Ames Assay And Toxicology

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Ames Assay And Toxicology

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Ames Assay And Toxicology

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Question:
Discuss about the Ames Assay And Toxicology.

Answer:

Introduction
The Ames assay is a short-term test that uses bacteria as an endpoint for the identification of carcinogen. This test used bacteria to assess that whether the test chemical can mutate the DNA of the test organism. Positive result of this assay determines the toxic and mutagenic nature of the chemical. Bruce Ames of university of California, Berkeley, developed this test. Nowadays, it is one of the widely used assay in toxicology (Glatt et al., 2016).
This Assignment is going to discuss the process to perform the test, basic principles to detect the mutants. Furthermore, type of mutations that can be detected using the assay.
Procedure of Ames assay

Isolation of an auxotrophic strain of Salmonella typhimurium has been done. This strain lacks the histidine produce gene in its DNA. Therefore, it is called as auxotrophic.
A test suspension has been prepared in which, the histidine negative salmonella is mixed in a plain buffer with the test chemical. Small amount of histidine is also added to the mixture, as this will allow few of the bacteria to synthesize histidine from the colony. After that, only those bacteria will mutate their histidine-producing gene to start the synthesis of it.
A control solution is also prepared where; only salmonella is cultured in the broth, with small amount of histidine.
Both these suspensions are incubated at 37 degree Celsius for 20 minutes.
After that, these suspensions are spread on two different agar plates and further incubated for 48 hours at 37 degree Celsius.
After 48 hours, the places are extracted from the incubator and evaluated according to the colonies present in them (Roberts, James & Williams, 2014).

Detection of mutation
After the completion of the incubation period, the colonies present in both the plates are counted. The control plate shows minimal colony, as some bacteria have utilized the histidine added in the suspension and after that, they are unable to generate any histidine due to the lack of His gene.
On the other hand, the plate containing suspension of test chemical shows more colonies than that of the control plate. This is because, the test chemical helps to mutate the His negative gene present in the DNA and reverts it to its active form. Eventually, the bacteria, despite the lack of histidine added in the medium, start synthesizing it on their own (Liu et al., 2015).
General mutations that can be detected
Five general mutations that can be detected using Salmonella typhimurium in Ames assay are depend on the test strains of the bacteria. TA1535, TA1537, TA1538, TA100 and TA98. TA 1535 strain detects the base pair substitution mutation. Frame shift mutations that causes base pair deletion can be detected using TA1538 strain. Strain 1537 detects frame shift mutation due to base pair addition. TA 100 and 98 detects base pair substitution and frame shift mutations respectively (De Serres & Hollaender, 2012).
Advantage and disadvantage of the Ames test

The first advantage of this in vitro assay is cost effectiveness and less time required for the assessment. In vivo testing requires larger commitment of physical resources like in complex mammalian genotoxicity tests.
The other advantage of Ames test is easy workflow and less complexity. Working with in vivo culture can be hectic and needs lot of attention, as skipping any event can be harmful for the assay.
To detect the carcinogen, many other tests need to be done, which becomes complex if done in vivo. Therefore, in vitro testing such as Ames test are performed.
On the other hand, the disadvantage is reliability. Intact animal increases the reliability to the test.
Other limitation is related to the protective contribution is automatically taken into account for in vivo testing, which is absent for the in vitro assay.
In vitro culture cannot mimic the eukaryotic cell conditions hence, rat cells are added in the Ames test. However, to understand the effect of the toxin on humans, rat cells are also not reliable. Therefore, this one of the major drawbacks of Ames test, in comparison with mammalian genotoxicity tests.

References
De Serres, F. J., & Hollaender, A. (2012). Chemical mutagens: Principles and methods for their detection. Springer Science & Business Media. https://books.google.co.in/books?hl=en&lr=&id=5xbpBwAAQBAJ&oi=fnd&pg=PR26&dq=Mutagenesis+and+Genetic+Toxicology&ots=LOaXYpE2Tl&sig=mQW3erxNnN6gCpMJL_tAeAjM8Pw#v=onepage&q=Mutagenesis%20and%20Genetic%20Toxicology&f=false
Glatt, H., Sabbioni, G., Monien, B. H., & Meinl, W. (2016). Use of genetically manipulated Salmonella typhimurium strains to evaluate the role of human sulfotransferases in the bioactivation of nitro?and aminotoluenes. Environmental and molecular mutagenesis, 57(4), 299-311.
Liu, L., Chen, L., Floehr, T., Xiao, H., Bluhm, K., Hollert, H., & Wu, L. (2015). Assessment of the mutagenicity of sediments from yangtze river estuary using salmonella typhimurium/microsome assay. PloS one, 10(11), e0143522.
Roberts, S. M., James, R. C., & Williams, P. L. (2014). Principles of toxicology: environmental and industrial applications. John Wiley & Sons. https://books.google.co.in/books?hl=en&lr=&id=apTVBQAAQBAJ&oi=fnd&pg=PA197&dq=Mutagenesis+and+Genetic+Toxicology&ots=WNkA7nGNPP&sig=COI5rFZfbN5mcXmZUOuJb5JIuB0#v=onepage&q=Mutagenesis%20and%20Genetic%20Toxicology&f=false

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