Safety assessment of diammonium phosphate and urea used in the manufacture of cigarettes

A tiered testing strategy has been employed to evaluate the potential for new ingredients, tobacco processes, and technological developments to alter the mainstream smoke or biological activity that results from burning cigarette tobacco. The foundation of this evaluation strategy is comparative testing, typically including chemical and biological assessments. In the manufacture of cigarettes, diammonium phosphate (DAP) and urea have been historically used as ingredients added to tobacco, to reconstituted tobacco sheet, and to other processed tobaccos. As part of ongoing stewardship efforts, a toxicological assessment of cigarettes with and without DAP and urea was conducted. Chemical and biological analyses were conducted for test cigarettes added 0.5% DAP and 0.2% urea in the final blend and also for those added 1.0% DAP and 0.41% urea in the final blend compared to reference cigarettes without added DAP or urea. Principal components of this evaluation included a determination of selected mainstream smoke constituent yields, an Ames assay in Salmonella typhimurium strains TA98 and TA100, a sister chromatid exchange assay in Chinese hamster ovary cells, a 13-week inhalation study of mainstream cigarette smoke in Sprague–Dawley rats, and a 30-week dermal tumor-promotion evaluation of mainstream cigarette smoke condensate in SENCAR mice. Comparative evaluations demonstrated that the addition of DAP and urea to cigarettes at up to 1% and 0.41%, respectively, does not alter the biological activity compared to reference cigarettes without DAP or urea.     

Toxicological evaluation of cigarettes with two banded cigarette paper technologies

A tiered testing strategy has been employed to evaluate the potential of tobacco processes, ingredients, or technological developments to change the biological activity resulting from burning cigarette tobacco. The strategy is based on comparative chemical and biological testing. The introduction of banded cigarette papers in cigarettes to meet New York state "Fire Safety Standards for Cigarettes" constitutes an example of a technological development evaluated utilizing this tiered testing strategy that included a comparison of the chemical and biological effects of cigarettes with and without the banded cigarette paper technologies (BCPT) (representative of current marketed technologies). Specific testing included mainstream cigarette smoke chemistry studies; in vitro studies included genotoxicity (Ames and sister chromatid exchange) and cytotoxicity studies (neutral red); in vivo studies included a 13-week inhalation study in Sprague-Dawley rats and a 30-week dermal tumor promotion study in SENCAR mice. Collectively, data indicated that cigarettes with and without BCPT had a similar toxicological profile in this test battery.     

A summary of toxicological and chemical data relevant to the evaluation of cast sheet tobacco

A tiered testing strategy based on a comparative chemical and biological testing program has been developed to evaluate the potential of tobacco processes, ingredients, or other technological developments to change the biological activity that results from burning tobacco. Cast sheet tobacco is a specific type of reconstituted tobacco sheet that can be used in the manufacture of cigarettes. The comparative chemical and biological testing program was used to compare the mainstream smoke and cigarette smoke condensate (CSC) from a Reference cigarette that did not contain cast sheet to that collected from Test cigarettes containing cast sheet at a final blend level of either 10% or 15%. Testing included mainstream cigarette smoke chemistry studies, in vitro studies (Ames assay, sister chromatid exchange assay, and neutral red cytotoxicity assay), and in vivo toxicology studies (13-week rat nose-only inhalation assay and 30-week mouse dermal tumor promotion assay). Certain statistically significant differences were observed in the chemical and biological studies when the Reference cigarette was compared to each of the Test cigarettes. However, when viewed collectively, the chemical and biological studies demonstrated that inclusion of cast sheet up to 15% in the final blend did not increase the inherent biological activity of mainstream cigarette smoke or CSC.     

Selenium-mediated reduction in the mutagenicity of cigarette smoke

Cigarette smoke contains carcinogens and mutagens and affects the health of smokers. Recently, increased research has proven the potentially protective activity of selenium (Se) against heavy metal toxicity, cancer, and other health disorders. Accordingly, we have proposed the fortification of tobacco with Se to develop safer cigarettes. As a start in evaluating any biological effects of added Se, we have determined the mutagenicity of inhaled, mainstream (MS) cigarette smoke condensate (CSC), with and without Se, in the preincubation assay of the Ames test. Initially, it was shown that Se, as sodium selenite, was not mutagenic at high concentrations (up to 80 micrograms/plate) with strains TA1538 and TA1978. Subsequently, the effects of different levels of Se, added to MS CSC, were examined with TA98, TA100, and TA1538. On the average, addition of 10 micrograms Se produced mutagenicity reductions of about 50%. Higher levels of added Se yielded further reductions. Cigarette sidestream (SS) smoke, collected between puffs, was also tested. Again, Se added to SS-CSC gave similar reductions, confirming its antimutagenic effect for both mainstream and sidestream smoke.     

