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Environmental Mutagenesis 4:181-189 (1982)

Anaphase Aberrations: A Measure of Genotoxicity in MutagenTreated Fish Cells R. M. Kocan, M. L. Landoit, and K. M. Sabo

Department of PathologH School of Medicine (R.M.K., K.M.S.) and School of Fisheries (M. L. L.), University of Washington, Seattle

Rainbow trout gonad cells (RTG-2) were cultured for various lengths of time in the presence of several classes of known mutagenic chemicals and several related compounds that possessed n o known mutagenic/carcinogenic activity. During the course of exposure the cells were examined for the presence of abnormalities in the chromosome arrangement of anaphase figures during mitosis. Untreated and solvent-treated (dimethylsulfoxide-treated) cells exhibited a background abnormality rate of 12% with only minor chromosomal defects being observed. This was also true for those cells exposed to naphthol and anthracene, two chemicals with no proven mutagenic or carcinogenic activity. Conversely, significant increases in the frequency of anaphase aberrations were produced in cells treated with N-methyl- N’-nitro-N-nitrosoguanidine, benzo(a)pyrene, 9-aminoacridine and mitomycin-C. These abnormalities were also far more complex and extensive than those observed in the control and nonmutagen-treated cells. Many species of fish have extremely small and numerous chromosomes, making resolution of chromosome defects such as sister chromatid exchange and deletions more difficult than in most mammalian diploid cells, which generally have larger and fewer chromosomes. Exami- nation of cells during anaphase eliminates the need to observe each chromosome separately as well as the need to produce well-spread metaphase chromosomes. Since the sensitivity of anaphase aberrations to known mutagenic/carcinogenic compounds appears to be quite high in trout cells and since hundreds of suitable cells are available for analysis, this may be an appropriate alternative or addition to some of the more standard chromsosome macrolesion tests developed in mammalian systems.

Key words: genoloxicily, fish chromosomes, mutagens, carcinogens, anaphase

INTRODUCTION

Although t h e field of genetic toxicology had its origins in studies on submamma- lian species [Brusick, 19801, mammalian and microbial species have become the main- stay of the short-term i n vitro mutagenicity tests tha t have been extensively developed and widely used to measure t h e mutagenic potential of a wide range of agents. Promi-

Received October 21, 1981; revised and accepted December 11, 1981.

Address reprint requests to Dr. Richard M. Kocan, Department of Pathology SM-30, University of Washington, Seattle WA 98195.

0192-2521/82/0402-0181%03.00 0 1982 Alan R. Liss, Inc.

182 Kocan, Landolt, and Sabo

nent among these are the Salmonella mutagen assay developed by Ames et a1 [1975], tests to detect chromosomal macrolesions such as sister chromatid exchange (SCE) [Latt, 1974; Stetka and Wolff, 1976a,b; Latt et al, 1977; Kligerman, 19791, micronucleus formation [Schmid, 1976; Maier and Schmid, 19761, and chromosomal aberrations [Ziemba-Zoltowska et al, 1980; Edwards et al, 1980; Tsuda, 1981; van Kesteren- van Leeuwen and Natarajan, 19801, as well as tests to detect microlesions such as DNA repair [Lieberman et al, 1971a,b; Viceps-Madore and Mezger-Freed, 19781 and direct cellular mutagenesis [Mankovitz et al, 1974; Arlett et al, 1975; Chinchar and Sinclair, 1978; Jacobs and DeMars, 19781. Since each procedure has its advantages and weak points, it has been suggested that a battery of tests be used to evaluate the mutagenic potential of substances [Nichols et al, 1977; Sobels, 19771. Since chromosomes can be examined in any living system that is undergoing mitosis or meiosis, this type of procedure can be used with embryos, regenerating tissue, lymphocytes, or cell culture.

We have chosen to evalutate anaphase aberrations using cell cultures as a source of chromosomes and mitotic figures. Cell cultures were chosen for use because cells from a diversity of species are currently available and because many others can be started from species of significance to research needs. This last consideration is im- portant because it allows investigators to study cells from species most likely to be af- fected by the substance(s) under study, thereby lending more significance to the results of a battery of tests.

Owing to the aquatic orientation of the research in our laboratory, we chose fish cells as a suitable cell type and adapted the anaphase preparation methods described by Nichols et a1 [1972, 19771 for use with an in vitro poikilothermic system. From among the many fish cell lines available, we initially chose two lines on the basis of their ability either to metabolize promutagens to mutagens [Diamond and Clark, 19701 or to undergo forward mutations when exposed to known mutagenic chemicals [Kocan et al, 19811.

