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COPY NO.
IMPERIAL TOBACCO LIMITED
RESEARCH LABORATORY
MONTREAL
RESTRICTED
PROGRESS REPORT
RESEARCH DEPARTMENT
July - December, 1976.
ISSUED BY: Mr. R.S. Wade
DISTRIBUTION:
Copy No.- 1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Library
Library
Mr. R.M. Gibb
Mr. R.S. Wade
Dr. T.A. Smith
Mr. S.M. Candlish
Dr. G.W. Boswall
Dr. S.J. Green
Dr. S.J. Green
Dr. S.J. Green
Dr. S.J. Green
Dr. S.J. Green
Dr. S.J. Green
Dr. D.G. Felton
Dr. D.G. Felton
Dr. C.I. Ayres6/
Dr. I.W. Hughes
Dr. R.A. Sanford
Dr. R.A. Sanford
Mr. R.D. Nicholls
OROUP 1111 D CINT111
le-I'd 4 APR IW7
. ........... .
DATE ISSUED: March 14, 1977.
21. Mr. R.D. Nicholls
22. Dr. M.H. Bilimoria
23. Dr. P.J. Dunn
24. Mr. E.R. Freiesleben
25. Mrs. J. Johnson
26. Mr. A.F. El Naggar
27. Mr. R.L. Rice
28. Mr. M.P. Scherbak
29. Mr. L. Bouchard
30. Mr. H.C. Roubicek
31. Miss R.R. Smith
32. Mr. B.J. Stirling
33. Mr. C.J. Brown
34. Mr. E.P. Gage
35. Mr. W.A. Gray
36. Mr. A. Kalhok
37. Mr. N.D. Maclennan
38. Mr. W.J. Ross
39. Mr. A. Schaffer
40. Mr. C. Warren
00
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INDEX
Page Code Title Researcher(s)
a Summary of Research Department activities.
I T-0433 Pilot Plant and Laboratory
Reproducibility. G.W.B.
2 T-0575 Delhi Research Station Samples for
Industry G.W.B.
Collaborative Evaluation - 1975.
5 T-0758 The Manufacture of Cigars 'or Smoking
Tests G.W.B.
of Tobacco fro. the L'Assomption Experimental
Farm - 1971.
7 T-0770 Cigar Tobacco Production Committee's
G.W.B.
Mechanization and Curing Experiments Cory4ucted
at the L'Assomption Experimental Farm.-
10 T-0773 Smoking Test of Experimental Cigar
Filler G.W.B.
Selections Grown on Two Private FarM5 under
the Direction of the Montcalm Cooperative -
1974.
11 T-0774 The Manufacture of Cigars for Smoking
Tests of G.w.B.
Tobacco from the L'Assomption Experimental Farm -
1974.
13 T-0775 Domestic Cigar Tobacco Evaluation.
G.W.B.
14 T-0807 Flue-cured and Air-cured Tobaccos Grown
an the G.W.B.
Imperial Leaf Tobacco Company Farm.
16 T-0820 Study of Methods to Promote Earlier
Maturing of G.W.B.
Flue-cured Tobacco.
18 T-4900 Leaf Research - Flue-cured Field
Experiment - G.W.B.
1971.
21 T-6420 Mechanization of Tobacco Production.
G.W.B.
23 T-7708 Microbial Mutagenicity Tests. M.H.S.
24 T-7709 Changes in Bronchial Epithelium and
Other Tissue s J.J.
Induced by Cigarette Smoke. M.H.B.
1. Effects of Tobacco Smoke on Tracheo-
bronchial Epithelium. The Incorporation
of Precursors into Glycoproteins.
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INDEX (Cont'd.)
Page Code Title Researcher(s)
2. Effect of Cigarette Smoke Exposure
on Lung AHH in Guinea Pigs
Maintained on a Lettuce-free Diet.
3. rnduction of AHH in Rodent Kidney
and Mutagenesis in Salmonella
Typhimurium TA-98.
30 T-8077
& Studies of Human Smoking Behaviour. P.J.D.
T-7051 &
E.R.F.
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SUMMARY OF IESEARCH DEPARTMENT ACTIVITIES
JULY - DECEMBER 1976
This report covers the research activities in the
areas of
Agrology, Biochemistry 6 Microbiology and Smoking
Behaviour.
