In vitro assay development

Agarwal et al. 1978), the quantification of these specific enzymes may indicate that exposure to endosulfan has occurred. Blood tests, such as decay curves for aminopyrine in plasma, which are semiquantitative indices of liver enzyme induction, have been used successfully in the past to demonstrate enzyme induction in pesticide-exposed workers. Because numerous chemicals found at hazardous waste sites also induce these hepatic enzymes, these measurements are not specific for endosulfan exposure. However, measurements of enzyme activity, together with the detection of the parent compound or its metabolites in tissue or excreta, can be useful indicators of exposure. All of these potential biomarkers require further verification in epidemiological studies. Further studies with focus on the development of methods to separate and measure the estrogenicity of endosulfan in in vitro assays would be valuable since these assays are more sensitive and discriminative than other conventional biomarkers. Preliminary results have been presented by Sonnenschein et al. (1995). 

The yeast reporter gene assays not only assess for the interaction of the chemical with the hormone receptor, but also the ability of that receptor-chemical ligand interaction to activate the hormone DNA response element. It should be realized, however, that most of these systems have been developed with human and mammalian hormone receptors and differences in ligand potencies can occur between different animal species. A comprehensive review of in vitro assays for measuring estrogenic activity, and some of the issues of comparability, is provided by Zacharewski (1997). 

There is a continuing interest in the development of biomarker assays for use in environmental risk assessment. As discussed elsewhere (Section 16.6), there are both scientific and ethical reasons for seeking to introduce in vitro assays into protocols for the regulatory testing of chemicals. Animal welfare organizations would like to see the replacement of toxicity tests by more animal-friendly alternatives for all types of risk assessment—whether for environmental risks or for human health. 

The study of DMPK has changed from a descriptive to a much more predictive science [3]. This is driven by great progress in bioanalytics, development of in vitro assays and in silica modeling/simulation, and a much better basic understanding of the processes. Thus, and fortunately, ADME-related attrition has lowered from around 40% in 1990 to around 10% in 2005 [13]. 

Although the pressure to screen large numbers of compounds quickly has led to the rapid development of in silico and in vitro assays, the sheer number and complexity of the processes involved in determining the disposition of any particular compound means that in vivo studies are still required to provide assurance that the important processes are modeled with sufficient accuracy [4-6], and indeed, that the potential contribution of processes for which there are no good in vitro models (e.g., biliary secretion) are adequately assessed. 

Schwartz, Z., Ornoy, A., and Soskolne, W.A. An in vitro assay of bone development using fetal long bones of mice morphological studies, ActaAnat. (Basel), 124, 197, 1985. 

There has been considerable progress in developing in vitro tests (a.25). The most success has been in local effects testing in biologically less complex systems, such as testing for skin and eye irritation. In contrast, efforts to develop in vitro tests to evaluate potential systemic toxicants have been hindered by the complexity of the systems involved, and it seems likely that a battery of different in vitro assays will be needed. 

There may be situations which warrant an assessment of carcinogenic potential, but immunogenicity and species specificity may preclude a two-year rodent bioassay. It may be necessary to develop in vitro assays to address a particular concern. For example, growth factors which may have the potential to support or stimulate the growth of transformed cells should be assessed for their ability to promote growth of either malignant or normal cells. 

The test is based on an in vitro assay of the uptake of the dye, neutral red (NR), in Balb/c 3T3 fibroblasts. It was developed to detect the phototoxicity induced by the combined interaction of the test substance and light of the wavelength range from 315 to 400 nm, the so-called UVA. The cytotoxicity is evaluated in the presence (+UVA) or absence (-UVA) of UVA light exposure, after application of a nontoxic dose of the compound. The cytotoxicological impact is assessed via the inhibition of the fibroblasts to take up the vital dye NR (NR is a weak cationic dye, penetrating easily into the cell membrane by a nonionic diffusion and accumulates in the lysosomes) one day after the initial treatment. Normally, healthy cells may incorporate and bind NR. Alterations of the cell surface or the lysosomal membranes, however, lead to a decreased uptake and binding of the dye. 

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