Aorta to coronary artery saphenous vein grafts

Mutation Research 560 (2004) 57–67

Comet assay using mullet (Mugil sp.) and sea catfish (Netuma sp.) erythrocytes for the detection of genotoxic pollutants in aquatic environment Vanessa Moraes de Andrade a,∗ , Thales R.O. de Freitas a , Juliana da Silva a,b a

Departamento de Genética, Instituto de Biociˆencias, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Bento Gonçalves 9500, CP 15053, CEP 91501-970, Porto Alegre RS, Brazil b PPG em Ensino de Ciˆ encias e Matemática, Universidade Luterana do Brasil (ULBRA), Canoas RS, Brazil Received 27 October 2003; received in revised form 29 January 2004; accepted 11 February 2004

Abstract The development of comet assay for aquatic organisms is of particular relevance in light of the importance of coastal fisheries to several countries around the world. Two of the most common fish species native to southern Brazil are the gray mullet (Mugil sp.) and sea catfish (Netuma sp.) for which we have produced a standardized comet assay using whole erythrocytes taken from samples of these fish. We investigated the potential of the comet assay for monitoring genotoxicity in mullet and sea catfish and made a preliminary investigation of the baseline levels of DNA damage in the erythrocytes of samples of these fish from non-polluted areas as well as assessing the in vitro sensitivity of erythrocyte exposed to 2, 4 and 8 × 10−5 M of methyl methanesulfonate (MMS) for 1, 2, 6 and 24 h at 25 and 37 ◦ C. Our results show that there was an increase in baseline DNA damage at higher temperatures and that the amount of MMS-induced DNA damage also increased at higher temperatures and that there was a clear dose/time response to treatment with MMS. To assess the possibility of using fish for environmental biomonitoring we also used the comet assay to investigate the in vitro genotoxic effect of MMS on whole blood cells from human donors and found a clear concentration-related effect at all exposure times, findings which agree with those of other workers. This study demonstrates the potential application of the comet assay to erythrocytes of mullets and sea catfish. However, these findings also suggest that temperature could alter both baseline DNA damage in untreated animals and in vitro cell sensitivity towards genotoxic pollutants. © 2004 Elsevier B.V. All rights reserved. Keywords: Single cell gel (SCGE)/comet assay; Fish; DNA damage; Mullet (Mugil sp.); Sea catfish (Netuma sp.); Biomonitoring

1. Introduction Aquatic environmental pollution is a serious and growing problem [1], with the increasing number of ∗ Corresponding author. Tel.: +55-51-33166733; fax: +55-51-33167311. E-mail address: [email protected] (V.M. de Andrade).

industrial, agricultural and commercial chemicals in the aquatic environment having led to various deleterious effects on organisms [2]. The impact of toxic materials on the integrity and functioning of cellular DNA has been investigated in many organisms under field conditions [3]. Several biomarkers have been utilized as tools for both the detection of exposure to genotoxic pollution and the effects of such pollution, such biomarkers including the presence of DNA adducts,

1383-5718/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2004.02.006

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chromosomal aberrations, DNA strand breaks and micronuclei measurements. In fish, blood erythrocytes are mainly used as sentinel markers of genotoxic exposure [3–7]. Aquatic organisms, such as fish, accumulate pollutants directly from contaminated water and indirectly by feeding on contaminated aquatic organisms [1]. The gray mullet (Mugil sp.) and the sea catfish (Netuma sp.) are estuarine-dependent fish which can be found from shallow estuaries to deep offshore water. These species have been used in pollution studies because they pass their early life stages near the shore and the adults feed mainly on detritus [8], it being known that marine sediments are a sink for anthropogenic contaminants and may act as a source of pollution for bottom-dwelling organisms [9]. The alkaline single cell gel electrophoresis assay, or comet assay, is a rapid, simple and sensitive procedure to quantify DNA lesions in individual cells for environmental monitoring [7,10–14]. The development of the comet assay for aquatic organisms, including fish, is of particular relevance in light of the importance of coastal fisheries to several countries [8,15,16], especially those which have estuarine regions which are subject to pollution [17]. The gray mullet and sea catfish are two of the most common fish species native to southern Brazil, and in this paper we describe a comet assay adapted to these fish which will make possible future biomonitoring of estuarine regions. 2. Materials and methods 2.1. In vitro MMS exposure Gray mullet (Mugil sp.) and sea catfish (Netuma sp.) were caught in a non-polluted area (Armazém lagoon, Rio Grande do Sul, Brazil) and blood samples collected from the cardiac vein of the mullets and the caudal vein of the sea catfish using heparinized syringes. Whole blood of 4–12 mullets and the same number of sea catfish were used for the experiments, the blood samples being diluted 1:120 (v/v) with RPMI 1640 medium and used immediately. The blood was exposed in vitro for 1, 2, 6 or 24 h at 25 and 37 ◦ C to 2, 4 or 8 × 10−5 M of the alkylating agent methylmethane sulfate (MMS; CAS 66-27-3/Sigma) by adding ap-

