Table of Contents
The aspect of safety assessment involves relevant procedures of substances’identificationthat can induce mutations.The chemical substances that induce mutations can be potentially harmful to the germ line, which canresultinto fatalmutation effects to future generations, such as fertility problems.Chemicals that can cause mutagenic reactions can alsoinducecancer, afeaturethat has fueled several mutagenicity testing programs.Mutations can occur as a result ofpointor gene mutations whichinvolvemodification of a single base, insertion or deletion of a few bases, as large rearrangements or deletions, as loss or gain offullchromosomes, rearrangements or breaks in the chromosome. Mutations of the genesare easily assessedin 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 identifyingharmfulsubstances that can lead to genetic damage, and,subsequently,cause gene mutations. Thistestuses several strains of Salmonella, containing pre-existing mutations thatmakebacteria not capable of synthesizing thenecessaryamino acid, known as histidine; therefore, not being able togrowand form colonies without the presence of this amino acid. The gene’sfunctioncan be restored by new mutations occurring at the location of the pre-existing gene mutations or near these genes, which then allow thecellto 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 testisusuallyreferredto as a reversion assay (Dearfield & Moore 2005).
The strains of Salmonella subjected to thistest,exhibit different forms ofmutationin arangeof genes found in the histidine operon. The role of these mutations is to be responsive to the mutagens thatfunctionthrough different mechanisms. These strainswere also engineeredwithadditionalmutations, in order to become increasinglysensitiveto a wide range of substances. The Salmonella / microsome mutagenicitytestwasinitiallydesignedto detect mutagenesis induced by chemical means. The use of the test in the past several yearshas been acknowledgedby the scientific community, as well as government corporations and agencies. Thetesthas gainedthe worldwideaccess 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 thehighpredictive value obtained from rodent carcinogenicity, following a mutagenic response.
Every strain of the Ames’ tester has undergone construction with a differentformoflesionsituated on the histidine operon. Additionally, the Ames tester strains contain several other mutations, whichraisethesensitivityof these strains to the various types of chemical mutagens. The RFA mutation occurring in every strain results into alipolysaccharidelayer, which forms a coating on the surface of the bacteria, thus, making the permeability of the bacterial wallincrease, and allows for large molecules topass. A deletion mutation occurs in all the Ames tester strains, except for TA102, and, subsequently, removes the repairmechanismfor accurate precision, therefore, enabling therepairofadditionalDNA lesions by the DNAmechanismthat isproneto errors (Mortelman & Zeiger 2000).Want an expert to write a paper for you Talk to an operator now
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, acrucialelementthat is useful in making thetestof bacterial mutagenicity useful is the incorporation of an exogenous activationsystemfor the mammalian metabolism. The assessment of the mutagenic potential of the cigarette smokeis usually basedon 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 toexplorethe Ames Salmonella/ microsome assay in detecting mutagenic potential of cigarette smoke.
The tester strainswill be preparedby the frozen stock culturesbeing grownovernight in nutrient broth at 37oC. 1 ml of each testerstrainculturewill be paced into a colour coded cuvette for reading in the spectrophotometer, at a wavelength of 650 nm. The readingswill be madeagainst a blank sample containing pure nutrient broth, and the optical densities recorded on a datasheet.Eight tubes containing 0.9mlof sterile distilled waterwill be preparedfor eachstrainto be used in theassayfor serial dilutions.There is one hundredμlof each dilution, 10-4 onto a nutrient agar plate, and a disposable sterile spreader for spreading on top of the agar plate. Thiswill be repeatedfor the rest of the dilutions. The plates will then be incubated for 24 hours after which they be read manually,especiallyfor the plates containing over 300 colonies. Thiswill be followedby preparation of the top agar tubes using the plate incorporationmethodand bottles using the pre-incubation method. The cofactormixwill be orderedfrom Molecular Toxicology Incorporated, USA, where themixis obtainedfrom the post-mitochondrial fraction of the mammalian liver, S9. This is useful for metabolic activation in +S9 assays. Before the day of testing, media platesVogel-Bonner would be removedfrom the cold room to another room, in order toadjustto room temperature overnight and labeled. The assayprocedureinvolving the plate incorporationmethodwillbeginwith melting the top agar tubes at 121°C for 15 minutes, in the autoclave, then transferred to the 47°C incubator for temperaturemoderationand also to prevent the agar from setting. G-6-P and NADPwill be addedto the co-factormixand then filtered through a 0.2μm sterile filter unit.
The commercially available S9 liver homogenate will then be added to the co-factormixand kept in ice during the testing process. 0.5mlof the cofactormixwill be addedto each of the S9 sterility tubes from the heating block, and immediately vortexed to mix the contents of the tube beforebeing pouredinto a labeled Vogel Bonner nutrient agar plate. A hundredμlof each strain of bacteriawill be addedto tubes in theblockand tested with16 negative controls (DMSO), 8 at the beginning of theassayand 8 at the end. 0.5ml of the co-factormixwill be addedto each of these tubes using a sterile dispenser beforebeing pouredto the corresponding labeled Vogel-Bonner nutrient agar plate. Thiswill be repeatedfor the TPMextracttubes. This procedurewill be repeatedfor each of the bacterial strains followed by checking the sterility of each assay component.
For the pre-incubation method, the sterilitycheckwill be doneon the co-factor mix before the start of the assay. 100 μl of each strain and the same amount of the test substancewill be addedthe correspondingculturetubes using a Gilson pipette. An aliquot of 0.5ml of theappropriateco-factormixwill be addedto each tube, and tested against a negative and positiveculturetubes followed by incubation. The culture tubes will then be transferred to a laminar flow cabinet, where 2.5ml of molten top agarwill be addedto each tube, vortexed and immediately poured onto a corresponding, labeled Vogel-Bonner nutrient agar plate. The plateswill be allowedtosetbeforebeing invertedand wrapped using cling film beforebeing incubatedovernight at 37oC.
The number of bacterial colonieswill be scoredand 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 assaywill be regardedas valid, if means, negative control count falls within thein-house historical ranges; positive control chemicals’induceclearincreases in reverting numbers confirming theactiveS-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 colonieswill be countedusing electronic counters orhandcounters, and expressed as reverting colonies per plate. Hand counting will beapplicablewhen there will be the presence of precipitate at high dose levels on the plates.In case all the plates are used, including the negative andpositivecontrol plates are from a single experiment, then countingwill be doneat the same time, because the cells formed on the plate will continue with thedivisiontoproducethe additionalmutant colonies withadditionalstoragetime.Toxicitywill be examinedwith the presence of precipitate by assessing thebackgroundlawn using a 40X dissecting microscope for thinning. From the results thatwill be obtainedfrom the experiment, a minimum fold increase, usually, 2-3 fold over the solvent controlwill be setto serve as a cut off between a non-mutagenic and mutagenic response. The only weakness of this approach occurs,especiallywhen themeanplate 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 betoosensitivefor the Salmonella strains containing averagely high reversion frequencies, for example, TA97, TA100 and TA102. It might also behighlysensitive for chemicals showinglowreversion frequencies, such as TA1537 and TA1535. By using the Ames Salmonella assay, this study willdemonstratethepossibilityto analyze the mutagenic potential fromnativecigarette smoke in a direct way, andobtaindose dependent revertants induction. Thiswill be achievablefrom the treatments with the bacterial strains, such as TA98 and TA100 (Zeiger, 1998).