ORGANIC POLLUTANTS: An Ecotoxicological Perspective - Chapter 13 pot

Biomarker assays provided the essential evidence that adverse effects on populations, communities, and ecosystems were being caused by envi-ronmental levels of particular chemicals.. The

Part

Further Issues

and Future Prospects

The first part of this text dealt with basic principles determining the distribution and effects of organic pollutants in the living environment The second focused on major groups of organic pollutants, describing their chemical and biological prop-erties and showing how these propprop-erties were related to their environmental fate and ecological effects Attention was given to case histories, especially to long-term studies conducted in reasonable depth and detail, which illustrate how some of these principles work out in practice in the complex and diverse natural environment The importance of these case studies should be strongly emphasized because, despite the shortcomings of many of them and the often only limited conclusions that can be drawn from them, they do provide insights into what happens in the “real world” as opposed to the theoretical one represented by the model systems that are necessarily employed during the course of risk assessment Consideration of these “case histo-ries” can give valuable guidelines with regard to the development of improved new ecotoxicity tests and testing systems (e.g., microcosms and mesocosms)

Since the recognition in the 1960s of the environmental problems presented by some persistent organochlorine insecticides, there have been many restrictions and bans placed upon these and other types of organic pollutants in western countries These restrictions have been in response to perceived environmental problems posed

by an individual compound or classes of compounds As we have seen, some restric-tions/bans have been followed, in the shorter or longer term, by the recovery of populations that were evidently adversely effected by them Such was the case with various species of predatory vertebrates following restrictions on organochlorine insecticides, or on dog whelks and other aquatic mollusks following restrictions on

the use of organotin compounds Thus, in more recent times, some relatively clear-cut cases of pollution problems associated with particular compounds or classes of compounds appear to have been resolved—at least in more developed countries of the world where there have been strong initiatives to control environmental pollution Consequently, in developed countries, there has been much less evidence for the existence of such relatively clear-cut pollution problems in recent years On the other hand, concern has grown that there may be more insidious long-term effects that have thus far escaped notice Interest has grown in the possible effects of mixtures

of relatively low levels of contrasting types of pollutants, to which many free-living organisms are exposed In the extreme case, it has been suggested that ecotoxicology might be regarded as just one type of stress, alongside others such as temperature, disease, nutrients, etc (Van Straalen 2003)

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 Disruptors,” and “Neurotoxicity and Behavioral Effects,” will all provide examples of the study of complex pollution problems The concluding chapter will attempt to look into the future What changes are we likely to see in pollution caused by organic compounds and in the regulatory systems designed to control such pollution? What improvements may there be in testing pro-cedures having regard for ethical questions raised by animal welfare organizations? Can ecotoxicity testing become more ecologically relevant? Can more information

be gained by making greater use of field studies?

Pollution Problems

13.1 INTRODUCTION

In the second part of the text, attention was focused on particular pollutants or groups

of pollutants Their chemical and biochemical properties were related to their known ecotoxicological effects Sometimes, with the aid of biomarker assays, it has been possible to relate the responses of individuals to consequent effects at the level of population and above Biomarker assays provided the essential evidence that adverse effects on populations, communities, and ecosystems were being caused by envi-ronmental levels of particular chemicals The examples given included population

declines of raptors due to eggshell thinning caused by p,pb-DDE, and decline or

extinction of dog whelk populations due to imposex caused by tributyl tin (TBTs) These were relatively straightforward situations where much of the adverse change was attributable to a single chemical In other cases, as with the decline of raptors in the U.K., effects were related to one group of chemicals, in this case the cyclodienes Since these events, there have been extensive bans on certain chemicals, and there is less evidence of harmful effects due to just one chemical or group of related chemi-cals Interest has moved toward the possible adverse effects of complex mixtures of chemicals, sometimes of contrasting modes of action, often at low levels