MATERIALS AND METHODS Cells

Two continuous cell lines, rainbow trout gonad (RTG-2) and bluegill fry (BF-2), were obtained from the National Fisheries Research Center (US Department of the In- terior, Seattle, Washington) in their 80-100 passage generation. These are well-charac- terized cells that have been under cultivation for nearly 20 years and that are available from the American Type Culture Collection, Rockville, Maryland [Wolf and Quimby, 1962, 1%6]. Both lines were shown to be free of mycoplasma by the method of Russell et a1 [1975]. Throughout the experiments RTG-2 cells were maintained at 18°C and BF-2 at 25°C. Stock cultures were passaged every week by splitting 1:3 (RTG-2) or 1:6 (BF-2). Cells used for experiments were taken from cultures that were confluent but still undergoing cell division as determined by the presence of mitotic figures when viewed with an inverted phase contrast microscope.

Culture Medium and Conditions Eagle’s minimal essential medium supplemented with 10% fetal bovine serum

(MEM-lo), glutamine (100 mM), nonessential amino acids (1 mM), sodium bicarbonate (8.9 mM), penicillin (15 units/ml), and streptomycin (15 pg/ml) was used throughout the experiments. The appropriate pH (7.1-7.3) was maintained by cultivation of the cultures in a 5% CO, atmosphere.

Fish Cell Genotoxicity 183

Experimental Procedures Cells were removed from the culture vessel with versene/trypsin, diluted 1 :3, and

then plated in a small volume onto alcohol-cleaned sterile glass slides in square petri plates (three slides per plate). After allowing approximately 1 hr for the cells to settle and attach, the plates were flooded with sufficient medium to cover the slides. The cells were then allowed to grow at their appropriate temperature for 6-8 hr, at which time they were exposed to the substance being studied. Three slides from each chemical and concentration were removed after 24 and 48 hr. These were fixed and stained according to the protocol described by Nichols et al [1977] and microscopically examined with a 470 x lens. A minimum of 100 anaphases per slide and two slides for each test concentration were examined (200 anaphases minimum at each time and concentration) and the percentage of normal and aberrant anaphase figures were recorded.

Each experimental set of cells was accompanied by a set of untreated cells and a set treated with spectrophotometric-grade dimethylsulfoxide (DMSO), the solvent used in all experiments. Throughout the experiments an attempt was made to classify the various types of anaphase aberration observed on the basis of the description given by Nichols et al [ 19771, but because of the extent and complexity of the chromosome damage from some treatments this was not always possible.

Test Chemicals Four polycyclic aromatic hydrocarbons (PAH), one aromatic amine, one nitrosa-

mide, and one fungal toxin were used in an attempt to perturb the normal anaphase con- figuration of chromosomes during mitosis. These were: Benzo(a)pyrene (B(a)P; Ald- rich Chemical Co., Milwaukee, Wisconsin), 3-methylcholanthrene (3-MC; Eastman Kodak Co., Rochester, New York), anthracene (Anth; Sigma Chemical Co., St. Louis, Missouri), 1 -naphthol (Naph; Fisher Scientific Co., Fair Lawn, New Jersey), 9-aminoacridine (9-AA, Sigma Chemical Co.), N-methyl-N’-nitro-N-nitrosoguanidine (MNNG; Sigma Chemical Co.), mitomycin-C (mit-C; Sigma Chemical Co.). With the exception of mit-C, which was dissolved in phosphate-buffered saline (PBS), each test compound was dissolved in DMSO to a final concentration of 1 mg/ml and further diluted with DMSO or culture medium to make the final working solutions. The chemicals were protected from light when not being used, and where necessary they were kept frozen until the stock solution was prepared. Control cultures were treated with DMSO at a final concentration of 0.5%, the highest concentration used in any of the experiments.

RESULTS Pilot experiments with the two cell types initially chosen indicated that the cells

capable of metabolizing promutagens exhibited more anaphase abnormalities in the presence of these compounds, thus making them a more sensitive and suitable system for this initial research effort. Comparison of the mixed function oxygenase activity in the two cell types by means of fluorometric methods [Nebert and Gelboin, 19681 showed that RTG-2 produced 724 picomoles of 3-hydroxybenzo(a)pyrene after 18 hr in- cubation with 1 pg/ml B(a)P, but BF-2 cells produced 0 picomoles. This agrees with the findings of Diamond and Clark [1970]. We therefore chose the RTG-2 cell line for the remainder of this study because it was capable of converting promutagens to their geno- toxic forms.


