EVALUATION OF BASAL MICRONUCLEUS FREQUENCY AND HEXAVALENT CHROMIUM EFFECTS IN FISH ERYTHROCYTES

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1 Environmental Toxicology and Chemistry, Vol. 20, No. 6, pp , SETAC Printed in the USA /01 $ EVALUATION OF BASAL MICRONUCLEUS FREQUENCY AND HEXAVALENT CHROMIUM EFFECTS IN FISH ERYTHROCYTES CLARICE TORRES DE LEMOS,* PATRÍCIA MILAN RÖDEL, NARA REGINA TERRA, and BERNARDO ERDTMANN Programa de Pesquisas Ambientais, Divisão de Biologia, Fundação Estadual de Proteção Ambiental Henrique Luis Roessler, Avenida Dr. Salvador França, 1701, , Porto Alegre, RS, Brazil Departamento de Genética, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves 9500, Prédio , CEP , Cx Postal 15053, Porto Alegre, RS, Brazil (Received 15 May 2000; Accepted 31 October 2000) Abstract Hexavalent chromium (Cr [VI]) genotoxicity was studied using fish micronucleus analysis in peripheral blood erythrocytes from Pimephales promelas, the fathead minnow. Forty-five- to 60-d-old fish were used to assess the spontaneous level of genotoxic damage. The genotoxic effect of Cr (VI) obtained from potassium dichromate (K 2 Cr 2 O 7 ) in tests performed for 7-, 14-, and 21-d exposure periods was estimated. Significant micronucleated erythrocyte (MNE) induction was detected in fish exposed for 7 d to 2.5 mg/l of Cr (VI), and induction decreased after 21 d of exposure. The results suggest a handling effect in fish manipulated compared to those not manipulated, thus demonstrating the importance of including parallel negative controls in experimental design. Basal levels of MNE are reported, providing laboratory values for future assay quality control. The importance of determining the period with the highest expression of the genotoxic effects in this assay system was also confirmed. Keywords Micronucleus Fish erythrocytes Hexavalent chromium Pimephales promelas INTRODUCTION Among the most frequently applied cytogenetic analyses, micronucleus assay is a fast, sensitive indicator of structural alterations, DNA loss, and numerical chromosomal abnormalities [1,2]. Several assays are possible using this endpoint, involving different organisms and tissues for the evaluation of clastogenicity and aneugenicity both in vivo and in vitro. Several authors have recommended the use of in vivo assays to improve the characterization of genotoxic effects of chemicals [3,4] or to increase the level of evidence regarding their potential risk to humans [5]. Genotoxic studies using cytogenetic analysis in fish have demonstrated the sensitivity of these organisms to chemical and environmental biomonitoring [6 8]. Fish have been used in several biochemical and toxicological studies (e.g., developmental, carcinogenic, and teratogenic), both in vitro and in vivo [9 11]. Among fish species used in toxicological assays, the fathead minnow (Pimephales promelas Rafinesque [Teleostei, Cyprinidae]) has been widely used in acute and chronic tests [12 14]. Therefore, it was advantageous to evaluate its sensitivity for genotoxicity studies as well. This study evaluated the spontaneous level of genotoxic damage shown by a population of P. promelas as assessed through the micronucleus test in peripheral blood erythrocytes. The genotoxic effect of hexavalent chromium (Cr [VI]) derived from potassium dichromate (K 2 Cr 2 O 7 ), a substance widely used in acute and chronic toxicity tests for quality control of aquatic organism sensitivity, was also estimated. In the chromate condition (i.e., oxyanion), Cr (VI) can easily penetrate cell membranes using the same type of sulfate anion transport system [15,16]. The toxic action results from its strong oxidative effect on membrane phospholipid proteins and nucleic acids [17]. * To whom correspondence may be addressed (labbiofepam@pro.via-rs.com.br). Genotoxic activity occurs through intracellular reduction to Cr (III), the most stable form of chromium ion. The Cr (VI) intracellular reductants produce reactive intermediates such as Cr (V) and Cr (IV). During this process, free-radical species such as active oxygen forms are generated that can react with cellular macromolecules, including DNA [18,], forming Cr- DNA adducts, single-strand breaks, and DNA cross-links [20]. The DNA-associated chromium is usually found in the trivalent form [21]. Several cellular components are considered to be chromium reductants, among them hepatic glutathione, cysteine, ascorbic acid, riboflavin, and nicotinamide adenine dinucleotide phosphate dependent flavoenzymes, such as microsomal cytochrome P450 [16,22,23], DT-diaphorase, hydrogen peroxide (H 2 O 2 ), and the mitochondrial electron transport chain [24]. In addition, Cr (VI) has been demonstrated to be