Quantifying Tobacco Mutagenicity through Ames Test

Quantification of a Mutagen: Tobacco By Selenia Lopez November 30, 2012 Section 44 Abstract: Tobacco is commonly used and kills millions until this day. Tobacco is a potential mutagen due to all the chemicals added. The spot overlay Ames test was conducted to test at what concentration of tobacco was it at the most mutagenic. The hypothesis of this experiment was as the concentration of tobacco increases, the growth of bacteria increases. The control for this experiment had a UV positive and a UV negative. Four different tobacco concentrations, Salmonella Typhimurium of strain 1538 were incubated for 24-72 hours to observe bacterial growth.

At a 100% bacterial growth was at its greatest number of colonization and at 5% the mutagen was at its least. These results reflected that tobacco has the ability to grow without histidine making it a mutagen and at which concentration was it the most mutagenic. Intro: A mutagen is a substance which increases the frequency of mutation in a plant or animal population, which can lead to a variety of consequences or alterations in the DNA structure (Ligorio, Izzotti, Pulliero, Arrigo 2011). Salmonella being a mutagen can cause mutations such as substitution, insertion, deletion and frame shift depending on the strain.

S. typhimurium carries a defective gene making it unable to synthesize histidine from its culture medium. Some types of mutations can be reversed with the gene regaining its function. Tobacco having lots of chemicals with possibility of being mutagenic is known to kill an estimated six million people worldwide each year and drains $500 billion annually. It can be consumed as a pesticide and in the form of nicotine tartrate. It is sometimes used in some medicines, but most commonly used as a drug. The use of Ames test is based on the assumption that any substance that is mutagenic. For this eason the FDA uses the Ames test to screen many chemicals to measures the mutagenic strength in bacterial cells (FDA 2012). In this experiment to test whether tobacco is mutagenic and if so at what concentration has the greatest bacterial growth. The spot overlay Ames test was conducted. Though it’s a cheap version of the Ames test, the result were still compatible. The hypothesis of this experiment was as the concentration of tobacco increases, the growth increases. The greatest growth should occur in the 100% concentration and the least in the 5% concentration. Methods and materials:

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The control for the experiment was an Agar plate that had a UV positive reactant with a known mutagen, and UV negative reactant that hasn’t been reactant with anything. With a micro pipette that amounted to 250ul, strain TA 1538 of Salmonella was Obtained and placed on to the Agar plate and spread with a sterilized rod. The four paper discs that have been soaked in the 5%, 25%, 50%, and 100% of tobacco concentration were placed spaced apart on the plate. The plate was sealed and place it in the incubator at 37 degree for 24-72 hours. Results: Table 1: The table shows the concentration of mutagens to the number of colonies observed.

Concentrations | Colonies | 100% | 39 | 50% | 13 | 25% | 8 | 5% | 2 | The control resulted in the UV positive having growth and the UV negative having none at all. At a 100% bacterial growth was at its greatest number of colonization at 39. At 5% the mutagen was at its least with only 2 colonies. Discussion: The mutagenic effect of the chemical has caused many bacteria to regain the ability to grow without histidine in tobacco, causing the formation of the colonies seen around the disc (Pounikar and Dawande 2010). Not only is a tobacco a mutagen, but colony growth increased as the concentration increased.

The hypothesis was supported according to the data show in the table. Even though the disc were slightly shifted. The numbers were still attainable. The 100% concentration had the highest bacterial colonization and the 5% concentration had the least as predicted. In future experiments, more trials can be done. In order to make sure the results will remain the same every time; or using other mutagens to see how they react in the 100% concentration. This type of experiment can also be useful in finding out whether smoking tobacco or chewing tobacco is more mutagenic or if the different brands make a difference on how mutagenic they can be.

Works Cited Asiatic Journal of Biotechnology Resources: Pounikar, R and Dawande, A. Y. (2010). Detection of potential carcinogens by Ames test. Doi: 01: 57-64. Department of Health Sciences: Ligorion M, Izzotti A, Pulliero A, and Arrigo P. (2011) Mutagens interfere with microRNA maturation by inhibiting DICER. An in silico biology analysis. Doi: 10. 1016 www. FDA. gov: Li Y, Yan J, Bishop M, Jones MY, Watanabe F, Biris AS, Rice P, Zhou T, Chen T. (2011) Genotoxicity evaluation of titanium dioxide nanoparticles using the Ames test and Comet assay.

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