Biomarkers assays

A biomarker is here defined as a biological response to an environmental chemical at the individual level or below, which demonstrates a departure from normality. Responses at higher levels of biological organization are not, according to this definition, termed biomarkers. Where such biological responses can be readily measnred, they may provide the basis for biomarker assays, which can be nsed to stndy the effects of chemicals in the laboratory or, most importantly, in the field. There is also interest in their employment as tools for the environmental risk assessment of chemicals. 

Some biomarkers only provide a measure of exposure others also provide a measure of toxic effect. Biomarkers of the latter kind are of particular interest and importance and will be referred to as mechanistic biomarkers in the present text. Some mechanistic biomarker assays directly measure effects at the site of action as described in Section 2.4 (see Chapter 4, Table 4.2, for examples). Inhibition of acetylcholinesterase is one example. Others measure secondary effects on the operation of nerves or the endocrine system (examples given in Table 4.2 and Chapters 15 and 16). 

The concept of biomarkers is illustrated in Figure 4.4. As the dose of a chemical increases, the organism moves from a state of homeostasis to a state of stress. With further increases in dose, the organism enters first the state of reversible disease, and eventually the state of irreversible disease, which will lead to death. In concept, all of these stages can be monitored by biomarker assays (lower part of conceptual diagram). 

Some biomarker responses provide evidence only of exposure and do not give any reliable measure of toxic effect. Other biomarkers, however, provide a measure of toxic effects, and these will be referred to as mechanistic biomarkers. Ideally, biomarker assays of this latter type monitor the primary interaction between a chemical and its site of action. However, other biomarkers operating down stream from the original toxic lesion also provide a measure of toxic action (see Figure 14.3 in Chapter 14), as, for instance, in the case of changes in the transmission of action potential... 

Examples of biomarker assays operating at different levels are given in Table 4.2. The recent development of omics technology should provide strong support to this approach (Box 4.3). Microarray analysis, for instance, can give a time-related sequence of gene responses that relate to the cellular changes of toxicity. 

In predicting the effects of a pollutant on population growth rate, the effects of the chemical on the values of t, I, and n are of central interest. Chemical residue data and biomarker assays that provide measures of toxic effects are relevant here because they can, in concept, be used to relate the effects of a chemical upon the individual organism to a population parameter such as survivorship or fecundity (Figures 4.5 and 4.6). Examples of this are discussed in the second part of the text, including the reduction of survivorship of sparrow hawks caused by dieldrin (Chapter 5), the... 

The development of models incorporating biomarker assays to predict the effects of chemicals upon parameters related to r has obvious attractions from a scientific point of view and is preferable, in theory, to the crude use of ecotoxicity data currently employed in procedures for environmental risk assessment. However, the development of this approach would involve considerable investment in research, and might prove too complex and costly to be widely employed in environmental risk assessment. 

Biotic indices that are relatively simple and inexpensive to apply can be very useful for identifying environmental problems caused by pollutants. Serious effects of pollutants can cause departures from normal profiles. The problem is, however, identifying which pollutants—or which other enviromnental factors—are responsible for significant departures from normality. This dilemma illustrates well the importance of having both a top-down and a bottom-up approach to pollution problems in the field. Chemical analysis and biomarker assays can be used to identify chemicals responsible for adverse changes in communities detected by the use of biotic indices. 

The more difficult thing is to develop models that can, with reasonable confidence, be used to predict ecological effects. A detailed discussion of ecological approaches to risk assessment lies outside the scope of the present text. For further information, readers are referred to Suter (1993) Landis, Moore, and Norton (1998) and Peakall and Fairbrother (1998). One important question, already touched upon in this account, is to what extent biomarker assays can contribute to the risk assessment of environmental chemicals. The possible use of biomarkers for the assessment of chronic pollution and in regulatory toxicology is discussed by Handy, Galloway, and Depledge (2003). 

Another issue is the development and refinement of the testing protocols used in mesocosms. Mesocosms could have a more important role in environmental risk assessment if the data coming from them could be better interpreted. The use of biomarker assays to establish toxic effects and, where necessary, relate them to effects produced by chemicals in the field, might be a way forward. The issues raised in this section will be returned to in Chapter 17, after consideration of the individual examples given in Part 2. 

