Ames Express™ Strains
Similar to the Ames strains, the Ames ExpressTM strains also carry specific mutations in the histidine biosynthesis pathway. As a result, the Ames ExpressTM strains are histidine-dependent and will not survive in a histidine deficient nutrient.
The EBPI Ames ExpressTM strains are unique to the Ames strain as these strains also express either human P450 1A2 or GST-theta enzymes internally which allows for the bio-activation of xenobiotic molecules into DNA reactive species in the absence of a S9 mix. The ability to induce revertant mutation through bio-activation makes these Salmonella strains the ideal biomarker in assessing mutagenic properties in test samples. As a result, exposure and incubation of the EBPI strains with a test sample followed by selection for the revertant mutants in a histidine deficient background allows researchers to assess the rate of revertant mutation which will reflect the level of mutagenicity of the test sample.
Benefits of the Ames ExpressTM Strains
- The system expresses recombinant human proteins rather than rat liver extract allowing better correlation to human health.
- Sequestering of mutagens by lipid components from S9 lysate mix is no longer a concern
- Short-lived reactive DNA mutagens have a increased chance of detection within this Ames test due to the internal P450 1A2, P450 1A1, P450 2E1 and GST T1-1bio-activation processes
Internal Human P450 1A2 Bio-Activation Ames Test
Expression of P450 1A2 and NADPH-P450 reductase
| TA1538 | -1 Frameshift | Recombinant Expression of P450 1A2 |
| TA98 | -1 Frameshift | Recombinant Expression of P450 1A2 |
| TA100 | Base-Pair | Recombinant Expression of P450 1A2 |
Internal Human P450 1A1 Bio-Activation Ames Test
Expression of P450 1A1 and NADPH-P450 reductase
| TA100 | Base-Pair | Recombinant Expression of P450 1A1 |
Internal Human P450 2E1 Bio-Activation Ames Test
Expression of P450 2E1 and NADPH-P450 reductase
| Base Pair | Recombinant Expression of P450 2E1 |
Internal Human GST Bio-Activation Ames Tests
Expression of GSTT1-1
| TA1535 | Base-Pair | Recombinant Expression of GST-T1-1 |
| TA100 | Base-Pair | Recombinant Expression of GST-T1-1 |
SOS-Express™ Test
Product No. 5031-1A2
The SOS ChromoTest kit is based on a novel genetically engineered E.coli, which measures the primary response of a cell to genetic damage as discussed above. The SOS-Express P450 1A2 bacterial stains contain a bicistronic protein expression vector (p4501A2-red), whcih contains human cytochrome P450 1A2 and human NADPH-P450 reductase cDNA. The recombinant expression of these two proteins helps in the bioactivation of drug and foreign compounds into DNA-reactive mutagens.
SOS-Express™ Test
Product No. 5031-GST T1-1
The SOS ChromoTest kit is based on a novel genetically engineered E.coli, which measures the primary response of a cell to genetic damage as discussed above. The SOS-Express GST T1-1 is a Phase 2 enzyme responsible for attaching glutathione conjugate molecules to reactive sites on a toxicant. Certain classes of compounds produce reactive metabolites upon conjugation with GST. Haloalkanes, organic thiocyanates, nitrosoguanides vicinal dihaloalkanes and quinones all produce reactive GST metabolites that cause mutagenicity and genotoxicity.
UMUc-Express™ Test
Product No. 5021-P450 1A2
The umu test uses the genetically engineer Salmonella typhimurium TA1535 [pSK1002] umuC which are exposed to different concentrations of the samples to be tested as indirect measure of DNA damage, or genotoxicity. The UMU-Express P450 1A2 bacterial stains contain a bicistronic protein expression vector (p4501A2-red), whcih contains human cytochrome P450 1A2 and human NADPH-P450 reductase cDNA. The recombinant expression of these two proteins helps in the bioactivation of drug and foreign compounds into DNA-reactive mutagens.
UMUc-Express™ Test
Product No. 5021-GST T1-1
The umu test uses the genetically engineer Salmonella typhimurium TA1535 [pSK1002] umuC which are exposed to different concentrations of the samples to be tested as indirect measure of DNA damage, or genotoxicity. Like the SOS-Express strains, the UMU-Express strain also carries the GST T1-1; it is a Phase 2 enzyme responsible for attaching glutathione conjugate molecules to reactive sites on a toxicant. Certain classes of compounds produce reactive metabolites upon conjugation with GST. Haloalkanes, organic thiocyanates, nitrosoguanides vicinal dihaloalkanes and quinones all produce reactive GST metabolites that cause mutagenicity and genotoxicity.