Establishing the effects of combinations of chemicals in the field is no simple mat-ter There are many cases where adverse effects at the level of population or above have been shown to correlate with levels of either individual pollutants or combina-tions of pollutants, but the difficulty comes in establishing causality, in establish-ing that particular chemicals at the levels measured in environmental samples are actually responsible for observed adverse effects This question has already been encountered in the studies of pollution of the Great Lakes of North America by

deter-mined by chemical analysis may contribute to population declines, including short-age of food, habitat change, disease, and climatic change—and other pollutants that have not been analyzed Such factors may very well correlate with the measured pol-lutant levels, especially where comparison is simply being made between the popula-tions in one or two polluted areas and a “reference” population in a “clean” area In badly polluted areas, there may be elevated levels of other pollutants in addition to those determined by chemical analysis, and these may have direct effects upon the species being studied, or indirect ones by causing changes in food supply

13.2 MEASURING THE TOXICITY OF MIXTURES

As explained earlier, toxicity testing of pesticides and industrial chemicals for the purposes of statutory environmental risk assessment is very largely done on single

minute proportion of the combinations of pollutants that occur in the natural envi-ronment can be tested for their toxicity This dilemma will be discussed further in Section 13.3 A different and more complex situation exists, however, in the real world; mixtures of pollutants are found in contaminated ecosystems, in effluents discharged into surface waters, for example, sewage and industrial effluents, and in waste waters from pulp mills The tests or bioassays employed here usually measure the toxicity expressed by mixtures, and investigators are presented with the prob-lem of identifying the contributions of individual components of a mixture to this toxicity Simple toxicity tests/bioassays often establish the presence of toxic chemi-cals without identifying the mechanisms by which toxicity is expressed The issue

is further complicated by the possibility that naturally occurring xenobiotics, such

as phytoestrogens taken up by fish, may contribute significantly to the toxicity that

is measured

In the simplest situation, chemicals in a mixture will show additive toxicity If envi-ronmental samples are submitted for both toxicity testing and chemical analysis, the toxicity of the mixture may be estimated from the chemical data, to be compared with the actual measured toxicity As explained earlier for the estimation of dioxin

may be expressed relative to that of the most toxic component (toxic equivalency fac-tor or TEF) Using TEFs as conversion facfac-tors, the concentration of each component can then be converted into toxicity units (toxic equivalents or TEQs) the summation of which gives the predicted toxicity for the whole mixture Often, the estimated toxicity

of mixtures of chemicals in environmental samples falls short of the measured toxicity Two major factors contribute to this underestimation of toxicity: first, failure to detect certain toxic molecules (including natural xenobiotics), and second, the determination

by analysis of chemicals that are of only limited availability to free-living organisms,

as when there is strong adsorption in soils or sediments In the latter case, analysis overestimates the quantity of a chemical that is actually available to an organism Potentiation (synergism) between pollutants can also contribute to the underestimation

of toxicity when making calculations based on chemical analysis (see Doi, Chapter 12

pollutants present in environmental samples are subjected to a fractionation procedure

in an attempt to identify the main toxic components By a process of elimination, tox-icity can then be tracked down to particular fractions and compounds

The advantages of combining toxicity testing with chemical analysis when deal-ing 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 toxic-ity of mixtures Of particular interest are bioassays that incorporate mechanistic

Dealing with Complex Pollution Problems 245

These can be used alone or in combination with standard toxicity tests, and some of them identify the mechanisms responsible for toxic effects, thus indicating the types

of compounds involved

INTEGRATED BIOMARKER APPROACH TO

MEASURING THE TOXICITY OF MIXTURES

A very large number of toxic organic pollutants, both manmade and naturally occur-ring, exist in the living environment However, they express their toxicity through a much smaller number of mechanisms Some of the more important sites of action of

to measuring the toxicity of mixtures of pollutants is to use appropriate mechanistic

biomarker assays for monitoring the operation of a limited number of mechanisms of toxic action and to relate the responses that are measured to the levels of individual chemicals in the mixtures to which organisms are exposed (Peakall 1992, Peakall and Shugart 1993) Such an approach can provide an index of additive toxicity of mixtures, which takes into account any potentiation of toxicity at the toxicokinetic level (Walker 1998c) Mechanistic biomarkers can be both qualitative and quantita-tive; they identify a mechanism of toxic action and measure the degree to which it operates Thus, they can provide an integrated measure of the overall effect of a group of compounds that operate through the same mechanism of action Where the mechanism of action is specific to a particular class of chemicals, it can be related to the concentrations of components of a mixture which belong to that class

