APPLICATION TO THE CANADIAN TOBACCO MANUFACTURES COUNCIL FOR A CRART-IN-AlID
TITLE: "Cigarette Smoke Exposure and Inherent Protective Mechanisms In
Mammalian Lung.
APPLICANTS: M.H. Bilimoria, Pathology Instutute, McGill University
D.J. Ecobichon, Dept. Pharmacology and Therapeutics, McGill
University
There is considerable evidence that protection of tissues from to ty
due to reactive intermediates is afforded by the tissue stores of glutatht-one
(GSH, L-r -glutamyl-cycteinyl-glycine). This nucleophilic agent. in conj@mc-
tion with cy-tosolic S-aryl and -alkyl transferases, det@oxifies the reactive
intermediates (free radicals. epoxides) by forming a water-soluble, nontoxic
and readily excretable conjugated product (1-5). The levels of this endo.:!
genous compound in body tissues, of the order of 0.5-10 mM, would suggest:a
significant role for CSH. While this role @as been extensively explored in
conjunction with biotransformation pathways in the liver and to some extelt
the kidney, other tissues, including the lung, have been largely ignored..
The concentration of CSH In rat lung Is approximately 2.0 mM or about
20% of that found in the liver (6). The presence of this level of a sc
of reactive metabolites and free radicals would si4gest that CSH could pL Ly an
active role in the detoxification of reactive (per se or activated in vivo)
intermediates in the lung. The enzymes which catalyze the conjugative re ction
with a wide variety of foreign chemicals are glutathione S-transferases
(EC 2.5.1.18). Several glutathione transferases (CST) have been Identified In
ian tissues in the mitochondria, microsomes. and cytoplasm and have en
shown to have broad, overlapping substrate specificities (7,8). Studies have
measured the levels of CST in mammalian J=g, showing that the activity
I
approximately 15-50% of that found In mammalian liver (9-13). Studies ofthe
BATCo document for Province of BritiSh Columbia 15 April 1999

pulmonary enzymes necessary to maintain a balance of reduced GSH arA o
dized form (GSSC, glutathione disulphide). the CSH-reductase and perox ase.,
have not been extensive (14). The importance of trace amounts'of selen um
(Se) to the activity of the CSH-peroxidase has been recognized, this me al
being an integral part of the GSH-enzyme complex (15). In the tobacco
industry, efforts have been made to increase the selenium content of tobacco
as one possible way of reducing the potential hazard 6f smoking since S' has
been demonstrated to significantly Inhibit both carcinogens and mutagend,
presumably by enhancing the further detoxification of reactive intermediates
to excretable non-toxic products (16-19). Recent studies have demonst,
ri ted
I
that approximately 4_% of the Se in cured tobacco could be transferred io
smoke following pyrolysis (20).
In addition to GSH, the lung contains other potent scavengers of
reactive metabolites and free radicals, namely ascorbic acid and the am@no
acid cysteine. It has been demonstrated,in in vitro systems, that cigarette
smoke will react with cysteine and ascorbic acid, producing cystine a-A.:
dehydroascorbic acid respectively, known antioxidant properties of these
agents (21-23). It is also known that the serum levels of ascorbic acid
are lower in smokers than in non-smokers (24,25). In addition, glucuxror@yl-
transferases are present In the lung. .again being involved In the conjiMation.
of hydroxy metabolites into water-soluble, excretable, non-toxic compo S.,
These enzymes have been studied in guinea pig and rat lung (26).
Given the fact that the lung would appear to have some capability 8f
protecting itself by the presence of antioxidant substances and essential
catalyzing enzymes, the questions which we would like to raise and to "swer
include
How stable are these enzyme-cofactor protective mechanisms in @he
BATCo document for Province of BritiSh Columbia 15 April 1999

lung in the presence of cigarette smoke?
2) What capacity do these protective mechanisms have for preventing
tissue toxicity?
EXPERIMENTAL
To study the influence of different concentrations of cigarette smole
on the levels of CSH, ascorbic acid, cysteine, CST, the CSH-reductase,
GSH peroxidase and UDP-glucuronyltransferases of maimnalian lung, we propOe
the following experimental protocols using two rodent (rat, hamster) andi
two nonrodent (guinea pig, rabbit) species, the B-AT-Mason inhalation
instrument and cigarettes prepared from blends of Canadian flue-cured
tobacco.
The first ques animals (males
and females of eac. of cigarette
smoke for optimal I puffs of smoke
followed by euthana :@entobarbital
overdose) and remov Iowing parame4rs,
(i) ascorbic a(
(ii) CSH and GSS ired by the
F-11man reac
(iii) GSH S-aryll -dinitrobenzene
and 2,4-dich
(iy) CSH S-alkyltransferase activity using 1,2-epoxy-3-(p-nItrophen)xy)
propane as the substrate (30)
(v) GSH-peroxidase activity using cumene hydroperoxide as the subs4ate
(151
(vi) CSH-reducta3e activity, examining the rate of oxidation of
NADPH by CSSG (31)
(vii) UDP-glucuronosyltransferase using alpha-naphthol or p-nitrophen@ol
L4
CO'
BATCo document for Province of BritiSh Columbia 15 April 1999

