Enzymes preparation, mutagenicity

The mutagenic activity of the extracts was assayed using Salmonella typhimurium strain TA 1535, as described by McCann et al. (58). Ten ul of fish extract were spot-tested without addition of an S9-enzyme preparation (57). MNNG (20 ug/plate) was used as a positive control. 

Several other principles should guide the construction of a battery. Importantly, the tests should be reliable and of clear biologic significance ( ). This means that they should truly measure what they purport to measure and that the end point should have conceptual relevance to mutagenicity or carcinogenicity. Secondly, a battery should seek to maximize the metabolic parameters provided by all tests. As an example, tests with intact cell metabolism should be included to extend the metabolism obtained with the commonly used exogenous subcellular preparations. This may be of particular importance in view of the artifactual enhancement of activation over detoxification that is known to be characteristic of enzyme preparations ( 5, ). Moreover, the DNA adducts formed by activation through mic-... 

This test uses specially constructed bacteria to detect reverse mutations. Each tester strain has enhanced sensitivity and selectivity for the classes of compounds it will pick up. Therefore, several tester strains are generally used to maximize the possibility of detecting a mutagenic compound. It is well known now that many compounds, for example polycyclic aromatic hydrocarbons, must undergo some kind of enzymatic or chemical modification before becoming reactive. Therefore, in many cases an enzyme preparation (commonly called S9) is added to the chemical in order to ensure conversion to a chemically-reactive species. 

The isoamylase enzyme preparation that was evaluated is obtained by pure culture fermentation of Pseudomonas amyloderamosa. The Committee reviewed toxicological data on this enzyme at its fifty-fifth meeting (Annex 1, reference 149) as part of the safety assessment of trehalose. The Committee at that meeting concluded that the available data on isoamylase (i.e. a study of acute toxicity, a 13-week study of toxicity and a bacterial mutagenicity assay) did not raise any safety concern. 

Perhaps the most widely used assay for mutagenic potential is the Ames test (Ames etal., 1973, 1975 Ames, 1979). In this test the compound under investigation is studied for its ability to bring about a reverse mutation in bacteria. The bacterial tester strains are selected to detect two types of mutation base-pair substitution (TA 1535, TA 100) and frameshift (TA 1537, TA 1538, TA 98). Generally, the assay is carried out in the presence and absence of an enzyme preparation (S-9) isolated from rodent liver in order to study the mutagenicity of potential metabolites. 

The conflicting set of results from bacterial mutagenicity tests is probably due to a lack of specific metabolic enzymes in microsomal preparations which were present in intact hepatocytes. 

To determine the mutagenic potential of nonaqueous liquids as measured by the Ames SaZmoneZ/a/mammalian-enzyme assay, the following protocol is recommended for the sample preparation. In step 1, the desiccator assay is performed on the neat material. The desiccator assay allows the detection of volatile mutagens (such as chlorinated solvents) that are often missed in the plate incorporation and pre-in-cubation assays (16, 17). In addition, a suspension of the neat material (20 mg/mL) is prepared by ultrasonication (5 min at room temperature) in high-purity DMSO (18, 19) and tested in the normal plate incorporation assay as well as in a pre-incubation Ames assay (20). The pre-in-cubation assay allows the detection of certain mutagens, such as dimethylnitrosamine, that require additional time for activation by mammalian or bacterial enzymes. A positive response in any of these three assays indicates the presence of mutagenic components, and the evaluation process is completed. 

Studies of CuZnSOD derivatives prepared by site-directed mutagenesis are also providing interesting results concerning the SOD mechanism. For example, it has been shown that mutagenized derivatives of human CuZnSOD with major differences in copper-site geometry relative to the wild-type enzyme may nonetheless remain fully active.Studies of these and similar derivatives should provide considerable insight into the mechanism of reaction of CuZnSOD with superoxide. 

This article describes our subsequent studies which have demonstrated that the direct-acting mutagenicity of diesel particle extracts is largely attributable to nitroaromatic compounds, including both mononitro-PAH compounds and their multlsubstituted derivatives. These studies have also examined the processes by which mammalian enzymes in the rat liver S9 preparation modify the activity of these mutagens. The use of normal and nitroreductase-deficient bacteria in the Salmonella assay has been instrumental in all of these studies. 

The mutagenicity of the dinitropyrenes in the Salmonella/S9 assay was markedly reduced by NADPH-dependent enzyme activity. However, when the cytosol and microsome enzymes in the S9 preparation were separated, the activity increased in assays containing cytosol enzymes and decreased in the presence of... 

Figure 6. The effects of cytosol and microsome enzymes from a rat liver S9 preparation on the mutagenicity of nitropyrenes and diesel particle extracts. Loss of activity is also described as a percent decrease in the residual direct-acting mutagenicity.

---