propriate concentrations of MMS dissolved in Hanks’ Balanced Salt Solution (HBSS/Sigma) to blood previously diluted with RPMI media such that the specified MMS concentrations were achieved. For a negative control we used blood without the addition of MMS solution but incubated under the same conditions. As an internal control we used human peripheral blood taken from 4 to 20 individuals which we treated in the same way, with and without the addition of MMS. 2.2. Comet assay The alkaline comet assay for human and fish blood cells was conducted according to published methods [7,14] except that because of the unique characteristics of the material investigated (blood from different species of fish) the pre-electrophoresis (unwinding) and electrophoresis conditions needed to be adapted in order to obtain minimum DNA damage in the control group (no MMS treatment) and maximum sensitivity in the blood samples treated with MMS. In brief, 5 ␮l of each diluted blood sample was added to 95 ␮l of 0.75% (w/v) low melting point agarose and the mixture added to a microscope slide pre-coated with 1.5% (w/v) of normal melting point agarose and covered with a coverslip. The slide was briefly placed on ice for the agarose to solidify and the coverslip carefully removed and the slide immersed in lysis solution (2.5 M NaCl, 100 mM EDTA and 10 mM Tris, pH 10.0–10.5) containing freshly added 1% triton X-100 and 10% dimethyl sulfoxide (DMSO) for at least 1 h at 4 ◦ C. Subsequently, the slides were incubated in freshly made alkaline buffer (300 mM NaOH and 1 mM EDTA, pH > 13) for 10 min for DNA unwinding, and electrophoresed in the same buffer. The electrophoresis conditions were 20 min at 300 mA and 20 V (0.7 V/cm) for mullet blood cells and 15 min at 270 mA and 25 V (0.9 V/cm) for sea catfish blood cells. All of these steps were carried out under dim indirect light. Following electrophoresis slides were neutralized in 400 mM Tris (pH 7.5) and the DNA stained with a solution containing 2 ␮g/ml ethidium bromide and covered with a coverslip. The slides were coded for blind analysis and analyzed immediately. To verify that the electrophoresis conditions were adequate, negative and positive human blood controls were included for each electrophoresis run. For the

V.M. de Andrade et al. / Mutation Research 560 (2004) 57–67

positive controls, 50 ␮l of whole human blood was mixed with 13 ␮l of a solution of MMS in HBSS to give a final MMS concentration of 8×10−5 M and the mixture incubated for 2 h at 37 ◦ C and the sample subjected to electrophoresis using the methods described in the literature for human blood [7,14]. The result of each electrophoresis was considered valid only if the negative and positive controls yielded negative and positive results, respectively. To calculate image length (IL), 100 randomly selected cells (50 from each of two replicate slides) from

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each sample were assessed using a fluorescence microscope fitted with a BP546/12 nm excitation filter and a 590 nm barrier filter and an eyepiece micrometer (1 unit ≈5 ␮m at 400× magnification) to estimate length in micrometers of the comet image (nuclear region + tail). Leukocytes were scored for human blood and erythrocytes for fish blood. To calculate a damage index (DI), cells were visually allocated into five classes according to tail size (0 = no tails and 4 = maximum-length tails) which resulted in a single DNA damage score for each sample

Table 1 Detection of DNA damage in blood leukocytes of human donors exposed to methyl methanesulfonate (MMS) in vitro for different exposure times and doses at 37 ◦ C MMS concentration (M) Comet analysisa

Exposure times of human leukocytes at 37 ◦ C 1 (h)

2 (h)

6 (h)

24 (h)

0 (Control)

