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HT60120112 I i Reprinted from O.iginr of .ffuman Canen it 19" Cold Spnng Harbor Laboratary t Genetic Regulation of Susceptibility to Polycyciic-hydrocarbon-induced Tumors in the Mouse R.E.Kourf Department of Biodiendeat oneotoay MICroulologlcai aeaoetdteu Botltoede. elarytantl 20016 D. W. N~ l7drmoomemst Phermacobqy Braneq Naeonfll Inadtute of Child Health am Human Dovelopment National U1lntutee ef Fleelth, BBtrt9Nde. Maryland 2007A Recent epidemiotogic data have creauid a growing concern that environmental chemicals may be responsible for a considerable proportion of all htsman cancer. The purpose of this paper is to provide a curreat review of the mem- brane-botmd monooxygenase systems which metabolize most environmental caecinogeas, to deseribe in detail the regulation of some of these enzymes in the mouse experimettital model system. and to demonsttate an association be- tween the mtuine Ah locus and +: increased susceptibility towatd chemical ttmtorigeoesis, drug toxicity. and mutagenesis. The possible extrapolation of some of these results to the httman, with respect to chemical carcinogenesi3. will also be dscussed briefly. TN8 MoiYOOXYSiENASB sYSTHMs vumeroua xenobioncs. including many chemical carcinogens. am so hydto- phobic that they would remain in the body indefinitely were it not for Phase-I and Phase-II dtug-metaboli7ing enzymes ( Williams 1959). During Phose-I metabolism. one or more polar groups (such as bydroxyl) are introduced into the parent molecufe. thereby presenting the Phasedi coajugating eozvmes (e.g•. UDP gluctnonosyl traosferase) with a substrate. The conjugates are slt®ciently polar to be exereted from the cell aad from the body. The clnoehrome P--t50-mediatedt monooxygenases are a coUeetive eit- t The eomettclatuev for the variouf torms of .ytoehrome P-t50 is currently medeqnete coroidering teat four or mwa foran AM d[umeuiehable by eteeavpt+oredc ( Hsugeu'as el. 19761 or uomunaettemical f'firomas•et•a6 1976) roehrt kquat. The e•entyaI bettet an- dersrandiag of ehemieal and eatdpic properties (Ryan eCii. 1975: Hauaes?'et A 1975) thould. in m+a permmit a aaaer suimbte eomeacitume to be deWsed. TLe Smet ttnudincm of tbe reduced hamopro[ein.CCL-ampkx from tlver mietoaomn is very etoae rosqq am for cytoChtome P-tsli i Ryan eflat,I97e1. bu6 hvw nanhapatk tiwn i Poiaed V at. 19"a1 and tufne cdturp i l)mena ad,PVeben 1975n1. it rangp between 414 and .ta6 am folloanoe polyeyeGc hydroearbon Pte 'Meat. For these reasom. we preter P-448 for liver mteroaomes and P,aS0 for noohaparic ussuee and aB-eultnm experimeou. Since this rcport deals pnncnpally with tumors m luag and stm. the nomenelattre ':ytochrome P1. a30" is used dhrourbout. % I /77

H 190120113 / \ 812 li, tC KouM and o. Y/. Nooort ample of Phase-1 enzymes. These membrane-bound multicomponent enzyme systems, requiring molecular oxygen and NADPH (Mason 1957), ate known to metabolize pofycyclic hydrocarbons, such as benzo(a)pyrene (BP) (ubiquitous in city smog, cigarette smoke, and charcoal-cooked foods), 3- methylcholanthrene (MC). and btphenyl; halogenated hydrocarbons, such as polvchlorioated and polybrominated biphenyls, iasecticides, and ingredients in soaps and deodorants; strong mutagens, :uch as N-met6yl-N'-nitro-N-aitro- sognanidine and nitrosamines; aminoato dyes and di.aw compounds; N- acetylarylamines and nitrofurans; numerous aromatic amimes, such as those found in hair dyes. nitro aromadw. and hetetocyclics: wood retpeaes: epoxides: carbamates; alkyl halides; safrole derivatives; certain fungal toxins and andbiotics; many of the chemotherapentic agents used to