10146351 6
M, a %I, , snot..
sso
I)nnanen
V.d. 4, No
.6
C,r, 101 0 W. h, the 3tnerwan S tin, For I harma vk g, awl l .frrimemnl Tlxnlemiss pnmrd in cS..t .
LOCALIZATION OF NICOTINE-14C, COTININE-14C, AND
NICOTINE-1'-N-OXIDE-14C IN TISSUES OF THE MOUS E
WILLIAM J . WADDELL AND CAROLYN MARLOWE
Oetarrmenr td Phemaeolegy. Gdrge of Madicse . Unrrerstry or Kentucky
(Raesrrod January t2 . 1976, and --d form Jury 26, 1976 )
ABSTRACT
P
The distributions of nicotine-"C (2'- or metlryl-habeled), catinino s'C . and motion-l'N-acidh-1'C wan studied by whole-body autoradiography in mice and in pardoned rabbit hangThsrcompounds wine administered after iv, an . to. or by inhalation . Bonk sections were Nan
Incubated with nicatom-"C and cotlnkq-"C in vitro. The distributions were compared in a
black (C57BL/8J1, brown (C571JJL and this (A/FJI strain and in CD-1 germ-free mica
After admimistraios of nicotktart'C it two. radioactMty wax beslize in all strains in bronchi .
next rumens., salivary glad, Hader, plawl live, kidney. stomach, spleen, patresas, bites
time. bona get@daddar . and adrenal machine. In the pigmantb strains, it was also localized In
matatin in the eye, bran, and halt. Radloaeti'eity, did not atsumnlats in de bronchi of late .
term fetuses or an 1-day-old newborn but was ptssent in the 2-day-old and older . Cetinutes'C and molter-1'-N-oxide-1`C, after In administration, did not localise in bronchi or nasal
muooaa at short time intervals shier bej .etian. Frown xRione incubated it vrrro did not
accunndate site., nieatvu-11C or eotbrkte-s'C in the bronchi : whereas the affinity for meltnin wen unchanged. Incubation of fresh, nnnfroeen moue king and peruaion of rabbit lung in
vitro with nicotita-14C produced the same locsteration of radioactivity in bronchi an that seen
shr in rA- Wrtdmistraion . Preheatbng the frozen sections no lungs in a microwave oven oefore incubation did not change the taelizaion of radioactivity in the bronchi or other masons .
The localisation of radioactivity in bronchi may be dal to rtmtebaism, active transport r
binding of nicotine ; this mechanism is destroyed by freezing the tissue .
A remarkable, intense localization of radioactivity has been reported preliminarily in melanin .
bronchi, and nasal mawsa of whote•body autoradiographs after-peremerai administration of [Nmirhyf-"Clnicotise to minx (1-5)- Other basic
drugs also have been reported to bind strongly to
melanin in the eye of mice (6) : whereas specific
uptake in bronchi has been reported for progesterone (7, 8). pregnenolone (8), aldosterone (9), coninone (10), hydrocortisone (10) . diethylstilbestro l
Thin hrn tieattan wa supportM by ,M Uruw+ry4 f
(II), ascorbic acid (12), ooscapine (13), dimethylmercury (14), and 17P-hydroxyl-4,4,17-trimethylandrost-5-en[23-d[isoxazole which is an
inhibitor of steroid metabolism (7) . The potential
significance of these localizations in bronchi and
melanin has prompted continuing attempts to
elucidate the mechanisms. Comparison with the
distribution of cotinine--"C and nicotine-I'-Noxde-"C, metabolites of nicotine, and incubation
of nicotine-"C or cotinine-"C with whole-body
sections and lung pieces in vitro have allowed
further inferences- It appears that the localization
of drugs in melanin might be a nonspecific binding
of a basic compound to an acidic protein . The
localization of nicotine-'•C in bronchi may be due
to active transport, binding, or metabolism of the
compound to metabolites other than cotinine or
nicotine- I'-N-oxide.
Kemrrcky Tobacco, and Heamr Rasaaen asittea Porno No.
KTAB-057 . PrNimirvry opens of this work ware peaane .d
a rte Foam, Am.wai C¢nseranes of the KanNeky Tooaow
and stealth Revaech IrWiuta . 1871 and at be Annul
masone. of one Fsdareien .1 American Soeietws lam
ExPMimertal .'glary and MMdicke, 7674 and 1976 .
Sad saprid Inquests tot Wtaiam J . Waddak, M.O.
0.partnem of Pharmarolopr . School of Madicire, Mimesite of Loo evitls, Louavion, Kentucky 40201 .
