Classifications

• • A—HUMAN NECESSITIES
  • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
  • A24D—CIGARS; CIGARETTES; TOBACCO SMOKE FILTERS; MOUTHPIECES FOR CIGARS OR CIGARETTES; MANUFACTURE OF TOBACCO SMOKE FILTERS OR MOUTHPIECES
  • A24D3/00—Tobacco smoke filters, e.g. filter-tips, filtering inserts; Filters specially adapted for simulated smoking devices; Mouthpieces for cigars or cigarettes
  • A24D3/06—Use of materials for tobacco smoke filters
• • A—HUMAN NECESSITIES
  • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
  • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
  • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
  • A24B15/18—Treatment of tobacco products or tobacco substitutes
  • A24B15/28—Treatment of tobacco products or tobacco substitutes by chemical substances
  • A24B15/30—Treatment of tobacco products or tobacco substitutes by chemical substances by organic substances
• • A—HUMAN NECESSITIES
  • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
  • A24D—CIGARS; CIGARETTES; TOBACCO SMOKE FILTERS; MOUTHPIECES FOR CIGARS OR CIGARETTES; MANUFACTURE OF TOBACCO SMOKE FILTERS OR MOUTHPIECES
  • A24D3/00—Tobacco smoke filters, e.g. filter-tips, filtering inserts; Filters specially adapted for simulated smoking devices; Mouthpieces for cigars or cigarettes
  • A24D3/06—Use of materials for tobacco smoke filters
  • A24D3/14—Use of materials for tobacco smoke filters of organic materials as additive

Definitions

• the invention relates to a cigarette filter containing rosemary extract and a method of reducing DNA damage caused by various harmful agents in cigarette smoke. More specifically, the present invention relates to the use of said filter to reduce DNA damage caused by benzo (a)pyrenes in human cells by the reduction of the human lung benzo (a) pyrene diol epoxide-dG (BPDE-dG) adduct.
• BPDE-dG pyrene diol epoxide-dG
• BPDE-dG human lung benzo (a)pyrene diol epoxide-dG (BPDE-dG) adduct concentrates in bronchial cells. This adduct now is recognized as a critical event in tumorigenesis by benzo(a)pyrenes. Cigarette smoke is a significant contributor to BPDE-dG formation. Tobacco use is by far the most widespread link between exposure to known carcinogens and death from cancer, and is therefore a model for understanding mechanisms of cancer induction.
• Benzo (a) pyrene (BP) is a highly carcinogenic polycyclic aromatic hydrocarbon (PAH) present in emission exhausts, charbroiled food and in small quantity in cigarette smoke, typically less than 10 ng per cigarette.
• PAH polycyclic aromatic hydrocarbon
• BP is one of more than 60 carcinogens in cigarette smoke that is involved in the aetiology of lung cancer. It is metabolically activated into benzo (a)pyrene-7 , 8-diol-9 , 10-epoxide (BPDE) which reacts with. DNA predominantly at the N 2 -position of guanine to produce primarily N 2 -guanine lesions, e.g. benzo(a)pyrene-7, 8-diol-9 , 10-epoxide-N 2 -deoxyguanosine (BPDE- dG) adduct.
• BPDE-DNA adducts in human tissues has been conclusively established and BPDE-dG adduct concentrated exclusively in bronchial cells and thus implicated in the initiation of human lung cancer.
• Rosemary ⁇ Rosmarinus officinalis Labiatae herb and oil, rosemary extracts, carnosic acid and carnosol are commonly used as spice and flavoring agents in food processing for their desirable flavor and high antioxidant activity.
• BP is considered to be a significant carcinogen involved in lung cancer induction in smokers and, as is shown in this study, reactive oxygen species contribute substantially in the formation of the critical lung tumorigenic adduct. While it is both critical to prevent addiction to tobacco and to enhance the efficacy of smoking cessation and reduction programs, these approaches have had little impact.
• the prevention of the formation of BPDE-dG adduct is one approach which may lead to decreasing lung cancer risk in addicted smokers .
