Tobacco Documents Online

(-.raiw.,pteneai.. Vol.4 Nd,4'. ly...l'll; , 4'N. 1'all Short Cornrrtunicatittn Inhibition of endogenous nitrosation of proline in rats by lyophilized beer constituents Brigitte PiKnatellit, Rene Scriban°, Genlyd Ucscotest and ffelmul BarLscht.' 4ntcrnational'Agency for Research on Canccr, Unit of En- vironmentaliCarcinogens and Host Factors, ISO cours Aibcrt-Thomas, F-69372 Lyon Cedex 08, =Ecole Nationale Supcricurc des Industries Agricoles et Alimentaires, 150 rue de I'Universite„59509'Douaii,and 3University of Lyon I, Laboratory of Organic Chcmistry,ll, ERA 689 CNRS, F-69622 Villcurbannc Ccdcx, Franee- 'cccivcd on~24 December 1982; accepted on 21 February 1983) Abstract Various amounts of lyophiliud beer were adminislered''to rats dosed with proline and sodium nitrite. N-Nitrosoproline that ingredients presenlin Ihis widely consumed beverage in- different brands; the effects in vitro were most pronounced at pH below 4. The highest inhibitory effect was with beers with a high total polyphenolic contentL Our results demonstrate inhibited in a dose-dependent fashion by Iyophilized beers of solutions. Both in vivo and in vitro nitrosation of proline was determined afler 15-min incubation of the same precursor (NYRO) excreted in the 24-h urine was monitored as an index of endogenous nitrosalion. In vitro formation of NPRO was hihif endogenous nitrosation. !' only recently: a reductiornin the mutagcnioilytesulting from effects on nitrosation of such polyphenolics have been studied beverages, including tea, fruit juice and beer. The modifying widely and are commonly consumed in foodstuffs and More recently; we demonstrated''catalysis of the nitrosation of proline in vivo by rcsorcinol and catechin and inhibition by ehlorogcnic acid (9- 10). Polyphenolic compounds occur which either inhibit or catalyse N-nitrosation in vitro (3-8). •mpounds (I). Their rate of formation is affectcd'by'a mber of chemicals (2), including phenolic compounds, source of exposure of the general population to N-nitroso Endogenous nitrosation of amino precursors, either in- gcsacd or occurring in the body, may be the largest' single ~ nitrosation of mcthylurca by coffee and tea cxtracts has been il reportcd;, (d I); similarly, lannins present in tea and other bcvcragcs have been shown to inhibit the nitrosation' of secondary alkyl amines (12); and Nakamura and Kawaba(a (13) have reportcd''a catalytic effect of green tea extracts on this rcaction, A variety of phcnoiic compounds are also present in beer (14); their structure and reiative concentrations depend on the composition of the raw material (barley and hops) used, Ihe type of brewing process and the procedures that may have 'L. w/NMn .1MreFt\MNItTMY .tNfJd hl" aJdrlSlCd. •:~Mireaarwrn.: .NI'R( l, N-nitrtW qr~Aine: 1111 Itir.. 1 tJ., uai,rJ. Fnr4rwl. ' been applied to remove excess phenoiic compounds from the beer, to avoid haze formation. As the catalytic and inhibitory effects of polyphenolics depend on their chemical strupure, pH and the relative concentrations of precursors and modify-. ing agents, we studied the effect of polyphenots occurring in commercial beer on the rate of nitrosation of prolinc in vitro and in rats in vivo. l r The methods used were previously described in detai (9,10 15,16). The effects of modifying agents (i.e., Iyophilized beer) were determined on urinary excretion or in vitro formation of N-nitrosoproline (NPRO)', measured''as an index