L j. BATCo document for Legal Senriees : Health Canada 20 May 1999 RESPONSES OP mm mmc, RENhL p ~W~ ~ ~~a~R1~ HYOROXYUSE PatMt OPOEEE 10 ClcM(ETTE SF(OKE" S~NULY t'slaB the 1,-I,:. iuhalation rrsten Ibich releases 1 prccirely-Palibrrted dilution of robaeco note into a ~dlr, sie end r11l rats, guinea pila, bm ~1 I·biL vera cxppsed ~ 6~ q~ rob e~u~t~ 4d m ioduce rae rerl a (10 puffs of 11:i dilat~~ of sob fr· cigarettes pre- erred Ira 1 blend of Canadian fluMllred eobrceos), The tissue ql tto- e~boP bdloVLle (hEE) activity 1 3erlnred in 9,000 f supernatanrs of hl- 6lrrcs Of lung, liver and kidnepb hours folloving qolna, elllreete sob was found to be 1 !otent iOdnCer of a, dilutions II biP u 1:40 inducing LhiS Cptypr in ra". ~dncl. PrBd rilrPc, I~d Ipcies differences in ~~~1~ were Oa~d~ induction up to I-fold, Ma observed onlv in lung and kidney of rl~ and fenzle rats, ln bamsters and tcrb~l, 4 P vee induced in both sad but onl!renal P in f~ll~taP. Innb and fale guinea p4s, only thcrenal P was induced soile I-fPld, Bepatic hEE vas not inducible in Iq of the splcila studied. me ln$sL of changes ~ hEE in a responsive IplEil and tissoeIa) could be 1 valuable tecbnlque for the quantitative evaluation Pt bio~iol effects of low E#amtionl If cigarette ate. tlraatcd It the Il'h ~1 II(E$ of rle Bocieg or Ilrieolq~, Ir drllr, IdlisLa, March 12-U, 1979, and to be subitted to TOXleOlOclI for p~liclrion~ N BATCo document for legal Services : Health Canada 26 May 1999 INTRODUCTIOS In the course of our studies, ve have already reported the tave! finding that uhen raPdom-bred male guinea pfgs are exposed to undiluted cigarette smoke in an ishalation machine designed in this laboratory [1), pulmoaarg a:yl hydrocarbon hpdrmylase (1IlW) was wt induced while renal 12Dt as [2,3! Ue also reported hi when Sprague-Davleg rats were exposed to rmo~e ender the same conditions, there vas a marked increase 4 both pulmdaary and renar aBB actlvi:ies, mwe organs and species specificities encouraged os to Mdert~e a guanrirari~e aldy of the induction of ABB in the tissues of several species of small laboratory animals, as ve nov have at our disposal the B.-A.T, inhala- rion system [y, in which the animal 15 held in a di~ber prwiding a nose~nlg exposure to 1 greciselg·callbratcd dilutirm of dsaretre smoh, these results are presented vlth the hope that ve will be able to extend these guaatitat!oe studies to other organs and species in the near future, -2- BATCo document for Legal Services : Health Canada 20 May 1999 _ ~Bms m raaons hpClq~tll 1111I II111Pd fllt SPTleL~IV~(I Iltl (300-0005), da·trd guineI pilr (300-1001) Iud Sp~ golden biters (5C1001) 9r~ mek~ld from Canadian Breeding Ferns, ltd., st. ealrlnt, Pue,, while ~e nongoliIIl gcrbils (50- ;001) vere obtlhed from hblebroo~ Fern, Inc,, lje$t Brodkfield, PII, Cigsrettes ~e cigarettes used in this study were dbar filrcrsl end ~4 nade frap s blend of e~dLn flue-cured toblccol, Exoo~Pre to citaette rob The I,d,r,~lPn iuhelatioo system vas employed in this study [4j ~ Ihe ~aoh concertrItiou veB generated by dravioI 35 mi lafp of Z seconds dantiop into I dlllt!r chrher, diluting this s~lcd~ I h~ palupe of sir end the expelling it into 1 central exposure hba, AniPaL vere restrained in cyUndricel ch~crd with their noses exPoaed contiml$ to the smoke in the crttll cYa. Par the dose rlsprl empu, rats pere exposed to 5 to db puffs It diIutioos of 1:5, 1:10, l:tO ·d 1:10, 5~ c~an~~til· in the exposure chanber re regularly monitored By sampling on Carhridge filter pads. control aninal vere kept in the chdctr to simlate the stress felt ~Lr exposure COPdltll~ preparation of tissues 81 ·oke exposure Is urrhd out letveen I and ~ e.m., after whioh the animals II transferred to dleir cages, and food, but not Pter, II YlrMn, The animals vere sacrificed It an optkl time of 6 hours later by an as~olr of labPul (~bptt] [3], pi tissues Ire CicUt r~d, plated a lee, veighed and homogenized in 3 volures of cold 0,15 I Ie1~,05 h Tris-BCl buffer, pH 7,5, usin8 1 rollaon homogeniter (Irbh Instnnents, ttd.)