• the invention relates to a cigarette filter containing rosemary extract and a method of reducing DNA damage caused by harmful agents in cigarette smoke. More specifically, the present invention provides for the use of said filter to reduce DNA damage caused by benzo (a)pyrenes in human cells by the reduction of the human lung benzo (a)pyrene diol epoxide-dG
• (BPDE-dG) adduct It has been found that the amount of (-)-antl- BPDE-dG adduct increases linearly with concentration of cigarette smoke in the presence of (+) -BP-7 , 8-diol . Catalase and superoxide dismutase inhibit its formation by more than 80%.
• cigarette smoke increases dose dependently the formation of (-) -anti-BPDE-dG and decreases the CYPs dependent formation of (+) -syn-BPDE, the adduct.
• Cigarette smoke thus may in this way responsible for the formation of the critical lung tumorigenic adduct.
• modified cigarette filter containing rosemary extract decreases by more than 70% of the BPDE-dG adducts level due to the cigarette smoke in MCF-7 cells. This discovery, I believe is a significant advance in decreasing lung cancer risk in addicted smokers .
• FIG. 1 Principal metabolic pathway and DNA binding of the carcinogen benzo (a)pyrene.
• Benzo (a)pyrene is a tobacco carcinogen that may be converted in vivo enzymatically or by oxygen reactive species to yield DNA-reactive dihydrodiol epoxides.
• Stereoselective generation of the mutagenic (+)-r- 7, t-8-dihydroxy-t-9, 10-oxy-7, 8,9, 10-tetrahydro-BP [ (+) -anti- BPD ⁇ ] from (-) -BP-7 , 8-dihydrodiol is catalyzed by cytochrome- P450-dependent monooxygenases (P450) or reactive oxygen species .
• P450 cytochrome- P450-dependent monooxygenases
• Figure 3 (a) Stereochemistry of BP-7, 8-diol epoxidation by peroxyl radicals and cytochrome P450 to reactive species (ai2ti-BPDE and syn-BPDE) that can bind to DNA, and (b) acid hydrolysis of DNA to BP-tetrols measured in this study.
• the hydrolysis of (-) -anti-BPDE-dG and (-)-syn-BPDE leads to the formation of BP-tetrol 1-2 and BP-tetrol II-l which however are -unstable and are converted into BP-tetrol I-land BP-tetrol II-2.
• BPDE-dG binding in MCF-7 cells after exposure to BP 2.5 ⁇ M or BP 2.5 ⁇ M + CS solution (dilution 20 times) for the time indicated. Cigarette smoke solution was added the last 2 hours during the exposure to BP (Scheme 1) .
• Analysis of BPDE-dG was performed as described in Materials and Methods . Values represent the means of two independent experiments with -4-6 HPDC runs plus Standard. The BPDE-dG value were 11.7 ⁇ 0.5 (mean ⁇ s.d.) Dg of adducts per mg DNA after 12 hours of incubation and 17.6 ⁇ 0.4, 26.1 ⁇ 0.9 after 18 hours and 24 hours respectively.
• the CYP spontaneous metabolism increased theses values to 17.2 ⁇ 0.5, 27.8 ⁇ 0.8 and 42.2 ⁇ 1.0 two hours after the reference time at 12, 18 and 24 hours respectively.
• the addition of cigarette smoke solution (CSS) induced a much more dramatic change during the same two hours period leading to a final BPDE-dG value of 36.9 ⁇ 1.2, 56.7 ⁇ 0.9 and 80.2 ⁇ 1.2 (mean ⁇ s.d.) Dg of adducts per ⁇ ig after 12, 18 and 24 hours respectively.
• the standard CSS system includes 2 ml calf thymus DNA (3 mg/ml), 600 ⁇ l 30 ⁇ M (+) BaP-7 , 8-diol and 5 ml of diluted CSS with PBS (1:19) at pH 7.4, and was incubated at room temperature fir 2h. Each value was obtained from three independent experiments performed in duplicate. The average error was about 12% in each duplicate experiment.
• the BPDE-dG value of the standard was 56 ⁇ 6.3 (mean ⁇ s.d.) adducts per mg DNA. 5.
• Cigarette smoking is causally associated with a large number of human cancers. Tobacco use is by far the most widespread link between exposure to known carcinogens and death from cancer, and is therefore a model for understanding mechanisms of cancer induction.