of nitrosa- tion. NPRO was analysed by'gas chromatography coupled with a thermal energy analyser as its methyl ester and N-nitrosopipecolic acid methyl ester was used as internal stan- dard. Four different brands of commercially available beer tiR. I..lnnucnee or.lyophitiized bcas (brandf A and B) on the nitrosarinn of proline in virro ua function of pH of reaafon medium. The.amyseontained 4 mmot/I sodium.nitrite rogeth'er.withlyophiliud heer type A(6.25.mVni reaaion medium) or type B(s mVmt fextFon medium) either in the abser..r or t!~ . for.A, A ror B) or.suppkmenled wuh 100 mmoVl proGne( is for A, A for 01. Control asuys (0) were carried itul in Ihe absen.e d lyophitiml tKCr and ~comained 4 ~mmot/t sodium nitrire toaeherr with IOnD mmalAL pndine.. For all.the ac.ays, precursors were di4sotvad',in citric attid.ilratetiuffer off variable pH. , NPRO formed (nma//q was mnwral after, 13 nan of itwvtia• INm at J7*C: Broken.knes represna the dineretan Mw.en nte anramts of NI'R() formed with and wirhoul wpPknenoiuK dteh•uphiir..fMn.A f---. or It .1 with.lu0 mmuVl rrotine. .~ .~.~~. -:.,: .. ~ C S

N.)'iy~nvlclli.r! uA' T.ble 1. lulh,r[wr.ur4ylqlltilibedllkNy,vmlde. (A9oU1 - Ihe nitnv,atemu1laalmern wlrn.atNlqm rala.rn vrco. liyu.mwm. IulnlhaY 1 (n:wkgr-mll. Notle 2lilak8rmrnd , lwrdine) " ~ .~)(ll:akl{mund~ _ Ininnoe). . .... . . .. . • 1.3: 0:5(S) 4 (Conuol)~. ~-...~A (6.25) :: A (6.25) " . . + C (6.23) C (6:23) NI'Rtt hunad - . 1'rohile ~~ Nitritt /n vlw1 ..tnm_I/2•fhl I'rr ral ~~ I'er I/rl Pnad H Inlubr meun a St) ~- of addkd liwl` ~. - (number of rals) pndinc° . ' 09 t 2I'(b) ------ ~~ . (IUI:Nmp (4 rmol). D (6.25) ~. . ~ .,. . .~.. + , .. 4.0 : 1.845) ~ . 7.0 O(6:25)~~ _ + 11.0 t 6;7(10)" ~~ O (3:12) + 2.0 : 0.9(5) D (3.12) + + ~I8.7 : 3.6 (S)• • D~(t.56) 3.0 * 0.8~.(S) ~. . ,.'* ~:... :..~ . O(1.56) - •38.2:~13J(S)' D(0.78) + 2.8:~ 0.8(4) O (0.78) + $r.9 * 46.1 (S)" D (0.39) 2J * 0:7 (3) D (0.39) + + 63.8 * 38.0 (31 (designated A to D) were lyophilized. Their contents of total polyphenols (including anthocyans)4 anthocyans and proline (mg/I of beer in parentheses) were, respectively: A(203; 38; 315), B(162; 18;,288):,C (98; 13; 107) and D(22dC 53, 426). Polyphcnols were analysed according to the method cited in (17); anthocyans by that in (18); and proline by'an automatic amino acid analyser. The inhibitory qffects of lyophilized beer specimens A and 0 on the formation of NPRO from proline and nitrite in vifro, as a function of the pH of the reaction medium, are shown in Figure I. Because all the beers contained some pro- line, inhibitian was measured by supplementing the lyophiliz- ed specimens with an excess amount of proline. Beer sample A inhibited nitrosation of proline at pH 2.1- 3.3 by 97-87% and samplc 13 by 91 -63%. When no exugcnous prolinc wa.s add.d, the level of, NPRO was only 4-204. oflhc expected 492 71.9 * 3S_3 (25) 70.6 0 + ~- . 2.0 : 0.4(S): 6.7 + 8.7~f 4.6, (10) • ~~ 2.6. :~ t.2~(S)~ 12.2 a 4.9.(10) :' " . LS t 1.015) 14.8 79. 19.3 : 9.8(S) " (.9 : 0.7(S) 17.2 19.1. : 7.3 (10) " 9.6 86 16.7 35.2 51.7 61.5 73 In wr.n (nnd/85 min)"-- __ TwaN-_I'er Itlfrmttl ap Inhihi. . of~.adtktl'~ . tion` .. proline" . . 1.9 ,- - 7.9 9.8 27 12.8 3.7 28.0 243 84.2 152.3 .