Susceptibility of male and female Japanese medaka (Oryzias latipes ...

Front. Environ. Sci. Eng. 2013, 7(1): 77–84 DOI 10.1007/s11783-012-0466-z

RESEARCH ARTICLE

Susceptibility of male and female Japanese medaka (Oryzias latipes) to 2,4,6-trichlorophenol-induced micronuclei in peripheral erythrocytes Nannan LIU, Mei MA (✉), Yiping XU, Jinmiao ZHA, Kaifeng RAO, Zijian WANG State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2012

Abstract 2,4,6-trichlorophenol (2,4,6-TCP) is a widespread probable human carcinogen and has been proven to have genotoxicity in in vitro assays. However, little genotoxicity information and no micronuclei induction data for 2,4,6-TCP is available from in vivo tests, especially for sex-specific differences. Following a preliminary test, a piscine peripheral erythrocyte micronucleus assay was conducted on medaka (Oryzias latipes) after a 28-day exposure to 2,4,6-TCP. In the present study, the mean micronuclei (MNC) frequencies of all of the groups increased in a dose-dependent manner, which indicated the potential genotoxicity of 2,4,6-TCP. Moreover, males were found to be more susceptible compared with females after a 28-day exposure to 2,4,6-TCP in all of the dosed groups above 10 μg$L–1. This is the first report on the potential of micronuclei induction and a sexsusceptible effect in the peripheral erythrocytes of mature fish after 2,4,6-TCP in vivo exposure. Keywords 2,4,6-trichlorophenol, genotoxicity, Japanese medaka, piscine micronucleus test in peripheral erythrocytes, gender difference

1

Introduction

2,4,6-trichlorophenol (2,4,6-TCP) is a priority pollutant that is widely used as a preservative, insecticide, herbicide, bactericide, or as an intermediate in the production of high chlorinated phenols [1]. This chlorophenolic compound is a toxic environmental pollutant and ubiquitously found in air, water, and soil. 2,4,6-TCP is carcinogenic in animals, causes lymphomas and leukemia in male F344 rats, and Received January 18, 2012; accepted September 9, 2012 E-mail: [email protected]

induces hepatocellular carcinomas in B6C3F1 mice via oral exposure after long-term treatment at high doses [2]. Because of the lack of available human carcinogen data and sufficient evidence in animals, 2,4,6-TCP has been classified as a Group B2 (probable human carcinogen) by the United States Environmental Protection Agency [3]. Recently, the occurrence of this hazardous chemical has received increased public concern. A relatively wide range of 2,4,6-TCP concentrations has been reported in bodies of water [4–6] and water supply systems [7–9]. Evidence has confirmed the absorption, persistence, and bioaccumulation of 2,4,6-TCP in the tissues of rats [10], fish, and other aquatic species [11]. The deleterious effects of 2,4,6-TCP may directly affect the safety of drinking water and bodies of water and are closely associated with environmental public health issues. Humans are potentially exposed to 2,4,6-TCP in occupational or contaminated environments through inhalation, ingestion, and dermal contact [12]. In human urine samples, studies have demonstrated that 2,4,6-TCP has been detected in the range of 0.3 to 2369 μg $L–1 [13–17] and has been identified in human blood [17] and breast milk [18] samples. Pollutants, such as 2,4,6-TCP, enter the aquatic environment and produce a series of detrimental effects on individual, population, community and ecological system of aquatic organisms. Genotoxicity plays an important role in assessing these deleterious effects. Among all of the genotoxicity assays, the detection of micronuclei, formed by the exclusion of whole chromosome or chromosomal fragments from the main nuclei, is a commonly applied biomarker to investigate cytogenotoxic damage at the chromosomal level. The piscine micronucleus assay is a sensitive and effective tool for evaluating genotoxicity in the aquatic environment because it does not rely on the karyotypic characteristic of the species [19–22]. When fish are exposed to a

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Front. Environ. Sci. Eng. 2013, 7(1): 77–84