-4-
as the substrate (32)
(ix) pulmonary AHH (33)
1hese enzymatic activities, etc.. will be measured following exposure
and will be compared with control, sham-exposed (held in restraint cones I
but exposed to air in chamber for the same length of time) animals to as ess
the overall effects of smoke on pulmonary tissue homogenates and fractio
(microsomes, cystosol). Having ascertained whether or not there is an
effect on the natural antioxidant chemicals or enzymatic activities, we
shall proceed to the second phase.
B/@- To study the capacity of the protective mechanisms to prevent injury!
I
dose response curves for the above-measured parameters will be establish fd.
Groups (n=5) of animals (two rodent and two nonrodent, males and females
will be exposed for an optimal time period (300 seconds - 60 puffs) to 10'5,
1: 10, 1: 20 and 1: 40 dilutions of freshly-generated smoke from cigarettes 1.
prepared from Canadian flue-cured tobacco. The animals will be euthaniz d
immediately after exposure and the lungs will be removed for assay of th@
above biochemical parameters. In addition, estimates of exposure will bi
assessed by spectrophotometric or gravimetric measurement of total particu-
late matter collected on Cambridge filter pads (1.0 micron pore) and by
estimation of the carboxyhemoglobin levels.
If the above exposure levels all cause maximum biological effects, tt
will be necessary to change cigarettes and use "extra-mild" and "ultra-mild"*
filtered cigarettes as we have done previously to establish dose-effect
relationships for pulmonary AHH (34). In this manner, we hope to establ Lsh
what "load", of cigarette smoke is necessary before the inherent protecti re
mechanisms begin to falter and/or become reduced in efficiency.
c/- Given that a dose-effect relationship can be established between smo@e
(-n
LJ_4
L.J.4
BATCo document for Province of British Columbia 15 April 1999

concentration and antioxidant reduction or enzyme inhibition,
set of experiments, using optimal exposure conditions to produce these I
effects, will be conducted to examine the rate at which the protective
antioxidants OSH. ascorbate) can be regenerated or synthesized in pulmonary
tissue. While not directly related to this study, Ecobichon has examin-
the hepatic and renal depletion and resynthesis of CSH in the rat, mous
hamster, guinea pig. and has found that in all, except the guinea pig, H
resynthesis.was well under way by 8 hr following depletion with diethyl!
-male-ate (33). The guinea pig was extremely slow in replacing tissue stores
of CSH, such levels not being achieved by 24 hr post-treatment.
Having selected an optimal smoke concentration and duration of exposure
to elicit a biological effect, groups of animals ( n-- 6, four species, malps
and females) will be exposed to deplete CSH and/or ascorbate, etc. and will
I
either be euthanized immediately for assay or will bd held for different
post-treatment intervals (2, 4, 8, 12 and 2-4 hr) and euthanized for analysis
of the above listed parameters (or selected functions) to ascertain when)
recovery is complete. It Is hoped that, as In the experiments conducted,'
i i
In the livers and kidneys, a rate of GSH and ascorbate synthesis can be
determined (33). If specific dose-related effects are seen for specific,;
enzymatic activities, the reversal of these effects will also be measure@
t
on appropriate pulmonary tissue preparations.
The biochemical aspects of the above experiments will be carried out In
the laboratory of Dr. Ecobichon where most of these methods are currently
operational. A technician familiar with these techniques will be requir I
and Miss PaH. Cameron, who has worked in this laboratory for some It years
will be hired on the grant for this purpose. The exposure chamber and
B-AT-Nason apparatus is housed in the laboratory of Dr. Billmoria. Dr.
one
BATCo document for Province of British Columbia 15 April 1999

-6-
Bilimorla's collaboration will Involve the planning of the experiments
exposure of the animals as well as consultation about the results and t eir
interpretation about the results and the preparation of manuscripts and
papers for publication
BATCo document for Province of British Columbia 15 April 1999

