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Mutagenesis, Vol. 18, No. 1, 73-76, January 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press

Application of a sea urchin micronucleus assay to monitoring aquatic pollution: influence of sample osmolality

Kyoko Saotome1,3 and Makoto Hayashi2

1 Yokohama City Institute of Health, 1-2-17 Takigashira, Isogo-ku, Yokohama 235-0012, Japan and 2 Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have improved our sea urchin micronucleus assay for aquatic samples and used it to evaluate marine pollution. We found that the water samples we had collected for 2 years from the Tokyo bay coast near Tokyo, an industrial megalopolis, were positive due to the water samples being hypo-osmotic rather than to chemical pollutants. The evidence was as follows: (i) the osmolality and salinity of the samples were about half that of sea water; (ii) the micronucleus frequency induced in the water sample decreased to the control level when the osmolality was increased to that of sea water; (iii) artificial sea water diluted with distilled water induced micronuclei dilution-dependently. Since micronucleus induction in the sea urchin assay is influenced by sample osmolality, the osmolality must be adjusted to that of sea water for the assay and osmotic pressure must be considered when evaluating water pollution.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Monitoring the aquatic environment is an important activity to prevent aquatic pollution caused by chemical contamination. We have used as monitoring techniques chemical analysis of water or aquatic organisms and acute and chronic toxicity assays on different life stages of aquatic organisms (fish and invertebrates) (Kobayashi, 1993Go). In the last decade, we have used monitoring by genotoxicity assays that measure gene mutations in bacteria (Kinae et al., 1985Go; Hashizume et al., 1992Go) or DNA damage in aquatic organisms (Sasaki et al., 1997Go). Recently, the development of micronucleus (MN) assays for several aquatic organisms, including fish (Carrasco et al., 1990Go; De Flora et al., 1993Go; Al-Sabti and Metcalfe, 1995Go; Hayashi et al., 1998Go), amphibians (Jaylet et al., 1986Go; Fernandez et al., 1993Go) and molluscs (Wrisberg, 1991Go; Burgeot et al., 1995Go) have provided another feasible approach to monitoring the aquatic environment.

Sea urchin bioassays have been widely used to evaluate the marine environment (Wilson and Armstrong, 1961Go; Okubo and Okubo, 1962Go; Oshida et al., 1981Go; Kobayashi, 1993Go). Since sea urchins are distributed throughout the world and some species live under rocks at the low tide line, we can easily sample them throughout the year by collecting different species. We can also obtain eggs and sperm, fertilize in vitro and easily synchronize their development. Though cytogenetic abnormalities have been reported (Vacquier and Brachet, 1969Go; Hose et al., 1983Go; Pagano et al., 1983Go) by which chromosome aberrations may occur in sea urchins, their detection is difficult because of smallness in size (1–3 µm) (Colombera, 1974Go; Saotome, 1982Go, 1987Go, 1989Go, 1991Go). In a previous paper (Saotome et al., 1999Go), we reported on the MN assay in sea urchin blastulae and demonstrated that model mutagens [mitomycin C (MMC), vinblastine and 1-ß-D-arabinofuranosylcytosine (AraC)] could induce MN dose-dependently and suggested application of the assay to the aquatic environment.

In the present study, we report on improvements to the MN assay and application of the assay to sea and river water. We also clarify several problems in the field application of the assay.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
We used MMC (Kyowa Hakko Kogyo, Tokyo, Japan) dissolved in artificial sea water (ASW) (Jamarin U; Jamarin Laboratory, Osaka, Japan) as the positive control. We tested model chemicals that are potential water pollutants, i.e. captan as a pesticide, triphenyltin chloride (min. 95%) as an anti-growth substance, 2-mercaptobenzothiazole as a vulcanization surfur accelerator and Trp-P-1 (acetate form) and 2-acetamidofluorene as polyaromatic hydrocarbons adsorbed to blue cotton. They were purchased from Wako (Osaka, Japan) and dissolved in dimethyl sulfoxide for use. Acetylcholine chloride was obtained from Wako.