A modified Ames assay reveals the mutagenicity of native cigarette mainstream smoke and its gas vapour phase

The evaluation of the mutagenic activity of cigarette smoke is mostly based on studies with condensates or extracts in the standard Ames assay. These samples only insufficiently reflect the composition of the actual generated aerosol. Therefore, such atmospheres should be analysed in their native composition to gain a real signal of its mutagenic capacity. Based on the technical difficulties of testing native air contaminants, there are no accepted methods for effective exposure of bacteria under such conditions. Therefore, we established a new experimental approach for direct exposure of bacteria in a modified CULTEX^® system (Patent no. DE 19801763/PCT/EP99/00295) connected to a smoking machine. This allowed us to investigate the mutagenic activity of native mainstream smoke of the research cigarette K2R4F by exposure of Salmonella Typhimurium strains. In comparison to studies using the plate incorporation assay, the direct exposure of bacteria to smoke on the agar surface enhances contact to the aerosols. By using this modification of the Ames assay, we demonstrate that it is possible to analyse the effects of native whole smoke and the gas vapour phase of cigarettes directly and achieve a dose-dependent induction of revertants. In a number of experiments, the treatment of strains TA98 and TA100 with whole smoke and the gas vapour phase of K2R4F cigarettes resulted in the induction of revertants dependent on the dilution of smoke and the number of cigarettes smoked. Our alternative procedure of exposing bacteria directly to gases and complex mixtures offers new possibilities in the field of inhalation genotoxicology for the evaluation of genotoxicity in the Ames assay.     

Comparative studies on the genotoxic activity of mainstream smoke condensate from cigarettes which burn or only heat tobacco

The in vitro genotoxic activity of mainstream cigarette smoke condensate (CSC) from cigarettes which heat but do not burn tobacco was compared to that of CSC from cigarettes which burn tobacco. CSCs from five cigarettes were compared. Three of the cigarettes [the Kentucky reference research cigarette (1R4F), a commercially available ultra-low tar brand (ULT) and a commercially available ultra-low tar menthol brand (ULT-menthol]) burn tobacco while two of the cigarettes [a regular (TEST) and a menthol (TEST-menthol]) heat tobacco. CSC from all cigarettes were collected by identical standard techniques, which involved collecting mainstream smoke particulate matter on Cambridge filter pads under FTC smoking conditions. The pads were extracted with DMSO, and the CSCs obtained [10 mg total particulate matter (TPM)/ml DMSO] were evaluated at identical concentrations in an in vitro genetic toxicology test battery. CSCs from 1R4F, ULT, and ULT-menthol cigarettes were mutagenic in Ames bacterial strains TA98, TA100, TA1537, and TA1538 in the presence of metabolic activation (S9 from Aroclor-induced rat liver) but negative in strain TA1535. In the absence of metabolic activation, 1R4F, ULT, and ULT-menthol CSCs were not mutagenic except for a weak response in strain TA1537 for the 1R4F and ULT CSCs. TEST and TEST-menthol CSCs were nonmutagenic in all five bacterial strains, both with and without metabolic activation. CSCs from 1R4F, ULT, and ULT-menthol cigarettes were positive in the CHO-chromosomal aberration assay and in the CHO--sister chromatid exchange assay both with and without metabolic activation while TEST and TEST-menthol CSCs were negative in both assays, either with or without metabolic activation. CSCs from 1R4F, ULT, and ULT-menthol cigarettes were weakly positive in inducing DNA repair in cultured rat hepatocytes while TEST and TEST-menthol CSCs were negative in this assay. All five CSCs were nonmutagenic in the CHO-HGPRT assay both with and without metabolic activation. CSCs from the 1R4F, ULT, and ULT-menthol cigarettes were cytotoxic in the CHO-HGPRT assay, both with and without metabolic activation, while TEST and TEST-menthol CSCs were not cytotoxic under either condition. These results demonstrate that mainstream CSCs from the TEST and TEST-menthol cigarettes are neither genotoxic nor cytotoxic under conditions where CSCs from 1R4F, ULT, and ULT-menthol cigarettes are genotoxic and/or cytotoxic in a concentration-dependent manner.     