Number of individuals per group Image length (␮m) Damage index Damage frequency (%)

20 21.5 ± 3.7 6.8 ± 8.0 5.3 ± 5.6g

20 22.2 ± 3.4 4.9 ± 6.1 3.5 ± 3.8

4 23.3 ± 6.6 0.5 ± 1.0 0.5 ± 1.0

16 21.1 ± 4.9 13.0 ± 11.3b,e,h 10.2 ± 9.8e,h

2 × 10−5

Number of individuals per group Image length (␮m) Damage index Damage frequency (%)

16 22.4 ± 5.8 36.5 ± 21.9j 30.1 ± 16.3j

16 24.6 ± 8.4 67.4 ± 43.2b,j 51.4 ± 20.2c,j

4 25.8 ± 8.8 92.0 ± 62.5b,i 50.2 ± 18.5k

16 29.6 ± 16.6d ,f ,k 104.6 ± 32.4d ,f ,k 55.1 ± 15.0d ,k

4 × 10−5

Number of individuals per group Image length (␮m) Damage index Damage frequency (%)

20 27.8 ± 11.4k ,m 89.2 ± 52.3k ,l 61.8 ± 20.2k ,m

20 34.4 ± 14.3d ,k ,n 163.5 ± 58.6d ,k ,m 83.2 ± 13.1c,k

4 35.3 ± 13.2b,i 216.2 ± 63.3d ,j,l 74.5 ± 10.7j

16 34.1 ± 18.1d ,k 158.7 ± 41.6d ,k ,l 86.2 ± 12.2d ,k ,m

8 × 10−5

Number of individuals per group 4 4 Image length (␮m) 36.8 ± 10.0k ,m 50.6 ± 9.1c,k ,n Damage index 194.2 ± 30.1k ,m 342.7 ± 19.5b,k ,m Damage frequency (%) 85.5 ± 3.0k ,m 100.0 ± 0.0b,k ,l,o

4 No viable cells No viable cells No viable cells

4 No viable cells No viable cells No viable cells

a Image length (IL), damage index (DI)—0 = no damage; 400 = maximum damage, and damage frequency (DF) represent the mean values obtained from an average of 100 leukocytes per sample. b Significant in relation to 1 h at P < 0.05. c P < 0.01. d P < 0.001. e Significant in relation to 2 h at P < 0.05. f P < 0.01. g Significant in relation to 6 h at P < 0.05. h P < 0.01. i Significant in relation to the control at P < 0.05. j P < 0.01. k P < 0.001. l Significant in relation to 2 × 10−5 M dose at P < 0.05. m P < 0.01. n P < 0.001. o Significant in relation to 4 × 10−5 M dose at P < 0.01. In all cases significance was tested using the Kruskall–Wallis test. Significance in respect of the control refers to significance in the same column, all other significances refer to the same row.

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(in the case of the piscines representing one fish), and consequently for each group (i.e. MMS concentration at a specified time and temperature) studied. Thus, the damage index (DI) of the group could range from 0 (completely undamaged = 100 cells × 0) to 400 (maximum damage = 100 cells × 4) [18–20]. The frequency (DF in %) was calculated for each sample based on the number of cells with tail versus those without. 2.3. Statistical analysis Statistical analysis was performed using the Kruskall–Wallis test for comparison of image length, damage index and comet frequency with time, temperatures and MMS concentration and when necessary the Mann–Whitney U-test was used to compared means.

3. Results Table 1 presents the comet assay results for human leukocytes exposed in vitro to different concentrations of MMS for different exposure times at 37 ◦ C. The mean values for DNA IL, DI, and DF of the treated human leukocytes were significantly greater from the controls without MMS, except for the image length values for the 2 × 10−5 M concentration of MMS at 1–6 h, while at the highest MMS concentration (8 × 10−5 M) there were no viable leukocytes for scoring at 6 and 24 h. Comet analysis at different exposure times clearly related increasing concentration to DNA damage. The damage index of the 24 h control group was significantly higher than that of the 1, 2 and 6 h control groups and the damage frequency for the 24 h control group significantly higher than that of the 2 h and 6 h control groups and the 1 h control group had

Table 2 Detection of DNA damage in mullet erythrocytes exposed to methyl methanesulfonate (MMS) in vitro for different exposure times and doses at 25 ◦ C MMS concentration (M)

