Faculteit Dierengeneeskunde

In a series of interviews with researchers of Utrecht University, Utrecht Medical Center or other ULS-partners, we want to give more insight into current developments in the replacement, reduction and refinement of laboratory animal experimentation within these research facilities. Take a look at former interviews here.

April 2016

New in vitro assays for endocrine disruptor screening

Reproductive toxicity testing comprises the highest number of laboratory animal use. Therefore, new models would be very welcome to refine, reduce, and replace animal testing in this field. Dr. Roelofs developed and compared in vitro screening assays to predict the hazardous effects of endocrine disruptors, chemicals which can modulate the endocrine system in humans and other organisms.

Considering the fact that a lot of laboratory animals are used in the field of reproductive toxicity testing of chemicals, the need for in vitro assays is very high. Within REACH (Registration, Evaluation, Authorization and Restriction of Chemicals), around 65% of all laboratory animals are used to test chemicals for developmental and reproductive toxic effects in vivo1.

Endocrine disruptors

Chemical compounds that need to be tested for reproductive toxicity are for example flame retardants, pesticides, plasticizers, pharmaceuticals or phytoestrogens, also called “endocrine disruptors”, because of their ability to modulate the endocrine system in humans and other organisms. Well-known examples are the bisphenol analogues, used as plastics in water bottles and as coatings inside food and beverage cans, of which potential hazards like adverse effects on male fertility are presumed2. The exact working mechanisms of these chemicals are still unknown and therefore it is difficult to predict safe exposure levels. These and other industrial chemicals need to be evaluated on environmental safety and health, preferably with rapid in vitro screening assays. Moreover, in vitro assays can potentially reduce and replace animal testing.

New in vitro screening assays

Recently, dr. Roelofs defended her PhD thesis about newly developed in vitro models to predict the hazardous effects of endocrine disruptors. The research has been conducted at the Institute for Risk Assessment Sciences (IRAS) at Utrecht University, in collaboration with the National Institute for Public Health and the Environment (RIVM). Dr. Roelofs compared newly developed screening assays with already existing ones and determined the toxicological endpoints. Reproductive toxicity is a complex endpoint, therefore it is difficult to develop a mechanistic assay which can assess all relevant parameters at once.

Dr. Roelofs specifically looked at in vitro assays to screen potential endocrine disruptors for steroidogenesis toxicity. The first in vitro assay assesses enzyme activity with the use of porcine adrenal cortex microsomes (1) and the second involves mouse testicular Leydig tumor cells to determine male sex steroid profiles (2).

1. Porcine adrenal cortex microsomes

In this new assay, possible interactions between endocrine disruptor chemicals with steroidogenic enzymes are determined in the porcine adrenal cortex, which shows a highly similar physiology compared to humans and can easily be obtained. Specifically, microsomes were isolated out of porcine adrenals obtained from a Dutch slaughterhouse and used to measure the activity of the enzyme CYP17, playing an important role in steroidogenesis. The enzyme CYP17 is responsible for the conversion of progestagens to the most abundant circulating endogenous sex steroid precursor, DHEA, important in adult and fetal sexual development and functioning. Despite of its importance, a rapid screening assay to determine chemical interactions with possible effects on the CYP17 enzyme is not yet available.

To test the new assay, Dr. Roelofs compared its hormone production with the widely used human adrenocorticocarcinoma cell line H295R, showing a two times higher DHEA hormone production. So the new assay proved to be suitable to test compounds for effects on DHEA production and thus CYP17 enzyme activity.

The next step was to determine possible interactions of endocrine disruptors with the CYP17 enzyme, using the new assay. Therefore, dr. Roelofs used the isolated microsomes to screen 28 endocrine disruptors and possible effects on enzyme activity3. The result was that eight of the tested compounds showed an inhibitory effect on CYP17 enzyme activity in either one of the assays, of which seven could be identified by the new assay. Each tested compound can be analyzed in just two hours. In conclusion, this assay allowed for a rapid and relevant screening of endocrine disruptors for effects on CYP17 activity.

2. Murine Leydig cells

Mouse Leydig tumorigenic cells from the murine testes, provided by the University of Iowa (USA), were cultured in vitro and exposed to, for example, bisphenol analogues, to determine changes in secreted male steroid hormone levels4. Testicular steroidogenesis was altered under influence of all tested bisphenol analogues.

Dr. Roelofs compared the results of this assay also with the validated H295R cell line. The newly developed assay showed better predictability in a physiologically more relevant steroidogenic pathway for the human fetal testis. And this type of assay is very welcome, because current available in vitro assays are not covering the fetal testis. Therefore, this assay fills in one of the major gaps in reproductive toxicity testing.

Screening Battery

Despite the predictive capacity of these newly developed assays, Dr. Roelofs addresses that a single cell-based assay will never be able to fully replace an animal experiment. A better solution would be a battery approach in which multiple assays could be combined to cover crucial elements in reproductive development5. High-throughput assays which provide mechanistic insights are more cost-effective and could be used to make a preselection of chemicals, before even turning to animal testing.

Dr. Roelofs  work was made possible by funding of the International European Community’s Seventh Framework Programme  ChemScreen (www.chemscreen.eu), focused on finding alternative reproductive toxicity screening batteries, and by the Doerenkamp-Zbinden Foundation (www.doerenkamp.ch), supporting research focused on animal-friendly testing strategies to replace the use of domestic animal species in research. Dr. Roelofs is currently employed by the Teaching Academy Utrecht University.

1: Alternative approaches can reduce chemical use under REACH (2004)
2: Gore A.C. et al. (2015) Executive Summary to EDC-2: The Endocrine Society's Second Scientific Statement on Endocrine-Disrupting Chemicals. Endocr. Rev. 36 (6): 593–602.
3: Roelofs et al. (2013), The relevance of chemical interactions with CYP17 enzyme activity: assessment using a novel in vitro assay. Toxicol Appl Pharmacol. 268 (3): 309-17. 
4: Roelofs et al. (2015), Structural bisphenol analogues differentially target steroidogenesis in murine MA-10 Leydig cells as well as the glucocorticoid receptor. Toxicology 329 :10-20.
5: Piersma et al. (2013), Evaluation of an alternative in vitro test battery for detecting reproductive toxicants. Reprod. Toxicol. 38: 53-64.