Mutagenesis vol. 29 no. 3 pp. 215–219 Advance Access publication 11 March 2014

doi:10.1093/mutage/geu005

FISH in micronucleus test demonstrates aneugenic action of rotenone in a common freshwater fish species, Nile tilapia (Oreochromis niloticus)

Karina M. Melo1,2, Cesar K. Grisolia2, Julio C. Pieczarka1, Ludmilla R. de Souza2, José de Souza Filho2 and Cleusa Y. Nagamachi1,*

*To whom correspondence should be addressed. Tel: +55 91 32490820; Fax: +55 91 91446436; Email: [email protected] Received on July 6, 2013; revised on February 8, 2014; accepted on February 10, 2014

Aneuploidies are numerical genetic alterations that lead to changes in the normal number of chromosomes due to abnormal segregation during cell division. This type of alteration can occur spontaneously or as a result of exposure to mutagenic agents. The presence of these agents in the environment has increased concern about potential damage to human health. Rotenone, derived from plants of the genera Derris and Lonchocarpus, is a product that is used all over the world as a pesticide and piscicide. Before establishing its potential and efficiency for these purposes, it is essential to know more about the possible adverse effects that it may cause. The current work aimed to evaluate the mutagenic potential of rotenone using fish from the species Oreochromis niloticus, as well as to help in understanding its action mechanism. Our results showed the mutagenic potential of rotenone evidenced by increased formation of micronuclei and nuclear buds at low doses of exposure. The use of fluorescence in situ hybridisation technique made it possible to measure the aneugenic potential of the substance, probably due to its impairment of mitotic spindle formation. Introduction The presence of genotoxins in the environment has increasingly elicited studies from researchers to acquire more knowledge about environmental agents that damage human health. These studies are directed towards the evaluation of risk, establishment of a safe exposure level and understanding of the toxicity mechanisms (1). Among the genetic alterations caused by genotoxins are aneuploidies, which are alterations that are well known for playing an important role in carcinogenesis and in reproductive failure (2,3). It is known that cancer is a genetic disease, and many carcinogenic agents can start their process by means of aneuploidies. Among the factors that may cause aneuploidies, the most notable ones are defects in the mitotic machinery, microtubules and centrosomes (4). The agents capable of causing chromosomal losses are called aneugenics and consist of a class of compounds that should be evaluated (5). Rotenone is an isoflavonoid derived from leguminous plants of the Derris and Lonchocarpus genera. Worldwide, rotenone

Material and methods Characterisation of the animals and exposure doses The fish used in this study belong to the species O. niloticus and were supplied by the Federal District Pisciculture Technology Center (Centro de Tecnologia em Piscicultura), under constantly monitored sanitary conditions. The study was approved by the ethics committee at the Federal University of Pará (BIO036-12). Previously, the fish had been acclimatised in aquariums with filtered water, aeration and constant temperature (25°C ± 2) for 15 days. The physical–chemical parameters of the water, such as pH, conductivity, temperature and ammonia, were duly controlled, with constant renewal of the water. The fish were maintained in a glass aquarium in a constant environment with controlled light, at 14 h:10 h of light/dark. The rotenone exposure concentrations in O. niloticus were based on the CL50 value found by Mascaro

© The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: [email protected].

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Laboratório de Citogenética, Departamento de Genética, Instituto de Ciências Biológicas, Universidade Federal do Pará, Campus do Guamá, Av. Perimetral, sn., Guamá, 66075–900 Belém, PA-Brazil, 2Laboratório de Genética Toxicológica, Departamento de Genética e Morfologia, Instituto de Ciências Biológicas, Universidade de Brasília, Asa Norte, 70910-900 Brasília, DF-Brazil, Instituto de Ciências Biológicas, Universidade Federal do Pará, Campus do Guamá, Belem, Para.

