Yeast-based assays

Among Lamiaceae family plants, except 4/wg postii, none of the species gave hit in the yeast based assay. However, a few Salvia species (S. triloba, S. blepharo-chlaena, S. syriaca), and a few Sideritis species (S. aytachii, S. lycia) showed high inhibition against at least one or two tested yeasts among the three yeasts nsed in DNA damaging microtiter assay (Table 6.1). 

Several amino acids in AGSl within the nucleotidebinding/hydrolysis motifs differ from their counterparts in Ras, and mutation of these residues in Ras leads to constitutive activity (Barbacid 1987 Feig and Cooper 1988), suggesting that AGSl may be constitutively active. AGS 1 is active in yeast-based assays without any apparent stimuli, and AGS 1 purified from yeast extracts is largely GTP bound (Cismowski et al. 2000). It is presently unclear whether these amino acid variations are sufficient to render AGS 1 constitutively active in mammalian cells or how posttranslational modifications of AGS 1 influence ifs level of basal and stimulated activity. 

Traceability and Interlaboratory Studies on Yeast-based Assays for the Determination of Estrogenicity... 

Relative Estrogenic Potency Determined by Yeast-based Assays... 

Yeast-based Assays May Be Applied with Different Objectives... 

The aim of this chapter is to give a brief overview of yeast-based assays the comparability of the results and the different uses and future perspective. 

Reporter gene assays that measure ER binding-dependant transcriptional and translational activity. The yeast-based assays fall within this group. The vast majority of yeast-based assays are based on S. cerevisiae containing a stably transfected human estrogen, receptor (hER), although there are several variations in strain and detection method (NICEATM, 2002)... 

Another much used yeast-based assay is the yEGFP assay (first described by Bovee et al., 2004). In this assay recombinant yeast cells are constructed that express the human estrogen reporter a (ER a) and yeast enhanced green fluorescence protein), the estrogenic potential is then measured by a Fluorometer. 

RELATIVE ESTROGENIC POTENCY DETERMINED BY YEAST-BASED ASSAYS... 

It is not very scientific to calculate averages of results from two different methods, which also show a large variation between then, but from the point of illustration, it was found acceptable and these averages have been plotted in Figure 5.3.1, along with averages and standard deviations from Tables 5.3.1 and 5.3.2 on yeast-based assays. 

The figure shows that because the standard deviations are so large, a large part of the mean values of the non-yeast-based are included in one standard deviation of the results of the yeast-based assays. However, more than half the values are outside the range, showing that there is a low consistency between the results of the non-yeast and yeast-based assays. 

Figure 5.3.1 Relative estrogenic potential determined by yeast-based assays (diamonds) and...

Unfortunately, the study only reports the results from two of the four yeast-based assays, which are presented in Table 5.3.4. 

YEAST-BASED ASSAYS MAY BE APPLIED WITH DIFFERENT OBJECTIVES... 

Acceptable variation coefficients are determined by the application of the assay, for prescreening purposes with a sufficiently low limit of detection, it is acceptable to have high variation coefficients. But when trying to establish a correlation between chemical data and results from yeast-based assays a variation coefficient on the relative estrogenic potential of more than 100% is quite problematic. 

The second issue, when using the yeast-based assays as a prescreening for chemical analysis, is whether these limits of detection are low enough. Studies have shown that 1 ng/1 ethynyl estradiol cause induction of vitellogenesis in carp (Purdom etal., 1994), which means that the sample would have to be concentrated at least 1000 times and this often makes the sample toxic because of elevated levels of other compounds, e.g. LAS, which kills the yeast at high concentrations. 

Yeast-based assays show a lot of potential for various applications, but especially as a cost-reducing prescreening tool for chemical analysis in environmental monitoring. 

P-D-glucopyranosylstigmasta-5,25 diene were evaluated in the yeast based assay (Table 6.3), but none of them showed activity while the new diterpene ajugalaevigatic acid showed only a weak A2780 ovarian cytotoxic activity [11] among against a panel of cancer cell line tested. 

Commercial DDT and DDD contain o,p-DDT [ 1,1,1-trichloro-2-(c -chlorophenyl)-2-(p-chlorophenyl)] ethane and o,p-DDD [l,l-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)] ethane isomers ( 10-20%), which are chiral [28]. Investigations have demonstrated the oestrogenic activity of o,p-DDT in fish and mammals [29-34]. It is also known that the —)-o,p-DDT enantiomer is a more active oestrogen mimic than the (+)-enantiomer in rats [35, 36]. Subsequently, Hoekstra etal. [37] reported a yeast-based assay to assess the enantiomer-specific transcriptional activity of DDT with... 

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