Note: Descriptions are shown in the official language in which they were submitted.
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A CIGARETTE FILTER CONTAINING ROSEMA.RY EXTRACT AND
A METHOD OF REDUCING DNA DAMAGE CAUSED BY HARMFUL AGENTS
IN CIGARETTE SMOKE BY USE OF SAID FILTER
1. Field of the Invention
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.
2. Background
The 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. 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 N2-position of
guanine to produce primarily N2-guanine lesions, e.g.
ben.zo(a)pyrene-7,8-diol-9,10-epoxide-N2 -deo.xyguanosine (BPDE-
dG) adduct. The presence of BPDE-DNA adducts in human tissues
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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.
However, prior to the instant invention, there was no
recognition that the use of a rosemary extract in a cigarette
filter would reduce the DNA damage caused in human cells by
benzo(a)pyrenes specifically, the human lung benzo(a)pyrene
.diol epoxide-dG (BPDE-dG) adduct which adduct now is
recognized as a critical event in tumorigenesis by
benzo(a)pyrenes.
3. Summary of the inventioxti,
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.
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it has been found that the amount of (-)-anti- 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%.
[ruhen MCF-7 cells are treated for 2 hours with the (+) -Bp-7, 8-
diol, cigarette smoke increases dose dependently the formation
of (-)-anti-BPDE-dG and decreases the CYPs dependent formation
of (+)-syn-BPDE, the adduct.
I have treated cells for up to one day with
benzo(a)pyrene and then exposed them for 2 hours with
cigarette smoke. During these 2 hours, I have discovered that
there is twice the increase in the adduct formation in cells
treated with cigarette smoke in comparison to levels in non
treated cells due to CYPs activity. Thus, I have found that
cigarette smoke activates by reactive oxygen species which it
contains the second step of benzo(a)pyrene metabolic way
leading to the formation of BPDE-dG adduct.
Cigarette smoke thus may in this way responsible for the
formation of the critical lung tumorigenic adduct.
Finally, I have found that 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.
4. Brief Descriptiota. of the Figures, Scheme and Table:
Figure 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-
BPDE] from (-)-BP-7,8-dihydrodiol is catalyzed by cytochrome-
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P450-dependent monooxygenases (P450) or reactive oxygen
species. Subsequent reaction of this electrophilic
intermediate with genomic DNA produce stable adduct between
dihydrodiol epoxide and the exocyclic amino group of
guanosine. This kind of DNA lesion may be converted into
mutations within the following replication cycle unless repair
of this adduct is produced.
Figure 2. Results obtained by using a cell-free system
concomitant with DNA adduction: 6 mg calf thymus DNA in 2 ml
water was added to 5 ml CSS with different dilutions and
reacted for 2 hours at room temperature with (+)-BP-7,8-diol
(final concentration 3.6 jaM). DNA was hydrolyzed and the
released BP-tetrol I were measured as outlined in Materials
and Methods.
Figure 3. (a) Stereochemistry of BP-7,8-diol epoxidation
by peroxyl radicals and cytochrome P450 to reactive species
(anti-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 =I-1 which however
are unstable and are converted into BP-tetrol I-land BP-tetrol
II-2.
Figure 4. Results obtained using MFC-7 cells. 10 x 106
cells/150 cm2 flask in a total volume of 20 ml were treated for
2 hrs with (+)-BP-7,8-diol (0.2 -~aM) alone or in presence of
different dilutions of CSS DNA was isolated, hydrolyzed and
the released BP-tetrols were measured and the binding levels
determined as outlined in Materials and Methods. Two distinct
peaks were observed on the chromatograms corresponding to BP-
tetrol I and BP-tetrol II derived from (-)-anti-BPDE-dG and
(+)-syn-BPDE-dG respectively (refs. 32-34). Values represent
the means plus Std of two independent experiments with 3-4
HPLC runs. Figure 4a, upper panel, increases of
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(-)-ant.i-BPDE-dG adducts with CSS dilution; Figure 4b, lower
panel, increases of 1/(+)-s.yn-BPDE-dG adduct with CSS
dilution.
