Genetic effects of contaminant exposure — towards an assessment of impacts on animal populations

the Science of the T&al lbvirmment &.-J..ndblsd.9nkINur-“us-Wm. ELSEVIER

The Scienceof the Total Environment 191 (1996) 23-58

Genetic effects of contaminant exposure - towards an assessment of impacts on animal populations Paul D.N. Hebert*a,

Mary Murdoch

Luikerb

“Department of Zoology, University of Guelph, Guelph, Ontario, NIG 2W1, Canada ‘EVS Consultants, 195 Pemberton Avenue, North Vancouver, B.C. V7P 2R4, Canada

Received1 December1995;accepted13May 1996

Abstract

This review aims both to identify the potential risks to animal populations as a consequenceof exposure to genotoxins and to identify the techniquesmost useful in assessing these risks. Theseevaluations are complicated by the fact that contaminant exposure acts both to restructure naturally occurring genetic diversity and, when contaminants have mutagenic activity, to enhance the rate of introduction of new variation. There is now evidence that contaminant exposure often leads to change in the genetic attributes of natural populations. Short-lived organismsoften develop resistanceto contaminants, with only modest impacts on diversity in the balance of the genome,although massivemortality occurs during the gene replacement.Resistanceis, however, less likely to evolve in specieswith small population sizes, such as many wildlife species.Such specieswill experience population declines or extinction as the impact of contaminants on physiological systemsis not counteractedby genereplacements.Even when adaptation to exposureoccurs, populationsmay suffer diminished fitnessas a consequenceof the mutageniceffects of contaminants.The expressionof theseeffects range from an increase in the incidence of developmental abnormalities to shifts in chromosomal and gene structure. The assessment of this broad range of impacts can only be accomplishedwith a spectrum of analytical approaches. However, recent advancesin molecular and developmentalgeneticsare now making possiblethe detailed assessment of thesemutagenicimpactsin natural populations. Keywords:

Table

Nuclear genome;Mitochondrial genome;DNA; Genetic diversity; Mutagenesis;Contaminant exposure

of contents Page No.

Titlepage.................................................................. Abstract .._...............................................................

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Acknowledgements

* Corresponding 004%9697/96/$15.00

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author. 0 1996 Elsevier

PI2 SOO48-9697(96)05169-S

Science

B.V.

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reserved

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24 1.

2.

P.D.N. Introduction The nature

2.1. 2.2. 2.3. 2.4. 2.5. 3.

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............................................................. of the genome .....................................................

Genetic diversity in the mitochondrial genome ..................................... Genetic diversity in the nuclear genome ......................................... The modulators of genetic diversity - demography, selection and mutation. ................... Genetic impoverishment . causes and consequences ................................. The modulation of mutation: mutagens and DNA repair ..............................

Interactions

between

contaminant

exposure

and natural

genetic

diversity

..........................

3.1. Genetic variation in susceptibility to contaminants .................................. 3.2. Contaminant effects on nuclear diversity ........................................ 3.3. Contaminant effects on mitochondrial DNA diversity ................................ 4.

Mutagenic

4.1. 4.2. 4.3. 4.4. 4.5. 4.6. 5.

Monitoring

5 .l. 5.2. 5.3. 5.4. 6.

7.

........................................................

DNA

damage

and repair.

..............................................

DNAadducts ........................................................ DNA strand breakage ................................................... Unscheduled DNA synthesis ............................................... Sister chromatid exchange .................................................

Monitoring

6.1. 6.2. 6.3. 6.4.

exposure

Endogenous mutagens ................................................... Heavy metals as mutagens ................................................ Organics as mutagens ................................................... Inducing change in the genome - the contaminant connection ........................... Fitness reductions arising from the mutagenic effects of contaminants ....................... Screening for DNA damage - the status quo .....................................

the cytogenetic

Micronuclei Genome size Ploidy shifts Chromosomal

Monitoring

effects of contaminant

exposure

.................................

......................................................... shifts ..................................................... ......................................................... aberrations .................................................

mutagenesis

of the mitochondrial

genome

.....................................

7.1. Sensitivity of the mitochondrial genome to mutagens ................................. 7.2. Effects of mitochondrial mutations on fitness ..................................... 7.3. Monitoring mutagenesis in the mitochondrial genome ................................ 8.

Monitoring

8.1, 8.2. 8.3. 8.4. 8.5. 9.

mutagenesis

Approaches Monitoring Monitoring Monitoring Monitoring

Evaluating

the genetic

9.1. Monitoring 9.2. Monitoring 10.

Conclusions

of the nuclear

for determining the nuclear mutagenesis nuclear mutagenesis nuclear mutagenesis nuclear mutagenesis

genome

.........................................

sensitivity of the nuclear genome to mutagens .................. with morphological traits .............................. with natural reporter genes ............................. with transgenics ................................... with native genes ...................................

effects of contaminants

on animal

populations

............................

contaminant impacts at a population level ................................ contaminant impacts on DNA structure and sequence .........................

.............................................................

Appendix A: Advancing the evaluation of the genetic impacts of contaminant exposure ................ References

.............................................................

1. Introduction Studies of the biological effects of contaminant exposure have, over the past century, explored a broad range of impacts ranging from effects on survivorship to the assessment of more subtle physiological and genetic impacts. However, regulatory programs have had a more directed ontogeny. Initial programs (Environment Canada, 1971; 1972) focused solely on the assessment of the impacts of contaminants on survivorship. The first

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24 25 25 26 27 27 28 29 29 30 32 32 33 33 33 33 34 35 35 36 36 37 37 38 38 39 40 40 41 41 41 42 43 43 43 44 4.5 46 47 47 48 48 48 50

acute lethality studies examined vertebrates, but later efforts surveyed effects on a broader spectrum of organisms. Assessments of acute toxicity were later supplemented with studies designed to assess the effects of chronic exposure on growth or reproduction. Aside from these tests which were generally conducted on effluent samples, attempts were made to develop in situ indicators of impact. The earliest studies in this regard focused on the analysis of shifts in species composition. However, the discovery in the 1970’s of the impact of DDT

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exposure on avian reproductive physiology stimulated much effort towards the development of biomarkers for environmental risk assessment (McCarthy and Shugart, 1990; Peakell, 1992). Since then, it has become accepted that a range of biomarkers are required to provide a comprehensive indication of the biological effects of contaminant exposure (Depledge, 1989). While the analysis of physiological impacts is now well advanced (Colborn and Clement, 1992), similar assessment of the genetic impacts of contaminant exposure has been delayed. There is now a growing recognition of the need to develop biomarkers which allow examination of the genetic impacts of contaminant exposure (Dieter, 1993; LeBlanc, 1994; Fox, 1995). This recognition is leading to the emergence of a new discipline, genetic ecotoxicology, which seeks to ascertain the extent of chemical- or radiation-induced changes in the genetic composition of natural populations (Anderson et al., 1994). These studies aim to protect species from population declines linked to genetic damage from contaminant exposure. Decisions concerning the steps required to provide such protection are, however, complicated by the fact that contaminant exposure can both deplete naturally occurring variation and enhance the rate of introduction of new diversity. This review deals separately with the two distinct impacts of contaminant exposure on genetic diversity. Sections 2 and 3 explore the effects of contaminant exposure as a selective agent which can lead to the reduction of genetic variation in natural populations. In contrast, Sections 4-8 examine the mutagenic effects of contaminants including their role in both the induction of cytogenetic change and shifts in nucleotide sequence. The main focus is on animal populations, but information on microbes and plants is also included when it provides important insights into issues under analysis.

2. The nature of the genome The genetic material of all metazoan animals has two distinct components: the nuclear genome which typically consists of several billion base pairs, and the mitochondrial genome which contains N 16 500

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base pairs. The nuclear genome is biparentally transmitted and usually present in one diploid copy per cell while the mitochondrial genome is usually both maternally inherited (since mitochondria from sperm ordinarily fail to enter the egg) and present in > 1000 copies per cell (Wallace, 1992). Until very recently, studies of genetic diversity focused almost exclusively on the nuclear genome. All cytogenetic techniques, since they involve the examination of large blocks of DNA, focus on the nuclear genome rather than the diminutive mitochondrial genome which can only be visualized with electron microscopy. Early studies, involving the indirect survey of DNA sequence diversity through mutational analysis, also focused on the nuclear genome, in part because most characters influencing the visible phenotype are controlled by nuclear genes. However, with the development of techniques permitting the direct examination of DNA sequences, studies of the mitochondrial genome have expanded rapidly, and in some areas, rival (in extent) work on the nuclear genome. 2.1. Genetic diversity

in the mitochondrial

genome

The 16 500 base pair (bp) mitochondrial genome typically codes for two ribosomal and 22 transfer RNAs as well as for 13 polypeptides which are all involved in oxidative phosphorylation (Wallace, 1994). Although there are a large number of copies of the mitochondrial genome in each cell, these genomes are ordinarily genetically identical or homoplasmic. However, cases are known where two or more mitochondrial genomes co-occur, and individuals carrying such mixed mitochondrial populations are said to be heteroplasmic (Moritz et al., 1987). This latter variation can reflect either newly arisen mutations or variation existing in the pool of mitochondria present in the egg which gave rise to the individual (Casane et al., 1994). In a few species, the latter diversity arises as a consequence of the biparental transmission of mitochondrial DNA (mtDNA) (Zouros et al., 1992). Surveys of natural populations have shown that variation in both the size and nucleotide composition of mitochondrial genomes is common in most animal species (Moritz et al., 1987). Restriction enzymes have now been widely used to ascertain the amount of variation in DNA sequences of the mitochondrial genome. Although these studies typ-

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ically involve examination of < 5% of the genome, they have shown that substantial sequence variation exists in many natural populations, with each variant termed a hapiotype. The entire array of haplotypes in a population are often divisible into a smaller number of genetically divergent lineages; each consists of a numerically dominant haplotype and its mutational derivatives. Sequence diversity owes its origin to the incorporation of novel nucleotides during replication of the mitochondrial genome. Sequence variation is greatest in the non-coding region containing the origin of replication of the mtDNA genome, but the 37 functional genes in the mitochondrial genome also show varying levels of sequence variation. This variation in the extent of diversity is likely linked to the proportion of sites in each gene that can be modified without impacting its functionality (Lynch and Jarrell, 1993). Analysis of patterns of sequence variation have shown that much of the diversity in mitochondrial genes coding for enzymes is selectively neutral, as it involves changes in the third nucleotide position of codons that do not affect the amino acid composition of the gene product. However, other variation does affect fitness. Deleterious mutations include those which lead to the partial or complete loss of function in an enzyme or to the disruption in mRNA translation as a result of shifts in sequence of one of the tRNA or rRNA genes. Other variation in nucleotide sequence may enhance fitness, at least under some circumstances. Studies of size variation among mitochondrial genomes has revealed the presence of both deletions and duplications (Moritz et al., 1987). Individuals with deletions are typically heteroplasmic since deletion events usually disrupt mitochondrial function. Increases in genome size due to duplications are more common, and are often found in a homoplasmic state, suggesting that they have no major effect on fitness. Increases in length of the mitochondrial molecule are typically due to the presence of tandem repeats located near the origin of heavy strand replication (Barrette et al., 1994). These duplications ordinarily lead to less than a IOOO-bp increase in genome size, but in some molluscs they result in up to a 15 OOO-bp increase (LaRoche et al., 1990). Deletions show a more

diffuse distribution throughout the genome than do duplications. The origin of deletions seems linked to the chance occurrence of short (5- 13 bp) stretches of DNA with identical sequences throughout the mitochondrial genome (Mita et al., 1990). This sequence homology sets the stage for recombinational events that can lead to the excision of a segment of the genome. Although such deletions often lead to the loss of mitochondrial function, mutants have an advantage because they replicate more rapidly than normal mitochondria because of their smaller genomes. However, for cell survival, mitochondria carrying a deletion must ordinarily co-occur with normal mitochondria; so, heteroplasmy is the rule. 2.2. Genetic diversity in the nuclear genome