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

1 The inhibition of brain cholinesterase is a biomarker assay for

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 verte-brates, a typical scenario is as follows: sublethal symptoms begin to appear at 40–50% inhibition of cholinesterase, lethal toxicity above 70% inhibition

2 The anticoagulant rodenticides warfarin and superwarfarins are toxic

because they have high affinity for a vitamin K binding site of hepatic

measure the percent of vitamin K binding sites occupied by rodenticides However, the technology is not currently available to do that On the other hand, the measurement of increases in plasma levels of undercarboxylated clotting proteins some time after exposure to rodenticide provides a good biomarker for this toxic mechanism

3 Some hydroxy metabolites of coplanar PCBs, such as 4-OH and

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 roden-ticide is bound, plasma levels of thyroxin, and plasma levels of vitamin A

4 Coplanar PCBs, PCDDs, and PCDFs express Ah-receptor-mediated

tox-icity (Chapter 6, Section 6.2.4) Binding to the receptor leads to induction

of cytochrome P450 I and a number of associated toxic effects Again, toxic effects are related to the extent of binding to this receptor and appear to

be additive, even with complex mixtures of planar polychlorinated com-pounds Induction of P4501A1/2 has been widely used as the basis of a biomarker assay Residue data can be used to estimate TEQs for dioxin (see

Chapter 7, Section 7.2.4)

In addition to the foregoing, three further examples in this list (numbers 5–7) deserve consideration These are (5) interaction of endocrine disrup-tors with the estrogen receptor, (6) the action of uncouplers of oxidative phosphorylation, and (7) mechanisms of oxidative stress Until now only the first is well represented by biomarker assays that have been employed

in ecotoxicology

5 Interaction with the estrogen receptor (ER) has been important in the

devel-opment of biomarker assays for endocrine disrupting chemicals (EDCs), and

(including bioassays) already developed is reviewed by Janssen, Faber, and Bosveld (1998) A surprisingly diverse range of chemicals can act as agonists

or antagonists for the estrogen receptor, producing “feminizing” or

“mas-culinizing” effects These include o,pb-DDE, certain PAHs, PCBs, PCDDS,

PCDFs, alkylphenols, and naturally occurring phyto- and myco-estrogens

However, it should be borne in mind that some EDCs (e.g., o,pb-DDE, PCBs)

probably act through their hydroxymetabolites, which bear a closer resem-blance to natural estrogens than the parent compounds and, second, that oth-ers (e.g., alkylphenols) are only very weak estrogens

A number of biomarker assays have been developed for fish Apart from

a variety of nonspecific endpoints such as organ weight and histochemical change, vitellogenin synthesis has provided a specific and sensitive endpoint, which has been very useful for detecting the presence of environmental estrogens at low concentrations A number of different cell lines have been developed for use in bioassays for rapid screening of environmental sam-ples These include fish and bird hepatocytes, mouse hepatocytes, human mammary tumor cells, and yeast cells (Janssen, Faber, and Bosveld 1998) The endpoints include vitellogenin production, competitive binding to ER,

Dealing with Complex Pollution Problems 247

the activation of galactosidase, the generation of light through the interme-diacy of reporter genes, and the elevation of mRNA levels The diversity

of the available bioassays reflects the high profile that endocrine disruptors have been given in recent years Some of these assays are described in more detail in Section 13.5

