MATERIALS AND METHODS

Animals
Red-tailed tinfoil barb (P. altus) 27.61±5.17 g in body weight and 13.50±0.77 cm in total length, were purchased from a commercial hatchery in Bangkok, Thailand. Tap water was filtered with activated charcoal (Aquapur, thysen, FRG) to eliminate chemical contamination. The physicochemical characteristics of water were measured daily, according to the experimental procedures described in Standard Methods for the Examination of Water and Wastewater (APHA, 2005). Conductivity was measured with Hanna instruments Model 3 DiST WP (Hanna Instruments Inc., USA). The pH was measured with a Cyberscan 510 (Eutech Instruments Inc., USA) and the temperature was measured with a glass mercury thermometer. A 16:8 h light-dark cycle was maintained throughout.

Acclimatization to laboratory conditions for 7 days was done using dechlorinated tap water that had the following physicochemical characteristics: temperature = 29.0±1.0°C, pH = 6.6-7.0, total hardness = 68-80 mg L^-1 (as CaCO₃), alkalinity = 75-80 mg L^-1 and conductivity = 190-220 μmhos cm^-1. Chlorine residual and ammonia were below detection limits. Fish were fed twice a day with 37%-protein commercial fish food (Charoen Pokphand Group, Bangkok, Thailand). The quantity of food was 2% of the initial body weight per day.

Experimental Design
Fish (n = 40) were randomly assigned to four equally sized groups. Each fish was injected intraperitoneal 1 mL/100 g BW with dose selected through range-finding trials that produced fairly quantifiable changes in cytogenetic protocol used as follows: (1) distilled water; (2) group II, 0.05% CdCl₂.H₂O (Sigma, Germany, CAS No. 10108-64-2); (3) 0.05% CdCl₂.H₂O plus 0.05% AA (Sigma, Germany, CAS No.50-81-7) and (4) 0.05% AA. The injected fish were kept separately in the glass flow-through aquaria (50 x 50 x 120 cm) with continuous aeration were filled with 200 L of dechlorinated tap water whose physicochemical characteristics were the same as those described previously.

At different times (24, 48, 72 and 96 h), 2 fish of each group were anesthetized with 0.2 g L^-1 MS-222 (tricaine methan sulphonate, Sigma, Germany, CAS No.886-86-2), weighed and measured. The organs (blood, gill, liver and fin cells) were removed and prepared for micronuclei and nuclear abnormality tests.

Micronuclei (MN) and Nuclear Abnormality (NA) Tests: Giemsa staining
Peripheral blood samples were obtained by caudal vein puncture using a heparinized syringe. Blood was smeared immediately on clean grease free microscope slides, air dried for 12 h and then fixed in absolute ethanol for 20 min. Each slide was stained with 5% Giemsa solution for 30 min.

Gill arches were removed and placed into vials containing Carnoy’s fixative (3:1 methanol:acetic acid). Gills were transferred into 20% acetic acid solution for 15 min for tissue maceration. After this chemical maceration, epithelial cells were then scraped off the gills and placed on clean slides. After tissue clumps on slides were removed, slides were air-dried and stained with 5% Giemsa solution for 30 min.

A portion of liver tissues were removed and placed into vials containing Carnoy’s fixative. Small pieces of tissues were transferred into vials containing 45% acetic acid solution for 30 min for tissue maceration. After maceration, tissues were gently minced and filtered to obtain a cell suspension. The obtained cells were smeared on a clean slide, air-dried and stained with 5% Giemsa solution for 30 min.

The edge of caudal fins was cut at a depth of 2-3 mm and placed into vials containing Carnoy’s fixative. Small pieces of tissue were transferred into vials containing 45% acetic acid solution for 30 min for tissue maceration. Then tissues were gently minced and filtered to obtain a cell suspension. The obtained cells were smeared on a clean slide, air-dried and stained with 5% Giemsa solution for 30 min.

Three slides were prepared for each organ. From each slide 1000 cells were scored under 1000x magnification using a Nikon E600 light microscope and photographed using a Nikon DXM 1200 digital camera (Tokyo, Japan). Slides were scored by a single observer using blind review. Frequencies of micronucleated (MN) and nuclear abnormality (NA) cells were expressed per 1000 cells (%).

Micronuclei and Nuclear Abnormality Cells Scoring
Only the cells clearly isolated from the surrounding cells were scored. The criteria for the identification of MN were earlier described: (a) MN must be smaller than one-third of the main nuclei, (b) MN must be clearly separated from the main nuclei, (c) MN must be on the same plane of focus and have the same color. Cells having two nuclei with approximately equal sizes were considered as binucleates (Fenech et al., 2003). Nuclear abnormality shapes were scored into one of the following categories: blebbed nuclei (BL), lobed nuclei (LB), notched nuclei (NT) and binuclei (BN) (Carrasco et al., 1990). The result was expressed as the mean value (%) of the sum for all the individual abnormality observed.

