Standard Guide for Measuring the Presence of Planar Organic Compounds Which Induce CYP1A, Using Reporter Gene Test Systems (Withdrawn 2012)
The compounds that bind to the Ah-receptor and induce CYP1A have often been shown to be either more toxic or carcinogenic, or both, than other organic compounds. Dioxins, furans, and PCBs have been shown to bioconcentrate in exposed organisms and biomagnify in the food web (see Guide E 1023). Testing with birds, mammals, and fish species has shown that exposure to these compounds can produce physiological, reproductive and histopathological effects (12, 13). Concern for the possible contamination of water, food, wildlife, soil, and aquatic sediment from these compounds has led to the requirement for analytical chemical analyses of a great many environmental samples. Use of a screening tool such as this Human Reporter Gene System (HRGS) will allow identification of significantly contaminated samples. These methods will aid in the cost-effective separation of high priority samples from those that do not require further costly chemical characterization.
1.1 This guide covers the recommended guidelines for performing a test for presence of organic compounds that bind to the Ah Receptor and induce the CYP1A locus on the vertebrate chromosome. Under appropriate test conditions, induction of CYP1A is evidence that the cells have been exposed to one or more of these xenobiotic organic compounds that include dioxins, furans, coplanar PCBs, and several polycyclic aromatic hydrocarbons (PAHs). Detection of induction has been made simple and rapid by the stable integration of the firefly plasmid such that Ah-receptor binding results in the production of luciferase. Luciferase production is a function of both the potency of the compound(s) and the concentration. This type of Human Reporter Gene System (HRGS) has shown concentration-response relationships using 2,3,7,8-TCDD, 5 coplanar PCBs, and several polycyclic aromatic hydrocarbons (PAHs) (1-3). This guide describes test conditions under which solvent extracts of environmental samples (water, tissue, soil, or sediments) may be tested for the presence of CYP1A-inducing organic compounds.
1.2 The test procedures presented in this guide have been published previously (4, 5 ) and established as EPA Method 4425 (6). These references should be consulted to obtain details regarding the construction and maintenance of the cell line, and the response of the cells to various organic substances.
1.3 All laboratory health and safety procedures should be followed. This includes the use of glasses, gloves, and other protective clothing, when handling the reagents. Information on toxicity, handling procedures and waste procedures should be reviewed prior to use of all chemicals.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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