treat human cancer, most drugs; both endogenous and synthetic steroids; and other endogenous substrates, such as biogenic arnines, indolea, thyroxine. ethanol, and fatty acids (see Jerina and Daly 1974; Sims and Grover 1974; Heidel- berger 1975; Nebert et al. 1975; Thorgeirsson and YebeR 1977. for taent reviews). It is now generally accepted that during the detoxification of these com- pounds by Phase-f and Phase-II ettzymes, potent reactive intermediates, capable of binding covalentlv with cellular mactomolecules, can be formed. The association of the steady-siate levels of certain reactive intermediates with cancer, mutation, and toxicity is presendy of great research interest. THE Aft LOCUS Induced Alonooxygonasle AcNvltles AaBooiatad with Cytochrottt® P,-4Sa Induction Thete is now immttnological evideece in rat liver (Thomas et al. 1976) for at least six different forms of P-450. One of thtse forms. cyoochrome P,-00, is induced by polycyclic aromatic compcunds in B6= and other responsive mouse strains, but the induction of t5is form of cytochrome by polyeycac aromatic compounds is absent in liver and markedly decreased in lung, bowel, kadney, lymph nodes, sldn, bone marrow. pigmented epithelium of the eye, and ovary in D2' and other nonresponsive mouse strains (reviewed by Nebert et aL 1975; Thorgeirssoa and Neben 1977). This "tespoasive" ness" to aromatic hydrocarbons was designated (Nebert et a4 1972a; Greea 1973) the AAt locus: the alleles and AM Ah• denote the 116 and the D2 inbred strains. respeetively. Numerous studies (reviewed by Nebert et al. 1975) indicate that an im- portant product of the Ah (tegulatory) locus in mice is a cytosolic teceptor (Poland et aL 1976) capable of binding to certain polycyclic aromatic in- ducers (Fig. 1). Such a complex in some manner activates the structural gene(s), thereby leatiing to increases in enzymes that tnetabolisa these in- = The C9'BL. 6N. CSTBL. 61. and CJ7BL. 6Cam inbred mowe srcnm used iotes+ chanseablv. • The DBA.:Y, DBA. J. and DBA.'Cum inbred strams used mterehnngeably. I \

~-~------ ~- - -- OeneNG 8uoeepabllltr to Oaneer and the AA Lotaro ata H T® 0120 114 ( Rl+Ctmd Ir4CTrve o+TERMa1Isi[8 N100uCTS I CANCtR MUiAGfNLSB ioRiCnr eIRTN OBrCTS FTgurri 1 Simpli8ed scheme demonsuatprg the relationship of the Ah locus in the mouse to cancer. mutagenesie. twucity. and otrth defeces. ducers (and other polycyclic aromatic nonidduciag compounds). In addiaon to innocuous products, reactive tnetaboGtes may also be getterated. Induction of one or more forms of rytochrome P,-450 is associated with the induction of the numerous monooxygenase activities listed in Figure 2. How so many substrates with wtiry different eh,etrtical structures can be oxygenated by a single enzvme active site is not understood. One likely possibility is that thete are several (or many ) forms of cytochrome P,-tS0. It is apparent that different forms of P-t•50 generate different ratios of metabolites from the same substrate. For example. comparing MC versus phenobarbital as tt e inducer in rat liver. various groups have shown that hy- droxylatious may occur predorninanrlv in different chemical posidons on the molecule for such substrates as biphenyl. testosterone. =-acetylamino9uorene, bromobeatena n-hexane, and benm(a)pyrene (see Nebert and Felton 1976; ThotBeirsson and Nebert 1977, for fttttl"er discussion). Such differences in the metabolite profile of a polyrycUc hydrocarbon or other foreign com- pound also stout thrt important differences may exist in the nature of the iotermediates formed. DiHerences in the reat:dviry of these intermediate; might tberetore result in marked dissimilarides in the toxicity or careittogs- nicity of a given compound. Recent examples in the rat in which this hypothesis appears to be the case include bromobeaxeae toxidry (Zampaglione et al. 1973) and high blood pressure (Rapp and Dahi 197G). The relationship between geaeacally regulated differences in cytochromes P-t50 and stts- ceptibiGty to chemically induced cancers. mutaqenesis. and toxidty in the mouse is discussed in the remainder of this paper. ~

