Educator, Administrator, Innovator -- An Interview with Dr. Micheal Slattery

Transcript of interview with Dr. Micheal Slattery.https://scholars.fhsu.edu/ors/1251/thumbnail.jp

Ultrasensitive High-Resolution Mass Spectrometric Analysis of a DNA Adduct of the Carcinogen Benzo[<i>a</i>]pyrene in Human Lung

Benzo­[<i>a</i>]­pyrene (BaP), an archetypical polycyclic aromatic hydrocarbon, is classified as “carcinogenic to humans” and is ubiquitous in the environment, as evident by the measurable levels of BaP metabolites in virtually all human urine samples examined. BaP carcinogenicity is believed to occur mainly through its covalent modification of DNA, resulting in the formation of BPDE–<i>N</i>²-dG, an adduct formed between deoxyguanosine and a diol epoxide metabolite of BaP, with subsequent mutation of critical growth control genes. In spite of the liquid chromatography–mass spectrometry (LC–MS)-based detection of BPDE–<i>N</i>²-dG in BaP-treated rodents, and indirectly through high-performance liquid chromatography (HPLC)-fluorescence detection of BaP-7,8,9,10-tetraols released from human DNA upon acid hydrolysis, BPDE–<i>N</i>²-dG adducts have rarely if ever been observed directly in human samples using LC–MS techniques, even though sophisticated methodologies have been employed which should have had sufficient sensitivity. With this in mind, we developed a liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) methodology employing high-resolution/accurate mass analysis for detecting ultratrace levels of these adducts. These efforts are directly translatable to the development of sensitive detection of other small molecules using trap-based LC–ESI-MS/MS detection. The developed methodology had a limit of detection (LOD) of 1 amol of BPDE–<i>N</i>²-dG on-column, corresponding to 1 BPDE–<i>N</i>²-dG adduct per 10¹¹ nucleotides (1 adduct per 10 human lung cells) using 40 μg of human lung DNA. To our knowledge, this is the most sensitive DNA adduct quantitation method yet reported, exceeding the sensitivity of the ³²P-postlabeling assay (∼1 adduct per 10¹⁰ nucleotides). Twenty-nine human lung DNA samples resulted in 20 positive measurements above the LOD, with smoker and nonsmoker DNA containing 3.1 and 1.3 BPDE–<i>N</i>²-dG adducts per 10¹¹ nucleotides, respectively

Preferential Glutathione Conjugation of a Reverse Diol Epoxide Compared with a Bay Region Diol Epoxide of Benzo[\u3ci\u3ea\u3c/i\u3e]pyrene in Human Hepatocytes

Many studies have examined the relationship between polymorphisms in glutathione S-transferase genes and cancer in people exposed to polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (BaP), but the results to date have been modest. Missing from these studies has been an exploration of the formation of the appropriate glutathione conjugates in humans. We incubated human hepatocytes from 10 donors with racemic anti-BaP-7,8-diol-9,10-epoxide (BPDE), believed to be a major ultimate carcinogen of BaP, or with the noncarcinogenic reverse diol epoxide, racemic anti-BaP-9,10-diol-7,8-epoxide (rev-BPDE). Incubations were carried out for 12 or 24 h. We used high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring at m/z 464 → m/z 317 to analyze the incubation mixtures for the mercapturic acid products that would result from glutathione conjugation. The standard mercapturic acids were synthesized by reaction of BPDE or rev-BPDE with N-acetylcysteine. We obtained convincing evidence in human hepatocytes for mercapturic acid formation from rev-BPDE in all 10 samples, in amounts up to 17 pmol/ml. However, we could detect mercapturic acids from BPDE in only 1 of 10 samples (0.05 pmol/ml). Taken together with our similar previous results of analyses of phenanthrene metabolites in human hepatocytes and human urine, the results of this study indicate that conjugation of BPDE with glutathione is a minor pathway in humans, indicating that glutathione S-transferase genotyping is not an effective method for assessing risk of PAH-induced cancer in humans, at least with respect to the diol epoxide pathway of PAH carcinogenesis

New Levels of High Angular Resolution EBSD Performance via Inverse Compositional Gauss-Newton Digital Image Correlation

Conventional high angular resolution electron backscatter diffraction (HREBSD) uses cross-correlation to track features between diffraction patterns, which are then related to the relative elastic strain and misorientation between the diffracting volumes of material. This paper adapts inverse compositional Gauss Newton (ICGN) digital image correlation (DIC) to be compatible with HREBSD. ICGN works by efficiently tracking not just the shift in features, but also the change in their shape. Modeling a shape change as well as a shift results in greater accuracy. This method, ICGN HREBSD, is applied to a simulated data set, and its performance is compared to conventional cross-correlation HREBSD, and cross-correlation HREBSD with remapping. ICGN HREBSD is shown to have about half the strain error of the best cross-correlation method with a comparable computation time