Reference No.
C3.1
Research Conference, Canada August, 1982
REDUCTION OF EXC."KE MUT.NGENIC--TY
BY LOWERING THE P-%CT--::. CONTEUT OF TOBACCOS
T.G. @14tchell
Proteins are universal constituents of living calls and the content in
growing tobacco plants can be as -@!%:-ch as ' 35% of lamina dry weight (1).
Levels in cured tobacco are generally much lower as the concentration falls
in the period of leaf maturation (s ee Figure 1) and again during curing.
The content in cured tobacco is usually expressed as protein nitrogen:
average values would be 0.91 for ftne-curod and 1.5% for burJAV. EquiX&,-
Told lenvtprotain,,@figu=s.'.&xe@-.5.6%.-gnd,.9.4%-respectiveLY. Protein nitrogen as
determined an tobacco is, more accurately, acid-insoluble nitrogen - the
extent to which it all represents prctein is uncertain but is probably >901k.
Huch recent interest in prctein from tobacco has centred on its potential
use as a nutrient of high value. For this purpose, studies have shown
that to obtain good yields and extractability the tobacco must be harvested
well before normal maturation in readiness for curing. As a result, leaf
residue remaining after protein extraction has few of the attributes
associated with quality tobaccos.
The r*Aidual.@protain-content-voL-cared-;tobacco--is lassociated-11.1with=severa.4
negative attributes- of Of particular
concern is the claim by JTS that it is a maj,or-.precursa of-.the compounds.
causk - tne amino carbo-
.ng autagenic activity in 'tobacco smoke.,iqcindensat
lines (2). Pyrolysis studies on' .ndividual amino acids have shown the
formation of qVen6"... (3) and it is I lkely that it is also produced during
the - combustion'-of :,protein. Higher degrees of correlation nave been found
for total nitrogen than lkaloids, amino acids or nitrate with cyanide,
benzo( ci) ov re ne , phenols and tar (4). The main extra factor in "total
nitrogen' would be protein.
In view of the poexib.Le -major signilicancs- of. tobacco. protein, ;-n relation,
to smoke 'biological-,activityi-.c-&=pcoqv!@-@@of --work.--!iszrproposed-.,4withT'thei-
objective of - conf irming its of f ects -&nd, establishing methods for .-itxo,rewval -I.
or reduction in 'tobaccof The in it Lal phase of the prograrm-te would be
divided into two projects with separate objectives.
1. To A t- @ nn-'-, tobacco protein content
and smoke mutagenic activity.
This would involve the selecticn. of tobaccos with different protein
contents for cigarette manufacture to a common specification for
subsequent evaluation by the Ames test.
1h.
C.
cogs
-.C.
O 195! British Americati Tobacco C3. Lut This =-=sot I.-c copied or showri to -ujhoni@d rcmcas. Lei
on
BATCo document for Province of British Columbia 19 April 1999

2
2. -lo exarnine fwasibility-of-reducing-protein lev*l#using known or
erimental treatments.
It .%U:t be assumed that most known techniques for reducing protein concen-
trations or removing from tobacco will affect other components of the
tobacco also. It would, therefore, be desirable to produce samples
processed in different ways In order to con that the observed effect
was due to change in protein concentration and not from other incidental
effects.
Comparison of the following known treatments is proposed;
(a) Removal by precipitation from HLC liquid fraction or Cylproc treat-
ment - dependent on sa=mles of appropriate materials being produced
from north America, comv;rison with their own controls.
(b) Protcolytic enzyme treatment applied to cured tobacco to produce
approximately 50% reduction in protein N and increase in soluble N.
(c) Assessment of effects of sugars and acids as casing treatments, to
interact with protein an@ possibly modify combustion of the protein.
Treatawnts (b) and (c) could be undertaken here and would yield tobaccos
for cigarette manufacture and subsequent mutagenic activity assessment.
Evaluation of (a) would be dependent on samples of HLC being avail-able
f rem 'the USA, or on the progress of further work to increase the extraction
rate with Cylproc treatments.
A further exploratory area which requires consideration Is to examine
selective extraction technicues which might be applied to cured tobaccos.
RZFEMCES
1. Lowe, R.H. G Sheen, S.J. 1982. Bait. Tabak. 11, 161.
2. Yoshida D. Matsumoto T. 1-980. Cancer Letters 10, 141.
3. Chotyk, O.T. 16 Schlotzhauer, W-S. 1973. Bait. Tabak 7, 165.
4. Tee, T. C, Rathkamp, G. Hoffmann, D. 1973. Bait. Tabak. 7, 190.
C.
C.:;
%X
0 1982 British American Tob3cen Co. Ud. This must rux be copied or shown it, unAucharised pcrsors. (_@,i
BATCo document for Province of British Columbia 19 April 1999

