Identification of adducts formed in the reactions of 5'-acetoxy-N'-nitrosonornicotine with deoxyadenosine, thymidine, and DNA

N'-Nitrosonornicotine (NNN) is the most prevalent of the carcinogenic tobacco-specific nitrosamines found in all tobacco products. Previous studies have demonstrated that cytochrome P450-mediated 5'-hydroxylation of NNN is a major metabolic pathway leading to mutagenic products, but to date, DNA adducts formed by this pathway have been only partially characterized, and there have been no studies reported on adducts formed with bases other than dGuo. Because adducts with dAdo and dThd have been identified in the DNA of the livers of rats treated with the structurally related carcinogen N-nitrosopyrrolidine, we investigated dAdo and dThd adduct formation from 5'-acetoxyNNN (3), a stable precursor to 5'-hydroxyNNN (2). Reaction of 3 with dAdo gave diastereomeric products, which were identified by their spectral properties and LC-ESI-MS/MS-SRM analysis as N(6)-[5-(3-pyridyl)tetrahydrofuran-2-yl]dAdo (9). This adduct was further characterized by NaBH(3)CN reduction to N(6)-[4-hydroxy-4-(3-pyridyl)but-1-yl]dAdo (17). A second dAdo adduct was identified, after NaBH(3)CN treatment, as 6-[2-(3-pyridyl)pyrrolidin-1-yl]purine-2'-deoxyriboside (18). Reaction of 3 with dThd, followed by NaBH(3)CN reduction, gave O(2)-[4-(3-pyridyl)-4-hydroxybut-1-yl]thymidine (11). Adducts 9, 11, 17, and 18 were all identified by LC-ESI-MS/MS-SRM comparison to synthetic standards. The reaction of 3 with calf thymus DNA was then investigated. The DNA was enzymatically hydrolyzed to deoxyribonucleosides, and the resulting mixture was treated with NaBH(3)CN and analyzed by LC-ESI-MS/MS-SRM. Adducts 11, 17, and 18, as well as the previously identified dGuo adducts, were identified. The results of this study provide a more comprehensive picture of DNA adduct formation by the quantitatively important 5'-hydroxylation pathway of NNN and will facilitate investigation of the presence of these adducts in laboratory animals treated with NNN or in people who use tobacco products.     

The 5-HT1-like receptors mediating inhibition of sympathetic vasopressor outflow in the pithed rat: operational correlation with the 5-HT1A, 5-HT1B and 5-HT1D subtypes

1. It has been suggested that the inhibition of sympathetically-induced vasopressor responses produced by 5-hydroxytryptamine (5-HT) in pithed rats is mediated by 5-HT₁-like receptors. The present study has re-analysed this suggestion with regard to the classification schemes recently proposed by the NC-IUPHAR subcommittee on 5-HT receptors.
2. Intravenous (i.v.) continuous infusions of 5-HT and the 5-HT₁ receptor agonists, 8-OH-DPAT (5-HT_1A), indorenate (5-HT_1A), CP 93,129 (5-HT_1B) and sumatriptan (5-HT_1B/1D), resulted in a dose-dependent inhibition of sympathetically-induced vasopressor responses.
3. The sympatho-inhibitory responses induced by 5-HT, 8-OH-DPAT, indorenate, CP 93,129 or sumatriptan were analysed before and after i.v. treatment with blocking doses of the putative 5-HT receptor antagonists, WAY 100635 (5-HT_1A), cyanopindolol (5-HT_1A/1B) or GR 127935 (5-HT_1B/1D). Thus, after WAY 100635, the responses to 5-HT and indorenate, but not to 8-OH-DPAT, CP 93,129 and sumatriptan, were blocked. After cyanopindolol, the responses to 5-HT, indorenate and CP 93,129 were abolished, whilst those to 8-OH-DPAT and sumatriptan (except at the lowest frequency of stimulation) remained unaltered. In contrast, after GR 127935, the responses to 5-HT, CP 93,129 and sumatriptan, but not to 8-OH-DPAT and indorenate, were abolished.
4. In additional experiments, the inhibition induced by 5-HT was not modified after 5-HT₇ receptor blocking doses of mesulergine.
5. The above results suggest that the 5-HT₁-like receptors, which inhibit the sympathetic vasopressor outflow in pithed rats, display the pharmacological profile of the 5-HT_1A, 5-HT_1B and 5-HT_1D, but not that of 5-HT₇, receptors.

