Mutagenesis, Vol. 14, No. 4, 375-383,
July 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Anaphase aberrations in the embryos of the marine tubeworm Pomatoceros lamarckii (Polychaeta: Serpulidae): a new in vivo test assay for detecting aneugens and clastogens in the marine environment
Southampton Oceanography Centre, Empress Dock, Southampton SO14 3ZH, 1 Plymouth Marine Laboratory, Citadel Hill, Plymouth PL1 2PB and 2 School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK
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Abstract |
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The marine environment receives a wide variety of chemical inputs, many of which have the potential to damage DNA or interfere with the process of cell division. Here we describe a new assay based on the early embryo and larval stages of a planktonic spawning, tube dwelling marine worm, Pomatoceros lamarckii, which for experimental purposes has the advantage of producing large numbers of ripe gametes throughout the majority of the year. One of the most promising end-points is the use of dividing cells to detect anaphase aberrations such as lagging chromosomes, tripolar anaphases, acentric fragments and chromosome bridges. Apart from the reference mutagens mitomycin C and cyclophosphamide and the well-documented spindle poison colchicine, we tested the fungicide carbendazim, a primary metabolite of the fungicide benomyl, and thiabendazole, a pesticide and antihelminthic drug; both of which are known to act as aneugens in other test systems. In addition we tested sodium hypochlorite, a widely used oxidizing agent and disinfectant, di-butylphthalate, a commercial plasticizer and suspected aneugen, and sodium chloride, a recognized non-genotoxin. Significant increases in the frequency of anaphase abnormalities occurred with most test compounds at relatively low concentrations, confirming the sensitivity of the new assay. Sodium chloride yielded a negative response except at the highest non-relevant concentrations, where some chromatid stickiness was observed. In addition, the developmental consequences of exposure to these compounds were assessed in 4–8 cell embryos and at 48 h once the embryos had metamorphosed into free swimming larvae. Mitotic inhibition and anaphase aberrations were found to be a more sensitive indicator of genotoxic exposure than larval development, although there was a suggestion of a possible mechanistic link between aneugenicity/clastogenicity and larval fitness. The new test assay provides a rapid and inexpensive method for screening chemicals and effluents destined for release into the marine environment for potential gamete effects.
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Introduction |
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The marine environment receives a wide variety of chemical inputs, many of which have the potential to damage DNA, the genetic material. Other agents, while not affecting DNA directly, have the ability to interfere with the mitotic spindle apparatus, thereby leading to the loss or gain of single chromosomes (aneuploidy) or entire sets of chromosomes (haplo/polyploidy). Aneuploidy is found at high frequency in spontaneous abortuses in man (see for example Hassold, 1986
) and is an important cause of birth defects (e.g. Downs syndrome), while in somatic cells it is a characteristic of certain types of cancer (Levan and Mitelman, 1977; Tsutsui et al., 1983). Evidence of aneuploidy and some types of structural chromosomal rearrangement, namely chromosome bridges and acentric fragments, can be seen during cell division when two sets of chromosomes begin to migrate towards the cell poles, at anaphase. Anaphase abnormalities are an important category of effects, which, unlike some other genetic end-points (e.g. sister chromatid exchange) can have serious detrimental consequences to the carrier cell or organism (Dixon, 1983). For this reason there have been a number of attempts to adapt this method for genotoxicity screening and field monitoring (see for example Kocan et al., 1982; Hose and Puffer, 1983; Landolt and Kocan, 1984; Liguori and Landolt, 1985; Longwell et al., 1992; Anderson et al., 1994).