Materials cad Methods
1n Visa Studies. Adult male and female mitt weighing
20 25 g and pregnant mi- .eighing 25 35 g, of the
LOCALIZATION OF NICOTINE-•'C AND METABOLITES
C57111.!6.1 . CS7L/l, and A/IlJ strains, were oht,,cd
from Jackson Laboratories (Bar Isarbor, Me .). Nobn of
the day the vaginal plop was discnvercd was roamed as
the 03 day of pregnancy . One- to sewn-day-old male
amt female mice, wciphing 1 .5 5.0 p . w re delicc,cd
from a BAL@/c mother . .led with a CD-1 mate . Adult
female CD-I gum-free mice, weighing 25 30 p, ware
obtained from Charles River Bruding Labs . (Wilmington. Mass.) . Thou mice were maintained germ-free
until sacrifseed. The t.-(N-ntcrhtl-"Clmeotine bitaruatc
wus from Amcrsham/Scarce Corp. (Arlington Heights.
Ill.) . batch numbers 4 and 5. with a spccif r activity of
36 mCi/mmol . ~rrroridinr,2-'•ClNicalinc was obuined from New England Nuclear Corp . (Boston,
Mau.), tot 862-100. with a specifu activity of 3.9 mCi/
mmol. The L-[5-prr,ohdinonr'•C)cotiaine was symhe .
sized by Drs. Herbert McKennis Jr . and Edward R .
Bowman. Medical College of Virginia . Richmond, with
a specific activity of 40 MCi/moot. The (pyvro/lditr-2"Clnicmine-l'-N-oxide was obtained from New England Nuclear Corp .. lot 862.101, with a specific activity
of 3 .9 mCi/meal- The wmpoot were dissolved in
0 .94 (w/v) NaCI for iv (tail vein), sc or ip injection .
Nicotine-"C (2'- a me hyl-labekdf and mini-"C
were subjected to thin-layer chromatography on silica get
with absolute abanollamtonelbeazenclSS% NH,OH
(1 :8 ;1011, v/v), In this system the methybiabetcd nicotine
bad an Rr of 0.76. which corresponded to authentic
nicotine; a smell impority was obsessed with an Rr of
0.14- The [2'-'Y)nitoine was similar to the methyllabeled nicotine except that two very minor impurities
were observed with R, values of 0.38 and 0.50. Chrome.
53 1
wgraphy of the coining-'•C resealed a single radinauire
peak with an R, of 0.47. The nicutine-l'-N uxde-° C
c seitjeet d I t thin-la)nr thrum : tnpraphy nn silica gel
with n-hotamd/n-prop :rnot/2 M NII,OH (2:1 :1, v/v)
and foond to bane a major r dioactive spot with an R, of
0 .24. which correspumf,a) to the standard nicounc-I'-Novide . Three miner impuritks were detected with Rr
, .tuts of 0 .36, 0.47, and 0.63Prolonged storage of the IN-nrrriW-'•Clnieoline
«sutted in demmpositioo (X97 : punt, I. Porifcation
w accomplished by thin-layer chromatography : the
area corresponding to meuioc-"C was scraped off the
plate and extracted in absolute ethanol . This entrust was
acidified with 1ICI to form the dichloride salt . Chromatography of this purified nicotine-'•C revealed a single
radioactive spot. Purification was performed in .all
batches just before each experiment .
inhalation of [N-arrrkri-••Cjnimt toe, convened to the
free base by addition of two equivalents of NoON, was
accomplished by insertion of a double-valved . nonrebreathing device containing the drug via a tracheotomy
inc moose anesthetized with 150 mg of phenobarbital per
kg ip.
Table I shows the schedule of adult mire injected iv
with the IN-maskrl-"Clnicotiae, the doses of the drug
administered . and the time interval between injection and
freezing of the animal . To the mica retaining 1 .0. 1 .4 mg
of mentor per kg iv. the isotopic solution was administered over a period of 4 min and the sacrifice tier was
determined from the end of the injection in freezing of
the mouse The lower dotes of nicotine (0 .12-0.60
mg/kg) were we11 tolerated and could be injected rapidly .
11 .1 1
TABLE I
Srhrdtdr of adr/i mirr ln/etred imr¢warruslr will IN-orrritf-'•C) nicotine for s-k¢fr-b¢de aurwadiaaropk r
Strain
C57BL/6J
CS7L/J
A/Hel
CD-I (germ-fee)
See
Male
Nonpregoaes female
Nonpregnant female
t23-day pregnant
143day pregnant
16 .5-day pregnant
Main
Nonpregnant female
173-day pregnant
Male
Nonpregnant female'
Nonpregnant Inmate
Nonprcgnam female
155-day pregnant
Nonprcgnam fanale
Dose
malks
010
0.60
1 .0-1,3
)3
13
12
020
1 .1-11
0 .15,
0.20
0.12
0.30 0 .60
1 .2-14
1 .4
0,17
Interval bawvm Injentoa sad Fneamg'
6 nun. 20 min
30 tee
2 min, 6min, 20 min, 60 min
burin
S min. 20 min, 60 mi n
6 Mi. . 20 mi n
6 min, 20 mi n
15 sec- 2min, 6 min, 20 mi n
13 tee, 45 sin, 2 min,bmi .. 20 mi n
20mis
6 mi n
5 see, 15 we. 30 sec
2 min. 6 min, 20 min
2mi.. 20 min
5 nest . IS sea . I min. 2 min, 20 mi n
Doses of 1 .0- 1,4 mg/kg were injected aver an interval of 4 min ; sacrifice time wus determined from nod of
injection to fir wing .
