Brock Matter

GLUTATHIONE TRAPPING TO MEASURE MICROSOMAL OXIDATION OF FURAN TO CIS-2-BUTENE-1,4-DIAL

Furan is a liver carcinogen and toxicant. Furan is oxidized to the reactive dialdehyde, cis-2-butene-1,4-dial, by microsomal enzymes. This reactive metabolite readily reacts with glutathione nonenzymatically to form conjugates. A high-performance liquid chromatography-electrochemical method for the detection of cis-2-butene-1,4-dial-glutathione (GSH) conjugates in microsomal preparations was developed to measure the extent of furan metabolism to cis-2-butene-1,4-dial in vitro. Previously unobserved mono-GSH reaction products of cis-2-butene-1,4-dial were detected in addition to the already characterized bis-GSH conjugates. Chemical characterization of these compounds indicated that the α-amino group of glutathione had reacted with cis-2-butene-1,4-dial to form a thiol-substituted pyrrole adduct. The analytical method was used to estimate the extent of furan oxidation in rat liver microsomes from untreated or acetone-pretreated F344 rats as well as in human P450 2E1 Supersomes. Our results confirm that cytochrome P450 2E1 can catalyze the oxidation of furan to cis-2-butene-1,4-dial. However, the data are also consistent with the involvement of other P450 enzymes in the oxidation of furan in untreated animals. This assay will be a valuable tool to explore tissue and species differences in rates of furan oxidation.

Quantitative High-Performance Liquid Chromatography−Electrospray Ionization Tandem Mass Spectrometry Analysis of Bis-N7-Guanine DNA−DNA Cross-Links in White Blood Cells of Cancer Patients Receiving Cyclophosphamide Therapy

Cyclophosphamide (CPA) is a DNA alkylating agent widely used in cancer chemotherapy. CPA undergoes metabolic activation to phosphoramide mustard and nornitrogen mustard (NOR) which alkylate the N-7 position of guanine in DNA to produce N-[2-(N7-guaninyl) ethyl]-N-[2-hydroxyethyl]-amine (G-NOR-OH) monoadducts and N,N-bis[2-(N7-guaninyl) ethyl] amine cross-links (G-NOR-G). G-NOR-G cross-links are strongly cytotoxic and are thought to be responsible for the biological activity of CPA. In the present work, an isotope dilution high-performance liquid chromatography-electrospray ionization (positive ion) tandem mass spectrometry (HPLC-ESI(+)-MS/MS) methodology was developed to accurately quantify G-NOR-G adducts in human blood. In our approach, DNA extracted from white blood cells (5-20 microg) is spiked with an internal standard of [(15)N(10)]-G-NOR-G and subjected to thermal hydrolysis to release G-NOR-G adducts from the DNA backbone. Following solid phase extraction, G-NOR-G conjugates are quantified by capillary HPLC-ESI-MS/MS in the selected reaction monitoring mode. The application of the new methodology is demonstrated for DNA extracted from blood of three cancer patients receiving 50-60 mg/kg of intravenous CPA. The highest numbers of G-NOR-G adduct (up to 18 adducts per 10(6) normal nucleotides) were observed 4-8 h following CPA administration, followed by a gradual decrease over time, probably due to adduct hydrolysis, DNA repair, and white blood cell death. This methodology will be useful for future investigations of the interindividual differences for CPA-induced DNA-DNA cross-linking.

Mapping structurally defined guanine oxidation products along DNA duplexes: influence of local sequence context and endogenous cytosine methylation.