-7-
REFERENCES
1) O.W. Griffith and A. Meiiter. Proc. N.A.S. 76, 5606 (1979).
2) G.J. Smith and G. Litwack. InReviews in Biochemical Toxicology.
Hodgson, J.H. Bend and R.M Philpot (Eds.) Elseview/North-Holl
Vol. 2, pp. 1-48 (1980).
3) D.J. Reed and P.W. Beatty. Ibid. pp. 213-242 (1980).
4) A. Meister. Trends in Biochem. Scis. 6. 231 (1981).
3) A. Meister. Science 220, 470 (1983).
6) M.S. Moron, J.W. Deplerre and B. Mannerv!Lk. Biochim. Blok..j.. Acrla
-582, 67 (1979).
7) W.H. Habig, M.J. Pabst and W.B. Jakoby. J. Biol. Chem. 249, 7130
(1974).
8) T. Freidberg, P. Bentley, P. Staslecki, H.R. Glott, D. Raphael an4
F. Oesch. J. Biol. Chem. 254, 12028 (1979).
9) C. Guthenberg and B. Mannervik. BBRC 86. 1304 (1976).
10) J.W. Depierre and M.S. Moron. Pharmacol. Res. Commun. 11, 421 (I 79).
11) J. VanCantfort, L. Kanil, J.E. Grielen, H.R. Glott a-rid, F. Oesch.
Biochem. Pharmacol. 28, 455 (1979Y.
12) H. Mukhtar and E. Bresnick. Chem-Blol. Interact. 15, 59 (1976).
13) S.J. Stohs, W.A. Al-Turk and C.R. Angle. Blochem. Pharmacol. @-i
2113 (1982)-
14) A. Meister and S.S. Tate. In 'Glutathione: Metabolism and Funct on"
I.M. Arias and W.B. Jakoby (Ed.). Raven Press, N.Y. (1976) pp-1
-604.
5.59
15) J.H. Prohaska and H.E. Ganther. -J. Neurochem. 27. 1379 (1976).
16) L.W. Wattenberg. J. N.C.i. 6o, ii (tq?8)-
17) J.H. Thompson and P.J. Becci. J. N.C.I. 1299 (1980).
18) 'M.14. Jacobs. Prev. Med.
362 (1983).
19) C. Adams, S. Martin and J. Milner. Fed. Proc. M, 790 (1980).
20) O.T. Chortyk, J.F. Chaplin and W.S. Schlotzhauer. J. Agric. Food;;
ehen.. 2. 64 (1984).
21) G. Green. Nature 194, 284 (1962).
22) chung-6, 3-2 C::)
M.H. Bilimoria and M.A. Nisbet. Beltrage zur Tabakfors
(1971).
-DIN.
NJ
BATCo document for Province of BritiSh Columbia 15 April 1999

-8-
23) C. Leuchtenberger and R. Leuchtengerger. Brit. J. Exp. Pathol. L8.
625 (1977).
24) 0. Pelletier. Int. J. Vitamin and Nut. Res. i6, 147 (197-7).
25) A.B. Kallner, D. Hartmann and D.H. Horning. Am. J. Clin. Nut.
1347 (1981).
26) A. Aitio. Xenobiot. 1, 13 (1973).
27) V. Zannoni. M. Lynch, S. Goldstein and P. Sato. Biochem. Med. Ii,
41 (1974).
28) G.M. Banke, X.L. Cheever, F.E. Mirer and S.D. Murphy.. Toxicol.
Appl. Pharmacol. 28, 97 (1974).
29) C.-H. Kuo, K. Maita, S.D. Sleight and J.B. Hock. Toxicol. Appl.
Pharmacol. 67, 78 (1983).
30) M. Younges, B. Schlichting, R. and C.-P. Siegers. Pharmacol. Res'.
Commun. 12, 115 (1980).
31) 1. Carlberg and B. Mannervik. J. Biol. Chem. 250, 5475 0975)-
32) G.W. Lucier, O.S. McDaniel and H.B. Matthews. Arch Biochem. Biop@ys.
14j, 320 (1971).
33) M.H. Bilimoria and D.J. Ecobichon. Toxicology 15, 83 (1980).
34) M.H. Bilimoria and D.J. Ecobichon. Toxicology 12, 20 (1981).
.5
35) D.J. Ecobichon. Can. J. Physiol. Pharmacol. 62. Submitted for
publication (1984).
(-fly
BATCo document for Province of BritiSh Columbia 1 5 April 1999

-9-
BUDGET: 1984-1985
I. Technician - Miss P. H. Cameron, B.Sc.
Tech. III 22,000.00
Fringe Benefits 3. 74o. oc i
II. Materials and Supplies
a) Chemicals - including CSSG, GSH, ascorbic
acid, NADPH, purified reductase for
peroxidase assa@
buffer salts alone
cost $345 U.S, for 100 mg)
+ Se assays 5,000-00
b) Animals
males and females of
W rats
(11) rabbits
(iii) guinea pigs 6,000.00
Day care costs and housing and
shipment 11000.00
c) Other - xeroxing, photographic preparation
and manuscript costs 500-00
$38-240-00
IL
LN
BATCo document for Province of British Columbia 15 April 1999