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is used as a pesticide and piscicide, and it is one of the main strategies in the combat and prevention of pests that arise in aquiculture tanks. In Brazil, this compound has been used by indigenous people to anesthetise fish and make fishing easier; indeed, this practice continues to be common among many Amazon river dwellers (6–8). Recent studies have linked exposure to rotenone with reproduction of anatomic, neuropathologic and behavioural characteristics of Parkinson’s Disease (9,10). Furthermore, studies on mutagenics show an increase in micronucleus formation after treatment with the pesticide in cell culture, leading researchers to suspect a possible action of rotenone on the mitotic spindle, which causes errors in chromosome segregation (4,11,12). Therefore, it is extremely important to clarify the action mechanism and adverse effects that rotenone can cause in the organism. In this article, we used a number of techniques to evaluate the genotoxic effects of rotenone and to assist in understanding its mechanism of action, using the fish species Oreochromis niloticus as test organism. The micronucleus test is a method that allows detection of aneugenic and clastogenic agents that cause chromosomal breaks and loss, respectively, during cell divisions, resulting in fragments surrounded by a membrane bond, which form a small nucleus visualised after cell division (13). The fluorescence in situ hybridisation (FISH) technique, when used in cells with micronuclei (MN), is a methodology that allows the two mechanisms of MN formation to be differentiated. This type of approach has been employed by other authors (14,15), but this is the first work done using the FISH technique with fish as the test organism, which can contribute strongly to genotoxicity studies, thanks to the possibility of a large study after controlled exposure of an organism to the substance. Furthermore, in view of the action of the metabolite in the body as a whole, the use of experimental models can also be valuable, because it may lead to greater understanding of the effect of a compound on the organism. This may result in a different response from those observed in cell cultures, due to the interaction with enzymes during metabolism (8). In addition, to determine the genotoxic potential of rotenone, we used two other methods to assist in understanding the mechanism of action of rotenone: evaluation of the apoptotic index and DNA fragmentation by flow cytometry.

K. M. Melo et al. et al. (16). The three first concentrations were 25, 50 and 75% of this value. However, none of these concentrations showed any sign of toxicity in the fish used in this study; next, three concentrations above CL50 were tested. For each concentration of rotenone, a total of eight animals (males and females) were used, weighing on average 10 g, distributed in glass aquariums containing a volume of 50 l. After 72 h of exposure, blood was collected by cardiac puncture, with a heparinised syringe.

Evaluation of apoptosis index. Previous analyses of exposure to rotenone at the three lowest concentrations did not show any sign of toxicity in the exposed fish. Next, we tested three concentrations that were higher than these (0.25 mg/l, 0.3125 mg/l and 0.375 mg/l), and for these we evaluated the apoptosis index. Blood was collected and 10 µl was diluted in 500 µl of bovine fetal serum; 50 µl of this suspension was mixed in 15 µl of acridine orange (3.0/µl). The slides were covered with coverslips and analysed under an Axiophot Zeiss fluorescence microscope with ×400 magnification, using a 510–530 nm filter. Cells were considered normal when they had rounded, well-defined green nuclei. Cells with fragmented green nuclei were considered apoptotic (18). One thousand cells per fish were counted and among them the number of cells in apoptosis was quantified. DNA fragmentation assay.  The DNA fragmentation assay was carried out with fish exposed to three concentrations of rotenone (0.006 mg/l, 0.2185 mg/l and 0.375 mg/l). The collected cells were fixed in ethanol 70% at 4°C and stored until the procedures were carried out. Subsequently, the cells were washed with phosphate-buffered saline, incubated with 50  µg/ml RNase A  for 30 min at 37°C and marked with 50 µg/ml propidium iodide for 30 min at room temperature (19). Ten thousand events per individual were quantified in a CyFlow® flow cytometer (Partec, Germany) and analysed by Windows™ FlowMax® software. Fragmented DNA was identified as the sub-G1 population (DNA content < 2n), and the fragmentation percentage was established considering the total number of events. Fluorescence in situ hybridisation For the FISH technique, SATA probes (GenBank entries: EF438158) specific to the centromeric region of fish from the species O. niloticus were used, as described by Ferreira and Martins (20) (Figure 1A). The FISH technique was applied in experiments carried out at concentrations of exposure to rotenone that showed a statistical difference in the formation of MN in relation to the control (0.06 mg/l, 0.125 mg/l, 0.187 mg/l, 0.25 mg/l). Two substances were used as positive control: cyclophosphamide (50 mg/kg i.p.) and colchicine (0.5 mg/kg i.p.). Cyclophosphamide is a clastogenic agent indicated as a positive control (OECD, 1997). In the FISH technique it is also necessary to use an aneugenic agent; in this work, colchicine was used, as recommended by several authors (21,22). After removing blood, blood smears were prepared and fixed in 100% ethanol for 10 min. The slides were stored at room temperature until it was time to apply the technique. The slides with the blood smears were submitted to a process of digestion of the cytoplasm in pepsin (10%), which varied from 10 to 40 min depending on the slide preparation time; older slides are more resistant and therefore require longer exposure to pepsin. After the pepsin, the slides were washed in 2× SSC for 5 min each and dehydrated in alcohol. The slides remained in an oven at 60°C for 1 or 2 h or overnight at 37°C. Denaturation of chromosomal DNA was done in formamide 50%/2× saline sodium citrate (SSC) for 1 min and 30 s at 70°C. Hybridisation mixtures containing 3 μl of probe and 10 μl of hybridisation buffer were denatured (65°C for 15 min) and placed on slides that had already been denatured. A glass coverslip was placed over the slide to spread