Comet analysisa

0 (Control)

Exposure times of mullet erythrocytes at 25 ◦ C 1 (h)

2 (h)

6 (h)

24 (h)

Number of mullets per group Image length (␮m) Damage index Damage frequency (%)

8 24.7 ± 11.7 152.2 ± 31.5 54.4 ± 9.5

8 24.0 ± 11.7 138.6 ± 45.7 51.5 ± 13.1

12 27.9 ± 13.0 192.1 ± 77.0 66.5 ± 15.9e

3b 33.9 ± 9.6d ,f 308.0 ± 14.0 90.0± 2.6d ,f ,i

2 × 10−5

Number of mullets per group Image length (␮m) Damage index Damage frequency (%)

4 30.5 ± 12.8 231.2 ± 113.6 73.2 ± 29.2

8 30.3 ± 11.7j 241.0 ± 75.4j 80.1 ± 16.3k

12 41.7 ± 9.3c,g,l 361.5 ± 35.0c,f ,k 97.5 ± 2.4f ,k

3b 40.8 ± 1.8e,j 400.0 ± 0.0c,e,h,j 100.0 ± 0.0c,f ,j

4 × 10−5

Number of mullets per group Image length (␮m) Damage index Damage frequency (%)

4 35.9 ± 8.8k 332.7 ± 32.2k 92.7 ± 7.1j

4 37.6 ± 10.9l 322.2 ± 60.7k 87.7 ± 12.4k

8 46.3 ± 11.0c,l 362.0 ± 88.7l 93.5 ± 17.9l

4 No viable cells No viable cells No viable cells

a Image length (IL), damage index (DI)—0 = no damage; 400 = maximum damage, and damage frequency (DF) are mean values obtained from an average of 100 erythrocytes per mullet. b Reduced sample-size due to technical reasons. c Significant in relation to 1 h at P < 0.05. d P < 0.01. e Significant in relation to 2 h at P < 0.05. f P < 0.01. g P < 0.001. h Significant in relation to 6 h at P < 0.05. i P < 0.001. j Significant in relation to the control at P < 0.05. k P < 0.01. l P < 0.001. In all cases significance was tested using the Kruskall–Wallis test. Significance in respect of the control refers to significance in the same column, all other significances refer to the same row.

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a significantly higher damage frequency as compared to the 6 h control group. There was a general increase in the DNA damage values with increased exposure time (24 > 6 > 2 > 1 h) at all MMS concentrations with the observed values being somewhat higher than expected, statistically significant differences being shown in Table 1. The comet assay data for mullet and sea catfish erythrocytes exposed to different MMS concentrations at different exposure times and temperatures are summarized in Tables 2–5 and illustrated in Figs. 1–3. Table 2 shows the results of experiments with mullet erythrocytes at 25 ◦ C. The damage frequency shows that significant DNA damage occurred in the 24 h control group as compared to the 1, 2 and 6 h control groups and there was significantly more damage at 6 h than at 2 h, although the amount of damage was less

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than at 24 h. This is supported by the image length values for the control group, which were significantly greater at 24 h than at 1 and 2 h. At both MMS concentrations (2 and 4 × 10−5 M), image length, damage index and damage frequency were significantly greater at all exposure times in relation to the control for that exposure time, with the exception of the group exposed to 2×10−5 M MMS for 1 h. After 24 h exposure to 2 × 10−5 M MMS the damage index and damage frequency values were significantly higher than at 1 h, while at 2 h not only were damage index and damage frequency values significantly lower than at 24 h but the image length values were also significantly lower. For the 6 h group, however, the only significant relationship with the 24 h group was that the damage index was significantly lower than at 24 h. The image length and damage index at 6 h for the 2 × 10−5 M MMS

Table 3 Detection of DNA damage in sea catfish erythrocytes exposed to methyl methanesulfonate (MMS) in vitro for different exposure times and doses at 25 ◦ C MMS concentration (M)

Comet analysisa

0 (Control)

Number of sea catfish per group Image length (␮m) Damage index Damage frequency (%) Number of sea catfish per group Image length (␮m) Damage index Damage frequency (%) Number of sea catfish per group Image length (␮m) Damage index Damage frequency (%)

2 × 10−5

4 × 10−5

Exposure times of sea catfish erythrocytes at 25 ◦ C 1 (h)

