Genetic and Molecular Ecotoxicology: A Research Framework Susan Anderson,1 Walter Sadinski,1 Lee Shugart,2 Peter Brussard,3 Michael Depledge,4 Tim Ford,5 JoEllen Hose,6 John Stegeman,7 William Suk,8 Isaac Wirgin,9 and Gerald Wogan0 1Lawrence Berkeley Laboratory, Berkeley, California; 2Oak Ridge National Laboratory, Oak Ridge, Tennessee; 3University of Nevada Reno, Reno, Nevada; 4University of Plymouth, Plymouth, UK; 5Harvard University, Cambridge, Massachusetts; 6 CCidental College, Los Angeles, California; 7Woods Hole Oceanographic Institute, Woods Hole, Massachusetts; 8National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina; 9New York University Medical Center, Tuxedo, New York; 10Massachusetts Institute of Technology, Cambridge, Massachusetts Participants at the Napa Conference on Genetic and Molecular Ecotoxicology assessed the status of this field in light of heightened concerns about the genetic effects of exposure to hazardous substances and recent advancements in our capabilities to measure those effects. We present here a synthesis of the ideas discussed throughout the conference, including definitions of important concepts in the field and critical research needs and opportunities. While there were many opinions expressed on these topics, there was general agreement that there are substantive new opportuni- ties to improve the impact of genetic and molecular ecotoxicology on prediction of sublethal effects of exposure to hazardous substances. Future studies should emphasize integration of genetic ecotoxicology, ecological genetics, and molecular biology and should be directed toward improving our understanding of the ecological implications of genotoxic responses. Ecological implications may be assessed at either the population or ecosys- tem level; however, a population-level focus may be most pragmatic. Recent technical advancements in measuring genetic and molecular responses to toxicant exposure will spur rapid progress. These new techniques have considerable promise for increasing our understanding of both mechanisms of toxicity on genes or gene products and the relevance of detrimental effects to individual fitness. - Environ Health Perspect 102(Suppl 12):3-8 (1994) Key words: genetic, molecular, ecotoxicology, biomarkers, genotypes, genotoxic, global pollution, populations, ecosystems, biodiversity, hazardous substances

Introduction informative diagnostic and prognostic Definition techniques available. Second, interest in The Napa Conference on Genetic and the preservation of biodiversity has height- We propose that genetic ecotoxicology Molecular Ecotoxicology provided an ened concerns that effects of hazardous may best be defined as: opportunity to assess the field of genetic substances on genetic variability are not "The study of chemical- or radiation- and molecular ecotoxicology in light of well understood. Third, there has been induced changes in the genetic material of important recent developments. First, there rapid advancement of new molecular natural biota. Changes may be direct alter- is a growing sense of urgency regarding approaches for analysis of gene structure ations in genes and gene expression or global pollution, which challenges us to and function as well as improved validation selective effects of pollutants on gene fre- assess subtle sublethal effects with the most of more classical techniques such as cytoge- quencies." netic analyses. These developments set the Thus, this definition attempts to stage for new research emphasizing broad encompass direct DNA damage, epigenetic This article synthesizes findings of the Napa goals and applications across levels of bio- effects, and changes in gene pools attribut- Conference on Genetic and Molecular Ecotoxicology logical organization. Thus, these concepts able to toxicant exposure. In the past, held 12-15 October 1993 in Yountville, California. must be integrated into a more compre- genetic ecotoxicology has been identified This conference was supported primarily by the National Institute of Environmental Health Sciences hensive, contemporary definition of genetic primarily with the study of direct DNA (NIEHS) Superfund Basic Research Program (W. Suk, ecotoxicology. damage. Semantic problems could stem Program Director). Additional support was provided The goals of this article are to identify a from efforts to expand the definition of NIEHS by the University of California, Berkeley, a response to embrace many sorts Superfund Program Project (M. Smith, Principal more inclusive identity and modern genotoxic Investigator, S. Neal, Project Administrator) and by an framework for future research in genetic and of molecular change. Therefore, we antici- award from the Pew Scholars in Conservation and molecular ecotoxicology. We present the pate that the proposed definition will serve the Environment (S. Anderson, Principal Investigator). consensus of the group at the Napa as a focus for future debate. Only recently We are indebted to the many participants in the con- ference whose voices are expressed throughout this Conference, but also highlight the diversity have attempts been made to place the con- review. of opinion on key topics. We present the sequences of "pollutant-induced changes in Address correspondence to Dr. Susan Anderson, following: a proposed definition of the field, genetic material" in a broader, more envi- Bldg. 70-193A, Lawrence Berkeley Laboratory, 1 Cyclotron Road, Berkeley, CA 94708. Telephone its potential relevance, key goals, core chal- ronmentally relevant context (1). (510) 486-4654. Fax (510) 486-5401. lenges, and a framework for future research.

