Potential Uses of Sea Urchin Embryos for Identifying Toxic Chemicals: Description of a Bioassay Incorporating Cytologic, Cytogenetic and Embryologic Endpoints Jo Ellen Hose

VANTUNA Research Group, Occidental College, 1600 Campus Road, Los Angeles, CA 90041, USA Key words: genotoxicity; teratogenicity; aquatic toxicology; hazard assessment; embryos; sea urchin.

A method for evaluating pollutant genotoxicity, embryotoxicity and teratogenicity using sea urchin embryos has been developed and was tested using benzo(u)pyrene (BP). Initial results suggested that the bioassay may be a sensitive indicator of pollutant toxicity and mutagenicity since several endpoints can be simultaneously assessed. The bioassay is rapid, inexpensive and appears applicable to a variety of toxicants and delivery methods. The test is based upon the standard 48 h sea urchin development assay and incorporates cytologic-cytogenetic analysis of embryos. Following toxic exposure of gametes, fertilization success is assessed. Embryos then develop for 48 h at which time survival and teratogenesis are evaluated. A subsample of embryos is stained and dissociated into monolayers and mitotic configurations are examined using light microscopy. Embryo mitotic rates are used as an indicator of overall embryonic health. Cytotoxic effects are concomitantly evaluated. Genotoxicity is measured using two methods: (1) anaphase aberration analysis, a technique which assesses abnormalities in the chromosome configurations (such as bridges and fragments) as the groups of chromosomes move to opposite poles and (2) micronucleus formation, a procedure examining the incidence of smaller, secondary nuclei composed of whole chromosomes or chromatid fragments. These two measurements preclude the need to examine individual chromosomes for deletions and exchanges, a laborious process in most aquatic organisms which possess numerous relatively small chromosomes. This genotoxicity-teratogenicity test appears promising for laboratory evaluations of individual substances or of complex chemical mixtures as well as for environmental monitoring of nearshore areas. The standard development assay has been used to screen pharmaceuticals and environmental contaminants and some recent investigations have included mitotic aberration analysis. Experiments in our laboratory suggest that the genotoxicity-teratogenicity test may be a feasible approach to field monitoring. Mutagen loads of spawning adult urchins could be assessed by conducting cytologic-cytogenetic analysis of resulting embryos although initial studies suggest that this method is less sensitive than direct embryo exposures.

INTRODUCTION In recent years aquatic toxicity tests have assumed increasing importance in assessing health effects of environmental pollutants. Toxicologists, particularly genetic toxicologists, are investigating the possibilities of using highly sensitive aquatic species as animal models for screening the biological activities of substances. In addition, aquatic toxicity tests which employ local species can yield direct results regarding the biological impact of a pollutant on an economicalIy o r ecologically important organism. Much of the current toxicology research is directed toward evaluating pollutants for genetic or reproductive toxicity. A number of routine mammalian mutagenicitytests have been attempted using aquatic species. Of these, sister chromatid exchange (SCE), which measures reciprocal exchange of segments between chromatids,’ is the most widely studied and has been successfully used with several species of marine and freshwater fishes?-4 as well as marine invertebrate^?-^ However, the biological significance of SCE induction is unclear’ and the method is extremely laborious for cold-water organisms which have low mitotic

rates or for species which possess large numbers of small c h r o m ~ s o m e sSimilar .~ restrictions exist for the examination of chromosomes for aberrations.”-” Other mutagenicity tests such as the production of ouabain-resistant mutants’* involve cultured cells and are limited to use with fishes since continuous cell lines have not yet been established for aquatic invertebrates. The dominant lethal test, a measure of all genetic alterations that produce death of o f f ~ p r i n g ’ and ~ the oyster or sea urchin development testsl4-I6 are the major embryotoxicity-teratogenicity assays used in aquatic toxicology. The objective of this report is to describe an inexpensive rapid bioassay procedure which can be used as a genotoxicity-teratogenicity screening method for individual substances or complex environmental mixtures. Although the technique is applicable to a large number of commercially or ecologically important aquatic species, the sea urchin was selected because of its prior use in pollutant bioassays, its widespread availability and the ease with which it can be artificially spawned and reared. The sea urchin embryo test (SET) is based upon the standard 48 h sea urchin development assay15’l6 and incorporates cytologic-cytogenetic analysis of embryos.” The two geno-

CCC-0260-437X/85/0005-0245 $05.00 0 Wiley Heyden Ltd, 1985

JOURNAL OF APPLIED TOXICOLOGY,VOL. 5,NO. 4,1985

245

J. E.HOSE

toxicity measurements included in the cytogenetic assessment are (1) anaphase aberration analysis, an examination of abnormalities in chromosome configurations as the groups of chromosomes move to opposite poles and (2) micronucleus formation, a measurement of the incidence of smaller, secondary nuclei composed of whole chromosomes or chromatin fragments. These two procedures eliminate the need for resolution of individual chromosomes and are suitable for use with a wide range of fishes and invertebrates, most of which possess large numbers of relatively small chromosomes. Cytotoxicity, survival and teratogenesis are also evaluated in the SET, providing the simultaneous assessment of several directly comparable endpoints. A secondary objective is to describe experimental results of the SET and related sea urchin development bioassays and to suggest potential applications of the test. At this time, the SET has been evaluated with only one compound, the environmental mutagen benzo(a)pyrene (BP)18*l 9 and has yielded promising results. Other researchers have utilized the survival and teratogenicity portions of the bioassay to screen pharmaceuticals, environmental contaminants, and complex effluents. The SET may also be used to monitor levels of environmental mutagens or teratogens in the field" although initial work suggests that this application may be less sensitive than is direct embryo exposure.