genotoxic agent, this exposure can induce cytogenotoxic damage on multiple tissues and organs. Compared with other tissues [23–25], the piscine micronucleus assay in peripheral erythrocytes has been successfully developed as a convenient and concise tool to estimate the cytogenotoxic potential because complex cell preparation is avoided. Importantly, the genotoxicant acts on hemopoietic organs to produce micronucleated erythrocytes, which may occur as circulating micronucleated erythrocytes in peripheral blood samples that are associated with the species-specific presence or absence of micronuclei removal after peripheral circulation [26]. This observation reveals that the piscine peripheral erythrocyte micronucleus assay is an appropriate and effective tool to monitor genotoxicity after subchronic or chronic exposure [27]. Moreover, fish are easily maintained in laboratories or in situ and thus, are excellent organisms to simulate the responses of higher vertebrates, which include the in vivo assessment of potential toxicant genotoxicity and carcinogenicity in humans [28]. The aim of this study was to evaluate the genotoxicity of 2, 4, 6-TCP. Although a negative response was obtained in the Ames Salmonella/microsome test [29], 2, 4, 6-TCP clearly showed mutagenicity in Saccharomyces cerevisiae MP-1 [30] and Bacillus subtilis [31,32]. The cytogenetic effect of 2, 4, 6-TCP was also investigated in mammalian organisms. Smaller litter sizes, higher stillbirth rate, heavier liver and spleen weights of offspring were observed in the Sprague Dawley rats that were given 2, 4, 6-TCP in drinking water compared with the untreated group [33], which indicated that 2, 4, 6-TCP was possibly genotoxic in mammals. The use of mammalian cell lines for in vitro assays may provide further evidence for the genotoxicity of 2, 4, 6-TCP. Specifically, the comet assay conducted on human leukemic cell line (HL)-60 cells confirmed a DNA damage effect [31], and micronucleus and chromosome aberration tests in Chinese hamster ovary and V79 cells have demonstrated chromosomal damage activity of 2, 4, 6-TCP [34]. At present, little genotoxicity information and no micronuclei induction data for 2, 4, 6TCP are available via in vivo fish tests. Only a 10-day exposure of 2, 4, 6-TCP at 5 μg$L–1 in zebrafish has been reported to elevate point mutations in the P53 gene in liver [35]; whether 2, 4, 6-TCP can induce chromosomal damage or induce gender-specific effects in fish is unknown. As a native freshwater teleost in East Asia, Japanese medaka have been extensively studied in genomic studies [36,37] and exhibit sex susceptible effects [38] and therefore, are an excellent model organism [36] in laboratory research [23,39,40] and in situ studies. The micronucleus assay in peripheral erythrocytes of medaka might be a suitable and successful tool to assess the potential genotoxic properties of stimulations from various external environments. It is important to assess the cytogenotoxic activity of 2, 4, 6-TCP using the micronucleus assay in peripheral erythrocytes of medaka.

Consequently, we examined genotoxic potency and for gender-specific differences by measuring the micronuclei (MNC) frequencies of medaka peripheral erythrocytes following 2, 4, 6-TCP exposure. In this study, we have described a modified protocol for the medaka micronucleus assay using peripheral erythrocytes in males and females and evaluated the potential influence of varying body parameters (i.e., body weight and body length) on MNC frequencies to complement the genotoxicity data for risk assessment.

2

Materials and methods

2.1

Chemicals

2, 4, 6-TCP (purity = 98%) was purchased from ACROS Organics, USA. A stock solution of 2, 4, 6-TCP was prepared in high performance liquid chromatographygrade dimethyl sulfoxide (DMSO) and mixed with distilled water. The final solvent concentration in the exposure water did not exceed 0.001% (v/v). 2.2

Experimental fish

Japanese medaka fish were raised in aquariums in dechlorinated tap water equipped with an aeration system (pH 7.2–7.6; hardness 44.0–61.0 mg$L–1 CaCO3) at a constant temperature (251°C) with a photoperiod of 16∶8 h (light : dark). Medaka were cultured and maintained for more than five generations in our laboratory and fed twice daily while feces were removed daily. 2.3

Exposure and experimental design

Before the experiment, fish were acclimatized in aerated glass aquariums (18 L) with dechlorinated tap water. Sexually mature fish that were from the same batch and had similar length (36.61.5 mm) and body weight (0.460.04 g) were used in the experiments after a 2week acclimatization period. A preliminary experiment was designed to determine the predicted no effect concentration of 2, 4, 6-TCP in medaka (n = 60) that were divided equally into 0, 10, 30, 100, 300 and 1000 μg $L–1 exposure groups for 7 days in circular glass beakers in which the water was changed on the next day. In this study, we administered a single collection with a continuous-flow system exposure because previous research data have verified that continuous dose exposure with a single sampling at a steady-state was more efficient than a single dose exposure with multiple samplings at various times in in vivo erythrocyte micronucleus studies [41]. Subsequently, the stock solutions were dosed to glass mixing vessels with a peristaltic pump at a rate of 0.5 mL$min–1 and diluted with dechlorinated tap water in a flow-through system at a rate of 3 L$h–1. Medaka (males:females = 1∶1)