The broad application of allozyme analysis since 1965 has provided a good overview of the extent of genetic diversity in natural populations. These studies have shown that allozyme variation is present at approximately one third of all gene loci, which can be assayed using this methodology and that individuals are heterozygous at - 7% of their gene loci (Nevo et al., 1984b). These average values conceal the presence of substantial divergence among species in their level of variation. There is substantial variation even among fairly closely related taxonomic groups. For example, amphibian species are on average heterozygous at twice as many loci as are species of fish (Ward et al., 1992). Trophic status also appears to effect genetic diversity. For example, large mammalian predators have substantially lower levels of genetic variation than other mammals (Merola, 1994). Since 1985 studies of genetic diversity in natural populations have been further extended by analyses of DNA sequence variation (Avise, 1994). These studies have not only confirmed the prevalence of variation at genes coding for structural and enzymatic proteins (Mitton, 1994; Karotam et al., 1995) but have also revealed extraordinarily high levels of variation in non-coding DNA (Yashi et al., 1994). For example, variation arising from the dispersion of small segments of repetitive DNA throughout the genome is so high that each individual has a distinct phenotype - giving rise to the term DNA fingerprinting (Jeffreys et al., 1985; 1990; Burke and Bruford, 1987).

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2.3. The modulators of genetic diversity graphy, selection and mutation

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Individuals of a single species, except those reproducing asexually, are rarely if ever genetically identical. The lack of identity reflects the prevalence of variation in DNA sequences, as well as diversity in chromosome structure or number. Species possess varying levels of genetic diversity that often seems linked to their breeding system or to demographic variation. For example, large-bodied species typically have both smaller population sizes and lower levels of genetic diversity than small-bodied species (Ward et al., 1992). Among species of similar size, levels of genetic diversity may vary as a consequence of demographic history. For example, the cheetah (O’Brien et al., 1983; 1987) the northern elephant seal (Bonnell and Selander, 1974; Hoezel et al., 1993) and the Florida panther (Roelke et al., 1993) have much lower levels of genetic diversity than most other large mammals, apparently linked to their recent passage through severe population bottlenecks (Menotti-Raymond and O’Brien, 1993). A well-developed theoretical framework exists which makes it possible to estimate the likely depletion of genetic variation in response to a collapse in population size (Wright, 1978). This work has established that direct census information on population size is typically insufficient to predict the loss in variation. Instead, the extent of depletion depends upon effective population size, which is determined by both gender ratios and variance in breeding success among individuals (Wright, 1938). It has also been shown that brief intervals of population collapse lead to a much less severe attrition of diversity, than those which are sustained (Nei et al., 1975; Templeton, 1980). Levels of genetic diversity can also be depleted as a result of selection. Species exposed to strong selection pressures either become extinct or adapt by increasing the frequency of genetic variants that enhance fitness. In some cases, adaptation can be accomplished by a single gene substitution as, for example, in some cases of insecticide resistance (Devonshire and Sawicki, 1979). In such cases, so long as recombination is substantial, the gene replacement does little to disturb variation in the balance of the genome. However, where the re-

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sponse to selection is more complex, involving shifts in allele frequencies at many loci, the level of genetic diversity in the entire genome can be diminished (Guttman and Dykhuizen, 1994). This loss of variation results from the process of ‘hitchhiking’ in which whole chromosome segments are driven to homozygosity through selection of genetic variants embedded in them. Populations affected by this process are much like those whose genetic diversity has been stripped by a population collapse. While selection and population bottlenecks act to reduce variation, the erosion of diversity is counterbalanced by a number of mechanisms. New variation in chromosome numbers can arise from centromeric fragmentation and fusion, or from the failure of chromosomes to separate at meiosis leading to aneuploids or polyploids. Novel gene arrangements can arise through deletions, duplications, inversions or translocations. Finally, shifts in DNA sequence are induced by an array of chemical and physical agents as well as by errors inherent in the process of DNA replication. Because of the degeneracy of the genetic code, some of this variation in DNA sequence has no effect on the gene product, resulting in variation which is likely to be neutral with respect to fitness. However, other variants, including the bulk of shifts in chromosoma1 structure and those where DNA sequence changes induce change in the gene product, have an effect on fitness. 2.4. Genetic impoverishment quences

-

causes and conse-

Studies have shown that levels of genetic diversity are impoverished in many endangered species relative to closely related taxa with higher abundance (Avise, 1994). In most, if not all of these cases, the lack of diversity is a consequence rather than the cause of the endangered status, reflecting the attrition of variation which accompanied population bottlenecks. Moreover, in cases such as the elephant seal, the limited genetic diversity did not restrain a recovery in population size once mortality factors were controlled (Hoezel et al., 1993). Despite the success of some invariant species, the need to maintain heterozygosity remains one of the key goals of conservation genetics (Soule and Wilcox, 1980; Soul&, 1987). The desirability of

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maintaining variation is supported by evidence suggesting that genetic impoverishment has two negative effects that may constrain population persistence. There is firstly good evidence to suggest a linkage between genetic impoverishment and susceptibility to pathogens and parasites. The strongest support for this relationship has come from studies showing that genotypically diverse sexual species are much less sensitive to pathogens than closely related but genetically invariant asexuals (Ladle, 1993). These results have been reinforced by other more anecdotal studies. For example, a viral pathogen that produced 60% mortality among genetically invariant cheetahs induced only 1% mortality in other felids with higher levels of genetic diversity (O’Brien and Evermann, 1988). In addition to the pathogen susceptibility which accompanies a loss of variation, the transition from genetic diversity to impoverishment leads to an intrinsic reduction in fitness. Termed inbreeding depression, this reduction in fitness is linked to decreases in growth rate, survivorship and fertility (Charlesworth and Charlesworth, 1987; Lacy et al., 1993). The most detailed understanding of inbreeding depression has come from selection experiments. These experiments aim to alter a specific character state by the breeding of a small number of individuals showing the highest expression of that trait. Such selection experiments have shown that inbreeding depression becomes more severe with the passage of generations and can finally lead to the non-viability of the selected line. While the importance of inbreeding depression has been confronted most directly in experimental studies, there is also evidence of its importance in nature. For example, males from inbred populations of lions have a much higher incidence of sperm defects than their outbred conspecifics (O’Brien and Evermann, 1988). Similarly, inbred pairs of the bird Parus major fledge fewer offspring than their outbred counterparts (Greenwood et al., 1978), and inbred mice and song sparrows have lower fitness than their outbred counterparts (Potts et al., 1994; Keller et al., 1994; Jimenez et al., 1994). The reduction in fitness arising through inbreeding is likely due, in part, to the increased frequency of individuals that are homozygous for deleterious recessives. In addition, the diminished overall heterozygosity of inbred lines

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may itself lead to a fitness reduction if, on average, heterozygotes are more fit than homozygotes. Although the relative contributions of these factors to inbreeding depression are unclear, it is known that the sensitivity of individual species to its effect varies by more than two orders of magnitude (Ralls et al., 1988; Hedrick and Miller, 1992). Such variation might reflect, in part, the past demographic history of a species as prior bouts of inbreeding may have largely cleansed the genome of deleterious recessives (Lande and Schemske, 1985). As a general rule, however, it appears that even those species, such as the cheetah, which have little genetic diversity appear to be more threatened by habitat loss than by their diminished levels of variation (Caughley, 1994; Merola, 1994; May, 1995). 2.5. The modulation of mutation: mutagens and DNA repair It has been recognized for > 70 years that physical and chemical agents can induce both changes in chromosome structure and gene mutation. Work on these effects in animal populations initially focused on Drosophila (Muller, 1927) and then on mice (Russell, 195 1). Despite these early scientific studies, the first comprehensive evaluation of mutagenic effects through governmental regulation began just 20 years ago with the in vitro analysis of revertant mutations at the histidine locus in Salmonella (Ames et al., 1973). These studies ofmutagenicity were soon supplemented by tests examining shifts in DNA structure as revealed through analysis of micronuclei and sister chromatid exchange (ASTM, 1994). Over the past decade, advances in DNA sequencing have made it possible to directly examine shifts in DNA structure as a consequence of mutagenic exposure under both laboratory and natural settings. The interpretation of these studies on shifts in DNA structure and sequence has been aided by the rapidgrowthofinformationconcerningDNArepair and replication. It is now known that DNA damage is repaired by three distinct systems in most organisms. The mismatch repair system, because of its ability to recognize newly synthesized strands of DNA, plays a key role in the correction of errors introduced during DNA replication (Modrich, 1994). By comparison, DNA excision repair takes aleadintheremovalofDNAadducts(Sancar, 1994),

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while transcription-coupled repair is most active for genes which are undergoing transcription (HanaWalt, 1994). Although the general details of DNA repair are understood, there is a need for much more information concerning the extent to which repair activity is modulated by DNA damage. Work on Escherichia coli has shown that DNA damage results in the induction of repair systems. The yet A regulon, which controls the expression of 20 genes collectively termed the SOS response (Walker, 1985), is best known, but other assemblages of genes important in the repair of damage are induced by other mutagens such as oxidative damage (Holbrook and Fornace, 1991). Although similar systems have been studied less deeply in eukaryotes, much evidence exists for the presence of similar inducible defences. The presence of inducible defences suggests that there should not be any simple linkage between exposure to a mutagenic agent and the incidence of mutations. There is, for example, good evidence that chronic exposure to a mutagen resulting in a specific total lifetime dosage produces a lower incidence of mutations than does a similar acute dose (Russell, 1989). While this pattern of impact is expected if inducible defences are important, their existence complicates the prediction of responses. The generality of conclusions is further constrained by divergence amongst organisms in the exact mechanism of DNA repair. There are, for example, important differences between the excision repair systems of prokaryotes and eukaryotes (Sancar, 1994). Although less dramatic, differences in repair systems also exist among organisms with more limited evolutionary divergence such as amongst different orders of mammals. This variation suggests, on one hand, that the results of mutagenicity studies on single model systems must be interpreted with caution. On the other hand, the increased mechanistic understanding of DNA repair also promises to provide guidance in the selection of systems most useful for gaining a broad understanding of the mutagenic effects of contaminant exposure.