MATERLALS AND METHODS ~~~~~~~~

~~~

~

~

~

~

The sea urchin development bioassay has been previously developed by researchers in Japan;' Sweden" and Italy.I6 The exposure procedure used in this sea urchin embryo test (SET) is based on the 48-h sea urchin development assay of Oshida et a1.15 Gametes and embryos are exposed to control seawater, a solvent control if necessary, and a series of at least four pollutant concentrations. Fertilization success was monitored 15 min subsequent to the addition of sperm to the eggs by simply removing an aliquot of the eggs and checking for the presence of a fertilization membrane. Embryos were reared at constant temperature (15 "C for the purple sea urchin, Strongylocentrotus purpuratus) with continuous stirring and aeration for 48 h at which time untreated embryos have completed gastrulation. The number of replicates in each treatment group is dependent upon the statistical confidence limits desired; we have used groups of three to four replicates with reproducible results. At the end of the development period (which may be modified to account for speciesspecific differences in rearing temperature and development rates), the numbers of embryos present and gastrulation abnormalities were recorded in a volumetric subsample. Embryo survival was calculated as the difference between the number present 15 min after introduction of sperm to the eggs and the number present at 48 h. For cytologiccytogenetic analysis, 48-h embryos were preserved in a 1: 10 dilution of neutralized formalin or 3 : 1 alcohol-acetic acid (Carnoy's f i ~ a t i v e ) . 'Specimens ~ may be stored in formalin for several years prior to use, but embryos in Carnoy's fixative should be analyzed within a few days. Mitotic aberration test. In the SET, genotoxicity was assessed using the anaphase aberration test and the micro246 JOURNAL OF APPLIED TOXICOLOGY,VOL. 5, N0.4,1985

nucleus test. Mitotic aberration analysis has been previously described for mammalian cells in metaphase and anaphase" and for fish cells in anaphase? A modification of Longwell and Hughes' methodI7 was used for sea urchin embryos.'' Formalin-preserved embryos were placed on a clean glass microscope slide, the excess formalin removed with tissue paper and embryos were carefully covered with a minimal amount of 45% acetic acid. After 5 min, the excess acetic acid was removed with tissue paper and a few drops of aceto-orcein stain (1 9 parts standard aceto-orcein [Sigma Chemical Co., St. Louis, Mo.] and one part concentrated propionic acid) applied in the same manner. After the embryos became darkly stained, a coverslip was applied and the embryos compressed into monolayers. Prepared slides must be used within 1-2 days since stained chrornosomes darken with time. Embryo monolayer preparations were examined for cytologic and cytogenetic abnormalitiesg' l 7 using the 60x or l O O x objective of a light microscope. Some investigators use a green filter to highlight the orcein-stained mitotic configuration^.^^ Cells were observed for the following cytologic irregularities (Fig. 1) (definitions are from references 17 and 24): Pycnosis - nucleus shrunken to a dense, structureless mass of chromatin. Karyolysis - dissolution of cell nucleus. Karyorrhexis - fragmentation of nucleus. Abortive mitoses - abnormal chromosome configurations unable to complete their mitoses. Pleomorphism - occurrence of widely different cellular forms. Sticky chromosome bridge - telophase bridges consisting of more than chromosome. Dedifferentiation - appearance of large, undifferentiated cells in an advanced embryo. Premature differentiation - appearance of advanced cell differentiation in an early embryo. Giant cell formation - large containing multiple, normal-sized nuclei. than a more laborious cell-by-cell assessment of cytologic abnormalities, we recommend scoring presence/ absence or approximate percentage of cells per embryo with each aberration. For each anaphase configuration, the following possible aberrations were recorded (Fig. 2) (definitions are from references 9 and 22): Stray chromosome - chromosome left at equator or outside of spindle during mitosis. Lagging chromosome - chromosome which lags behind the main body of chromosomes. Acentric fragment - chromosomal fragment left at equator during cell division. Attached fragment - chromosomal fragment that lags behind main body of chromosomes and appears to be attached by a thin strand of chromatin. Translocation bridge - chromatin bridge stretched between the two groups of anaphase chromosomes. Side-arm bridge - pseudochiasma. Unequal chromosome distribution - chromosome nondisjunction. Multipolar spindle- tri- or multipolar anaphase spindle.

SEA URCHIN BIOASSAY

Pycnosis

(3 Sticky Chromosome Bridge

Figure 1. Types of cytologic abnormalities which may be present in toxicant-treated embryos.

Stray Chromosome

Lagging Chromosome

Acentric Fragment

Attached Fragment

Unequal Chromosome Distribution

@ (es 9 u

@

63

Translocation Bridge

-

Side arm

Bridge

n

(el

(a i .!Z<$.*.

Micronucleus Formation

.~.&..a.. ;;jp i&d

.. '.-:c'

<>;::?y.%

v,.:<;-

2.

Multiple Micronuclei ( Mult inucleated Cel I)

@ Q

n

Multipolar Spindle

W

Figure 2. Types of cytogenetic aberrations which may be present in toxicant-treated embryos.

JOURNAL OF APPLIED TOXICOLOGY,VOL. 5,NO.4,1985

247

J. E. HOSE

The mitotic aberration rate was expressed as the percentage of mitotic configurations with at least one aberration. These aberrations were visible during anaphase/telophase. It is suggested that the number of cells in prophase, prometaphase and metaphase also be noted. Since each mitosis has been recorded, an estimate of the number of mitosing cells per embryo (mitotic index) would be available. Dividing the mitotic index by the average number of cells per embryo (which can be obtained from a mean of approximately five embryos) yielded the mitotic rate. For a typical 48 h purple sea urchin gastrula containing about 1000 cells, from 8 to 12 mitotic configurations were commonly observed. Micronucleus test. While scanning the embryo preparation for cytologic or cytogenetic abnormalities, cells were simultaneously assessed for the presence of: (1) Micronucleus formation - extracellular fragments of chromatin which form a secondary n u ~ l e u s . ' ~ ( 2 ) Multiple micronuclei (multinucleated cell) - cell containing from two to 15 micronuclei.