Statistical Analysis
All data were expressed as means±SD. A two-way analysis of variance was used to determine the significance of micronuclei and nuclear abnormalities test. The least-significant difference (LSD) was used for determination of significant differences at p<0.05.

RESULTS

Normal erythrocyte, approximately diameter 7 μm, was contained mainly elliptical nuclei (Fig. 1A). The small non-refractile circular or ovoid particle lying in the cytoplasm and resembling a nucleus with respect to staining properties was considered as micronuclei. The size of the micronuclei varied to some extent (between 1/25th and 1/5th that of nuclear size) but the number was always one. The position of the micronuclei in the cytoplasm also varied, some located very near to the nucleus or some located vary far even at the periphery of the cell (Fig. 1B). Some of the nuclei clearly deviated from their normal shape and were either blebbed (BL, Fig. 1C), lobed (LB, Fig. 1D), notched (NT, Fig. 1E) and binucleated cells (BN, Fig. 1F). All abnormalities of nuclei were scored. In briefly, cells with two nuclei were considered as binucleates. The two nuclei should be approximately equal size, staining pattern and staining intensity, within the same cytoplasmic boundary. Blebbed nuclei presented a relatively small evagination of the nuclear membrane, which contained euchromatin. Evaginations larger than the blebbed nuclei, which could have several lobes, were classified as lobed nuclei. Nuclei with depth into a nucleus were recorded as notched nuclei.

Fig. 1:	Photomicrographs of erythrocytes with normal nucleus (A); with micronuclei (B) and with nuclear abnormalities: blebbed nuclei (C); lobed nuclei (D); notched nuclei (E); binuclei (F) of P. altus

Fig. 2:	Photomicrographs of gill cells with normal nucleus marked by arrows (A) and with micronuclei indicated by MN (B-C) of P. altus

Fig. 3:	Photomicrographs of liver cells with normal nucleus (A) and with micronuclei indicated by MN (B) of P. altus

Fig. 4:	Photomicrographs of fin cells with normal nucleus (A) and with micronuclei indicated by MN (B) of P. altus

Normal gill cell approximately diameter 4-5 μm was contained mainly elliptical nuclei (Fig. 2A). Similarly to MN in periphery blood, micronuclei of gill cells were small non-refractile circular or ovoid particle lying in the cytoplasm and resembling a nucleus with respect to staining properties (Fig. 2B-C). Liver cells with normal nucleus and with micronuclei were shown in Fig. 3A-B, respectively. Fin cells with normal nucleus and with micronuclei were shown in Fig. 4A-B, respectively.

Table 1:	Frequencies (%) of micronuclei in erythrocytes, gill, liver and fin cells of P. altus exposed to different time and treatments (mean±SD)
Cont. = Control group; Cd = Cadmium group; Mix = Cadmium plus ascorbic acid; AA = Ascorbic acid, a = The mean difference was significant in row when compared the control group (p≤0.05), b = The mean difference was significant in row when compared the cadmium group (p≤0.05), c = The mean difference was significant in row when compared the mix group (p≤0.05), * = The mean difference was significant in column when compared the erythrocytes (p≤0.05), # = The mean difference was significant in column when compared the gill cells (p≤0.05)

Table 2:	Frequencies (%) of nuclear abnormalities cells in erythrocytes of P. altus exposed to different time and treatments (mean±SD)
Cont. = Control group; Cd = Cadmium group; Mix = Cadmium plus ascorbic acid; AA = Ascorbic acid, a = The mean difference was significant in row when compared the control group (p≤0.05), b = The mean difference was significant in row when compared the cadmium group (p≤0.05), c = The mean difference was significant in row when compared the mix group (p≤0.05)

The frequencies of MN observed in various treated and control fish have been summarized in Table 1. It is evident that MN was not significantly induced in the control group at 24, 48, 72 and 96 h. Otherwise; the differences were statistically significant in treated group when compared with those of the control group at all the time intervals. The frequencies of MN were significant highest number in fish treated with Cd alone. However, when both Cd and AA were conjointly administered, the MN appeared to be reduced to some extent, the difference being statistically significant at 48 h. The frequencies of NA observed in various treated and control fish have been summarized in Table 2. Similarly to MN, the frequencies of NA were also significant greater number in fish treated with Cd. On the other hand, the MN frequencies in erythrocytes were the most sensitive to the treatments and could provide valuable information than those in gill, liver and fin cells. When NA in erythrocytes were analyzed separately, it was observed that the frequency of each abnormality shapes were found in the following order: NT > LB >BN > BL. Results of MN and NA revealed the gradually increased with the time up to 48 h in all cases and decreased to some extent at 72 and 96 h.

DISCUSSION

MN is cytoplasmic chromatin masses with the appearance of small nuclei that arise from chromosome fragments or intact whole chromosomes lagging behind in the anaphase stage of cell division. Their presence in cells is a refection of structural and/or numerical chromosomal aberrations arising during mitosis (Heddle et al., 1991). MN and NA tests in fish are generally performed in enucleated peripheral blood erythrocytes mainly due to its technical feasibility. In most studies short-term exposure period ranging between 24 and 96 h were reported to be sufficient to induce micronuclei and erythrocytes have been reported to be a sensitive biomarker of genotoxicity (Heddle et al., 1991; Ayllon and Garcia-Vazquez, 2000).