• Benzo(a)pyrene is a highly carcinogenic polycyclic aromatic hydrocarbon (PAH) present in emission exhausts, charbroiled food and in small quantity in cigarette smoke, typically less than 10 ng per cigarette.
• PAH polycyclic aromatic hydrocarbon
• BP is one of more than 60 carcinogens in cigarette smoke that is involved in the aetiology of lung cancer. It is metabolically activated into benzo (a)pyrene-7, 8-diol-9, 10-epoxide (BPDE) which reacts with DNA predominantly at the 3ST 2 -position of guanine to produce primarily M 2 -guanine lesions, e.g. benzo (a)pyrene-7 , 8-diol- 9 , 10-epoxide-N 2 -deoxyguanosine (BPDE-dG) adduct .
• BPDE-DNA adducts in human tissues has been conclusively established and BPDE-dG adduct concentrated exclusively in bronchial cells and is thus implicated in the initiation of human lung cancer.
• This carcinogen is metabolized by phase I enzymes to a large number of metabolites including phenols, arene oxides, quinones, dihydrodiols , and diol epoxides.
• An overview of BP metabolic way leading to the formation of (+) -artti-BPDE-dG adduct is presented in Fig. 1.
• the ultimate carcinogen (+) -anti-BPDE is formed, from BP by two rounds of cytochrome P450-mediated oxidation.
• the first step of this oxidation leads preferentially to (-) -7, 8-dihydro-7, 8-dihydrobenzo (a)pyrene [(-)BP -7,8-diol].
• the diol is further oxidised primarily to the highly mutagenic (+)-r-7, t-8-dihydroxy-t-9 , 10-oxy- 7, 8, 9, 10-tetrahydro-BP [(+) -anti-BPDE] .
• ROS from the cigarette smoke may be in part responsible for the increased BPDE-dG adduct formation.
• My invention provides (i) a means for determining the relative contribution of ROS in cigarette smoke on the activation of BP-7,8-diol in comparison with cytochrome P450; (ii) a means for establishing whether cigarette smoke's ROS promotes the carcinogenic process by contributing to the metabolism of BP-7,8-diol resulting in an increase in the formation of the critical lung BPDE-dG; (iii) a filter containing a scavenger of cigarette free radicals to significantly decrease the formation of BPDE-dG ; and (iv) use of said filter to significantly decrease the function of BPDE- dG adducts .
• Proteinase K (EC 3.4.21.64, from Tritirachium album) was purchased from Sigma (St. Louis, MO), RNase Tl (EC
• Phosphate-buffered saline contained 3.0 mM KCl, 1.5 mM KH 2 HPO 4 , 140 mM NaCl, 8.0 mM Na 2 HPO 4 , (pH 7.4), HPLC-grade water,
• HPLC High-performance liquid chromatography
• Cigarette Smoke/PBS Solution SCS
• This aqueous solution named cigarette smoke solution (CSS) was reacted immediately with exogenous DNA or added to MCF-7 cells in culture in the presence of benzo (a)pyrene or its proximate metabolite (+) -BaP-7 , 8-diol .
• benzo (a)pyrene or its proximate metabolite (+) -BaP-7 , 8-diol were used (see below) .
• the human mammary carcinoma cell line MCF-7 was grown in 150-cm 2 cell culture flasks in a total volume of 20 ml minimal essential medium E- MEM supplemented with 10% FCS, 15 mM Hepes buffer, and antibiotics (200 units/ml penicillin, 200 ⁇ g/ml streptomycin, and 25 ⁇ g/ml ampicillin) . Cells were maintained and treated at 37 0 C in 5% CO 2 /95% air atmosphere. After MCF-7 cells had covered 90% of the surface area of the flasks, (2-3 days after splitting of a confluent culture) , the medium was replaced with 20 ml of fresh medium containing 10% serum. Twenty four hours later, near-confluent cells e.g.
• DNA Preparation and. Hydrolysis DNA isolation from MCF-7 cell pellets was carried out by treatment with RNase, proteinase K, salting procedure (31) and chloroform. Briefly, the cell pellets were resuspended in EDTA-sodium dodecyl sulfate (SDS) buffer [10 mM Tris buffer, 1 mM Na 2 EDTA, 1% SDS (w/v) , pH 8] incubated for 1 h at 37°C with RNase Tl (2000 U/ml) and RNase A (DNase free; 100 ⁇ g/ml) on a shaker (100 rpm) .