6 Uncouplers of oxidative phosphorylation Oxidative phosphorylation of

ADP to generate ATP is a function of the mitochondrial inner membrane

of animals and plants Compounds that uncouple the process are general biocides, showing toxicity to animals and plants alike For oxidative phos-phorylation to proceed, a proton gradient must be built up across the inner mitochondrial membrane The maintenance of a proton gradient depends

on the inner mitochondrial membrane remaining impermeable to protons Most uncouplers of oxidative phosphorylation are weak acids that are lipo-philic when in the undissociated state Examples include the herbicides dinitro ortho cresol (DNOC) and dinitro secondary butyl phenol (dinoseb), and the fungicide pentachlorophenol (PCP) The proton gradients across inner mitochondrial membranes are built up by active transport, utilizing energy from the electron transport chain that operates within the membrane The direction of the gradient falls from the outside of the membrane to the inside (Figure 13.1) The dissociated forms (conjugate bases) of the weak acids combine with protons on the outside of the membrane to form undis-sociated lipophilic acids, which then dissolve in the membrane and diffuse

outside of the membrane, they dissociate to release protons, and so act as proton translocators They run down proton gradients, and hence “uncouple”

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FIGURE 13.1 Uncouplers of oxidative phosphorylation.

oxidative phosphorylation, dissipating the energy that would otherwise have driven ATP synthesis The action of uncouplers can be measured in isolated mitochondria with an oxygen electrode that follows the rate of oxygen

Thus, the combined toxic action of mixtures of “uncouplers” can be studied

in isolated mitochondria Such studies can be used to investigate the signifi-cance of tissue levels of mixtures of, for example, substituted phenols, found

in tissues of animals after exposure to them in vivo

7 The participation of some OPs in redox cycling with consequent oxidative

stress It has become increasingly apparent that the toxicity of certain

com-pounds is due to their ability to facilitate the generation in tissues of highly

cause cellular damage including lipid peroxidation and DNA damage, and have been implicated in certain disease states such as atherosclerosis and some forms of cancer (Halliwell and Gutteridge 1986) Because they are so unstable, they are difficult or impossible to detect Proof of their existence depends upon indirect evidence The appearance of characteristic products

of oxyradical attack (e.g., oxidized lipids, malonaldehyde from lipid per-oxidation, and oxidative adducts of DNA), and the induction of enzymes involved in their destruction (e.g., superoxide dismutase, catalase, and per-oxidase) can all provide evidence for the presence of oxyradicals and give some indication of their cellular concentrations

These highly reactive species can be generated as a consequence of the presence of certain organic pollutants, such as bipridyl herbicides and aromatic nitro compounds (Figure 13.2) Taking as examples the herbicide paraquat (Hathway 1984), and nitro-pyrene (Hetherington et al 1996), both can receive single electrons from reductive sources in the cell to form unstable free radicals These radicals can then pass the electrons on to molecular oxygen to form superoxide anion, with regeneration of the original molecule Thus, a cyclic process is established, the net effect being to transfer electrons from a reductive source to oxygen with generation of an oxyradical Once formed, superoxide can undergo further reactions to form hydrogen peroxide and the highly reactive hydroxy radical The toxicity of paraquat to plants and animals is believed to be due, largely or entirely, to cellular damage caused by oxyradicals In the case of plants, these radicals attack the photosynthetic system (see Hassall 1990)

In animals, toxic action is mainly against Type 1 and Type II alveolar cells, which

There is evidence that mechanisms other than the production of free radicals of nitrogen-containing aromatic compounds are important in the case of pollutants Refractory substrates for cytochrome P450, such as higher chlorinated PCBs, may facilitate the release into the cell of active forms of oxygen (e.g., the superoxide ion)

by, in effect, blocking binding sites for substrates to be oxidized and thereby deflect-ing activated oxygen produced by the heme nucleus The unused activated oxygen may then escape from the domain of the cytochrome P450 in the form of superoxide

to cause oxidative damage elsewhere in the cell

Dealing with Complex Pollution Problems 249

At the time of writing, the toxicity of oxyradicals generated by the action of pol-lutants is highly topical because of the relevance to human diseases It is not an easy subject to investigate because of the instability of the radicals and the different mechanisms by which they may be generated Hopefully rapid progress will be made

so that monitoring the effects of oxyradicals will make an important contribution

to the growing armory of mechanistic biomarkers for the study of environmental effects of organic pollutants

