DNA Adducts from <i>N-</i>Nitrosodiethanolamine and Related β-Oxidized Nitrosamines in Vivo: ³²P-Postlabeling Methods for Glyoxal- and <i>O</i>⁶-Hydroxyethyldeoxyguanosine Adducts

The mechanism by which environmentally prevalent N-nitrosodiethanolamine (NDELA) and related 2-hydroxyethyl- or other β-oxidized nitrosamines initiate the carcinogenic process has remained obscure. 32P-Postlabeling assays for the pH sensitive glyoxal-deoxyguanosine (gdG) and the O6-2-hydroxyethyldeoxyguanosine (OHEdG) DNA adducts have been developed as probes in this mechanistic investigation and used in both in vitro and in vivo experiments. The ready cleavage of the glyoxal fragment from gdG at pH 7 and greater has required methods of optimization in order to achieve a detection limit of 0.05 μmol/mol of DNA. Nuclease P1 treatment enhances the detection of gdG adducts but does not increase the detection limit for OHEdG. For OHEdG, best results were achieved using fraction collection from HPLC (0.3 μmol/mol of DNA). Using radiochemical methods, both adducts could be detected either by HPLC or 2D TLC. NDELA, N-nitrosomorpholine (NMOR), N-nitrosomethyethanolamine (NMELA), and N-nitrosoethylethanolamine (NEELA) all produce both gdG and OHEdG adducts in rat liver DNA in vivo and are called bident carcinogens because fragments from both chains of the nitrosamine are incorporated into DNA. N-Nitroso-2-hydroxymorpholine (NHMOR), a metabolite of NDELA and NMOR, generates gdG in DNA in vitro and in vivo. gdG DNA adducts were found in the range 1.1−6.5 μmol/mol of DNA. OHEdG DNA adducts were produced from equimolar amounts of nitrosamines in rat liver in vivo over the range 4−25 μmol/mol of DNA and in the order NMELA > NEELA > NDELA > NMOR. Deuterated isotopomers of NDELA showed a marked isotope effect on DNA OHEdG adduct formation. α-Deuteration markedly decreased OHEdG adduct formation while β-deuteration had the opposite effect. These data support the hypothesis that NDELA and related nitrosamines are activated by both enzyme mediated α-hydroxylation and β-oxidation. The formation of OHEdG adducts from NDELA requires α-hydroxylation of the 2-hydroxyethyl chain, and formation of gdG necessitates a β-oxidation as well. The bident nature of these carcinogens may explain why they are relatively potent carcinogens despite the fact that major proportions of doses are excreted unchanged

DNA Guanine Adducts from 3-Methyl-1,2,3-oxadiazolinium Ions

The reaction of 3-methyl-1,2,3-oxadiazolinium tosylate 10, a close model for a putative reactive intermediate in the carcinogenic activation of ethanol nitrosamines such as (2-hydroxyethyl)methylnitrosamine 1, with various guanine derivatives, including acycloguanosine 12, deoxyguanosine, deoxyguanosine monophosphate, and cyclic guanosine monophosphate, various DNA oligomers, and calf-thymus DNA has been examined to determine whether this compound methylates and hydroxyethylates guanine residues as proposed. In all of the transformations, 7-(2-(methylnitrosamino)ethyl)guanine (14) is the major product, following acidic hydrolysis, and exceeds the formation of 7-methylguanine by ratios ranging from 4:1 to 48:1, depending upon the guanine bearing substrate. O6-(2-(Methylnitrosamino)ethyl)deoxyguanosine (20) was prepared from the Mitsunobu coupling of 1 and a protected deoxyguanosine derivative. 20 is not produced in the reaction of 10 and deoxyguanosine and decomposes to 1 and guanine upon mild acid treatment, suggesting possible neighboring group participation in its facile hydrolytic cleavage. All of the major products from the reaction of 10 and 12 have been characterized, including the direct alkylation product, 7-(2-(methylnitrosamino)ethyl)acycloguanosine (13), and N2-(2-(methylnitrosamino)ethyl)guanine, which was independently synthesized. Elucidation of the reactions of DNA with 10 and other electrophiles was facilitated by the development of both partial and total enzymatic hydrolysis assays utilizing 32P-5‘-labeled DNA oligotetramers containing one of each base type and HPLC with radiometric detection. The partial hydrolysis assay gives information as to the type of base being modified, and the total hydrolysis assay permits a determination of the number of adducts produced for a given base. The assays permit a comparison between reactions where the same type of base adduct could be expected. Comparisons of the reactions of ethylene oxide and 10 using this methodology showed that 10 does not hydroxyethylate guanine in DNA