Mice anesthetized with phenobarbital before nicotine adminntration .
Mouse inject d ip with 6 mg of unlutwkd nicotine per kg 20 min before -6w-live nicutine .
• Mice err iethed<d with pentobarbital before nicotine administration.
530
Source: http://industrydocuments.library.ucsf.edu/tobacco/docs/qmvc0212
E
O`
O
'. C
1.0146351 7
LOCALIZATION OF NICOTINE-"C AND METABOLITES 53 3
532 WADDELL AND MARLOWE
\tt.v 1 .• I, tdk,l ualon , nr I,
.tt ., m1. .1i .a, „, -n(--I 4 '
-0-111
. :,
ansatwu-,I Ii .11111.1 0-111
:, t
I li xr he, ,; r tg lt epr er prnlaharhn%1 Ml n ll ,I„ 311 toil', +t 111, 1„ ,, .
n holm., ...o 1114, :k{nnm .tratr„n . {n a,uen, n, 1„ tho', r-', ih, . w ., . unhoawul. ..net the ,n tic s w ;, IH'. .. . . I .., ra t
1v Ills w'hah reeei,id th, 1l-metier 'C tm .rrunc ,, . h
whole-lurj, . :t tun ; v.lima „r ,It . . Ifn. : : . ..1
,. mi- -11 ni ;aota'J s . mI Iouen 211 nun later . (Inc
reach tangs in :, I n,,m l endtn :r.,cr 1,a.m mewled 7, it,
`+Jn11 mount' of tacit of the1 kn,r sect,-, w . an (V2211
t ku
. .m :,Jolt (57rl1 e .l lan, ::tle I,I m;rlg. ,
:1
tr.5•da,
pregnant t't7) ,J (1 .15 n,F' lg, and ;, 1-. 4- . and
7•d.,, -„IJ m,uv cacti nunrd 1 .1 n cLo •\ I- and
`-Jan-„kl r . ode roamed VI mrA4 in and note
l,II,J , .,in nlater . One Icmal 1dtel rnun
.
pre e:ned ,:h : arge done of unlafwted ran, p . One
mterou :,,c,non .l'he Ito we h.anng had been ca .,htrdted
in the . I :rh„r:nnr, t ., he ,ujiwi.nt w„ dear,,, :/1 ht .t ., .
ehomeai :relit-, I„r Iasi: Sch,dn,;eno .e 17„-1 geld
phn .ph .+t .ne llN„n in, Hh„Ic-bend, .e . men . .
Prrtused kuhkil Lunt : 1hr Ion;, I,,nn roc 4„
fn . ,1 mu w I he A/H.J urain u,, admim .ICnJ the
nuhoaatt A.-une he .halulinm: the mouse w .r al .
Zealand H hie rabbi., each ,. e,Nh,.v ghoul + kF . seer,
periu ..d in ncgau,c-pros
. hrc .,thmg ch:n,,bcr nnh
the teohnxtu. dc .cntvd h: \ .,r,ch•r and high:-, i,+,
(Inc pair of long, sea . h-rfuud ,. i,h hl„nd .nnta,ntnr In
taint It, inhale the -a,,- (or 41, in and hen aher
hrteil, he/tile fncnrryt . The co6ntne-'K w , njooted It
MCI 144 sett or P1.1 MI v
unlabeled niambic . The other pair of Ian;, Ha, nerlu .od
of one moot of each at the three strain .. C-5761 ./al .
C57LN, and A/HeJ: the mine w re Lilted n min Inter
witb the same amount at nVCatine .'•C wnhaat a , addoJ
-labeled Altar pert-an lee 2u min. the pump.
were -lapped, th, bkhd allawcd to drain . and the lung,
cut free from the apparole, and D,uen ,-u, the
The Ulriufrdxx•'-"C-Inicutirx wes injeeud t into one
male arpt one female of the C57BL/6f and the A/H .1
strain.: all laser mitt w sc Gunn 6 in later . The
C570L/6I mice receised a does of 1 .3 mg/kg and the
,l'He) re eived 1 .9 Mg/kg: approairriatel1 3 in was
required to inject the inatopk solution . The cwcrifwt tim e
uv det ermined from the end of the injection . The Iprrrnlidnrr•1-"Cjnieatine-t Noside r injected 'r into
one mate and one Icmate of the C57BL/6J strain and
frozen 1 2 min: it was also given is to I .a mates and two
femaks of the A/H J urain : one male and ono female
ten frozen at 6 min and one pair was franc at I hr . The
dose of IN. compound to air mice wa, approsimateh 4
mF/kg end w injected over about a I-min imcn'al .
Larch of the mice were Ynestheti,ed tightly with ether just
hefure freumg hI immersion in Dr, Iceili- .c The,
were then prttcssd for whole-b.rda autorad, .gr .rhl h,
the tither; teehntyur. which dun not aiuw thawing at
contact with an, sateen- 115. 16) .
