The aspect of safety assessment involves relevant procedures of substances’ identification that can induce mutations. The chemical substances that induce mutations can be potentially harmful to the germ line, which can result into fatal mutation effects to future generations, such as fertility problems. Chemicals that can cause mutagenic reactions can also induce cancer, a feature that has fueled several mutagenicity testing programs. Mutations can occur as a result of point or gene mutations which involve modification of a single base, insertion or deletion of a few bases, as large rearrangements or deletions, as loss or gain of full chromosomes, rearrangements or breaks in the chromosome. Mutations of the genes are easily assessed in bacteria or other systems of the cell, while the damage on the chromosome of mammalian cells can be evaluated by observing the cell’s chromosome for rearrangements or breaks under magnification (Mortelman & Zeiger 2000).
The assay for Salmonella typhimurium or microsome is a widely used short term assay on bacteria for identifying harmful substances that can lead to genetic damage, and, subsequently, cause gene mutations. This test uses several strains of Salmonella, containing pre-existing mutations that make bacteria not capable of synthesizing the necessary amino acid, known as histidine; therefore, not being able to grow and form colonies without the presence of this amino acid. The gene’s function can be restored by new mutations occurring at the location of the pre-existing gene mutations or near these genes, which then allow the cell to produce histidine. The new mutations occurring in the cells of the bacteria can grow and form colonies without the presence of histidine. This explains why the test is usually referred to as a reversion assay (Dearfield & Moore 2005).
The strains of Salmonella subjected to this test, exhibit different forms of mutation in a range of genes found in the histidine operon. The role of these mutations is to be responsive to the mutagens that function through different mechanisms. These strains were also engineered with additional mutations, in order to become increasingly sensitive to a wide range of substances. The Salmonella / microsome mutagenicity test was initially designed to detect mutagenesis induced by chemical means. The use of the test in the past several years has been acknowledged by the scientific community, as well as government corporations and agencies. The test has gained the worldwide access to an initial screen in the determination of the potential of new drugs and chemicals that can cause mutagenesis. This is for the basic reason of the high predictive value obtained from rodent carcinogenicity, following a mutagenic response.
Every strain of the Ames’ tester has undergone construction with a different form of lesion situated on the histidine operon. Additionally, the Ames tester strains contain several other mutations, which raise the sensitivity of these strains to the various types of chemical mutagens. The RFA mutation occurring in every strain results into a lipolysaccharide layer, which forms a coating on the surface of the bacteria, thus, making the permeability of the bacterial wall increase, and allows for large molecules to pass. A deletion mutation occurs in all the Ames tester strains, except for TA102, and, subsequently, removes the repair mechanism for accurate precision, therefore, enabling the repair of additional DNA lesions by the DNA mechanism that is prone to errors (Mortelman & Zeiger 2000).
The Ames assay of Salmonella or microsome mutagenicity has evolved over the past years, from the initial screening of several mutants of histidine, resulting to the identification, and selection of mutants that contain high sensitivity to reversion, by a number of chemical mutagens. Since bacteria can not metabolize chemicals through the cytochrome P450, as is the case with vertebrates, such as mammals, a crucial element that is useful in making the test of bacterial mutagenicity useful is the incorporation of an exogenous activation system for the mammalian metabolism. The assessment of the mutagenic potential of the cigarette smoke is usually based on the studies, which extracts in the Ames Salmonella assay. The use of this assay can enable the investigation of the mutagenic potential of mainstream cigarette smoke by exposure to Salmonella typhimurium strains (Aufderheide & Gressmann 2007). This proposal seeks to explore the Ames Salmonella/ microsome assay in detecting mutagenic potential of cigarette smoke.
