Identification of 4‑(3-Pyridyl)-4-oxobutyl-2′-deoxycytidine Adducts Formed in the Reaction of DNA with 4‑(Acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone: A Chemically Activated Form of Tobacco-Specific Carcinogens

[Image: see text] Metabolic activation of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1) and N′-nitrosonornicotine (NNN, 2) results in the formation of 4-(3-pyridyl)-4-oxobutyl (POB)-DNA adducts, several of which have been previously identified both in vitro and in tissues of laboratory animals treated with NNK or NNN. However, 2′-deoxycytidine adducts formed in this process have been incompletely examined in previous studies. Therefore, in this study we prepared characterized standards for the identification of previously unknown 2′-deoxycytidine and 2′-deoxyuridine adducts that could be produced in these reactions. The formation of these products in reactions of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc, 3), a model 4-(3-pyridyl)-4-oxobutylating agent, with DNA was investigated. The major 2′-deoxycytidine adduct, identified as its stable cytosine analogue O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]-cytosine (12), was O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxycytidine (13), whereas lesser amounts of 3-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxycytidine (14) and N(4)-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxycytidine (15) were also observed. The potential conversion of relatively unstable 2′-deoxycytidine adducts to stable 2′-deoxyuridine adducts by treatment of the adducted DNA with bisulfite was also investigated, but the harsh conditions associated with this approach prevented quantitation. The results of this study provide new validated standards for the study of 4-(3-pyridyl)-4-oxobutylation of DNA, a critical reaction in the carcinogenesis by 1 and 2, and demonstrate the presence of previously unidentified 2′-deoxycytidine adducts in this DNA

DNA Adduct Formation from Metabolic 5′-Hydroxylation of the Tobacco-Specific Carcinogen <i>N</i>′‑Nitrosonornicotine in Human Enzyme Systems and in Rats

<i>N</i>′-Nitrosonornicotine (NNN) is carcinogenic in multiple animal models and has been evaluated as a human carcinogen. NNN can be metabolized by cytochrome P450s through two activation pathways: 2′-hydroxylation and 5′-hydroxylation. While most previous studies have focused on 2′-hydroxylation in target tissues of rats, available evidence suggests that 5′-hydroxylation is a major activation pathway in human enzyme systems, in nonhuman primates, and in target tissues of some other rodent carcinogenicity models. In the study reported here, we investigated DNA damage resulting from NNN 5′-hydroxylation by quantifying the adduct 2-(2-(3-pyridyl)-<i>N</i>-pyrrolidinyl)-2′-deoxyinosine (py-py-dI). In rats treated with NNN in the drinking water (7–500 ppm), py-py-dI was the major DNA adduct resulting from 5′-hydroxylation of NNN <i>in vivo</i>. Levels of py-py-dI in the lung and nasal cavity were the highest, consistent with the tissue distribution of CYP2A3. In rats treated with (<i>S</i>)-NNN or (<i>R</i>)-NNN, the ratios of formation of (<i>R</i>)-py-py-dI to (<i>S</i>)-py-py-dI were not the expected mirror image, suggesting that there may be a carrier for one of the unstable intermediates formed upon 5′-hydroxylation of NNN. Rat hepatocytes treated with (<i>S</i>)- or (<i>R</i>)-NNN or (2′<i>S</i>)- or (2′<i>R</i>)-5′-acetoxyNNN exhibited a pattern of adduct formation similar to that of live rats. <i>In vitro</i> studies with human liver S9 fraction or human hepatocytes incubated with NNN (2–500 μM) demonstrated that py-py-dI formation was greater than the formation of pyridyloxobutyl-DNA adducts resulting from 2′-hydroxylation of NNN. (<i>S</i>)-NNN formed more total py-py-dI adducts than (<i>R</i>)-NNN in human liver enzyme systems, which is consistent with the critical role of CYP2A6 in the 5′-hydroxylation of NNN in human liver. The results of this study demonstrate that the major DNA adduct resulting from NNN metabolism by human enzymes is py-py-dI and provide potentially important new insights into the metabolic activation of NNN in rodents and humans

Developing a Biased Unmatched Bivalent Ligand (BUmBL) Design Strategy to Target the GPCR Homodimer Allosteric Signaling (cAMP over β-Arrestin 2 Recruitment) Within the Melanocortin Receptors.