genotoxic in various in vitro and in vivo test systems [7,17,20,22,23,25 28]. These observations justify the analysis of potassium dichromate from a genotoxic point of view to define a positive control for environmental genotoxicity studies using the fish P. promelas. MATERIALS AND METHODS Organisms Fish of the species P. promelas aged from 45- to 60-d-old (juvenile form, before sexual maturation) and measuring approximately mm were obtained from SITEL- CORSAN cultures (Sistema Integrado de Tratamento de Efluentes Líquidos Companhia Rio-Grandense de Saneamento, Triunfo-RS, Brazil) or from Bioensaios Análises e Consultoria Ambiental (Porto Alegre-RS, Brazil). Fish were maintained in water with a hardness of 100 mg/l of CaCO 3 before the genotoxicity study. Therefore, fish were acclimated to the test conditions (i.e., deionized tap water, reconstituted to a hardness of 40 mg/l of CaCO 3 ) before treatment with Cr (VI). 1320

2 Cr (VI) micronucleus induction in fish Environ. Toxicol. Chem. 20, The use of this reconstituted water is important to evaluate the individuals under conditions similar to those found in the water resources of the State of Rio Grande do Sul, Brazil, because we intend to use this assay in future water-quality studies. To evaluate spontaneous genotoxic levels, 90 of the 117 control individuals were used as negative controls, parallel to the Cr (VI) exposures, and were handled using the same regime. In addition, 27 of the 117 control individuals from the culture acclimated to the hardness condition of the test were also used. Exposure The Cr (VI) was obtained as potassium dichromate (analytical grade 99.5%, CAS ; Merck, Darmstadt, Germany), prepared as a fresh stock solution in distilled water for each assay, and dissolved in reconstituted water (i.e., test medium). The assays were carried out in 2-L test medium aquaria, with six fish per exposure period (7, 14, and 21 d) randomly distributed. Media were renewed every 48 h to minimize alterations caused by metabolization and presence of catabolites. Aquaria were covered to avoid medium evaporation leading to alterations in chromium concentration. Artemia sp. was provided every day as food after defrosting and washing to eliminate excess salt. Each exposure period was accompanied by its respective negative controls (i.e., reconstituted water). Slide preparation Smears were prepared with peripheral blood collected through caudal section. Fixation was achieved using absolute methanol for 10 min, followed by staining in 10% Giemsa solution (v/v) for the same period. Micronucleus analysis Micronucleus analysis was performed with peripheral blood erythrocytes according to the following criteria: size smaller than one-third of the main nucleus with no refraction, similar staining, and separation from and no bridge link to the main nucleus. Preliminary tests with P. promelas at concentrations of 1.0, 1.5, 2.0, and 2.5 mg/l of potassium dichromate were performed to evaluate the sensitivity of organisms and to estimate the most effective concentration for use in the main experiments. Three fish were used for each dilution, and two exposure periods (7 and 14 d) as well as 2,000 cells/fish were analyzed. In the other experiments, five fish in each exposure period were evaluated whenever no problem with individuals or smears was detected during the test. Statistical analysis Results were compared statistically using Student s t test after the data were transformed into x x 0.5, where x is the number of cells with micronuclei detected among the total number of cells analyzed. This procedure is recommended for data showing a Poisson distribution. RESULTS AND DISCUSSION Evaluation of basal level of micronuclei The evaluation of individuals not exposed to the toxic substance was important, because to our knowledge, no data about background micronuclei frequency in this species have been reported. Micronucleated erythrocyte (MNE) frequency was Fig. 1. Micronucleated erythrocytes (MNE), verified in 2,000 cells/ fish, of 117 nontreated individuals from two groups: A, individuals from culture, not manipulated; B, individuals from negative control exposure, handled as the exposed fish for 7, 14, and 21 d. analyzed in 117 control individuals. Twenty-seven of these, identified as group A, were removed from the cultures and acclimated to the reconstituted test water. Another 90 individuals, identified as group B, were used as parallel negative controls and handled in the same manner as the treated organisms (7, 14, and 21 d) but without exposure to potassium dichromate. The mean MNE in group A was 0.52 (standard deviation, 1.25; range, 0 5). In group B, the mean MNE was 2.01 (standard deviation, 2.49; range, 