By division of the number of cells per embryo into the total number of micronucleated cells per embryo, the micronucleation rate could be calculated. To obtain reliable cytogenetic aberration rates, in our experience an adequate number of embryos per treatment is about 20. However, should a precise confidence interval be needed, the number of replicates may be determined statistically. ~~

~

RESULTS Developmental effects. Sea urchin gametes and embryos were exposed to environmental concentrations of BP ranging from 0.5 to 50 p/L (Table 1). By 48 h, dissolved BP concentrations were 0.5 p / L BP (initial concentrations of 0.5-10 pg/L BP) or 2.0 pg/L BP (initial concentration of 5 pg/L BP). No effect was observed on fertilization success. However, significantly fewer (P< 0.05) embryos treated with at least 1 pg/L BP had completed gastrulation

at 48 h than had solvent-treated embryos. Several developmental defects such as failure to undergo cleavage, retarded development and cytolysis were observed in BP-treated embryos.I8 Cytogenetic effects. Purple sea urchin (S. purpurants) gametes and embryos were exposed to environmental levels of BP in the SET.18 Following treatment with from 0.5 to 50 pg/L BP, fertilization success was unaffected. However, developmental abnormalities were observed in 48 h embryos exposed to initial BP concentrations of 1.0 to 50 pg/L relative to solvent (ethanol)-treated control embryos (Table 2). Genotoxic effects, as evidenced by increased anaphase aberration rates, were significant at the lowest BP dose tested, 0.5 pg/L. Chromosomes or acentric frag ments outside the spindle apparatus and translocation bridges (Fig. 3) accounted for most of the observed mitotic aberrations. Micronucleus induction and cytologic abnormalities were present only at treatment levels of 1.O pg/L BP and greater. Results show that the mitotic index and the incidence of aberrant anaphases were the two most sensitive measurements examined. The incidence of anaphase aberrations was highly correlated with the log BP dose (r = 0.98, P < 0.01). Similarly, the total number of aberrant mitoses per embryo (Fig. 4) was also related to the log BP dose (r = 0.98, P < 0.01). This is in contrast to other embryologic, cellular and cytogenetic effects whose distributions lacked a dose-response relationship. The micronucleation rate, total cytologic abnormalities and the percentage of normal gastrulae were roughly similar between treatments of 1.O and 50 pg/L BP. Cell division was equally inhibited at all BP concentrations. Results obtained following paternal treatment of purple sea urchin with BP were similar to those described above." Sperm from urchms injected 7 days earlier with 0, 20 or 100 mg BP/kg was used to fertilize control eggs. BP induced sperm abnormalities, notably small or amorphous heads and a slight but not statistically significant increase in abnormal gastrulae was also noted (Table 2). As in the previous experiment, the most sensitive measurements examined were the mitotic rate and the anaphase aberration rate. Each of these indices was significantly affected at both

Table 1 . Embryologic, cytologic and cytogenetic effects of benzo(a)pyrene (BP) in the purple sea urchin. Four replicates were tested at each concentration and 40 embryos per concentration were used for cytologic-cytogenetic analysis. From'* Initial BP concentration (/lg/LI Seawater control (0.0) Ethanol control

(0.0) 0.5 1 .o 5.0 10.0 50.0

% Normal gastrulae

% Fertilized X +- S.E.

X

91 + 3

94 t 2

95 + 2 90 5 3

84 ? 2 84 + 5 77 ?la 73 t3' 73+la 72 r2'

93 + 2 93 t2 84 + 4 89 r5

+ S.E.

a Significantly different from ethanol control, P

Number of mitoses/embyro X +- S.E. X

10.3 t 0.6 9.4 + 0.5 5.6 t 0.4' 6.1 +0.4' 6.2 r0.4' 6.2 +0.4' 5.7 + 0.4'

< 0.05

248 JOURNAL OF APPLIED TOXICOLOGY, VOL. 5, NO. 4,1985

% Mitoses cytogenetic abnormalities

Number of micronucleated cells/embryo

X

Total cytologic abnormalities

0.49

0.00

0

0.53 4.91' 9.84' 12.50' 15.73' 21.49'

0.00 0.00 0.32 0.38 0.38 0.35

0 0 18 5 11 5

SEA URCHIN BIOASSAY

Table 2. Embryologic, cytologic and cytogenetic effects of parental exposure to benzo(a)pyrene (BP) in the purple sea urchin. Treated sperm were used to fertilize untreated eggs. Three replicates of each treatment group were used and 36 embryos/treatment were used for cytologic-cytogenetic analysis. From" Treatment group

Normal gastrulae k S.E.

X Number of mitoses/embryo

Control 20 mg BP/kg lOOmgBP/kg

75+7 66 2 2 58r10

10.2?0.1 9.9 ? 0 . l 8 7.1 +O.la

X

r S.E.

a Significantly different from control, P

% Mitoses cytogenetic abnormalities

X

Number of micronucleated cells/embryo

Total cytologic abnormal it ies

1.63 3.90a 7.03'

1 .o 3.1 44.0'

0 1 5a

< 0.05

Figure 3. Monolayer preparation from a 48 h sea urchin embryo showing an anaphase configuration with a translocation bridge (arrow). Several other mitotic figures are evident. Aceto-orcein.

2374X.