Unlike some other types of chromosome assay (e.g. metaphase analysis), anaphase analysis is easily transferred between species without the need for detailed knowledge of karyotype. Furthermore, the method lends itself to environmental screening because it is cheap and relatively simple to perform. Previously, we presented evidence which showed that embryos of the mussel (Mytilus edulis) originating from a chemically polluted dock had significantly higher levels of aneuploidy compared with those from an uncontaminated site (Dixon, 1982). Marine invertebrate embryos are a popular choice of research material in genetics studies since they represent one of the few dependable sources of dividing cells. A serious drawback with the mussel, a popular sentinel species in marine pollution research for many years (Bayne, 1976), is the limited period of time each year when embryos are available for study. In an attempt to overcome this problem, we turned to the intertidal tubicolous polychaete Pomatoceros lamarckii, which shares a number of important chromosomal features in common with Mytilus (namely chromosome size, medium number of chromosomes and sensitivity to mutagens), while it lacks the high degree of gametogenic synchrony which typifies the other species. A closely related species, Pomatoceros triqueter, lives subtidally down to depths of >600 m and represents an equally useful organism for benthic environment studies (Dixon et al., 1998). A supply of ripe gametes can usually be obtained simply by removing the animals from their calcareous tubes using blunt forceps, which stimulates them to release their gametes outside the normal spawning season; in the UK, Pomatoceros spawns naturally between July and August, after which time there may be a temporary shortage of gametes, which may last for 1–2 months. In this respect, Pomatoceros is the complete opposite of Mytilus, whose gametes are only available for a short time interval in the spring. The two sexes are distinguishable by their abdomen colour; mature females are rose-pink or orange while males are greenish yellow or cream coloured (there is some evidence of protandrous hermaphroditism in Pomatoceros, but since there is no overlap between the two sexual phases this does not present a problem in the present context). In this paper we describe an adaptation of the anaphase assay applied to P.lamarckii. Based on the results of a series of acute laboratory exposure experiments, using a variety of known or suspected genotoxins, we demonstrate both the sensitivity and specificity of the test assay, which supports its applicability to marine genotoxicological testing.
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Materials and methods |
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Animal collection and husbandry
Aggregations of P.lamarckii growing on the undersides of small rocks were collected from the shallow sublittoral zone at Tinside, Plymouth Hoe, in southwest Devon, UK (a clean site). Animals were maintained at 15°C in a closed seawater circulation system of total volume 60 l, which was changed weekly. They were fed a daily ration of Marine Liquifry (Interpet, Dorking, UK) at a rate of 5 drops/10 l. Fresh, filtered (0.45 µm) Eddystone seawater (FSW) was used throughout this study.
Adult worms (10–15 mm body length, 20–50 mm tube length) were detubed using stout forceps and placed in clean watch glasses where they released gametes (see Figure 1). Usually two or three ripe females were sufficient for five experimental treatments. Only fresh gametes were used in the experiments. Only animals releasing large, even sized primary oocytes (unfertilized eggs) or active spermatozoa, without any evidence of large numbers of immature stages, were selected for the experiments. Great care was taken to avoid mixing sperm and oocytes prior to the experiment, but there was some evidence that fertilization may sometimes occur internally, in which case those batches of oocytes containing fertilized eggs or embryos were rejected.
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Experimental procedure
Primary oocytes at a concentration of ~1000/ml were exposed to the test compounds in 10 ml volumes of FSW at 15°C for 3 h prior to fertilization. The following compounds were investigated: mitomycin C (MMC) and cyclophosphamide (CP), direct and indirect acting mutagens, respectively; colchicine (Colch), a spindle poison and possible carcinogen; thiabendazole (TBZ), an antihelminthic drug and possible aneugen (Marrazini et al., 1994); carbendazim (CZ), an antimitotic fungicide and possible mutagen (see for example Gillet and Roubaud, 1983
); sodium hypochlorite (NaOCl), an oxidizing agent and disinfectant; dibutylphthalate (DBP), a plasticizer of the phthalate group (of which some, e.g. diethylhexylphthalate, have been shown to be aneugenic; Parry et al., 1984); and a non-genotoxic control, sodium chloride (NaCl) (see for example Scott, 1986). All the chemicals were obtained from the Sigma Chemical Co. Stock solutions were made up immediately before each experiment. Where necessary, the solubility of the test compounds was enhanced by ultrasonication. After the initial 3 h pre-fertilization treatment, the oocytes were fertilized by the addition of fresh, active sperm at a concentration of ~106/ml (as determined initially by haemocytometer counts). When <95% fertilization was recorded in the FSW control, the experiment was terminated.
Embryo development was monitored at intervals. A subsample of embryos was fixed in 3 changes of cold Carnoy's fixative (methanol:glacial acetic acid, 3:1) at 2 h 15 min post-fertilization. After staining with aceto-orcein stain (see Dixon, 1982), using a moderate heat source to control the staining reaction, 100 embryos were scored for fertilization and/or development rate, based on the presence of polar bodies/cleavage divisions. A further sample was taken later to coincide with the 4–8 cell transition (~20% 8 cell stage, 3–3.5 h after first adding sperm), which optimized the proportion of cells at anaphase. Using these later stage embryos, 2x100 anaphase cells were scored for the presence of aneugenic effects (lagging chromosomes/multipolar anaphases) and clastogenicity (bridges and fragments). An additional 100 embryos were scored for other cellular and chromosomal effects, including asynchronous cell divisions and haplo/polyploidy (Dixon, 1985).
