PetroLabs

Nitration Assay

Background: The Nitration Assay is a sensitive method for quantifying the relative amounts of polycyclic aromatic compounds (PACs) in fossil fuel-derived complex mixtures, combustion products, and a wide range of related environmental samples. The method relies upon the extreme sensitivity of Ames Test bacterial strain TA98 to nitroPAC-mediated mutagenicity, and the relative ease with which many PACs can be chemically nitrated. Because the assay measures the mutagenic potency of chemically nitrated derivatives, rather than of the parent PACs themselves, it cannot provide a direct measure of the potential carcinogenicity of test materials.

How it works: The first step in the procedure is to chemically nitrate the test material, usually by reacting it for 30 minutes with concentrated nitric acid at 80ºC. It should be noted that such harsh reaction conditions are not needed for the PACs of greatest biological interest, among them the EPA priority pollutants, in that unalkylated and lightly alkylated compounds are almost instantly nitrated at room temperature. The more rigorous treatment used here ensures that compounds more resistant to derivatization react, thereby increasing the sensitivity of the method. But even under such severe reaction conditions, it has been shown that only PACs are derivatized, a feature of the method that confers its near absolute chemical selectivity. Once the nitration reaction is complete, the samples are cooled to room temperature and extracted with methylene chloride. A measured volume of the extract is removed to a new test tube, the methylene chloride stripped off, and the resulting residue reconstituted into dimethyl sulfoxide (DMSO) for Ames testing. The version of the Ames Test used for this purpose is the so-called "non-activated assay," a feature of the method made possible because the tester strain used (TA98) possesses the endogenous nitroreductase enzymes necessary for activation of nitroPAC to their mutagenic forms, making addition of exogenous metabolizing mixture (S-9) unnecessary. This aspect of the test confers its far higher sensitivity relative to S-9-dependent assays like the Modified Ames Test, in that DNA-adducting species are generated inside the bacteria and need not be transported through the cell wall to reach their ultimate DNA target. Moreover, the fact that nitroaromatics are the only S-9-independent mutagens likely to be found in typical test materials means that the method is not only chemically, but biologically selective for PACs.

Endpoint: The endpoint of the Nitration Assay is directly analogous to that of the Modified Ames Test, i.e. the slope of the dose-response curve for mutagenicity. In the Nitration Assay, it is termed the Nitration Mutagenicity Index (NMI).

Interpretation of NMI: As mentioned above, NMI does not in a direct way predict carcinogenic potential. Instead it is used as a means of comparing the relative PAC contents of various compositionally-related complex mixtures, in much the same way IP 346 and other analytical methods are sometimes used. However, because the mutagenicity measured in both the Modified Ames Test and the Nitration Assay is directly proportional to PAC concentration, it should be possible to develop correlation curves between the two endpoints for refinery streams with closely similar compositional profiles.

Applications: The Nitration Assay is applicable to any sample containing PACs. It is particularly useful for environmental or biomonitoring samples where either sample size or PAC concen-tration is too low for standard methods such as the Modified Ames Test and Method IP 346.

Logistics: See Modified Ames Test.

Work Product: We will work with sponsors to tailor studies to their individual needs.