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Fig. 1.  Application of FISH technique in MN to differentiate clastogenic and aneugenic agents. (A) SATA probe markers in centromeres of all chromosomes of the species O. niloticus; (B) Normal nucleus marked with SATA probe; (C) C − MN; (D) C + MN (indicated by arrow); (E) C − NB; (F) C + NB. the probe and sealed with flexible glue. The slide was put in a dark chamber and incubated in an oven at 37°C for 48 h. After hybridisation, washes were carried out at 40°C, in formamide (50%), 2× SSC and 4× tween for 5 min each. The probes were marked indirectly with biotin, and signal detection was done with Avidin-CY3. For this, 0.4  μl of Avidin-CY3 dissolved in 200 μl of 4× tween was spread on each slide, covered with a plastic coverslip and incubated in an oven at 37°C for 20 min. After this time, each slide was washed in 4× tween; the excess was run-off and a drop of 4ʹ,6-diamino-2-phenylindole with anti-fade was added. The slides were covered with a glass coverslip and sealed with varnish. Analysis of the technique was done by observing the cells containing micronucleus under an Axiophot Zeiss fluorescence microscope with ×1000 magnification. The MN containing positive centromere marking (C + MN) were considered lost whole chromosomes, and those with negative centromere (C − MN) to be the result of chromosomal breaks. The presence (C + NB) or absence (C − NB) of centromeric signals was also observed in the nuclear abnormalities denominated NBs (Figure 1B–F). Statistical analysis For statistical analysis the non-parametric Mann–Whitney U-test was used, considering the significance level to be P  0.05).

Table III.  Frequency of Index of apoptosis in erythrocytes of O. niloticus, after exposure to rotenone at the three highest doses

DNA fragmentation DNA fragmentation was seen in all evaluated groups. However, there was a reduction in DNA fragmentation in the tested rotenone concentrations, and this was statistically significant for the two highest doses compared with the control group (Table IV). Fluorescence in situ hybridisation Table  V shows results for FISH, differentiating the presence and absence of centromeres in the MNs (C+ and C−), by using SATA DNA probes, which are capable of evidencing C + MN. Both C + MN (50%) and C − MN (50%) were responsible Table I.  Frequency of MN in erythrocytes of O. niloticus, after exposure to different concentrations of rotenone Treatment