After the anaphase samples were taken, the remaining embryos were divided into two portions. The first was washed several times in clean FSW to remove any extraneous test compound and then allowed to continue development for a total of 48 h, after which the larvae were fixed in 4% formalin for a larval bioassay. The other portion was allowed to continue development in the presence of the test compound without washing. Embryos, larvae and prepared slides were analysed and photographed under a Zeiss Photomicroscope III, using phase contrast and a green filter to enhance contrast.
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Results |
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Effects on fertilization rate
The percentage of fertilized embryos at 2 h 15 min is shown in Figure 2A and B
. MMC had no detectable effect on the percentage fertilized even at 1.2x10–4 M, the highest dose tested. Similarly, CP had no detectable effect on the percentage of oocytes fertilized until the highest (lethal) dose of 1x10–2 M was reached. The remaining test compounds produced varying degrees of reduced fertilization rate with increasing concentration. The high toxicity of NaOCl and DBP proved problematical, since there was a marked reduction in the yield of anaphases with these compounds. The non-genotoxic control (NaCl) caused a marked reduction in fertilization rate at a concentration of 0.1 M, equivalent to a salinity of 40.8 p.p.t. (normal sea water has a salinity of 33–35 p.p.t.); in its natural habitat Pomatoceros is more likely to encounter hypo- rather than hyper-saline conditions (see Lyster, 1965) since it is found occasionally at the mouths of estuaries and in rock pools where it can be subjected to low salinity conditions. It was apparent from these findings that fertilization success is not a consistent indicator of chemical insult. The comparative toxicities of the different test compounds are shown in Figure 2C. It can be clearly seen that in terms of effects on fertilization, NaOCl proved highly toxic, CZ, TBZ, DBP and Colch displayed intermediate toxicities and CP and NaCl were the least toxic compounds tested.
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When stained with aceto-orcein, a characteristic germinal vesicle (clear zone) was seen inside primary oocytes, which contained one or more nucleoli. Chromosomes were visible within the germinal vesicle as either late prophase I (diakinesis stage) or metaphase I meiotic. Individual female worms differed in their oocyte stage (diakinesis or metaphase I) at the time of release; note that this applies to artificial spawning conditions.
Anaphase aberrations
Examples of normal and abnormal anaphases are shown in Figure 3. The levels of anaphase aberrations for a number of different test compound concentrations are presented in Figure 4. The clastogenic effects we observed were mainly chromosome bridges and acentric fragments, but there was also some evidence of chromosome shattering at high dose levels of MMC and NaOCl.
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In order to analyse the data statistically, the mean frequencies of aneugenicity and clastogenicity were combined to give a total abnormality frequency for each treatment. After the data had been square root transformed to reduce the heterogeneity of variances, a one-way ANOVA with multiple comparison (Fisher's LSD test) was applied.
In all cases the frequency of abnormality for the negative controls was <4% (mainly lagging chromosomes). MMC induced the highest levels of anaphase aberrations, with the highest dose giving a mean of almost 80 abnormalities/100 embryos scored. In contrast to the other compounds, MMC produced a high proportion of acentric fragments, in addition to a large number of aneugenic effects. Colch produced a significant response above that of the negative control at the two highest doses with a definite bias towards aneugenicity, although the total frequency of aberrations was only a fraction of that seen for MMC. A significant dose–response effect was also seen for TBZ, with the bias being towards lagging chromosomes and away from clastogenicity, suggesting that its mode of action was mainly via spindle disruption. CZ, a metabolite of the fungicide benomyl, yielded a significant increase in aberrations only at the highest dose tested (1x10–6 M). In contrast, NaOCl failed to produce a significant increase in aberration frequency but had a marked effect on fertilization rate. The indirect acting mutagen CP produced a significant increase in aberration frequency at the two highest doses, of which approximately one third was attributable to clastogenicity. Exposure to DBP resulted in a significant increase in aberration frequency at concentrations at and above 1x10–7 M, with the highest dose (1x10–5 M) giving 30% aberrant cells of which the majority were attributable to aneugenicity. Finally, the NaCl results, while not being statistically significant, showed a trend suggestive of a dose-related increase. However, the effects recorded for NaCl were probably not typical anaphase aberrations, instead they could be attributed to chromosome stickiness caused by osmotic stress.
