COPY NO. ... .......
IMPERIAL TOBACCO LIMITED
RESEARCH LABORATORY
MONTREAL
RESTRICTED
PROGRESS REPORT
RESEARCH DEPARTMENT
January June 1977
ISSUED BY: Mr. R.S. Wade DATE ISSUED: August 5.
1977.
DISTRIBUTION:
Copy No.: 1. Lib rary 21. Mr. A.F. El Naggar
2. Mr. R.M. Gibb 22. Mr. E.R. Freiesleben
3. Mr. R.S. Wade 23. Dr. G.W. Boswall
4. Mr. S.M. Candlish 24. Mr. L. Bouchard
5. Dr. T.A. Smith 25. Mr. H. Roubicek
6. Dr. S.J. Green 26. Miss R.R. Smith
7. Mr. E. R ittershaus 27. Mr. B.J. Stirling
8. Dr. F. Seehofer 28. Mrs. J. Johnson
9. Dr. W.B. Fordyce 29. Mr. R.L. Rice
10. Dr. D.G. Felton 30. Mr. A. Schaffer
H. Dr. D.G. Felton-- 31. Mr. E.P. Gage
12. Dr. C.1. Ayres 32. Mr. W.A. Gray
13. Dr. I.W. Hughes 33. Mr. A.1. Kalhok
14. Dr. R.A. Sanford 34. Mr. W.J. Ross
15. Dr. R.A. Sanford 35. Mr. N.D. Maclennan
16. Mr. R.G. Nicholls 36. Mr. C.J. Brown
17. Mr. R.G. Nicholls 37. Mr. C. Warren
18. Dr. M.H. Bilimoria
19. Dr. P.J. Dunn
20. Mr. M.P. Scherbak
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INDEX
Page Code Title Researcher(s)
a Summary of Research Department Activities.
I T-0575 Delhi Research Station Samples for
Industry G.W.B.
Collaborative Evaluation.
2 T-0758 The Manufacture of Cigars for Smoking
Tests G.W.B.
of Tobacco from the L'Assomption Experimental
Farm - 1971.
3 T-0770 Cigar Tobacco Production Committee's
G.W.B.
mechanization and Curing Experiments Conducted
at the L'Assomption Experimental Farm.
4 T-0774 The Manufacture of Cigars for Smoking
Tests G.W.B.
of Tobacco from the L'Assomption Experimental
Farm - 1974.
5 T-0820 Study of Methods to Promote Earlier
Maturing G.W.B.
of Flue-Cured Tobaccos.
7 T-4900 Leaf Research - Flue-Cured Field
Experiment - G.W.B.
197).
8 T-6420 Mechanization of Tobacco Production.
G.W.B.
11 T-7709 Changes in Bronchial Epithelium and
Other J.J. &
Tissues Induced by Tobacco Smoke. M.H.B.
1. Effects of Tobacco Smoke on Tracheo-
bronchial Epithelium. The Incorporation
of Precursors into Glycoproteins.
2. Bronchial Epithelia] Permeability.
16 T-7708 Microbial Mutagenicity Tests in Tobacco
and m.H.B.
Health Research.
18 T-7710 Biochemical Effects of Tobacco Smoke
Exposure M.H.B.
in Experimental Animals. J.J.
1. Metabolle Activation of Ary) Amines
by Rodent Kidney and Lung Preparations.
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Page Code Title Researcher(s)
2. Metabolic Activation of Aryl Amines
by Dog Kidney.
3. Papers accepted for publication.
21 T-8077 Studies in Human Smoking Behaviour.
P.J.D.
T-7051
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a
SUMMARY OF RESEARCH DEPARTMENT ACTIVITIES
JANUARY - JUNE 1977
This report covers the research activities in the
areas of
Agrology. Biochemistry & Microbiology and Smoking
Behaviour.
Cooperative work with Agriculture Canada
Experimental Stations
continues in evaluating promising new varieties,
pesticides and tobacco
production practices. As a consequence of this
work the licensing of a
new cultivar L64-201 as a commercial cigar filler
variety has been
requested recently by the L'Assomption
Experimental Farm. At the Delhi
Research Station, the testing of insecticides has
resulted in having
Pirimor and Lannate-L registered and recommended
for controlling aphids
on tobacco. These products are now available to
replace Thiodan which is
no longer recommended or registered for use on
tobacco because its residues
in the cured tobacco have tended to be higher than
desirable. The C.T.K.C.
sponsored program at the Imperial farm which has
been active in studying
the mechanization of harvesting and curing
flue-cured tobacco in recent
years is producing this summer about 25 acres of
tobacco. This tobacco
will be used in testing the Johnson Cross-Flow
modular kiln and evaluating
additional instrumentation placed on bulk and
forced-air kilns. The Cross-
Fio. kiln has been modified somewhat in an effort
to correct some of the
problems which were identified in 1976 in
obtaining a uniform cure throughout
each module. Additional instrumentation has been
added to the bulk kilns
and to the forced-air (TCS-100) kiln. With these
instruments the kilns
should be supplied with optimum combinations of
temperatures and humidities
throughout the various stages of the flue-curing
process. Redesigned heat
exchangers have also been produced to reduce the
fuel consumption during
flue-curing.