~ lertcs ocre eentrihlcd at 9,0001 far U minutes and the superpatants obtained vere used for hioohenicsl studies, -3- O BMCo docunsnt for Legal Sonl#s : Health Canada 20 May 189) 1Sflr 3f Y ~et~itr ~I )aedre d ~edlq c d· ai E ·Ind b Idar d Crlbpin Bj ,,,d o I··n Y i dr doa p$r~L·. DrtlIll ~ dr ?rr ,~, bl~~ Lie been lacr~d ~1· ~et14 letcnlna~i~ The d~id I~ct prIeedurI MI ~1P~'i al· Sratistial IrltJis ~ ~~Q~I ~ol~ ~r uJed o I~df be r~dsrkll ~~er~ d 3(O.DS vis p~ap II de ~~ fr liPlfLPee, ·4- BATCo document for legal Services : Health Canada 20 Yay 1999 RTSJITS in rat kidnev Dose-response r~7'e · the dose-response relationship Do the bash of palimiPary experiments, bervelP hRR a~:~i!! oi rar;ddnPy and th ~aoer of puffs (or duratipl of darermined st calibrated dilutions of 1:jl exposure) of c~arette smoke uas 1:!0, 1:10 and i:IO, Ihe results obtained are ItLbcnted In Figure 1, in mean of analyses of ~issuLs from a ~ini- which each paint is derived from the nun ci Eix i~di~~ual sni~dls, F!a this fi$ute !t vill be clearly seen ~ha~ ASs iPd~c~iPn is dependent both on rL duration d exposure and the ~noh conlen- Jarion. Brll inercases in activity were pbsened early iaUo~ng exp0wl to only a icy gaffs ai a 1:i dilulioo oi snoira, and a more gradual fnereare to a 1:40 dilutioa~ There also appears being found~fo~~ving prolonged exposure ~e inhaled smoke, maximum peak ~D be an ~pc~ response of the tissue to IwelJ pi linl ac?l~ity 1,00W above too:rol being oboe~ed at all rmdre eonrcn- trarions rested. IaducliDP of ~ in tissPes of four species described ab~ve, it Ms decided to snlec~ Frw the deserespDPge Nrves 1C pd:a at 1:I dilution. ior a ogrte~aric sweg of ~B~ induction in lung, lidnep and Il~er o[ a a~LI. oi srml! lab~raro~ rprciea-mtr, h~srert, gu~ra plgl apd gerlilr me'e'panse, of plm~a~, real and hepsde P ci eiL and fnale syrian baplrero, SpragUrOauley rats, noagollrn $erbilJ and r~nd~-br~d pin~s pigs are shown in Figure 2, Each bar sbaw the mean activity and standard de~ia~~on of aealy4Pr tw Ilmini~ of sir iadi~id~ ,imais. Ilnr~ed tissue differences in bad 111 Isl as veil as irr iPdu~- ibilfryl Save Seen obserPed betveen sexes and Jpeeies. y9le h~trrs had about 21 times more IHB in the Iridncy than the fe~aalL of the spPcirr 90 other Ipe;ies Ehou~d.:Be high levels of renat iiBB noted in -5- O BATCo document for Legal Services : Health Canada 20 May 1999 male hamsters~ I simi~r sea difference vas also obsmed vith respect to ABB levels in rat liver, the nles phcving about 5 bes as ~ch activity as the f~ales. Up ta a L-fbid induction ei ~H~ was ablervd in 4 4 and L~idnq of male and fc~ale rats, while a 5·iold induction of this mzgme was obsencd in the Iridnrg of pinu l~s~ In the case ~i the hmu~e~, ~he long was fntuc- !ble in both sexes, while the kidney Ms inducible only in the female of the species. In~eralliPpBI onlg tL log wee inducible in pale and iePale gerbils, the kidney being mreseo~asi~e in both senes, ~F~ was net indsible In the liver of either eer oI aPy pi the species studied, -6- BATCo document for Legal Services : Health Canada 20 May 1999 ___ DISL~USSION Of ~he:eceatll developed expoNTe aysr~ns for studying tobacco woke rolirolo$y [d,[ii, the one descr.;bed by Bwca 14]t appPur to be the Post :eli- Iblr. Using :bir Pachino, 3incs and bir colleagues haPe cPmple~rd awe ucellent dosimetrie