0 14). The modal number of cells with micronuclei was zero in the two groups studied. The data showed a Poisson distribution (Fig. 1). A statistically significant difference (p 0.001) was observed between the groups of untreated fish (A and B), suggesting an effect of handling. Handling stress in the individuals exposed to different treatment periods (7, 14, and 21 d) was evaluated, and significant increases in cells with micronuclei frequency (p 0.05) after 14 d of exposure compared to 7 d were found. No significant difference was found between the 7- and 21-d and between the 14- and 21-d periods, respectively, indicating that the differences observed in the 14-d exposure could be related to handling or to differentiated individuals present in this sample. At all periods evaluated (7, 14, and 21 d), handled individuals showed a statistically significant difference from those of the nonmanipulated group A (p 0.05). These results show that although the larger number of individuals evaluated allowed characteristic frequency determination in healthy individuals, the use of parallel controls is necessary to evaluate the effect of test conditions. The use of individuals from the culture, acclimated to test conditions, allows checking of the results of the parallel negative control. It is also appropriate for estimating individual health conditions. The mean values reported for P. promelas in the present study are within the observed basal level range for other fish species, as verified in an extensive review on genotoxic studies using fish [8]. Those authors found that among the teleost fish evaluated, when considering only individuals submitted to exposure conditions similar to those used in the present study, the micronuclei frequency observed in erythrocytes of negative control individuals ranged from zero to 14. Definition of potassium dichromate concentration The results obtained for different potassium dichromate concentrations during the preliminary test are given in Table

3 1322 Environ. Toxicol. Chem. 20, 2001 C. Torres de Lemos et al. Table 1. Micronucleated erythrocytes (MNEs) observed in 2,000 cells/fish exposed to different K 2 Cr 2 O 7 concentrations and in the parallel negative controls (C ) for 7 and 14 d K 2 Cr 2 O 7 C 7d 2.0 mg/l 2.5 mg/l 14 d 1.0 mg/l 1.5 mg/l 2.0 mg/l 2.5 mg/l MNE Range Mean SD a Mean SD b c c a Absolute value; SD standard deviation. b Transformed value: MNE 0.5. c p No physiological effects on fish at the concentrations and exposure periods studied were observed. The substance concentration adequate for genotoxic testing is defined as half the minimum concentration that led to detectable physiological disturbances in the organisms during the preliminary toxicity test [29]. The chromium concentrations used were chosen based on results from acute sensitivity tests in which 4 mg/l caused skin loss in fish (Bioensaios Análises e Consultoria Ambiental, unpublished data). Data show micronuclei frequencies significantly higher than those of controls in the 2.5- mg/l aquaria, which is equivalent to 0.88 mg/l of Cr (VI), after 7- and 14-d exposures. No significant difference was found between the genotoxic effects of exposure during the two periods at this concentration. The 7- and 14-d exposure periods for observations of genotoxic effects were defined based on studies that showed the ability of the organism to remove cells damaged in 7-d exposures when exposed to stressors for a longer period [30]. The results observed during the preliminary tests led us to use a 2.5 mg/l solution of potassium dichromate in the following tests, because it was possible to identify its genotoxic effect after 7 and 14 d. A 21-d period was also included to assess a possible decrease in the effect studied. Genotoxicity Evaluation Table 2 shows the total number of micronucleated erythrocytes induced by potassium dichromate (2.5 mg/l) in fish exposed for 7, 14, and 21 d in six tests totaling 53 individuals and 106,000 cells scored. Means and standard deviations for each treatment are presented as absolute and transformed values. The statistical significance of the results compared to the parallel negative controls (C ) is also presented for each exposure period. Fish treated with potassium dichromate showed variable responses for induction of micronucleated erythrocytes compared to the negative control. This variability is indicated by the high standard deviations found. Genotoxic response variation, such as that observed in P. promelas, has been reported for rainbow trout (Oncorhynchus mykiss) exposed to polluted river water [30]. Factors such as age and female hormonal status can influence these responses [8]. For this reason, we used fish with