There are some assays in fish aimed to evaluate the differential sensitivity of diverse species to different chemicals (Allon and Gardia-Vazquez, 2000). But in most cases the method of exposure has been intraperitoneal injection as described in this study and the previous reports, for the example, cadmium significantly induced micronuclear expression in brown trout, S. trutta (Sanchez-Galan et al., 1999) and rainbow trout, O. mykiss (Castano et al., 1998). In the present study, the peak values for MN and NA in erythrocytes have been obtained after 48 h. Similar time-dependent effects were also previously reported in fish erythrocytes exposed to textile mill effluent (Cavas and Ergene-Gozukara, 2003) and cadmium chloride (Ayllon and Garcia-Vazquez, 2000). Pollution by heavy metals is an important problem due to their stable and persistent existence in the environment. It is well known that heavy metals interferes the regular chromosome segregation during cell division mainly by inhibition of polymerization of actin tubules, an essential structure of the mitotic spindle (Miura and Imura, 1987). It was suggested that the mechanism of Cd genotoxicity is mainly conditioned by single strand breaks in DNA through the direct cadmium-DNA interactions as well as by the action of incision nucleases and/or DNA-glycosylase during DNA repair (Privezentsev et al., 1996). Correspondingly, most of the toxic chemicals that produce genotoxic effects have been known to form reactive oxygen species as well as electrophilic free-radical metabolites that interact with DNA to cause disruptive changes. It has been suggested that during the heavy metal exposure, electrophilic ions and radicals are produced, interacting with nucleophilic sites in DNA and leading to breaks and other related damage in the latter.

Further, it would be revealed from the results of this study that Cd produced genotoxic effects. Interestingly the injection of ascorbic acid appeared to modulate the genotoxic effects of Cd at all time intervals. The exact mechanism which ascorbic acid minimizes the genetoxic of Cd is not known. However, it is known that ascorbic acid has marked nucleophilic properties it might intercept reactive electrophilic metabolites produced by Cd, thereby preventing their attack on nucleophilic sites on DNA and hence blocking adduct formation (Liehr et al., 1989). Otherwise ascorbic acid is an anti-oxidant which might inhibit the oxidative metabolism of Cd and thus could prevent the production of mutagenic electrophilic metabolites (Goncharova and Kuzhir, 1989). Also as part of a redox buffer system ascorbic acid can scavenge harmful free radical metabolites or reactive oxygen species (Sato et al., 1990). Thus, the general protective effect of ascorbic acid observed against Cd induced genotoxicity could actually be accomplished through one or many of these inhibition mechanisms.

In the present study, the MN and NA tests were carried out on the different types of cells. On the erytrocytes, those are the most commonly used for these tests in the fish. On the gill cells, those are more directly exposed to environmental contaminants. Since genotoxic activation of xenobiotics usually occurs in the liver, the hepatic MN and NA assays are also thought to be a sensitive tool for detecting genotoxic responses. The hepatic micronuclei technique is rarely used in fish and there are only several studies available in the literature (Cavas et al., 2005; Al-Sabti, 1995). In the present study, the MN frequencies in erythrocytes were the most sensitive to the treatments and could provide valuable information than those in gill, liver and fin cells. There are contradictory results about the sensitivity of these cells in the MN and NA tests. Cavas et al. (2005) reported that gill and liver cells were found to be more sensitive tissues than peripheral blood cells. The reason for this is their higher mitotic index values. The same situation might also be considered for fin cells. Arkhipchuk and Garanko (2005) reported the highest increases of cells having micronuclei were observed in blood after fish exposure to cadmium ions and in the fin tissue after the copper impact. In the present study, there was a general tendency of occurrence of notched and lobed types in higher frequencies. An analysis revealed spontaneous frequencies of nuclear abnormalities in erythrocytes were found in the following order: NT > LB > BN > BL. Thus, our results seem to be in agreement with previous studies (Cavas and Ergene-Gozukara, 2003; Mallick and Khuda-Bukhsa, 2003).

In conclusion, the results presented in this study show that the efficacy of ascorbic acid in reducing genotoxicity in fish associated with acute exposed to cadmium. Otherwise, they showed the sensitivity and suitability of micronuclei and nuclear abnormality frequencies in erythrocytes for pollution biomarkers. Our data indicated that the fish micronucleus test and the new parameter in erythrocytes as the incidence of abnormal shape nuclei are the valid technique for monitoring genotoxic effects induced by heavy metals.

ACKNOWLEDGMENTS

This study was funded by the Thailand Research Fund (TRF) and the Commission on Higher Education: the New Researchers Grant 2006 and in part by Mahidol University International College and Faculty of Science, Mahidol University.