Environmental Health Perspectives 3 ANDERSON ETAL.

Relevance potentially some of the most important ardous substances in the environment. Hazardous substances are distributed widely sublethal impacts of exposure to genotoxic Biomarkers alone could be used to provide in ecosystems due to diverse human activi- substances (8). As the state of the art of historical information on individuals and ties such as energy usage, industrial enter- research in ecotoxicology advances, we populations affected by hazardous exposure, prises, agriculture, and activities of the hopefully will be able to document more identify species at risk due to exposure, defense industry. The long-term ecological such effects and improve our capabilities to derive cleanup targets for site remediation, effects of these substances, as well as effects predict effects on populations. assess effects ofcontaminants on the genetic of increasing ultraviolet-B (UV-B) radiation Examples of changes in gene pools diversity of populations, and measure as a consequence of ozone depletion, are attributable to pollutant exposure are avail- effects in sentinel species as potential indi- largely unknown. For example, ecosystem able in an underappreciated, diverse litera- cators of human exposure to chemicals pre- protection is based largely on assessments of ture. Well-known examples, such as those sent at Superfund sites. lethality in model organisms rather than on of industrial melanism, pesticide tolerance, an understanding of toxic effects in situ on and tolerance to metals in numerous organ- Goals and Challenges natural populations. Similarly, damage isms have been reviewed (9). Recently We propose that the overall goal of genetic assessments ofcatastrophic events such as oil attention has been directed toward the ecotoxicology is best described as follows: spills have, until recendy (2), relied on mor- effects of pollutants on gene pools of fish "To assess, predict, and prevent signifi- tality censuses of exposed animals and have (10) and natural microbial communities cant radiation- or chemical-induced genetic not often considered sublethal genotoxicity. (11). New molecular capabilities should and epigenetic damage in populations." Although ozone depletion has received heighten the impact ofsuch research. Practically, attaining this goal will be enormous international attention, only a We now recognize that pervasive global demanding, in part because "significant few studies have examined the damaging pollution requires that the effects of haz- genetic damage" is difficult to define and effects of increased UV-B on DNA of wild ardous substances on biodiversity be well- therefore difficult to measure, and because organisms, even in the affected Antarctic studied. Several lines of evidence have our ability to predict the consequences of ecosystem (3). In agricultural settings shown that pollutants cause decreased such change is inadequate. throughout the world, genotoxic com- genetic variability in populations and that There are at least two possible definitions pounds are used as pesticides and herbicides, such decreases are detrimental to popula- of "significant genetic damage." The first yet we know that some of these substances tion viability (10,12). Others (13-16) definition is based on a population-level can elicit adverse genetic and epigenetic have raised the concern that pollutants focus: responses in nontarget populations (4). could affect evolutionary processes. Thus, Definition 1. Significant genetic dam- Additionally, global mining practices have it is vital that we consider research in age is defined as altered genetic structure or mobilized tons ofhazardous mutagenic met- genetic ecotoxicology as crucial to conserv- function in individuals that ultimately als such as arsenic, mercury, nickel, and ing the genetic variability contained within results in decreased population abundance chromium into the environment where they ecosystems (17). How can we predict the or irreversible changes in the genetic vari- are now potentially serious toxic threats. loss of variability? How can we optimize ability within gene pools. The ecological effects of these mining activi- restorations? How can we communicate This definition is attractive for several ties remain poorly understood. that there is no going back once genetic important reasons. The population is the In the human health arena, the conse- variation has been eliminated? level at which evolutionary processes are quences of exposure to genotoxic sub- It is