The water samples were collected at two points off the coast near Tokyo (Yume-no-Shima), an industrial megalopolis, during 1995–1997. The samples were stored at –80°C until use.

For the MN assay, we used the sea urchin Hemicentrotus pulcherrimus (2n = 42) collected from the Misaki Marine Biological Station, University of Tokyo. We used ASW throughout the work. We obtained eggs and sperm by injecting 0.1 ml of 0.01 M acetylcholine chloride into the body cavity of mature adults. We diluted the sperm and added it to egg suspensions. When the fertilized eggs were incubated at 18°C for about 5 h, they cleaved eight times and reached the early blastula stage (about 256 blastomeres). We used blastulae for assay after we squashed them lightly and confirmed that there were about 256 blastomeres.

We conducted the MN assay as previously reported (Saotome et al., 1999Go) with the following modifications: (i) 24-well microtiter plates, (ii) 2500 embryos/well, (iii) 1 ml sample/well, (iv) a high cell density, (v) examination of 4000 cells and (vi) MMC as a positive control. We placed 1 ml of ASW (control) or test sample and 50 µl of the blastulae suspension (50 000/ml sea water) in microtiter plates and left them at 18°C overnight. The early blastula stage was the best for treatment and the gastrula stage for preparation to obtain the highest MN frequency, as already reported (Saotome et al., 1999Go). The MN frequency was low in early cleaving embryos owing to a lack of the G phase, while MN were easily identified in gastrulae, which have a G phase and a small amount of cytoplasm. When the control embryos reached the gastrula stage (had divided at least twice), we transferred the suspensions to microtubes and centrifuged them for 5 min at 300 g. We then, suspended the embryos in 1 M urea and dissociated them by pipetting. We fixed the dissociated cells with a methanol:acetic acid (9:1) mixture and washed them twice with fresh fixative. We made a condensed cell suspension and placed a drop of it on a glass slide and permitted it to air dry. We stained the preparations with 0.1% acridine orange in Sørensen's phosphate buffer or a standard pH 6.9 solution (Horiba Ltd, Tokyo, Japan) for 2 min and then rinsed them several times with the buffer or the solution. We examined 4000 cells at a magnification of 600x with the aid of a fluorescence microscope equipped with a B filter. We measured osmolality with an Advanced Cryomatic Osmometer Model 3C2 and the salt concentration with a digital Pefractometer Pallet 100.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The six modifications used in the present study (see Materials and methods) improved our assay (Saotome et al., 1999Go) in that we were able to simultaneously handle many samples, decrease the sample volume, effectively examine many cells and increase the sensitivity of the assay. When the embryos were treated with MMC in this system, the MN frequency increased proportional to the concentration at 0–20 µg/ml (Figure 1Go).



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  		Fig. 1. . MN frequency induced in sea urchin embryos by MMC.

 
Based on a statistical review of historical data, the probability of <=1.25{per thousand} was less than 0.05. Therefore, we decided to judge MN induction in this paper as follows: (i) a MN frequency of <=1.25{per thousand} was negative, (ii) a MN frequency >2.5{per thousand} was positive and (iii) a MN frequency between 1.25 and 2.5{per thousand} was inconclusive. Accordingly, MMC clastogenicity at 0.6 µg/ml was inconclusive and at 1.3, 2.5, 5.0, 10.0 and 20.0 µg/ml was positive (Figure 1Go).

In a preliminary study we tested MN induction by the model chemicals at several concentrations. Embryos treated at lower concentrations showed delayed gastrulation and those treated at higher concentrations developed to blastulae having abnormal blastocoels with excess cells. The developmental toxicity caused by the chemicals was similar to that previously reported for MMC, vinblastine and AraC (Saotome et al., 1999Go). Table IGo shows MN induction at the lowest concentration of pollutants that caused developmental toxicity, except for 2-mercaptobenzothiazole, which was inconclusive but induced abnormal nuclei at a frequency of 13.0{per thousand}. The MN frequency induced by the chemicals increased dose-dependently, ranging from 5.7{per thousand} for captan to 26.5{per thousand} for 2-acetamidofluorene. The embryos developed to pluteus with MN when they did not suffer severe developmental damage, but when their development was abnormal, they had irregular nuclei and not MN.


