[예방약학실험11] Ames test

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1. Ames test

Ames test는 박테리아를 이용하여 화학물질의 돌연변이 유발 가능성을 시험하는 실험 방법입니다. 이 실험에서는 히스티딘 생합성 능력이 결여된 Salmonella typhimurium TA100 균주를 사용합니다. 이 균주는 히스티딘 보충 없이는 생존할 수 없으므로, 돌연변이 유발 물질에 노출되면 히스티딘 생합성 능력을 회복하여 생존할 수 있게 됩니다. 이를 통해 화학물질의 돌연변이 유발 가능성을 확인할 수 있습니다.
2. Salmonella typhimurium TA100

Salmonella typhimurium TA100은 Ames test에 사용되는 균주입니다. 이 균주는 히스티딘 생합성 능력이 결여되어 있으며, 수선 효소가 손상되어 있습니다. 따라서 돌연변이 유발 물질에 노출되면 히스티딘 생합성 능력을 회복하여 생존할 수 있게 됩니다. 이를 통해 화학물질의 돌연변이 유발 가능성을 확인할 수 있습니다.
3. Compound K

Compound K는 이번 실험에서 시험 대상 물질입니다. Ames test 결과에 따르면, Compound K는 돌연변이 유발 가능성이 있는 것으로 나타났습니다. 실험 결과에서 Compound K가 포함된 샘플에서 일부 well에서 색 변화가 관찰되었는데, 이는 Compound K가 돌연변이를 유발하여 히스티딘 생합성 능력을 회복한 균주가 증식했기 때문입니다. 추가 실험을 통해 Compound K의 농도에 따른 돌연변이 유발 정도를 확인할 필요가 있습니다.
4. S9 fraction

S9 fraction은 간세포에서 추출된 것으로, 대사 효소를 함유하고 있습니다. Ames test에서 S9 fraction은 시험 물질을 대사시키는 역할을 합니다. 실험 결과에서 Compound K가 포함된 샘플에서 색 변화가 관찰되었지만, S9 fraction이 포함된 샘플에서는 색 변화가 관찰되지 않았습니다. 이는 Compound K가 간에서 대사된 후에는 돌연변이 유발 활성이 없어지는 것을 의미합니다.
5. Bromocresol purple

Bromocresol purple은 Ames test에서 사용되는 지시약입니다. 이 지시약은 pH 6.8 이상에서 보라색, pH 5.2 이하에서 노란색을 띱니다. Ames test에서 돌연변이가 유발되면 히스티딘 생합성 능력이 회복된 균주가 증식하여 산을 생성하게 되고, 이에 따라 노란색으로 변하게 됩니다.

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1. Ames test

The Ames test is a widely used bacterial reverse mutation assay that is considered a reliable and efficient method for evaluating the mutagenic potential of chemical compounds. It utilizes genetically modified strains of Salmonella typhimurium bacteria that are deficient in the ability to synthesize the amino acid histidine, making them unable to grow on histidine-deficient media. When exposed to a mutagenic compound, these bacteria can revert to a histidine-producing state, allowing them to grow on the selective media. The Ames test is valuable in assessing the genotoxicity of substances, as mutations in the bacterial genome can indicate the potential for a compound to cause genetic damage in higher organisms, including humans. The test is relatively simple, cost-effective, and can provide rapid results, making it a widely adopted tool in the field of toxicology and drug development. While it does not directly measure carcinogenicity, the Ames test serves as a useful screening method to identify potential mutagens and guide further, more comprehensive investigations.
2. Salmonella typhimurium TA100

Salmonella typhimurium TA100 is a widely used bacterial strain in the Ames test, a standard assay for evaluating the mutagenic potential of chemical compounds. This strain is genetically engineered to be deficient in the ability to synthesize the amino acid histidine, rendering it unable to grow on histidine-deficient media. When exposed to a mutagenic substance, the TA100 strain can undergo a reverse mutation, restoring its ability to produce histidine and allowing it to grow on the selective media. The TA100 strain is particularly sensitive to base-pair substitution mutations, making it a valuable tool for detecting a wide range of mutagenic compounds. Its use in the Ames test has contributed significantly to our understanding of chemical genotoxicity and has become a standard method in regulatory toxicology and drug development. The reliability and reproducibility of the TA100 strain have made it an essential component of the Ames test, providing a robust and well-characterized system for assessing the mutagenic potential of various substances.
3. Compound K