not possible to assess genotoxic manifest; thus this definition provides a stances have been researched intensively for risks in detail in all species and popula- theoretical context for examining genetic decades. In contrast, the level of scrutiny tions. Thus, model species will continue to alterations. The development of approaches directed towards genetic ecotoxicology has be highly important in establishing the to associate effects on individuals with been more fragmented. This situation is mechanistic basis and validation of diag- effects on the population continues (16). somewhat surprising given the potential for nostic probes, and disclosing phylogenetic Many of these are discussed below and in animals in ecosystems to serve as sentinels differences in resistance or susceptibility. papers within this issue (2,8). Thus, it is for effects in humans. Applying such knowledge to analyses of tempting to embrace a population-level Nevertheless, documentation of geno- sentinel species may then indicate poten- focus for describing significant genetic toxic effects in ecosystems attributable to tial hazards to other members of an ecosys- damage. hazardous substances has emerged (5). This tem and even to humans (18,19). Studies A broader, but more unwieldy, has recently included reports of tumors in of ras oncogenes in winter flounder (20), definition of "significant genetic damage" aquatic species (6), truncated age classes in molluscan germinomas (21) and responses embraces an ecosystem-level focus: populations exhibiting increased incidence to Ah-receptor agonists in diverse verte- Definition 2. Significant genetic dam- of tumors (7), damage assessments linking brates (22) all indicate a similarity of toxic age is defined as altered genetic structure or genotoxic effects of oil spills and subse- mechanisms in many groups of animals function in individuals that ultimately quent mortality in commercial fish species and suggest that effects on sentinel species results in disruption of an ecosystem and (2), and findings implicating epigenetic might indicate effects on other species as loss ofecosystem functional diversity. effects as the cause of endocrine and repro- well. It is vital that we continue to deter- This approach encompasses a more ductive disruption in a range ofspecies (4). mine the associations between responses in holistic view recognizing the connectivity Undoubtedly, however, other effects sentinel species and potential effects in among ecological processes and the scale at remain undetected. It is easier to detect humans. which ecological effects occur. However, tumors in an animal than to characterize Further research in genetic ecotoxicol- many of the ecosystem-level interactions long-term alterations of its reproductive ogy could provide numerous benefits remain poorly defined and-difficult to mea- potential. Yet, reproductive alterations are directed at improved management of haz- sure, making predictions beyond popula-

4 Environmental Health Perspectives GENETIC AND MOLECULAR ECOTOXICOLOGY tions difficult at best. Thus, it is much more A conceptual model was formulated Hopefully, an improved understanding difficult to superimpose this somewhat based upon the general consensus that more of mechanisms of effect can be coupled to vague concept of significant genetic damage research should be focused on linkages molecular characterizations of genotypic onto an already amorphous paradigm. between mechanisms and effects across lev- diversity at specific loci, with the intent of Nevertheless, critical environmental chal- els of biological organization (Figure 1). determining genotypic susceptibility to lenges, such as ecosystem restoration, dictate Thus, the model begins with biomarker exposure and resultant effects on genotypic that genetic ecotoxicologists broaden their responses and ends with populations. It diversity in populations. We can then perspective to include thinking about the includes three types of genotoxic syndrome attempt to understand what altered DNA enormous complexity of ecosystems. This as primary examples of outcomes of or reduced reproductive success in intoler- approach provides a stimulus to do that. genotoxic exposure: malignant tumors, ant individuals means in terms of the Differences in opinion regarding the decreased reproductive success, and altered resiliency, and hence persistence, of popu- appropriate definition of "significant genotypic diversity. We use these examples lations. Thus, advances in molecular tech- genetic damage" will