Mutagenic effects of tributyltin and inorganic lead ( Pb II ) on the fish H . malabaricus as evaluated using the comet assay and the piscine micronucleus and chromosome aberration tests

Genotoxicity studies on toxic metals and their organic compounds are very important, especially so in the investigation of the effects of these compounds on the aquatic environments where they tend to accumulate. The use of endemic aquatic organisms as biological sentinels has proved useful to environmental monitoring. We assessed the mutagenic potential of tributyltin (TBT) and inorganic lead (PbII) using samples of the fish Hoplias malabaricus (commonly called traíra) using the comet assay and the piscine micronucleus and chromosome aberration tests. Eighteen H. malabaricus were acclimatized in three individual aquariums, each containing six fish, six fish being exposed to 0.3 μg/gof body weight (bw) of TBT, six to 21 μg/gbw of PbII and six being used as controls. Exposure to TBT and PbII was achieved by feeding the fish every five days with Astyanax (a small fish that is part of the normal diet of H. malabaricus) which had been injected with solutions of TBT, PbII or with water (the control group). After two months the H. malabaricus were sacrificed and their peripheral blood collected and subjected to the comet and micronucleus assays, the chromosome aberration assay being conducted using kidney-tissue. Although the comet assay showed now mutagenic effects at the lead concentrations used but encountered results with TBT, the micronucleus and chromosome aberrations assays both indicated that TBT and PbII are potentially mutagenic (p < 0.01), the micronucleus assay showing morphological alterations of the nucleus.


Introduction
The aquatic environment is the ultimate destination for almost all urban, industrial and agricultural wastes.Because of their real or perceived potential as mutagens or carcinogens, xenobiotic chemicals have often been the focus of attention, frequent pollutants of marine and/or fresh waters being inorganic lead (PbII) and organic tin compounds (Pain, 1995;Johnson, 1998;Alzieu, 2000;Godwin, 2001).
Organic tin compounds such as tributyltin (TBT) are used for many purposes, principally in antifouling paints and the plastics industry (Alzieu, 2000), several reports strongly suggesting that TBT can adversely affect the physiology of not only mammals (Mizuhashi et al., 2000) but also marine organisms (Alzieu, 1991(Alzieu, , 2000;;Santos et al., 2000).In spite of the fact that tin can bind to DNA no direct damage has yet been demonstrated, although some studies have shown apoptosis in mouse timocytes and hepatocytes exposed to different concentrations of TBT (Yamanoshita et al., 2000).
Lead has been in use for thousand years, although the toxic effects of lead compounds were only recognized less than hundred years ago.Inorganic and organic lead enters the environment in several ways (Pain, 1995;Godwin, 2001) and can have diverse effects on organisms, including mimicking essential elements (e.g.calcium, magnesium, iron and zinc) with consequent effects on enzymes containing SH groups (Jennette, 1981), increased incorporation of erroneous nucleotides (Johnson, 1998) and the accumulation of free radicals due to alteration of the oxidative processes of cells and effects on repair mechanisms in which lead has been implicated as a co-carcinogen (Fracasso et al., 2002).
This paper reports the results a study of the neotropical fish Hoplias malabaricus in which this fish was exposed to TBT and PbII and the effects of the exposure assessed using the comet assay and the piscine micronucleus and chromosome aberration tests.

Methodology
The fish we chose as a model was Hoplias malabaricus [Bloch, 1794] (Trahira, traíra in Portuguese), a neotropical piscivorous fish witn a wide geographic distribution range, from Panamá to the rio Colorado in south of Argentina.This fish has the advantage that it is easily fed under laboratory conditions and, because it devours its pray whole, can be easily exposed to xenobiotics by feeding with a prey species previously injected with the material under investigation, a further advantage being that this species has 40 relatively large bi-armed chromosomes which makes studying the genetic effects of metals easier.
In our study, we used 18 adult H. malabaricus reared in captivity at a fish farm (Fazenda Canguiri, Universidade Federal do Paraná, Curitiba, Brazil) which were divided into three groups of six fish, one group being exposed to tributyltin (TBT), one group to lead (PbII) and one group be kept unexposed as a control group.Each group was housed in a separate aquarium and acclimatized to a water temperature of 22 °C (≈ pH 7.6) and a 12 h light/dark photoperiod for 60 days before starting the experiments.
We exposed our H. malabaricus to TBT and PbII by feeding them with Astyanax (a small fish that forms part of the normal diet of Hoplias) which had been artificially contaminated by intraperitoneal injection of tri-n-butyltin or lead (Aldrich Chemical Co) in such a way as to produce a final concentration of 21µg PbII/g of body weight (bw) or 0.3 µg TBT/g bw in the H. malabaricus after a suitable feeding schedule.The feeding schedule consisted of offering each H. malabaricus one Astyanax each day for four days over a period of 13 feed-cycles (about two months), Astyanax injected with TBT or PbII being fed to the appropriate experimental groups and Astyanax injected with water only being fed to the control group.Four days after the last feed-cycle the H. malabaricus were sacrificed after being anesthetized with 3-aminobenzoic acid ethyl ester (Sigma, São Paulo-SP, Brasil), blood being collected from the caudal vein for the comet assay and the piscine micronucleus test and kidney tissue for the chromosome aberration test.

