In recent years, humans are exposed to various carcinogenic agents, artificial colors like Green #3, Red#3, Citrus red #2, Saccharin, Etc.The present study emphasis is made to know the effect of these common agents in particular with reference to artificial food color Allura red AC (E 129) (FD & C # 40)- orange / red food dye on DNA damage using Cat Fish Clarias batrachus as an model organism. The methodology will be to understand direct damage to nucleus by Micronuclei test and Chromosomal damage by alkaline single-cell gel electrophoresis (COMET) ASSAY. The freshwater fish catfish Clarias batrachus was used for specificity genotoxic indicators micronucleus assay and (comet) assays. The fish was exposed to 1g/L of Allura red AC (E 129) (FD & C # 40) for a period of 7, 14, 28, and 35 days ectodermally. The blood sample was tested for genotoxicity. The results revealed DNA damage through the micronuclei and alkaline single-cell gel electrophoresis (comet) assays. Hence it is concluded that the usage of food color containing Allura red AC (E 129) (FD & C # 40) - orange / red food dye may be toxic at genetic level if the usage is prolonged.
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Keywords: Allura RedÂ AC (E129), micronucleus assay, COMET assay, Clarias batrachus
Food was fresh and natural in the early ages. Due to population explosion there was a demand for increase in productivity of consumer goods which made an impact on producers to add more chemical compounds (food additives) to increase the level of yield and to make it look more fresh and good. Food additives show various symptoms of threats on long term and short term exposures3.
So both acute and chronic exposures produce permanent changes to affected organs2. Hence genotoxic studies are needed to study the effect of any chemical compound or substance. Micronuclei test is the most widely used assay due to its proven correctness for fish species4. Micronuclei presence in cells is due to the structural and numerical chromosomal aberrations arising during mitosis.
The Single Cell Gel Electrophoresis is a responsive technique for the detection of DNA damage at the level of individual cell6. The comet assay under alkaline conditions (pH>13) can detect DNA damage, i.e. single strand breakage or DNA cross-links and incomplete excision repair even7. DNA lesions have been detected by inducing chemical mutagens using Comet assay. Fishes are generally used as toxicity indicators. Many authors advise fish as the best animal model for these studies8, 9.
The present study is made to know the effect of these common agents in particular with reference to artificial food color Allura red AC (E 129) (FD & C # 40) - orange / red food dye on DNA damage using Cat Fish Clarias batrachus as model organism.
MATERIALS AND METHODS
Healthy specimens of Clarius batrachus were bought from Ranipettai. Fishes of same age and size which hatched from the same lot of eggs were collected and were transported to the Environmental Biotechnology laboratory for acclimatization. And experiment was conducted in CO2 & Green Technology Center of VIT University1.
Acclimatization was done by stocking fishes in a rectangular cement tank (4m x 6m x 3m), which was soap washed, disinfected with potassium permanganate and thoroughly rinsed thrice former to filling with water. Fish were acclimatized to laboratory condition for a night, before being used for experiments. No symptoms of disease were noticed during succeeding periods. During adaptation, the stock was maintained at normal temperature and fed once daily with fish food (In the form of balls). Water was regularly changed for every 24hours and well aerated in order to reduce any accumulation of excretory products and to make sure whether there is sufficient oxygen supply to fish. Feeding was stopped for 48 hours before the experiments were started so as to keep the experimental animals in same metabolic condition1.
The glass-aquaria of 50 L capacity which were cleaned, was filled with clean water were used for genotoxical studies. 1g/L i.e. 1g of Allura red AC (E 129) (FD & C # 40) - orange / red food dye was dissolved in 1 liter of water and was used in the present study. A control (50 fish) without Allura red AC (E129) was maintained simultaneously. Experiment was conducted for 35 days. 5 fishes were randomly selected from control and experimental aquaria at regular intervals (weekly) i.e. (7, 14, 21, 28, and 35 days) and blood was collected for genotoxicological studies.
Collection of blood
Always on Time
Marked to Standard
Blood was drawn from the caudal vein by using plastic disposable syringe fitted with 22 gauge needle which was already moisture with heparin,(Beparin, heparin sodium, IP 2000 IU ml-1, derived from intestinal mucosa containing 0.15 percentage w/v cholesterol IP preservative) an anticoagulant. Blood collected from treatment and control was expelled into the separate heparinized centrifuge tubes and was placed immediately in the refrigerated condition. This blood sample was used for determination of all the parameters5.
Two drop of blood sample was taken from catfish and a smear was made on glass slide. The slide was then fixed with methanol for 15 minutes and air dried. The next day it was stained with 15% giemsa solution for 10 minutes and then the slide was dehydrated in alcohol and cleaned in xylene. Slides were mounted in DPX for observing under the microscope1.
Single cell gel electrophoresis (Comet assay)
The glass slide was dipped in 1% normal melting agar for first layer and allowed for getting set for 5min at 4oC. Fish blood (containing cells) was added to 80Âµl of 0.65% low melting agar in PBS (Dissolve the following in 800ml distilled water. NaCl -8g, KCl-0.2g, Na2HPO4 -1.44g, KH2PO4 -0.24g, Adjust pH to 7.4. Adjust volume to 1L with additional distilled water. Sterilize by autoclaving). This was transferred to the slide to produce the final layer. After solidification of agar the slide was kept in the cold lyses solution (2.5M of NaCl, 100mM of EDTA, 10mM of Tris-HCl, pH-10, 1% of sodium sarcosinate, 1% of Triton X-100, 10% of DMSO, pH 10) at 4oC overnight in dark. The slide were taken gently and placed in horizontal gel box containing electrophoresis buffer (1mM of Na2EDTA, 300mM of NaoH, and adjust pH to 13) for about 20 minutes. The electrophoresis was performed in cold water bath. After electrophoresis the slide was washed three times in neutralization buffer (Tris-HCl buffer of pH 7.5). Then the slide was being photographed using gel documentation1.
The statistical analysis was made individually on each sample and the mean value of five individual observations were taken for each observation.
The results of the present study showed that the food color Allura RedÂ AC (E129) (FD&C Red #40) - orange / red food dye is even potential enough to induce cancer on a long term exposure.
The thorough examination of the blood smears showed the evidences of micronuclei in treated fish (cat fish) (Fig 1).
Fig 1. Micronuclei test result of the exposed test organism Cat fish Clarias batrachus
Single cell gel electrophoresis
The test showed remarkable results as there was significant DNA damage in the blood cells. The level of damage was differing from week to week based on the time of exposure. The pictures below depict the level of damage in regular time intervals.
7th day 14th day 28th day 35thday
Fig 2: Pictures of single cell gel electrophoresis test taken in weekly intervals Clarias batrachus
Food colors or additives are widely used in edible items mostly, by manufacturers to make it look fresher and to make it more appetizing. Though it succeeds in playing its role well it causes major damages in the DNA3. The results of present study prove the above fact to be evident. Since both the genotoxicological studies i.e. micronucleus test and single cell gel electrophoresis (COMET) assay had given positive results. It is advisable to reduce the intake of these kinds of food additives in our day to day lives. The basic concept of the comet assay is that the presence of single strand break DNA fragments and moves them from the nucleoid core towards the anode, thus resulting in 'Comet' formation11. It is a very sensitive test and helps in getting remarkable results in relative studies.
Food additives like Allura RedÂ AC (E129) (FD&C Red #40) - orange / red dye used in food items might make the food more appealing and could taste better but its exclusive property of causing DNA damage should be taken into consideration and should be banned from usage for human welfare.
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