Mutagenesis Advance Access originally published online on June 14, 2005
Mutagenesis 2005 20(4):291-295; doi:10.1093/mutage/gei039
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Published by Oxford University Press on behalf of the UK Environmental Mutagen Society 2005
Genotoxic damage in zebra fish (Danio rerio) by arsenic in waters from Zimapán, Hidalgo, Mexico
Chemical Investigation Center, Independent University from Hidalgo State (UAEH), highway Pachuca-Tulancingo km 4.5, University City, Pachuca, Hidalgo, México, C.P. 42076
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Abstract |
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The induction of micronuclei in gill cells of zebra fish (Danio rerio) maintained in calcium–magnesium bicarbonated waters from a reference well and ‘Zimapán 5’ well, the latter with an arsenic (As) content ranging from 0.395 to 0.630 p.p.m., was studied. The specimens were studied during 180 days in three separated lots: in reference well-water (negative control), in reference water to which was added 5 mg/l As5+ (positive control); and in water from ‘Zimapán 5’ well, with 65 specimens/lot. In waters an As concentration diminution was observed with time, whereas in fish there was an increase. After 30 days there was an As diminution in water from positive control of 1092.65 p.p.b. (36.42 p.p.b./day), whereas in fish it had increased to 523.81 p.p.b. (17.46 p.p.b./day). For the water from ‘Zimapán 5’ well, there was a diminution of 211.40 p.p.b. (7.04 p.p.b./day), and in fish there was an increase of 74.73 p.p.b. (2.49 p.p.b./day). In relation to micronucleus frequency in gill cells, at the end of 180 days in the negative control there was a spontaneous generation of 0.8 micronuclei/1000 cells, in the positive control there was a micronucleus frequency 163.5 times greater than in the negative control, whereas for the fish exposed to ‘Zimapán 5’ well-water the micronucleus frequency was 56.25 times greater than in the negative control. Taken together these results demonstrate the genotoxicity to Danio rerio of As in the well water.
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Introduction |
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Arsenic (As) is a carcinogen to which numerous human groups around the world are exposed (1
). It is absorbed by the lung, gastrointestinal system and skin, it crosses the placental barrier and it concentrates to a significant extent in the fetus (2). In Hidalgo, Mexico, the State Management of the Water National Commission has recognized the high arsenic content in the water from Zimapán wells. The total As concentration in the ‘Zimapán 5’ well-water (Z5WW) varies from 0.395 to 0.630 p.p.m. (3,4). For this reason, the Zimapán population is at risk of health problems related to exposure to this non-metal (5). Zimapán, Hidalgo, Mexico is located between 20° 57' and 20° 34' north and between 99° 33' and 99° 12' west (Figure 1). The ‘Zimapán 5’ well is located at Mineros street No. 40, Llano Norte Colony in the Zimapán's municipal head.
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Fish have been widely used as models for monitoring toxic chemicals in aquatic environments (6
,7). Baez (8) exposed healthy tilapias (Oreochromis niloticus) to 0.00, 0.05 and 15.73 p.p.m. of additional As (administered as arsenic trioxide) in the Z5WW over 96 h and a concentration increase was found of the nonmetal in whole fish of 435, 465 and 10 044 p.p.b. (dry basis), respectively. McGeachy and Dixon (9) determined the average time for the equilibrium loss in the trout Oncorhynchus mykiss exposed to 60 and 120 mg/l arsenate to 15°C, which was 210 and 34.8 h with an As concentration in fish of 8100 and 8600 p.p.b. in humid base. Naqvi et al. (10) exposed adult crayfish Procambarus clarkii to 0.5, 5 and 50 p.p.m. of the herbicide monosodium methanearsonate in the water over 8 weeks at the end of which the total As concentration in fresh tissue was of 930, 4290 and 4600 p.p.b., respectively.
Among other techniques, the micronucleus test is very applicable to fish. The micronuclei [MNS] form by the loss of acentric fragments or whole chromosomes that are not incorporated in the brother nucleus during mitosis, by apoptosis, or phagocytosis, among other mechanisms (11). Cavas and Ergene-Gözükara (12) exposed the fish Oreochromis niloticus to 5, 10 and 20% of a textile effluent over 9 days, obtaining an increase in the micronucleus frequency in gill cells with the increase of the dose and time.
In human studies, Basu et al. (13) investigated individuals exposed to arsenic through drinking ground water (containing 368.11 µg/l of As) in West Bengal, India, and found a statistically significant increase in micronucleus frequency in oral mucosa cells, urothelial cells and lymphocytes (5.15, 5.74 and 6.39/1000 cells, respectively) when compared with unexposed controls (0.77, 0.56 and 0.53/1000 cells, respectively). Gonsebatt et al. (14) studied micronuclei in exfoliated epithelial cells obtained from the oral mucosa and urine samples in exposed inhabitants from Santa Ana, where drinking water has 408.17 µg/l As, finding a significant increase in micronucleus frequency in oral and urinary epithelial cells (males being more affected than females) as well as a higher number of micronucleated oral cells in individuals with skin lesions.