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HTE0120-116 t7saatlo 9uceepttbllltp to Caneier and the Ah d.oew ala \ AHHI Induction as an Indicator ot Phenotype at the Ah Loctro Survey of lnb.ed.Yloute Srrainr The AHH assay (Fig. 3) Lc a reliable. simple, and very sensidve assessment of aromatic hydrocarbon responsiveness following treatment of animals with polycyclic hydrocarbon inducets. Using AFDt induction as an indicator of phenotype at the Ah locus, several laboratories have found (Table 1) that about half, or slightly more than half, of all inbred mouse strains examined a-a responsive (as are wild taice, random-btod mice. and about 20 inbred strains of tats tested [unpubLl), and the remaining mouse 3trais,s ate tton- respousive. During the past 60 or 70 years of developing these inbred mouse strains, there has evolved asable mutation whereby certain strains lack (either quantitatively or qualitatively ) tae gene product of the A h loctts, the cytosolic receptor molecule (PolandetaL 1976). Diferenr Types of Genetfe E:presston Induction of AHH activity and cytochrome F',-t50 by MC is expressed almost exdosively as an autosomal dominant trait among otfspring of the appropriate crosses between B6 and D2 inbred strains (Nebert and Gielen 1972; Nebert et aL 1972b; Thomas et al. 1972; Gielen et al. 19,72) and as an additive trait among offspring of the appropriate crosses between the C3a and D2 inbred strains (Thomas and Hutmn 1973; Robinson et al. 1974). On the other hand, the tack of induction of AHH activity and P,-450 by MC is expressed as an autosomal dominant trait among offspring of the appropriate crosses be- tvveen C37BL/6N and AKR,'N parent saains ( Robiason et aL 1974). The simplest gertetic model to explain mostL but not a0, of the data indudes a minimum of six alleles and two loci (Robinson et aL 1974; and reviewed by Nebert et al. 1975). However. for all intents and purposes, we may re- qatd genetic expression at the Ah locus in offspring from the appmpriete crosses batween B6 and D2 strains and between C3 and D2 strains, respec- tively, as dominant and addidve. Secause of the di.ainct phenotypes generated in progeny from the F, x D2 backcross and the F: geaeration, we can evaluate the possible importancr of steadv-state levels of reactive intermedimes in the mechanism of chemically induced catcinogettesis, mutagenesis, or Plgtav 2 Chemical eeactiom reptoemutg induced monooxygemse "aetivities" atr sociaoed with cytochrome(s) P,-t50 induction and the Ahe a0ele in the mouse. Nca shown is UDP gluctuonosyl nnsferase activity with 4qrethyl- urnbetliferone as substrate (Owens and Vebast 1973b), a membrane-bouad. metabolically cootuinated enayme whose induction apptmes to be closely correlated Wtth the Ahb auele. Only the substrate and the maJor product am shown. The activities are lieted in chronolog;tml oMar of publicat(on ( Nehert and Gielen 1972; Nebert et aL 197J; Robinson and Netierr 1974: Thorgeys- son et aL 1975; Atlaa and Nebart 1976: Burke et aL 1977; T,Sorgeiraaon et al., this volume). + Arpl hydroarbon (bmzofalpyrenel hydroxqla,e (EC 1.14.1s.2). SThe C3H,HeN. C3H/HeJ. C34i.fMai. and C3HjfC1Nm inbred strains used inter. cttangeably.