Nannan LIU et al. Susceptibility of male and femal Japanese medaka to 2,4,6-TCP induced micronuclei

were randomly exposed to nominal concentrations of 10, 30, 100, 300, 1000 μg$L–1 and DMSO [0.001% (v/v)] as a solvent control in continuously aerated glass aquariums (18 L) for 28 days. There were 20 fish in each of the aquariums that contained approximately 16 L of water. Only 5 male and 5 female fish were used in the micronuclei assay; the remaining 10 fish were used for another test. Two duplicates were carried out for the treatment and control groups. 2.4

Micronucleus assay

The fish were sacrificed after a 28-day exposure. Blood samples from each group were collected by cutting the medaka tails and taking blood in a heparinized microcapillary tube. The micronucleus assay in piscine erythrocytes was conducted following a modified version of the previously mentioned protocol [27]. The peripheral blood erythrocytes from each fish were dropped onto three clean slides that were flattened by other slides to produce an evenly distributed blood smear, treated with a fixative (methanol) for 15 min at room temperature, air-dried, stained with 10% Giemsa in a phosphate buffer (PBS), washed twice with PBS and mounted. Micronucleus scoring was conducted on the cells that had been spread onto clean slides and air-dried. The slides were blindly coded and scored by one observer. For micronuclei analysis, approximately 5000 erythrocytes on a single slide were observed at a 1000 magnification under a light microscope. This study was conducted using a modified micronucleus assay protocol that used peripheral erythrocytes of Japanese medaka with approximately 15000 cells counted per fish. The frequencies of clearly outlined and typically shaped micronuclei in the peripheral blood erythrocytes were observed. The criteria, which were introduced by Fenech [42], specified that scorable cells should be separate, easily distinguished and of approximately equal size. Moreover, only micronucleus (MN) isolated from the main nucleus and stained in the same color as the main nucleus with an area less than 1/3 of the nucleus could be scored. 2.5

Statistical analysis

The MNC frequency was calculated as the incidence rate per 1000 erythrocytes. Statistical analyses were performed using SPSS v11.0 (SPSS Inc., Chicago, IL, USA), and the figures were drawn with Origin 8.0. The mean differences in MNC frequency between the solvent control and exposure groups or between males and females in each group were compared by one-way analysis of variance (ANOVA) and the Least Significant Difference test. Partial correlation coefficients were calculated between the body parameters (i.e., body weight and body length) and MNC frequencies.

3

79

Results and discussion

In the present study, the medaka micronucleus assay via blood smear analysis was conducted in peripheral erythrocytes to monitor the susceptibility of males and females to 2, 4, 6-TCP. In this study, no dead fish were observed in the preliminary experiments, which thereby excluded possible lethal concentrations for the following experimental design. Consequently, 1000 μg$L–1 of 2, 4, 6TCP was assigned as the highest concentration in a 28-day exposure. The mean MNC frequencies of medaka regardless of sex exhibited dose-dependent increases throughout the study. However, significant differences in average MNC frequencies were observed in males compared with females that were exposed to concentrations greater than 10 μg$L–1 of 2, 4, 6-TCP. The average MNC frequency (meanS.D.) in the duplicate solvent control groups was 0.860.08 ‰. The control groups included 10 males with a mean body length of 37.90.9 mm and a mean body weight of 0.470.03 g and 10 females with a mean body length of 37.10.8 mm and a mean body weight of 0.460.03 g. No significant difference in frequencies between males and females of the solvent control group was observed. Subsequently, the frequencies for the individual males and females in the solvent control group against the body length and the body weight were plotted separately in Fig. 1. The MNC frequencies of the individual males in the solvent control group varied within a range of 0.8 ‰–1.0 ‰, whereas the range for the individual females varied between 0.7 ‰ and 0.9 ‰. No obvious correlation between body lengths and MNC frequencies by gender was observed; the adjusted r2 values (P values) for males and females were – 0.12 (0.88) and 0.15 (0.15), respectively. Similarly, independence was found in MNC frequencies that were plotted against the body weight. The adjusted r2 values (P values) for males and females were – 0.08 (0.59) and – 0.06 (0.52), respectively. The MNC frequencies in the 2, 4, 6-TCP-treated groups were very significantly increased (P < 0.01) compared with the solvent control group. The body length and body weight of the 100 exposed fish (50 males and 50 females) were recorded. In particular, the mean body length and body weight of male were 37.01.6 mm and 0.470.05g, while those of female were 35.91.9 mm and 0.450.06 g, respectively. We considered whether body weight and body length were possible influencing factors. Body weight and body length were insignificantly different in all of the dosed groups. Further partial correlation analysis between MNC frequencies and body factors (weight and length) was conducted in all fish or by gender in all of the groups. Pearson correlation coefficients are presented in Tables 1– 3). Body weight and body length were positively correlated in all fish and male and female groups, whereas