2 (h)

8 20.7 ± 4.2 10.4 ± 5.3 4.0 ± 2.9 4 20.7 ± 3.1 15.5 ± 13.1 9.2 ± 9.9 4 24.6 ± 9.8 67.5 ± 75.8i,l 28.0 ± 20.8j

8 20.7 10.8 4.7 8 22.6 44.4 24.1 4 42.7 314.5 92.7

± 4.1 ± 12.7 ± 7.2 ± 6.9i ± 14.6i ± 11.1i ± 11.2b,k ± 69.7k ,m ± 7.3b,k

6 (h)

24 (h)

12 20.6 ± 4.6 8.5 ± 7.8 2.3 ± 2.6 12 31.5 ± 11.8c,e,j 172.6 ± 123.1c,e,j 71.2 ± 27.9c,f ,j 8 48.3 ± 5.1c,k ,l 383.0 ± 20.4c,k ,l 99.7 ± 0.5c,k ,l

4 22.3 ± 7.2 33.7 ± 25.9 14.0 ± 10.0 4 48.2 ± 5.6d ,f ,i 380.0 ± 40.0d ,f ,h,i 99.5 ± 1.0d ,g,i 4 No viable cells No viable cells No viable cells

a Image length (IL), damage index (DI)—0 = no damage; 400 = maximum damage, and damage frequency (DF) represent the mean values obtained from an average of 100 erythrocytes per sea catfish. b Significant in relation to 1 h at P < 0.05. c P < 0.01. d P < 0.001. e Significant in relation to 2 h at P < 0.05. f P < 0.01. g P < 0.001. h Significant in relation to 6 h at P < 0.01. i Significant in relation to the control at P < 0.05. j P < 0.01. k P < 0.001. l Significant in relation to 2 × 10−5 M dose at P < 0.05. m P < 0.01. In all cases significance was tested using the Kruskall–Wallis test. Significance in respect of the control refers to significance in the same column, all other significances refer to the same row.

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Table 4 Detection of DNA damage in mullet erythrocytes exposed to methyl methanesulfonate (MMS) in vitro for different exposure times and doses at 37 ◦ C MMS concentration (M)

Comet analysisa

0 (Control)

Exposure times of mullet erythrocytes at 37 ◦ C 1h

2h

6h

24 h

Number of mullets per group Image length (␮m) Damage index Damage frequency (%)

8 27.9 ± 10.8 170.9 ± 28.7 73.9 ± 8.8

8 32.9 ± 12.7 221.1 ± 36.5b 81.5 ± 10.0

4 No viable cells No viable cells No viable cells

4 No viable cells No viable cells No viable cells

2 × 10−5

Number of mullets per group Image length (␮m) Damage index Damage frequency (%)

4 39.7 ± 7.6c 366.5 ± 5.2d 97.5 ± 1.7c

4 39.7 ± 6.5 373.2 ± 17.2c 99.5 ± 1.0d

4 No viable cells No viable cells No viable cells

4 No viable cells No viable cells No viable cells

4 × 10−5

Number of mullets per group Image length (␮m) Damage index Damage frequency (%)

8 39.9 ± 9.8d 332.1 ± 49.7d 97.4 ± 3.2d

8 45.2 ± 7.4b,e 380.5 ± 31.1b,e 99.6 ± 0.7e

4 No viable cells No viable cells No viable cells

4 No viable cells No viable cells No viable cells

8 × 10−5

Number of mullets per group Image length (␮m) Damage index Damage frequency (%)

4 47.5. ± 6.5e 373.5 ± 26.1e 99.5 ± 1.0e

4 53.4 ± 4.1b,e,f ,g 395.5 ± 9.0e 99.0 ± 2.0d

4 No viable cells No viable cells No viable cells

4 No viable cells No viable cells No viable cells

a Image length (IL), damage index (DI; 0 = no damage; 400 = maximum damage), and damage frequency (DF) represent mean values obtained from an average of 100 erythrocytes per mullet. b Significant in relation to 1 h at P < 0.05. c Significant in relation to control at P < 0.05. d P < 0.01. e P < 0.001. f Significant in relation to 2 × 10−5 M dose at P < 0.01. g Significant in relation to the 4 × 10−5 M dose at P < 0.05. In all cases significance was tested using the Kruskall–Wallis test. Significance in respect of the control refers to significance in the same column, all other significances refer to the same row.