The evaluation of tobacco samples produced by the
Delhi Research
Station and the L'Assomption Exoerimental Farm is
an ongoing activity
for Imperial and other manufacturers concerned
with the production and
marketing of cigarettes and cigars. This work
enables the manufacturers
to be ful ly aware of the smoking quality and
other attributes of
tobaccos from new varieties and new tobacco
production practices before
new
they are recommended to the tobacco growers. At
L'Assompti a
cigar cultivar L64-201 is producing a high yield
of good quality leaf
and it is likely to be submitted for licensing as
a commercial variety in
the near future. The Delhi Research Station
continues to identify some
promising insecticides for the control of
hornworms and aphids. Tobaccos
so treated have acceptable smoking quality and if
residue levels are
acceptable some new chemicals could be submitted
for registration for use
on tobacco in 1977. The C.T.M.C. sponsored program
at the Imperial farm
included the construction and operation of a
Johnson Cross-Flow Modular
kiln as well as an intensive study of bulk kilns
to identify the key
factors which may require special attention so as
to improve operating
efficiency.
The assistance given by two of our scientists to
the C.T.M.C.
sponsored research program at McGill has
progressed satisfactorily
during the period under review. one part of the
program has been directed
towards a study of biochemical changes in the
mucous secreted by tracheal
cells upon exposure of rats to cigarette smoke.
The other part of the
program is a study of the induction (increase) of
the aryl hydrocarbon
4 :1
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b
hydroxylase (AHH) group of enzymes in various
organs of rats and guinea
pigs following exposure of the ani.als to
cigarette smoke. It is believed
that both in humans and animals several forms of
AHH can be induced, some
of which may be responsible for detoxification
while others for enhancing
the carcinogenicity of toxicants. The Ames test,
employing mutants of
Salmonella tyDhimurium, is being Used to get an
insight into the type of
AHH induced by tobacco smoke. The value of this
test, both in research
and in screening studies. lies in the fact that an
excellent correlation
has been obtained between mutagenicity in this
bacterial test and
carcinogenicity in mammals.
Differences in the induclion of AHH between the
rat and the guinea
pig, and also among different organs have been
noted. The work is being
reported in detail in scientific journals. The
longer term objective of
these projects is to be able to utilize the
knowledge garined from these
biochemical studies of the effects of smoke on the
resplratory tract first
on animals and secondly with human tissues for the
purpose of developing
relatively quick tests for the screening of
tobacco products.
The current emphasis on research into smoking
behaviour is the
utilization of the measurement of carbon monoxide
in the exhaled air of
smokers as a means of determining the relative
amounts of smoke taken
into the lungs by the smokers and whether or not
these amounts change
with the smoking of different types of cigarettes.
Preliminary results
indicate lower alveolar carbon monoxide resting
levels when cigarettes
of a higher nicotine level were being smoked.
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T-0433 Pilot Plant and Laboratorv Reoroducibilitv
Personnel: G.W. Boswall, S. Webster, 4.A. Gray,
R.R. Smith,
B.J. Stirling.
Objective: To determine the reliability and
reproducibility of smoke
data from cioarettes, containing either unaged or
well
aged tobacco, manufactured from the same tobaccos
by
several pilot plant operations over a Period of
about
three years.
Status: Single grade cigarettes were
manufactured,..from four pilot
plant operations using grades D, H, AF aud BF from
both
the 1967 and 1970 Ontario crops. The total elaosed
time
between the first and fourth pilot plant
Operations was
almost three years. After each operation the fresh
cigarettes were analyzed and repeat analyses were
carried
out on retained cigarettes from previous
operations. The
analytical tests have been completed but the
evaluation of
the accumulated data has presented some
statistical problem
which have now been sorted out, and the data are
now being
subjected to analysis of variance.
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T-0575 Delhi Research S:ation Samoles for Industry
Collaborative Evaluation - 1975.
Personnel: G.W. Boswall, B.J. Stirling, R.R.
Smith, H. Roubicek and
S. Webster.
Objective: To evaluate and compare cigarettes made
from varieties and
other flue-cured tobaccos produced under various
experi-
mental conditions by the Delhi Research Station in
1975.
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
treat-
ments 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 treatments considered for
inclusion in
the Ontario Ministry of Agriculture and Food
Publication 298
"Tobacco Production Recommendations".
The samples submitted by the Station from the 1975
crop to
the cigarette manufacturing companies are listed
below
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together with the manufacturer of each group of
samples.
The samples produced by each manufacturer were
distributed
to the other three manufacturers and all
cigarettes were
evaluated independently by the four companies.
