Progress 07/15/15 to 07/14/17
Outputs
Target Audience: The primary stakeholders are the testing laboratories for the quality and safety of foods. Changes/Problems: None. What opportunities for training and professional development has the project provided?The postdoctoral student, Pushkar Kulkarni, underwent training and professional development. How have the results been disseminated to communities of interest?By this publication: Shao, G., Agar, J., Giese, R. W. (2017) Cold-Induced Aqueous Acetonitrile Phase Separation: A Salt Free Way to Begin QuEChERS, J. Chromatogr. A 1506, 128-133. doi.org/10.1016/j.chroma.2017.05.045 What do you plan to do during the next reporting period to accomplish the goals?The phase 1 project has been completed.
Impacts
What was accomplished under these goals?
A1. Non-Technical Summary We were funded by the NIFA Exploratory Research Program for the project, "Comprehensive Assay for Carcinogens in Food". The basic idea of the project was to detect carcinogenic chemicals (carcinogens) universally in foods in a single procedure. There are three steps in the proposed method: (1) extract carcinogens from the food; (2) activate the procarcinogens metabolically to their corresponding genotoxic (DNA-reactive) products (ultimate carcinogens) in the presence of a Bait reagent that traps these latter products covalently; and (3) detect the resulting carcinogen-Bait conjugates by mass spectrometry. Two types of Bait reagents have been studied. The first type comprises a mass tag to make carcinogen-Bait conjugates very sensitive in the mass spectrometer, along with a nucleophilic moiety to react with genotoxic chemicals (essentially all organic genotoxic chemicals are electrophilic). We selected a cationic xylyl (Cmass tag for the first type of Bait, since it can provide detection at the amol level; it is an anchimeric-assisted neutral loss cationic tag. The second Bait is DNA. Following is the progress that we have made. We will first discuss the type 1 category of Baits (CAX-Baits, of which there are four), and then the type 2 Bait (DNA) A2. Progress Details A2a. Strategy for Developing a CAX-Bait Reagent We chose to compare the performance of four types of CAX-Bait reagents in order to select an optimum one for our assay of carcinogens in foods. The four criteria for selection of the best CAX-Bait were: (1) ease of synthesis; (2) efficiency of reaction with model genotoxic chemicals; (3) ability of the Bait to capture a diversity of electrophilic chemicals; and (4) stability of the Bait-carcinogen conjugates. A2b. Bait I: N2-CAX-G The guanine (G) nucleobase of DNA is a prominent target for electrophilic chemicals in general, so we selected two CAX-guanine Baits for study. In the first one ( Bait I) the CAX mass tag is attached to the N2 position of guanine, leaving the other nitrogen atoms along with the O6 and C8 atoms of guanine available for reaction with an electrophilic chemical. We prepared Bait I and tested it by reaction with benzyl bromide as a model genotoxic chemical. The reaction was efficient and yielded a stable product with a strong response in the mass spectrometer. The actual site of attachment of the benzyl moiety to the Bait is unknown; the site shown is arbitrary. Shortcomings of Bait I: its synthesis is tedious, and it lacks an ability to capture aldehyde carcinogens efficiently or at all. A2c. Bait II: C8-CAX-G In this Bait reagent, the CAX mass tag is attached to the C8 position of guanine. Also, a biotin moiety is incorporated to facilitate the isolation of a corresponding CAX-guanine-carcinogen product. While Bait II can capture aldehydes, reaction of the resulting Schiff bases with sodium borohydride would be necessary to stabilize the products by reduction. Otherwise Bait II basically has the same advantages and disadvantages as Bait I. A2d. Bait III: CAX-Hydrazide This bait (CAX-Hydrazide, abbreviated CAX-H) comprises a CAX mass tag attached to the C4-position of phenylhydrazide. It is easy to prepare, and reacts efficiently with benzyl bromide to give a stable product with a strong response in the mass spectrometer. This encouraged us to evaluate Bait III further by reacting it with a mixture of model carcinogens including benzyl bromide. Some representative, model