Livestock Research for Rural Development 30 (8) 2018 Guide for preparation of papers LRRD Newsletter

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Acute cytotoxicity of the young Nile tilapia (Oreochromis niloticus) injected by aflatoxin B1

N Srikacha, L Neeratanaphan1, S Jongyotha2, U Tengjaroensakul3 and B Tengjaroenkul

Faculty of Veterinary Medicine, Khon Kaen University, Thailand
btengjar@kku.ac.th
1 Faculty of Science, Khon Kaen University, Thailand
2 Khon Kaen Inland Fisheries Research and Development Center, Thailand
3 Faculty of Science, Chiang Mai University, Thailand

Abstract

The objective of this study was to investigate acute toxicity of the Nile tilapia (Oreochromis niloticus) injected by aflatoxin B 1 (AFB1). Young tilapia were randomly allocated into 5 treatments: 1) control treatment; 2) negative control treatment (intraperitoneally injected with 0.3 ml of 25% dimethyl sulfoxide (DMSO)); and 3)-5) toxin treatments (intraperitoneally injected with AFB 1 at concentrations of 300, 600 and 1,200 ppb, respectively). The results revealed that at higher toxin concentrations, the number of metaphase cells was greater decreased as well as the percentage of chromosomal aberration (CA) increased. AFB1 causes structural CA. There were six types of CA in this study included SCG (single chromatic gap), DCG, ISCG, D, F and SCB. The most frequent CA found in AFB 1 exposed to Nile tilapia was SCG, which is one of the most sensitive biomarker to detect the level of severity of the cytotoxicity. This study can be concluded that acute AFB1 exposure causes cytotoxic effect in tilapia. Furthermore, the AFB1 contamination in the feed should be a concern because of its potential effects on fish health.

Keywords: aberration, aflatoxins, chromosome, cytotoxicity, tilapia


Introduction

Market demand for a healthy protein food source for human, especially fish becomes increase. Nile tilapia culture at high density is a strategy in order to cope with that demand (FAO 2016). Feed and its qualities are key success factors for the fish growth, immunity and disease resistance. Thailand locates in tropical with relatively warm and high humidity which is suitable for fungal growth, and faces mycotoxins contamination problem in feed ingredients (Wu et al 2011; Bbosa et al 2013). Mycotoxins are metabolic substances produced by fungi that can cause diseases and death in humans and animals (Sahoo et al 2001; Matejova et al 2016). A previous survey suggested that more than one-third of grains is contaminated with mycotoxins in the Asia-Pacific region (Binder et al 2007). In several occurrences, to present, feed raw materials were reportedly detected with aflatoxins (AF). AF are the most common toxin produced by Aspergillus flavus, A. parasiticus. and A. niger . Among these mycotoxins, aflatoxin B1 (AFB1) is generally accepted as the most potent agent among AF (Sahoo and Mukherjee 2001). The symptoms of aflatoxicoses in fish were loss of appetite, reduction of growth performance, liver damages, high mortality as well as cancer (Tuan et al 2002; Binder et al 2007; Gowda et al 2008; Deng et al 2010; Tengjaroenkul et al 2013; Selim et al 2014). Furthermore, AFB1 can induce chromosomal aberration (CA) and DNA defects in human, rat and certain aquatic creatures (Rooney 2001; Hassan et al 2010). As most reports on CA related to the aflatoxins were chronic exposure by fed the toxin contaminated diets for several weeks or months (Tuan et al 2002; Deng et al 2010; Selim et al 2014). Acute effects of the toxin were revealed limitedly. Therefore, acute aflatoxicoses in the young Nile tilapia by intraperitoneal injection of AFB1 at different concentrations was conducted to investigate its cytotoxicities.