Murine lung response to kaolin conveyed by cigarette smoke

Summary The effects of chronic (3 to 8.5 months) smoke inhalation from cigarettes laced with 0.1, 1.0 and 10.0 mg kaolin (hydrated aluminum silicate) per gram of tobacco on the morphological integrity of lungs and the pulmonary macrophage population were evaluated in young and old male C57BL/6 mice. Lacing procedures, monitored by determining aluminum content in acid-digested aliquots of tobacco via atomic absorption spectrometry (AAS), proved to be uniform. Amounts of aluminum in right lungs of young mice evaluated by AAS and of kaolin assessed by electron diffraction and polarizing light microscopy were larger in mice which inhaled smoke from cigarettes laced with more kaolin. A more pronounced increase in lung parenchymal tissue and decrease of alveolar space was observed in old mice subjected to smoke from cigarettes containing higher doses of kaolin than in similarly treated young animals. Concomitant with the above, the lung macrophage population did not increase as markedly in response to smoke inhalation in old mice nor did it increase in as clear a dose-response fashion to kaolin as it did in young animals. Further, the degree of ultrastructural alteration of the phagocytes in the old mice suggested impaired cell function. Plate-like material resembling kaolin crystals was most conspicuous in lung macrophages of mice which inhaled largest amounts of kaolin. Manifestations of abnormal aggregates of lymphocytes and macrophages correlated with kaolin dose inhaled in old mice but not in young animals. The reported observations indicate that 1) kaolin gains access to lungs via cigarette smoke inhalation, 2) macrophages are important in maintaining pulmonary homeostasis and 3) the inorganic compound kaolin appears to impede macrophage function, resulting in potentiation of lung abnormalities.     

A modified CULTEX® system for the direct exposure of bacteria to inhalable substances

Increasing attention is being paid to the impact on human health of inhaled gaseous compounds and complex mixtures such as cigarette smoke. The evaluation of the genotoxicity of such materials is mostly based on experiments with model substances or mixtures and condensates in the standard Ames assay. Due to the methodological difficulties of testing air contaminants in their natural gaseous or aerosolised state, there are no generally accepted concepts and techniques for effective exposure of bacteria under such conditions. Therefore, we established a novel experimental approach using an exposure device based on the cell exposure system CULTEX®. This allows us to investigate chemically and physically unchanged atmospheres like mainstream cigarette smoke by exposing bacteria of Salmonella typhimuriumstrains directly on the surface of culture media. The CULTEX® exposure device can be connected to gas or aerosol generating systems. The introduction of this exposure device in the field of inhalation genotoxicology offers new test strategies for the in vitro evaluation of a wide range of inhalable substances in both laboratory and ambient situations. A patent was applied for this technical solution.     

Comparative studies of the mutagenicity of environmental tobacco smoke from cigarettes that burn or primarily heat tobacco

The mutagenicity of particulate matter concentrated from environmental tobacco smoke (ETS) from a prototype cigarette that primarily heats tobacco was compared to that of four popular commercially available cigarettes that burn tobacco. ETS was generated by six individuals simultaneously smoking 1 cigarette each in a 20-min time period in a 45 m³ environmental chamber operated in the static mode (without ventilation). Respirable suspended particles (RSP) were collected on polytetrafluoroethylene (PTFE) filters at a flow rate of 3 LPM for 120 min. Less ETS-RSP (86–90%) was emitted by the prototype tobacco-heating cigarette than by the tobacco-burning cigarettes. RSP was extracted from the filters by sequential sonication in acetone and dichloromethane. The acetone extract was dried under nitrogen and the dichloromethane filtrate was added and then dried to obtain ETS-RSP for testing. Mutagenicity was assessed in the microsuspension modification of the Ames Salmonella/microsome assay with strains TA98 and YG1024 in the presence of 5% S9 metabolic activation. The results show that the mutagenic activity of RSP from the prototype cigarette was reduced by 75–83% on a per-mg basis when compared to the commercially available cigarettes and was reduced by 96–98% when calculated as revertants/m³ air under identical smoking conditions. Environ. Mol. Mutagen. 31:169–175, 1998 © 1998 Wiley-Liss, Inc.     

