Genotoxicity screening of organic chemicals using DNA and LC-MS.

摘要

Genotoxic compounds form covalently bound adducts with DNA, a process which could lead to mutagenesis and carcinogenesis. In this dissertation, methods that can be used to screen organic chemicals for genotoxicity were developed. The first method employs DNA-polyion films, neutral thermal hydrolysis of these films, and liquid chromatography-mass spectrometry (LC-MS) to quantify DNA adducts of molecules that interact directly with DNA. Neutral thermal hydrolysis provides a simple sample preparation procedure for N7-guanine and N3-adenone adducts because these adducts are selectively ejected from the DNA chain, resulting in a clean sample matrix enriched in nucleobase adducts. Coupling neutral thermal hydrolysis with LC-MS provides sensitive methods to detect and quantitate DNA adducts, and structural information is provided by MS. Neutral hydrolysates of DNA-polyion films used in voltammetric genotoxicity screening sensors were analyzed by LC-MS. Results showed that concentrations of N7-guanine DNA adducts with methyl methane sulfonate and styrene oxide increased with incubation time with the same trends as found for sensor response, allowing for the estimation of DNA damage rates. In addition, an approximate standardization procedure for neutral thermal hydrolysis was developed and validated that avoids need for a pure standard, and should be useful in cases where nucleobase adduct standards are unavailable.; The second method mimics the toxicity pathway in mammalian liver. Protein biocatalysts and DNA were assembled layer-by-layer on carbon cloth. These biocatalyst-DNA films were incubated with molecules that are "metabolized" by the biocatalyst, and resulting DNA damage detected by LC-MS/MS. For proof of concept, myoglobin (Mb) was used as the biocatalyst in the film, and the influence of suspected human carcinogen styrene was evaluated. H2O2 activated Mb, which converts styrene to styrene oxide, mimicking the action of cytochrome P450. The in situ generated SO reacted with DNA on the film. After incubation, the films were subjected to neutral thermal hydrolysis, and the hydrolysate was analyzed for the presence adducts using CapLC-MS/MS with an on-line pre-concentration column. Adducts were detected in films incubated in styrene and H2O 2 allowing estimation of relative kinetics within ∼1 min of reaction. Similar procedure was used to detect DNA adducts of benzo[a]pyrene.