studier iwolviPg calculation of cigarette dose epui~allPrs from dose levels achieved in several species and have epep considered an ecRr 10kTtOP iIoP chase anipall r~udiu Io puanti[iearion of erposure in h~s~ [10,111. E~Drure of tobacco Emo~e her been shown to result in i~due~io~ of ~B in the long and kidney at several znlmals species 12,3,12-10]. The Pain ptjl::~ve of pur IP~CLllipa~ILmS MI to determine if ue could dereft and ~Nre a tissue biochemieal eifcct of smPh exposure employing t~is Inhda:iPo spta in which highly diluted n~3ite could be Employed, ~he cazy~e bHB Ms selected because it uay be impoIran in cancer on account of its role in the production of higblg reactive clKcrophlles frw lolycyclic hydrocarbons [11116] ~ The induction of I as a leasure of tobacco rs~Pte ~oxie!ty pr well as an fnden: of food-screening for safety esseswent i~aoe already beeP suggested 12,11,18]. ht cigarette smoi~e is a potent inducer of this eo~yme is shown by the fact that dilutions as high as 1:40 induced :jir Polyme in the rat Wdney. Ihp aePllent dose~-rapcmse rrB:i~bhi~ seen with rat reral ~gB soggtJted eLt similar dosrrapoPse studies should be undertaken using other labPralorg animals in order to identify the mcst senS- itivc olgb0s BOd speeieb. 'Ihe lungs of pacs and ienelaa of three Ipecies (hamsters, rat and gerbF1) responded positively to inhaled tobacco sPoice, the induction of dgB baing two to3-foldhigher tbancontrol letnls, IncoPtra~t roPur arUcr study, dep- resioa of guinea pig lung lgB was rot observed [3], This Pay be due to the fact that the guinea pigs were erpsed to diluted rather tban ucdilufa woke, -7- BATCo document for Legal Services : Health Canada 20 May 1999 and coaseguently, marked diill:enr ~ancellrraticns of pPlwlially reactive caaounds. ID the new deliverT. syrra, exposure to undiluted smoke is ippossele because transfer to the eypcs~e chamber depends upon at least a 1:5 dilution of the smoke. Rapid dilution oi ssoi~e is considered important in order to limit particle growth [I] · Onip particles betuern 0,25-3.0 1; are considered ~spor~nt in causing lung damage [.91. It is also posaalt that the rapid d~i~ ution of the smoke, while jreventing particle growth., results Inmore rapid chemical oxidatian which would not occur when uPdiluted s~dre Is used, rt is of interest to speculate why the ABII of certain tissues uas pot induced following exposure to tobacco smoke. Of the four species studied, only the renal activities of ale and female rats and guinea pigs wre induced, results which agree with those reported in the literature IZ,U,IIJ. Bepatie ~I~ of all species failed to respond folloving e~posure a did the enzyme in male and female guinea pig lung and male hamster i~dney, The tissues that do not respond to tobacco gnoite erpopnre possessed high basal levels of ARE Imliire responsive tissues invhich lov:e~elscf P veredereered. Those tissues uieh high basal i~B~ activitP ~sa~ be much more sensitive to inducing agents in the ewironent, the enzgmt being already marimalry induced. While it has been shovn that hepatic hpa can be induced by polpcyclle hgdrocatboos such as benza(o)pyrene, dib~nz(P,h)anthracene and chrysene, the concentrations of these chemicals in diluted aokemap not behigh enough to initiate response in the tissues capable of rapidly degrading the [20,11]. For example, the hovll polgcyclle antic hydrocarbons present in cigarette 8mole, which include powerful carcinogens ouch as bmzo(~)pJrme, dibeaz(s,h)anrhraccne and 5-Plelhgl- clnysene, add up to less than 0.5 Ilg par cigarette [221. In addition, the con- centration of potential inducing agents reaching the liver would be further diluted by a widespread