Observations at the chromosome level were frequently correlated with cellular and embryonic effects. Treated embryos could be visibly divided into two categories, one comprised of essentially normal embryos and the other of smaller, less differentiated, malformed individuals (Fig. 5). The grossly malformed test embryos containing numerous anaphase aberrations were composed of high proportions of pycnotic, karolytic and multinucleated cells. These embryos contained from 600 to 900 cells as opposed to normal embryos composed of 1000-1300 cells.

0.5

1

5

10

50

BENZO(A)PYRENE CONCENTRATION IN PPB

Figure 4. Relationship between the log benzo(a1pyrene (BPI dose and the number of mitotic aberrations observed in 48 h sea urchin embryos exposed to EP. r3df = 0.98, P < 0.01.

treatment levels. Similar types of aberrations (chromosomes outside spindle and translocation bridges) were most prevalent in treated embryos. Micronuclei and cytologic abnormalities (sticky bridges, pycnosis and karyolysis) were observed only at the higher BP dosage.

DISCUSSION Developmental effects. Although many aquatic species are suitable for genotoxicity-teratogenicity tests (oyster, Crassostrea gigas;14 green abalone, Haliotis fulgensf6 mussel, Mytilus edulis f > polychaete worms, Neanthes sp.: sea urchins, Strongylocentrotus sp., Lytechinus sp., Paracentrotus sp., Sphaerechinus sp., Arbacia sp. and Psammechinus sp. (see Table 3 for references); rainbow trout, Salmo gairdnerif7*28 ?ilapia, Tilapia mossambica f 9 and JOURNAL OF APPLIED TOXICOLOGY, VOL. 5, NO. 4,1985 249

J. E.HOSE

Figure 5. ( a ) Monolayer preparation of control sea urchin gastrula containing between 1000 and 1300 cells. Aceto-orcein. 84X. ( b ) Monolayer preparation of small, deformed sea urchin gastrula resulting from paternal treatment w i t h 100 mg benzo(a)pyrenelkg. Embryo contains between 600 and 900 cells and few mitotic configurations are evident. 84 X. Aceto orcein.

Table 3. Selected previous research from sea urchin development bioassays Chemical type Neurochemicals Metals Antiseptic Inhibitor of D N A synthesis Wastewater Polycyclic aromatic hydrocarbons Oil dispersants Styrene and derivatives Tobacco smoke condensates

250 JOURNAL OF APPLIED TOXICOLOGY, VOL. 5,NO. 4,1985

Developmental effect

+ + + + +

+I-

+ +

+

Reference

56 16,36,37 57 58 15 18,19,31,59 60 61 62

SEA URCHIN BIOASSAY

mudminnows, Umbra sp. and Nothobranchius SP?~~'), the majority of the data has been obtained from sea urchins (Table 3). Developmental effects have been elicited in sea urchins by antagonists of neurotransmitters; heavy metals such as cadmium, chromium and arsenic; nalidixic acid, an inhibitor of DNA synthesis; and municipal sewage effluents. The direct-acting mutagen, plovidone-iodine, was teratogenic in sea urchins, while the teratogenicity of polycyclic aromatic hydrocarbons, which are indirect-acting mutagens, was dependent upon the method and timing of exp~sure.''~~~ One of the strengths of the sea urchin embryo test is that it yields directly comparable data from the chromosomal, cellular and embryonic levels of organization and can thus be used to link sublethal responses and lethality. The sea urchin embryo system is relatively simple and allows all cells to be evaluated. The SET can also be used to determine the most sensitive index of toxicity to which further testing should be directed. A recent study suggests that results from the in vivo fish cyogenetic assay are predictive of long term biological effect^.^' In order to properly interpret the significance of the chromosomal macrolesions and cytologic aberrations measured in the SET, some discussion of their origin and significance is necessary. Anaphase aberrations. Mitotic aberrations are usually results of chromosome breakage or spindle damage" (Fig. 6 ) . A chromosome bridge is formed via an asymmetrical chromosome interchange or chromatid intra- or interchange. Side-arm bridges or pseudochiasmata, however, can be induced by abnormal chromosome stickiness as well as chromosomal breakage. When chromosomal bridges break during anaphase, fragments can remain attached to the main body of chromosomes. Acentric fragments are formed by chromatid or chromosome breakage during metaphase. Nondisjunctions (lagging or stray chromosomes) can result from either spindle or chromosome damage. Chromosome nondisjunctions or multipolar spindles can cause aneuploidy. While chromosome errors are lethal if they occur before gastrulation, postgastrulation lethality is dependent upon the number of affected cells and their ontogenetic fates.33 Criticism of the mitotic aberration technique has been raised regarding the possibility of aberrations being produced as the result of pressure distortion artifact.% Low rates of mitotic aberrations have been reported in control fish and sea urchin embryo Observations of similar types of anaphase aberrations in fixed and living cultures of rainbow trout gonad (RTG-2) cellsz3 and in trout embryos3* do not support the pressure artifact argument. Mitotic aberration analysis has been used in a few published studies with sea urchins, although no methods were given and it is unclear whether aberration analysis was confined to anaphase/telophase configurations or if metaphase figures were included. Increased mitotic aberration rates were observed in the sea urchins Paracentrotus lividus and Sphaerechinus granularis exposed to mercury and arsenic.36i37Arsenic also elicited cytotoxic responses.36 Cadmium-exposed Psammechinus microtubercalatus embryos failed to exhibit genotoxic effects.16 Although a few data are available at this time regarding the validity of cytologic-cytogenetic analysis using aquatic species as a screening method for genotoxins or teratogens,