continue. In the in the following discussion to illustrate the nology have contributed greatly to a new meantime, the definitions provided here predictive potential of biomarkers and an synthesis in genetic ecotoxicology that inte- could serve as focal points for debate. interdisciplinary framework that emphasizes grates key response parameters along a con- Another key barrier to the overarching linkages in genotoxicological research. tinuum from DNA to populations. It is goal of genetic ecotoxicology is that despite Ideally, biomarkers that indicate molec- also likely that future gains in genetic eco- recent technological advances, the useful- ular or genetic change would have predic- toxicology will depend increasingly on ness of biomarkers of genotoxicity for pre- tive value related to organismal fitness. these techniques. This realization is critical dicting effects on fitness and populations is Biomarkers are measured in individuals when considering the linkages depicted in in need of further validation. The follow- and are thus less obviously related to effects our conceptual model (Figure 1). ing section addresses research needs for that are measured on populations, but it is Potential relationships between bio- enhancing the use ofbiomarkers. apparent that there are strong associations markers of genotoxicity and malignant between changes in frequencies of bio- tumors have been suggested by research Research Framework marker responses in individuals and higher- conducted on fish and in the area of The evolving conceptual and technical order effects (8). human health. In addition, parallels in the foundation of genetic ecotoxicology calls In any discussion of the predictive sequence from metabolic activation of pro- for research that encompasses a broad view capacity of biomarkers, the potential carcinogenic sites of adduct formation in of the consequences of genotoxic exposure. importance of new molecular techniques DNA, and mutations in oncogenes and Ultimately, we need to test hypotheses must be emphasized. Researchers in genetic suppressor genes, have been established in focused on mechanisms and effects across ecotoxicology have begun to capitalize on mammalian and nonmammalian verte- levels of biological organization, from that the rapidly advancing technology in molec- brates (18). Thus, cancer represents an of DNA to populations and, ideally, even ular biology (7,15,23). As more critical identifiable disease that can be linked to to ecosystems. Such a synthesis undoubt- genes are identified, and the functions of chemical exposure, and for which present edly will require more interdisciplinary col- their products are understood, studies to mechanistic understanding has provided laboration, including cooperative efforts elucidate mechanisms of effect can be molecular markers. between molecular biologists, ecotoxicolo- greatly accelerated. In addition, there are However, sublethal effects on reproduc- gists, population geneticists, and commu- opportunities to understand the signifi- tive success might be far more pervasive, and nity ecologists to forge comprehensive cance of structural alterations in genes in not yet appreciated fully by genetic ecotoxi- studies of genetic alterations in wild popu- terms of protein function in processes criti- cologists. Research has been conducted on lations. cal to survival or reproductive success. linkages between biomarkers ofearly biolog-

Altered structure and Early biological effect function Mutations Cell death Population level Altered gene expression Altered gene expression consequences Biologically effective dose Aneuploidy Fertility decline Genotoxic syndrome Altered genotypic diversity DNA adducts Oncogene activation Protein dysfunction Malignant tumors Altered age class structure DNA-protein crosslinks SCE Endocrine disruption Decreased reproductive success Decreased population Pyrimidine dimers Micronuclei Gene amplifications Altered genotypic diversity abundance and distribution Anaphase aberrations Development abnormalities Population extinction Translocations Physiological impairment Lethality t t t Duration of exposure and relationship of effects to life history strategies DNA repair Cell regeneration Duration of sensitive Mean sensitivity of individuals Compensatory gene expression Sensitivity of gametogenic stages in population, including by gametogenic stages Synchrony of gametogenesis age class Severity of (background) biotic and abiotic factors

Figure 1. Possible ramifications of genotoxic exposure.