With regard to the C.T.M.C. sponsored research
program at McGill,
Dr. Hogg, the grantee, will be accepting a new
post at the University of
British Columbia on September Ist. and will
continue his work there on
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b
bronchial epithelia] permeability and tight cell
junctions. The
C.T.M.C. will continue to provide some support for
his projects.
Dr. Witschi of the University of Montreal,
consultant on the Parts of
the program carried out by Dr. Bilimoria and Ms.
Johnson, left at the
end of June to take up a position at Oak Ridge
National Laboratories
in Tennessee. The loss of these two very capable
researchers is
unfortunate but Dr. Bilimoria and Ms. Johnson will
be continuing their
work at McGill under the direction of Dr.
Richardson, Chairman of
Pathology. There will also be periodic
consultation with Dr. Hogg
as well as occasionally with Dr. Witschi.
During the first part of the period under review,
development of
a bioassay based on the inhibition of glycoprotein
synthesis of the
mucous-secreting system of the tracheal bronchial
tissues by tobacco
smoke was pursued. Although interesting changes in
the incorporation of
leucine were observed, this project was put aside
in order that the
technique of measuring changes in the permeability
of bronchial
epithelium using a radioimmunoassay employing
horseradish peroxidase
could be learned before Dr. Hogg's departure.
Early indications are
that the technique shows a smoke-related dose
response.
Good progress has been made on the project on aryl
hydrocarbon
hydroxylase (AHH), one of the microsomal enzymes
responsible for the
metabolism of drugs and toxicants. This enzyme
system is responsible
for converting inert polycycl;c hydrocarbons to
highly reactive inter-
mediates, which then react with critical
macromolecules such as DNA.
This could ultimately lead to mutation and cancer.
Inability of cigarette smoke to induce AHH in
guinea pig lung
was considered an observation important enough to
merit publication in a
toxicology journal. Similarly, the observation
that exposure to cigarette
smoke can induce renal AHH in rodents, and that
this induction resulted in
increased mutagenic activity in the Ames test, led
to another publication
in this same journal. This increased mutagenicity
shown by tissue that
has been obtained from cigarette smoke-exposed
animals is highly
significant on account of the excellent
correlation obtained between .1th.
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carcinogenicity in animals and mutagenicity in
micro-organisms. This
work is being extended to include higher mammals,
including man, some
results of which, in the dog, are described in
this report.
The technique of monitoring alveolar carbon
monoxide levels in
exhaled air of smokers has proven useful to
distinguish patterns of
smoking forspecific changes in product design.
Lower alveolar carbon
monoxide resting levels were observed when
cigarettes of higher nicotine
yield were smoked, indicating that the higher
nicotine delivery probably
caused a lesser degree of inhalation. When the
product was changed to
give equivalent nicotine delivery with a lower
pressure drop, the smokers
did not decrease the effort with which the puffs
were taken. This
resulted in increased nicotine intake into the
mouth and also somewhat
higher alveolar resting CO levels. Studies to
further explore the
relationship between smoking patterns, deliveries
and inhalation are
continuing.
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T-0575 Delhi Research Station Samples for Industry
Collaborative Evaluation.
Personnel: G.W. Boswall, B.J. Stirling, R.R.
Smith, H. Roubicek.
Objective: To evaluate and compare cigarettes made
from varieties and
other flue-cured tobaccos produced under various
experimental
conditions by the Delhi Research Station.
Status: The Delhi Research Station produces
numerous tobacco samples
each year to evaluate the merits of different
varieties, modified
cultural and curing practices, and new
agricultural chemicals
such as pest control products for the production
of flue-cured
tobacco. A select number of samples from the more
promising
varieties and treatments are made available to the
four
Canadian cigarette manufacturers for their
evaluation of smoking
quality. Only when samples reach or surpass
certain minimum
chemical and physical parameters and have
acceptable smoking
quality, are new varieties or treat-ents
considered for inclusion
in the Ontario Ministry of Agriculture and Food
Publication 298
"Tobacco Production Recommendations".
The tobacco samples submitted by the Station from
the 1976
crop to the cigarette manufacturing companies are
listed below
together with the manufacturers of each group of
samples.