Comet assay
For the comet assay, also known as the single-cell gel test (SCGE), we used the procedure of Singh et al. (1988), with a few modifications.Clean microscope slides were prepared by covering them with a thin layer of 0.75% (w/v) NMP agarose (Gibco), two slides being prepared for each fish.For each slide, 10 µL of whole fish blood was diluted in 1 mL of bovine calf serum and 10 µL of this mixture placed in 120 µL of LMP agarose gel (Gibco) which was then spread on the base layer of the slide and a cover slip added, the agarose gel being allowed to solidify by placing the slide in a refrigerator for 10 min.When the gel had solidified, the coverslips were gently removed and the slide placed in a lysis solution at 4 °C for 1 h to remove cellular proteins.After lysis, the slide was placed in an electrophoresis box containing an alkaline solution (pH > 13) for 25 min at 4 °C and then subjected to electrophoreses at 25 V, 300 mA, for 25 min, after which the slide was removed, neutralized, fixed in absolute ethanol and stained with 20 µL of 10 µL/mL ethidium bromide.Comets were scored using a Zeiss epifluorescence microscope.For each fish, 100 cells were visually analyzed according to the method of Kobayashi et al. (1995), the comets being classified as having: no detectable DNA damage and no tail (class 0); a tail with a length less than the diameter of the nucleus (class 1); a tail between one and two nuclear diameters long (class 2); a tail longer than twice the diameter of the nucleus (class 3).Apoptotic cells that presented totally fragmented nuclei were not scored (Speit and Hartmann, 1999).Differences between fish treated with TBT and PbII and untreated control fish were tested using the nonparametric Kruskal-Wallis test.

Piscine micronucleus test (PMT)
For each fish, two microscope slides were prepared by drawing a drop of fish blood over the slide to form a thin smear which was air-dried for 24 h, fixed with absolute methanol (Merck) for 1 h and stained for 20 min with 5% (w/v) Giemsa in pH 6.8 phosphate buffer (Merck).For each fish, 4000 erythrocytes were examined under 1000X magnification and scored for the presence of both typical micronuclei and nuclear alterations manifested as changes in the normal elliptic shape of the nuclei (Ayllon and Garcia-Vazquez, 2000).These two features were considered as nuclear abnormalities and scored together, frequencies being tested for significance using the χ 2 test.

Chromosome aberration test (CAT)
Chromosome preparations were obtained by the short term culture of kidney cells (Fenocchio et al., 1991), 30 metaphase plates from each individual being scored for alterations such as the number of breaks or gaps and the level of de-condensation.Observed frequencies for treated and controls were tested using the χ 2 test.

Results and Discussion
The comet assay showed a significant increase of tailed nucleoids in the erythrocytes of fishes treated with PbII, while fish treated with TBT showed a lower frequency of comets than untreated controls (Figure 1).
The spontaneous (or basal) frequency of micronuclei (MN) in fish is normally very low and in our study no significant differences were observed in the PMT between unexposed control fish and fish exposed to TBT and PbII.However, statistically significant differences were seen between the frequency of erythrocytes with altered nuclear morphology as compared to conserved nuclear morphology (Table 1, Figure 2 a,b,c).
The number of micronuclei in fish cells is a highly variable feature and different authors report different numbers of spontaneous micronuclei for the same species (Gustavino et al., 2001), although various authors (Hooftman and de Raat, 1982;Hose et al., 1987;Ayllon and Garcia-Vazquez, 2000) have suggested that variations in the shape of nucleous could represent an alternative approach for detecting genotoxicity.In spite of the fact that a correlation between nuclear abnormalities and genotoxic effects has not yet been established, preliminary observations strongly suggest that such morphological alterations could be a manifestation of the effects of xenobiotics.It has been suggested that in genotoxicity studies with fish models both, alterations in nuclear morphology and micronuclei number, should be considered when using the PMT (Ayllon and Garcia-Vazquez, 2000;Hose et al., 1987).
Analysis of chromosome aberrations using the CAT showed that TBT and PbII promote structural alterations (e.g.gaps and breaks) but no numerical changes were observed (Table 2), although de-condensation of chromatin on chromatid arms (Figure 3) was observed.
Chromosome aberration test have not been widely used in fish due to the characteristics of their karyotypes, although H. malabaricus appears to be an appropriate biological model because this species has only about 40 medium sized bi-armed chromosomes.In our experiments, we frequently observed chromatin de-condensation which we scored as chromosome aberrations (Table 2, Figure 3).
All the end-points analyzed in this paper show that PbII significantly increases DNA damage, principally by inducing single strand breaks that could possibly initiate double strand breaks, as result of the inactivation or alteration of repair mechanisms (Obe et al., 2002).nucleoids in the comet assay.Although there is no completely satisfactory explanation for these observations TBT has also given ambivalent results in some other studies, Yamanoshita et al. (2000) having reported that, depending on concentration, TBT can either cause apoptosis or protect against it.To resolve these questions new assays using different concentrations of TBT are needed.This paper is the first to present results based on the use of three end-points to investigate the effects of toxic metals on an endemic neotropical fish, our data indicating that H. malabaricus represents a good experimental model for genotoxicity studies.

Figure 1 -
Figure 1 -Absolute number of comets found in each treatment.

Figure 3 -
Figure 3 -Most frequent chromosome aberrations.Thin arrows indicate chromatid breaks and thick arrows chromatin de-condensation.

Table 1 -
Number of cells with micronuclei and morphological alterations in their nuclei.

Table 2 -
Absolute number of chromosomal alterations found in each treatment.

Table 3 -
Number of cells in each comet assay class.
*Significant by the Kruskal-Wallis test at p < 0.01.