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  		Table I. . MN induction by five pollutants at the lowest concentration showing developmental toxicity
 
We applied the sea urchin MN assay to sea water and monitored the marine environment. Though samples from several collection points were negative in the MN assay (data not shown), the embryos treated with the water sample from Tokyo Bay showed acute developmental toxicity. When we diluted the samples 1- to 16-fold with ASW, acute developmental toxicity was not affected until 2- to 16-fold. From December 1995 to March 1997 we collected water samples four times and assayed the samples diluted 2-, 4- or 8-fold with ASW. Figure 2Go shows the values for samples diluted 2-fold, which showed the same tendency as those diluted 4- and 8-fold. The water samples from Tokyo Bay induced MN at frequencies ranging from 3.8 to 24.8{per thousand}; these values were high compared with the frequency (5.8{per thousand}) induced by MMC (10 µg/ml), the positive control. These results could suggest that Tokyo Bay water was always clastogenic, although the extent varied with the year of collection.



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  		Fig. 2. . MN frequency (bars), osmolality (closed circles) and salinity (open circles) in the water samples collected from Tokyo Bay 1995–1997. Samples were diluted 1:1 with ASW; a and b designate different collecting points. ASW is the negative control and MMC is the positive control.

 
Since the MN frequencies induced by Tokyo Bay water seemed too high to be due to chemical pollutants or synergistic effects produced by a complex mixture of several chemicals, we considered other possibilities. The pH of the water samples was 7.8, the same as ASW, and increased to 8.2 when they were diluted 2-fold with ASW. In our system, development was normal from pH 5.7 to 9.5. We kept the temperature constant, so osmotic pressure and salinity seemed the likely problem, especially since several rivers empty into Tokyo Bay, diluting it with fresh water. The osmolality of the water samples ranged from 576 mOsm/kg H2O in December 1995 to 313 mOsm/kg H2O in March 1997 (Figure 2Go), whereas that of the ASW was 927 mOsm/kg H2O. The salinity of the water samples we collected ranged from 21{per thousand} in December 1995 to 11{per thousand} in March 1997 (Figure 2Go), whereas that of the ASW was 32{per thousand}. The osmolality corresponded to the salinity. When we adjusted the osmolality of the March 1997 water sample to that of sea water, it no longer induced MN (Figure 3aGo). When we diluted ASW with distilled water (DW) at ratios of 9:1, 8:2, 7:3, 6:4 and 5:5 the MN frequency increased relative to concentration at 9:1 and 8:2 (Figure 3bGo) and developmental toxicity was observed at 7:3, 6:4 and 5:5.



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  		Fig. 3. . MN induction by water samples from Tokyo Bay versus ASW. (a) Decrease in MN frequency induced by the March 1997 sample following adjustment of its osmolality to that of sea water. (b) MN frequency induced by ASW diluted with distilled water (DW) 9:1 and 8:2.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
We here report an improvement of the sea urchin embryo MN assay for environmental aquatic samples. We were able to handle 24 samples simultaneously and speedily by using 24-well microtiter plates instead of 35 mm dishes, which we had used in our previous study (Saotome et al., 1999Go). Although we examined 4000 rather than 1000 cells to determine MN frequency, it took only ~15 min when we used a high density cell preparation that permitted analysis of 50–100 cells in one microscope field.

The MN frequency increased directly with MMC concentration (Figure 1Go). The spontaneous MN frequency was almost 0.6{per thousand}. The MN frequency induced by MMC at 10 µg/ml was 6–9{per thousand}, which was a little lower than that (10–20{per thousand}) obtained by the previous version of the assay (Saotome et al., 1999Go). Because we increased the number of cells analyzed, we could decrease the minimum detectable concentration of MMC from 6.0 to 0.6 µg/ml. Although we used embryos at a concentration of 2500/ml in our system, this concentration may be high compared with that (30/ml) in aquatic invertebrate embryo toxicity tests (mussel and oyster). However, we confirmed that embryos developed normally to the pluteus at that concentration and we could obtain 107–108 eggs easily from one female.