standardized ages (before sexual maturity) to reduce the risk of variation caused by these factors. The results for individual animals, pooled according to time of exposure to Cr (VI), are shown in Figure 2. This procedure was possible because the variances among individuals showed no significant difference (p 0.05). Considering a larger number of exposed individuals, the response was significant (p 0.001) for a 7-d exposure. The effect observed during the 14- d period for 2.5 mg/l of potassium dichromate, despite not being statistically different from the negative control because of the large deviation, was not significantly reduced in relation to the 7-d period according to the means found in these two exposures. No significant micronucleus induction occurred during the 21-d exposure. These data agree with those of De Flora et al. [30], who demonstrated the ability of organisms to eliminate damaged cells after longer exposures. Itoh and Shimada [31], in a bone marrow cell study, estimated the influence of metallothionein in decreasing DNA damage induced by Cr (VI). This metalloprotein can scavenge free hydroxyl and superoxide radicals resulting from intracellular transformations of Cr (VI) to Cr (III). These radicals play an acknowledged role in producing toxicity. The authors observed the protective effect of this protein in animals treated with a metallothionein inductor (BiNO 3 ) before administration of Cr (VI). This pretreatment suppressed micronucleus induction compared to the assay with metal alone. The Table 2. Micronucleated erythrocytes (MNEs) verified in 2,000 cells/fish exposed to K 2 Cr 2 O 7 (2.5 mg/l) and in the parallel negative controls (C ) for 7, 14, and 21 d K 2 Cr 2 O 7 7d 14d 21d C 7d 14d 21d Fish MNE Range Mean SD a Mean SD b c a Absolute value; SD standard deviation. b Transformed value: MNE 0.5. c p

4 Cr (VI) micronucleus induction in fish Environ. Toxicol. Chem. 20, Fig. 2. Micronucleated erythrocytes (MNEs; mean standard deviation) found in fish exposed to K 2 Cr 2 O 7 (2.5 mg/l) and in the parallel negative controls (C ) for 7, 14, and 21 d. The vertical bars represent the standard deviation. authors also detected a decrease in micronuclei using two consecutive doses of Cr (VI) (i.e., K 2 CrO 4 ), suggesting that the first treatment with Cr (VI) would have induced the metalloprotein, reducing the effects in this experiment compared to those with only one dose. A similar mechanism could influence the decrease in genotoxic effect observed with P. promelas exposed for 21 d to a potassium dichromate concentration of 2.5 mg/l. During the preliminary tests (Table 1), P. promelas showed a decrease in the genotoxic effect over a 14-d period for a lower Cr (VI) concentration (2.0 mg/l). These data may indicate that for lower metal concentrations, a sufficient amount of metalloprotein to protect the cells is obtained in a shorter period of time. Lu and Yang [24] reported another mechanism to explain the decrease in damage induced by Cr (VI) and established a CHO cell strain selected for resistance to Cr (VI), progressively increasing CrO 3 doses in which the LC50 was approximately 25-fold that of parental cells. An extensive characterization of these resistant cell strains in relation to various possible markers that could justify the observed tolerance did not show any difference compared to nonresistant parental cells, but a difference was found in sulfate anions and Cr (VI) absorption, which were greatly reduced in chromium-resistant cells. The authors concluded that chronic exposure to low chromium doses could induce defects in the membrane anion transport system. They also stated that the system observed in CHO cells appears to be similar to the anion exchanger reported in erythrocytes [32]. On the other hand, Das and Nanda [6] suggested an inhibiting effect on cellular division, with subsequent hindrance of the passage of affected cells into the bloodstream, to explain the decreased effect observed in MNEs of fish exposed to paper mill effluents over longer periods of exposure. These authors used one-, two-, and three-month exposures, and they observed increases during the first month and decreases during the second and third months. Because of the variety of mechanisms involved in the production of genotoxic damage induced by Cr (VI) and its reduction to the trivalent form, which is found in DNA cross-links and adducts, the participation of several steps in the decrease of this damage at the cytoplasmic and DNA level cannot be excluded. The genotoxic effect of Cr (VI) was also evaluated in the goldfish (Carassius auratus gibelio) [7], in which significant increases of MNE frequencies were found in fish exposed for 7 d to 