F3.gure 5. BPDE-dG binding in MCF-7 cells after exposure
to BP 2.5 pM or BP 2.5 pM + 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 HPLC 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.) ^g of adducts
per mg after 12, 18 and 24 hours respectively.
Figure 6. BPDE-dG binding in MCF-7 cells after exposure
to BP 2.5 M or BP 2.5 M + CS solution obtained from standard
filter and filter containing rosemary extract. The cigarette
smoke solution was added for the last 2 hours during the
exposure to BP for the time indicated (Scheme 1). Analysis of
BPDE-dG was performed as described in Materials and Methods.
The HPLC runs show that there is only one peak on
chromatograms which correspond to BP-tetrol I derived from
(+)-anti-BPDE-dG. Values represent the means of two
independent experiments with 4-6 HPLC runs plus Std.
Scheme 1. MCF-7 cells were treated with BP for 12 hrs
and 18 hrs following with cigarette smoke for another 2 hours
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together with BP (experiments A and B respectively). The goal
of this experiment was to activate at different times the BP
to produce BP-7,8-diol and after treat the cells with
cigarette smoke and follow its effect during the last 2 hours.
The control represents cells treated only with BP.
Table 1. Effect of scavengers on BPDE-dG level caused by
cigarette smoke in a cell-free system
% BPDE-dG relative
Treatment to standard CS
Standard CSS 100
+ Superoxide Dismutase SOD (20 ug) 16
+ Catalase (4 pg) 12
+ Inactivated Catalase (4ug) 100
Romarin filtered CSS instead Standard CSS 42
The standard CSS system includes 2 ml calf thyinus DNA (3
mg/ml), 600 ~a.l 30 uM (+) 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.
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5. Detailed Description
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 (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. 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 N2-position of guanine to produce
primarily NZ-guanine lesions, e.g. benzo(a)pyrene-7,8-diol-
9,10-epoxide-N2-deoxyguanosine (BPDE-dG) adduct.
The presence of 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 (+)-anti-BPDE-dG
adduct is presented in Fig. 1.
In more detail, 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]. Numerous studies have
clearly identified the [(+)-anti-BPDE] as the primary
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carcinogenic metabolite of BP exhibiting enhanced mutagenic
activity in vitro and in vivo. Most previous studies of
genetic variation in metabolism of lung carcinogens have
focused on metabolic activation by various cytochromes P450s,
although expression of these enzymes in lung is generally
low). The activation of BP-7,8-diol by lung epithelial cells
is not caused solely by classic CYPs/GSTs dependent
biotransformation processes, but also involves several
metabolic routes other than CYPs. These include,
lipooxygenase, lipid-peroxidation products and peroxidase-
dependent pathways, COX-1'and COX-2.
Increasing evidence suggests the causal significance of
tobacco free radicals in lung cancer induction in smokers.
Each puff of smoke forms over 10 trillion free radicals
present in smoke, which may contribute to both tumor
initiation as well as promotion of various forms of human
cancer caused by repeated attacks from ROS on cellular
macromolecules. The major free radical species are postulated
to be an equilibrium mixture of semiquinones, hydroquinones
and quinones. It is suggested that this free radical complex
causes redox cycling that generates superoxide anion from
molecular oxygen and leads to the formation of hydrogen
peroxide and hydroxyl radical. These reactive species cause
DNA nicking and single-strand breaks in DNA of cultured
rodent and human cells. Quinone-associated redox cycling may
also be involved in these effects; hydroquinone and catechol
are believed to play a major role.
I have discovered that cigarette smoke can activate by
its oxygen generated radicals the second step of the BP
metabolic- pathway leading to the formation of BPDE-dG adduct,
presumably by metabolism of the formed in the cells (-)-BP-
7,8-diol to (+)-r-7,t-8-dihydroxy-t-9, 10-oxy-7,8,9,10-
tetrahydro-BP [ (+) -a.nti-BPDE] (Fig. 1).