Examination of 1,800 untreated and solvent-treated anaphases from six separate experiments resulted in a base line of 85-90% normal anaphase cells (x = 12% aberrant cells, SD f 3.89), whereas the cultures treated with the five known mutagendcar- cinogens all had a substantial increase in the number of aberrant anaphase figures (Fig. 1). Based on t test comparisons of treated and control cells, a mean aberration value of 16-18% was significant at the 0.05 level. The majority of abnormal anaphases in the control cultures exhibited only a small lagging or displaced chromosome fragment (Fig. 2B). Treatment of the cultures with each of the test compounds, with the exception of anthracene and naphthol, resulted in increases in the percentage of aberrant anaphase figures and also in the complexity of the alteration (Fig. 2C,D,E,F). Table I summarizes the percentage of each type of aberration seen in several treatment groups. These values are somewhat inaccurate, since many of the cells actually had multiple defects. Neither of the negative control compounds, naphthol nor anthracene, produced an increase in the number of abnormal anaphase figures even though they were present in concentrations up to that which was inhibitory to cell proliferation (20 and 30pg/ml, respectively) based on the absence of mitotic figures at these concentrations.

00 .:.:.:

...

24 48 Exposure time

(hours)

end. .I .2.5 I .I I .I .5 I I 4 8 16 5 101530 5 1020 I 5 10 MNNG 9-AA E(a)P 3-MC MIT-C ANTH NAPH p g l m l pglrnl pg/ml pg/ml ng/rnl pg/ml pg/ml

Fig. 1. Rainbow trout cells were exposed to five known mutagens/carcinogens and two related compounds with no known mutagenic/carcinogenic activity. All five of the active compounds produced abnormalities in the anaphase chromosome structure compared to the untreated controls and cells exposed to related but nonmutagenic compounds. Each bar represents the abnormalities observed in uw) anaphase figures, except that the control represents 1,800 anaphase cells. MNNG, N-methyl-N’-nitro-N-nitrosoguanidine, a nitrosamide; 9-AA. 9-aminoacridine, an aromatic amine; B(a)P, benzo(a)pyrene; 3-MC, 3-methylcholanthrene; Anth, anthracene; Naph, naphthol-polycyclic aromatic hydrocarbons; and mit-C, mitomycin-C, a fungal toxin with antitumor activity.


Fig. 2. Anaphase aberrations observed in cultured fish cells following exposure to several classes of organic mutagens/carcinogens. (A) Normal anaphase; (B) lagging chromosome; (C) multiple defects; lagging fragments, lagging chromosomes, and displaced fragments; (D) attached fragments; (E) side-arm bridge; and (F) multipolar anaphase with chromosome fragments.


TABLE I. Types of Anaphsse Aberrations Seen in Mutagen-Treated RTG2 Cells

Attached Acentric Number of fragments fragments Bridges Side-arm bridges Multipolar cells

Treatment cells scored (To) (To) (TO) (To)

Controla 500 3.6 4.6 2.6 0.6 0.6 9-AA 200 18.5 9.0 13.5 0.5 1 .o Wa)P 200 13.5 8.5 7.5 0.5 1 .O MNNG loo 29.0 28.0 9.0 3.0 1 .O 3-MC 100 2s .O 24.0 5.0 0 0

aControl = untreated and DMSO-treated. 48 hr; 9-AA = 9-aminoacridine, 1 &ml, 24 hr; B(a)P = benzo(a)pyrene, 1 &ml, 24 hr; MNNG = N-methyl-N'-nitro-N-nitrosoguanidine, 1 &ml, 24 hr; 3-MC = 3-methylcholanthrene, 4 &ml, 48 hr.

Both of the mutagenic/carcinogenic polycyclic aromatic hydrocarbons (B(a)P and 3-MC) produced elevations in the number of abnormalities observed and both were more effective after 48 hr than after 24 hr, presumably because of the time required for metabolism to the active mutagenic form to occur. The 48 hr exposure to B(a)P in- creased the aberrations by a factor of 10 over that observed after 24 hr and was toxic or inhibitory to cell division at or above 5 pg/ml.

The direct-acting mutagens (MNNG, mit-C, and 9-AA) were also effective in in- creasing the number of observed abnormalities. MNNG and 9-AA did not show as dramatic an increase in effectiveness at 48 hr as did the polycyclic aromatics, presumably owing to their immediate effect on the cells and short life span in culture medium. Mit- C, the antitumor fungal product, produced a dramatic increase in abnormalities and required only nanogram amounts to produce the effect, whereas all other compounds were present in approximately microgram/milliliter concentrations. With the exception of 9-AA, which inhibited cell division at concentrations above 1 pg/ml, each of the mutagenic compounds inhibited mitosis after 48 hr exposure to the highest concentration tested.

DISCUSSION

Several classes of organic mutagens/carcinogens were shown to produce significant cytogenetic changes in the form of anaphase aberrations in cultured trout cells. Both direct-acting mutagens and promutagens, which require metabolism to their active intermediate form, were capable of producing abnormal anaphase figures and they ap- peared to do so in proportion to their concentration and length of exposure to the test cells.