the method should be further developed since it appears to be sensitive' and is rapid, inexpensive and does not require cytologists to review the embryo preparations.% Hose et a]." noted that for BP, anaphase aberration rates of sea urchin embryos were of comparable sensitivity to two of the more widely used aquatic genotoxicity tests, the induction of SCE's and chromosomal aberrations in fishes. On the basis of their in vivo studies, Kocan et al.' observed that the overall sensitivity of the fish anaphase aberration test was similar to those of SCE analysis and the Ames Salmonella/microsome test. Because of its high sensitivity, the in vitro anaphase aberration test was recommended by the Ad Hoc Committee of the Environmental Mutagen Society and the Institute for Medical Research to be included in any battery of mutagenicity tests3' Micronucleus formation. After completion of mitosis, extranuclear chromatin fragments can fuse with the main nucleus or form micronuclei?' A few micronuclei are shown in Fig. 5. In cases of more complex mitotic aberrations, multiple micronuclei may be formed. Multinucleate cells contain from two to 15 micronuclei. Cells with more than 15 micronuclei are termed pulverized,a but cannot be easily distinguished from karyorrhectic cells and thus are counted in the SET classification scheme as karyorrhectic. Since only a portion of the chromatin fragments observed during mitosis persist as micronuclei, the micronucleus test is less sensitive than anaphase aberration analysis. However, because recording micronucleus formation during cytologic-cytogenetic analysis requires little extra time, the embryo micronucleus assay would function as a confirmatory test. The mouse micronucleus test is a developed test for carcinogenicity' with comparable specificity (93%) and predictive value (92%) to the Ames test!' Due to its relatively low sensitivity, the mouse micronucleus test is useful as a secondary screening test or confirmatory test for carcinogenicity!' Micronucleus induction has recently been used as an indicator of genetic damage in fetal mouse43 and embryonic fisha cells. According to a report by the U.S. Environmental Protection Agency's Gene-Tox micronucleus formation and chromosomal breakage (or lbss) have never been shown to occur independently of one another in any dividing cell population. Cytologic effects. A number of cytologic abnormalities may be observed in embryos as a result of toxic insults.33 Nuclear pycnosis, karyolysis and karyorrhexis imply irreversible cellular injury. Abortive mitoses were observed in BP-treated sea urchin embryos in which the prophase chromosomes appeared hypochromatic and swollen. Numerous sticky chromosome bridges were found in BPtreated fish and urchin embryos and are suggestive of severe toxic damage.33 These ranged in appearance from relatively slender chromosomal bridges resembling, but thicker than, translocation bridges to hyperchromatic dumbell-shaped configurations in which all the chromosomes were adherent. All mitotic figures containing sticky bridges are considered by LongwellM to be abortive. Cells from BPtreated sea urchin and fish embryos were strikingly pleomorphic. One particularly intriguing observation was that of giant cells containing multiple, normally-sized nuclei in treated urchin gastrulae. These multinucleate cells which appear to form following fusion of several cells may result from cell membrane changes.47 Cell dedifferentiation, the JOURNAL OF APPLIED TOXICOLOGY, VOL. 5, NO. 4,1985

251

J. E. HOSE

I

MUTAGEN TREATMENT

@ t

1

Chromatid or Chromosome Breakage

Mitosis

(3

Mitotic Aberration

/

Side-arm Bridge

1

fuse with nucleus

Acentric Fragment

Translocation or

Stray or Lagging Chromosome

\rez::lted

Normal Daughter

Micronucleus

Cel I

Formation

fuse with nucleus

J

Normal Daughter Cell

loss of fragment

J/

Normal Daughter Cel I

,

/

remain isolated

Micronucleus Formation

attached fragment

Normal

Daughter

Cel I

Figure 6. Formation and fate of cytogenetic aberrations induced in mutagen-treatedembryos.

primitive undifferentiated appearance of cells from a latestage embryo, is a response to severe stress leading to cellular tissue disorganization or d i ~ i n t e g r a t i o n . ” . Alterna~~ tively, aberrant premature differentiation of cells in earlystage embryos might also be observed. Toxicological considerations. Several questions arise when using aquatic embryos to evaluate the toxicity of chemicals which require metabolic activation. First, no data are available regarding the capacity of sea urchin embryos to metabolize xenobiotic compounds. Mixed function o x y g e n a ~ eand ~ ~ aryl sulfataseS0 activities have been detected in adult sea urchins and aryl [benzo(u)pyrene] hydrocarbon hydroxylase activity has been demonstrated in embryos of two fish specie^.^^*^^ Genotoxicity has been 252 JOURNAL OF APPLIED TOXICOLOGY, VOL. 5 , NO. 4,1985

induced by direct-acting compounds in mouses3 and fish4 embryos. Therefore, the genotoxic effects observed in BPtreated sea urchin gastrulae are presumed to be indirect evidence of the presence of monooxygenase activity. With the exception of the incidence of anaphase aberrations, all other embryologic, cytogenetic and cytologic indices exhibited a threshold of response occurring at dissolved BP levels of 0.5-1 .O&/L. These results suggest Lhat (1) all types of mitotic aberrations may not produce similar cytologic consequences, (2) some degree of breakage repair may be possible, (3) a threshold exists for cytologic and embryologic damage to become manifest, and/or (4) the observed biological effects may r.eflect a threshold for BP solubility in seawater.