H T2012011' ( 816 R. E. xoert end D. w. Ner,.n BEN20(ajPYRENE NAOPM I NADM Meer 02 MICROSOMES DtMY0R00qLS ~ ~~OUC aENZO(a]PYRENE QUINONES tyNVDROxY8E~1ZO(a)PYRENp PQLYHYOROxY ctnerutidtTED v+tooutnTs tiOVALENTLY BOUND tRguro 3 Current coocept of the A-HH activity. The substrate BP is oxygenated to arene oxides which rearrmge nonen,zymically to phenols or are oxygenated to phenolic derivatives by direct oxygen insertion (see lerina and Daly 1974: !Vebert et al. !975. for further discussion). The 3- aed 9-pheools have the strongest fluorescence in alkali (llolder et a4 1975). Other oxygenated denvanves of BP. including dihydtodiols and quinones. are not measured by this assay. One unEr of AEIIi activity is de9ned (Neben and Gielen 1972) as that amount of enzyme catalyzing per rnia at 37°C the formation of hydrox. ylated product causing fluoresxnce equivalent to that of I pmole of 3- hydroxybenxolalpytene. (Reprinted, with parmiasioa, from Nebett aad Felton 1976.) toxicity among siblings in the satne litter or among individuals sharing the same uterus. This genetic probe is a particularly powerful experimental model system in the research ateas of pharmacology, toxicology, teratology, and chemical carcinogenesis because the test compounds studied often cause undesirable side effects (e.g., sedation, malnutntion. hormonal imbalance) that ate hard to distin,;,iish from specific pharmucologie, toxicologtc, or carcinogenic eBects of the compounds. Simifarftp in Genetic Espretsioh between Liver and Otker Ttrsues The genetic regulation of an induced enzyme in one cissue need not be the same as that in other tissues. Hoarever, the induction of AHH, and several other monooaygenase activities as well (Poland et al. 1974; Nebert et al. 1975), appears to have similar genetic expression in all tissues examined (Gielea et el. 197?; and reviewed by Thotgeirsson and Nebert 1977).13ere ate difi1itatlties in a ve.q cate:fel gtmetit analysis of this point bccause most responsive "control" mice have, in fact, slightly induced AHH activity In many dstues, which can be lowered by changing the diet (Wanenberg 1975). Hence the slighdy higher hepatic and pulmonary 4FIIi activities seen in the responsive "rnntrol" B6 and C3 mice (Fig. 4) may rtflect the enzyme ac- tivities slightly induced by environmeatal factors. One can see in Figure 4 that induced .kHH in ( B6D2 ) F, liver or lung is

a--- - _..~ -_- H T ®01201 1 E ( Gamtia Suseptl6alty to Canar end tua AN I owti att' Tabh I Determmatioo of Geeedc Reapoe5iveeess or vonrespooarveoess at the Ah Locas in Various Strains and Substram ot the Houaa Mousa.Nus+eurcrdos Retponave Non- (nered rcsponsivr srrmnr serotns ReMenee C37BL/6N DBA/2N Nebert and Gielm (1972); Nebers-et aL (1972b) C37BLJ6J DBA/7J ibotaae et al. (1974) CS71JJ DBA/ 17 C3H/HeJ AJCR/J DF.II 1mJ BALB/eJ RF/ J CBAlJ LP/J A/J ST/bJ A/HeJ SiL/J SM/J SWR/J PG'1 AiU/Ssl SBC.' ) ReJ NZW/BLN Gieleaetal.-(1972) NZB/BLN C3Hi HeN AKIt/ N Nebert et al. (1973) BALB/cAeN BRSUN't'IN CBA/NN A/HaN STR/N NH/LwN P/JN AJJN C57BL 6CUm Kouti et al. I 1973b ) C3H/ tMai B10.BR/J C58i1 BALB/cCr CS7BL/ loScSa SEA/Gal C3HeB/FeJ C3H/ IiCum AKXL-t8A DBA/2Cum Kouri ec al. (1973a) NZB/ BLNJ Poland and Cilover (1975) Koui(1976) AdCXL-38 Naugea ec al. (1976); Atlas et al. (1976b) sligltlly less than that in 86 liver or lung. '11se apptoxianatdy 50:50 bimodal disuibudon among (B6D2)F1 x D2 offspring and approximately 25:75 oonrespoosive:respoasive bimodality among the (B6D2)Fs geaeration can be seea clearly in both liver and lung of MC-treared anima6. The additive eapsesdon of AHH induction is seen in the liver (and lung; data not illus- traoed) of (C3D2)F, individuals; hoaever, the approximately 50:50 iater- mediate:high bimodelity among the (C3D2)F, x C3 ofsprin•g, the ap-