3. Interactions between contaminant exposure and natural genetic diversity

The first studies examining contaminant

effects on

29

natural populations focused on cases involving the development of resistance to specific heavy metal or organic contaminants. Most of this early work focused on polygenic characters, but by 1975, allozyme analysis made possible two approaches to the direct assessment of contaminant impacts on variation at single gene loci. Laboratory studies investigated the extent of fitness variation among allelicvariants at allozyme loci in response to defined contaminant exposure, while field studies examined the impacts of contaminant exposure on genotypic diversity in natural populations. Work on allozyme markers is now being supplemented by studies examining contaminant effects on both nuclear and mitochondrial DNA sequence diversity. 3.1. Genetic variation in susceptibility to contaminun ts The most detailed investigations on the evolution of resistance to contaminant exposure have examined plant populations growing on mine spoil heaps containing high concentrations of several heavy metals (Macnair, 1987). This work has shown that, in less than a century, some plant species, especially perennials, evolved resistant forms (Antonovics et al., 1971; Shaw et al., 1987). However, as only 10% of the local flora typically colonizes polluted sites, it appears that many species are either unable to adapt or are competitively pre-empted from colonization. Genetic analysis has indicated that a variety of physiological and morphological shifts are involved in this development of resistance. As well, these studies have shown that heavy metal resistance is not generic. Instead, plants must develop independent mechanisms permitting survival when exposed to each metal (Antonovics et al., 1971). The development of resistance comes at a cost; metal tolerant plants are competitively inferior to non-resistant individuals in the absence of heavy metals (Pitelka, 1988). The introduction of synthetic herbicides and pesticides subsequently provided an opportunity to study the evolution of resistance to organic contaminants. Study of these systems has now established the frequent development of resistance in both plant (Macnair, 1987) and invertebrate (McKenzie and Batterham, 1994) populations over intervals of just a few years. In fact, > 500

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species of insects are now known to have developed resistance to one or more insecticides (Georghiout and Lagunes-Tejeda, 1991). In contrast to the situation with heavy metals, where resistance is ordinarily polygenic, only one or a few genes are typically involved with herbicide (Erickson et al., 1985; Pitelka, 1988) and insecticide (McKenzie and Batterham, 1994) resistance, although exceptions are known (Groeters, 1995; Tabashnik, 1995). In some cases the physiological basis of resistance is well understood. For example, broad resistance to insecticides in both the peach aphid (Devonshire and Sawicki, 1979; Field et al., 1988) and mosquitoes (Mouchbs et al., 1986; 1990) has been linked to duplications of esterase loci which produce an enzyme that degrades these compounds. Similarly, resistance to heavy metals in Drosophila is the result of duplications of the gene coding for metallothionein (Maroni et al., 1987). Although most work on the response of animal populations to contaminant exposure has focused on the evolution of pesticide resistance, a few studies have examined the response to other pollutants. These investigations have shown that populations from sites with elevated levels of metals and organics are often more tolerant of acute exposure to the contaminant than are populations from clean sites (Vinson et al., 1963; Baker et al., 1985). These descriptive studies fail, however, to discriminate between tolerance arising from physiological acclimation and from genetic change (Klerks and Weis, 1987; Weis and Weis, 1989). Both processes are clearly important. Animals exposed to heavy metals show a gradual increase in their tolerance as a result of their enhanced synthesis of metal-binding metallothioneins (Pascoe and Beattie, 1979; Dixon and Sprague, 1981a,b), while the tolerance of plants is often enhanced by the synthesis of phytochelatins (Grill et al., 1985). Other studies have, however, demonstrated the presence of genetic differences in resistance among conspecific populations. Populations of mosquitofish have, for example, been identified with genetically determined variation in tolerance to phenol (Angus, 1983) and DDT (Andreason, 1985) and populations of rodents have developed resistance to control agents such as endrin (Webb and Horsfall, 1967) and warfarin.

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Similarly, populations of benthic invertebrates including oligochaetes (Klerks and Levinton, 1987; 1989), crustaceans (Brown, 1976; 1978; Fraser et al., 1978) and annelids (Bryan, 1976) show genetic variation in tolerance to heavy metals. In the case of one cladoceran crustacean, a > lOOO-fold difference in tolerance to cadmium was detected among genotypes (Baird et al., 1989; 1990). Most studies on the evolution of resistance provide no sense of the incidence of resistant genotypes in the original population (but see Macnair, 1987). Was, for example, the frequency of resistant individuals closer to 10e2 or 10-6? The answer to this question is undoubtedly situation-dependent, since resistance is a quantitative trait. Even so, studies are needed to clarify the relative contributions of genetic adaption and physiological acclimation to resistance, especially in the cases of metal tolerance. 3.2. Contaminant

effects on nuclear diversity

Most work examining the effects of contaminant exposure on natural variation has involved the study of allozyme loci. This approach has largely been one of necessity as, until recently, this was the only single-locus variation which could be readily assayed in nature. Although some studies have examined the consequences of thermal (Nevo et al., 1977; Smith et al., 1983) or organophosphate (Mortimer and Hughes, 1991) exposure, most allozyme work has examined the impacts of heavy metals on allozyme diversity in aquatic organisms. These analyses were motivated, in part, by the relatively detailed information available on metal exposure in aquatic ecosystems. However, the focus on heavy metal contamination also has a mechanistic justification, as prior work has shown that the activity of many enzymes can be inhibited by metal ions (Webb, 1966). This inhibition apparently arises as a consequence of the participation of specific metallic cofactors in many enzyme-mediated reactions, which can be competitively inhibited by other metals (Lavie and Nevo, 1982; Nevo et al., 1983; Chagnon and Guttman, 1989a,b). The most intensive examination of the effects of contaminants on the genetic structure of natural populations has been carried out on benthic

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invertebrates from the Mediterranean. Several studies have now established the presence of variation in genetic composition among conspecific populations from sites with differing pollution regimes (Nevo et al., 1978; Battaglia et al., 1980; Gillespie and Guttman, 1989; Patarnello et al., 1991). Efforts to verify the involvement of selection in the causation of this variation have led to experimental studies of genotypic survival following contaminant exposure. Initial work indicated that genotypes at the phosphoglucomutase locus in a crustacean showed variable survivorship in the presence of mercury (Hg) (Nevo et al., 1981). Subsequent work also indicated that mercury selected against rare homozygotes at the phosphoglucose isomerase (Pgi) locus in each of five gastropod species (Lavie and Nevo, 1986a). Later studies showed that the patterning of genotypic frequencies in natural populations of these species corresponded to expectations (i.e. mercury-resistant genotypes peaking in frequency at a site with this contaminant (Nevo et al., 1984a,b; 1987)). Similar investigations into the effects of other heavy metals on allozyme variation showed that selection by cadmium favoured homozygotes at the Pgi locus in each of five Mediterranean gastropods (Lavie and Nevo, 1986a,b). In contrast, zinc and copper selected against the Pgi and phosphoglucomutase (Pgm) homozygotes, respectively, in one of these species (Lavie and Nevo, 1982). Later studies, exploring the joint effects of cadmium and mercury exposure, indicated that different genotypes were favoured by the mixture of contaminants than those expected from the study of single contaminants (Lavie and Nevo, 1986b,c; 1987; Ben-Shlomo and Nevo, 1988). The most recent work, which has involved the examination of multi-locus as opposed to single-locus variation, has suggested the pervasive effect of selection as evidenced by the differential survivorship of specific multi-locus genotypes (Lavie and Nevo, 1988; Nevo, 1991). A second major study has examined the effects of heavy metal pollution on genetic diversity in mosquitofish. Again, evidence has been obtained to suggest genotypic differences in sensitivity to pollution. Diamond et al. (1989) identified differences in survivorship among genotypes at three of

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eight allozyme loci following mercury exposure. A similar study examining the effects of arsenate exposure revealed fitness differences among genotypes at two of these eight loci (Newman et al., 1989), while Chagnon and Guttman (1989b) found survivorship differences at two of three loci following cadmium exposure. The study found the least tolerant genotypes tended to be rare homozygotes, a result similar to that obtained in earlier work on marine gastropods (Lavie and Nevo, 1986a). Particularly detailed work has now been directed towards efforts to understand the effects of mercury on variation at the glucosephosphate isomerase-2 locus, which shows a diallelic (@i-2’*, Gpi-21°0) polymorphism. An experimental study of individuals from one population of mosquitofish showed that Gpi-238 homozygotes had a lower survivorship following Hg exposure than the other two genotypes at this locus (Diamond et al., 1989). Efforts to provide a mechanistic explanation for this observation produced a surprising result; the Gpi-238 enzyme seems less sensitive to Hg inhibition than the Gpi-21°0 enzyme (Kramer et al., 1992; Kramer and Newman, 1994). The likelihood that variation at the Gpi-2 locus has any simple relationship to Hg resistance has also been diminished by two other observations. Firstly, the analysis of Hg tolerance among fish from two natural populations, including the one used in the original study, showed no fitness differences among genotypes at the Gpi-2 locus (Diamond et al., 1991). The same study also showed a more rapid time to death in fish from the population exposed to Hg contamination than in fish from the control site suggesting that adaptation to Hg has been minimal. A subsequent investigation (Lee et al., 1992) provided evidence that the interpretation of earlier studies is clouded by both the presence of familial differences in susceptibility to mercury and by the tendency of groups of related individuals to aggregate. Collectively, the results from mosquitofish and Mediterranean invertebrates suggest that genotypic frequencies at many allozyme loci are impacted by contaminant exposure. There is, however, no simple pattern of response. Each heavy metal induces specific survivorship variation among the array of genotypes at a locus and

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the impact of contaminant mixtures cannot be predicted from their individual effects. Indeed, there is evidence that the genotypic response to a specific contaminant can vary among populations of a single species (Patarnello et al., 1991; Diamond et al., 1991; Lee et al., 1992). The incidence of these fitness differences have undoubtedly been exaggerated by the reliance on experimental protocols that involve a large number of concordant tests without subsequent correction for table-wide significance (Hochberg, 1988). There is, however, a much more critical issue in relation to the interpretation of these differences. The usefulness of allozymes as indicators of contaminant exposure requires the identification of loci showing consistent responses to a specific contaminant (Diamond et al., 1991). Yet, past studies have not been designed to discriminate between direct fitness effects at the locus under study and indirect fitness effects resulting from selection at linked loci. The likelihood of detecting selection which arises as a consequence of such selection can be minimized by carrying out tests on individuals from a large number of different populations rather than just one or two. In the absence of this safeguard, it should be concluded that all fitness effects arise through linked variation. Therefore, single-locus information will be capricious and provide no direct information on selection. The study of such variation has, however, provided indirect evidence of the impact of selection induced by contaminant exposure. As all past studies have shown that populations exposed to contaminants retain high levels of allelic diversity, it is apparent that selection is not, however, strong enough to purge populations of their diversity. 3.3. Contaminant effects on mitochondrial DNA diversity Only a single study has so far examined the relationship between mtDNA diversity in natural populations and exposure to contaminants. Murdoch and Hebert (1994) showed that haplotype diversity in populations of brown bullheads was lower at contaminated sites in the Great Lakes than at neighbouring control sites. Populations at contaminated sites often showed fixation for sin-

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gle haplotypes, a result which would be expected if these populations had undergone bottlenecks which eroded much of their natural diversity. Because of its matriarchal, haploid mode of inheritance, the effective population size for mtDNA is only l/4 that of nuclear genes; thus, mtDNA is a more sensitive indicator of past population collapses. For the same reason, the recovery of mtDNA diversity as a result of immigration following a population collapse occurs more slowly than at nuclear loci.

