Immunoassays for proteins alkylated by nicotine-derived N-nitrosamines

Polyclonal antibodies recognizing the pyridyloxobutyl (POB) moiety of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were produced in rabbits immunized either with POB-bovine albumin or POB-Sepharose. The POB intermediates necessary to modify the protein were generated by alkaline (pH 9.0) treatment of the synthetic precursor 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone. In a competitive enzyme linked immunoabsorbent assay (ELISA), 70 pmole NNK inhibited 50% of the binding of the anti-POB antibodies to POB-protein absorbed on microtiterplates. This 50% inhibition varied from 70 pmole to 200 nmole using a series of NNK analogues, depending on the integrity of the POB moiety. Immunological techniques initiated in this study detect NNK-protein conjugates or measure the quantity of POB groups liberated upon alkaline or acid treatment of NNK modified protein.     

白色念珠菌感染与口腔癌的关系

口腔癌是头颈部最常见的癌症之一,严重影响患者生存质量和生活水平。白色念珠菌是口腔中最常见的机会性致病真菌,当宿主免疫功能低下时表现出致病性,容易引起念珠菌感染。近年来研究发现白色念珠菌感染与口腔癌关系密切,本文对口腔癌患者白色念珠菌感染的流行病学特点,以及白色念珠菌感染对口腔癌发生发展的影响及其机制研究进行综述。通过回顾相关文献发现:口腔癌患者白色念珠菌感染风险增加,白色念珠菌感染可能通过损伤口腔上皮、产生致癌物质、触发慢性炎症及辅助性T细胞17免疫反应等机制促进口腔癌的发生发展。然而目前这些机制的研究仍比较表浅,缺乏充足的直接证据,未来仍需进行大量研究,以期进一步明确白色念珠菌的促癌机制,为防治口腔癌提供新思路。     

Dose-response study of the carcinogenicity of tobacco-specific N-nitrosamines in F344 rats

Summary Tobacco and tobacco smoke contain relatively high amounts of four tobacco-specific N-nitrosamines. Of these, N-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N-nitrosoanatabine (NAT) were bioassayed at three dose levels by subcutaneous injections into male and female F344 rats in 60 subdoses amounting in total to 9, 3, and 1 mmol/kg. Compared with the solvent control group (trioctanoin), both NNN and NNK induced significant numbers of tumors of the nasal cavity (P<0.01) at all three dose levels in both male and female rats. Significant numbers of tumors were also induced by NNK in the lung at all three dose levels and in the liver at the highest dose level (P<0.05). In addition to nasal tumors NNN also induced esophageal tumors at a significant rate in male rats at the high and medium dose levels and in female rats at the high level (P<0.05); NAT was inactive at the three doses tested. Bioassays at lower dose levels as well as biochemical studies are strongly indicated for NNN and NNK since these nitrosamines occur in relatively high amounts in both chewing tobacco and tobacco smoke.Abbreviations HPLC high performance liquid chromatography - NAB N-nitrosoanabasinc - NAT N-nitrosoanatabine - NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone - NNN N-nitrosonornicotine Dedicated to Professor Hermann Druckrey on the occasion of his 80th birthdayThis is No. XXVII of A Study of Tobacco Carcinogenesis. The study is supported by U.S. National Cancer Institute Grant CA-29580We thank Ms. Maria Nicholais and Mr. Joel Reinhardt from the Research Animal Facility for their excellent technical assistance     

Intake of Volatile N—nitrosamines and Their Ability to Exogenously Synthesize in the Diet oif Inhabitants from High—risk Area of Esophageal Cancer in Southern China

Objective Nan'ao County in Guandong Province is a high-risk area of esophageal cancer in Southern China. Of the suspected etiological factors in the environment, N-nitrosamines and their precursors have received the greatest attenfion. Methods Sixty samples of the diet ingested by the inhabitants were collected and detected for volatile N-nitrosamines and their precursors. Five Nnitrosamines detected by Gas Chromatography-Thermal Energy Analyzer were N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosopyrrolidine, N-nitrosopiperidine and N-ditrosomethyl-benzylamine. Results The average content of 5 volatile N-nitrosamines in the diet was 312.0 μg/kg (median). The daily intake of the nitrosamines was 286.5 μ tg/head/day. Only the ability to exogenously synthesize N-nitrosopiperidine was powerful among 5 volatile N-nitrosamines. By a computerized stepwise regression analysis and curve fitting, we studied the correlation among the nitrosamines, the precursors and the major food items in the samples. Conclusion It demonstrated that a relatively high content of volatile N-nitrosamines was present in the diet collected in the area.     

The mutagenicity of dialkyl nitrosamines in the salmonella plate assay

N-nitrosodimethylamine (DMN) is not mutagenic in the standard Salmonella plate incorporation assay (Ames test) in the presence of an in vitro metabolic activation system (S-9) derived from rat liver. When the S-9 was derived from Aroclor- or phenobarbital-induced mouse or hamster liver or from uninduced hamster liver, mutagenic activity was observed. Increasing the amount of S-9 above the usual maximum level of 50 μ1 per plate increased the mutagenic response. Similarly, the mutagenicity of N-nitrosodiethylamine (DEN) and N-nitrosodi(n-butyl)amine (DBN) was greater in the presence of hamster liver S-9 than when mouse or rat liver was used. Data are also presented indicating that the ability of rat liver S-9 to mediate the mutagenic activity of DMN in the “preincubation” assay is due to the fact that the various components are present in this assay at several times the concentrations attained in the standard plate incorporation assay.     