Mean and SD of fMN

Control (filtered water) Cyclophosphamide i.p. (50 mg/kg) Colchicine i.p. (0.5 mg/kg) Rotenone (0.06 mg/l) Rotenone (0.125 mg/l) Rotenone (0.187 mg/l) Rotenone (0.25 mg/l) Rotenone (0.3125 mg/l) Rotenone (0.375 mg/l)

0.0041 (±0.0117) 0.0744 (±0.722)a 0.1914 (±0.2612)a 0.1115 (±0.0582)a 0.1473 (±0.1106)a 0.1009 (±0.0847)a 0.0492 (±0.0348)a 0.0332 (±0.0590) 0.0468 (±0.0527)

SD: standard deviation. Three thousand cells were analyzed per individual. Statistically different compared with control.

a

Treatment

Mean and SD of apoptotic index

Control (filtered water) Rotenone (0.25 mg/l) Rotenone (0.3125 mg/l) Rotenone (0.375 mg/l)

0 0.05 (±0.0926) 0.0883 (±0.1728) 0.1 (±0.1528)

SD: standard deviation. One thousand cells were analysed per individual.

Table IV.  DNA fragmentation in blood cells of O. niloticus, after exposure to rotenone Treatment

Mean and SD of fragmented DNA (%)

Control (filtered water) Rotenone (0.06 mg/l) Rotenone (0.2185 mg/l) Rotenone (0.375 mg/l)

47.92 (±10.96) 35.14 (±16.73) 32.78 (±5.45)a 21.50 (±5.89)a

SD: standard deviation. Ten thousand events per individual were quantified. a Statistically different compared with control.

Table V.  Result of FISH analysis with SATA probe for O. niloticus DNA in MNs induced after exposure to rotenone, cyclophosphamide and colchicine Treatment

Number of MN analyzed

C + MN

C − MN

Control (filtered water) Cyclophosphamide i.p. (50 mg/kg) Colchicine i.p. (0.5 mg/kg) Rotenone (0.06 mg/l) Rotenone (0.125 mg/l) Rotenone (0.187 mg/l) Rotenone (0.25 mg/l)

32 104

16 (50%) 48 (46.15%)

16 (50%) 56 (53.85%)

100 100 100 101 100

92 (92%) 76 (76%) 77 (77%) 78 (77.23%) 78 (78%)

8 (8%) 24 (24%) 23 (23%) 23 (22.77%) 22 (22%)

Table II.  Frequency of NA in erythrocytes of O. niloticus, after exposure to different concentrations of rotenone Treatment

Mean and SD of fAN

Control (filtered water) Cyclophosphamide i.p. (50 mg/kg) Colchicine i.p. (0.5 mg/kg) Rotenone (0.06 mg/l) Rotenone (0.125 mg/l) Rotenone (0.187 mg/l) Rotenone (0.25 mg/l) Rotenone (0.3125 mg/l) Rotenone (0.375 mg/l)

0.1724 (±0.1711) 0.5665 (±0.3708)a 0.8884 (±0.5897)a 1.1865 (±0.5913)a 1.1466 (±0.7026)a 0.9428 (±0.6810)a 0.3197 (±0.2400) 0.2031 (±0.3682) 0.3496 (±0.4922)

SD: standard deviation. Three thousand cells were analyzed per individual. Statistically different compared to control.

a

Table VI.  Result of FISH analysis with SATA for O. niloticus DNA in NB induced after exposure to rotenone, cyclophosphamide and colchicine Treatment

Number of NB analyzed

C + NB

C − NB

Control (filtered water) Cyclophosphamide i.p. (50 mg/kg) Colchicina i.p. (0.5 mg/kg) Rotenone (0.06 mg/l) Rotenone (0.125 mg/l) Rotenone (0.187 mg/l) Rotenone (0.25 mg/l)

23 108

17 (73.91%) 69 (63.89%)

6 (26.09%) 39 (36.11%)

105 107 109 100 105

86 (81.90%) 90 (84.11%) 86 (78.90%) 76 (76%) 80 (76.19%)

19 (18.1%) 17 (15.89%) 23 (21.10%) 24 (24%) 25 (23.81%)

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exposed to rotenone in relation to the negative control, but this difference was only significant in the four lowest concentrations. There was also a statistical difference between the individuals exposed to the two substances used as positive control (cyclophosphamide and colchicine) compared with the negative control group. The result for NAs showed potential cytotoxicity of rotenone at low concentrations too. The NAs found in the three lowest concentrations were statistically significant in relation to the negative control. There was also a statistical difference between positive and negative controls (Table II).