     

BRCA1 alterations are associated with endometriosis,but BRCA2 alterations show no detectable endometriosis risk: a study in Indian population

Purpose

To investigate the role of genetic variations and expression alterations of BRCA1 and BRCA2 genes in the pathophysiology of endometriosis.

Methods

A genetic association study was conducted in 573 endometriosis cases and 490 controls of Indian origin. We genotyped 13 selected promoter SNPs of BRCA1 gene and 2 selected promoter SNPs of BRCA2 gene by PCR-sequencing analysis. In addition, to better understand genetic contributions to the pathophysiology of endometriosis, the expression pattern of BRCA1 & 2 was analyzed in the eutopic endometria of endometriosis cases and controls by western-blot and immunohistochemical analysis.

Results

Our results revealed significant association between BRCA1 rs71361504 (−/GTT) SNP and endometriosis risk in Indian women (P < 0.0001), while the remaining SNPs of both BRCA1 & 2 genes showed no difference between cases and controls. Western-blot and immunohistochemical analysis revealed significantly decreased BRCA1 expression levels in eutopic endometria of patients compared with controls (P < 0.05). Furthermore, nuclear BRCA1 was frequently lost compared with cytoplasmic BRCA1 in eutopic endometria of patients. Expression of BRCA2 did not differ between patients and controls.

Conclusions

BRCA1 rs71361504 SNP may modify the endometriosis risk in Indian women. In addition, decreased expression of BRCA1 may play an important role in the pathophysiology of endometriosis. The analysis of BRCA1 genetic variants and/or expression might help to identify patients at high risk for disease outcome.

Electronic supplementary material

The online version of this article (doi:10.1007/s10815-014-0379-9) contains supplementary material, which is available to authorized users.     

Extra adrenal pheochromocytoma of urinary bladder

Pheochromocytoma of urinary bladder is rare disease causing a hypertension, palpitation, throbbing headache & hemorrhage induced by voiding. This case is reported because of rarity of this disease in urinary bladder.     

Heat Shock Protein–Peptide Complexes, Reconstituted In Vitro, Elicit Peptide-specific Cytotoxic T Lymphocyte Response and Tumor Immunity

Heat shock protein (HSP) preparations derived from cancer cells and virus-infected cells have been shown previously to elicit cancer-specific or virus-specific immunity. The immunogenicity of HSP preparations has been attributed to peptides associated with the HSPs. The studies reported here demonstrate that immunogenic HSP–peptide complexes can also be reconstituted in vitro. The studies show that (a) complexes of hsp70 or gp96 HSP molecules with a variety of synthetic peptides can be generated in vitro; (b) the binding of HSPs with peptides is specific in that a number of other proteins tested do not bind synthetic peptides under the conditions in which gp96 molecules do; (c) HSP–peptide complexes reconstituted in vitro are immunologically active, as tested by their ability to elicit antitumor immunity and specific CD8⁺ cytolytic T lymphocyte response; and (d) synthetic peptides reconstituted in vitro with gp96 are capable of being taken up and re-presented by macrophage in the same manner as gp96– peptides complexes generated in vivo. These observations demonstrate that HSPs are CD8⁺ T cell response–eliciting adjuvants.     

Intrathyroidal thymic tissue in children: Avoiding unnecessary surgery

Background/Purpose

Intrathyroidal thymic tissue may be misinterpreted as a thyroid lesion in children, leading to invasive tests or resection. We sought to describe the characteristic imaging features of these lesions and to evaluate the safety of non-operative management.