Mutagenicity of Soy Sauce as Determined by the Ames Test

Abstract
A number of substances called mutagens are known to cause mutations in the genetic code of DNA. The goal of this experiment was to expose whether soy sauce contains mutagenic properties that could revert auxotrophic Salmonella typhimurium​ ​ TA1538 to phototrophic Salmonella typhimurium​ TA1538. To determine the mutagenicity of soy sauce, the Ames test was performed; it is a common test of a substance’s ability to induce mutations in DNA. This experiment uses bacteria in the auxotrophic form to see how many colonies will revert to the phototrophic form of the bacteria. In the assay, water is used as a negative control, 4NOP is used as a positive control, and soy sauce is used as the experimental group. The calculated reversion rates were 3.73-7 ×10-8​ ​ for the negative control, 4.007×10-8​ ​ for the experimental group, and 1.229 ×10​ ​ for 4NOP.

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The reversion rate of the bacteria introduced to soy sauce was nearly identical to that of the one introduced to water, which was the negative control, and it was much lower than the reversion rate of the bacteria introduced to the positive control of 4NOP. Therefore, these results indicate that soy sauce is not a mutagen and is relatively harmless for human consumption. However, the nucleotide BLAST search revealed a 14 nucleotide deletion(Picture 4&5) with an E value of .000, meaning the deletion was most likely not due to chance and likely attributed to the addition of soy sauce to the sample.
Introduction
The mutagenicity of soy sauce, a popular Asian garnish, has been debated for some time. Understanding if soy sauce is a mutagen and how it affects the human body is important to consumers all over the world. In the scientific community, there have been conflicting results in regards to this matter. In one experiment, the chemical HMF, a fragrant enhancer of soy sauce, was found to have mutagenic properties, creating breaks in the DNA of Salmonella bacteria. This experiment found that nitrosatable precursors, found commonly in soy sauce, could become mutagenic and carcinogenic under gastric conditions (Hiramato K. et al, 1996). On the other hand, another experiment found that soy sauce was, in fact, the opposite; soy sauce was found to be an antioxidant and that it had anticarcinogenic effects. In this experiment, soy sauce was introduced to the diet of mice, and the mice with this diet had a decrease in the number of forestomach neoplasms (Benjamin H., 1991). Antioxidants such as soy sauce are in a class of foods that neutralize free carcinogenic radicals. There is also an argument that mutagenic properties of soy sauce may not be the cause of cancer, but methylation patterns from different types of diets may cause cancer. This would mean that soy sauce itself is not carcinogenic (Goldman R. and Shields P., 2003).
To prove the mutagenicity of a substance, the Ames test is a common assay used to identify the ability of the substance to damage or change DNA. The Ames test does this by using the Salmonella typhimurium ​ ​bacteria, which already has a mutation causing the protein histidine not to be produced by the bacteria. This makes Salmonella typhimurium ​ ​an auxotroph, meaning it cannot produce the nutrients it needs to survive. In the Salmonella strain used, S. typhimurium​ TA1538, there was a deletion mutation that created the lack of histidine and an LPS defect, allowing the mutagen to enter the cell by weakening the cell membrane. An experimental substance is then added to the bacteria, and if the bacteria then continues to grow then the new colonies are recognized as revertant colonies, because the experimental substance caused a mutation allowing the bacteria to become prototrophic and make its own histidine (Mortelmans K and Zeiger E., 2000). In addition to this, electrophoresis can be used to distinguish the changed DNA by pulling it through gel pores of small sizes utilizing a positive anode that attracts the negatively charged DNA backbone. To further prove a mutation, a BLAST search compares the actual nucleotide sequences of the original DNA and those of the DNA affected by the experimental substance to observe any mutations that occurred.
Methods
A series of dilutions were conducted to an overnight culture of histidine S. typhimurium​ TA1538. The number of colony-forming units (CFU) were determined using the total number of colonies per ml plated and the total dilution factor. The original culture contained 2 x 10-8 9​ CFU/mL. A serial dilution was performed to produce a final plate containing 5 x 10​ ​ CFU/mL.
The Ames test was then conducted to test the genotoxicity of soy sauce, 4NOP, and water. Three agar plates were made by vortexing an overnight culture of S. typhimurium​ TA1538 in order to produce a uniform distribution of cells in the media. 100 uL of the overnight culture were put into a tube of top agar that was in a hot water bath. The tube was vortexed to distribute the bacteria, then the agar was poured into a minimal glucose plate. 10 uL of
Salmonella was added to a test plate of agar containing a small amount of histidine and 4NOP as the positive control. 4NOP is the positive control because it is a well known mutagen, that causes DNA damage (Asgard R., 2013). 10 uL of Salmonella was added to a second test plate of agar containing a small amount of histidine, along with soy sauce as the experimental group. Salmonella was added to a test plate of agar containing a small amount of histidine, plus 10 uL water as the negative control. The plates were inverted and incubated at 37 degrees Celsius for 48 hours. The amount of revertant colonies, a result of a cell being able to synthesize its own histidine, were observed and recorded to determine if a mutation occurred. The number of revertant colonies in the negative and positive controls was compared to the number of revertant colonies in the experimental to determine if soy sauce is a mutagen. The reversion frequency was calculated for each group.