carcinogens are detected simultaneously in this way: acetaldehyde; chloroacetaldehyde; benzoquinone; benzaldehyde, and styrene oxide. We then applied Bait III to an extract of cooked hamburger, and several aldehydes were detected. We were particularly pleased with the performance of the Bait III, although the hydrazone products of aldehydes would need to be processed under nonacidic conditions. A2e. Bait IV: CAX-Aniline This Bait comprises a CAX mass tag attached to the C4-position of aniline. While this Bait is easy to prepare, its reaction with benzyl bromide is slower than that of Bait III. Nevertheless it forms stable products after reduction with sodium cyanoborohydride that give a strong response in the mass spectrometer Bait IV provides an option for detecting aldehyde carcinogens in a way that should be more rugged, as needed, than Bait III, due to stability of the reduced products with Bait IV under both acidic and basic conditions A2f: New Kind of QuEChERS. The QuEChERS (quick, easy, cheap, effective, rugged, safe) procedure is widely employed for extraction of pesticides and other contaminants in foods, as has been reviewed (Rejczak and Tuzimski, 2015; Han, et al., 2016). We set up a modified QuEChERS procedure in which the initial phase separation is accomplished by a cold-induced phase separation rather than by adding salt, potentially making it easier to subject the sedimented matrix to further extraction for more complete isolation of carcinogens (Shao and Giese, 2017). The method was applied to salmon and beef. A QuEChERS procedure (many variations are in practice) is a good choice for extracting carcinogens from foods for detection. A2g. Melt-Labeling Assay for DNA adducts We have set up a convenient assay for detection of DNA adducts that will meet the needs of this project for DNA as the type 2 Bait. We refer to this assay as a "melt-labeling assay". The method relies on the preferential melting (as by increasing the temperature) of local DNA structure around a structure-destabilizing adduct (many DNA adducts weaken or distort local DNA structure; nucleotide excision repair relies on this property). The melted zone, including labeling-susceptible adducts there, is preferentially labeled with a mass tag (we have used CAXto date; "cationic xylyl bromide") A2h. Detection of Benzoquinone DNA adducts by Melt-Labeling with CAX-B. Quinones are a class of food carcinogens. A benzoquinone-exposed sample of DNA (100 μg) containing a known concentration of BQ-C (referring to BQ-dCMP; about 23 adducts in 106 nucleotides) was subjected to melt-labeling with CAX-B. the previously unknown adduct, BQ-hmC (referring to BQ-hydroxymethyl-dCMP) was detected at S/N of 70. Considering that mC is 4% of C in DNA, and assuming that hydroxymethyl-deoxycytosine is 1% of methyldeoxycytosine, then the level of BQ-hmdCMP being detected at observed S/N = 70 in the lower mass spectrum corresponds to 4 BQ-hmdCMP in 109 nucleotides. Thus, CAX-B melt labeling/LC-MALDI-TOF/TOF-MS can be very sensitive. A3. References Cited 1. De Jongh, H., Berthuis, R. K., Vies, R. O., Barrett, C. B., Ord, W. O. (1962) Investigation of the factor in ground peanut meal responsible for "Turkey X disease", Biochim. Biophs. Acta 65, 548-551. Han, L., Sapozhnikova, Y., Lehotay, S. J. (2016) Method validation for 243 pesticides and environmental contaminants in meats and poultry by tandem mass spectrometry coupled to low-pressure gas chromatography and ultrahigh-performance liquid chromatography, Food Control. 66, 270-282. doi: org/10.1016/foodcont.2016.02.019 Rejczak, T., Tuzimski, T. (2015) A review of recent developments and trends in the QuEChERS sample preparation approach, Open Chem. 13, 980-1010. doi: 10.1515/chem-2015-0109 Shao, G., Agar, J., Giese, R. W. (2017) Cold-Induced aqueous acetonitrile phase separation: A salt -free way to begin quick, easy, cheap, effect, rugged, safe, J. Chromatogr. A 1506,128-133. doi:10.1016/j.chroma.2017.05.045
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2017
Citation:
Shao, G., Agar, J., Giese, R. W. (2017) Cold-Induced Aqueous Acetonitrile Phase Separation: A Salt-Free Way to Begin QuEChERS, J. Chromatogr. A 1506, 128-133. doi.org/10.1016/j.chroma.2017.05.045.
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