SAMPLE CIGARETTE MANUFACTURER
15 Varieties Benson and Hedges
6 Fungicides Irperial Tobacco
2 Herbicides
3 Maturities
7 Insecticides ~acdonald Tobacco
3 Nematicides
13 Samples (containing Rothmans
sheet from chopped
whole tobacco plants 1974 Crop)
Except for the untreated control in each
experiment.
all samples are generally identified only by code
and
identity of the treatments are made available only
after
the results from all collaborators have been
submitted
to the Research Station. The results of the
evaluation
by the Imperial smaking panel for many of the
samples
were included in Laboratory Report No. 130L issued
October 15, 1976, and an Addendum to Lab. Report
No. 130L
covering results for the variety samples will be
issued
early in 1977. Similar reports were issued by the
other
companies and results were discussed by all
collaborators
at a meeting on October 27, 1976 and the
distribution of
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1976 samples among the four manufacturers for
cigarette
making and smoke evaluation was agreed upon.
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T-0758 The Manufacture of Cigars for Smoking Tests
of Tobacco
from the L'Assomotion Experimental Farm - 1971.
Personnel: G.W. Boswall, S. Webster, R.R. Smith,
H. Roubicek
Objective: To evaluate the subjective smoking
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,
pajrt~icularly in
plant breeding, and now we acceDt 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.
Further-
more, the priorities of cigar product development
work
leave our smoking panel very little available time
for
L'Assomption samples. When it can accomDdate
outside
samples, only those of highest priority such as
from the
"Cigar Mechanization and Curing Experiment -
Project
T-0770" are considered.
As stated in our previous progress report cigars
from
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the Topping and Spacing Experiment have not been
evaluated
by the smoking panel because samples from projects
of
higher priority created a back log of smoking
tests, but
these tests are now approaching completion.
Consequently
cigars from the Topping and Spacing Experiment may
be
submitted t6 the smoking panel in the near future.
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T-0770 Cigar Tobacco Production Committee's
Mechanization and
Curina Exoeriments Conducted at the L'Assomotion
Experimental Farm
Personnel: G.W. Boswall, R.R. Smith, H. Roubicek,
S. Webster
Objective: To evaluate the subjective smoking
properties of cigar
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 Soci6td
Cooperative de
Montcalm, 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 mod-
ern production techniques (mechanization) and the
dollar
returns that farmers experience from other crops.
The
committee instituted an experimental program to
modern-
ize the harvesting and curing of cigar filler
which, if
successful, would hopefully encourage farmers to
resume
cigar tobacco production. The Quebec Department of
Agriculture imported two mechanical harvesting
machines
and two bulk kilns were erected at the
Experimental Farm
in 1974.
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From the various samples produced in 1975, ten
were
selected for subjective smoking tests.
CS-1 -A RH 211, conventional curing.
CB-1-B L64-201, .1
CB-2-A RH 211 , bl ack ol asti c covered ool e
barn.
CB-2-B L64-201, " .1 1.
CB-34 1-20 L64-201, 2nd. bulk cure.
CB-3-B 21-40 RH 211,
CB-4-8 1-20 L64-201,
CB-4-B 21-40 RH 211,
CB-4-C 1-20 L64-201, 3rd. bulk cure.
CB-4-C 21-40 M 211, " 1.
The Imperial smoking panel considered the
conventionally
cured variety RH 211 similar to the conventionally
cured
strain L64-201. Within the RH 211 comparison all
curing
procedures were subjectively acceptable. Within
the L64-
201 comparisons the conventional cure and the
second bulk
cure (CM) were rated as equally acceptable, while
the
other three L64-201 cures were progressively down
graded
because of slightly increased irritation (CM),
increased
irritation and poorer taste (C82B) and
significantly in-
creased irritation and less acceptable sidestream
aroma
and taste (34C).
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The program was continued at the L'Assomption
Experimental
Farm in 1976 and the more promising samples from a
yield
and leaf quality standpoint will be selected by
the com-
mittee when it meets early in 1977.
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10.
T-0773 Smoking Test of EXDerimental Cigar Filler
Selections
Grown on Two Private Farms under the Direction of
the
Montcalm Cooperative - 1974.
Personnel: G.W. Boswall , S. Webster, R.R. Smith,
H. Roubicek.
Objective: To compare the subjective smoking
properties of a cigar
filler selection L64-201 and the popular variety
RH211.
Status: The two cigar filler selections L64-169
and L64-201,
both a cross of Parfume d'Italie X Ottawa 705,
have been
compared in several tests over the past fiw years.
Now
L64-169 will be dropped from tests on private farm
because of its slightly inferior yield and
tendency to
be more subject to pole rot than L64-201.