- YC'xoups of OD V1 :rats(300 * SO 8 body weiBht) were faaal for 20h before starting Iheexperiments. Precursor solutioru werenra•Pared in ctilrii xidatitrate buffer, -fi , H 2.5. Ran were Biven 0:5 ml of eitha proline solulion (control eaporimenl 41 or of a sollttion o lyot+hida.ed beer (d'ufferent 6rands A lo D) at thc doses indir.ated C. . nbout or. with suppiememed :protine (experiments S- 22). immedialellV. followed by an administration of 0.3. ml of a. fmhly prelwad nitrite solution by su>rnach sube. !n vkra; precursor solutions were insvbated lal )7'C for 15 minn in buffered solution, pH 2.5 ((na4 volume, I nnl); NF~Vpt for.experimeno 4, differensa bet- .een mean amount of NPRO excrned in 24-.h urine (or formn) in IS min) in IAepresence and in the absence of proline added to the lyophiliad twer samPlt. Meart , amoumofNPROecaTaedin24hurine(orformalin ISmin)wascakvlaledby.subinctin8thebac4groundlevd.ofNPRO,r•fienratsra•eivedsodiumnpriteonly (experimem 3), from the amount of NPRO Imasural in ex(xaiment e. Relilive to the amount ofNPRO in expcriment 4.'IMan of Ihreeexperilnents.'Wikncon tot was applied for statistical eWaluati6n of difference beaween NPRO emcreted in experiment 4 vernv tha/ in experirnentsin which lyophilim/ bcers were in8wed ~. . toqeher with pdine. ' '., p <0.01:. ", P <0.02: ", ta statiainl 3i8nifiwnce. . . . . . . -. . ~~ . . value for sample A and I I-40"l. for sample 0, confirming that beer constituents inhibit the nitrosation of naturally , ocurring proline in beer. __ The influence of increasing concentrations of lyophilized beers (samples A(o D) on the nitrosation of prolinc in vifro alt pH 2.5 is shown in Fagure 2. NPRO formation was reduced with increasing conccntralions of all four samples of lyophilized beer in a non-lincar fashion. At the lowest concentration (equivalent 80 0.4 ml of bcer per ml of reaction medium), inhibition was 28-33%; and, a( the highest eon- centration (equivalent to 6.25 ml of beer per nil of reaction mcdium), inhibition ranged between 92 and 97q.; at in- lermcdiate cont cntrations of lyophilized hccrs;. their in- hibitory effects dccrcascd in the follawing artk•r: It > 1), A > C. AI(hough Ihc differences were relativcly srnall, sampk (' 11:N1 Ihc lowcs( corwttiuratiaN nf total ptdy(slk•Iluls (and an•

. Inhlhili.n of endagemws witruertibn by.Iyupkill2ed lirer notion that polyphcnolics may be predominantly responsible ( for the inhibitory effects, a competitive reaction of other (rapidly) nitrosatable (amino) compounds present in beer, with nitrite, cannot be ruled out. Therefore, experiments in this laboratory are in progress (a) to identify the component(s) in ' 0 75 lyophilizcd beer that are responsible for inhibiting nitrosa- 0 tion, (b) to measure the amount of total N-nitroso com- d poundi (19) formed after nitrosation of beer samples A to D. z As the method is applicablelo human subjects (15), studies t are under way to examine the modifying effect of beer on the 050 ° nitrosalion of proline after ingestion of this amino acid, with ' V vegetable juice as a source of nitrate. Using the same pro- L" tocol, vitamin C and vitamin E have been shown to inhibit Q this nitrosation reaction in one human volunteer (15.20). J 0.25 ¢ Acknowledgements 0 1 2 3 4 AMOUNTOFLYOPHILIZEOBEER. (oq.ml/n>treactionmedium) Iig. 2. InRuence of vairying.amounts of lyophidizod hoer (brands A to D).on the ninnsation of proline in viiroat pH2:3- The aceiys contained 4 mmol/1 xtdium nitrile together with varying amounts (mt orf ber/mt raetimnmafiuim) of lyophilind beers (brands A.to D) in the absence of or with 100 mmol/I prolincdissolved in citric a.idcitrate buffer at pH 2.3. A control assay eamed out in the absence of