The molecular basis for induction of human cancers by tobacco specific nitrosamines

Cellular DNA damage that is misrepaired or not repaired, constitutes a necessary, although not sufficient prerequisite for induction of cancer. For carcinogenic oral snuffs with extremely high concentrations of tobacco specific nitrosamines (TSNA) the DNA adduct levels predicted from animal experiments exceed those found in "unexposed" individuals. On the other hand, and supported by extensive Swedish epidemiological data, no significant increase of TSNA-induced DNA damages can be anticipated in humans from the use of low-nitrosamine oral snuffs. The extrapolated adduct concentrations are orders of magnitude lower than those found in the corresponding human tissues, a discrepancy that is difficult to account for by species differences. Furthermore, in exposed subjects the observed increment in the background levels of pyridyloxobutyl(POB)-hemoglobin adducts - a relevant indicator for TSNA activation - lie in a range predicted by rodent data. When based on the same type of tissues this provides justification for extrapolating rates of TSNA induced adduct formation from animals to humans. A TSNA exposure that does not affect the background level of pro-mutagenic DNA lesions should be considered as "virtually safe". The high background concentrations of methylated and POB-DNA adducts in "unexposed" humans must be ascribed to other sources than tobacco.     

Evaluation of the mutagenic effects of myosmine in human lymphocytes using the HPRT gene mutation assay

The minor tobacco alkaloid myosmine is implicated in DNA damage through pyridyloxobutylation similar to the tobacco-specific nitrosamines (TSNA). In contrast to TSNA, occurrence of myosmine is not restricted to tobacco. Myosmine is genotoxic to human cells in the comet assay. In this study, the mutagenic effect of myosmine was evaluated using the cloning hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation assay. Four hour exposure of isolated peripheral blood lymphocytes from 14 subjects homozygous for the Leu84 wild-type of the O⁶-methylguanine-DNA-methyltransferase (MGMT) gene to 1 mM of myosmine increased mutant frequency from 0.73 ± 0.58 × 10⁻⁶ in control to 1.14 ± 0.89 × 10⁻⁶ lymphocytes (P < 0.05). These new data further confirm the mutagenic effects of myosmine.     

Metabolism of a glucuronide conjugate of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in rats

 Besides 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), [4-(methylnitrosamino)-1-(3-pyridyl)but-1-yl]-β-O-d-glucosiduronic acid (NNAL-Glu) is another important metabolite of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) which has been detected in the urine of tobacco users and non-smokers heavily exposed to sidestream cigarette smoke. In order to evaluate the toxicological significance of NNAL-Glu formation and excretion, the metabolism of [5-³H]-NNAL-Glu was studied in rats. Five male F344 rats were administered 3.7 mg/kg [5-³H]-NNAL-Glu by i.v. injection and the metabolites in urine analysed by HPLC. More than 90% of the radioactivity was excreted in urine within the first 24 h. Unchanged NNAL-Glu accounted for 81.2±3.1% of the total radioactivity; the remaining part of the dose appears to be deconjugated resulting in the urinary excretion of NNAL (3.6±1.7%) and its α-hydroxylation (11.5±2.2%) and N-oxidation (3.6±1.6%) products. The presence of α-hydroxylation products of NNAL-Glu in urine suggests that this NNK metabolite may be activated in vivo to carcinogenic intermediates. Received: 25 April 1994 / Accepted: 29 June 1994     

Smokeless tobacco use and risk of cancer of the pancreas and other organs

Little information is available on the role of tobacco, alcohol and diet in the survival of upper aero digestive cancers. Our study analysed the survival of 931 laryngeal and hypopharyngeal cancer patients, enrolled in a population based case-control study conducted at 5 centres in southeast Europe during 1979-1982. Age at the time of diagnosis and site of origin of tumour were observed to be predictors of the survival. Cigarette smoking, and to a limited extent alcohol drinking, before the diagnosis of tumour seem to influence the overall survival whereas high intakes of vegetables and vitamin C were observed to favourably affect the prognosis. For mortality from upper aerodigestive cancer protective effects of high intakes of vegetables, fibres and vitamin C were observed. Our results support the hypothesis that there is a role for dietary intervention to improve survival of laryngeal and hypopharyngeal cancer patients.     

Chemical,physical, and in vitro characterization of research cigarettes containing denicotinized tobacco

The use of very low nicotine tobacco cigarettes is currently being investigated as a possible harm reduction strategy. Here, we report the smoke chemistry, toxicity, and physical characteristics of very low nicotine cigarettes that were made using blended tobacco processed through a supercritical CO₂ fluid extraction, which resulted in elimination of 96% of nicotine content (denicotinized (denic) tobacco). Three types of test cigarettes (TCs) were manufactured with tobacco filler containing 100% denic tobacco (TC100), 50% denic tobacco and 50% unextracted tobacco (TC50/50), and 100% unextracted tobacco (TC0). Mainstream smoke (MS) was generated for measurement of 46 analytes and cytotoxicity and mutagenicity determination. Analysis of physical characteristics of TCs demonstrated they were well made with <5% variability among cigarettes for most parameters measured. We observed significant changes in the levels of smoke constituents, including decreases in formaldehyde, nitrosamines, and phenol, and increases in aliphatic hydrocarbons, aliphatic nitrogen compounds, aromatic amines, halogen compounds, and metals. Use of denic tobacco resulted in changes in the chemical composition of MS, but these changes did not modify biological activity as measured in the mutagenicity and cytotoxicity assays.