4. Mutagenic

exposure

Populations are exposed to a range of physical (Drobetsky et al., 1995; Newcombe, 1971) and chemical agents (Singer and Kusmierek, 1982) which can induce shifts in chromosomal morphology or nucleotide sequence. Chemical agents can be partitioned into endogenous mutagens, created as a byproduct of normal metabolic pathways, and exogenous mutagens, including organic and metallic contaminants which interact with DNA. This distinction is blurred by the fact that the mutagenic activity of some exogenous compounds is greatly enhanced by biochemical pathways in the organism (Buhler and Williams, 1988). This metabolic activation process, which occurs as a result of enzyme-mediated reactions, results in the synthesis of more polar and hence more easily excreted compounds. Such compounds are typically more reactive with DNA than their antecedents. Although this metabolic activation is primarily catalyzed by the mixed function oxygenase system, the nature of the reaction products are situation-dependent. Hence, species exposed to a similar spectrum of contaminants may experience differing mutagenic exposures. While most earlier schemes were directed towards the simple binary categorization (mutagenic, non-mutagenic) of compounds, existing data suggest that this effort is misguided (Mendelsohn et al., 1992; Brusick, 1994). Instead of being separable into two groups, compounds show continuous variation from those with no mutagenic activity to those mutagens that induce a broad spectrum of effects.

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4.1. Endogenous mutagens The oxidative phosphorylation pathway is the most potent source of endogenous mutagens. This pathway provides a steady infusion of oxygen radicals including superoxide (Miquel, 1992). Cells are able to reduce the extent of damage through the action of superoxide dismutase which converts superoxide (O,.) to H,O, which is itself subsequently eliminated by catalase and peroxidase. However, prior to its enzymatic degradation, H,O, can react with 0,. to produce the highly reactive hydroxyl radical (.OH). The latter can induce a variety of changes in DNA structure including both OH adducts and ring-opening products. As well, two-electron oxidation reactions arising from cytochrome P-450 give rise to bulky adducts of base structures (Malins and Gunselman, 1994). 4.2. Heavy metals as mutagens A broad range of heavy metal ions (including chromium, copper, iron and nickel) are known to be mutagenic (Chen and Thilly, 1994; Reid et al., 1994). As most of these ions do not form covalent adducts with DNA, their mutagenic effect is likely exerted via the generation of oxygen-free radicals. Reactive oxygen species, such as the superoxide ion or hydrogen peroxide, arising through normal cellular processes interact with metal ions to form .OH radicals. There is now growing evidence that mutational spectra produced by exposure to metal ions are similar to those arising from exposure to oxygen-free radicals. For example, reactive oxygen species induce a high proportion of C -+ T transitions similar to those produced by metal ions such as Cut 2 and Ni+ 2 (Reid et al., 1994). However, salts of other metals including platinum and chromium appear to exert their mutagenic effects through the production of either DNAprotein crosslinks or interstrand crosslinks with DNA (Kohn, 1983). 4.3. Organics as mutagens A broad spectrum of organic compounds interact with DNA to enhance rates of mutation (Singer and Kusmierek, 1982). Some of these compounds are base analogues that are incorporated into DNA strands during repair or replica-

33

tion, while others alter the structure of existing nucleotides through, for example, the addition of an alkyl group. In both of these cases, the modifications result in base pair substitutions. Other organic molecules lead to the addition of single base pairs during DNA repair or replication, resulting in frameshift mutations that inevitably destroy gene functionality. The specificity of these effects means that studies of mutational spectra can in principle be employed to deduce the agent responsible for an increase in mutation rate. 4.4. Inducing change in the genome - the contaminant connection Exposure to contaminants can have two diametrically opposed effects on the nature and extent of genetic diversity present in natural populations. On one hand, contaminants act to strip the gene pool of diversity, as both a direct consequence of population collapses and as an indirect consequence of strong selection arising from contaminant exposure. On the other hand, those contaminants with mutagenic activity act to enhance genetic diversity by increasing mutation rates. There is, however, no simple balancing of effects. Variation stripped from the genome through exposure to contaminants is largely neutral or selectively favoured, while most newly introduced variation is deleterious. As a result, exposure to contaminants is likely to diminish fitness over time, but the extent of this fitness reduction depends upon the relative importance of xenobiotic compounds as agents of selection and mutation in comparison with natural environmental variables. Human activities have led to both the concentration of naturally occurring mutagenic agents, such as ionizing radiation and heavy metals, and to the synthesis of novel mutagenic agents. The discharge of these compounds into the environment results in the broad exposure of organisms. As with the physiological effects of contaminants, it is likely that some animal species have a far higher exposure to these substances than do human populations. Thus, they serve as ideal targets for studies which aim to quantify mutagenic effects. However, until recently, there was little opportunity to exploit this situation, because

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Measure Concentrationsof Exposure

Contaminants MetabolJtes DNA Adducts

G-i h4icronuclei .Q z Sister Chromatid Exchange i! Sa3 6 -2

Aberrations Aneuploidy Poly@oidy

In vitro / irk viva mutagenesis

.

Fig. 1. Quantifying the exposure and effects of mutagenic substances on animal populations.

methodologies were not available that could be used on natural populations. These constraints are now being eroded by both a growing repertoire of tests and a more detailed understanding of how contaminants exert their mutagenic effects. 4.5. Fitness reductions arising from the mutagenic effects of contaminants All animal

species are exposed to a range of

mutagens of both anthropogenic and natural origin. Fig. 1 summarizes the techniques available to analyze both the exposure and effects of these substances. The extent of exposure to mutagens undoubtedly varies among taxa. However, the nature of this variation may not be simple, While organic contaminants reach peak concentrations in the highest trophic levels, it is possible that the most

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potent mutagenic effects occur as a result, for example, of metallic ions that show little biomagnification. Determinations of exposure are further complicated by the fact that most quantification techniques require the analysis of a specific compound or its metabolites, requiring both the a priori recognition and priorization of mutagenic substances. In contrast to the chemical-specific nature of assessments on exposure, effect studies integrate the impacts of all mutagenic activity. Effect analysis is itself complicated by the fact that mutagenic exposure can lead to impacts ranging from gross shifts in nuclear morphology to single nucleotide alterations in a gene. This complexity has been addressed through the development of a number of test protocols; however, most of these tests focus on somatic tissues. This latter focus is in a sense paradoxical, as the effects of mutagenic change in somatic tissues are often delayed until late in life and result in little decline in fitness. By contrast, mutations in gametic tissues often lower the viability of gametes, embryos or neonates and lead to a direct reduction in fitness. Studies on human population have, for example, established that most miscarriages involve embryos with a cytogenetic abnormality. Clearly, increases in the incidence of such abnormalities above a certain level could jeopardize the survival of populations. Although studies on gametic tissue may be desirable, analysis has focused on somatic tissues for technical reasons. In practice, so long as the two types of tissue show strong covariation in their response to mutagenic exposure, this approach has no undesirable effect. 4.4. Screening for DNA damage - the status PO Programs are now in place in most countries to evaluate the mutagenic potential of new compounds. Japan couples the analysis of mutation rates in bacteria with the examination of chromosomal aberrations in mammalian tissue culture (Sofuni, 1993). Canada employs the same pair of tests, but it also includes in vivo cytogenetic analysis for chemicals whose structural properties or planned volumes of synthesis indicate a particular exposure risk (Clayson et al., 1993). The United

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States employs three tests: the standard bacterial assay and in vivo tests for both mutagenic and cytogenetic effects (Auletta et al., 1993). The discovery of mutagenic activity in one of these standard assays ordinarily elicits the use of supplementary techniques to gain a better sense of mutagenic risk. While these screening programs should minimize the introduction of new chemicals with potent mutagenic activity into the marketplace, they are insufficient to guarantee that risks to animals are minimal. Perhaps most importantly the tests are carried out on single compounds; complex mixtures and transformations in nature may provoke an unexpected mutagenic response. Past efforts to gauge these risks have been constrained by the fact that most standard tests of mutagenicity are difficult or impossible to employ in natural settings. Recent advances in the field of environmental mutagenesis are now making it apparent that this gap in understanding will soon be addressed. The most spectacular advances have resulted from the exploitation of new techniques in molecular genetics. These techniques have greatly simplified the acquisition of direct information on DNA sequence diversity providing a basis for the direct quantification of DNA damage. Assessments of the mutagenic impacts of contaminants are now examining both the mitochondrial and nuclear genomes.

5. Monitoring

DNA damage and repair

Exposure to contaminants can lead to both the modification of nucleotides and to the physical disruption of DNA strands. Four approaches have been developed to examine the extent of such damage to bulk DNA. Three of these methods involve the analysis of shifts in DNA structure or patterns of replication. The first of these techniques, the study of DNA adducts, provides an indication of the extent of base pair modification as a consequence of contaminant exposure, while the second approach determines the extent of breakage in DNA strands. The third approach, analysis of unscheduled DNA synthesis, provides an indirect measure of the extent of DNA damage

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and repair, as indicated by the incorporation of radiolabelled nucleotides during those phases of the cell-cycle when DNA is not undergoing replication. In contrast to these methods which involve studies at the DNA level, studies of sister chromatid exchange rely on the visual inspection of metaphase chromosomes to examine variation in rates of strand breakage and reunion. 5.1. DNA

adducts

The study of DNA adducts is motivated by the fact that many environmental contaminants are thought to exert their genotoxic effects through covalent binding with DNA. The analytical methods employed in these studies aim to quantify shifts in the pattern and abundance of DNA adducts. The most widely used technique, 32Ppostlabelling, has the advantage of both high sensitivity (1 adduct/lO’ nucleotides) and a partial ability to discriminate among adducts (Randerath et al., 1981; Gupta et al., 1982). The use of this technique has shown that experimental exposure to contaminants, such as benzo[a]pyrene, induces the formation of specific adducts. Moreover, these studies have established that DNA adducts produced by a single exposure often persist for several months (Stein et al., 1993; Holbrook et al., 1992). The first efforts to use 32P-postlabelling to survey adduct formation in fish populations produced evidence of an association between contaminant exposure and the abundance of adducts (Dunn et al., 1987; Varanasi et al., 1986; 1989). However, more recent investigations have revealed an important complication; DNA adducts frequently occur at substantial concentrations in populations unexposed to contaminants. Data interpretation is complicated by the fact that the concentrations of these endogenous adducts often show substantial seasonal variation peaking typically prior to reproduction (Garg et al., 1992). Kurelec et al. (1989) found that endogenous adducts were sufficiently common to obscure any impact from contaminant exposure in several Yugoslavian fish species. Studies on other fish species have, however, revealed linkages between contaminant exposure and adduct concentrations (Collier et al., 1993; McCain et al., 1992). Endo-

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genous adducts appear particularly common in mammals (Stein et al., 1994), and because of their presence, it has been impossible to demonstrate any association between contaminant exposure and adducts (Ray et al., 1991; Holbrook et al., 1992). The chemical characterization of DNA using combined gas chromatography-mass spectrometry and infrared spectroscopy has recently provided another approach for the analysis of DNA damage. This approach allows recognition of OH adducts as well as damage leading to ring openings, as opposed to the bulky adducts identified through 32P-postlabelling. The two studies using this technique demonstrated a clear association between DNA damage and contaminant exposure (Malins and Haimanot, 1991; Malins and Gunselman, 1994) in marine fishes. Individuals exposed to contaminants had up to 1% of their nucleotides modified; this ratio is 2000 times higher than that for bulky nucleotides (Maccubbin, 1994). The application of this approach to the study of mammal populations where 32P-postlabelling has failed to show an effect of contaminant exposure seems particularly worthwhile. Aside from the diverse causation of DNA adducts, their impact on DNA replication and function remains uncertain. It is, for example, unknown if adducts are differentially excised from the coding and non-coding regions of the genome. As most of the DNA in higher organisms does not appear to have a coding function (CavalierSmith, 1985), it is possible that adducts are restricted to or at least more abundant in the non-coding segments of the genome, where their phenotypic impact would be negligible. 5.2. DNA

strand breakage

The DNA molecules in each cell must undergo continuous maintenance to sustain their integrity. Several of the key mechanisms in this repair process involve the degradation of a short stretch of DNA leading to a transitory break in one DNA strand. The incidence of such strand breaks may be enhanced both as a direct result of contaminant exposure or as an indirect effect of the excision of adducts induced by the contaminants (Shugart and Theodorakis, 1994). Two ap-