In %'1em Stadies. Mato C57BLitt mice that had
coed or ipiccnans were Jruzen and sea oned a.
described ahner . The a frozen-dried. whole-bod, actions
It n,,I aim it, the ha,r . .tni nu. i l„I lie hest. . :u all nut I1,•n' .J . alter tnlcittu I
2i . lIt aIhin I trues . \ . 110 and C II- I . arc
deso,d al o,ehutin . no raJ,oacticih wa, seen in
theca silo, m those ,train,. T here nuts no thltnrcnee
,n d,stributton between the perm-free and the con,entional mow . (lie ().
The tetu,e . in the preencmt. pigmcmcsi Train
.u all slave, of gestation studied had intense .e-
EYE BRAIN
eontolonon, of rtdalacti, it, to the melanin of the
a, earl) a, (I min alter,, administnGon of th e
impound u, the mother Iiig .4) . Little rtdhtaeti,il, ntuhl he detected in the fetuses set the mice with
sacriiiee tints, of 2 lien or less : hauc%cr . by I. min
seluctisc uptake w ohxn'ed in the Ih .S-day
fetus . tl o h the s ,cption of the crc. all fetal tissue
eumenir:ttions were less than that in maternal
blood . Untlorni distribution of radioaeii,iry t o
BRONCHI STOMACH SPLEEN KIDNEY
interatomic stage in sodium earhasrmc,h,l <ulielos .
with Dry In/hexane . Autarudtugraphs were prepared in
the tame manner a, the ahok-hod, .e rinn ,
HARDER'S GLAND SALIVARY GLAND LIVER INTESTIN E
Results
In Vein . .hirorine . The typical distribution of
radioactivitx seen in the whole-bods autoradiographs after administration of )N .,,,gt/ii'l•"Cj
nicotine is shown in fig . I . High coneentratiunf
were seen in melanin in the eye . bronchi . sah,arr
gOnd . Iiver, kidney . stomach. Harder' ., gland . and
pancreas . The isfets of the pancreas show intense
amounts at activity . Increased a+neenliauon+ were
also seen in moll mucvsa . pallbladder . bone,
adrenal medulla, walls of blood ve . scls, and epi.
thelium of (he trachea and esophagus . In most of
the animals, the red pulp t,f the spleen had large
concentrations . and at later time imcr,als the connc-ubated 15 min at -mm empera, arc in 25 mt of
tents of the intestine had sipnifteani amounts rat
0.9' t\d I .on,aining either 2 ,Ci ni carmine-°C. or
radioacttony . At time inten'al, up to h mil, . there
Irani 6'.J w, . 4 .5 Xi of nteotine ."C . The sue tin . Here
were large amounts of radiuact icil' in the beano
than gendy hurled un l:imwipe, and all,rwed to dr, in
but after lr min the amount rapidh decreased Hiih
rM1 t, mcrntght before gluing aga,nr X-ray titre .
liar, ('5761 tat mine were killed h . locating rho
residual aclis ito primarih in the Ittpptteampu . .
-eels: the lungs wore rumored ..d .--J in one of thin'
At 15 se: and at all time infer,al, thereafter ftddifferent . yu. , of fnz,ing . inrutuung in tad, 0o,
hrwinF iv administration of nicotine . the accumu.otetton ._ and nsv.'titmiaF- aeyuonee n., I Ir .t, Ian;
lation I . the . .It of the bronchi Was intense . \i 5
Pie— were in ..Rnrti. IN— vmhdded nd nn/en, the n
sec the brunch could not be discerned hecaue of
cocooned. Inc-'-druid . and placed , \-r tit.the intense radioaar sir in blood in the lungs . No
n
who pine.
ti no. 2. rh same as no. I . oscepllhat
difference., could Ir. detected in the atnuradioof lung wan: franc- and thawul baton ,neuhating in
graphs after odmirn,wr :n .orI of either the purified
: tong
udn,aa„'r s„fauon . In ,eyacn,a no. 3, rho pre e
or unpurifted niclutinc-"C .
s n,nnntd d. Inuen. and van„nod heh+re ,nouh,ttiu,
The nN) difference among the (our strain ,
Th:a,. .rutum r tilt I~ err.: ai t rant .,mprra s n
14 n„ a to' . *,,C I c. .u . 1 4 s 01' n n char the n.,, ptpn,u-sued cities Cf'BL, rat and
Son hero ,uhain added earner no ,,h.r- C57L ;J . re,eaied .-..vac a :cumulatiur. .f rod -•
FIG . L A prier el an aulwadionrph of n rmafe CS7BL/N -rouser fins- 1 min eject Jrieirrrfon of J J nr)r aJ
IA'-nrrr,ri'I-"C)nirorirrr per kg .
00 hito areas correspond to radioactivity .