Sample
6 Insecticide Samples
5 Nematicide Samples
2 Fungicide Samples
3 Growth Regulator Samples
12 Variety Samples
Cigarette Manufacturer
Imperial Tobacco
Benson & Hedges
Rothmans
Rothmans
Macdonald Tobacco
Most of these cigarette samples have been made and
distribvted
to each company for an evaluation of smoking
quality.
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T-0758 The Manufacture of Cigars for Smoking Tests
of Tobacco
from the L'As5omotion Experimental Farm - 1971.
Personnel: G.W. Bos.all, R.R. Smith, H. Roubicek.
Objective: To evaluate the subjective smokina
properties of cigar tobaccos
from various plant breeding, topping and spacing,
and curing
experiments conducted at L'Assomption.
Status: Until a few years ago we undertook to
evaluate the smoking
quality of the majority of experimental cigar
tobacco samples
produced at L'Assomption. However, the
Experimental Farm
program has been greatly expanded, particularly in
plant
breeding, and now we accept as our main
responsibility the
manufacture of samples into cigars and leave the
smoking
tests of varieties, strains and selections to a
panel operated
by L'Assomption and composed of members recruited
throughout
the Agriculture Canada Organization. Furthermore,
the priorities
of cigar product development work leave our
smoking panel very
little available time for L'Assomption samples.
However, the
Imperial smoking panel has just completed all the
twenty eight
tests involved in the Topping and Spacing
Experiment but the
results have not yet been issued as a memorandum.
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T-077D Cigar Tobacco Production Committee's
Mechanization and
Curing Experiments Conducted at the L'Assomption
Experimental Farm
Personnel: G.W. Boswall, R.R. Smith, H. Roubicek.
Objective: To evaluate the subjective smoking
properties of cicar filler
samples harvested and cured by various methods.
Status: Cigar filler production in Quebec has been
decreasing
dramatically in recent years. Consequently a Cigar
Tobacco
Production Committee composed of representatives
of the
cigar manufacturers, the Socitt6 Cooperative de
Montcaim,
and the Canada and Quebec Departments of
Agriculture, was
formed in 1973 to study the situation. It became
obvious
that cigar tobacco lacks both the modern
production techniques
(mechanization) and the dollar returns that
farmers experience
from other crops. The committee instituted an
experimental
program to modernize the harvesting and curing of
cigar filler
which, if successful, would hopefully encnurage
farmers to
resume cigar tobacco production. The Quebec
Department of
Agriculture imported two mechanical harvesting
machine!~ and
two bulk kilns were erected at the Experimental
Farm in 1974.
Each year the more promising cigar tobacco samples
that have
been produced are selected for cigar manufacture
for smoking
tests. Results of these smoking tests indicate
that all
curing methods, including the use of forced air
with controlled
temperature and humidity which can complete a cure
in 7 to 8
days, are acceptable to the smoker.
Eleven samples from the 1976 crop have been
selected for
smoking tests and these samples are being
processed at the
Joliette leaf plant in preparation for cigar
manufacture.
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T-0774 The Manufacture of Cigars for Smoking Tests
of Tobacco
from the L'Assomption ExDerimental Farm - 1974.
Personnel: G.W. Boswall, H. Roubicek.
Objective: To evaluate the subjective smoking
properties of cigar tobaccos
from various varieties and various plant breeding
and cultural
experiments.
Status: The samples selected by the L'Assomption
Experimental Farm fo,
manufacture into cigars by Imperial Tobacco have
been processed
into short filler and the seven s-mples of cigars
from the
preliminary variety test were forwarded to
L'Assomption in
1976. The rest of the samples were manufactured
recently and the
cigars will be evaluated by the smoking panels
indicated below.
SAMPLES EVALUATED BY
7 Varieties (preliminary test) L'Assomption panel
14 Varieties (final test) L'A5somption panel
24 Germ Plasm entries L'Assomption panel
8 Topping and Spacing Imperial panel
Experiment
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5.
T-o820 Study of methods to Promote Earlier
Katuring of
Flue-Cured Tobaccos.
Personnel: G.W. Boswall, S. Webster, 8.J.
Stirling-
Objective: To evaluate and compare the cultural
practices required to
achieve early maturity and the completion of
harvest by
student-t before they return to classes.
Status: The 1976 experiment was confined to the
Virginia 115 variety
which was grown in five different plots ranging in
size from
0.5 to 4 acres. The tobacco seedlings were
produced in the
greenhouse at two different rates of seeding and
three
different procedures were used for hardening the
seedlings
prior to planting in the field. Additional
experimental
factors included the date of planting and the time
and height
of topping. These large plots were machine
harvested and
cured in bulk kilns. In addition samples obtained
by hand
priming small plots were cured and retained for
sorting into
grades and analyses for nicotine and sugars. All
plots were
successfully harvested by September 10. It appears
that the
date of planting and the topping practice had more
effect on
the grade and chemical composition of the tobacco
than did the
different greenhouse procedures. The data are
being
incorporated in a report covering the work
sponsored by the
CTMC on the Imperial Farm in 1976.