DNA oxidation by reactive oxygen species is nonrandom, potentially leading to accumulation of nucleobase damage and mutations at specific sites within the genome. We now present the first quantitative data for sequence-dependent formation of structurally defined oxidative nucleobase adducts along p53 gene-derived DNA duplexes using a novel isotope labeling-based approach. Our results reveal that local nucleobase sequence context differentially alters the yields of 2,2,4-triamino-2H-oxal-5-one (Z) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (OG) in double stranded DNA. While both lesions are overproduced within endogenously methylated MeCG dinucleotides and at 5′ Gs in runs of several guanines, the formation of Z (but not OG) is strongly preferred at solvent-exposed guanine nucleobases at duplex ends. Targeted oxidation of MeCG sequences may be caused by a lowered ionization potential of guanine bases paired with MeC and the preferential intercalation of riboflavin photosensitizer adjacent to MeC:G base pairs. Importantly, some of the most frequently oxidized positions coincide with the known p53 lung cancer mutational “hotspots” at codons 245 (GGC), 248 (CGG), and 158 (CGC) respectively, supporting a possible role of oxidative degradation of DNA in the initiation of lung cancer.

Epicatechin-rich cocoa polyphenol inhibits Kras-activated pancreatic ductal carcinoma cell growth in vitro and in a mouse model

Activated Kras gene coupled with activation of Akt and nuclear factor‐kappa B (NF‐κB) triggers the development of pancreatic intraepithelial neoplasia, the precursor lesion for pancreatic ductal adenocarcinoma (PDAC) in humans. Therefore, intervention at premalignant stage of disease is considered as an ideal strategy to delay the tumor development. Pancreatic malignant tumor cell lines are widely used; however, there are not relevant cell‐based models representing premalignant stages of PDAC to test intervention agents. By employing a novel Kras‐driven cell‐based model representing premalignant and malignant stages of PDAC, we investigated the efficacy of ACTICOA‐grade cocoa polyphenol (CP) as a potent chemopreventive agent under in vitro and in vivo conditions. It is noteworthy that several human intervention/clinical trials have successfully established the pharmacological benefits of cocoa‐based foods. The liquid chromatography (LC)–mass spectrometry (MS)/MS data confirmed epicatechin as the major polyphenol of CP. Normal, nontumorigenic and tumorigenic pancreatic ductal epithelial (PDE) cells (exhibiting varying Kras activity) were treated with CP and epicatechin. CP and epicatechin treatments induced no effect on normal PDE cells, however, caused a decrease in the (i) proliferation, (ii) guanosine triphosphate (GTP)‐bound Ras protein, (iii) Akt phosphorylation and (iv) NF‐κB transcriptional activity of premalignant and malignant Kras‐activated PDE cells. Further, oral administration of CP (25 mg/kg) inhibited the growth of Kras‐PDE cell‐originated tumors in a xenograft mouse model. LC–MS/MS analysis of the blood showed epicatechin to be bioavailable to mice after CP consumption. We suggest that (i) Kras‐driven cell‐based model is an excellent model for testing intervention agents and (ii) CP is a promising chemopreventive agent for inhibiting PDAC development.

Quantitative analysis of trihydroxybutyl mercapturic acid, a urinary metabolite of 1, 3-butadiene, in humans

1,3-Butadiene (BD) is a known human carcinogen present in cigarette smoke and in automobile exhaust, leading to widespread exposure of human populations. BD requires cytochrome P450-mediated metabolic activation to electrophilic species, e.g. 3,4-epoxy-1-butene (EB), hydroxymethyl vinyl ketone (HMVK), and 3,4-epoxy-1,2-diol (EBD), which form covalent adducts with DNA. EB, HMVK, and EBD can be conjugated with glutathione and ultimately excreted in urine as monohydroxybutenyl mercapturic acid (MHBMA), dihydroxybutyl mercapturic acid (DHBMA), and trihydroxybutyl mercapturic acid (THBMA), respectively, which can serve as biomarkers of BD exposure and metabolic processing. While MHBMA and DHBMA have been found in smokers and nonsmokers, THBMA has not been previously detected in humans. In the present work, an isotope dilution HPLC-ESI(-)-MS/MS methodology was developed and employed to quantify THBMA in urine of known smokers and nonsmokers (19-27 per group). The new method has excellent sensitivity (LOQ, 1 ng/mL urine) and achieves accurate quantitation using a small sample volume (100 μL). Mean urinary THBMA concentrations in smokers and nonsmokers were found to be 21.6 and 13.7 ng/mg creatinine, respectively, suggesting that there are sources of THBMA other than exposure to tobacco smoke in humans, as is also the case for DHBMA. However, THBMA concentrations are significantly greater in urine of smokers than that of nonsmokers (p < 0.01). Furthermore, THBMA amounts in human urine declined 25-50% following smoking cessation, suggesting that smoking is an important source of this metabolite in humans. The HPLC-ESI(-)-MS/MS methodology developed in the present work will be useful for future epidemiological studies of BD exposure and metabolism.