Why use the Ames ExpressTM Strains for Bioactivation?
The body encounters a multitude of non-essential foreign substances on a daily basis. Many of these compounds are lipophilic (fat-loving) and unreactive, but favor absorption across biological membranes and storage in fatty tissues. Lipophilic molecules are difficult to excrete and must undergo enzymatic conversions to increase their hydophilic (water-loving) characteristics, and favor excretion. These processes, called biotransformations, are carried out by multiple enzymes, found primarily in the liver, which work in conjunction to this end.
In general, biotransformation is a two step process, although this is not always the case. Phase 1 reactions are carried out primarily by the family of enzymes called cytochrome P450 (CYP 450). The enzymes attach or expose a functional group on a toxicant which increases the hydrophilic character of the molecule, but also provides a reactive site for subsequent reactions. Phase 2 processes attach large water soluble conjugates to the newly-formed functional group. This blocks the reactivity produced by Phase 1 processes, and further increases the hydrophilicity of the molecule to strongly favor excretion.
Although the biotransformation system is highly efficient, under conditions of increased exposure or environmental stresses, reactive metabolites escape metabolic control and can react with a biomolecule like a protein or DNA. These adducts (addition products) can cause cytotoxicity and mutagenicity and may result in carcinogenicity and cell death.
Mechanistically, many known toxicants rely on bioactivation pathways to produce deleterious effects. The incorporation of metabolic enzyme systems into mutagenicity assays has significantly increased the biological relevance and sensitivity of these test systems to multiple classes of carcinogens.
At EBPI, we have engineered the expression of 4 human metabolic enzymes (CYP P450 1A2, CYP P450 1A1, CYP P450 2E1 and the GST T1-1) into our Ames test bacteria, which eliminates the need to add rat liver enzymes (S9 fraction) to the experiment. S9 fractions have been the traditional method to include bioactivation into these types of assays, but suffer from limitations that we believe are overcome with this expression system.
P450 1A2 is a Phase 1 enzyme found primarily in the hepatic tissue and is responsible for metabolizing polyaromatic hydrocarbons (PAHs), aromatic amines and nitro aromatics. It performs a variety of chemical reactions on several different xenobiotic classes and is a key contributor to the mechanistic mutagenicity of well known carcinogens like benzo(a)pyrene (B[a]P), PhIP and 4-aminobiphenyl.
P450 1A1 is a Phase 1 enzyme that performs many oxidative activation reactions on a wide range of polycyclic aromatic hydrocarbons (PAHs) found in combustion processes, cigarette smoke, industrial activity and natural resource development. Although it is capable of performing heteroatom hydroxylation reactions similar to the P450 1A2, hydroxylation and epoxidation reactions on conjugated aromatic systems make up the bulk of activation reactions performed by this enzyme.
P450 2E1 is a Phase 1 enzymes that preforms important oxidation activation reactions on a number of haloalkanes and some PAHs, but is more importantly involved as the primary activator for a family of molecules called nitrosamines. Nitrosamines are important carcinogens formed from a variety of processes including the acid catalyzed combination of nitrite and amines as well as a byproduct from disinfection of drinking water sources by UV light.
GST T1-1 is a Phase 2 enzyme responsible for attaching glutathione conjugate molecules to reactive sites on a toxicant. GST acts primarily as a detoxification enzyme and the products are, by enlarge, excreted without incident. However, certain classes of compounds produce reactive metabolites upon conjugation with GST. Haloalkanes, organic thiocyanates, nitrosoguanides vicinal dihaloalkanes and quinones all produce reactive GST metabolites that cause mutagenicity. These reactions are important mutagenic pathways for several prominent pollutants and can serve as biomarkers for the presence of polychlorinated alkanes and alkenes in waste water effluent and drinking water samples.
The Ames ExpressTM line of bacterial strains are developed for EBPI's newly developed Modified Ames ISO Kit, though can also be inserted into the Traditional Ames Test as well as EBPI's widely used Muta-ChromoPlate Kits.