Compound K, also known as 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol, is a metabolite of the ginsenoside Rb1, which is a major active component of ginseng. Compound K has been the subject of extensive research due to its potential therapeutic properties, particularly in the areas of cancer, inflammation, and metabolic disorders. One of the key features of Compound K is its ability to exhibit anti-cancer effects through various mechanisms, such as inducing apoptosis, inhibiting cell proliferation, and suppressing angiogenesis in various cancer cell lines. Studies have shown that Compound K can effectively target and inhibit the growth of various types of cancer, including breast, prostate, colon, and lung cancer. Additionally, Compound K has demonstrated anti-inflammatory properties, which may be beneficial in the management of chronic inflammatory conditions. Furthermore, Compound K has been investigated for its potential in improving metabolic health. Some studies have suggested that Compound K may have the ability to enhance insulin sensitivity, regulate glucose homeostasis, and potentially alleviate symptoms associated with metabolic disorders, such as type 2 diabetes. Overall, Compound K is a promising natural compound with a wide range of therapeutic applications, and continued research in this area may lead to the development of novel treatments or adjuvant therapies for various health conditions.
4. S9 fraction

The S9 fraction is a crucial component in the Ames test and other in vitro genotoxicity assays. It is a metabolic activation system derived from the liver of rodents, typically rats or hamsters, that provides a source of metabolic enzymes to mimic the biotransformation of chemicals that may occur in the human body. The S9 fraction contains a mixture of cytochrome P450 enzymes, as well as other phase I and phase II metabolic enzymes, which can metabolize certain chemicals into more reactive or genotoxic metabolites. This is particularly important for the Ames test, as many compounds require metabolic activation to become mutagenic and induce reverse mutations in the bacterial strains used in the assay. The inclusion of the S9 fraction in the Ames test allows for the evaluation of both direct-acting mutagens and pro-mutagens, which require metabolic activation to exert their genotoxic effects. This is a significant advantage of the Ames test, as it provides a more comprehensive assessment of the mutagenic potential of chemical compounds. The use of the S9 fraction in the Ames test and other in vitro assays has been extensively validated and is considered a reliable approach to simulate the metabolic processes that may occur in the human body. It is an essential component in the assessment of chemical safety and the development of new drugs and other products.
5. Bromocresol purple

Bromocresol purple is a pH indicator dye that is commonly used in various analytical and biological applications. In the context of the Ames test, bromocresol purple plays a crucial role in the detection of bacterial growth and the identification of revertant colonies. In the Ames test, the bacterial strains used, such as Salmonella typhimurium TA100, are genetically modified to be histidine-dependent. When these bacteria are exposed to a mutagenic compound, they may undergo a reverse mutation, restoring their ability to synthesize histidine and allowing them to grow on histidine-deficient media. Bromocresol purple is added to the growth media in the Ames test to facilitate the visualization and enumeration of these revertant colonies. The dye changes color in response to changes in pH, with a purple color indicating a basic pH and a yellow color indicating an acidic pH. As the bacteria grow and metabolize the available nutrients, they produce acidic byproducts, causing a localized decrease in pH around the colonies. This pH change is detected by the bromocresol purple dye, which turns yellow, allowing the revertant colonies to be easily identified and counted. The use of bromocresol purple in the Ames test is a simple yet effective way to enhance the sensitivity and reliability of the assay. By providing a clear visual cue for the presence of revertant colonies, bromocresol purple contributes to the overall accuracy and reproducibility of the Ames test, which is a widely accepted and standardized method for evaluating the mutagenic potential of chemical compounds.