Bioassays dose-response curve

The hormone itself can introduce complexity into bioassays. Many hormones must now be seen and understood not as chemical entities but as chemical pathways where hormonal activity is distributed across a number of chemical species. The more we learn about the pharmacological properties of members of a pathway, the more we are realizing that each one has a mix of common and unique properties. The practical point is that we must be careful about which hormone we choose to drive our bioassays. A hormonal chemical pathway may contain sinks as well as sources. Metabolism and uptake of a hormone can introduce significant distortions into bioassays. All of these factors leave their fingerprints on dose-response curves, and a pharmaceutical researcher developing a new bioassay has to learn to read the signs. 

So far, we have reviewed the various ways in which complex dose-response curves in intact-tissue bioassays can be the result, the pharmacological resultant, of two or more interacting activities. Now, if all that these bioassays achieved was to blur and obscure the underlying activities, they would have to give way to the newer, analytically simpler assays based on chemistry and biochemistry. However, the beauty of intact-tissue bioassays is that they are analytically tractable by using families of dose-response curves and appropriate mathematical models, the complexity of intact hormone-receptor systems can, indeed, be interpreted. Bioassay allows them to be studied as systems in ways denied to simple biochemical assays. 

The more classical approach to assess the presence of marine biotoxins in seafood is the in vivo mouse bioassay. It is based on the administration of suspicious extracted shellfish samples to mice, the evaluation of the lethal dose and the toxicity calculation according to reference dose response curves, established with reference material. It provides an indication about the overall toxicity of the sample, as it is not able to differentiate among individual toxins. This is a laborious and time-consuming procedure the accuracy is poor, it is nonspecific and generally not acceptably robust. Moreover, the mouse bioassay suffers from ethical implications and it is in conflict with the EU Directive 86/609 on the Protection of Laboratory Animals. Despite the drawbacks, this bioassay is still the method of reference for almost all types of marine toxins, and is the official method for PSP toxins. 

Guinea Pig Bioassay of GT-1 and GT-2. At less than nanogram concentrations, extracts 6T-1 and GT-2 produced an enhancement of histamine stimulation of the ileal preparation. At nanogram concentrations or larger, both caused an inhibition and hence a shift of the dose response curve (Figure 3). Replotting these data for GT-1 and GT-2 into a Michaelis-Menten format (Figure 4) indicates that the action of GT-1 and GT-2 fractions are... 

TCDD TEQ/g sediment, the 30X diluted extract gave a DR CALUX response of 34.9 6.1 pg 2,3,7,8-TCDD TEQ/g sediment. In general, an effect of dilution on the total DR CALUX TEQ content in sediment samples is observed. Although the exact nature for this observation is not known, it is hypothesized that this is due to the presence of various compounds in sediment extracts showing variable affinity toward the Ah receptor. Dose-response curves in the DR CALUX bioassay of individual compounds have been studied and showed obvious differences (Hosoe et al, 2002) both in maximum response and slope of the curve fit. 

In this type of curve, if log dose is plotted against the percentage response, the shape of the curve remains unchanged. This log dose-response curve is useful in bioassays. 

To test the validity of the bioassay itself we prepared a diet containing increasing amounts of rotenone, a compound derived from isoflavones and thus chemically not far removed from the soybean phytoalexins. Results in this case followed exactly the expected dose response curve (Table VII). Both survival and weight gain of larvae were drastically affected by increasing concentrations of rotenone. This experiment showed that the bioassay would be capable of detecting toxic effects of the phytoalexins on the soybean looper larvae, if such effects were acute. It showed also that the detoxification mechanisms in the soybean looper, a rather polyphagous insect, may permit it to adequately overcome the antibiotic effect of the isoflavonoid phytoalexins, but not that of the isoflavone rotenone. 

Fig. 5.2 Dose-response curve for 2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD) in the CALUX bioassay. The concentration (expressed as TCDD) can subsequently be determined by comparing the response obtained with a sample extract with the calibration...

An in vitro bioassay can be designed in several ways, but requires statistical validity. A one point assay is not valid. The bioassay should be designed to consider factors that introduce variability, and the analysis should test such variability. A measurement series of a test sample should be compared to an equivalent series of the reference material, carefully considering the comparisons between the linear portions of the dose-response curves (Mire-Sluis et al., 1996). To test validity of a bioassay inter- and intra-assay variability should be considered in both preparation, and in the case of multiwell plates, the variability between each plate. To reduce the positional effect in plate tests, it is advisable to distribute the points on the curves randomly and also to include a reference standard in each plate (Gaines-Das and Meager, 1995). One of the most widely used techniques to validate a bioassay s performance is to include internal duplicates. The data arising from the comparison can be important in assessing the test s variability. 

DeLean A, Munson PJ, Rodbard D (1978) Simultaneous analysis of families of sigmoidal curves Application to bioassay, radioligand assay, and physiological dose-response curves. Am J Physiol 235 E97-E102... 

Overall, cancer risk assessment involves the four steps of hazard identification, dose-response, exposure assessment, and risk characterization. The dose-response curve established for cancer potency derivation for a chemical is based on evaluation of data on the carcinogenicity and dose-response characteristics of the chemical. The pharmacokinetics and mechanistic data evaluation (e.g., genotoxic or nongenotoxic) and a dose-response review of all adequate bioassays are conducted to determine, if target dose estimates or a dose-response model different from the default may be suggested. 

The RPF method is based on dose addition and assumes that the chemicals in a mixture share a common toxic mode of action this means that when tested in the same bioassay, the dose response curves of each component should be similarly shaped. The components in this similarity group are assumed to be true toxicologic clones of each other and have... 

Figure 12 Composite dose-response data for rat and mouse drinking water bioassays. Vinyl acetate induced squamous cell carcinoma of the oral cavity, esophagus, and forestomach. The tumor incidence was greatest for the oral cavity. These data, which include male and female rats and mice, illustrate the sharp break in the dose-response curve with clear evidence for a practical threshold. Data are from Bogdanffy et al. (65), Maltoni et al. (46), and JBRC (45).

A wide range of environmental samples have been evaluated for dioxin-like compounds with piscine cell line bioassays (Table 5). In nearly all studies some samples have been found to be positive. These results have been converted to bioassay-derived TCDD equivalent concentrations220, and different methods for doing this from the dose-response curves have been developed38 208. When the same samples have been applied to bioassays with mammalian and fish cell lines, the results have been broadly similar104 219 220. However, a few individual samples have sometimes been observed to differ33 220. In most studies, chemical analysis of the... 

Figure 8. Log dose-response curve for the effects of epinepherine and norepinepherine on the isolated cat spleen bioassay system. (Reprinted with permission from Ref. 11.)...

Figure 2. Anti-angiogenesis in the Chick Embryo Bioassay. Dose response for saccharide concentration (in yg in 10 yl sol.) with H-cortisone ( 60 yg in 10 yl sol.). For heparins the shaded area represents past experience bars represent results with fj-cyclodextrin tetradecasulfate. Curve A and B, see text.

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