Edinburgh Napier University

  "There is a paucity of knowledge relating to the effects of ENM on the male reproductive system. Thus, there is a clear requirement for development of knowledge on the area. The aim of this PhD, based within EU-FP7 MARINA project, is therefore to investigate toxicity of ENM to the male reproductive system. With a focus on development of non-animal testing methods in line with the 3Rs principle, this will make use of established male reproductive cell lines, then to compare results to tissues collected from studies conducted in vivo. Thus, the ultimate aim is to develop robust and reliable in vitro test methods for the prediction of engineered nanomaterial (ENM) toxicity.

A panel of commonly used engineered nanomaterials were selected as representative of the field - Titanium Dioxide, Zinc Oxide, Silver, Silica Dioxide, Carbon Nanotubes and Cerium Dioxide. Leydig cells, present within the testicular intertsititum, are responsible for production of androgens which act on both local and systemic targets and are key to normal function of the male reproductive system. For in vitro testing, the mouse leydig cell, TM3, has been selected as a representative cell line (Mather, 1980, Mather et al., 1982). The selected assays reflect toxicity endpoints from basic cytotoxicity, to functional markers of cell protein expression, and cellular integrity and uptake within key target cells of the male reproductive system. During the work programme, the potential for interference of ENM with the assays will also be considered at each stage.

As the TM4 (Sertoli) cell line is being investigated at Edinburgh Napier University in parallel to the work being undertaken within this degree programme, conditioned media treatments from TM3 cells exposed to ENM will also be applied to TM4 cells to identify whether there is any ‘cross talk’ between the cell types by way of release of secondary metabolites. This will establish whether future work could include setting up a co-culture of the cells for more sophisticated analysis of ENM toxicity in vitro.

Toxicity testing will also be undertaken with primary cells isolated from animals, using the same panel of tests for the TM3 cell line. In addition, tissue from the reproductive organs of animals exposed to the ENM panel in vivo will also be analysed for morphological changes and using the same markers of protein expression to establish effects on function of the organ. These investigations will form a point for comparison to the TM3 cell line’s responses to exposure to the panel of ENM, and will act as a validation for its ability to predict toxicity.

The output of this work programme aims to contribute to both building the knowledge base relating to male reproductive toxicity from ENM in vitro, and to presenting a useful alternative model for predicting toxicity from engineered nanomaterials, in line with the 3Rs principle for reduction, refinement and replacement of animals within testing
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