Additional file 1 of A causal relationship between panic disorder and risk of alzheimer disease: a two-sample mendelian randomization analysis

Supplementary Material

DataSheet_1_Comprehensive assessment of HF-rTMS treatment mechanism for post-stroke dysphagia in rats by integration of fecal metabolomics and 16S rRNA sequencing.docx

BackgroundThe mechanism by which high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) improves swallowing function by regulating intestinal flora remains unexplored. We aimed to evaluate this using fecal metabolomics and 16S rRNA sequencing.MethodsA Post-stroke dysphagia (PSD) rat model was established by middle cerebral artery occlusion. The magnetic stimulation group received HF-rTMS from the 7th day post-operation up to 14th day post-surgery. Swallowing function was assessed using a videofluoroscopic swallowing study (VFSS). Hematoxylin-eosin (H&E) staining was used to assess histopathological changes in the intestinal tissue. Intestinal flora levels were evaluated by sequencing the 16S rRNA V3-V4 region. Metabolite changes within the intestinal flora were evaluated by fecal metabolomics using liquid chromatography-tandem mass spectrometry.ResultsVFSS showed that the bolus area and pharyngeal bolus speed were significantly decreased in PSD rats, while the bolus area increased and pharyngeal transit time decreased after HF-rTMS administration (p ConclusionThese findings suggest a synergistic role for the gut microbiota and fecal metabolites in the development of PSD and the therapeutic mechanisms underlying HF-rTMS.</p

Hydrophilic, Pro-Drug Analogues of T138067 Are Efficacious in Controlling Tumor Growth In Vivo and Show a Decreased Ability To Cross the Blood Brain Barrier

The novel anticancer compound T138067 is an irreversible inhibitor of tubulin polymerization. Amides 3−6 were synthesized using standard methodologies and determined to be significantly less lipophilic than T138067 based on logP calculations. Tubulin polymerization and [3H]-T138067 competition assays revealed that these amides are pro-drugs for parent aniline 2. Amides 3−5 showed no detectable signs of crossing the blood brain barrier, while amide 6 was found in extremely small amounts (12 ng/g of brain tissue). Aniline 2, which was formed in vivo from these amides, was found in significantly smaller amounts (approximately 20 to >5000 times) in the brain than when 2 was administered directly. The in vivo efficacy of amide 6 approached that of T138067 and was better tolerated when administered to athymic nude mice bearing MX-1 human mammary tumor xenografts

Synthesis, in Vitro Covalent Binding Evaluation, and Metabolism of ¹⁴C‑Labeled Inhibitors of 11β-HSD1

In this letter, we reported the design and synthesis of three potent, selective, and orally bioavailable 11β-HSD1 inhibitors labeled with ¹⁴C: AMG 456 (<b>1</b>), AM-6949 (<b>2</b>), and AM-7715 (<b>3</b>). We evaluated the covalent protein binding of the labeled inhibitors in human liver microsomes in vitro and assessed their potential bioactivation risk in humans. We then studied the in vitro mechanism of <b>2</b> in human hepatocytes and the formation of reactive intermediates. Our study results suggest that <b>1</b> and <b>3</b> have low potential for metabolic bioactivation in humans, while <b>2</b> has relatively high risk

Figure S2 from CD47-blocking Antibody ZL-1201 Promotes Tumor-associated Macrophage Phagocytic Activity and Enhances the Efficacy of the Therapeutic Antibodies and Chemotherapy

CD47 expression and phagocytosis by ZL-1201</p

Figure S4 from CD47-blocking Antibody ZL-1201 Promotes Tumor-associated Macrophage Phagocytic Activity and Enhances the Efficacy of the Therapeutic Antibodies and Chemotherapy

Fc-dependent synergy of ZL-1201</p

Figure S3 from CD47-blocking Antibody ZL-1201 Promotes Tumor-associated Macrophage Phagocytic Activity and Enhances the Efficacy of the Therapeutic Antibodies and Chemotherapy

In vitro phagocytosis by ZL-1201</p

Figure S1 from CD47-blocking Antibody ZL-1201 Promotes Tumor-associated Macrophage Phagocytic Activity and Enhances the Efficacy of the Therapeutic Antibodies and Chemotherapy

ZL-1201 binding to CD47</p