• SDS EDTA-sodium dodecyl sulfate
• proteinase K (300 ⁇ g/ml) was added and the incubation continued overnight at 37 0 C. After digestion, 6M NaCl was added to have final concentration of IM followed by a centrifugation at 10000 g. DNA in the supernal was precipitated with 2 vol. ethanol, washed with 70%, 100% ethanol, ether, dried and dissolved in 10 mM Tris buffer. Again, KNase A (100 ⁇ g/ml) and RNase Tl (2000 U/ml) were added and the solution incubated at 37 0 C for 1 h. followed by proteinase K (100 ⁇ g/ml) for another 2 hours at 37°C.
• the solution was extracted once with chloroform, centrifuged and the solution was made IM NaCl.
• DNA was precipitated with 2 vol. cold ethanol.
• the portion of DNA to be hydrolyzed was rinsed with 100% ethanol to remove unbound BP-tetrols.
• the DNA, free of unbound BP-tetrols, was dissolved in water and the DNA concentration was determined by A 2 go nm ..
• the purity was ascertained by the ratios at A 2 6o/A 28 o and A 26 o / A 23 o.
• the amount of DNA for analysis was hydrolyzed as described previously by incubation at 90 0 C for 4 hour in a final concentration of 0.1 N HCl. This releases tetrols (Fig. 3) from BPDE-DNA adducts with > 90% recovery.
• the volume of the hydrolysate for injection was made 700 ⁇ l containing 5-10 ⁇ g DNA.
• BPDE-Bp-dG adduct level The adduct levels were determined by HPLC-FD as previously described [32,33] using r-7 , c-9 , t-8 , t-10-tetrahydroxy-7 , 8 , 9 , 10- tetrahydrobenzo (a)pyrene (BP-tetrol Il-l) as an internal standard [34] .
• the hydrolysate was loaded onto a Latex pre- column module (HD-Germany) containing 5 ⁇ m Ci ⁇ reverse-phase materiel (Nucleosil 100) equilibrated with 10% MeOH and washed for 20 min with 12 ml 10% MeOH.
• BP tetrol 1-1 trans-anti-BP-tetrol
• BP-tetrol II-l trans-syn-BP-tetrol
• internal standard 36.9 min
• BP tetrol II-2 cis-syrc-BP-tetrol
• the detection limit was 0.5 pg of BP tetrol 1-1 and BP-tetrol II-l .
• the level of each BP-tetrol was determined by using a standard curve generated from the fluorescence peak area of authentic BP-tetrol standard analyzed just before the analysis of MCF-7 samples.
• the BP-tetrol-I-1 detected is derived after hydrolysis of (+) -antl-BPDE-DNA adduct.
• the hydrolysis of (- ) -antl-BPDE-dG leads to the formation of BP-tetrol 1-2, which however is unstable and is converted in BP-tetrol 1-1 (Pig. 3) (38).
• the level of the formed (-) -anti-BPDE-dG was measured by the quantity of BP-tetrol 1-1 found on HPLC runs. Based on the finding that BPDE reacting with DNA produce primarily BPDE-N 2 -dG (7), I assumed that BP-tetrol-I-1 level corresponds to this of BPDE-N 2 -dG.
• the HPLC runs were quantitatively reproducible, and variability between the two assays was lower than 5%.
• BP molecular signature The mechanism of mutagenesis by BP is sufficiently well defined and used as a "molecular signature" to establish the causal nature between particular genetic events in development of tumors and carcinogenic exposure (the "smoking gun”).
• the "BP molecular signature” has major implication for pinpointing the tobacco smoke as the cause of human lung cancer, and for the elaboration of specific strategies to minimize tobacco smoking, or introduce preventive measures .
• Specific agents used in cancer chemoprevention appear to act by inhibiting carcinogen damage to DNA, mutagenesis, tumor promotion and/or tumor progression.
• CS is an aerosol of complex chemical composition containing both organic and inorganic compounds, of which 4800 have been identified so far. Both vapor phase and particulate phase of smoke are known to possess free radicals.