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proaches are in common use to quantify the incidence of strand breakage: the alkaline unwinding assay (Shugart, 1988) and agarose gel electrophoresis (Theodorakis et al., 1994). Both approaches exploit the fact that as strand breaks increase in frequency, the DNA strand comprising a single chromosome is, in effect, sheared into ever smaller fragments. Hence, by quantifying the size of DNA fragments, the incidence of strand breakage can be inferred. The alkaline unwinding assay is based on the fact that, at high pH, denaturation of the double helix is initiated at single strand breaks. By ascertaining the rate of strand degradation, it is possible to infer the number of strand breaks. The second approach uses gel electrophoresis to ascertain variation in the average size of DNA fragments which can be used to infer the incidence of strand breakage (Theodorakis et al., 1994). Moreover, by running both normal and denaturing gels, it is possible to compare the incidence of single- and doublestrand breakages. Use of these assay techniques on fish (Di Giulio et al., 1993), turtles (MeyersSchone et al., 1991) and invertebrates (Nacci and Jackim, 1989) have shown that strand breaks are generally elevated in organisms exposed to both organic contaminants and radionucleotides. 5.3. UnscheduledDNA synthesis

Studies of unscheduled DNA synthesis can only effectively be carried out on tissue cultures, and most prior work has focused on cell lines of mammals and especially those of rat hepatocytes (Swierenga et al., 1991a). There has, however, been an effort to extend the number of study systems and success has recently been reported in developing a fish assay (Ali et al., 1993). Regardless of the cell line, studies of unscheduled DNA synthesis monitor variation in the incorporation of radiolabelled nucleotides through autoradiography or scintillation counting. The latter technique, although more rapid, has the disadvantage of measuring the incorporation of nucleotides through both unscheduled DNA synthesis and normal DNA replication. This problem is particularly serious in cell lines with a high mitotic index; the fixation of nucleotides through unscheduled DNA synthesis can be swamped by regular DNA

37

synthesis. There have recently been efforts to develop hybrid in viva/in vitro systems which allow test animals to be exposed to a specific contaminant after which cells are isolated from a specific organ and rates of DNA repair are measured in vitro (Madle et al., 1994). As a general analytical approach, unscheduled DNA synthesis suffers from the fact that it measures repair rather than damage and hence reveals more about an organism’s response to DNA damage than about the extent of damage induced by exposure. 5.4. Sister chromatid exchange

Studies of sister chromatid exchange rely upon the differential labelling of chromatids to permit the recognition of exchanges of DNA between them. In practice, labelling is accomplished through the brief exposure of cell lines or organisms to 5-bromodeoxyuridine, an analogue of thymidine, which is incorporated during DNA replication. Because this compound quenches the fluorescence of some DNA-specific stains, its differential presence can be used to both distinguish sister chromatids and track exchange events. It is now believed that sister chromatid exchanges arise as a consequence of breaks in DNA strands near replication forks followed by a fusion of sister strands rather than the original strands. Contaminants which increase the rate of sister chromatid exchange may do so by blocking movement of the replication fork (Tucker et al., 1993). The interpretation of results is complicated by the fact that 5bromodeoxyuridine itself increases the rate of sister chromatid exchange. Hence, efforts to examine the effect of a specific contaminant rely upon the demonstration of its role in increasing the rate of exchange above control levels (Tucker et al., 1993; Pesch, 1990). Because of the joint need for exposure to 5-bromodeoxyuridine and subsequent isolation of metaphase chromosomes, most work on sister chromatid exchange has focussed on mammalian cell lines (Tucker et al., 1993). Recent work has, however, attempted to monitor variation in sister chromatid exchange under natural conditions, using marine polychaetes (Anderson et al., 1990; Pesch, 1990), fish (Hooftman and Vink, 1981; Vigfusson et al., 1983) and birds (Ellenton and

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McPherson, 1983a,b). As a general approach, measurements of sister chromatid exchange suffer from the disadvantage of lacking any clear linkage to fitness. In fact, since sister chromatids are genetically identical, the reciprocal exchange of chromosomal segments can have no direct effect on fitness. There is an obvious need to establish that a surrogate relationship exists between the frequency of sister chromatid exchanges and other genetic damage. While such a relationship has been established for in vitro work on mammalian cell cultures (Carrano et al., 1978; 1982), there is a need to generalize these results.

6. Monitoring the cytogenetic effects of contaminant exposure The extent of cytogenetic damage induced by contaminant exposure can be quantified most simply through the analysis of shifts in the incidence of micronuclei or through the study of shifts in genome size distribution (Bickham, 1994). Alternatively, detailed cytogenetic studies can document variation in chromosome number or structure (Bickham, 1994). Studies of micronuclei and genome size variation can be carried out on any organism, although analysis is simplified when the genome size of the species under study is moderately large. In principle, this means that all vertebrates, as well as many invertebrates, are potential targets for analysis. In contrast, the study of ploidy shifts demands that chromosomes be large enough for enumeration and that cells be captured at the appropriate stage (metaphase) of the cell cycle. The cytogenetic requirements for work on structural variation (aberrations) are even more stringent, as chromosomes must be large enough to permit their individual morphological characterization (Alink, 1982; Pesch, 1990). As a result of these restrictions, studies of the latter type are restricted to species which have been both the target of detailed karyological analysis and possess a modest number of large chromosomes. Most past studies of the cytogenetic effects of contaminant exposure have involved in vitro studies on mammals. There has, however, been a

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growing effort to expand the range of organisms studied using these techniques. There is also a realization that in vivo studies are critical to overcome the uncertainty concerning risks to natural populations. Despite progress in the development of new protocols, there is evidence that both intra- and interspecific variation in susceptibility to cytogenetic damage is often large enough to complicate the interpretation of results (Leonard et al., 1982; Natarajan et al., 1982). 6.1. Micronuclei

As their name suggests, micronuclei are masses of DNA (resembling small nuclei) found in the cytoplasm, rather than being contained within the nuclear membrane (Jaylet and Zoll, 1990; Di Georgio et al., 1994). Micronuclei form when acentric chromosome fragments are unable to attach to a spindle fiber during cell division or when an intact chromosome is excluded from the nucleus because of flawed cell division. Hence, micronuclei may be a consequence of either chromosomal breakage or dysfunction of the spindle mechanism (ASTM, 1994). These two types of micronuclei can be distinguished with centromeric probes (Natarajan, 1994), and there is evidence that genotoxins can be differentiated by whether they induce chromosomal breakage or loss (Chen et al., 1994a,b). Although mammals have been the target of most work, a growing number of studies have investigated the incidence of micronuclei in aquatic organisms including fish (Jaylet and Zoll, 1990; Williams and Metcalfe, 1992; Al-Sabti, 1992), amphibians (Jaylet and Zoll, 1990; Fernandez et al., 1993), bivalves (Wrisberg et al., 1992) and echinoderms (Hose and Puffer, 1983). Collectively, these studies have shown that the frequency of micronuclei varies both among natural populations (Wrisberg et al., 1992) and seasonally (Fernandez et al., 1993). These latter effects are often striking; Hughes and Hebert (1991) report 5-fold seasonal shifts in the incidence of micronuclei in a marine fish. The incidence of micronuclei is typically ascertained in tissues with a high mitotic index (e.g. erythrocytes) but rates of cell division can also be stimulated in tissues that normally have low rates of division (Williams and Metcalfe,

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1992). The cells under study are stained with a DNA-specific dye, and the incidence of ‘accessory’ nuclei is determined in a sample of N 5000 cells. However, results are sensitive to variation in the rate of cell turnover, especially if exposure to contaminants is intermittent. Therefore, protocols have been developed to permit recognition of cells which have undergone division within a specified period of time. One of the most useful of these methods involves the administration of cytocholasin which blocks the final stage of cell division. Cells which have divided following its application are binucleate. By restricting analysis to this category of cells, the prevalence of micronuclei formation can be analyzed over a specific interval of time (Al-Sabti, 1994; Di Georgio et al., 1994). As more information on micronuclei formation in non-mammalian systems has been gathered, it has become apparent that a number of factors constrain the usefulness of this test. The most serious constraint relates to the difficulty in discriminating shifts in nuclear morphology due to contaminant exposure from those due to other factors (Carrasco et al., 1990). The micronucleus test was first used to examine anucleate mammalian erythrocytes for fragments of nuclear DNA; a system where the recognition of micronuclei was straightforward. In contrast, the blood cells of other organisms including amphibians, birds, fish, reptiles and invertebrates are nucleated. The presence of a nucleus complicates recognition of micronuclei because the nuclear membrane itself often shows irregularities that mimic micronuclei. Moreover, the incidence of such irregularities can be increased by viral disease and nutritional deficiencies (Carrasco et al., 1990). In addition, work on mammals suggests caution in the interpretation of variation since the incidence of micronuclei varies between genders and strains, and even when these factors are standardized, the sensitivity of the test is limited (Styles et al., 1983). Former studies of the relationship between contaminant exposure and the incidence of micronuclei reinforce the difficulties in application of this test. One of the most careful studies found no evidence of a linkage between contaminant expo-

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sure of fish populations and the frequency of micronuclei (Carrasco et al., 1990). Other studies reported up to a 3-fold increase in micronuclei between heavily contaminated and pristine sites (Hose et al., 1987; Metcalfe, 1988) but they failed to exercise sufficient control over the age and gender of organisms or to examine the possible role of disease and nutritional status in accounting for this variation. Collectively, these studies suggest the need for the careful design and interpretation of studies aiming to determine the effect of contaminant exposure on the incidence of micronuclei. 6.2. Genome size shifts

While studies of micronuclei monitor the incidence of nuclear fragmentation, the analysis of genome size variation permits integration of all agents which shift the amount of nuclear DNA. The determination of genome sizes is accomplished through the use of a DNA-specific stain, whose fluorescent or light-absorptive properties permit quantification of DNA content. While several microdensitometry systems can be used to quantify genome size, only flow cytometry makes possible determinations on very large numbers of cells and is sensitive enough to detect a 2-3X shift in genome size (Watson, 1987). Flow cytometry has shown that the nuclei of cells from a single individual usually show little variation, except for those cells undergoing replication or those with elevated DNA content due to endopolyploidy. Nucleated blood cells are an ideal target for analysis, as they lack endopolyploidy and are easily collected in large quantities. Genome size variation in these cells typically approximates a normal distribution, with diversity among nuclei reflecting variation in dye acquisition and instrument error. Efforts to examine the effects of environmental contaminants on genome size involve comparisons of the extent and nature of variation in control and impacted populations. Evidence of impact can be registered through increased variance in genome sizes (Otto et al., 1981) or through deviations from normality that reflect mosaicism in nuclear size due to the loss or duplication of a chromosome (Bickham et al., 1988).