Viewing the foregoing examples overall, the first five all involve interaction between organic pollutants and discrete sites on proteins, one of them the active site of an enzyme, the others being “receptors” to which chemicals bind to produce toxicological effects Knowledge of the structures and properties of such receptors facilitates the development of QSAR models for pollutants, where toxicity can be predicted from chemical parameters (Box 17.1) Indeed, new pesticides are some-times designed on the basis of models of this kind For example, some ergosterol synthesis inhibitor fungicides (EBIs) that can lock into the catalytic site of P450s have been discovered by following this approach Interactions such as these are essentially similar to the interactions of agonists and/or antagonists with receptors

in pharmacology

The last two examples do not belong in the same category, there being no discrete single binding site on a protein Uncouplers of oxidative phosphorylation operate across the inner mitochondrial membranes, their critical properties being the ability

to reversibly interact with protons and their existence in the uncharged lipophilic state after protons are bound Oxyradicals can, in principle, be generated by a variety

of redox systems in differing locations, which are able to transfer single electrons to oxygen under cellular conditions The systems that carry out one electron reduction

of nitroaromatic compounds and aromatic amines have yet to be properly elucidated

R =

Paraquat CH3N

3-Nitropyrene

NCH3 NCH3Free radical

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+ +

CH3N+

FIGURE 13.2 Superoxide generation by 3-nitropyrene and paraquat.

Neither of these mechanisms of toxic action is susceptible to the kind of QSAR analysis referred to earlier, the employment of which depends on knowledge of the structure of particular binding sites

PATHWAYS OF EXPRESSION

When chemicals have toxic effects, the initial molecular interaction between the chemical and its site of action (receptor, membrane, redox system, etc.), is followed

by a sequence of changes at the cellular and whole-organism levels that eventually lead to the appearance of overt symptoms of intoxication Until now, discussion has been focused upon mechanisms of toxicity, that is, on the primary interaction of toxic chemicals with their sites of action As we have seen, biomarker assays such as the measurement of acetylcholinesterase inhibition can monitor this initial interac-tion in a causal chain that leads to the overt expression of toxicity Such mechanistic biomarkers are specific for particular types of chemicals acting at particular sites By contrast, other biomarkers that measure consequent changes at higher levels of orga-nization, for example, the release of stress proteins, damage to cellular organelles, and disturbances to the nervous system or endocrine system are less specific, and can, in principle, provide integrated measures of the effects of diverse chemicals in

a mixture operating through contrasting mechanisms of action It is possible, there-fore, to measure the combined effects of chemicals working through different modes

of action if these effects are expressed through a common pathway (e.g., the nervous system or the endocrine system) that can be monitored by a higher-level biomarker assay For example, two chemicals may act on different receptors in the nervous system, but they may both produce similar disturbances such as tremors, hyperexcit-ability, and even certain changes in the EEG pattern, all of which can be measured

by higher-level biomarker assays

When moving from the primary toxic lesion to the knock-on effects at higher levels of organization, the higher one goes, the harder it becomes to relate mea-sured effects to particular mechanisms of toxic action Thus, it is advantageous to use combinations of biomarkers operating at different organizational levels rather than single biomarker assays when investigating toxic effects of mixtures of dissimilar compounds; it becomes possible to relate initial responses to higher-level responses

in the causal chain of toxicity Although they often do not give clear evidence of the mechanism of action, higher-level biomarker assays (e.g., scope for growth in mol-lusks, or behavioral effects in vertebrates) have the advantage that they can give an integrated measure of the toxic effects caused by a mixture of chemicals

Taken together, combinations of biomarker assays working at different organiza-tional levels can give an “in-depth” picture of the sequence of adverse changes that follows exposure to toxic mixtures, when compounds in the mixture with different modes of action cause higher-level changes through a common pathway of expres-sion Two prime examples are (1) chemicals that cause endocrine disruption, and (2) neurotoxic compounds To illustrate these issues further in more depth and detail,