MELANIN IN MENINGE S
NASAL MCICOS A
,darn rn. nr rn:n u Pnnr or an a,.uuudioeruph o, a fe+me 07111, r✓ ,n„mr leer-, n mu, wen , .
inirrtiwr set l r n+e al (~-nerrh, l-"( ;r .,.ern, . per Ag a
e . rre.p,md to radtuactii ttr
Source: http://industrydocuments.library.ucsf.edu/tobacco/docs/qmvc0212
10146351 8
534
WADDELL AND MARLOWE
NASAL EPITHELIUM SITE OF INJECTION BRONCHI VERTEBR A
LOCALIZATION OF NICOTINE-"C AND METABOLITES 53 5
sat the p.rttcrv al tit-trthutum Ik-twrcn fits muanuc or ot .n sat : ut,,na th,a recctscd If,, mcauns
-'et tmn 1,,, 1-, ,.i :rtfi :e 4hg . nr .
PITUITARY SALIVARY GLAND TRACHEA GALL BLADDER LIVER INTESTINE
Fly: . 3 . A print ujan ourorddiograph f . fenlalr CD-1 gernt-hrr ntovar i u :en 20 -,in Otter rr injrrrion o!
IN-Orrrbci-,•Cl rricoriee .
White areas cerreRrortd to radiouetivity.
HARDER`S GLAND BRONCHI LIVER SPLEEN I LETS IN PANCREA S
lit. Jutuhuoon of Ip rrr.,Gifin.' . -^Cfnfcmnc
. the uuns es that it the me11111-lahcicl matcrwl wnh u esccpttnn . I lw hnrnhnmon u( radio,
aalttrs to the hn.tchi wa, not n . mtenate : ., that
suds the methyl-labeled n,eaii e .
l , .mint an,l The distrthu.an of cutinop-'•C o,ts renrarkahli unit . ... . .
and. in part -lit . ctmmne did not uceomutate in
dre bronchiar nasal rnuc.sa . The eoncentrai ion at
radtttactiv .s in melanin was snmcwhat less than
that with nivutine . The distribution of nicotinel'-h
:-oxide-1'C at 6 min is shown in fig- 7 . Radioactivity was not localized in bronchial or nasal
epithelium at this time interval . Howcver, at I hr
ntncssortby difference betty cco file di .trihuti„n of
mcotnw-"( and c.aininc-'•C when Irurrn-deed .
whole-h,ds -'lions were incubated with these
c .mtpound . of i itru . Both compounds were localt,ed in ntelamn in the etc . but wither accumulated
to the hrainshi . Heaimg the sections in the microu .n en did nor after the distribution pattern,
for either cumpound .
Lune 7istrn•. Fresh pieces of whole lung incuhatcd with nicotine-"C' but without addhisrnal
nonrudwaelive nicotine accumulated raduraci,ilv in the bronchi (fig . It); this is xttuenec no . I described in Methods. Furthermore- heating the
pieces of lung for 20 sec in the microwave oven did
not prevent the accumulation- although it was decreased somewhat . Fresh pieces of whole lung
incubated with rtieutine-14C containing an additional 47 rrmol 17 .6 mgt of nonradioactive nice-
there was a definite increase in the uptake in the
bronchial and nasal epithelium . tine did not accumulate radioactivity in the
In Attu . Whole-body Sertioirr . There was no . bronchi .
BRAIN
TRACHEA BRONCHUS VERTEBRAE INTESTIN E
SALIVARY GLAND BONE STOMACH FETAL EYE INTESTIN E
i-rt, . s . ,4 galas sat as amamdiryer nit a/anregaanr Ci7BL/A1 rsto .er.• ar ro .1 tor: sat rrrraii„n mre lrv : rn t ,nin afar
by aSstnirrrorion .112 mx tatIA-nrerkd-'•Clnirorinr per kit .
SALIVARY GLAND THYMUS HEART LIVER STOMACH URIN E
lnttnsc r+diuaetiaify can be seen in the fetal ere . White areas correspond w raduraaivity .
fetal tissues was observed in the late-term fetuses
of mothers infected sc or is with nicotine 20 min
Wore sacrifice . Again . there was the remarkable
uptake in the eye and a slightly decreased uptake
in the central nervous system . By 20 min the fetal
cancYntration was approximately equal to that of
maternal bitted . In nine of the fetuses at am time
could any increased affinity ire seen in the bronchi .
Small . radioactive spots in the lung were idemifed tat correspond to blood.
The pattern of.distribution after inhalation of
nicotine-"C is shown in fig . 5 . Intense radioactivity w is found in the bronchi- ntfactort and septal
epithelium. -inc . and contents of the stomach
The salivary gland, Harder', gland. adrenal
medulla . kidney . thymus. hrain . hone . spleen .
liver . and intestine all showed concentrations of
radioactivity creaser than blood .
The neonatat mice were selectively accumulating intense concentrations of radioactivirs in the
bronchi ac early as 2 day . ptstpartum after ip
administration of nicotine-'tC . but none out seen
there at 1 day postpartum . At 3 data of age the
nasal mucosa, adrenal, hone. liter, kidney . aaii .
vary gland, and urine showed increased uptake,
most of the radioactivity in the adrenal appaarcd
to he in a zone hctween the Jr, Il ., and curter .