Smoke from traditional commercial, harm reduction and research brand cigarettes impairs oviductal functioning in hamsters (Mesocricetus auratus) in vitro

BACKGROUND: Cigarette smoke from 2R1 research brand cigarettes and specific toxicants in smoke inhibit oviductal functioning. Our purpose was to test the hypothesis that smoke from commercial cigarettes, including harm reduction cigarettes, inhibits oviductal functioning and to measure the concentration of previously identified toxicants in smoke from research and commercial cigarettes. METHODS: Mainstream (MS) and sidestream (SS) smoke solutions from two research, six traditional commercial and three harm reduction brands were tested in vitro using an oviductal assay that measures ciliary beat frequency, oocyte retrieval rate and smooth muscle contraction. RESULTS: Generally, smoke from each brand of cigarette was inhibitory in the three oviductal bioassays. SS, the major component of environmental tobacco smoke, was usually more inhibitory than MS, the smoke inhaled by active smokers. Nine cigarette toxicants, previously shown to be highly inhibitory in the oviductal bioassays, were quantified in MS and SS. 4-Methylpyridine, which was inhibitory by itself in picomolar doses, was present in the highest concentration in MS and SS solutions from all brands tested. In general, toxicant concentrations were higher in SS than in MS solutions. CONCLUSIONS: These data show that commercial brands of cigarettes, including harm reduction cigarettes, contain toxicants that inhibit biological processes in the oviduct and could affect reproductive outcomes.     

Volatile N-nitrosamines in mainstream cigarette smoke: occurrence and formation

Summary Analytical data is presented for the occurrence of the three major volatile N-nitrosamines in cigarette tobacco and mainstream smoke, namely N-nitrosodimethylamine (NDMA), N-nitrosoethylmethylamine (NEMA) and N-nitrosopyrrolidine (NPYR) as well as for the occurrence of the corresponding precursor amines and non-volatile N-nitrosamino acids in tobacco. Experimental studies using the C20 reference cigarette show that NPYR present in mainstream smoke results from direct transfer of preformed NPYR (ca. 1.5%), decarboxylation of N-nitrosoproline in tobacco (ca. 10%), pyrolytic nitrosation of pyrrolidine in tobacco (ca. 37%) and concerted decarboxylation/nitrosation of proline (ca. 52%) during tobacco pyrolysis.Abbreviations DMA dimethylamine - EMA ethylmethylamine - iNEP isonipecotic acid - NDEA N-nitrosodiethylamine - NDMA N-nitrosodimethylamine - NDMMOR N-nitroso-2,2-dimethylmorpholine - NEMA N-nitrosoethylmethylamine - NEP N-nitrosonipecotic acid - NMPA 4-(N-nitrosomethylamino)butyric acid - NMPA 3-(N-nitrosomethylamino)propionic acid - NPIC N-nitrosopipecolic acid - NPIP N-nitrosopiperidine - NPRO N-nitrosoproline - NPYR N-nitrosopyrrolidine - NSAR N-nitrososarcosine - PIC pipecolic acid - PIP piperidine - PYR pyrrolidine - TEA thermal energy analyser - TSNA tobacco-specific N-nitrosamines     

Comparative tumor promotion assessment of e‐cigarette and cigarettes using the in vitro Bhas 42 cell transformation assay

In vitro cell transformation assays (CTA) are used to assess the carcinogenic potential of chemicals and complex mixtures and can detect nongenotoxic as well as genotoxic carcinogens. The Bhas 42 CTA has been developed with both initiation and promotion protocols to distinguish between these two carcinogen classes. Cigarette smoke is known to be carcinogenic and is positive in in vitro genotoxicity assays. Cigarette smoke also contains nongenotoxic carcinogens and is a tumour promoter and cocarcinogen in vivo. We have combined a suite of in vitro assays to compare the relative biological effects of new categories of tobacco and nicotine products with traditional cigarettes. The Bhas promotion assay has been included in this test battery to provide an in vitro surrogate for detecting tumor promoters. The activity of an electronic cigarette (e‐cigarette; Vype ePen) was compared to that of a reference cigarette (3R4F) in the promotion assay, using total particulate matter (TPM)/aerosol collected matter (ACM) and aqueous extracts (AqE) of product aerosol emissions. 3R4F TPM was positive in this assay at concentrations ≥6 µg/mL, while e‐cigarette ACM did not have any promoter activity. AqE was found to be a lesssuitable test matrix in this assay due to high cytotoxicity. This is the first study to use the Bhas assay to compare tobacco and nicotine products and demonstrates the potential for its future application as part of a product assessment framework. These data add to growing evidence suggesting that e‐cigarettes may provide a safer alternative to traditional cigarettes. Environ. Mol. Mutagen. 58:190–198, 2017. © 2017 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.     

