Dose-response bioassay

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.

An inadequate intake in the diet of those food chemicals that are essential nutrients results in health risks. Indeed these risks are by far the most important in terms of the world s population where malnutrition is a major public health problem. But, unlike the toxic chemicals, they would show a very different dose-response if they were subject to similar animal bioassays. At very low doses there would be a high risk of disease that would decrease as the dose was increased, the curve would then plateau until exposure was at such a level that toxicity could occur. Figure 11.2 shows this relationship which is U- or J-shaped rather than the essentially linear dose-response that is assumed for chemicals that are only toxic. The plateau region reflects what is commonly regarded as the homeostatic region where the cell is able to maintain its function and any excess nutrient is excreted, or mechanisms are induced that are completely reversible. 

Plant physiologists and other biological scientists also have their important role to play in allelopathy. They must devise suitable bioassays to detect the suspected allelopathic compounds, follow the biological activity of the individual and associated chemicals, develop activity profiles for identified chemicals, and determine the conditions (dose/response) for chemicals to arrive at the threshold levels. They must also determine which chemicals contribute... 

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. 

Phenol has been tested in animals for carcinogenicity by the oral and dermal routes, but results are equivocal. In a chronic NCI cancer bioassay (NCI 1980), a significant incidence of tumors (pheochro-mocytomas of the adrenal gland, leukemia, or lymphomas) occurred only in male rats exposed to the lowest dose level (2,500 ppm, 277 mg/kg/day) of phenol but not in male or female mice or male rats exposed to a higher dose level (5,000 ppm, 624 mg/kg/day). Since tumors occurred only in males in one of the two species tested, and since a positive dose-response relationship was not established, this study does not provide sufficient evidence to conclude that phenol is carcinogenic when administered by the oral route. Dermal application of phenol has been shown to result in tumors in mice phenol is a tumor promoter when it is applied after the application of the tumor initiator DMBA (Boutwell and Bosch 1959 Salaman and Glendenning 1957 Wynder and Hoffmann 1961). However, this effect occurs at dose levels of phenol that produce severe skin... 

Many toxicologists are concerned about possible misinterpretation of bioassay results when the MTD (the highest bioassay dose) has turned out to produce serious toxicity as well as a tumor response. They contend that the excessive toxicity that somehow decreased... 

The lowest Cancer-Effect-Levels (CELs) in the NCI (1978) bioassay are the closes that caused hepatocellular carcinoma in rats (4 mg/kg/day) and mice (52 mg/kg/day) (Table 2-1, Figure 2-1). Using the dose-response data for the hepatocellular carcinoma in rats, EPA (1980, 1988a) derived and verified an oral slope factor (q1 ) of 8.0 x 10 (mg/kg/day) 1 for 1,2-diphenylhydrazine. Using this slope factor, the doses associated with upper-bound lifetime cancer risk levels of 10 to 10 are calculated to be 1.3 x 10 to 1.3 x 10 mg/kg/day, respectively (Figure 2-1). 

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. 

Further data on the effects of chronic inhalation exposure to 1,4-dichlorobenzene would be useful, especially because chronic exposures to 1,4-dichlorobenzene in the air, in the home, and the workplace are the main sources of human exposure to this chemical. Any further testing of the effects of chronic exposure to 1,4-dichlorobenzene via the oral route should probably be done at lower levels of 1,4-dichlorobenzene than those that have already been used in the NTP (1987) bioassay, and should focus on dose-response relationships involving the hepatic, renal, hematopoietic, central nervous system, and metabolic pathways. Data on the effects of chronic dermal exposure to 1,4-dichlorobenzene may be useful if dermal absorption and systemic distribution of 1,4-dichlorobenzene can be demonstrated from toxicokinetic studies, since chronic dermal exposure to 1,4-dichlorobenzene occurs as a result of bathing and showering in drinking water that contains low levels of this chemical in many U.S. communities. 

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. 

Many toxicity studies, especially long-term bioassays carried out to determine potential carcinogenicity, use high-dose levels (e.g., maximum tolerated dose), and consequently, any hormetic response would be missed. To be properly evaluated, more doses and a wider dose response would have to be investigated. 

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...

For any hazardous substance, estimates of the relationship of dose to response in humans are based on either animal or human data. For example, only about 20 of the approximately 300 chemical carcinogens regulated by EPA have dose-response relationships based on human data from epidemiologic studies the remainder are based on animal bioassays. In contrast, the dose-response relationships for radiation are based primarily on the results of human epidemiologic studies. 

Experimental compounds were evaluated in a dose-response format. Azoxystrobin or other fungicides that have both protective and curative activity were used as a standard, and a solvent control was used in every study. The number of disease lesions per leaf was used to determine the ability of the test compounds to prevent infections. The size of the lesions was used to determine the curative activity of compounds. Each fungicide concentration is replicated four times and the experiment is repeated at least once. This bioassay does not differentiate between direct effects on the fungus and indirect effects through induction of plant defenses. However, if a compound is much more active in this in vivo assay than than the in vitro microtiter assay, induction activity is indicated. 

Risk Assessment. The model provides an approach to estimating doses of MEHP to the testes of the rat following oral doses of DEHP and might be useful for internal dose-response assessment of rat bioassay data in which the toxicity end point of interest is testicular toxicity. However, such uses of the model, or other potential uses in risk assessment, have not been evaluated. 

Additional bioassays in animals do not seem necessary. Further research on dose-response relationships for the many biochemical effects of peroxisome proliferators leading to liver cancer in rodents, identification of specific thresholds, and potential reversibility, would be informative only if an extrapolation model for cancer was deemed appropriate in spite of profound differences between human and rodent responses. 

---