From this age and older there w, ;, i,nte difference
OLFACTORY, SEPTAL EPITHELIUM BRONCHI LIVER
1-tt,<
en
toooieaJh .eraph,Ir..ne
high
rr,ttr,Jl -nrrro+ ; 'C- Jnxr.rirtr h .
t.rhar .rti,xr.
t . The d . rr hull n of r d t 'n, i h h .,te-tr,d n-„n is N An cnlarrctn .nt shining the tntenw•+
.cummatu,r mtit tP .. ,n nd pat cplhet ,t . C. .1 -1 gcme '.v the rrmartaMc ailmtn of the brnneitt
\4 itriy -nca, o--pond to radi,tac.s tt . .
Source: http://industrydocuments.library.ucsf.edu/tobacco/docs/qmvc0212
10146351 9
536
WADDELL AND MARLOWE
LOCALIZATION OF NICOTINE-"C AND METABOLITE S
EYE BRAIN SITE OF INJECTION BRONCHU S
53 7
NON-HEATED HEATE D
BRONCHI
BRONCHI
Flo, b . Prints on, uwnrudio,rvp.4s instil In-oh fun, pi"- inru/arsS iu a aafioe snAuuon runnninnr
iY-mrtltrl---Clni..r/ne bruaw tree-trio and sect testing isryurnre nu . I in irrrhrsds t.
PHARNYX SALIVARY GLAND LIVER INTESTIN E
It . . 6 . .s print of as aurawdto,roph of an arhino 4dar-W innate f ozen 211 min obit c adminislrariun of
)fore rhti-"C)niraitor .
White areas correspond to radioactivity .
BRAIN BRONCHUS
EYE
STOMACH
KIDNE Y
HEART LIVER INTESTINAL WALL
.tendiutraph of a malt A/ii estate tenets 6 ink, oiler iv adno*iurarios, of
Fro . 7 . .4 print of an ae
nOs,iinr'('-\ aside-r"C.
tfhitc areas correspond to radioactivity .
Freezinc and thawing the pieces of lung before
the incubation (sequence no . 2 in Methods) comp)etel, prevented the accumulation of radioactis .
ity in the bronchi . Heating of these pieces after
freezing did not alter the puucm of distribution .
Embedding and freezing the pieces of lung he.
lore sectioning and incubating (sequence no . 3)
radioactivit,s in the perfuscd rabbit lungs showed
the same intense accumulation in the bronchial
epithelium as that seen in the mouse in vivo. Furthermore, the transition from smaller bronchi,
where the radioactivity accumulated . to the lamer
bronchi . where it did not accumulate . could be
cleans discerned (fig . 4) .
also compkteis prevented the uptake of radioactivin in the bronchi . Heating had no effect nn
this distribution, These results were the same for
all time intervals between death of the animal and
incubation in the radioactive solution : the shortest
onto, intcnat was I h r
Rabbit Lung Perfusion . The distribution of
The cart} reports on the distribution of nicotine ."C Isy whole-texly autnradiography failed t,,
mention the remarkable accumulation in bronchi
and melanin (20 :21 . The hq' . .oncentration in
bronchi may be seen in thee. imoraCiueruph's.
Discussion
Intense radmacuvity can be seen in the bronchi of the nonheafed lung tissue halt print) and some uptake . although
decreased. in the lour o issue which had been heated ina micronasr oxen (right print) . yfhoe areas correspond to
tadtoaakit) ,
although it was not identified . These animals had
been pretreated with hexobarbital to decrease the
toxicity of the large done of nicotine . The rapid
uptake in the brain with rapid decline after about
6 min was one focus of the earlier report (20) .
Also . uptake into ganglia (21) and the islets of
the pancreas (23) was well described . In the
present report no attempt was made to discern
ganglia . since Appelgren et al . (21)had thoroughly
described this phenomenon .
An earlier report on the sholr-bads suloradi .
ography of cotinine-aH in mix: allowed some
prneruliealions which are consistent with those
reported in this stud) (241 . However, tritium. is
not nearly as good an isotope as is "C for wholehody autoradiographi : energies higher than that
of tritium are needed to expose X-ray film .
The locus in the prescn : report on the bronchi
is prompted in part by the pussibilin of time cUnneciwn between the accumulation of nicotine at
ihis site and the incidence of bronehogenic carcinoma. Other substances which enter the bud)
through inhalation may also be accumulated here .
and knowledge of the mechanism of accumulation
could hots prophylactic or therapcuio ; applications .
The intense accumulation in the bronchi after
nicotine-'•C was seen at all time intervals. including 15 sec after parentcral administration . and
in all strains . The mechanism of this process
pratides fur both a rapid and persistent uptake .
