Induction of Cancer in Zebra Fish Using Simple Carcinogen

 

Reena Rajkumari B^1*, Sneha A², Rhea F². Anusha S²

¹Assistant Professor Senior Integrative Biology Department School of Bio -Sciences and Technology, VIT University, Vellore – 632014 Tamilnadu, India

²School of Biosciences and Technology, VIT University, Vellore.

 

ABSTRACT:

Benzene is a very common air pollutant and is known to be a carcinogen. It causes cancer in people who are exposed to benzene vapors over a very long period of time. Cancer is one of the leading causes of death, causing millions of death every year. Zebra fish is a very promising experimental model for drug testing and cancer studies. In this experiment benzene was used as a carcinogen in zebra fishes to check if it induces cancer in the fishes. These cancer induced fishes may be used for drug discovery and testing for cancer therapy. Different concentrations of benzene were injected into the zebra fishes, and the fishes were monitored over a period of 14 days. The fishes were observed for presence of any cancerous growth, and their weights were checked in order to determine any increase in mass. The results were inconclusive as no tumor growth was observed. This could be due to the short time over which they were monitored or the concentration of benzene being used may be low. May be if the time period, the period of exposure and the concentration of the benzene were increased, some significant results might be observed. 

 

 

 

INTRODUCTION:

Malignant tumours have led to the cause of death in industrial countries. Most of the tumours show either initial or acquired resistance to chemotherapy and hence are very difficult to treat when detected in advanced stages and in the case of relapse. Due to the wider use of intensive chemotherapy, the prognosis for childhood cancer has improved remarkably however, the outcomes for patients with relapsed cancer remain poor.  This highlights the need for the development of novel tools¹ to understand the mechanisms underlying cancer progression and for screening of potential therapeutics. 

The mouse is the standard experimental model for infectious and non-infectious studies.

 

Despite the mouse being frequently used as a model for human disease, several aspects of urine biology limit its routine use in largescale genetic and therapeutic screening. Many researchers who have an interest in an embryologically and genetically tractable disease model have now turned to zebrafish.  Zebrafish life cycle allows easy access to all developmental stages, the optical clarity of embryos and larvae allow real-time imaging of developing pathologies. They are easy to handle compared to mouse and they grow into maturitywithin months.² Zebra fish can be used in Complicated mutagenesis and screening strategies on a large scale with aminimumeconomy which is not possible inwithother vertebrate systems. Zebrafish models are used in wide variety of human diseases.³ Zebra fish and humans show striking similarity in hematopoietic development.⁴ Zebra fish have cognates of all human adult blood lineages (including T and B lymphocytes).⁵ Hence it is conceivable thatdrugs which show hematopoietic effects in zebra fishmight have similar effects on human cells.⁶ Most of the tumours induced in zebrafish are by carcinogens, tumour Cell lines or oncogenic transgenes.⁷ They demonstrate the ability to invasive growth and metastasis and they can also be successfully transplanted into either syngeneic or immunosuppressed recipients. These features are also a characteristic feature for zebrafish models of leukemia caused by transgenic oncogenes specifically when expressed in lymphocyte progenitor cells.⁸

 

Figure 1: Shows the basic structure of banzene

 

Benzene is a colourless fluid which is highly flammable Figure.1. It is a carcinogen. Benzene can be commonly found as a pollutant in the environment. It is used for production of many industrial compounds. Benzene is one of the major pollutants in the metropolitan cities. Its major source is emissions from motor vehicles, forest fire, burning wood and waste products and even tobacco contains appreciable amount of benzene.⁹ All this gets accumulated in the environment. An average man is exposed to benzene every day. Most people begin to smell benzene in air at approximately 60 parts per million (ppm) and recognize it as benzene at 100 ppm. Some people taste benzene in water at 0.5– 4.5 ppm. One part per million is approximately equal to one drop in 40 gallons. Those exposed the most are mainly the workers and tobacco smokers (is present in cigarette smoke). Prolonged exposure to benzene causes toxic effects in humans as well as animals. Every increase in dose of benzene increases the cancer risk.^9,10 Long term exposure to benzene may also lead to cancer. Benzene is also known to be genotoxic to human cells. It increases the incidences of mutational changes resulting in mutations. Benzene has been known to cause cancer in many animal models such as rats and mice. In this paper the carcinogenic effect of benzene was tested using zebrafish, a model organism. . 

 

MATERIALS AND METHODS:

Animal model:

For the experiment 30 zebra fishes (Daniorerio) were obtained from a local breeder. Fishes of both the sexes were used for the study. Adult fishes were selected for thetumour induction. All the fishes were weighed beforethe experiment. The aquarium was maintained at 28 ˚C and the fishes were fed 70.6mg of tetramin tropical flakes thrice a day. The water in the aquarium was replaced every 7 days.

 

Carcinogen:

Out of the many environmental carcinogens, benzene was selected for this experiment as it is known to cause cancer both in humans as well as animals. 