Fig. 1. Comparisons between the mean damage index values for mullet erythrocytes at 25 and 37 ◦ C.

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Table 5 Detection of DNA damage in sea catfish erythrocytes exposed to methyl methanesulfonate (MMS) in vitro for different exposure times and doses at 37 ◦ C MMS concentration (M)

Comet analysisa

0 (Control)

Exposure times of sea catfish erythrocytes at 37 ◦ C 1h

2h

6h

24 h

Number of sea catfish per group Image length (␮m) Damage index Damage frequency (%)

8 23.6 ± 8.5 53.7 ± 31.8 24.5 ± 14.2

8 25.7 ± 11.5 61.4 ± 43.3 22.7 ± 13.8

4 28.8 ± 12.3 125.5 ± 83.2 49.2 ± 19.6

4 No viable cells No viable cells No viable cells

2 × 10−5

Number of sea catfish per group Image length (␮m) Damage index Damage frequency (%)

4 42.7 ± 10.8f 313.2 ± 36.1f 90.0 ± 7.2f

4 45.9 ± 7.0f 353.2 ± 45.9f 96.5 ± 5.2f

2b 49.6 ± 2.8 396.0 ± 5.7 100.0 ± 0.0

4 No viable cells No viable cells No viable cells

4 × 10−5

Number of sea catfish per group Image length (␮m) Damage index Damage frequency (%)

8 39.3 ± 13.0g 266.7 ± 124.8g 83.7 ± 18.1g

8 37.2 ±13.7f 277.1 ± 124.7f 86.6 ± 16.6f

3b 50.0 ± 0.0 400.0 ± 0.0e 100.0 ± 0.0e

4 No viable cells No viable cells No viable cells

8 × 10−5

Number of sea catfish per group Image length (␮m) Damage index Damage frequency (%)

4 33.3 ± 10.8e 205.0 ± 19.9e 90.2 ± 4.5f

4 48.0 ± 15.5c,g 312.7 ± 30.1c,e 96.5 ± 3.8d ,f

4 No viable cells No viable cells No viable cells

4 No viable cells No viable cells No viable cells

a Image length (IL), damage index (DI; 0 = no damage; 400 = maximum damage), and damage frequency (DF) are mean values obtained from an average of 100 erythrocytes per catfish. b Reduced sample-size due to technical reasons. c Significant in relation to 1 h at P < 0.05. d P < 0.001. e Significant in relation to the control at P < 0.05. f P < 0.01. g P < 0.001. In all cases significance was tested using the Kruskall–Wallis test. Significance in respect of the control refers to significance in the same column, all other significances refer to the same row.

Fig. 2. Comparisons between the mean damage index values for sea catfish erythrocytes at 25 and 37 ◦ C.

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Fig. 3. Mean damage index values for mullet and sea catfish erythrocytes exposed in vitro at 25 ◦ C to 2 × 10−5 M methyl methanesulfonate (MMS) and control erythrocytes unexposed to MMS.

group were significantly higher than at 1 h, while image length, damage index and damage frequency at the same MMS concentration were all significantly higher at 6 h than at 2 h. For the 4 × 10−5 M MMS group image length was the only significant parameter with relation to time, being significantly greater at 6 h than at 1 h. At this concentration there were no viable cells after exposure for 24 h. Except for the 1 h 2 × 10−5 M MMS group and the 24 h 4 × 10−5 M MMS group (no viable cells), those groups exposed to MMS for the same time were all significantly more damaged than the controls without MMS (Table 2, columns). Table 3 presents the comet assay data for sea catfish erythrocytes at 25 ◦ C. In comparison with the control there was a significant increase image length, damage index and damage frequency for the 2 × 10−5 M MMS concentration after exposure for 2, 6 and 24 h, while for the 4 × 10−5 M MMS concentration there was also an increase in the same three factors (IL, DI and DF) except that at this concentration it was at exposure times of 1, 2 and 6 h and there was no increase in image length in the 1 h group and no viable cells in the 24 h group. As compared to the 2 × 10−5 M MMS concentration, the 4 × 10−5 M concentration produced