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I have also discovered that this activation is at least
twice higher than this obtained with CYPs machinery.
Further, I have discovered that ROS from the cigarette
smoke may be in part responsible for the increased BPDE-dG
adduct formation.
r have also found that a filter containing a formulated
rosemary powder can reduce considerably the BPDE-dG level due
CS oxygen generated radical, and that my discoveries can be
used for cigarette filters which reduce the formation of
carcinogenic BPDE-dG adduct in bronchial epithelial cells.
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.
7. Examples
Chemicals. Proteinase K (EC 3.4.21.64, from Trit.frachiu.rn
album) was purchased from Sigma (St. Louis, MO), RNase T1 (EC
3.1.21.3, from Aspergillus oryzae) and RNase (DNase free,
heterogeneous mixture of ribonucleases from bovine pancreas)
were obtained from Boehringer Mannheim (Mannheim, Germany).
Phosphate-buffered saline (PBS) contained 3.0 mM KC1, 1.5 mM
KH2HPO4, 140 mM NaCl, 8.0 mM Na2HP04, (pH 7.4), HPLC-grade water,
MeOH, ether and ethanol for spectroscopy from E. Merck,
Darrnstadt, Germany. If not stated otherwise, the others
chemicals were purchased from Sigma (L'Isle d'Abeau Chesnes,
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France), Boehringer Tngelheim (Heidelberg, Germany) and
Boehringer Mannheim (Mannheim, Germany). All BP metabolite
standards were obtained from National Cancer Institute,
Chemical Carcinogen Reference Standard Repository Ma.dwest
Research Institute (Kansas City, MO).
Apparatus. High-performance liquid chromatography (HPLC)
was carried out with a Hewlett-Packard high pressure isocratic
and gradient systems (Waldborn, Germany) equipped with a
Shimadzu RF-lOAXL fluorescence detector linked to a Hewlett-
Packard integrator.
Preparation of Cigarette Smoke/PBS Solution (CSS).
Smoking was performed according to Pryor et al without the
Cambridge filter. Essentially the same smoke collecting
method has been used earlier by Nakayama et al. The smoke
from burning 8 cm of one cigarette (Marlboro) during 3.8 min
with the help of constant vacuum generated from a water pump
was bubbled through 10 ml of phosphate-buffered saline (PBS)
solution which traps both the gas-phase and tar cigarette
smoke chemicals. As there were no water-insoluble tar
compounds present on the walls of the wash bottles, a major
part of the water-soluble compounds from the smoke of a single
cigarette was contained in the 10 ml PBS solution. 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. Different dilutions of CSS were
used (see below).
Incorporation of a Rosemary Powder Extract into the
Cigarette Filter. The filter of the conventional cigarette
was removed and 40 mg of a rosemary powder extract, was
introduced in the place free from the filter near to the
cigarette itself. After this operation, the filter was
reinstalled. The effect of this filter was evaluated by mass
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spectrometry. Briefly the cigarette smoke was bubbled in an
organic solution containing 3,3,5,5-tetramethyl-l-pyrroline-N-
oxide (TMPO) a spin trap adduct. The amount of hydroxyl
radical adduct was then quantified using liqui.d
chromatography, mass spectrometry. Under the smoking
conditions used a 30% decrease in the hydroxyl radical was
observed.
Reaction of Exogenous DNA with (+)-BP-7,8-Diol in
Presence of Diluted C3.garette Smoking Solution (CSS). 2 ml
calf thymus DNA (3 mg/ml) was added to 5 ml diluted 20 times
CSS and reacted for 2 hours at room temperature with (+)-Bp-
7,8-diol (final concentration 3.6 pM) according to the
following reaction:
DNA + [(+)-BP-7,8-dio17+CSS-> (-)-anti-BPDE-N2-dG
The level of the resulting (-)-anti BPDE-dG adduct was
measured (see below). As control an experiment without CSS
was performed.