Asynchrony and haplo/polyploidy
Other types of abnormality, including asynchronous division and haplo/polyploidy, were observed following exposure to Colch, TBZ and CZ. Under normal conditions, cell division remains synchronized in Pomatoceros embryos up to and including the 8 cell stage. In asynchronous embryos, however, one or more cells were found to be prematurely out of phase with the rest. TBZ (Figure 5A) and CZ (Figure 5B) had the most dramatic effect, with up to 45% of embryos showing asynchronous division. Colch treatment resulted in only a small increase in asynchrony and haplo/polyploidy and then only at the highest dose (results not shown). TBZ induced a dramatic increase in polyploidy at the highest concentration (Figure 5A). All the polyploids observed during this study were tetraploid.
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Larval bioassay
Normal 48 h old free swimming, trochophore larvae can be identified by the presence of prototrochal cilia and a complete internal gut structure, as well as having a characteristic shape and regular size; abnormal larvae were typically smaller and irregular in shape (Figure 6A and B
). Results of the 48 h larval bioassay, with and without washing at the 4–8 cell transition, are presented in Figure 7A and B. With all compounds, the overall effect of increasing concentration was to elicit a marked reduction in normal development, both with and without washing (except CP non-rinsed). However, the removal of the toxicant at the 4–8 cell stage did have the effect of increasing the frequency of normal development in some cases, namely MMC, NaOCl, CZ and DBP. In contrast, Colch and TBZ showed little difference between the washed and non-washed treatments. The threshold concentrations of each compound that resulted in <90% normal larvae (with and without washing) can be seen in Figure 7C. In keeping with the anaphase assay findings, NaOCl was by far the most toxic of all the compounds tested. In contrast, CP was the least toxic. The effect of removing the toxicant at the 4–8 cell stage clearly demonstrated that a transient exposure to a toxicant could lead to abnormalities later on in development.
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A pseudo-divisional stage, which superficially resembled a 64 cell embryo/larva, was sometimes spawned together with normal oocytes, usually at a frequency <1% (Figure 6C
). It has already been mentioned that a small proportion of female worms released eggs that were already fertilized, which was attributed to sperm entering their tubes from trickle spawning male individuals in the same tank. However, the pseudo-divisional stage was found to lack cell nuclei and the only chromatin was in the form of 12 condensed metaphase I bivalents, indicating an abnormal oocyte (Pomatoceros has a diploid chromosome number of 2n = 24; see Dixon et al., 1998). These pseudo-embryos were at first thought to be a symptom of a parasite infestation, but recent investigations have shown that normal metaphase I oocytes have a tendency to form this type of aberrant body after they have remained unfertilized for several hours. In the present context, these effete oocytes, which appear to involve the unscheduled production of internal cell membranes, did not represent a serious complication and were omitted from the analysis. This effect has also been seen with high doses of certain chemicals, e.g. benzene and toluene (Dixon, unpublished results). |
Discussion |
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The tube worm genus Pomatoceros is well suited to genotoxicity studies because of its wide geographical and eurybathic distribution (inhabiting both shallow and deep water benthic environments), the production of ripe gametes throughout most of the year and its tolerance of laboratory conditions (a closely related species, Pomatoceros caeruleus, is found in the southern hemisphere). In the UK, two species of Pomatoceros are found: P.lamarckii, which occurs low down on rocky shores in the shallow sublittoral zone, and P.triqueter, which lives subtidally down to depths of
600 m. While the tubes are similar, with the characteristic dorsal keel which gives the group the common name of keel worms, the two species can be distinguished morphologically, once removed from their tube, by the shape of the operculum (Zibrowius, 1968). Recently, we have shown that these two species also have small but significant karyotypic differences (Dixon et al., 1998). Over the past two decades, the marine mussel Mytilus edulis has become a popular subject in ecotoxicological studies. However, due to its low rate of cell division and a highly synchronized gametogenic cycle, the mussel is not an ideal candidate for cytogenetic studies; although Parry and colleagues have had considerable success using the mussel as an efficient bioaccumulator of mutagens from low concentrations in seawater, both under field and laboratory conditions (for a review see Kadhim, 1990). One drawback with Pomatoceros is the problem of being unable to separate the worm tubes from their substratum; this means that a population needs to be found where the worms are growing on stones, small boulders or other easily transportable substrates. This problem may apply more to P.lamarckii than P.triqueter, which can be dredged at depth in large numbers attached to shell debris.