In 1977, because the Imperial Farm uses a Roanoke
Automatic
harvester, the emphasis in the field plots is to
try various
procedures which may help to increase the
proportion of
desirable and saleable leaf in a crop by
minimizing the amount of
foreign material, increasing the nicotine level,
and identifying
procedures which produce plants and leaves whose
physical
characteristics are suitable for mechanical
harvesting and
random leaf bulk curing.
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T-0820 (Cont'd.)
Apart from the varieties involved (Virginia 115,
Delhi 76
and Synthetic D-57) a novel procedure is to be
used which
calls for the removal of approximately the 4
bottom leaves
about the middle of July. This -will permit the
automatic
harvester to be operated above the ground level
f.,r the
first priming and thus reduce the amount of sand
and weeds in
the harvested tobacco. The fewer leaves (14-15) on
the plants
may contribute to a higher nicotine content and
should permit
the complete harvesting of the crop in four
primings. Separate
identical small plots in four replications have
beem-establ i shed
for hand harvesting and curing in small chambers
at the Delhi
Research Station. These small samples should
provide reliable
yield, grade, and chemical data.
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T-4900 Leaf Research - Flue-Cured Field Experiment
- 1971.
Personnel: G.W. Boswall, B.J. Stirling, R.R.
Smith.
Objective: To systematically evaluate th,-.
relationships between the
grading characteristics of flue-cured tobacco and
the
physical, chemical, and tar producing properties.
Status: A field experiment was conducted on the
Company farm in
1971 as the laboratory's initial attempt to
establish
correlations between the chemical, physical and
tar
production properties of flue-cured grades and the
con-
ventional grading criteria such as colour,
blemish, grain,
body etc. As reported in our previous progress
report it
was decided in 1974 to send all the accumulated
analytical
data to Imperial Tobacco Limited, Bristol for
statistical
analysis and interpretation. A draft of a
statistical report
was received from Bristol early in 1977. Dr.
Springali, who
conducted the statistical study and authored the
report,
visited us in May. He discussed with
representatives of our
R. & D. Dept. and our Leaf organization the
details of the
experiment and his interpretation of the results
of the
statistical studies he made on the accumulated
data. For all
involved in the experiment this interchange of
information
provided a better understanding of the scope and
limitations of
the experiment. With the additional information
obtained during
his visit, Dr. Springall will add to and modify
some aspects of
his original draft and forward a final edition to
us in the
near future.
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T-6420 mechanization of Tobacco Production
Personnel: G.W. Boswall , B.J. Stirling, R.R.
Smith and H. Roubicek.
Objective: To investigate the various forms of
mechanization that
might be adapted to tobacco production and reduce
its manual
labor requirement.
Status:_ Although the Roanoke Automatic harvester
has been used quite
successfully in these studies for the past few
years, random
leaf orientation has been identified a- causing
some curing
problems. Therefore a large part of our farm
resources is
being devoted to studying additional
instrumentation for kilns
to improve their ability to provide the optimum
combinatiorsof
temperatures and humidities throughout the curing
process.
A 'CrDSS-flow kiln' which could lead to more
complete mechaniz-
atiort of harvesting and curing is being tested
again in 1977.
The plans for 1977 may be outlined as follows:
LEAF CURING
A. Bulk Kilns
Curing tests run on the CTMC farm in 1975 and 1976
indicate
that the flue-curing p ocess can be simplified to
a predictable
science rather than the present "art" acquired by
experience.
The objectives in carrying out this work can be
stated as
follows:
(1) to be able to provide sufficient
instrumentation, kiln
modifications, and curing program information to
enable
the average farmer to become an efficient cureman
with
minimum instruction.
(2) to be able to make the most efficient use of
energy in
the curing process.
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9.
T-6420 (Cont'd.)
Forced Air Kiln (TCS-100)
In an attempt to assist the majority of Ontario
tobacco
producers, tests will be conducted on the existing
forced
air kiln located on the C.T.M.C. experimental
farm. These
tests are aimed at tobacco curing energy
requirements and
quality improvements. Initial work will involve
optimum
environmental control during the curing process.
This work
will parallel the work on bulk curing.
Heat Exchanqer
Primary heat exchanger evaluation revealed
promising results
during the 1976 test period. A report has been
prepared and
was distributed in March, 1977.* Further testing
with heat
exchangers is required to gain confidence in the
findings of
1976. Design alterations will be made in an effort
to over-
come the problems encountered in 1976. Tests will
be conducted
on bulk and forced air curing systems.