Cell Culture Conditions and Treatment. The human mammary
carcinoma cell line MCF-7 was grown in 150-cm2 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/mi streptomycin,
and 25 g/ml ampicillin). Cells were maintained and treated
at 37 C in 5% C02/95% 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.
more than 90% of cells in GO/G1 phase were treated with DMSO
alone or with carcinogen (see below) dissolved in DMSO and
cigarette smoke/PBS (CSS see above). The final concentration
of DMSO did not exceed 0.1% of the total incubation volume.
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Control samples included in each incubation set were treated
with DMSO alone.
a) Treatment of MCF-7 CeI.Is with (+) -BP-7, 8-DioZ and
Cigarette Smoke. Cells were treated for 2 hours with (+)-BP-
7,8-diol (0.2 M) alone or in presence of different dilutions
of CSS. The (+)-BaP-7,8-diol was activated by ROO generated
from the CS and cell CYP's to form (-)-anti-BPDE-dG and (+)-
syn-BPDE-dG respectively. Their levels were measured by the
formation of BP-tetrol 1-1 and BP-tetrol 11-2 (see below and
Fig. 3).
b) Time/Dose Exposure Experiments with BP. To
characterize time/dose exposure to BP and BPDE-dG level, cells
(10x106 cells /150 cm2 flask, total volume of 20 ml) were
treated with medium containing final concentration of 1.25,
2.5 and 5.0 M each for 6,12,18 and 24 hours (two
flasks/dose/time point). BPDE-dG adduct formed in the cells
increase linearly in a dose-and time-dependent manner to as
was shown also by others (30). On the basis of the res-ults
obtained we choose 2.5 M as working concentration for BP.
c) Treatment of MCF-7 Cells with BP and Cigarette Smoke.
To see the effect of CS concentration, the experience A of
scheme N 1 was performed with different CSS dilutions (1:79;
1:39; 1:19; 1:9 vol/vol). On the basis of the results
obtained from this experience I choose 1:19 (vol/vol) dilution
as working CS concentration. Thus, the cells (see above "Cell
Culture and Treatment") were treated with BP (2.5 M) and CSS
(dilution 1:19 vol/vol) according to Scheme 1(See Figure 6-
Scheme 1).
All incubation sets were repeated 2-3 times with
duplicate samples. At the end of treatment, cells were
examined microscopically for morphological changes, then
harvested by trypsinization with 0.05% trypsin-EDTA (0.05%
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trypsin, 0.14 M NaCl, 3 mM KC1, 0.1 M Na2HP04r 1.5 mM KaHPOq,
0.5 mM EDTA). After addition of an equal volume of inedium
containing 10% FCS, the cells were centrifuged at 1000 g,
washed three times with PBS, and the cell pellet then stored
frozen at -20 C. The viability of the cells treated with BP
and cigarette smoke or (+)-BP-7,8-diol and cigarette smoke was
roughly 90% at the time of harvesting as determined by a
Trypan blue exclusion assay. The doses used did not show any
cytotoxicity as measured by lactate dehydrogenase activity
assay (ELISA Kit, Boehringer, Mannheim).
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 Na2EDTA, 1% SDS
(w/v), pH 8] incubated for 1 h at 37 C with RNase T1 (2000
U/ml) and RNase A (DNase free; 100 U,g/ml) on a shaker (100
rpm). Then proteinase K (300 1.a.g/ml) was added and the
incubation continued overnight at 37 C. After digestion, GM
NaCl was added to have final concentration of 1M 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, RNase A (100 U.g/ml) and RNase T1 (2000 U/ml) were added
and the solution incubated at 37 C for 1 h. followed by
proteinase K(Z00 ug/ml) for another 2 hours at 37 C. The
solution was extracted once with chloroform, centrifuged and
the solution was made 1M NaCI. 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 A260 The purity was
ascertained by the ratios at A260iA280 and A260iA230 . The amount
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of DNA for analysis was hydrolyzed as described previously by
incubation at 90 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 tixl containing 5-10 ug DNA.