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Studies on populations of the rodent Peromyscus have shown increased variance in genome sizes in areas heavily impacted with organic and metallic contaminants (McBee and Bickham, 1988). A similar increase in genome size variation was observed in studies on turtle populations exposed to radionuclides (Bickham et al., 1988; Lamb et al., 1991). The latter studies also provided evidence of mosaicism in nuclear size, but there was no significant increase in the frequency of mosaics at contaminated sites. The latter case emphasizes that genome size variation is a natural occurrence and indicates the need to carry out studies that provide a sufficient basis to demonstrate the impact of contaminants as opposed to natural variation in genome size. 6.3. Ploidy shifts

When chromosomes are sufficiently large to permit their direct enumeration, it is possible to ascertain more directly the nature of the ploidy changes responsible for shifts in genome size. In the case of aneuploidy, these shifts involve the loss or gain of single chromosomes; while in polyploidy, an entire haploid chromosome set is involved. As polyploidy is extremely rare in animals, excepting asexuals, most work has focused on the process of aneuploidy. Aneuploid cells arise from the non-disjunction of homologous chromosomes during cell division, but little is known about the cause of this aberrant behaviour. Aneuploid individuals typically have low fitness. For example, in humans, aneuploidy for sex chromosomes leads to Klinefelter’s or Turner’s syndrome, while Down’s syndrome is a well known example of somatic aneuploidy. Many other somatic aneuploids appear to be lethal resulting in spontaneous abortions or early infant deaths (Sheu et al., 1990; Mailhes and Marchetti, 1994). Both physical and chemical agents have been shown to induce nondisjunction events in a dose-dependent fashion, producing an increased incidence of aneuploidy. For example, the prevalence of somatic aneuploids is increased in humans exposed to ionizing radiation (Ohtaki et al., 1994). Heavy metals and hydrocarbons have similarly been shown to greatly increase the frequency of aneuploid cells in freshwater snails (Barsiene, 1994).

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As the incidence of aneuploidy is determined by chromosomal counts on metaphase cells, its study depends on the reliable enumeration of chromosomes. The identification of missing or duplicated chromosomes has, in the past, often been constrained by the morphological similarity of nonhomologous chromosomes. However, the use of differential staining techniques such as G/C banding and fluorescent chromosome-specific probes (‘chromosome painting’) have now largely overcome this difficulty (Fenech and Ford, 1993; Eastmond et al., 1994). Previous work has shown that the incidence of aneuploidy in human populations varies with age and gender (Ohtaki et al., 1994). Also, differences in the incidence of aneuploids in response to a specific chemical challenge have been detected between males and females (Mailes and Marchetti, 1994). This variability, coupled with the effects of exposure interval and dosing methodology, act to contribute to the complexity of data interpretation (Mailes and Marchetti, 1994). 6.4. Chromosomal

aberrations

The study of chromosomal aberrations involves the examination of individual chromosomes for the deletion, duplication or rearrangement of its normal gene array (Swierenga et al., 1991b). Although most chemically-induced aberrations appear to form during DNA synthesis (Natarajan, 1994), those induced by ionizing radiation may arise at any time of the cell cycle. Many studies of chromosomal aberrations have involved in vitro analyses on cell lines (CHO, V79) from the Chinese hampster or from human lymphocytes (Swierenga et al., 1991b), but in vivo studies have been conducted on the same species (Anderson et al., 1990) as well as on fish (Kligerman et al., 1975; Prein et al., 1978; Hooftman, 1981; Hooftman and Vink, 1981). Studies are initiated by exposing organisms or cell lines to a contaminant followed by the addition of colchicine to capture dividing cells at metaphase. Chromosome preparations are then stained and the incidence of chromosome aberrations is quantified. Work of this sort has established that some chemical mixtures do produce an increase in the incidence of chromosome aberrations (Sofuni et al., 1985; Wilcox and

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Williamson, 1986). However, the incidence of aberrations is also affected by cell type (Wilcox and Williamson, 1986) and there is also evidence of individual to individual variation in sensitivity (Natarajan et al., 1982). Because of the rather stringent cytogenetic requirements, relatively few studies have examined the effects of contaminant exposure on the incidence of chromosomal aberrations in nature. However, McBee et al. (1987), in a careful study of two rodent species, showed higher levels of karyological damage in populations at a contaminated site than at nearby control sites. Their work also indicated a differential response for these two taxa, but it was unclear whether this reflected varying sensitivity or exposure.

7. Monitoring genome

mutagenesis of the mitochondrial

7.1. Sensitivity of the mitochondrial genome to mutagens Studies have suggested that the mitochondrial genome is far more exposed to mutagenic damage than is the nuclear genome. Work on polycyclic aromatic hydrocarbons has shown 50-600-fold higher binding levels of these contaminants with mitochondrial than nuclear DNA (Allen and Coombs, 1980; Backer and Weinstein, 1980; Niranjan et al., 1982). This difference is likely due to several factors. Firstly, the enzymes which metabolize these compounds are located in the mitochondrial membrane, bringing them into proximity with mitochondrial DNA (mtDNA). As well, the high lipid content of these membranes serves to ensure the accumulation of organic contaminants in the mitochondria. Finally, mtDNA lacks proteins such as histones which help to protect nuclear DNA from damage (Wallace, 1992). Aside from its higher exposure to organic contaminants, mtDNA is also in contact with oxygen-free radicals, such as superoxide, generated during oxidative phosphorylation (Richter et al., 1988). Also, since metallic contaminants exert their mutagenic effect through interaction with oxygen-free radicals, the mitochondrial genome is differen-

41

tially exposed to their effects. Comparative studies of DNA have confirmed the greater impact of oxidative damage on nucleotides in mtDNA rather than nuclear DNA, and they have shown that the extent of this damage increases with age (Mecocci et al., 1993; Shigenaga et al., 1994). Given this high level of exposure to mutagens, one would expect rates of mutation in the mitochondrial genome to be elevated above those in the nucleus, unless it was counterbalanced by the presence of more effective repair mechanisms. In fact, mitochondrial DNA appears to possess more limited repair capabilities than the nuclear genome. It does, for example, lack the ability to repair pyrimidine dimers (Clayton et al., 1974). In addition, the rapid turnover rates of mitochondria ensures that the mitochondrial genome undergoes more cycles of replication than the nuclear genome. 7.2. Effects of mitochondrial mutations on fitness As information on the prevalence of mitochondrial mutations has accumulated, it has been recognized that they play a much more prominent role in human disease than previously supposed (Howell, 1994). Mitochondrial mutations have now been shown to be responsible for a number of neuromuscular and neurological diseases (Lestienne, 1992; Wallace, 1992). These diseases occur as a result of both deletions and point mutations in the mitochondrial genome (Holt et al., 1988). Deletions ordinarily occur in a heteroplasmic state, as they result in dysfunctional mitochondria, whose persistence depends upon the survival of their host cell whose energy needs are met by normal mitochondria. The same pattern exists for point mutations that disrupt oxidative phosphorylation through the production of non-functional enzymes or the disruption of mRNA translation as a result of shifts in tRNA structure (Lauber et al., 1991). Deletions involved in the production of human disease involve the excision of up to 8 kb of the mitochondrial genome (Zhang et al., 1992; Linnane et al., 1992). Studies of individuals affected with mitochondrial disease have established that

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deleted molecules are most frequent in cells which cease division early in life, such as neural and muscle cells, but are rare in those sustaining rapi,d rates of division such as the stem cells for blood cell production. For example, Holt et al. (1988) found that deleted mitochondrial DNA molecules were common in muscle cells but absent from leukocytes of individuals with mitochondrial myopathies. These differences suggest that selection is able to cleanse actively dividing cells of defective mtDNA molecules. Although these mitochondrial diseases show the expected pattern of matrilineal inheritance, many pedigrees show expression in only one generation. This suggests the rapid rise in frequency of defective mitochondrial molecules. Several studies have shown that offspring from a single mother can show great variation in the ratios of defective/normal molecules suggesting bottlenecking during oogenesis (Koehler et al., 1991; Linnane et al., 1992). Mitochondrial diseases often also show variable age of onset and degree of expression due to stochastic shifts in the frequency of normal and mutant mtDNA molecules in various tissues of affected individuals. There is now clear evidence that the incidence of mitochondrial mutations increases with age, and that such mutations are likely involved in both the general decline in energy production and in late onset neurological diseases (Arnheim and Cortopassi, 1992; Ames et al., 1993). 7.3. Monitoring genome

mutagenesis

in the mitochondrial

Despite the circumstantial evidence to suggest that mtDNA should be prone to mutations, the first studies to examine this issue found little evidence of intraindividual diversity. For example, the analysis of sequence diversity among multiple copies of a specific mitochondrial gene (COIII) isolated from red blood cells of single humans showed virtually no diversity (Monnat and Loeb, 1985). On average, only one sequence change was detected per 50 000 base pairs, and this lack of intraindividual sequence variation, even when mitochondria were isolated from different tissues, was confirmed in later work (Monnat and Reay, 1986). More recent studies have confirmed that

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exposure to mutagenic agents induces a surprisingly low number of sequence changes in mtDNA isolated from tissue cultures (Mita et al., 1988). This sequence stability has not been explained; it is possible that damaged mitochondrial genomes are either unable to replicate or are degraded by DNAase. In contrast to this stability with respect to nucleotide substitutions, there is evidence for the prevalence of large deletions (up to 2000 bp) in the mitochondrial genome. These deletions invariably occur in a heteroplasmic condition with each one usually comprising < 0.1% of the mtDNA molecules in a cell (Corral-Debrinski et al., 1992). As the detection of these length variants relies on a polymerase chain reaction (PCR)-based assay that selectively favours the amplification of those mtDNA molecules with the largest deletions, there is not yet a good understanding of the number of different deletions present in an individual (Linnane et al., 1992; Arnheim and Cortopassi, 1992). Individual deletions vary in abundance among tissues. Frequencies are highest in tissues, such as the heart and brain, whose cells have ceased division. There is also a clear increase in the abundance of deletions as individuals age (Piko et al., 1988; Arnheim and Cortopassi, 1992; Corral-Debrinski et al., 1992; Gadaleta et al., 1992). As deletions are most common in tissues with the highest oxidative demand, they are thought to arise from oxidative damage produced jointly by radicals generated through both normal cell metabolism and by exposure to mutagenic agents (Ames et al., 1993). Given that mitochondrial deletions accumulate during the lifetime of an individual, one might expect the similar accumulation of deletions across generations. Yet there is much evidence that this is not the case because juveniles have a far lower incidence of deleted mitochondrial DNA than their maternal parents. The mechanism responsible for this cleansing of the genome is not clear - Linnane et al. (1992) have suggested that selection against deleted molecules is accomplished by the bottlenecking of mitochondria during oogenesis followed by the selective mortality of those eggs which inherit a high proportion of deleted mitochondrial molecules.

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mutagenesis of the nuclear genome

8.1. Approaches for determining the nuclear genome to mutagens

the sensitivity

of

Although the first useful test for quantifying mutations in the nuclear genome was developed > 65 years ago (Muller, 1927; Russell, 1951), analyses of mutagenicity have benefited greatly from recent advances facilitating the examination of DNA sequence diversity. Much recent work has been directed towards the study of mutation rates at gene loci where mutants can be recognized with a simple screening process (reporter genes). By coupling the rapid isolation of mutants and analysis of DNA sequence diversity among them, it is not only possible to quantify the incidence of mutants but also to ascertain the nature of sequence shifts involved in them. Studies of mutation rates at natural reporter genes invariably involve the analysis of a single somatic tissue, as work is typically carried out on either tissue cultures or on blood cells. These mutations are, in a sense, irrelevant since they have no effect on the fitness of the individual carrying them. However, work on reporter genes is motivated by the probability that mutation rates at these loci are a surrogate for systemic variation in mutation rates in both somatic and gametic tissues. Studies of mutation rates are now also being addressed through the use of organisms with transgenic reporter genes, such as lat. These transgenic organisms provide the advantage of permitting tissue-specific assessments of mutagenicity. Mutational spectra, the pattern of DNA alterations induced by exposure to a specific mutagen, vary substantially among mutagenic agents. However, at present, such spectra are typically ascertained by exposing a large number of cells to a mutagen and, subsequently, analyzing sequence shifts in a large number of mutants for a particular gene (up to 20 000). This screening process has now been greatly facilitated by the joint use of denaturing-gradient gel electrophoresis and high fidelity PCR (Chen and Thilly, 1994).