Other metals also induce micronuclei; Kapka et al. (15) examined 67 children aged nine from Silesia who were exposed to lead, with an average blood concentration of 4.8 mg/dl, compared with 2.5 mg/dl in the controls and found a micronucleus frequency of 2.8 versus 1.0, respectively. Joardar and Sharma (16) administered mice with different oral doses of MnSO4 and KMnO4 over a period of 3 weeks and observed that the frequencies of chromosomal aberrations and micronuclei in bone marrow cells were increased significantly by both salts and that the clastogenic effects were directly related to the concentrations used.
The general objective of this study was to evaluate the genotoxic effect induced by As from Zimapán ‘drinking water’ using zebra fish (Danio rerio) as a biomonitor. Our specific objective was to determine the micronucleus frequency in gill cells.
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Materials and methods |
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Physicochemical and microbiological characterization of well waters
Monthly samplings from Zimapán 5 well and from a reference well, located in University City (Pachuca, Hidalgo, Mexico), [UAEHWW], were made from February 2003 to January 2004. Physicochemical and microbiological analyses were made according to the norm NOM-201-SSA1-2002 (17
). For the metals and As determination, the samples were preserved in 3% nitric acid. The metal analysis was made by emission spectroscopy with plasma inductive connection (ICP) in Perkin–Elmer equipment (18,19). The As determination was made by atomic absorption spectroscopy with hydride generation, previous prereduction with ascorbic acid to 25%, KI to 25% and HCl conc. (20,21).
Exposure of Danio rerio to dibasic sodium arsenate (Na2HAsO4·7H2O)
For this study zebra fish, 4 months old with an average weight of 0.2 g were obtained from a pet shop. Sixty-five animals were placed in each of three lots; one for the negative control (UAEHWW without As addition), another for the positive control (with an As initial concentration of 5055.96 p.p.b. of total As, added to the UAEHWW as dibasic sodium arsenate (Baker analyzed lot 6 41 366)), and finally the lot with the Z5WW (with an As initial concentration of 451.40 p.p.b.). During 180 days fish were maintained at 26°C, with permanent aeration, 20 liter water per lot, feeding with Tetramin dry flakes twice a day (22,23). The first total water change was after 30 days and thereafter every 4 days.
The total As concentration in waters and in fish (excluding gills, based on dry weight) was determined on days 0, 1, 2, 3, 15, 30, 60, 90, 120, 150 and 180 by atomic absorption spectroscopy with hydride generation, previous prereduction with ascorbic acid to 25%, KI to 25% and HCl conc. Before the prereduction the sampled water aliquots were preserved in nitric matrix to 3% and each dry fish was digested with concentrated nitric acid in a microwave oven.
Micronucleus frequency in gill cells
The micronucleus frequency on days 0, 1, 2, 3, 15, 30, 60, 90, 120, 150 and 180 was determined using five fish for each time and treatment, staining with 30% Giemsa solution (the stock solution diluted 3:7 in Sorenson's buffer) and later scoring 1000 cells/slide under 1000x magnification (12,23). Two entire gills per fish were extracted for each slide, because the gills were very small and it was necessary to have sufficient tissue for the score. The following criteria were used: (i) micronuclei should be >1/3 diameter of the main nucleus, (ii) they should be on the same plane of focus, (iii) they should have the same color, texture and refraction as the main nucleus, (iv) they should have oval or round shape and (v) they should be clearly separated from the main nucleus.
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Results |
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Characterization of waters from ‘Zimapán 5’ and UAEH wells
The results of water characterization appear in Table I (averages from February 2003 to January 2004).
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In all cases results of 3 retorts/month and 12 months are reported (n = 36). Parameters such as pH, conductivity, dissolved total solids, total solids, nitrates, nitrites, chlorides, among others, are higher in the UAEHWW than in the Z5WW, which shows that the first is saltier but it fulfills the established parameters for the Mexican norm for water for human consumption (24
). In general, all the evaluated parameters are below the permissible limits established by the norm. Both cases had calcium–magnesium bicarbonated waters, indicating that the UAEHWW had greater calcium content than magnesium.
Evaluation of As bioaccumulation in zebra fish
The As exposures began in September 2003 and finished in April 2004, with 6 months of evaluation. The results are shown in Table II.
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A concentration decrease in As was observed for the positive control and the Z5WW waters, with a simultaneous increase in As levels in the fish. After 30 days 47.94% of the As lost from the positive control water had bioaccumulated in the fish, the remainder was eliminated by excretion in the sediment. Similarly 35.35% of the As lost from the Z5WW after 30 days had bioaccumulated in the fish. Figure 2 shows the diminution of As concentration in waters and Figure 3 shows the increase in the As concentration in fish.