Our initial examination of cultured RTG-2 cells revealed a spontaneous background of abnormal anaphase figures of approximately 10- 15%. Of these, the majority was lagging chromosomes or chromosome fragments, which would not likely pose a hazard to the cells involved, since each daugter cell would receive a normal complement of chromosomes. Occasionally a nondisjunction, known to be deleterious to the cells since one daughter cell receives one too few chromosomes following cell division, was observed. Essentially all of the abnormalities were classifiable by the scheme described by Nichols et al [ 19771. This classification consists of 1) acentric fragments, which are chromosomal fragments left at the equator during cell division; 2) attached fragments, which


are chromosome fragments that lag behind the main body of chromosomes and appear to be attached by a thin strand of thread-like chromatin; 3) chromsome bridges, which are units of chromatin stretched between the two groups of anaphase chromosomes; 4) side-arm bridges or pseudochiasma; and 5 ) tri- or multipolar anaphase figures.

The aberrations described are macrolesions that can be observed both in anaphase and metaphase, with standard cytological techniques. Some aberrations have no known counterpart in metaphase and may result in cell mortality following mitosis. Any alteration in chromosomal number or loss of chromosomal fragments would likely result in cell death [Dewey et al, 19711.

The mechanism(s) by which the compounds we studied exert their effects are not known. Most of these compounds bind to a number of cellular components including DNA [Brookes and Lawley, 1964; Goshman and Heidelberger, 19671. It may be that by modifying the structure of one strand or part of one strand of DNA within a chromosome, the relationship during mitosis is disturbed. Damage to the spindle apparatus has also been shown to have modifying effects capable of altering the final chromosomal arrangement. It is likely that the mechanism of modification differs with each class of compounds tested.

The lack of any increase in chromosomal lesions in the cells treated with naphthol and anthracene, the nonmutagenic polycyclic aromatic compounds, suggests that the lesions may be linked to each chemical’s capacity to produce mutations and not specifi- cally to their chemical class. Each of the PAHs that showed activity produced a higher rate of anaphase damage and at lower concentrations after 48 hr exposure. Although this suggests the necessity for metabolic activation, it will be necessary to perform metabolic inhibition studies with these cells to confirm this unconditionally.

This cytogenetic procedure is attractive for aquatic animal testing because many lower vertebrates and invertebrates have extremely small chromosomes, making most of the standard cytogenetic techniques unsuitable owing to poor resolution of chromosomes and chromatids. In some instances these problems have been overcome by use of cells from species that have either larger than average chromosomes or relatively few chromosomes compared to other fish. Kligerman and Bloom [1976], Barker and Rack- ham [1979], Kligerman [1979], and Stromberg et a1 [1981] successfully used fresh- and saltwater species as sources of chromsomes for mutagen testing by the sister chromatid exchange method. Pesch and Pesch [1980] were able to adapt this same method to the chromosomes of a polychaete worm, while Kocan et a1 [1979,1981] succeeded in using fish cells for mutagen testing by showing that cytotoxicity and the production of ouabain-resistant mutants were possible in cultured cells derived from several species of fish. The use of fish chromosomes was likewise extended to wild fish embryos by Long- well and Hughes [1980] when they described anaphase abnormalities in mackerel embryos that had been exposed to an oil spill.

The in vitro system described here is a cytogenetic measure of chromosome damage and is comparable to the Ames test and the SCE test in its sensitivity for detecting the various classes of compounds that we tested. The RTG-2 cells used in this system have the advantage of possessing a highly active mixed function oxygenase system, thus eliminating the need to incorporate microsomes derived from unrelated species into the cultures. It is also inexpensive and rapid, making it suitable as a substitute for other tests when aquatic ecosystem testing is being considered. Although this anaphase test procedure has not been extensively used in mutagen/carcinogen testing, it was given a high


priority as an in vitro test procedure by the Ad Hoc Committee of the Environmental Mutagen Society and the Institute for Medical Research [Nichols et al, 19721. In addition, induction in our laboratory of similar aberrations in the chromosomes of fish embryos suggests that it will prove useful as an in vivo test system.

It appears likely that with the large number of test procedures available, fish and invertebrate cells can be successfully used for mutagen/carcinogen testing of aquatic contaminants and as model systems for the study of diseases of higher animals.

ACKNOWLEDGMENTS

The authors wish to thank D. Mulcahy and A.C. Fox, National Fisheries Re- search Center, Seattle, Washington, for supplying the cell lines and Ms. Val Munzlinger and Ms. Virginia Wejak for secretarial assistance.

This work was supported by National Institutes of Health grants HL-03174 and ES-02 190 and National Oceanic and Atmospheric Administration (OMPA) grant NASORAD00053.

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