endocrine disruption, retinoids

Here, Alberto Mantovani discusses how Vitamin A joins endocrine disruption, an increasingly crucial issue

Endocrine disruption continues to be a matter of high concern, and a subject of intensive activities at the public, political, regulatory and academic levels. It is a concern for the public, that requests adequate measures to protect health and environment now and for the future; it is a concern for enterprises as well, as a number of products (e.g. agrochemicals) will be banned and safer alternatives have to be sought.

Test guidelines for endocrine disrupters

Currently, available regulatory test guidelines relevant to the identification of endocrine disrupters are largely limited to estrogen, androgen, thyroid and steroidogenesis (EATS) pathways. These pathways are crucial for organism well-being, yet, the endocrine network is definitely more complex than that. Thus, there is an increasing interest and need to develop test methods, biomarkers, and Adverse Outcome Pathways (AOPs), for identification and evaluation of endocrine disrupters in addition to the EATS pathways.

The retinoid system is involved in fundamental life processes, including important roles in regulating reproduction, embryofetal development, as well as lipid homeostasis in mammals. Everything depends on dietary intake of vitamin A: a complex metabolic machinery controls the uptake of dietary forms of vitamin A (retinyl esters, β-carotene etc.), and their transformation into signalling retinoic acids; the extra- and intra-cellular retinoic acid concentrations in tissues and along life-stages are strictly controlled in vertebrates and invertebrates, as the retinoid system is evolutionarily conserved.

The system is regulated by nuclear receptors, just alike steroid hormones, the retinoic acid receptors, RARs and RXRs: the retinoic acids are the in situ synthesized receptor ligands, with hormone-like activities. There is evidence for direct control of at least 27 genes by retinoic acid-liganded receptors, In addition the RXRs can dimerise also with other nuclear receptors including the PPARs (peroxisome proliferator activated receptor), which are critical targets for such major pollutants as phthalates and perfluoroalkyl compounds. The dimerisations result in hormonal cross-talk, which should be considered as a key element when investigating endocrine disruption-related AOP. Most important, the retinoid system is susceptible to environmental agents.

To date, identified AOP involving the retinoic acid signalling mainly concern congenital anomalies, such as neural tube and axial defects, and cleft palate, These AOPs indicate that inappropriate activation of RAR (mediating nuclear receptor response to retinoic acid), as well as inhibition of CYP 26 (involved in degrading retinoic acid) or Raldh (involved in synthesising retinoic acid from its precursor retinal) can alter the cellular metabolism and/or spatiotemporal distribution of retinoic acid, which in turn can result in irreversible developmental effects.

Retinoid system perturbations

Concentrations of retinoids in tissues as well as circulatory levels are affected by such chemicals as organochlorine pesticides, dioxins, PCBs, brominated flame retardants and organotins. Retinoid system perturbations can occur through different molecular events and can have functional impact on multiple organs. For instance, cleft palate can be induced by the triazole fungicides through CYP 26-mediated RAR activation; other developmental toxicity pathways may result from an indirect disruption of retinoic acid signalling, such as from the activation of the aryl hydrocarbon receptor (the “dioxin receptor”). The possible relevant AOP extend beyond the, however important, congenital anomalies; metabolic dysfunction is considered a high-rank health concern for which the retinoic acid involvement is recognised. In addition, retinoid signalling is important in the development and function of the ovary, the testis and other female and male reproductive tissues, hence, for the whole field of reproductive toxicology. Thus, it is likely that mechanisms related to retinoic acid would feature prominently in the toxicology stage of the next years.

While research proceeds, the risk assessors and risk managers do face a crucial question: how to exploit this growing body of evidence, in order to specifically capture perturbations of the retinoid system with respect to endocrine disruption as well as in regulatory toxicology in general? How to set screening tests and characterize appropriate points of departure? A preliminary list of tools to identify chemicals that disrupt retionoid signalling may include RXR and RAR reporter assays, CYP 26 activity (in vitro screens), and adipocyte differentiation (in vitro functional assay). Weight gain, adipose tissue mass, lipid accumulation, retinoid level measurements in serum and liver represent endpoints that can be used in regulatory in vivo studies; while they are non-specific per se, a pattern of endpoints may provide a phenotype highly suggestive of retinoic pathway disruption in vivo. Obviously, the induction of craniofacial defects and/or cleft palate in prenatal developmental toxicity tests would trigger a strong suspicion about the presence of retinoid-related mechanisms.

What will the project do?

Stemming from the accumulation of data and questions, a project to analyse the needs and benefits of incorporating the retinoid system into the OECD Testing Guideline programme was included on the work plan of the OECD already in 2015. The retinoid system project was jointly proposed and initiated by the Swedish Chemicals Agency and the European Commission.

The project aims to explore, in the existing literature, if and how diverse types of toxicities and health outcomes over the life course might be linked to retinoid system alterations. The goal is to map the existing knowledge in detail and to identify knowledge gaps on the retinoid system in biology and toxicology. This validated knowledge basis will be used as a starting point for the development of screening and testing methods in the OECD Test Guideline programme and to support the development of retinoid system-relevant AOPs. The project is progressing through the consultation of a broad representation of regulatory and academic scientists; a finalized Detailed Review Paper (DRP) is foreseen by the end of 2020. The DRP will include chapters on male and female reproduction, neurodevelopmental disorders, as well as skeletal and craniofacial malformations.

For further reading, I reccommend the following open-access documents: Nordic Co-operation “Retinoids in Mammalian Reproduction, with an Initial Scoping Effort to Identify Regulatory Methods”, 20/04/2020, and Elise Grignard, Helen Håkansson, Sharon Munn, (2020) “Regulatory needs and activities to address the retinoid system in the context of endocrine disruption: The European viewpoint”. Reproductive Toxicology, 93: 250-58.

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Istituto Superiore di Sanità
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