discdbutt0o in the body's tissues following aborption frw the lung. -8- BATCo document for Legal Services : Health Canada 20 May 1999 Frw our studies it is dear that sex and species differences vill have to~be taken into considesarioo in selecting animals for studying tobacco smoke toxicology. In conclusion, it may be stated that good control wer the nev inhalation system has been daonsaated in studying a relevant bio- ehenical effect of tobacco sooke in o~erimeatal animals. -9- BATCo document for Legal Services : Health Canada 20 May 1999 __ IE~CB i. A.S. 5ipani, I. Iooue end J,C, apll, kb~ ~CLt,, 31 (1974) 75. 2. ~.H~ I~~ria, J~ Johnson, J~C. PdIt end 3.P, WiEachil Toxieol, hppl, ~~col., 11 (1977) 119~ 3, ~.H, lil~i~, J, Johnson, J,C. 8~~ ~ B,P, Witsehi, Toxiool, bepl, phenweol., 11 (1977) 433, 4, J,I. bm, I0~Cpl0~l 6 (1976) 189, 5. L,IJ, Lttdcrl, f,L, Lcong rd I,J, 5~~, Cnncst Pr,, 21 (1962) 1120, 6, D.U. Lbert end B,V, Celboin, J, 8ial, Ohs,, 243 (1968] 6242, 7, Y,N, hlbfd~el 81Dchon. J,, 67 (1957) 423. 8. R, 1~, la, biIa. Blt~~, 2 (1975) 11. 9. W,L, bdda, D~A~ Crwaia, W,E. OaIbelr, ~,1. Cuc~in, J.R, Stokclg ed j, Pendr!cir, brch. Environ. 81Lh,, 3] (1979) 64, 10. 1. 1~, J,t. 8aen, 1,i. ~il:a ad P.C.)~ Ltta, rdcoloa, 6 (1976) MI. Il. 1. ne, ~ctlol~l (1977) 180~ 12, 1.1. vcIeh, I~ loh end I,B, erp, lIlo 5fi,, 10 (1971) 215. 13~ J.r, ~at end J. 61ala, bt. f. Grit, 19 (1917) ~31~ 14. P. 1Pf~, 111, G~un. Cha. ?lthoI, Ph;uMcol,, 17 (1977) 101, 15. J.I. ~~n, Clnecr Pee., 30 (1970) 159. 16. D.P, Tb~a, 8, Ill~l I.T.1. LI, 1, levin, I.B, ConncJ end P,W, Jorine, hoc, NetI, bed. 8ci., U,S., 73 (1976) 3581, II. P,J, ~~ J.P, leer, 'ToxiooI, 4pl, ~ha~Ycol,, 36 (1976) 331, 18. r. Pityse end I, SnBLnto, b8rie, Ibl. Qa., /3 (1979) 833, 19. E,1, YInder ed 1, tofranP, fLceo ed robeooo Sleb: 8hld1le in Prpui- nene;ll Oueinogenesie, bondsie Iral, Islort, 1967, p,91, 20, I,H, eonncl, lturascoI, Pm,, 19 (1967) lr~~ 21. B.t. Oalboin, Ib, Clneer LI,, 10 (1967) 1, 22. 6, i. WInder end Q, Ioffrmnn, P1 Is ~1. f, nad., 300 (1979) 194. -IO- B#Ca doelment for legJ tenter : H,~t~ Caaldl 20 rey 1999 IEC~S nCre 1. Pe dosc-ralonle rcIationship bca 1~1 hydrocarbon bdrqltst (g) activity of rat lddney and the ntnber of pfi~ Ior duration of glare) of cigarette eo~e at calibttted dilotioDa d 1:1, 1:10, 1:20 and 1:40, PeY ut~lBI measured It 6 krs after aposllre to the r~a. Each Point nhoWa the nan Df dqliblC aualPseS of ~IPC from 6 individual ~lr0~ Figure 2, The rclps*br of lung, renal ;md 4~ Y Oi eontrol (C) and spoke-exposed (T) ~t and fale Syrian hamsterr, bprllue Davley rats, Mong- olian babils and randorbrcd guinea Pil'~ hninal vere ~lpd to 10 pdfs of 1 1:5 dilution of cigarette ·o~e while c~ol animals re haled in the ch~crs for the ~( length of tine hnt breathed ro· dr. Each blr 8has the lean actMg and standard deviation of analylea froo 6-9 individual anipals. ~L Isttr~~ (') indicates enzymatic activity significantly different (p(0,05) from coP~ol~aloea, -11- N BATCo document far legal Senlces : Health Canada 20 May 1999 FIGURE 1 Time (Seconds) 0 50 100 200 300 400 3.0 r i~l:lo o 2.0 .,·c· E t > P I c 1:20 r u I E I~ 't IO I ~n 1.0 1:40 I I ii mO 10 20 30 ·40 50 60 70 80 Number of Clgorotto Puffs BATC6 document for legal Services : Health Canada 20 May 1999 FIGURE 2 Lung IM"'' Ok""l' Homder RPI Gerbil I~rtuine~~iB ~za cC LEI,O VIL Q:ii I I· r 0 C~Et~CTCI m C f C i C C Kidney Hamster Gorbil Guinea Pig mlo 1.0 oB b E 1.0 r2 "O C r D C i t C i 0 i C C i C i Liver . Hamster Rat Gerbil Guinea Pig !50 a40 F;30 ~4 20 ,10 o C I C C i t i C T t i C i C BATCo document for legal Services : Health Canada 20 May 1999 P N W O BATCo document for Legal Services : Health Canada 20 Hay 1999