Peakall, D.B. (1992). Animal Biomarkers as Pollution Indicators—A wide-ranging account of biomarker assays in higher animals. 

Thus, it is often not possible to measure the combined effects of members of one group of pollutants with a single mechanistic biomarker assay. The situation... 

The toxicology of PCBs is complex and not fully understood. Coplanar PCBs interact with the Ah-receptor, with consequent induction of cytochrome P4501A1/2 and Ah-receptor-mediated toxicity. Induction of P4501A1 provides the basis of valuable biomarker assays, including bioassays such as CALUX. Certain PCBs, for example, 3,3, 4,4 -TCB, are converted to monohydroxymetabolites, which act as thyroxine antagonists. PCBs can also cause immunotoxicity (e.g., in seals). 

PCDDs and PCDEs, together with coplanar PCBs, can express Ah-receptor-mediated toxicity. TCDD (dioxin) is used as a reference compound in the determination of TEFs, which can be used to estimate TEQs (toxic equivalents) for residues of PHAHs found in wildlife samples. Biomarker assays for Ah-receptor-mediated toxicity have been based on the induction of P450 lAl. TEQs measured in field samples have sometimes been related to toxic effects upon individuals and associated ecological effects (e.g., reproductive success). 

Tributyltin compounds used as antifouling agents on boats have had serious toxic effects upon many mollusks, including populations of oysters and dog whelks. Females of the latter species developed a condition known as imposex, which rendered them infertile and caused local extinction of the population in shallow coastal waters. Imposex provides the basis for a valuable biomarker assay. 

Induction of P450 lAl/2 provides the basis for biomarker assays for PAHs and other planar organic pollutants, such as coplanar PCBs, PCDDs, and PCDFs. 

The measurement of inhibition of brain AChE is a valuable biomarker assay for OPs and carbamates and is not jnst an index of exposure. Being an assay based on... 

This third part of the book will be devoted mainly to the problem of addressing complex pollution problems and how they can be studied employing new biomarker assays that exploit new technologies of biomedical science. Chapter 13 will give a broad overview of this question. The following three chapters, The Ecotoxicological Effects of Herbicides, Endocrine Disrupters, and Neurotoxicity and Behavioral Effects, will all provide examples of the study of complex pollution problems. 

The advantages of combining toxicity testing with chemical analysis when dealing with complex mixtures of environmental chemicals are clearly evident. More useful information can be obtained than would be possible if one or the other were to be used alone. However, chemical analysis can be very expensive, which places a limitation on the extent to which it can be used. There has been a growing interest in the development of new, cost-effective biomarker assays for assessing the toxicity of mixtures. Of particular interest are bioassays that incorporate mechanistic... 

Four examples will now be given of such mechanistic biomarker assays that can give integrative measures of toxic action by pollutants, all of which have been described earlier in the text. Where the members of a group of pollutants share a common mode of action and their effects are additive, TEQs can, in principle, be estimated from their concentrations and then summated to estimate the toxicity of the mixture. In these examples, toxicity is thought to be simply related to the proportion of the total number sites of action occupied by the pollutants and the toxic effect additive where two or more compounds of the same type are attached to the binding site. 

The inhibition of brain cholinesterase is a biomarker assay for organophosphorous (OP) and carbamate insecticides (Chapter 10, Section 10.2.4). OPs inhibit the enzyme by forming covalent bonds with a serine residue at the active center. Inhibition is, at best, slowly reversible. The degree of toxic effect depends upon the extent of cholinesterase inhibition caused by one or more OP and/or carbamate insecticides. In the case of OPs administered to vertebrates, a typical scenario is as follows sublethal symptoms begin to appear at 40-50% inhibition of cholinesterase, lethal toxicity above 70% inhibition. 

Some hydroxy metabolites of coplanar PCBs, such as 4-OH and 3,3 4,5 -tet-rachlorobiphenyl, act as antagonists of thyroxin (Chapter 6, Section 6.2.4). They have high affinity for the thyroxin-binding site on transthyretin (TTR) in plasma. Toxic effects include vitamin A deficiency. Biomarker assays for this toxic mechanism include percentage of thyroxin-binding sites to which rodenticide is bound, plasma levels of thyroxin, and plasma levels of vitamin A. 

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