Materials and methods

Aflatoxins preparation

A. parasiticus strain NRRL 1999 was spiked in autoclaved Jasmin rice and incubated at 25C for 7 days. Then, the dried contaminated rice weighed 20 g was ground and mixed with 10 ml distilled water, 10 g hyflo-supercel and 100 ml chloroform using stirrer at 150 rpm for 30 min. The mixture was filtered through filter paper No. 4. For toxin extraction, the filtered solution was poured into glass column contained with 2.5 g florisil, later consecutively poured with 5 ml chloroform, 20 ml methanol and the mixture of acetonitril and distilled water (ration 49:1). The final fraction of the solution was collect to detect AFB1 concentration using ELISA test kit. (Uganda National Bureau of Standards 2009; Selim et al 2014)

Fish and experimental design

Ninety young tilapia fish weighed approximately 8 g were randomly allocated into 5 treatments: 1) control treatment; 2) negative control treatment (intraperitonally injected with 0.3 ml of 25% dimethyl sulfoxide (DMSO)); and 3)-5) toxin treatments (intraperitoneally injected with AFB1 at concentrations of 300, 600 and 1,200 ppb, respectively). The experiment was conducted in 3 replications. Six experimental fish in each replication were reared in aerated 30 L plastic bucket at Aquatic Laboratory, Faculty of Veterinary Medicine, Khon Kaen University, Thailand. Water quality was controlled following aquaculture standard procedure. All protocols were reviewed and approved in accordance with laboratory animal standards of Khon Kaen University recommended by the Guide for the Care and Use of Thai Laboratory Animals Ethic Committee (El-Barbary and Mohamed 2014).

Chromosome preparation

Kidney was a white blood cell riched organ for chromosomal collection in this in vivo cytotoxicity study (Chen and Ebeling 1968; Nanda et al 1995). The experimental fish were injected with 0.05% colchicine into abdominal cavity at 1 ml per 100 g body weight. The fish was then placed in the aerated bucket for one hour. After that, the kidney was removed out of the body, and cut entirely into small pieces before being mixed with 0.075 M potassium chloride (KCl). After discarding all large pieces of tissue, the suspended lymphocyte cells in 8 ml KCl is incubated for 30 minutes at room temperature, and centrifuged at 1,500 rpm for 10 minutes. Then the supernatant was discarded before being fixed in fresh cool fixative (3 methanol: 1 acetic acid). The fixative solution was gradually added up to 8 ml before centrifuging again at 1,500 rpm for 10 minutes and then discarded the supernatant. The fixation process was repeated 3-4 times until supernatant was cleared. After that, each tube was concentrated to 2 ml and dropped 10 cm high on a clean and warm glass slide by micropipette (20 L per slide for metaphase cell count and 2 drop per slide for metaphase cell photograph),and left the slide to dry at room temperature. The slide was conventional staining by 20% Giemsa’s solution at pH 6.8 for 30 minutes.

Assessment of metaphase cell counts and chromosomal aberrations

Cell counts were performed on mitotic metaphase cells under a light microscope. Total number of the metaphase cell of each replicate per 20 L was counted. One hundred clear and numerical metaphases were selected from each replication for CA assessment (CAA). CAA were identified as the follow: single chromatid gap (SCG), double chromatid gap (DCG), isochromatid gap (ISCG), deletion (D), fragmentation (F) and single chromatid break (SCB). The CA results were recorded and analyzed. The fundamental number of chromosome arm was obtained by assigning a value of two arms of metacentric, submetacentric, telocentric and acrocentric chromosomes. All parameters were used in karyotyping according to international standards (Darwish and Mosallam 2014).

Statistical analysis

The percentages of CA and averages of total metaphase cell counts were analyzed using one way ANOVA and Turkey’s honest significant difference (HSD) test. The difference of each concentration of AFB 1 was analyzed using a paired t-test. All statistical tests were conducted at a 95% confidence level.


Results and discussion

Karyotype

The Nile tilapia karyotype in control treatment is in accordance with standard cytogenetic database (Rooney 2001), and diploid chromosome number (2n) used in this study was 2n=44, and the fundamental number (NF) was 58. The types of chromosomes were presented as 2 large acrocentrics, 2 small submetacentrics, 10 small acrocentrics, and 30 small telocentrics in consistence with Supiwong et al (2013) who studied chromosomal characteristic of the Nile tilapia from mitotic and meiotic cell division by T-lymphocyte cell (Figure 1).