Therefore
only L64-201 and RH211 were grown on two private
farms
under the direction of the cooperative. The
tobacco of
the same variety from both farms was combined and
cigar
samples were manufactured. The smoking tests have
been
completed and no significant differences were
perceived.
On that basis L64-201 would appear to be as
subjectively
acceptable as RH211.
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T-0774 The Manufacture of Cigars for Smoking Tests
of
Tobacco from the L'Assomption Experimental Farm -
1974.
Personnel: G.W. Boswall, S. Webster, H. Roubicek.
Objective: To evaluate the subjective smoking
properties of cigar
tobaccos from various varieties and various plant
breed-
ing and cultural experiments.
Status: The samples selected by the L'Assomption
Experimental
Farm for ffanufacture into cigars by Imperial
Tobacco
have been processed into short filler and f~e
seven
samples of cigars from the preliminary variety
test have
been forwarded to L'Assomption. The rest of the
samples
will be manufactured in due course and evaluated
by the
smoking panels indicated below.
SAMPLES EVALUATED BY
7 Varieties (preliminary test) L'Assomption panel
14 Varieties (final test) L'Assomption panel
24 Germ Plasm entries L'Assomption panel
8 Topping and Spacing Experiment Imperial panel
The manufacture of these samples has been delayed
due to
tight production schedules at Joliette where an
additional
cigar blend had to be prepared to serve as a
master control
for the L'Assomption smoking panel because of the
small
amount of tobacco in the designated control for
each experi-
ment. This master control blend has now been
prepared and
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the manufacture of the cigars is scheduled for
February
1977.
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T-0775 Domestic Ciqar Tobacco Evaluation
Personnel: G.W. Boswall and H. Roubicek
Objective: To determine the acceptability of
Comstock (RH211)
tobacco produced in the Shawville district of
Quebec.
Status: The required blends of cigar tobacco from
the Shaw-
ville and St. Jacques districts were requested
from
Joliette. Cigars will be manufactured and
evaluated
by the smoking panel during the first few months
of
1977.
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14.
T-0807 Flue-cured and Air-cured Tobaccos Grown on
the
Imoerial Leaf Tobacco Comoany Farm.
Personnel. G.W. Boswall, S. Webster, R.R. Smith,
H. Roubicek.
Objective: To evaluate various varieties and types
of tobacco
grown on small replicated plots on the Imperial
Leaf
Tobacco Company Farm.
Status: After Dr. B. Povilaitis, a geneticist,
retired from the
Delhi Research Station, he accepted a seasonal
position
at the Imperial Farm to conduct some small -0-lot
experi-
ments. These experiments involved some f1be-cured
tob-
accos of special interest to our leaf personnel
and some
air-cured types which are not being studied by
government
experimental farms in Canada. The first year
(1973) only
four flue-cured and four air-cured varieties or
selections
were grown for observation, but Dr. Povilaitis
also under-
took some plant breeding, made some variety
crosses and
produced the hybrid seed. The following year the
flue-
cured plots included four crosses he produced to
introduce
a yellow character into the two popular varieties
(Delhi
34 and Virginia 115) grown in Canada. Our leaf
buyers
were also interested in having some burley,
Maryland,
cigar and dark air-cured samples produced, so
several
variety comparison plots of each type of tobacco
were grown.
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Agronomic, chemical and some smoking evaluations
were
carried out on most of the more promising samples.
Sev-
eral Nicotiana Rustica varieties were also grown
to ob-
serve plant characteristics and to determine their
poten-
tial as nicotine producers. Reports on the data
accumulated
from the 1973 and 1974 samples are now in
preparation and
work on the evaluation of the 1975 samples is
continuing.
None of these activities were completed during the
last
half of 1976.
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T-0820 Study of Methods to Promote Earlier
Maturinq of
Flue-Cured Tobacco.
Personnel: G.W. Boswall, S. Webster, R.R. Smith.
Objective: To evaluate.and compare the cultural
practices required to
achieve early maturity and the completion of
harvest by
students before they return to classes.
Status: In 1975 at the Imperial farm, cultural
practices,
suggested by the Delhi Research Station, were
incorporated
into the production of tobacco in an "early
..'maturity plot",
and for comparison tobacco was also produced in a
."control
plot" following procedures commonly used in
Ontario. The
objective of completing the harvest of the early
maturity
plot while student labor was still available was
realized
but at a substantial financial loss in comparison
to the
control. The early maturity culture resulted in a
yield
reduction of about 360 lbs. per acre and coupled
with
immaturity and lower quality, particularly in the
two upper
primings, a financial loss of about $380. per
acre. Full
details of this experiment have been incorporated
in a
report entitled "A Report of CTMC Sponsored
Activities
Conducted on the Imperial Leaf Tobacco Company
Farm in 1975"
which was issued August 2. 1976.