lyophiliied bar, contained 4 nwnoVl sodium nitrite together with 106 mmoUt proline dissolved in citric acid,eiirate buffer at pH 2.5: NPRO formed (rmol/I)) wass mnsure.d after 15 min of incubation ~ at 37'C.. The differences between the amounts of NPRO (rmoV1) formed in the presence and absence of 100 mmol/I proline are ptated for each beeetested A(O): B(L%): C( O) and D( o): The rdative yickl of NPRO (right-hand ordinate) wu calculated by,taking the yield of the vomrd experiment (absence of lyoph'ilizod beer. Itl) u1. .. ...... _, thocyans). We assume, therefore, that this type of compound may be responsible for the more pronounced inhibition of samples B. D and A, all of which had about two times more polyphenolics than sample C. The effect of lyophilized beers on nitrosation of proline C was also examined in rats in vivo (Tabie 1). The precursors administered were sodium nitrite, proline and Iyophilized beer (specimens A to D). Experiments I-4 served as control experiments; % inhibition of formation of NPRO in vivo was calculated in experiments 5-22 on the basis of NPRO form- cd in experiment 4. As studied in detail for sample D, inhibi- tion of NPRO formation was dependent on the amount of lyophilized beer ingested (experiments 7-10, 13-22). The pattern of inhibition of nitrosation in vivo was very similar to that observed in vitro, although the magnitude of the effects was smaller: all four beer specimens inhibited nitrosalion in vivo by 75-90e1o;'specimen C'again exerted the most feeble inhibiting effect. For the highest concentrations of (he four beers tested; the inhibitory effects were shown to be statistically significant (Pable 1). In conclusion, the results presented in this study demon- slrate that beers containing larger amounts of total poly- phenolics including anlhocyans inhibit nitrosation of proline in vivo and in vitro more strongly than other beers. The in- hibitory effec(s were shown to be concentralion-dependent. In gencral, there was good agreement between data obtained in virrn and in rats in vivo, confirming the results of earlier wudics (10,16). Altlmugh (hcsc data are consistent with the The authomswish to thank Dr Gary Mahoo for advice on the statistical calculations. Mr M.Benard (ENSIA) forr providing and analysing beer specimens. Mr 1.C.Bireiat for the treatment of animals. Ms E.Hesehine for editorial assistance and Ms Y.Gnnjard for secretarial help. TTiswort was supported in part by Contract no.930 498from the.DNfytion Gtntrak I la Reelierche Scientifque et Teetinique (D.G:RS.T.), France. The TEA detector was provided on loan by the National Cancer Inttitute of the United Swa under Contract NOl CP 33 713. References I. Coordinating Committee for Scientific and Technical Assessment of . Environmental Pollutants (1978) Nitnqnfa An Emvirionmeqtal AsFEmwtsnf - NaitonalAadenryofStienoa,.Washitgton,DC. . . .~•r" _ •.2. Doujlass.,M.L, Katiemff.B.L. Arderson.G.A. and Cheng.M.C,p, (1978): The dlenisay of niuosa+oine formaeian, :Jubition and datsuninn, .;'A J. Soc. Cosrnef. Ctienr.. 29, 381,G06. - 3. Gny.lil- and DuBan,LR. (1973), Inhibition of N-nitrosamine formation - in tnodd %od'systerns, J. Food 3r.i., 40. 981-961. 4.. Davies,R. and McWeeney;D.J. (1977), Catalytic effect of rc(rophenots onN.nitrosamineforenacion,Natsve.266,657-65tt .,. ,. ' S. Davies.R.,Dennis,M.1.,Massey.R.C.andMeWeeney,D.J.