Tobacco smoke: Unraveling a controversial subject

Cigarettes are a modern and industrial form of tobacco use and obviously involve more than just tobacco. A multitude of physical processes and chemical reactions occur inside the burning zone of a cigarette. Cigarette smoke is an aerosol of liquid droplets (the particulate phase) suspended within a mixture of gases and semi-volatile compounds. Two kinds of smoke with different composition and properties are produced during smoking: mainstream smoke inhaled by the smoker and sidestream smoke, which is released into the environment between puffs from the lit end of the cigarette. Several techniques and modifications have altered the design of the cigarette during the last 50 years and changed smoke composition, with the effect of lower tar and nicotine smoke yields. An enormous amount of research has been done since the 1950s on smoke composition. With regard to the numerous toxic or carcinogenic constituents identified in tobacco smoke, there is a strong focus in the industry and with the authorities on the over 40 compounds, called "Hoffmann analytes". The yields of tar and nicotine in mainstream smoke of a cigarette brand as printed on the pack are measured with smoking machines under highly standardized conditions. Yields must comply with regulatory limits set in a number of countries. Smoking by machine is different from the smoking behavior of humans. There is a growing movement to develop more "realistic" methods to estimate smoke yields. But it is unclear whether alternative smoking regimens are more representative of human smoking behavior and provide better predictions of human exposure. Tobacco smoke has strong biological and toxicological effects in vitro and in vivo. There is an obvious need for developing a unified and validated testing approach particularly for the assessment of additives and the evaluation of new potentially reduced exposure products (PREPs). This paper gives a comprehensive overview of cigarette design, the composition and toxicity testing of smoke, and the way machines and people smoke - with links to the more detailed literature.     

Evaluation method for the cytotoxicity of cigarette smoke by in vitro whole smoke exposure

An in vitro whole smoke (WS) exposure method was established to evaluate the toxicological effects of fresh cigarette smoke using the VITROCELL^® system associated with the neutral red uptake (NRU) cytotoxicity assay. The VITROCELL^® system is a newly representative culture and exposure system for in vitro studies of gases or complex mixtures. The impacts of two factors on cytotoxicity measurements of cigarette smoke were investigated using this WS exposure system. The factors include synthetic air exposure and optimal time to perform the NRU assay after smoke exposure. Results showed that synthetic air exposure used in the system did not significantly alter cell survival; 24 h after smoke exposure appeared to be an optimal time-point to assess the cytotoxicity of cigarette smoke. A clear dose–response relationship between smoke exposure and cell viability was demonstrated using this system, and the evaluation method was sensitive to distinguish the differences in smoke-induced cytotoxic effects from different cigarettes. In addition, we tried converting the values of EC₅₀ from WS exposure testing into the values in unit used in total particulate matter (TPM) testing for a purpose of comparison, and the data indicate that the cytotoxicity of smoke measured by WS exposure is greater than that measured by TPM exposure.     

Statistical analysis of in vitro data for risk assessment – Exemplified for a case of Ames test data

Ames test data of experiments with smoke of six cigarette types were used for dose-response analysis and for derivation of a measure of mutagenic potency. Each cigarette type had been tested using a smoking machine and four dilutions of the smoke of each of seven cycles (one to seven cigarettes). Three plates had been exposed per cigarette number/smoke dilution combination and three control plates had been simultaneously exposed to clean air with each set of smoke-exposed plates. It was the aim of the statistical analysis to determine the slopes of dose-response relationships of various cigarette types and to compare them using statistical tests. Basically, the following procedure is recommended: (1) calculate a dose measure on the basis of the number of smoked cigarettes per cycle and dilution air flow. (2) Use the absolute count values of the individual plates as effect variable. (3) Describe the dose-response relations of the individual cigarette types on the basis of all available data with a polynomial model by means of Poisson regression analysis accounting for overdispersion. (4) Identify the linear dose-response region using the likelihood ratio test and restrict the data set to this region. (5) Use the slope of the linear model in the restricted data set as the basis of the mutagenicity measure. (6) Compare the slope for the individual cigarette type with the slope for a reference cigarette by means of multivariate Poisson regression using the likelihood ratio test and accounting for overdispersion. It is finally recommended to express the mutagenic potency as percentages related to the reference cigarette K2R4F. This type of cigarette was set here equal to 100%; the following values are then obtained for some commercially available cigarette types: type A 25%, type B 90%, type C 119%, type D 13%, type E 59%. The differences are statistically significant.     

