,
Cancer Letters, 43 (1988) 173- 177
Elsevier Scientific Publishers Ireland Ltd.
so.,.,
,
Contribution of polycyclic aromatic compounds to the
carcinogenicity of sidestream smoke of cigarettes evaluated by
implantation into the lungs of rats
G . G r i m m e r a , H . Bruneb, G . Dettbarna, K.-W. Naujack", U. Mohrc a n d
R. Wenzel-Hartungb
biological lnstitute for Environmental Carcinogens, 0-2070 Grosshansdorf, bVaselinwerk, Biological Laboratory,
0-2000 Hamburg 11 a n d clnstitutefor Experimental Pathology, Medical School, 0.3000 Hannouer 61 (F.R.G.)
(Received 8 J u n e 1988)
(Revision received 22 August 1988)
(Accepted 31 August 1988)
non-aromatic material and 2- and 3-ring
PAHs .
Summary
Particles and semivolatiles from sidestream
smoke of cigarettes smoked on a smoking
machine were collected by a filter combination
consisting of a glass fibre filter and silanized
polystyrene beads. The extract of the glass
fibre filter was separated by a Sephadex LH-20
column chromatography into a fraction containing non-aromatic material plus polycyclic
aromatic compounds (PAC) with 2 and 3 rings
and a fraction consisting of PAC with 4 and
more rings. T o evaluate the carcinogenicity,
both fractions as well as the semivolatiles were
implanted into the lungs of Osborne- Mendel
rats at a dose level o f one cigarette per animal
and compared with three dose levels of
benzo[a]pyrene (BaP). The most pronounced
carcinogenic effect o f the sidestream smoke
(100 ng BaP per cigarette) was caused by the
fraction containing polycyclic aromatic hydrocarbons (PAH) with 4 and more rings (5
carcinomas of the lungs/35 rats). This fraction
represents ony 3 . 5 % by weight o f the total
sidestream smoke condensate. By contrast,
the semivolatile material did not provoke any
tumors. Only a small contribution to the total
carcinogenicity (1 carcinoma o f the lungs/35
rats) was observed for the fraction containing
0304-3835/88/$03.50
Keywords: polycyclic aromatic compounds;
carcinogenicity; sidestream smoke.
Introduction
From studies on chronic exposure to passive
smoking is concerned IARC [7] summarized:
ing from 1.1to 3.4, has been reported [I-51.
Epidemiological studies o n passive smoking
have been recently surveyed by Preston-Martin et al. [6].
As far a s the carcinogenic effect of passive
smoking is concerned IARC (7) summarized:
"Knowledge of the nature of sidestream and
mainstream smoke, of the materials absorbed
during "passive" smoking, and of the quantitative relationships between dose and effect that
are commonly observed from exposure to carcinogens leads to the conclusion that passive
smoking gives rise to some risk of cancer".
Thus it seemed worthwhile to investigate to
what extent well-known carcinogens present in
tobacco smoke, such a s PAH, azaarenes and
other PAC, may contribute to the carcinogenicity of the condensed sidestream smoke.
O 1988 Elsevier Scientific Publishers Ireland Ltd
Published and Printed in Ireland
Our experiments were started under the
working hypothesis that the carcinogenic activity is partly caused by a fraction containing
PAHs with 4 and more rings, as in the case of
gasoline engine exhaust [8,9] and diesel
exhaust [ l o ] , flue gas condensate from coalfired furnaces [I].] as well as mainstream
smoke condensate of cigarettes as published
by Dontewill et a]. [ E l .
To this end, particles and semivolatiles from
sidestream smoke of cigarettes smoked on a
smoking machine were collected by a filter
combination, as previously described [13].
The carcinogenic potency of the semivolatiles
as well as the non-aromatic and aromatic fractions of the particles trapped on a glass fibre filter were evaluated in Osborne Mendel rats by
implantation of the material obtained from a
single cigarette into the animals' lungs. The
three parts of the sidestream smoke were
compared with various dose levels of BaP. A
mixture trioctanoin and beeswax was used as
the vehicle. In the case of the PAH-containing
fraction, a sixfold dose level (corresponding to
6 cigarettes) was also applied.
Materials and methods
Collecting procedure
A single cigarette (German blend) was
smoked on a smoking machine. RM 1/G R
58.2 (Fa. Borgwaldt, Hamburg), according to
DIN-regulation. The sidestream smoke, particles and semivolatiles were collected in the filter system (see Figs. l and 2) under slightly
reduced pressure (air flow velocity about 2 0
mm/s). Figure 2 shows the filter system, consisting of a silicon-bound glass fibre particle filter and a subsequent filter for trapping
semivolatiles comprised of silanized polystyrene beads (Porapak PS, 60-80 mesh). This
material retains low boiling substances with
> 126OC (e.g. n-octane) completely at room
temperature even in the nanogram range. The
trapped substances could be extracted quantitatively and impurity-free from so-laden material. The sidestream material (particles and
semivolatiles) contained about 100 ng BaP/
Fig. 1. Collecting arrangement for particles and semi-volatiles in
main and sidestream smoke. 1. Filter system; 2, to vacuum
pump; 3, to smoking machine; 4, plain grind.
Porapok
PS
Fig. 2. Filter system for collection of particles (silanized glass fibre
filter) and semi-volatiles (Porapak PS) .
cigarette. Data on the concentrations of several
other PAC have been reported previously
[13].
Five cigarettes were smoked on a single filter
system; 11.8 mg semivolatiles/cigarette (increase in weight of the Porapak PS/filter per
cigarette) and 7 . 2 6 mg particles (glass fibre filter) were obtained.