After consultation with personnel of the Delhi
Research
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Station and our leaf men, it was decided to
continue this
proJect in 1976, but some modified cultural
procedures
have been incorporated into the program. Five
plots of
Virginia 115 tobacco were produced and cured
successfully.
The leaf has been graded and subjected to chemical
ana-
lysis for total alkaloids and reducing sugars.
These
data are being compiled and will be included in
the report
on the work conducted at the Imoerial farm in
1976. The
program which will be followed in 1977 will be
decided in
consultation with the Delhi Research Station and
the CTMC.
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18.
T-4900 Leaf Research - Flue-Cured Field Experiment
- 1971.
Personnel: G.W. Boswall, B.J. Stirling, R.R.
Smith.
Objective: To systematically evaluate the
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-ayid
the
conventional grading criteria such as colour,
blemish,
grain, body etc. For this purpose, the two most
commonly
grown varieties were emoloyed, namely Delhi 34 and
Virginia 115; two degrees of maturity at harvest
time
were built into the experimental design and each
treatment
was replicated threefold in the field. The tobacco
was
harvested by individual leaf position and, in the
process,
an attemot was made to maintain a fairly uniform
interval
of approximately 7 days between the two degrees of
maturity,
which were designed to be slightly imriature (i.e.
3-4 days)
and slightly overmature. 240 samples (2x2x3x2O) of
approx-
imately 200 leaves were harvested and cured in
conventional
kilns. Finally, the three most experienced company
graders
sorted each of these samples, on a leaf by leaf
basis, into
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a blemish (cleanliness) category and then resorted
these
into a colour category, both of which were based
on pre-
determined scales developed specifically for this
experi-
ment. The last requirement of the graders was to
assign
a rating for arain and body and a company grade to
each of
the colour/61emish subsamples that evolved from
the sort-
ing process. In excess of2200samples were obtained
from
the experiment. Nicotine, reducing sugar and
bulking den-
sity measurements were made on all available
samples.
Smoke delivery measurements, utilizing the ground
tobacco
technique, were limited to the samples frop bne
field
replicate for each variety.
Examination of the various means of acquiring the
statis-
tical expertise necessary to analyze our data
resulted in
the decision to engage Imperial Tobacco (G.B. &
I.) Bristol,
England to statistically analyse and interpret the
data.
Our raw analytical data (chemical, physical, and
tar) were
forwarded to Mr. W. Paige, Bristol, early in 1974
but the
energy crisis eXDerienced in Great Britain at that
time
delayed the work. Early in 1976 4r. Paige visited
us and
reported that considerable data processing had
been done.
From his discussions with us in Montreal he
obtained fur-
ther details of the experimental procedures
employed in
the project which are a necessary prerequisite to
deciding
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20.
what further statistical analyses should be
performed.
Such details also aid in the valid interpretation
of
the statistical outout. The report of the
statistical
analyses, although expected the latter part of
October,
was not received in 1976. A draft of the
statistical
report was deceived in Montreal on January 18,
1977.
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T-6420 Mechanization of Tobacco Production
:)ersonnel: G.W. Boswal I , S. Webster, 3.J.
Stirling, R.R. Smith,
and H. Roubicek.
Objective: To investiqate the various forms of
mechanization that
might be adapted to tobacco production and reduce
its
manual labor requirement.
Status: For 1975 a prototype mechanical bulk
curing rack filling
device was designed, constructed and evaluated at
the
Imperial farm. This device is intended to_y~educe
the
labor requirement of bulk curing to that if the
johnson
Modular System and to afford better curing of
mechani-
cally harvested tobacco by achieving optimum
density and
distribution of tobacco in the curing racks. The
device
performed well in most respects, and required 2
men for
its operation instead of the 4 men normally used
in man-
ually filling bulk racks at the kiln.
A prototype bulk rack emptying, desanding, and
tobacco
inspection station was constructed and used in
1975. The
device was efficient in emptying racks and quite
effective,
if operated slowly, in removing large amountsof
sand from
the first primino whose leaves, because of their
proximity
to the soil, have a characteristically high sand
content.
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BATCo document for Province of British Columbia 23
April 1999


Because of the requirement of leaf sorting, the
process was
slow and labor intensive for all primings. The
operation
of the rack filling device and the rack emptying
device
has been described in a report entitled "A Report
on
CTMC Sponsored Activities Conducted on the
Imperial Leaf
Tobacco Company Farm in 1975", issued August 2,
1976.