Understanding the functional relevance of G protein-coupled receptor (GPCR) homodimerization has been limited by the insufficient tools to assess asymmetric signaling occurring within dimers comprised of the same receptor type. We present unmatched bivalent ligands (UmBLs) to study the asymmetric function of melanocortin homodimers. UmBLs contain one agonist and one antagonist pharmacophore designed to target a melanocortin homodimer such that one receptor is occupied by an agonist and the other receptor by an antagonist pharmacophore. First-in-class biased UmBLs (BUmBLs) targeting the human melanocortin-4 receptor (hMC4R) were discovered. The BUmBLs displayed biased agonism by potently stimulating cAMP signaling (E

A Direct in Vivo Comparison of the Melanocortin Monovalent Agonist Ac-His-DPhe-Arg-Trp-NH2 versus the Bivalent Agonist Ac-His-DPhe-Arg-Trp-PEDG20-His-DPhe-Arg-Trp-NH2: A Bivalent Advantage

Bivalent ligands targeting putative melanocortin receptor dimers have been developed and characterized in vitro; however, studies of their functional in vivo effects have been limited. The current report compares the effects of homobivalent ligand CJL-1-87, Ac-His-DPhe-Arg-Trp-PEDG20-His-DPhe-Arg-Trp-N

Comprehensive High-Resolution Mass Spectrometric Analysis of DNA Phosphate Adducts Formed by the Tobacco-Specific Lung Carcinogen 4‑(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, <b>1</b>) is a potent lung carcinogen in laboratory animals and is believed to play a key role in the development of lung cancer in smokers. Metabolic activation of NNK leads to the formation of pyridyloxobutyl DNA adducts, a critical step in its mechanism of carcinogenesis. In addition to DNA nucleobase adducts, DNA phosphate adducts can be formed by pyridyloxobutylation of the oxygen atoms of the internucleotidic phosphodiester linkages. We report the use of a liquid chromatography–nanoelectrospray ionization–high-resolution tandem mass spectrometry technique to characterize 30 novel pyridyloxobutyl DNA phosphate adducts in calf thymus DNA (CT-DNA) treated with 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc, <b>2</b>), a regiochemically activated form of NNK. A ¹⁵N₃-labeled internal standard was synthesized for one of the most abundant phosphate adducts, dCp­[4-oxo-4-(3-pyridyl)­butyl]­dC (CpopC), and this standard was used to quantify CpopC and to estimate the levels of other adducts in the NNKOAc-treated CT-DNA. Formation of DNA phosphate adducts by NNK <i>in vivo</i> was further investigated in rats treated with NNK acutely (0.1 mmol/kg once daily for 4 days by subcutaneous injection) and chronically (5 ppm in drinking water for 10, 30, 50, and 70 weeks). This study provides the first comprehensive structural identification and quantitation of a panel of DNA phosphate adducts of a structurally complex carcinogen and chemical support for future mechanistic studies of tobacco carcinogenesis in humans

Methyl DNA Phosphate Adduct Formation in Rats Treated Chronically with 4‑(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and Enantiomers of Its Metabolite 4‑(Methylnitrosamino)-1-(3-pyridyl)-1-butanol

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a powerful lung carcinogen in animal models and is considered a causative factor for lung cancer in tobacco users. NNK is stereoselectively and reversibly metabolized to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which is also a lung carcinogen. Both NNK and NNAL undergo metabolic activation by α-hydroxylation on their methyl groups to form pyridyloxobutyl and pyridylhydroxybutyl DNA base and phosphate adducts, respectively. α-Hydroxylation also occurs on the α-methylene carbons of NNK and NNAL to produce methane diazohydroxide, which reacts with DNA to form methyl DNA base adducts. DNA adducts of NNK and NNAL are important in their mechanisms of carcinogenesis. In this study, we characterized and quantified methyl DNA phosphate adducts in the lung of rats treated with 5 ppm of NNK, (<i>S</i>)-NNAL, or (<i>R</i>)-NNAL in drinking water for 10, 30, 50, and 70 weeks, by using a novel liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method. A total of 23, 21, and 22 out of 32 possible methyl DNA phosphate adducts were detected in the lung tissues of rats treated with NNK, (<i>S</i>)-NNAL, and (<i>R</i>)-NNAL, respectively. Levels of the methyl DNA phosphate adducts were 2290–4510, 872–1120, and 763–1430 fmol/mg DNA, accounting for 15–38%, 8%, and 5–9% of the total measured DNA adducts in rats treated with NNK, (<i>S</i>)-NNAL, and (<i>R</i>)-NNAL, respectively. The methyl DNA phosphate adducts characterized in this study further enriched the diversity of DNA adducts formed by NNK and NNAL. These results provide important new data regarding NNK- and NNAL-derived DNA damage and new insights pertinent to future mechanistic and biomonitoring studies of NNK, NNAL, and other chemical methylating agents

