Genotoxicity Tests
Testing for toxicity in the environment has expanded beyond simply addressing acute agents that cause high level effects to sensitive organisms. Testing for specific cellular effects from low levels exposures as a part of assessment regimes is quickly gaining regulatory importance to address challenges in the types and quantities of compounds being introduced into the environment from human activity. Liberal water use, climate change, population growth, pharmaceutical disposal and new industrial methods for natural resource extraction are changing natural ecosystems and affecting both human and animal exposures. Microcontaminants and enzymatic metabolites are combining in complex ways to produce largely unknown and constantly changing mixtures of toxicants present in our testable areas. Unfortunately, traditional assessment strategies do not adequately represent new exposure situations, and testing directly for sub-chronic human health endpoints like genotoxicity, which can become problematic over life-time exposure scenarios, are currently not considered by regulatory bodies beyond a few European countries.
Worldwide, agencies are recognizing the importance to adopt new strategies that include genotoxicity testing and mixture analysis for waste water because hundreds of thousands of new chemicals are being introduced into the environment, unintended by-products and transformation products of these chemicals arising from human metabolism and excretion as well as environmental breakdown products are also present and not accounted for, and chemical mixtures can have adverse additive or cumulative effects on organisms, although individual components of the mixture are below effective levels.
Current technological advancements in water testing are enabling cost effective and rapid measurements to test for sub-chronic endpoints like genotoxicity. EBPI have developed several test systems to improve water assessment by exploiting bacterial SOS responses for high throughput analysis. These assays use modified bacteria that have the induction of a SOS DNA response gene coupled to a gene that expresses a measurable colorimetric endpoint. The degree of induction of the SOS response can be quantified by the degree of colour change, and correlates with the amount of DNA damage in the cell from toxicant exposure. These assays conform to international ISO standards and were suggested for incorporation into legislation for waste water effluent testing as part of the OSPAR commission (2002). They are currently mandated in Europe as part of the water framework directive (WFD). The bacteria employed for these tests are sensitive to some compounds that test negative in the traditional Ames assays, and are ideal for individual screening experiments, or used in conjunction with other mutagenicity assays.
All our genotoxicity assays can be coupled with S9 fraction addition when using traditional methods for toxicant bioactivation. However, EBPI also offers our recently developed UMU-Express and SOS-Express assays which have been genetically engineered to express either the human CYP 450 1A2, CYP 450 1A1 or the GST T1-1 enzymes. The test methods are similar to traditional UMU and SOS assays, but they show marked improvement in sensitivity, biological relevance and environmental screening ability without the need to add extraneous metabolic systems. These assays offer the next step in rapid genotoxic evaluation with increased human health and environmental implications.
SOS-ChromoTest™ Kit
The SOS ChromoTestTM kit is based on a novel genetically engineered E.coli, which measures the primary response of a cell to genetic damage. In just a few hours, the kit provides a clear, completely objective measurement of the genotoxicity of a sample by a simple visual qualitative evaluation of the degree of DNA damage the cell experiences by observing the colour obtained.
UMU-ChromoTest™ Kit
EBPI has developed the umu-c test into the UMU-CHROMOTEST, a simple procedure based upon the International Organization for Standardization protocol ISO 13829 (Water Quality-Determination of the genotoxicity of water and waste water using the umu-test) which can be performed easily in a non-Specialized laboratory.
Sedi-Tox™ Direct Sediment Genotoxicity Test
7021 umuC Direct Contact Sediment Genotoxicity Test
The UMU-ChromoTest™ (umuC assay) employs Salmonella typhimurium TA 1535 [pSK 1002] bacteria in which the SOS DNA repair response umuC gene has been linked to the β gal gene responsible for producing the β-galactosidase enzyme. The degree of DNA damage repair using the SOS gene pathway is directly linked to the production of β-galactosidase, which is measured by the enzyme’s reaction with a yellow chromogen. Strain TA 1535 [pSK 1002] contains both the rfa mutation, which produces a defective, more permeable cell membrane, and the uvrB mutation, which eliminates the accurate excision DNA repair mechanism, resulting in more repair by error-prone mechanisms. The results from this assay agree very closely with traditional Ames mutagenicity tests, with the added advantage of only using a single bacterial strain.
The assay is based on the Organization of Standardization Protocol ISO 13829.
7031 SOS Direct Contact Sediment Genotoxicity Test
The SOS line of assays are designed for rapid detection of genotoxicity or DNA damage and utilizes the synthesis of the B-galactosidase enzyme whose gene (LacZ) is connected to an SOS promoter. Once a lesion has been detected, the SOS promoter is induced to start the transcription of the SOS genes and the LacZ gene is transcribed as well. This gene produces an enzyme which is assayed using a simple colour change. The degree of colorimetric expression is quantified and given as the SOS-inducing potency (SOSIP). Although the SOS tests also correlate well with the Ames assays, they have been shown to be slightly less sensitive, although they also report less false-positive results.