Figure 1. Metaphase cells (A) and karyotype of Nile tilapia with 44 chromatids (B).
Metaphase cell count

After total number of the metaphase cell of each treatment per 20 L was counted, the results revealed that at higher toxin concentrations, the number of metaphase cells was lower. The metaphase cell numbers of the control and negative control treatments were similar (80.33-80.67), while the fish cell exposed to AFB 1 (31.00-33.33) were reduced significantly compared with control treatment (P<0.05)(Table 1). Injection of AFB1 is the reason for an occurrence of the reduction of lymphocyte metaphase cell. This probably because AFB1 reduces protein and DNA content by inhibition of several important metabolic pathways in the cells, and leading to a functional failure involving lymphocyte production in the head kidney, a major blood production of fish (Al-Terehi et al 2013; Bbosa et al 2013). Moreover, AFB1 also disrupts normal working process and loss of control over cellular growth and cell division in kidney (Vermal 2004). In addition, El-Barbary and Mohamed (2014) reported that AFB 1 likely cause lymphocytolytic due to the reduction of globulin levels in fish, and support the decrease of lymphocyte metaphase cell demonstrated in this study.

Types and percentages of CA

The results showed that the percentages of total CA in fish treated with AFB1 were more than of those observed in the control treatment (Table 2). The tendency of CA was depended upon concentration of AFB 1. The total percentages of cells with CA exposed to each concentration of AFB1 at 300, 600 and 1,200 ppb for 72 hours were 10.334.04%, 15.994.00% and 26.342.52 %, respectively, compared with the control and the negative control treatment as 3.331.15 and 5.670.58%, respectively (Table 2). Statistical analyses revealed that the total percentages of cells with CA were significant (P<0.05) differences between fish treated with AFB 1 and control treatment, except for the AFB1 300 ppb treatment. In this study there are six types of CA found in the lymphocytes including SCG, DCG, ISCG, D, F and SCB(Figure 2).

The SCG was the most CA revealed in this study. The percentage of SCG of cells with CA were significant (P<0.05) differences between fish treated with AFB1 and control treatment, except for the AFB1 300 ppb treatment. Moreover the percentage of DCG, ISCG, SCB, D and F increased slightly as concentrations of AFB1 increased. The AFB 1 1,200 ppb treatment was significantly (P<0.05) different in D as compared with control and AFB1 300 ppb treatment.

As acute AFB1 injection at 1,200 ppb, the fish demonstrated 20% aberrations of the metaphase cells, with SCG as the most found aberration and significantly different from other concentrations (P<0.05). These results were similar to previous studies reported that AFB1 could induce cytogenetic effects such as SCB, SCG and D in human and animals. Hassan et al (2010) reported that tilapia fed diet contaning 1.5 ppm AFB1 for 3 weeks (subacute exposure) revealed chromosomal abnormality more than 20%, with 6 types of the CA. Chromosomal break was the most observed aberration, following by centromeric attenuation, centric fusion and Gap (single- and Isochromatid-), whereas the control treatment demonstrated chromosomal defects approximately 3.0%, relatively lower than the control in this study (approximately 3.33%). However, The aberration in the control treatment was below 5%, is generally accepted. The aberrations investigated in the control was likely due to chemical effect of colchicine (a chemical help inhibition of mitotic cells in the metaphase stage by blocking function of mitotic spindle)(Ew 1965; Takeshita et al 2016). DMSO has been extensively used for its excellent solvent properties. El-Barbary and Mohamed 2014 dissolved AFB1 in 25% DMSO as the vehicle to intraperitoneally injection in the Nile tilapia, and they report that DMSO did not cause the toxicity. Moreover, DMSO is considered to be not mutagenic for bacteria and drosophila. Aye et al 2010 revealed that DMSO could not induce chromosomal abnormalities because DMSO did not induce both DNA strand-breaks and micronuclei in CHO cells in that study.