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43

nuclear mutagenesis with morpho-

The three most commonly used direct assays for mutagenicity (Ames test, Drosophila sex-linked recessive lethal, mouse specific-locus) each permit an assessment of the mutagenicity of compounds in a laboratory setting. The Ames test involves an in vitro assay of mutagenicity through the measurement of revertant mutations at the histidine locus in the bacterium Salmonella. Before exposing the bacteria, compounds are mixed with mammalian microsomal oxidases to produce substances that would be generated in vivo (Maron and Ames, 1983; Gee et al., 1994). The other two tests have the advantage of permitting an in vivo assessment of mutagenicity. The Drosophila test quantifies the incidence of mutations with a recessive lethal effect and exploits the fact that - 20% of the genome in this species is X-linked (Lee et al., 1983). The test involves exposing male Drosophila to a suspected mutagen and capturing, through genetic finesse, the X chromosomes which they transmit to their female progeny. Each captured chromosome is subsequently tested for the presence/absence of a recessive lethal. Recessive lethals generated through exposure to mutagenic agents can be recognized because male zygotes receiving a Xchromosome with a recessive lethal die prior to the adult stage. This test has the advantage of examining the joint frequency of occurrence of mutations at - 800 loci on the X-chromosome. The specific-locus test involves exposing a homozygous wild-type mouse to a suspected mutagen and then mating it to an individual homozygous for recessive alleles at each of seven loci affecting phenotype. In the absence of mutation, the offspring of this cross are all phenotypitally wild-type; any offspring derived from a gamete carrying a mutation shows expression of a mutant phenotype. A recent modification of this test examines the incidence of mutations at allozyme loci rather than at those controlling the external phenotype (Lewis et al., 1991). Aside from these two tests, which examine hereditary changes in germ-line DNA, tests are also available to assay mutagenic impacts in somatic tissue. The mouse spot test involves the examination of mice

44

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for coat colour mutations. The mutations arise early enough in embryogenesis to result in a patch of pelage with deviant coloration (Russell and Major, 1957). A similar test is available for Drosophila, but in this case, the incidence of shifts in eye colour is assessed. Although each of these tests are useful for examining the mutagenic effects of contaminants under laboratory conditions, none can be directly applied in nature. There have, however, been efforts to develop indirect applications of these tests to permit an assessment of the mutagenic exposure of animal species. The most common of these approaches involves the extraction and concentration of contaminants from animals and the subsequent assay of the mutagenic potency of these extracts through, for example, the Ames test (Ellenton et al., 1983b; Rodriguez-Ariza et al., 1992; Romero et al., 1992; Marvin et al., 1993). 8.3. Monitoring reporter genes

nuclear mutagenesis with natural

Reporter genes are loci which can be easily screened for mutational events. Work of this sort has, so far, been restricted to mammals. Specifically, most efforts have been directed towards the analysis of genes directing the synthesis of three enzymes involved in nucleotide salvage pathways: APRT (adenine phosphoribosyl transferase), TK (thymidine kinase) and HPRT (hypoxanthine guanine phosphoribosyl transferase). Although the APRT and TK genes are smaller than the HPRT gene (2.6 and 10 kb versus 33 kb), they are less easily analyzed because of their autosomal inheritance (Cariello and Skopek, 1993). As HPRT is X-linked, only a single functional copy exists per cell due to male hemizygosity and Xchromosome inactivation in females. Hence, a single mutational event in the HPRT gene can alter the phenotype of the cell carrying it. Although the enzyme is not critical for growth, HPRT mutants can be selected because of their resistance to the otherwise toxic effects of purine analogues such as 6-thioguanine (Cariello and Skopek, 1993). Assessments of mutation rates usually involve the collection of T lymphocytes from the blood. Clonal lines of T lymphocytes can be established in vitro and screened for mutations

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by adding 6-thioguanine to the culture medium. This analytical protocol has the great advantage of permitting the assessment of mutagenic impacts on the HPRT locus in natural situations (Albertini et al., 1985; Albertini, 1994). Studies on the APRT locus are, by comparison, restricted to the in vitro exposure of tissue cultures to compounds suspected of having mutagenic activity. Mutations are recognized in a similar fashion, but the study must be carried out on a tissue culture line which has the gene in hemizygous condition (Bradley and Letanovec, 1982). Similar studies on TK employ a mouse lymphoma cell culture which has only a single functional copy of the gene (Applegate et al., 1990). Rates of mutation in lymphocyte or tissue cultures that have been exposed to contaminants are compared with spontaneous mutation rates which normally average, for example, - 4 x 10 - 6 at the HPRT locus. Laboratory studies have shown clear dose-response relationships with up to 40-fold increases in the incidence of mutants above controls for a number of mutagenic agents at both the APRT and HPRT loci (Zimmer et al., 1991). Detailed studies of these mutations have shown that most involve single nucleotide alterations occurring at a large number of sites throughout the genes (Cariello and Skopek, 1993). It has been suggested that these hemizygous reporter genes may be poor indicators of certain classes of mutational events (such as large deletions or those that arise via recombinational events between homologues). This criticism seems valid as work on the TK locus has shown that N 50% of the mutations induced by some mutagens at this locus involve deletional events (Applegate et al., 1990). Although analyses at the HPRT locus may fail to detect all mutational events, they do make it possible to examine shifts in the incidence of mutations under natural conditions. Studies comparing individuals exposed to specific environmental contaminants and control subjects have revealed significant increases in the incidence of mutations at the HPRT locus in response to exposure. These differences are, however, far less than those observed when tissue cultures are challenged with high levels of mutagens (Zfold in nature versus 40-fold in the lab). Nonetheless,

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significant increases in HPRT mutation rates have now been detected in response to exposure to radon (Bridges et al., 1991), ethylene oxide (Tates et al., 1991) and polycyclic aromatic hydrocarbons (Perera et al., 1993). Recent studies have aimed to extend the number of gene loci which can be examined through in vivo studies by exploiting the use of allele-specific antibodies (Langlois et al., 1986; Tates et al., 1989). Antisera have, for example, been developed against three mutants in the globin beta chain (Marafante et al., 1991). As the spontaneous frequency of these globin variants is only 1 x 10 - *, a large number of cells must be scored to detect an elevation in the frequency of mutations above background. A similar approach has been used to examine the loss of activity in naturally occurring glycophorin A variants. These studies focus on analysis of individuals heterozygous for the two common alleles (M,N) at this locus: and use flow cytophotometry of dye-coupled antibodies to identify erythrocytes that have lost activity of either the M or the N allele (Langlois et al., 1986). Survivors of the atomic bomb at Hiroshima show a 6-fold higher incidence of mutations at glycophorin A than do control individuals (Langlois et al., 1987). However, a later study of individuals exposed to polycyclic aromatic hydrocarbons failed to detect a significant increase in mutation rates at this locus (Perera et al., 1993).

8.4. Monitoring nuclear mutagenesiswith transgenies

Transgenic organisms contain a segment of foreign DNA which has been stably integrated into their genome. In the case of transgenics designed for mutagenicity studies, the foreign DNA permits both the simple assessment of mutation rate and easy recovery of the target sequence. One line of rats and two of mice (Muta-Mouse, Big Blue) are now available for mutagenicity assays (Mirsalis et al., 1994). All three transgenics employ a portion of the lac operon from E. coli for the assessment of mutagenicity and employ a lambda (1) phage shuttle vector to aid in its recovery and subsequent evaluation (Gossen et al., 1989; Kohler et al., 1990).

45

Assays for mutagenicity are carried out by exposing transgenic organisms to suspected mutagens and then waiting long enough to permit DNA replication before sacrificing the test animal. Total DNA is then extracted from one or more of its tissues and the transgenic DNA is recovered by exposing it to 3, phage protein extracts. These proteins recognize and excise the integrated shuttle vector from the host DNA and subsequently encapsulate it to produce infectious viral particles. These viruses are then used to infect a strain of E. coli lacking a complete lac operon, which is subsequently plated on a medium containing B-galactoside that allows the growth of bacteria infected by the phage. A visual assay permits the discrimination of those bacteria which received a mutant lac gene from the phage from those with a normal lac gene. For example, with the Muta-Mouse assay the frequency of mutations is determined by simply dividing the number of mutant white plaques by the number of wild-type blue plaques (Malling and Burkhart, 1992). There is now good evidence of a positive relationship between dose and the incidence of mutations following exposure to an array of mutagens with up to 75fold increase above background (Myhr, 1991; Gossen et al., 1989). Malling and Burkhart (1992) have suggested that the test is much less sensitive than mouse specific-locus assays. These assays show up to a lOOO-fold increase in mutation rate following chemical exposure, but no studies have yet examined this issue directly (Provost et al., 1992). Following their recognition, individual mutants can be sequenced. Work of this type has shown that the sequence shifts in lac genes isolated from mice are very different from those which occur when E. coli itself is exposed to the same mutagen (Burkhart and Malling, 1993). These differences are likely due to the differing DNA repair systems of these organisms. While further study is needed to clarify their limitations, it is clear that transgenics represent a major advance for the in vivo assessment of mutagenicity. They possess a great advantage over natural reporter genes, as mutagenic impacts can be assessed in all tissues. Perhaps more importantly, they provide a more rapid and cost-effec-

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tive opportunity to obtain both quantitative information on mutation rates and information on the sequence shifts induced by a mutagen. On the negative side, since the synthesis and maintenance of transgenic strains leads to inbreeding, it is possible that their mutation response will differ from that of an ‘average’ individual of their parent species. There is, however, much opportunity to enhance the sophistication of transgenics. For example, their value in the assessment of risks to human health would be extended if transgenics contained not only reporter/recovery inserts but also the array of human genes involved in the metabolism of mutagenic substances and in the repair of DNA damage so that experimental animals metabolized xenobiotics more like humans (MacGregor, 1994). There is also the possibility of developing new transgenics which might be employed to monitor mutagenicity in natural settings. For example, transgenic fish might be used to monitor mutagenicity in aquatic environments. Such appraisals might involve either the deployment of caged individuals or the deliberate release of transgenics through stocking programs. 8.5. Monitoring genes

nuclear

mutagenesis

with native

The most direct method for ascertaining mutagenic effects involves comparison of the incidence of nucleotide changes in the nuclear genomes of control and exposed individuals. Somatic mutation rates might, for example, be determined by comparing the sequence similarity of a battery of genes isolated from a number of different tissues. In the absence of mutation, all sequences should be identical. Such assessments require both the isolation of target DNA sequences and a subsequent determination of their sequence similarity. Until recently, these analyses required the cloning of individual genes and their analysis via direct sequencing or restriction analysis. The few studies which have been attempted using this approach have shown that spontaneous shifts in DNA sequence occur at a frequency of less than one change in 100 000 base pairs per generation. Given this low frequency of change, the detection of even a lo-fold increase in mutation rates requires the analysis of - 200 000 bp of DNA in