K. M. Melo et al.

Discussion

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Exposure levels of rotenone were based on the work of Mascaro et  al. (16), which determined the LC50 of the substance for this fish species. However, in our study, even at concentrations above the LC50, there was no evidence of toxicity in exposed individuals; only one death was observed at the highest concentration. This disagreement in the results can be attributed to the action of other substances present in the product used by the authors. The rotenone used by Mascaro et al. (16) consisted of a powder obtained from the roots of Derris spp., which had a 5% rotenone content. In the current work, commercial rotenone was used, with a 95% degree of purity. It may be that other compounds influenced higher toxicity, and the harmful effect observed in the aforementioned study may be attributed not only to the active effect of the compound, but to the formulation of the product as a whole, since it contains solvents and adjuvants. The mixture of different compounds may result in a biological response that is different to the one expected from the action of the contaminants alone (24). As regards investigation of rotenone’s mutagenic potential, some studies have already been carried out using the MN test. However, all of them were done in cell culture. This is the first in vivo work using fish as test organism with the FISH technique combined with the conventional MN test, using probes that mark centromeric regions. The results found may help in understanding the process of MN formation in relation to rotenone’s mechanism of action. This evaluation is particularly important in differentiating an agent as clastogenic or aneugenic (25). To test the efficiency of the method, two mutagenic agents, cyclophosphamide and colchicine, known to be clastogenic and aneugenic, respectively, were used. A statistically significant increase in MNs was observed in both treatments in relation to the control. In the treatment with cyclophosphamide, there was little difference between the presence and absence of markers in the MNs. Although this agent is known for its clastogenic action, being an alkylating agent of DNA, results similar to those found in the this study were also found by Schuler et  al. (26) after a metabolically competent cell culture was exposed to cyclophosphamide, indicating that the compound has clastogenic as well as aneugenic action after metabolic activation. As our study used in vivo exposure, it is probable that in this case the cyclophosphamide also presented this differentiated action due to a metabolisation process. In the treatment with colchicine the number of C + MN markers was much higher than the number of C − MN markers in the exposed individuals, confirming the aneugenic action of the substance, which has already been described by other authors (21,27,28). These data confirm the sensitivity of this experimental protocol in detecting clastogenic and aneugenic agents. The MN data showed rotenone’s genotoxic effects on groups exposed to low concentrations of the substance. At the same concentrations the FISH technique was also carried out and showed a higher number of C + MN than C − MN markers, indicating rotenone’s aneugenic action. Previous investigations have alerted researchers to the suspicion that rotenone acts on mitotic spindle fibres, thus jeopardising their correct segregation after cell division and leading to MN formation by whole chromosomes being lost (4,11,12). However, the results found in this work showed mutagenic action from rotenone only at the lowest doses of exposure. One explanation for this fact may be related to the blocking of cell division at high exposure doses. MN are formed during cell