Table 1. The effect AFB1 on number of kidney metaphase cell exposed to various AFB1 concentrations for 72 hours.

Treatment

Number of metaphase cell
(cell/20 L)

Control

80.332.52b

Negative control

80.6711.37b

AFB1 300 ppb

33.333.21a

AFB1 600 ppb

33.3310.97a

AFB1 1,200 ppb

31.0012.29a

p

<0.001

# Data are mean SD. Different letters in the same column indicate significantly different (P<0.05).

Furthermore, Al-Terehi et al (2013) reported that Albino rat fed AFB 1 1 ppm for 15 days, and the rat demonstrated total of 53.65% CA such as chromatid break, sticky, aneuploidy, and ring, with approximatelly 6.56% CA in the control. Ghaly et al (2010) showed chromosomal problem in Swiss albino rat in the control treatement, with the revealation of 7 CA (P<0.001) after fed AFB1 1 ppm for 2 weeks. Effects of acute exposure of the AFB1 may differ due to a range of sensitivities among animals to the toxin, and also by several factors, including concentration, exposure time, breed, age, sex, metabolic mediators produced (especially epoxide) during detoxification of each species (Eaton and Groopman 1994). AFB1 is one of mycotoxins causes genetic mutation because its effect directly or indirectly on the DNA, RNA and protein (Darwish and Mosallam 2014). AFB1 can bind to DNA to cause its structural aberration. DNA damage is the major effect of AFB 1 including apurinic site, DNA breaks and DNA adduct. Apurinic site, the loss of purine base in DNA (depurination), cause DNA structural aberration, strand scission and block DNA replication that associated with CA (Bailey et al 1996). In addition AFB1 increase reactive oxygen species (ROS)(free radicals) which induce different damages to DNA, RNA and protein. Free radicals cause DNA strand break (single strand break and double strand break), if it is not properly repaired can lead to genomic instability as well as CA and apoptosis (Al-Terehi et al 2013; Bbosa et al 2013).

Table 2. The effect AFB1 on Chromosomal aberration (CA) in kidney metaphase cell after exposed for 72 hours.

Treatment

Percentage of CA

Total
CA

SCG

DCG

ISCG

SCB

D

F

Control

3.00a±1.00

0.00a±0.00

0.00a±0.00

0.00 a±0.00

0.00a±0.00

0.33 a ±0.58

3.33a±1.15

Negative control

4.67a±1.53

0.00a±0.00

0.00a±0.00

0.00 a±0.00

0.00a±0.00

1.00 a ±1.00

5.67a±0.58

AFB1 300 ppb

6.00a±2.65

0.33a±0.58

1.00a±0.00

0.67 a±0.58

0.33a±0.58

2.00 a ±1.00

10.33ab±4.04

AFB1 600 ppb

11.33b±2.52

0.33a±0.58

1.00a±1.00

0.33 a±0.58

1.33ab±0.58

1.67 a ±0.58

15.99b±4.00

AFB1 1,200 ppb

20.00c±1.00

0.67a±1.15

1.67a±1.53

0.67 a±1.15

2.00b±1.00

1.33 a ±1.53

26.34c±2.52

p

<0.001

0.682

1.25

0.534

0.005

0.357

<0.001

# Data are percentage ±SD. Different letters in the same column indicate significantly different (P<0.05). SCG = single chromatid gap, ISCG = isochromatid gap, DCG= double chromatid gap, SCB =single chromatid break, D = deletion and F = fragmentation



Figure 2. Normal chromosome (control treatment)(A). Six types of CA (arrows) found in kidney metaphase
cell exposed to AFB1, including single chromatid gap (B), isochromatid gap (C), double
chromatid gap (D), single chromatid break (F), Deletion (F), fragmentation (G).


Conclusion


Acknowledgement

This research was funded by Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen, Thailand, and.Research Group on Toxic Substances in Livestock and Aquatic Animals


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Received 20 April 2018; Accepted 18 July 2018; Published 1 August 2018

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