Assay, in vitro

It is important to understand some limitations of in vitro artificial membrane and cell permeability assays when the data is used for compound selection and structure permeability relationship study. Therefore, before moving into the structure permeability relationship section, limitations of in vitro assays are discussed in this section. There are a number of other limitations of in vitro models which should be considered [3, 70[. 

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Finally, some amphiphilic sweeteners, eg, aspartame, saccharin, and neohesperidin dihydrochalcone, have been shown to be capable of stimulating a purified G-protein direcdy in an in vitro assay (136). This suggests some sweeteners may be able to cross the plasma membrane and stimulate the G-protein without first binding to a receptor. This type of action could explain the relatively longer response times and the lingering of taste associated with many high potency sweeteners. 

Many groups throughout the world tried to repeat the experiments using similar in vitro assays and other test systems, but without success. Current experimental data suggest additive interactions do occur between EDs, but the issue of interactive effects, and synergism in particular, will undoubtedly remain a topic of intense debate for some time to come. 

For additional evaluation of the effect of hydrophobization and the molecular weight of the polymers on the biological immuno-stimulating activity, we investigated the ex vivo cytokine (interIeukin-6 [IL-6], and tumor necrosis factor [TNFj-inducing activity from human peripheral whole blood cells of hydrophobized polymers by use of fractionated poly(M A-CDA) with narrow poly-dispersity. Since this assay uses the intact human cells, it shows more accurate results than in vitro assay using cultured cell line [25]. 

Investigations of the aromatase inhibiting effects in in vitro assays gave no indicahon of an endocrine response after incubation with mono-, di-, or trioctyltin (Cooke, 2002). 

Mirzadeh H, Khorasani MT, and Sammez P. Laser surface modification of polymers A novel technique for the preparation of blood compatible materials-II In vitro assay. Iranian Polym, 1998, 7, 5. 

As discussed in the Endocrine Effects section, endosulfan has shown weak estrogenic properties in some in vitro assays, but no such properties could be confirmed in studies in vivo. 

Genotoxic Effects. Endosulfan has been evaluated for genotoxicity in a variety of in vivo and in vitro assays. As summarized in Tables 2-4 and 2-5, the results of these assays have been positive and negative, but the majority of mutagenicity tests reported positive results. Certain studies were unsatisfactory, as indicated below. 

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). 

Following from the above, behavioral assays, which can be relatively simple and cost-effective, can be very useful as primary screens when testing chemicals for their neurotoxicity in the context of medical toxicology (see Dewar 1983, Atterwill et al. 1991, and Tilson 1993). Where disturbances of behavior are identified, subsequent more specific tests, including in vitro assays, may then be performed to establish... 

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. 

Stronkhurst, J., Leonards, P., and Murk, A.J. (2002). Using the dioxin receptor Calux in vitro assay to screen marine harhour sediments for a dioxin-hke mode of action. Environmental Toxicology and Chemistry 21, 2552-2561. 

Experiments in cell-free in vitro assays have shown that formation of a PNA triplex invasion complex with a homopurine motif on the template strand results in the steric hindrance of RNA polymerases, leading to complete arrest of further... 

Figure 11.2 A decision tree, based on an associated inflnence diagram, can help organize and integrate information about risks and the way in which research work buys better information that allows choice of the options most likely to succeed. This example describes the relationship between in silico predictions and in vitro assay results for the same compound structures.

NAGEL s c, VOM SAAL F s, WELSHONS w V (1999) Developmental effects of oestrogenic chemicals are predicted by an in vitro assay incorporating modification of cell uptake by serum. J Steroid Biochem Mol Biol. 69 343-57. 

Pellegrini, N. et al.. Total antioxidant capacity of plant foods, beverages and oils consumed in Italy assessed by three different in vitro assays, J. Nutr. 133, 2812, 2003. 

Fraser, P.D. and Sandmann, G., In vitro assays of three carotenogenic membrane-bound enzymes from Escherichia coli transformed with different crt genes, Biochem. Biophys. Res. Commun. 185, 9, 1992. 

Identification of proteins that bind to Z-DNA added one further step to the establishment of the presence of Z-DNA in vivo and its possible biological role. Herbert and Rich [22] demonstrated an in vitro assay system where one type of double-stranded RNA adenosine deaminase, called DRAD-binding Z-DNA. There are evidences that topoisomerase II from Drosophila, hiunan and calf thymus recognizes a number of DNA shapes, including Z-DNA [34,35]. Bloomfield and coworkers [36] have found that the condensation of plasmids is enhanced by Z-DNA conformation in d(CG)n repeats. The information related to B-Z transition [31], the effect of ligands on it [28,29] and X-ray crystal structure data [37,38] appear to suggest that the possible biological role of this polymorphic form of DNA will be soon established. 

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]. 

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