The 1976 program at the Imoerial farm involved the
con-
struction and operation of a Johnson Cross-Flow
Modular
Kiln, and the study of ways and means of improving
the
operation of bulk kilns so as to determine-,What
modifications
and additional instrumentation might be required
to enable
the average farmer to become an efficient cureman
with mini-
mum instructions. The results of these activities
are
being incorporated into a report by Mr. R.
Minshull the
engineer employed by the CTMC.
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BATCo document for Province of British Columbia 23
April 1999


23.
T-7708 Microbial Mutaaenicity Tests
Personnel: M.H. Bilimoria
Obiecti~e: To use microbial mutagenicity tests for
the detection
of potential mutagens and carcinogens in smoke and
for
comparing tobacco products.
Status: No further work has been done on this
project during the
period under review because of the concentration
of effort
on the program being pursued at Mc Gill University
and described
under T-7709.
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BATCo document for Province of BritiSh Columbia 23
April 1999


24.
T-7709 Changes in Bronchial Eoitheliun and Gthe.-
Tissues Induced
by Cigarette Smoke.
Personnel: J. Johnson, M.H. Bilimoria, H.P.
Witsch, J.C. Hogg.
Obiective: To establish biochemical parameters of
tcoacco-soke
induced lesions in the bronchial tree arc
pulmonary
parenchyma. It is hoped that one or several
parameters
can be defined which may then serve as a
short-term
screening te'st for the evaluation of product
improvement.
To maximize the relevance of such a test to the
effects
of smoking on humans, priority was giver, to
studies of
the tracheobronchial tree, over those involving
lung
tissues.
Status: 1. Effects of Tobacco Smoke on
Tracheobronrhial EDithel;um.
The Incorporation of Precursors into
GI-ycoproteins.
In a previous report it was stated that tobacco
smoke
might possibly affect the tracheobronchial
epithelium
in ways that are biochemically and morphologically
similar
to Vitamin A deficiency. It will be recalled that
Vitamin A deficiency causes a decrease in the
biosynthesis
of certain glycopeptides pre-sent in goblet cells,
as well
as neoplastic transformation resembling that
induced by
chemical carcinogens such as benzopyrene. Knowing
the
latter to be a component of tobacco smoke, it was
proposed that acute smoke exposure could
conceivably
interfere with giycoprotein synthesis in a similar
fashion.
The method used has been described in detail in
the
previous Progress Report. Briefly, it consists of
excising tracheas from control (sharr.-smoked) and
smoked
rats, mincing them and incubating them for 3 hours
with
a radioactive precursor. Subsequently, the protein
is
extracted, dried, weighed and counted. The
incorporation
of radioactivity in terms of Specific Activity
(dpm/mg
Prot.) is determined.
BATCo document for Province of BritiSh Columbia 23
April 1999


25.
T-7709 (cort'd)
Our first experiments utilized glucosemine-C 14 as
radioactive precursor. We have already shown that
there are no significant differences in the incor-
poration of glucosamine-C14 between control rats
and
rats that have been exposed to cigarette smoke
from
5 cigarettes and which were sacrificed immediately
after or 24 hours after exposure.
We therefore proceeded to measure incorporation
when
rats were exposed to smoke from the same number of
cigarettes but sacrificed at 48 hours and 72 hours
after
smoke exposure. In some instances rats that were
sacrificed at 72 hours, were exposed to smoke from
5
cigarettes daily, making a total exposure.to smoke
of
15 cigarettes. However, at no time were-
significant
differences seen in incorporation of radioactivity
between control and smoke-exposed animals. These
results
presumably indicate that at these levels of smoke
exposure,
changes in metabolic activity of the tracheal
epithelium
as regards glucosamine-C14 are either not
affected, or
else too small to be detected by this method of
analysis.
Researchers have shown that small but measurable
and
significant changes in the number of goblet cells,
which
are believed to be the only source of
glycoproteins in
the tracheobronchial tree, occur only after as
long as
12 weeks of smoke exposure. Inasmuch as it is
presumed
that biochemical changes precede morphological
changes, it
would appear that the exposures to cigarette smoke
described above provide an insufficient insult to
cause
detectable differences in rates of incorporation
of
glucosamine-C14. It is therefore proposed to
determine
if changes in glycoprotein synthesis can be
measured after
a longer exposure of 5-7 days.