Extraction
Glass fibre filters (14 filters corresponding to
7 0 cigarettes) were extracted with dichloromethane and subsequently with benzene. Solvents were evaporated in a rotatory
evaporator. Porapak P S of 1 4 filters were
extracted with acetone.
Separation of the residue of the glass fibre
filters
Chromatography on Sephadex LH-20 (50
g) was performed by elution with propanol-2
(300 ml, fraction 1; 7 0 x 1 6 . 2 mg) , and subsequently with acetone (500 ml) and methanol
(250 ml) (fraction 2 , 7 0 x 1 . 0 6 mg)
containing PAH with 4 and more rings. Details
of the method have been described elsewhere
[141.
For a sixfold dose level of the PAH-fraction,
350 cigarettes were smoked and worked up in
the same way.
Preparation of the implantation material
For implantation (e.g. 7 0 animals) the material to be tested (e.g. semivolatiles, 7 0 x 11.8
mg) was dissolved in acetone and heated to
about 50°C. T o the warm solution, beeswax
(70 x 3 2 mg) and trioctanoin (70 x 5 . 2 3
mg) were a d d e d , and the solvent was cautiously evaporated : (1)at 70°C under normal
pressure and subsequently (2) at 45OC and 2 0
Torr for 1 min. The final material had a melting
point of 52-56OC; fraction 1 (70 x 1 6 . 2
mg), beeswax (70 x 2 5 mg), trioctanoin ( 7 0
x 8.8 mg); fraction 2 (70 X 1 . 0 6 m g ) ,
beeswax ( 7 0 x 2 3 . 9 mg), trioctanoin ( 7 0 X
2 5 mg) .
Control groups
A mixture of beeswax and trioctanoin was
used as the vehicle. An untreated group and a
group that received the vehicle only served as
controls.
BaP (CAS : 50-32-8) purified by sublimation
(200°C,
bar) subsequently recrystallized
Table 1. Tumor incidence and survival time of rats after treatment with semivolatiles and fractions of the particle phase
from sidestream smoke.
Material
n
Dose
(mg)
Median survival
time in weeks
(range)
PAH-free material
and PAH 2 + 3 rings
PAH 4 and more rings
PAH 4 and more rings
(sixfold)
Semivolatiles
(gaseous phase)
BaP
BaP
BaP
Control (vehicle)
Control (untreated)
"1squamous cell carcinoma; 1 bronchiolar/alveolar adenocarcinoma
No. of carcinomas
Carcinoma
incidence
(%)
from toluene served as the positive control
(0.3, 0.1 and 0 . 0 3 mg BaP/animal) .
Animals
Inbred female Osborne-Mendel rats were
selected for the long term experiment according to Stanton et al. [15] and Hirano et al.
1161. The animals were obtained from the
Small Animals Section, Veterinary Resources
Branch, Division of Research Service, National
Institute of Health, Bethesda, MD. At the
beginning of the study the mean body weight
of the 3-month-old animals was 2 5 0 g. The
animals were housed under conventional conditions, 3 animals each per hanging plastic
Macrolon cage (type III), each containing
sterilized granulated wood bedding (Altromin,
C o . , Lage/Lippe, F.R.G .) . The implantation
technique was carried out a s described by
Stanton et al. [15].
Results and discussion
Table 1 presents the median survival time
and the number of lung tumors provoked by
various fractions of the sidestream smoke condensate at a dose level of o n e cigarette. In the
case of the fraction containing PAH with more
than three rings, a higher (sixfold) dose level
was additionally applied to a small number of
animals.
The fraction containing PAH with more
than 3 rings, although only 3 . 5 % by weight of
the total sidestream-smoke ( 2 8 . 8 6 mg) of a
single cigarette, induced a high carcinoma
incidence ( 1 4 . 3 % ) . These results lead to the
conclusion that most of the carcinogenicity of
sidestream smoke is caused by its content of
PAC consisting of 4 and more rings. By contrast, the semivolatiles of the sidestream smoke
provoked n o tumors at all. A small carcinogenic effect was produced by the particle phase
without the PAC with 4 and more rings. The
results are summarized in Fig. 3 .
T o provoke a comparable carcinoma incidence, 0.03 to 0 . 1 mg of pure BaP must be
applied, assuming that pure BaP alone exhibits
the same potency as BaP in a complex mix-
Fig. 3. Carcinoma incidence of the fractions of sidestream (semivolatiles and particulates)
ture. Provided that there is a linear doseresponse relationship in the dose range tested,
a dose of about 0 . 0 6 mg of BaP simulates the
effect of the PAC-fraction from sidestream
smoke of a single cigarette, which contains
about 100 ng of BaP. This means that only
0 . 1 7 % of the total carcinogenicity of the sidestream smoke PAC-fraction can be attributed to
BaP present in the smoke. Therefore, it may
be assumed that the carcinogenic effect results
from the mixture of PAC with 4 and more
rings. O n the other hand, cocarcinogenic or
synergistic effects (by induction of PAH-activating enzymes such a s monooxygenases)
within the fraction of PAC with 4 and more
rings cannot be excluded. Obviously, n o such
effect was found to arise from the non-aromatic fraction in the particles.
Acknowledgement
Das diesem Bericht zugrunde liegende Vorhaben wurde mit Mitteln des Bundesministers
fiir Forschung und Technologie unter d e m
Kennzeichen HDA3-01HK774 gefordert. Die
Verantwortung fiir den Inhalt dieser
Veroffentlichung liegt bei den Autoren.
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