Ii has been reported to persist for I month after a
single dose (3) The only animal, that did not
Source: http://industrydocuments.library.ucsf.edu/tobacco/docs/qmvc0212
accumulate the material in the bronchi were the
fetuses in ottrnu and the i-d v oId newborn . Lindquist and Ultberg (5) reported that in full-term
fetuses of the NMR I stram a selective accumulation could be seen in the larynx . trachea, and
bronchi, and in melanin . However. in our experi .
ments the 16 .5. or 17.5-day-old fetus of the
strains we used showed no increased affinity in the
respirator) tract at 6 or 20 min after iv or sc ad .
ministration of nicotine to the mother . In the fetal
lung all radlnactnr spots were identified to be
blswd and were clear[) not in the wall of the
bronchi . Net increased concentration could be seen
in the bronchi of the 1-day-old mouse . However .
by 2 days postpartum the bronchi actively accumulate the radioactivity . Possibilities consistent
with our observations are that metabolism, active
trartspott . ur binding substances in the bronchi in
the strains we used ore not developed until the 2m?
day utter birth. Marked strain dilferensrs exist or,
the age of appearance of activity for drug metabolism (25) .
Studies with other compounds that accumulate
in the bronchi sugges that metabolism in this tissue is responsible for the accvmutation (7 141, It
is well known that compounds are metabo)itcd in
homogenized lung tissue, but the cells responsible
have not been identified, Type It alveolar epithelial cells and bronchial epithelial cells hate been
suggested (runt mdtrest evidence iu b, the acute
cells (26) Hrstachemical esidcncc of met ahsslism
S. both akeolar C?t and bronchial epithelia! s-eih
has been presented (210 . Vie hat, bw'n unable tt
10146352 0
538
WADDELL AND MARLOW E
TRANSITIONAL JUNCTIO N
BRONCH I
Fa. . 9 . A Prier of an --f"diatvw'h of robbh lrnlr, a*irk „we Pedaled for 20 min „drh broad conrainin;
(N-mnk,1-"C}+ricaine Ff., unlabeled itkuriee .
Recd-aivor can be well in the smaller bronchi but no uptake is observed in the larger bronchi . White areas
eorresryrnd to radkesct,v,l ,
repeat the experiments with benzo(alpyrene (271
tine in both truatnicnts The (Ptrrrdidinv-=-'•C{
of the (N-melhrl-'•CJnicatine . This is consistent
with the total blocking accomplished by the very
large concentration of unlabeled nicotine in the in
vitro experiments .
The experiments with sections and lung in vitro
demonstrate three important poims . First of all .
the process of freezing the tissue destroys its ability to accumulate radioactivity in bronchi . Second .
the large difference in specific activity between
the nicotine and cotinine used in viiv may account
for the lack of apparent uptake of connive in vivo .
This had to be demonstrated in virro because the
specific acn'ity of the eotinine-"C was the highest aratfahle, and if the nicotine-"C had been
diluted with unlabeled nicotine to the specific
activity of the cotinine-"C . the dose would have
been man times the lethal done of nicotine . Since
nicotine-"C diluted to the same specific activity
as the codeine-'C did not accumulate in the
bronchi of fresh . unfrozen lung in vitro, it i, not
pov,ihle to conclude why cutinine does not ac vmulat, in. bronchi . Thirdh, heating the section, in
the microwave oven does not destroy the uptak .
nicotine had about one-tenth the specific activity
mechanism . This was somewhat surprising . but
and question the likelihood of alveolar cells being
act ive in metabolism . Our experiments with germ.
free mice rule out an implication of a mechanism
related to the presence of bacteria : affinity by
macraphages is thus unlikely . If the extent of
accumulation of radioactivity in bronchial epithelium and liver is indicative of the extent of metabolic activity at these sites . the bronchial epiihrlium has a much higher rate of metabolism than
liver . Dilution of bronchial epitbehum with other
lung tissue when the whole organ is homogenized
would reduce the apparent metabolic activity of
lung it, less than that of liver . This would give a
misleoding estimate of the rate in the bronchi .
The accumulation of radioactivity in the
hr-chi was somewhat less after udministration of
(P ru•didinv2-''Cjnicotine or (N-merh34•'•CJ
nicotine after pretreatment with unlabeled nicntinc than it was after administration of (,'t-rtterht1"CJnieatine atone . These decreases can he accounted for by the blocking from unlabeled nr:o-
LOCALIZATION OF NICOTINE . "C AND METABOLITE S
w;ta:,ur . that c,tudnr,m, which dr .u .rv 11"r
enruer . J,• hat dc,tnn th .. ntr .hani .m . I rccnrn•
rho a„tic, , . ou3r . dcwu .uy . .
Srncc the mctahohtc•, -it anal flout nc-t \ .ast,le d ., not .te vututatc In the hnm.lu an nasal
c•pithehwn at tr o ,n . one intcrpret .uirvu it the
upl :dc- al radioa,i oitc :nirr :,dnnni,tr :u ran +r1
mantas-"(r , nrctalrobl' c . nu .o n 11-41tm
h, other anup„ural, at this r,n . . rNwnuac- 1"•!\asIde is a major nick; tn .lnc m lone t_Jut : o+.abraIion of radioactivity in hronchial and nasal epdhvIium at I hr that not at o olio) alter administaoion
of this oxide could be due to reduction 0l" the -ids
back to nicotine- which then IocaLaed in this site,
Other possibilities for accumulation in these epithelial cello include active transport or binding of
nicotine.