3
Tobacco Prctein and Smoke Mutagenic Activity : Proposed Outline
Programme of Work and its Interactions.
Phase I -
Objective : To validate the hypothesis that mutagenic activity of smoke is
directly related to the protein content of the tobaccos.
Activity : Examination of cigarettes made from tobaccos differing in
protein content, using the Ames test to measure mutagenic
act-".'i ty.
Main staff involved : Dr. E.D. Massey, Dr. G.A. Few.
Timing : 1982 - 1983.
Phase 2.
Objective To establish whether increases in proteins, amino acids or
sugars, by addition to the blend, cause changes in smoke
muticenic activity.
Activity Pretaration and testing of a range of cigarettes with varying
ratios of nitrogenous and carbohydrate components obtained by
selected additions to the blend tobaccos. Where necessary,
isolation of tobaccos - specific compounds may be required in
order to examine their effect.
Main staff involved : Dr. E.D. Massey, Dr. G.A. Few.
T-Ining : 1983.
Phase 3.
Objective : To establish, by laboratory scale trials, routes to processes
for reducing the protein content of tobacco.
Activitv : Exam-,nation of possible techniques for reducing the protein
content of tobacco, including enzymes solvents and different
processes such as HLC, Cylproc.
Mai-
staff involved : A.H. Other + CONTRACT RESEARCH
1983 - 94.
Phase 4.
Objective To ;reduce cigarettes using tobaccos reduced in protein content
by cne or more of the techniques identified in Phase 3.
Activity Application of processes to larger quantities of tobacco for
cigarette manufacture.
mair staff involved t A.N. Other + Process Staff.
Timing : Late 1983 84.
C 19S2 British A.-rican Tobacco Co. Ltd. This must not be cep" or sj"wn tc. Una"thadsw Pcnons.
@-c
%C-
CD
BATCo document for Province of British Columbia 19 April 1999

4
Phase
Objective : To develoo co=ercially acceptable cigarettes with reduced
smoke mutagenic activity.
Main staff involved : Dr. E.D. Massey, A.N. Other,
Process staff, Product D*velopment
staff.
Timing Late 184 onwards dependent on progress of 3 and 4.
0 1982 Bntish Amemcan Tobacco Ca. L.-, This must not b* copicd or shown Ic- unhorsed persons.
BATCo document for Province of British Columbia 19 April 1999

KAIN WORK ACT 1V IT I ES AND PR:NCIPAL RESEARCH INVOLVEIENTS.
1982 1983 1984
PHASE I
Dr. E.D. Hassey 0.3 1 1
1(83 only)
Dr. G.A. Few 0.1 I
PHASE 2.
all Massay 0.3
Dr. E.D .
Dr. G.A. Few 0.1 I
IM
PHASE 3.
A-11. Other (0.5)
+
Contract Research
PHASE 4.
A.N. Other (0.5)
Process Staff
(Dr. E. D. Hassey)
PHASE 5.
IProcess Staff
IA.N. Other
I?.D. Staff
Dr E.D. Hassey
C_-
C:
0 1982 British American Tobacco Co. L:J. This mus. not be cooed or shown to unaudionsed persons.
BATCo document for Province of British Columbia 19 April 1999

0
0
CL
0
0
C
3 FLOW DIAGRAM
CD
G
Work Phases and Interactions, Ancillary/Support Service Requirements.
CONTRACT
RESEARCH
n
0
t
VALIDATION PROTEIN REMOVAL APPLICATION/
(PHASE 1) LAB. SCALE ASSESSHEN DEVELOPMENT
(PHASE 3) (PHASE 4) (PHASE 5)
AI)DITIVES
IIROTKTN/VEPTIDE/ CIGARETTE CIGARETTE
(PHASE 2) AMINO ACID MANUFACTURE MANUFACTURE
IWASUREMENT
CIGARETTE CAS CAS
to H&HUFALTURF I
> CIGARETTE CAS
CAS MANUFACTURE
14ATIIS & STATS ? CAS
to

FIG. I
RELATIVE PROTEIN CONTENT IN RELATION TO TIME CF GROWTH
rIN
100-
so.
GO- -X KY 14
NC 95
z
40. a. >
0. M
20-
31@7 7@ 8 14/8 2i/8 2i/8 4@9 1119
DATE OF SAMPLING
DATA DERIVED FRO14 INFORMATION IN REF 1
X_
C=
BATCo document for Province of British Columbia 19 April 1999