Endogenous cytosine methylation and the formation of carcinogen carcinogen–DNA adducts

All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine ((Me)C, X = Me in Scheme 1). The same sites (e.g. p53 codons 157, 158, 245, 248, and 273) are mutational hotspots in smoking induced lung cancer, suggesting that methylated CG dinucleotides may be preferentially targeted by the reactive metabolites of tobacco carcinogens. We employed a stable isotope labeling HPLC-ESI-MS/MS approach to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diolepoxide metabolites of bay region polycyclic aromatic hydrocarbons, e.g. benzo[a]pyrene diol epoxide (BPDE). In contrast, cytosine methylation was protective against O(6)-guanine alkylation by tobacco tobacco-specific nitrosamines, e.g. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), To investigate the mechanisms behind these effects, a series of structural analogs of (Me)C were prepared, and their effects on reactivity of base the paired dG towards BPDE was examined. We found that the presence of the C-5 substituent on cytosine influences the reactivity of its partner guanine towards BPDE and modifies the stereoisomeric composition of the resulting N(2)-BPDE-dG adducts.

Comparative metabolism of furan in rodent and human cryopreserved hepatocytes.

Furan is a liver toxicant and carcinogen in rodents. Although humans are most likely exposed to furan through a variety of sources, the effect of furan exposure on human health is still unknown. In rodents, furan requires metabolism to exert its toxic effects. The initial product of the cytochrome P450 2E1-catalyzed oxidation is a reactive α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). BDA is toxic and mutagenic and consequently is considered responsible for the toxic effects of furan. The urinary metabolites of furan in rats are derived from the reaction of BDA with cellular nucleophiles, and precursors to these metabolites are detected in furan-exposed hepatocytes. Many of these precursors are 2-(S-glutathionyl)butanedial-amine cross-links in which the amines are amino acids and polyamines. Because these metabolites are derived from the reaction of BDA with cellular nucleophiles, their levels are a measure of the internal dose of this reactive metabolite. To compare the ability of human hepatocytes to convert furan to the same metabolites as rodent hepatocytes, furan was incubated with cryopreserved human and rodent hepatocytes. A semiquantitative liquid chromatography with tandem mass spectrometry assay was developed for a number of the previously characterized furan metabolites. Qualitative and semiquantitative analysis of the metabolites demonstrated that furan is metabolized in a similar manner in all three species. These results indicate that humans may be susceptible to the toxic effects of furan.

High throughput HPLC–ESI−-MS/MS methodology for mercapturic acid metabolites of 1,3-butadiene: Biomarkers of exposure and bioactivation