(t978),Sooe effects of, phenol and thid-nitrosatioo on N:nitrosamine forrnation, in Walker.E.A., Cattegnaro,M., Griciuae,L and Lyk,R.E. (eds.), Pnviroe- menrof Asperu ojN-Nifraro Cornpoundt /URC Scienryle Plrbficmions Na 191, International Agency for Research on Cancer, Lyon. France, pp. 183-197. s. walker.E.A., Pignaoellt.e. and Cas,egnaw,M. p979). Catalytic effect of --" pnitrosophenol on the.nitrosation of dialhylamine, J. ASric. Food. CLem., 27, 393-396. . . . ., , -~ 7.: Pignaldli-B., Friesen,M.D. and Wa1kv,E.A. (1980).The rok of phawls . in catalysis of nNrosaminc formation, in. Walker,E.A., Gni~iute,L,. Casteg- naro,M. and BOrssOnyi,M. (edi.), N.Nitrmo CorrrpoundW Analyst; Forrrra- don and Orsvnrnoe (URC Sctientirir A.bllriotionr No. 31). International Asency..for Rooreh on Canoer. Lyon,Franoe, pp.93-109... S. W alker.E.A., PignatelG,B. and Friesat,M. (1982).'IBe role of phenoh in . aulysis of niorosamine formation, J. Sri. Food Atx-, 33, 81-88. V 9. Pignatelti,B., Bhlziat,J.C.,O'NeiI1,I1K. and Banich,H.(1982),. O Catalrtic role of~some phenolic substanees in endogenmu formation of N-nitroso compounds, in Bartsch,H, O'Nei1t,1.K., Castegnaro,M. and Okada.M. (eds.), N-Nhro.so. Compounds: Occurrener and Biolosirnf £f- OJecrr ffARC Scienry/ic ptrbfecarionr No. I11, International Agency for O Research on Cancer, Lyon, France. pp. 413-e23. ,_ 10. Pijnatelli,B., Bhlxiat,l.C., Descores,G: and Bansch,H..(19821. ~. Catalysis of niirosation bs vitro and in sivo in rats by cataciiin and resorcinol ,, and inhibition by chlorogenic aeid, Careinogenesir„3, I01S-1049.. r~. (1. Sticfi.H.F.. Rosin,M.P. and Bryson.L (1982). Inhibition of mutagenici- _ 2 ly of a model nllrosalion reaction by naturally oRTnring pltenoaa3, noffee and 1 ~ Ia, Mwat. RQ., 95, 119-1 7l. - 12. Bogovski.P.. cauegtwro,M., Pwweli.B. and Walker.E.A. ( ~ The inhibiting effea of urmins an the formation of nitmsamines, in ski,P.. Preutsmann.R.. and Wdka,E.A. (eds.), N;Kareroa Co+nporrndt Gt1 Analyyir andl formation ffARC Srienrj~rc Publitmdans No. J1. pp. 127-129 l. 13. . Nakamura,M. and Kawabata,T. (1981), Effect of.Japanese.green tea aa airrosaraine formation in vdio, J. Food Sri., rk, 706)07. - 14. Dadic,M.. Van Ghehrws,l.E.A...and Valyi.Z. (19M). Cltmuta6 constf- ,..r :". _. tuents of NyIon•fi6.beer adsorbate. 1. Prdiminary tnvaztigatitxn and annw ey.rmtbsn earems, J/na. Breuirg. 7r+, 2G7-2g11..

rti~Pli . ~ .~;~. R. W rnaelnt rt af. . 16., Ohshinn,H., Ifhtziat,J.C: anJ: Ifartseh,H. f19g~2). Mhnilnring nnlK, f1MTrkr.r., 41. J6SI(-)662. IS.. (RiJtlinw,ll. and Ua.nech,H:..(19g1). Ouamitalivc utimatinn of en. Jnganws nitnxation in man Ay moniuxing N•nornsn(woline eMrtaal in the N•nunnamimr a.ida e.cretal in the urine and ifttrs of ran as% an imfa of en- .. rf~1f IWMTNNlIIS No.. 91,. Internalional Agency fa Researdi on Camn.. Walka,E.A.(ed..).N-NibratoCornprwndrinrheFnvironnrenrffARCSiirn- uf total na~v.datik nitlrasamines in microgram amounts, in Dogovski,P. and I t9. Wallicn;C.l... I-'ueggk.D.G. and Lunt,T.G: (197q, The dctnminaticm 1g. IX Clerck,l. (1762). Cirus rk. Hrnc.eriR Vol. 2. pp. 61gb39. .l'uiv.K rk la. Itrasserie, 7.urKh, pp. U'63 (7.7); . ,-17.. liurqran.UreweryCawentiom((97S): m AnirIMYY. Jnl fdiIMMr, RCYUe ' ..1h1/tllMMlx nltnl.NalMm, CkNlintwYl", 3. Hf•12t1. Vimmin C/Asnrbir. Arir1l AnnlicJ'Scinkr Publichers. Lominn nnd New in riwr foxnutian of nitrosamines, in Counse11,1.N. and. Hornig,D:H: (afs.h ' 21/. (Mnhimm.H. andlllart*dr.H. (19g1). The innueke of vitamin C on the ;`I.ynn. Frake. (t(r..22-25. 't.ct~,`'` _ '. Jcrsey. 1kP. 213-224:.. ; j1 '