significantly higher damage index values at 1, 2 and 6 h and significantly higher image length and damage frequency values at 6 h. Comparing different MMS exposure times for each dose, at 2 × 10−5 M MMS DNA damage in terms of increased image length, damage index and damage frequency was statistically higher at 6 and 24 h than at 1 and 2 h, with the damage index values being significantly higher at 24 h than at 6 h. For the 4×10−5 M MMS concentration the image length and damage frequency values were statistically greater at 2 h than at 1 h, while the image length, the damage index and damage frequency were statistically higher at 6 h than at 1 h. Table 4 shows the results of the mullet erythrocyte experiments at 37 ◦ C. In this experiment no differences were observed between the different exposure times and it was impossible to assess the 6 and 24 h exposure times because no viable cells survived at any of the MMS concentrations nor in the control groups without MMS. In addition, at this temperature almost all the cells (>97%) showed DNA damage irrespective of the MMS concentration to which they had been exposed. The damage index value for the control at 2 h was statistically higher than at 1 h. When compared with the

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controls, DNA damage values were significantly increased at all MMS concentrations with the exception of the image length values for the 2 × 10−5 M MMS concentration at 2 h exposure time. There was no statistical differences between the MMS concentrations, except for the fact that the image length value for the 2 h 8 × 10−5 M MMS concentration group was statistically higher than for the 2 × 10−5 M and 4 × 10−5 M MMS groups. Table 5 shows the results for sea catfish erythrocytes at 37 ◦ C. In this species at this temperature and the longest exposure time (24 h) it was also not possible to asses either the erythrocytes exposed to MMS or the controls because there were no viable cells, which was also the case for the 6 h group exposed to 8 × 10−5 M MMS. Blood samples exposed to 2, 4 or 8 × 10−5 M MMS contained statistically more damaged cells than those which had not been exposed to MMS. The image length, damage index and damage frequency values for the 8×10−5 M MMS concentration were significantly higher at 2 h than they were at 1 h. The damage index values at 25 and 37 ◦ C for mullets are shown in Fig. 1 and for sea catfish in Fig. 2. The comparisons for the mullets shown in Fig. 1 is for the 1 and 2 h exposure time groups, significant values being observed for the 1 and 2 h for the 2 × 10−5 M concentration and for the 2 h control group with damage being greater at 37 ◦ C than 25 ◦ C. Fig. 2 shows the results for sea catfish erythrocytes exposed at 25 ◦ C and 37 ◦ C, from which it can be seen that there was a significant increase in damage at 37 ◦ C when compared to the controls at 25 ◦ C and there were also significant differences between MMS concentrations with damage being greater at 37 ◦ C than at 25 ◦ C. Fig. 3 shows a comparison between the damage index values for the control group without MMS and for the 2 × 10−5 M MMS concentration group for mullet and sea catfish erythrocytes at 25 ◦ C, from which it can be seen that, with the exception of the 24 h MMS group, there are significantly more damaged mullet than sea catfish erythrocytes.

4. Discussion The comet assay may prove to be a powerful tool for measuring the relationship between DNA damage and the exposure of aquatic organisms to genotoxic

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pollutants. This assay offers considerable advantages over the other cytogenetic methods (e.g. the presence of chromosome aberrations, sister chromatid exchange and the micronucleus test) used for the detection of damage to DNA because in the comet assay the cells do not need to be mitotically active [21]. We have standardized the Comet assay for whole blood erythrocytes of mullets and sea catfish, two of the most common species native to southern Brazil. This study provides data which will be useful for future work involving the biomonitoring of regions where mullets and sea catfish occur and is important because the assessment of genotoxic effects is crucial to any comprehensive study of contaminants in aquatic environments. The genotoxic effect of MMS in the comet assay has previously been demonstrated in whole blood cells from human donors, making it possible to compare our results with fish and evaluate their suitability for biomonitoring. In this study we used MMS as a model for alkylation damage because alkylating agents are thought to be the most potent and abundant genotoxic agents found in aquatic environments [22]. When human leukocytes were exposed to different MMS concentrations for different times we found a clear concentration-related effect which was valid for all exposure times, our results being in agreement with previously published data [23,24]. We also found that in leukocytes which were not exposed to MMS (the negative control group) showed significantly more DNA damage at 24 h than at 1, 2 and 6 h, although this damage was still within the 10% considered normal for negative controls in this type of assay [25]. No viable cells were found after 6 and 24 h exposure to the highest MMS concentrations, probably due to the toxic effect of this compound on cells, similar results on MMS cell toxicity having been obtained by Rank and Jensen [26]. We applied the comet assay to mullet and sea catfish blood samples exposed and unexposed to different MMS concentrations at 25 and 37 ◦ C with the aim of evaluating the sensitivity of these fish in relation to temperature. The data obtained with our in vitro treatments of mullet and sea catfish erythrocytes showed an increasing amount of DNA damage when the temperature increased. Papers regarding the influence of temperature on DNA damage in fish are rare, although Buschini et al. [21] reported an increasing amount of

