In Vitro Ocular Toxicology-The Way Forward

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In Vitro Ocular Toxicology-The Way Forward

Monica Berry, University of Bristol Published: June 4, 2009
About the Author(s)
Born in the old East (Romania), educated in Israel (BSc in Zoology and MSc in Entomology at Tel Aviv University), and the UK (PhD in Zoology at the University of Bristol). As a research in vitro toxicologist in Ophthalmology I focussed on the human ocular surface and detection of low toxic effects. To represent the salient features of the target tissue, constructs have evolved from primary cell monolayers, intact and wounded, to 3D cultures including a number of cell types. I am also keen to include endpoints that reflect intercellular communication, additional to cell survival and proliferation. My other research interest is the tear film and ocular surface physiology, and especially the role of mucins in health and disease.

Monica Berry, PhD Senior Research Fellow Academic Unit of Ophthalmology
University of Bristol
Bristol BS2 8AE, UK

I argue that there has been some stasis in the field, partly because research into new tests for in vitro toxicology has not been prioritised by academic sponsors, and partly because industrial sponsors focused (too narrowly in my view) on models that seemed a priori promising or that quickly led to prevalidation. European legislation has, in a way, forced cosmetic companies to adopt this attitude, and this urgent need for validated tests is both pushing the field forward and a barrier to the development of tests that keep pace with scientific developments.

Why this gloom? I have recently viewed a number of posters at a leading Ophthalmic meeting, posters that were very similar to those presented at in vitro toxicology meetings some twenty years ago. Monolayers of cells, a single measure of “toxicity”, and claims that while correct for the particular experimental conditions, might prove unsafe for other materials and conditions. Recently published papers present an epithelial model that has long been used by the cosmetic industry – Skinethic corneal epithelium –as a novel construct for in vitro toxicology. The much used MTT test is applied, in conjunction with histochemical measures of cell cycle. No dynamic measures, no early or late measures of toxicity, no measures of recovery from injury. Others use a rabbit corneal cell line and MTT, for a short test. To more fully appraise the effects, sometimes subtle, of a new product, dynamic outcomes and assessments of recovery are necessary. Neither are new directions in ocular toxicology, but progress on these roads has been slight. (Grumpy says mea culpa!)

We have become used to surrogate endpoints, and surrogate experimental constructs. It has become routine to use cell lines – they do minimize variability in experiments – without reference to the target population for whose integrity and health the test aims to protect. Is an immortalized epithelium a good model for the very young or the very old cornea? Would a person with, say, dry eyes experience a different level of toxicity than someone with a “normal” ocular surface? (Normal is quite difficult to define. Here I used the term to denote an ocular surface that can respond to changes in the external environment and return to homeostasis). It might be time to revisit the choices of endpoints and constructs in the light of new technical abilities, and more so, because of health and safety responsibilities that drive toxicology.

Lately, it seems, some of the most popular ocular surface cell lines we use have become more erratic. It is imperative that new cell lines are developed and tested for their similarities to human corneal or conjunctival cells. Now we need to define the word similar. Perhaps the most important similarity is the cellular response to a particular type of stimulus. Cellular response is defined as a change in state or activity of a cell, in terms of movement, secretion, enzyme production, gene expression, etc., as a result of a particular stimulus type. Careful choice of the type of stimulus would make full use of previous work on common toxicants. This is no other than the mechanistic argument, applied not only to endpoints but to ways of building the experimental set-up.

We can now use the enormous amount of genomic and proteomic data that awaits mining to extract information pertinent to cell death, wound healing and recovery from stress. Addressing processes and their regulation as relevant to the eye give us not only a time scale to observe responses but also gene-products and pathways to use as end points. There certainly is a lot of research activity in these areas: we would benefit from an interest group focused on the eye.

Toxicology, and in vitro toxicology especially, is necessarily interdisciplinary. The importance of this feature cannot be overstated. Take an easy example, fashionable now and of growing importance as an environmental, medical and life-style factor: nanoparticles. To understand why they might be different to their mico- and macro- counterparts, involves physical chemistry and material science. At the nitty-gritty of experiment the toxicologist needs to determine, among others, the dosage and state of the material at presentation to the tissue culture model. And rather than fishing for any (possibly surrogate) change in the state of the cell, finding an endpoint that is relevant involves knowledge of transport to and into the cell, compartmentalisation therein, and re-release in the intercellular space. I see interdisciplinarity growing in importance in in vitro toxicology, and an increased use of biophysical and biomathematical tools in everyday testing.

I am arguing against one-test-fits-all. (Not a new argument, either.) Objectivity need not equate with blindness, on one hand, nor with confidence in the stated ingredients, on the other. By analogy to products whose characteristics signify severe harm and need not be tested, a hierarchy of tests needs to be drawn to asses which level of testing a new entity requires, because of its ingredients and their known interactions with the target organ, including blood barriers, mucosal protection, metabolic processing and immune effects. It is the test strategy and tight, well controlled protocols, together with highly accurate experimentation that regulatory agencies should sanction, rather than grey uniformity.

In vitro toxicology is the future, and not only for the cosmetic industry. It is incumbent on us to make this future safe for users, and make our discipline good science. We are dealing with models, necessarily a simplification of a complex and multi-related biology. A clear statement of advantages and limitations of a particular construct and testing strategy should be far more convincing than a panacea-of-the-moment going to pre-validation. To gain respect from colleagues and the public in general, we need to get away from tests that are immutable.

©2009 Monica Berry

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