division and can only be observed after the whole daughter cell process has finished, i.e., when a cell cycle has been completed. Thus, the failure to observe MNs would occur because the cell had not reached the next cycle of cell division. If this is true, rotenone seems to act in a similar process to the action mechanism of colchicine, which weakens the structure of the mitotic spindle, hampering its polymerisation and, consequently, its link to the chromosome. On some occasions, at high concentrations, colchicine leads to its complete disintegration, preventing the cell cycle from continuing and, in this case, making the cell return to the interphase with double the DNA content (29). It is also known that rotenone blocks complex I of the respiratory chain, leading to an increase in the formation of reactive oxygen species that induce cell death after cell culture is exposed to rotenone (30–33). Indeed, an alternative explanation for the occurrence of MNs in only the lower doses would be that, when higher doses are used, rotenone could be causing some cell disturbances and consequently a delay in the cell cycle. Therefore, in this work, the apoptosis index was also evaluated at the three highest exposure doses, although there was no induction of apoptosis in any of the tested doses. In addition, the evaluation of DNA fragmentation by flow cytometry showed reduced damage to the DNA in individuals exposed to rotenone, and these data support the idea that MN formation after exposure to rotenone may indeed be due to its action at the level of mitotic spindle fibres, interfering directly in the cell cycle. Thus, an increase in the formation of MN and the absence of DNA fragmentation after exposure to rotenone show that the cell appears to act in two different manners: one inhibits complex I of the respiratory chain and the other impairs the formation of the mitotic spindle during cell division, causing aneuploidy. Problems in the polymerisation of the microtubules can also lead to other nuclear alterations, such as the formation of irregular nuclei and NBs. In fact, the increase in NA frequency may also have been caused by this action of rotenone at the level of spindle fibres. Although nuclear abnormalities are generally used as biomarkers of genotoxicity and genomic instability, the mechanism by which they are formed is still the subject of controversy. Bombail et  al. (34) suggested that formation of NAs occurs when a certain quantity of DNA undergoes delayed mitosis, so that the resulting nucleus does not show the oval morphology considered normal, but presents instead protuberances or inlets. Disturbances of the nuclear genome caused by DNA amplifications, or alterations in the patterns of condensation and architecture of the chromatin, may lead to instabilities that can be manifested by means of nuclear abnormalities and MNs (35). One of the NAs, the NB, consists of small inclusions similar to a MN, but which continue to be physically linked to the nucleus. Some authors suggest that they may be the result of the DNA amplification process, which is selectively localised on the periphery of the nucleus, itself eliminated by budding (36). Lindberg et al. (37) used centromeric and telomeric DNA probes from the FISH technique to observe the presence or absence of the sign of NBs in folate-deficient cultures. As most of the analyzed buds did not show markers, the authors proposed that these NBs might be the result of interstitial fragments of chromosomes. In contrast, the data obtained by FISH in the current work showed that most NBs showed C + NB. It is therefore probable that the NB formation mechanism occurs in accordance with the mechanism of action of the exposure substance. In the case of rotenone, it is plausible to say that the NBs are being formed due to the delay of one or more chromosomes

Aneugenic action of rotenone

in relation to the others because of the malformation of spindle fibres during mitosis. Based on the data produced, the results found in this work confirm the aneugenic potential of rotenone. They alert us to the possible risks that continuous exposure to this substance may cause, since even at low doses/concentrations it presents strong mutagenic potential for aquatic species. Funding

Acknowledgements This study is part of the Master’s dissertation of KMM, who was a recipient of a CAPES Master’s Scholarship. The authors are grateful to Dr Cesar Martins (UNESP, Botucatu), who generously provided the SATA probe that enabled the FISH technique to be applied to MN. The authors are also grateful to Adauto Lima Cardoso for his indispensable help during the adjustment of the FISH technique in MN. Conflict of interest statement: None declared.

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This work was supported by the Pará Foundation for Supporting Science in the National Excellence in Research Program (PRONEX-FAPESPA 011/2008); the National Council for Scientific and Technological Development (CNPq 306989/2009-3; 307071/2009-0); the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES, Master scholarship); the Federal University of Pará (PROPESP); and the University of Brasília.

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FISH in micronucleus test demonstrates aneugenic action of rotenone in a common freshwater fish species, Nile tilapia (Oreochromis niloticus).

Aneuploidies are numerical genetic alterations that lead to changes in the normal number of chromosomes due to abnormal segregation during cell divisi...
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