Determinations of BPDE-LVZ-dG adduct level. The adduct
levels were determined by HPLC-FD as previously described
[32,33] using r-7,c-9,t-S,t-10-tetrahydroxy-7,8,9,10-
tetrahydrobenzo(a)pyrene (BP-tetrol II-1) as an internal
standard [34]. The hydrolysate was loaded onto a Latex pre-
column module (HD-Germany) containing 5l,un C1B reverse-phase
materiel (Nucleosil 100) equilibrated with 10% MeOH and washed
for 20 min with 12 ml 10% MeOH. Subsequently, the pre-column
was switched by a Valco Instruments switching valve to flow
over a 4.6 mm x 25 cm 5 um C1S reverse-phase (Nucleosil 100)
analytical column (Alltech GmbH, Unterhaching, Germany). The
products obtained by hydrolysis were eluted with the following
MeOH/H20 gradient: 50%, 0-17 min; 50 to 60%, 17-32 min; 60%,
32-42 min; 60 to 100%, 42-57 min. Retention times of the BP
tetrols were: BP tetrol I-1 (trans-anti-BP-tetrol) (35.2 min);
BP-tetrol II-1 (trans-syn-BP-tetrol), internal standard (36.9
min); BP tetrol II-2 (cis-syn-BP-tetrol)(42.3 min).
Fluorescence was assessed at an excitation wavelength of 344
nrn and emission wavelength of 398 nm. As I did not detect the
formation of BP tetrol II-1 in separate analysis of MCF-7
samples, I used it as internal standard (2 pg added to each
HPLC run) for verification of the relative retention time.
The detection limit was 0.5 pg of BP tetrol I-1 and BP-tetrol
II-1. 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 (+)-anti-BPDE-DNA adduct. The hydrolysis of (-
)-anti-BPDE-dG leads to the formation of BP-tetrol 1-2, which
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however is unstable and is converted in BP-tetrol I-1 (Fig. 3)
(38). Thus, the level of the formed (-)-anti-BPDE-dG was
measured by the quantity of BP-tetrol I-1 found on HPLC runs.
Based on the finding that BPDE reacting with DNA produce
primarily BPDE-N2-dG (7), I assumed that BP-tetrol-I-1 level
corresponds to this of BPDE-N2-dG. The level of BPDE bound to
MCF-7 DNA was quantified in duplicates. The adduct level was
calculated from the equitation 1 pmol/mg DNA / 3.125 = 1
adduct per 106 nucleotide. The HPLC runs were quantitatively
reproducible, and variability between the two assays was lower
than 5%.
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.
I have explored the relative role of CS on the
biotransformation of BP-7,8-diol to BPDE capable of forming
stable DNA adduct in human cells. Numerous studies have
demonstrated that stable PAH-DNA adducts can lead to mutations
through mis-incorporation of nucleotides or deletion. 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. While the gas phase
radicals are generally short-lived the radicals in the
particulate phase are relatively stable and consist of a
hydroquinone, semiquinone, quinone complex, this complex is an
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active redox system capable of reducing molecular oxygen to
produce superoxide, eventually leading to hydrogen peroxide
and hydroxyl radicals. In addition, 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).
The Effect of Cigarette Smoke on (+)-anti.-BPDE-dG Using a
Cell-Free System Concomitant with DNA Adductiorn. To elucidate
the mechanism of BPDE-dG formation dependent from active
oxygen generated from cigarette smoke, I looked for this
adduct in cell-free in vitro system concomitant with DNA
adduction. The CSS solution containing the gas-phase and tar
cigarette smoke radicals was immediately reacted with DNA in
the presence of (+)-BP-7,8-diol (see protocol above). The
results from this experiment show that CS can oxidize the (+)-
BP-7 , 8 diol to (-)-anti-BPDE which in turn form the (-)-anti-
BPDE-dG adduct. The amount of (-)-anti-BPDE-dG increased
linearly and dose dependently (See Fig. 2).