80


Fig. 1 Micronucleus frequency DMSO-treated medaka in the solvent control group plotted against body length (A) and body weight (B)

MNC frequencies were negatively correlated with body length in all fish groups. The results were not concordant with laboratory findings that reported after irradiation [23]. However, similar findings were reported regarding metal Table 1

ion accumulation in mosquito fish [22]. These findings simply indicate that body weight and body length should be considered when conducting the micronucleus assay. In addition, strictly controlled experimental conditions, such as temperature, sex ratio, age, and feeding conditions, require much attention. A change in the micronucleus frequency of the medaka peripheral erythrocytes was used as an indicator for 2, 4, 6TCP genotoxic potency. Following a 28-day exposure, the mean MNC frequencies of all of the dosed groups were higher compared with that of the corresponding solvent control group and were dose dependent throughout the study. Significant differences in the MNC frequencies between the solvent control and the 2, 4, 6-TCP-treated groups as well as among all of the groups were found. The rate of micronuclei erythrocytes increased in a dosedependent manner after 2, 4, 6-TCP exposure (Table 2). Accordingly, a significant relationship between 2, 4, 6TCP exposure concentrations and MNC frequencies was measured by ANOVA. However, results from the medaka peripheral erythrocyte micronucleus test with gender differences have been rarely reported. Therefore, gender-specific genotoxicity was evaluated by employing a piscine micronucleus assay in peripheral erythrocytes of medaka after 2, 4, 6-TCP exposure. In groups that were treated with exposures greater than 10 μg$L–1, 2, 4, 6-TCP induced micronuclei in males and females at different rates, with males being more sensitive than females. The gender-specific differences were compared in males and females of the same treatment groups. The rate (mean S.D.) of the aberrant cells was calculated from the number of micronuclei per 1000 erythrocytes or the number of micronuclei per 1000 erythrocytes in an individual fish divided by the group mean of the number of micronuclei per 1000 erythrocytes for the solvent control fish. Significant differences between the female and male fish in all of the dosed groups excluding the solvent control and 10 μg$L–1 groups were observed (Fig. 2). Moreover, the extent of the concentration-dependent effects was magnified with an increase in exposure concentration. The males exhibited higher micronuclei frequencies than the females when the exposure level of 2, 4, 6-TCP was greater than 10

Pearson correlation coefficients were studied in all fish from all treatment groups between micronuclei frequencies and body factors (weight

and length) total body weight total body weight

total body length *

micronucleus frequency

1.000

0.575

0.053

P=.

P = 0.000

P = 0.565

1.000

– 0.236*

P=.

P = 0.009

total body length

micronucleus frequency

1.000 P=.

*

Note: Correlation is significant at the 0.05 level (2-tailed).


81

Table 2 Pearson correlation coefficients were studied in male fish from all treatment groups between micronuclei frequencies and body factors (weight and length). male body weight

male body weight

male body length

male micronucleus frequency

1.000

0.571*

– 0.027

P=.

P = 0.000

P = 0.836

1.000

– 0.375*

P=.

P = 0.003

male body length

male micronucleus frequency

1.000 P=.

*

Note: Correlation is significant at the 0.05 level (2-tailed)

Table 3 Pearson correlation coefficients were studied in female fish from all treatment groups between micronuclei frequencies and body factors (weight and length) female body weight

female body weight

female body length

female micronucleus frequency

1.000

0.548*

0.078

P=.

P = 0.000

P = 0.552

1.000

– 0.285*

P=.

P = 0.027

female body length

female micronucleus frequency

1.000 P=.

*

Note: Correlation is significant at the 0.05 level (2-tailed)

μg$L–1. A linear regression analysis of the variables demonstrated that the difference between the exposure concentrations and gender-specific micronuclei induction ratio in males and females was significant. The ascent rates of the MNC frequencies were 1.21 and 1.82 for females and males, respectively. Statistical analyses were calculated with a trend test that demonstrated the adjusted r2 values (P values) for males and females in the linear regression were 0.97 (0.002) and 0.98 (0.001), respectively. Males were found to be more susceptible than females after a 28-day 2, 4, 6-TCP exposure greater than 10 μg$L–1. Results obtained in present genotoxicity study indicated that males were more susceptible than females in accordance with a previously reported cancer test in which 2, 4, 6-TCP was found to act on the hematopoietic system to induce lymphomas and leukemia tumors in male