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BATCo document for Province of BritiSh Columbia 23
April 1999


26.
T-7709 (cont'd)
Incorporation Of leuc;ne has been shown to
decrease in
rat tracheal epithelium after 3 days smoke
exposure.
Experiments were therefore performed to see if
this
effect could be duplicated in this laboratory.
Preliminary
experiments involved measuring the kinetics of the
system
with leucine. The same experimental conditions
were
found to prevail to obtain maximum incorporation
of
leucine is with glucosamine, i.e. 3 hours
incubation
at 370C with at least I lim radioactive leucine.
To find
out whether the incorporation of leucine could be
inhibited,
cycloheximide, a known inhibitor of protein
synthesis was
administered in vivo to rats 90 minutes before
sacrificing.
Incorporation was found to be inhibited by about
50%.
Having established that the system could-&e
inhibited,
rats were exposed to tobacco smoke from standard
flue-
cured cigarettes. Rats were sacrificed
immediately, 3,
24 and 48 hours after smoking. Results show that
the
incorporation of leucine was inhibited by about
35.8%
immediately after smoking but no inhibition was
seen at
3, 24 or 48 hours.
Future experiments will involve exposing rats to
more
acute smoking regimes. Rats will be exposed to the
smoke
of 5 cigarettes/day for time periods ranging from
4-7 days,
after which the incorporation of leucine will be
measured.
If significant changes in glycoprotein synthesis
are found
to occur after these exposure times, efforts will
be made
to apply the findings for use as a routine
bioassay which
might possibly be effective in differentiating
tobacco
types.
BATCo document for Province of British Columbia 23
April 1999


T-7709 (cont'd)
2. Effect of Ciqarette Smoke Exposure on Lung AHH
in
Guinea Pigs Maintained on a Lettuce-free Diet
When we exposed guinea pigs to smoke from regular
flue-
cured cigarettes, we found that instead of
observing an
increase in the levels of AHH in the lung, we
obtained
a distinct depression, which persisted up to about
12
hours after smoke exposure. In all studies
reported thus
far with -other species such as rats, hampsters
and mice,
exposure to smoke always resulted in induction of
this
enzyme. Before we could conclude that this as a
real
species difference, we carried out several
experiments
to confirm that our observation was real. First,
we
checked out our exposure system by showing that
Sprague-
Dawley rats when similarly exposed
showeq,-..induction of
AHH in the lung. Next we tested other known
inducers of
AHM such as Aroch)or 1254, 3-methyicholanthrene,.
and 3,4-
benzopyrene, and found that only 3,4-benzopyrene
gave
increased activity of AHH in guinea pig lung.
Arochlor
treatment resulted in a depression of lung AHH
similar to
that observed with smoke. Lastly, we explored the
possibility that the guinea pig diet was
responsible for
our seeing no induction of AHH in guinea pig lung.
In this laboratory guinea pigs are maintained on
diet which
includes fresh lettuce to supply the vitamin C
that these
animals cannot make for themselves. It is known
that
vegetables such as lettuce are good inducers of
AHH in
the lung. If now the lungs of these animals, fed
lettuce,
are fully induced, exposure to smoke will not
result in
any further increase in AHH activity. Thus, it
appeared
important that we carry out our smoke exposure
using guinea
pigs maintained on a lettuce free diet. but given
vitamin C
in the drinking water. When we did this, we found
that
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BATCo document for Province of BritiSh Columbia 23
April 1999


28.
T-7709 (cont'd)
cigarette smoke still failed to induce AHH in the
lung,
3 hours after smoke inhalation. Once again, we
obtained a distinct depression of AHH activity in
the
lung at 3 hours after smoke inhalation. These
results,
coupled with our observation that guinea pig
kidney is
inducible by cigarette smoke, clearly show that
cigarette
smoke does not induce AHH in the lung of the
guinea pig
and that this negative result is indeed a case of
a true
species difference.
3. Induction of AHH in Rodent Kidney and
Mutagenesis in
Salmonella Typhimurium TA-98
Epidemiological data suggest that cigarette
smoking is
associated with an increased mortality from
bladder cancer.
This could be due to the fact that carcinogenic
compounds
inhaled with and absorbed from cigarette smoke
reach the
kidney before they are eliminated. On reaching the
kidney they could result in induction of aryl
hydrocarbon
hydroxylase, an enzyme with a possible central
role in
cancer. Thus, a study to determine if exposure of
small
laboratory animals to tobacco smoke would induce
renal
AHH, appeared an attractive proposition from the
point of view
of a short-term test, and was carried out.