Previously it was found that large amounts of active
oxygen such as H202 and 02` were generated from cigarette smoke
after trapping the smoke in PBS. This active oxygen generated
from cigarette smoke might be responsible for the observed
formation of (-)-anti-BPDE-dG. To verify this, I checked the
effect of catalase and superoxide dismutase (SOD) on the (-) -
anti-BPDE-dG produced, and found that the both enzymes
inhibited the formation of the adduct. Inactivated catalase
showed no effect (Table 1). From these results I concluded
that cigarette smoke can oxidize (+)-BP-7,8-diol, thus forming
(-)-ant.i-BPDE-dG, and that such capacity can be explained
mainly by the action of oxygen generated from cigarette smoke.
The Effect of Cigarette Smoke on (-)-anti-BFDE-dG Adduct
Formed in MCF-7 Cells Treated with (+)-BP-7,8-diol. Two
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independent pathways have been shown to participate in the
metabolism of BP-7,8-diol to BPDE (Fig.3). The cytochrome
P450 dependent metabolism of the (+)-enantiomer leads
preferentially to (+)-syn-BPDE whereas the pathway involving
haem-containi.ng proteins in conjunction with a peroxide (e.g.
lipid peroxide) preferentially results in (-)-anti-BPDE. The
(-)-BP-7,8-diol on the other hand, may be metabolized by both
pathways and results in the formation of (+)-anti-BPDE, the
ultimate form of BP, and (-)-syn-BPDE. The different pathways
can be distinguished by HPLC analysis since the tetrols
derived from anti- and syn-BPDE respectively are clearly
separated under my conditions.
To investigate further the role of cigarette smoke
dependent epoxidation of (+)-BP-7,8-diol leading to the
formation of (-)-anti-BPDE that form with DNA (-)-anti-BPDE-dG
adduct, human mammary cell line MCF-7 was used. The reason
for which I used MCF-7 cells to see the effect of cigarette
smoke ROS on the activation of (+)-BP-7, 8 diol was that these
cells have little peroxidase activity. The cells were treated
with the (+)-BP-7,8-diol, a stereochemical probe which can
distinguish the adducts formed by ROS and CYPs dependent ways
(Fig. 3). Two distinct peaks were observed on the
chromatograms corresponding to BP-tetrol I and BP-tetrol II
derived from (-)-anti-BPDE-dG and (+)-syn-BPDE-dG respectively
(refs. 32-34). Cigarette smoke increased linearly and dose
dependently the ROS dependent formation of (-)-anti-BPDE-dG
(Fig. 4a) and decreased the CYPs dependent formation of (+)-
syn-BPDE-dG adduct measured by the formation of BP-tetrol II.
This decrease is also dose dependant and the inverse of DNA
adducts increased linearly with cigarette smoke concentration
(4b). The inhibitory effect of CS on CYPs dependent formation
of (+)-syn-BPDE-dG and increased formation of (-) -anti-sPDE-dG
adduct confirm the role of the oxygen generated from the
cigarette smoke in the formation of (-)-anti-BPDE-dG adduct.
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Other studies showed that induced CYPs activity was
impaired by the oxidative challenge. The mechanism underlying
such a phenomenon could be a down-regulation of cytochrome
P4501A1 gene. Having little peroxidase activity may lead MCF
cells under "stress" conditions to increased DNA damage and
reduced repair capacity. Consequently this may cause an
increase of BPDE-DNA adducts independently from BP-7,8-diol
activation.