50 and 100 ng/ml of this metal. In the present study, 884 ng/ml of Cr (VI) were used, which denotes higher resistance of P. promelas to the action of this compound. A review on the sensitivity of fish species to 200 compounds indicated P. promelas as the most resistant species in practically all available comparisons for acute toxicity [12]. For this reason, we employed very young animals (45 60 d old) to study the most sensitive phase, which allowed us to obtain sufficient peripheral blood for smears. A Cr (VI) dose similar to that of the present study was used in mouse bone marrow micronucleus assays with no genotoxic effect []. Those authors used a dose of 1.0 mg/l, which could be ingested by humans (0.05 mg/l is the allowable limit for drinking water according to the World Health Organization and the Environmental Protection Agency, as cited by the International Agency for Research on Cancer [25]). They also used a dose of the substance greater than the palatability limit for rodents (20 mg/l), besides a dose of 5 mg/l in the drinking water of the animals for 48 h, or administered 24 and 48 h before sacrifice for another group, with negative responses for genotoxicity in all treatments. On the other hand, positive results for micronucleus induction were found in newt larvae exposed to Cr (VI) in doses very similar to that used in this study (1 mg/l) [28]. The possibility of damage caused by Cr (VI) is increased in aquatic organisms, because the exposure routes are different from those observed in rodents. Fish exposed to potassium dichromate showed a high frequency of cases in which no blood extraction was possible (.8%, 22/111), because blood flow was not enough for a smear, reducing the total number of test subjects studied. This occurred more frequently in animals subjected to the Cr (VI) test substance than in controls (1.9%, 2/104). Erythropoiesis inhibition has been suggested as a possible consequence of exposing fish to industrial effluents [6]. The toxic effects of Cr (VI) on the blood-forming organs have also been cited in humans [18]. According to our data, it can be concluded that the assay using P. promelas for micronucleus analysis allowed the detection of Cr (VI) genotoxicity, suggesting the value of this species for environmental genotoxic monitoring. It was also possible to demonstrate the importance of the parallel negative control submitted to the same test conditions, and of the knowledge regarding the species micronucleus basal level to construct historical laboratory data with which to control assay quality. The importance of determining the period with the highest effect of induced genotoxicity was also shown. The time must be sufficient for expression of genotoxicity, but not long enough to allow the organism to develop a defensive system to survive. Some mechanisms can lead to a negative response in the micronucleus test using Cr (VI), such as (1) protection by metalloproteins, (2) selection of cells/animals resistant to the toxic agent during a long period of chronic exposure, and (3) a toxic agent that can kill or inhibit cell divisions. Therefore, it is important to establish the proper time to maximize the test sensitivity. In laboratory bioassays, when conditions can be controlled, we believe that Cr (VI) could be used as a positive control for this fish species. The present study of Cr (VI) genotoxicity in P. promelas suggests 7 d as the best time period for expression of micronuclei in erythrocytes compared with 14- and 21-d periods. Acknowledgement K.D. Scherer and G.D. Souza provided technical assistance with the assay work, and J.E.A. Silva helped us in the laboratory activities. SITEL-CORSAN and Bioensaios Análises e

5 1324 Environ. Toxicol. Chem. 20, 2001 C. Torres de Lemos et al. Consultoria Ambiental provided the test fish. This research was supported by Fundação Estadual de Proteção Ambiental Henrique Luis Roessler, PADCT-FINEP, and Fundação de Amparo à Pesquisa do Rio Grande do Sul. REFERENCES 1. Heddle JA, Hite M, Kirkhart B, Mavournin K, Macgregor JT, Newell GW, Salamone MF. 83. The induction of micronuclei as a measure of genotoxicity. U.S. Environmental Protection Agency Report. Gene-Tox Program. Mutat Res 123: Heddle JA, Cimino MC, Hayashi M, Romagna F, Shelby MD, Tucker JD, Vanparys PH, MacGregor JT. 91. Micronuclei as an index of cytogenetic damage: Past, present, and future. Environ Mol Mutagen 18: Dearfield KL, Auletta AE, Cimino MC, Moore MM. 91. Considerations in the U.S. Environmental Protection Agency s testing approach for mutagenicity. Mutat Res 258: Lohman PHM, Mendelsohn ML, Moore DH II, Waters MD, Brusick DJ, Ashby J, Lohman WJA. 92. 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