• the radicals in the particulate phase are relatively stable and consist of a hydroquinone, semiq ⁇ inone, quinone complex
• this complex is an active redox system capable of reducing molecular oxygen to produce superoxide, eventually leading to hydrogen peroxide and hydroxyl radicals.
• at least 60 different CS carcinogens have been implicated in tumor initiation and promotion; the most potent carcinogens agent contained in CS are BP and NNK (4- (methylnitrosamino) -1- (3-pyridyl) -1- butanone) .
• (-) -BP-7 8-diol
• the different pathways can be distinguished by HPLC analysis since the tetrols derived from antl- and syrz-BPDE respectively are clearly separated under my conditions .
• MCF-7 The Effect of cigarette Smoke on BPDE-dG Adduct Formed in Cells Treated wit!.. BP.
• MCF-7 cells have high CYPlAl enzyme activity for the metabolic activation of BP leading to the formation of (-) -BP-7, 8-diol and consequently to ( +) -anti- BPDE-dG (Fig. 1) .
• the level of adduct formation at 6 hours was considerably lower than that observed after 12 and 24 hours of exposure.
• the cells were treated for 12 and 18 hours with BP to induce the formation of (-)-BP-7,8 diol which is substrate for
• ⁇ -) -Bp-7 , 8-diol is the absence of BP-tetrol II derived from syn-BPDE on HPLC runs which precursor is ( +) -BP-7 , 8-diol
• BP-tetrol I derived from ( +) -eueri-BPDE-dG.
• the difference between cells treated with CSS and those non treated (controls) is presented on Fig. 5.
• the cell line used in this study maintained the capability to lower the CYPlAl expression after oxidative challenge by CS.
• the suppression of cytochrome P450 presumably lowers activation of BP to (-) -BP-7 , 8-diol and ( +) -eueri-BPDE.
• the increased difference by CS is due to the increased metabolism of (-)-BaP-7,8 diol by ROS generated from CS.
• Rosemary ⁇ Rosmarinus officinalis Labiatae herb and oil are commonly used as spice and flavoring agents in food processing for its desirable flavor and high antioxidant activity.
• Topical application of rosemary extract, carnasol or ursolic acid to mouse skin inhibited the covalent binding of benzo (a)pyrene to epidermal DNA, tumor initiation by 7 , 12-dimethylbenz (a) antracene (DMBA), TPA-induced tumor promotion, ornitine decarboxylase activity and inflammation.
• Rosemary extracts were proved to be efficient not only in the promotion phase but also in the initiation phase.
• Rosemary extracts, carnosic acid and carnosol strongly inhibit phase I enzyme, CYP 450 activities and induce the expression of the phase II enzyme, glutathione S-transferase (GST) and cjuinone reductase activities.
• Carnosol inhibits nitric oxide (NO) production in activated macrophage .
• the antioxidant property had been referred to as the mechanistic basis of their protective effects.
• Fig. 6 The results presented in Fig. 6 were obtained when the MCF-7 cells were treated with BP.
• Two groups of experiments were performed (A and B) .
• the cells were treated with BP for 12 and 18 hours respectively following with CSS from the two filters for another 2 hours together with BP (Scheme 1) .
• Scheme 1 To evaluate the CYPs dependent increase of the adduct during these last 2 hours, two controls for each group were performed : 12 and 14 hours for group A, 18 and 20 hours for group B.
• the CSS from the standard filter double the binding level obtained for 14 and 20 hours.
• rosemary filter strongly impedes the increase obtained by the standard filter, more than 70% in the two groups (Fig. 6) . ) .
• the modified filter scavengers ROS and consequently decreases the activation of (-) -BP-7, 8-diol (Fig. 3) .
• rosemary powder may have also other mechanisms to reduce BPDE-dG formation.
• whole rosemary extract (6 ⁇ g.ml "1 ) also inhibits CYPlAl activity and DNA adduct formation by 80% after 6 hours co-incubation with 1.5 ⁇ M BP in human bronchial epithelial cells (BEAS-2B) .
• using filters which decrease the amount of the free radicals to reduce the formation of the critical tumorogenic adduct are a significant benefit for the addicted smokers .
• My inventive rosemary cigarette filter therefore is a promising candidate for chemopreventive programs with the aim to reduce BPDE-dG in bronchial epithelial cells.

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