B. Cross-Flow Kiln
A problem which has been recognized in recent
years is the
general poor quality of random cured leaf coming
from farms
which use automatic tobacco harvesting machines
regardless of
the type of kilns (traditional, TCS-100, or bulk)
that are
used. The Cross-Flow kiln developed by Dr. W.H.
Johnson at
N.C.S.U. is believed to have the potential to
provide the
necessary conditions to produce a good quality
cure of random
leaf, and additionally could be adapted to provide
a more
complete mechanization of the tnbacco harvesting
and curing
operations while maintaining a quality cured
product.
"Reducing Energy For Curing Tobacco in Ontario" by
R.G. Winfield and
Ron Minshall for Ontario Ministries of Agriculture
and Food, and Energy,
Toronto, Ontario, and Canadian Tobacco
Manufacturers' Council, Montreal,
Quebec. Xh.
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10.
T-6420 (Cont'd.)
The Cross-Flow kiln was installed, evaluated at
reduced
throughput, and modified to some extent in 1976
but it
requires additional modifications for the 1977
season and
it should be operated -at full capacity in order
to realistically
evaluate its performance. The following
modifications will
be made prior to the 1977 harvest season.
(a) Modules: The 1977 module design will be such
that
it overcomes the problems experienced in 1976.
(b) Heat Exchanger: The cross flo. curing system
w.-s
initially designed to incorporate the use of a
heat
exchanger in an effort to minimize the curing
energy
requirements. The heat exchanger tested in this
system in 1976 was found to be inadequate and for
this reason, a modified unit will be tested in
1977.
(c) Reorder Equipment: As yet, the efforts aimed
at fast
reorder have not been totally successful. It is
planned
to use the cross flow as a test kiln for further
work
in this area. Plans are aimed at testing various
water
sprays and environmental conditions.
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T- 7709 Changes in Bronchial Epithelium and Other
Tissues Induced
by Tobacco Smoke.
Personnel: J. Johnson, M.H. Bilimoria, M.P.
Witschi, J.C. Hogg.
Objective: To establish biochemical parameters of
tobacco-smoke
induced lesions in the bronchial tree and
pulmonary
parenchyma. It is hoped that one or several
parameters
can be defined which may then serve as a
short-term
screening test for the evaluation of product
improvement.
To maximize the relevance of such a test to the
effects
of smoking on humans, priority was given to
studies of
the tracheobronchial tree, over those involving
lung
tissues.
Status: 1. Effects of Tobacco Smoke on
Tracheobronchial Epithelium.
The Incorporation of Precursors into
Glycoproteins.
Work continued on the incorporation of radioactive
precursors into tracheobronchial glycoproteins.
These
experiments were designed to develop a bioassay
based on
the inhibition of glycoprotein synthesis of the
mucous-
secreting system of the tracheobronchial tissues
by
tobacco smoke.
It has previously been shown in this laboratory
that
tobacco smoke caused an inhibition of
incorporation of
leucine into glycoproteins when rats were exposed
to
tobacco smoke from 5 standard flue-cured
cigarettes Aen
sacrificed immediately after smoking but not at 3,
24 or
48 hours after. It has also been shown that smoke
was
ineffective in causing significant changes in
glycoprotein
synthesis with this smoking regime when
glucosamine was
used as precursor.
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T-7709 (cort'd. )
12.
Based on the findings of other researchers who
have
shown that significant changes in qoblet cell
number
occur only after as long as 12 eeks of smoke
exposure,
it as decided to increase our smoke exposure to 5
cigarettes/day for 4-7 days.
Experiments with glucosamine, leucine and fucose
were
thus carried out using this increased level of
smoke
exposure. One group of rats was exposed to smoke
from 5
cigarettes daily for 4 consecutive days and
sacrificed
24 hours after smoking while another was exposed
to the
same number of cigarettes daily for 5 consecutive
days
and sacrificed 72 hours after smoking. Again only
leucine incorporation showed any significant
changes at
this increased smoke dosage. Interestingly,
leucine
incorporation showed significant increases both at
4 and
5 days of smoke exposure. Incorporation of
glucosamine
and of fucose was unaffected by the increased
smoke
exposure.
Due to the departure in the near future of both
Drs. Hogg
and Witschi, the work was interrupted at this
point, in
order that the technique and methodology of the
project
"Bronchial Epithelial Permeability" (described
below),
could be learned while the expertise was still
available.
It is hoped to resume this project when the time
and
opportunity become possible.
2. Bronchial Epithelia) Permeability.
Studies conducted by Hogg and co-orkers and
supported by
the Canadian Tobacco manufacturer's Council, have
shown
that cigarette smoke and other irritants are
capable of
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13.
