Use of a New Enzyme Extraction System to Improve the Sensitivity of SOS/umu Test and Application to Environmental Samples

Abstract

The purpose of this study was to find a better enzyme extraction reagent for the SOS/umu test to replace the conventional one (the combination of sodium dodecyl sulfate (SDS) and Z-buffer), which has the disadvantage of denaturing b-galactosidase leading to decreased measurement sensitivity. By adopting a microplate system, the performance of the umu test using BugBuster Master Mix, a commercially available enzyme extraction reagent, was compared with that using the conventional reagent for detecting the genotoxicity of known mutagens as well as environmental samples. BugBuster Master Mix was found to increase the detection sensitivities of the selected genotoxins and environmental water samples, due to the fact that it doesn't denature b-galactosidase. The result of this study showed that BugBuster Master Mix could be a better enzyme extraction reagent for umu test.With increasing numbers of chemical substances being discharged into the environment, evaluation and monitoring of the impacts of these chemicals to the water environment and human health is becoming more and more important (Ohe et al. 2004). Rapid and sensitive bioassay systems have been developed for detecting environmental genotoxins as a whole based on the ability of these substances to cause DNA damage (Monarca et al. 2004).Among different bioassays for the detection of environmental mutagens, the SOS/umu test, which monitors the expression of one of the SOS genes (umuC gene) by measuring b-galactosidase activity (Oda et al. 1985), has attracted vast attention because of its simplicity, short test time and low requirement for disinfection (Hamer et al. 2000). By employing a single Salmonella typhimurium tester strain (S. typhimurium TA1535/pSK1002), this bioassay system allows the detection of SOS mutagenesis after treatment with DNA-damaging agents (Michel 2005). As the standardized method of ISO (ISO/CD 13829) (ISO 2000) and DIN (DIN 38415-3) (DIN 1996), the SOS/umu test has been extensively employed to detect genotoxicity in different media including airborne particles (Oda et al. 2004), tap water (Shen et al. 2003), reclaimed water (Wu et al. 2010) and industrial wastewater (Dizer et al. 2002).Enzyme extraction is one of the important steps in the SOS/ umu test to detect the induced activity of b-galactosidase. In comparison with toluene, the frequently used protein extraction detergent, sodium dodecyl sulfate (SDS), demonstrated its advantage in shortening test time and increasing induced bgalactosidase activity (Whong et al. 1986), and the combination of SDS and Z-buffer (Miller 1972) has been widely used as the enzyme extraction system for the SOS/umu test. However, SDS treatment has been reported to denature bgalactosidase (Demeo et al. 1988;El Mzibri et al. 1996;Whong et al. 1986) and may result in poor protein recovery (De Mey et al. 2008), which can decrease the measurement sensitivity of b-galactosidase activity.

Protein:Protein Interactions in the Cytoplasmic Membrane Influencing Sugar Transport and Phosphorylation Activities of theE. coliPhosphotransferase System

“…During incubation, samples were removed for measurements of OD 600nm and β-galactosidase activities after being appropriately diluted. To measure β-galactosidase activities, 0.8 ml of Z-buffer containing β-mercaptoethanol (2.7 μl/ml) and SDS (0.005%) were mixed with 0.2 ml of sample and 25 μl of CHCl 3 in test tubes [55]. The tubes were vortexed twice (each for 10 seconds) at a constant speed and incubated in a 37 °C water bath until equilibration.…”

Qualitative and quantitative assessment of genotoxins usingSRRzlysis reporter under the control of a newly designed SOS responsive promoter inEscherichia coli

“…Due to the report that SDS treatment may result in the denaturation of b-galactosidase, 19 a more effective and commercially available protein extraction detergent, BugBuster Master Mix containing nuclease and lysozyme, was applied to the SOS Chromotest assay. 20 Mitomycin C (MMC) and methyl methanesulfonate (MMS) are direct-acting alkylating agents that may induce SOS response in E. coli through forming cross-links to adjacent guanines in DNA. 21,22 MMC may strongly induces the SOS response, while MMS showed lower mutagenic activities in the SOS/umu-test.…”

Development of a high‐throughput genotoxicity assay using Umu test strains expressing human cytochrome P450s and NADPH‐P450 reductase and bacterial O‐acetyltransferase

“…As lacZ gene, which is encoded by β‐galactosidase, is fused with umuC , the induction of umuC and the occurrence of DNA damage by genotoxic chemicals can be detected as β‐galactosidase activity. This test is used for high‐throughput genotoxicity screening of environmental chemicals and candidate pharmaceutical compounds [International Organization for Standardization, ; Oda, 2004; Yasunaga et al, ; Allinson et al, ; Tian et al, ], requiring only a small amount of test substance, and producing results in a short time.…”

“…A microplate‐based umu test can be used to evaluate a large number of samples for genotoxicity screening since it is technically simple and requires only a small amount of test samples. Accordingly, an umu ‐microplate test method using the typical strains Salmonella typhimurium TA1535/pSK1002 and NM2009 (overexpressing O ‐AT) along with S9 mix has been developed [Reifferscheid et al, ; Oda et al, ; Yan et al, ; Tian et al, ]. For the Ames test, high‐throughput Ames test have been developed as well [Sui et al, ].…”