Volume 102, Supplement 12, December 1994 5 ANDERSON ETAL. ical effect and either altered structure and incorporate a broader spectrum of mecha- specificity. Due to technological advances function or decreased reproductive success nisms by which genetic variability can be at the molecular level, it is conceivable to (8). In contrast, little has been done to reduced, including toxicant exposure. be able to identify specific critical genes or assess relationships between biomarkers of Thus, in terms of understanding the most gene products such as the p53 gene or biologically effective dose and any measure critical effects of genotoxic exposure in cytochrome P450 enzymes (30,31), to of reproductive success. However, evidence nature, there is considerable need for relate genotypes at these loci to fitness, and that relates alterations in reproductive suc- genetic ecotoxicologists and population subsequently to relate fitness to overall het- cess to changes at the population level (2) geneticists to join efforts. erozygosity at these loci in the population. indicate the need for such markers. The principles underlying research on To facilitate documentation of whether Numerous specific avenues of research effects of genotoxicants on genotypic diver- or not these more specific techniques and could strengthen the use of biomarkers to sity are not new (Figure 2). In a highly het- relationships can be important and useful, predict reproductive effects. For example, erozygous population, there are likely to be perhaps collaborative, multilevel studies promising future research might include certain genotypes that are more sensitive to with classic model systems should be measures of alterations in specific genes genotoxic exposure than others. This is undertaken. These studies could serve as which are known to result in dysfunctional especially true if this population is het- prototypes for research with less well-stud- gametes or abnormal embryonic develop- erozygous at loci that are both critical to ied organisms and broaden the technical ment. In general, there is a need for studies fitness and susceptible to toxicant-induced scope of theoreticians and empiricists alike. of linkages between exposure to contami- structural alterations. Genotoxic exposure Eventually, models could be formulated nants, increases in frequencies of biomark- can act as a selective force by eliminating that predict effects of genotoxicants on ers, and reduced reproductive success with sensitive genotypes, or by reducing the wild populations. a select array of contaminants, biomarkers, number ofoffspring that they contribute to There are a number of other issues that and species. One means of accomplishing the next generation. The result is a reduc- must be considered regardless of the partic- this goal is the increased application ofbio- tion in the total genetic variation within ular effect of genotoxic exposure under marker techniques directly in germ cells that population or a shift in genotypic fre- scrutiny. These include: a) concentrations and embryos. More comprehensive docu- quencies. It is generally accepted that of some toxicants in the environment will mentation of such relationships is vital in genetic variation provides the requisite flex- likely increase on a global scale; b) toxi- order to address questions of whether bio- ibility for a population to persist in the face cants occur as complex mixtures; and c) markers of sublethal DNA damage are of variable biotic and abiotic selective chemical compounds are often present at indicative ofeffects on fitness. forces over time. Reduced variation can low, yet possibly toxic concentrations. There are obstacles to addressing these thus lead to increased rate of extinction. Most studies thus far have examined effects needs, due primarily to the difficulty of Research into toxic effects on genotypic from the perspective of high-dose, acute measuring biomarkers and reproductive diversity can be conducted in a variety of exposures to single toxicants. We know success in exposed feral populations, but we ways. One of the potentially most produc- very little about effects of chronic exposure posit that more could be done with those tive approaches would be to: a) use bio- to lower levels of multiple mutagens that populations and species that are experimen- markers to identify individuals sensitive to are pervasive and persistent in the environ- tally tractable (24) and more should be exposure (29); b) determine the genotypes ment. As global pollution problems gain attempted with those that are not. There is of these individuals at critical loci; c) deter- more attention, we will also be forced to an urgent need to assess genotoxic impacts mine the genotypes oftolerant individuals at determine the significance of low-level across taxa in wild populations under envi- these same loci; d) determine whether cer- responses in comparison to background ronmentally realistic conditions (25,26). tain alleles at these loci conferred greater fit- levels of factors that might elicit these Nondestructive biomarkers are vitally ness and, ideally, why that was the case; and responses. This represents an exact parallel important in this regard, especially with then e) determine the frequencies of geno- to the important debate over food muta- respect to sampling of threatened or endan- types at these loci in exposed and unexposed gens in human health risk assessment. In gered populations (27). populations. This might be particularly easy ecotoxicology, little has been done to Alterations in genotypic diversity are to address at the microbial level. address this problem (32,33). another potential effect of exposure to Similar approaches have been pursued The heritability of toxicant-induced genotoxicants. Not many genetic ecotoxi- by examining suites of allozymes in mutations and the importance of muta- cologists have studied effects of toxicant exposed and unexposed populations (10). tional load have not been well-studied. exposure on gene pools (10,16). The major improvement that the suggested Remarkably, theoretical treatments of fixed Unfortunately, not many population approach offers over past efforts is one of mutations and mutational load have not geneticists, in whose realm studies of geno- typic frequencies usually fall, have consid- ered the effects of toxic pollution on Population level genetic variability either. In addition, the consequences Altered genotypic diversity role and magnitude of interindividual vari- Genotype offers selective Altered range of phenotype ability is poorly understood (16,28). It is Sensitive and tolerant advantage or Altered age class structure time to recognize that, at a minimum, individuals disadvantage with Decreased population respect to toxic exposure abundance genetic ecotoxicologists are interested in and distribution conserving populations of currently or Population extinction potentially exposed species and therefore must think beyond effects on individuals. Likewise, population geneticists must Figure 2. Potential genotoxic effects on genotypic diversity.