DNA content of cultured mammalian cells exposed to smoke and smoke fractions from cigarettes containing tobacco or NSM,a tobacco substitute.

The effects of smoke and smoke fractions from tobacco and a substitute smoking material (NSM) on the DNA content of mammaliam cells in culture were measured. Tobacco smoke caused significant (P less than 0.001) changes in the DNA content of all the mammalian cells exposed compared with controls. NSM smoke did not have a significant effect on the DNA content of the exposed cells (P less than 0.95). Smoke from blends of NSM and tobacco caused changes in DNA content in proportion to the amount of tobacco in the mixtures. Condensate from cigarettes containing tobacco or blends of tobacco and NSM caused significant (P less than 0.001) changes in DNA content of mammalian cell populations in culture, whereas equal weights of condensate from NSM alone or NSM containing nicotine did not cause significant changes (P less than 0.05). NSM produces 28% of the weight of condensate per cigarette in comparison with tabacco and would, therefore, be expected to be far less biologically active than tobacco. Filtered smoke from cigarettes containing tobacco caused significant (P less than 0.001) changes in the DNA content of mammalian cells in culture. These changes were quantitatively similar to those caused by whole smoke suggesting that the gas phase of cigarette smoke is biologically more reactive than the particulate phase. The filtered smoke from the substitute smoking material NSM did not cause significant (P less than 0.95) changes in DNA content of cultured mammalian cells. Filtered smoke from blends of NSM and tobacco caused changes in DNA content in proportion to the amount of tobacco in the mixture.     

Evaluation of DNA damage in mice topically exposed to total particulate matter from mainstream and sidestream smoke from cigarettes and bidis

The genotoxic potential of total particulate matter (TPM) from mainstream smoke (MS) and sidestream smoke (SS) of Indian smoking products, namely cigarettes and bidis, as well as a brand of US cigarettes, was studied by determining the levels of bulky aromatic DNA adducts in mouse tissues. TPM from MS or SS of various smoking products [equal weights (2.5 mg) or the amount derived from equal (0.25) cigarette/bidi] was applied topically to mouse skin once a day for four consecutive days and adduct levels were determined in DNA from skin and lung by (32)P-post-labelling analysis. Relatively higher levels of bulky aromatic DNA adducts were noted in mouse skin treated with MS from a single Indian non-filter (INF) cigarette when compared with MS of a single bidi (with about half the product weight and one-quarter the tobacco compared with a cigarette), while comparable adduct levels were noted with SS from these two products. Considering the differences in the yields of constituents of tobacco smoke from the different products analyzed, the genotoxic potential of INF, Indian filter king (IFK) and American filter (AF) cigarettes as well as bidis was determined by topically applying an equal amount of TPM (rather than equal product-derived TPM). SS-derived TPM from all the products showed relatively higher levels of total polycyclic aromatic hydrocarbons and induced relatively higher levels of bulky aromatic DNA adducts than those derived from MS. The data indicate that TPM (MS + SS) from cigarettes appears to be more genotoxic than that from bidis and the contribution of tendu leaf (a non-tobacco bidi wrapper) to the generation of bulky aromatic DNA adducts appears to be significant, particularly in SS of bidis. Topical pretreatment with curcumin decreased the levels of TPM-derived adducts while pretreatment with dietary turmeric failed to show such protection.     