T-7709 (cont'd.)
changing bronchial epithelial permeability
allowing large
molecules to penetrate to the submucosa and thence
into
the systemic circulation. These studies were done
using
an electronic microscopic tracer technique. More
recently,
these researchers have shown that this effect can
also be
readily monitored using a simple radioimmunoassay.
The
technique consists of instilling an antigen
(horse-radish
peroxidase - HRP) into the tracheobronchial tree
and
measuring its appearance in the blood plasma by
radio-
immunciassay.
The actual measurement of HRP in the plasma
utilizes the
technique of solid-phase radioimmunoassay in
antibody
coated disposable polystyrene tubes. The principle
is
based on the ability of antibody to strongly
adsorb to
the tube surface thereby providing sites for
competitive
binding of labelled (tracer) and unlabelled forms
of
antigen. When both tracer and uniabe)led antigen
are
introduced into such coated tubes they
competitively bind
to the antibody sites. After binding takes place,
and the
free tracer antigen is removed from the tubes, the
amount
of bound tracer can be obtained by counting the
radioactivity
remaining in the tube. Because of the competitive
binding,
the amount of bound tracer varies inversely with
the total
antigen present. The method is sensitive such that
nanogram
quantities of antigen can be measured.
This technique has been applied to smoke-exposed
guinea-
pigs in the following way:
Groups of guinea pigs weighing 300-400 gms. were
separated
into groups of 6-8 and exposed to smoke or were
sham-smoked
(controls). During exposure the animals were
lightly
anaesthetized with Nembutal, tracheostomized and a
cannula
placed in the carotid artery for collection of
blood
samples. A test dose of HRP (2 mi. of 5 mg/mI HRP)
as
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BATCo document for Province of BritiSh Columbia 23
April 1999


T-7709 (corit'd.)
14.
instilled into the tracheobronchial tree via the
tracheostomy tube and 4 blood samples were
collected over
a period of 20 minutes at 0, 10, 15 and 20 minute
intervals.
At the 20 minute time point, test animals were
then exposed
to various levels of smoke and blood samples taken
at 25,
30, and 40 minutes after exposure. Sera
(containing HRP)
from the blood samples were then collected by
centrifug-
ation and subjected to the radioimmunoassay
described above.
The rate of appearance of HRP into the blood prior
to
exposure to smoke was thus compared to its
appearance
following exposure and the ratio of these slopes
was used
as a measure of epithelial damage.
Four groups of guinea-pigs were used, viz. a
control group
(shamr-smoked with 20 puffs air), two groups which
were
exposed to 5 puffs and to 20 puffs smoke from
standard flue-
cured tobacco cigarettes. and a fourth group which
was
exposed to 20 puffs smoke from cigarettes made
from non-
tobacco material (saw-dust obtained from white
pine wood).
Our results show a tendency toward a dose response
with
tobacco smoke, with 5 puffs cigarette smoke
showing a slight
but insignificant increase in appearance of HRP
after
exposure. Sera from animals exposed to 20 puffs
tobacco
smoke showed a significant increase in rate of
entry of
HRP. Samples from animals exposed to 20 puffs of
non-tobacco
smoke also showed a significant increase in HRP
similar to
that obtained with 20 puffs tobacco smoke, while
control
samples showed exactly the same rate of appearance
of HRP
in blood before and after sham-smoking.
Research has shown that the appearance in blood of
an
electron microscopic tracer administered to
bronchial
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BATCo document for Province of British Columbia 23
April 1999


T-7709 (cont'd.)
epi thel ium is associated with a penetration of
the tracer
through the epithelia] barrier as analysed by
electron
microscopy. Using this tracer technique,
morphological
changes were detected in all areas of the
tracheobronchial
tree after doses of tobacco smoke from 5
cigarettes or more.
However, in the present study electron microscopy
of tissue
specimens from trachea and bronchi from all 4
groups failed
to show any morphological changes. It is thus
presumed
that the levels of smoke exposure (5 puffs and 20
puffs).
while showing increased rates of appearance of HRP
after
exposure, are insufficient to cause detectable
morphological
changes. Further experiments are therefore
underway, in
which animals are exposed to tobacco smoke
continuously
from 20-40 minutes, with the smoking machine
delivering
5 puffs/minute of smoke. In this way the animals
will
receive an almost continuous stream of smoke from
4-5
cigarettes over 20 minutes. Hopefully, both a
larger
and more significant rate of leakage of HRP will
be
detected with concomitant detection of
morphological
changes as visualized by electron microscopy.
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BATCo document for Province of BritiSh Columbia 23
April 1999


16.
T-7708 Microbial Mutaqenicity Tests in Tobacco and
Health Re5e~rch.
Personnel: M.H. Bilimo,ja.