Random bred guinea pigs and rats were exposed to
smoke
from 5 regular flue-cured cigarettes, and an equal
number
were put through the stress of shad smoking.
Levels of AHH
were measured immediately after, and at
1,3,6,12,24 and
48 hours after smoke exposure. A significant
increase
of AHH was observed 1-3 hours after exposure. Rats
showed
peak enzyme activity at 6 hours, while guinea pigs
showed
peak values at 12 hours. In both rats and guinea
pigs,
enzyme levels had fallen to basal levels
forty-eight hours
after smoke exposure.
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BATCo document for Province of BritiSh Columbia 23
April 1999


T-7709 (cont'd)
In comparing the capacities of 9,000 g
supernatants to
activate 2-anthramine to a mutagen for Salmonella
typhimurium TA-98, a marked species difference was
observed. Thus, although the 9,000 g supernatant
from
normal guinea pig kidney had a lower level of AHH
than
had the corresponding rat kidney preparation, the
guinea
pig preparation was about five times more active
in
activating 2-anthramine to a mutagen for S.
typhimurium
TA-98. We then tested renal 9,000 g supernatants
from
smoke exposed animals, at the time of peak
induction of
AHH (12 hours after exposure for guinea pigs and 6
hours
for rats). Kidney supernatants from smoke-exposed
guinea
pigs were considerably more active than ~-*re
supernatants
from control animals. No difference was~observed
between
rats exposed to tobacco smoke and controls, even
though
the AHH activity in the 9,000 g supernatant was 10
times
as high. These results are interesting in that
they
suggest a species difference with respect to the
type of
AHH present, and should lead the way to a relevant
short-
term test for tobacco smoke toxicity. A paper on
this will
be published in a 1977 issue of Toxicology and
Applied
Pharmacology.
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BATCo document for Province of BritiSh Columbia 23
April 1999


3 c).
T-8077, T-7051 Studies of Human Smoking Behaviour
Personnel: P.J. Dunn and E.R. Freiesleben
Objective: To study the real effects on smokers of
modifying smoking
products in order to reduce intake of harmful
substances.
Status: The microprocessor unit has been completed
and is capable
of transforming the analog signals transmitted
from the
transducer into binary code which is computed into
individual and average puffing responses of
pressure drop,
duration and interval as well as velocity and
volume
measurements generated from the present slave
smoker
apparatus.
Consultations have taken place with
Ontar'.0"Research
Foundation as well as Dept. of
Mechanical-Engineering,
McGill University, with regard to the assessment
of the
present slave smoker equipment and to alternative
mouth
piece cigarette holders and valve controls for
smoking
duplication. It is hoped that changes in these
components
can be accommodated within the remaining slave
smoker
instrumentation.
A comparative study of rebreathing as well as
breath-holding
as techniques for collecting alveolar CO samples
has
demonstrated that the former leads to more
accurate results
with fewer problems in sample collection. Using
this
technique, it was demonstrated that alveolar CO
uptake
correlated very well (r ' 0.98) when controlled
concen-
trations of CO had been sham-smoked. This study
was
further verified with the observation of a good
correlation
between alveolar CO and a limited number of COHb
concentrations.
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BATCo document for Province of BritiSh Columbia 23
April 1999


T-8077, T-7051 An infra-red CO analyzer
(Bendix-Bosch CO Analyzer)
has been ordered to replace the present gas
chromato-
graphic technique for CO determinations; it is a
much
faster technique with accuracy adequate for
alveolar
determinations.
A groLpof 4 Players Filter Regular cigarette
smokers
have been monitored for changes in smoking
parameters and
alveolar CO levels with a cigarette of 30%
increased
nicotine (through addition of nicotine citrate).
Relatively
few differences in smoking parameters were
observed, but
noticeable and consistently lower alveolar CO
resting
levels were noted with this product change. An
equivalent
study on du Kaurier Regular smokers is presently
being
undertaken. With the completion of this study,
more
conclusive evidence of the effects of nic9tine on
alveolar
CO levels will be available.
It is hoped to follow up this du Maurier study by
monitor-
ing the effects, on the same smokers of changed
pressure
drop of cigarettes which deliver equivalent
nicotine levels.
In order to better appreciate the effect of
variations in
human smoking on the transfer of CO, tar, and
nicotine, a
study is presently underway monitoring changes in
these
3 components under extremes of velocity
conditions. It is
hoped that the results of this study along with
lung CO
data will aid in better understanding of both lung
and mouth
intake of CO and nicotine.
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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