Then the genomic DNA was isolated by using InstaGene Matrix to bind to extracellular debris. An isolated bacterial colony from the experimental mutagen plate is suspended in 1 mL of water. The tube was centrifuged for 2 minutes at 6,000 rpm. A pellet formed and the supernatant was removed. 200 uL of InstaGene Matrix was added to the pellet and incubated for 20 minutes at 56 degrees celsius. The tube was vortexed for 10 seconds after incubation and placed in a boiling water bath for 8 minutes. Then the mixture was vortexed again for 10 seconds and spun at 6,000 rpm for 6 minutes. Next, DNA amplification was performed using Polymerase Chain Reaction Amplification (PCR). The supernatant was placed in a fresh tube and then 20 uL was transferred to the well along with 20 uL of PCR master mix containing primers, buffer, dNTPs, Taq polymerase, and magnesium ions. The mixture was placed in a thermal cycler where the reaction undergoes 40 cycles of amplification for approximately 3 hours. The molecular changes that confer the revertant phenotype will be identified.
Following the PCR, the now amplified DNA sample was run through gel electrophoresis. 5 uL of loading dye was added to a well which causes the DNA to sink into the well and allows for its progress to be tracked. 20 uL of the DNA was loaded into the well with agarose red, which allowed the DNA to be visualized by making it fluorescent under UV light. The gel was run at 220 volts for 30 minutes. The method separated the DNA according to size.
Lastly, the online search program BLAST determined the nucleotide sequence and mutations in the DNA sample. The original auxotrophic sequence of the his S. typhimurium​ TA1538 and the revertant sequence of the histidine S. typhimurium​ ​ TA1538 with soy sauce were compared to determine the mutations. An “Expect Value” was assigned to the alignment in order to determine whether an alignment as good as or better than that found by BLAST would be expected to occur due to chance.
Results
All three media plates produced revertant colonies, and we calculated the CFU/mL from the overnight culture to be 3.7433×10​9​. The experimental plate, soy sauce, produced fifteen-8 revertant colonies with a reversion frequency of 4.007×10​ . The negative control, water,​ -8 produced fourteen revertant colonies with a reversion frequency of 3.73×10​ ​. The positive -7 control, 4NOP, produced forty-six revertant colonies with a reversion frequency of 1.229×10​ (Pictures 1-3).
The Electrophoresis gel was conducted to separate DNA by size to see if the DNA had reverted to its phototrophic state, but it did not show any distinguishable results (picture 6); however, the BLAST search revealed a 14 nucleotide long deletion with an E value of zero, which means the deletions were most likely not due to chance (Picture 4 and 5).
Discussion
Out of all the plates with varying experimental conditions, the positive control, containing 4NOP, yielded the greatest reversion frequency of 1.229×10​-7​. The positive control containing the most revertant colonies reaffirms that 4NOP is a known mutagen of his- S. typhimurium​ TA1538. The plate with the sterile water served as the negative control with a-8 reversion frequency of 3.73×10​ ​. The fourteen revertant colonies in the negative control plate were results of contamination and/or spontaneous mutations. Fifteen revertant colonies were found on the experimental plate that contained soy sauce with a reversion frequency of 4.007× 10​-8​. Based on the data of revertant colonies alone, there is not enough evidence from the Ames test to support that soy sauce is mutagenic. The presence of the colonies could be due to contamination and/or spontaneous mutations. On the contrary, the BLAST search showed a 14 nucleotide deletion attributed to the presence of soy sauce. This supports the conclusion that soy sauce is a mutagen.
The contradicting results in this experiment mirror the contradicting findings of the scientific world. For example, H, Benjamin et al, found that soy sauce contains antioxidant substances that actually inhibit carcinogenesis. This study found that soy sauce was beneficial to overall health (Benjamin H. et al, 1991). However, in a very similar experiment, the Ames test rendered mutagenic results for soy sauce in a Salmonella typhimurium ​ ​TA100 strain. This study concluded that soy sauce should only be used only when necessary because of its possible adverse effects (Growther L. et al, 2008).
There are many possible reasons for this ambiguity in the results from different experiments. In the experiment that concluded mutagenic properties of soy sauce, two brands of soy sauce- Koikuchi and Honjozo- were bought from a local market in Tokyo, Japan. In the same experiment, electrophoresis was used to prove the DNA breaking activity of soy sauce (Hiramato K. et al, 1996). In the experiment that concluded that soy sauce had anticarcinogenic properties, the soy sauce was freshly obtained from the Kikkoman Company in Walworth, Wisconsin. Also, the experiment added soy sauce to the diets of rats to prove the anticarcinogenic effects of soy sauce (Benjamin H. et al, 1991). There are many different factors that could have impacted the outcome of their experiments. The freshness of the soy sauce could affect its properties as well as the ingredients used to make the soy sauce. They also used two very different methods to prove their conclusions. To prove the mutagenicity of soy sauce, the different components of soy sauce would have to be tested.
References
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Benjamin, H., Storkson, J., Nagahara, A., & Pariza, M. W. Inhibition of benzo(a)pyrene-induced mouse forestomach neoplasia by dietary soy sauce. Cancer Research.​ ​ 1991. 51​ ​(11), 2940. ​ 0>​ . Accessed 2019 Nov 11.
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Figures

Picture 4​ The BLAST program showed 14 deletions in the bacteria introduced to soy sauce. The deletion is shown by the dash marks that represent the missing nucleotides from the sequence of the bacteria sample introduced to soy sauce.

Picture 5​ This is an image from the BLAST program also showing the 14 deletions pictured by the red line.

Picture 6 ​Results from Gel Electrophoresis