6 Environmental Health Perspectives GENETIC AND MOLECULAR ECOTOXICOLOGY considered the potential effects of toxicant organisms that are at a less sensitive age or increasing availability of reliable diagnostic exposure. For example, contaminants may with species from an exposed community tools will greatly improve our ability to alter the timescales in which mutational that are not representative of effects within assess the sublethal effects of exposure to load accumulates and thus alter the relative that community. Ideally, we would want to hazardous substances. We must envision significances of natural selection and drift know the mechanisms dictating develop- their promise for addressing these problems as evolutionary forces. This is especially mental or life-history sensitivity. These and identify the most urgent directions for true regarding asexually reproducing and sorts of determinations could also help future research. However, a broader view hermaphroditic species, as well as very establish a more genuine, and general, use- of the ecological consequences of exposure small populations of animals with sexual fulness ofindicator or sentinel species. to hazardous substances, one that encom- reproduction. In addition, contaminants In addition to the practical concerns of passes a multidisciplinary focus, is also may affect the demographic features of assessing genotoxicity in the present and needed. These concepts will be important populations, and these changes are not nec- predicting it in the future, attempts to in at least five critical areas of research, essarily considered in existing theory. reverse toxicity from the past will present including restoration ecotoxicology, effects Multigeneration effects of toxicant challenging opportunities for genetic eco- of global environmental change, effects of exposure have only recently come to the toxicologists, as restorations of toxic sites contaminants on the genetic diversity of forefront of scientific and public thinking, are a prominent goal. Restorations illus- populations, multilevel assessments of the due largely to concerns over pesticides that trate the importance of establishing firm predictive capability of biomarkers, and act as estrogen analogues (4). These com- linkages between effects on individuals and linkages between molecular changes in ani- pounds can induce epigenetic effects that effects on populations and of determining mal sentinels of environmental toxicity and are expressed in progeny of exposed par- how specific tools can be employed to implications for human health. To succeed ents. The effects are not only a function of answer critical questions. For example, will in establishing a more multidisciplinary dose, but also the developmental stage dur- we be able to predict which individuals will focus, we must improve the predictive ing which exposure occurred. Both multi- have maximum fitness in restored popula- capability of biomarker techniques with generation genetic and epigenetic effects tions? It is within such applied contexts respect to assessing effects on populations. must be considered more thoroughly if we that the predictive value of biomarkers, An understanding of molecular mecha- are to comprehend what is surely a broad molecular characterizations of genetic sen- nisms will provide a foundation for the use range ofgenotoxic effects. sitivity, and hypothesized susceptibilities of of biomarkers in this regard, but we also Some of the more obvious factors that relatively invariable gene pools to extinc- need to understand mechanisms ofeffect at could modify genotoxic effects are pre- tion, could get their severest tests. all levels of biological organization. sented in the conceptual model (Figure 1). Eventually, we must understand better Ultimately, understanding of the linkages Perhaps one of the more important ones to how toxicant-induced changes in DNA between molecular and biochemical consider is the differential sensitivities of affect populations and communities (17). responses to toxic exposure and effects on developmental stages in gametes and individual fitness, populations, and ecosys- embryos, as well as differential sensitivities Conclusions tems must be integrated with theories from among species with different life history Techniques in genetic ecotoxicology are in population genetics and ecology to form a strategies. It is important that we under- a rapidly evolving state such that reliable broader model for assessing and predicting stand whether effects can be underesti- tools are now available for addressing more effects ofenvironmental pollution. mated if studies are conducted with complex environmental problems. The

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