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Micronucleus frequency
The micronucleus frequency in gill cells of the fish maintained in the negative control water for 180 days was 0.8 micronuclei/1000 cells. On the other hand, the positive control water produced a frequency 163.5 times greater than in the negative control, whereas for the fish in the Z5WW an increase in frequency 56.25 times greater than was observed in the negative control, along with other nuclear abnormalities. Results are shown in Figures 4 and 5, and Table III.
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Discussion |
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The UAEHWW was saltier than Z5WW, both waters were calcium–magnesium bicarbonated and in general they were below the official norm limits for water for human consumption. However, the latter showed an As concentration almost 20 times greater than that allowed by norm NOM-127-SSA1-1994 (24
). In relation to the bioaccumulation, there was an As concentration increase in fish and a diminution in waters with time. After 30 days a diminution of As concentration in water of 1092.65 p.p.b. (36.42 p.p.b./day) was observed for the positive control, whereas in fish (excluding gills) it increased to 523.81 p.p.b. (17.46 p.p.b./day). Also the Z5WW showed a diminution of 211.40 p.p.b. (7.04 p.p.b./day) and the fish had an increase of 74.73 p.p.b. (2.49 p.p.b./day). The increase in As concentration in fish exposed to the positive control was much more rapid than in those exposed to Z5WW, with a bioaccumulation of 47.94 and 35.35%, respectively, of the As lost from the water.
After 180 days of exposure the As concentration in fish (excluding gills) from Z5WW treatment was 397.76 p.p.b. (dry basis) which is near the value reported by Baez (8) of 435 p.p.b. (dry basis) for whole fish (Oreochromis niloticus) exposed over 96 h to the same water. On the other hand, the As concentration of 4051.93 p.p.b. in fish from the positive control water was similar to the value reported by Naqvi et al. (10) of 4290 p.p.b. in fresh tissue of crayfish Procambarus clarkii exposed to 5 p.p.m. of monosodium methanearsenate herbicide in water over 8 weeks.
As shown in Figures 2 and 3, the negative slopes showing As diminutions in waters, with values of –33.847 and –6.5916 p.p.b./day, correspond to the positive control and Z5WW treatments, respectively. On the other hand, the As bioaccumulation in zebra fish can be expressed as a rate of 23.614 and 2.2077 p.p.b./day for fish from positive control and Z5WW treatments, respectively. This is proportional to the As initial concentration in waters which was 11.2 times greater for the positive control water than for Z5WW, demonstrating that the bioaccumulation is linear.
In relation to the genotoxicity there was an increase in micronucleus frequency with time, which was greater in fish treated with the positive control than in fish treated with Z5WW, accompanied in both cases, by other nuclear abnormalities as well. With both treatments As promoted cell turnover at sublethal concentrations, with an increase in micronucleus frequency of 0.7398 and 0.2561 micronuclei/day (Figure 5), respectively, a ratio of 2.88:1.
The micronucleus frequency of 10.2/1000 cells at 30 days in fish treated with Z5WW is similar to that of 10.16/1000 cells reported by Cavas and Ergene-Gözükara (12) in gill cells at 3 days of exposing the fish O.niloticus to 10% of a textile effluent. However, the frequency of 4.4 micronuclei/1000 cells after 15 days of the same treatment is similar to 5.15 micronuclei/1000 cells found by Basu et al. (13) in oral mucosa cells from individuals exposed to 368.11 µg/l of As in drinking ground water.
It is important to mention that the Z5WW had greater concentrations of manganese (330 µg/l) and lead (12 µg/l) than the UAEHWW, as both manganese (16) and lead (15) also induce micronuclei. Although these exceeded the permissible maximum limit (NOM-127-SSA1-1994) by 2.0 and 1.2 times, they do not compare with the As which exceeded the permissible limit by 19.2 times; from this, we conclude that this non-metal is the cause of the major genotoxic damage.
In conclusion, the results of this study demonstrate the significant genotoxic effects of As bioaccumulation in zebra fish gill cells.
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Acknowledgments |
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We are grateful to Dr Armida Zúñiga Estrada for allowing us to make the microbiological analysis and part of the physicochemical analysis in the State Laboratory of Public Health from Hidalgo. We are also grateful to Dr Juan Carlos Gaytan Oyarzun for having allowed us to use the genetics laboratory to conduct the biological studies of this investigation.
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Notes |
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* To whom correspondence should be addressed. Fax: +0177171 72133; Email: BARO670528{at}yahoo.com.mx
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References |
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Received on February 15, 2005; revised on April 15, 2005; accepted on April 19, 2005.
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