Given the generally low numbers of dividing cells in adult marine invertebrates, attention has turned towards their germ cells and embryos/larvae. Anaphase analysis has been the subject of a number of studies using the early reproductive stages of marine invertebrates and fishes (see for example Hose and Puffer, 1983; Kocan et al., 1985). In recent times there appears to have been a decline in its popularity partly due to the advent of new techniques (e.g. sister chromatid exchange and fluorescence in situ hybridization). Unlike some of the newer end-points, the consequences of aneuploidy to the carrier cell are unambiguous. Furthermore, the anaphase assay has the advantage that it can be easily transferred between species and cell types without any need for a detailed knowledge of the karyotype, specific probes, etc. Furthermore, anaphase analysis has the potential for application under field conditions. For example, recently Stiles et al. (1991) showed that embryos of the hard clam (Mercenaria mercenaria) from highly industrialized areas exhibited more irregularity in chromosome number and greater frequency of larval abnormality than animals from clean sites. The majority of human aneuploidies are lethal, i.e. individuals having incomplete karyotypes will die at an early stage in embryonic development (Hook, 1983), however, a small proportion of abnormal embryos survive to maturity with reduced viability (Mikkelson, 1977). Kocan et al. (1985) demonstrated, using flow cytometry, an unequal distribution of DNA in trout cell lines and embryos following mutagen exposure that was transferable between several generations of cells, suggesting that anaphase aberrations can lead to heritable genetic defects in aquatic species.
The findings of the Pomatoceros assay, in terms of mutagenicity/aneugenicity assessment, are summarized in Table I, together with the published results for a range of other aquatic and non-aquatic assays. There was a high degree of concordance between our findings and those obtained with these other in vitro and in vivo methods, both in terms of sensitivity and absolute determination of genotoxic potential. Of particular interest, given the shortage of available information, were the findings for CZ and DBP, which both showed clear evidence of aneugenicity in the Pomatoceros assay. CZ is a widely used, proprietary horticultural fungicide which has recently replaced benomyl, previously shown to be aneugenic, on the grounds that it is a less hazardous primary metabolite. Dibutylphthalate is a widely used plasticizer. Both these compounds have the potential to contaminate the coastal marine environment via waste disposal and surface water run-off.
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Sodium chloride was included in this study to determine whether anaphase aberrations could be induced by processes other than those associated with aneugen/mutagen exposure. Compared with mammalian cells, extremely high concentrations of NaCl were required to produce any effect, which was not surprising given the euryhaline (wide salt tolerant) nature of the test species (Lyster, 1965
; Oglesby, 1969). Those few anaphase abnormalities which were recorded following salt treatment were not of the same type as was observed for the other compounds. Instead, these consisted mainly of sticky or tangled chromosomes rather than the typical anaphase bridges and fragments and were clearly artefactual in origin and the result of hypersaline stress.
A useful feature of the Pomatoceros aneugenicity assay is the ability to monitor the effects of toxicant exposure on embryo and larval development. In most cases there was a positive correlation between the results of the anaphase assay and the frequency of abnormal 48 h old larvae (rinsed samples), which suggests a possible mechanistic link between aneugenicity/clastogenicity and larval fitness. However, mitotic inhibition and anaphase aberrations were found to be a more sensitive indicator of genotoxin exposure than larval development. This finding agrees with previous investigations (e.g. Hose et al., 1983) and is consistent with the general lack of specificity in the larval bioassay response.
Over the years, larval bioassays, particularly those applied to bivalve molluscs, have become recommended procedures for water quality testing (e.g. American Society for Testing and Materials, 1980). Apart from the limitations imposed by seasonality in reproduction, bivalve (and echinoderm)-based bioassays tend to be expensive both in terms of time and manpower. Furthermore, larval bioassays are vulnerable to bacterial contamination, which may necessitate the undesirable and routine use of antibiotics. Apart from its ease and low cost, an important advantage with the Pomatoceros assay is that it can be carried out in extremely small volumes (10 ml), which, in the case of embryos, yields a result in only a few hours, thereby enabling a greater number of treatments to be screened in a given time, without serious risk of microbial contamination.
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Acknowledgments |
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Part of this work was carried out during a UK-NERC studentship (GT04/98/275/MS) awarded to J.T.W.
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Notes |
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3 To whom correspondence should be addressed. Tel: +44 1703 596014; Fax: +44 1703 596247; Email: drd{at}soc.soton.ac.uk
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References |
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Received on November 10, 1998; accepted on March 5, 1999.
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