F344 rats [2] and not female rats. Although equivocal reports have been released on the differential sensitivity of males and females to the genotoxic effects of X-ray irradiation and polychlorinated naphthalene exposure [23,28,40], all of these indicate that gender difference might be an essential factor in investigating the deleterious effects. Particularly, more attention should be paid to genetic sex-determined species, such as the medaka, which act in the same manner as mammals. 2, 4, 6-trichlorophenol is a widely detected toxicant that is resistant to biodegradation [43]. Acute and sub-chronic toxicity tests have been conducted on Chinese local species to derive the predicted no effect concentration (PNEC) for 2, 4, 6-TCP. These findings demonstrate that fish can serve as highly sensitive organisms to assess the toxicity of 2, 4, 6-TCP [44]. Moreover, 2, 4, 6-TCP has been determined to accumulate in fish, disturb fish’s normal behavior,

Table 4 Micronuclei frequencies in peripheral erythrocytes of medaka in control and 2, 4, 6-trichlorophenol-treated fish for 28 days exposure concentration /(μg$L–1) Solvent control

No. of fishes observed

No. of cells counted

frequencies of micronucleated erythrocytes/ ‰ (MeanS.D.)

frequencies of micronuclei/ ‰ (MeanS.D.)

20

300539

0.860.07a

0.860.08a

b

10

20

300520

1.860.21

1.860.21b

30

20

300623

3.380.55c

3.370.55c

d

100

20

300503

4.200.55

4.220.56d

300

20

300745

5.550.89e

5.560.90e

1000

20

300719

7.761.62f

7.771.62f

Note: The letters a, b, c, d, e, and f indicate differences among all of the groups (P < 0.01)

82


4

Fig. 2 The micronuclei frequencies of peripheral erythrocytes in medaka exposed to varying nominal concentrations of 2, 4, 6trichlorophenol. The meansS.D. were recorded as the number of micronuclei per 1000 erythrocytes in an individual fish divided by the mean micronuclei frequency ( ‰) of the solvent control group. The dashed line represents the mean micronuclei frequency of the solvent control in males and females. The vertical bars indicate the gender-specific induction ratio of the mean micronuclei frequency of the treated group compared with the solvent control, in which the asterisk (*) indicates a significant difference between males and females of the same exposure group at P£0.05

influence enzyme activities, and even cause mortality. Genotoxicity to tested organisms is dependent largely on the mode of action of the genotoxic compound. Medaka are increasingly utilized in genotoxicity and carcinogenesis studies because of their excellent sensitivity to xenobiotic compounds [45] and relatively high levels of monooxygenase activity and cytochrome P450 compared to that of other fish [46]. Moreover, metabolic activation by cytochrome P450 may play a key role in the toxicity and carcinogenicity of 2,4, 6-TCP. When Aroclor 1254induced rat liver S9 fractions were included in the test system, 2,4, 6-TCP was shown to undergo NADPHdependent, cytochrome P450-catalyzed oxidation to yield 2,6-dichloro-1, 4-benzene diol [47]. In contrast, 2,4,6-TCP also has been regarded as an important substrate for horseradish peroxidase (HRP) and shown to undergo hydroperoxide-dependent, HRP-catalyzed oxidative dechlorination to 2, 6-dichloro-1, 4-benzoquinone [48]. 2,4,6-TCP has also demonstrated a distinct connection between its toxicity and active oxygen species, and it has been further proven that active oxygen species contribution to the formation of micronuclei and the observed DNA damage [47]. These mechanisms of genotoxicity may partly explain the gender-specific genotoxic effects observed in the piscine micronucleus assay in peripheral erythrocytes of Japanese medaka after 2, 4, 6-TCP exposure.

Conclusions

In summary, the present genotoxicity study clearly demonstrates that 2, 4, 6-TCP induces a dose-dependent increase in micronuclei in peripheral erythrocytes of medaka. The strength of the relationship between the body parameters and MNC frequencies were evaluated by Pearson’s correlation. No correlation between MNC frequencies and body weight was observed, which was in contrast to a negative correlation between MNC frequencies and body length in all of the groups. Moreover, differential sensitivity indicated that males were more susceptible than females to the genotoxicity of 2, 4, 6-TCP exposure. Results reveal that 2, 4, 6-trichlorophenol has a cytogenotoxic effect in peripheral erythrocytes of medaka by the piscine micronucleus assay, and a sex difference should be considered according to the environmental pollution class. Further research is needed to investigate various genotoxicants for additional evidence. Acknowledgements This work was supported by the Environmental Protection National Commonwealth Research Project (No. 200909040), the National Natural Science Foundation of China (Grant No. 20921063), and International Scientific and Technological Cooperation Projects by the Ministry of Science and Technology of China (No. 2009DFA91920).

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