SEA URCHIN BIOASSAY

Other problems involve the solubility and bioavailability of toxicants in aqueous systems. Actual doses of lipophilic substances often differ substantially from those which are added. Levels of degradable or lipophilic compounds decline rapidly, necessitating periodic monitoring. Implicit in this statement is the problem of relating biological effects to a changing pollutant concentration. Since there are no data to document a critical period for BP toxicity during sea urchin development, biological effects were related to initial BP levels. In fish embryos, toxicity appears to be correlated with body burdens of BP and its metabolites.% Therefore, assessment of pollutant tissue concentrations may be desirable for determination of toxic effects and if measured in conjunction with soluble pollutant levels would provide information regarding bioconcentration potential. Applications of the sea urchin embryo test. As described, the SET is useful for screening individual substances or complex environmental mixtures for toxicity, genotoxicity and teratogenicity. Alternatively, by using embryos from adults collected from polluted areas, the SET can be used for monitoring the toxic potential of nearshore waters. Paternal exposure to BP produced chromosome macrolesions and cellular toxicity but equivocal embryonic effects. Low incidences of mitotic aberrations were elicited by high paternal BP doses, 7% aberrations at 100 mg BP/kg, compared to those obtained from embryonic exposures,

21% aberrations at 50 pg BP/L. Thus, although the use of the SET to monitor parental mutagen loads is feasible, it appears to be a less sensitive method than is direct embryo exposure. Of course, complementary field validations are essential before the SET can be used to reliably estimate the toxicity of environmental samples. The sea urchin development test is also amenable to more sophisticated cytogenetic methods to pinpoint chromosomal lesions. Metaphase analysis of treated embryos can be accomplished by incorporating methods similar to those described by Nichols er al.22 Sister chromatid exchange rates could be measured using a modification of the procedure developed for mussel (Myrilus edulis) embryos.' Because the molecular biology of the sea urchin embryo has been extensively studied,55 this alternative species is well suited to investigating mechanisms of toxicity.

Acknowledgments The author thanks Drs A. Longwell o f National Marine Fisheries Service, Milford, Connecticut and M. Landolt and R. Kocan of the University o f Washington for their invaluable demonstration and discussion of embryo cytogenetic analysis. Embryo bioassays were performed by Mr P. Oshida and Mr S. Bay of the Southern California Coastal Water Research Project. A previous draft o f the manuscript was reviewed by V. Ligouri and Dr H . Puffer. The manuscript was typed b y Waheedah Muhammad. This work was presented at the 5 t h Annual Meeting of the Society of Environmental Toxicology and Chemistry (SETAC), Arlington, Virginia, USA, in November 1984.

REFERENCES 1.

2.

3. 4.

5.

6.

7. 8. 9. 10. 11.

International Commission for Protection Against Environmental Mutagens and Carcinogens. Screening strategy for chemicals that are potential germ-cell mutagens in mammals. Mutat. Res. 114. 117-177 (1983). A. D. Kligerman and S. E. Bloom, Sister chromatid differentiation exchanges in adult mudminnows (Umbra limi) after in vivo exposure t o 5-bromodeoxyuridine. Chromosoma 56, 101-1 09 ( 1976). C. J. Barker and B. D. Rackham, The induction of sisterchromatid exchanges in cultured fish cells (Ameca splendens) by carcinogenic mutagens. Mutat. Res. 68, 381-387 (1979). P. T. Stromberg, M. L. Landolt and R. M. Kocan, Alterations in the Frequency of Sister Chromatid Exchanges i n Flatfish from Puget Sound, Washington Following Experimental and Natural Exposure t o Mutagenic Chemicals, p. 43. NOAA Technical Memorandum, Office of Marine Pollution Assessment, Boulder, Colorado (1981). G. G. Pesch and E. C. Pesch, Neanthes arenaceodentata (Polychaeta Annelida), a proposed cytogenetic model for marine genetic toxicology. Can. J. Fish Aquat. Sci. 37, 12251228 (1980). D. R. Dixon and K. R. Clarke, Sister chromatid exchange: A sensitive method for detecting damage caused by exposure t o environmental mutagens in t h e chromosomes of adult Mytilus edulis. Mar. Biol. Lett. 3, 163-172 (1982). F. L. Harrison and I. M. Jones, A n in vivo sister-chromatid exchange assay in the larvae of the mussel Mytihs edulis: Response t o 3 mutagens. Mutat. Res. 105.235-242 (1982). I. F. H. Purchase, A n appraisal of predictive tests for mutagenicity. Mutat. Res. 99, 53-71 (1982). R. M. Kocan, L. M. Landolt and K. M. Sabo, Anaphase aberrations: A measure of genotoxicity in mutagen-treated fish cells. Environ. Mut. 4, 181-189 (1982). A. E. Prein, G. M. Thie, G. M. Alink, J. H. Koeman andC. L. M. Poels, Cytogenetic changes i n fish exposed t o water of the river Rhine. Sci. Tot. Environ. 9, 287-291 (1978) A. P. Krishnaja and M. S. Rege, Induction of chromosomal aberrations i n fish Boleophthalmus dussumeri after exposure in vivo t o mitocycin C and heavy metals mercury, selenium and chromium. Murat. Res. 102,71-82 (1982).

12. R. M. Kocan, M. L. Landolt, J. Bond and E. P. Benditt, ln vitro effect of some mutagens/carcinogens on cultured fish cells. Arch. Environ. Contam. Toxicol. 10, 663-671 (1981). 13. U. H. Ehling, L. Machemer, W. Buselmaier, J. Dycka, H. Frohberg, J. Kratochvilova, R. Lang, D. Lorke, D. Muller, J. Peh. G. Rohrborn, R. Rolls, M. Schulze-Schencking and H. Wiemann, Standard protocol for the dominant lethal test on male mice. Arch. Toxicol. 39, 173-185 11978). 14. C. E. Woelke, Development of a Water Receiving Quality Bioassay Criterion Based on the 48-hour Pacific Oyster (Crassostrea gigas) Embryo, Technical Report No. 9, p. 93. Washington Department of Fisheries (1972). 15. P. S. Oshida, T. K. Goochey and A. J. Mearns, Effects of municipal wastewater on fertilization, survival, and development of the sea urchin, Strongylocentrotus purpuratus. In Biological Monitoring o f Marine Pollutants. pp. 389-402, ed. by F. J. Vernberg, A. Calabrese, F. P. Thurberg and W. B. Vernberg. Academic Press, New York (19811. 16. G. Pagano, A. Esposito and G. G. Giordano, Fertilization and larval development in sea urchins following exposure of gametes and embryos t o cadmium. Arch. Environ. Contam. TOX~CO ~. 11,47-55 ( 1 982). 17. A. C. Longwell and J. B. Hughes, Cytologic, cytogenetic, and development state o f Atlantic mackerel eggs from sea surface waters of the New York Bight, and prospects for biological effects monitoring with ichthyoplankton. J. Cons.