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control and exposed individuals. Coupling this large amount of sequencing with the effort required to clone specific DNA fragments meant that this approach was not feasible. Over the past decade, several major technical advances have overcome the primary obstacles to the direct analysis of sequence diversity. The need for the time-consuming step of cloning DNA fragments has now been largely overcome by PCRbased amplification of DNA. This technique provides a simple rapid method for the isolation of short segments (500-2000 bp) of DNA needed for analysis. PCR primers are now available to isolate DNA from a number of genes. Simplifying the isolation of DNA through PCR was an important first step, but it was also necessary to develop a technique permitting the rapid discrimination of DNA strands carrying a mutation from their much more common wild-type counterparts. Several techniques are now in use which exploit the fact that the melting properties of DNA are so extremely sequence dependent, that single base pair substitutions can be recognized (Dianzani et al., 1993). Current methods couple gel electrophoresis with gradients of temperature (Wartell et al., 1990) or denaturant (Fischer and Lerman, 1983) to expose variation in the melting properties (and hence sequences) of DNA fragments N 500 base pairs in length. While early work with denaturing gradient gel electrophoresis suggested that only one half of the mutants could be detected, resolution has now improved to N 100% (Sheffield et al., 1989). These approaches allow sequencing efforts to be directed towards the small percentage of DNA strands which actually possess a mutational change. As such, these studies are making it possible to generate information on the mutational spectra induced by specific mutagens (Cariello and Thilly, 1986; Cariello et al., 1991). It is particularly exciting that, in contrast to many of the classical approaches to the study of mutagenicity, there are no restraints in applying these new methods to the study of natural populations. Aside from monitoring sequence shifts in coding genes, some work is now examining the generation of new alleles at mini- and microsatellite loci. These loci, which are interspersed through-

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out the genome of all eukaryotes, show up to a 1% mutation rate per generation (Jeffreys et al., 1988;, 1994). These changes typically involve a shift in the number of nucleotide repeats rather than their sequence, apparently reflecting slippage events during DNA replication. The study of these loci is attractive because of their high incidence of mutations, but their utility is limited by two factors. Firstly, their transmission genetics is complex. For example, the incidence of slippage mutations is strongly dependent on the number of repeats with the probability of mutation rising dramatically once a size threshold is exceeded. Secondly, and more importantly, the linkage between slippage mutations which create new microsatellite loci and nucleotide substitutions in the coding regions of the genome is uncertain.

9. Evaluating the genetic effects of contaminants on animal populations Contaminant exposure has complex effects on the genetic attributes of natural populations. On one hand, contaminant exposure acts to both disrupt epigenesis and erode the genetic diversity in natural populations. On the other hand, contaminant exposure can, through its mutagenic effects, elevate the rate of introduction of new cytogenetic and allelic variants. The analysis of the first effects requires study of both developmental end-points and of patterns of genetic diversity in natural populations. In contrast, the analysis of the second effects requires the assessment of genetic diversity in the somatic cells of single individuals or among familial members. In developing studies which aim to broadly investigate the genetic impacts of contaminant exposure on natural populations, two issues are paramount. Firstly, such studies are only worthwhile when populations can be assigned to different exposure regimes and when the exchange of individuals is low enough to ensure the genetic integrity of populations over a period of several generations. Secondly, it is apparent that no single analytical approach can provide an appraisal of the range of potential effects. The balance of this section examines those techniques most appropri-

41

ate for inclusion in efforts to survey the genetic impacts of contaminant exposure on natural populations. 9.1. Monitoring contaminant impacts at a population level The most subtle genetic effects of contaminant exposure appear to lie in their disruption of developmental pathways. It is, for example, increasingly recognized that developmental stability is reduced by a variety of stressors (Leary and Allendorff, 1989; Parsons, 1990). These impacts are reflected at a phenotypic level in two ways: through increasing deviations from perfect bilateral symmetry and by an increase in the incidence of deviant phenotypes (Graham et al., 1993a,b). While phenodeviants, such as crossbills in cormorants, have attracted the broadest public attention, the greatest opportunity for future work lies in the analysis of developmental asymmetry. The interpretation of such studies is complicated by the effect of other factors (such as inbreeding) on asymmetry but its technical ease suggests the value of including such work as one component of any comprehensive effort to gauge the impact of contaminants on natural populations. Contaminant exposure can also induce change in the genetic composition of natural populations. Where contaminant exposure induces mortality, genetic diversity can be reduced either as an indirect consequence of population bottlenecking and the subsequent stochastic loss of variation, or, where genetically-based resistance is present, through selection. Past work has shown that populations exposed to contaminants often show shifts in their genetic composition. However, work on allozyme loci suggests that these impacts are rarely, if ever, sufficient to deplete genetic diversity. Although shifts in gene frequency are common, the response at single loci often varies among sites. This suggests that these changes are an indirect consequence of selection rather than providing a single-locus monitor of the effect of contaminant exposure. Only in the case of insecticide resistance has it been possible to establish a strict causal relationship between contaminant exposure and gene replacements. Given the greater sensitivity of the mitochondrial genome to bottle-

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necks, future work should focus on the study of shifts in the patterning of variation in this segment of the genome. 9.2. Monitoring contaminant structure and sequence

impacts

on DNA

As described in the earlier section of this paper, a broad range of techniques have been developed to examine the effects of contaminants on DNA structure. Many of these techniques are, however, of very limited value in the assessment of impacts on natural populations. However, the simplest of the techniques, micronucleus analysis, is broadly applicable to natural populations. Moreover, in those few organisms where it has been possible to employ a comprehensive array of tests, it has been shown that the induction of micronuclei served as a good surrogate for other cytogenetic effects (Mendelsohn et al., 1992). There is, at present, no single well-established methodology which permits the appraisal of contaminant effects on DNA sequence stability. However, it is apparent that the direct analysis of sequence shifts in native genes is now technically feasible (Cotton, 1989). It seems likely that the first large-scale studies of this type will focus on mtDNA because of the apparent sensitivity of this segment of the genome to mutagens and because of the ease with which multiple copies of a specific gene can be isolated in a single individual or from a mother and her progeny. These direct sequencing studies will have the enormous advantage of being applicable to any species. Moreover, as more primers develop, it will also be possible to extend analyses to nuclear loci.

ten challenged because of their indirect linkage to population processes. Hence, regulatory programs must seek to include elements which meet both scientific and societal scrutiny. The ideal test is one which not only provides a basis for early intervention but also elicits high public concern. An array of techniques are now available to monitor the genetic effects of contaminant exposure. As an exception to general rules, those tests which aim to gauge impacts at a populational level, such as studies of genetic diversity or developmental asymmetry, seem an unlikely touchstone for public concern. By comparison, tests of a more reductionist&z nature, particularly those which ascertain the direct mutagenic effects of contaminant exposure, seem likely to generate more societal interest. The development of this concern stems from the broad public recognition of the potential role of mutagenesis in both the induction of tumours and the causation of birth defects. Given this strong public concern and recent methodological advances, it is apparent that priority should be given to appraisal of the mutagenic impacts of contaminant exposure on natural populations.

Acknowledgements The synthesis of this review was supported by the Canadian Wildlife Service and by the Natural Sciences and Engineering Research Council of Canada. We thank Dr. G. Fox for both stimulating this project and critiquing earlier versions of this paper. Drs. S. Anderson, J. Bickham, T. Crease, M. Depledge, S. Guttman and A. Hilliker provided helpful comments.

10. Conclusions Societal concerns in relation to contaminant exposure have traditionally focused on impacts of a graphic nature, such as massive mortality or sustained population declines of animals. While there is broad consensus that such impacts are unacceptable, efforts to develop more subtle indicators of exposure, which might allow the preemption of serious impacts, have met with public resistance. Paradoxically, pre-emptive tests are of-

Appendix A Advancing the evaluation of the genetic impacts of contaminant exposure General

There is a need to target a small number of species for intense genetic analysis. Where possible, these taxa should have been the prior subject

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of the Total

and

other

identify a small number of model species and carry out studies to ascertain the impact of disease, nutritional status, age and gender on the incidence of micronuclei.

on natural diversity

There is now good evidence that the selective effects of contaminant exposure are neither sufficiently harsh, or perhaps that impacted populations are sufficiently protected from gene exchange, to elicit a substantial reduction in nuclear diversity. The analysis of mitochondrial DNA diversity provides a superior option, because this segment of the genome is particularly sensitive to population bottlenecks and selective sweeps. As well, the recovery of diversity is much slower than for nuclear genes, especially when females are philopatric. B. DNA DNA

damage and repair

adducts

There is a need to develop methods which permit the discrimination of endogenous adducts from those arising through exposure to environmental contaminants. Methods are also required which permit the comparison of adduct abundance in coding and non-coding regions of the genome. DNA

strand breakage

The use of gel electrophoresis, coupled with sophisticated densitometry systems offers the greatest potential for determination of variation in strand lengths as a consequence of contaminant exposure. Sister chromatid

exchange

Studies are required to verify the linkage between the incidence of sister-chromatid exchanges and other genetic changes which more directly impact fitness. C. Cytogenetic

impacts

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49

Luiker

Genome size shifts

There is a need for much more detailed analysis of the causation of variance in genome sizes. The reliance of flow cytometry on absorbance of DNA-specific dyes means that any factor which results in differential condensation of DNA can impact apparent genome size. Ploidy shiftslchromosomal

aberrations

Efforts should be made to extend work from mammalian systems to invertebrate systems, such as orthopterans, which possess more easily studied karyotypes. D. Mutagenesis

(general)

Mutations are rare events whose detection relies on screening large amounts of DNA for sequence diversity. At present, analysis requires sequence comparisons for large numbers of relatively small DNA fragments. The study of mutation rates would be greatly advanced if it were possible to either examine diversity in a much larger number of molecules or in much larger DNA fragments. Hence, either of the following methodological advances would greatly facilitate the detection of diversity. 1. Methods now available, such as denaturing gradient gel electrophoresis or single stranded conformational polymorphism analysis, permit the recognition of a single base pair change in 500 bp of DNA. The ability to detect a single bp substitution in 10 kb (or better yet 100 kb) would enormously advance our ability to assess mutational impacts on natural populations. 2. Alternatively, there is a need to develop a technique which would permit the bulk screening of a large number (e.g. 106) DNA molecules for variation at a specific nucleotide site.

Micronuclei

Work on micronuclei could be advanced by exploiting image analysis systems to automate and quantify irregularities in nuclear morphology for a large number of cells. There is also a need to

Monitoring

mutation

rates in transgenics

The deployment of transgenic organisms into both control and impacted environments will provide a basis for assessing mutational impacts

50

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in nature. Concerns in relation to the release of transgenics might be dealt with through the use of caged organisms or their sterilization before release. Monitoring

mutation

rates in native genes

Even given current restrictions on the recognition of mutations, it is now feasible to screen DNA sequence diversity in lo7 base pairs. Because they are more easily isolated, mitochondrial genes are the preferred target for studies which aim to determine the effects of contaminants on mutation rates in nature. Work should aim to determine the extent of sequence diversity in multiple copies of a gene isolated from single individuals. By comparing variation in the incidence of mutations among individuals from control and impacted sites, it will be possible to gauge the mutational impact of contaminant exposure.

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