The tester strains will be prepared by the frozen stock cultures being grown overnight in nutrient broth at 37oC. 1 ml of each tester strain culture will be paced into a colour coded cuvette for reading in the spectrophotometer, at a wavelength of 650 nm. The readings will be made against a blank sample containing pure nutrient broth, and the optical densities recorded on a datasheet. Eight tubes containing 0.9 ml of sterile distilled water will be prepared for each strain to be used in the assay for serial dilutions. There is one hundred μl of each dilution, 10-4 onto a nutrient agar plate, and a disposable sterile spreader for spreading on top of the agar plate. This will be repeated for the rest of the dilutions. The plates will then be incubated for 24 hours after which they be read manually, especially for the plates containing over 300 colonies. This will be followed by preparation of the top agar tubes using the plate incorporation method and bottles using the pre-incubation method. The cofactor mix will be ordered from Molecular Toxicology Incorporated, USA, where the mix is obtained from the post-mitochondrial fraction of the mammalian liver, S9. This is useful for metabolic activation in +S9 assays. Before the day of testing, media plates Vogel-Bonner would be removed from the cold room to another room, in order to adjust to room temperature overnight and labeled. The assay procedure involving the plate incorporation method will begin with melting the top agar tubes at 121°C for 15 minutes, in the autoclave, then transferred to the 47°C incubator for temperature moderation and also to prevent the agar from setting. G-6-P and NADP will be added to the co-factor mix and then filtered through a 0.2μm sterile filter unit.
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The commercially available S9 liver homogenate will then be added to the co-factor mix and kept in ice during the testing process. 0.5 ml of the cofactor mix will be added to each of the S9 sterility tubes from the heating block, and immediately vortexed to mix the contents of the tube before being poured into a labeled Vogel Bonner nutrient agar plate. A hundred μl of each strain of bacteria will be added to tubes in the block and tested with16 negative controls (DMSO), 8 at the beginning of the assay and 8 at the end. 0.5ml of the co-factor mix will be added to each of these tubes using a sterile dispenser before being poured to the corresponding labeled Vogel-Bonner nutrient agar plate. This will be repeated for the TPM extract tubes. This procedure will be repeated for each of the bacterial strains followed by checking the sterility of each assay component.
For the pre-incubation method, the sterility check will be done on the co-factor mix before the start of the assay. 100 μl of each strain and the same amount of the test substance will be added the corresponding culture tubes using a Gilson pipette. An aliquot of 0.5ml of the appropriate co-factor mix will be added to each tube, and tested against a negative and positive culture tubes followed by incubation. The culture tubes will then be transferred to a laminar flow cabinet, where 2.5ml of molten top agar will be added to each tube, vortexed and immediately poured onto a corresponding, labeled Vogel-Bonner nutrient agar plate. The plates will be allowed to set before being inverted and wrapped using cling film before being incubated overnight at 37oC.
The number of bacterial colonies will be scored and recorded for each of the culture plates. Contaminations occurring in any of the scoring plates will also be recorded, and the plates discarded depending on the magnitude of contamination. The assay will be regarded as valid, if means, negative control count falls within the in-house historical ranges; positive control chemicals’ induce clear increases in reverting numbers confirming the active S-9 preparation, or when there is less than 5% of the plates lost during contamination (Aufderheide & Gressmann 2007).
Projected Results/ Discussion
After removal of plates from the incubator, the formed colonies will be counted using electronic counters or hand counters, and expressed as reverting colonies per plate. Hand counting will be applicable when there will be the presence of precipitate at high dose levels on the plates. In case all the plates are used, including the negative and positive control plates are from a single experiment, then counting will be done at the same time, because the cells formed on the plate will continue with the division to produce the additional mutant colonies with additional storage time. Toxicity will be examined with the presence of precipitate by assessing the background lawn using a 40X dissecting microscope for thinning. From the results that will be obtained from the experiment, a minimum fold increase, usually, 2-3 fold over the solvent control will be set to serve as a cut off between a non-mutagenic and mutagenic response. The only weakness of this approach occurs, especially when the mean plate count changes by a few revertants, thus, causing a difficulty in distinguishing a chemical mutagen from a non-mutagen (Cariello & Piegorsch 1996). However, the minimum fold increase may be too sensitive for the Salmonella strains containing averagely high reversion frequencies, for example, TA97, TA100 and TA102. It might also be highly sensitive for chemicals showing low reversion frequencies, such as TA1537 and TA1535. By using the Ames Salmonella assay, this study will demonstrate the possibility to analyze the mutagenic potential from native cigarette smoke in a direct way, and obtain dose dependent revertants induction. This will be achievable from the treatments with the bacterial strains, such as TA98 and TA100 (Zeiger, 1998).
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