~-------- --------_:.-...-----==_... .~._---- HTM0120119 N.66 ~ ps t.0 1.5 10 23 unrts AHwa WET wBCNr ~ -- -'N~ABI F, 1 -_AI,_ :a'Sb i Fa841 _-Am- Nr3Bi MC- ®5 TRFJITED 02 uvsR CONTROLJ ee _ & 1 02 1 rLN~S1 75 LUW G CONTRO 55 N.~o m N-a MC- e5 "t-I TREATED ~ H.a F, . 9e N°te 02 0 R.03 N~ I 0 600 t.Z00 18m 2a00 ] G00 NMRS ANWaq NpCR090tw1L Fnal'6N 0 ts 70 a e0 VWM AHM¢ WW WW -- _ ~ N-ae I ~ F. . N-G, F,+q ro~l l _ I F. { - y t y-M l o wo tiD t.5C0 2,4011 SC05 wWrs iueveq Naat09oMlu FNOtEN ~ 0 t5 70 4 79 UNIfS aNNta W!T vY[IOKr ROUM $ Genetic variattoe in hepadc (rop) and pulmonary (+mEddle) AHIH activity in co5trol and MC-+reated o05pring from approprvate ctostes betweea B6 and D2 inbred strains and hspatic AHH activity (boao.n) ia cnetrol and MC- ttuated o8:aprmg from apptopriete ctcmes between C3 and D2 inbred straine. H3,togtanm for liver sampla r5presen t specific AHH activity in coat[nl mice and in mice treated iotrapedtooeaUy 24 hr beforehand nrith NC (100 mg/kg body avt): •coanol9 readved cors oil introp5ntoneaUy. For lung samples. the mice received inttattachea)ly. 24 hr beforaband. 300 gg MC. vA 10 Ed of 0.2% gelatio--0.8S°.6 NaCI: controls received the sterile vftde a)one. The mice weighed between I S and 20 g. The number of mice es5mined in. dh7daally is gtvaa at the right for eacb gr.oup. BIG l

i H T®0120120 Genstlo Sueeepdb111tp lo Cancer and the Aa Locus 919 LUNG ~ --do DAYS AFTER SINGLE TREATMENT OF OTAATR/4CMEAl_ NMC Flqere S Kineties of hepauc and pulmonary ANH induction followiag inaravaeheal admimatranon of MC. 500 pg in 20 r•l of 0.2% gelatin-0.8345, NaCI, to B6. f86D2)F,. and D2 mice. The mice weighed between 15 and 20 g. Fach symbol reprwsents the maan of four animals determined individually. (For further dmils. see Kouri et al. 1976.) proximatel,v 50:50 low:intermediete bimodality among the IC3D2)F, x D2 offaprmg, and th: approumately 25:50::5 low: intermediate: high trimodal distribution among the (C3D2)F_ generadon that can be teen easily in the liver is not easily demonstrable iu, the lung because of relatively small diI !erenoes berween these groups and relatively large variation among pulmonary AEIIi( in these MC-treated animals. The differences in hepatic and oonhepatie induction of AFIIi activity (and presumably crnxhrome[s] P,-4S0) are apparent among B6, (B6D2)FI, and D2 mice as a functioo of both time (Fig. 5) and dose of inducer (Fig. 6). A responsive polyrydic-hyd[ooarhon-treated mouse is therefore subject to both quantitative and qualitative increases in the steady-state levels of certain raastive intermediates because ef both an increase in cytocheome(s) P,4.SO content and an increased P,-4S0/P-150 ratio in numerous tissues. The relative contiant of P,-4SO, compared with other forms of P-ASO. may be especiaBy large (e,g., ratios of 10:1 or S0:1) in tisstte.a such as skin and lung but never n.aches even a 1:1 ratio in liver (ICahl et al. 1976). This relatively large change in the profile of cytochrome(s) P-4S0, compared avith smaller increments of chenge in epoxide hydrarase (Scbmassmann et al. 1976), UDP glucu,toaosyl nansferase (Nemoto and Gelboin 1976). and epoxide-glutathione S-transfetase (J. R. Bead, J. S. Felton. J. N. Ketley, W. ). Jakoby and D. W. Nebert unpubl. ) activities with BP as substrate, might be a factor in explatmng why polycyclic hydrocarbons cause tumors in sltin and lung but rarely in liver.

oM~ SOC- SOo- _yEa ! 4 zoo~~ - .oo ~ - 3 - so.--, 8!0 ~ Pgne e Spedfio AKi activity in Inrar. kidney, bowe6 and lung of 86. ID2. or (B6D2) F, mice ea a(nne- tiun of the dosage of P-oapliltfio- flavotte. Braeken deeote standard devietiod Esch symbol mpriosiats ehe meaa of Bve or, six individual determiaatiotu per gtoup. All raice und were iounatum females of idenacal age. .UI detetmioa- eiooa of atzyme aet rvity in a given twme were perfurmed in the same usay on the tame day. (Re- ptioted with permission. from Viwa et al. 1975.) H1®0120121