Hes1在烟草诱导人支气管上皮细胞恶性转化过程中的作用

目的:探讨转录因子发状分裂相关增强子1(hairy and enhancer of split,Hes1)在香烟烟气凝集物(cigarette smoke condensate,CSC)诱导永生化人支气管上皮细胞BEP2D恶性转化中的作用。方法:CSC(1L空气中点燃1支香烟)慢性染毒BEP2D细胞至第70代,软琼脂集落形成实验检测CSC诱导的细胞恶性转化表型;采用RT-PCR和Western blot法检测各代细胞的Hes1表达;MTT法、细胞集落形成实验和流式细胞术检测Notch通路阻断剂DAPT或脂质体转染Hes1-siRNA对CSC染毒BEP2D细胞增殖与凋亡的影响。检测吸烟大鼠外周小气道组织中Hes1的表达;采用免疫组化法和RT-PCR法检测非小细胞肺癌组织及正常气道组织中Hes1的表达。结果:第70代BEP2D细胞具备恶性转化表型;Hes1在CSC染毒BEP2D细胞中的表达总体呈逐渐增高的趋势;DAPT和Hes1-siRNA均能通过下调Hes1显著抑制第70代BEP2D细胞的增殖,诱导其凋亡;Hes1在卷烟烟气暴露大鼠气道黏膜1月和6月组的表达较同期对照组显著增高;吸烟显著诱导肺癌组织和正常气道表达Hes1。结论:Hes1可能通过促进凋亡与增殖失衡,参与吸烟诱导的肺癌发生。     

Comparative bone marrow clastogenicity of eigarette sidestream, mainstream and recombined smoke condensates in mice

The chromosome-damaging effects of cigarette sidestream (SS)and mainstream (MS) smoke condensates and a mixture of thesewere compared in 8-week-old NMRI mice by intraperitoneal administration.Each filtered commercial brand of cigarette was smoked by asmoking machine under the standard conditions, and the separatelycollected SS and MS smoke condensates were extracted with acetone/methanolas described elsewhere. The extracts were tested before andafter treatment of animals with an enzyme inducer (Aroclor 1254)or inhibitor (Metyrapone). Increased formation of micronucleiwithin polychromatic erythrocytes (PCEs) of femural bone marrow30 h after injection of the extracts was regarded as being dueto a clastogenic effect. Regardless of the type of smoke extractinjected, the increased formation of micronuclei was found tobe dose dependent. The SS smoke condensate induced 29% moremicronuclei than the MS smoke condensate, the difference beingsignificant (P < 0.01). The overall clastogenicity of a 1:1 mixture of SS and MS smoke condensates was not substantiallydifferent from the activity of either SS or MS smoke condensatealone. Pretreatment of animals with Aroclor clearly enhancedthe differences between the number of micronucleated PCEs causedby SS versus MS smoke condensate; SS smoke condensate induced50% more micronuclei than did MS smoke condensate (P < 0.001).Pretreatment of mice with Metyrapone did not modify appreciablythe induction of micronuclei by either type of smoke. Theseresults are discussed with reference to our previous data involvinginhalation experiments and the recent issue of passive smoking.     

DNA adduct formation in mice following dermal application of smoke condensates from cigarettes that burn or heat tobacco.

A prototype cigarette that heats tobacco (test cigarette), developed by R.J. Reynolds Tobacco Company, has yielded consistently negative results in several in vivo and in vitro genetic toxicology tests. The objective of the present study was to evaluate the potential of cigarette smoke condensate (CSC) from the test cigarette to induce DNA adducts in mouse tissues and compare the results with those obtained with CSC from a reference tobacco-burning cigarette (1R4F). CD-1 mice were skin-painted with CSC from reference and test cigarettes three times a week for 4 weeks. The highest mass of CSC applied was 180 mg "tar" per week per animal for both reference and test cigarette. DNA adducts were analyzed in skin and lung tissues using the 32P-postlabeling method with the P1 nuclease modification. Distinct diagonal radioactive zones (DRZ) were observed in the DNA from both skin and lung tissues of animals dosed with reference CSC, whereas no corresponding DRZ were observed from the DNA of animals dosed with either test CSC or acetone (solvent control). The relative adduct labeling (RAL) values of skin and lung DNA from reference CSC-treated animals were significantly greater than those of the test CSC-treated animals. The RAL values of the test CSC-treated animals were no greater than those of solvent controls. The negative results in DNA adduct assays with test CSC are consistent with all previous results of in vivo and in vitro genetic toxicology testing on this cigarette and provide additional evidence that smoke condensate from the test cigarette is not genotoxic.