and it helps to solve a number of practical challenges that are associated with the use of hydrophobic compounds in aquatic exposure scenarios. Overall, we developed a new approach allowing us to advance the zebrafish embryo test for use in the assessment of estrogen disrupting compounds.

Maternal metabolic disorders and fertility: importance of lipotoxicity on oocyte, sperm and embryo - Karolien Desmet (19/11/2019)

​Karolien Desmet

  • 19 november 2019
  • Promotoren: Prof. Jo Leroy en Prof. Peter Bols

Abstract

Lipolysis-associated maternal metabolic disorders, like obesity and diabetes type II in women and negative energy balance in dairy cows, are a major risk factor for subfertility. These altered metabolic states are reflected in the follicular and oviductal micro-environment with emphasis on elevated concentrations of non-esterified fatty acids (NEFAs). Although information is available about the toxic effects of elevated NEFA concentrations on oocyte and embryo developmental competence, there remains a lack of information regarding the underlying mechanisms. In this dissertation, the bovine model was used to study the relation between lipolysis-linked maternal metabolic disorders and fertility.

We showed that elevated NEFA concentrations during either in vitro oocyte maturation or early embryo development impact the resultant blastocysts’ transcriptomic and epigenetic profiles. This disturbance in epigenetic reprogramming may render the embryo susceptible to changes during further development. We observed that good quality blastocysts produced from metabolically compromised oocytes have defective post-hatching development after embryo transfer to a recipient cow. These embryos were retarded in growth, exhibited disturbed metabolism and transcriptomic profile, and displayed a reduced capacity to signal its presence to the mother.

Also the oviductal environment plays a vital role in the functionality of and interaction between the oocyte and sperm cells. We showed that although sperm functionality was affected under lipolytic conditions, adequate numbers of viable sperm cells retained their fertilizing ability. In contrast, the fertilization process was affected as well as subsequent embryo development.

Metabolic disorders and sub-/infertility are both increasing in men and women. The combination of having both obese parents on fecundity has received minimal attention although most couples share lifestyle and thus obesity in both persons is more likely to occur. We investigated if metabolic conditions of couples seeking assisted reproductive services in an IVF-clinic could be linked to embryo quality and metabolism. Aberrations in follicular fluid, female and male waist circumference had an effect on embryo quality and metabolism. Combining BMI of both parents led to an association between parental BMI and embryo metabolism. Only male BMI was associated with pregnancy outcome.

We concluded that lipolytic conditions cause subfertility through impairment of oocyte maturation, fertilization and embryo development. We also substantiated an effect of the parents’ metabolic health on quality and metabolism of their embryos in a human setting. In characterizing these effects, we may increase the awareness of the importance of parental metabolic health in relation to fertility and carryover effects persisting in the offspring.

The use of zebrafish feeding trials for the safety assessment of genetically modified crops - Isabelle Gabriëls (06/06/2019)

​Isabelle Gabriëls

  • 6 juni 2019
  • Promotoren: Prof. Dries Knapen, Prof. Marc De Loose en Dr. Lucia Vergauwen

Abstract

In the EU, a mandatory part of the safety assessment of genetically modified crops (GMOs) consists of a 90-day rodent feeding trial. As a relevant addition to the current assessment strategy, our aim was to develop a complementary zebrafish feeding trial specifically designed to address a number of shortcomings of existing test methods, using maize as a case study.

First, to ensure the use of nutritionally balanced experimental diets, we investigated whether substitution of a non-GM maize into the zebrafish diet causes component-related effects. High dietary maize percentages resulted in an overall disturbed uptake and processing of carbohydrates, influencing processes such as growth rate, and affected mRNA transcriptional liver patterns of genes involved in key metabolic processes. Balancing the physiological tolerance of the fish and achieving a toxicologically relevant dose of the GMO, 15% was selected as the optimal maize substitution level to be used for all experiments.

Next, a subchronic zebrafish feeding trial was developed for the toxicological safety assessment of GMOs. Endpoints included general health parameters, effects on energy metabolism, and reproductive effects including transgenerational effects. As a key aspect of our experimental design, we investigated the importance of different non-GM comparators. Our results highlight the importance of distinguishing between potential effects caused by the transformation process (i.e., the process of genetically modifying the plant) and those caused by the presence of the transgenic event itself. Indeed, most differences (in general health endpoints as well as transcriptional liver profiles) were identified between the conventional wild type control (i.e., the original genetic starting material) and null segregant (i.e., the plant progeny created during the transformation process but lacking the transgenic event) on the one hand, and between wild type and GM maize on the other hand, while differences between GM maize and null segregant were limited. To assess the toxicological relevance of biological responses, we further optimized a method allowing the interpretation of results relative to the natural response variation, i.e., the normal biological range in endpoint responses of the fish. We established natural response variation datasets for various zebrafish endpoints and discussed equivalence testing as a conceptual approach using examples originating from the subchronic/transgenerational feeding trial.

Overall, this work forms a strong foundation for advancing zebrafish feeding trials and, to facilitate the development of further strategies, main conclusions and recommendations have been summarized in a Guidance Document for the safety evaluation of GM crops using the zebrafish model.