A Direct in Vivo Comparison of the Melanocortin Monovalent Agonist Ac-His-DPhe-Arg-Trp-NH₂ versus the Bivalent Agonist Ac-His-DPhe-Arg-Trp-PEDG20-His-DPhe-Arg-Trp-NH₂: A Bivalent Advantage

Bivalent ligands targeting putative melanocortin receptor dimers have been developed and characterized in vitro; however, studies of their functional in vivo effects have been limited. The current report compares the effects of homobivalent ligand CJL-1-87, Ac-His-DPhe-Arg-Trp-PEDG20-His-DPhe-Arg-Trp-NH₂, to monovalent ligand CJL-1-14, Ac-His-DPhe-Arg-Trp-NH₂, on energy homeostasis in mice after central intracerebroventricular (ICV) administration into the lateral ventricle of the brain. Bivalent ligand CJL-1-87 had noteworthy advantages as an antiobesity probe over CJL-1-14 in a fasting-refeeding in vivo paradigm. Treatment with CJL-1-87 significantly decreased food intake compared to CJL-1-14 or saline (50% less intake 2–8 h after treatment). Furthermore, CJL-1-87 treatment decreased the respiratory exchange ratio (RER) without changing the energy expenditure indicating that fats were being burned as the primary fuel source. Additionally, CJL-1-87 treatment significantly lowered body fat mass percentage 6 h after administration (<i>p</i> < 0.05) without changing the lean mass percentage. The bivalent ligand significantly decreased insulin, C-peptide, leptin, GIP, and resistin plasma levels compared to levels after CJL-1-14 or saline treatments. Alternatively, ghrelin plasma levels were significantly increased. Serum stability of CJL-1-87 and CJL-1-14 (<i>T</i>_1/2 = 6.0 and 16.8 h, respectively) was sufficient to permit physiological effects. The differences in binding affinity of CJL-1-14 compared to CJL-1-87 are speculated as a possible mechanism for the bivalent ligand’s unique effects. We also provide in vitro evidence for the formation of a MC3R-MC4R heterodimer complex, for the first time to our knowledge, that may be an unexploited neuronal molecular target. Regardless of the exact mechanism, the advantageous ability of CJL-1-87 compared to CJL-1-14 to increase in vitro binding affinity, increase the duration of action in spite of decreased serum stability, decrease in vivo food intake, decrease mice’s body fat percent, and differentially affect mouse hormone levels demonstrates the distinct characteristics achieved from the current melanocortin agonist bivalent design strategy

Benzene uptake in Hookah smokers and non-smokers attending Hookah social events: regulatory implications.

BackgroundBenzene is a human hematotoxicant and a leukemogen that causes lymphohematopoietic cancers, especially acute myelogenous leukemia. We investigated uptake of benzene in hookah smokers and non-smokers attending hookah social events in naturalistic settings where hookah tobacco was smoked exclusively.MethodsWe quantified S-phenylmercapturic acid (SPMA), a metabolite of benzene, in the urine of 105 hookah smokers and 103 non-smokers. Participants provided spot urine samples the morning of and the morning after attending an indoor hookah-only smoking social event at a hookah lounge or in a private home.ResultsUrinary SPMA levels in hookah smokers increased significantly following a hookah social event (P &lt; 0.001). This increase was 4.2 times higher after hookah lounge events (P &lt; 0.001) and 1.9 times higher after home events (P = 0.003). In non-smokers, urinary SPMA levels increased 2.6 times after hookah lounge events (P = 0.055); however, similar urinary SPMA levels were detected before and after home events, possibly indicating chronic exposure to benzene (P = 0.933).ConclusionsOur data provide the first evidence for uptake of benzene in hookah smokers and non-smokers exposed to hookah tobacco secondhand smoke at social events in private homes compared with their counterparts in hookah lounges. Hookah tobacco smoke is a source of benzene exposure, a risk factor for leukemia.ImpactBecause there is no safe level of exposure to benzene, our results call for interventions to reduce or prevent hookah tobacco use, regulatory actions to limit hookah-related exposure to toxicants including benzene, initiate labeling of hookah-related products, and include hookah smoking in clean indoor air legislation