The Effect of Cigarette Smoke osn BPIaE-dG Adduct Formed in
Cells Treated with BP. Previous studies with MCF-7 cell
cultures revealed that these cells possess inducible P4501B1
and P4501A1 activity. The presence of P450 catalyzed
metabolic turnover of BP and the absence of detectable
peroxidase activity in MCF-7 cells, allowed the evaluation of
the role of cigarette smoke oxygen radicals on BP activation
in human cell cultures. MCF-7 cells have high CYP1A1 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. After treatment with 2.5 M BP for 6 hours
approximately 2000 pg adducts per mg DNA were formed, whereas
more than 11000 pg and more than 20000 pg adducts per mg DNA
were present after 12 hours and 24 hours respectively
(Wilcoxon Rank Sum Test gives p=0.0022). I
The cells were treated for 12 and 18 hours with BP to
induce the formation of (-)-BP-7,8 diol which is substrate for
ROS. Indirect confirmation for the preferentially formation of
(-)-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
(Fig.3). The cells were then exposed for 2 hours with CSS of
cigarette smoke together with BP. The HPLC runs show that
there is only one peak on chromatograms which correspond to
BP-tetrol I derived from (+)-ant.i-BPDE-dG. The difference
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between cells treated with CSS and those non treated
(controls) is presented on Fig. 5. As mentioned above the
cell line used in this study maintained the capability to
lower the C'YP1A1 expression after oxidative challenge by CS.
The suppression of cytochrome P450 presumably lowers
activation of BP to (-)-BP-7,8-diol and (+)-anti-BPDE. Thus
the increased difference by CS is due to the increased
metabolism of (-)-BaP-7,8 diol by ROS generated from CS.
Wilcoxon Rank Sum Test gives p=0.0022 for treated with CS vs
controls for 14 hours and 20 hours respectively. Recently
Dramatic damage by BP of DNA occurs in human bronchial
epithelial cells forming BPDE-dG adduct which could be
considered as "critical" for the initiation of human lung
cancer bronchial epithelial cells. Thus, active oxygen
species generated in cigarette smoke could play an important
role in the formation of this "critical" adduct in bronchial
epithelial cells (Fig. 1).
The Effect of Filter Containing Rosemary Extract on the
Formation of BPDE-dG Adduct. 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 quinone reductase activities.
Carnosol inhibits nitric oxide (NO) production in activated
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macrophage. The antioxidant property had been referred to as
the mechanistic basis of their protective effects.
With the aim of removing free radicals and reactive
oxygen species in the cigarette smoke a small amount of
rosemary powder was incorporated in a standard filter (see
Materials). The decrease in free radicals in the condensate
induced by the filters incorporating a rosemary extract was
estimated by the quantitation of the hydroxyl radical content
of CSS with a spin trap (TMPO) using LC-ESI-MS/MS. Under the
smoking conditions used a 30% decrease in the hydroxyl radical
was observed. Due to the efficiency of this filter to reduce
the level of the free radicals in cigarette smoke, as compared
to a comparable standard Marlboro filter without the additive,
I compared the effect of CS passed through this filter in
comparison to the standard filter on the formation of BPDE-dG
using MCF-7 cells.
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). 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.
However, the 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). Aside from the reduction of the free radicals, rosemary
powder may have also other mechanisms to reduce BPDE-dG
formation.
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Using whole rosemary extract (6 g.ml-1) also inhibits
CYP1A1 activity and DNA adduct formation by 80% after 6 hours
co-incubation with 1.5 pM BP in human bronchial epithelial
cells (BEAS-2B). Thus, 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.
it will be understood that the above description of the
present invention is susceptible to various modifications,
changes and adaptations, and the same are intended to be
comprehended within the meaning and range of equivalents of the
appended claims. The most obvious modification, for example, is
the use of various gel materials as the electroresponsive
composition of matter.
It will thus be seen that the objects set forth above,
among those made apparent from the preceding description, are
efficiently attained and, since certain changes may be made in
carrying out the above method (process) without departing from
the spirit and scope of the invention, it is intended that all
matter contained in the above description shall be interpreted
as illustrative and not in a limiting sense.
It is also to be understood that the following claims are
intended to cover all of the generic and specific features of
the invention herein described and all statements of the scope
of the invention which, as a matter of language, might be said
to fall there between.
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