 

METHOD:

Benzene was dissolved in ethanol to prepare an initial stock solution of 1000 ppm. From the stock solution, concentrations in the range of 5, 10 and 20 ppm benzene solution was prepared by diluting with phosphate buffer saline (PBS). The zebra fishes were divided into 4 groups, each group comprising 3 males and 3 females respectively. Each group was exposed with the different concentration of the benzene solution as shown in the table 1. The benzene exposure was accomplished through intra-peritoneal injections. For the control group, a solution of PBS and ethanol in the ratio of 199:1 was used.

 

Table 1: The dosage and number of fishes used for the carcinogenesis testing of benzene

Group	Dosage of benzene (in ppm #)	Number of Adult Zebra fishes
		Male	Female	Total
I	5	3	3	6
II	10	3	3	6
III	20	3	3	6
IV	0 (control)	3	3	6
Total		12	12	24

 

 

Figure 2: Insulin injections used for intraperitoneal injections of the fishes

 

Zebra fishes were made immotile by lowering the temperature to less than 12°C, by adding ice slowly to the water and measuring with a thermometer. The fish was placed on a sponge which had a cleavage to hold the fish from slipping away.  Insulin syringes were used to inject the fish Figure.2.

 

The fishes were monitored for 14 days for any visible signs of tumor. The fishes were weighed twice a day to record any change in the weight. 500ml of normal water is placed in a beaker; the weight of the beaker with water is recorded. In the same beaker the zebra fish was added and the weight of the beaker with water and zebra fish was measured. The zebra fishes were anesthetized, without the use of any chemicals, by immersion in a pre-weighed beaker of cold water (12°c) for less than a minute as temperature drop, the fish slows down and the weight can be measured. The difference between the two weights gives the weight of the zebra fish. The weight of the fish before and after induction was compared which gives the weight of the tumor if present.

 

RESULT:

As benzene is a carcinogen, benzene should have caused cancer in the zebra fishes. Over the two weeks, the fishes were monitored after injection, no significant changes or presence of tumours was visible. The weights of the fishes before and after induction were checked in order to determine any notable growth or increase in weight. Table 2 shows the results obtained. But, no changes were seen. No tumor growth or weight gain was observed in any of the fishes.

 

Table 2: The results after intraperitoneal injection of benzene

 

CONCLUSION:

Benzene is a very common pollutant and a chemical commonly used in laboratory. If the compound is proved to be carcinogenic to zebra fishes, tumor growth can be observed in them. Zebra fishes are widely used in cancer studies and the carcinogens available are often expensive. This could form a cheap and less harmful compound for inducing cancer and used for various drug discovery experiments.

 

Benzene being a carcinogenic air pollutant needs to be kept under check. As, it is carcinogenic to humans it is important to determine the concentration at which it is harmful. This experiment could help determine the toxic dose of benzene. The experiment could be conducted with higher concentrations of benzene as no tumors were seen with the concentrations (5-20 ppm). This study concludes that the benzene concentrations were low, as they were not toxic to the fishes. In future studies, the time duration and the number of exposures could be changed from single to multiple. A constant exposure to benzene might cause the desired effects.

 

REFERENCES:

1.       Hideshima T, Anderson KC. Molecular mechanisms of novel therapeutic approaches for multiple myeloma. Nat Rev Cancer. 2002; 2(12): 927-937.

2.       Lieschke GJ, Currie PD. Animal models of human disease: zebrafish swim into view. Nat Rev Genet. 2007; 8(5): 353-367.

3.       Shive HR. Zebrafish Models for Human Cancer. Vet Pathol. 2013; 50(3): 468-482.

4.       Dodd  a, Curtis PM, Williams LC, Love DR. Zebrafish: bridging the gap between development and disease. Hum Mol Genet. 2000; 9(16): 2443-2449.

5.       Langenau DM, Ferrando AA, Traver D, et al. In vivo tracking of T cell development, ablation, and engraftment in transgenic zebrafish. Proc Natl Acad Sci U S A. 2004; 101(19): 7369-7374.

6.       Stern HM, Zon LI. Cancer genetics and drug discovery in the zebrafish. Nat Rev Cancer. 2003; 3(7): 533-539.

7.       Mizgirev I V., Revskoy S. A new zebrafish model for experimental leukemia therapy. Cancer Biol Ther. 2010; 9(11): 895-903.

8.       Goessling W, North TE, Zon LI. New waves of discovery: Modeling cancer in zebrafish. J Clin Oncol. 2007; 25(17): 2473-2479.

9.       Cronkite EP, Drew RT, Inoue T, Hirabayashi Y, Bullis JE. Hematotoxicity and carcinogenicity of inhaled benzene. Environ Health Perspect. 1989; 82(15): 97-108.

10.     Maltoni C, Ciliberti A, Cotti G, Conti B, Belpoggi F. Benzene, an experimental multipotential carcinogen: Results of the long-term bioassays performed at the Bologna Institute of Oncology. Environ Health Perspect. 1989; 82(2): 109-124.

 

 

 

Accepted on 20.12.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(2):628-630.