Objective: Tc use microbial mutagenicity tests
(including DNA repair
tests) for the detection of potential mutagens and
carcinogens in smoke, and for comparing tobacco
products.
Status: Several years ago we had used quite
extensively the DNA
repair test Hevised by Rosenkranz to determine the
possible
mutagenicity of pure compounds known to occur in
tobacco
smoke, as well as several smoke condensates. This
study
revealed that several compounds such as cyanide,
acrolein,
catechol, pyrogallol, hydroquinone and nicotine,
known to
occur in tobacco smoke, could damage DNA, and
therefore,
possibly, were mutagenic.
We are now interested in finding whether these
compounds which
appear to damage DNA, as shown by the Rosenkranz
test, are
also mutagenic in the Ames test. A detailed study
of nicotine,
in which we employed metabolic activation systems
prepared from
several rodent tissues, showed that it was not
mutagenic in the
Ames test. We examined hydroquinone next, mainly
as a result
of a request by the International Agency for
Research on Cancer
to cite our hydroquinone study in a monograph
being prepared
by this W.H.O. body. To detect the mutagenicity of
hydro-
quinone in the Ames test, we employed metabolic
activation
systems prepared from rat liver, lung and kidney,
and employed
the 3 strains of S. typhimurium, viz., TA-98,
TA-100 and TA-1537,
recommended by Ames. All these metabolic
activation systems
failed to convert hydroquinone to utagenic
intermediates for
the S. typhimurium strains referred to above.
Even though compounds such as nicotine and
hydroquinone, which
indicate possible damage to DNA, are negative in
the Ames test,
interest continues in the DNA repair test. This is
understandable
since any compound damaging DNA should be
considered hazardous.
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BATCo document for Province of BritiSh Columbia 23
April 1999


T-7708 (Cont'd.)
Further, since the Ames test is valuable for
detectina
compounds with possible cancer initiating
activity, it is
suggested that the DNA repair test also picks up
cocarcinogens
and tumour promoting agents as indicated by the
positive effect
of several phenolic compounds, which have been
shown to be
either cocarcinogenic or tumour promoters in
animal tests. In
any case a battery of these short-term tests is
recommended
in detecting the possible
mutagenicity/carcinogenicity of
chemical agents.
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BATCo document for Province of BritiSh Columbia 23
April 1999


T- 7710 Biochemical Effects of Tobacco Smoke
Exposure in
Experimental Animals.
Personnel: M.H. Bilimoria, J. Johnson, H.P.
Witschi, J.C. Hoag.
Objective: To identify biochemical parameters that
will serve as reliable
indicators of tissue damage produced by inhalation
of tobacco
smoke.
Status: 1. Metabolic Activation of Aryl Amines by
Rodent Kidney
and Lunq Preparations.
We exposed random bred guinea pigs and
Sprague-Dawley
rats to cigarette smoke and found that renal AHH
activity
increased considerably 6-12 hours after exposure.
We
considered such a study interesting Fron, the
point of view
of devising short-term tests for tobacco smoke,
since aryl
hydrocarbon hydroxylase (AHH) plays an important
role in
the metabolic activation of polycycl;c
hydrocarbons to
mutagens, and ultimate carcinogens. Furthermore,
epidemiological studies suggest a link between
cigarette
smoking and increased mortality from bladder
cancer.
When we prepared 9,0009 supernatants from these
rodent
kidneys, we found that while we could activate
2-anthramine
to intermediates mutagenic for S. typh;murium
TA-98, we
could not activate 3,4-benzo ict) pyrene or
tobacco smoke
condensate to give mutagenic intermediates for
this strain.
This was reminiscent of the 9,000g supernatants
prepared
from rodent lungs which too, could activate
2-anthramine,
but not 3,4-benzo (al pyrene or tobacco smoke
condensate.
Consequently, it was of interest to find out
whether other
aryl amines were similarly activated by rodent
kidney and
lung preparations. Both lung and kidney
preparations from
these two rodents fai I to activate both a and
3-naphthylamines
to mutagenic intermediates for TA-98 at
concentrations up to
50 uy/plate. However, when we examined
2-ani.ofl~orene, we
found that guinea pig kidney 9,0009 supernatant
could activate CZ)
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BATCo document for Province of BritiSh Columbia 23
April 1999


19.
T-7710 (Cont'd.)
this compound to mutagenic intermediates for TA-98
just
as it did 2-anthramine. Inability to activate the
naphthylamines was a surprise, since
B-naphthylamine is
a well known bladder carcinogen. However, more
work will
have to be done before any conclusions can be
drawn
regarding the nature of the enzymes involved in
the
activation of aryl amines, or perhaps, even the
validity
of the Ames test.