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DNA damage in the cells of the mussel Dreissena polymorpha at increasing temperatures and Chapple et al. [27,28] observed an increase in the average levels of heat-shock proteins (and protein damage) involved in the development of tolerance to extreme temperature in mussels. Our results show that high temperatures can also increase baseline DNA damage, with such damage being about 10% in sea catfish at 25 ◦ C and more than 20% at 37 ◦ C (Tables 3 and 5), although with mullets there was no great difference between the baseline DNA damage level at either 25 or 37 ◦ C with the damage frequency being in excess of 50% (Tables 2 and 4). These results are supported by the data given in Figs. 1 and 2, which compare the damage index values for the same experimental parameters. A disadvantage in applying the comet assay to mullets has been the high background levels of variation (which also occurs in other aquatic organisms [29]), and which in some instances has been attributed to apoptosis [30]. This hypothesis appears to be consistent with our results because we observed highly fragmented nuclei showing complete disintegration of the head region as well as no viable cells in some groups. Taken as a whole, our data suggests that mullets are more sensitive than sea catfish under in vitro conditions. A clear dose-time response occurred in respect to the MMS treatment used in our study to model alkylation damage. For both the mullets and sea catfish when the doses of MMS were increased, there was an increase in the DNA damage at both temperatures. The control values for our in vitro studies with fish blood were relatively high compared to the control values for the human blood samples. The differences observed after MMS treatment seem to be related to the different metabolic responses of the two fish species, similar differences between different species to the same agent having been demonstrated by other workers [11]. Furthermore, when we compared both species at thewell-established temperature of 25 ◦ C and at a low MMS concentration (Fig. 3) the mullets had a baseline and exposed level of DNA damage statistically higher than that of the sea catfish, showing that the sea catfish are more suitable for biomonitoring studies. This agrees with the view put forward in the review by Cotelle and Ferard [11] who suggested that flatfish (which, like sea catfish, are sediment-feeders)

are especially suitable and recommended for biomonitoring studies. While guidelines relating to the use of the comet assay have been published for mammalian genotoxicology [14], no standardized protocol currently exists for aquatic organism. As pointed out above, and also in the overview of marine invertebrate methodology by Dixon et al. [31], variations in protocol can lead to major differences results. Thus, any descriptive method involving aquatic animals has an important role in improving the comet assay and applying it to aquatic biomonitoring. Our study provides recommendations regarding a sensitive and reproducible protocol and the potential application of the comet assay to mullet and sea catfish erythrocytes for hazard assessment and biomonitoring. In addition, the comparison of data on the exposure of mullet and sea catfish erythrocytes to different doses of MMS at different exposure times and at different temperatures has shown that temperature, an important environmental factor, can influence both the baseline genetic damage and sensitivity towards a genotoxic agent. The main implication of our data is that any interpretation of biological parameters in environmental studies with these species should be made with caution and that it is essential to distinguish the influence of natural fluctuation such as seasonal temperature change from anthropogenic stressors or pollutants while it is also always very important to include a good control in every biomonitoring study.

Acknowledgements The authors thank Loreci da Silva and his father, Mr. Loro da Silva, and Juliano Silveira for help during the field work. We also thank the Centro de Estudos Costeiros, Limnológicos e Marinhos of the Federal University of Rio Grande do Sul (CECLIMARUFRGS). References [1] Y.F. Sasaki, F. Izumiyama, E. Nishidate, S. Ishibashi, S. Tsuda, N. Matsusaka, N. Asano, K. Saotome, T. Sofuni, M. Hayashi, Detection of genotoxicity of polluted sea water using shellfish and the alkaline single-cell gel electrophoresis (SCE) assay: a preliminary study, Mutat. Res. 393 (1997) 133–139.

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