1,3-Butadiene (BD) is an important industrial and environmental carcinogen present in cigarette smoke, automobile exhaust, and urban air. The major urinary metabolites of BD in humans are 2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene/1-(N-acetyl-L-cystein-S-yl)-2-hydroxybut-3-ene (MHBMA), 4-(N-acetyl-L-cystein-S-yl)-1,2-dihydroxybutane (DHBMA), and 4-(N-acetyl-L-cystein-S-yl)-1,2,3-trihydroxybutyl mercapturic acid (THBMA), which are formed from the electrophilic metabolites of BD, 3,4-epoxy-1-butene (EB), hydroxymethyl vinyl ketone (HMVK), and 3,4-epoxy-1,2-diol (EBD), respectively. In the present work, a sensitive high-throughput HPLC-ESI(-)-MS/MS method was developed for simultaneous quantification of MHBMA and DHBMA in small volumes of human urine (200 μl). The method employs a 96 well Oasis HLB SPE enrichment step, followed by isotope dilution HPLC-ESI(-)-MS/MS analysis on a triple quadrupole mass spectrometer. The validated method was used to quantify MHBMA and DHBMA in urine of workers from a BD monomer and styrene-butadiene rubber production facility (40 controls and 32 occupationally exposed to BD). Urinary THBMA concentrations were also determined in the same samples. The concentrations of all three BD-mercapturic acids and the metabolic ratio (MHBMA/(MHBMA+DHBMA+THBMA)) were significantly higher in the occupationally exposed group as compared to controls and correlated with BD exposure, with each other, and with BD-hemoglobin biomarkers. This improved high throughput methodology for MHBMA and DHBMA will be useful for future epidemiological studies in smokers and occupationally exposed workers.

Abstract 1697: A study on the reactivity of activated polycyclic aromatic hydrocarbons (PAH) with guanines base pared to C-5 substituted cytosines

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC All CG dinucleotides within exons 5-8 of the human p53 tumor suppressor gene contain endogenous 5-methylcytosine (MeC). Guanine residues within these sites (e.g. codons 157, 158, 245, 248, and 273) are the major mutational hotspots for smoking induced lung cancer, suggesting that MeC mediates the reactivity of neighboring guanine bases towards tobacco carcinogens. Previous studies have shown that the reactivity of a carcinogenic diol epoxide metabolite of the human carcinogen, benzo[a]pyrene (B[a]P), towards the exocyclic amino group of guanine, is increased when guanine is base paired to MeC as compared to unmethylated cytosine. In the present study, the structural basis for the enhanced reactivity of BPDE towards MeC: G base pairs was investigated using a stable isotope labeling approach and a series of MeC structural analogs, including 5-ethyl-dC, 5-propyl-dC, N4-ethyl-dC, 5-chloro-dC, 5-bromo-dC, 5-iodo-dC, 5-propynyl-dC, difluorotoluene, pyrrolo-dC, phenylpyrrolo-dC, and diaminonaphthyl-derived nucleoside. Synthetic DNA duplexes derived from the frequently mutated region of the p53 tumor suppressor gene (5′-CCCGGCACCCGC[15N3, 13C1-G]TCCGCG-3′, from exon 5) were prepared containing [15N3, 13C1]-labeled guanine opposite C, MeC, or nucleobase analogs. Circular dichroism (CD) and UV melting studies have shown that C-5 substituents on cytosine do not disrupt the structure and stability of the DNA duplex. Following treatment with (±)-anti-benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide [(±)-anti-BPDE], (-)-anti-benzo[a]pyrene-s-7,t-8-dihydrodiol-t-9,10-epoxide [(-)-anti-BPDE], or related PAH diol epoxides, 5-methyl chrysene diol epoxide, benzo[c]phenanthrene diol epoxide, benzo[g]chrysene diol epoxide, and benzo[a,l]pyrene diol epoxide, and enzymatic hydrolysis of the adducted DNA to 2′-deoxynucleosides, the amounts of stereoisomeric N2-guanine adducts formed at the labeled site were determined by capillary HPLC-ESI+-MS/MS. We found that the presence of 5-methylcytosine and nucleobases with extended aromatic systems increases the reactivity of the partner guanine towards BPDE and other PAH diolepoxides, while 5-fluoro-dC and 5-iodo-dC lead to a decreased reactivity. Furthermore, the presence of C-5-cytosine analog modifies the stereoisomeric composition of the resulting adducts. Low temperature fluorescence and molecular docking studies reveal that the presence of MeC and unnatural base analogs with extended aromatic systems facilitate the formation of the pre-covalent BPDE-DNA complexes which place BPDE in a favorable orientation for trans attack by the N2 position of guanine. These results provide a mechanistic insight into the origins of increased reactivity of PAHs towards MeCG dinucleotides. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1697.