2. Metabolic Activation of Aryl Amines by Doq
Kidney
One of the aims of our biological research
programme at
McGill University is to find out if conclusions
drawn from
our rodent studies are applicable to higher
mammals
including man. As a step in this direction we
decided to
find out whether dog kidney can activate aryl
amines to
mutagenic intermediates for S. typhimurium TA-98.
The next
step would be to examine human kidney in such
studies.
In the case of one dog kidney sample that was made
available
from another experiment in progress in the
Pathology Department,
we have succeeded in obtaining a 9,000g
supernatant that can
activate 2-anthramine, but not 3,4-benzo(a)pyrene
(even up to
200 ug/plate tested), to intermediates mutagenic
for
S. typhimurium TA-98. Relatively high
concentrations of
2-anthramine (UP to 50 ug/plate) had to be used
before a
Positive response was obtained with dog kidney. In
contrast,
using the guinea pig preparation only I ug/plate
produced a
very large number of revertants of S- typhimurium
TA-98. We
hope to repeat this observation and extend it to
human kidney
samples.
3. Papers accepted for publication.
0) M.H. Bilimoria, J. Johnson, J-C. Hogg and H.P.
Witschi.
Pulmonary Eryl hydrocarbon hydroxylase: Tobacco
smoke-
exposed guinea pigs. Toxicol. Appl. Pharmacol.,
1977.
In press.
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BATCo document for Province of British Columbia 23
April 1999


20.
(ii) Idem, Activation of 2-aminoanthracene to
muraoenic
intermediates by guinea piq and rat microsomal
enzymes following tobacco smoke inhalation.
Ibid., 1977. In press.
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BATCo document for Province of BritiSh Columbia 23
April 1999


21
T-8077 Studies in Human Smoking Behaviour.
T-7051
Personnel- P.J. Dunn.
Objective: To study the real effects on smokers of
modifying smoking
products in order to reduce the intake of harmful
substances.
Status: An evaluation of the present (Freiri)
slave smoking equip-
ment has demonstrated that this equipment design
cannot be
altered to duplicate the puffing rates of human
smoking.
As a consequence Ontario Research Foundation has
been engaged
to develop an improved smoking duplicator which
will operate
in conjunction with the present differential
transducers.
Tandburg recording equipment and the
microprocessor built by
McGill Univ-rsity. This new design will
incorporate a mouth-
piece very similar to that of GR & DC Southampton
and may
assist in co-operative and/or comparative studies.
A report describing the changes in smoking
parameters as well
as alveolar CO concentrations of 4 Players Filter
Regular
smokers with a cigarette of 30% increased nicotine
yield has
been completed and it is hoped to be submitted for
publication
in the near future. A similar study including 4 du
Maurier
Regular smokers is presently being put into report
form.
A comparative study has been completed where a
group of 7 smokers
has been monitored with a cigarette constructed to
yield
equivalent nicotine with 30% lower pressure drop
relative to
the normal smoking product. From the analysis of
butts
collected through the course of this study, it was
noted that
all smokers consistently took more nicotine fro.
both cigarettes
when smoking freely as opposed to using the
cigarette holder
and monitoring instrumentation. There was a 56%
increase in
mouth nicotine intake with the decreased pressure
drop cigar-
ette. Although this cigarette delivered 29% less
CO under
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BATCo document for Province of BritiSh Columbia 23
April 1999


22.
T-8077 (cont'd.)
T-7051
standard conditions than the normal cigarette, the
alveolar
CO concentrations of all smokers decreased by only
16t with
this product change. A report will be forthcoming
after
complete data analysis.
In order to better appreciate the effect of
ventilated
cigarettes on human smoking behaviour, a study has
commenced monitoring the smoking parameters and
resting
alveolar CO levels of smokers with their normal
ventilated
cigarette as well as a product offering less
ventilation and
thus higher nicotine delivery. A complementary
study has
also been planned with a group of smokers of
milder unventilated
cigarettes changing to cigarettes of varying
degrees of venti-
iation.
To demonstrate the effect of variations in human
smoking on
the transfer of CO, tar and nicotine, extremes in
velocity
conditions were carried out on standard smoking
machine
apparatus. It has been found that under conditions
of con-
stant puff volume and puff number, TPM reaches a
maximum and
then decreases steadily with increasing velocity.
Under these
same conditions, nicotine delivery is inversely
related to puff
velocity. Levels of CO, on the other hand,
increase and then
level off with increasing puff velocity. In order
to appreciate
whether these results were related to specific
cigarette design,
a similar study is being conducted on cigarettes
of different
T/N ratios.
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BATCo document for Province of British Columbia 23
April 1999


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BATCo document for Province of British Columbia 23
April 1999


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BATCo document for Province of British Columbia 23
April 1999