The accumulation of both nicotine-"C and
cotinine-"C in melanin occurred under all conditions studied in this report, It would appear that
this is a simple binding of a basic compound to an
acidic protein . Although the accumulation in the
melanin in the meninges of the brain is dramatic .
there is no reason to believe that this has any direct
effect on the action of nicotine in the brain . There
was no detectable difference in the extent of uptake of nicotine-'C in the black (C570Lf6J) or
brown (C57L/J) strains. Only the are albino
human would offer a comparable analogy to the
albino mice (A/HeJ and CD-1) .
The nasal mucosa also had an intense accumulation of radioactivity from oicourte-'•C . 144•
have not included this tisouc in the systematic
study because of the difficulty in handling the
tissue in the various procedures ,
Aeknowfedgnnenta . The author, are indebted to
Drs . Herbert McI' Ennis Jr . and Edward R . Bnwman Inn the synthesis of the cotinive-"C . We
,,,,h alit, to acknowledge the etcelleni technica l
,sr,tancc of his . Bevcriy Bruckner . The rabbit
lung perfusions were done in collaboration with
Dr . Harry bustenbauder. College of Pharma,-s,
L ni,'enin of Kentucky . the are indebted to him
amf his ,, .,,rant,. Mr, . Barbara Hsgedorn and
hits. Shirley h. arren for their splendid cooper-atrun . The germ-free mie,: were injected and main,
tamed with the invaluable assistance of Dr . Helmut Gordon and Mr. Geza Bruckner .
Rrreraacn
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1 197 .4 ,
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9
1196'1 .
A . . \ppetc,en and L. Hamer,,, .I, rY Pharnra,'ad.
7u,trrarf . tL xh U97?t-
10. A . Hanneren. L . Hanson . S . is . Sidstrand . and S
L, fibers, .411 Endurrnrul . 47.95(191 .11 .
t I . G . B<'ngn it and S . halbert:, Arrn E"ndurrurot dt .
$6 t
t L . Haermarstr0m . Aria Phrriut. Stand, 70 (Suppt
289) . 11196h1.
f?r 1 . E. idanpuen•Hciktita Lee J . Plrornm,Yl . L 2h
119b7) .
t4 . 9 . Ostlund, Arra Pharnrared . Patient. 27 (SUM],
I t . 1119691.
15 . S. Ulthcrg, .4t,to Radial
. SoPpl, It&. 1 (19 .44).
16. W. J . Waddell and C Mao-t . Slam TeehnoI. 41.
61 (1w.9) .
17 . A, C. E . Purse . "Histochemisu, ." Vol . L 3rd ed..
pp 1342. 1144 . Liule . Brown, Brwon, f969,
I6 1 . F . A . hfeld .owo and L. U . blown, 'Staining
Mcthada, H,uiolucic and Hislovhemic-al ." p IM .
Paul B. H,wher . Now 'tort . 19M1.
Iv . It . N . \eimeicr and C . Bingham . Lit, S,r . Pon 11,
It, 907 t19111.
lu. E . Huns,on and C . G. Sehmitcrtow, J . Phanuurrrt .
E.rp . Thee. 1-17.91 t1962t .
it, L : L . Appelgren. E . Hansom, and C . G, School—
to-. Acre Phvriol . Scald, 59, 3 .141 11963).
= . C . G . Sehmnerto. and E Hanson. in "Tobaece
A1Laloid . and Related CumM,und,' al 5 . can
Lufc, . -d I. .-"5 . Macmillan . Sc„ Sork . I96i .
'? . P . Stan .
. and It T151w, J . Lulu.„-,d . Sx. 21
19-?r .
24. L R . birwman. E . Hanasrm, L II Trunhult H
Stctsennas. In,, and C . G . Sehm vvif;rw" J . Phan
rnc%d Exp . Tbrr . W . 301 ttsMi .
13 31 i it addctt and G . C, 11ar1UVe- ,n "I'errnatal
Pharm,:cab1!" !B. L . hurt,,,, vd .t . Pr 1l9 2b,.
}cudem,c Plea.. Nec }'ter t . tb'fi .
:h . 1. is . Bend- Cl, 1.. it, H,inks and T . L. Cram . Ur,re
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. t. . Leone
. J . H],r. .,'hrrrr .
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anal S D . Ganp .uti . Hi, r,. blur . J . 3, 117 11171,,
N. H. 31,1, c.n,,. Jr . I h 7urnhull, and I It ,
Bow elan. J. 4Yt . C in n . . .},., . 79, n 3t,` a 1 ,3' I .
Source: http://industrydocuments.library.ucsf.edu/tobacco/docs/qmvc0212
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Source: http://industrydocuments.library.ucsf.edu/tobacco/docs/qmvc0212
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