The potential pollutants of the aquatic environment induced MN in the sea urchin (Table IGo) at concentrations that were high compared with those detected in environmental samples; for example 0.0009–0.0017 µg/ml 2-mercaptobenzothiazole was detected in Tokyo Bay (Sasaki et al., 1995Go) but >0.6 µg/ml was required for MN induction. The ability to detect current levels may be low in this system. These pollutants, however, rarely exist singly but as complex mixtures. In complex mixtures, two or more pollutants may interact synergistically to induce MN, and this assay would then be appropriate for overall evaluation of water contamination.

When we applied the assay to sea water, the samples from Tokyo Bay induced MN at a high frequency (Figure 2Go). The collecting area is near reclaimed land, where golf courses exist and rivers empty and where halogenated hydrocarbons (such as trichloroethylene, tetrachloroethylene, 1,1,1-trichloroethane and bromodichloromethane, 0.00013–0.00022 µg/ml), pesticides (such as diazinon, 0.0002 µg/ml) and other chemicals (such as 2-mercaptobenzothiazole, 0.0009–0.0017 µg/ml) have been detected (Sasaki et al., 1995Go). We therefore thought at first that serious pollution was responsible for our positive results. The concentrations of these chemicals, however, seemed too low to explain MN induction by these chemicals. We found that it was the hypotonicity of the sample water that was responsible and that hypo-osmotic ASW also induces MN (Figure 3bGo). Thus osmotic adjustment may be necessary when this assay is applied to field samples of sea water.

Osmotic pressure is an influential factor in genotoxicity assays. Chromosome aberrations are detected in V79 Chinese hamster cells (Nowak, 1989Go) and human lymphocytes (Kalweit et al., 1990Go) treated under hypotonic culture conditions. Hyper-osmotic solutions also induce chromosome aberrations (Ishidate et al., 1984Go). The MN formed under non-physiological osmotic pressure may be derived from chromosomal aberrations as follows: lowered osmolality could cause lysosomal damage leading to endonuclease release (Nowak, 1989Go) and hyper-osmotic medium could affect chromatin structure and/or enzyme activities, altering the efficiency and accuracy of DNA replication or repair (Galloway et al., 1987Go).

When we evaluate water pollution in the field we must pay attention to other physiological factors besides osmotic pressure, including pH, temperature and dissolved oxygen concentration, because these factors affect larval survival or the development of various invertebrate species when they are non-physiological (Pechenik, 1987Go). Also, high and low pH induce chromosome aberrations in cultured Chinese hamster ovary cells (Morita et al., 1989Go) and hyperthermia induces MN in mice (Asanami and Shimono, 1998Go). Induction of MN by high or low oxygen concentration has not been sufficiently investigated yet. We must also consider the combined effect of two or more of these factors (Brenko and Calabrese, 1969Go; Pechenik, 1987Go).

This assay can be adapted to river water by adjusting its osmolality to that of sea water with Jamarin U. Althhough we have assayed river water samples from polluted and populated areas, we have not yet obtained any positive results.

In this study to evaluate sea water in the field, we assayed water samples using embryos and sea urchins raised in the laboratory, but we also think it important to assay using target cells from embryos or adult sea urchins obtained in the field, as was done with fish (Hayashi et al., 1998Go). Since collecting embryos is difficult, developing a MN assay for adult sea urchins is the next important problem to solve.


   Acknowledgments
 
The authors are grateful to the staff of the Misaki Marine Biological Station, University of Tokyo, for kindly supplying materials. We also thank Dr Wakata of Yamanouchi Seiyaku (KK) for kindly allowing us to use the osmometer. This research was supported by a grant from the Environmental Agency of Japan.


   Notes
 
3 To whom correspondence should be addressed at present address: Division of Environmental Policy, Environmental Protection Bureau, Yokohama City, Minato-cho 1-1, Naka-ku, Yokohama 231-0017, Japan. Tel: +81 45 671 4103; Fax: +81 45 641 3580; Email: ki00-saotome{at}city.yokohama.jp Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received on April 29, 2002; revised on September 9, 2002; accepted on September 9, 2002.


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