179,275-291 (1980). 18. J. E. Hose, H. W. Puffer, P. S. Oshida and S. M. Bay, Developmental and cytogenetic abnormalities induced in the purple sea urchin by benzo(a1pyrene. Arch. Environ. Contam. Toxicoi. 12,319-325 (1983). 19. J. E. Hose and H. W. Puffer, Cytologic and cytogenetic anomalies induced in purple sea urchin embryos (Srrongylocentrotus purpuratus S.) by parental exposure t o benzo(a)pyrene. Mar. Biol. Lett. 4, 87-95 (1983). 20. N. Kobayashi, Marine pollution bioassay b y using sea urchin eggs in the inland Sea of Japan (The Seto-Naikai). Pub/. Set0 Mar. Lab. 19, 359 (1972). 21. B. E. Hagstrom and S. Lonning, The effects of Esso Corexit 9527 on the fertilizing capacity of spermatozoa. Mar. Poll.

JOURNAL

OF APPLIED TOXICOLOGY, VOL. 5, NO. 4,1985 253

J. E. HOSE Bull. 8, 136-138 (1977). 22. W. W. Nichols, R. C. Miller and C. Brandt, In vitro anaphase and metaphase preparations i n mutagen testing. I n Handbook of Mutagenicity Test Procedures, pp. 225-233, ed. by B. J. Kilbey, M. Legator, W. Nichols and C. Ramel. Elsev~er/North Holland, New York (1977). 23. R. M. Kocan, School o f Fisheries, University of Washington, Seattle, WA 98195, personal communication. 24. A. Osol, Blakiston's Pocket Medical Dictionary, 3 r d ed., p. 964. McGraw-Hill Co., New York (1973). 25. W. Schmid, The micronucleus test. Mutar. Res. 31, 9-15

(1975). 26. J. E. Hose, unpublished research. 27. R. 0.Sinnhuber, J. D. Hendricks, J. H. Walesand G. B. Putnam,

Neoplasms in rainbow trout, a sensitive animal model for environmental carcinogenesis. Ann. N. Y. Acad. Sci. 298,

398-408 (1977).

28. J. B. Hannah, J. E. Hose, M. L. Landolt, 6. S. Miller, S. P. Felton and W. T. Iwaoka, Benzo(a)pyrene-induced morphologic and developmental abnormalities i n rainbow trout (Salmo gairdneri R.). Arch. Environ. Confam. Toxicol. 11, 727734 (1982). 29. A. A. Wardhaugh, Dominant lethal mutations in Tilapia mossambica (Peters) elicited by myeleran. Mutat. Res. 88, 191-196 (1981). 30. R. N. Hooftman, The induction of chromosome aberrations in Nofobranchius rachowi (Pisces: Cyprinodontidae) after 31. 32.

33.

34.

35.

36.

37.

38.

39.

40. 41.

42.

treatment with ethyl methanesulphonate or benzo(a1pyrene. Murat Res. 91, 347-352 11981). E. de Angelis and G. G. Giordano, Sea urchin egg development under the action of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene. Cancer Res. 34, 1275-1280 (1974). V. M. Liguori and M. L. Landolt, Anaphase aberrations: A n in vivo measure of genotoxicity. I n Proceedings from U.S. Environmental Pro tection Agency Symposium on ShorfTerm Genetic Bioassays in the Evaluation of Complex Environmental Mixtures, vol. I V , in press. A. C. Longwell, In the Wake of the Argo Mercharw, proceedings of a symposium held January 11-13, pp. 116-125. Center for Ocean Management Studies University of Rhode Island, Kingston, Rhode Island (1978). A. D. Kligerman, The use of aquatic organisms t o detect mutagens that cause cytogenetic damage. I n Radiation Effects on Aquatic Organisms, pp. 241-252, ed. By N. Egami, Japan Scientific Society Press, Tokyo/University Park Press, Baltimore (1980). J. E. Hose, J. B. Hannah, D. DiJulio, M. L. Landolt, B. S. Miller. W. T. lwaoka and S. P. Felton, Effects of benzo(a)pyrene on early development of flatfish. Arch. Environ. Contam. Toxicol. 11. 167-171 (1982). G. Pagano, A. Esposito, P. Bove, P. M. de Angelis, A. Rota, E. Vamvakinos and G. G. Giordano, Arsenic-induced developmental defects and mitotic abnormalities in sea-urchin development. Murat Res. 104,351-354 (1982). G. Pagano, P. 8ove. M. de Angelis, A. Esposito, A. Rota and G. G. Giordano, Mercury-induced developmental defects and mitotic aberrations i n sea urchin development. Mutat. Res. 97,210 (1982). J. A. Beardmore, C. J. Barker, B. Battaglia. R. J. Berry, A. C. Longwell, J. F. Payne and A. Rosenfield, The use of genetical approaches t o monitoring biological effects of pollution. J. Cons. 179, 299-305 ( 1980). A d Hoc Committee of the Environmental Mutagen Society and the Institute for Medical Research, Chromosome methodologies in mutagen testing. Toxicol. Appl. Pharmacol. 22, 269-275 ( 1 972). R. W. Kapp, Jr and B. E. Eventoff, Mutagenicity of dimethylsulfoxide (DMSO): In vivo cytogenetics study in the rat. Teratog. Carcinog. Mutag. 1, 141-145 (1980). F. J. De Serres and J. A. Ashby (eds), Evaluation of ShortTerm Tesfs for Carcinogens, Report of the International Collaborative Programme, Elsevier/North Holland, Amsterdam (1981). 1. F. H. Purchase, Appraisal o f the merits and shortcomings of t e s t s of mutagenic potential. I n Mechanisms of Toxicity and Hazard Evaluation: Proceedings of the Second International Congress of Toxicology, pp. 105-1 19, ed. by B. Holmstedt,

254 JOURNAL OF APPLIED TOXICOLOGY, VOL. 5, N0.4,1985

R. Lauwerys, M. Mercier and M. Roberfroids. Elsevier, Amsterdam (1980). 43. L. Henderson, R. Cole, J. Cole, H. Cole, Z. Aghamohammedi and T. Regan, Sister-chromatid exchange and micronucleus induction as indicators of genetic damage in maternal and foetal cells. Mutar. Res. 126,47-52 11984). 44. J. E. Hose, J. 8. Hannah, H. W. Puffer and M. L. Landolt, Histological and skeletal abnormalities in benzo(a)pyrenetreated rainbow t r o u t alevins. Arch. Environ. Contam. Toxicol.

13,675-684 (1984). 45. J. A. Heddle, M. Hite, B. Kirkhart, K. Mavournin, J. T. MacGregor, G. W. Newel! and M. F. Salamone. The induction of micronuclei as a measure o f genotoxicity. Mutat. Res. 123, 61-118 (1983). 46. A. C. Longwell, National Marine Fisheries Service, Middle Atlantic Coastal Fisheries Center, Milford, Connecticut, personal communication. 47. A. Parkinson and S. Safe, A r y l hydrocarbon hydroxylase induction and i t s relationship t o the toxicity of halogenated aryl hydrocarbons. Toxicol. Environ. Chem. Rev. 4, 1-46

11981). 48. H. Rosenthal and D. F. Alderdice, Sublethal effects of environmental stressors. natural and pollutional, on marine fish eggs and larvae. J. Fish. Res. Board Can. 33, 2047-2065 (1976). 49. R. F. Lee, Mixed function oxygenases (MFO) in marine invertebrates. Mar. Biol. Left 2,87-105 (1981). 50. C. C. Malins and W. T . Roubal, A r y l sulfate formation in sea urchins (Strongylocenfrotus droebachiensis) ingesting marine algae (Fucus districhus) containing 2,6-dimethylnaphthalene. Environ. Res. 27,290-297 ( 1 982). 51. R. L. Binder and J. J. Stegeman, Induction of aryl hydrocarbon hydroxylase activity in embryos of an estuarine fish (Fundulus heteroclitus). Biochem. Pharmacol. 29,949-951 ( 1980). 52. R. L. Binder and J. J. Stegeman, Basal levels a n d i n d u c t i o n 3 hepatic aryl hydrocarbon hydrosylase activity during the embryonic period of development in brook trout. Biochem. Pharmacol. 32, 1324-1327 (1983). 53. S. M. Galloway, P. E. Perry, J. Meneses. D. W. Nebert and R. A. Pedersen, Demonstration by sister chromatid exchange of genetic differences i n benzo(a)pyrene metabolism in cultured mouse embryos at early gestational age. Proc. Nat. Acad. Sci. ( 1 980). 54. 0. L. Winkler, K. L. Duncan, J. E. Hose and H. W. Puffer, Effects o f benzo(a)pyrene on the early development of California grunion, Leuresfhes tenuis (Pisces, Atherinidae) Fish. Bull. U.S.A. 81,473-481 (1983). 55. E. H. Davidson, 6 . R. Hough-Evans and R. J. Britten, Molecular biology of the sea urchin embryo. Science 217, 17-26

(1982). 56. M. A. Landau, G. A. Buznikov, A. S. Kabankin, N. A. Teplitz

and P. E. Chernilovskaya, Sensitivity of sea urchin embryos t o cytotoxic neuropharmacological drugs, the correlations between activity and lipophilicity of indole and benzole derivatives. Comp. Biochem. Physiol. 69C, 359-366 ( 1 981). 57. P. G. Rosenkranz, D. R. Gordon, W. T. Speck and H. S. Rosenkranz, Developmental abnormalities in the develop ment of the American sea urchin induced b y povidine-iodine, a widely used vaginal antiseptic. Mutat Res. 77, 387-390

(1980). 58. S. J. Czinn, W. T. Speck and H. S. Rosenkranz, Abnormalities

in the development of the American sea urchin induced by nalidixic acid. Murat Res. 91, 119-121 (1981). 59. M. P. Ceas, Effects of 3-4 benzopyrene on sea urchin egg development. Acta Embryol. Exp. 3, 267-272 (1974). 60. B. E. Hagstrom and S. Lonning, The effects of Esso Corexit 9527 o n the fertilizing capacity of spermatozoa. Maf. Poll. Bull. 8,136-138 (1977). 61. G. Pagano, A. Esposito, G. G. Giordano and 8 . F. Hagstrom, Embryotoxic and teratogenic effects of styrene and i t s derivatives on sea urchin development. Scand. J. Work €nviron. Health 4, 136-141, supplement 1 (1977). 62. H. Bresch, H. R . Spielhoff, U. Mohr and H. Barkemeyer, Use of the sea urchin egg for quick screen testing of the biological activities of substances. I. Influence of fractions of a tobacco smoke condensate on early development. Proc. Soc. Exp. Biol. Med. 141, 747-752 (1972). Received 16 November 1984; accepted 5 April 1985